UNIVERSIDADE FEDERAL DE MINAS GERAIS INSTITUTO DE CIÊNCIAS BIOLÓGICAS DEPARTAMENTO DE MORFOLOGIA PROGRAMA DE PÓS-GRADUAÇÃO EM BIOLOGIA CELULAR

Transcrição

1 UNIVERSIDADE FEDERAL DE MINAS GERAIS INSTITUTO DE CIÊNCIAS BIOLÓGICAS DEPARTAMENTO DE MORFOLOGIA PROGRAMA DE PÓS-GRADUAÇÃO EM BIOLOGIA CELULAR TESE DE DOUTORADO PROLIFERAÇÃO E MORTE CELULAR DURANTE A FOLICULOGENESE DE Prochilodus argenteus DA BACIA DO RIO SÃO FRANCISCO. RALPH GRUPPI THOMÉ Belo Horizonte 2012

2 Ralph Gruppi Thomé Proliferação e morte celular durante a foliculogênese de Prochilodus argenteus da bacia do rio São Francisco. Tese de doutorado apresentada ao Programa de Pós graduação em Biologia Celular da Universidade Federal de Minas Gerais, como requisito para a obtenção do título de Doutor. Belo Horizonte Instituto de Ciências Biológicas - UFMG 2012

3 Esta tese foi realizada no Laboratório de Ictiohistologia - Departamento de Morfologia e Centro de Microscopia Eletrônica (CEMEL) - Instituto de Ciências Biológicas da Universidade Federal de Minas Gerais. ORIENTADORA: PROFA. DRA. ELIZETE RIZZO CO-ORIENTADOR: PROF. DR. NILO BAZZOLI COLABORADOR: DR. YOSHIMI SATO APOIO FINANCEIRO E INSTITUCIONAL: - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); - Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); - Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG); - Convênio CEMIG GT CODEVASF.

4

5 Aos meus pais: Ronaldo e Eliene.

6 Não há nada como um sonho para se criar um futuro. Victor Hugo

7 AGRADECIMENTOS Aos meus orientadores, Profª. Dra. Elizete Rizzo e Prof. Dr. Nilo Bazzoli pela paciência, confiança e orientação na minha jornada científica, e principalmente pela amizade; Ao Prof. Dr. Yoshimi Sato, pelos ensinamentos transmitidos e contribuição na obtenção do material biológico; Ao meu amigo e grande parceiro de pesquisa Fabrício Flávio Theóphilo Domingos; Aos amigos do Laboratório de Ictiohistologia, por tornarem os dias mais agradáveis e pela grande contribuição para o desenvolvimento deste trabalho, em especial a Mônica; À coordenação do Curso de Pós-graduação em Biologia Celular, na pessoa da Profª. Dra. Denise Carmona; Aos professores do Departamento de Morfologia, em especial a Profª. Dra. Gleydes Gambogi Parreira; Aos professores do Curso de Graduação EAD - Ciências Biológicas; Aos amigos da Biologia Celular; Aos amigos do Programa de Pós-graduação em Zoologia de Vertebrados PUC-Minas; Aos amigos da EAD-UFMG; Aos amigos e companheiros da época da graduação na PUC e UFMG; Aos amigos da Universidade Federal de São João Del Rei, Prof. Dr. Hélio Batista dos Santos, Profª. Dra. Rosy Iara M. Azambuja, Prof. Dr. Stenio Nunes Alves e Prof. Dr. Rodrigo Ribeiro Resende; Aos amigos do glorioso time de veteranos do curso de Ciências Biológicas-UFMG; À minha amada família pelo apoio incondicional; À Ludmila, pelos momentos de ajuda, compreensão e principalmente pelo carinho; A Deus, por permitir que eu pudesse acertar e errar junto com todas as pessoas que passaram pela minha vida.

8 O passado é história, o futuro é mistério, e o hoje é uma dádiva. Por isso, é chamado de presente. Provérbio chinês

9 SUMÁRIO Resumo...i Abstract...iii Lista de abreviaturas...v Lista de tabelas...vi Lista de figuras...vii 1- INTRODUÇÃO E JUSTIFICATIVA DESENVOLVIMENTO DOS FOLÍCULOS OVARIANOS REGRESSÃO OVARIANA APÓS DESOVA APOPTOSE E PROLIFERAÇÃO CELULAR O RIO SÃO FRANCISCO ESPÉCIE DE ESTUDO OBJETIVOS OBJETIVO GERAL OBJETIVOS ESPECÍFICOS MATERIAIS E MÉTODOS AMOSTRAGEM HISTOLOGIA TUNEL IMUNOFLUORIMETRIA INDIRETA IMUNOHISTOQUÍMICA WESTERN-BLOT TESTE COLORIMÉTRICO MORFOMETRIA ANÁLISES ESTATÍSTICAS RESULTADOS ARTIGO PUBLICADO: DISTRIBUTION OF LAMININ Β2, COLLAGEN TYPE IV, FIBRONECTIN AND MMP-9 IN OVARIES OF THE TELEOST FISH. 20

10 4.2 ARTIGO PUBLICADO: APOPTOSIS, CELL PROLIFERATION AND VITELLOGENESIS DURING THE FOLLICULOGENESIS AND FOLLICULAR GROWTH IN TELEOST FISH DISCUSSÃO CONCLUSÕES REFERÊNCIAS BIBLIOGRÁFICAS ANEXOS ANEXO I: DUAL ROLES FOR AUTOPHAGY DURING FOLLICULAR ATRESIA IN FISH OVARY ANEXO II: AUTOPHAGY AND APOPTOSIS INTERPLAY DURING FOLLICULAR ATRESIA IN FISH OVARIES: A MORPHOLOGICAL AND IMMUNOCYTOCHEMICAL STUDY

11 i Resumo Estudos sobre a reprodução de teleósteos fornecem informações básicas sobre o desenvolvimento de gametas e possibilitam o melhor manejo de populações de peixes em ambientes naturais e em sistemas de cultivo. A formação, o crescimento, bem como a involução dos folículos ovarianos após desova são acompanhados por remodelação da matriz extracelular tanto quanto por proliferação e morte celular programada. Essas alterações morfológicas podem influenciar a maturação gonadal e a esteroidogenese. O presente trabalho teve como objetivo analisar remodelação da matriz extracelular, a proliferação e a morte celular durante o desenvolvimento e a regressão dos folículos ovarianos de Prochilodus argenteus. Para isso, o estudo foi realizado em duas etapas. Inicialmente, avaliou-se a foliculogênese de peixes mantidos em cativeiro e submetidos à desova induzida por hipofisação. Durante a involução dos folículos pós-ovulatórios, observou-se a queda dos níveis plasmáticos de 17β estradiol após a desova que pode ter contribuído para a intensa apoptose de células foliculares. Pela análise imunohistoquímica, laminina β2 e colágeno tipo IV foram identificados como os principais constituintes da membrana basal. A perda de integridade da membrana basal devido à lise dos seus principais constituintes coincidiu com o aumento da apoptose folicular. Os resultados indicaram que a enzima metaloproteinase da matriz 9 está envolvida na maturação final e na involução dos folículos pós-ovulatórios. Na segunda etapa, avaliou-se a proliferação e morte celular durante a foliculogenese, além da vitelogenese de P. argenteus capturados em dois trechos do rio São Francisco: trecho 1 (S1) logo após a barragem de Três Marias, e trecho 2 (S2) na confluência do rio São Francisco com o rio Abaeté. Tanto o desenvolvimento dos folículos quanto o sucesso na reprodução e a frequência de atresia folicular são influenciados por fatores ambientais que atuam no eixo hipotálamo-hifófise-gonada. Em S1, baixos níveis de oxigênio dissolvido foram registrados no período chuvoso. A temperatura da água foi maior em S2, independentemente do período analisado. Em S1, os ovários continham ovócitos menores, alta atividade enzimática de caspase-3 e apoptose, baixos valores de IGS e PCNA, como também uma menor expressão de catepsina-d, quando comparados às fêmeas capturadas em S2. No período seco, a análise da freqüência relativa das

12 ii estruturas ovarianas mostrou apenas ovogônias e ovócitos perinucleolares em peixes de ambos os trechos. Por outro lado, no período chuvoso, a frequência relativa de ovogônias e ovócitos perinucleolares diminuiu e dos ovócitos vitelogênicos aumentou em S2. Folículos pós-ovulatórios foram observados apenas em S2, enquanto folículos atrésicos ocorreram em maior frequência em S1. Em conclusão, nossos resultados sugerem que a estrutura e composição da matriz extracelular, e os níveis plasmáticos de 17β estradiol estão relacionados à apoptose, e desempenham papel importante durante o desenvolvimento folicular e recuperação ovariana pós-desova. Além disso, mostraram que a apoptose, proliferação celular e catepsina-d podem ser utilizados como biomarcadores de impacto ambiental. PALAVRAS-CHAVE: Prochilodus argenteus, foliculogenese, matriz extracelular, apoptose, proliferação celular, vitelogenese, rio São Francisco.

13 iii Abstract Studies about of reproduction of teleosts provide informations about the understanding of the development of gametes and tenable better management of wild populations. The formation and growth of the ovarian follicles and the involution of postovulatory follicles are accompanied by changes in the extracellular matrix, proliferation and cell death. These changes may influence the follicular maturation and steroidogenesis. The major goal of this work was to analyze the extracellular matrix remodeling, proliferation and cell death during development and regression of ovarian follicles of Prochilodus argenteus. The present work was performed in two steps. Initially, we evaluated the folliculogenesis of fish kept in captivity and subjected to induced spawning. The drop in plasma 17β estradiol levels after spawning could be contributed to the intense apoptosis observed during postovulatory follicle involution. By immunohistochemical analysis, laminin β2 and collagen type IV were identified as the major constituents of the basement membrane. The loss of integrity of the basement membrane occurred due to lyses of the major constituents, and coincides with increased follicular apoptosis. The integrity of the basement membrane could be important for the survival of follicular cells. Furthermore, the matrix metalloproteinase 9 results suggest that this enzyme is involved in final oocyte maturation and regression of postovulatory follicles. In the second phase, folliculogenesis of P. argenteus was evaluated by quantification of ovarian structures, proliferation and cell death, and the vitellogenesis of fish caught in two sites of the São Francisco River, site 1 (S1) after the Três Marias dam, and site 2 (S2) at the confluence of the São Francisco River with the Abaeté River. Development of ovarian follicles, success in reproduction and frequency of follicular atresia are influenced by environmental factors that act in the hypothalamic-gonadal-hypophysal axis. In S1, low levels of dissolved oxygen were registered in the rainy period. The water temperature was higher in the São Francisco River immediately after the confluence with the Abaeté River (S2), regardless of the period. In S1, the ovaries showed smaller oocytes, high caspase-3 enzymatic activity and apoptosis, lower GSI and PCNA expression, as well as a lesser quantity of cathepsin-d when compared to females captured from S2. Regarding relative frequency of ovarian structures, in the dry

14 iv period, only oogonia and perinucleolar oocytes were found in fish ovaries from both sites. On the other hand, in the rainy period, the relative frequency of oogonia and perinucleolar oocytes decreased and the vitellogenic oocytes increased in S2. Postovulatory follicles were observed only in S2, whereas atretic follicles occurred at a higher frequency in S1. In conclusion, our results suggest that the structure and composition of the extracellular matrix, and plasma levels of 17β estradiol related to apoptosis, play an important role during the follicular development and post-spawning involution in teleost fishes. Besides, our results showed that evaluation of apoptosis, cell proliferation and cathepsina-d can be used as biomarkers of environmental impact. KEYWORDS: Prochilodus argenteus, folliculogenesis, extracellular matrix, apoptosis, cell proliferation, vitellogenesis, São Francisco River.

15 v Lista de abreviaturas 17β-20α DHP - 17β, 20α dihidroxi-4-pregnen-3-one BMP15 - fator morfogenético de ossos 15 Cat-D catepsina-d DA dopaminérgicos DAB diaminobenzidina DNA ácido desoxirribonucleico E2-17β estradiol FSH hormônio folículo estimulante GDF9 - fator de crescimento e diferenciação 9 GH - hormônio do crescimento GnRH hormônio liberador de gonadotrofinas GV vesícula germinativa GVBD quebra da vesícula germinativa IGF - fator de crescimento semelhante à insulina IGS índice gonadossomatico LH hormônio luteinizante MIS - esteroides indutores da maturação MMP - metaloproteinase da matriz MPF fator promotor da maturação PAS ácido periódico de Schiff PCNA - antígeno nuclear de proliferação celular PGC células germinativas primordiais RNA ácido ribonucleico TGF-β - família do fator transformador de crescimento beta TIMP - inibidor tecidual de metaloproteinase TNF - fator de necrose tumoral UHE - usina hidrelétrica

16 vi Lista de tabelas Tabela 1: Anticorpos primários utilizados para imunohistoquímica de ovários de P. argenteus...17 Tabela 2: Primary antibodies, supliers, and dilutions (Thomé et al., 2010 Artigo 1-Table 1) Tabela 3: Biometric, gonadosomatic index and morphometric analysis of P. argenteus females captured in two sites of the São Francisco River: S1 immediately following the Três Marias Dam and S2 in the confluence with Abaeté River (Thomé et al., 2012 Artigo 2-Table 1) Tabela 4: Abiotic parameters of the water in two sites of the São Francisco River: S1 immediately following the Três Marias Dam and S2 in the confluence with Abaeté River (Thomé et al., 2012 Artigo 2-Table 2)

17 vii Lista de figuras Figura 1: Esquema comparativo de desenvolvimento folicular entre vertebrados...2 Figura 2: Resumo do controle hormonal da foliculogenese em peixes...3 Figura 3: Hipótese para o destino das células foliculares em peixes teleósteos ovíparos após desova...7 Figura 4: Modelo proposto para relação entre autofagia e apoptose na atresia folicular..8 Figura 5: Prochilodus argenteus Spix & Agassiz, Figura 6: Mapa de localização da área de estudo no rio São Francisco...16 Figura 7: Histological sections of P. argenteus ovaries stained with toluidine blue (a, b, d), Gomory trichrome (c), hematoxylin-eosin (e) and periodic acid-schiff (PAS)- hematoxylin (f, g) (Thomé et al 2010 Artigo 1-Fig 1) Figura 8: Immunohistochemical for laminin β2 (a d), collagen type IV (e) and MMP-9 (f i) in ovarian follicles of P. argenteus. (Thomé et al., 2010 Artigo 1-Fig 2) Figura 9: Immunohistochemical reaction for actin (b, f) and histological sections of spawned ovary in P. argenteus stained with Gomory trichrome (a, e), hematoxylineosin (c) and periodic acid-schiff (PAS)-hematoxylin (d, g, h) (Thomé et al., 2010 Artigo 1-Fig 3)...25 Figura 10: Immunohistochemical reaction for laminin β2 (a, b, e), collagen type IV (c, d), fibronectin (h, i) and MMP-9 (f, g) in postovulatory follicles of P. argenteus (Thomé et al., 2010 Artigo 1-Fig 4)...25 Figura 11: Immunostained area of laminin β2, collagen type IV and MMP-9 of the postovulatory follicles in P. argenteus (Thomé et al., 2010 Artigo 1-Fig 5)...26 Figura 12: TUNEL-positive reaction during follicular development and postovulatory regression in P. argenteus (Thomé et al., 2010 Artigo 1-Fig 6).26 Figura 13: Follicular apoptosis index in resting (O1), ripe (O4) and postovulatory follicles = POF (0, 48 and 96 h) in P. argenteus. (Thomé et al., 2010 Artigo 1-Fig 7).26

18 viii Figura 14: Plasma 17β estradiol level concentrations in ovaries in resting, ripe and postspawning = PS (0, 48 and 96 h) in P. argenteus (Thomé et al., 2010 Artigo 1-Fig 8) Figura 15: Histological sections of P. argenteus ovaries stained with hematoxylin and eosin (A and D) and Gomori trichrome (B, detail in B and C) (Thomé et al., 2012 Artigo 2-Fig 1)...35 Figura 16: Relative frequency of oogonia (Oo), perinucleolar (PNF), previtellogenic (PVF), vitellogenic (VF), atretic (AF) and postovulatory follicles (POF) of P. argenteus captured in two sites of the São Francisco River: S1 immediately following the Três Marias Dam and S2 in the confluence with Abaeté River (Thomé et al., 2012 Artigo 2-Fig 2) Figura 17: Immunohistochemistry for PCNA in ovarian sections of the P. argenteus from São Francisco River: S1 immediately following the Três Marias Dam and S2 in the confluence with Abaeté River (Thomé et al., 2012 Artigo 2-Fig 3) 36 Figura 18: TUNEL-positive reaction in ovaries of P. argenteus captured in two sites from the São Francisco River: S1 immediately following the Três Marias Dam and S2 in the confluence with Abaeté River (Thomé et al., 2012 Artigo 2-Fig 4)...37 Figura 19: Immunohistochemistry for cathepsin-d in ovarian sections of the P. argenteus from São Francisco River: S1 immediately following the Três Marias Dam (A) and S2 in the confluence with Abaeté River (B) (Thomé et al., 2012 Artigo 2-Fig 5).38 Figura 20: Schematic summary for possible formation and development of the ovarian follicles in teleosts under environmental influence (Thomé et al., 2012 Artigo 2-Fig 6)

19 1- INTRODUÇÃO E JUSTIFICATIVA O Brasil ocupa o primeiro lugar do mundo no número de espécies de peixes de água doce (McAllister et al., 1997), com quase 2600 já descritas (Buckup et al., 2007). Os Characiformes e Siluriformes, respondem por cerca de 70% de todas as espécies de peixes neotropicais, seguidos pelos grupos Gymnotiformes, Perciformes e Cyprinodontiformes, compondo a ictiofauna brasileira de água doce (Vari e Malabarba, 1998). Além desses cinco grupos, há outros que individualmente não são representativos, mas quando combinados compõem um grande número de espécies (Godinho et al., 2010). Além da grande diversidade da nossa ictiofauna, os peixes de água doce possuem papel de destaque na pesca comercial e esportiva representando uma rica fonte de proteínas (Sato e Godinho, 2003). Por isso, o entendimento do comportamento reprodutivo dos peixes, através da análise da maturação gonadal, é importante para a melhoria da gestão dos recursos da pesca e conservação das espécies nativas. De fato, estudos sobre a biologia reprodutiva e da cascata de eventos moleculares na gametogênese e fecundação são considerados parâmetros importantes na conservação da biodiversidade (Wildt e Wemmer, 1999). 1.1 DESENVOLVIMENTO DOS FOLÍCULOS OVARIANOS Os ovários dos teleósteos são revestidos por túnica albugínea que emite septos para o interior do órgão formando lamelas ovulígeras nas quais se encontram ovogônias e folículos ovarianos em diferentes fases de desenvolvimento (Bazzoli, 2003). Os folículos ovarianos de peixes são constituídos de ovócito e suas camadas envoltórias: zona pelúcida, células foliculares e teca conjuntiva (Grier et al., 2009). Em seu sentido mais amplo, o termo ovogênese se refere ao processo pelo qual as células germinativas primordiais (PGCs) tornam-se ovócitos aptos a serem fertilizados. A ovogênese pode ser descrita através de seis etapas principais: (1) a formação das PGCs, (2) diferenciação da PGCs em ovogônias, (3) diferenciação de ovogônias em ovócitos que entram em meiose, (4) crescimento dos ovócitos, em prófase I da meiose, (5) retomada da meiose na maturação final ovocitária, e (6) extrusão do ovócito durante ovulação e consequente desova (Patiño e Sullivan, 2002; van den Hurk e Zhao, 2005).

20 Nos ovários de mamíferos, a divisão mitótica das células germinativas ocorre por completo antes do nascimento, com isso o número de ovócitos a ser liberado é limitado ao número de folículos primordiais que serão gerados durante a embriogênese. Ao contrário, em muitos vertebrados com alta fecundidade como os peixes teleósteos, o número de ovócitos pode ser infinito (Figura 1). Nos ovários de peixes e de anfíbios adultos, células germinativas mitóticas com características citológicas de ovogônias são observadas e, assim, a manutenção do estoque destas células permite a formação de ovócitos maduros continuamente ao longo da vida (Nakamura et al., 2010; 2011). Figura 1: Esquema comparativo de desenvolvimento folicular em vertebrados (Adaptado de Nakamura et al., 2011). A foliculogênese é regulada hormonalmente pelo eixo hipotálamo-hipófisegonada. Em peixes teleósteos, estímulos ambientais são convertidos em informações sensoriais no cérebro culminando com a liberação pelo hipotálamo de hormônios liberadores de gonadotropinas (GnRH) que via corrente sanguínea chegam à adenohipófise, que sintetiza e libera hormônios gonadotrópicos: hormônio folículo estimulante (FSH) e hormônio luteinizante (LH). O FSH e o LH encontram seus respectivos receptores nas gônadas, que respondem secretando hormônios esteroides: 17β estradiol (E2), promotor da proliferação mitótica de ovogônias e da vitelogenese, e 17β, 20α dihidroxi-4-pregnen-3-one (17β-20α DHP), que promove o reinício da meiose das células germinativas e maturação folicular final (Yaron e Levavi-Sivan, 2011) (Figura 2).

21 Figura 2: Resumo do controle hormonal da foliculogênese em peixes (Adaptado de Yaron e Levavi-Sivan, 2011). Na ovogênese de mamíferos os ovócitos não aumentam de tamanho, diferente do observado em teleósteos, onde os ovócitos crescem enquanto o ciclo celular está bloqueado em diplóteno na prófase meiótica (Nagahama, 1994). Nesta fase, os ovócitos dos teleósteos se desenvolvem dentro de folículos ovarianos, estabelecidos quando células foliculares associadas a uma membrana basal e células tecais envolvem completamente o ovócito (Patiño e Sullivan, 2002; Grier et al., 2009). O FSH estimula as células especiais da teca a sintetizar testosterona, que será convertida em E2 pelas células foliculares através da ação da enzima P450 aromatase. No fígado, o E2 estimula os hepatócitos a produzirem vitelogenina e coreogenina, que são levadas pela corrente sanguínea até o ovário, onde participam da formação do vitelo e da zona pelúcida durante a maturação ovocitária (Senthilkumaran et al., 2004). O vitelo acumula no citoplasma do ovócito que aumenta de diâmetro. Assim o FSH regula a secreção de E2 e a incorporação de vitelogenina dentro dos ovócitos (Yaron e Levavi-Sivan, 2011) (Figura 2a). Os ovócitos vitelogênicos podem permanecer estacionados na prófase meiótica por tempo variado, começando o processo de maturação final após estímulos ambientais, quando enfim a meiose é retomada. O hipotálamo libera GnRH, com ou sem inibição por dopaminérgicos (DA), e a adenohipófise responde aumentando a

22 quantidade de LH na circulação sanguínea. Após a ligação do LH aos seus receptores nas células foliculares, o folículo ovariano inicia o processo de maturação final, começando com a produção dos esteroides indutores da maturação (MIS), por exemplo, a 17β-20α DHP. A ligação de MIS aos seus receptores na membrana plasmática do ovócito é seguida pela ativação do fator promotor de maturação (MPF) (Figura 2b). O processo de maturação do ovócito é reconhecido morfologicamente pela migração da vesícula germinativa (GV) em direção ao pólo animal e a quebra da vesícula germinativa (GVBD), o envelope nuclear rompe-se e a cromatina compactada na metáfase irá permitir a fertilização (Yaron e Levavi-Sivan, 2011). Dentre todas as células da linhagem germinativa as ovogônias tem a maior proporção núcleo/citoplasma. Os núcleos das ovogônias variam de ovais a esféricos, com fina cromatina e nucléolo único. O citoplasma destas células apresenta mitocôndrias, ribossomos e escasso retículo endoplasmático e complexo de Golgi (MacMillan, 2007). Nos ovários de peixes em início de desenvolvimento, são observadas histologicamente três fases distintas de células germinativas: ovogônias isoladas envolvidas por células somáticas que expressam Sox9b, ovogônias ligadas por pontes citoplasmáticas formando aglomerados de células, ninhos de ovogônias, e ovócitos em estágio inicial de desenvolvimento. O gene Sox9 foi descrito inicialmente em mamíferos estando envolvido com desenvolvimento gonadal de machos, particularmente na diferenciação da célula de Sertoli. O Sox9b é um ortólogo funcional em teleósteos, sendo expresso durante o desenvolvimento gonadal nas células de suporte como as células foliculares (Nakamura et al., 2011). O núcleo das ovogônias está circundado por várias mitocôndrias esféricas e nuage, a qual é uma estrutura granular e eletrondensa sem limite de membrana formada por proteínas associadas a ácido ribonucleico (RNA) (Santos et al., 2006). Três tipos principais de desenvolvimento de ovócitos foram descritos para os peixes (Wallace e Selman, 1981): (a) sincrônico, todos os ovócitos desenvolvem e ovulam ao mesmo tempo, (b) grupo-sincrônico, pelo menos duas populações de ovócitos podem ser reconhecidas no ovário durante todo período reprodutivo, e (c) assincrônica, ovócitos em diferentes fases de desenvolvimento estão presentes nos ovários, sem dominância. Visando o sucesso reprodutivo, os teleósteos desenvolveram diferentes estratégias de desova frente às diversas variações ambientais. Na desova total, associada a ambientes com notáveis variações sazonais, o desenvolvimento de ovócitos é grupo-sincrônico e os ovócitos são liberados durante um curto período. A desova

23 múltipla ou parcelada está associada a ambientes lênticos, onde o desenvolvimento de ovócitos é assíncrônico e eles são liberados em lotes durante um prolongado período reprodutivo (Bazzoli, 2003; Lubzens et al., 2010). O desenvolvimento folicular pode ser dividido em três fases: crescimento primário, crescimento secundário e maturação final (Lubzens et al., 2010). O crescimento primário é caracterizado por uma intensa proliferação de organelas, acúmulo de RNA materno no citoplasma e diferenciação das células foliculares e tecais. Nesta fase, ocorre também o início da formação da zona pelúcida. No ooplasma justanuclear, a proliferação de organelas membranosas ocorre em área conhecida como corpo de Balbiani ou núcleo vitelínico. Durante este período, os ovócitos aumentam em tamanho e a basofilia citoplasmática torna-se mais intensa (Quagio-Grassioto et al., 2011). Grandes quantidades de glicoproteínas são sintetizadas na fase final do crescimento primário e são incorporados em vesículas formadas na periferia do ooplasma, chamadas de alvéolos corticais (Patiño e Sullivan, 2002). Os alvéolos corticais liberam seu conteúdo durante a reação cortical no espaço perivitelínico, sendo responsáveis pelo endurecimento da zona pelúcida dificultando a polispermia durante a fertilização (Tyler e Sumpter, 1996). Vários fatores de crescimento têm sido relacionados com a regulação do crescimento primário. O fator de crescimento e diferenciação 9 (GDF9) e o fator morfogenético de ossos 15 (BMP15) são membros da família do fator transformador de crescimento beta (TGF-β) e expressos em ovócitos. Em mamíferos, ambos desempenham um papel importante no crescimento do folículo ovariano primordial (Gilchrist et al., 2004; Juengel et al., 2004;. Moore e Shimasaki., 2005). Estes dois fatores, GDF9 e BMP15, também foram relacionados com o crescimento primário de folículos ovarianos de teleósteos (Lubzens et al., 2010). O sistema do fator de crescimento semelhante à insulina (IGF) regula uma variedade de processos celulares incluindo crescimento, proliferação, sobrevivência, migração e diferenciação (Wood et al., 2005). Em vertebrados, o IGF1 é sintetizado principalmente no fígado sob influência do hormônio do crescimento (GH), e recente estudo sugere que a regulação da função ovariana pelo sistema IGF é conservado na evolução animal (Reinecke, 2010). A expressão do sistema IGF, incluindo peptídeos, receptores e proteínas de ligação, tem sido demonstrada em gônadas de peixes, como tilápia, carpa, salmão, seabream, zebrafish, robalo e esturjão (Perrot et al., 2000; Radaelli et al., 2010). Alguns estudos sugerem que IGF1 está envolvido na regulação do crescimento, diferenciação e

24 reprodução através do estímulo a proliferação celular e inibição da apoptose e o IGF2 na foliculogênese durante a maturação final ovocitária (Kagawa et al., 1995; Perrot et al., 2000; Radaelli et al., 2010). Recentemente, além do IGF1 e IGF2 foi descrita a existência de uma terceira forma de IGF denominada IGF3, exclusiva de peixes e associada à reprodução de machos (Berishvili et al., 2010). O crescimento secundário ou fase vitelogênica é caracterizado pela captação de vitelogenina. A vitelogenina é uma glicofosfolipoproteína sintetizada no fígado e carreada para o ovário, chegando ao folículo ovariano pela vascularização da teca, passando pelos espaços intercelulares das células foliculares, pelos canais da zona pelúcida até alcançar a membrana plasmática ovocitária. A vitelogenina se liga ao seu receptor, é internalizada por endocitose seletiva e entra em contato com as enzimas lisossomais, como a catepsina-d (Cat-D), que degradam a vitelogenina em moléculas menores, lipovitelina e a fosfovitina, que darão origem ao vitelo (Patiño e Sullivan, 2002; Carnevali et al., 2006). A expressão gênica da cat-d ocorre em ovócitos de tamanhos diferentes durante o crescimento secundário, especialmente na fase inicial da vitelogenese (Carnevali et al., 2008) e tem sido sugerida como biomarcador de desregulação endócrina por estar envolvida no processo de formação do vitelo durante a maturação do ovócito, desempenhando um papel fundamental para o sucesso da reprodução de peixes (Carnevali e Maradona, 2003). O processo de maturação final é caracterizado pela redução ou interrupção da endocitose de vitelogenina, retomada da meiose e quebra da vesícula germinativa culminando com a ovulação e desova (Lubzens et al., 2010). 1.2 REGRESSÃO OVARIANA Após a desova, os ovários apresentam folículos pós-ovulatórios e atrésicos que involuem progressivamente até sua recuperação total do ovário, para retornar a fase de repouso e iniciar novo ciclo reprodutivo (Bazzoli, 2003). Os folículos pós-ovulatórios são remanescentes dos folículos ovarianos constituídos de células foliculares, membrana basal e teca conjuntiva (Grier et al., 2007). Diferentemente do corpo lúteo de mamíferos os folículos pós-ovulatórios de peixes ovíparos não apresentam atividade hormonal, sendo estruturas transitórias após desova (Selman e Wallace, 1989; Drummond et al., 2000; Santos et al., 2005). Nos vertebrados, grande número de folículos ovarianos que são recrutados para o desenvolvimento não completam a maturação e por isso, degeneram sendo reabsorvidos

25 no processo conhecido como atresia folicular (Saidapur, 1978). Nos teleósteos, os ovócitos vitelogênicos que não foram ovulados durante o período reprodutivo tornam-se atrésicos e são lentamente reabsorvidos durante a regressão ovariana pós-desova (Rizzo e Bazzoli, 1995; Miranda et al., 1999; Santos et al., 2005). Fatores intrínsecos e extrínsecos, incluindo a hipofisectomia, irradiação, substâncias anti-gonadotróficas, estresse térmico, confinamento, jejum prolongado e outros podem induzir atresia folicular em ovários de peixes e diminuir o potencial reprodutivo das espécies (Saidapur, 1978; Nahum et al., 1996). Alguns estudos indicam que a apoptose é o principal mecanismo envolvido na eliminação de células somáticas e germinativas em folículos atrésicos de aves e mamíferos (Andreu-Vieyra e Habibi, 2000; Hussein, 2005). Em teleósteos, a morte celular programada parece desempenhar um papel secundário durante a atresia folicular (Santos et al., 2008a). Estudo recente indicou que a apoptose e autofagia podem agir cooperativamente, nos ovários de peixes, e uma dupla função, na sobrevivência e na morte celular, foi atribuída à autofagia para garantir uma regressão ovariana mais eficiente após a desova (Thomé et al., 2009 Anexo I) (Figura 3). Nos folículos pós-ovulatórios a taxa de apoptose das células foliculares aumenta rapidamente após desova, enquanto que no folículo atrésico inicialmente a autofagia é predominante e a taxa de apoptose só aumenta na fase final do processo (Santos et al., 2005; Thomé et al., 2006; Santos et al., 2008a). Figura 3: Hipótese para o destino das células foliculares em peixes teleósteos ovíparos após desova (Adaptado de Thomé et al., 2009 Anexo I).

26 Durante a atresia folicular, células foliculares hipertrofiadas fagocitam o vitelo em degeneração durante a reabsorção do folículo (Santos et al., 2008a). Estudos realizados em ovários de Oncorhyncus mykiss sugerem que a atresia folicular envolve a degradação de proteínas vitelínicas mediada por enzimas lisossomais (Wood e Van Der Kraak, 2003). Dentre as hidrolases ácidas presentes no compartimento lissosomal, destaca-se a cat-d que é uma protease aspártica da superfamília das pepsinas e está envolvida em muitos processos fisiológicos, tais como degradação, apoptose e autofagia (Zaidi et al., 2008). Entre as proteínas envolvidas na autofagia, beclin-1 induz a nucleação de uma vesícula com dupla membrana chamada vacúolo autofágico (Maiuri et al., 2007, 2010). Em células autofágicas, o conhecimento sobre a organização do citoesqueleto de actina e seu papel na biogênese do vacúolo autofágico ainda é escasso (Reggiori et al., 2005). A interação de beclin-1 com a proteína anti-apoptótica bcl-2 é essencial na determinação do destino das células e no crosstalk entre autofagia e apoptose (Klionsky, 2007). Em teleósteos, proteínas autofágicas (beclin-1, Atg, LC3, Lamp) e apoptóticas (bcl-2, bcl-xl, bax e bid) podem ser ativadas de maneira coordenada dependendo do estágio da atresia folicular, estabelecendo uma relação entre autofagia e apoptose (Figura 4) (Morais, 2009; Morais et al., 2012 Anexo II). Figura 4: Modelo proposto para relação entre autofagia e apoptose na atresia folicular. (Adaptado de Morais et al., 2012 Anexo II).

27 1.3 APOPTOSE E PROLIFERAÇÃO CELULAR Em condições normais, o funcionamento do ovário é dependente de remodelações cíclicas com formação, desenvolvimento e involução dos folículos, tornando o ovário um excelente modelo para estudo dos eventos de desenvolvimento e regressão tecidual (Bagavandoss, 1998). O crescimento folicular e a ovulação são acompanhados por alterações na matriz extracelular, como também ocorre na involução dos folículos pósovulatórios. Essas alterações podem influenciar na maturação folicular, na sobrevivência das células foliculares e na esteroidogênese. Células de organismos multicelulares são influenciadas pelo microambiente onde estão inseridas através de interações com células vizinhas e com a matriz extracelular (Pinkse et al., 2004). Os componentes da matriz extracelular dos ovários têm sido caracterizados em várias espécies de mamíferos, tais como bovinos, ovinos, eqüinos, murinos e primatas, sendo mais comumente estudados: colágeno tipo I, colágeno tipo IV, laminina e fibronectina (Berkholtz et al., 2006a). O colágeno tipo IV interage por meio de seus domínios terminais para formar uma rede extracelular flexível e plana. A laminina é uma glicoproteína que participa na adesão das células à lâmina basal, enquanto que a fibronectina é importante nos processos de adesão, migração, crescimento e diferenciação celular (Rodgers et al., 2003). As interações célula-matriz exercem grandes efeitos na regulação gênica, estrutura do citoesqueleto, diferenciação e crescimento celular (Frisch e Francis, 1994). Em mamíferos, a membrana basal é importante para a sobrevivência celular atuando como fator anti-apoptótico em células epiteliais (Pullan et al., 1996). A sobrevivência das células tronco-epidermais é garantida pela interação célula-matriz, uma vez que os queratinócitos que expressam integrina e aderem ao colágeno tipo IV não entram em apoptose (Tibério et al., 2002). O desprendimento da célula de sua matriz extracelular promove a desfosforilação da quinase de adesão focal resultando na ativação do efeito cascata das caspases induzindo a apoptose (Frisch e Francis, 1994; Pinkse et al., 2004). O equilíbrio entre regeneração e degradação da matriz extracelular é mantido, em parte, pela ação de enzimas proteolíticas extracelulares que são secretadas por células locais. Dentre estas se destacam as metaloproteinases da matriz (MMPs), as quais dependem da ligação do Ca 2+ ou Zn 2+ para sua atividade. Atualmente, a família das MMPs é composta por 25 tipos agrupados em 4 classes: colagenases (MMP-1, -8, -13), gelatinases (MMP-2 e MMP-9), estromalisinas e as MMPs típicas de membranas (MT- MMPs) (Sternlicht e Werb, 2001). Três mecanismos básicos atuam para assegurar o

28 controle das MMPs: secreção da forma inativa (pro-enzima), confinamento por receptores de superfície celular e inibidores teciduais de metaloproteinases (TIMPs). O aumento de TIMPs atua coordenando as ações de MMPs, regulando a localização e extensão da remodelação da matriz extracelular durante o processo ovulatório em mamíferos (Curry e Osteen, 2003). As MMPs desempenham um importante papel no processo ovulatório de diferentes grupos de vertebrados, atuando na ruptura folicular, fragmentação da membrana basal e fibras conjuntivas associadas ao folículo ovariano (Curry e Osteen, 2003; Ogiwara et al., 2005). Em teleósteos, a distribuição e a ação proteolítica das MMPs foram descritas em fígado, músculos, brânquias e gônadas (Matsui et al., 2000; Lodemel et al., 2004; Ogiwara et al., 2005). Durante a foliculogênese e crescimento folicular, o balanço entre os sinais proliferação e morte celular determinam o destino do folículo ovariano para ovulação ou para atresia folicular (Krysko et al., 2008). O antígeno nuclear de proliferação celular (PCNA), é uma proteína nuclear não histônica que apresenta 36 kda em mamíferos (Bravo e Graf, 1985; Bravo e MacDonald-Bravo, 1987). O PCNA é um importante regulador do ciclo celular, muito conservado entre as espécies, atua como co-fator da DNA-polimerase delta e está envolvido nos processos de reparo de DNA (Bravo et al., 1987). O PCNA é encontrado na matriz nuclear, começando a ser sintetizado na fase G1, atingindo os mais altos níveis durante a fase S e diminuindo na fase G2 e mitose (Kurki et al., 1988). Os primeiros estudos sobre proliferação celular utilizaram basicamente dois métodos de avaliação da proliferação celular: a contagem de figuras de mitose em cortes histológicos utilizando a morfologia como parâmetro de identificação (Beaumont e Mandl, 1962) e a contagem após a incorporação dos marcadores timidina triciada ou BrdU (Dolbeare, 1995). Atualmente, além dos métodos citados, a expressão de PCNA tem sido utilizada como ferramenta útil para a identificação da proliferação celular durante a gametogênese e embriogênese de mamíferos e peixes (Leung et al., 2005; Hutt et al., 2006; Loppion et al., 2008). O PCNA foi detectado nas células foliculares de zebrafish durante o crescimento dos folículos ovarianos (Korfsmeir, 2002) e em células da teca de folículos atrésicos de Prochilodus argenteus e de Leporinus taeniatus (Santos et al., 2008a). A apoptose é um processo fisiológico importante para manter um número apropriado de células no tecido. É muito conservado durante a evolução e requer uma especializada maquinaria envolvendo uma família de proteases denominadas de

29 caspases (Kerr et al., 1972). As caspases ativam endonucleases dependentes de íons Ca 2+ /Mg 2+, que clivam o DNA em regiões internucleossômicas, gerando fragmentos múltiplos de pares de bases nucleotídicas (Huettenbrenner et al., 2003). Células em apoptose apresentam perda de junções de adesão célula-célula e célula-matriz extracelular, retração celular, condensação da cromatina na periferia do envoltório nuclear e fragmentação celular em corpos apoptóticos que são fagocitados por células vizinhas ou fagócitos sem desencadear reação inflamatória (Hsueh et al., 1994). Alterações do citoesqueleto têm sido amplamente relatadas em células apoptóticas nas quais a forma da célula e sua ancoragem são dependentes da reorganização dos filamentos de actina (Korsnes et al., 2007). A apoptose pode ser induzida por diferentes estímulos extrínsecos, reunidos em dois grupos de acordo com seus mecanismos de ação. No primeiro grupo, estão os membros da família do fator de necrose tumoral (TNF) e o Fas/Fas-Ligante. No segundo grupo encontram-se indutores de injúrias no DNA, alterações hormonais, substâncias tóxicas e perda de ancoragem celular (McConkey, 1998). Fatores intrínsecos, relacionados com alterações nas mitocôndrias também podem desencadear apoptose. Dentre eles, a família Bcl-2 é composta por proteínas intracelulares que ajudam a regular a ativação das pro-caspases. Alguns membros dessa família, como a própria bcl-2 e bcl-x, inibem a apoptose, enquanto outros como bad e bax promovem a ativação da apoptose. A inibição do fator anti-apoptótico da via bcl-2 induz apoptose em células foliculares de folículos atrésicos de mamíferos, aves e peixes (Hsueh et al., 1994; Johnson et al., 1996; Johnson, 2003; Morais et al., 2012 Anexo II). Em teleósteos, o aumento significativo de apoptose foi utilizado como bioindicador de ambientes aquáticos impactados expostos a diferentes tipos de xenobióticos, i.e., substâncias químicas estranhas a um organismo ou sistema biológico prejudicando desde a função hepática até a taxa reprodutiva do peixe (Janz et al., 1997; Piechotta et al., 1999; Janz et al., 2001; Weber et al., 2001; Weber et al., 2002). A importância da apoptose na dinâmica reprodutiva de peixes teleósteos foi investigada na seleção e recrutamento de folículos para a vitelogênese em O. mykiss (Janz e Van Der Kraak, 1997), no desenvolvimento dos folículos ovarianos de Carassius auratus e O. mykiss (Wood e Van Der Kraak, 2001) e durante a involução de ovários após desova de Astyanax bimaculatus (Drummond et al., 2000), L. taeniatus (Santos et al., 2005) e Prochilodus costatus (Thomé et al., 2006).

30 1.4 O RIO SÃO FRANCISCO O rio São Francisco, considerado como o rio da integração nacional, descoberto em 1502, tem esse título por ser o caminho de ligação do Sudeste e do Centro-Oeste com o Nordeste. Suas águas são utilizadas principalmente para geração de energia, irrigação, abastecimento urbano e industrial. Desde as suas nascentes, na Serra da Canastra, em Minas Gerais, até sua foz, na divisa de Sergipe e Alagoas, ele percorre km, atravessando regiões diversificadas, tanto em relação às condições climáticas e topográficas, quanto à diversidade social (fonte: Desta forma, a bacia é tradicionalmente dividida em quatro segmentos: alto, médio, submédio e baixo São Francisco. O alto compreende da nascente até Pirapora, numa extensão de 630 km; o médio com km estende-se até Remanso, o submédio até Cachoeira de Paulo Afonso com 686 km e o baixo até a foz no Oceano Atlântico (Paiva, 1982). As maiores usinas hidrelétricas (UHE), em área alagada ou em potência, encontram-se na calha principal do rio. Apenas a UHE de Três Marias, foi construída no alto São Francisco, enquanto as demais: Sobradinho, Itaparica, Moxotó, o complexo Paulo Afonso e Xingó ocupam o terço inferior do rio. Em conjunto, elas têm capacidade de geração de MW, mas que inundaram cerca de Km 2 de áreas férteis (Godinho e Godinho, 2003). Os barramentos apesar de importantes para o desenvolvimento econômico provocam alterações graves e irreversíveis no regime hidrológico natural dos rios, alterando também a qualidade dos hábitats e a dinâmica de toda a biota. As espécies mais afetadas são aquelas que realizam longas migrações e que necessitam de diferentes hábitats para completar seu ciclo de vida (Agostinho et al., 2008). A UHE de Três Marias foi construída em 1961 para produção de energia elétrica, regularização do rio e controle de cheias. Em 1980, a Estação de Hidrobiologia e Piscicultura da Companhia de Desenvolvimento dos Vales do São Francisco e Parnaíba (CODEVASF) foi estabelecida as margens da represa de Três Marias, com o objetivo de desenvolver tecnologia de reprodução induzida destinada à produção de alevinos para repovoamento, visando à preservação da ictiofauna nativa, além de pesquisas científicas (Sato et al., 2003). A represa de Três Marias, apresenta 100 mil hectares de área inundada, 21 x 10 9 m 3 de água e estratificação térmica no verão, sendo a água do hipolímnio 2 a 3 o C mais fria de novembro a fevereiro no período reprodutivo das espécies migradoras brasileiras (Sato et al., 2005). Logo, a jusante da UHE de Três Marias, o rio São Francisco apresenta apenas pequenos tributários (córregos e riachos)

31 que não restabelecem o perfil físico-químico natural da água. No entanto, alguns desses pequenos tributários (Barreiro grande, Consciência, Espírito Santo) recebem efluentes industriais sem tratamento (Ribeiro, 2010). O rio Abaeté é o primeiro tributário de médio porte caracterizado pelo fundo rochoso e várias corredeiras e cachoeiras, desaguando cerca de 34 km após a barragem de Três Marias. O rio Abaeté minimiza o impacto da barragem apresentando condições favoráveis à reprodução de várias espécies de peixes migradores de importância econômica (Sato et al., 2005). 1.5 ESPÉCIE DE ESTUDO A curimatã-pacu Prochilodus argenteus Spix & Agassiz, 1829 (Figura 5), endêmico da bacia do rio São Francisco, é uma das espécies predominantes na pesca da região de Três Marias, podendo alcançar mais de 10 kg de peso corporal (Reis et al., 2003; Sato e Godinho, 2003). De hábito alimentar iliófago, a curimatã-pacu alimenta-se de lodo com finos materiais particulados depositados no fundo do rio, podendo ser um bom modelo biológico para análise da contaminação com metais e outros resíduos químicos depositados no sedimento dos rios. A espécie não desova em ambientes lênticos (Arantes et al., 2011) e é usualmente submetida à reprodução induzida na Estação de Hidrobiologia e Piscicultura de Três Marias, visando o repovoamento do reservatório (Sato et al., 1996). Apresenta desova total, ovos livres, desenvolvimento embrionário rápido e não possui comportamento de cuidado parental (Sato et al., 2003). Realiza migrações reprodutivas na estação chuvosa, geralmente de novembro a janeiro, no rio São Francisco e em seus afluentes, principalmente no rio Abaeté (Sato e Godinho, 2003). Figura 5: Exemplar de Prochilodus argenteus Spix & Agassiz, 1829 com cerda de 45cm de comprimento total.

32 2- OBJETIVOS 2.1 OBJETIVO GERAL Analisar o desenvolvimento e a regressão dos folículos ovarianos da curimatãpacu Prochilodus argenteus, em condições naturais e de confinamento, com ênfase nos processos de remodelação da matriz extracelular, proliferação e morte celular. 2.2 OBJETIVOS ESPECÍFICOS - Avaliar a foliculogênese em condições de confinamento; - Analisar a distribuição espacial e temporal de MMP-9, laminina, fibronectina e colágeno tipo IV em ovários pré e pós-desova; - Relacionar alterações da membrana basal e níveis de E2 com a apoptose em ovários pré e pós desova; - Analisar a distribuição espacial e temporal do citoesqueleto de actina em folículos pós-ovulatórios; - Analisar e comparar a distribuição dos folículos ovarianos em diferentes fases de desenvolvimento em dois trechos do rio São Francisco, a jusante da UHE de Três Marias; - Analisar proliferação e morte celular das células germinativas e foliculares, além da vitelogenese, através da atividade da caspase-3 e expressão de PCNA e cat-d.

33 3- MATERIAIS E MÉTODOS 3.1 AMOSTRAGEM Os animais utilizados no presente trabalho foram obtidos em duas etapas. Na primeira etapa, utilizaram-se 20 exemplares de P. argenteus nos seguintes estádios: repouso, maturação e desovado. Os peixes foram capturados no rio São Francisco e mantidos em confinamento na Estação de Hidrobiologia e Piscicultura de Três Marias. Para obtenção dos ovários desovados os peixes foram submetidos à desova induzida por hipofisação. Na segunda etapa, fragmentos de ovários de P. argenteus que foram capturados em dois trechos do rio São Francisco (Figura 6): 25 exemplares logo a jusante da barragem de Três Marias (S1) e 20 exemplares na confluência com rio Abaeté (S2). Os fatores físico-químicos da água (ph, temperatura da água, oxgênio dissolvido e condutividade) foram determinados com sonda Horiba modelo W-22XD durante a coleta sempre pela manhã, com amostragem da margem esquerda, centro e margem direita do rio. O projeto seguiu os princípios éticos de experimentação com animais de acordo com o Colégio Brasileiro de Experimentação Animal (COBEA) e foi aprovado pelo Comitê de Ética em Experimentação Animal da UFMG (CETEA protocolo nº: 073/08). Trecho 2 Trecho 1 Trecho 2 Trecho 1 Figura 6: Mapa do rio São Francisco a jusante da UHE de Três Marias (Adaptado de Sato et al., 2005).

34 3.2 HISTOLOGIA Amostras de ovários fixadas em líquido de Bouin por 6 horas a temperatura ambiente foram incluídas em parafina ou em glicol metacrilato (JB-4 Polysciences). Secções histológicas foram coradas com hematoxilina-eosina, tricrômico de Gomori ou azul de toluidina-borato de sódio. Algumas amostras foram submetidas à técnica histoquímica do PAS e contra-coradas com hematoxilina. 3.3 TUNEL Amostras de ovários fixadas em paraformoldeído 4% em tampão fosfato 0,1M ph 7,3 por 24 horas a 4 C foram incluídas em parafina, seccionados e submetidos à técnica de TUNEL in situ utilizando o Kit TdT FragEL Calbiochem ou Kit Apoptag Millipore para a identificação da fragmentação do DNA das células em apoptose. Para isso, incubaram-se as secções histológicas com mistura da enzima terminal deoxynucleotides transferase (TdT) e deoxinucleotídeos conjugados com biotina em câmara úmida a 37 C por 3 h. Em seguida, aplicou-se solução de estreptavidina conjugada com peroxidase em câmara úmida a temperatura ambiente por 45 min. A reação da peroxidase foi revelada com diaminobenzidina (DAB) durante 2 min à temperatura ambiente. As lâminas foram contra-coradas com hematoxilina. Para controle-negativo, uma das lâminas não recebeu a mistura contendo a enzima TdT e deoxinucleotídeos. Para a permeabilização e inativação de peroxidase endógena foram utilizadas proteinase K e água oxigenada 3% respectivamente. Foram consideradas como células apoptóticas aquelas que apresentaram reação TUNEL-positiva e alterações celulares como retração citoplasmática e/ou fragmentação semelhante a corpo apoptótico. 3.4 IMUNOFLUORIMETRIA INDIRETA Amostras de sangue (1,0 5,0 ml) de exemplares com ovários em repouso, maturação e desovado (0, 48 e 96 horas) foram coletadas através de punção da veia caudal. As amostras foram transferidas para tubos de ensaio e mantidas até coagulação para em seguida serem centrifugadas (10-15 min a 800 rpm). Alíquotas do soro foram mantidas a -20ºC e a determinação dos níveis de E2 foi realizada através de imunofluorimetria indireta (kit AutoDELFIA Estradiol Perkin Elmer).

35 3.5 IMUNOHISTOQUÍMICA Amostras de ovários fixadas em DMSO/metanol ou paraformaldeído foram incluídas em paraplast (Sigma) e seccionadas e submetidas a imunohistoquímica. Foram utilizados os anticorpos primários descritos na tabela 1. Utilizou-se pré-tratamento para recuperação antigênica com proteinase K a temperatura ambiente ou pré-tratamento com tampão citrato ph 6,0 a 95 C por 30 min. Após lavagem com PBS, utilizou-se tampão (BSA 2%) para bloqueio de reações inespecíficas e H 2 O 2 3% para bloqueio da peroxidase endógena. As secções foram incubadas com o anticorpo primário overnight a 4 C. Após lavagem com PBS, os cortes foram incubados com anticorpo secundário conjugado com biotina (Kit LSAB- DAKO) e a reação foi revelada com DAB. Para as análises em microscopia confocal (Carl Zeiss LSM 510) os cortes de tecido foram incubados com anticorpo secundário ALEXA 488 anti-coelho (1:200) e ALEXA 568 anti-camundongo (1:200) e para marcação do núcleo das células utilizou-se DAPI (1:500). Para controle negativo, uma das lâminas não recebeu anticorpo primário. Tabela 1. Anticorpos primários da Santa Cruz Biotechnology utilizados para imunohistoquímica de ovários de P. argenteus. Antígeno Natureza Diluição MMP-9 Monoclonal- camundongo 1:10 Colágeno IV Policlonal- coelho 1:100 Laminina β2 Policlonal- coelho 1:50 Fibronectina Policlonal- coelho 1:25 Actina Monoclonal- camundongo 1:100 PCNA Policlonal- camundongo 1:80 Cat-D Policlonal- coelho 1: WESTERN-BLOT Com o objetivo de determinar a especificidade dos anticorpos, uma vez que foram utilizados anticorpos de mamíferos, e avaliar a expressão das proteínas de interesse, 100 mg de amostra do tecido foram lisados com tampão de lise + inibidores de proteases. O homogeneizado foi centrifugado a g por 30 min, e o sobrenadante contendo as proteínas foi mantido. As proteínas foram adicionadas em gel de eletroforese a 12% e posteriormente transferidas para membrana de nitrocelulose. Após a transferência, as

36 reações inespecíficas foram bloqueadas com BSA 1%. A membrana foi incubada por 20 min com os anticorpos primários. Depois de três lavagens com tampão PBST, a membrana foi incubada por mais 10 min com anticorpo secundário conjugado com peroxidase. A reação foi revelada pela adição de 3 3 diaminobenzidina em PBS contendo cloronaftol, metanol e água oxigenada. Todos os testes foram feitos em triplicatas e a densidade das bandas obtidas foi estimada utilizando-se do ImageJ Software. 3.7 TESTE COLORIMÉTRICO Para identificar a atividade enzimática da caspase-3 em ovários foi utilizado o kit de teste colorimétrico para Caspase-3 da R&D Systems. As amostras do tecido foram pesadas e lisadas com 0.5 ml do tampão de lise celular. O homogenizado foi centrifugado por 1 min a g. O sobrenadante foi mantido e incubado com substrato colorimétrico da caspase-3 (DEVD-pNA) a 37 C por 2 h. O nível da atividade da caspase-3 foi diretamente proporcional a reação de cor. 3.8 MORFOMETRIA Foi realizada contagem de células apoptóticas (TUNEL-positivas), área imunomarcada (Software ImagePro-Plus), medida do diâmetro ovocitário e frequência de estruturas ovarianas. 3.9 ANÁLISE ESTATÍSTICA Os testes estatísticos foram realizados no software GraphPad InStat.

37 4- RESULTADOS Os resultados obtidos no presente trabalho foram publicados nos seguintes artigos: THOMÉ R.G., SANTOS H.B., SATO Y., RIZZO E., BAZZOLI N., DISTRIBUTION OF LAMININ Β2, COLLAGEN TYPE IV, FIBRONECTIN AND MMP-9 IN OVARIES OF THE TELEOST FISH. JOURNAL OF MOLECULAR HISTOLOGY. 41, THOMÉ R.G., DOMINGOS F.F.T., SANTOS H.B., MARTINELLI P.M., SATO Y., RIZZO E., BAZZOLI N., APOPTOSIS, CELL PROLIFERATION AND VITELLOGENESIS DURING THE FOLLICULOGENESIS AND FOLLICULAR GROWTH IN TELEOST FISH. TISSUE AND CELL. 44,

38 J Mol Hist (2010) 41: DOI /s ORIGINAL PAPER Distribution of laminin b2, collagen type IV, fibronectin and MMP-9 in ovaries of the teleost fish Ralph Thomé Hélio Batista dos Santos Yoshimi Sato Elizete Rizzo Nilo Bazzoli Received: 28 April 2010 / Accepted: 15 July 2010 / Published online: 4 August 2010 Ó Springer Science+Business Media B.V Abstract Extracellular matrix in the ovarian follicle has been characterised for several mammalian species but there are no reports that describe the immunolocalisation of the extracellular matrix elements, matrix metalloproteinases, and its relation to plasma 17b estradiol levels and follicular apoptosis during the teleost s reproductive cycle. The present study used immunohistochemistry to characterise the distribution of laminin b2, collagen type IV, fibronectin and matrix metalloproteinases-9 (MMP-9). The TUNEL in situ technique was used to quantify apoptosis and indirect immunofluorimetric to determine plasma 17b estradiol levels. The TUNEL-positive reaction associated with morphological features exhibited follicular apoptosis. During postovulatory follicle involution, the drop in plasma 17b estradiol levels after spawning contributed to the intense apoptosis observed. By immunohistochemical analysis, laminin b2 and collagen type IV were identified as the major constituents of the basement membrane. The loss of integrity of the basement membrane occurred due to lyses of the major constituents, and coincides with increased follicular apoptosis. The integrity of the basement membrane is important for the survival of follicular cells. Furthermore, the MMP-9 results suggest that this enzyme is R. Thomé H. B. dos Santos E. Rizzo Universidade Federal de Minas Gerais, Belo Horizonte, Brazil Y. Sato Estação de Hidrobiologia e Piscicultura de Três Marias (CODEVASF), Três Marias, Brazil N. Bazzoli (&) Programa de Pós graduação em Zoologia de Vertebrados (PUC-minas), Belo Horizonte, Brazil bazzoli@pucminas.br involved in final oocyte maturation and regression of postovulatory follicles. Fibronectin was observed on the surface of follicular cells of the postovulatory follicle in P. argenteus, this being important for maintaining normal cell adhesion to extracellular matrix. In conclusion, our results suggest that the structure and composition of the extracellular matrix, and plasma 17b estradiol levels related to apoptosis, play an important role during the follicular development and post-spawning involution in teleost fishes. Keywords Apoptosis 17b estradiol Extracellular matrix Matrix metalloproteinases Ovarian follicle Introduction Teleost reproduction presents many original features, such as spawning types and parental behaviour, sensitivity to environmental factors, and specific features of the gametogenesis, such as the duration of the vitellogenesis, and egg morphology (Jalabert 2005). Their ovaries are surrounded by a connective tunica that issues numerous septa projecting into the ovarian lumen, called ovigerous lamellae, where nests of oogonia can be found, as well as follicles at different stages of development. Post-spawning, postovulatory and atretic follicles are gradually reabsorbed until the ovaries are fully recovered, which return to the resting stage to start a new reproductive cycle (Drummond et al. 2000; Santos et al. 2005, Thomé et al. 2009). During the reproductive cycle, the ovarian tissue is constantly remodelling, with extensive cell proliferation and differentiation, besides changes of extracellular matrix from the onset of the follicular development until involution of tissues after ovulation (Curry and Osteen 2003). Among the processes and factors involved in tissue remodelling stand 123

39 216 J Mol Hist (2010) 41: out apoptosis, changes in hormone levels and degradation of extracellular matrix in contact with the cells. Apoptosis is a physiological, genetically programmed process that is important for tissue homeostasis in multicellular organisms and requires specialised machinery involving proteases known as caspases (Kerr et al. 1972; Takle and Andersen 2007). The activation of endonucleases, and subsequent cleavage of DNA, is a definitive step toward apoptosis, but there are many factors that can play a role in earlier stages to inhibit the entire apoptotic process, such as variation in hormones levels, modifications of the cytoskeleton or loss of cell anchorage (Frisch and Francis 1994; Janz et al. 1997; Kulms et al. 2002). In mammalian ovaries, many studies indicate that 17b estradiol can act as an anti-apoptotic factor for granulosa cells (Guthrie et al. 1995; Chun et al. 1996; Narkar et al. 2006). The relationship between follicular apoptosis and plasma 17b estradiol levels in the reproductive dynamics of teleosts has been investigated in a few species, for example Oncorhyncus mykiss (Janz et al. 1997) and Chalcalburnus tarichi (Kaptaner and Ural 2006). Cytoskeletal changes have been extensively reported in apoptotic cells in which alterations of cell shape and anchorage are dependent on reorganisation of actin filaments and focal adhesion contacts (Korsnes et al. 2007). In relation to cell anchorage, the detachment of the cell of its extracellular matrix promotes the dephosphorylation of focal adhesion kinase resulting in activation of caspase cascade leading to apoptosis (Frisch and Francis, 1994). The extracellular matrix cell interactions influence gene regulation, cytoskeleton structure, differentiation and many aspects of cell growth (Irving-Rodgers and Rodgers 2005). The balance between degradation and regeneration of the extracellular matrix is maintained, in part, by the action of extracellular proteolytic enzymes that are secreted by local cells. Most of these enzymes are matrix metalloproteinases (MMPs), which depend on the binding of Ca 2? or Zn 2? for their activity (Sternilicht and Werb 2001). During oogenesis, the extensive changes in the ovarian extracellular environment have been largely attributed to the action of MMPs (Curry and Osteen 2003). The MMPs play an important role in the ovulatory process in different vertebrate groups, acting in the follicular rupture, fragmentation of the basement membrane and connective fibres of the follicle (Curry and Osteen 2003; Ogiwara et al. 2005). The role of MMPs in the dynamics of the reproductive cycle for teleosts has been few investigated. The expressions of gelatinase A (MMP-2) and B (MMP-9) were detected in the ovaries of Oryzias latipes (Matsui et al. 2000; Ogiwara et al. 2005). The follicular growth and ovulation as well as the involution of postovulatory follicles of ovarian follicles are accompanied by changes in the extracellular matrix. These changes can influence the follicular maturation, the cell survival, and steroidogenesis. However, there are no studies that identify the cells responsible by the MMPs synthesis and distribution of elements of the extracellular matrix in teleosts. In this context, this study used immunohistochemistry to characterise the distribution of laminin b2, collagen type IV, fibronectin and MMP-9 in follicular development until involution post-spawning. Follicular apoptosis was assessed by TUNEL in situ, and the plasma 17b estradiol levels by immunofluorometry. Materials and methods Animals A total of 20 females of the Prochilodus argenteus, a commercial fish from the São Francisco river basin, were utilised. The followed reproductive stages were assessed: resting (n = 3), ripe (n = 4), recently post-spawning (n = 5), 48 h post-spawning (n = 4) and 96 h postspawning (n = 4). Since P. argenteus does not naturally spawn in captivity, ripe females (36.8 ± 0.3 cm total length; 0.6 ± 0.05 kg body weight) were induced to spawn in order to obtain spawned ovaries. The fish received two doses of crude carp pituitary extract (CCPE) with a 12 h interval between doses according to methodology previous established for the species (Sato et al. 1996). Following spawning, females were kept in concrete tanks with a continuous water flow at 24 C until 96 h post-spawning. Fishes were anaesthetised with tricaine methanosulfonate (Sigma 0.17 g/l) for data collection. Histology Ovarian samples were fixed in Bouin s fluid for 6 h at room temperature. The samples were subsequently embedded in paraffin or glycol methacrylate, sectioned with 5 lm thickness, and stained with hematoxylin-eosin (HE), Gomori trichrome or toluidine blue-sodium borate (BT). Some sections were also processed for the periodic acid-schiff (PAS) reaction and counterstained with hematoxylin. TUNEL in situ reaction Ovarian samples were fixed in 4% paraformaldehyde solution in 0.1 M phosphate buffer (PBS) at ph 7.3 for 24 h, embedded in paraffin, sectioned with 6 lm thickness and submitted to TUNEL assay by using the TdT FragEL DNA fragmentation detection kit (Calbiochem). The sections were briefly incubated with terminal deoxynucleotidyl transferase (TdT) and biotin-conjugated deoxinucleotides for 3 h at 37 C, followed by streptavidin conjugated with peroxidase for 45 min in a humidified chamber at room temperature. Finally, the reaction was developed with 123

40 J Mol Hist (2010) 41: diaminobenzidine (DAB) for 2 min and followed by counterstaining with hematoxylin. For the negative control, the treatment with TdT labelled-deoxinucleotides was omitted. For inactivation of endogenous peroxidase and permeabilisation of cell membranes, hydrogen peroxide 3% and proteinase K were used, respectively. Immunohistochemistry For the detection of MMP-9, collagen type IV, laminin b2, fibronectin and actin (Table 1), ovarian samples were fixed in DMSO/methanol (2:8), embedded in paraplast (Sigma) and sectioned with 6 lm thickness. For antigen recovery, two methods were used: enzymatic with proteinase K for 10 min at room temperature for laminin b2 and fibronectin and heat-induced epitope retrieval with sodium citrate buffer (10 mm Sodium Citrate, 0.05% TWEEN 20, ph 6.0) for 30 min at 95 C for MMP-9 for collagen type IV and actin. After washing in phosphate-buffered saline (PBS), the sections were incubated with 3% hydrogen peroxide in methanol to inactivate endogenous peroxidase and subsequently with blocking buffer (BSA 2%? TRI- TON X-100? TWEEN 20) for 30 min to allow permeabilisation and block unspecific staining. Primary antibodies were applied to the sections overnight at 4 C. For immunohistochemical staining, two methods were used: immunoperoxidase detection with horseradish peroxidase (HRP) kit LSAB 2 System HRP (DakoCytomation, Carpinteria, CA., USA) visualised by diaminobenzidine (DAB) counterstained with hematoxylin and immunofluorescent detection with secondary antibodies ALEXA 488 anti-rabbit (1:200) and ALEXA 568 anti-mouse (1:200) and DAPI (1:500) were used for DNA labelling. A negative control was performed by omitting treatment with the primary antibody. Morphometric analyses The follicular apoptosis index (apoptotic cells/total cells in the follicle) was established in histological sections subjected to the TUNEL technique. The means of the follicular apoptosis index (AI) values were calculated by using 30 follicles at each stage of follicular development: resting, ripe, recently post-spawning, 48 and 96 h post-spawning. The TUNEL-positive cells that also presented morphological alterations were identified as apoptotic cells (Santos et al. 2005). The immunostained area of laminin b2, collagen type IV and MMP-9 was calculated by digital image analysis using ImagePro-Plus (Media Cybernetics, Silver Spring, MD). Images were captured through an Axiostar Carl Zeiss microscope fitted with a Sony Cyber-shot DSC- W1 camera. A series of 15 random images of postovulatory follicles from different specimens were taken for each immunostained parameter to obtain a mean value for statistical comparison. The brown stain was selected, a colour mask was made, and then applied, equally, to all images and measurements were taken. Only the percentage of the postovulatory follicle correspondent to immunostained area was computed. Plasma 17b estradiol levels Samples of blood ( ml) from females at resting, ripe and recently post-spawning, 48 and 96 h post-spawning were collected through caudal vein puncture. Aliquots of the serum were kept at -20 C and the 17b estradiol levels were determined by indirect immunofluorimetric assay using the Estradiol AutoDELFIA Ò test (PerkinElmer Waltham, Massachusetts, USA) with sensitivity of 13.6 pg/ ml. Statistics Statistical analysis was performed using GraphPad InStat software, version 3.05 (San Diego, CA., USA). Values were expressed as means ± SEM, and results were considered significant at the 95% confidence interval. The Kruskal Wallis test followed by the Dun s test was used to compare the means of the follicular apoptosis index. The ANOVA followed by the Tukey s test were used to compare to 17b estradiol levels, and also to the immunostaining reactions. Graphics were produced using GraphPad Prism software, version Table 1 Primary antibodies, suplliers, and dilutions Antibodies Clone Dilution Supplier Antigen recovery Anti-MMP-9 (2C3) monoclonal-mouse 1:10 sc Heat-induced epitope retrieval Anti-collagen type IV (H-234) polyclonal-rabbit 1:100 sc Heat-induced epitope retrieval Anti-laminin b2 (H-300) polyclonal-rabbit 1:50 sc Enzymatic Anti-fibronectin (H-300) polyclonal-rabbit 1:25 sc-9068 Enzymatic Anti-actin (C-2) monoclonal-mouse 1:100 sc-8432 Heat-induced epitope retrieval 123

41 218 J Mol Hist (2010) 41: Results Follicular development In the resting ovaries of P. argenteus, the ovigerous lamellae showed oogonia nests and perinucleolar follicles, with oocytes presenting basophilic cytoplasm, a central vesiculous nucleus and several peripheral nucleoli (Fig. 1a, b). These oocytes were surrounded by squamous follicular cells and thin connective theca (Fig. 1c). During advanced maturation the ovaries showed vitellogenic follicles in the ovigerous lamellae, characterised by oocytes filled with acidophilic yolk globules, a central nucleus and also surrounded by squamous follicular cells (Fig. 1d, e). The ovaries reached full maturation characterised by displacement of the nucleus of vitellogenic oocytes towards the micropyle (Fig. 1e). The PAS-positive reaction (Fig. 1f, g) was detected in zona radiata, granulations between the yolk globules, basement membrane, and eosinophilic granulocytes. The development of the zona radiata was initiated in the advanced perinucleolar stage, becoming a thick layer in the vitellogenic oocyte. During follicular development, the follicular cells were supported by a thin and continuous basement membrane. Immunolabelling for extracellular matrix proteins, laminin b2 (Fig. 2a d) and collagen type IV (Fig. 2e) were detected in the basement membrane of the oogonia nests, ovigerous lamellae and ovarian follicles during the different development stages. In resting ovaries, the MMP-9 was detected in the muscle cells of arteries, although in the oogonia nests and perinucleolar follicles, the MMP-9 showed no immunoreactivity (Fig. 2f). In the vitellogenic follicles, the MMP-9 labelling was observed in zona radiata inner, micropyle cell, and some follicular cells (Fig. 2g i). Fig. 1 Histological sections of P. argenteus ovaries stained with toluidine blue (a, b, d), Gomory trichrome (c), hematoxylin-eosin (e) and periodic acid-schiff (PAS)-hematoxylin (f, g). a, b Ovary in resting stage with perinucleolar follicles (O1 and O2) and oogonia (Oo) with pre-follicular cells (PF). c Perinucleolar follicle surrounded by squamous follicular cells (arrows) and thin connective theca (CT). d Ovary in advanced stage of maturation with a predominance of vitellogenic follicles (O4). e Nucleus (N) displaced towards the micropyle (M) during final oocyte maturation. f Histochemical PASpositive reaction in perinucleolar follicle with a thin zona radiata (ZR) and basement membrane (BM). g Histochemical PAS-positive reaction in vitellogenic follicle with a zona radiata (ZR) thickened and thin basement membrane (BM), eosinophilic granulocytes (arrowhead). Bars 20 lm (a c, e, f and g), 100 lm (d) Fig. 2 Immunohistochemical for laminin b2 (a d), collagen type IV (e) and MMP-9 (f i) in ovarian follicles of P. argenteus. Laminin b2 was detected through immunofluorescent-green (a) and immunoperoxidase (b d) and collagen type IV immunofluorescent-green (e) in the basement membrane of the oogonia nests (c, e), ovigerous lamellae (d) and ovarian follicles (a, b). The MMP-9 was detected in the muscle cells (MC) of arteries in resting ovary, and in zona radiata inner (ZRI), micropyle cell (M), and some follicular cells (arrows) in the vitellogenic follicles (O4). BM = basement membrane; Oo = oogonia nests; LO = ovigerous lamellae; O1 and O2 = perinucleolar follicles; ZR = zona radiata. Bars 20 lm 123

42 J Mol Hist (2010) 41: Postovulatory follicular regression In recently spawned ovaries, postovulatory follicles had a large, irregular lumen covered by a layer of hypertrophied follicular cells (Fig. 3a) with an exuberant peripheral ring of actin (Fig. 3b) and a poorly developed connective theca with eosinophilic granulocytes (Fig. 3c). The follicular cells of postovulatory follicles, recently spawned, were supported in thin and continuous basement membrane (Fig. 3d). During the regression of postovulatory follicles, the follicular cells returned to the squamous shape with a dense nucleus (Fig. 3e) and disorganised actin cytoskeleton (Fig. 3f). Gradually, the connective theca became thicker and the follicular lumen was occluded (Fig. 3e). During the postovulatory regression (48 and 96 h), the basement membrane became diffuse, discontinuous and with a degradation aspect (Fig. 3g, h). Other basement membranes were also very evident in the theca of the postovulatory follicles and supporting endothelium of blood vessels. Continuous labelling to laminin b2 (Fig. 4a, b) and collagen type IV (Fig. 4c) was detected in the basement membrane of the recently spawned postovulatory follicles. Both proteins showed discontinuous labelling in the postovulatory follicles with 48 h and 96 h post-spawning (Fig. 4d, e). In the postovulatory follicles, the MMP-9 was detected in the follicular cells, increasing in intensity in 48 h. In the theca, cells looking like smooth muscle cells of Fig. 3 Immunohistochemical reaction for actin (b, f) and histological sections of spawned ovary in P. argenteus stained with Gomory trichrome (a, e), hematoxylin-eosin (c) and periodic acid-schiff (PAS)-hematoxylin (d, g, h). a Postovulatory follicle recently spawned, showing a wide convoluted lumen and hypertrophied follicular cells. b Postovulatory follicle recently spawned, showing with an exuberant peripheral ring of actin (red) in follicular cells and nucleus (blue). c Eosinophilic granulocytes (arrowhead) in postovulatory follicles. d Histochemical PAS-positive reaction in recently spawned postovulatory follicle with a thin basement membrane (BM) and eosinophilic granulocites (arrowhead). e Postovulatory follicle, 96 h after spawning, showing a partially occluded follicular lumen, squamous follicular cells and thick connective theca. f Postovulatory follicle 96 h after spawning with disorganised actin cytoskeleton (red). Histochemical PAS-positive reaction in postovulatory follicle 48 h (g) and 96 h (h) after spawning evidenced by the fragmentation of the basement membrane (BM). L = lumen; arrows = follicular cells. Bars 20 lm Fig. 4 Immunohistochemical reaction for laminin b2 (a, b, e), collagen type IV (c, d), fibronectin (h, i) and MMP-9 (f, g) in postovulatory follicles of P. argenteus. Laminin b2 was detected through enzymatic methods (a, b, e) and collagen type IV fluorescentgreen methods (c, d) in the basement membrane (BM) of the postovulatory follicles (POF). Fibronectin was detected through fluorescent-green methods, (h, i) in the basement membrane (BM) and surface of follicular cells (arrows). The MMP-9 was detected in follicular cells (arrows), theca cells looking like smooth muscle cells (TC) and eosinophilic granulocytes (arrowhead) of the postovulatory follicles (f) and 48 h after spawning (g). BV = blood vessel. Bars 20 lm (b i), 100 lm (a) 123

43 220 J Mol Hist (2010) 41: Fig. 5 Immunostained area of laminin b2, collagen type IV and MMP-9 of the postovulatory follicles in P. argenteus. Note the decrease of immunostained area for laminin b2, collagen type IV until 96 h after spawning, while the MMP-9 immunostained area showed a peak at 48 h post-spawning. Values represent means ± SEM. Asterisks indicate that means differ significantly (P [ 0.05) Fig. 7 Follicular apoptosis index in resting (O1), ripe (O4) and postovulatory follicles = POF (0, 48 and 96 h) in P. argenteus. Note the peak of the apoptotic index at 48 h post-spawning. Values represent means ± SEM (n = 30 follicles at each time point). Different letters indicate that means differ significantly (P [ 0.05) Fig. 6 TUNEL-positive reaction during follicular development and postovulatory regression in P. argenteus. a Perinucleolar follicle (O2). b Vitellogenic follicle (O4). c Recently spawned postovulatory follicle. d Postovulatory follicle 48 h post-spawning. e Postovulatory follicle 96 h post-spawning. L = lumen; arrows = follicular cells; arrowheads = theca cells. Bars 20 lm postovulatory follicles and eosinophilic granulocytes also showed immunoreactivity for MMP-9 (Fig. 4f, g). Fibronectin in connective tissue and basement membrane of endothelium was immunolocalised. In the postovulatory follicles the fibronectin was detected on the basement membrane, surface of follicular cells, and connective tissue (Fig. 4h, i). Morphometric analyses of the immunostained laminin b2 and collagen type IV in the postovulatory follicles showed that both proteins decreased gradually from zero to 96 h and MMP-9 presented a statistical peak at 48 h (Fig. 5). Apoptosis and plasma 17b estradiol levels Reaction TUNEL-positive that allowed to evidence of the DNA fragmentation (Fig. 6a e) associated with morphological characteristics, such as condensation of the Fig. 8 Plasma 17b estradiol level concentrations in ovaries in resting, ripe and post-spawning = PS (0, 48 and 96 h) in P. argenteus. Note a peak of the plasma 17b estradiol levels in ripe followed by abrupt drop soon after spawning (0 h). Values represent means ± SEM. Different letters indicate that means differ significantly (P [ 0.05) chromatin, cell shrinkage and fragmentation similar to apoptotic bodies, allowed the apoptosis identification in the follicular and theca cells. The apoptosis follicular index (Fig. 7) increased from perinucleolar follicles to postovulatory follicles with 48 h post-spawning, decreasing with 96 h post-spawning but did not show a significant statistical difference between 48 and 96 h (P \ 0.05). The plasma 17b estradiol levels (Fig. 8) increased statistically from resting to ripe, and decreased abruptly soon after post-spawning. Moreover, the plasma 17b estradiol levels in the females with 48 and 96 h post-spawning do not show a statistical difference when compared to females in the resting stage (P \ 0.05). Discussion This study reported for the first time the immunolocalisation of laminin b2, collagen type IV, fibronectin and MMP- 123

44 J Mol Hist (2010) 41: and their relation to plasma 17b estradiol levels and follicular apoptosis during the remodelling of the ovarian follicles for teleosts before and post-spawning. Immunohistochemical studies in fish have been made using mammalian antibodies, based on evolutionary conservation of proteins (Ortiz-Delgado and Sarasquete 2004; Sharma and Kinsey 2006; Câmara-Pereira et al. 2009; Santos et al. 2009). Although the development and morphological characteristics of ovarian follicles before and after the spawning of P. argenteus are similar to those of other Prochilodontidae such as Prochilodus costatus (Thomé et al. 2006), Prochilodus scrofa (Romagosa et al. 1985) and other teleosts (Grier et al. 2007), the follicular remodelling and molecules involved in these processes had not yet been analysed. The morphological characteristics of the follicular cells can be different during oocyte maturation depending on the species. In P. argenteus, similar to most Characiformes, follicular cells remained squamous throughout oocyte development (Quagio-Grassiotto and Guimarães 2006), unlike some Siluriformes which show follicular cells that are squamous in the perinucleolar follicles and prismatic in vitellogenic follicles (Santos et al. 2006). In the post-spawning ovary of the P. argenteus, the follicular cells become prismatic, but returned to the squamous form at the end of the process of involution of the postovulatory follicles, similar to observed in Astyanax bimaculatus (Drummond et al. 2000) and Leporinus taeniatus (Santos et al. 2005). In the present study, an exuberant ring of actin in follicular cells of postovulatory follicles, recently spawned, was observed probably supporting the prismatic shape of these cells and may be related to the first stage of apoptosis. In fact, according to Ndozangue-Touriguine et al. (2008), in the first stage of apoptosis, the actin is reorganised into a peripheral ring, making an association with myosin leading to contraction and formation of blebs, facilitators of the interaction with phagocytic cells. On the other hand, the disorganisation of actin filaments observed in follicular cells of postovulatory follicles of the P. argenteus with 96 h post-spawning may be related to the final step of apoptosis, probably due to depolymerisation of actin. This supposition is reinforced by reports that observed the dismantlement of the apoptotic cells into apoptotic bodies after actin depolymerisation (Korsnes et al. 2007; Ndozangue-Touriguine et al. 2008). Moreover, caspase-mediated cleavage of tensin contributes to the disruption of actin organisation and interrupts extracellular matrix-mediated survival signals through phosphatidylinositol 3-kinase (Kook et al. 2003). The TUNEL reaction has been widely used for the labelling of fragmented DNA of apoptotic cells in teleost fishes (Drevinick et al. 2006; Santos et al. 2009), amphibians (Hensey and Gauta 1998), reptiles (Lang et al. 2002), birds (Ito et al. 2002) and mammals (Svoltys et al. 2000). In the present study, the follicular apoptosis was also identified by TUNEL reaction in follicular and theca cells of the perinucleolar, vitellogenic and postovulatory follicles. During the follicular ovarian development in P. argenteus, low values of the apoptosis index indicated that apoptosis has a physiological role, controlling cell numbers and eliminating unwanted cells. In contrast, high values of the apoptosis index were observed in stress conditions compromising the follicular ovarian viability (Drevinick et al. 2006). In the postovulatory follicles of the P. argenteus, the apoptosis index increased progressively until two days post-spawning and decreased thereafter, suggesting that apoptosis contributes greatly to the removal of follicular cells in postovulatory follicles, which has also been observed in other teleost fishes (Drummond et al. 2000; Kaptaner and Ural 2006; Santos et al. 2009; Thomé et al. 2009). During follicular development, the survival of follicular cells ensures that the oocyte can ripen and become suitable for reproduction. The testosterone produced in special cells in the theca is converted by follicular cells into 17b estradiol, responsible for stimulating the hepatocytes to produce vitellogenin and zona radiata proteins (Senthilkumaran et al. 2004). In the present study, plasma 17b estradiol levels showed their highest values in the advanced maturation of the ovarian follicle, supporting the oocyte viability. In this sense, studies in ovarian follicles cultured from rainbow trout, O. mykiss, showed that the addition of 17b estradiol in ovarian follicle incubation caused suppression of apoptotic DNA fragmentation compared to the control without hormone (Janz and Van Der Kraak 1997). The abrupt drop of the plasma 17b estradiol levels soon after P. argenteus spawned, could have influenced the increase of follicular apoptosis until 48 h post-spawning, similarly reported in C. tarichi (Kaptaner and Ural 2006). Together, these results suggest that high levels of the plasma 17b estradiol levels can act as an anti-apoptotic factor during follicular development and oocyte maturation. In addition, the decrease of the 17b estradiol levels post-spawning could be contribute to the increase of the apoptosis during postovulatory regression stage, i.e., 48 h post-spawning of the P. argenteus. Eosinophilic granulocytes were widely dispersed in the stroma and ovarian follicles of P. argenteus in all stages of postovulatory follicles involution. Eosinophils, neutrophils, and basophils showed immunoreactivity to MMP-9 in mammalian reproductive tissue (Hulboy et al. 1997; Suzukawa et al. 2006). Probably, the activity of MMP-9 in granulocytes can be related to cell migration, facilitating the passage of these cells by the extracellular matrix of theca connective (Korpos et al. 2009). Leukocytes were 123

45 222 J Mol Hist (2010) 41: also observed in ovaries of other teleosts (Drummond et al. 2000; Grier et al. 2007) which may be involved in the phagocytosis of apoptotic bodies, but their contribution to tissue remodelling in the ovaries of teleosts is not well understood. Basement membranes of ovarian follicles, both before and after the spawning of P. argenteus, were evidenced by the PAS-positive reaction. The PAS technique also revealed other basement membranes, associated with theca cells that looked like smooth muscle cells, and endothelium coinciding with the observations of Van Wezel et al. (1998) in ovarian cattle follicles. Immediately after P. argenteus spawned, the smooth muscle cells showed immunoreactivity to MMP-9. According to Jalabert (2005) these cells can be related to follicular lumen occlusion, and actively participate in the follicular reorganisation, breaking down the collagen fibre framework of the theca. In P. argenteus, laminin b2 and collagen type IV were identified as the major constituents of the basement membrane of the ovarian and postovulatory follicles. The integrity of the basement membrane, evidenced by the continuous labelling of the lamimin b2 and collagen type IV, allowed for the development of ovarian follicles, results also observed in the ovaries of mammals (Rodgers et al. 2003; Berkholtz et al. 2006a). In addition, the cellbasement membrane interaction allowed an increase of 17b estradiol production by follicular cells, similarly observed in mammals (Berkholtz et al. 2006b). In sheep, the association of integrin a6b1 of the granulosa cell with laminin from the basement membrane promoted the increased production of estradiol that acts like an anti-apoptotic factor (Le Bellego et al. 2002). In the present study, discontinuous labelling to the laminin b2 and collagen type IV in basement membrane of the postovulatory follicles at 96 h post-spawning indicates that the degradation of basement membrane is due to lysis of the major constituents. The loss of integrity of the basement membrane coincided with the increase of follicular cell apoptosis, suggesting its influence on cell survival of postovulatory follicles. In the postovulatory follicles of P. argenteus, morphological alterations of the basement membrane, and decrease of integrin b1 and collagen type IV may contribute to the increase of follicular cell apoptosis (Santos et al. 2009). A study conducted in another Prochilondontidae, P. costatus, also indicated a relationship between structural changes in the basal membrane and occurrence of apoptosis in follicular cells (Thomé et al. 2006). In cultured mammary cells, an intact basement membrane supports the differentiated cell phenotype and suppresses apoptosis, whereas degradation of the basement membrane leads to a loss of cell function and the induction of apoptosis (Boudreau et al. 1996). Fibronectin was immunolocalisated in follicular cells of postovulatory follicles of P. argenteus, suggesting that these cells can be responsible by fibronectin synthesis. In fact, fibronectin, and laminin has been demonstrated as extracellular matrix protein synthesized by follicular cells (Carnigie 1990; Zhao and Luck 1995). In the present study, fibronectin was also observed as a delicate labelling in the basement membrane of postovulatory follicles of P. argenteus, a fact also related to those in antral and secondary follicles of mice (Berkholtz et al. 2006a). The labelling of fibronectin on the surface of follicular cells of the postovulatory follicle of P. argenteus can be explained by the fact that the fibronectin domains have to interact with collagen type IV and integrin from the cell surface, it being important for maintaining normal cell adhesion to extracellular matrix (Rodgers et al. 2003). According to Iwahashi et al. (2000) the collagen type IV detected in connective theca may be involved with the organisation of the extracellular fibronectin. This interaction between collagen type IV and fibronectin can act in cell migration that occurs during the late remodelling of postovulatory follicles of P. argenteus. In this study, involvement of MMP-9 in tissue remodelling was analysed, and the results showed that this enzyme is present in vitellogenic and postovulatory follicles. The MMPs are related to ovulation in different vertebrate groups including fish, and may be responsible for fragmentation of the zona radiata and the extracellular matrix of connective theca (Curry and Osteen 2001, Smith et al. 2002; Ogiwara et al. 2005). In the ovaries of P. argenteus, the fragmentation of the basement membrane in the postovulatory follicles with 48 and 96 h post-spawning may be related to the synthesis of MMP-9 by follicular cells detected by immunohistochemistry. Similarly, Matsui et al. (2000) identified, through analysis of mrna expression (PCR), MMP-9 in the postovulatory tissue of O. latipes, highlighting the importance of this enzyme in the involution of postovulatory follicles. In conclusion, our results indicated that the structure and composition of the extracellular matrix, and the plasma 17b estradiol levels related to apoptosis play an important role during the follicular development and post-spawning involution in teleost fishes. The integrity of the basement membrane is important for the survival of follicular cells, and loss of integrity coincides with increased follicular apoptosis. Furthermore, the MMP-9 could be involved in final oocyte maturation and regression of postovulatory follicles. The ovary of the teleost fishes proved to be a useful system for studies about extracellular matrix-cell interactions and ovarian morpho-physiology. Acknowledgment This work was supported by CNPq, FAPEMIG, CAPES and CODEVASF. 123

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48 Tissue and Cell 44 (2012) Contents lists available at SciVerse ScienceDirect Tissue and Cell j our na l ho me p age: Apoptosis, cell proliferation and vitellogenesis during the folliculogenesis and follicular growth in teleost fish R.G. Thomé a, F.F.T. Domingos a, H.B. Santos b, P.M. Martinelli a, Y. Sato c, E. Rizzo a, N. Bazzoli d, a Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, P.O. Box 1686, CEP Belo Horizonte, Minas Gerais, Brasil b Universidade Federal de São João Del Rei, Av. Sebastião Gonç alves Coelho, 400, CEP Divinópolis, Minas Gerais, Brasil c Estaç ão de Hidrobiologia e Piscicultura de Três Marias (CODEVASF), P.O. Box 11, CEP Três Marias, Minas Gerais, Brasil d Programa de Pós-graduaç ão em Zoologia de Vertebrados (PUC-minas), Av. Dom José Gaspar 500, CEP BeloHorizonte, Minas Gerais, Brasil a r t i c l e i n f o Article history: Received 19 August 2011 Received in revised form 4 November 2011 Accepted 8 November 2011 Available online 6 December 2011 Keywords: Apoptosis PCNA Cathepsin-D P. argenteus São Francisco River a b s t r a c t Aiming to better understand folliculogenesis, this study evaluated cell death and proliferation of ovarian cells, besides cathepsin-d expression in Prochilodus argenteus captured in two sites of the São Francisco River downstream from the Três Marias Dam, Brazil. In the site immediately following the Dam (S1), low levels of dissolved oxygen were registered in the rainy period. The water temperature was higher in the São Francisco River immediately after the confluence with the Abaeté River (S2), regardless of the period. In S1, the ovaries showed smaller oocytes, high caspase-3 enzymatic activity and apoptosis, lower cells in proliferation and GSI, as well as a lesser quantity of cathepsin-d when compared to females captured from S2. Regarding relative frequency of ovarian structures, in the dry period, only oogonia and perinucleolar oocytes were found in fish ovaries from both sites. On the other hand, in the rainy period, the relative frequency of oogonia and perinucleolar oocytes decreased and the vitellogenic oocytes increased in S2. Postovulatory follicles were observed only in S2, whereas atretic follicles occurred at a higher frequency in S1. Our results showed that apoptosis, cell proliferation and cathepsina-d evaluation can be used as biomarkers of environmental impact Elsevier Ltd. All rights reserved. 1. Introduction In most teleost fish, at the onset of each reproductive cycle a fraction of oogonia start a series of mitotic divisions for renewal or enter meiosis becoming oocytes, which develop inside the ovarian follicles (Grier et al., 2009). The basic structure of the ovarian follicle is established when the prefollicular cells associated at oocyte with the basal membrane surrounded (Patiño and Sullivan, 2002; Thomé et al., 2010). Throughout folliculogenesis, the survival of the follicular cells ensures the oocyte growth and maturation (Quagio- Grassiotto and Guimarães, 2006). The follicular development can be divided into three stages: primary growth, secondary growth, and oocyte final maturation (Selman and Wallace, 1989; Tyler and Sumpter, 1996). The primary growth includes the perinucleolar stage and the formation of cortical alveoli. The vitellogenesis occurs during secondary growth, and is characterised by the accumulation of yolk globules in the ooplasm (Grier, 2000; Patiño and Sullivan, 2002). During the vitellogenesis, lysosomal enzymes such as cathepsin-d cleave the vitellogenin into smaller molecules which will form the yolk (Carnevali et al., 2006). The cathepsin-d Corresponding author. Tel.: address: bazzoli@pucminas.br (N. Bazzoli). gene expression occurs during oocyte secondary growth, mainly in the initial stage of vitellogenesis (Carnevali et al., 2008). During folliculogenesis and follicular development, the balance between the proliferation and cell death signals determines the fate of the ovarian follicles ovulation or follicular atresia (Krysko et al., 2008). Programmed cell death or apoptosis is an important physiological process to maintain an appropriate number of cells in the tissue (Kerr et al., 1972), and may be triggered by several factors, including heavy metals and thermal stress (Drevinick et al., 2006; Tseng et al., 2006). Apoptosis is conserved during evolution and requires specialised cell machinery involving a family of proteases called caspases, which are found in teleosts (Takle and Andersen, 2007). These enzymes are divided as initiator caspase-2, -8, -9, -10 and effector caspase-3, -6, -7 (Fischer et al., 2005). The role of apoptosis in the teleost reproduction has been investigated in the selection and recruitment of follicles for vitellogenesis (Janz and Van Der Kraak, 1997), development of ovarian follicles (Wood and Van Der Kraak, 2001), and ovarian involution after spawning (Drummond et al., 2000; Thomé et al., 2006; Santos et al., 2008). The proliferating cell nuclear antigen (PCNA) is a 36 kda nonhistone protein, also known as cyclin (Bravo and Graf, 1985; Bravo and MacDonald-Bravo, 1987). During the cell cycle, PCNA is synthesised at the G1 stage, it reaches its highest levels during stage S and decreases during G2 and mitosis (Kurki et al., 1988). Some studies /$ see front matter 2011 Elsevier Ltd. All rights reserved. doi: /j.tice

49 R.G. Thomé et al. / Tissue and Cell 44 (2012) have used the PCNA expression of as a useful tool for identifying cell proliferation during gametogenesis and embryogenesis of vertebrates (Hutt et al., 2006; Leung et al., 2005; Loppion et al., 2008). The immunohistochemical reaction for PCNA has been detected in follicular cells during follicle growth (Korfsmeier, 2002; Quagio- Grassiotto et al., 2011) and in theca cells of atretic follicles (Santos et al., 2009). Studies about the reproductive biology and the cascade of molecular events in gametogenesis and fecundation are considered important parameters in conservation of biodiversity (Wildt and Wemmer, 1999). Fish are sensitive to environmental factors and present diverse reproductive strategies, which imply different types of oocyte development and spawning (Jalabert, 2005). Despite the differences in the synchronism of growth and egg formation, morphological and physiological characteristics of oocyte development are common to most teleosts (Lubzens et al., 2010). The curimatã-pacu Prochilodus argenteus is a species of commercial interest and has an illiophagous feeding habit (Sato and Godinho, 2003). However, in recent years, stocks of this species have reduced in several regions of the São Francisco River basin due to fishing individuals below the size limit established by Brazilian legislation for marketing in the form fillets fish (Godinho et al., 2003). Despite its commercial and ecological importance, there are few studies on natural populations of P. argenteus. It is known that the species presents altered levels of sex steroids and does not reproduce immediately downstream of the Três Marias Dam, only reproducing successfully after the confluence of tributaries (Sato et al., 2005; Arantes et al., 2010). However, there are no studies that related the cell proliferation, apoptosis and cathepsin-d during the folliculogenesis with the reproduction success of the curimatã-pacu in nature. In this context, the present study aims to evaluate proliferation and cell death, besides cathepsin-d expression in P. argenteus, in order to propose biological parameters for conservation, sustainable commercialisation and to contribute to the understanding of the follicular development mechanism in teleost fish. 2. Materials and methods 2.1. Study area In the Três Marias region the dry and rainy periods are well defined. The driest quarter corresponds to the months of June, July and August and the rainiest quarter to the months of November, December and January (ONS, 2009). The study analysed fish from two sites of the São Francisco River downstream from the Três Marias Dam: site 1 (S1) immediately following the dam (18 11 S, W) and site 2 (S2) in the confluence with the Abaeté River (18 02 S, W). The temperature of the water and the dissolved oxygen concentration were recorded during the sampling in the morning, using a Horiba model W-22XD instrument Sampling A total of 45 P. argenteus females were sampled in sites S1 (n = 25) and S2 (n = 20) from 2008 to The following biometric data were recorded for each specimen: total length (TL), body weight (BW), gonad weight (GW) and, additionally, the gonadosomatic index (GSI = 100GW/BW) was calculated Histology and morphometry Ovary samples of each specimen were fixed in Bouin s fluid for 12 h, embedded in paraffin, sectioned at 5 m thickness and stained with hematoxylin-eosin or Gomori trichrome for histological and morphometric analyses. The diameters of 100 perinucleolar follicle taken from each site/dry period and 100 vitellogenic follicles taken from each site/rainy period were measured in histological ovarian sections using a micrometric ocular attached to a light microscope. Only intact oocytes with low retraction and which had sections passing through the nucleus were measured. The relative frequency of oogonia (Oo), perinucleolar (PNF), previtellogenic (PVF), vitellogenic (VF), postovulatory (POF) and atretic (AF) follicles, was determined from histological ovarian sections of 8 specimens from each site during the peak of the dry and rainy periods TUNEL assay Ovary samples (n = 5/site in dry period) were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer ph 7.3 for 24 h at 4 C, embedded in paraffin, sectioned at 4 m thickness and subjected to the in situ TUNEL technique for detection of DNA fragmentation of the cells in apoptosis (ApopTag Plus Peroxidase In situ Millipore, USA). In order to achieve this, histological sections were incubated with a mixture of the enzyme terminal deoxynucleotide transferase (TdT) and biotinylated deoxynucleotide conjugates in a humid chamber at 37 C for 3 h. Then, a solution of streptavidin peroxidase conjugate in a humid chamber at room temperature was applied for 45 min. The peroxidase reaction was revealed with diaminobenzidine (DAB) for 2 min at room temperature. The slides were counter-stained with hematoxylin. For negative control, one of the slides did not receive the mixture of TdT and deoxynucleotide. For permeabilisation and blocking of endogenous peroxidase, proteinase K and hydrogen peroxide 3% were used respectively before the TUNEL reaction Caspase-3 colorimetric assay In order to detect the enzymatic activity of caspase-3 in ovaries, the colorimetric test from R&D Systems was used. Ovary samples (n = 10/site in dry period) were weighed and lysed with 0.5 ml of the cell lysis buffer. The homogenate was centrifuged for 1 min at 10,000 g. The supernatant was kept and incubated with substrate of caspase-3 (DEVD-pNA) at 37 C for 2 h. The absorbance of each sample at 405 nm was recorded and the caspase-3 activity level was directly proportional to the colour reaction Immunohistochemistry Ovary samples (n = 5/site in dry period to PCNA; n = 5/site in rainy period to cathepsin-d) were fixed in Bouin s fluid, embedded in paraplast (Sigma) and sectioned at 4 m thickness. For the antigen retrieval, we used a pre-treatment with citrate buffer ph 6.0 at 95 C for 30 min. After washing with PBS, a buffer with BSA 2% + Triton X Tween 20 was used to block the non-specific reactions and tissue permeabilisation, and 3% H 2 O 2 for blocking of endogenous peroxidase. The sections were incubated with primary antibody overnight at 4 C PCNA (PC10 anti-mouse by Santa Cruz Biotechnology, 1:80) and cathepsin-d (H-75 anti-rabbit by Santa Cruz Biotechnology, 1:25). The sections were incubated with secondary antibody together with biotin (LSAB-DAKO kit) and the immunohistochemistry reaction was revealed with DAB. For negative control, one of the slides did not receive primary antibody Western blotting In order to determine the specificity of the antibodies used in immunohistochemistry and to evaluate their expression, 100 mg of the ovary samples (n = 5/site in dry period to PCNA; n = 5/site in rainy period to cathepsin-d) were lysed with lysis buffer with protease inhibitors. The homogenate was centrifuged at 120,000 g for 30 min and the supernatant, containing the proteins, was utilized.

50 56 R.G. Thomé et al. / Tissue and Cell 44 (2012) Table 1 Biometric, gonadosomatic index and morphometric analysis of P. argenteus females captured in two sites of the São Francisco River: S1 immediately following the Três Marias Dam and S2 in the confluence with Abaeté River. N Dry period Rainy period S1 S2 S1 S TL (cm) ± 1.14 a ± 1.57 a ± 1.53 a ± 3.88 a BW (kg) 0.56 ± 0.05 a 0.77 ± 1.57 a 1.29 ± 0.13 a 1.56 ± 0.38 a GSI (%) 0.18 ± 0.02 a 0.23 ± 0.03 a 3.96 ± 1.38 a 8.84 ± 2.53 b PN1 ( m) ± c0 a ± 4.34 b PN2 ( m) ± 3.40 a ± 4.90 b VF ( m) ± a ± b Data obtained were expressed as means ± SEM. Different letters indicate significant differences between sites (p < 0.05). TL, total length; BW, body weight; GSI, gonadossomatic index;, follicle diameter, PN1, initial perinucleolar; PN2, advanced perinucleolar; VF, vitellogenic follicle. The proteins were added to 12% electrophoresis gel transferred to a nitrocellulose membrane and subsequently submitted to blocking of the non-specific reaction with 1% BSA. Following, the membrane was incubated in Snap-id system for 20 min with the primary antibodies PCNA (PC10 anti-mouse by Santa Cruz Biotechnology, 1:300) and cathepsin-d (H-75 anti-rabbit by Santa Cruz Biotechnology, 1:200). After washing with PBS with Tween 20 buffer, the membrane was incubated for another 10 min with peroxidase secondary antibody (anti-igg Sigma, 1:500). The reaction was revealed by the addition of DAB in PBS containing chloronaphthol, methanol and hydrogen peroxide. All tests were performed in triplicate, and the bands density obtained was estimated using Image J Software Statistical analyses The data was presented as means ± standard error (SEM). In order to compare the sites S1 and S2, T student s test was used followed by Welch s correction, with p < Results There was no significant statistical difference between the females captured in S1 and S2 in relation to size and weight of P. argenteus (40.2 ± 7.8 cm total length; ± g body weight). The GSI was higher in S2, with a significant statistical difference during the rainy period. The diameters of the perinucleolar follicles and vitellogenic follicles were significantly higher in S2 (Table 1). The abiotic parameters of water of the São Francisco River, such as, temperature, oxygen dissolved, ph and conductivity presented a significant statistical difference between S1 and S2 in both periods. In addition, in the rainy period was observed that the water temperature was 1.1 C higher in S2, and a wide variation for the dissolved oxygen between the sites (Table 2) Ovarian follicles morphology In the dry period, the ovaries presented ovigerous lamellae with oogonia nests, prefollicular cells, and perinucleolar follicles. The oogonia presented an ovoid nucleus, fine chromatin and a single nucleolus. In perinucleolar follicles, the oocytes had basophilic cytoplasm, a large nucleus with thin chromatin and peripheral nucleoli enclosed by squamous follicular cells and thin connective theca (Fig. 1A). In the rainy period, the ovigerous lamellae contained vitellogenic follicles with evident zona radiata, ooplasm filled with yolk globules and a central nucleus (Fig. 1B), as well as perinucleolar and previtellogenic follicles, the latter with the characteristic cortical alveoli. After spawning, the ovaries showed postovulatory follicles, which presented a single layer of prismatic follicular cells and a poorly developed connective theca (Fig. 1C). Non-ovulated vitellogenic follicles in the process of atresia were characterised by the fragmentation of the zona radiata, yolk globules liquefaction and follicular cells hypertrophy (Fig. 1D). The relative frequency of the oogonia and the ovarian follicles in different developmental stages varied during the sampling period (Fig. 2). In the dry period, only oogonia and perinucleolar follicles were observed in both sites. However, in the rainy period, the frequency of oogonia and perinucleolar follicles decreased in S2 while the frequency of vitellogenic follicles increased, and a rare occurrence of previtellogenic follicles was also registered. The postovulatory follicles were observed only in S2, while atretic follicles were registered at a higher frequency in S Proliferation and cell death In S1 specimens, the immunostaining for PCNA was predominant in the oogonia, whereas in S2 it was observed in oogonia, perinucleolar oocytes and prefollicular and follicular cells (Fig. 3A D). In Western blotting, the antibody for PCNA revealed a conspicuous band of approximately 36 kda. Analysis of the bands intensity showed that PCNA expression was significantly high in the females in S2 (Fig. 3E). The TUNEL assay allowed the identification of apoptotic cells with typical DNA fragmentation (Fig. 4A D). In S1, oogonia and follicular cells were TUNEL-positives, whereas in S2 this staining was registered in the ovigerous lamellae cells and in some follicular cells. The colorimetric assay of caspase-3 in ovaries of P. argenteus showed significantly high enzymatic activity in S1 (Fig. 4E). Table 2 Abiotic parameters of the water in two sites of the São Francisco River: S1 immediately following the Três Marias Dam and S2 in the confluence with Abaeté River. Parameters Dry period Rainy period S1 S2 S1 S2 Temperature ( C) 22.5 ± 0.01 a 22.8 ± 0.10 b 22.9 ± 0.04 a 24.0 ± 0.01 b Dissolved oxygen (mg/l) 7.1 ± 0.09 a 7.4 ± 0.05 b 3.5 ± 0.13 a 8.6 ± 0.03 b ph 6.7 ± 0.01 a 7.3 ± 0.01 b 6.2 ± 0.01 a 5.6 ± 0.02 b Conductivity 49.8 ± 0.10 a 67.4 ± 0.16 b 53.8 ± 0.09 a 24.6 ± 0.25 b Data obtained were expressed as means ± SEM with ranges between parentheses. Different letters indicate significant differences between sites (p < 0.05).

51 R.G. Thomé et al. / Tissue and Cell 44 (2012) Fig. 1. Histological sections of P. argenteus ovaries stained with hematoxylin and eosin (A and D) and Gomori trichrome (B, detail in B and C). (A) Ovary with oogonia (Oo), prefollicular cells ( ), initial perinucleolar follicles (PN1), advanced (PN2) and squamous follicular cells ( ); (B) vitellogenic follicles (VF) filled with yolk globules. The detail shows the micropliar cell (M) and squamous follicular cells ( ); (C) postovulatory follicles (POF) with prismatic follicular cells ( ); (D) atretic follicles (AF) with hypertrophied follicular cells ( ) and liquefied yolk globules. Bars = 50 m (A D) and 20 m (detail in B) Cathepsin-D In vitelogenic oocytes, the immunostaining of this lysosomal enzyme was observed between the yolk globules of the vitellogenic oocytes in both sites (Fig. 5A and B). In S1, the immunostaining for cathepsin-d was also found in the follicular cells of the vitellogenic follicles. In Western blotting, the band detected by the antibody cathepsin-d showed intense around 55 kda, with density significantly high in fish ovaries from S2 during the rainy period (Fig. 5E). 4. Discussion Fig. 2. Relative frequency of oogonia (Oo), perinucleolar (PNF), previtellogenic (PVF), vitellogenic (VF), atretic (AF) and postovulatory follicles (POF) of P. argenteus captured in two sites of the São Francisco River: S1 immediately following the Três Marias Dam and S2 in the confluence with Abaeté River. This study presented a morphological and molecular analysis of the folliculogenesis and ovarian follicles growth in P. argenteus, captured in different environmental conditions in the São Francisco River. In site S1, close to the Três Marias Dam, where the water temperature and dissolved oxygen are not favorable to cell proliferation

52 58 R.G. Thomé et al. / Tissue and Cell 44 (2012) Fig. 3. Immunohistochemistry for PCNA in ovarian sections of the P. argenteus from São Francisco River: S1 immediately following the Três Marias Dam and S2 in the confluence with Abaeté River. (A and C) (S1) and (B and D) (S2) with staining of the oogonia (Oo), prefollicular cells ( ), initial perinucleolar follicles (PN1), advanced (PN2) and follicular cells ( ). (E) Representative Western blotting and respective graphic representation of the image analysis of the PCNA expression in S1 and S2. Bars = 50 m (A and B) and 20 m (C, D and detail in D).

53 R.G. Thomé et al. / Tissue and Cell 44 (2012) Fig. 4. TUNEL-positive reaction in ovaries of P. argenteus captured in two sites from the São Francisco River: S1 immediately following the Três Marias Dam and S2 in the confluence with Abaeté River. (A C) (S1) labelled in oogonia and follicular cells. (D) (S2) labelled follicular cells and ovigerous lamellae cells. (E) Graphical representation of the enzyme activity of caspase-3 in S1 and S2. Bars = 50 m (A and B) and 20 m (Detail in A, C and D). and vitellogenesis resulting in the impairment of the reproduction of P. argenteus. In site S2, in the confluence of the São Francisco River with the Abaeté River, the environmental conditions were favorable for gonadal maturation and spawning of this species. Different environmental parameters can interfere in fish reproduction, however the temperature and dissolved oxygen are indicated as the most important modulators of reproductive activity (Alvarenga and Franç a, 2009; Wu et al., 2003).

54 60 R.G. Thomé et al. / Tissue and Cell 44 (2012) Fig. 5. Immunohistochemistry for cathepsin-d in ovarian sections of the P. argenteus from São Francisco River: S1 immediately following the Três Marias Dam (A) and S2 in the confluence with Abaeté River (B), with staining in follicular cells and between the yolk globules in the vitellogenic follicles (VF) and follicular cells ( ). (C) Representative Western blotting and respective graphical representation of the image analysis of the cathepsin expression in S1 and S2. Bars = 20 m. In the rainy period, the GSI values of the females captured in S2 were significantly higher than the ones captured from S1. In females, the GSI usually varies throughout the reproductive cycle and reflects the energetic investment for reproduction (Guerrero et al., 2009). In this study, the variation in GSI values observed for fish from both sites may be related to differences in the process of selection and growth of the ovarian follicles, which is reflected in follicle diameter, relative frequency of oogonia and ovarian follicles. However, ovarian disruption related to impacted environments may be associated with lower GSI values during gonadal maturation (Louiz et al., 2009), such as detected in S1 in this study. In the present study, the perinucleolar follicles of fish from S1 were significantly smaller than S2. Sex steroids and growth factors such as growth and differentiation factor 9 (GDF9) and bone morphogenetic factor 15 (BMP15) are involved in primary growth of ovarian follicles (Lubzens et al., 2010). During secondary growth, vitellogenic follicles were formed, which, in ovaries of fish captured in S1, presented lower cathepsin-d expression. Our results, associated with the low estradiol values (Arantes et al., 2010), could explain the smaller diameter of the vitellogenic follicles in ovaries of fish from S1. The relative frequency of oogonia and ovarian follicles in different developmental stages evidenced the successful reproduction of the curimatã-pacu in the rainy period when the environmental conditions of the São Francisco River were favorable for folicullogenisis. In S2, the reduction in the number of oogonia in the rainy period coincided with a high frequency of vitellogenic follicles. In S1, the recorded numbers of oogonia changed little throughout the periods studied and a lower frequency of vitellogenic follicles was observed in the rainy period. Throughout the reproductive cycle, follicular development should be primary growth followed by secondary growth (Selman and Wallace, 1989; Tyler and Sumpter, 1996), as observed in S2. But these distinctive steps of gametogenesis do not always proceed as successive events (Nakamura et al., 2011). During the rainy period, postovulatory follicles were observed only in S2 and the relative frequency of atretic follicles was higher in S1. Postovulatory follicles are biomarkers of reproductive success and are used to determine time and frequency of spawning in the reproductive cycle of teleosts (Sato et al., 2005). On the other hand, follicular atresia is a process of non-ovulated vitellogenic follicle resorption, and a high rate of atresia indicates failure in reproduction (Arantes et al., 2010, 2011). In P. argenteus ovaries, the PCNA presented a molecular weight of 36 kda, as observed in mammals (Chapman and Wolgemuth, 1994). In the testis of the Anguilla japonica, and the Scyliorhinus canicula, besides the 36 kda a second band of 32 kda was also detected by Western blotting (Miura et al., 2002; Loppion et al., 2008). Due to the difficulty of production and commercialisation of specific antibodies for each fish species, the immunohistochemical studies for this species have been made using mammalian antibodies, based on evolutionary conservation of antigens, and the Western blotting is used to confirm the specificity of the reaction. In fish captured in S2, a higher PCNA expression was identified through immunostaining, especially in the oogonia nuclei, prefollicular cells, perinucleolar oocytes and follicular cells. Immunostaining in the oogonia is associated with DNA synthesis, which is also described in cells of spermetogenic lineage of fish (Miura et al., 2002), whereas staining in the oocytes nuclei, is also reported in primordial follicles of mammals (Oktay et al., 1995), may be explained by the role PCNA plays in repairing and

55 R.G. Thomé et al. / Tissue and Cell 44 (2012) Fig. 6. Schematic summary for possible formation and development of the ovarian follicles in teleosts under environmental influence. (A) When the environmental conditions are favorable to reproduction, we observed that the oogonia tend to meiosis, initiating folliculogenesis with high follicular cell proliferation and apoptosis rate is low. In contrast, the oogonia tend to mitosis for self-renewal, and the apoptosis rate is high. (B) The follicular growth also depends of the favorable conditions. In this case, we observed the tendency to vitellogenic follicle formation with an increase of the cathepsin-d expression favoring the oocyte maturation, and consequently, the number of postovulatory follicles (POF) increases. revising synthesised DNA, ensuring the success of follicular development. Once folliculogenesis starts, the prefollicular and follicular cells proliferate to support oocyte growth (Quagio-Grassiotto et al., 2011). In the dry period, only oogonia and perinucleolar follicles were observed in both sites. Despites the oogonia have been identified in different germ cell types in germinal cradles (Nakamura et al., 2010) herein it was considered as one cell type. The proliferation or apoptosis of germ cells during the gonadal sex differentiation, formation of germinal cradles, affect the adult fertility (Saito et al., 2007). In S1, were observed a higher frequency of apoptosis and lower frequency of cell proliferation. In S2, the relationship between apoptosis and cell proliferation was reversed to the one in S1, with fewer apoptotic cells and higher cellular proliferation, leading to a larger number of vitellogenic follicles in the rainy period. These results are corroborated by in vitro studies (Janz and Van Der Kraak, 1997) showing that the increase in apoptosis implies the decrease in the number of vitellogenic follicles. During follicular development, a low apoptosis rate of the follicular cells may be considered physiological, controlling the appropriate number of cells and eliminating unwanted cells (Thomé et al., 2010). However, high values of apoptosis were observed in unfavorable conditions, compromising the follicular viability (Drevinick et al., 2006), similar to what observed in fish from S1. In this study, the presence of cathepsin-d in the ooplasm of vitellogenic follicles was observed in fish captured in both sites, with a higher quantity in S2. In P. argenteus ovaries, a band of 55 kda molecular weight corresponding to cathepsin-d was detected by Western blotting, whereas in the liver of Gadus morhua, a band of approximately 40 kda was found (Wang et al., 2007). Cathepsin-D expression has been suggested as a biomarker of endocrine disorder since it is involved in the process of yolk formation during oocyte maturation, having a fundamental role in the fish reproductive success (Carnevali and Maradona, 2003). The immunostaining for cathepsin-d observed in the follicular cells of vitellogenic follicles in S1 fish may be related to the destination of these follicles: ovulation or follicular atresia. During follicular atresia, follicular cells hypertrophy and operate on the phagocytosis of the yolk globules (Santos et al., 2009). Studies performed on ovaries of the Oncorhyncus mykiss suggest that follicular atresia involves vitellinic protein degradation mediated by lysosomal enzymes (Wood and Van Der Kraak, 2003). The water parameters of the São Francisco River in the two sites analysed in this study make these sites a natural laboratory for the study of fish reproductive biology. Thus, we propose a model for assessment of folliculogenesis and follicular growth in teleost fish (Fig. 6). In favorable environments, as we observed in S2 of the present study, the oogonia are stimulated to enter meiosis and to start folliculogenesis. This hypothesis, the prefollicular cells proliferate and differentiate into follicular cells which continue to proliferate, while is observed a low occurrence of apoptosis and enzyme activity of caspase-3. On the other hand, in unfavorable environments, as we observed in S1 of the present study, there are mitotic divisions of the oogonia and few meiosis, low cell proliferation and a lot of apoptosis (Fig. 6A). In the hypothesis related to follicular growth (Fig. 6B) in the favorable environments, a larger number of follicles developed to become filled with yolk globules with increased cathepsin-d expression, promoting oocyte maturation and, consequently, spawning. Differently, in unfavorable environments, the few vitellogenic follicles which were formed entered into follicular atresia. Therefore, our findings indicate impairment of the folliculogenesis and ovarian follicle development in fish when the physiochemical waters parameters are unfavorable. Moreover, the

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57 5- DISCUSSÃO Estudos sobre o comportamento reprodutivo de peixes teleósteos, por meio da análise do desenvolvimento gonadal, fornecem informações valiosas para a melhoria da gestão de recursos pesqueiros e conservação das espécies nativas. Os peixes habitam uma grande variedade de ambientes aquáticos resultando em numerosas adaptações conhecidas como estratégias reprodutivas, que implicam em diferentes tipos de desenvolvimento ovocitário e de desova (Jalabert, 2005). Apesar disso, há evidências de que as características morfo-fisiológicas do desenvolvimento ovariano são comuns a maioria dos teleósteos (Lubzens et al., 2010). A curimatã-pacu, P. argenteus, reúne características que o tornam um excelente modelo para estudos de diferentes aspectos em biologia do desenvolvimento (Sato et al., 2003; Santos et al., 2008a,b; Thomé et al., 2009 Anexo I; Arantes et al., 2011; Morais et al 2012 Anexo II) tanto quanto de alterações decorrentes de impacto ambiental (Sato et al., 2005; Arantes et al., 2010). O presente trabalho foi realizado em 2 etapas. Inicialmente, avaliou-se a foliculogênese de P. argenteus utilizando fêmeas mantidas em cativeiro e submetidas à desova induzida por hipofisação. Nesta etapa, foi observada a formação do folículo ovariano com o estabelecimento da membrana basal, a relação das proteínas de matriz extracelular e os níveis de E2 com a taxa de apoptose, pré e pós desova. Na segunda etapa, avaliou-se a foliculogênese, através da quantificação de estruturas ovarianas, proliferação e morte celular, de peixes capturados em dois trechos do rio São Francisco, trecho 1 (S1) logo após a barragem de Três Marias e trecho 2 (S2) na confluência do rio São Francisco com o rio Abaeté. A análise das variações do índice de apoptose folicular e E2 nos períodos estudados sugerem que além do papel no desenvolvimento folicular, o E2 pode atuar como fator anti-apoptótico para as células foliculares e germinativas. A brusca queda dos níveis de E2 observada no ovário recém desovado pode ter contribuído para o aumento progressivo no índice de apoptose folicular em fêmeas mantidas em cativeiro. No mesmo sentido, foram encontrados baixos níveis de estradiol em fêmeas de P. argenteus capturadas no rio São Francisco logo após a barragem de Três Marias (Arantes et al., 2010), local correspondente ao S1 do presente trabalho, e onde ocorreram maior atividade enzimática da caspase-3 e incidência de células apoptóticas. Estudos com folículos ovarianos pré-ovulatórios de trutas arco-íris, O. mykiss, a adição de E2 no meio de incubação causou supressão da fragmentação do DNA relacionado

58 com a apoptose em comparação com o meio sem o hormônio (Janz e Van der Kraak, 1997). No presente estudo, a marcação descontínua de laminina β2 e colágeno tipo IV na membrana basal dos folículos pós-ovulatórios com 96 horas após desova indicaram que a degradação da membrana basal deve-se à lise de seus principais constituintes. A perda da integridade da membrana basal foi acompanhada pelo aumento do índice de apoptose folicular e queda do estradiol, demonstrando sua influência na sobrevivência celular em folículos pós-ovulatórios. Estudo realizado em outro Prochilondontidae, P. costatus, também indicou relação entre alterações estruturais na membrana basal e a ocorrência de apoptose nas células foliculares (Thomé et al., 2006). Em células do epitélio mamário de camundongos, a membrana basal intacta manteve os fenótipos celulares diferenciados e também atuou como fator anti-apoptótico, e sua degradação resultou em perda de função e apoptose (Boudreau et al., 1996). Em ovinos, a associação de integrina α6β1 de células granulosas com a laminina da membrana basal garantiu o aumento da produção de E2 atuando como fator anti-apoptótico (Le Belego et al., 2002; Berkholtz et al., 2006b). No presente estudo, analisou-se a participação da MMP-9 na remodelação tecidual e os resultados obtidos mostraram que esta enzima está presente nos folículos vitelogênicos e pós-ovulatórios. As MMPs estão relacionadas com a ovulação em diferentes grupos de vertebrados incluindo peixes, podendo ser responsável pela fragmentação da zona pelúcida e da matriz extracelular da teca (Curry e Osteen, 2001; Ogiwara et al., 2005). A maior fragmentação da membrana basal nos folículos pósovulatórios com 96 horas após desova pode estar relacionada com a síntese de MMP-9 detectada pela imunohistoquímica nas células foliculares. No mesmo sentido, MMP-9 foi identificada nos tecidos pós-ovulatórios de O. latipes, através da análise do mrna (Matsui et al., 2000), mostrando a importância desta enzima na involução dos ovários após desova. A fibronectina e a laminina podem estar relacionadas com o desenvolvimento folicular estimulando a proliferação e sobrevivência das células granulosas em folículos ovarianos de ovinos (Huet et al., 2001). Em fêmeas de P. argenteus mantidas em cativeiro a formação dos folículos ovarianos foi acompanhada pela imunomarcação de laminina β2, colágeno tipo IV e fibronectina. Nos peixes capturados no rio São Francisco, em S2, foi registrada intensa proliferação celular durante a foliculogênese, quantificada pela maior expressão proteica de PCNA, e observada especialmente pela

59 imunomarcação com PCNA de núcleos de ovogônias, células pré-foliculares, ovócitos perinucleolares e células foliculares. A imunomarcação nas ovogônias pode estar associada à síntese de DNA, fato também descrito em células da linhagem espermatogênica de peixes (Miura et al., 2002). Uma vez iniciada a foliculogênese, as células pré-foliculares e foliculares proliferam para acompanhar o crescimento do ovócito, apoiadas em uma membrana basal contínua. Em peixes, várias funções são atribuídas às células foliculares, entre elas, metabolismo e transporte de moléculas para o ovócito (Quagio-Grassioto e Guimarães, 2006). Além disso, a testosterona produzida nas células especiais da teca é convertida pelas células foliculares em E2, que estimula os hepatócitos a produzirem vitelogenina e coreogeninas (Senthilkumaran et al., 2004). Os valores do IGS de fêmeas P. argenteus capturadas em S2 foram maiores se comparados àqueles de S1 com diferença estatística na estação chuvosa, corroborando estudos prévios (Arantes et al., 2010). O IGS em teleósteos normalmente varia ao longo do ciclo reprodutivo e reflete o investimento energético para a reprodução (Guerrero et al., 2009). No presente estudo, as variações dos valores de IGS observadas entre os peixes caputados em S1 e S2, podem estar relacionadas com diferenças no processo de seleção e crescimento dos folículos ovarianos, refletidas em diâmetro ovocitário, freqüência relativa de ovogônias e folículos ovarianos, além da proliferação ou morte celular. Por outro lado, disfunções ovarianas relacionadas com ambientes impactados podem estar associadas à redução dos valores de IGS em todas as fases do ciclo reprodutivo (Louiz et al., 2009). Os folículos pós-ovulatórios foram observados em fêmeas submetidas à desova induzida como naquelas capturadas em S2, mas a freqüência relativa de folículos atrésicos foi maior em peixes capturados em S1. Os folículos pós-ovulatórios são indicativos histológicos do sucesso reprodutivo sendo utilizados para determinar o tempo e a freqüência de desova durante o ciclo reprodutivo dos teleósteos (Sato et al., 2005), e também, como o relatado no presente trabalho, são ótimos modelos de estudo da interação célula-matriz extracelular. Por outro lado, a atresia folicular é um processo de reabsorção de folículos ovarianos, e uma elevada taxa de atresia, tal como observado em peixes de S1 do presente trabalho, é característica de ambientes impactados (Arantes et al., 2010). A regressão ovariana após a desova de peixes teleósteos é um modelo experimental adequado para o estudo da morte celular programada (Thomé et al., 2009 Anexo I). Nesta fase, os ovários apresentam folículos pós-ovulatório e atrésicos, que são

60 reabsorvidos durante a regressão ovariana. Os folículos pós-ovulatórios são rapidamente reabsorvidos durante a recuperação do ovário após a estação reprodutiva (Drummond et al., 2000; Santos et al., 2005; Thomé et al., 2006), enquanto que a regressão dos folículos atrésicos é um processo fisiológico prolongado, variando de 4 a 7 meses em teleósteos neotropicais de água doce (Miranda et al., 1999). Assim, mecanismos diferentes de morte celular podem estar envolvidos nos eventos de eliminação de folículos pós-ovulatórios e atrésicos. Nos ovários pós-desova de P. argenteus submetidos à desova induzida, foi observado um anel conspícuo de actina em células foliculares de folículos pósovulatórios recem formados, que pode estar relacionado com a primeira etapa da apoptose. De fato, na primeira etapa de apoptose, os filamentos de actina são reorganizados em um anel periférico, fazendo uma associação com a miosina levando à contração e formação de bolhas, facilitadores da interação com células fagocíticas (Ndozangue-Touriguine et al., 2008). Por outro lado, a desorganização dos filamentos de actina observada em células foliculares de folículos pós-ovulatórios com 96h pósdesova pode estar relacionado com a etapa final da apoptose, provavelmente devido a despolimerização de actina. Esta suposição é reforçada pela desorganização das células apoptóticas em corpos apoptóticos (Korsnes et al., 2007; Ndozangue-Touriguine et al., 2008). Além disso, clivagem da tensina, fosfoproteína citoplasmática associada aos locais de adesão, mediada por caspase contribui para ruptura da organização do citoesqueleto e interrompe os sinais de sobrevivência celular (Kook et al., 2003). Durante a atresia folicular de três espécies de Characiformes, o citoesqueleto de actina do ooplasma sofre mudanças associadas com a degradação do vitelo, enquanto ele permanece preservado nas células foliculares (Morais et al., 2012 Anexo II). Na levedura Saccharomyces cerevisiae, o citoesqueleto de actina é necessário para o recrutamento da via Cvt para a formação de pré-autofagossomos (Reggiori et al. 2005). Tomados em conjunto, estes dados sugerem que o citoesqueleto de actina poderia estar envolvido na formação do fagossomo após a endocitose do vitelo acumulado no ovócito vitelogênico, bem como na nucleação dos vacúolos autofágicos. Assim, a manutenção do citoesqueleto de actina poderia ser um sinal de sobrevivência para as células foliculares durante fases inicial e avançada da atresia. No presente estudo, observou-se a presença de cat-d no ooplasma de ovócitos vitelogênicos de peixes capturados nos dois trechos do rio São Francisco, sendo em maior quantidade, quando avaliada por Western blot, registrada em S2. Marcação

61 imunohistoquímica para cat-d observada nas células foliculares de folículos vitelogênicos em peixes de S1 pode estar relacionada com o destino destes folículos: para ovulação ou atresia folicular. Durante a atresia folicular as células foliculares hipertrofiam-se e atuam na fagocitose de glóbulos de vitelo (Santos et al., 2008a). A cat- D também foi detectada nas células foliculares em todas as fases de regressão de folículos atrésicos, enquanto as células tecais não apresentaram imunorreatividade (Morais et al., 2012 anexo II). Observações histológicas feitas em ovários de truta arcoíris indicaram que a atresia folicular envolve degradação proteolítica das proteínas do vitelo mediada por enzimas lisossômicas (Wood e Van Der Kraak, 2003). A expressão de cat-d tem sido sugerida como biomarcador de desregulação endócrina por estar envolvida no processo de formação do vitelo durante a maturação do ovócito, desempenhando um papel fundamental para o sucesso da reprodução de peixes (Carnevali e Maradona, 2003). A cat-d observada em grânulos citoplasmáticos durante os primeiros estágios de atresia folicular, anterior à formação de vacúolos autofágicos, está envolvida na proteólise de vitelo e desencadeamento de autofagia (Morais et al., 2012 Anexo II). Sendo assim, as células foliculares dos folículos atrésicos de peixes em S1 poderiam estar se preparando para o processo de atresia sintetizando enzimas lisossômicas como a cat-d. Em resumo, os resultados obtidos do presente estudo fornecem novos conhecimentos sobre formação, crescimento e remodelação dos folículos ovarianos durante a maturação gonadal de P. argenteus da bacia do rio São Francisco. Além disso, estes dados sugerem que proliferação e morte celular, tanto quanto a expressão proteica de PCNA, caspase-3 e cat-d possam ser utilizados como biomarcadores de impacto ambiental. Considerando que foram registrados altos níveis de contaminação por metais pesados, principalmente cádmio e zinco, no rio São Francisco (Ribeiro, 2010), torna-se importante investigar alterações nos padrões normais de desenvolvimento utilizando biomarcadores que reflitam os impactos ambientais causados por atividades antropogênicas.

62 46 6- CONCLUSÕES 1) A estrutura e composição da matriz extracelular dos ovários e os níveis plasmáticos de E2 estão relacionados com a taxa de apoptose desempenhando papéis importantes durante o desenvolvimento folicular e involução dos folículos pós-ovulatórios em peixes teleósteos. 2) A integridade da membrana basal é importante para a sobrevivência das células foliculares, e a perda da integridade pela lise dos seus principais constituintes, laminina-β2 e colágeno IV, coincide com apoptose de células foliculares. 3) A MMP-9 pode estar envolvida na maturação final ovocitária e regressão de folículos pós-ovulatórios. 4) O ovário dos peixes teleósteos provou ser um sistema útil para estudos sobre interações célula-matriz extracelular e morfo-fisiologia ovariana. 5) A foliculogênese e crescimento de folículos ovarianos de peixes sofrem influência do ambiente, sendo importantes para estudos sobre impacto ambiental, manejo e reprodução de peixes. Baixa temperatura associada a baixos níveis de oxigênio dissolvido pode comprometer o desenvolvimento folicular. 6) A cat-d participa da vitelogênese, podendo ser utilizada como biomarcador de impacto ambiental principalmente se associado a distúrbios hormonais, além de estar envolvida no processo de atresia folicular.

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69 53 MORAIS R.D.V.S., THOMÉ, R.G.; LEMOS, F.S.; BAZZOLI, N.; RIZZO, E. (2012). Autophagy and apoptosis interplay during follicular atresia in fish ovary: A morphological and immunocytochemical study. Cell Tissue Res. in press. MOURA D.F,; MARTINS, Y.S.; SANTOS, G.B.; BAZZOLI, N. (2011). Unusual pattern of adult male testis in a gonochore fish, Satanoperca pappaterra. J. Applied Icth. 27(3): NAHUM, R.; BEYTH, Y.; CHUN, S.Y.; HSUEH, A.J.; TSAFRIRI, A. (1996). Early onset of deoxyribonucleic acid fragmentation during atresia of preovulatory ovarian follicles in rats. Biol. Reprod. 55: NAGAHAMA, Y. (1994). Endocrine regulation of gametogenesis in fish. Int. J. Dev. Biol. 38: NAKAMURA, S.; KOBAYASHI, K.; NISHIMURA, T.; HIGASHIJIMA, S.; TANAKA, M. (2010). Identification of germline stem cells in the ovary of the teleost. Science 328: NAKAMURA, S.; KOBAYASHI, K.; NISHIMURA, T.; TANAKA, M. (2011). Ovarian germline stem cells in the teleost fish, medaka (Oryzia latipes). Int. J. Biol. Sci. 7: NDOZANGUE-TOURIGUINE, O.; HAMELIN, J.; BREÀRD, J. (2008). Cytoskeleton and apoptosis. Biochem. Pharmacol. 76: OGIWARA, K.; TAKANO. N.; SHINOHARA, M.; MURAKAMI, M.; TAKAHASHI, T. (2005). Gelatinase A and membrane-type matrix metalloproteinases 1 and 2 are responsible for follicle rupture during ovulation in the medaka. PNAS. 102: PAIVA, M.P. (1982). Grandes represas do Brasil. Editerra. 304p. PATIÑO, R.; SULLIVAN, C.V. (2002). Ovarian follicle growth, maturation, and ovulation in teleost fish. Fish. Physiol. Biochem. 26: PERROT, V.; MOISEEVA, E.B.; GOZES, Y.; CHAN, S.J.; FUNKENSTEIN, B. (2000). Insulin-like growth factor receptors and their ligands in gonads of a hermaphroditic species, the gilthead seabream (Sparus aurata): expression and cellular localization. Biol. Reprod. 63: PIECHOTTA, G.; LACORN, M.; LANG, T.; KAMMANN, U.; SIMAT, T.; JENKE, H. S.; STEINHART, H. (1999). Apoptosis in dab (Limanda limanda) as possible new biomarker for anthropogenic stress. Ecotoxicol. Environ. Saf. 42(1):

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74 58 8- ANEXOS Os seguintes artigos foram escritos durante o período de elaboração do presente trabalho e contribuem para o melhor entendimento do tema desenvolvido: THOMÉ R.G., SANTOS H.B., ARANTES F.P., DOMINGOS F.F.T., BAZZOLI N., RIZZO E. (2009). DUAL ROLES FOR AUTOPHAGY DURING FOLLICULAR ATRESIA IN FISH OVARY. AUTOPHAGY 5:1, MORAIS R.D.V.S., THOMÉ R.G., LEMOS F.S., BAZZOLI N., RIZZO E. (2012). AUTOPHAGY AND APOPTOSIS INTERPLAY DURING FOLLICULAR ATRESIA IN FISH OVARY: A MORPHOLOGICAL AND IMMUNOCYTOCHEMICAL STUDY. CELL AND TISSUE IN PRESS

75 8.1 THOMÉ ET AL., 2009 ANEXO I 59

76 8.2 MORAIS ET AL., 2012 ANEXO II 63

77 [Autophagy 5:1, ; 1 January 2009]; 2009 Landes Bioscience Article Addendum Dual roles for autophagy during follicular atresia in fish ovary Ralph G. Thomé, Helio B. Santos, Fabio P. Arantes, Fabrício F.T. Domingos, Nilo Bazzoli and Elizete Rizzo* Departamento de Morfologia; Instituto de Ciências Biológicas; Universidade Federal de Minas Gerais; Belo Horizonte, Brasil Key words: autophagy, apoptosis, follicular atresia, follicular cell, ovary, teleost fish Autophagy, a highly conserved catabolic program for degrading proteins and organelles, is essential for cell and tissue homeostasis. Primarily, this process has a cytoprotective role under nutrient deprivation, but several stress stimuli can induce autophagy and, thus, distinct programmed cell death (PCD) pathways can be actived when stress is not abolished. Fish ovaries are a suitable experimental model system for studying the mechanisms of PCD due to the presence of postovulatory and atretic (i.e., nonovulated) follicles, which follow different routes after spawning. Apoptosis of the follicular cells is the major mechanism responsible for the rapid resorption of the postovulatory follicles. Recently, we investigated the contribution of PCD during follicular atresia in two species of freshwater fish. In contrast to mammals, this study revealed that follicular apoptosis is not a major process for deletion of follicular cells in atretic follicles. Furthermore, we detected autophagic vacuoles containing degenerating organelles increasing through follicular atresia in both species. In this addendum, we propose a hypothesis for follicular cell removal during ovarian regression in oviparous fish. In this model, autophagy could have dual roles in follicular atresia. Thus, fish ovaries after breeding are suitable models for studying the interactions among the different cell death pathways. Programmed cell death (PCD) is a genetically regulated active process essential for the maintenance of tissue development and homeostasis, controlling cell numbers and eliminating unwanted cells. 1 Apoptosis or type I cell death is a well-characterized PCD pathway that affects single cells detached from neighboring cells and the basement membrane. 2 Autophagy, a highly regulated catabolic process, can either be involved in the turnover of long-lived proteins and organelles or can specifically target damaged organelles. 3,4 The crosstalk between apoptosis and autophagy is complex and involves *Correspondence to: Elizete Rizzo; Departamento de Morfologia; Instituto de Ciências Biológicas; Universidade Federal de Minas Gerais; UFMG; C. P.486; Belo Horizonte MG, Brasil; Tel.: ; Fax: ; ictio@icb.ufmg.br Submitted: 10/03/08; Revised: 10/22/08; Accepted: 10/30/08 interaction with Bcl-2 family proteins. 1,3 In several conditions autophagy constitutes a stress adaptation to avoid cell death, whereas in other settings it is an alternative pathway to cell demise, known as autophagy-mediated or type II programmed cell death. 5 The major effectors in type I PCD are caspases that active Ca 2+ /Mg 2+ - dependent endonucleases, which cleave the DNA into fragments of base pairs. 2 Cathepsins or proteasomal proteins are active primarily in type II PCD, whereas caspases may be involved in later events. 6 The prosurvival function of autophagy has been very well characterized under nutrient and growth factor deprivation, but the precise role of this catabolic process is uncertain in dying cells. 4,5 Follicular atresia is a hormonally controlled degenerative process by which vertebrate ovarian follicles lose their integrity and are eliminated prior to ovulation. Several studies indicate that apoptosis is the major mechanism for elimination of the granulosa cells during follicular atresia and luteolysis in mammalian ovary. 7,8 In teleost fish, follicular atresia is frequently associated with environmental stress or changes in hormonal levels Although atretic follicles are important indicators of environmental impact, the mechanisms of follicular atresia are not yet fully understood in teleosts. In our investigations, we used fish ovaries as an experimental model system for studying PCD after spawning. In this period, postovulatory and atretic follicles are reabsorbed progressively during ovarian remodeling. In oviparous fish, postovulatory follicles are transitory structures with no contribution to the synthesis of steroid hormones. They are excellent morphological indicators of reproductive success, and they degenerate quickly after spawning. 12,15,16 In contrast, the regression of atretic follicles is a prolonged physiological process varying from four to seven months duration in neotropical freshwater fish. 13 Studies with different fish species showed that apoptosis is an essential process in ovarian regression, contributing mainly to deletion of the follicular cells in the postovulatory follicles. 12,14-16 However, a similar mechanism has not been established for atretic follicles. 13,15,17 In a recent study, we investigated the contribution of PCD during follicular atresia in two species of freshwater fish Prochilodus argenteus and Leporinus taeniatus. 17 These species do not spawn spontaneously in captivity, and a widespread process of follicular regression occurs in their ovaries after the reproductive period. In this study, we observed degeneration of the zona pellucida and the yolk, which were engulfed by hypertrophied follicular cells (Fig. 1A) as has also been reported in several other species. 13,15,17 Unlike mammals, follicular cells of teleosts are important for proteolysis and resorption of the yolk during most follicular atresia. Moreover, evidence in goldfish 2009 Landes Bioscience. Do not distribute. Previously published online as an Autophagy E-publication: Addendum to: Santos HB, Thomé RG, Arantes FP, Sato Y, Bazzoli N, Rizzo E. Ovarian follicular atresia is mediated by heterophagy, autophagy and apoptosis in Prochilodus argenteus and Leporinus taeniatus (Teleostei: Characiformes). Theriogenology 2008; 70: ; PMID: ; DOI: /j.theriogenology Autophagy 117

78 Autophagy during follicular atesia in fish ovary Figure 1. Ovarian regression in Prochilodus argenteus and Leporinus taeniatus. (A) Yolk (Y) and zona pellucida (ZP) degeneration and engulfment (arrow) by hypertrophied follicular cells (FC) during follicular atresia. (B) TUNEL-positive reaction in follicular cells and apoptotic body (arrowhead). (C) Follicular cell undergoing apoptosis with loss of cell adhesion (asterisk), surface blebbing (B) and nuclear fragmentation (double arrow) in postovulatory follicle. (D) Follicular cell undergoing intense autophagic activity with lysosomes (L) and autophagic vacuoles (AV) with degenerating organelles. (E) Large autophagic vacuole with cytoplasmic material and degenerating mitochondria (M). (F) Multivesicular body (MB) and myelin-like structure (MF) in follicular cell. (G) Ladder pattern of apoptotic DNA by agarose gel eletrophoresis in different times after spawning. Scale bars: (A and B): 10 μm, (C and D): 1 μm, (E and F): 0.5 μm. and rainbow trout indicated that apoptosis could not be a prominent early event in the onset of follicular atresia. 18,19 In our studies, apoptosis has been detected in the follicular cells by the TUNEL assay that showed DNA fragmentation (Fig. 1B), and also by other major morphological hallmarks of apoptosis (Fig 1C): chromatin condensation in a crescent pattern underlying the nuclear envelope, cell shrinkage, loss of cell-cell interactions, detachment from basement membrane, and cell fragmentation in apoptotic bodies. The ladder pattern of the apoptotic DNA was also revealed by agarose gel electrophoresis (Fig 1G) during postspawning regression in P. argenteus ovaries. 16 Through morphometry, we recently detected an apoptosis rate (%) increasing progressively in follicular cells of postovulatory follicles until three days and decreasing thereafter. 14,16 In contrast, follicular apoptosis increased only during late follicular atresia, which occurred about four to six months after breeding. 17 Thus, we believe that apoptosis contributes greatly to the removal of follicular cells in postovulatory follicles, but alternative mechanisms of PCD could also be active in the follicular atresia. Autophagic vacuoles with degenerating organelles were abundant in follicular cells (Fig. 1D and E) during yolk removal in P. argenteus and L. taeniatus. 17 Large vacuoles filled with cytoplasmic material and organelles, such as mitochondria, ribosomes and endoplasmic reticulum are features of macroautophagy, which is associated with autophagic or type II PCD. 3,6 Multivesicular bodies and myelin-like structures (Fig. 1F) are also commonly detected during follicular atresia in teleosts by electron microscopy. 13,17 In support of a prodeath function for autophagy during follicular atresia, we have detected immunohistochemical labeling for caspase 3 in the follicular cells of postovulatory follicles of P. argenteus, whereas atretic follicles were not labeled during advanced regression. 17 The absence of caspase 3 activation is usually viewed as a requisite in order to categorize an alternative cell death pathway. 4 In mammals and birds, studies on follicular atresia have focused on the apoptosis of granulosa cells, but autophagic cell death has not been ruled out. 20 Both types of PCD have also been reported to act cooperatively to efficiently eliminate the degenerated nurse cell during Drosophila virilis oogenesis. 21 In addition to a probable contribution in PCD, autophagy is primarily a survival mechanism in starving cells usually utilized to generate both nutrients and energy for maintaining cell viability. 4 Regarding follicular atresia in fish ovary, we believe that autophagy might have a prosurvival role for follicular cells as long as they are actively involved in oocyte removal. The interaction of Beclin 1 with the antiapoptotic protein Bcl-2 is crucial in determining cell fate and the crosstalk between autophagy and apoptosis. 22 However, the relationship among the PCD types still needs to be investigated in fish ovary after spawning. In conclusion, we propose a hypothesis for follicular cell removal during ovarian regression in oviparous fish (Fig. 2). In this model, autophagy could play dual roles in response to follicular atresia. Acknowledgements We are grateful to Dr. Yoshimi Sato for his valuable contribution during experimental procedures in Hydrobiology and Hatchery Station of Três Marias-CEMIG GT/CODEVASF. The work was supported by grants from FAPEMIG, CNPq and CAPES Landes Bioscience. Do not distribute. 118 Autophagy 2009; Vol. 5 Issue 1

79 Autophagy during follicular atesia in fish ovary References 1. Degterev A, Yuan J. Expansion and evolution of cell death programmes. Nat Rev Mol Cell Biol 2008; 9: Huettenbrenner S, Maier S, Leisser C, Polgar D, Strasser S, Grusch M, Krupitza G. The evolution of cell death programs as prerequisites of multicellularity. Mutat Res 2003; 543: Maiuri MC, Zalckvar E, Kimchi A, Kroemer G. Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol 2007; 8: Debnath J, Baehrecke EH, Kroemer G. Does autophagy contribute to cell death? Autophagy 2005; 1: Mizushima N, Levine B, Cuervo A, Klionsky MDJ. Autophagy fights disease through cellular self-digestion. Nature 2008; 451: Lockshin RA, Zakeri Z. Apoptosis, autophagy and more. Int J Biochem Cell Biol 2004; 36: Hussein MR. Apoptosis in the ovary: molecular mechanisms. Hum Reprod Update 2005; 11: Makrigiannakis A, Coukos G, Blaschuk O, Coutifaris C. Follicular atresia and luteolysis evidence of a role for N-cadherin. Ann N Y Acad Sci 2000; 900: Janz DM, McMaster ME, Weber LP, Munkittrick KR, Van Der Kraak G. Recovery of ovary size, follicle cell apoptosis, and HSP70 expression in fish exposed to bleached pulp mill effluent. Can J Fisher Aquat Sci 2001; 58: Sato Y, Bazzoli N, Rizzo E, Boschi MB, Miranda MOT. Influence of the Abaeté River on the reproductive success of the neotropical migratory teleost Prochilodus argenteus in the São Francisco River, downstream from the Três Marias dam, Southeastern Brazil. Riv Res Appl 2005; 21: Drevnick PE, Sandheinrich MB, Oris JT. Increased ovarian follicular apoptosis in fathead minnows (Pimephales promelas) exposed to dietary methylmercury. Aquat Toxicol 2006; 79: Drummond CD, Bazzoli N, Rizzo E, Sato Y. Postovulatory follicle: a model for experimental studies of programmed cell death or apoptosis in teleosts. J Exp Zool 2000; 287: Miranda ACL, Bazzoli N, Rizzo E, Sato Y. Ovarian follicular atresia in two teleost species: a histological and ultrastructural study. Tissue Cell 1999; 31: Thomé RG, Santos HB, Arantes FP, Prado PS, Domingos FFT, Sato Y, Bazzoli N, Rizzo E. Regression of post-ovulatory follicles in Prochilodus costatus Valenciennes, 1850 (Characiformes, Prochilodontidae). Braz J Morphol Sci 2006; 23: Santos HB, Rizzo E, Bazzoli N, Sato Y, Moro L. Ovarian regression and apoptosis in the South American teleost Leporinus taeniatus Lutken (Characiformes, Anostomidae) from the São Francisco basin. J Fish Biol 2005; 67: Santos HB, Sato Y, Moro L, Bazzoli N, Rizzo E. Relationship among follicular apoptosis, integrin β1 and collagen type IV during early ovarian regression in the teleost Prochilodus argenteus after induced spawning. Cell Tissue Res 2008; 332: Santos HB, Thomé RG, Arantes FP, Sato Y, Bazzoli N, Rizzo E. Ovarian follicular atresia is mediated by heterophagy, autophagy and apoptosis in Prochilodus argenteus and Leporinus taeniatus (Teleostei: Characiformes). Theriogenology 2008; in press. 18. Wood AW, Van Der Kraak G. Apoptosis and ovarian function: novel perspectives from the teleosts. Biol Reprod 2001; 64: Wood AW, Van Der Kraak G. Yolk proteolysis in rainbow trout oocytes after serum-free culture: evidence for a novel biochemical mechanism of atresia in oviparous vertebrates. Mol Reprod Dev 2003; 65: Krysko DV, Diez-Fraile A, Criel G, Svistunov AA, Vandenabeele P, D Herde K. Life and death of female gametes during oogenesis and folliculogenesis. Apoptosis 2008; 13: Figure 2. Hypothesis for the follicular cell fate in oviparous fish ovary after breeding. In the postovulatory follicle, apoptosis or type I cell death is the major cell death pathway, which contributes greatly to follicular cell removal, although possible involvement of autophagy is not rejected. In the follicular atresia, autophagy is the primary response in order to maintain viability of the follicular cell. However, some cells may also commit to die by apoptosis, independent or dependentcaspase. Another role in cell clearance is also possible for autophagy, when phagocytes are not sufficient for removal of the whole cell remains. The crosstalk between apoptosis and autophagy would have a cytoprotective effect leading to survival of the follicular cell during oocyte deletion. Autophagic vacuoles accumulated in follicular cells during late atresia induce autophagy-mediated or type II cell death. Otherwise, apoptosis could be triggered for more efficient removal of the follicular cells in follicular atresia. 21. Velentzas AD, Nezis IP, Stravopodis DJ, Papassideri IS, Margaritis LH. Mechanisms of programmed cell death during oogenesis in Drosophila virilis. Cell Tissue Res 2007; 327: Klionsky DJ. Autophagy: from phenomenology to molecular understanding in less than a decade. Nat Rev Mol Cell Biol 2007; 8: Landes Bioscience. Do not distribute. Autophagy 119

80 Cell Tissue Res (2012) 347: DOI /s REGULAR ARTICLE Autophagy and apoptosis interplay during follicular atresia in fish ovary: a morphological and immunocytochemical study R. D. V. S. Morais & R. G. Thomé & F. S. Lemos & N. Bazzoli & E. Rizzo Received: 21 October 2011 / Accepted: 11 January 2012 / Published online: 8 February 2012 # Springer-Verlag 2012 Abstract Follicular atresia in fish ovary provides an interesting model for studying autophagy and apoptosis. In order to improve knowledge of the mechanisms regulating ovarian regression, we investigated the immunolocalisation of various proteins involved in the complex network of autophagy and apoptosis. Females of three species of freshwater fish maintained in captivity were sampled after the reproductive period and the main events of follicular atresia were assessed by histology: splits in the zona radiata, yolk degradation and reabsorption, hypertrophy of the follicular cells, accumulation of autophagic vacuoles, closing of the follicular lumen and thickening of the theca. The interplay of apoptosis and autophagy was analysed by TUNEL in situ and by immunocytochemistry for caspase-3, bax, bcl-2, beclin-1 and cathepsin-d. During early and advanced stages of follicular regression, the actin cytoskeleton was well developed and labelling for bcl-2 and cathepsin-d were pronounced in the follicular cells at a stage when they were intensively involved in yolk phagocytosis. Immunofluorescence for beclin-1 was prevalent in the follicular cells, punctate labelling often surrounding autophagic vacuoles during the advanced stage of follicular regression, a critical This study was supported by grants from Brazilian Research Foundations: FAPEMIG, CNPq and CAPES. R. D. V. S. Morais : R. G. Thomé : F. S. Lemos : E. Rizzo (*) Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, C.P. 486, Belo Horizonte, Minas Gerais, Brasil ictio@icb.ufmg.br N. Bazzoli Programa de Pós-Graduação em Zoologia de Vertebrados, Pontifícia Universidade Católica de Minas Gerais, PUC Minas, Belo Horizonte, Minas Gerais, Brasil step towards cell death. TUNEL-positive reaction and immunostaining for bax and caspase-3 demonstrated the participation of apoptosis in late follicular regression. Overall, this study provides evidence that autophagic and apoptotic proteins are activated in a coordinated fashion depending on the stage of follicular regression, with interplay between autophagy and apoptosis being essential in determining the fate of the cell during follicular atresia in fish ovary. Keywords Bax. Beclin-1. Bcl-2. Caspase-3. Cathepsin-D. Follicular atresia. Fish Introduction In vertebrates, a large number of ovarian follicles that are recruited for development do not complete maturation, degenerate and are reabsorbed in a process known as follicular atresia. In fish, the vitellogenic follicles that are not spawned during the reproductive period become atretic and are then slowly reabsorbed during ovarian regression after spawning (Rizzo and Bazzoli 1995; Miranda et al. 1999; Santos et al. 2005). Intrinsic and extrinsic factors, including hyphophysectomy, irradiation, anti-gonadotropic substances, thermal stress, confinement and starvation, induce follicular atresia in fish ovary and decrease the reproductive potential of the species (Saidapur 1978; Nahum et al. 1996). Several studies indicate that apoptosis is the main mechanism involved in the elimination of somatic and germ cells in atretic follicles of birds and mammals (Andreu-Vieyra and Habibi 2000; Hussein 2005). However, this programmed cell death appears to play a secondary role during follicular atresia in teleost fish (Santos et al. 2008; Thomé et al. 2009).

81 468 Cell Tissue Res (2012) 347: Apoptosis is a well-characterised programmed cell-death pathway that affects single cells detached from neighbouring cells and the basement membrane. Highly conserved during evolution, apoptosis requires specialised machinery that involves proteases known as caspases (Djavaheri- Mergny et al. 2010). Furthermore, the Bcl-2 protein family, including the anti-apoptotic members, bcl-2 and bcl-x L and also the pro-apoptotic members bax and bad, are central regulators of apoptosis by connecting signals of survival and cell death that are generated within or outside the cell (Adams and Cory 2001; Borner 2003). Evidence obtained from various models supports the idea that multiple programs of cell death can be triggered depending on the circumstances (Assunção Guimarães and Linden 2004; Degterev and Yuan 2008). Under stress or cellular damage, including starvation, oxidative stress, nutrient deprivation and the withdrawal of growth factors, autophagy is primarily induced to provide the energy necessary to support changes in metabolism, thus ensuring the survival of the cell; however, under some conditions, autophagy can also lead to apoptosis and/or alternative pathways of cell death (Maiuri et al. 2007; Moreau et al. 2010). Macroautophagy (hereafter referred to as autophagy) is a complex catabolic program, which is genetically regulated and evolutionarily conserved from yeast to humans, for the recycling of cytoplasmic organelles and long-lived or malfunctioning proteins through the lysosomal machinery (Cao and Klionsky 2007; Kang et al. 2011). Among the lysosomal hydrolases, an aspartic protease of the superfamily of pepsins, cathepsin-d, has a central role in protein turnover and tissue remodelling and also participates in programmed cell death (Zaidi et al. 2008; Carmona-Gutiérrez et al. 2011). The main morphological changes occurring during autophagy include the formation of the autophagosome (hereafter referred to as the autophagic vacuole) and the collapse of cytoplasmic organelles, whereas the nuclear structure remains apparently unaffected (Assunção Guimarães and Linden, 2004; Maiuri et al. 2007; Kroemer et al. 2009). Autophagic vacuoles are well established as being formed at random sites in the cytoplasm and then trafficked along microtubules in a dynein-dependent fashion towards the microtubule-organising centre for lysosomal degradation (Jahreiss et al. 2008). However, knowledge of the organisation of the actin cytoskeleton in autophagic cells and whether actin plays a direct role in the biogenesis of the autophagic vacuole remains scarce (Reggiori et al. 2005). Among the autophagic proteins, beclin-1, a product of tumour suppressor gene Beclin-1, the mammalian orthologue of yeast Atg6, is part of the class III phosphatidylinositol 3-kinase (PI3K) complex of the endoplasmic reticulum and induces the nucleation of a double-membrane vesicle, the autophagic vacuole (Maiuri et al. 2007, 2010). Furthermore, beclin-1 interacts with bcl-2, an anti-apoptotic protein, playing a crucial role in the relationship between autophagy and apoptosis (Zhou et al. 2010). Interestingly, recent studies have shown that the caspase-mediated cleavage of beclin-1 inactivates beclin-1-induced autophagy and enhances apoptosis in several cellular types (Cho et al. 2009; Djavaheri- Mergny et al. 2010; Wirawan et al. 2010). The crosstalk between apoptosis and autophagy is complex, since in some circumstances, autophagy is a form of adaptation to stress by suppressing apoptosis, whereas under other conditions, it can induce an alternative pathway to cellular demise, known as autophagic cell death (Mizushima et al. 2008; Maiuri et al. 2010). Although enhanced autophagy has been associated with cell death in many cases, these reports have failed to demonstrate a causal role for autophagy in initiating a killing pathway (Fimia and Piacentini 2010). In fish ovary, previous studies have indicated that both apoptosis and autophagy act cooperatively and a dual role has been attributed to autophagy to ensure more efficient ovarian regression after spawning (Thomé et al. 2009). In order to improve knowledge of the mechanisms regulating ovarian regression, the aim of this study has been to investigate, by immunocytochemistry, the localisation of proteins involved in autophagy and apoptosis pathways during follicular atresia in fish with various reproductive strategies. Materials and methods Animals Adult females of three characiform fish species (Table 1) kept in a hatchery for 2 3 years in southeastern Brazil (18 12 S, W) were used. During stocking, the fish were maintained in a 200-m 2 tank at a stocking rate of 1 kg fish per 7 m 2, supplied with a continuous flow of water from the Três Marias Reservoir and fed daily with a commercial diet containing 22% crude protein. After the reproductive period (November to February), non-spawned females were killed by transversal section of the spinal cord following the ethical principles established by the Brazilian College of Animal Experimentation (COBEA, Our research was approved by the Ethics Committee of Animal Experimentation (CETEA - protocol 071/2008) at the Federal University of Minas Gerais (UFMG), Brazil. Histology Samples of the middle region of the ovary were fixed in Bouin s fluid for 6 h at room temperature, embedded in Paraplast, sectioned at a thickness of 5 μm and stained with haematoxylin and eosin (HE) for analyses of the main events of atretic follicles regression.

82 Cell Tissue Res (2012) 347: Table 1 Biometric data and reproductive strategy of the characiform fishes used Species Number Total length (cm) Body weight (g) Reproductive strategy Astyanax bimaculatus (Linnaeus, 1758) ± ±6.6 Sedentary Leporinus obtusidens (Valenciennes, ± ±70.2 Migratory 1837) Prochilodus argenteus Spix and Agassiz, ± ±142.2 Migratory TUNEL in situ reaction Ovarian samples were fixed in 4% paraformaldehyde solution in 0.1 M phosphate buffer at ph 7.3 for 24 h at 4 C, embedded in paraplast, sectioned at a thickness of 5 μm and submitted to TUNEL (terminal transferase-mediated dutp nick-end labelling) assay by using the ApopTag kit (Millipore). Before reaction, the sections were washed in phosphate-buffered saline (PBS) and treated with proteinase K for 20 min for tissue permeabilisation followed by 3% H 2 O 2 for 30 min to inactivate endogenous peroxidase. Next, the sections were incubated with terminal deoxynucleotidyl transferase (TdT) and digoxigenin-conjugated deoxynucleotides for 1 h at 37 C and then with anti-digoxigenin solution conjugated with peroxidase for 30 min in a humidified chamber at room temperature. After the sections had been washed with PBS, the reaction was developed with diaminobenzidine (DAB) for 6 min at room temperature followed by counterstaining with haematoxylin. For the negative control, the treatment with TdT-labelled deoxynucleotides was omitted. Immunocytochemistry Ovarian samples were fixed in 4% paraformaldehyde solution, embedded in Paraplast, sectioned at a thickness of 5 μm and submitted to immunocytochemistry reactions for the detection of the main proteins of the autophagic and apoptotic pathways (Table 2). For antigen recovery, sections Table 2 Primary antibodies supplied by Santa Cruz Biotechnology, with dilution and antigen recovery as used in the imunohistochemical reactions Antibodies Dilution Antigen recovery Actin (C-2) monoclonal mouse, sc :100 Heat-induced Bcl-2 (N-19) polyclonal rabbit, sc-492 1:50 No recovery Bax (P-19) polyclonal rabbit, sc-526 1:100 Heat-induced Beclin-1 (H-300) polyclonal rabbit, 1:100 Heat-induced sc Caspase-3 (E-8) monoclonal mouse, 1:200 Heat-induced sc-7272 Cathepsin-D (H-75) polyclonal rabbit, sc :25 Heat-induced were boiled in 10 mm sodium citrate with 0.05% Tween 20 at ph 6.0 for 30 min. After being washed in PBS, the sections were incubated with 3% H 2 O 2 in methanol to inactivate endogenous peroxidase. For permeabilisation and to block unspecific staining, blocking buffer (2% bovine serum albumin+0.05% Triton X % Tween 20) with 6% goat serum was used for 45 min. Next, primary antibody was applied to the sections overnight in a humidified chamber at 4 C. For immunoperoxidase, sections were washed with PBS and incubated with biotinylated secondary antibody (LSAB 2 System HRP, DakoCytomation) for 2 h and then with peroxidase-conjugated streptavidin for 45 min; immunostaining was revealed with DAB and the sections were counterstained with haematoxylin. For immunofluorescence, the sections were incubated with a secondary antibody, namely anti-rabbit IgG conjugated with Alexa fluor 488 (1:200) or anti-mouse IgG conjugated with Alexa fluor 568 (1:200). Nuclear DNA labelling was performed by propidium iodide (1:500) or 4,6-diamidino-2-phenyl-indole (DAPI; 1:500). For the negative control, the treatment with the primary antibody was omitted. Sections were examined with a confocal laser microscope. Results Morphological hallmarks of follicular atresia Ovaries of the three fish species showed perinucleolar and vitellogenic follicles and atretic follicles in various stages of regression. Healthy vitellogenic follicles exhibited a thick zona radiata, squamous follicular cells and a thin connective theca (Fig. 1a). Follicular atresia was subdivided into three stages, following the main morphological events of the regression of the vitellogenic follicles: early, advanced and late. In early atresia, the yolk globules lost their integrity becoming liquefied, the zona radiata exhibited splits and the follicular cells hypertrophied progressively (Fig. 1b). Advanced follicular atresia was characterised by highly columnar follicular cells, which were in intense phagocytic activity to engulf the remains of the yolk and zona radiata (Fig. 1c). At this stage, autophagic vacuoles were gradually formed and became numerous in the follicular cells when most of the yolk had been reabsorbed (Fig. 1d). In the late

83 470 Cell Tissue Res (2012) 347: Fig. 1 Main events of ovarian follicular atresia in three fish species: Leporinus obtusidens, Prochilodus argenteus, Astyanax bimaculatus. Histological sections stained with haematoxylin and eosin. a Healthy vitellogenic follicle showing thick zona radiata (ZR), squamous follicular cells (FC) and intact yolk globules (Y). b Zona radiata (ZR) with splits and yolk being degraded (Y) during early atresia. c Yolk reabsorption (Y) by hypertrophied follicular cells (FC) and vascularised theca (T) during advanced follicular atresia. d Follicular cells (FC) with autophagic vacuoles (star); the follicular lumen (L) is partially occluded and the theca (T) is thicker during advanced follicular atresia. e The theca (T) is fibrous, thickened and vascularised (BV) during late follicular regression. f Connective scar at the end of follicular regression. Bars 30 µm stage of follicular atresia, the follicular lumen was gradually occluded, the follicular cells were less numerous, some granulocytes invaded the follicular area and the theca became fibrous, thickened and vascularised (Fig. 1e), forming a connective scar at the end of follicular regression (Fig. 1f). Analyses of ovaries of the three species by immunofluorescence showed an actin cytoskeleton around the yolk globules in healthy vitellogenic follicles and a thin labelling in squamous follicular cells (Fig. 2a-c). During follicular atresia, actin reactivity was reduced in the degenerating oocyte and strong labelling was evident in the hypertrophied follicular cells (Fig. 2d-f). Apoptosis During follicular atresia, apoptosis was shown by the DNA fragmentation (TUNEL-positive reaction) associated with morphological features such as condensation of the chromatin, shrinkage and cell fragmentation to form apoptotic bodies. During early and advanced stages of regression, TUNEL-positive cells were rarely observed (Fig. 3a). In contrast, follicular and thecal cells in apoptosis were more frequently registered at the late stage of follicular regression (Fig. 3b). Positive-reaction for caspase-3 revealed the execution of apoptosis during late follicular atresia (Fig. 3c).

84 Cell Tissue Res (2012) 347: Fig. 2 Immunofluorescence of actin cytoskeleton (red) in health vitellogenic follicles (a-c) andin follicular cells during follicular atresia (d-f) inp. argenteus. Nuclei are stained with DAPI (blue). Note that yolk globules showed unspecific fluorescence (blue). Bars 30 µm Scarce reaction for caspase-3 was observed during early follicular atresia. Immunofluorescence analyses demonstrated bcl-2 protein in the cytoplasm of the cells during all stages of follicular regression. In the early stage of atresia, strong staining was observed in the granulocytes and thecal cells (Fig. 3d, e). During the advanced stage, the hypertrophied follicular cells were mainly labelled for bcl-2 protein (Fig. 3f), whereas at the late stage of regression, the remaining cells were still positive for bcl-2 (Fig. 3g). In perinucleolar follicles, the follicular cells were also labelled for bcl-2 (data not shown). In the ovaries of all the species analysed, the immunostaining for the protein bax was characterised as points scattered throughout the cytoplasm. In the early and advanced stages of atresia, weak and diffuse staining was detected in the follicular cells and autofluorescence was found in the yolk globules and in the yolk engulfed by the follicular cells (Fig. 3h). At the late stage of follicular atresia, the labelling for bax was prominent in the cytoplasm of follicular cells, granulocytes and thecal cells (Fig. 3i). Autophagy The expression pattern of cathepsin-d was dependent on the stage of regression of the atretic follicle. During early follicular atresia, a minor immunoreaction was observed among the yolk globules but was more evident in cytoplasmic granules (probably phagolysosomes that engulfed the yolk) of the follicular cells (Fig. 4a-b). At the advanced stage of follicular regression, diffuse immunostaining was scattered in the cytoplasm of the hypertrophied follicular cells, mainly around autophagic vacuoles (Fig. 4c-e). Finally, in the late stage, the remaining cells showed weak immunostaining (Fig. 4f). Theca cells were not immunoreactive for cathepsin-d. Immunofluorescence for beclin-1 protein was prevalent in the follicular cells as points spread within the cytoplasm and often surrounding autophagic vacuoles during the advanced stage of follicular regression (Fig. 5a-c). Thecal cells were also labelled in this phase. After reabsorption of the yolk, the reactivity for beclin-1 was less prominent (Fig. 5d), whereas during the late stage of follicular regression, the remaining cells showed weak immunostaining (Fig. 5e). Interplay between apoptosis and autophagy Double-labelling for beclin-1 and caspase-3 was performed in the ovaries of the three fish species. In the early stage of atresia, beclin-1 was localised mainly in the follicular cells, whereas reaction for caspase-3 was discrete (Fig. 6a). During the advanced stage of atresia, the double-immunostaining showed evident expression of caspase-3, even though the signal for beclin-1 was still high (Fig. 6b-d). At the late stage of atresia, the reaction for caspase-3 was prominent in the remaining cells of the atretic follicles (Fig. 6e-f). Discussion This study has shown, for the first time, the immunolocalisation of bcl-2, bax, caspase-3, beclin-1 and cathepsin-d

85 472 Cell Tissue Res (2012) 347: Fig. 3 Apoptosis during ovarian follicular atresia in three fish species: L. obtusidens, P. argenteus, A. bimaculatus. Nuclei are stained with haematoxylin (a, b) or propidium iodide (red, c-h). a TUNEL-positive follicular cells (arrow) in early atresia. b Numerous TUNEL-positive cells (arrows) in late atresia. c Immunoreaction for caspase-3 in late atresia. d, e Intense immunofluorescence to bcl-2 in granulocyte (arrows) and theca (T) cells during early atresia; zona radiata (ZR) was not reactive. f Hypertrophied follicular cells (FC) showing immunoreaction to bcl-2. g Remains of theca cells (T) with labelling for bcl-2. h Immunofluorescence to bax with weak staining in follicular cells during early atresia. i Prominent immunoreaction for bax in the cytoplasm of follicular cells, granulocytes and thecal cells (T) in late atresia. Bars 30 µm (a, b), 20 µm (c, e h), 10 µm (d) during regression of vitellogenic follicles of three fish species and suggests that an interplay occurs between autophagy and apoptosis during follicular atresia, with a balance between signals of survival or death being essential in determining the fate of the follicular cells (Table 3, Fig. 7). In insect ovaries, both apoptosis and autophagy operate synergistically in order to achieve a rapid and efficient clearance of cells that are no longer needed, resulting in undisturbed oocyte maturation (Nezis et al. 2006; Velentzas et al. 2007a, b; Mpakou et al. 2011). Although freshwater fish exhibit various reproductive strategies, the major events of follicular atresia are similar in the species analysed in the present study and in the others reported previously (Rizzo and Bazzoli, 1995; Miranda et al. 1999; Santos et al. 2005, 2008). In addition, the imunocytochemical reactions for autophagic and apoptotic proteins also show a similar pattern of molecular expression among species, suggesting that follicular atresia is not directly related to the reproductive strategy of the species, since A. bimaculatus performs multiple spawnings throughout

86 Cell Tissue Res (2012) 347: Fig. 4 Immunoreaction to cathepsin-d revealed via peroxidase during ovarian follicular atresia. Nuclei are stained with haematoxylin. a, b Follicular cells (FC) showing cytoplasmic granules (arrows) stained in early atresia (Y yolk, ZR zona radiata). c Advanced follicular atresia with intense reabsorption of the yolk (Y). d, e Follicular cells (FC) with diffuse labelling around autophagic vacuoles (stars). f Late follicular atresia with the remaining cells showing weak staining for cathepsin-d. Bars 30 µm the year, whereas L. obtusidens and P. argenteus are total spawners and their reproduction is synchronised with the rainy season (Bazzoli 2003). On the basis that autophagic and apoptotic pathways are highly conserved in evolution and with the support of the morphogical and immunocytochemical results of the present study, we can now suggest that ovarian follicular atresia follows a general pattern of regression and is regulated by common mechanisms in fish with external fertilisation. The actin cytoskeleton that we have observed in the ooplasm of healthy vitellogenic follicles in this study might be related to support of the yolk globules, according to the data reported for P. argenteus with regard to induced spawning by hypophysation (Arantes et al. 2011). During follicular atresia, the actin cytoskeleton of the ooplasm undergoes changes associated with the degradation of the yolk, whereas it remains preserved in follicular cells. In the yeast Saccharomyces cerevisiae, the actin cytoskeleton is required for the recruitment of the Cvt cargo pathway to the preautophagosomal structure and for the correct cycling of Atg9 (Reggiori et al. 2005). Taken together, these data suggest that, in the fish investigated in the present study, actin might be involved in phagosome formation to engulf the yolk accumulated in the oocyte and in the nucleation of autophagic vacuoles. Thus, the maintenance of the actin cytoskeleton might be a survival signal for the follicular cells during early and advanced atresia. In contrast, the prominent ring of actin that has been observed in follicular cells of recently spawned postovulatory follicles is probably related to apoptosis (Thomé et al. 2010). Indeed, in the first stage of apoptosis, the cytoskeleton of actin is reorganised into a peripheral ring, making an association with myosin leading to contraction and

87 474 Cell Tissue Res (2012) 347: Fig. 5 Immunofluorescence to beclin-1 (green) during ovarian follicular atresia. Nuclei are stained with propidium iodide (red). a, b Follicular cells (FC) are immunoreactive in early atresia (ZR zona radiata). c Hypertrophied follicular cells (FC) showing intense labelling for beclin-1 (inset higher magnification of follicle cell undergoing hypertrophy). d Advanced follicular atresia, with intense immunostaining for beclin-1in theca (T) and follicular cells (FC). e Discrete labelling in theca cells (T) during late follicular atresia. Bars 20 µm (a, c e), 10 µm (b) the formation of blebs (Ndozangue-Touriguine et al. 2008). This is a facilitator of the interaction with phagocytic cells and might be associated with late follicular atresia when the remaining follicular cells undergo apoptosis. Studies on zebrafish embryos have shown homology amongthebcl-2familymembersinfishandmammals (Kratz et al. 2006); however, the role of the Bcl-2 family in fish ovaries remains unclear. In the present study, we have detected the expression of bcl-2 protein during all phases of the regression of atretic follicles, predominantly in follicular cells at the stage when they exhibit intense phagocytic activity. Indeed, bcl-2 is best known for preventing apoptosis by binding to bax or bak thereby avoiding the release of cytochrome c from mitochondria (Borner 2003; Zhou et al. 2010). In vitro, human granulosa cells show high levels of bcl-2, providing evidence that this protein plays an important role in the suppression of apoptosis (Sasson and Amsterdam 2002). On the other hand, the reactivity for bax detected in the present study is greater in the follicular cells in the late stage of follicular regression, simultaneously

88 Cell Tissue Res (2012) 347: Fig. 6 Double-labelling for beclin-1 (green) and caspase-3 (red) during ovarian follicular in L. obtusidens. Nuclei are stained with DAPI (blue). a Follicular cells (FC) with weak immunostaining for caspase-3, in contrast to strong reaction for beclin-1 during early atresia (Y yolk). b-d Labelling for beclin- 1(b) and caspase-3 (c) and the merged image (d) in advanced atresia. e Double-staining for beclin-1 and caspase-3, with predominance for caspase-3 in late atresia. f Reaction for caspase-3 was prominent in the remaining cells of the atretic follicles. Bars 20 µm Table 3 Expression pattern of proteins of the autophagic and apoptotic pathways during follicular atresia in the ovary of three fish species (+ weak immunostaining, ++ moderate immunostaining, +++ intense immunostaining) Protein Early atresia Advanced atresia Late atresia Bcl Bax Caspase Cathepsin-D Beclin with the TUNEL- and caspase-3-positive reactions, which confirm the execution of apoptosis. Similarly, a comparison of healthy and atretic follicles has demonstrated an increased expression of bax in granulosa cells in atretic follicles of rats, quail and humans (Andreu-Vieyra and Habibi 2000). Taken together, the bcl-2 and bax detected in the ovaries of this study indicate the involvement of the Bcl-2 protein family in the regulation of follicular atresia in fish ovary. In the present study, cathepsin-d has been detected in the follicular cells at all stages of follicular regression, whereas