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Estrogens and Regucalcin in Testicular Apoptosis and Sperm Function: “A Matter of Life and Death”

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Estrogens and Regucalcin in Testicular Apoptosis and Sperm Function: “A Matter of Life and Death” UNIVERSIDADE DA BEIRA INTERIOR Ciências da Saúde Estrogens and regucalcin in testicular apoptosis and sperm function: “a matter of life and death” Sara Carina de Lima Correia Thesis for Doctoral Degree in Biomedicine (3rd cycle of studies) Supervisor: Sílvia Cristina da Cruz Marques Socorro, PhD Co‐supervisor: José Eduardo Brites Cavaco, PhD Co‐supervisor: Anna Maria Margaretha van Pelt, PhD Covilhã, October 2014 ii Ao Carlos e à minha mãe. “O caminho faz‐se caminhando” mas sempre com alguém do nosso lado. iii Agradecimentos Quero aqui expressar os meus agradecimentos a todos aqueles que contribuíram para a realização deste trabalho. Em primeiro lugar, quero agradecer aos meus orientadores Professora Doutora Sílvia Cristina da Cruz Marques Socorro, Professor Doutor José Eduardo Brites Cavaco e Professora Doutora Ans van Pelt pela orientação científica durante a realização deste trabalho, pelo apoio, motivação e disponibilidade demonstrados. Em especial, à Professora Sílvia pelos e‐mails fora de horas, noites mal dormidas e por toda a amizade. Aos meus colegas e amigos dos laboratórios de investigação da Faculdade de Ciências da Saúde, Cátia Vaz, Margarida Gonçalves, Luís Pedro Rato, Ricardo Marques, Henrique Cardoso, Marília Figueira, Inês Gomes e Catarina Ferreira. Com animação tudo se torna mais fácil. Em especial, à Cátia e à Margarida por toda a amizade e momentos de confidências. A todas as pessoas do Centro de investigação em Ciências da Saúde. Não podendo deixar de referir a Sofia Duarte e a Margarida Carrilho pelo apoio e boa disposição. Ao Carlos que viveu intensamente esta tese talvez tanto quanto eu e esteve presente em todos os momentos (mesmo quando à distância). Sem dúvida que esta tese tinha de ser dedicada a ti. Sem ti tudo não seria mais difícil, seria impossível. Agradeço com carinho aos meus pais, Ana Maria e Joaquim Magalhães por estarem sempre presentes servindo como exemplo e apoiando. À minha sobrinha Raquel que a dada altura do Doutoramento me disse: “Titi não te preocupes, vai tudo correr bem”. Ao meu pequenino, o meu sobrinho Santiago cujos olhinhos sorriem e fazem nascer uma vontade imensa de lutar. À minha irmã, ao meu cunhado e aos meus avós por todo o apoio e carinho transmitido ao longo destes anos. À Universidade da Beira Interior e a todas as pessoas que conheci que de certo modo deixaram algo. Por último, gostaria de agradecer à FCT por financiar o meu doutoramento (SFRH/BD/60945/2009). iv v Publications in International Peer-Reviewed Journals Included in the thesis: Correia S, Cardoso HJ, Cavaco JE and Socorro S. Estrogens as apoptosis regulators in mammalian testis: angels or devils? Expert Reviews in Molecular Medicine (submitted, under revision). IF: 6.62 Correia S, Alves MR, Cavaco JE, Oliveira PF and Socorro S. Estrogenic regulation of testicular expression of SCF and c‐kit: implications in germ cell survival and male fertility. Fertility and Sterility (2014) 102(1):299‐306. IF: 4.174 Correia S, Alves MG, Oliveira PF, Alves MR, van Pelt AMM, Cavaco JE, Socorro S. Transgenic overexpression of regucalcin leads to suppression of thapsigargin‐ and actinomycin D‐induced apoptosis in the testis by modulation of apoptotic pathways. Andrology (2014) 2(2):290‐298. IF: 3,294 Correia S, Oliveira PF, Guerreiro PM, Lopes G, Alves MG, Canário AVM, Cavaco JE, Socorro S. Sperm parameters and epididymis function in transgenic rats overexpressing the Ca2+‐binding protein regucalcin: a hidden role for Ca2+ in sperm maturation? Molecular Human Reproduction (2013) 19(9):581‐9. IF: 4.542 Laurentino S, Correia S, Cavaco JE, Oliveira PF, Sousa M, Barros A, and Socorro S. Regucalcin, a calcium‐binding protein with a role in male reproduction? Molecular Human Reproduction (2012) 18 (4): 161‐170. IF: 4.542 Other publications during the PhD: Figueira MI, Cardoso HJ, Correia S, Maia CJ and Socorro S. Hormonal regulation of c‐KIT receptor and its ligand: implications for human infertility? Progress in Histochemistry and Cytochemistry DOI: 10.1016/j.proghi.2014.09.001. IF: 5.909 Cardoso HJ, Figueira MI, Correia S, Vaz CV and Socorro S. The SCF/c‐KIT system in the male: survival strategies in fertility and cancer. Molecular Reproduction and Development DOI:10.1002/mrd.22430. IF: 2.812 Vaz CV, Maia CJ, Marques R, Gomes IM, Correia S, Alves MG, Cavaco JE, Oliveira PF, Socorro S. Regucalcin is an Androgen‐Target Gene in the Rat Prostate Modulating Cell‐Cycle and Apoptotic Pathways. The Prostate (2014) 74:1189‐1198. IF: 3.843 Oliveira PF, Alves MG, Martins AD, Correia S, Bernardino RL, Silva J, Barros A, Sousa M,Cavaco JE, Socorro S. Expression pattern of G protein‐coupled receptor 30 in human seminiferous tubular cells. General and Comparative Endocrinology (2014) 201:16‐20. IF: 2.823 vi Correia S and Socorro S. New Emerging Androgenic Actions in the Regulation of Sperm Production and Function. Theriogenology Insight (2014) 4(1):33‐48. Alves MG, Martins AD, Vaz CV, Correia S, Moreira PI, Oliveira PF, Socorro S. Metformin and male reproduction: effects in Sertoli cell metabolism. British Journal of Pharmacology (2014) 171(4):1033‐42. IF: 4.99 Marques R, Maia C, Vaz C, Correia S, Socorro S. The diverse roles of calcium‐binding protein regucalcin in cell biology: from tissue expression and signalling to disease. Cellular and Molecular Life Sciences (2014) 71(1):93‐111. IF: 5.615 Laurentino S, Pinto P, Correia S, Cavaco JE, Canário AVM, Socorro S. Structural variants of sex steroid hormone receptors in the testis: from molecular biology to physiological roles. OA Biotechnology (2012) 1 (2):4. Laurentino SS *, Correia S *, Cavaco JE, Oliveira PF, Rato L, Sousa M, Barros A, and Socorro S. Regucalcin is broadly expressed in male reproductive tissues and is a new androgen‐target gene in mammalian testis. Reproduction (2011) 142: 447‐456. IF: 3,451 *both authors contributed equally vii viii Resumo A espermatogénese é um processo complexo e rigorosamente coordenado, através do qual milhares de espermatozóides são produzidos diariamente nas gónadas masculinas. Para além de realizarem esta função, os testículos dos mamíferos são um órgão endócrino, e produzem as hormonas esteróides sexuais necessárias para a estimulação do processo espermatogénico e para o normal desenvolvimento do fenótipo masculino. Nos últimos anos, os estrogénios têm sido identificados como importantes reguladores da função reproductiva masculina, no entanto, a sua função na espermatogénese não tem encontrado unanimidade entre os investigadores. Alguns estudos sugerem que os estrogénios actuam como factores de sobrevivência para as células germinativas, enquanto outros, associam a acção destas hormonas à apoptose testicular e à diminuição do número de células germinativas. Para além disso, os alvos moleculares subjacentes aos efeitos dos estrogénios no testículo dos mamíferos, levando à sobrevivência ou à morte celular, permanecem por esclarecer. A regucalcina (RGN) é uma proteína de ligação ao cálcio (Ca2+) que tem um papel importante na homeostase da concentração intracelular deste ião, e para a qual foi sugerida uma função na espermatogénese. A RGN foi identificada em diversos tecidos do tracto reproductivo masculino, sendo descrita a sua regulação pelos androgénios no testículo, bem como, a sua regulação pelos estrogénios em células de próstata de rato e humana. No entanto, são totalmente desconhecidos os efeitos dos estrogénios nos níveis de expressão da RGN no testículo. É de salientar ainda a função que tem vindo a ser atribuída à RGN ao nível do controlo da sobrevivência celular e apoptose, assim como, as suas propriedades antioxidantes. Apesar de se reconhecer que o controlo rígido da apoptose e do stress oxidativo são de importância fulcral para a função testicular, a função da RGN no testículo permanece desconhecida. É durante a passagem pelo epidídimo que os espermatozóides sofrem um processo de maturação que lhes permite adquirir motilidade progressiva e a capacidade de fertilizar o oócito. Apesar de ter sido descrita a existência de um gradiente de concentração de Ca2+ ao longo do epidídimo, os efeitos deste ião no funcionamento do epidídimo encontram‐se insuficientemente explorados. A presente tese tem como objectivo principal elucidar a relação existente entre os efeitos estrogénicos e a apoptose das células germinativas, incluindo igualmente os efeitos reguladores destas hormonas esteróides na expressão testicular da RGN. Em segundo lugar, será explorado o papel da RGN na regulação da apoptose e do stress oxidativo no testículo, assim como, na maturação dos espermatozóides no epidídimo. Uma dose de 100 nM de 17β‐ estradiol (E2), mimetizando as concentrações de estrogénios encontradas no testículo de pacientes inférteis, culminou na apoptose das células germinativas e na diminuição da proliferação, o que aconteceu simultaneamente com alterações na expressão do sistema SCF/c‐kit. O tratamento com E2 foi ainda acompanhado por um aumento da expressão da RGN, o que sugere que os níveis desta proteína podem estar aumentados como um mecanismo ix de prevenção da apoptose induzida pelo E2. Relativamente ao papel da RGN na modulação da apoptose testicular, observou‐se que esta tem um papel importante na prevenção da morte celular induzida por estímulos nocivos. Mais ainda, verificou‐se que a sobreexpressão da RGN conduz a um aumento da protecção contra o stress oxidativo no testículo, o que confirma o papel citoprotector desta proteína. Os resultados obtidos demonstraram também a relevância da manutenção dos níveis de Ca2+ no lúmen do epidídimo e
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