Espermatogênese in vitro: desenvolvimento de um modelo e avaliação do papel da relaxina
Arquivos
Data
2014
Autores
Pimenta, Maristela Taliari 
Orientadores
Lazari, Maria de Fatima Magalhaes
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
O papel de relaxina na reproducao masculina ainda e incerto. O knockout para relaxina em machos diminui a maturacao espermatica e estudos de nosso laboratorio mostraram que relaxina e seu receptor RXFP1 sao expressos em celulas de Sertoli de rato e relaxina promove a proliferacao das celulas de Sertoli em cultura, o que pode afetar a espermatogenese indiretamente. Relaxina tambem poderia afetar a espermatogenese diretamente, ao interagir com seu receptor RXFP1 detectado em espermatides alongadas. Para investigar o papel de relaxina na espermatogenese, desenvolvemos uma cocultura de celulas extraidas de testiculos de ratos de 7 dias de idade, que apresentam, como celulas germinativas, somente celulas tronco espermatogoniais (SSCs) e espermatogonias indiferenciadas. As celulas foram cultivadas em Matrigel, previamente tratado para remover componentes difusiveis que afetam a diferenciacao de celulas germinativas. Apos cultivo por 48h, na ausencia ou presenca de 0,5% de soro fetal bovino (FBS) ou relaxina humana H2 100 ng/mL (RLN), as celulas foram mantidas, por 24h, em meio livre de suplemento e cultivadas, por 48 ou 120h adicionais, na ausencia de suplementacao. A diferenciacao das celulas germinativas foi analisada por imunocitoquimica, imunofluorescencia e citometria de fluxo, apos 2, 5 ou 8 dias (D2, D5 e D8). A razao entre celulas germinativas e somaticas (G/S) caiu drasticamente de D2 para D5, e esta queda nao foi afetada por FBS, mas foi parcialmente impedida por RLN, que preservou a proporcao de celulas germinativas, especialmente das populacoes 2C e 4C (espermatogonias e espermatocitos). Alem disso, RLN aumentou a proporcao de celulas somaticas 4C. A expressao genica e proteica dos seguintes marcadores de diferentes estagios da espermatogenese foi analisada por PCR quantitativo ou citometria de fluxo: Plzf (espermatogonias indiferenciadas), c-kit (espermatogonias diferenciadas), Sycp3 (celulas em meiose) e Odf2 (celulas pos-meioticas). Em D5, RLN aumentou significativamente a expressao genica de c-Kit e Odf2 e a proteica de PLZF e c-KIT. RLN aumentou o numero de celulas ODF2-positivas, na imunocitoquimica. Alem disso, relaxina favoreceu a organizacao das celulas testiculares em estruturas semelhantes a tubulos, aumentou o numero de celulas mioides e sua organizacao ao redor dos tubulos, e aumentou a expressao de caderina 2 e β-catenina, que participam de juncoes celula-celula. O silenciamento da relaxina endogena com siRNA impediu a organizacao das celulas testiculares em cultura e alterou o balanco entre autorrenovacao e diferenciacao das celulas germinativas. Estes achados sugerem que a relaxina pode desempenhar um papel na espermatogenese, por aumentar o numero de celulas germinativas pre e pos-meioticas, e favorecer a organizacao das celulas testiculares
The role of relaxin in male reproduction is still unclear. The knockout of relaxin decreases sperm maturation, and studies from our laboratory have shown that relaxin and RXFP1 are expressed in rat Sertoli cells and relaxin stimulates Sertoli cell proliferation, which could indirectly affect spermatogenesis. Relaxin may directly affect spermatogenesis through interaction with RXFP1 previously detected in elongating spermatids. To investigate a role of relaxin in spermatogenesis, we developed a coculture of cells extracted from the testis of 7dayold rats, which only contains spermatogonial stem cells and undifferentiated spermatogonia. Cells were plated on Matrigel, which was depleted of diffusible components that could interfere with germ cell differentiation by incubation with growth medium. Cells were cultured for 48 h in the absence or presence of 0.5% fetal bovine serum (FBS) or 100 ng/mL H2 relaxin (RLN). Cells were then kept for 24 h in supplementfree medium, and cultured for additional 48 or 120 h in the absence or presence of FBS or RLN. Cell number and differentiation were analyzed by immunocytochemistry, confocal microscopy and flow cytometry, after 2, 5 or 8 days (D2, D5 and D8, respectively). The ratio between germ and somatic cells (G/S) fell drastically from D2 to D5, and this fall was not affected by FBS, but it was partially prevented by RLN, which preserved especially 2C and 4C populations of germ cells (spermatogonia and permatocytes). RLN also increased 4C population of somatic cells. The gene and protein expression of the following markers of different stages of spermatogenesis was analyzed by quantitative PCR and flow cytometry: Plzf (undifferentiated spermatogonia), ckit (differentiated spermatogonia), Sycp3 (meiosis) and Odf2 (post meiosis). RLN tended to increase gene expression of Plzf and significantly increased gene expression of cKit in D5. RLN significantly increased gene expression of ckit and Odf2 in D5. RLN increased both PLZF and cKIT protein expression in D5, and increased the number of ODF2positive cells in immunocytochemistry assay. Furthermore RLN favored organization of cells in tubularlike structures in 2Dculture, increased the number of myoid cells and their arrangement in the periphery of the tubular like structures, and increased the number of cadherin 2 and βcatenin positive cells, possibly favoring cellcell junctions. The knockdown of relaxin impaired meiotic progression and significantly increased gene expression of the premeiotic genes Plzf and cKit. In addition, the knockdown affected cellular organization, impairing the arrangement of myoid cells around the tubular structures. These findings suggest that RLN plays a role in spermatogenesis by increasing the number of premeiotic and post meiotic germ cells, and favoring the arrangement of testicular cells.
The role of relaxin in male reproduction is still unclear. The knockout of relaxin decreases sperm maturation, and studies from our laboratory have shown that relaxin and RXFP1 are expressed in rat Sertoli cells and relaxin stimulates Sertoli cell proliferation, which could indirectly affect spermatogenesis. Relaxin may directly affect spermatogenesis through interaction with RXFP1 previously detected in elongating spermatids. To investigate a role of relaxin in spermatogenesis, we developed a coculture of cells extracted from the testis of 7dayold rats, which only contains spermatogonial stem cells and undifferentiated spermatogonia. Cells were plated on Matrigel, which was depleted of diffusible components that could interfere with germ cell differentiation by incubation with growth medium. Cells were cultured for 48 h in the absence or presence of 0.5% fetal bovine serum (FBS) or 100 ng/mL H2 relaxin (RLN). Cells were then kept for 24 h in supplementfree medium, and cultured for additional 48 or 120 h in the absence or presence of FBS or RLN. Cell number and differentiation were analyzed by immunocytochemistry, confocal microscopy and flow cytometry, after 2, 5 or 8 days (D2, D5 and D8, respectively). The ratio between germ and somatic cells (G/S) fell drastically from D2 to D5, and this fall was not affected by FBS, but it was partially prevented by RLN, which preserved especially 2C and 4C populations of germ cells (spermatogonia and permatocytes). RLN also increased 4C population of somatic cells. The gene and protein expression of the following markers of different stages of spermatogenesis was analyzed by quantitative PCR and flow cytometry: Plzf (undifferentiated spermatogonia), ckit (differentiated spermatogonia), Sycp3 (meiosis) and Odf2 (post meiosis). RLN tended to increase gene expression of Plzf and significantly increased gene expression of cKit in D5. RLN significantly increased gene expression of ckit and Odf2 in D5. RLN increased both PLZF and cKIT protein expression in D5, and increased the number of ODF2positive cells in immunocytochemistry assay. Furthermore RLN favored organization of cells in tubularlike structures in 2Dculture, increased the number of myoid cells and their arrangement in the periphery of the tubular like structures, and increased the number of cadherin 2 and βcatenin positive cells, possibly favoring cellcell junctions. The knockdown of relaxin impaired meiotic progression and significantly increased gene expression of the premeiotic genes Plzf and cKit. In addition, the knockdown affected cellular organization, impairing the arrangement of myoid cells around the tubular structures. These findings suggest that RLN plays a role in spermatogenesis by increasing the number of premeiotic and post meiotic germ cells, and favoring the arrangement of testicular cells.
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Citação
PIMENTA, Maristela Taliari. Espermatogênese in vitro: desenvolvimento de um modelo e avaliação do papel da relaxina. 2014. 140 f. Tese (Doutorado em Ciências) – Escola Paulista de Medicina, Universidade Federal de São Paulo. São Paulo, 2014.