RESUMO
To determine the function of germ cell nuclear factor (GCNF) in female reproduction, we generated an oocyte-specific GCNF knockout mouse model (GCNF(fl/fl)Zp3Cre(+)). These mice displayed hypofertility due to prolonged diestrus phase of the estrous cycle and aberrant steroidogenesis. These reproductive defects were secondary to a primary defect in the oocytes, in which expression of the paracrine transforming growth factor-beta signaling molecules, bone morphogenetic protein 15 (BMP-15) and growth differentiation factor 9 (GDF-9), were up-regulated in GCNF(fl/fl)Zp3Cre(+) females at diestrus. This was a direct effect of GCNF, as molecular studies showed that GCNF bound to DR0 elements within the BMP-15 and GDF-9 gene promoters and repressed their reporter activities. Consistent with these findings, abnormal double-oocyte follicles, indicative of aberrant BMP-15/GDF-9 expression, were observed in GCNF(fl/fl)Zp3Cre(+) females. The Cre/loxP knockout of GCNF in the oocyte has uncovered a new regulatory pathway in ovarian function. Our results show that GCNF directly regulates paracrine communication between the oocyte and somatic cells by regulating the expression of BMP-15 and GDF-9, to affect female fertility.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fertilidade/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Receptores de Superfície Celular , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Proteína Morfogenética Óssea 15 , Células CHO , Cricetinae , Proteínas do Ovo/metabolismo , Feminino , Regulação da Expressão Gênica , Fator 9 de Diferenciação de Crescimento , Integrases/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Biológicos , Membro 1 do Grupo A da Subfamília 6 de Receptores Nucleares , Oócitos/citologia , Ovário/citologia , Ovário/metabolismo , Transgenes , Proteínas Virais/metabolismo , Zona Pelúcida/metabolismo , Glicoproteínas da Zona PelúcidaRESUMO
Levonorgestrel (LNG), a 19-nor-testosterone derivative, is widely used in contraceptive formulations. This compound does not bind to the estrogen receptor (ER), but it shows estrogen-like effects under in vivo and in vitro conditions. The estrogenicity of LNG may be attributed to its bio-transformation into non-phenolic metabolites. In this study, the ability of A-ring reduced LNG metabolites to activate transcription via an estrogenic mechanism of action, including differences between ER alpha and ER beta subtypes, were investigated. Transactivation assays were performed in HeLa cells transfected with expression vectors for ER alpha and ER beta and an estrogen-responsive reporter gene. Cells were also transfected with expression vectors for both progesterone receptor (PR) isoforms (A or B). As expected, the tetrahydro derivatives of LNG (3 alpha,5 alpha- and 3 beta,5 alpha-LNG) showed significantly lower PR-mediated transcriptional activities through both isoforms when compared with progesterone (P(4)) and LNG. In contrast, the 3 beta,5 alpha-tetrahydro derivative resulted in a significant activation of estrogen-dependent gene transcription. This effect was selectively confined to the ER alpha, since little if any activity could be observed with the ER beta and no antagonistic activities were demonstrated. This study provides structural and molecular clues for the well documented in vitro and in vivo intrinsic estrogenicity of 19-nor-testosterone-derived progestins and ligand requirements for ER alpha recognition.