miR-27b-3p inhibits estrogen secretion of goose granulosa cells by targeting CYP1B1 through the AMPK signaling pathway.

Although miR-27b-3p has been evidenced to regulate the proliferation, apoptosis, and differentiation of a variety of mammalian cell types, its actions and mechanisms on ovarian cell steroidogenesis remains largely unknown in both mammalian and avian species. In this study, we aimed to determine the expression profiles of miR-27b-3p in granulosa cell layers during goose ovarian follicle development and to reveal its actions on estrogen (E2) secretion of goose granulosa cells as well as the underlying regulatory mechanisms. It was observed that miR-27b-3p was ubiquitously expressed throughout follicle development but exhibited much higher levels in hierarchical- than in prehierarchical follicles. In cultured granulosa cells from the fourth through second largest preovulatory (F4-F2) follicles of goose, up- and downregulation of miR-27b-3p by using its mimic and inhibitor significantly decreased and increased E2 secretion, respectively. Meanwhile, the mRNA levels of STAR and CYP19A1 were significantly reduced while those of CYP11A1 and 3ßHSD were elevated in the mimic-transfected granulosa cells. By comparison, downregulation of miR-27b-3p enhanced the mRNA levels of STAR but had no significant effects on those of CYP19A1, CYP11A1, and 3ßHSD. Results from bioinformatic prediction and luciferase reporter assay demonstrated that CYP1B1 was a downstream target of miR-27b-3p. Although the siRNA-mediated downregulation of CYP1B1 did not significantly change E2 secretion by goose granulosa cells, it reduced the mRNA levels of STAR and CYP19A1 as well as those of LKB1 and AMPKα, which are involved in the AMPK signaling pathway. Taken together, these data suggest that miR-27b-3p plays an inhibitory role in E2 secretion by goose F4-F2 granulosa cells, at least in part, by targeting CYP1B1 through the AMPK signaling pathway.

Molecular characterization, expression profile and transcriptional regulation of the CYP19 gene in goose ovarian follicles.

Cytochrome P450 Family 19 (CYP19) is a crucial enzyme to catalyze the conversion of androgens to estrogens. However, the regulatory mechanism of goose CYP19 gene remains poorly understood. The present study attempted to obtain the full-length coding sequence (CDS) and 5'-flanking sequence of CYP19 gene, to investigate its expression and distribution profiles in different sized follicles, and to analyze the transcriptional regulatory mechanism of CYP19 gene in goose. Results showed that its CDS consisted of 1512 nucleotides and the encoded amino acid sequence contained a classical P450 structural domain. Homology analysis showed that there were high homologies of nucleotide and amino acid sequences between goose and other avian species. Its promoter sequence spanned from -1925 bp to the transcription start site (ATG) and several transcriptional factors were predicted in this region. Further analysis from luciferase assay showed that the luciferase activity was the highest spanning from -118 to -1 bp by constructing deletion promoter reporter vector. In addition, result from quantitative real-time polymerase chain reaction indicated that the mRNA level of CYP19 gene were highly expressed in theca layer of the fifth largest follicle, and the cellular location was in the theca externa cells by immunohistochemistry. Taken together, it could be concluded that the transcription activity of CYP19 gene was activated by transcriptional factors in its proximal region of promoter to promote the synthesis of estrogens, regulating the selection of pre-hierarchical into hierarchical follicle in goose.