Determination of the roles of GREM1 gene in granulosa cell proliferation and steroidogenesis of hen ovarian prehierarchical follicles

Gremlin genes are known members of the DAN family of bone morphogenetic protein (BMP) antagonists, but their functions and regulatory mechanisms in ovarian follicular development of chicken remain unknown. The current study was designed to investigate the mRNA expression patterns of gremlin1 gene (GREM1) and its protein location in the follicles sampled, and to explore the biological effect of GREM1 on the prehierarchical follicular development. This work revealed that chicken GREM1 mRNA exhibits a constant expression level across all the prehierarchical follicles (PFs) from 1-4 mm to 7-8 mm in diameter, and the preovulatory follicles (from F6 to F1) by using RT-qPCR (P > 0.05). The GREM1 protein is predominantly expressed in the oocytes and granulosa cells (GCs) of the PFs by immunohistochemistry. Furthermore, our data demonstrated that siRNA-mediated knockdown of GREM1 in the GCs resulted in a significant reduction in cell proliferation (P < 0.001); conversely, overexpression of GREM1 in the GCs led to a remarkable increase in cell proliferation (P < 0.001). Interestingly, the expression levels of proliferating cell nuclear antigen (PCNA) and cyclin D2 (CCND2) mRNA and proteins were notably increased when GREM1 expression was upregulated in the GCs (P < 0.01), however, the expression levels of CYP11A1 and StAR were markedly downregulated (P < 0.01). The current results showed that GREM1 gene plays a stimulatory role in GC proliferation during growth and development of the prehierarchical follicles in vitro but an inhibitory role in GC differentiation and steroidogenesis of the hen ovary follicles. (C) 2020 The Authors. Published by Elsevier Inc.