Regulation of AMH, AMHR-II, and BMPs (2,6) Genes of Bovine Granulosa Cells Treated with Exogenous FSH and Their Association with Protein Hormones

Abstract

Anti-Mullerian hormone (AMH) is an important reproductive marker of ovarian reserve produced by granulosa cells (GCs) of pre-antral and early-antral ovarian follicles in several species, including cattle. This hormone plays a vital role during the recruitment of primordial follicles and follicle stimulating hormone (FSH)-dependent follicular growth. However, the regulatory mechanism of AMH expression in follicles is still unclear. In this study, we compared the expression of <i>AMH, AMHR-II, BMP2, BMP6, FSHR,</i> and <i>LHCGR</i> genes during follicular development. In-vitro expression study was performed with and without FSH for <i>AMH, AMHR-II, BMP2,</i> and <i>BMP6</i> genes in bovine GCs which were isolated from 3-8 mm follicles. Association among the mRNA expression and hormone level was estimated. GCs were collected from small (3-8 mm), medium (9-12 mm) and large size (13 to 24 mm) follicles before, during onset, and after deviation, respectively. Further, mRNA expression, hormones (AMH, FSH, and LH), apoptosis of GCs, and cell viability were detected by qRT-PCR, ELISA, flow cytometry, and spectrophotometry. <i>AMH, AMHR-II, BMP2,</i> and <i>FSHR</i> genes were highly expressed in small and medium follicles as compared to large ones. In addition, the highest level of AMH protein (84.14 ± 5.41 ng/mL) was found in medium-size follicles. Lower doses of FSH increased the viability of bovine GCs while higher doses repressed them. In-vitro cultured GCs treated with FSH significantly increased the <i>AMH, AMHR-II,</i> and <i>BMP2</i> expression levels at lower doses, while expression levels decreased at higher doses. We found an optimum level of FSH (25 ng/mL) which can significantly enhance <i>AMH</i> and <i>BMP2</i> abundance (<i>p</i> < 0.05). In summary, <i>AMH, AMHR-II,</i> and <i>BMP2</i> genes showed a higher expression in follicles developed in the presence of FSH. However, lower doses of FSH demonstrated a stimulatory effect on <i>AMH</i> and <i>BMP2</i> expression, while expression started to decline at the maximum dose. In this study, we have provided a better understanding of the mechanisms regulating <i>AMH, AMHR II,</i> and <i>BMP2</i> signaling in GCs during folliculogenesis, which would improve the outcomes of conventional assisted reproductive technologies (ARTs), such as superovulation and oestrus synchronization in bovines.