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 AMH, AMHR-II, BMP2, BMP6, FSHR, and LHCGR genes during follicular development. In-vitro expression study was performed with and without FSH for AMH, AMHR-II, BMP2, and BMP6 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. AMH, AMHR-II, BMP2, and FSHR 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 AMH, AMHR-II, and BMP2 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 AMH and BMP2 abundance (p < 0.05). In summary, AMH, AMHR-II, and BMP2 genes showed a higher expression in follicles developed in the presence of FSH. However, lower doses of FSH demonstrated a stimulatory effect on AMH and BMP2 expression, while expression started to decline at the maximum dose. In this study, we have provided a better understanding of the mechanisms regulating AMH, AMHR II, and BMP2 signaling in GCs during folliculogenesis, which would improve the outcomes of conventional assisted reproductive technologies (ARTs), such as superovulation and oestrus synchronization in bovines.

The Factors and Pathways Regulating the Activation of Mammalian Primordial Follicles in vivo

“…The anti-Mullerian hormone (AMH) is a glycoprotein of the TGF-β superfamily and is produced by the granulosa cells of pre-antral and early antral ovarian follicles in several species (Umer et al, 2019). It is generally accepted that AMH inhibits the activation of mammalian primordial follicles (Shahrokhi et al, 2018).…”

Cellular and Molecular Adaptation of Bovine Granulosa Cells and Oocytes under Heat Stress

“…Estradiol plays an important role in the development of ovarian follicles, oocyte maturation, and endometrial proliferation. GCs have specific receptors for the gonadotropins FSH and LH [54], insulin-like growth factor (IGF) [58], and anti-Müllerian hormone (AMH) [59,60]. Any disturbance of GC quality and their proliferation capability may also have indirect effects on the development of the follicle and may disrupt oocyte maturation and, consequently, result in impaired embryo development with unsatisfactory pregnancy outcome [61].…”

“…HS results in intracellular ROS accumulation, causing oxidative stress [88] and apoptosis [89], which subsequently lead to a decline in fertility [31,90]. In response, the activation of fork head box O3 (FOXO3) and kelch-like ECH associated protein 1 (KEAP1) under HS protects cells from oxidative stress by upregulating antioxidant enzymes superoxide dismutase 2 (SOD2) and catalase (CAT) [60,91,92]. In Saccharomyces cerevisiae and quail, genes from the glutathione peroxidase family were also shown to be induced under HS [93,94].…”

MiR-214-3p promotes proliferation and inhibits estradiol synthesis in porcine granulosa cells

“…There are three types of estrogen, the most active of which is estradiol [7]. During the synthesis of estradiol, FSH (follicle-stimulating hormone) receptors produced by GCs bind to FSH from the pituitary gland, which activates the FSH signaling pathway and increases the expression of related enzymes (e.g., CYP11A1, a cytochrome P450) and promotes estradiol synthesis [8,9]. FSH can interact with receptors in the surface membranes of GCs, activate adenylyl cyclase and subsequently increase intracellular cAMP levels.…”