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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.
摘要：
抗苗勒管激素（AMH）是一些物种的窦前和窦前卵巢卵泡的颗粒细胞（GC）产生的卵巢储备的重要生殖标记，包括牛。这种激素在原始卵泡的募集和卵泡刺激素（FSH）依赖性卵泡生长中起着至关重要的作用。然而,AMH在卵泡中表达的调控机制尚不清楚。在这项研究中,我们比较了AMH的表达，AMHR-II,BMP2,BMP6,FSHR,和卵泡发育过程中的LHCGR基因。在有和没有FSH的情况下进行AMH的体外表达研究，AMHR-II,从3-8毫米卵泡中分离的牛GC中的BMP2和BMP6基因。估计mRNA表达与激素水平之间的关联。从小（3-8毫米）收集GCs，中等（9-12毫米）和大尺寸（13-24毫米）卵泡之前，在发病期间，偏差后，分别。Further,mRNA表达，激素（AMH，FSH,和LH），GCs凋亡，通过qRT-PCR检测细胞活力，ELISA,流式细胞术,和分光光度法。AMH,AMHR-II,与大卵泡相比，BMP2和FSHR基因在小卵泡和中等卵泡中高度表达。此外,AMH蛋白水平最高(84.14±5.41ng/mL)出现在中等大小的卵泡中.较低剂量的FSH增加了牛GC的活力，而较高剂量的FSH抑制了它们。用FSH处理的体外培养的GCs显著增加AMH，AMHR-II,和较低剂量的BMP2表达水平，而表达水平在较高剂量时降低。我们发现FSH的最佳水平（25ng/mL）可以显着提高AMH和BMP2丰度（p<0.05）。总之,AMH,AMHR-II,BMP2基因在FSH存在下发育的卵泡中表达较高。然而,较低剂量的FSH对AMH和BMP2表达有刺激作用，而表达在最大剂量开始下降。在这项研究中,我们对调节AMH的机制有了更好的了解，AMHRII,和卵泡发生期间GCs中的BMP2信号传导，这将改善传统辅助生殖技术（ART）的结果，如牛的超排卵和发情同步。
