Abstract：
The Tianzhu white yak, a globally rare species, holds immense value as a source for yak materials. While the Fas/FasL pathway is pivotal in granulosa cells apoptosis, its precise molecular workings remain enigmatic. This study endeavours to decipher the role of follicle-stimulating hormone (FSH) in suppressing ovarian granulosa cells (GC) apoptosis in the Tianzhu white yak. Utilizing advanced cell culture techniques, we employed the MTT method, flow cytometry, fluorescence labelling and RT-PCR to investigate the apoptotic effects of FSH on yak GCs. Our results reveal that FSH\'s inhibitory effect on GC apoptosis follows a normal distribution pattern, peaking at an FSH concentration of 100 ng/mL with an apoptosis inhibition rate of 89.31%. When serum was withdrawn, an FSH concentration of 2 × 106 ng/mL reduced apoptosis by 72.84%. Annexin V-FITC staining revealed membrane invaginations, bubble and protrusion formation on the cell surface, and alterations in membrane structure and cell morphology. Flow cytometry analysis further demonstrated that FSH administration prior to early granulosa cell apoptosis had a more profound effect than during gradual apoptosis, both showing a suppressive effect on early follicular granulosa cell apoptosis. A transcription-level analysis conducted 3 h prior to serum withdrawal, with the addition of 100 ng/mL FSH, revealed intricate regulations in the expression of Fas/FasL. Notably, we observed a gradual increase in FasL expression over time, yet the presence of FSH effectively down-regulated FasL expression to baseline levels, without notable changes in Fas expression. Immunocytochemical analysis further confirmed the presence of both Fas and FasL on the cell membrane, nucleus and cytoplasm, with varying intensities depending on the duration of FSH treatment. Our findings suggest that FSH may suppress the apoptotic pathway in follicular primarily by down-regulating FasL expression, indicating that Fas-regulated mitochondrial pathways play a more prominent role compared to death receptor pathways. This study offers a fresh perspective on the mechanism underlying follicular atresia in Tianzhu white yaks and lays a solid theoretical foundation for the expansion of this endangered species\' population.
摘要：
天祝白牦牛,一种全球稀有物种，作为牦牛材料的来源具有巨大的价值。虽然Fas/FasL通路在颗粒细胞凋亡中起关键作用，其精确的分子工作原理仍然是个谜.本研究试图破译促卵泡激素（FSH）在抑制天祝白牦牛卵巢颗粒细胞（GC）凋亡中的作用。利用先进的细胞培养技术，我们采用了MTT法，流式细胞术,荧光标记和RT-PCR研究FSH对牦牛GCs的凋亡作用。我们的结果表明，FSH对GC凋亡的抑制作用遵循正态分布模式，在FSH浓度为100ng/mL时达到峰值，凋亡抑制率为89.31％。当血清被撤出时,FSH浓度为2×106ng/mL时，细胞凋亡减少72.84％。膜联蛋白V-FITC染色显示膜内陷，在细胞表面形成气泡和突起，以及膜结构和细胞形态的改变。流式细胞术分析进一步表明，在颗粒细胞凋亡早期之前给予FSH比在逐渐凋亡期间具有更深远的影响。对早期卵泡颗粒细胞凋亡均有抑制作用。在血清戒断前3小时进行转录水平分析，添加100ng/mL的FSH，揭示了Fas/FasL表达的复杂调控。值得注意的是,我们观察到FasL表达随时间逐渐增加，然而，FSH的存在有效地将FasL表达下调至基线水平，Fas表达没有显著变化。免疫细胞化学分析进一步证实细胞膜上存在Fas和FasL，细胞核和细胞质，不同的强度取决于FSH治疗的持续时间。我们的研究结果表明，FSH可能主要通过下调FasL表达来抑制卵泡凋亡途径，表明与死亡受体途径相比，Fas调节的线粒体途径发挥着更突出的作用。这项研究为天祝白牦牛卵泡闭锁的机制提供了新的视角，并为该濒危物种种群的扩大奠定了坚实的理论基础。
