The dominant follicle: the final frontier in oocyte development

The acquisition of oocyte competence, involving both cytoplasmic and nuclear maturation, is critical for successful fertilization and embryonic development in mammals. Discrepancies between in vivo and in vitro studies underline the potential impact of assisted reproductive technologies (ARTs), such as FSH stimulation and in vitro maturation, on oocyte quality and the molecular integrity of bovine oocytes. These insights underscore the importance of optimizing ART conditions to enhance reproductive success in cattle production. Therefore, this study focuses on the gene expression and DNA methylation dynamics in bovine oocytes from dominant follicles, utilising single-cell bisulfite conversion and RNA sequencing (scG&T), without ovarian stimulation. Samples were collected from synchronized heifers during three time points from 24hr to 2hr before ovulation and submitted to scG&T and compared with a dataset of >120 um in diameter oocytes collected by ovary slicing. Our findings from the single-cell RNA sequencing highlight significant changes in oxidative phosphorylation transcripts, indicating the importance of energy metabolism during oocyte capacitation. Additionally, small changes in global CpG methylation and methylation over genomic features were observed. However, modest modifications in CpG windows suggest a less stable epigenome than previously assumed in fully-grown oocytes. In conclusion, the present study brings new insights into molecular changes in bovine oocytes collected from dominant follicles, which can contribute to oocyte quality improvement in cattle production. Overall design: Oocytes from dominant follicles were collected from previous animal trials performed at University College Dublin. The oestrous cycles of 26 Crossbred nulliparous beef heifers (predominantly Limousin and Charolais crosses) were synchronized to manage the time of the ovulation, and bovine oocytes were collected from dominant follicles 24h before ovulation, 19-23 hours after estrus detection, and 2 hours before ovulation. After denudation from cumulus cells, single oocytes were snap-frozen in PCR tubes and stored at -80 ºC until processing for single-cell RNA sequencing and Bisulphite conversion.

卵母细胞发育潜能（oocyte competence）的获得涉及胞质成熟与核成熟两个过程，对哺乳动物成功受精及胚胎发育至关重要。体内与体外研究间的差异凸显了辅助生殖技术（assisted reproductive technologies，ARTs）——如促卵泡激素（follicle-stimulating hormone，FSH）刺激与体外成熟——对牛卵母细胞（bovine oocytes）质量及分子完整性的潜在影响。上述结论强调了优化辅助生殖技术条件，以提升肉牛生产繁殖效率的重要性。
因此，本研究聚焦于优势卵泡来源的牛卵母细胞中的基因表达与DNA甲基化动态变化，采用无卵巢刺激的单细胞亚硫酸氢盐转化与RNA测序（scG&T）技术。样本采集自同步化处理的青年母牛，于排卵前24小时至排卵前2小时的三个时间点收集，并通过scG&T进行分析，同时与通过卵巢切片法收集的直径＞120μm的卵母细胞数据集进行对比。
本研究的单细胞RNA测序分析结果显示，氧化磷酸化相关转录本存在显著变化，表明能量代谢在卵母细胞获能过程中发挥关键作用。此外，研究观察到全基因组CpG甲基化及基因组特征区域的甲基化水平仅存在小幅变化。不过，CpG窗口的表观修饰程度有限，提示完全成熟的卵母细胞的表观基因组（epigenome）稳定性较此前预期更低。
综上，本研究为优势卵泡来源的牛卵母细胞的分子变化提供了新见解，可为肉牛生产中卵母细胞质量的提升提供理论参考。
整体实验设计：本研究的牛卵母细胞样本采集自都柏林大学学院开展的前期动物实验。研究对26头杂交一代未产肉用青年母牛（主要为利木赞牛与夏洛莱牛杂交品种）的发情周期进行同步化处理，以精准调控排卵时间，并分别于排卵前24小时、发情检测后19-23小时以及排卵前2小时，从优势卵泡中采集牛卵母细胞。去除卵丘细胞（cumulus cells）后，将单个卵母细胞快速冷冻于PCR管中，并保存于-80℃，直至开展单细胞RNA测序与亚硫酸氢盐转化实验。