Comparative transcriptome profile analysis of granulosa cells from porcine ovarian follicles during early atresia

At various stages of ovarian follicular development, more than 99% of follicles will be eliminated through a degenerative process called atresia. The regulatory mechanisms of atresia have been elucidated to some extent, involving hormones, growth factors, cytokines, and other factors. However, the stimuli initiating atresia in follicular granulosa cells remain unknown. In this study, we isolated the granulosa cells from porcine ovarian follicles (3–5 mm diameter) divided into healthy follicles (HFs) and early atretic follicles (EAFs). We applied high-throughput RNA sequencing to identify and compare differentially expressed genes (DEGs) between HFs and EAFs. A total of 31,694 genes were detected, of which 21,806 were co-expressed in six samples, and 243 genes (<i>p</i> &lt; 0.05; FDR &lt; 0.05) were differentially expressed (DEGs), including 123 downregulated and 120 upregulated in EAFs. GO analysis highlighted hormone metabolism, plasma membrane localization, and transporter activity. The pathway analysis indicated that 51 DEGs, involved in steroidogenesis, cell adhesion molecules, and TGF-beta signaling pathways, were highly related to atresia. Additionally, the interaction network of DEGs (<i>p</i> &lt; 0.01; FDR &lt; 0.05) using STRING highlighted LHR, ACACB, and CXCR4 as central nodes. In summary, this transcriptome analysis enriched our knowledge of the shifted mechanisms in granulosa cells during early atresia and provided novel perspectives into the atresia initiation.

在卵巢卵泡发育的各个阶段，超过99%的卵泡会通过一种名为闭锁（atresia）的退行性过程被清除。卵泡闭锁的调控机制已在一定程度上得到阐明，涉及激素、生长因子、细胞因子等多种调控因子。然而，启动卵泡颗粒细胞发生闭锁的上游刺激因素仍不明确。本研究从猪卵巢直径3~5mm的卵泡中分离颗粒细胞，并将其分为健康卵泡（healthy follicles, HFs）与早期闭锁卵泡（early atretic follicles, EAFs）两组。我们采用高通量RNA测序技术，鉴定并比较了HFs与EAFs之间的差异表达基因（differentially expressed genes, DEGs）。本研究共检测到31694个基因，其中21806个在6个样本中共同表达；另有243个基因（p<0.05；错误发现率（FDR）<0.05）为差异表达基因（DEGs），其中在EAFs中下调基因123个、上调基因120个。基因本体（Gene Ontology, GO）富集分析结果显示，差异表达基因显著富集于激素代谢、质膜定位以及转运蛋白活性等功能条目。通路富集分析显示，参与类固醇生成、细胞黏附分子以及转化生长因子β（TGF-β）信号通路的51个DEGs与卵泡闭锁密切相关。此外，借助STRING数据库构建DEGs（p<0.01；FDR<0.05）的蛋白互作网络后发现，LHR、ACACB与CXCR4为该网络的核心节点。综上，本转录组分析深化了我们对早期闭锁阶段颗粒细胞内调控机制转变的认识，并为卵泡闭锁启动机制的研究提供了全新视角。