Transcriptome Profiling of the Theca Interna from Bovine Ovarian Follicles during Atresia

The theca interna is a specialized stromal layer that envelops each growing ovarian follicle. It contains capillaries, fibroblasts, immune cells and the steroidogenic cells that synthesize androgens for conversion to estradiol by the neighboring granulosa cells. During reproductive life only a small number of follicles will grow to a sufficient size to ovulate, whereas the majority of follicles will undergo regression/atresia and phagocytosis by macrophages. To identify genes which are differentially regulated in the theca interna during follicular atresia, we undertook transcriptome profiling of the theca interna from healthy (n = 10) and antral atretic (n = 5) bovine follicles at early antral stages (<5 mm). Principal Component Analyses and hierarchical classification of the signal intensity plots for the arrays showed primary clustering into two groups, healthy and atretic. A total of 543 probe sets were differentially expressed between the atretic and healthy theca interna. Further analyses of these genes by Ingenuity Pathway Analysis and Gene Ontology Enrichment Analysis Toolkit software found most of the genes being expressed were related to cytokines, hormones and receptors as well as the cell cycle and DNA replication. Cell cycle genes which encode components of the replicating chromosome complex and mitotic spindle were down-regulated in atretic theca interna, whereas stress response and inflammation-related genes such as TP53, IKBKB and TGFB1 were up-regulated. In addition to cell cycle regulators, upstream regulators that were predicted to be inhibited included Retinoblastoma 1, E2 transcription factor 1, and hepatocyte growth factor. Our study suggests that during antral atresia of small follicles in the theca interna, arrest of cell cycle and DNA replication occurs rather than up- regulation of apoptosis-associated genes as occurs in granulosa cells.

卵泡内膜层（theca interna）是包裹每个正在发育的卵巢卵泡的特化间质层。该层包含毛细血管、成纤维细胞、免疫细胞，以及负责合成雄激素、供邻近颗粒细胞转化为雌二醇的类固醇生成细胞。在生殖生命周期中，仅有少量卵泡能生长至足够体积并发生排卵，而绝大多数卵泡会发生退化/闭锁，并被巨噬细胞吞噬清除。

为鉴定卵泡闭锁过程中卵泡内膜层内差异调控的基因，我们对早期窦状阶段（直径<5mm）的健康（n=10）与窦状闭锁（n=5）牛卵泡的内膜层开展了转录组表达谱分析。针对芯片的信号强度图进行主成分分析与层级聚类后，结果显示样本可被分为健康与闭锁两个主要聚类簇。闭锁与健康卵泡内膜层间共计有543个探针集呈现差异表达。

通过IPA（Ingenuity Pathway Analysis）与基因本体富集分析工具包软件对上述基因进行进一步分析后发现，多数表达基因与细胞因子、激素、受体，以及细胞周期和DNA复制相关。编码复制染色体复合物与有丝分裂纺锤体组分的细胞周期基因在闭锁卵泡内膜层中呈下调表达，而TP53、IKBKB、TGFB1等应激反应与炎症相关基因则呈上调表达。

除细胞周期调控因子外，被预测受到抑制的上游调控因子还包括视网膜母细胞瘤1（Retinoblastoma 1）、E2转录因子1（E2 transcription factor 1）以及肝细胞生长因子。本研究表明，在小型卵泡窦状闭锁过程中，卵泡内膜层会出现细胞周期阻滞与DNA复制抑制，而非如颗粒细胞那样上调凋亡相关基因。