Comparative analysis among different species reveals that androgen receptor is a core transcriptional factor regulating the chicken follicular selection and ovulation [RNA-Seq 2]

The differences in reproductive processes at the molecular level between viviparous and oviparous animals are evident, and the site in the ovary that synthesizes sex hormones (androgens and oestrogens) and the trends for enriching sex hormones during follicle development in chickens are different from those in mammals, suggesting that the effect of sex hormones on follicle development in chickens is probably different from that in viviparous animals. To explore the specific role of androgen receptors (ARs) on chicken follicular development, we matched the correspondence of follicular development stages among chickens, humans, cows and identified chicken-specific genes related to follicle development (GAL-SPGs) by comparing follicle development-related genes and their biological functions among species (chickens, humans, and cows). A comparison of the core transcription factor regulatory network of granulosa cells (or ovaries) based on super enhancers among species (chicken, human, and mouse) revealed that AR is a core transcriptional regulator specific to chickens. In vivo experiments showed that inhibition of AR significantly reduced the number of syf (selected stage follicles) in chickens and decreased the expression of GAL-SPGs in F5 follicles, while in vitro experiments showed that inhibition of AR expression in chicken granulosa cells (GCs) significantly down-regulated the expression levels of GAL-SPGs, indicating that AR could regulate follicle selection through chicken-specific genes related to follicle development. A comparison among species (77 vertebrates) of the conserved genomic regions, where chicken super-enhancers are located, revealed that the chicken AR super-enhancer region is conserved in birds, suggesting that the role of AR in follicle selection may be widespread in birds. In summary, we found that AR can regulate follicle selection through chicken-specific genes related to follicle development, which also emphasizes the important role of AR in follicle selection in chickens and provides a new perspective for understanding the unique process of follicle development in chickens. Overall design: RNA extracted from primary granulosa cells transfected with AR-specific siRNA (treat) and nonsense siRNA (control) were sent to Novogene Co., Ltd. (Beijing, China) for library construction, and sequencing. All samples performed paired-end 150 bp sequencing on the Illumina Nova platform.

胎生动物与卵生动物在分子层面的生殖过程差异显著，且鸡卵巢中合成性激素（雄激素（androgens）与雌激素（oestrogens））的位点，以及卵泡发育过程中性激素的富集趋势，均与哺乳动物存在差异，提示性激素对鸡卵泡发育的调控作用可能不同于胎生动物。
为探究雄激素受体（androgen receptors, ARs）在鸡卵泡发育中的具体作用，本研究匹配了鸡、人、牛三者的卵泡发育阶段对应关系，并通过比较不同物种（鸡、人、牛）的卵泡发育相关基因及其生物学功能，筛选得到鸡特异性卵泡发育相关基因（GAL-SPGs）。
通过跨物种（鸡、人、小鼠）基于超级增强子（super enhancers）的颗粒细胞（granulosa cells）或卵巢核心转录因子调控网络比较分析，发现AR是鸡特异性的核心转录调控因子。
体内实验显示，抑制AR可显著降低鸡的选择期卵泡（selected stage follicles, syf）数量，并下调F5卵泡中GAL-SPGs的表达水平；体外实验则证实，抑制鸡颗粒细胞（granulosa cells, GCs）中AR的表达，可显著下调GAL-SPGs的表达量，表明AR可通过鸡特异性卵泡发育相关基因调控卵泡选择过程。
通过对77种脊椎动物的保守基因组区域进行跨物种比较，发现鸡AR超级增强子所在区域仅在鸟类中保守，提示AR在卵泡选择中的调控作用可能广泛存在于鸟类类群中。
综上，本研究发现AR可通过鸡特异性卵泡发育相关基因调控卵泡选择过程，这一发现既强调了AR对鸡卵泡选择的重要调控作用，也为理解鸡卵泡发育的独特机制提供了新视角。
整体实验设计：将转染AR特异性小干扰RNA（siRNA，处理组）与无义siRNA（对照组）的原代颗粒细胞提取的总RNA，送至诺禾致源公司（Novogene Co., Ltd.，中国北京）进行文库构建与测序。所有样本均在Illumina Nova测序平台（Illumina Nova platform）上完成双端150bp测序。