NR2F2 Regulation of Fetal Leydig Cell Differentiation: Insights into Differences of Sex Development [multiome]

Testicular fetal Leydig cells are specialized for androgen production during embryogenesis. Testosterone is essential for regulating sex differentiation, spermatogenesis, and fertility. Deficiencies in Leydig cell differentiation can lead to various disorders of sex development and male reproductive conditions, such as ambiguous genitalia, hypospadias, cryptorchidism, and infertility. Understanding the differentiation of fetal Leydig cells is essential for comprehending male sexual differentiation, reproductive health, and fertility. Fetal Leydig cells originate from proliferating progenitor cells in the gonadal interstitium, marked by genes like Arx, Pdgfra, Tcf21, Wnt5a, and Nr2f2. However, the precise mechanisms governing the transition from interstitial cells to Leydig cells remain elusive. Through integrated approaches involving animal models and multiomics, we have demonstrated that fetal Leydig cells originate from a Nr2f2 positive non-steroidogenic interstitial cell population. NR2F2 promotes progenitor cell fate while suppressing Leydig cell differentiation. Moreover, embryonic deletion of Nr2f2 in mouse testes resulted in disorders of sex development, including reduced testicular size, Leydig cell hypoplasia, cryptorchidism, and hypospadias. Collectively, our findings highlight the critical role of Nr2f2 in orchestrating the transition from interstitial cells to Leydig cells during testicular development. Homozygous Nr5a1-cre mice were crossed with homozygous Rosa-tdTomato female. Whole testes were collected from E14.5 embryos in PBS and immediately frozen in liquid nitrogen for storage.睾丸胎儿型莱迪希细胞（Testicular fetal Leydig cells）是胚胎发育阶段专司雄激素生成的特化细胞。睾酮对于调控性别分化、精子发生与生育能力至关重要。莱迪希细胞（Leydig cell）分化缺陷可引发多种性别发育异常及男性生殖系统病症，包括外生殖器性别不清、尿道下裂、隐睾症以及不育症。阐明胎儿型莱迪希细胞的分化机制，对理解男性性别分化、生殖健康及生育能力具有重要意义。胎儿型莱迪希细胞起源于性腺间质中的增殖性祖细胞，该类细胞可通过Arx、Pdgfra、Tcf21、Wnt5a及Nr2f2等基因的表达特征进行标记。然而，调控间质细胞向莱迪希细胞转化的精确分子机制仍未阐明。本研究通过整合动物模型与多组学（multiomics）分析策略，证实胎儿型莱迪希细胞起源于一群表达Nr2f2的非类固醇生成间质细胞群。NR2F2可促进祖细胞命运定向，同时抑制莱迪希细胞的分化进程。此外，在小鼠睾丸中进行胚胎期Nr2f2基因敲除，可诱发性别发育异常表型，包括睾丸体积缩小、莱迪希细胞发育不全、隐睾症及尿道下裂。综上，本研究结果明确了Nr2f2在睾丸发育过程中调控间质细胞向莱迪希细胞转化的关键作用。将纯合Nr5a1-cre小鼠与纯合Rosa-tdTomato雌性小鼠进行杂交。从胚胎第14.5天（E14.5）的胚胎中分离完整睾丸，置于磷酸盐缓冲液（phosphate buffered saline, PBS）中后立即放入液氮中冻存备用。