NR2F2 Regulation of Fetal Leydig Cell Differentiation: Insights into Differences of Sex Development [RNA-Seq]

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. Three E14.5 testes (control or COUP-TFII KO) were pooled per sample. RNA was extracted using the Arcturus™ PicoPure™ RNA Isolation Kit (Applied Biosystems, KIT0204). Briefly samples were dissociated in 100 ml of in extraction buffer with a microtube homogenizer and incubated at 42°C for 30 minutes. Posteriorly samples were snap frozen for at least one hour. RNA was precipitated with 100 ml of 70% ethanol and loaded into a pre-conditioned column. Samples were centrifuged and the column was washed with Wash Buffer 1, followed by a step of DNA removal using the RNase-Free DNase Set (Qiagen, #79254). Samples were washed twice with Wash Buffer 2 and the columns were centrifuged to eliminate residual buffer. Samples were eluted in 12 ml of elution buffer.