H3K36me2 CUT&Tag-seq of Sertoli cells from control and cko mice
Sertoli cells are pivotal in supporting germ cell development and are the primary source of retinoic acid (RA), thereby facilitating the testicular microenvironment and spermatogenesis within the seminiferous tubules. Nevertheless, the molecular mechanisms and essential factors that regulate the Sertoli cell-dependent RA pathway remain to be fully elucidated. Herein, we demonstrate that histone demethylase KDM2A in Sertoli cells plays an essential role in facilitating spermatogenesis by modulating the RA signaling pathway.We first examined spatio-temporal expression patterns of KDM2A in testicular tissue from normal individuals and patients with idiopathic non-obstructive azoospermia (iNOA) through reanalysis of publicly available single-cell RNA sequencing datasets. To investigate its role in Sertoli cells, we constructed Sertoli cell-specific conditional knockout (cKO) of KDM2A mouse model and characterized the phenotype of KDM2A cKO mice. To explore the underlying mechanisms, we performed multi-omics analysis (RNA-seq, IP-MS, CUT&amp;RUN, CUT&amp;TAG) of purified Sertoli cells and in vitro cell knockdown assays. RA injection experiment was also used to assess the phenotypic rescue effects.The ablation of KDM2A in mouse Sertoli cells results in male infertility, characterized by impaired spermatogonial differentiation, defective meiosis, compromised cell adhesion, and disrupted blood-testis barrier (BTB) function within the seminiferous tubules. Multi-omics analysis revealed that KDM2A interacts with various splicing factors and regulates the alternative splicing of RA-related molecules, such as RAI14 and CYP2D22. Many differentially expressed genes involved in cell-cell adhesion and RA signaling were identified upon KDM2A depletion in Sertoli cells. KDM2A was found to directly target Zeb1 and fine-tune H3K36me2 at the Zeb1 promoter, thereby regulating the expression of RA synthases (including ALDH1A2 and ALDH1A3) in Sertoli cells. Notably, supplementation with exogenous RA could partially rescue defective spermatogenesis in Kdm2a cKO mice.This finding reveals that KDM2A, which is expressed by Sertoli cells, functions as an essential epigenetic regulator required for male fertility. Furthermore, it provides a potential therapeutic approach for treating male infertility caused by dysfunctional Sertoli cell-derived testicular microenvironments.