CUT&RUN seq of P5 Sertoli cells from control and kdm2a cko testes
Spermatogenesis is a highly regulated process involving intricate cellular interactions within the testicular microenvironment, particularly between germ cells and Sertoli cells. Sertoli cells play a pivotal role in supporting germ cell development, maintaining the blood-testis barrier, and facilitating the overall architecture of the seminiferous tubules. Dysregulation of these processes can lead to male infertility, making it essential to elucidate the molecular mechanisms underpinning the function of Sertoli cells. The histone demethylase KDM2A plays an important role in germ cells, but its specific contributions to Sertoli cells remain inadequately characterized. This study investigated the role of Sertoli cells in the testicular microenvironment through a conditional knockout (cKO) model of Kdm2a, driven by Amh-Cre. Compared to control mice, cKO mice exhibited significant testicular size reduction and impaired spermatogonia differentiation, ultimately leading to infertility due to the absence of elongated spermatids. Morphological analyses revealed abnormal adhesion and migration of spermatogonia and sertoli cells, along with compromised blood-testis barrier (BTB) integrity in cKO testes. RNA-seq of primary Sertoli cells isolated at P5 and P14 indicated that differentially expressed genes are enriched in pathways related to cell adhesion, cytoskeletal organization, and retinoic acid (RA) signaling. Mass spectrometry identified interactions between KDM2A and various splicing factors, regulating the alternative splicing of RA-related molecules Rail4 and Cyp2d22. Notably, exogenous RA supplementation in cKO mice at P3 significantly increased the number of differentiating spermatogonia and pre-leptotene spermatocytes, resulting in the appearance of elongated spermatids from previously stalled round spermatids. CUT&amp;RUN sequencing demonstrated that Zeb1 is a direct target of KDM2A and knockdown of Zeb1 in primary Sertoli cells led to aberrant expression of RA synthases (Aldh1a2 and Aldh1a3). Dual-luciferase assays suggested that KDM2A may regulate RA synthesis via Zeb1, thereby maintaining normal spermatogonia differentiation and spermatid elongation processes. These findings underscore the critical role of Kdm2a in the testicular microenvironment for proper spermatogenesis.