Introduction

Gonocytes differentiate to spermatogonial stem cells (SSCs), which helps maintain spermatogenesis throughout life. We previously showed that lysine (K)-specific demethylase 5A (Kdm5a) expression is higher in undescended testes than in normal testes of juvenile rats, indicating that Kdm5a regulates early spermatogenesis via histone H3K4 modification (J Urol., 2014). Here, we aimed to elucidate the function of Kdm5a in gonocyte differentiation.

Methods

Mouse spermatogonial GC-1 cells were harvested 24 h after transfection with pEZ-M03 (control), Gfp expression vector, or Kdm5a-Gfp expression vector. Western blot was then performed to assess H3K4 methylation status, and quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed for 13 genes related to SSC development (Esr1, Esr2, Gdnf, Gfra1, Kit, Klf4, Myc, Nanog, Neurog3, Pgr, Pou5f1, Ret, Thy1). Next, chromatin immunoprecipitation (ChIP) assay was performed to clarify the association between SSC-related genes and H3K4 methylation status. Further, we performed microarray analysis to clarify the underlying regulatory mechanisms of Kdm5a overexpression.

Results

Kdm5a expression was significantly higher and H3K4me3 expression significantly lower in GC-1 cells overexpressing Kdm5a. Quantitative RT-PCR revealed that of the 13 genes related to SSCs development, the expression of Esr2, Neurog3, Pou5f1, Ret, and Thy1 was significantly higher in GC-1 cells overexpressing Kdm5a. ChIP assay revealed that H3K4me3 was markedly enriched at the Ret promoter. Microarray analysis showed high expression of Tet1 (fold change = 19.4), Btc (19.4), and Scml (16.4), and low expression of Wnt1 (108.2), Sox6 (26.7), and Sox8 (26.4). These genes are reportedly associated with epigenome and gonadal development.

Conclusions

Since Kdm5a removes the methyl groups from methylated H3K4, which is associated with transcriptional repression, it was suggested that Kdm5a suppresses Ret expression via epigenetic regulation. It was previously reported that Ret colocalizes with Gfra1 in SSCs, and Gdnf signaling via the RET tyrosine kinase/Gfra1 receptor complex is required for SSC self-renewal. In this study, it is suggested that SCC differentiation from gonocytes is inhibited by the action of Kdm5a on germ cells via suppression of Ret expression.

Funding

none