Abstract
Self-renewal and differentiation of spermatogonial stem cells (SSCs) are the foundation of spermatogenesis throughout a male's life. SSC transplantation will be a valuable solution for young male patients to preserve their fertility. As SSCs in the collected testis tissue from the patients are very limited, it is necessary to expansion the SSCs in vitro. Previous studies suggested that histone methyltransferase ERG-associated protein with SET domain (ESET) represses gene expression and is essential for the maintenance of the pool of embryonic stem cells and neurons. The objective of this study was to determine the role of ESET in SSCs using in vitrocell culture and germ cell transplantation. Cell transplantation assay showed that knockdown of ESET reduced the number of seminiferous tubules with spermatogenesis when compared with that of the control. Knockdown of ESET also upregulated the expression of apoptosis-associated genes (such as P53, Caspase9, Apaf1), whereas inhibited the expression of apoptosis-suppressing genes (such as Bcl2l1, X-linked inhibitor of apoptosis protein). In addition, suppression of ESET led to increase in expression of Caspase9 and activation of Caspase3 (P17) as well as cleavage of poly (ADP-ribose) polymerase. Among the five ESET-targeting genes (Cox4i2, spermatogenesis and oogenesis Specific Basic Helix-Loop-Helix 2, Nobox, Foxn1 and Dazl) examined by ChIP assay, Cox4i2 was found to regulate SSC apoptosis by the rescue experiment. BSP analyses further showed that DNA methylation in the promoter loci of Cox4i2was influenced by ESET, indicating that ESET also regulated gene expression through DNA methylation in addition to histone methylation. In conclusion, we found that ESET regulated SSC apoptosis by suppressing of Cox4i2 expression through histone H3 lysine 9 tri-methylation and DNA methylation. The results obtained will provide unique insights that would broaden the research on SSC biology and contribute to the treatment of male infertility.
Highlights
spermatogonial stem cells (SSCs) transplantation was established in 1994.9,10 Donor SSCs can be transplanted into recipient mice and initiate the process of spermatogenesis
The expression of ESET was gradually increasing during testis development, in parallel to the global level of H3K9me[3] (Figures 1b and c)
We further examined the distribution of ESET and H3K9me[3] in SSCs by co-immunofluorescence staining
Summary
SSC transplantation was established in 1994.9,10 Donor SSCs can be transplanted into recipient mice and initiate the process of spermatogenesis. The histone methyltransferase ERG-associated protein with SET domain (ESET) ( known as SETDB1 or KMT1E) represses gene expression through H3K9me2/3.28,29 ESET has shown to be vital for the maintenance of embryonic stem cells (ESCs).[30] ESET plays essential roles both in the maintenance of articular cartilage and survival of neurons.[31,32] As conventional ablation of the Eset gene leads to embryonic lethality,[33] the functions of ESET in spermatogenesis have not been examined extensively. The results obtained will further broaden the research on SSC biology and provide new idea for the treatment of male infertility
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