Gene expression dynamics during the gonocyte to spermatogonia transition and spermatogenesis in the domestic yak

Background:Spermatogenesis is a cellular differentiation process that includes three major events:mitosis of spermatogonia,meiosis of spermatocytes and spermiogenesis.Steady-state spermatogenesis relies on functions of spermatogonial stem cells(SSCs).Establishing and maintaining a foundational SSC pool is essential for continued spermatogenesis in mammals.Currently,our knowledge about SSC and spermatogenesis is severely limited in domestic animals.Results:In the present study,we examined transcriptomes of testes from domestic yaks at four different stages(3,5,8 and 24 months of age)and attempted to identify genes that are associated with key developmental events of spermatogenesis.Histological analyses showed that the most advanced germ cells within seminiferous tubules of testes from 3,5,8 and 24 months old yaks were gonocytes,spermatogonia,spermatocytes and elongated spermatids,respectively.RNA-sequencing(RNA-seq)analyses revealed that 11904,4381 and 2459 genes were differentially expressed during the gonocyte to spermatogonia transition,the mitosis to meiosis transition and the meiosis to post-meiosis transition.Further analyses identified a list of candidate genes than may regulate these important cellular processes.CXCR4,a previously identified SSC niche factor in mouse,was one of the up-regulated genes in the 5 months old yak testis.Results of immunohistochemical staining confirmed that CXCR4 was exclusively expressed in gonocytes and a subpopulation of spermatogonia in the yak testis.Conclusions:Together,these findings demonstrated histological changes of postnatal testis development in the domestic yak.During development of spermatogonial lineage,meiotic and haploid germ cells are supported by dynamic transcriptional regulation of gene expression.Our transcriptomic analyses provided a list of candidate genes that potentially play crucial roles in directing the establishment of SSC and spermatogenesis in yak.

Dynamic changes in the expression of apoptosis-related genes in differentiating gonocytes and in seminomas

Apoptosis is an integral part of the spermatogenic process, necessary to maintain a proper ratio of Sertoli to germ cell numbers and provide an adequate microenvironment to germ cells. Apoptosis may also represent a protective mechanism mediating the elimination of abnormal germ cells. Extensive apoptosis occurs between the first and second postnatal weeks, at the point when gonocytes, precursors of spermatogonial stem cells, should have migrated toward the basement membrane of the tubules and differentiated into spermatogonia. The mechanisms regulating this process are not well-understood. Gonocytes undergo phases of proliferation, migration, and differentiation which occur in a timely and closely regulated manner. Gonocytes failing to migrate and differentiate properly undergo apoptosis. Inadequate gonocyte differentiation has been suggested to lead to testicular germ cell tumor （TGCT） formation. Here, we examined the expression levels of apoptosis-related genes during gonocyte differentiation by quantitative real-time polymerase chain reaction, identifying 48 pro- and anti-apoptotic genes increased by at least two-fold in rat gonocytes induced to differentiate by retinoic acid, when compared to untreated gonocytes. Further analysis of the most highly expressed genes identified the pro-apoptotic genes Gadd45a and Cycs as upregulated in differentiating gonocytes and in spermatogonia compared with gonocytes. These genes were also significantly downregulated in seminomas, the most common type of TGCT, compared with normal human testicular tissues. These results indicate that apoptosis-related genes are actively regulated during gonocyte differentiation. Moreover, the down-regulation of pro-apoptotic genes in seminomas suggests that they could represent new therapeutic targets in the treatment of TGCTs.

Epidermal growth factor receptor pathway substrate 8 (Eps8) expression in maturing testis

Aim: Although epidermal growth factor receptors are expressed in the testes, whether they signal through epidermal growth factor receptor pathway substrate 8 (Eps8) is unknown. Here we evaluated the expression pattern of Eps8 in the maturing testis. Methods: The expression of Eps8 was analysed by Northern blotting, immunocy-tochemistry and Western blotting in primary Sertoli cell cultures and in testicular tissue of rodents. Results: Eps8 is specifically expressed in gonocytes, Leydig and Sertoli cells of the neonatal rats and in Leydig and Sertoli cells of the adult rats and mice. Although gonocytes express Eps8, no signal was found in prepubertal or mature spermatogonia and the expression level of Eps8 in Sertoli cells increases with age. No regulation of Eps8 expression in primary immature rat Sertoli cells by Follicle stimulating hormone (FSH) was detected by Western blotting. Conclusion: Eps8 seems to be involved in the growth factor-controlled regulation of cell proliferation and differentiation in the seminiferous epithelium. Eps8 is a possible marker for gonocytes and in Sertoli cells it could be involved in crosstalk with other growth factor pathways.