Abstract
Spermatogonial stem cells (SSCs) are undifferentiated cells that are required to maintain spermatogenesis throughout the reproductive life of mammals. Although SSC transplantation and culture provide a powerful tool to identify the mechanisms regulating SSC function, the precise signalling mechanisms governing SSC self-renewal and specific surface markers for purifying SSCs remain to be clearly determined. In the present study, we established a steady SSC culture according to the method described by Shinohara's lab. Fertile progeny was produced after transplantation of cultured SSCs into infertile mouse testis, and the red fluorescence exhibited by the culture cell membranes was stably and continuously transmitted to the offspring. Next, via advanced mass spectrometry and an optimized proteomics platform, we constructed the proteome profile, with 682 proteins expressed in SSCs. Furthermore bioinformatics analysis showed that the list contained several known molecules that are regulated in SSCs. Several nucleoproteins and membrane proteins were chosen for further exploration using immunofluorescence and RT-PCR. The results showed that SALL1, EZH2, and RCOR2 are possibly involved in the self-renewal mechanism of SSCs. Furthermore, the results of tissue-specific expression analysis showed that Gpat2 and Pld6 were uniquely and highly expressed in mouse testes and cultured SSCs. The cellular localization of PLD6 was further explored and the results showed it was primarily expressed in the spermatogonial membrane of mouse testes and cultured SSCs. The proteins identified in this study form the basis for further exploring the molecular mechanism of self-renewal in SSCs and for identifying specific surface markers of SSCs.
Highlights
Spermatogenesis is an intricate and coordinated process by which thousands of spermatozoa are produced per second throughout the 24-hr day within the testis
Like other adult self-renewing tissues that rely on stem cells for the replenishment of differentiated cells at a constant rate or more rapidly after toxic injury, continual spermatogenesis is dependent on an adult tissue-specific stem cell population called spermatogonial stem cells (SSCs)
The results indicated that SALL1, EZH2 and RCOR2 might be important for SSC selfrenewal and maintenance of pluripotency
Summary
Spermatogenesis is an intricate and coordinated process by which thousands of spermatozoa are produced per second throughout the 24-hr day within the testis. With regard to SSC culture, in 2003, the Japanese research team comprising Kanatsu-shinohara et al succeeded in the long-term culture of SSCs from neonatal mouse testis, and named these cells germline stem (GS) cells These cell lines continued to proliferate for at least 2 years and restored fertility to congenitally infertile recipient mice following transplantation into seminiferous tubules [5]. We established and compared the proteome profiles of MEF (mouse embryonic fibroblast) feeder cells and mouse SSCs co-cultured with MEFs; we identified 682 proteins expressed in SSCs. Further bioinformatics analysis showed that among these proteins were several molecules that are known to be regulated in SSCs. Based on this, several nucleoproteins and membrane proteins were chosen for further investigation, to identify the highly expressed proteins, and thereby elucidate the molecular mechanism of self-renewal and determine more specific surface markers of SSCs
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