Establishment of a proteome profile and identification of molecular markers for mouse spermatogonial stem cells

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.

Deficiency of X‐ray repair cross‐complementing group 1 in primordial germ cells contributes to male infertility

“…On the next day, floating cells were transferred to another plate and then passaged 2 or 3 times before use. The SSC culture medium was prepared as described in Zhou et al (17).…”

Strawberry Notch 1 (SBNO1) promotes proliferation of spermatogonial stem cells via the noncanonical Wnt pathway in mice

“…Moreover, in our previous study, we showed that stromal interaction molecule 1 (STIM1) is required for testicular cord formation via gonad culture. Besides testicular culture, cultured SSCs are a proven stable platform for exploring SSC development 192526. For example, in our previous work, we established a steady SSC culture method and fertile progeny were produced after transplantation of cultured SSCs into infertile mouse testis.…”

“…For each experiment, at least eight explants were pooled from the testes of 3 mice in each group. For SSC culture, long-term culture of mouse SSCs was established in accordance with a previous described protocol 19. Briefly, dissociated testicular cells were cultured overnight on a 0.2% ( w/v ) gelatin-coated tissue culture plate (2 × 10 5 cells per 3.8 cm 2 ).…”

Cellular nucleic acid-binding protein is vital to testis development and spermatogenesis in mice

“…Since Sato et al first obtained functional sperm in cultured neonatal mouse testes (Sato et al 2011), this culture method has been widely used as a platform for the mechanistic understanding of spermatogenesis by several researchers (Chen et al 2014, Zheng et al 2014b. As for SSC culture, it is also a stable platform for exploring the development of spermatogonial stem cells and molecular mechanisms of spermatogenesis (Zhou et al 2015, Kanatsu-Shinohara et al 2016, Helsel et al 2017, Wang et al 2017. However, future studies investigating the detailed molecular mechanism of CNBP in testes using conditional knockout mouse models are warranted.…”

“…Long-term culture of mouse SSCs was established in accordance with a previous described protocol (Zhou et al 2015). Briefly, testis from 2-day neonatal mice was digested with 1 mg/mL collagenase (type IV; Sigma-Aldrich) for 15 min, followed by 0.25% trypsin EDTA (Invitrogen) with 1.4 mg/mL DNase (Sigma-Aldrich) for 10 min.…”