Marker expression reveals heterogeneity of spermatogonia in the neonatal mouse testis

Abstract

Prospermatogonia transition into type A spermatogonia, which provide the source for the spermatogonial stem cell (SSC) pool. A percentage of these type A spermatogonia then differentiate to enter meiosis as spermatocytes by ~P10. It is currently unclear when these distinct populations are initially formed in the neonatal testis, and when the expression of markers both characteristic of and required for the adult undifferentiated and differentiating states are established. In this study, we compared expression of known spermatogonial cell fate markers during normal development and in response to the differentiation signal provided by retinoic acid (RA). We found that some markers for the undifferentiated state (ZBTB16/PLZF and CDH1) were expressed in nearly all spermatogonia from P1 through P7. In contrast, differentiation markers (STRA8 and KIT) appeared in a subset of spermatogonia at P4, coincident with the onset of RA signaling. GFRA1, which was present in nearly all prospermatogonia at P1, was only retained in STRA8/KIT− spermatogonia. From P4 through P10, there was a great deal of heterogeneity in the male germ cell population in terms of marker expression, as markers characteristic of the undifferentiated (except GFRA1) and differentiating states were co-expressed through this interval. After P10, these fate markers diverged to mark distinct populations of undifferentiated and differentiating spermatogonia, and this pattern was maintained in the juvenile (P18) and adult (P>60) testis. Taken together, these results reveal that the spermatogonia population is heterogeneous during the first wave of spermatogenesis, and indicate that neonatal spermatogonia may not serve as an ideal substitute for studying the function of adult spermatogonia.

Ectopic POU5F1 in the male germ lineage disrupts differentiation and spermatogenesis in mice

“…Recently Niedenberger et al . proposed a model in which GFRA1 marks the first SSCs, while GFRA1 negative cells represent the differentiated spermatogonia that constitute the first wave of spermatogenesis (Niedenberger et al 2015). Given the normal presence of endogenous POU5F1 in gonocytes and undifferentiated spermatogonia we predicted that the SSC pool would be established normally despite the presence of cOCT4;Vasa-Cre.…”

“…Then we tested whether the reduction in total germ cell number could be explained by a block in differentiation in cOct4;Vasa-Cre mice. Differentiation can be visualized by the onset of STRA8 expression (Zhou et al 2008, Niedenberger et al 2015). While STRA8+ cells were clearly less frequent in cOCT4;Vasa-Cre, and the fraction of STRA8+ germ cells tended to be lower, the difference was not significantly significant (Fig.…”

“…9H–N). In wildtype mice GFRA1 and STRA8 immunostaining are generally expected to be in mutually exclusive subsets of spermatogonial populations – namely the undifferentiated and differentiated spermatogonia, respectively (Niedenberger et al 2015). However, in cOct4;Vasa-Cre mice at dpp 6 we frequently observed GFRA1+ cells that were also STRA8+ (Fig.…”

Rhox13 is required for a quantitatively normal first wave of spermatogenesis in mice

“…Images were captured as described above. IIF was performed as before (Busada, et al 2015a, Busada, et al 2014, Busada, et al 2015b, Niedenberger, et al 2015). Briefly, 4% PFA-fixed and O.C.T.-embedded 5 µm frozen sections were incubated in blocking buffer for 30 min at room temperature and then incubated in primary antibody for 1 h at room temperature.…”

“…Spermatogenesis begins in the neonatal mouse testis with the transition of quiescent prospermatogonia into stem, progenitor, and differentiating type A spermatogonia (Busada and Geyer 2015, Drumond, et al 2011, Niedenberger, et al 2015, Yang and Oatley 2014, Yoshida, et al 2006). These three types of spermatogonia provide the foundation for the ‘first wave of spermatogenesis’ that occurs during the first ~6 weeks of postnatal life, and after that the spermatogonial stem cells (SSCs) supply subsequent waves during steady-state spermatogenesis.…”