Marker expression reveals heterogeneity of spermatogonia in the neonatal mouse testis

Prospermatogonia transition to 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 as to 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 is 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 expression of markers, 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 juvenile (P18) and adult (P>60) testes. 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.