To date, in the human seminiferous epithelium, only six associations of cell types have been distinguished, subdividing the epithelial cycle into six stages of very different duration. This hampers comparisons between studies on human and laboratory animals in which the cycle is usually subdivided into 12 stages. We now propose a new stage classification on basis of acrosomal development made visible by immunohistochemistry (IHC) for (pro)acrosin. IHC for acrosin gives results that are comparable to periodic acid Schiff staining. In the human too, we now distinguish 12 stages that differ from each other in duration by a factor of two at most. B spermatogonia are first apparent in stage I, preleptotene spermatocytes are formed in stage V, leptonema starts in stage VII, and spermiation takes place at the end of stage VI. A similar timing was previously observed in several monkeys. Stage identification by way of IHC for acrosin appeared possible for tissue fixed in formalin, Bouin fixative, diluted Bouin fixative, Cleland fluid, and modified Davidson fixative, indicating a wide applicability. In addition, it is also possible to distinguish the 12 stages in glutaraldehyde/osmium-tetroxide fixed/plastic embedded testis material without IHC for acrosin. The new stage classification will greatly facilitate research on human spermatogenesis and enable a much better comparison with results from work on experimental animals than hitherto possible. In addition, it will enable a highly focused approach to evaluate spermatogenic impairments, such as germ cell maturation arrests or defects, and to study details of germ cell differentiation.

Spermatogenesis is maintained by a pool of spermatogonial stem cells (SSCs). Analyses of the molecular profile of SSCs have revealed the existence of subsets, indicating that the stem cell population is more heterogeneous than previously believed. However, SSC subsets are poorly characterized. In rodents, the first steps in spermatogenesis have been extensively investigated, both under physiological conditions and during the regenerative phase that follows germ cell damage. In the widely accepted model, the SSCs are type Asingle (As) spermatogonia. Here, we tested the hypothesis that As spermatogonia are phenotypically heterogeneous by analyzing glial cell line-derived neurotrophic factor (GDNF) family receptor α1 (GFRA1) expression in whole-mounted seminiferous tubules, via cytofluorimetric analysis and in vivo colonogenic assays. GFRA1 is a coreceptor for GDNF, a Sertoli cell-derived factor essential for SSC self-renewal and proliferation. Morphometric analysis demonstrated that 10% of As spermatogonia did not express GFRA1 but were colonogenic, as shown by germ cell transplantation assay. In contrast, cells selected for GFRA1 expression were not colonogenic in vivo. In human testes, GFRA1 was also heterogeneously expressed in Adark and in Apale spermatogonia, the earliest spermatogonia. In vivo 5-bromo-2′-deoxyuridine administration showed that both GFRA1+ and GFRA1− As spermatogonia were engaged in the cell cycle, a finding supported by the lack of long-term label-retaining As spermatogonia. GFRA1 expression was asymmetric in 5% of paired cells, suggesting that As subsets may be generated by asymmetric cell division. Our data support the hypothesis of the existence of SSC subsets and reveal a previously unrecognized heterogeneity in the expression profile of As spermatogonia in vivo. Disclosure of potential conflicts of interest is found at the end of this article.

In mice and other mammals, spermatogenesis is maintained by spermatogonial stem cells (SSCs), a cell population belonging to undifferentiated type A spermatogonia. In the accepted model of SSC self-renewal, Asingle (As) spermatogonia are the stem cells, whereas paired (Apaired (Apr)) and chained (Aaligned (Aal)) undifferentiated spermatogonia are committed to differentiation. This model has been recently challenged by evidence that As and chained (Apr and Aal), undifferentiated spermatogonia are heterogeneous in terms of gene expression and function. The expression profile of several markers, such as GFRA1 (the GDNF co-receptor), is heterogeneous among As, Apr and Aal spermatogonia. In this study, we have analysed and quantified the distribution of GFRA1-expressing cells within the different stages of the seminiferous epithelial cycle. We show that in all stages, GFRA1+ chained spermatogonia (Apr to Aal) are more numerous than GFRA1+ As spermatogonia. Numbers of chained GFRA1+ spermatogonia are sharply reduced in stages VII–VIII when Aal differentiate into A1 spermatogonia. GFRA1 expression is regulated by GDNF and in cultures of isolated seminiferous tubules, we found that GDNF expression and secretion by Sertoli cells is stage-dependent, being maximal in stages II–VI and decreasing thereafter. Using qRT-PCR analysis, we found that GDNF regulates the expression of genes such as Tex14, Sohlh1 and Kit (c-Kit) known to be involved in spermatogonial differentiation. Expression of Kit was upregulated by GDNF in a stage-specific manner. Our data indicate that GDNF, besides its crucial role in the self-renewal of stem cells also functions in the differentiation of chained undifferentiated spermatogonia.

The human spermatogonial compartment is essential for daily production of millions of sperm. Despite this crucial role, the molecular signature, kinetic behavior and regulation of human spermatogonia are poorly understood. Using human testis biopsies with normal spermatogenesis and by studying marker protein expression, we have identified for the first time different subpopulations of spermatogonia. MAGE-A4 marks all spermatogonia, KIT marks all B spermatogonia and UCLH1 all Apale-dark (Ap-d) spermatogonia. We suggest that at the start of the spermatogenic lineage there are Ap-d spermatogonia that are GFRA1^High, likely including the spermatogonial stem cells. Next, UTF1 becomes expressed, cells become quiescent and GFRA1 expression decreases. Finally, GFRA1 expression is lost and subsequently cells differentiate into B spermatogonia, losing UTF1 and acquiring KIT expression. Strikingly, most human Ap-d spermatogonia are out of the cell cycle and even differentiating type B spermatogonial proliferation is restricted. A novel scheme for human spermatogonial development is proposed that will facilitate further research in this field, the understanding of cases of infertility and the development of methods to increase sperm output.

In mammals, spermatogenesis is maintained by spermatogonial stem cells (SSC). In their niche, SSC divide to self‐maintain and to produce a transit‐amplifying population that eventually enters the meiotic cycle to give rise to spermatozoa. The low number of SSC and the lack of specific markers hinder their isolation and enrichment. Stem cells in several adult tissues can be identified by using their verapamil‐sensitive Hoechst dye‐effluxing properties, which define the characteristic “side population” (SP). Here we show, by multicolor flow cytometric analysis, that immature mouse testis contains a “side‐population” (T‐SP), which is Sca‐1 ^pos , Ep‐CAM ^pos , EE2 ^pos , α6‐integrin ^pos , and αv‐integrin ^neg . A 13‐fold enrichment in SSC activity was observed when sorted T‐SP cells from ROSA 26 mice were transplanted in busulfan‐treated mouse testis. Whereas an incomplete range of spermatogenic stages was encountered two months after transplantation of unsorted testicular cells, the transplantation of T‐SP cells generated all associations of mouse germ cells representing the full range of spermatogenic stages. These data suggest that Hoechst staining and cell sorting might provide a novel approach to SSC enrichment in mammals.

Several mRNAs coding for a cAMP-specific phosphodiesterase (ratPDE3/IVd) with divergent 5' regions have been detected in mammalian cells. To determine the physiological significance of these differences, the expression of these mRNAs and the properties of the corresponding proteins were investigated. At least three mRNA species derived from the ratPDE3/IVd gene (ratPDE3.1, ratPDE3.2, and ratPDE3.3 mRNAs) are present in Sertoli and thyroid cells and in brain. Expression of ratPDE3.1 and ratPDE3.2 but not ratPDE3.3 mRNAs was dependent on hormone stimulation. The ratPDE3.2 and ratPDE3.3 mRNA variants were translated into polypeptides with immunochemical and biochemical properties identical to the native cAMP phosphodiesterases (PDEs) found in the Sertoli cell and thyroid FRTL-5 cells. Incubation of the recombinant PDEs with the catalytic subunit of the cAMP-dependent protein kinase in a cell-free system caused the phosphorylation and activation of the ratPDE3.3 protein variant. Under the same experimental conditions, ratPDE3.1 and ratPDE3.2 protein variants were neither phosphorylated nor activated by the cAMP-dependent protein kinase. Similar results were obtained by stimulating cells expressing the three recombinant PDE variants with dibutyryl cAMP. These findings demonstrate that the ratPDE3/IVd gene codes for PDE forms subject to different regulations.