Elena Vicini

Identification of Spermatogonial Stem Cell Subsets by Morphological Analysis and Prospective Isolation

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. STEM CELLS 2009;27:3043–3052

Identification and enrichment of spermatogonial stem cells displaying side-population phenotype in immature mouse testis

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‐1pos, Ep‐CAMpos, 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.

Distribution of GFRA1-expressing spermatogonia in adult mouse testis

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.

17-β-estradiol elicits genomic and non-genomic responses in mouse male germ cells

Estrogens have been postulated to exert a detrimental effect on spermatogenesis in vivo. Since mouse male germ cells express estrogen receptors, we have investigated whether molecular pathways are activated by estrogen stimulation of these cells. Our results demonstrate that estrogen receptor β is expressed in mitotic and meiotic male germ cells as well as in the spermatogonia derived GC‐1 cell line. By using this cell line, we show that 17‐β‐estradiol triggers activation of a transcriptional response that requires a functional estrogen receptor. Moreover, GC‐1 cells respond to estrogens by transiently activating a signal transduction pathway that impinges on the mitogen‐activated protein kinases (MAPK) ERK1 and ‐2. A similar dose‐dependent transient activation of ERKs was also observed in primary mouse spermatocytes in culture. Activation by the estrogen was specific because other steroids such as progesterone and dihydrotestosterone were ineffective and because it could be blocked by the selective inhibitor of the ERK pathway and by competitive inhibitors of the estrogen receptor. Finally, we observed that 17‐β‐estradiol does not affect spontaneous or induced apoptosis in cultured mouse spermatocytes, indicating that the apoptotic effects observed in vivo require additional testicular components.

Novel Stage Classification of Human Spermatogenesis Based on Acrosome Development

ABSTRACT 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.

Beta-chemokine receptors 5 and 3 are expressed on the head region of human spermatozoon

Induction of human sperm chemotaxis is an established phenomenon, though signaling systems physiologically involved have not been identified. Recently, it has been demonstrated that RANTES is present in the follicular fluid and that this molecule is a chemoactractant for human spermatozoa. However, the presence of ß‐chemokine receptors on human spermatozoa has never been reported. By cytometric, Western blotting and immunofluorescence analysis, we demonstrate the presence of CCR5 and CCR3 on ejaculated spermatozoa from healthy subjects. CCR5 was detected in the periacrosomal region of the sperm surface, whereas CCR3 was also present in the postacrosomal cap. Individual variability was observed on CCR5 and CCR3 positive sperm percentages. Presence of Δ32+/− mutation was demonstrated in two subjects expressing CCR5 in half of the ejaculated spermatozoa. Our findings represent the missing information in favor of the possibility that ß‐chemokines and their receptors are involved in sperm chemotaxis. Identification of molecular mechanisms of sperm chemotaxis may allow us to identify predictive parameters of sperm fertilizing ability in hypofertile or infertile subjects. Finally, both CCR5 and CCR3 expressed on the sperm cell surface may be involved in HIV‐1 adhesion to spermatozoa, thus allowing these cells to perform as virion cellular carriers during sexual transmission of HIV‐1 infection.

The niche-derived glial cell line-derived neurotrophic factor (GDNF) induces migration of mouse spermatogonial stem/progenitor cells.

In mammals, the biological activity of the stem/progenitor compartment sustains production of mature gametes through spermatogenesis. Spermatogonial stem cells and their progeny belong to the class of undifferentiated spermatogonia, a germ cell population found on the basal membrane of the seminiferous tubules. A large body of evidence has demonstrated that glial cell line-derived neurotrophic factor (GDNF), a Sertoli-derived factor, is essential for in vivo and in vitro stem cell self-renewal. However, the mechanisms underlying this activity are not completely understood. In this study, we show that GDNF induces dose-dependent directional migration of freshly selected undifferentiated spermatogonia, as well as germline stem cells in culture, using a Boyden chamber assay. GDNF-induced migration is dependent on the expression of the GDNF co-receptor GFRA1, as shown by migration assays performed on parental and GFRA1-transduced GC-1 spermatogonial cell lines. We found that the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP) is specifically expressed in undifferentiated spermatogonia. VASP belongs to the ENA/VASP family of proteins implicated in actin-dependent processes, such as fibroblast migration, axon guidance, and cell adhesion. In intact seminiferous tubules and germline stem cell cultures, GDNF treatment up-regulates VASP in a dose-dependent fashion. These data identify a novel role for the niche-derived factor GDNF, and they suggest that GDNF may impinge on the stem/progenitor compartment, affecting the actin cytoskeleton and cell migration.

An Orchestrated Intron Retention Program in Meiosis Controls Timely Usage of Transcripts during Germ Cell Differentiation

Summary Global transcriptome reprogramming during spermatogenesis ensures timely expression of factors in each phase of male germ cell differentiation. Spermatocytes and spermatids require particularly extensive reprogramming of gene expression to switch from mitosis to meiosis and to support gamete morphogenesis. Here, we uncovered an extensive alternative splicing program during this transmeiotic differentiation. Notably, intron retention was largely the most enriched pattern, with spermatocytes showing generally higher levels of retention compared with spermatids. Retained introns are characterized by weak splice sites and are enriched in genes with strong relevance for gamete function. Meiotic intron-retaining transcripts (IRTs) were exclusively localized in the nucleus. However, differently from other developmentally regulated IRTs, they are stable RNAs, showing longer half-life than properly spliced transcripts. Strikingly, fate-mapping experiments revealed that IRTs are recruited onto polyribosomes days after synthesis. These studies reveal an unexpected function for regulated intron retention in modulation of the timely expression of select transcripts during spermatogenesis.

Identification of side population cells in mouse primordial germ cells and prenatal testis.

In mammals, the stem cells of spermatogenesis are derived from an embryonic cell population called primordial germ cells (PGCs). Spermatogonial stem cells displaying the “side population” (SP) phenotype have been identified in the immature and adult mouse testis, but noting is known about the expression of the SP phenotype during prenatal development of germ cells. The SP phenotype, defined as the ability of cells to efflux fluorescent dyes such as Hoechst, is common to several stem/progenitor cell types. In the present study, we analyzed and characterized the Hoechst SP via cytofluorimetric analysis of disaggregated gonads at different time points during embryonic development in mice. To directly test the hypothesis that the SP phenotype is a feature of germ cell lineage, experiments were performed on transgenic animals expressing enhanced green fluorescent protein (EGFP) under the control of the Oct4 promoter, to identify early germ cells up to PGCs. We found that prenatal gonads contain a fraction of SP cells at each stage analyzed, and the percentage of cells in the SP fraction decreases as development proceeds. Surprisingly, more than 50% of the PGCs displayed the SP phenotype at 11.5 dpc (days post coitum). The percentage of germ cells with the SP phenotype decreased steadily with development, to less than 1% at 18.5 dpc. Cytofluorimetric analysis along with immunocytochemistry performed on sorted cells indicated that the SP fraction of prenatal gonads, as in the adult testis, was heterogeneous, being composed of both somatic and germ cells. Both cell types expressed the ABC transporters Abcg2, Abcb1a, Abcb1b and Abcc1. These findings provide evidence that the SP phenotype is a common feature of PGCs and identifies a subpopulation of fetal testis cells including prospermatogonia whose differentiation fate remains to be investigated.

Expression of the adaptor protein m-Numb in mouse male germ cells

Numb is an adaptor protein that is asymmetrically inherited at mitosis and controls the fate of sibling cells in different species. The role of m-Numb (mammalian Numb) as an important cell fate-determining factor has extensively been described mostly in neural tissues, particularly in progenitor cells, in the mouse. Biochemical and genetic analyses have shown that Numb acts as an inhibitor of the Notch signaling pathway, an evolutionarily conserved pathway involved in the control of cell proliferation, differentiation, and apoptosis. In the present study, we sought to determine m-Numb distribution in germ cells in the postnatal mouse testis. We show that all four m-Numb isoforms are widely expressed during postnatal testis development. By reverse transcriptase-PCR and western blot analyses, we further identify p71 as the predominantly expressed isoform in germ cells. Moreover, we demonstrate through co-immunoprecipitation studies that m-Numb physically associates with Ap2a1, a component of the endocytotic clathrin-coated vesicles. Finally, we employed confocal immunofluorescence microscopy of whole mount seminiferous tubules and isolated germ cells to gain more insight into the subcellular localization of m-Numb. These morphological analyses confirmed m-Numb and Ap2a1 co-localization. However, we did not observe asymmetric localization of m-Numb neither in mitotic spermatogonial stem cells nor in more differentiated spermatogonial cells, suggesting that spermatogonial stem cell fate in the mouse does not rely on asymmetric partitioning of m-Numb.