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1.  SALL4 Expression in Gonocytes and Spermatogonial Clones of Postnatal Mouse Testes 
PLoS ONE  2013;8(1):e53976.
The spermatogenic lineage is established after birth when gonocytes migrate to the basement membrane of seminiferous tubules and give rise to spermatogonial stem cells (SSC). In adults, SSCs reside within the population of undifferentiated spermatogonia (Aundiff) that expands clonally from single cells (Asingle) to form pairs (Apaired) and chains of 4, 8 and 16 Aaligned spermatogonia. Although stem cell activity is thought to reside in the population of Asingle spermatogonia, new research suggests that clone size alone does not define the stem cell pool. The mechanisms that regulate self-renewal and differentiation fate decisions are poorly understood due to limited availability of experimental tools that distinguish the products of those fate decisions. The pluripotency factor SALL4 (sal-like protein 4) is implicated in stem cell maintenance and patterning in many organs during embryonic development, but expression becomes restricted to the gonads after birth. We analyzed the expression of SALL4 in the mouse testis during the first weeks after birth and in adult seminiferous tubules. In newborn mice, the isoform SALL4B is expressed in quiescent gonocytes at postnatal day 0 (PND0) and SALL4A is upregulated at PND7 when gonocytes have colonized the basement membrane and given rise to spermatogonia. During steady-state spermatogenesis in adult testes, SALL4 expression overlapped substantially with PLZF and LIN28 in Asingle, Apaired and Aaligned spermatogonia and therefore appears to be a marker of undifferentiated spermatogonia in mice. In contrast, co-expression of SALL4 with GFRα1 and cKIT identified distinct subpopulations of Aundiff in all clone sizes that might provide clues about SSC regulation. Collectively, these results indicate that 1) SALL4 isoforms are differentially expressed at the initiation of spermatogenesis, 2) SALL4 is expressed in undifferentiated spermatogonia in adult testes and 3) SALL4 co-staining with GFRα1 and cKIT reveals distinct subpopulations of Aundiff spermatogonia that merit further investigation.
doi:10.1371/journal.pone.0053976
PMCID: PMC3543410  PMID: 23326552
2.  Human spermatogonial stem cells: a possible origin for spermatocytic seminoma 
International journal of andrology  2011;34(4 Pt 2):e296-e305.
In mammals, spermatogenesis is maintained throughout life by a small subpopulation of type A spermatogonia called spermatogonial stem cells (SSCs). In rodents, SSCs, or Asingle spermatogonia, form the self-renewing population. SSCs can also divide into Apaired (Apr) spermatogonia that are predestined to differentiate. Apaired spermatogonia produce chains of Aaligned (Aal) spermatogonia that divide to form A1 to A4, then type B spermatogonia. Type B spermatogonia will divide into primary spermatocytes that undergo meiosis. In human, there are only two different types of A spermatogonia, the Adark and Apale spermatogonia. The Adark spermatogonia are considered reserve stem cells, whereas the Apale spermatogonia are the self-renewing stem cells. There is only one generation of type B spermatogonia before differentiation into spermatocytes, which makes human spermatogenesis less efficient than in rodents. Although the biology of human SSCs is not well known, a panel of phenotypic markers has recently emerged that is remarkably similar to the list of markers expressed in mice. One such marker, the orphan receptor GPR125, is a plasma membrane protein that can be used to isolate human SSCs. Human SSCs proliferate in culture in response to growth factors such as GDNF, which is essential for SSC self-renewal in mice and triggers the same signaling pathways in both species. Therefore, despite differences in the spermatogonial differentiation scheme, both species use the same genes and proteins to maintain the pool of self-renewing SSCs within their niche. Spermatocytic seminomas are mainly found in the testes of older men, and they rarely metastasize. It is believed that these tumors originate from a postnatal germ cell. Because these lesions can express markers specific for meiotic prophase, they might originate form a primary spermatocyte. However, morphological appearance and overall immunohistochemical profile of these tumors indicate that the cell of origin could also be a spermatogonial stem cell.
doi:10.1111/j.1365-2605.2011.01199.x
PMCID: PMC3146023  PMID: 21790653
spermatogonial stem cells; human; mouse; spermatocytic seminoma
3.  NEUROG3 Is a Critical Downstream Effector for STAT3-Regulated Differentiation of Mammalian Stem and Progenitor Spermatogonia1  
Biology of Reproduction  2012;86(5):164.
ABSTRACT
Spermatogenesis relies on coordinated differentiation of stem and progenitor spermatogonia, and the transcription factor STAT3 is essential for this process in mammals. Here we studied the THY1+ spermatogonial population in mouse testes, which contains spermatogonial stem cells (SSC) and non-stem cell progenitor spermatogonia, to further define the downstream mechanism regulating differentiation. Transcript abundance for the bHLH transcription factor Neurog3 was found to be significantly reduced upon transient inhibition of STAT3 signaling in these cells and exposure to GDNF, a key growth factor regulating self-renewal of SSCs, suppressed activation of STAT3 and in accordance Neurog3 gene expression. Moreover, STAT3 was found to bind the distal Neurog3 promoter/enhancer region in THY1+ spermatogonia and regulate transcription. Transient inhibition of Neurog3 expression in cultures of proliferating THY1+ spermatogonia increased stem cell content after several self-renewal cycles without effecting overall proliferation of the cells, indicating impaired differentiation of SSCs to produce progenitor spermatogonia. Furthermore, cultured THY1+ spermatogonia with induced deficiency of Neurog3 were found to be incapable of differentiation in vivo following transplantation into testes of recipient mice. Collectively, these results establish a mechanism by which activation of STAT3 regulates the expression of NEUROG3 to subsequently drive differentiation of SSC and progenitor spermatogonia in the mammalian germline.
STAT3 signaling regulates expression of NEUROG3 in stem and progenitor spermatogonia to control differentiation, and this pathway is suppressed by signaling from the self-renewal factor GDNF.
doi:10.1095/biolreprod.111.097386
PMCID: PMC3364931  PMID: 22378757
differentiation; NEUROG3; progenitor spermatogonia; spermatogonia; spermatogonial stem cell; STAT3
4.  Loss of Etv5 Decreases Proliferation and RET Levels in Neonatal Mouse Testicular Germ Cells and Causes an Abnormal First Wave of Spermatogenesis1 
Biology of Reproduction  2009;81(2):258-266.
Mice that are ets variant gene 5 (ETV5) null (Etv5−/−) undergo the first wave of spermatogenesis but lose all spermatogonial stem cells (SSCs) during this time. The SSC loss in Etv5−/− mice begins during the neonatal period, suggesting a role for ETV5 in SSC self-renewal during this period. Herein, we show that Etv5 mRNA was present in perinatal mouse testis and that ETV5 was expressed in fetal Sertoli cells and by germ cells and Sertoli cells during the neonatal period. Transplantation of Etv5−/− germ cells failed to establish spermatogenesis in W/Wv mice testes, indicating that germ cell ETV5 has a key role in establishment or self-renewal of transplanted SSCs. The SSC self-renewal is stimulated by glial cell-derived neurotrophic factor (GDNF) acting through the RET/GDNF family receptor alpha 1 (GFRA1) receptor complex in SSCs. Immunohistochemistry, quantitative PCR, and laser capture microdissection revealed decreased RET mRNA and protein expression in spermatogonia of neonatal Etv5−/− mice by Postnatal Days 4–8, indicating that disrupted GDNF/RET/GFRA1 signaling may occur before initial spermatogonial stem/progenitor cell decrease. Etv5−/− spermatogonia had reduced proliferation in vivo and in vitro. Decreased cell proliferation may cause the observed decreases in the number of type A spermatogonia (Postnatal Day 17) and daily sperm production (Postnatal Day 30) in Etv5−/− mice, indicating quantitative impairments in the first wave of spermatogenesis. In conclusion, ETV5 is expressed beginning in fetal Sertoli cells and can potentially have effects on neonatal Sertoli cells and germ cells. In addition, ETV5 has critical effects on neonatal spermatogonial proliferation, which may involve impaired signaling through the RET receptor.
Neonatal expression of ETV5 in mouse testis is essential for spermatogonial stem cell maintenance and normal spermatogenesis.
doi:10.1095/biolreprod.108.075200
PMCID: PMC2849825  PMID: 19369650
ERM; first wave; GDNF; GFRA1; Sertoli cells; spermatogenesis; spermatogonial stem cells; testis
5.  Glial Cell Line-derived Neurotrophic Factor Regulation of Genes Essential for Self-renewal of Mouse Spermatogonial Stem Cells Is Dependent on Src Family Kinase Signaling*S 
The Journal of biological chemistry  2007;282(35):25842-25851.
Self-renewal and differentiation by spermatogonial stem cells (SSCs) is the foundation for continual spermatogenesis. SSC self-renewal is dependent on glial cell line-derived neurotrophic factor (GDNF); however, intracellular mechanisms stimulated by GDNF in SSCs are unknown. To investigate these mechanisms we utilized a culture system that maintains a mouse undifferentiated germ cell population enriched for self-renewing SSCs. In these cultures mRNA for the transcription factors Bcl6b, Erm, and Lhx1 are up-regulated by GDNF and decreased in its absence. The expression of all three molecules was further identified in undifferentiated spermatogonia in vivo. Using small interfering RNA to reduce expression and transplantation to quantify stem cell numbers, Bcl6b, Erm, and Lhx1 were shown to be important for SSC maintenance in vitro. Next, GDNF was shown to activate both Akt and Src family kinase (SFK) signaling in SSCs, and culture of SSCs with inhibitors to Akt or SFKs followed by transplantation analysis showed significant impairment of SSC maintenance in vitro. Apoptosis analysis revealed a significant increase in the percentage of apoptotic cells when Akt, but not SFK, signaling was impaired, indicating that multiple signaling pathways are responsible for SSC self-renewal and survival. Biochemical and gene expression experiments revealed that GDNF up-regulated expression of Bcl6b, Erm, and Lhx1 transcripts is dependent on SFK signaling. Overall, these data demonstrate that GDNF up-regulation of Bcl6b, Erm, and Lhx1 expression through SFK signaling is a key component of the intracellular mechanism for SSC self-renewal.
doi:10.1074/jbc.M703474200
PMCID: PMC4083858  PMID: 17597063
6.  PAX7 expression defines germline stem cells in the adult testis 
The Journal of Clinical Investigation  2014;124(9):3929-3944.
Spermatogenesis is a complex, multistep process that maintains male fertility and is sustained by rare germline stem cells. Spermatogenic progression begins with spermatogonia, populations of which express distinct markers. The identity of the spermatogonial stem cell population in the undisturbed testis is controversial due to a lack of reliable and specific markers. Here we identified the transcription factor PAX7 as a specific marker of a rare subpopulation of Asingle spermatogonia in mice. PAX7+ cells were present in the testis at birth. Compared with the adult testis, PAX7+ cells constituted a much higher percentage of neonatal germ cells. Lineage tracing in healthy adult mice revealed that PAX7+ spermatogonia self-maintained and produced expanding clones that gave rise to mature spermatozoa. Interestingly, in mice subjected to chemotherapy and radiotherapy, both of which damage the vast majority of germ cells and can result in sterility, PAX7+ spermatogonia selectively survived, and their subsequent expansion contributed to the recovery of spermatogenesis. Finally, PAX7+ spermatogonia were present in the testes of a diverse set of mammals. Our data indicate that the PAX7+ subset of Asingle spermatogonia functions as robust testis stem cells that maintain fertility in normal spermatogenesis in healthy mice and mediate recovery after severe germline injury, such as occurs after cancer therapy.
doi:10.1172/JCI75943
PMCID: PMC4153705  PMID: 25133429
7.  The Niche-Derived Glial Cell Line-Derived Neurotrophic Factor (GDNF) Induces Migration of Mouse Spermatogonial Stem/Progenitor Cells 
PLoS ONE  2013;8(4):e59431.
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.
doi:10.1371/journal.pone.0059431
PMCID: PMC3632550  PMID: 23613711
8.  Regulation of Mouse Spermatogonial Stem Cell Differentiation by STAT3 Signaling1 
Biology of Reproduction  2010;83(3):427-433.
Homeostasis of many tissues is maintained by self-renewal and differentiation of stem cells. Spermatogenesis is one such system relying on the activity of spermatogonial stem cells (SSCs). Several key regulators of SSC self-renewal have been identified, yet knowledge of molecules that control SSC differentiation is undefined. In this study, we found that transient impairment of STAT3 signaling enhances SSC self-renewal in vitro without affecting general spermatogonial proliferation, indicating an alteration in the balance of SSC fate decisions that inhibited differentiation. Confirming this observation, short hairpin RNA-mediated stable reduction of STAT3 expression in cultured SSCs abolished their ability to differentiate beyond the undifferentiated spermatogonial stage following transplantation into recipient testes. Collectively, these results demonstrate that STAT3 promotes the differentiation of SSCs. In contrast, STAT3 plays a central role in maintaining self-renewal of mouse embryonic stem cells, and STAT signaling is essential for self-renewal of male germline stem cells in Drosophila.
Impairment of STAT3 signaling altered spermatogonial stem cell fate decisions, leading to an increased rate of self-renewal and blockade of spermatogonial differentiation in mouse seminiferous tubules.
doi:10.1095/biolreprod.109.083352
PMCID: PMC2924805  PMID: 20505165
differentiation; spermatogonial stem cell; STAT3
9.  Endocrine Regulation of Spermatogonial Stem Cells in the Seminiferous Epithelium of Adult Mice 
BioResearch Open Access  2012;1(5):222-230.
Abstract
A balance between self-renewal and differentiation of spermatogonial stem cells (SSCs) is required to maintain sperm production throughout male life. The seminiferous epithelium is organized into stages of spermatogenesis based on the complement of germ cell types within a tubular section of the testis. The stages exist in close physical proximity and foster diverse phases of germ cell development despite exposure to a similar endocrine milieu that supports coordinated spermatogenesis. The objective of the current study was to identify the population dynamics of SSCs in vivo. We hypothesized that SSC populations and their niches are specifically distributed across the mature seminiferous epithelium in the mouse testis. To test this hypothesis, we conducted stem cell transplantation of germ cells obtained from stage-specific clusters of seminiferous tubules representing areas of high responsiveness to follicle-stimulating hormone (IX–I), androgen (II–IV), and retinoid (V–VIII) signaling. Similarly, we analyzed the expression of genes linked with SSC activity in these groups of stages. No stage-specific differences in the colonization efficiency or the colony number were detected after SSC transplantation, indicating that SSCs are equally distributed across all stages of the seminiferous tubule. In contrast, SSCs obtained from donor stages IX–IV established larger donor-derived colonies due to increased colony expansion. SSCs originating from different stages have varying degrees of stem cell activity in vivo, a notion consistent with Gdnf, Ret, and Bcl6b expression data. These results support the conclusion of a stage-specific, microenvironment-regulating SSC self-renewal and suggest the presence of a transit-amplifying population of undifferentiated spermatogonia in vivo.
doi:10.1089/biores.2012.0259
PMCID: PMC3559217  PMID: 23514745
gamete biology; spermatogenesis; spermatogonial stem cells; stem cells; testis
10.  The POU Domain Transcription Factor POU3F1 Is an Important Intrinsic Regulator of GDNF-Induced Survival and Self-Renewal of Mouse Spermatogonial Stem Cells1 
Biology of Reproduction  2010;82(6):1103-1111.
Continual spermatogenesis relies on a pool of spermatogonial stem cells (SSCs) that possess the capacity for self-renewal and differentiation. Maintenance of this pool depends on survival of SSCs throughout the lifetime of a male. Response to extrinsic stimulation from glial cell line-derived neurotrophic factor (GDNF), mediated by the PIK3/AKT signaling cascade, is a key pathway of SSC survival. In this study, we found that expression of the POU domain transcription factor POU3F1 in cultured SSCs is up-regulated via this mechanism. Reduction of Pou3f1 gene expression by short interfering RNA (siRNA) treatment induced apoptosis in cultured germ cell populations, and transplantation analyses revealed impaired SSC maintenance in vitro. POU3F1 expression was localized to spermatogonia in cross-sections of prepubertal and adult testes, implying a similar role in vivo. Through comparative analyses, we found that expression of POU5F1, another POU transcription factor implicated as essential for SSC self-renewal, is not regulated by GDNF in cultured SSCs. Transplantation analyses following siRNA treatment showed that POU5F1 expression is not essential for SSC maintenance in vitro. Additionally, expression of NODAL, a putative autocrine regulator of POU5F1 expression in mouse germ cells, could not be detected in SSCs isolated from testes or cultured SSCs. Collectively, these results indicate that POU3F1, but not POU5F1, is an intrinsic regulator of GDNF-induced survival and self-renewal of mouse SSCs.
Reduction of POU3F1 expression in mouse SSCs impairs their self-renewal and survival in vitro, whereas similar reduction of POU5F1 expression has no effect.
doi:10.1095/biolreprod.109.083097
PMCID: PMC2874496  PMID: 20181621
GDNF; POU3F1; POU5F1; self-renewal; spermatogonial stem cell
11.  Molecular dissection of the male germ cell lineage identifies putative spermatogonial stem cells in rhesus macaques 
Human Reproduction (Oxford, England)  2009;24(7):1704-1716.
BACKGROUND
The spermatogonial stem cell (SSC) pool in the testes of non-human primates is poorly defined.
METHODS
To begin characterizing SSCs in rhesus macaque testes, we employed fluorescence-activated cell sorting (FACS), a xenotransplant bioassay and immunohistochemical methods and correlated our findings with classical descriptions of germ cell nuclear morphology (i.e. Adark and Apale spermatogonia).
RESULTS
FACS analysis identified a THY-1+ fraction of rhesus testis cells that was enriched for consensus SSC markers (i.e. PLZF, GFRα1) and exhibited enhanced colonizing activity upon transplantation to nude mouse testes. We observed a substantial conservation of spermatogonial markers from mice to monkeys [PLZF, GFRα1, Neurogenin 3 (NGN3), cKIT]. Assuming that molecular characteristics correlate with function, the pool of putative SSCs (THY-1+, PLZF+, GFRα1+, NGN3+/−, cKIT−) comprises most Adark and Apale and is considerably larger in primates than in rodents. It is noteworthy that the majority of Adark and Apale share a common molecular phenotype, considering their distinct functional classifications as reserve and renewing stem cells, respectively. NGN3 is absent from Adark, but is expressed by some Apale and may mark the transition from undifferentiated (cKIT−) to differentiating (cKIT+) spermatogonia. Finally, the pool of transit-amplifying progenitor spermatogonia (PLZF+, GFRα1+, NGN3+, cKIT+/−) is smaller in primates than in rodents.
CONCLUSIONS
These results provide an in-depth analysis of molecular characteristics of primate spermatogonia, including SSCs, and lay a foundation for future studies investigating the kinetics of spermatogonial renewal, clonal expansion and differentiation during primate spermatogenesis.
doi:10.1093/humrep/dep073
PMCID: PMC2698327  PMID: 19336441
spermatogonial stem cells; Adark spermatogonia; Apale spermatogonia; xenotransplantation; primate
12.  The pluripotency factor LIN28 in monkey and human testes: a marker for spermatogonial stem cells? 
Molecular Human Reproduction  2012;18(10):477-488.
Mammalian spermatogenesis is maintained by spermatogonial stem cells (SSCs). However, since evidentiary assays and unequivocal markers are still missing in non-human primates (NHPs) and man, the identity of primate SSCs is unknown. In contrast, in mice, germ cell transplantation studies have functionally demonstrated the presence of SSCs. LIN28 is an RNA-binding pluripotent stem cell factor, which is also strongly expressed in undifferentiated mouse spermatogonia. By contrast, two recent reports indicated that LIN28 is completely absent from adult human testes. Here, we analyzed LIN28 expression in marmoset monkey (Callithrix jacchus) and human testes during development and adulthood and compared it with that in mice. In the marmoset, LIN28 was strongly expressed in migratory primordial germ cells and gonocytes. Strikingly, we found a rare LIN28-positive subpopulation of spermatogonia also in adult marmoset testis. This was corroborated by western blotting and quantitative RT–PCR. Importantly, in contrast to previous publications, we found LIN28-positive spermatogonia also in normal adult human and additional adult NHP testes. Some seasonal breeders exhibit a degenerated (involuted) germinal epithelium consisting only of Sertoli cells and SSCs during their non-breeding season. The latter re-initiate spermatogenesis prior to the next breeding-season. Fully involuted testes from a seasonal hamster and NHP (Lemur catta) exhibited numerous LIN28-positive spermatogonia, indicating an SSC identity of the labeled cells. We conclude that LIN28 is differentially expressed in mouse and NHP spermatogonia and might be a marker for a rare SSC population in NHPs and man. Further characterization of the LIN28-positive population is required.
doi:10.1093/molehr/gas025
PMCID: PMC3457707  PMID: 22689537
LIN28; pluripotency; primate; spermatogonial stem cell; testis
13.  Spermatogonial Stem Cells Derived from Infertile Wv/Wv Mice Self-Renew In Vitro and Generate Progeny Following Transplantation1 
Biology of Reproduction  2009;81(2):293-301.
Loss-of-function mutation of the Kit gene causes a severe defect in spermatogenesis that results in infertility due to the inability of its cognate ligand, KIT ligand (KITL), to stimulate spermatogonial proliferation and differentiation. Although self-renewal of mouse spermatogonial stem cells (SSCs) depends on glial cell line-derived neurotrophic factor (GDNF), there is no unequivocal evidence that SSCs with a KIT deficiency can self-renew in vivo or in vitro. In the testis of Wv/Wv mice, in which the KIT tyrosine kinase activity is impaired, spermatogonia with SSC phenotype were identified. When Wv/Wv spermatogonia were cultured in an SSC culture system supplemented with GDNF in a 10% O2 atmosphere, they formed clumps and proliferated continuously. An atmosphere of 10% O2 was better than 21% O2 to support SSC self-renewal. When Wv/Wv clump-forming germ cells were transplanted into testes of infertile wild-type busulfan-treated mice, they colonized the seminiferous tubules but did not differentiate. However, when transplanted into the testes of infertile W/Wv pups, they restored spermatogenesis and produced spermatozoa, and progeny were generated using microinsemination. These results clearly show that SSCs exist in Wv/Wv testes and that they proliferate in vitro similar to wild-type SSCs, indicating that a functional KIT protein is not required for SSC self-renewal. Furthermore, the results indicate that a defect of KIT/KITL signaling of Wv/Wv SSCs does not prevent spermatogonial differentiation and spermatogenesis in some recipient strains.
KIT-deficient spermatogonial stem cells can self-renew in culture and can generate functional spermatozoa when transplanted into suitable recipients.
doi:10.1095/biolreprod.109.075960
PMCID: PMC2849820  PMID: 19369648
germline stem cells; growth factors; spermatogenesis; spermatogonial stem cells; testis
14.  Dynamic Changes in EPCAM Expression during Spermatogonial Stem Cell Differentiation in the Mouse Testis 
PLoS ONE  2011;6(8):e23663.
Background
Spermatogonial stem cells (SSCs) have the unique ability to undergo self-renewal division. However, these cells are morphologically indistinguishable from committed spermatogonia, which have limited mitotic activity. To establish a system for SSC purification, we analyzed the expression of SSC markers CD9 and epithelial cell adhesion molecule (EPCAM), both of which are also expressed on embryonic stem (ES) cells. We examined the correlation between their expression patterns and SSC activities.
Methodology and Principal Findings
By magnetic cell sorting, we found that EPCAM-selected mouse germ cells have limited clonogenic potential in vitro. Moreover, these cells showed stronger expression of progenitor markers than CD9-selected cells, which are significantly more enriched in SSCs. Fluorescence-activated cell sorting of CD9-selected cells indicated a significantly higher frequency of SSCs among the CD9+EPCAMlow/- population than among the CD9+EPCAM+ population. Overexpression of the active form of EPCAM in germline stem (GS) cell cultures did not significantly influence SSC activity, whereas EPCAM suppression by short hairpin RNA compromised GS cell proliferation and increased the concentration of SSCs, as revealed by germ cell transplantation.
Conclusions/Significance
These results show that SSCs are the most concentrated in CD9+EPCAMlow/- population and also suggest that EPCAM plays an important role in progenitor cell amplification in the mouse spermatogenic system. The establishment of a method to distinguish progenitor spermatogonia from SSCs will be useful for developing an improved purification strategy for SSCs from testis cells.
doi:10.1371/journal.pone.0023663
PMCID: PMC3156235  PMID: 21858196
15.  UTF1, a Putative Marker for Spermatogonial Stem Cells in Stallions 
PLoS ONE  2014;9(10):e108825.
Spermatogonial stem cells (SSCs) continuously undergo self-renewal and differentiation to sustain spermatogenesis throughout adulthood in males. In stallions, SSCs may be used for the production of progeny from geldings after cryopreservation and therapy for infertile and subfertile stallions. Undifferentiated cell transcription factor 1 (UTF1) is a putative marker for undifferentiated spermatogonia in humans and rats. The main purposes of this study are to determine the following: 1) changes in the expression pattern of UTF1 at various reproductive stages of stallions, 2) subpopulations of spermatogonia that express UTF1. Testicular samples were collected and categorized based on the age of the horses as follows: pre-pubertal (<1 yr), pubertal (1–1.5 yr), post-pubertal (2–3 yr), and adult (4–8 yr). Western blot analysis was utilized to determine the cross-activity of the UTF1 antibody to horse testes tissues. Immunohistochemistry was conducted to investigate the UTF1 expression pattern in germ cells at different reproductive stages. Whole mount staining was applied to determine the subpopulation of UTF1-positive spermatogonia. Immunohistological analysis showed that most germ cells in the pre-pubertal and pubertal stages were immunolabeled with UTF1, whereas only a few germ cells in the basal compartment of the seminiferous tubule cross-sections of post-pubertal and adult tissues were UTF1-positive. No staining was observed in the Sertoli or Leydig cells at any reproductive stages. Whole mount staining showed that As, Apr, and chains of 4, 8, 16 Aal spermatogonia were immunolabeled with UTF1 in the post-pubertal stallion tubule. Isolated single germ cells were also immunolabeled with UTF1. In conclusion, UTF1 is expressed in undifferentiated spermatogonia, and its antibody can be used as a putative marker for SSCs in stallions.
doi:10.1371/journal.pone.0108825
PMCID: PMC4182753  PMID: 25272017
16.  Spermatogonial Stem Cell Niche and Spermatogonial Stem Cell Transplantation in Zebrafish 
PLoS ONE  2010;5(9):e12808.
Background
Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis, and reside within a specific microenvironment in the testes called “niche” which regulates stem cell properties, such as, self-renewal, pluripotency, quiescence and their ability to differentiate.
Methodology/Principal Findings
Here, we introduce zebrafish as a new model for the study of SSCs in vertebrates. Using 5′-bromo-2′-deoxyuridine (BrdU), we identified long term BrdU-retaining germ cells, type A undifferentiated spermatogonia as putative stem cells in zebrafish testes. Similar to rodents, these cells were preferentially located near the interstitium, suggesting that the SSC niche is related to interstitial elements and might be conserved across vertebrates. This localization was also confirmed by analyzing the topographical distribution of type A undifferentiated spermatogonia in normal, vasa::egfp and fli::egfp zebrafish testes. In the latter one, the topographical arrangement suggested that the vasculature is important for the SSC niche, perhaps as a supplier of nutrients, oxygen and/or signaling molecules. We also developed an SSC transplantation technique for both male and female recipients as an assay to evaluate the presence, biological activity, and plasticity of the SSC candidates in zebrafish.
Conclusions/Significance
We demonstrated donor-derived spermato- and oogenesis in male and female recipients, respectively, indicating the stemness of type A undifferentiated spermatogonia and their plasticity when placed into an environment different from their original niche. Similar to other vertebrates, the transplantation efficiency was low. This might be attributed to the testicular microenvironment created after busulfan depletion in the recipients, which may have caused an imbalance between factors regulating self-renewal or differentiation of the transplanted SSCs.
doi:10.1371/journal.pone.0012808
PMCID: PMC2942835  PMID: 20862221
17.  SOHLH1 and SOHLH2 coordinate spermatogonial differentiation 
Developmental biology  2011;361(2):301-312.
Spermatogonial self-renewal and differentiation are essential for male fertility and reproduction. We discovered that germ cell specific genes Sohlh1 and Sohlh2, encode basic helix-loop-helix (bHLH) transcriptional regulators that are essential in spermatogonial differentiation. Sohlh1 and Sohlh2 individual mouse knockouts show remarkably similar phenotypes. Here we show that SOHLH1 and SOHLH2 proteins are co-expressed in the entire spermatogonial population except in the GFRA1+ spermatogonia, which includes spermatogonial stem cells (SSCs). SOHLH1 and SOHLH2 are expressed in both KIT negative and KIT positive spermatogonia, and overlap Ngn3/EGFP and SOX3 expression. SOHLH1 and SOHLH2 heterodimerize with each other in vivo, as well as homodimerize. The Sohlh1/Sohlh2 double mutant phenocopies single mutants, i.e., spermatogonia continue to proliferate but do not differentiate properly. Further analysis revealed that GFRA1+ population was increased, while meiosis commenced prematurely in both single and double knockouts. Sohlh1 and Sohlh2 double deficiency has a synergistic effect on gene expression patterns as compared to the single knockouts. SOHLH proteins affect spermatogonial development by directly regulating Gfra1, Sox3 and Kit gene expression. SOHLH1 and SOHLH2 suppress genes involved in SSC maintenance, and induce genes important for spermatogonial differentiation.
doi:10.1016/j.ydbio.2011.10.027
PMCID: PMC3249242  PMID: 22056784
Spematogonia; Sohlh1; Sohlh2; differentiation; stem cell
18.  Development of Quantitative Microscopy-Based Assays for Evaluating Dynamics of Living Cultures of Mouse Spermatogonial Stem/Progenitor Cells1 
Biology of Reproduction  2012;87(4):90.
ABSTRACT
Spermatogonial stem cell (SSC) self-renewal and differentiation are required for continuous production of spermatozoa and long-term fertility. Studying SSCs in vivo remains challenging because SSCs are rare cells and definitive molecular markers for their identification are lacking. The development of a method for propagating SSCs in vitro greatly facilitated analysis of SSCs. The cultured cells grow as clusters of a dynamic mixture of “true” stem cells and differentiating progenitor cells. Cells in the stem/progenitor culture system share many properties with spermatogonia in vivo; however, to fully exploit it as a model for spermatogonial development, new assays are needed that account for the dynamic heterogeneity inherent in the culture system. Here, assays were developed for quantifying dynamics of cultures of stem/progenitor cells that expressed histone-green fluorescent protein (GFP). First, we built on published results showing that cluster formation in vitro reliably predicts the relative number of SSCs. The GFP-based in vitro cluster assay allows quantification of SSCs with significantly fewer resources than a transplantation assay. Second, we compared the dynamics of differentiation in two experimental paradigms by imaging over a 17-day time frame. Finally, we performed short-term live imaging and observed cell migration, coordinated cell proliferation, and cell death resembling that of spermatogonia in the testes. The methods that we present provide a foundation for the use of fluorescent reporters in future microscopy-based high-throughput screens by using living spermatogonial stem/progenitor cultures applicable to toxicology, contraceptive discovery, and identification of regulators of self-renewal and differentiation.
Live imaging of cell proliferation and cell death in spermatogonial stem/progenitor cell cultures elucidates properties of cluster formation.
doi:10.1095/biolreprod.112.101717
PMCID: PMC3507545  PMID: 22933516
cell death; differentiation; germ cell; green fluorescent protein; lentivirus; live imaging; spermatogenesis; spermatogonial stem cell; undifferentiated spermatogonia
19.  Paracrine Mechanisms Involved in the Control of Early Stages of Mammalian Spermatogenesis 
Within the testis, Sertoli-cell is the primary target of pituitary FSH. Several growth factors have been described to be produced specifically by Sertoli cells and modulate male germ cell development through paracrine mechanisms. Some have been shown to act directly on spermatogonia such as GDNF, which acts on self-renewal of spermatogonial stem cells (SSCs) while inhibiting their differentiation; BMP4, which has both a proliferative and differentiative effect on these cells, and KIT ligand (KL), which stimulates the KIT tyrosine-kinase receptor expressed by differentiating spermatogonia (but not by SSCs). KL not only controls the proliferative cycles of KIT-positive spermatogonia, but it also stimulates the expression of genes that are specific of the early phases of meiosis, whereas the expression of typical spermatogonial markers is down-regulated. On the contrary, FGF9 acts as a meiotic inhibiting substance both in fetal gonocytes and in post-natal spermatogonia through the induction of the RNA-binding protein NANOS2. Vitamin A, which is metabolized to Retinoic Acid in Sertoli cells, controls both SSCs differentiation through KIT induction and NANOS2 inhibition, and meiotic entry of differentiating spermatogonia through STRA8 upregulation.
doi:10.3389/fendo.2013.00181
PMCID: PMC3840353  PMID: 24324457
primordial germ cells; spermatogonial stem cells; spermatogenesis; meiosis; growth factors; paracrine control; signal transduction; gene expression
20.  Indirect Effects of Wnt3a/β-Catenin Signalling Support Mouse Spermatogonial Stem Cells In Vitro 
PLoS ONE  2012;7(6):e40002.
Proper regulation of spermatogonial stem cells (SSCs) is crucial for sustaining steady-state spermatogenesis. Previous work has identified several paracrine factors involved in this regulation, in particular, glial cell line-derived neurotrophic factor and fibroblast growth factor 2, which promote long-term SSC self-renewal. Using a SSC culture system, we have recently reported that Wnt5a promotes SSC self-renewal through a β-catenin-independent Wnt mechanism whereas the β-catenin-dependent Wnt pathway is not active in SSCs. In contrast, another study has reported that Wnt3a promotes SSC self-renewal through the β-catenin-dependent pathway, as it can stimulate the proliferation of a spermatogonia cell line. To reconcile these two contradictory reports, we assessed Wnt3a effects on SSCs and progenitor cells, rather than a cell line, in vitro. We observed that Wnt3a induced β-catenin-dependent signalling in a large subset of germ cells and increased SSC numbers. However, further investigation revealed that cell populations with greater β-catenin-signalling activity contained fewer SSCs. The increased maintenance of SSCs by Wnt3a coincided with more active cell cycling and the formation of germ cell aggregates, or communities, under feeder-free conditions. Therefore, the results of this study suggest that Wnt3a selectively stimulates proliferation of progenitors that are committed to differentiation or are in the process of exiting the SSC state, leading to enhanced formation of germ cell communities, which indirectly support SSCs and act as an in vitro niche.
doi:10.1371/journal.pone.0040002
PMCID: PMC3386240  PMID: 22761943
21.  Spermatogonial stem cells in higher primates: are there differences to those in rodents? 
Reproduction (Cambridge, England)  2009;139(3):479-493.
Spermatogonial stem cells (SSCs) maintain spermatogenesis throughout the reproductive life of mammals. While Asingle spermatogonia comprise the rodent SSC pool, the identity of the stem cell pool in the primate spermatogenic lineage is not well established. The prevailing model is that primate spermatogenesis arises from Adark and Apale spermatogonia, which are considered to represent reserve and active stem cells, respectively. However, there is limited information about how the Adark and Apale descriptions of nuclear morphology correlate with the clonal (Asingle, Apaired Aaligned), molecular (e.g., GFRα1, PLZF) and functional (SSC transplantation) descriptions of rodent SSCs. Thus, there is a need to investigate primate SSCs using criteria, tools, and approaches that have been used to investigate rodent SSCs over the past two decades. SSCs have potential clinical application for treating some cases of male infertility, providing impetus for characterizing and learning to manipulate these adult tissue stem cells in primates (nonhuman and human). This review recounts the development of a xenotransplant assay for functional identification of primate SSCs and progress dissecting the molecular and clonal characteristics of the primate spermatogenic lineage. These observations highlight similarities and potential differences between rodents and primates regarding the SSC pool and the kinetics of spermatogonial self-renewal and clonal expansion. With new tools and reagents for studying primate spermatogonia, the field is poised to develop and test new hypotheses about the biology and regenerative capacity of primate SSCs.
doi:10.1530/REP-09-0255
PMCID: PMC2895987  PMID: 19880674
Spermatogonial stem cells; primates; Adark; Apale; spermatogenesis; xenotransplantation; clonal expansion
22.  Signaling molecules and pathways regulating the fate of spermatogonial stem cells 
Microscopy research and technique  2009;72(8):586-595.
Spermatogenesis is the process that involves the division and differentiation of spermatogonial stem cells (SSCs) into mature spermatozoa. SSCs are a subpopulation of type A spermatogonia resting on the basement membrane in the mammalian testis. Self-renewal and differentiation of SSCs are the foundation of normal spermatogenesis, and thus a better understanding of molecular mechanisms and signaling pathways in the SSCs is of paramount importance for the regulation of spermatogenesis and may eventually lead to novel targets for male contraception as well as for gene therapy of male infertility and testicular cancer. Uncovering the molecular mechanisms is also of great interest to a better understanding of SSC aging and for developing novel therapeutic strategies for degenerative diseases in view of the recent work demonstrating the pluripotent potential of the SSC. Progress has recently been made in elucidating the signaling molecules and pathways that determine cell fate decisions of SSCs. In this review, we first address the morphological features, phenotypic characteristics, and the potential of SSCs. And then we focus on the recent advances in defining the key signaling molecules and crucial signaling pathways regulating self-renewal and differentiation of SSCs. The association of aberrant expression of signaling molecules and cascades with abnormal spermatogenesis and testicular cancer are also discussed. Finally we point out potential future directions to pursue in research on signaling pathways of SSCs.
doi:10.1002/jemt.20698
PMCID: PMC2766659  PMID: 19263492
spermatogonial stem cells; signaling pathways; renewal; differentiation
23.  Asymmetric Distribution of UCH-L1 in Spermatogonia Is Associated With Maintenance and Differentiation of Spermatogonial Stem Cells 
Journal of cellular physiology  2009;220(2):460-468.
Asymmetric division of germline stem cells in vertebrates was proposed a century ago; however, direct evidence for asymmetric division of mammalian spermatogonial stem cells (SSCs) has been scarce. Here, we report that ubiquitin carboxy-terminal hydrolase 1 (UCH-L1) is expressed in type A (As, Apr, and Aal) spermatogonia located at the basement membrane (BM) of seminiferous tubules at high and low levels, but not in differentiated germ cells distant from the BM. Asymmetric segregation of UCH-L1 was associated with self-renewal versus differentiation divisions of SSCs as defined by co-localization of UCH-L1high and PLZF, a known determinant of undifferentiated SSCs, versus co-localization of UCH-L1 low/− with proteins expressed during SSC differentiation (DAZL, DDX4, c-KIT). In vitro, gonocytes/spermatogonia frequently underwent asymmetric divisions characterized by unequal segregation of UCH-L1 and PLZF. Importantly, we could also demonstrate asymmetric segregation of UCH-L1 and PLZF in situ in seminiferous tubules. Expression level of UCH-L1 in the immature testis where spermatogenesis was not complete was not affected by the location of germ cells relative to the BM, whereas UCH-L1-positive spermatogonia were exclusively located at the BM in the adult testis. Asymmetric division of SSCs appeared to be affected by interaction with supporting somatic cells and extracelluar matrix. These findings for the first time provide direct evidence for existence of asymmetric division during SSCs self-renewal and differentiation in mammalian spermatogenesis.
doi:10.1002/jcp.21789
PMCID: PMC2732714  PMID: 19388011
24.  Haploinsufficiency of the paternal-effect gene Dnmt3L results in transient DNA hypomethylation in progenitor cells of the male germline 
STUDY QUESTION
How does haploinsufficiency of the paternal-effect gene Dnmt3L affect DNA methylation establishment and stability in the male germline?
SUMMARY ANSWER
Reduced expression of DNMT3L in male germ cells, associated with haploinsufficiency of the paternal-effect gene Dnmt3L, results in abnormal hypomethylation of prenatal germline progenitor cells.
WHAT IS KNOWN ALREADY
The DNA methyltransferase regulator Dnmt3-Like (Dnmt3L) is a paternal-effect gene required for DNA methylation acquisition in male germline stem cells and their precursors. In males, DNMT3L deficiency causes meiotic abnormalities and infertility. While Dnmt3L heterozygous males are fertile, they have abnormalities in X chromosome compaction and postmeiotic gene expression and sire offspring with sex chromosome aneuploidy. It has been proposed that the paternal effects of Dnmt3L haploinsufficiency are due to epigenetic defects in early male germ cells. DNA methylation is an essential epigenetic modification essential for normal germ cell development. Since patterns of DNA methylation across the genome are initially acquired in prenatal male germ cells, perturbations in methylation could contribute to the epigenetic basis of the paternal effects in Dnmt3L+/− males.
STUDY DESIGN, SIZE, DURATION
This is a cross-sectional study of DNA methylation in Dnmt3L+/+ versus Dnmt3L+/− male germ cells collected from mice at 16.5 days post-coitum (dpc), Day 6 and Day 70 (n = 3 per genotype, each n represents a pool of 2–20 animals). Additionally, DNA methylation was compared in enriched populations of spermatogonial stem cells (SSC)/progenitor cells from Dnmt3L+/+ and Dnmt3L+/− males following ∼2 months in culture.
MATERIALS, SETTING, METHODS
DNA methylation at intergenic loci along chromosomes 9 and X was examined by quantitative analysis of DNA methylation by real-time polymerase chain reaction at the time of initial acquisition of epigenetic patterns in the prenatal male germline (16.5 dpc) and compared with patterns in early post-natal spermatogonia (Day 6) and in spermatozoa in mice. DNA methylation status at CpG-rich sites across the genome was assessed in spermatogonial precursors from Day 4 male mice using restriction landmark genomic scanning.
MAIN RESULTS AND THE ROLE OF CHANCE
At 16.5 dpc, 42% of intergenic loci examined along chromosome 9 and 10% of those along chromosome X were hypomethylated in Dnmt3L heterozygotes. By Day 6 and in spermatozoa, germ cell DNA methylation was similar in heterozygous and wild-type mice. DNA methylation stability of acquired patterns in wild-type and Dnmt3L+/− SSC/progenitor cell culture was analyzed at numerous loci across the genome in cells cultured in vitro and collected at passages 6–28. While the methylation of most loci was stable in culture over time, differences at ∼1% of sites were found between Dnmt3L+/− and Dnmt3L+/+ cultures.
LIMITATIONS, REASONS FOR CAUTION
Evaluation of DNA methylation in SSCs can only be performed after a period of culture limiting the investigation to changes observed during culture when compared with DNA methylation differences between genotypes that could be present at the beginning of culture establishment.
WIDER IMPLICATIONS OF THE FINDINGS
The DNA methylation defects described here in prenatal male germline progenitor cells and SSC culture are the earliest epigenetic perturbations yet identified for a mammalian paternal-effect gene and may influence downstream epigenetic events in germ cells at later stages of development. Together, the results provide evidence of a ‘window’ of susceptibility in prenatal male germ cell precursors for the induction of epimutations due to genetic perturbations and, potentially, in utero environmental exposures.
STUDY FUNDING/COMPETING INTEREST(S)
Canadian Institutes of Health Research (CIHR) provided funding for J.M.T. (MOP229913) and M.C.N. (MOP86532). The authors have no conflicts of interest to declare.
doi:10.1093/humrep/des395
PMCID: PMC3695691  PMID: 23159436
epigenetics; DNA methylation; Dnmt3L; spermatogonial stem cells; paternal effect
25.  Characterization, cryopreservation and ablation of spermatogonial stem cells in adult rhesus macaques 
Stem cells (Dayton, Ohio)  2007;25(9):2330-2338.
Spermatogonial stem cells (SSCs) are at the foundation of mammalian spermatogenesis. While rare Asingle spermatogonia comprise the rodent SSC pool, primate spermatogenesis arises from more abundant Adark and Apale spermatogonia and the identity of the stem cell is subject to debate. The fundamental differences between these models highlight the need to investigate the biology of primate SSCs, which have greater relevance to human physiology. The alkylating chemotherapeutic agent, busulfan, ablates spermatogenesis in rodents and causes infertility in humans. We treated adult rhesus macaques with busulfan to gain insights about its effects on SSCs and spermatogenesis. Busulfan treatment caused acute declines in testis volume and sperm counts, indicating a disruption of spermatogenesis. One year after high-dose busulfan treatment sperm counts remained undetectable and testes were depleted of germ cells. Similar to rodents, rhesus spermatogonia expressed markers of germ cells (VASA, DAZL) and stem/progenitor spermatogonia (PLZF and GFRα1), and cells expressing these markers were depleted following high-dose busulfan treatment. Furthermore, fresh or cryopreserved germ cells from normal rhesus testes produced colonies of spermatogonia, which persisted as chains on the basement membrane of mouse seminiferous tubules in the primate to nude mouse xenotransplant assay. In contrast, testis cells from animals that received high-dose busulfan produced no colonies. These studies provide basic information about rhesus SSC activity and the impact of busulfan on the stem cell pool. In addition, the germ cell depleted testis model will enable autologous/homologous transplantation to study stem cell/niche interactions in nonhuman primate testes.
doi:10.1634/stemcells.2007-0143
PMCID: PMC3593092  PMID: 17585169
Busulfan; chemotherapy; infertility; spermatogenesis; spermatogonial stem cells; xenotransplantation

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