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1.  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
2.  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
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.  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
5.  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
6.  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
7.  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
8.  H3K27 Demethylase, JMJD3, Regulates Fragmentation of Spermatogonial Cysts 
PLoS ONE  2013;8(8):e72689.
The spermatogonial stem cell (SSC) compartment is maintained by self-renewal of stem cells as well as fragmentation of differentiating spermatogonia through abscission of intercellular bridges in a random and stochastic manner. The molecular mechanisms that regulate this reversible developmental lineage remain to be elucidated. Here, we show that histone H3K27 demethylase, JMJD3 (KDM6B), regulates the fragmentation of spermatogonial cysts. Down-regulation of Jmjd3 in SSCs promotes an increase in undifferentiated spermatogonia but does not affect their differentiation. Germ cell-specific Jmjd3 null male mice have larger testes and sire offspring for a longer period compared to controls, likely secondary to increased and prolonged maintenance of the spermatogonial compartment. Moreover, JMJD3 deficiency induces frequent fragmentation of spermatogonial cysts by abscission of intercellular bridges. These results suggest that JMJD3 controls the spermatogonial compartment through the regulation of fragmentation of spermatogonial cysts and this mechanism may be involved in maintenance of diverse stem cell niches.
doi:10.1371/journal.pone.0072689
PMCID: PMC3744460  PMID: 23967333
9.  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
10.  Gfra1 Silencing in Mouse Spermatogonial Stem Cells Results in Their Differentiation Via the Inactivation of RET Tyrosine Kinase1 
Biology of reproduction  2007;77(4):723-733.
Spermatogenesis is the process by which spermatogonial stem cells divide and differentiate into sperm. The role of growth factor receptors in regulating self-renewal and differentiation of spermatogonial stem cells remains largely unclear. This study was designed to examine Gfra1 receptor expression in immature and adult mouse testes and determine the effects of Gfra1 knockdown on the proliferation and differentiation of type A spermatogonia. We demonstrated that GFRA1 was expressed in a subpopulation of spermatogonia in immature and adult mice. Neither Gfra1 mRNA nor GFRA1 protein was detected in pachytene spermatocytes and round spermatids. GFRA1 and POU5F1 (also known as OCT4), a marker for spermatogonial stem cells, were co-expressed in a subpopulation of type A spermatogonia from 6-day-old mice. In addition, the spermatogonia expressing GFRA1 exhibited a potential for proliferation and the ability to form colonies in culture, which is a characteristic of stem cells. RNA interference assays showed that Gfra1 small interfering RNAs (siRNAs) knocked down the expression of Gfra1 mRNA and GFRA1 protein in type A spermatogonia. Notably, the reduction of Gfra1 expression by Gfra1 siRNAs induced a phenotypic differentiation, as evidenced by the elevated expression of KIT, as well as the decreased expression of POU5F1 and proliferating cell nuclear antigen (PCNA). Furthermore, Gfra1 silencing resulted in a decrease in RET phosphorylation. Taken together, these data indicate that Gfra1 is expressed dominantly in mouse spermatogonial stem cells and that Gfra1 knockdown leads to their differentiation via the inactivation of RET tyrosine kinase, suggesting an essential role for Gfra1 in spermatogonial stem cell regulation.
doi:10.1095/biolreprod.107.062513
PMCID: PMC2911237  PMID: 17625109
differentiation; Gfra1; Gfra1 knockdown; mouse; spermatogonial stem cells; testis
11.  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
12.  Foxo1 is required in mouse spermatogonial stem cells for their maintenance and the initiation of spermatogenesis 
The Journal of Clinical Investigation  2011;121(9):3456-3466.
Spermatogonial stem cells (SSCs) capable of self-renewal and differentiation are the foundation for spermatogenesis. Although several factors important for these processes have been identified, the fundamental mechanisms regulating SSC self-renewal and differentiation remain unknown. Here, we investigated a role for the Foxo transcription factors in mouse spermatogenesis and found that Foxo1 specifically marks mouse gonocytes and a subset of spermatogonia with stem cell potential. Genetic analyses showed that Foxo1 was required for both SSC homeostasis and the initiation of spermatogenesis. Combined deficiency of Foxo1, Foxo3, and Foxo4 resulted in a severe impairment of SSC self-renewal and a complete block of differentiation, indicating that Foxo3 and Foxo4, although dispensable for male fertility, contribute to SSC function. By conditional inactivation of 3-phosphoinositide–dependent protein kinase 1 (Pdk1) and phosphatase and tensin homolog (Pten) in the male germ line, we found that PI3K signaling regulates Foxo1 stability and subcellular localization, revealing that the Foxos are pivotal effectors of PI3K-Akt signaling in SSCs. We also identified a network of Foxo gene targets — most notably Ret — that rationalized the maintenance of SSCs by the Foxos. These studies demonstrate that Foxo1 expression in the spermatogenic lineage is intimately associated with the stem cell state and revealed what we believe to be novel Foxo-dependent mechanisms underlying SSC self-renewal and differentiation, with implications for common diseases, including male infertility and testicular cancer, due to abnormalities in SSC function.
doi:10.1172/JCI57984
PMCID: PMC3163967  PMID: 21865646
13.  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
14.  Spermatogonial Stem Cell Markers and Niche in Equids 
PLoS ONE  2012;7(8):e44091.
Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis and are located in a highly dynamic microenvironment called “niche” that influences all aspects of stem cell function, including homing, self-renewal and differentiation. Several studies have recently identified specific proteins that regulate the fate of SSCs. These studies also aimed at identifying surface markers that would facilitate the isolation of these cells in different vertebrate species. The present study is the first to investigate SSC physiology and niche in stallions and to offer a comparative evaluation of undifferentiated type A spermatogonia (Aund) markers (GFRA1, PLZF and CSF1R) in three different domestic equid species (stallions, donkeys, and mules). Aund were first characterized according to their morphology and expression of the GFRA1 receptor. Our findings strongly suggest that in stallions these cells were preferentially located in the areas facing the interstitium, particularly those nearby blood vessels. This distribution is similar to what has been observed in other vertebrate species. In addition, all three Aund markers were expressed in the equid species evaluated in this study. These markers have been well characterized in other mammalian species, which suggests that the molecular mechanisms that maintain the niche and Aund/SSCs physiology are conserved among mammals. We hope that our findings will help future studies needing isolation and cryopreservation of equids SSCs. In addition, our data will be very useful for studies that aim at preserving the germplasm of valuable animals, and involve germ cell transplantation or xenografts of equids testis fragments/germ cells suspensions.
doi:10.1371/journal.pone.0044091
PMCID: PMC3429436  PMID: 22937157
15.  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
16.  Cyclical and Patch-Like GDNF Distribution along the Basal Surface of Sertoli Cells in Mouse and Hamster Testes 
PLoS ONE  2011;6(12):e28367.
Background and Aims
In mammalian spermatogenesis, glial cell line-derived neurotrophic factor (GDNF) is one of the major Sertoli cell-derived factors which regulates the maintenance of undifferentiated spermatogonia including spermatogonial stem cells (SSCs) through GDNF family receptor α1 (GFRα1). It remains unclear as to when, where and how GDNF molecules are produced and exposed to the GFRα1-positive spermatogonia in vivo.
Methodology and Principal Findings
Here we show the cyclical and patch-like distribution of immunoreactive GDNF-positive signals and their close co-localization with a subpopulation of GFRα1-positive spermatogonia along the basal surface of Sertoli cells in mice and hamsters. Anti-GDNF section immunostaining revealed that GDNF-positive signals are mainly cytoplasmic and observed specifically in the Sertoli cells in a species-specific as well as a seminiferous cycle- and spermatogenic activity-dependent manner. In contrast to the ubiquitous GDNF signals in mouse testes, high levels of its signals were cyclically observed in hamster testes prior to spermiation. Whole-mount anti-GDNF staining of the seminiferous tubules successfully visualized the cyclical and patch-like extracellular distribution of GDNF-positive granular deposits along the basal surface of Sertoli cells in both species. Double-staining of GDNF and GFRα1 demonstrated the close co-localization of GDNF deposits and a subpopulation of GFRα1-positive spermatogonia. In both species, GFRα1-positive cells showed a slender bipolar shape as well as a tendency for increased cell numbers in the GDNF-enriched area, as compared with those in the GDNF-low/negative area of the seminiferous tubules.
Conclusion/Significance
Our data provide direct evidence of regionally defined patch-like GDNF-positive signal site in which GFRα1-positive spermatogonia possibly interact with GDNF in the basal compartment of the seminiferous tubules.
doi:10.1371/journal.pone.0028367
PMCID: PMC3235125  PMID: 22174794
17.  Imatinib has deleterious effects on differentiating spermatogonia while sparing spermatogonial stem cell self renewal 
Imatinib mesylate is among a growing number of effective cancer drugs that provide molecularly targeted therapy; however, imatinib causes reproductive defects in rodents. The availability of an in vitro system for screening the effect of drugs on spermatogenesis would be beneficial. The imatinib targets, KIT and platelet-derived growth factor receptor beta (PDGFRB), were shown here to be expressed in “germline stem” (GS) cell cultures that contain spermatogonia, including spermatogonial stem cells (SSCs). GS cell cultures were utilized to determine whether imatinib affects SSC self renewal or differentiation. GS cells grown in imatinib retained self renewal based on multiple assays, including transplantation. However, growth in imatinib led to decreased numbers of differentiated spermatogonia and reduced culture growth consistent with the known requirement for KIT in survival and proliferation of spermatogonia. These results build upon the in vivo studies and support the possibility of utilizing GS cell cultures for preclinical drug tests.
doi:10.1016/j.reprotox.2010.12.056
PMCID: PMC3103660  PMID: 21295132
Spermatogonial stem cells; self renewal; differentiation; imatinib mesylate; spermatogonia; fertility; drug test
18.  Isolation of undifferentiated and early differentiating Type A spermatogonia from Pou5f1-GFP reporter mice 
Limited understanding of the mechanisms underlying self-renewal and differentiation of spermatogonial stem cells hampers our ability to develop new therapeutic and contraceptive approaches. Mouse models of spermatogonial stem cell development are key to developing new insights into the biology of both the normal and diseased testis. Advances in transgenic reporter mice have enabled the isolation, molecular characterization, and functional analysis of mouse Type A spermatogonia subpopulations from the normal testis, including populations enriched for spermatogonial stem cells. Application of these reporters both to the normal testis and to gene-deficient and over-expression models will promote a better understanding of the earliest steps of spermatogenesis, and the role of spermatogonial stem cells in germ cell tumor.
doi:10.1007/978-1-61779-436-0_3
PMCID: PMC3314309  PMID: 22144234
Reporter mice; undifferentiated spermatogonia; Pou5f1; green fluorescent protein; fluorescence-activated cell sorting
19.  Cell-cycle-dependent Colonization of Mouse Spermatogonial Stem Cells After Transplantation into Seminiferous Tubules 
Abstract
Spermatogonial stem cells (SSCs) migrate to the niche upon introduction into the seminiferous tubules of the testis of infertile animals. However, only 5–10% of the transplanted cells colonize recipient testes. In this study, we analyzed the impact of cell cycle on spermatogonial transplantation. We used fluorescent ubiquitination-based cell cycle indicator transgenic mice to examine the influence of cell cycle on SSC activity of mouse germline stem (GS) cells, a population of cultured spermatogonia enriched for SSCs. GS cells in the G1 phase are more efficient than those in the S/G2-M phase in colonizing the seminiferous tubules of adult mice. Cells in the G1 phase not only showed higher expression levels of GFRA1, a component of the GDNF self-renewal factor receptor, but also adhered more efficiently to laminin-coated plates. Furthermore, this cell cycle-dependency was not observed when cells were transplanted into immature pup recipients, which do not have the blood-testis barrier (BTB) between Sertoli cells, suggesting that cells in the G1 phase may passage through the BTB more readily than cells in the S/G2-M phase. Thus cell cycle status is an important factor in regulating SSC migration to the niche.
doi:10.1262/jrd.2013-083
PMCID: PMC3958584  PMID: 24256919
Cell cycle; Sertoli cells; Spermatogenesis; Stem cells; Transplantation
20.  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
21.  Repression of kit Expression by Plzf in Germ Cells▿  
Molecular and Cellular Biology  2007;27(19):6770-6781.
Male mice lacking expression of Plzf, a DNA sequence-specific transcriptional repressor, show progressive germ cell depletion due to exhaustion of the spermatogonial stem cell population. This is likely due to the deregulated expression of genes controlling the switch between spermatogonial self-renewal and differentiation. Here we show that Plzf directly represses the transcription of kit, a hallmark of spermatogonial differentiation. Plzf represses both endogenous kit expression and expression of a reporter gene under the control of the kit promoter region. A discrete sequence of the kit promoter, required for Plzf-mediated kit transcriptional repression, is bound by Plzf both in vivo and in vitro. A 3-bp mutation in this Plzf binding site abolishes the responsiveness of the kit promoter to Plzf repression. A significant increase in kit expression is also found in the undifferentiated spermatogonia isolated from Plzf−/− mice. Thus, we suggest that one mechanism by which Plzf maintains the pool of spermatogonial stem cells is through a direct repression of kit expression.
doi:10.1128/MCB.00479-07
PMCID: PMC2099235  PMID: 17664282
22.  Undifferentiated Primate Spermatogonia and their Endocrine Control 
The biology of spermatogonial stem cells is currently an area of intensive research, and contemporary studies in primates are emerging. Quantitative regulation of sperm output by the primate testis appears to be exerted primarily on the transition from undifferentiated to differentiating spermatogonia. This review examines recent advances in our understanding of the mechanisms governing spermatogonial renewal and early differentiation in male primates, with a focus on the monkey. Emerging revisions to the classic view of dark and pale type A spermatogonia as reserve and renewing spermatogonial stem cells, respectively, are critically evaluated, and essential features of endocrine control of undifferentiated spermatogonia throughout postnatal primate development are discussed. Obstacles in gaining a more complete understanding of primate spermatogonia are also identified.
doi:10.1016/j.tem.2010.03.001
PMCID: PMC2896565  PMID: 20359909
23.  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
24.  Identification of Glial Cell Line-Derived Neurotrophic Factor-Regulated Genes Important for Spermatogonial Stem Cell Self-Renewal in the Rat1 
Biology of Reproduction  2009;81(1):56-66.
Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis throughout the life of a male. Because SSCs of many species can colonize the mouse testis, and glial cell line-derived neurotrophic factor (GDNF) is responsible for stimulating SSC self-renewal in rodents, we reasoned that molecular mechanisms of SSC self-renewal are similar across species. GDNF-regulated genes have been identified in mouse SSCs; however, downstream targets of GDNF are unknown in other species. The objective of this work was to identify GDNF-regulated genes in rat SSCs and to define the biological significance of these genes for rat SSC self-renewal. We conducted microarray analysis on cultured rat germ cells enriched for SSCs in the presence and absence of GDNF. Many GDNF-regulated genes were identified, most notably, Bcl6b and Etv5, which are important for mouse SSC self-renewal. Bcl6b was the most highly regulated gene in both the rat and mouse. Additionally, we identified three novel GDNF-regulated genes in rat SSCs: Bhlhe40, Hoxc4, and Tec. Small interfering RNA treatment for Bcl6b, Etv5, Bhlhe40, Hoxc4, and Tec resulted in a decrease in SSC number, as determined by transplantation, without a change in total cell number within the culture. These data indicate that, like in the mouse SSC, Bcl6b and Etv5 are important for rat SSC self-renewal, suggesting that these genes may be important for SSCs in all mammals. Furthermore, identification of three novel GDNF-regulated genes in the rat SSC extends our knowledge of SSC activity and broadens the foundation for understanding this process in higher species, including humans.
Bcl6b, Etv5, Bhlhb2, Hoxc4, and Tec are regulated by glial cell line-derived neurotrophic factor and important for spermatogonial stem cell self-renewal in the rat.
doi:10.1095/biolreprod.108.075358
PMCID: PMC3093986  PMID: 19339709
adult stem cells; germline; growth factors; microarray; rat; self-renewal
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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