MiRNA-20 and mirna-106a regulate spermatogonial stem cell renewal at the post-transcriptional level via targeting STAT3 and Ccnd1.

Studies on spermatogonial stem cells (SSCs) are of unusual significance because they are the unique stem cells that transmit genetic information to subsequent generations and they can acquire pluripotency to become embryonic stem-like cells that have therapeutic applications in human diseases. MicroRNAs (miRNAs) have recently emerged as critical endogenous regulators in mammalian cells. However, the function and mechanisms of individual miRNAs in regulating SSC fate remain unknown. Here, we report for the first time that miRNA-20 and miRNA-106a are preferentially expressed in mouse SSCs. Functional assays in vitro and in vivo using miRNA mimics and inhibitors reveal that miRNA-20 and miRNA-106a are essential for renewal of SSCs. We further demonstrate that these two miRNAs promote renewal at the post-transcriptional level via targeting STAT3 and Ccnd1 and that knockdown of STAT3, Fos, and Ccnd1 results in renewal of SSCs. This study thus provides novel insights into molecular mechanisms regulating renewal and differentiation of SSCs and may have important implications for regulating male reproduction.

Isolation, characterization, and culture of human spermatogonia.

This study was designed to isolate, characterize, and culture human spermatogonia. Using immunohistochemistry on tubule sections, we localized GPR125 to the plasma membrane of a subset of the spermatogonia. Immunohistochemistry also showed that MAGEA4 was expressed in all spermatogonia (A(dark), A(pale), and type B) and possibly preleptotene spermatocytes. Notably, KIT was expressed in late spermatocytes and round spermatids, but apparently not in human spermatogonia. UCHL1 was found in the cytoplasm of spermatogonia, whereas POU5F1 was not detected in any of the human germ cells. GFRA1 and ITGA6 were localized to the plasma membrane of the spermatogonia. Next, we isolated GPR125-positive spermatogonia from adult human testes using a two-step enzymatic digestion followed by magnetic-activated cell sorting. The isolated GPR125-positive cells coexpressed GPR125, ITGA6, THY1, and GFRA1, and they could be cultured for short periods of time and exhibited a marked increase in cell numbers as shown by a proliferation assay. Immunocytochemistry of putative stem cell genes after 2 wk in culture revealed that the cells were maintained in an undifferentiated state. MAPK1/3 phosphorylation was increased after 2 wk of culture of the GPR125-positive spermatogonia compared to the freshly isolated cells. Taken together, these results indicate that human spermatogonia share some but not all phenotypes with spermatogonial stem cells (SSCs) and progenitors from other species. GPR125-positive spermatogonia are phenotypically putative human SSCs and retain an undifferentiated status in vitro. This study provides novel insights into the molecular characteristics, isolation, and culture of human SSCs and/or progenitors and suggests that the MAPK1/3 pathway is involved in their proliferation.

Age affects gene expression in mouse spermatogonial stem/progenitor cells.

Spermatogenesis in man starts with spermatogonial stem cells (SSCs), and leads to the production of sperm in approximately 64 days, common to old and young men. Sperm from elderly men are functional and able to fertilize eggs and produce offspring, even though daily sperm production is more than 50% lower and damage to sperm DNA is significantly higher in older men than in those who are younger. Our hypothesis is that the SSC/spermatogonial progenitors themselves age. To test this hypothesis, we studied the gene expression profile of mouse SSC/progenitor cells at several ages using microarrays. After sequential enzyme dispersion, we purified the SSC/progenitors with immunomagnetic cell sorting using an antibody to GFRA1, a known SSC/progenitor cell marker. RNA was isolated and used for the in vitro synthesis of amplified and labeled cRNAs that were hybridized to the Affymetrix mouse genome microarrays. The experiments were repeated twice with different cell preparations, and statistically significant results are presented. Quantitative RT-PCR analysis was used to confirm the microarray results. Comparison of four age groups (6 days, 21 days, 60 days, and 8 months old) showed a number of genes that were expressed specifically in the older mice. Two of them (i.e. Icam1 and Selp) have also been shown to mark aging hematopoietic stem cells. On the other hand, the expression levels of the genes encoding the SSC markers Gfra1 and Plzf did not seem to be significantly altered by age, indicating that age affects only certain SSC/progenitor properties.

Nodal signaling via an autocrine pathway promotes proliferation of mouse spermatogonial stem/progenitor cells through Smad2/3 and Oct-4 activation.

Spermatogenesis is the process that involves the division and differentiation of spermatogonial stem cells into spermatozoa. However, the autocrine molecules and signaling pathways controlling their fate remain unknown. This study was designed to identify novel growth factors and signaling pathways that regulate proliferation, differentiation, and survival of spermatogonial stem/progenitor cells. To this end, we have for the first time explored the expression, function, and signaling pathway of Nodal, a member of the transforming growth factor-beta superfamily, in mouse spermatogonial stem/progenitor cells. We demonstrate that both Nodal and its receptors are present in these cells and in a spermatogonial stem/progenitor cell line (C18-4 cells), whereas Nodal is undetected in Sertoli cells or differentiated germ cells, as assayed by reverse transcription-polymerase chain reaction, Western blots, and immunocytochemistry. Nodal promotes proliferation of spermatogonial stem/progenitor cells and C18-4 cells, whereas Nodal receptor inhibitor SB431542 blocks their propagation as shown by proliferation and bromodeoxyuridine incorporation assays. Nodal knockdown by RNA interference results in a marked increase of cell apoptosis and a reduction of cell division as indicated by terminal deoxynucleotidyl transferase dUTP nick-end labeling and proliferation assays. Conversely, overexpression of Nodal leads to an increase of cell proliferation. Nodal activates Smad2/3 phosphorylation, Oct-4 transcription, cyclin D1, and cyclin E expression, whereas SB431542 completely abolishes their increase. Together, Nodal was identified as the first autocrine signaling molecule that promotes proliferation of mouse spermatogonial stem/progenitor cells via Smad2/3 and Oct-4 activation. This study thus provides novel and important insights into molecular mechanisms regulating proliferation and survival of spermatogonial stem/progenitor cells.

Effect of high intratesticular estrogen on global gene expression and testicular cell number in rats.

BACKGROUND: The identification of estrogen receptors alpha and beta and aromatase in the testis has highlighted the important role of estrogens in regulating spermatogenesis. There is a wealth of information on the deleterious effects of fetal and neonatal exposure of estrogens and xenoestrogens in the testis, including spermiation failure and germ cell apoptosis. However, very little is known about gene transcripts affected by exogenous estradiol exposure in the testis. The objective of the present study was to unveil global gene expression profiles and testicular cell number changes in rats after estradiol treatment. METHODS: 17beta-estradiol was administered to adult male rats at a dose of 100 micrograms/kg body weight in saline daily for 10 days; male rats receiving only saline were used as controls. Microarray analysis was performed to examine global gene expression profiles with or without estradiol treatment. Real time RT-PCR was conducted to verify the microarray data. In silico promoter and estrogen responsive elements (EREs) analysis was carried out for the differentially expressed genes in response to estradiol. Quantitation of testicular cell number based on ploidy was also performed using flow cytometry in rats with or without estradiol treatment. RESULTS: We found that 221 genes and expressed sequence tags (ESTs) were differentially expressed in rat testes treated with estradiol compared to the control; the microarray data were confirmed by real time RT-PCR. Gene Ontology analysis revealed that a number of the differentially expressed genes are involved in androgen and xenobiotic metabolism, maintenance of cell cytoskeleton, endocytosis, and germ cell apoptosis. A total of 33 up-regulated genes and 67 down-regulated genes showed the presence of EREs. Flow cytometry showed that estradiol induced a significant decrease in 2n cells (somatic and germ cells) and 4n cells (pachytene spermatocytes) and a marked increase in the number of elongated and elongating spermatids. CONCLUSIONS: This study provides a novel insight into the molecular basis for spermiation failure and apoptosis caused by 17beta-estradiol and it also offers new mechanisms by which adult exposure to environmental estrogens can affect spermatogenesis and fertility.

Spermatogonial stem cells: mouse and human comparisons.

Spermatogonial stem cells (SSCs) have unique characteristics in that they produce sperm that transmit genetic information from generation to generation and they can be reprogrammed spontaneously to form embryonic stem (ES)-like cells to acquire pluripotency. In rodents, it is generally believed that the A-single (A(s)) is the stem cell population, whereas the A-paired (A(pr)) and A-aligned (A(al)) represent the progenitor spermatogonial population. The A(1) to A(4) cells, intermediate, and type B spermatogonia are considered differentiated spermatogonia. In human, very little information is available about SSCs, except for the earlier work of Clermont and colleagues who demonstrated that there are two different types of A spermatogonia, the A(dark) and A(pale) spermatogonia. The A(dark) spermatogonia were referred to as the reserve stem cells, whereas the A(pale) were considered the renewing stem cells. In this review, we outline several spermatogonial renewal schemes for both rodents and primates, including man. We also compare phenotypic markers for spermatogonia/spermatogonial stem cells in rodents and humans and address SSC potential and therapeutic application.

Wnt signaling promotes proliferation and stemness regulation of spermatogonial stem/progenitor cells.

Spermatogonial stem cells (SSCs) self-renew throughout life to produce progenitor cells that are able to differentiate into spermatozoa. However, the mechanisms underlying the cell fate determination between self-renewal and differentiation have not yet been delineated. Culture conditions and growth factors essential for self-renewal and proliferation of mouse SSCs have been investigated, but no information is available related to growth factors that affect fate determination of human spermatogonia. Wnts form a large family of secreted glycoproteins, the members of which are involved in cell proliferation, differentiation, organogenesis, and cell migration. Here, we show that Wnts and their receptors Fzs are expressed in mouse spermatogonia and in the C18-4 SSC line. We demonstrate that WNT3A induces cell proliferation, morphological changes, and cell migration in C18-4 cells. Furthermore, we show that beta-catenin is activated during testis development in 21-day-old mice. In addition, our study demonstrates that WNT3A sustained adult human embryonic stem (ES)-like cells derived from human germ cells in an undifferentiated stage, expressing essential human ES cell transcription factors. These results demonstrate for the first time that Wnt/beta-catenin pathways, especially WNT3A, may play an important role in the regulation of mouse and human spermatogonia.

Small RNA molecules in the regulation of spermatogenesis.

Small RNA molecules (small RNAs), including small interfering RNAs (siRNAs), microRNAs (miRNAs), and piwi-interacting RNAs (piRNAs), have recently emerged as important regulators of gene expression at the post-transcriptional or translation level. Significant progress has recently been made utilizing small RNAs in elucidating the molecular mechanisms regulating spermatogenesis. Spermatogenesis is a complex process that involves the division and eventual differentiation of spermatogonial stem cells into mature spermatozoa. The process of spermatogenesis is composed of several phases: mitotic proliferation of spermatogonia to produce spermatocytes; two meiotic divisions of spermatocytes to generate haploid round spermatids; and spermiogenesis, the final phase that involves the maturation of early-round spermatids into elongated mature spermatids. A number of miRNAs are expressed abundantly in male germ cells throughout spermatogenesis, while piRNAs are only present in pachytene spermatocytes and round spermatids. In this review, we first address the synthesis, mechanisms of action, and functions of siRNA, miRNA, and piRNA, and then we focus on the recent advancements in defining the small RNAs in the regulation of spermatogenesis. Concerns pertaining to the use of siRNAs in exploring spermatogenesis mechanisms and open questions in miRNAs and piRNAs in this field are highlighted. The potential applications of small RNAs to male contraception and treatment for male infertility and testicular cancer are also discussed.

Gdnf upregulates c-Fos transcription via the Ras/Erk1/2 pathway to promote mouse spermatogonial stem cell proliferation.

Glial cell line-derived neurotrophic factor (GDNF) plays a crucial role in regulating the proliferation of spermatogonial stem cells (SSC). The signaling pathways mediating the function of GDNF in SSC remain unclear. This study was designed to determine whether GDNF signals via the Ras/ERK1/2 pathway in the C18-4 cells, a mouse SSC line. The identity of this cell line was confirmed by the expression of various markers for germ cells, proliferating spermatogonia, and SSC, including GCNA1, Vasa, Dazl, PCNA, Oct-4, GFRalpha1, Ret, and Plzf. Western blot analysis revealed that GDNF activated Ret tyrosine phosphorylation. All 3 isoforms of Shc were phosphorylated upon GDNF stimulation, and GDNF induced the binding of the phosphorylated Ret to Shc and Grb2 as indicated by immunoprecipitation and Western blotting. The active Ras was induced by GDNF, which further activated ERK1/2 phosphorylation. GDNF stimulated the phosphorylation of CREB-1, ATF-1, and CREM-1, and c-fos transcription. Notably, the increase in ERK1/2 phosphorylation, c-fos transcription, bromodeoxyuridine incorporation, and metaphase counts induced by GDNF, was completely blocked by pretreatment with PD98059, a specific inhibitor for MEK1, the upstream regulator of ERK1/2. GDNF stimulation eventually upregulated cyclin A and CDK2 expression. Together, these data suggest that GDNF induces CREB/ATF-1 family member phosphorylation and c-fos transcription via the Ras/ERK1/2 pathway to promote the proliferation of SSC. Unveiling GDNF signaling cascades in SSC has important implications in providing attractive targets for male contraception as well as for the regulation of stem cell renewal vs. differentiation.

Expression profiling of purified male germ cells: stage-specific expression patterns related to meiosis and postmeiotic development.

Gene expression profiling was performed using the National Institute on Aging 15,000-cDNA microarray to reveal the differential expression pattern of 160 genes between meiotic pachytene spermatocytes and postmeiotic round spermatids of the mouse. Our results indicate that more genes are expressed in spermatids than in spermatocytes. Genes participating in cell cycle regulation and chromatin structure and dynamics are preferentially expressed in spermatocytes, while genes for protein turnover, signal transduction, energy metabolism, and intracellular transport are prevalent in spermatids. This suggests that a switch of functional requirement occurs when meiotic germ cells differentiate into haploid spermatids. Concordant expression patterns were obtained when quantitative real-time polymerase chain reaction was performed to verify the microarray data. Interestingly, the majority of the differentially expressed genes were underrepresented in mitotic type A spermatogonia, and they were preferentially expressed in the testis. Our results suggest that an even higher proportion of the mouse genome is devoted to male gamete development from meiosis than was previously estimated. We also provide evidence that underscores the advantage of using purified germ cells over whole testes in profiling spermatogenic gene expression to identify transcripts that demonstrate stage-specific expression patterns.

Structural characterization and expression studies of Dby and its homologs in the mouse.

In spite of recent evidence showing the importance of DBY (DEAD-box RNA helicase Y) in spermatogenesis in human, the biologic role of its homolog Dby (also known as Ddx3y) in the mouse is less clear. The present study aims at characterizing the molecular structure of Dby and comparing its expression with its X- and autosome-linked homologs in embryonic gonads and developing germ cells in mice. Molecular cloning by rapid amplification of 3'-cDNA ends showed that the Dby gene in the mouse gives rise to 2 transcripts that differ only in the length of the 3'-untranslated region as a consequence of the use of alternative polyadenylation signals. Measurement by quantitative real-time polymerase chain reaction showed that both transcripts were ubiquitously expressed and were present in male germ cells and Sertoli cells. They were more abundant in type A spermatogonia compared with pachytene spermatocytes and round spermatids. Expression of Dby in the embryonic gonad increased from day 10.5 and reached a peak at day 17.5. The expression level of Dby decreased after birth and remained low in adult male gonads. Although the level of expression of Dby was much lower than its X chromosome homolog, Ddx3 (also known as Ddx3x) in all samples examined, the pattern of expression of the 2 genes was comparable. In contrast, their autosomal homolog, D1Pas1(also known as PL10), was predominantly expressed in pachytene spermatocytes and round spermatids. This result is in accord with meiotic sex chromosome inactivation in that Dby and Ddx are replaced in pachytene spermatocytes by their autosomal retroposon. These observations indicate that unlike DBY in humans, the role of Dby in spermatogenesis is less obvious in the mouse and its biologic activity may be replaced by that of Ddx3 and D1Pas1.

Role of glial cell line-derived neurotrophic factor in germ-line stem cell fate.

The overall goal of this study is to unravel the role(s) played by glial cell line-derived neurotrophic factor (GDNF) in the fate of spermatogonial stem cells. There is great interest in the biology of spermatogonial stem cells, or A(single) spermatogonia, because of their importance in the treatment of infertility, the development of contraceptives, and the understanding of the etiology of testicular cancer, particularly seminoma. In the mouse, spermatogonial stem cells express GFRalpha-1, the receptor for GDNF, and respond to this growth factor in vivo and in vitro. GDNF is produced by the adjacent Sertoli cells, which are part of the germ-line stem cell niche in vertebrates. We specifically isolated GFRalpha-1-positive spermatogonia using an immunomagnetic bead technique. We then stimulated the cells with 100 ng/mL of rGDNF for 10 hours; unstimulated cells served as negative controls. Microarray analysis, immunocytochemistry, and Western blotting revealed that Numb, a regulator of the Notch pathway, is upregulated by GDNF in spermatogonial stem cells. There are indications that in rats, mice, and humans, the Notch pathway promotes spermatogonial differentiation. We observed that an increase in Numb expression is concomitant with Notch degradation in these cells. Thus, through Numb, GDNF might inhibit differentiation and allows the maintenance of the stem cell pool in the mouse seminiferous epithelium.

Expression of testicular germ cell genes identified by differential display analysis.

Using differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) we identified transcripts encoding for the RNA helicase mDEAH9, Ran binding protein 5 (RanBP5), and 3 novel complementary DNAs designated GC3, GC12, and GC14 in developing testicular germ cells. Sources of RNA for the initial DDRT-PCR screen were purified mouse type A spermatogonia, adult mouse wild-type testis, and W/W(v) mutant mouse testis. We identified cDNA fragments for mDEAH9, RanBP5, GC3, GC12, and GC14 in testis and type A spermatogonia samples from wild-type mice, but not in samples from the W/W(v) mouse testis. These same transcripts were absent in Northern blots of testis RNA from mice treated with busulfan 30 days prior, but were present in testis RNA from wild-type mice at 5, 15, 25, and 40 days of age. The mDEAH9 gene was expressed in many tissues, whereas RanBP5 and GC12 genes were expressed predominantly in the testis with much lower expression in other tissues. The expression of GC3 and GC14 were limited to the testis as evidenced by Northern blot and RT-PCR analyses. The mDEAH9 transcript was not detected in cultured interstitial cells but was found at low levels in cultured immature Sertoli cells, whereas the RanBP5, GC3, GC12, and GC14 transcripts were not detected in either cultured testicular interstitial cells or cultured Sertoli cells. RT-PCR analyses of isolated spermatogonia, pachytene spermatocytes, and round spermatids revealed that mDEAH9, RanBP5, GC3, GC12, and GC14 genes were expressed in all 3 cellular populations. In situ hybridization analyses of testis samples from 40-day-old mice localized expression of mDEAH9, RanBP5, GC3, GC12, and GC14 to the seminiferous tubules. RanBP5 expression appeared to be regulated during the cycle of the seminiferous epithelium, with the highest expression in stages III through VII. Expression of GC14 was greatest in the meiotic germ cell populations.

Establishment and characterization of neonatal mouse sertoli cell lines.

Sertoli cells isolated from 6-day postpartum mouse testes were conditionally immortalized with the simian virus 40 large tumor antigen gene (SV40-LTAg) under the control of a promoter inducible with ponasterone A, an analog of ecdysone. This strategy produced 2 cell lines, which exhibited mixed phenotypes. We first tested the conditional expression of the LTAg gene in the presence or absence of ponasterone A. The results showed that both cell lines expressed LTAg when the inducer was present in the culture media. When ponasterone A was removed, the majority of the cells died. After 60 generations, however, the continued expression of LTAg in the absence of the hormone indicated that unknown changes may have occurred in the genome of the cells. One of the cell lines was further subcloned, resulting in 7 new lines exhibiting a morphology resembling that of Sertoli cells in tissue culture. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on RNA collected from each cell line in order to determine which cells were phenotypically similar to Sertoli cells in vivo. All cell lines expressed the products of the Sertoli cell-specific genes stem cell factor (SCF) and sulfated glycoprotein-2 (SGP-2), in addition to alpha-inhibin, GATA-1, and steroidogenic factor-1. Further, the lines express growth and differentiation factors known to act upon germ cells in vivo and in vitro such as leukemia inhibitory factor (LIF), transforming growth factor beta (TGF-beta), and basic fibroblast growth factor (bFGF). Moreover, when used as feeder layers in cocultures, at least 2 of these lines are able to maintain the viability of type A spermatogonia for at least 7 days and to support the first steps of spermatogonial differentiation.

Identification of differentially expressed genes in mouse spermatogenesis.

Complementary DNA microarray and quantitative polymerase chain reaction were used as tools for discovering genes that are differentially expressed in the mouse under normal physiological conditions at distinctive stages of male germ cell development, that is, type A spermatogonia, pachytene spermatocytes, and round spermatids. By using this strategy, we identified a set of genes exhibiting differential expression patterns in spermatogenesis, suggesting that specific functions of the encoded products occurred during the developmental process. Among them were several genes previously not known to be active in testis, which signified undiscovered functional roles of these genes during spermatogenesis. Many of the genes identified were not previously characterized. This study highlights new targets for manipulation to unravel the molecular mechanism of spermatogenesis.

Cellular source and mechanisms of high transcriptome complexity in the mammalian testis.

Understanding the extent of genomic transcription and its functional relevance is a central goal in genomics research. However, detailed genome-wide investigations of transcriptome complexity in major mammalian organs have been scarce. Here, using extensive RNA-seq data, we show that transcription of the genome is substantially more widespread in the testis than in other organs across representative mammals. Furthermore, we reveal that meiotic spermatocytes and especially postmeiotic round spermatids have remarkably diverse transcriptomes, which explains the high transcriptome complexity of the testis as a whole. The widespread transcriptional activity in spermatocytes and spermatids encompasses protein-coding and long noncoding RNA genes but also poorly conserves intergenic sequences, suggesting that it may not be of immediate functional relevance. Rather, our analyses of genome-wide epigenetic data suggest that this prevalent transcription, which most likely promoted the birth of new genes during evolution, is facilitated by an overall permissive chromatin in these germ cells that results from extensive chromatin remodeling.

Trial of recombinant follicle-stimulating hormone pretreatment for GnRH-induced fertility in patients with congenital hypogonadotropic hypogonadism.

CONTEXT AND OBJECTIVE: The optimal strategy for inducing fertility in men with congenital hypogonadotropic hypogonadism (CHH) is equivocal. Albeit a biologically plausible approach, pretreatment with recombinant FSH (rFSH) before GnRH/human chorionic gonadotropin administration has not been sufficiently assessed. The objective of the study was to test this method. DESIGN AND SETTING: This was a randomized, open-label treatment protocol at an academic medical center. PATIENTS AND INTERVENTIONS: GnRH-deficient men (CHH) with prepubertal testes (<4 mL), no cryptorchidism, and no prior gonadotropin therapy were randomly assigned to either 24 months of pulsatile GnRH therapy alone (inducing endogenous LH and FSH release) or 4 months of rFSH pretreatment followed by 24 months of GnRH therapy. Patients underwent serial testicular biopsies, ultrasound assessments of testicular volume, serum hormone measurements, and seminal fluid analyses. RESULTS: rFSH treatment increased inhibin B levels into the normal range (from 29 ± 9 to 107 ± 41 pg/mL, P < .05) and doubled testicular volume (from 1.1 ± 0.2 to 2.2 ± 0.3 mL, P < .005). Histological analysis showed proliferation of both Sertoli cells (SCs) and spermatogonia, a decreased SC to germ cell ratio (from 0.74 to 0.35), and SC cytoskeletal rearrangements. With pulsatile GnRH, the groups had similar hormonal responses and exhibited significant testicular growth. All men receiving rFSH pretreatment developed sperm in their ejaculate (7 of 7 vs 4 of 6 in the GnRH-only group) and showed trends toward higher maximal sperm counts. CONCLUSIONS: rFSH pretreatment followed by GnRH is successful in inducing testicular growth and fertility in men with CHH with prepubertal testes. rFSH not only appears to maximize the SC population but also induces morphologic changes, suggesting broader developmental roles.

Isolation of human male germ-line stem cells using enzymatic digestion and magnetic-activated cell sorting.

Mammalian spermatogenesis is a process whereby male germ-line stem cells (spermatogonial stem cells) divide and differentiate into sperm. Although a great deal of progress has been made in the isolation and characterization of spermatogonial stem cells (SSCs) in rodents, little is known about human SSCs. We have recently isolated human G protein-coupled receptor 125 (GPR125)-positive spermatogonia and GDNF family receptor alpha 1 (GFRA1)-positive spermatogonia using a 2-step enzymatic digestion and magnetic-activated cell sorting (MACS) from adult human testes. Cell purities of isolated human GPR125- and GFRA1-positive spermatogonia after MACS are greater than 95%, and cell viability is over 96%. The isolated GPR125- and GFRA1-positive spermatogonia coexpress GPR125, integrin, alpha 6 (ITGA6), THY1 (also known as CD90), GFRA1, and ubiquitin carboxyl-terminal esterase L1 (UCHL1), markers for rodent or pig SSCs/progenitors, suggesting that GPR125- and GFRA1-positive spermatogonia are phenotypically the SSCs in human testis. Human GPR125-positive spermatogonia can be cultured for 2 weeks with a remarkable increase in cell number. Immunocytochemistry further reveals that GPR125-positive spermatogonia can be maintained in an undifferentiated state in vitro. Collectively, the methods using enzymatic digestion and MACS can efficiently isolate and purify SSCs from adult human testis with consistent and high quality. The ability of isolating and characterizing human SSCs could provide a population of stem cells with high purity for mechanistic studies on human SSC self-renewal and differentiation as well as potential applications of human SSCs in regenerative medicine.

Oocyte-like cells induced from mouse spermatogonial stem cells.

BACKGROUND: During normal development primordial germ cells (PGCs) derived from the epiblast are the precursors of spermatogonia and oogonia. In culture, PGCs can be induced to dedifferentiate to pluripotent embryonic germ (EG) cells in the presence of various growth factors. Several recent studies have now demonstrated that spermatogonial stem cells (SSCs) can also revert back to pluripotency as embryonic stem (ES)-like cells under certain culture conditions. However, the potential dedifferentiation of SSCs into PGCs or the potential generation of oocytes from SSCs has not been demonstrated before. RESULTS: We report that mouse male SSCs can be converted into oocyte-like cells in culture. These SSCs-derived oocytes (SSC-Oocs) were similar in size to normal mouse mature oocytes. They expressed oocyte-specific markers and gave rise to embryos through parthenogenesis. Interestingly, the Y- and X-linked testis-specific genes in these SSC-Oocs were significantly down-regulated or turned off, while oocyte-specific X-linked genes were activated. The gene expression profile appeared to switch to that of the oocyte across the X chromosome. Furthermore, these oocyte-like cells lost paternal imprinting but acquired maternal imprinting. CONCLUSIONS: Our data demonstrate that SSCs might maintain the potential to be reprogrammed into oocytes with corresponding epigenetic reversals. This study provides not only further evidence for the remarkable plasticity of SSCs but also a potential system for dissecting molecular and epigenetic regulations in germ cell fate determination and imprinting establishment during gametogenesis.

Signaling molecules and pathways regulating the fate of spermatogonial stem cells.

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.