Spermatogonial Stem Cells Activity Research Articles

Dynamic Changes in EPCAM Expression during Spermatogonial Stem Cell Differentiation in the Mouse Testis

BackgroundSpermatogonial 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 FindingsBy 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/SignificanceThese 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.

Wnt5a is a cell-extrinsic factor that supports self-renewal of mouse spermatogonial stem cells

The maintenance of spermatogonial stem cells (SSCs) provides the foundation for life-long spermatogenesis. Although glial-cell-line-derived neurotrophic factor and fibroblast growth factor 2 are crucial for self-renewal of SSCs, recent studies have suggested that other growth factors have important roles in controlling SSC fate. Because β-catenin-dependent Wnt signaling promotes self-renewal of various stem cell types, we hypothesized that this pathway contributes to SSC maintenance. Using transgenic reporter mice for β-catenin-dependent signaling, we found that this signaling was not active in SSCs in vitro and in most spermatogonia in vivo. Nonetheless, a pan-Wnt antagonist significantly reduced SSC activity in vitro, suggesting that some Wnt molecules exist in our serum-free culture system and contribute to SSC maintenance. Here, we report that Wnt5a promotes SSC activity. We found that Wnt5a-expressing fibroblasts supported SSC activity better than those not expressing Wnt5a in culture, and that recombinant Wnt5a stimulated SSC maintenance. Furthermore, Wnt5a promoted SSC survival in the absence of feeder cells, and this effect was abolished by inhibiting the Jun N-terminal kinase cascade. In addition, Wnt5a blocked β-catenin-dependent signaling. We detected the expression of Wnt5a and potential Wnt5a receptors in Sertoli cells and stem/progenitor spermatogonia, respectively. These results indicate that Wnt5a is a cell-extrinsic factor that supports SSC self-renewal through β-catenin-independent mechanisms.

The Yin and Yang of WNT4 Signaling in the Adult Gonad.

The WNTs are a family of secreted glycoprotein signaling molecules best known for the multiple roles that they play during embryonic development. WNT signaling first attracted the attention of the reproductive biology research community a little over a decade ago, when a groundbreaking study by Vainio et al. demonstrated that the ablation of Wnt4 in female mice causes partial sex-reversal and oocyte loss. More recently, the regulated expression of several WNTs and WNT signaling effectors has been shown to occur in adult male and female gonads. For instance, in mice, WNT4 expression persists in ovarian granulosa cells after birth, and is markedly induced during ovulation/luteinization, strongly suggesting a role for WNT4 in adult ovarian physiology. To define the function(s) of WNT4 in postnatal granulosa cells, we used a conditional gene targeting approach. We showed that Wnt4flox/-;Amhr2tm3(cre)Bhr/+ mice had reduced ovary weights, produced smaller litters, and had only a quarter of the normal number of healthy antral follicles at puberty. RTPCR analyses of Wnt4flox/-;Amhr2tm3(cre)Bhr/+ granulosa cells and cultured granulosa cells that overexpress WNT4 demonstrated that WNT4 regulates the expression of Star, Cyp11a1 and Cyp19, steroidogenic genes previously identified as downstream targets of the WNT signaling effector CTNNB1 (β-catenin). Decreased serum progesterone levels were found in immature, gonadotropin-treated Wnt4flox/-;Amhr2tm3(cre)Bhr/+ mice. Together, these data indicated that WNT4 is required for normal antral follicle development, and may act by regulating granulosa cell functions including steroidogenesis. In male mice, ourselves and others have shown that constitutive activation of CTNNB1 in the Sertoli cells of the Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ model results in progressive germ cell loss and sterility. More recently, we sought to determine if the latter phenotype could be due to a loss of spermatogonial stem cell (SSC) activity. Reciprocal SSC transplants between Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ and wild-type mice showed that Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ testes lose SSC activity over time, and cannot support colonization by donor SSCs. Microarray analyses showed that CTNNB1 induces the expression of genes associated with the female sex determination pathway in Sertoli cells, including Wnt4. We therefore tested the effects of exogenous WNT4 on SSC-enriched germ cell cultures, and found that WNT4 reduced germ cell numbers due to increased apoptosis and reduced SSC capacity to colonize recipient testes. Conversely, conditional inactivation of Wnt4 in the Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ model rescued spermatogenesis and male fertility, indicating that WNT4 is the major effector downstream of CTNNB1 responsible for germ cell loss. Taken together, our data indicate diametrically opposed functions for WNT4/CTNNB1 signaling in the adult female and male gonad: a powerful positive regulator of follicle development on the one hand, an equally powerful negative regulator of spermatogenesis on the other. (Supported by the Canadian Institutes of Health Research and the National Sciences and Engineering Research Council of Canada). (platform)

Wnt5a is a cell-extrinsic factor that supports self-renewal of mouse spermatogonial stem cells

The maintenance of spermatogonial stem cells (SSCs) provides the foundation for life-long spermatogenesis. Although glial-cell-line-derived neurotrophic factor and fibroblast growth factor 2 are crucial for self-renewal of SSCs, recent studies have suggested that other growth factors have important roles in controlling SSC fate. Because β-catenin-dependent Wnt signaling promotes self-renewal of various stem cell types, we hypothesized that this pathway contributes to SSC maintenance. Using transgenic reporter mice for β-catenin-dependent signaling, we found that this signaling was not active in SSCs in vitro and in most spermatogonia in vivo. Nonetheless, a pan-Wnt antagonist significantly reduced SSC activity in vitro, suggesting that some Wnt molecules exist in our serum-free culture system and contribute to SSC maintenance. Here, we report that Wnt5a promotes SSC activity. We found that Wnt5a-expressing fibroblasts supported SSC activity better than those not expressing Wnt5a in culture, and that recombinant Wnt5a stimulated SSC maintenance. Furthermore, Wnt5a promoted SSC survival in the absence of feeder cells, and this effect was abolished by inhibiting the Jun N-terminal kinase cascade. In addition, Wnt5a blocked β-catenin-dependent signaling. We detected the expression of Wnt5a and potential Wnt5a receptors in Sertoli cells and stem/progenitor spermatogonia, respectively. These results indicate that Wnt5a is a cell-extrinsic factor that supports SSC self-renewal through β-catenin-independent mechanisms.

Homologous Recombination in Rat Germline Stem Cells1

Spermatogonial stem cells (SSCs) are the only stem cells in the body with germline potential, which makes them an attractive target for germline modification. We previously showed the feasibility of homologous recombination in mouse SSCs and produced knockout (KO) mice by exploiting germline stem (GS) cells, i.e., cultured spermatogonia with SSC activity. In this study, we report the successful homologous recombination in rat GS cells, which can be readily established by their ability to form germ cell colonies on culture plates whose surfaces are hydrophilic and neutrally charged and thus limit somatic cell binding. We established a drug selection protocol for GS cells under hypoxic conditions. The frequency of the homologous recombination of the Ocln gene was 4.2% (2 out of 48 clones). However, these GS cell lines failed to produce offspring following xenogeneic transplantation into mouse testes and microinsemination, suggesting that long-term culture and drug selection have a negative effect on GS cells. Nevertheless, our results demonstrate the feasibility of gene targeting in rat GS cells and pave the way toward the generation of KO rats.

Effects of Chemotherapeutic Agents for Testicular Cancer on Rat Spermatogonial Stem/Progenitor Cells

Spermatogonial stem cells (SSCs) are responsible for the production of spermatozoa throughout adulthood and for the recovery of spermatogenesis following exposure to cytotoxic agents. Previously, we have shown that the combined administration of bleomycin, etoposide, and cisplatin (BEP) used in the treatment of testicular cancer causes impaired spermatogenesis and reduced sperm production in the rat. However, definitive evidence about the potential impact of such chemotherapy on SSCs is still lacking. The objective of this study was to determine whether chronic exposure to BEP treatment causes adverse effects on rat SSC activity. We first investigated the effects of BEP treatment on the clonal organization of undifferentiated spermatogonia by staining whole-mount preparations of rat seminiferous tubules for GFRA1 and ZBTB16 (previously known as PLZF), 2 established markers of undifferentiated spermatogonia. We found that BEP treatment drastically reduced the number of A-aligned spermatogonia while sparing A-single and A-paired cells from the effect. Next, we determined the SSC activity following BEP exposure. Adult transgenic rats carrying EGFP expression in the germ line were treated with BEP for 9 weeks, and SSCs were quantified using spermatogonial transplantation. We found that BEP treatment significantly decreased SSC numbers, which were restored to the control level after a 9-week recovery period. These results demonstrate that BEP treatment transiently affects the activity of rat SSCs.

Sertoli Cells Dictate Spermatogonial Stem Cell Niches in the Mouse Testis

Sustained spermatogenesis in adult males relies on the activity of spermatogonial stem cells (SSCs). In general, tissue-specific stem cell populations such as SSCs are influenced by contributions of support cells that form niche microenvironments. Previous studies have provided indirect evidence that several somatic cell populations and the interstitial vasculature influence SSC functions, but an individual orchestrator of niches has not been described. In this study, functional transplantation of SSCs, in combination with experimental alteration of Sertoli cell content by polythiouracil (PTU)-induced transient hypothyroidism, was used to explore the relationship of Sertoli cells with SSCs in testes of adult mice. Transplantation of SSCs from PTU-treated donor mice into seminiferous tubules of normal recipient mice revealed a greater than 3-fold increase in SSCs compared to those from testes of non-PTU-treated donors. In addition, use of PTU-treated mice as recipients for transplantation of SSCs from normal donors revealed a greater than 3-fold increase of accessible niches compared to those of testes of non-PTU treated recipient mice with normal numbers of Sertoli cells. Importantly, the area of seminiferous tubules bordered by interstitial tissue and percentage of seminiferous tubules associated with blood vessels was found to be no different in testes of PTU-treated mice compared to controls, indicating that neither the vasculature nor interstitial support cell populations influenced the alteration of niche number. Collectively, these results provide direct evidence that Sertoli cells are the key somatic cell population dictating the number of SSCs and niches in mammalian testes.

Techniques for Culturing Spermatogonial Stem Cells Continue to Improve

Techniques for Culturing Spermatogonial Stem Cells Continue to Improve

Fluorescence-activated cell sorting may allow separation of malignant leukemic cells from a testicular cell suspension

OBJECTIVE: Spermatogonial stem cell (SSCs) may have clinical application for treating male infertility (e.g., secondary to cancer treatment). However, with certain hematologic malignancies, there is concern about reintroducing malignant cells during transplantation. The objective of this study is to examine the feasibility of separating leukemic cells from a suspension of human testicular cells via fluorescence-activated cell sorting (FACS). DESIGN: Basic science research. MATERIALS AND METHODS: The expression of 22 cell surface markers was analyzed via flow cytometry on human testicular cell suspensions as well as on three different human leukemic cell lines. Human testis cells were sorted by FACS based on expression of selected markers and the resulting fractions were stained with SALL-4, a marker of stem and progenitor spermatogonia. In this manner, markers of interest were identified if 1) they did not stain malignant cells but did stain the population of SALL-4 positive cells, or 2) they stained all of the malignant cells but not the SALL-4 positive cells. RESULTS: FACs analyses indicate that SALL-4+ cells were in the Ep-CAM+ fraction of human testis cells. Ep-CAM was not expressed on the MOLT-4 T cell leukemia cell line. In contrast, HLA-ABC and CD49e were highly expressed on the MOLT-4 cell line, but were not expressed by SALL4+ human testis cells. CONCLUSION: SALL4+ human spermatogonia (Ep-CAM+, HLA-ABC-, CD49e-) and MOLT-4 leukemia cells (Ep-CAM-, HLA-ABC+, CD49e+) have distinct cell surface characteristics that might be exploited to remove malignant contaminants from potentially therapeutic SSCs. FACS experiments utilizing these cell surface markers to fractionate MOLT-4 spiked human testis cell suspensions are ongoing. We are awaiting transplant results to determine the relative SSC activity and malignant potential of each fraction.

Regulation of Mouse Spermatogonial Stem Cell Differentiation by STAT3 Signaling1

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.

Development of a Short-Term Fluorescence-Based Assay to Assess the Toxicity of Anticancer Drugs on Rat Stem/Progenitor Spermatogonia In Vitro1

In vitro culture of rodent spermatogonial stem cells (SSCs) has become an important asset in the study of mammalian SSC biology. Supported by added growth factors, SSCs divide in culture and form aggregates of stem/progenitor spermatogonia, termed clusters. Recent studies have shown that serial passaging of clusters results in long-term maintenance and amplification of the SSC pool and that this culture system can also be used for short-term semiquantification of SSC activity. Here, we report the development of an automated assay to assess the activity of rat stem/progenitor spermatogonia in vitro and its application for investigating the cytotoxicity of chemotherapeutic drugs on these cells. Cultures of EGFP-expressing rat spermatogenic cells allowed us to determine the number and two-dimensional surface area of clusters using an automated fluorescence imaging system, thereby providing quantitative data of SSC activity. Using this assay, we examined the germ cell toxicity of three drugs that are routinely used in testicular cancer therapy, namely, bleomycin, cisplatin, and etoposide, alone and in combination. All three drugs showed a significant and dose-dependent reduction of cluster number and surface area, indicating their adverse effects specific to spermatogonia. The inhibitory concentration at which cluster number and surface area are inhibited by 50% (IC(50)) was the lowest with etoposide and the highest with cisplatin, implying that etoposide was most toxic to spermatogonia in vitro. These results suggest that the SSC culture should provide an effective and efficient system to assess the germ cell toxicity of various drugs and chemical compounds.

Spermatogenesis After 1-Stage Fowler-Stephens Orchiopexy in Experimental Cryptorchid Rat Model

To evaluate the validity of 1-stage Fowler-Stephens orchiopexy, we performed this procedure for intra-abdominal testes in an experimental cryptorchid rat model and assessed postoperative spermatogenesis. Cryptorchidism in rats was induced by injecting flutamide into the abdomen of pregnant Sprague Dawley rats for 7 days (days 14 to 20 of gestation). Four-week-old cryptorchid rats were divided into the 4 groups of sham operation (group 1, 7 rats), orchiopexy only (group 2, 7), spermatic vessel ligation only (group 3, 7), and spermatic vessel ligation and orchiopexy (1-stage Fowler-Stephens orchiopexy, group 4, 7). The testes were removed 12 weeks after birth in all groups, and testicular weight, testicular histopathological findings and presence of sperm in the epididymis were examined. All operated testes had reticulated vessels on the tunica albuginea. There were no statistically significant differences in the testicular weight between groups 1 (mean +/- SD 0.47 +/- 0.04 gm) and 4 (0.30 +/- 0.19), suggesting that Fowler-Stephens orchiopexy did not induce significant testicular atrophy. However, seminiferous tubule diameters in group 4 were significantly smaller than in group 1 (p <0.001), and no sperm was observed in the epididymis of group 4 rats, suggesting that Fowler-Stephens orchiopexy reduced seminiferous tubule diameters and did not improve spermatogenesis. Although Fowler-Stephens orchiopexy is a good procedure to maintain testicular size and correct cosmetic deformity of an absent scrotum, it may not significantly contribute to the improvement of spermatogenesis.

Phenotypic Plasticity of Mouse Spermatogonial Stem Cells

BackgroundSpermatogonial stem cells (SSCs) continuously undergo self-renewal division to support spermatogenesis. SSCs are thought to have a fixed phenotype, and development of a germ cell transplantation technique facilitated their characterization and prospective isolation in a deterministic manner; however, our in vitro SSC culture experiments indicated heterogeneity of cultured cells and suggested that they might not follow deterministic fate commitment in vitro.Methodology and Principal FindingsIn this study, we report phenotypic plasticity of SSCs. Although c-kit tyrosine kinase receptor (Kit) is not expressed in SSCs in vivo, it was upregulated when SSCs were cultured on laminin in vitro. Both Kit− and Kit+ cells in culture showed comparable levels of SSC activity after germ cell transplantation. Unlike differentiating spermatogonia that depend on Kit for survival and proliferation, Kit expressed on SSCs did not play any role in SSC self-renewal. Moreover, Kit expression on SSCs changed dynamically once proliferation began after germ cell transplantation in vivo.Conclusions/SignificanceThese results indicate that SSCs can change their phenotype according to their microenvironment and stochastically express Kit. Our results also suggest that activated and non-activated SSCs show distinct phenotypes.

Identification of Glial Cell Line-Derived Neurotrophic Factor-Regulated Genes Important for Spermatogonial Stem Cell Self-Renewal in the Rat1

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.

Spermatogonial stem cells: characteristics and experimental possibilities

The continuation of the spermatogenic process throughout life relies on a proper regulation of self-renewal and differentiation of the spermatogonial stem cells. These are single cells situated on the basal membrane of the seminiferous epithelium. Only 0.03% of all germ cells are spermatogonial stem cells. They are the only cell type that can repopulate and restore fertility to congenitally infertile recipient mice following transplantation. Although numerous expression markers have been helpful in isolating and enriching spermatogonial stem cells, such as expression of THY-1 and GFRalpha-1 and absence of c-kit, no specific marker for this cell type has yet been identified. Much effort has been put into developing a protocol for the maintenance of spermatogonial cells in vitro. Recently, coculture systems of testicular cells on various feeder cells have made it possible to culture spermatogonial stem cells for a long period of time, as was demonstrated by the transplantation assay. Even expansion of testicular cells, including the spermatogonial stem cells, has been achieved. In these culture systems, hormones and growth factors are investigated for their role in the process of proliferation of spermatogonial stem cells. At the moment the best culture system known still consists of a mixture of testicular cells with about 1.33% spermatogonial stem cells. Recently pure SV40 large T immortalized spermatogonial stem cell lines have been established. These c-kit-negative cell lines did not show any differentiation in vitro or in vivo. A telomerase immortalized c-kit-positive spermatogonial cell line has been established that was able to differentiate in vitro. Spermatocytes and even spermatids were formed. However, spermatogonial stem cell activity by means of the transplantation assay was not tested for this cell line. Both the primary long-term cultures and immortalized cell lines have represented a major step forward in investigating the regulation of spermatogonial self-renewal and differentiation, and will be useful for identifying specific molecular markers.

Aging of Male Germ Line Stem Cells in Mice1

In the present study, we investigated the effect of aging on spermatogonial stem cells (SSCs) and on the testicular somatic environment in ROSA26 mice. First, we examined testis weights at 2 mo, 6 mo, 1 yr, and 2 yr of age. At 1 and 2 yr, bilateral atrophied testes were observed in 50% and 75% of the mice, respectively; the rest of the mice had testis weights similar to those of young mice. Next, we evaluated the number and the activity of aged SSCs using spermatogonial transplantation. Numbers of SSCs in atrophied testes decreased in an age-dependent manner to as low as 1/60 of those in testes of young mice. Numbers of SSCs in nonregressed testes were similar regardless of age. The colony length, which is indicative of the potential of SSCs to regenerate spermatogenesis, was similar with donor cells from atrophied testes of 1-yr-old mice and those from testes of young mice, suggesting that SSCs remaining in 1-yr atrophied testes were functionally intact. Colonies arising from SSCs derived from 2-yr atrophied testes were significantly shorter, however, indicating that both SSC numbers and activity declined with age. Finally, we transplanted donor cells from young animals into 1- and 2-yr atrophied testes. Although the weight of 2-yr testes did not change after transplantation, that of 1-yr testes increased significantly, indicating that 1-yr, but not 2-yr, atrophied testes are permissive for regeneration of spermatogenesis by SSCs from young mouse testes. These results demonstrate that both SSCs and somatic environment in the testis are involved in the aging process.

Expression patterns of cell-surface molecules on male germ line stem cells during postnatal mouse development

Spermatogonial stem cells (SSCs) are stem cells of the male germ line. In mice, SSCs are quiescent at birth but actively proliferate during the first postnatal week, while they rarely divide in adult, suggesting an age-dependent difference in SSC characteristics. As an approach to evaluate this possibility, we studied the expression pattern of cell-surface molecules on neonatal, pup, and adult mouse SSCs. Using immunomagnetic cell sorting, testis cells were selected for the expression of alpha(6) integrin, alpha(v) integrin, c-kit receptor tyrosine kinase (Kit), or a binding subunit of glial-cell-line-derived neurotrophic factor (GDNF) receptor, GFRalpha1. Selected cells were assayed for their stem cell activity using spermatogonial transplantation. The results showed that SSCs expressed alpha(6) integrin, but not alpha(v) integrin and Kit, regardless of age. The SSC activity in pup GFRalpha1(+) cells was higher than that in adult and neonatal cells, indicating that the expression pattern of GFRalpha1 varied age-dependently. To evaluate if SSCs show an age-dependent difference in their response to GDNF, we cultured highly enriched pup and adult SSCs with GDNF: we could not observe such an age-dependent difference in vitro. In addition, we failed to immunologically detect the expression of two types of GDNF receptor signaling subunits on SSCs. These results indicate that SSCs may change the expression patterns of cell-surface molecules during postnatal development, and suggest that GDNF receptor molecules may not be abundantly or specifically expressed in the in vivo population of mouse SSCs.

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

In mammals, spermatogenesis is maintained by spermatogonial stem cells (SSC). In their niche, SSC divide to self-maintain and to produce a transit-amplifying population that eventually enters the meiotic cycle to give rise to spermatozoa. The low number of SSC and the lack of specific markers hinder their isolation and enrichment. Stem cells in several adult tissues can be identified by using their verapamil-sensitive Hoechst dye-effluxing properties, which define the characteristic "side population" (SP). Here we show, by multicolor flow cytometric analysis, that immature mouse testis contains a "side-population" (T-SP), which is Sca-1pos, Ep-CAMpos, EE2 pos, alpha6-integrin pos, and alpha(v)-integrin neg. A 13-fold enrichment in SSC activity was observed when sorted T-SP cells from ROSA 26 mice were transplanted in busulfan-treated mouse testis. Whereas an incomplete range of spermatogenic stages was encountered two months after transplantation of unsorted testicular cells, the transplantation of T-SP cells generated all associations of mouse germ cells representing the full range of spermatogenic stages. These data suggest that Hoechst staining and cell sorting might provide a novel approach to SSC enrichment in mammals.

Germ cell failure is the cause of luxoid (Lu) mouse mutant defective spermatogenesis

Objective: Spermatogonial stem cells continue to renew and differentiate throughout the lifespan. Successful male reproduction requires proper regulation and balance of these two pathways. Disruptions of the mechanisms that affect spermatogonial stem cell activity can contribute to male infertility. The well known luxoid mouse mutation has been described as altering the balance between spermatogonial stem cell renewal and differentiation. It is unclear what testicular cell type is affected by the luxoid defect. This study determined whether the defect in spermatogenesis is due to a failure of the somatic component of the testis or due to an intrinsic problem of the germ line stem cell.

Retrovirus-mediated modification of male germline stem cells in rats.

The ability to isolate, manipulate, and transplant spermatogonial stem cells provides a unique opportunity to modify the germline. We used the rat-to-nude mouse transplantation assay to characterize spermatogonial stem cell activity in rat testes and in culture. Our results indicate that rat spermatogonial stem cells can survive and proliferate in short-term culture, although a net loss of stem cells was observed. Rat spermatogonial stem cells also were susceptible to transduction with a retroviral vector carrying a lacZ reporter transgene. Using a 3-day periodic infection protocol, 0.5% of stem cells originally cultured were transduced and produced transgenic colonies of spermatogenesis in recipient mouse testes. The level of transgenic donor-derived spermatogenesis observed in the rat-to-mouse transplantation was similar to levels that produced transgenic progeny in the mouse-to-mouse transplantation. This work provides a basis for understanding the biology of rat spermatogonial stem cells. Development of an optimal rat recipient testis model and application of these methods for germline modification will enable the production of transgenic rats, potentially valuable tools for evaluating genes and their functions. In addition, these methods may be applicable in other species where existing transgenic methods are inefficient or not available.