Culture conditions and single growth factors affect fate determination of mouse spermatogonial stem cells

Cell fate determination between self-renewal or differentiation of spermatogonial stem cells (SSCs) in the testis is precisely regulated to maintain normal spermatogenesis. However, the mechanisms underlying the process remain elusive. To address the problem, we developed a model SSC culture system, first, by establishing techniques to obtain enriched populations of stem cells, and second, by establishing a serum-free culture medium. Flow cytometric cell sorting and the SSC transplantation assay demonstrated that Thy-1 is a unique surface marker of SSCs in neonatal, pup, and adult testes of the mouse. Although the surface phenotype of SSCs is major histocompatibility complex class I(-) Thy-1(+) alpha 6-integrin(+) alpha v-integrin(-/dim) throughout postnatal life, the most enriched population of SSCs was obtained from cryptorchid adult testes by cell-sorting techniques based on Thy-1 expression. This enriched population of SSCs was used to develop a culture system that consisted of serum-free defined medium and STO (SIM mouse embryo-derived thioguanine and ouabain resistant) feeders, which routinely maintained stem cell activity for 1 wk. Combining the culture system and the transplantation assay provided a mechanism to study the effect of single growth factors. A negative effect was demonstrated for several concentrations of basic fibroblast growth factor and leukemia inhibitory factor, whereas glial cell line-derived neurotrophic factor and stem cell factor appeared to have a positive effect on stem cell maintenance. The stem cell enrichment strategies and the culture methods described provide a reproducible and powerful assay system to establish the effect of various environmental factors on SSC survival and replication in vitro.     

Stem cell and niche development in the postnatal rat testis

Adult tissue stem cells self-renew and differentiate in a way that exactly meets the biological demand of the dependent tissue. We evaluated spermatogonial stem cell (SSC) activity in the developing rat testis and the quality and accessibility of the stem cell niche in wild type, and two busulfan-treated models of rat pup recipient testes using an SSC transplantation technique as a functional assay. While our results revealed a 69-fold increase in stem cell activity during rat testis development from neonate to adult, only moderate changes in SSC concentration were observed, and stem cells from neonate, pup, and adult donor testes produce spermatogenic colonies of similar size. Analysis of the stem cell niche in recipient rat testes demonstrated that pup testes support high levels of donor stem cell engraftment when endogenous germ cells are removed or compromised by busulfan treatment. Fertility was established when rat pup donor testis cells were transplanted into fetal- or pup-busulfan-treated recipient rat pup testes, and the donor genotype was transmitted to subsequent generations. These results provide insight into stem cell/niche interactions in the rat testis and demonstrate that techniques originally developed in mice can be extended to other species for regenerative medicine and germline modification.     

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.     

Homing of mouse spermatogonial stem cells to germline niche depends on beta1-integrin

Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis. In a manner comparable to hematopoietic stem cell transplantation, SSCs colonize the niche of recipient testes and reinitiate spermatogenesis following microinjection into the seminiferous tubules. However, little is known about the homing mechanism of SSCs. Here we examined the role of adhesion molecules in SSC homing. SSCs isolated from mice carrying loxP-tagged beta1-integrin alleles were ablated for beta1-integrin expression by in vitro adenoviral cre transduction. The beta1-integrin mutant SSCs showed significantly reduced ability to recolonize recipient testes in vivo and to attach to laminin molecules in vitro. In contrast, genetic ablation of E-cadherin did not impair homing, and E-cadherin mutant SSCs completed normal spermatogenesis. In addition, the deletion of beta1-integrin on Sertoli cells reduced SSC homing. These results identify beta1-integrin as an essential adhesion receptor for SSC homing and its association with laminin is critical in multiple steps of SSC homing.     

E-cadherin can be expressed by a small population of rat undifferentiated spermatogonia in vivo and in vitro

Spermatogonial stem cells (SSCs) maintain gamete production in the testes throughout adult life by balancing self-renewal and differentiation. In vitro culture of SSCs is a crucial technique for gene manipulation of SSCs to generate transgenic animals, for transplantation of SSCs to restore male fertility for infertile man, and for generation of pluripotent stem cells from SSCs to differentiate into various cell lineages. Isolation of highly purified SSCs is an all-important component for development of these techniques. However, definitive markers for SSCs, which purify SSCs (100% enrichment), are unknown. SSCs of many species can colonize the mouse testis; thus, we reasoned that same molecules of SSCs are conserved between species. In mouse, undifferentiated spermatogonia express the surface marker E-cadherin. The hypothesis tested in this work was that E-cadherin (also known as CDH1) can be expressed by undifferentiated spermatogonia of rat testes. In this paper, cross-section immunohistochemistry and whole-mount immunohistochemistry of rat seminiferous tubules were conducted to show that E-cadherin-positive cells were small in number and there are single, paired, and aligned spermatogonia attached along the basement membrane. During in vitro culture period, the undifferentiated rat spermatogonial colonies co-expressed E-cadherin and glial-derived neurotrophic factor family receptor alpha-1 or E-cadherin and promyelocytic leukemia zinc finger. Data collected during the study demonstrate that E-cadherin is expressed by a small population of rat undifferentiated spermatogonia both in vivo and during in vitro culture period.     

GDNF family receptor alpha1 phenotype of spermatogonial stem cells in immature mouse testes

Spermatogonial stem cells (SSCs) are essential for spermatogenesis, and these adult tissue stem cells balance self-renewal and differentiation to meet the biological demand of the testis. The developmental dynamics of SSCs are controlled, in part, by factors in the stem cell niche, which is located on the basement membrane of seminiferous tubules situated among Sertoli cells. Sertoli cells produce glial cell line-derived neurotrophic factor (GDNF), and disruption of GDNF expression results in spermatogenic defects and infertility. The GDNF signals through a receptor complex that includes GDNF family receptor alpha1 (GFRA1), which is thought to be expressed by SSCs. However, expression of GFRA1 on SSCs has not been confirmed by in vivo functional assay, which is the only method that allows definitive identification of SSCs. Therefore, we fractionated mouse pup testis cells based on GFRA1 expression using magnetic activated cell sorting. The sorted and depleted fractions of GFRA1 were characterized for germ cell markers by immunocytochemistry and for stem cell activity by germ cell transplantation. The GFRA1-positive cell fraction coeluted with other markers of SSCs, including ITGA6 and CD9, and was significantly depleted of KIT-positive cells. The transplantation results confirmed that a subpopulation of SSCs expresses GFRA1, but also that the stem cell pool is heterogeneous with respect to the level of GFRA1 expression. Interestingly, POU5F1-positive cells were enriched nearly 15-fold in the GFRA1-selected fraction, possibly suggesting heterogeneity of developmental potential within the stem cell pool.     

Male germ line stem cells have an altered potential to proliferate and differentiate during postnatal development in mice

Spermatogonial stem cells (SSCs) continuously support spermatogenesis after puberty. However, accumulating evidence suggests that SSCs differ functionally during postnatal development. For example, mutant mice exist in which SSCs support spermatogenesis in the first wave after birth but cease to do so thereafter, resulting in infertility in adults. Studies using a retroviral vector have shown that the vector transduces pup SSCs more efficiently than adult SSCs, which suggests that pup SSCs divide more frequently. Thus, it is hypothesized that the SSCs in pup and adult testes have different characteristics. As an approach to testing this hypothesis in the present study, we investigated the proliferation kinetics of pup SSCs (6-9 days old) and their self-renewal/differentiation patterns for the first 2 mo after transplantation, and compared them to those of adult SSCs. Using serial transplantation, we found that the number of pup SSCs declined over the first week after transplantation. Thereafter, it increased ~4-fold by 1 mo and ~9-fold by 2 mo after transplantation, which indicates that pup SSCs continuously proliferate from 1 wk to 2 mo after transplantation. Compared to the proliferation of SSCs derived from adult intact testes, that of pup SSCs was lower at 1 mo but similar at 2 mo, indicating the delayed proliferation of pup SSCs. However, the pup SSCs regenerated spermatogenic colonies at 1 mo that were similar in length to those of SSCs from adult intact testes. Therefore, these results suggest that some functional differences exist in SSCs during postnatal development, and that these differences may affect the abilities of SSCs to self-renew and differentiate.     

Genetic analysis of the clonal origin of regenerating mouse spermatogenesis following transplantation

Spermatogonial transplantation provides a straightforward approach to quantify spermatogonial stem cells (SSCs). Because donor-derived spermatogenesis is regenerated in the form of distinct colonies, the number of functional SSCs can be obtained by simply counting the number of colonies established in recipient testes. However, this approach is legitimate only when one colony arises from one stem cell (one colony-one stem cell hypothesis). In this study, we evaluated the validity of this hypothesis. Two populations of donor cells were obtained from the testes of two transgenic mouse lines and mixed at a 1:1 ratio. Following transplantation of the cell mixture, donor-derived colonies were visualized and individually excised, and genomic DNA was extracted from each colony. Based on unique marker genes of the two transgenic lines, the genotype of the cells contained in a colony was examined by polymerase chain reaction. A colony was determined to be clonal when only one transgene was detected. The results showed that 100% and 90% of colonies were clonal when <5 and 19 colonies were formed per recipient testis, respectively. However, the clonality of colonies decreased as the colony number per recipient testis or the length of each colony increased. These results support the one colony-one stem cell hypothesis and demonstrate that spermatogonial transplantation provides a highly quantitative assay for SSCs; however, these conclusions are applicable under a defined transplantation condition.     

Germ line stem cell competition in postnatal mouse testes

Niche is believed to affect stem cell behavior. In self-renewing systems for which functional transplantation assays are available, it has long been assumed that stem cells are fixed in the niche and that ablative treatments to remove endogenous stem cells are required for successful donor engraftment. Our results demonstrate that enriched populations of donor stem cells can produce long-lasting spermatogenic colonies in testes of immature and mature, nonablated mice, albeit at a lower frequency than in ablated mice. Colonization of nonablated recipient testes by neonate, pup, and cryptorchid adult donor spermatogonial stem cells demonstrates that competition for niche begins soon after birth and that endogenous stem cells influence the degree and pattern of donor cell colonization. Thus, a dynamic relationship between stem cell and niche exists in the testis, as has been suggested for hematopoiesis. Therefore, similar competitive properties of donor stem cells may be characteristic of all self-renewing systems.     

Nanostructure and β1-integrin distribution analysis of pig's spermatogonial stem cell by atomic force microscopy

Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis and male fertility. However, spermatogenesis has direct links with some adhesion molecules on SSCs membrane. Β1-integrin (CD29) is such a kind of adhesion molecule and a biomarker of pig's SSCs. Therefore, quantitative characteristics of β1-integrin expression level in a single cell could help us to capture the signal switch and understand the mechanism of spermatogenesis. In this study, atomic force microscopy (AFM) was used to obtain the morphology and ultrastructure of SSCs at nanometer level, and the CD29 Ab-functionalized AFM tip was used to examine β1-integrin distribution on the cell membrane. There were many force-binding spots on about 50% of cell membrane binding to the CD29 Ab-functionalized AFM tip, and the mean bind rupture force was 283.63±12.56PN which was much larger than the non-specific average force 70.75±10.95PN. Meanwhile, β1-integrin on SSCs membrane was distributed non-uniformly, and there were some β1-integrins appeared to be expressed as 150-350 nm nanoclusters on the membrane. Our results discovered the structure of SSCs at nanometer level by AFM. The force between β1-integrin antigen-antibody interactions and the distribution of β1-integrin protein on SSCs membrane were also firstly demonstrated.     

Aging of male germ line stem cells in mice

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.     

Production of transgenic rats via lentiviral transduction and xenogeneic transplantation of spermatogonial stem cells

Spermatogonial stem cells (SSCs) continue to proliferate in the testis to support spermatogenesis throughout life, which makes them ideal targets for germline modification. Although recent success in the production of transgenic and knockout animals using SSCs has opened up new experimental possibilities, several problems, including the low efficiency of germ cell transplantation and poor fertility rates, remain to be resolved. In the present study, we took advantage of the xenogeneic transplantation to resolve these problems. Rat SSCs were transduced in vitro with a lentiviral vector that expressed enhanced green fluorescent protein (EGFP), and then transplanted into the testes of immunodeficient mice. The transduced rat SSCs produced EGFP-expressing spermatogenic cells, and microinsemination using these cells was used to produce transgenic rats, which stably transmitted the transgene to the next generation. Thus, xenogeneic transplantation is a powerful strategy for transgenesis, and smaller xenogeneic surrogates can be used for male germline modification using SSCs.     

胶质细胞源性神经营养因子引发精原干细胞自我更新的信号通路

胶质细胞源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF)是TGF-β超家族的一个相关成员。哺乳动物睾丸曲细精管内支持细胞分泌的GDNF,能促进精原干细胞(spermatogonial stem cells,SSCs)的自我更新与增殖。SSCs去分化诱导产生的多能干细胞已被广泛应用于再生医学领域,且SSCs在制作转基因动物、男性不育治疗和体外实施精子发生过程等方面,具有极大的应用价值。所以,GDNF引发SSCs自我更新的作用机理非常值得探索。通过对GDNF引发SSCs自我更新的信号通路进行系统梳理,我们发现了如下的作用过程:GDNF与GFR-α1特异性结合,活化Ret蛋白酪氨酸激酶,随后激活Ras/ERK1/2、PI3K-Akt和SFK信号通路,促进SSCs的自我更新;同时,在该过程中还存在信号通路间的交联对话现象。     

NOTCH signaling in Sertoli cells regulates gonocyte fate

Gonocytes (or prospermatogonia) are the precursors to spermatogonial stem cells (SSCs), which provide the foundation for spermatogenesis through their ability to both self-renew and generate daughter cells. Despite their relative importance, the regulatory mechanisms that govern gonocyte maintenance and transition to SSCs are poorly understood. Recently, we reported that constitutive activation of NOTCH1 signaling in Sertoli cells causes gonocyte exit from quiescence—the first suggestion of the potential role of this signaling pathway in the testis.

This Extra View will review what is known about NOTCH signaling, particularly in Sertoli cells and germ cells in the testes, by providing a background on germ cell biology and a summary of our recently published data on NOTCH1 signaling in Sertoli cells. We also describe additional data showing that aberrant proliferation and differentiation of gonocytes in response to constitutive activation of NOTCH1 signaling in Sertoli cells involves de novo expression of cell cycle proteins and a marked upregulation of the KIT receptor. These data further suggest that NOTCH signaling orchestrates a dynamic balance between maintenance and differentiation of gonocytes in the perinatal testis.     

Rac mediates mouse spermatogonial stem cell homing to germline niches by regulating transmigration through the blood-testis barrier

The homing ability of spermatogonial stem cells (SSCs) allows them to migrate into niches after being transplantated into infertile testes. Transplanted SSCs attach to Sertoli cells and transmigrate through the blood-testis barrier (BTB), formed by inter-Sertoli tight junctions, toward niches on the basement membrane. The most critical step is the passage through the BTB, which limits the homing efficiency to <10%. Here we demonstrated the involvement of Rac1 in SSC transmigration. Rac1-deficient SSCs did not colonize the adult testes, but they reinitiated spermatogenesis when transplanted into pup testes without a BTB. Moreover, a dominant-negative Rac1 construct not only reduced the expression of several claudin proteins, which comprise the BTB, but also increased SSC proliferation both in vitro and in vivo. Short hairpin RNA (shRNA) -mediated suppression of claudin3, which was downregulated by Rac inhibition, reduced the SSC homing efficiency. Thus, Rac1 is a critical regulator of SSC homing and proliferation.     

Identification of a novel bone marrow-derived B-cell progenitor population that coexpresses B220 and Thy-1 and is highly enriched for Abelson leukemia virus targets.

A novel stage in early B-lymphocyte differentiation has been identified in normal mouse bone marrow cells. Earlier work had demonstrated that bone marrow cells characterized by low levels of Thy-1 and lack of a panel of lineage markers (Thy-1lo Lin- cells) were highly enriched for pluripotent hematopoietic stem cells. In this paper, we present evidence that another bone marrow population, which expressed low levels of Thy-1 and coexpressed B220, a B-lineage-specific form of the leukocyte common antigen, contained early and potent precursors for B lymphocytes upon in vivo transfer to irradiated hosts. These Thy-1lo B220+ cells, comprising 1 to 2% of bone marrow cells, were enriched for large cells in the mitotic cycle; the population lacked significant pluripotent hematopoietic stem cell activity and myeloid-erythroid progenitors. Most strikingly, Thy-1lo B220+ cells represented a highly enriched population of bone marrow cells that could be targets of Abelson murine leukemia virus transformation. We propose that Thy-1lo B220+ bone marrow cells represent the earliest stage of committed lymphocyte progenitors, intermediate in differentiation between Thy-1lo Lin- pluripotent stem cells and, in the B lineage, Thy-1- B220+ pre-B cells.     

Effects of different feeder layers on short-term culture of prepubertal bovine testicular germ cells in-vitro

Spermatogonial stem cells (SSCs) are exceptional adult stem cells that transfer genes to new generations. This behavior makes them unique cells for the production of transgenic farm animals. However, this goal has been hampered by their spontaneous differentiation during in vitro culture. Therefore, the objective of this study was the evaluation of the effects of different feeders on in vitro short-term culture of prepubertal bovine testicular germ cells. The isolated cell suspensions containing SSCs were enriched by Bovine serum albumin (BSA) and gelatin and were cultured in the presence of Glial-derived neurotrophic factor (GDNF), Epidermal Growth Factor (EGF) and basic Fibroblastic Growth Factor (bFGF). After 7 d of culture, colonies were harvested and cultured on four different feeders, including SIM mouse embryo-derived thioguanine and ouabain resistant (STO), mouse embryonic fibroblast, bovine Sertoli cells (BSC) and on a laminin-coated plate. The number and area of colonies were measured at seven, 11 and 14 d post-culture. The expression of germ cells markers was detected using immunofluorescence and flow cytometry analyses on day 7, and quantitative real-time PCR at 14 d post-culture. Immunocytochemical staining revealed that colonies were positive for Dolichos biflorus agglutinin (DBA), Thy-1, Oct-4, c-ret, α6-integrin, β1-integrin and negative for c-kit. In addition, the number and area of those colonies formed on the STO feeder were significantly greater than the other groups. Relative expressions of Thy-1 in the STO and in BSC groups were significantly higher than other groups but expression of Oct-4 was highest in the laminin group compared to other groups. In conclusion, STO might be a suitable feeder layer for in vitro propagation of bovine testicular germ cells.     

精原干细胞的生物学特性

精原干细胞(spermatogonialstemcells,SSCs)是雄性生殖系干细胞,位于睾丸曲细精管基膜上,既具有自我更新潜能,又具有定向分化潜能,是自然状态下出生后动物体内在整个生命期间进行自我更新并能将基因传递至子代的唯一成体干细胞。自SSCs移植技术建立以来,有关其分离、鉴定、培养、冻存、转基因操作及移植等方面均已取得长足进步,使人们对其生物学特性有了更深入的了解。根据最近的相关进展,系统评述了SSCs的相关生物学特性,以期为该领域及其他干细胞研究提供借鉴。     

Spermatogonial stem cells in the testis of an endangered bovid: Indian black buck (Antilope cervicapra L.)

Numerous wild bovids are facing threat of extinction owing to the loss of habitat and various other reasons. Spermatogonial stem cells (SSCs) represent the only germline stem cells in adult body that are capable of self-renewal and that can undergo differentiation to produce haploid germ cells. SSCs can, therefore, serve as a useful resource for preservation of germplasm of threatened and endangered mammals. The Indian black buck (Antilope cervicapra L.) is a small Indian antelope that is listed as endangered by the Indian Wildlife Protection Act, 1972. Immunohistochemical analysis of testes tissues of black buck revealed the presence of spermatogonia that were specifically stained by lectin-Dolichos biflorus agglutinin (DBA). The expression of pluripotent cell-specific markers, NANOG and stage-specific embryonic antigen-1 (SSEA-1), was detected in spermatogonia. Interestingly, the expression of POU5F1 (OCT3/4) was absent from spermatogonia, however, it was detected in differentiating cells such as spermatocytes and round spermatids but not in elongated spermatids. The expression of NANOG protein was also present in spermatocytes but absent in round and elongated spermatids. Using the testis transplantation assay, stem cell potential of black buck spermatogonia was confirmed as indicated by the presence of colonized DBA-stained cells in the basal membrane of seminiferous tubules of xenotransplanted mice testis. The findings from this study suggest the presence of SSCs in the testis of an endangered bovid for the first time and open new possibility to explore the use of SSCs in conservation.