Functional analysis of stem cells in the adult rat testis

Adult stem cells maintain several self-renewing systems and processes in the body, including the epidermis, hematopoiesis, intestinal epithelium, and spermatogenesis. However, studies on adult stem cells are hampered by their low numbers, lack of information about morphologic or biochemical characteristics, and absence of functional assays, except for hematopoietic and spermatogonial stem cells. We took advantage of the recently developed spermatogonial transplantation technique to analyze germ line stem cells of the rat testis. The results indicate that the stem cell concentration in rat testes is 9.5-fold higher than that in mouse testes, and spermatogenic colonies derived from rat donor testis cells are 2.75 times larger than mouse-derived colonies by 3 mo after transplantation. Therefore, the extent of spermatogenesis from rat stem cells was 26-fold greater than that from mouse stem cells at the time of recipient testis analysis. Attempts to enrich spermatogonial stem cells in rat testis populations using the experimental cryptorchid procedure were not successful, but selection by attachment to laminin-coated plates resulted in 8.5-fold enrichment. Spermatogonial stem cells are unique among adult stem cells because they pass genetic information to the next generation. The high concentration of stem cells in the rat testis and the rapid expansion of spermatogenesis after transplantation will facilitate studies on stem cell biology and the introduction of genetic modifications into the male germ line. The functional differences between spermatogonial stem cells of rat vs. mouse origin after transplantation suggest that the potential of these cells may vary greatly among species.     

Expansion of murine spermatogonial stem cells through serial transplantation

Mammalian male germ cells might be generally thought to have infinite proliferative potential based on their life-long production of huge numbers of sperm. However, there has been little substantial evidence that supports this assumption. In the present study, we performed serial transplantation of spermatogonial stem cells to investigate if they expand by self-renewing division following transplantation. The transgenic mouse carrying the Green fluorescent protein gene was used as the donor cell source that facilitated identification and recollection of colonized donor germ cells in the recipient testes. The established colonies of germ cells in the recipient testes were collected and transplanted to new recipients. This serial transplantation of spermatogonial stem cells repopulated the recipient testes, which were successfully performed sequentially up to four times from one recipient to the next. The incubation periods between two sequential transplantations ranged from 55 to 373 days. During these passages, the spermatogonial stem cells showed constant activity to form spermatogenic colonies in the recipient testis. They continued to increase in number for more than a year following transplantation. Colonization efficiency of spermatogonial stem cells was determined to be 4.25% by using Sl/Sl(d) mice as recipients that propagated only undifferentiated type A spermatogonia in their testes. Based on the colonization efficiency, one colony-forming activity was assessed to equate to about 20 spermatogonial stem cells. The spermatogonial stem cells were estimated to expand over 50-fold in 100 days in this experiment.     

Long-term culture of mouse male germline stem cells under serum-or feeder-free conditions

Spermatogonial stem cells are the only stem cells in the body that transmit genetic information to the next generation. These cells can be cultured for extended periods in the presence of serum and feeder cells. However, little is known about factors that regulate self-renewal division of spermatogonial stem cells. In this investigation we examined the possibility of establishing culture systems for spermatogonial stem cells that lack serum or a feeder cell layer. Spermatogonial stem cells could expand in serum-free conditions on mouse embryonic fibroblasts (MEFs), or were successfully cultivated without feeder cells on a laminin-coated plate. However, they could not expand when both serum and feeder cells were absent. Although the cells cultured on laminin differed phenotypically from those on feeder cells, they grew exponentially for at least 6 mo, and produced normal, fertile progeny following transplantation into infertile mouse testis. This culture system will provide a new opportunity for understanding the regulatory mechanism that governs spermatogonial stem cells.     

Genetic and epigenetic properties of mouse male germline stem cells during long-term culture

Although stem cells are believed to divide infinitely by self-renewal division, there is little evidence that demonstrates their infinite replicative potential. Spermatogonial stem cells are the founder cell population for spermatogenesis. Recently, in vitro culture of spermatogonial stem cells was described. Spermatogonial stem cells can be expanded in vitro in the presence of glial cell line-derived neurotrophic factor (GDNF), maintaining the capacity to produce spermatogenesis after transplantation into testis. Here, we examined the stability and proliferative capacity of spermatogonial stem cells using cultured cells. Spermatogonial stem cells were cultured over 2 years and achieved approximately 10(85)-fold expansion. Unlike other germline cells that often acquire genetic and epigenetic changes in vitro, spermatogonial stem cells retained the euploid karyotype and androgenetic imprint during the 2-year experimental period, and produced normal spermatogenesis and fertile offspring. However, the telomeres in spermatogonial stem cells gradually shortened during culture, suggesting that they are not immortal. Nevertheless, the remarkable stability and proliferative potential of spermatogonial stem cells suggest that they have a unique machinery to prevent transmission of genetic and epigenetic damages to the offspring, and these characteristics make them an attractive target for germline modification.     

Commitment of fetal male germ cells to spermatogonial stem cells during mouse embryonic development

The continuous production of mammalian sperm is maintained by the proliferation and differentiation of spermatogonial stem cells that originate from primordial germ cells (PGCs) in the early embryo. Although spermatogonial stem cells arise from PGCs, it is not clear whether fetal male germ cells function as spermatogonial stem cells able to produce functional sperm. In the present study, we examined the timing and mechanisms of the commitment of fetal germ cells to differentiate into spermatogonial stem cells by transplantation techniques. Transplantation of fetal germ cells into the seminiferous tubules of adult testis showed that donor germ cells, at 14.5 days postcoitum (dpc), were able to initiate spermatogenesis in the adult recipient seminiferous tubules, whereas no germ cell differentiation was observed in the transplantation of 12.5-dpc germ cells. These results indicate that the commitment of fetal germ cells to differentiate into spermatogonial stem cells initiates between embryonic days 12.5 and 14.5. Furthermore, the results suggest the importance of the interaction between germ cells and somatic cells in the determination of fetal germ cell differentiation into spermatogonial stem cells, as normal spermatogenesis was observed when a 12.5-dpc whole gonad was transplanted into adult recipient testis. In addition, sperm obtained from the 12.5- dpc male gonadal explant had the ability to develop normally if injected into the cytoplasm of oocytes, indicating that normal development of fetal germ cells in fetal gonadal explant occurred in the adult testicular environment.     

Biological activity and enrichment of spermatogonial stem cells in vitamin A-deficient and hyperthermia-exposed testes from mice based on colonization following germ cell transplantation

Spermatogenesis is a complex process in which spermatogonial stem cells divide and subsequently differentiate into spermatozoa. This process requires spermatogonial stem cells to self-renew and provide a continual population of cells for differentiation. Studies on spermatogonial stem cells have been limited due to a lack of unique markers and an inability to detect the presence of these cells. The technique of germ cell transplantation provides a functional assay to identify spermatogonial stem cells in a cell population. We hypothesized that vitamin A-deficient (VAD) and hyperthermically treated testes would provide an enriched in vivo source of spermatogonial stem cells. The first model, hyperthermic treatment, depends on the sensitivity of maturing germ cells to high temperatures. Testes of adult mice were exposed to 43 degrees C for 15 min to eliminate the majority of differentiating germ cells. Treated donor testes were 50% of normal adult testis size and, when transplanted into recipients, resulted in a 5.3- and 19-fold (colonies and area, respectively) increase in colonization efficiency compared to controls. The second model, VAD animals, also lacked differentiating germ cells, and testes weights were 25% of control values. Colonization efficiency of germ cells from VAD testes resulted in a 2.5- and 6.2-fold (colonies and area, respectively) increase in colonization compared to controls. Hyperthermically treated mice represent an enriched source of spermatogonial stem cells. In contrast, the low extent of colonization with germ cells from VAD animals raises important questions regarding the competency of stem cells from this model.     

Pattern and kinetics of mouse donor spermatogonial stem cell colonization in recipient testes.

Recently a system was developed in which transplanted donor spermatogonial stem cells establish complete spermatogenesis in the testes of an infertile recipient. To obtain insight into stem cell activity and the behavior of donor germ cells, the pattern and kinetics of mouse spermatogonial colonization in recipient seminiferous tubules were analyzed during the 4 mo following transplantation. The colonization process can be divided into three continuous phases. First, during the initial week, transplanted cells were randomly distributed throughout the tubules, and a small number reached the basement membrane. Second, from 1 wk to 1 mo, donor cells on the basement membrane divided and formed a monolayer network. Third, beginning at about 1 mo and continuing throughout the observation period, cells in the center of the network differentiated extensively and established a colony of spermatogenesis, which expanded laterally by repeating phase two and then three. An average of 19 donor cell-derived colonies developed from 10(6) cells transplanted to the seminiferous tubules of a recipient testis; the number of colonized sites did not change between 1 and 4 mo. However, the length of the colonies increased from 0.73 to 5.78 mm between 1 and 4 mo. These experiments establish the feasibility of studying in a systematic and quantitative manner the pattern and kinetics of the colonization process. Using spermatogonial transplantation as a functional assay, it should be possible to assess the effects of various treatments on stem cells and on recipient seminiferous tubules to provide unique insight into the process of spermatogenesis.     

Long-term proliferation in culture and germline transmission of mouse male germline stem cells

Spermatogenesis is a complex process that originates in a small population of spermatogonial stem cells. Here we report the in vitro culture of spermatogonial stem cells that proliferate for long periods of time. In the presence of glial cell line-derived neurotrophic factor, epidermal growth factor, basic fibroblast growth factor, and leukemia inhibitory factor, gonocytes isolated from neonatal mouse testis proliferated over a 5-month period (>10(14)-fold) and restored fertility to congenitally infertile recipient mice following transplantation into seminiferous tubules. Long-term spermatogonial stem cell culture will be useful for studying spermatogenesis mechanism and has important implications for developing new technology in transgenesis or medicine.     

Homing efficiency and proliferation kinetics of male germ line stem cells following transplantation in mice

Stem cells in the male germ line (spermatogonial stem cells [SSCs]) are an important target for male fertility restoration and germ line gene modification. To establish a model system to study the biology and the applications of SSCs in mice, I used a sequential transplantation strategy to analyze the process by which SSCs colonize the stem cell niche after transplantation and to determine the efficiency of the process (homing efficiency). I further analyzed the proliferation kinetics of SSCs after colonization. The number of SSCs gradually decreased during the homing process, and only 12% of SSCs successfully colonized the niche on Day 7 after transplantation, but the number of SSCs increased by Day 14. Thus, homing efficiency of adult mouse SSCs is 12%. These results indicate that SSCs are rapidly lost upon transplantation and require approximately 1 wk to settle into their niches before initiating expansion. Using this SSC homing efficiency, I calculated that approximately 3000 SSCs exist in one normal adult testis, representing approximately 0.01% of total testis cells. Between 7 days and 1 mo after transplantation, SSCs proliferated 7.5-fold. However, they did not significantly proliferate thereafter until 2 mo, and only 8 SSCs supported one colony of donor-derived spermatogenesis from 1 to 2 mo. These results suggest that self-renewal and differentiation of SSCs are strictly regulated in coordination with the progress of an entire unit of regenerating spermatogenesis.     

Functional analysis of spermatogonial stem cells in Steel and cryptorchid infertile mouse models

Spermatogenesis is a complex and productive process that originates from stem cell spermatogonia and ultimately results in formation of mature spermatozoa. The stem cell undergoes self-renewal throughout life, but study of its biological characteristics has been difficult because a very small number (2 to 3 in 10(4) cells) exist in the testis and they can only be identified by function. Although the development of the spermatogonial transplantation technique has provided an assay system for stem cells, efficient methods to enrich stem cells have not been available. Here, we examined two infertile mouse models, Steel/Steel(Dickie)(Sl/Sl(d)) and experimental cryptorchid, as a source of testis cell populations enriched in stem cells. The Sl/Sl(d) testis showed little enrichment, which raises questions about how adult stem cell number is determined and about the currently accepted belief that adult stem cells are independent of Sl factor. The cells recovered from cryptorchid testes were enriched for stem cells 25-fold (colonies) or 50-fold (area) compared to wild-type testes. The cryptorchid condition does not affect stem cell activity, but eliminates almost all differentiated cells, and about 1 in 200 cells is a stem cell. Thus, cryptorchid testes provide an important approach for purification and characterization of spermatogonial stem cells.     

Anchorage-independent growth of mouse male germline stem cells in vitro

Spermatogenesis originates from a small number of spermatogonial stem cells that reside on the basement membrane and undergo self-renewal division to support spermatogenesis throughout the life of adult animals. Although the recent development of a technique to culture spermatogonial stem cells allowed reproduction of self-renewal division in vitro, much remains unknown about how spermatogonial stem cells are regulated. In this study, we found that spermatogonial stem cells could be cultured in an anchorage-independent manner, which is characteristic of stem cells from other types of self-renewing tissues. Although the cultured cells grew slowly (doubling time, approximately 4.7 days), they expressed markers of spermatogonia, and grew exponentially for at least 5 months to achieve 1.5 x 10(10) -fold expansion. The cultured cells underwent spermatogenesis following transplantation into the seminiferous tubules of infertile animals and fertile offspring were obtained by microinsemination of germ cells that had developed within the testes of recipients of the cultured cells. These results indicate that spermatogonial stem cells can undergo anchorage-independent, self-renewal division, and suggest that stem cells have the common property to survive and proliferate in the absence of exogenous substrata.     

浅谈精原干细胞研究进展

精原干细胞是雄性动物体内精子发生过程中起重要作用的精原细胞类型,不但具有干细胞特性,还能定向分化为雄性配子将自身基因传递给后代。除此之外,体外培养和鉴定精原干细胞为移植和转基因提供了基础。我们对精原干细胞的生物学特性、分离培养、鉴定、移植及精原干细胞介导的转基因进行简要概述。     

Primate spermatogonial stem cells colonize mouse testes

In mice, transplantation of spermatogonial stem cells from a fertile male to the seminiferous tubules of an infertile recipient male results in progeny with donor-derived haplotype. Attempts to extend this approach by transplanting human testis cells to mice have led to conflicting claims that no donor germ cells persisted or that human spermatozoa were produced in the recipient. To examine this issue we used the baboon, a primate in which testis cell populations of several ages could be obtained for transplantation, and demonstrate that donor spermatogonial stem cells readily establish germ cell colonies in recipient mice, which exist for periods of at least 6 mo. However, differentiation of germ cells toward the lumen of the tubule and production of spermatozoa did not occur. The presence of baboon spermatogonial stem cells and undifferentiated spermatogonia in mouse seminiferous tubules for long periods after transplantation indicates that antigens, growth factors, and signaling molecules that are necessary for interaction of these cells and the testis environment have been preserved for 100 million years of evolutionary separation. Because germ cell differentiation and spermatogenesis did not occur, the molecules necessary for this process appear to have undergone greater divergence between baboon and mouse.     

CD9 is a surface marker on mouse and rat male germline stem cells

Spermatogenesis is dependent on a small population of stem cells. Despite the biological significance of spermatogonial stem cells, their analysis has been hampered by their scarcity. However, spermatogonial stem cells can be enriched by selection with an antibody against cell-surface molecules. In this investigation, we searched for new antigens expressed on spermatogonial stem cells. Using the spermatogonial transplantation technique, we examined expression of the CD9 molecule, which is commonly expressed on stem cells of other tissues. Selection of both mouse and rat testis cells with anti-CD9 antibody resulted in 5- to 7-fold enrichment of spermatogonial stem cells from intact testis cells, indicating that CD9 is commonly expressed on spermatogonial stem cells of both species. Therefore, CD9 may be involved in the common machinery in stem cells of many self-renewing tissues, and the identification of a common surface antigen on spermatogonial stem cells of different species has important implications for the development of a technique to enrich stem cells from other mammalian species.     

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.     

Dorsomorphin Promotes Survival and Germline Competence of Zebrafish Spermatogonial Stem Cells in Culture

Zebrafish spermatogonial cell cultures were established from Tg(piwil1:neo);Tg(piwil1:DsRed) transgenic fish using a zebrafish ovarian feeder cell line (OFC3) that was engineered to express zebrafish Lif, Fgf2 and Gdnf. Primary cultures, initiated from testes, were treated with G418 to eliminate the somatic cells and select for the piwil1:neo expressing spermatogonia. Addition of dorsomorphin, a Bmp type I receptor inhibitor, prolonged spermatogonial stem cell (SSC) survival in culture and enhanced germline transmission of the SSCs following transplantation into recipient larvae. In contrast, dorsomorphin inhibited the growth and survival of zebrafish female germline stem cells (FGSCs) in culture. In the presence of dorsomorphin, the spermatogonia continued to express the germ-cell markers dazl, dnd, nanos3, vasa and piwil1 and the spermatogonial markers plzf and sox17 for at least six weeks in culture. Transplantation experiments revealed that 6 week-old spermatogonial cell cultures maintained in the presence of dorsomorphin were able to successfully colonize the gonad in 18% of recipient larvae and produce functional gametes in the resulting adult chimeric fish. Germline transmission was not successful when the spermatogonia were cultured 6 weeks in the absence of dorsomorphin before transplantation. The results indicate that Bmp signaling is detrimental to SSCs but required for the survival of zebrafish FGSCs in culture. Manipulation of Bmp signaling could provide a strategy to optimize culture conditions of germline stem cells from other species.     

Characterization of spermatogonial stem cell maturation and differentiation in neonatal mice

Initiation of the first wave of spermatogenesis in the neonatal mouse testis is characterized by the differentiation of a transient population of germ cells called gonocytes found in the center of the seminiferous tubule. The fate of gonocytes depends upon these cells resuming mitosis and developing the capacity to migrate from the center of the seminiferous tubule to the basement membrane. This process begins approximately Day 3 postpartum in the mouse, and by Day 6 postpartum differentiated type A spermatogonia first appear. It is essential for continual spermatogenesis in adults that some gonocytes differentiate into spermatogonial stem cells, which give rise to all differentiating germ cells in the testis, during this neonatal period. The presence of spermatogonial stem cells in a population of cells can be assessed with the use of the spermatogonial stem cell transplantation technique. Using this assay, we found that germ cells from the testis of Day 0-3 mouse pups can colonize recipient testes but do not proliferate and establish donor-derived spermatogenesis. However, germ cells from testes of Day 4-5 postpartum mice colonize recipient testes and generate large areas of donor-derived spermatogenesis. Likewise, germ cells from Day 10, 12, and 28 postpartum animals and adult animals colonize and establish donor-derived spermatogenesis, but a dramatic reduction in the number of colonies and the extent of colonization occurs from germ cell donors Days 12-28 postpartum that continues in adult donors. These results suggest spermatogonial stem cells are not present or not capable of initiating donor-derived spermatogenesis until Days 3-4 postpartum. The analysis of germ cell development during this time frame of development and spermatogonial stem cell transplantation provides a unique system to investigate the establishment of the stem cell niche within the mouse testis.     

Clonal origin of germ cell colonies after spermatogonial transplantation in mice

Spermatogenesis originates from a small number of spermatogonial stem cells that can reinitiate spermatogenesis and produce germ cell colonies following transplantation into infertile recipient testes. Although several previous studies have suggested a single-cell origin of germ cell colonies, only indirect evidence has been presented. In this investigation, we tested the clonal origin hypothesis using a retrovirus, which could specifically mark an individual spermatogonial stem cell. Spermatogonial stem cells were infected in vitro with an enhanced green fluorescence protein-expressing retrovirus and subsequently transplanted into infertile recipient mice. Live haploid germ cells were recovered from individual colonies and were microinjected into eggs to create offspring. In total, 45 offspring were produced from five colonies, and 23 (51%) of the offspring were transgenic. Southern blot analysis indicated that the transgenic offspring from the single colony carried a common integration site, and the integration site was different among the transgenic offspring from different colonies. These results provide evidence that germ cell colonies develop from single spermatogonial stem cells.