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Research Article |
Horizontal Medical Research Organization,3
Department of Molecular Genetics,4
Center for Medical Education,5 Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan
RIKEN,6 Bioresource Center, Ibaraki 305-0074, Japan
ABSTRACT
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.
developmental biology, gametogenesis, Sertoli cells, spermatogenesis, testis
INTRODUCTION
Spermatogenesis originates from a self-renewing population of spermatogonia called spermatogonial stem cells [1, 2]. These cells are the only stem cells in the body that can transmit genetic information to offspring, and they have enormous proliferative potential to sustain spermatogenesis throughout the life span of animals [3]. Spermatogonial stem cells are potentially useful in infertility treatments, animal transgenesis, or regenerative medicine because of their unique biological properties [4–7]. Despite their importance, the study of these cells has been hampered by the lack of a functional assay system to identify stem cells. Because stem cells can be identified only by their ability to self-renew, it is difficult, on the basis of morphological analysis, to distinguish these cells from spermatogonia that are committed to differentiate.
To overcome this problem, Brinster et al. reported a functional assay for spermatogonial stem cells in 1994 [8]. Dissociated donor testis cells produce colonies of spermatogenesis following transplantation into the seminiferous tubules of an infertile recipient testis. The transplanted stem cells migrate through the tight junction between Sertoli cells and proliferate on the basement membrane [9]. The colonies first expand horizontally on the basement membrane, but they start to differentiate vertically in the center of the colony and undergo meiotic division and spermiogenesis [9]. Stem cells are thought to increase their number at the margin of the colony, whereas they start to differentiate at the center. The growth of colonies continues for several months, and mature spermatozoa are observed 3 mo after transplantation [9]. The recipient animals eventually produce offspring from the donor cells [10]. By definition, only stem cells can initiate and maintain long-term spermatogenesis and produce these results. This transplantation technique provided a new opportunity for studying and manipulating spermatogonial stem cells.
Although the transplantation technique was pioneered more than a decade ago, one of the questions that remains to be answered is the clonal origin of germ cell colonies. The technique has been used for quantifying the number of stem cells [11, 12], but this has been based on the assumption that colonies are derived from single stem cells, which remains to be proven. The single-cell origin of germ cell colonies was supported by the linearity of the curve relating the number of germ cells transplanted to the number of colonies that developed in the recipient testis [13]. In another study, Zhang et al. transplanted the mixed testis cell populations from two different transgenic mice, excised the individual colony, and examined its origin by PCR analysis [14]. Their study revealed that only one genotype was present in the colony when there were less than 20 colonies in the testis. Although these studies lend support to the clonal origin of stem cell colonies, they do not eliminate the possibility that the colonies originated from two or more cells that had remained aggregated during the preparation of the testis cell suspension. In addition, PCR-based assays are often accompanied by problems in specificity and reproducibility, and different approaches are required to resolve this problem.
In this study, we attempted to test the hypothesis that colonies are derived from single stem cells using retroviral transduction of spermatogonial stem cells and microinsemination [7, 15, 16]. Retroviruses integrate into the genome randomly; they have been used extensively to mark stem cells clonally to trace their differentiation patterns in various stem cell systems [17–19]. Donor germ cells were transduced with enhanced green fluorescence protein (EGFP)-expressing retrovirus, and offspring were produced using germ cells that developed from EGFP-expressing colonies by microinsemination. The genotype of the resulting animals was determined to see whether each colony arose from a single stem cell.
MATERIALS AND METHODS
Animals and Transplantation
The Institutional Animal Care and Use Committee of Kyoto University approved all animal experimentation protocols. Donor testis cells were collected from 5- to 10-day-old C57BL6 (B6) male mice (Japan SLC, Shizuoka, Japan). Testis cells were dissociated using a previously described two-step enzymatic digestion method [20]. For transplantation, busulfan-treated C57BL/6 x DBA/2 F1 (BDF1) mice or congenitally infertile WBB6F1-KitW/KitW-v mice (designated W; both from Japan SLC) were used as recipients. BDF1 mice were used for germ cell transplantation at least 4 wk after the intraperitoneal injection of busulfan (44 mg/kg) at 4 wk of age [20]. W mice lack all stages of differentiating germ cells because of mutations in the gene that encodes KIT receptor tyrosine kinase [21]. For the microinjections, approximately 10 µl of the donor cell suspension were introduced into the seminiferous tubules of a BDF1 testis, whereas 2 µl were microinjected into a W testis [22]. Cells were microinjected through the efferent duct [20], and 75%–85% of the tubules in each recipient testis were filled.
Retrovirus Preparation
An overview of the experimental design is outlined in Figure 1. To introduce retroviral genes into spermatogonial stem cells, we used a recently reported in vitro retrovirus-mediated gene transfer technique [7], which led to the production of transgenic offspring after the transplantation of transduced stem cells into recipient testes. We used two types of vectors that express EGFP genes under different promoters. The pMy-internal ribosomal entry site (IRES)-EGFP vector (designated pMy) was a generous gift from Dr. T. Kitamura (Tokyo University, Japan). The pGpig vector was constructed from a replication-defective ecotropic retroviral vector pGen- [23]. An EGFP (Clontech, Palo Alto, CA) attached downstream of an encephalomyocarditis virus IRES sequence was inserted under phosphoglycerate kinase 1 (Pgk1) promoter (gift from Dr. T. Nomura, Kyoto University). Whereas the pMy vector depends on the modified long-terminal repeat (LTR) for EGFP expression, the LTR is inactivated in pGpig, so EGFP gene expression depends on the internal Pgk1 promoter. These viral vectors were previously used to transduce different kinds of stem cells [7, 24].
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Viral particles were produced by the transient transfection of packaging cells. The Plat-E packaging cell line was transiently transfected with retrovirus cDNA by Fugene6 (Roche Diagnostics, Tokyo, Japan) [25]. The supernatant, which was concentrated as previously described [26], was collected 2 days later. The final titer of the virus was 
109 or 108 colony-forming units/ml for pMy or pGpig, respectively.
Retroviral Infection of Spermatogonial Stem Cells
For the donor cells, we collected testes from 5- to 10-day-old B6 pups. The testes of this stage contain an enriched population of spermatogonial stem cells and have a higher frequency of retrovirus infection than those from mature testes [7, 22]. After dissociation, 1.2–1.8 x 107 testis cells were placed on mitotically inactivated mouse embryonic fibroblasts, and the mixture was centrifuged at 1750 x g for 1 h at 32°C in the presence of 4 µg/ml polybrene [27]. After a 1- to 2-day culture at 37°C in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS), the infected cells were washed twice with PBS, and 55%–95% of the input cells were harvested by trypsin digestion. The cells were suspended in DMEM supplemented with 10% FCS and transplanted into recipient testes. This technique allows competent donor cells to colonize into empty niches to permit the regeneration of donor stem cells [8]. Busulfan-treated adult BDF1 mice were used for the transplantation of pMy-infected cells, and congenitally infertile W pups were used for the transplantation of pGpig-infected cells. In a total of six experiments, approximately 1.4–2.1 x 106 pMy-infected cells were transplanted into BDF1 recipients, whereas 1.1–1.5 x 105 pGpig-infected cells were transplanted into W recipients. The viability of the cells was greater than 95% as determined by trypan blue exclusion.
Microinsemination
The seminiferous tubules of recipient testes were dissected under UV illumination, which allowed for the specific identification of donor cells because the recipient testis does not exhibit endogenous fluorescence. Fragments of EGFP-expressing seminiferous tubules were recovered, and each fragment was dissociated mechanically to collect the germ cells. We chose colonies that were longer than 1 mm to ensure sufficient recovery of germ cells. In these colonies, germ cells occupied 100% of the basal surface of the tubule. The number of colonies was quantified by counting the total number of colonies observed under a stereomicroscope. Microinsemination was performed as previously described by the intracytoplasmic injection into BDF1 oocytes [15]. After in vitro culture, two- or four-cell-stage embryos were transferred to the oviducts of Day 1 pseudopregnant Institute for Cancer Research (ICR) female mice (Japan SLC). Offspring were born by cesarean section on Day 19.5.
Southern Blot
Genomic DNA was isolated from the offspring by phenol/chloroform extraction, followed by ethanol precipitation [5]. Twenty micrograms of DNA were digested with indicated restriction enzymes and separated on a 1.0% agarose gel. DNA was transferred and blotted onto a nylon membrane (Hybond-N+; Amersham Biosciences, Buckinghamshire, UK). Hybridization was performed according to a conventional protocol. The full-length EGFP cDNA was used as a probe for hybridization. The membrane was hybridized for 16 h at 65°C with a 32P-labeled probe.
Statistical Analysis
Statistical analysis was performed using ANOVA.
RESULTS
Retroviral Infection of Spermatogonial Stem Cells In Vitro
To assess the single-cell origin of germ cell colonies, we used two retroviral vectors to mark spermatogonial stem cells. Although we failed to observe EGFP-positive colonies after transplantation of pMy-infected cells, we found that following the transplantation of pGpig-infected cells into W pup testes, the transplantation of cultured cells resulted in the colonization of all recipient testes (Table 1) and that EGFP-expressing colonies were observed (Fig. 2, A and B). The number of colonies ranged from 2 to 18 per testis.
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After mechanical dissociation of the fluorescent colonies (Fig. 2B), we observed EGFP-expressing germ cells and found that 60.9% ± 5.0% (mean ± SEM, n = 3) of the cells showed fluorescence under UV light. Round spermatids and spermatocytes are round cells without tail and could be identified by their morphology. They commonly show low nuclear/cytoplasmic ratio and characteristic nuclear appearance. Because of weak activity of the Pgk1 promoter in the later stage of spermatogenesis [28], EGFP expression in germ cells became weaker as the cells differentiated. Although round spermatids showed relatively strong fluorescence, only a portion of elongated spermatids showed weak fluorescence (Fig. 2, C and D). EGFP expression in spermatozoa was barely detectable. Nevertheless, these results indicated that pGpig-infected donor stem cells colonized the recipient testes and completed spermatogenesis.
Production of Transgenic Offspring by In Vitro Microinsemination
We chose two different W recipients that received a transplantation of pGpig-transduced testis cells to examine whether each colony was derived from a single stem cell. In total, six EGFP-positive colonies were removed from the recipient testes under UV light 20 wk after the transplantation of donor cells. The length and intensity of EGFP fluorescence varied among the six colonies, suggesting the different integration sites of the retrovirus. Of these six colonies, elongated spermatids with tails were recovered from five colonies after mechanical dissociation (designated colonies 1–5). These cells were generally nonmotile and were picked by micromanipulation for injection into BDF1 oocytes. In total, 212 embryos were constructed, and 162 embryos that reached the two- or four-cell stage were transferred into 11 pseudopregnant ICR mothers. Although 1 of the 11 recipients died after embryo transfer, the remainder gave birth to normal offspring by cesarean section at term. The number of offspring ranged from 4 to 19 for each group; 45 offspring were obtained in total (Fig. 2E).
To determine the presence of the retroviral vector in the genome, we recovered DNA from all the offspring and performed Southern blot analysis using an EGFP-specific probe. Genomic DNA was digested with Sca I or Dra I, both of which do not cleave within the probe. Thus, the EGFP probe hybridized with a single unique-size fragment from each retroviral integration. Of the 45 offspring, 23 (51.1%) contained the retroviral transgene. Transgenic offspring were produced from four of five colonies, and 33.3%–73.7% of the offspring from each colony contained the transgene (Table 2).
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Analysis of the offspring from colonies 2–4 showed that all the transgenic animals that were derived from the same colony showed bands of identical size by Sca I digestion (Fig. 3A). This relationship was conserved with Dra I digestion, indicating that they represent a common integration event. Furthermore, transgenic animals from different colonies showed bands of different sizes, indicating that they represent different integration events. The differences among these groups were statistically significant (P < 0.0001 by ANOVA). In contrast, offspring from colony 1 had three different patterns when they were analyzed by the same approach. Of the 19 total offspring, 8 of the 14 transgenic offspring had two bands (16.5 kb and 7.3 kb), and the remaining six offspring had only one band (two at 16.5 kb and four at 7.3 kb). A similar relationship was confirmed with Dra I digestion (Fig. 3B). These results show that two retroviral integrations occurred at different positions in this stem cell and that the two transgenes were transmitted independently by segregation during meiosis, eventually producing three patterns in the offspring. Taken together, our results indicate that the transgenic offspring from individual colonies were identical in stem cell origin.
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DISCUSSION
The colonization of spermatogonial stem cells into recipient testes has provided the first functional assay for spermatogonial stem cells [8]. It involves a complicated process of cell migration into the niche and subsequent expansion of stem cells and differentiation into progenitor cells. The process of stem cell colonization is apparently nonphysiological because stem cells migrate to the basal side and colonize a niche when they are injected into the adluminal side of seminiferous tubules. While germ cells normally differentiate from the basal part to the adluminal lumen [1, 2], the colonized stem cells could still perform spermatogenesis, producing gametes that are normal in appearance and function. Although several morphological studies were conducted on how stem cell colonies develop after transplantation [9, 29, 30], it remains unclear whether the colonies developed from clonal proliferation of a single stem cell.
In concept, our approach mirrors those previously taken to demonstrate the clonal origin of hematopoietic stem cells. It was shown in the early 1960s that transplanted hematopoietic cells produce spleen colonies after intravenous injection into lethally irradiated recipient mice [31]. To demonstrate their single-cell origin, the transplantation of mixed donor cells was attempted, and the results of these studies supported this hypothesis [32–34]. These studies, however, could not exclude the possibility that colonies are derived from aggregated donor cells. The most rigorous evidence of the clonal nature of colonies was provided by transplanting irradiated bone marrow cells [35]. Irradiation induced unique chromosomal damage to the donor cells, which served as a specific marker to trace the progenitor cells that developed from single cells. However, we did not apply this technique for two reasons. First, we speculated that radiation-induced chromosomal damages might influence the normal differentiation of germ cells. It has been known that chromosomal abnormalities are often found in infertile males [36]. Second, accurate cytological analysis of a small number of cells is technically difficult. In our previous study with single-colony analysis, only 104–105 cells could be recovered from single colonies [37].
We used a retroviral marking technique to overcome these problems. Retroviral marking of stem cells has been widely used to study the kinetics or fate of stem cells from several self-renewing systems, including the hematopoietic system [17–19]. Retroviruses can randomly integrate into the genome of donor cells, which allows for the clonal identification of stem cells. In the spermatogenic system, it was recently shown that the in vitro transduction of donor cells and subsequent transplantation into infertile recipients led to the labeling of spermatogonial stem cells [7, 38]. These studies showed that the unique marking of stem cells by a retrovirus is an effective method for tracing the dynamics of stem cells, which may be applicable to demonstrate the clonal origin of stem cell colonies. Another important advantage of the current approach is in vitro microinsemination [39, 40], which allows the development of offspring from a small number of germ cells that could be recovered from pieces of seminiferous tubules. Not only mature spermatozoa but also more immature germ cells, such as round spermatids or primary spermatocytes, can be used to fertilize eggs for offspring production [39, 40]. Because of these advantages, the technique has revolutionized conventional assisted reproduction techniques and is widely used to treat infertile animals and men [15, 16]. In this study, these techniques were combined and used as tools for the clonal analysis of germ cell colonies. This is in contrast to traditional approaches for lineage analysis of somatic cells, which depend on DNA analysis of small tissues or the retransplantation of colonies into secondary recipients. Microinsemination provided a unique and reliable method of analyzing the donor origin of germ cell colonies.
The results in the present study provide new evidence in support of the clonal origin of germ cell colonies. In the present experiments, the injected testes of the recipient mice were analyzed 4–5 mo after transplantation, which corresponds to approximately four spermatogenic cycles in mice [1, 2]. Therefore, differentiated progenitor cells, even if they were infected, would disappear, and only stem cell-derived colonies would be generated [9]. Although periodic infection during several days was necessary for infection of mouse and rat spermatogonia [7, 41], infection could be achieved even after overnight culture, suggesting that a combination of concentration and centrifugation is a more efficient method for spermatogonia transduction. However, the infection of stem cells was clearly demonstrated only with pGpig vector, and we did not observe EGFP signal after pMy infection. A possible explanation is that expression silencing occurred with pMy vector. Alternatively, the lower seeding efficiency of stem cells in adult recipients may not have allowed the colonization of the small number of transduced stem cells [22].
Overall, the efficiency of transgenic offspring production reached 50% and surpassed the results of previous studies (5%–30%) in which transgenic offspring were produced by natural mating [7, 38]. The current efficiency (50%) is likely the upper limit expected with this vector because the EGFP transgenic marker was expressed predominantly in the diploid stage and we could not distinguish elongated spermatids that carried the transgene for microinsemination. The efficiency could be improved by the use of a haploid promoter for EGFP expression. Partly because of this problem, we failed to produce transgenic offspring from colony 5 because of the low amounts of spermatozoa that were present in the colonies. Nonetheless, transgenic offspring were produced from four other colonies, which validated our approach for the genetic analysis of colony formation. Of the four colonies that produced transgenic offspring, three colonies (colonies 2–4) were shown to produce offspring with a consistently identical viral integration site. The pattern was specific to each colony and differed from one to another. The viral integration must have occurred at the stem cell level because differentiated progenitor cells, even if they were infected, would have differentiated and disappeared by the time of analysis. Therefore, these results clearly indicate that the transgenic progeny originated from different stem cells and that each infected stem cell underwent self-renewal division to produce committed progenitors bearing the same viral integration site.
In contrast, the Southern blot analysis of colony 1 showed that their transgenic offspring had three patterns of viral integration. However, further analysis using different restriction enzymes showed that the integration event in the offspring having a single band was identical to one of the two events that occurred in different offspring. It suggests that these patterns resulted from the segregation of two transgenes during meiosis; some of the progeny inherited only one of the transgenes, and others inherited two transgenes. Therefore, we can conclude that this result was produced by the two viral integrations that occurred at the diploid stem cell level, supporting the clonality for colony 1. Although multiple integrations of the transgenes do not generally pose this type of problem in the lineage analysis of somatic stem cell systems, the present case suggests that care should be taken for the interpretation of results when it is applied to germ-line cells; meiotic recombination may alter the distribution of viral integration sites.
Although our results suggest the clonal origin of spermatogonial stem cell colonies, they do not exclude the possibility that multiple stem cells exist in a niche [42]. Indeed, our retransplantation experiment previously showed that a single colony contains multiple stem cells after spermatogonial transplantation [37]. However, we still do not know how many stem cells maintain spermatogenesis, the normal life span of the spermatogonial stem cell, or their possible regulatory mechanisms. Although these questions are difficult to study directly in normal testes, approaches taken in this study, namely, retroviral marking and single colony analysis after transplantation, may provide a simple model system to dissect the complex processes of spermatogenesis. The use of EGFP will be particularly useful because it allows the vital analysis of stem cells and their progenitors. These types of studies will ultimately lead to a better understanding of the spermatogenesis mechanism.
ACKNOWLEDGMENTS
We thank Ms. A. Wada for her technical assistance.
FOOTNOTES
1 Supported by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan and by grants from CREST and the Human Science Foundation (Japanese) and also supported in part by Special Coordination Funds for Promoting Science and Technology from MEXT. 
2 Correspondence: Mito Kanatsu-Shinohara, Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto University 53 Shogoin, Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. FAX: 81 75 751 4169; mshinoha{at}virus.kyoto-u.ac.jp
Received: 30 January 2006.
First decision: 1 March 2006.
Accepted: 5 April 2006.
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locus by Stat5: implication for accessibility control by the IL-7 receptor. Immunity 1999 11:213-223[CrossRef][Medline]This article has been cited by other articles:
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