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Regular Article |
a School of Molecular Biosciences, Center for Reproductive Biology, Washington State University, Pullman, Washington 99164-4660
b Department of Physiology, School of Medicine, Southern Illinois University, Carbondale, Illinois 62901
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gamete biology, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Spermatogonial stem cell transplantation is a technique in which germ cells from a donor animal are injected into the seminiferous tubules of an azoospermic recipient [4, 5]. Following injection, the spermatogonial stem cells migrate or are translocated by Sertoli cells to the basal compartment of the seminiferous tubules. Stem cell mitosis resumes, and spermatogonia enter spermatogenesis and generate donor-derived spermatozoa [5]. This technique can be utilized to answer basic questions regarding spermatogenesis [6] and spermatogonial stem cell renewal [5] and to determine the testicular cell that is responsible for infertility [7, 8]. This technique can potentially employ sperm to transfer genetic information to offspring as well [9].
The spermatogonial stem cell is a difficult cell to study for a variety of reasons. It has no spermatogonial stem cell-specific markers, and only a small number of these cells reside in the testis [10]. Shinohara et al. [11, 12] have enriched for spermatogonial stem cells with the use of both in vitro and in vivo methods. Selecting testicular cells expressing 
- and ß-integrin, either by cell culture [11] or fluorescence-activated cell sorting [12], resulted in significant increases in germ cell colonization efficiency following transplantation. Likewise, transplanting germ cells from infertile donors resulted in enhanced colonization efficiency [13]. These studies indicate that the ratio of spermatogonial stem cells to total cells can be increased utilizing simple biochemical and physical characteristics. This research requires an assay to identify spermatogonial stem cells. Spermatogonial stem cell transplantation is a functional, in vivo system that provides definitive evidence for the presence of spermatogonial stem cells in a cell population.
Vitamin A deficiency in rodents results in multiple physiological effects [2]. One of these effects is a loss of mature germ cells in the testis, resulting in infertility. Interestingly, a single injection of retinol and the inclusion of vitamin A in the diet results in the resumption of spermatogenesis [14]. This indicates that spermatogonial stem cells remain in the testis of vitamin A-deficient (VAD) animals and are capable of initiating spermatogenesis under permissive conditions. Similarly, local testicular heating or hyperthermia exposure results in testicular regression due to germ cell apoptosis, in which most mature germ cells are sloughed into the lumen of the seminiferous tubules and removed [3]. Complete spermatogenesis resumes in the testes of these animals [15].
We hypothesized that these two in vivo models, VAD and hyperthermia, would provide an enriched source of spermatogonial stem cells. The germ cell transplantation technique was used to assess the enrichment of spermatogonial stem cells in the testes of these animals. Our results demonstrate that hyperthermia provides an enriched source of spermatogonial stem cells compared to untreated controls and VAD donors.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Protocols for the use of animals in these experiments were approved by the Washington State University Animal Care and Use Committee and were in accord with National Institutes of Health (NIH) standards established by the Guidelines for the Care and Use of Experimental Animals. Donor cells were recovered from the transgenic mouse line B6,129-TgR(Rosa26)26Sor, originally purchased from the Jackson Laboratory (Bar Harbor, ME) and maintained at Washington State University. These mice express the Escherichia coli LacZ gene during all stages of spermatogenesis which stain blue when incubated with 5-bromo-4-chloro-3-indoyl ß-D-galactoside (X-gal). Animals were housed in a standard animal facility with free access to food and water.
To generate VAD mice, Rosa26 female mice were fed a VAD diet (Teklad Trucking, Madison, WI) for at least 4 wk and bred with Rosa26 males. Male pups received the same diet until they became VAD. At the age of 14–16 wk, when body weight was slightly decreased, animals were killed for germ cell preparation. Control animals were fed a normal diet and killed at 14–16 wk.
Testicular hyperthermia-exposed mice were generated as previously described [16]. Briefly, adult Rosa26 males (age, 12 wk) were anesthetized with an i.p. injection of ketamine (0.1 mg/kg) and xylazine (0.05 mg/kg), and the testis was secured in the scrota and immersed for 15 min in a thermostatically controlled water bath at 43°C. Animals were killed for germ cell preparation 10–15 days following treatment. Control animals were treated in a similar way, except the water bath was 25°C.
Donor testes were enzymatically digested as described by O'Brien [17] with modifications. Briefly, testes were removed, immersed in Hanks buffer, and the tunica removed. Testes were transferred to tubes containing digestion media, which consisted of 0.5 mg/ml of collagenase type IV (Sigma, St. Louis, MO), 0.25 mg/ml of trypsin (Gibco BRL, Bethesda, MD), 0.05 mg/ml of DNase (Sigma) in Ca- and Mg-free Hanks buffer (pH 7.4). Testes were digested for 15 min by shaking at 33°C to dissociate tubules. The tube containing the tubule suspension was transferred to ice, and the tubules were allowed to sediment for 5 min. Supernatant was removed, fresh digestion media added, and the tubules digested for 15 min by shaking at 32–37°C. Following the second digestion, the cell suspension was gently pipetted for several minutes and then centrifuged at 500 x g for 4 min. Cells were resuspended in Hanks buffer containing 0.3 mg/ml of soybean trypsin inhibitor (Sigma), and the cell concentration was determined using a hemocytometer. The cell suspension was centrifuged again at 500 x g for 4 min and then resuspended in EKRB media [17] containing 0.03% trypan blue (Gibco BRL) at a concentration of 107 cells/ml.
Recipient Mice and Analysis
Donor cells were transplanted into testes of immunologically compatible B6,129 mice 4 wk following treatment with busulfan (40 mg/kg). A midline incision was made, and the testis was exposed. A small hole was made in the efferent bundle, and a glass needle containing 7 µl of the germ cell suspension was inserted into the rete testis. The cells were injected into the seminiferous tubules via the rete testis, the testis replaced, and the animal allowed to recover.
The testes of donor animals were analyzed at 3 and 6 wk following transplantation. Recipient mice were killed by cervical dislocation, and the testes were removed. Testes were fixed in 4% (v/v) paraformaldehyde in PBS (pH. 7.4) for 1 h on ice and washed twice in LacZ buffer (0.2 M sodium phosphate [pH 7.3], 2 mM MgCl2, 0.02% (v/v) NP-40, and 0.01% (v/v) sodium deoxycholate) for 30 min. Staining of ß-galactosidase-positive cells was achieved by incubating testes in LacZ stain solution (LacZ buffer containing 20 mM potassium ferricyanide, 20 mM potassium ferrocyanide, and 1 mg/ml of X-gal).
At 3 wk following transplantation, blue-staining donor colonies were counted. At 6 wk following transplantation, blue-staining donor colonies were again counted, and the total area of colonization was determined using NIH Image 1.62 (Bethesda, MD). Both the number of colonies and the total area of colonization were used to measure the degree of donor cell colonization. Statistical analysis was performed by Student t-test or ANOVA. All data are presented as the mean ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To obtain VAD Rosa26 transgenic animals, breeding pairs were fed a VAD diet for 4 wk. No effect on fertility or litter size was seen. The male offspring were maintained on a VAD diet until they reached their maximum weight (age, 
12–13 wk), and then body weight declined. When body weight declined, animals were considered to be VAD and were killed for germ cell transplantation. The average weight of testes from VAD animals was 26.5 ± 5.4 mg (n = 8), in contrast to 100.5 ± 5.7 mg (n = 8) for control testes (Table 1). The number of cells recovered from a VAD testis (2.15 x 106 cells) was 2.5% the number from a control testis (8.5 x 107 cells). Spermatogenesis was not present in the testis of VAD animals, and only spermatogonia and Sertoli cells were observed on the basement membrane (Fig. 1).
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The testes of adult Rosa26 animals were exposed to mild hyperthermia by immersing the scrota in a 43°C water bath for 15 min. A single exposure to heat causes degeneration and loss of germ cells through apoptosis. The loss of germ cells occurs rapidly, and by Day 10 following heat exposure, the majority of germ cells are absent (Fig. 1). Therefore, animals were killed 10–15 days following heat exposure for germ cell transplantation. The average weight of testes from hyperthermia-exposed animals was 52 ± 18.3 mg (n = 12), in contrast to 100.5 ± 5.7 mg (n = 8) for control testes (Table 1). The number of cells recovered from a hyperthermia-exposed testis was 6.25 x 106, which is 7.2% of the number recovered from a control testis (8.5 x 107 cells).
Hyperthermia-exposed testes and testes from VAD mice regress while maintaining a small population of germ cells. In addition, under the correct environmental conditions, spermatogenesis will resume, and germ cells will differentiate normally in these animals. We hypothesized that the testes from these mice would provide an enriched source of spermatogonial stem cells.
Transplantation of Germ Cells from VAD and Hyperthermia-Exposed Testes
Germ cells from VAD and hyperthermia-exposed testes were injected at a concentration of 107 cells/ml into the seminiferous tubules of busulfan-treated mice. A volume of 7 µl was injected into each testis. The extent of colonization was evaluated at 3 and 6 wk following transplantation. Testes were stained with X-gal, and the number of blue donor cell-derived colonies and the total colonized area were determined. Control donors for both experiments were adults maintained on a normal diet. Control donors for the hyperthermia-exposure experiment had their scrota secured and exposed to a 25°C water bath for 15 min. The number of colonies and the area of colonization were not different between the two control groups; therefore, these data were pooled.
With the use of these methods to determine colonization efficiency, we compared the extent of colonization of recipient testes following transplantation of germ cells from VAD and hyperthermia-exposed testes to that of controls. At 3 wk following transplantation, the number of colonies generated by germ cells from VAD and control mice was 14.3 ± 4.3 (n = 7) and 2.6 ± 0.8 (n = 6), respectively (Fig. 2). In addition, the number of colonies generated by germ cells from testicular hyperthermia-exposed donors was 22.56 ± 1.2 (n = 7) (Fig. 2). Therefore, germ cells from VAD donors generated a 5.5-fold increase in stem cell colonization, and germ cells from testicular hyperthermia-exposed donors improved colonization by a factor of 8.6. The total area of colonization was not determined for 3-wk samples due to the small size of the colonies.
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Enrichment of Stem Cells in VAD and Hyperthermia-Exposed Testes
At 6 wk following transplantation, the number of donor-derived colonies and the area of colonization were determined for animals injected with germ cells from VAD and hyperthermia-exposed testes. The number of blue colonies generated by germ cells from VAD mice was 10 ± 1.44 (n = 7), and the total area of colonization was 17.26 ± 1.85 mm2 (n = 7). The number of blue colonies generated by germ cells from hyperthermia-exposed testes and controls was 21.4 ± 1.9 (n = 7) and 4.0 ± 1.9 (n = 7), respectively. Additionally, the total area of colonization for hyperthermia-exposed and control donors was 52.8 ± 6.4 mm2 and 2.79 ± 0.5 mm2, respectively. These data are represented graphically in Figure 3. The number of blue colonies and the area of colonization by VAD donors were increased a factor of 2.5 and 6.2, respectively, when compared to controls (Fig. 3). Similarly, the number of blue colonies and the area of colonization were increased by a factor of 5.35 and 19, respectively, when cells from testicular hyperthermia-exposed samples were compared to controls (Figs. 3 and 4). The transplanted cells from VAD and hyperthermia-exposed testes generated normal-appearing spermatogenesis in recipient seminiferous tubules following transplantation (Fig. 5). Surprisingly, although VAD resulted in testes with greater regression (Table 1), cells from hyperthermia-exposed donors were more efficient in stem cell colonization by a factor of 2.1 for colony number and 3.0 for total area of colonization when compared to VAD donors.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An adult mouse testis contains 20 000–35 000 spermatogonial stem cells, representing 0.02%–0.035% of the total cells in the testis [10]. The number of stem cells in the mouse testis appears to undergo five to six doublings from birth to adulthood [18]. In the adult, spermatogonial stem cells must undergo self-renewal so that males can generate sperm throughout life [10]. Due to the small number of spermatogonial stem cells in the testis, identification of a specific spermatogonial stem cell marker has been difficult [11]. Development of the spermatogonial stem cell transplantation technique, which introduces germ cells into the testis of an azoospermic recipient, has led to new avenues of spermatogonial stem cell research [4, 19]. Stem cells injected into the seminiferous tubules of the recipient will colonize and initiate donor-derived spermatogenesis. This technique provides a functional assay to assess the presence, biological activity, and enrichment of spermatogonial stem cells [13].
Colonization data from the VAD mice donors raise interesting questions regarding the importance of vitamin A to the biology of spermatogonial stem cells. Vitamin A is essential for the maintenance of spermatogenesis [2], and VAD has been utilized as a model to investigate multiple aspects of spermatogenesis [14, 20]. In a VAD animal, the testis is characterized by a loss of differentiating germ cells. Spermatogenesis is arrested at the level of undifferentiated type A spermatogonia, before their differentiation into type A1 spermatogonia [10]. Thus, the seminiferous epithelium in VAD testes contains only undifferentiated spermatogonia, including spermatogonial stem cells. To generate VAD animals, adult females were put on a VAD diet for 3 wk before the addition of males. After the mice pups were born, lactating females were maintained on a VAD diet, and subsequent to weaning, male pups were maintained on a VAD diet until they were killed as germ cell donors. Therefore, male VAD donors were exposed to a VAD diet both during gestation and throughout their life span. Based on the degree of testicular regression observed in VAD animals (75% reduction in testicular wt) and the high number of type A spermatogonia in the testis of a VAD animal, we anticipated a high degree of stem cell enrichment as determined by transplantation. However, VAD donors improved the number of colonies by a factor of 2.5 and the area of colonization by a factor of 6.2. The number of cells recovered from a VAD testis (2.15 x 106 cells) was 2.5% of the number recovered from a control testis (8.5 x 107 cells). The enrichment of stem cells was approximately 5-fold, so the number of stem cells in a testis from a VAD animal is only approximately 12.5% of that in an adult control testis.
The low number of stem cells in the testis of a VAD animal has several possible explanations. First, spermatogonial stem cells may be sensitive to the low vitamin A concentration in VAD animals. As the vitamin A concentration decreases in the testis, spermatogonial stem cells may undergo cell death. Thus, a smaller population of stem cells is available to colonize the recipient testis following injection. Similarly, stem cells may lose the expression of factors that are essential for Sertoli cell recognition and translocation of stem cells to the basement membrane of the seminiferous tubule to initiate colonization following injection. The stem cells would then be removed from the seminiferous tubules with other cells that do not colonize.
Another possible explanation for the low colonization efficiency of stem cells from the testes of VAD animals is that the stem cells do not differentiate during important developmental stages in neonatal mice. Approximately 3 days following birth, gonocytes in the lumen of the developing seminiferous tubule migrate to the basal compartment of the tubule and initiate spermatogenesis [10]. Animals used as donors for the VAD experiments were being nursed by females being fed a VAD diet. Thus, during the critical time of spermatogenesis initiation at 0–5 days postpartum, germ cells in the developing seminiferous tubules were not exposed to vitamin A. Consequently, an important developmental signal may have been absent in these animals, leading to a significant reduction in the number of stem cells in the adult testes. Further experiments are underway to investigate the possible mechanism at work in these animals.
Germ cells from testicular hyperthermia-exposed animals injected into recipient animals improved colonization efficiency significantly when compared to controls and VAD donors. Similarly, Shinohara et al. [13] reported that germ cells from experimentally cryptorchid testes also improved colonization efficiency in transplantation experiments. That differentiating germ cells in testes are sensitive to heat exposure is well-known [3]. After a single exposure to 43°C for 15 min, spermatocytes undergo apoptosis and are eliminated from seminiferous tubules [16]. Experimental cryptorchidism, in which the testis is secured in the body cavity, also results in testicular regression due to testicular exposure to the core body temperature [3]. Spermatogenesis in cryptorchid animals and, presumably, in hyperthermia exposure is arrested at the point when undifferentiated A spermatogonia, specifically Aal spermatogonia, differentiate into A1 spermatogonia [21]. Following testicular hyperthermia exposure, spermatocytes undergo apoptosis and, along with differentiating spermatids, are sloughed into the lumen [16]. This is a rapid process; spermatocyte apoptosis initiates in 12–24 h following exposure and continues for several days. Thus, in hyperthermia-exposed testes, the only cells in the seminiferous tubules are spermatogonia and Sertoli cells. We selected 10–15 days following hyperthermia exposure as the time point at which to harvest donor cells from hyperthermia-exposed testes, because testicular regression was complete and histological examination indicated that the majority of differentiating germ cells were absent from the seminiferous tubule.
The colonization of germ cells from hyperthermia-exposed testes was 6- and 19-fold higher than that in controls based on the number of colonies and the area of colonization, respectively. The number of cells in a control testis (8.5 x 107 cells) was 14-fold greater than the number in a hyperthermia-exposed testis (6.25 x 106 cells). Therefore, the total number of stem cells in the hyperthermia-exposed testis was approximately the same as that in the adult control testis. This indicates that a 15-min treatment at 43°C did not affect stem cell viability. The same conclusion was made for stem cells that were transplanted from an experimental cryptorchid testis [13].
Hyperthermia exposure does not result in testicular regression or stem cell enrichment to the same extent as experimental cryptorchidism. Shinohara et al. [13] reported that the testis weight following experimental cryptorchidism was 41% of the control weight, whereas hyperthermia exposure resulted in a 50% reduction in testis weight. Histological observation of hyperthermia-exposed testes indicated that a small percentage of differentiating germ cells remain following exposure. This may be due to the short duration of the exposure (15 min) or to individual variation between animals. The presence of a small percentage of differentiating germ cells in the hyperthermia-exposure animals is a probable explanation for the lower enrichment that was observed in these experiments when compared to the experimental cryptorchidism germ cell transplantation data. However, hyperthermia exposure has several possible advantages over experimental cryptorchidism. First, the treatment does not require surgery. Second, testicular regression is more rapid, so cells can be obtained for experiments in weeks versus months. Lastly, long-term endocrine disruption will not be a factor, as may be the case in cryptorchidism. Both methods can be used with other species to enrich for stem cells.
Viewed collectively, results from spermatogonial transplantation experiments are shedding light on the biology of spermatogonial stem cells [13]. These results demonstrate that hyperthermia exposure does not affect stem cell biological activity, whereas the low number of stem cells in the VAD testis indicates that vitamin A has important roles in stem cell development or maintenance in mice. A similar result was found with germ cells from Sl mutants, suggesting a role for Sl factor in stem cell development [13]. The characterization and genetic manipulation of spermatogonial stem cells is an important end point that can be facilitated using in vivo models, such as hyperthermia exposure, for stem cell enrichment.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH grant (National Institute of Child Health and Human Development) HD 35494 to M.D.G. and HD 08577-02 to D.J.M. 
2 Correspondence: Michael D. Griswold, School of Molecular Biosciences, Washington State University, Box 644660, Pullman, WA 99164-4660. FAX: 509 335 9688; griswold{at}mail.wsu.edu
Accepted: December 3, 2001.
Received: July 30, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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for development or function. Endocrinology 2000; 141:1273-12766-integrin are surface markers on mouse spermatogonial stem cells. Proc Natl Acad Sci U S A 1999; 96:5504-5509This article has been cited by other articles:
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D. Jeong, D. J. Mclean, and M. D. Griswold Long-Term Culture and Transplantation of Murine Testicular Germ Cells J Androl, September 1, 2003; 24(5): 661 - 669. [Abstract] [Full Text] [PDF]
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