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Testis |
Department of Animal Sciences,4
School of Molecular Biosciences,5 Center for Reproductive Biology, Washington State University, Pullman, Washington 99164
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gamete biology, gametogenesis, sperm, spermatogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The establishment of a stable population of spermatogonial stem cells in the testis begins in the embryo as primordial germ cells (PGCs), which arise from the embryonic ectoderm and migrate to the genital ridge [9]. Sertoli cells differentiate and encompass the germ cells, and the first identifiable seminiferous cords appear while PGCs proliferate. PGCs differentiate into gonocytes that are mitotically quiescent until approximately 3 days after birth in rats and mice, when it appears that gonocytes can migrate to the basement membrane of the seminiferous tubule or remain in the lumen and degenerate. After migration to the basement membrane, it is believed that gonocytes differentiate into spermatogonial stem cells, initiate the first wave of spermatogenesis, or degenerate [10]. In the rat, from Day 0 to Day 4 postpartum there are two populations of gonocytes based upon appearance, round and pseuodopod [7]. The pseudopod subpopulation of gonocytes are capable of colonizing recipient nude mice testes and initiating donor-derived spermatogenesis following transplantation, whereas the round subpopulation does not have this capability. This characteristic appears to be specific to rat gonocytes; murine gonocytes do not have two distinct subpopulations. In mice, the spermatogonial stem cell population in the testis increases 39-fold from neonate to adult [5]. Shinohara and colleagues [11] reported the ability of germ cells from neonatal mouse pups (Days 0–2 postpartum) to colonize recipient mouse testes was limited, especially when compared with cryptorchid adult donors. The ability of gonocytes from neonatal mice to colonize and initiate donor-derived spermatogenesis and the specific age in which gonocytes differentiate into spermatogonial stem cells has not been investigated.
The spermatogonial stem cell transplantation technique is a straightforward assay for detecting the presence and biological activity of spermatogonial stem cells in a suspension of cells. We have used this system to evaluate the biological activity and enrichment of spermatogonial stem cells from vitamin A-deficient and testicular hyperthermia-treated mice to demonstrate unexpected differences in these sources of stem cells [4]. The goal of this study was to critically evaluate the ability of gonocytes and spermatogonial stem cells from neonatal mice to colonize and develop donor-derived spermatogenesis in recipient mice. We hypothesized that gonocytes represent an ideal source of cells to colonize recipient testes because of their primitive nature and ability to differentiate into stem cells. The spermatogonial stem cell transplantation technique provides an ideal assay to evaluate the timing of stem cell differentiation, with the potential to investigate factors necessary for the differentiation of spermatogonial stem cells in vitro.
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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 in accord with National Institutes of Health 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 ß-galactosidase gene in all stages of spermatogenesis, and cells stain blue when incubated with 5-bromo-4-chloro-3-indoyl ß-D-galactoside (X-gal). Therefore, germ cells from Rosa26 mice that colonize recipient testes can be identified by their blue color following staining with X-gal. Animals were housed in a standard animal facility with free access to food and water.
Breeding pairs of Rosa26 mice were monitored daily for litters, and the day of birth was considered Day 0. Groups of male pups from the same litter were used as donor animals. Donor testes were enzymatically digested as described by O'Brien [12], with modifications. Testes were removed and immersed in Hanks buffer, and the tunica was removed. Testes were transferred to tubes containing digestion medium, which consisted of 0.5 mg/ml collagenase type IV (Sigma, St. Louis, MO), 0.25 mg/ml trypsin (GibcoBRL, Bethesda, MD), and 0.05 mg/ml DNase (Sigma) in Ca-, Mg-free Hanks buffer, pH 7.4. Testes were digested by shaking for 15 min 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 medium was added, and the tubules were digested by shaking for 15 min at 37°C. Following the second digestion, the cell suspension was gently pipetted for several minutes and centrifuged at 500 x g for 4 min. Cells were resuspended in Hanks buffer containing 0.3 mg/ml soybean trypsin inhibitor (Sigma), and cell concentration was determined using a hemocytometer. The cell suspension was centrifuged at 500 x g for 4 min and resuspended in EKRB medium [12] containing 0.03% trypan blue (GibcoBRL) 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 were exposed. A small hole was made in the connective tissue enclosing the efferent tubules, and a glass needle containing 7 µl (70 000 cells) of the germ cell suspension was inserted into the rete testis through the efferent tubules. The cells were injected into the seminiferous tubules via the rete testis, the testis were replaced, and the animal was allowed to recover. All efforts were made to inject all 7 µl of the cell suspension into each recipient testis; however, when this was not possible the volume actually injected was determined and used when the recipient testis was evaluated for colonization.
The testes of recipient animals were analyzed 5–6 wk following transplantation. Recipient mice were killed by cervical dislocation, and the testes were removed. Testes were fixed in 4% 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% NP-40, and 0.01% 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 X-gal).
At 5–6 wk following transplantation, blue-staining donor colonies were counted and the total area of colonization was determined using NIH Image 1.62. Both the number of colonies and the total area of colonization were used to measure the degree of donor cell colonization. The number of colonies and total area of colonization were normalized for each recipient testis by accounting for the number of cells injected in each testis. A minimum of three transplantation experiments were conducted for each donor age. Statistical analysis was performed by Student t-test or ANOVA using the number of colonies in each recipient testis per 10 000 cells injected and the total area of colonization in each recipient testis per 10 000 cells injected as the basis of each calculation, respectively. All data are represented as mean ± SEM.
Histological Analysis
Histological analysis of samples was used to evaluate germ cells in donor testes and to identify the location and type of germ cell expressing ß-galactosidase in recipient testes. Testis samples not stained with X-gal were handled using standard techniques and counterstained with hematoxylin. Recipient testis samples stained with X-gal were passed through a series of ethanol solutions for complete dehydration and then washed twice for 2 h with Histo-Clear (National Diagnostics, Atlanta, GA) at room temperature. Samples were then transferred to liquid paraffin (55°C) and incubated overnight. Liquid paraffin was then changed, and samples were incubated at 55°C for an additional 2 h. Samples were then placed in blocks, and 6-µm sections were cut and placed on slides. Deparaffinization was accomplished with Histo-Clear to avoid fading of blue X-gal stain with xylenes. Sections were counterstained with eosin. Images were taken with a CoolSNAPcf digital camera (Media Cybernetics, Silver Spring, MD).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To determine the biological activity of germ cells from neonatal mice, donor testis cells from Rosa26 transgenic mice at Days 0, 1, 2, 3, 4, 5, 10, 12, and 18 postpartum were injected into the seminiferous tubules of busulfan-treated wild-type mice. In addition, germ cells from adult Rosa26 mice were injected into the testes of additional recipients for comparison purposes.
Histological analysis of the testes from Days 0, 3, and 6 postpartum show no differentiating germ cells in the seminiferous tubules (Fig. 1). Gonocytes present in the seminiferous tubules of testes from Day 0 mice were found in the center of the seminiferous tubule (Fig. 1A). By Day 3 postpartum, gonocytes had migrated to the basement membrane of the tubule, but a small number of gonocytes were still present in the center of the tubule (Fig. 1B). At Day 6 postpartum, the gonocytes had differentiated into type A spermatogonia, and no germ cells were visible in the center of the tubules (Fig. 1C). In contrast, at Day 10 postpartum the seminiferous tubules of the testis contained primary spermatocytes (Fig. 2A), and by Day 18 postpartum the most advanced germ cells were pachytene or secondary spermatocytes (Fig. 2B), whereas adult testes contain all types of differentiating germ cells (Fig. 2C).
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The number of cells recovered from the testes of Day 0 to adult animals demonstrates the increased complexity of the testis during development. A total of 0.5 x 106 ± 0.05 cells (n = 12) were recovered from the testes of mice at Days 0, 1, and 2 postpartum, respectively. A dramatic increase in the number of cells recovered occurred at Day 3 postpartum (1.05 x 106 ± 0.06; n = 4), increased slightly at Day 4 postpartum, and remained consistent for the next several days of development (1.3 x 106 ± 0.05 at Days 4, 5, and 7 postpartum, respectively; n = 11). By Day 10 of development, 2.0 x 106 ± 0.12 cells (n = 4) were recovered from the testis, and this number increased as testis development continued until animals became adults (8.25 x 107 ± 0.52; n = 3).
The analysis of busulfan-treated recipient testes 5–6 wk after transplantation demonstrated that germ cells from mice at Day 3 postpartum and younger contributed to low levels of colonization, as evident from the colonies of blue-staining cells (Fig. 3). These colonies were remarkably small, usually consisting of two or three closely associated cells. In contrast, colonies originating from germ cells from testes of Day 4 postpartum and older mice indicated spermatogonial stem cells from these mice formed stem cell niches capable of producing extended areas of donor-derived spermatogenesis (Fig. 4). Germ cells from adult mice testes had colonization patterns similar to those of germ cells from testes of Day 4 postpartum and older mice, albeit at lower levels (data not shown).
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The total number of donor-derived colonies increased dramatically when cells from the testes of Day 3 postpartum and older mice were used as donors (Fig. 5). There were 3.0 times as many colonies derived from testicular germ cells from Day 3 postpartum (16 ± 2.0 per 105 cells) mice than from Day 1 postpartum mice (5 ± 0.5 per 105 cells) or Day 2 postpartum mice (3.5 ± 0.43 per 105 cells). The number of donor-derived colonies originating when testicular germ cells from Day 4 and Day 5 postpartum mice (11.5 ± 0.1 and 20.5 ± 0.2 per 105 cells, respectively) were transplanted was not significantly higher than that for testicular germ cells from Day 3 postpartum mice, respectively (Fig. 5). A further increase in the number of donor-derived spermatogenic colonies was observed when germ cells from the testes of Day 10 and Day 12 postpartum mice were transplanted into busulfan-treated recipients (Fig. 5). Germ cells from the testes of Day 10 and Day 12 postpartum mice (50 ± 4.3 and 52 ± 3.9 per 105 cells, respectively) formed 10 times as many colonies as testicular germ cells from Day 1 postpartum mice and 3.5 times as many colonies as germ cells from Day 3 postpartum mice. Testicular germ cells from Day 28 postpartum mice, in which elongating spermatids are present, formed 25.5 ± 6.3 colonies per 105 cells. This number is significantly lower than the number of colonies from testicular germ cells of Day 10 and Day 12 postpartum mice. The number of colonies formed from Day 28 postpartum donors was not significantly different from the number of colonies from testicular germ cells of Day 5 postpartum mice. Thus, Day 12 postpartum pups are the source of spermatogonial stem cells that provides the highest number of spermatogonial stem cells in relation to the total number of differentiating germ cells in the testis when the testicular environment has not been altered by mutation, temperature, or nutrition.
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The total area of colonization generated by donor testicular germ cells from different postpartum ages was determined by comparing the total blue area in each recipient testis. The size of the colonies is indicative of the potential of colonizing cells to generate donor-derived spermatogenesis. Small areas of colonization indicate that donor cells survive in the recipient testes but do not form a stem cell niche. Germ cells from the testes of Day 0–3 postpartum mice formed very small colonies with little or no expansion beyond the initial site of colonization (Figs. 3 and 6). The area of colonization was much larger when testicular germ cells of Day 4 postpartum and older mice were used as donors (Fig. 6). Total colony area using testicular germ cells from Day 12 postpartum mice was 18 times larger than colony area when testicular germ cells from Day 1–3 postpartum mice were transplanted. These data and the data on the number of colonies formed from each donor indicate that the spermatogonial stem cell potential of germ cells changes significantly at approximately Day 3 postpartum in the mouse testis.
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Histological Evaluation of Recipient Testes
The significant difference in the ability of testicular germ cells from Day 1–3 postpartum mice to colonize and form extended areas of donor-derived spermatogenesis in recipient mice when compared with older animals could be attributed to the inability of cells from the older animals to form or find stem cell niches at the basement membrane of seminiferous tubules. To critically evaluate this possibility, we examined histological sections of recipient testes that were injected with testicular germ cells from Day 1–2 postpartum mice and older donors that had significantly higher levels of colonization. Blue-stained cells from the testes of donor Day 1 postpartum Rosa26 mice migrated to the basement membrane of the seminiferous tubule and appeared to form small cohorts of cells (Fig. 7A). No differentiating germ cells were present, indicating that these cells are dormant or lack the ability to initiate differentiation. However, these cells are located in the area of the seminiferous tubule consistent with the stem cell niche. Donor cells from the testes of Day 5 and Day 10 postpartum mice formed large colonies of differentiating germ cells that filled the recipient seminiferous tubules (Fig. 7, B and C). Therefore, the spermatogonial stem cells that establish these colonies reside in or develop a niche and are capable of initiating germ cell differentiation in recipient seminiferous tubules.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our findings that testicular germ cells from Day 4 postpartum and older mice are capable of initiating donor-derived spermatogenesis indicate that gonocytes must resume mitosis and migrate to the basement membrane of the seminiferous tubules before they differentiate into spermatogonial stem cells. At birth, most gonocytes are not dividing and are found at the center of the seminiferous tubules [14]. At Days 1.5–3 postpartum in mice, gonocytes resume mitosis, and migration of most gonocytes to the basement membrane of the seminiferous tubule resumes after Day 2 postpartum [14, 15]. Thus, the onset of gonocyte migration to the basement membrane of the seminiferous tubule does not occur at precisely the same time in development as does the resumption of gonocyte mitosis [15]. This process appears to be quite dynamic during the first several days postpartum. The relocation to the basement membrane initiates in a small number (1.4%) of gonocytes at Day 18.5 postcoitus. The percentage of gonocytes that have relocated to the basement membrane steadily increases from Day 0.5 postpartum (8.8%) to >60% by Day 2.5 postpartum. In contrast, incorporation of bromodeoxyuridine (BrdU) by gonocytes, indicating mitosis, does not begin until Day 1.5 postpartum [15]. By Day 2.5 postpartum, the majority of relocated gonocytes are BrdU positive (39.5 positive vs. 31.0 negative). Almost all gonocytes (87.4%) have relocated to the basement membrane by Day 4.5 postpartum, with a gradual increase in the percentage of these cells that are BrdU positive (51.5 % by Day 4.5 postpartum). Although the small percentage of gonocytes that fail to relocate to the basement membrane are BrdU positive (6.1%), it is generally accepted that all gonocytes that do not migrate to the basement membrane undergo apoptosis [14–16]. The difference in the timing of resumption of mitosis and relocation of gonocytes suggests that the regulation of these two cellular events is controlled by different mechanisms. In addition, it appears that migration and the resumption of mitosis does not indicate that gonocytes have differentiated into spermatogonial stem cells because testicular germ cells from Day 4 postpartum and older mice are sufficiently differentiated to form stem cell niches and donor-derived spermatogenesis (Fig. 6). Therefore, three cellular events, 1) migration to the basement member, 2) resumption of mitosis, and 3) differentiation into stem cells, that are controlled by different mechanisms occur for the formation of a spermatogonial stem cell population in the neonatal mouse testis.
The inability of testicular germ cells from the testes of Day 0–3 mice to initiate donor-derived spermatogenesis in recipients is in stark contrast to the situation in testicular germ cells of neonatal rats. In the rat, characterization of gonocytes isolated from Day 0–4 postpartum animals indicated that these cells can be divided into two populations, pseudopod and round, based on microscopic examination of morphology [7]. However, we were unable to identify distinct populations of gonocytes, such as pseudopod, in testicular germ cell preparations from Day 0–4 postpartum mice (data not shown). Transplantation of testicular rat germ cells from the pseudopod and round populations into the testes of azoospermic nude mice demonstrated that the pseudopod gonocytes had the ability to produce and maintain colonies of spermatogenesis following transplantation. However, the majority of the cells in the round population underwent apoptosis in vitro and did not form colonies when transplanted. The ability of the pseudopod population to establish male germ line stem cell activity may be associated with the presence of distinct cytoplasmic extensions that allow the cell to migrate to the basement membrane of the seminiferous tubule. Although the timing of spermatogonial stem cell differentiation is different in rats and mice, the seminiferous tubules in mice will support the differentiation of rat spermatogonial stem cells [7].
Histological examination of recipient testes injected with germ cells from Day 0–3 animals indicates that these cells migrate to the basement membrane of the seminiferous tubule (Fig. 7). However, these cells do not initiate donor-derived spermatogenesis, implying they do not form a stem cell niche. Thus, testicular germ cells from Day 0–3 postpartum mice migrate to the location where stem cell niches are found but do not differentiate into spermatogonial stem cells. These data suggest that the testicular stem cell niche is dependent not only on the recipient tubule environment but also on the donor cell and possibly interactions between germ cells and Sertoli cells. Transplantation of gonocytes that do not establish complete spermatogenesis represents an attractive model for comparison with stem cells that do establish a niche to investigate the cellular and environmental factors important in initiating spermatogenesis.
The final objective of this work was to determine the age of the donor that will produce the best colonization of recipient testes when the donor testis environment is not manipulated. The number of spermatogonial stem cells in the testis increases 39-fold from neonate to adult; however, this increase occurs at the same time germ cell maturation into spermatozoa is ongoing, creating a large number of differentiating germ cells [5]. We determined that the age of development for the highest number of donor-derived colonies in proportion to the number of cells injected was Day 12 postpartum (Fig. 5). After Day 12 postpartum, the accumulation of differentiating germ cells in the testis appears to outpace the formation of spermatogonial stem cell niches (Fig. 5). Therefore, the neonatal testis after Day 5 is an attractive source of enriched spermatogonial stem cells without the need of testicular environmental manipulation (i.e., cryptorchism, testicular hyperthermia, vitamin A deficiency, genetic mutations, etc.).
The application of the spermatogonial stem cell transplantation technique to other species is constantly expanding [17]. The ideal environment and donor cell type for each species may be different. The highest rates of fertility in mice are achieved with adult cryptorchid donor cells injected into the testes of mouse pups of the naturally occurring W/Wv mutant [5]. Similar donors or recipients may be difficult to obtain in other species or other situations. Therefore, continued application of the transplantation technique in investigations of spermatogonial stem cell biology and activity are essential for a better understanding of spermatogenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Derek J. McLean, Department of Animal Sciences, Washington State University, Box 646353, Pullman, WA 99164-6353. FAX: 509 334 4246; dmclean{at}wsu.edu
3 Current address: Women's Health Research Institute, Wyeth Research, Collegeville, PA 19426
Received: 10 March 2003.
First decision: 25 March 2003.
Accepted: 14 August 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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