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Biology of Reproduction 66, 944-949 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Functional Analysis of Stem Cells in the Adult Rat Testis1

Kyle E. Orwiga, Takashi Shinohara3,a, Mary R. Avarbocka, and Ralph L. Brinster2,a

a Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.

developmental biology, gamete biology, male reproductive tract, spermatogenesis, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Spermatogenesis is a highly organized and productive process that generates 107 mature sperm per gram of testis per day in the rat [1, 2]. Spermatogonial stem cells lie at the foundation of this process and, like other adult stem cells, have the ability to self-renew and generate progenitors that produce large numbers of differentiated progeny to maintain and/or regenerate spermatogenesis. Spermatogenesis is among the most productive of self-renewing systems, with approximately 12 amplifying divisions between the stem cell and the differentiated product [2]. Thus, a single stem cell in the rat testis can theoretically give rise to 4096 spermatozoa [2], and similar levels of production are found in hematopoiesis [3]. Modification of a single spermatogonial stem cell can therefore have a dramatic impact on the tissue.

Due, in part, to their potential for regenerating unhealthy or damaged tissues, stem cells have become the focus of intense investigation. It has been suggested that adult stem cells from various tissues and species might share molecular characteristics that could provide general insight into stem cell biology [4–8], and several recent studies have provided evidence in support of this view [6–8]. The hematopoietic stem cell has been the most extensively studied adult stem cell. Development of a functional assay, over 40 yr ago [9, 10], enabled investigators to identify biochemical markers [11], develop enrichment strategies, and eventually purify the hematopoietic stem cell [12, 13]. The male germ line is the only other adult self-renewing system for which a stem cell functional assay is available [14, 15]. A unique feature of the spermatogonial stem cell transplantation assay is that donor-derived spermatogenic colonies are believed to remain in close association with the transplanted stem cells that produced them, thus allowing quantitative and qualitative analysis of clones derived from individual donor stem cells [16]. Although neural and epidermal stem cells can be transplanted [17–20], these systems do not lend themselves to quantitative analysis of donor cell populations. However, these stem cell populations can be expanded in culture [21–23], a feature not available for other adult stem cell systems. Therefore, all self-renewing systems, each with its attendant attributes and limitations, contribute to the combined knowledge about adult stem cells.

The recent advent of a transplantation assay allowed the definitive identification of mouse spermatogonial stem cells based on their ability to produce spermatogenic colonies in mouse recipient testes [14, 15]. Using this assay, several markers and enrichment strategies for the male germ line stem cell in the mouse have been identified [24–26], providing a valuable resource for further genetic and biochemical characterization. It has been demonstrated that {alpha}6- and ß1-integrins are present on the mouse spermatogonial stem cell, which accounts at least in part for the ability of laminin selection to enrich testis cell populations for stem cell activity [24]. In contrast, the c-Kit receptor is absent or present in very low concentration on the stem cell [24, 26] but is expressed as spermatogonia differentiate [27].

Although many of the classical studies on spermatogenesis and testis development were performed in the rat, and considerable morphometric and physiologic data exist about the processes involved in this species, the spermatogonial transplantation assay has not been fully developed for the rat. Testis cell transplantations have been reported for rat to mouse [28], rat to rat, and mouse to rat [29], but these studies did not address the biochemical and physical characteristics of rat spermatogonial stem cells. Therefore, further development of the rat testis model to analyze adult stem cells is critical because the rat has several advantageous experimental characteristics. First, like the mouse, small size, short gestation, and large litter sizes make the rat amenable to laboratory investigations. Second, the rat provides more tissue than the mouse, a particular advantage in systems in which the cells of interest are rare and purification procedures yield only a small number of cells. Third, regeneration of spermatogenesis after irradiation or chemotherapy in humans resembles that in rats more than that in mice [30]. Finally, the rat testis might provide unforeseen advantages for the identification, isolation, and evaluation of adult stem cells that are not evident in the mouse or in other tissues.

The present investigation revealed important and unexpected advantages of the rat model for the examination of male germ line stem cells. Rat testes contain about 120-fold more spermatogonial stem cells than mouse testes. In addition, 8.5-fold enrichment of rat spermatogonial stem cells can be achieved by simple selection on laminin-coated plates. Therefore, the rich source of spermatogonial stem cells in the rat testis will provide a foundation for continued investigation and development of this model of adult stem cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Donor Animals and Cell Collection

Mouse donor testis cells were obtained from adult (16–17 wk old) mice from the transgenic line B6;129S-Gtrosa26 (designated ROSA26; The Jackson Laboratory, Bar Harbor, ME), which expresses the Escherichia coli lacZ (E. coli lacZ) transgene in all spermatogenic cell types, with ß-galactosidase localized in the cytoplasm (Fig. 1, A and C, [31]). Rat donor testis cells were obtained from adult (12–24 wk old) Sprague-Dawley rats carrying a fusion transgene composed of the mouse metallothionein I (MT) promoter driving the expression of the lacZ structural gene [32]. The MT-lacZ transgene is expressed in differentiating germ cells and some Sertoli cells and encodes a nuclear-localized ß-galactosidase protein (Fig. 1, B and D, [28]). For some experiments, donor rats were made cryptorchid by surgically removing the testes from the scrotum, severing the gubernaculum, and ligating the testis high in the abdominal cavity [29]. Expression of the lacZ transgene and production of the ß-galactosidase protein in donor testis cells allows the unequivocal identification of donor-derived spermatogenic colonies in recipient testes after staining with the substrate 5-bromo-4-chloro-indolyl ß-D-galactoside (X-gal [16]). Single-cell suspensions from rat and mouse donor testes were prepared by enzymatic digestion [14, 33, 34]. Cells for transplantation were suspended in Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) at concentrations of 1–2 x 107 cells/ml for wild-type rat and mouse and 2–3 x 107 cells/ml for cryptorchid rat.



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  		FIG. 1. Macroscopic and histologic appearance of mouse and rat adult donor testes. ROSA26 adult mouse testes (A and C) express the lacZ transgene in all stages of spermatogenesis (C), as demonstrated by blue staining in the presence of the X-gal substrate. MT-lacZ adult rat testes (B and D) express the lacZ transgene in germ cells (D). Nuclear fast red counterstain (C and D). Bar = 5 mm (A and B), 100 µm (C and D)

Laminin Selection

Laminin-coated plates (60 mm) were prepared as previously described [24, 25]. For each experiment, 4 x 107 adult wild-type rat testis cells were suspended in 8 ml DMEM containing 10% FBS and divided among four laminin-coated plates. Cells were incubated for 15 min at 37°C, followed by five washes with PBS to remove unbound cells. Attached cells were removed by trypsin (0.25%)-EDTA (1 mM) digestion for 5 min at 37°C, followed by strong pipetting. Cells were prepared for transplantation by resuspension in DMEM 10% FBS at concentrations of 0.72–0.83 x 107 cells/ml.

Recipient Mice and Transplantation Procedure

Rat and mouse donor testis cell populations were transplanted into immunologically compatible NCr Swiss nude (nu/nu; Taconic, Germantown, NY) recipient mice that were treated with busulfan (44 mg/kg, Sigma, St. Louis, MO) at 4–6 wk of age [14, 28, 35]. Busulfan-exposed recipient testes are virtually devoid of endogenous germ cells, and approximately 10 µl of donor testis cell suspension can be introduced per testis at the time of transplantation, about 6 wk after busulfan treatment. Recipient mice were anesthetized by Avertin injection (640 mg/kg, i.p.). The Animal Care and Use Committee of the University of Pennsylvania approved all experimental procedures in accordance with The Guide for Care and Use of Laboratory Animals of the National Academy of Sciences (Assurance no. A3079-0).

Analysis of Recipient Testes

Testes of recipient mice were collected 3 mo after donor cell transplantation, stained with X-gal to visualize donor-derived spermatogenesis [16], and analyzed by a computer-assisted imaging system [36]. Donor spermatogonial stem cells are defined by their ability to produce blue colonies of spermatogenesis in recipient testes, and each colony is thought to be clonally derived from a single spermatogonial stem cell [16, 36]. Differentiated germ cells cannot produce and sustain colonies of spermatogenesis, and endogenous germ cells do not express the lacZ transgene. Colony number and colony length were determined to evaluate donor-derived spermatogenesis in recipient testes. Because donor testis cell concentrations varied, colony number was normalized to 105 or 106 cells injected per testis.

Statistics

Our analyses sought to identify statistically significant differences between donor testis cell populations with regard to colony number and colony length. The outcomes are presented using means and SEMs, and log transformations were used to obtain approximately normal distributions. To account for the repeated measures on each animal (from left and right testes), generalized estimating equations methodology was used [37]. This methodology accounts for the correlation structure between the repeats and does not delete any animal having some missing repeats (as a repeated-measures ANOVA would do). The models examined the effects of donor and were adjusted for differences in experimental day. Models for each outcome were run using the original, untransformed data and the log-transformed data. Only the results of the original data are presented since the conclusions did not differ when using the log-transformed data. Pairwise comparisons were performed for the enrichment study and Bonferroni corrections were made to the P values to adjust for multiple comparisons.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Spermatogonial Stem Cell Activity in Rat and Mouse Donor Testis Cell Populations

Wild-type adult ROSA26 mouse and MT-lacZ rat donor testis cell populations were transplanted into immunodeficient NCr nude recipient mice to compare stem cell activity, by concentration (colonies per 106 cells injected) and colony size (colony length in millimeters). Each blue, donor-derived colony of spermatogenesis is thought to arise from a single transplanted stem cell, thus allowing clonal analysis of donor stem cell activity [16, 36]. Mouse donor testes weighed 78.8 ± 4.3 mg and contained 33.2 ± 7.0 x 106 cells per testis (Table 1). Rat donor testes were 24 times larger than mouse donor testes (1910.8 ± 74.6 mg) and contained 12.6-fold more cells (418.5 ± 31.2 x 106 cells, Table 1). Evaluation of recipient testes 3 mo after transplantation revealed that rat testes had a 9.45-fold higher concentration of spermatogonial stem cells than mouse testes (169.2 ± 17.1 vs. 17.9 ± 4.0 colonies per 106 cells injected, P <= 0.0001, Table 1; Fig. 2, A and B). Based on these results, the total number of functional stem cells per rat testis is calculated to be 70 810 [(169.2 colonies/106 cells) x (418.5 x 106 cells per testis)]. In contrast, our analysis detected 594 functional stem cells per testis in the mouse [(17.9 colonies/106 cells) x (33.2 x 106 cells per testis)]. Thus, the rat testis contains about 120 times more stem cells than the mouse testis (Table 1). Both rat and mouse donor cells produced complete spermatogenesis in recipient mouse testes, characterized by multiple germ cell layers and mature spermatozoa in the tubular lumen (Fig. 2, C and D)


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  		TABLE 1. Analysis of stem cells in mouse and rat testes.a



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  		FIG. 2. Macroscopic and histologic appearance of recipient testes transplanted with mouse and rat adult donor testis cells. NCr nude recipient mouse testes were stained with X-gal 3 mo after transplantation to visualize mouse (A and C) and rat (B and D) donor-derived spermatogenic colonies. Both mouse and rat donor testis cells generated complete spermatogenesis, characterized by multiple germ cell layers and mature spermatozoa, in recipient testes. Note that the lacZ stain is localized in the cytoplasm of ROSA26 mouse testis cells (C) and the nucleus of MT-lacZ rat testis cells (D). Nuclear fast red counterstain (C and D). Bar = 1 mm (A and B), 40 µm (C and D)

In addition to containing a higher concentration of spermatogonial stem cells, donor cell-derived colonies from rat stem cells were 2.75-fold larger than those from mouse stem cells at the time of analysis (7.72 ± 0.56 vs. 2.81 ± 0.29 mm, P <= 0.0001, Table 1). Therefore, the extent of spermatogenesis arising from rat donor testis cells 3 mo after transplantation is 26-fold greater than that from mouse donor testis cells (9.45-fold more colonies x 2.75-fold greater length per colony). During the 3-mo evaluation period, rat donor-derived colonies expanded an average of 85.8 µm/day (7.72 mm/90 days) compared with 31.2 µm/day for mouse colonies (2.81 mm/90 days). Spermatogenic colony growth rates of 45.9 µm/day and 27 µm/day have previously been reported in busulfan-treated recipients and irradiated mouse testes, respectively [16, 38]. A mouse testis contains 2 meters of seminiferous tubules and 2 x 106 Sertoli cells, or an average of 1 Sertoli cell per micrometer of length [39–42]. Therefore, spermatogonia in expanding spermatogenesis, derived from a rat donor stem cell, associate with about 86 new mouse Sertoli cells per day (86 µm/day x 1 Sertoli cell per 1 µm) or 1 Sertoli cell every 17 min. By comparison, spermatogonia in a mouse stem cell-derived colony associate with about 1 new Sertoli cell every 45 min.

Enrichment of Spermatogonial Stem Cells from Adult Rat Testes

Experimental cryptorchid surgery [25] and laminin-selection [24, 25] represent in vivo and in vitro methods, respectively, for the enrichment of mouse spermatogonial stem cell populations. Therefore, we tested similar enrichment strategies in the rat. Previous reports showed that exposure of the mouse testis to high core body temperature via the experimental cryptorchid procedure caused a reduction to about one third of control weight and a 40-fold reduction in testis cell number by 2 mo, which lead to a 25-fold enrichment of spermatogonial stem cells [25]. In the present study, cryptorchid rat testes were also about one third the weight of wild-type testes by 2 mo after the cryptorchid surgery (645 mg vs. 1910.8 mg, Table 1 and Fig. 3) and had a 50-fold reduction in cell number (8.4 x 106 vs. 418.5 x 106 cells per testis, Table 1 and Fig. 3). Most seminiferous tubules of cryptorchid donor rat testes were devoid of differentiating germ cells, similar to the pattern noted in the mouse ([25], Fig. 3, C and D). Consequently, one might expect a commensurate 25-fold enrichment of spermatogonial stem cells in the cryptorchid rat testis, provided that stem cells survive the exposure to core body temperature [25, 43]. Surprisingly, the cryptorchid rat testis was not enriched for spermatogonial stem cells, but rather contained a stem cell concentration similar to that found in wild-type testes (4.8 ± 1.0 vs. 7.7 ± 0.8 colonies per 105 cells injected, P = 0.74, Fig. 4A). Although cryptorchid rat testes were often edematous and appeared unhealthy, the surviving spermatogonial stem cells produced normal colonies of spermatogenesis, characterized by multiple germ cell layers and mature spermatozoa in the tubular lumen (Fig. 4, B and C).



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  		FIG. 3. Macroscopic and histologic appearance of cryptorchid and wild-type MT-lacZ rat testes. Cryptorchid rat testes (A) weighed 645 ± 43 mg, and 8.4 ± 0.78 x 106 cells were recovered per testis. A wild-type testis (B) is shown for comparison; the average weight and cell recovery from wild-type donor rat testes are shown in Table 1. Two months after the cryptorchid surgery, most seminiferous tubules contained only Sertoli cells, which were unstained (C) or stained (D). Wild-type testes exhibited complete spermatogenesis (E), characterized by multiple germ cell layers and mature spermatozoa in the tubular lumen. Testes were stained for ß-galactosidase activity with X-gal, sectioned, and counterstained with nuclear fast red (C and D) or eosin (E). Bar = 5 mm (A and B), 30 µm (C and D), 80 µm (E)



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  		FIG. 4. Evaluation of methods to enrich testis cell populations for spermatogonial stem cells. Wild-type, laminin-selected wild-type, and cryptorchid MT-lacZ rat testis cell populations were transplanted into NCr nude recipient mice. The degree of colonization from these donor cell populations 3 mo after transplantation is represented by the number of individual blue spermatogenic colonies per 105 cells transplanted (A). Values are means ± SEMs. B) NCr nude recipient of cryptorchid rat donor testis cells stained with X-gal 3 mo after transplantation. Histologic examination demonstrated complete spermatogenesis, characterized by multiple germ cell layers and mature spermatozoa (C). Nuclear fast red counterstain (C). Bar = 2 mm (B), 40 µm (C)

The spermatogonial stem cell niche is located on the basement membrane of the seminiferous tubule [44]. Because laminin is a major extracellular matrix constituent of the basement membrane [45], spermatogonial stem cells should bind to laminin. Indeed, a 5- to 7-fold enrichment of spermatogonial stem cells was demonstrated by selection of wild-type mouse testis cells on laminin-coated plates [25]. When wild-type rat testis cells were placed on laminin-coated plates, an 8.5-fold enrichment of spermatogonial stem cells occurred, compared with unselected wild-type controls (65.7 ± 11.8 vs. 7.7 ± 0.8 colonies per 105 cells injected, P <= 0.0002, Fig. 4A). The number of spermatogenic colonies produced by wild-type donors in this experiment was lower than that shown in Table 1 because the number of colonies per recipient testis was very high, which results in merging of colonies and an underestimation of stem cell number. However, since similar high colony numbers were observed for wild-type and laminin-selected groups in this experiment, they are directly comparable.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the absence of morphologic and/or biochemical criteria, a spermatogonial stem cell can only be definitively identified by its ability to produce and maintain spermatogenic colonies in a transplantation assay. Thus, the establishment of a transplantation assay for mouse spermatogonial stem cells [14, 15] enabled the identification of biochemical markers and development of enrichment methods that take advantage of immunologic or physical binding properties [24–26], surgical manipulation [25], and developmental characteristics [46]. There has been comparatively little progress in characterizing the rat spermatogonial stem cell by similar techniques, partly because of difficulties in developing a good rat recipient for transplantation [29]. However, in the absence of a suitable rat recipient, transplantation of rat testis cells into the seminiferous tubules of immunodeficient, busulfan-treated NCr nude mouse testes provides a valuable functional assay for rat spermatogonial stem cell activity [28]. Using the rat-to-mouse transplantation model, we demonstrated that the rat testis has both a high concentration and large number of spermatogonial stem cells, containing about 120-fold more functional stem cells than the mouse testis. Furthermore, rat stem cell-derived spermatogenic colonies grow to 2.75 times the size of mouse stem cell-derived colonies by 3 mo after transplantation (Table 1); thus, the extent of spermatogenesis from rat donor testis cells is more than 26-fold greater than that from mouse donor testis cells (9.5-fold more functional stem cells x 2.75-fold longer colonies). A previous report indicated that rat donor testis cells also colonized rat recipient testes more effectively than mouse donor testis cells [29]. However, the difference was difficult to quantify because the rat recipient model generated considerable endogenous spermatogenesis, which competes with donor cells, and few animals were involved [29].

The difference in stem cell concentration between rat and mouse wild-type testis cell populations determined in this study (9.5-fold) assumes that the transplantation efficiency is similar for the two donor species. To ascertain the spermatogonial stem cell transplantation efficiencies, the number of stem cells present in the donor testis before transplantation must be known. The number of spermatogonial stem cells per rat testis can be derived from classical studies of testis morphometry. In an extensive study quantifying type A spermatogonia in the seminiferous epithelium, Huckins [47] identified 0.44 type A isolated (Ais) spermatogonia per 23 Sertoli cells and 0.39 type A paired (Apr) spermatogonia per 23 Sertoli cells. There are 20–40 x 106 Sertoli cells per rat testis [48]. Assuming that the number of spermatogonial stem cells in the rat testis is represented by all of the Ais spermatogonia and one half of the Apr spermatogonia (some dividing spermatogonial stem cells would be identified as Apr spermatogonia), the rat testis contains about 830 000 spermatogonial stem cells [(0.635 stem cells per 23 Sertoli cells) x (30 x 106 Sertoli cells per testis)]. Therefore, the rat testis contains about 1 stem cell per 504 total cells [418.5 x 106 cells per testis (Table 1)/830 000 stem cells per testis]. We observed 70 810 functional spermatogonial stem cells per rat testis and therefore estimate the transplantation efficiency of rat spermatogonial stem cells into mouse testes to be 8.5% (70 810/830 000). This value is remarkably similar to the transplantation efficiency observed for mouse spermatogonial stem cells in this and previous studies. Morphometric studies of the adult wild-type mouse testis estimate that spermatogonial stem cells comprise about 1 in 5000 cells [41, 49]. Our analyses detected 1 stem cell (blue colony of spermatogenesis) per 55 866 mouse testis cells injected (106/17.9, Table 1), indicating a transplantation efficiency of 8.9% (5000/55 866), which is consistent with our previous results [16, 26, 36, 46]. Therefore, the rat-to-mouse difference in spermatogonial stem cell concentration appears not to be attributed to differences in transplantation efficiency, but must indicate that significantly more stem cells are present in the rat.

The development of methods for the isolation, enrichment, and/or purification of spermatogonial stem cells will be key to the identification of their biochemical and morphologic properties. Attempts to enrich this important adult stem cell from the rat testis using methods that were effective in the mouse met with mixed results. In the mouse, the experimental cryptorchid procedure results in death of most germ cells, leaving only undifferentiated spermatogonia, including stem cells, by 2 mo after surgery [50] and leads to a 25-fold enrichment of stem cells [25]. In contrast, the experimental cryptorchid procedure provided no enrichment of spermatogonial stem cells in rat testes, suggesting that male germ line stem cells in the rat are more sensitive to core body temperature than those in the mouse. It has been suggested that the absence of spermatogenesis in the rat testis leads to an imbalance between the endocrine and spermatogenic compartments, which is detrimental to germ cells [30] and/or somatic cells [51]. Furthermore, the rat testis is more susceptible than the mouse testis to permanent damage under these conditions [30]. Perhaps similar damage also extends to the spermatogonial stem cells of the cryptorchid rat testis.

Although the experimental cryptorchid procedure did not provide an effective means for the enrichment of spermatogonial stem cells in the rat, in vitro selection on laminin-coated plates resulted in 8.5-fold enrichment (Fig. 4A), which compares favorably with that in the mouse; in the mouse, laminin selection provides 5- to 7-fold enrichment [25]. Since {alpha}6- and ß1-integrins comprise a known receptor for laminin, and both integrins were individually identified on mouse stem cells [24], these will be ideal antigens to use for further enrichment of rat testis cell populations for spermatogonial stem cells. More importantly, the rat testis donor-to-mouse recipient transplantation assay described here will allow assessment of a wide range of possible rat spermatogonial stem cell markers.

Among adult stem cells, the male germ line stem cell has the unique ability to pass genetic information to the next generation, and it has now been demonstrated to provide an effective vehicle for the production of transgenic mice [52]. Testis cells from both mature and immature mice were transduced in vitro with a retrovirus carrying a reporter transgene. These cells were transplanted into recipient testes, where colonies of donor cell-derived spermatogenesis were generated, and the resulting sperm transmitted the transgene to progeny. The transgene remained stably integrated in the genome and was passed in Mendelian ratios for at least three generations without silencing of expression. While the production of transgenic mice has become routine [53], transgenesis through the male germ line might be easily translated to other species in which current transgenic methodologies are not as efficient. The results of the current study demonstrating the efficient colonization of recipient testes by rat spermatogonial stem cells suggest that these cells will provide a particularly effective vehicle for genetic modification of the germ line. The development of a good recipient model will be an essential prerequisite for the extension of this methodology to the rat and other species.


   ACKNOWLEDGMENTS
 
We thank H. Kubota and B.-Y. Ryu for critical review of the manuscript. We appreciate the assistance of C. Freeman and R. Naroznowski for animal maintenance and experimentation, C. Brensinger for statistical analyses, and J. Hayden for help with photography.


   FOOTNOTES
 
First decision: 8 November 2001.

1 T.S. was supported by the Japan Society for Promotion of Science. Microscopic sections were produced in the Institute for Human Gene Therapy, Cellular Morphology Core (5-P30-DK-47747-07). Financial support for the research was from the National Institute of Health (NICHD 36504); the Commonwealth and General Assembly of Pennsylvania; and the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation. Back

3 Current address: Department of Medical Chemistry, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto 606-8501, Japan Back

2 Correspondence: R.L. Brinster, School of Veterinary Medicine, University of Pennsylvania, 3850 Baltimore Ave., Philadelphia, PA 19104. FAX: 215 898 0667 Back

Accepted: November 15, 2001.

Received: October 25, 2001.


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TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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