| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
developmental biology, gametogenesis, male reproductive tract, spermatogenesis
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The spermatogonial transplantation technique, developed several years ago for the mouse [5, 6], was essential for the ultimately successful production of transgenic mice by genetic modification of the male germline. The functional transplantation assay allowed the quantitative characterization of spermatogonial stem cell activity in donor testis cell populations [7–9], evaluation of recipient mouse testis models [4, 9, 10], and development of efficient gene delivery methods [3, 4]. In addition, the transplantation technique provided the vehicle for introducing genetically modified spermatogonial stem cells into infertile recipient testes. Retroviral vectors have been used to introduce a lacZ reporter transgene into mouse pup or cryptorchid adult spermatogonial stem cells [3, 4]. Transplantation of the transduced stem cells into infertile W mutant mouse pup recipient testes resulted in the restoration of fertility and production of offspring, 4.5% of which carried and expressed the lacZ transgene. The transgene remained stably integrated in the genome and was passed in Mendelian ratios for at least three generations without silencing of expression [4].
This new transgenic technology complements existing methods and may have some important advantages. First, introduction of the transgene into spermatogonial stem cells virtually assures germline integration. Second, a single experimental recipient can generate several germline transgenics because each donor stem cell-derived spermatogenic colony is likely to represent a unique chromosomal integration. Third, concatamerization is not observed with retroviral transduction, which will facilitate the use of inducible expression vectors. Because the spermatogenic process is well conserved in mammals [11], this method might be adapted to other species where transgenic methods are inefficient or not available. When combined with the rapidly expanding repertoire of somatic cell gene therapy vectors, this technology will facilitate the generation of transgenic animal models for the functional characterization of genes.
Rats are widely used models of basic biology and human disease and could be used as a logical first step toward extending the new transgenic technique to other species. The availability of several inbred rat strains, abundant physiological, pharmacological, biochemical, and cellular data, and emerging genetic resources (the Rat Genome Program and the Rat Expressed Sequence Tag Program) provide an excellent foundation for the investigation of genes and their functions. Although many of the classical studies on spermatogenesis and testis development were performed in the rat, the spermatogonial transplantation assay has not been fully developed in this species. Production of transgenic rats by retrovirus-mediated modification of the male germline will require 1) identification of optimal donor testis cell populations that efficiently colonize recipient testes, 2) development of a good rat recipient testis model, and 3) determination of the best method for transducing rat spermatogonial stem cells.
In a recent comparative study, adult rat testes contained a 9.5-fold higher concentration of spermatogonial stem cells than did adult mouse testes, and rat donor-derived spermatogenic colonies grew to 2.75 times the length of mouse-derived colonies by 3 mo after transplantation into immunodeficient nude mouse recipient testes [12]. Therefore, the extent of spermatogenesis from adult rat testis cells is 26-fold greater than that from the same number of adult mouse testis cells (9.5-fold more colonies x 2.75-fold longer colonies). These data suggest that rat spermatogonial stem cells will also provide an effective vehicle for germline modification. There are only a few reports of rat spermatogonial transplantation into rat testes, and results from those studies were not quantitative and involved only a few animals [13–15]. These initial studies suggest that rat-to-rat spermatogonial transplantation is possible and results in donor-derived spermatogenesis in recipient testes, but rat recipient preparation is plagued by problems of high sensitivity to the toxic systemic effects of busulfan and incomplete removal of endogenous spermatogenesis [15]. Additional studies are necessary to develop an optimal rat recipient testis model. However, in the absence of a suitable rat recipient, xenogeneic transplantation to nude mouse testes provides a valuable assay to evaluate rat donor testis cell populations, culture conditions, and transduction methods [12, 16, 17].
The purpose of this investigation was to characterize spermatogonial stem cell activity in short-term rat testis cell cultures and to evaluate methods for retrovirus-mediated transduction of rat spermatogonial stem cells, using the rat-to-nude mouse transplantation assay as a functional end point. Our results indicate that rat spermatogonial stem cells are susceptible to retrovirus-mediated gene transfer during short-term culture, and transduction efficiency was similar to that reported for the mouse.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The first experiment was designed to assess spermatogonial stem cell activity in short-term cultures of rat pup testis cells. Donor cells were obtained from 12- to 15-day-old Sprague-Dawley (SD) rat pups carrying a fusion transgene composed of the mouse metallothionein I (MT) promoter driving expression of the Escherichia coli lacZ structural gene [18]. The MT-lacZ transgene encodes the ß-galactosidase protein and is expressed in germ cells and Sertoli cells, thus allowing the unequivocal identification of transgenic donor cells in the recipient testis after staining with the 5-bromo-4-chloro-indolyl ß-D-galactoside (X-gal) substrate. For the second set of experiments, wild-type SD rat pup (10–14 days old) testis cells were subjected to transduction with a Moloney murine leukemia virus-based retrovirus (Gen-pgkßgal) containing the phosphoglycerate kinase 1 (pgk) promoter and the lacZ reporter transgene [19]. For all treatments, donor cells were recovered from rat testes using a two-step enzymatic digestion procedure essentially as previously described [20, 21]. Seminiferous tubules were removed from the tunica albuginea and digested with collagenase type IV (1 mg/ml; Sigma, St. Louis, MO) in Hanks balanced salt solution (HBSS) for 10–15 min at 33°C with shaking. Dispersed seminiferous tubules were washed three times in HBSS to remove interstitial cells. Further digestion of isolated tubules with trypsin (0.5 mg/ml)/EDTA (1 mM)/DNase I (1.4 mg/ml) in HBSS followed by filtration through a nylon mesh (40 µm) produced a single cell suspension, which in the Day 10–15 rat pup is composed primarily of myoid cells, Sertoli cells, spermatogonia (including spermatogonial stem cells), and primary spermatocytes [22].
MT-lacZ rat testis cells were cultured (6.5 x 106 cells) over mitomycin C-treated SIM mouse embryo-derived thioguanine- and ouabain-resistant fibroblast cell line (STO) feeder cells (4.8 x 104 cells/cm2) with Dulbecco modified Eagle medium containing 10% fetal bovine serum (DMEM/FBS) in 25-cm2 flasks. A total of 13 x 106 cells were plated for each time point, harvested after 1, 3, and 5 days in culture, and suspended in 223, 168, and 70 µl DMEM/FBS, respectively. Donor cell concentrations were adjusted to minimize the possibility of colony merging in the transplantation assay and to facilitate quantitative assessment of stem cell activity in each donor population. Because results of previous results in the mouse suggested a dramatic decrease in stem cell concentration during short-term culture [4], the concentration of donor cells was adjusted to compensate for the expected loss. Results were normalized to colonies per 106 cells injected, the procedure currently used in mice [8, 9]. Freshly collected MT-lacZ donor testis cells were designated 0-day culture controls and were injected directly into recipient testes.
Viral Transduction
The Gen-pgkßgal vector was produced in GP+E-86 retrovirus packaging cells [23] maintained in DMEM/FBS (105 cells/cm2). Virus-conditioned medium was obtained from 48-h cultures of confluent retrovirus-producing cells, filtered (0.4 µm) to remove debris and floating cells, and added with polybrene (5 µg/ml) to donor testis cell cultures. Three different in vitro transduction procedures were evaluated, 2.75-h, overnight (O/N, 16 h), and 3-day (64 h) periodic infection. For all treatment groups, an aliquot of virus-conditioned medium was saved for each day of infection to determine the virus titer by NIH-3T3 cell transduction.
For the 2.75-h infection, SD donor testis cells were suspended in virus-conditioned medium (63.6 x 106 testis cells/8 ml), gassed with humidified air, and rotated at room temperature for 2.75 h. Following incubation, donor cells were washed three times with Dulbecco PBS and resuspended in 235 µl DMEM/FBS for transplantation. The extensive washing procedure was used to remove latent virus particles from the suspension prior to transplantation, decreasing the possibility that endogenous stem cells were transduced. This systematic approach provides insight into the dynamics of virus-mediated transduction in vitro and facilitates the establishment of optimal transduction conditions.
For the O/N infections, approximately 6.4 x 106 donor testis cells were plated per 25-cm2 flask over mitomycin C-treated STO feeder cells with virus-conditioned medium. A total of 25.2 x 106 and 39 x 106 donor testis cells were cultured for two replicate O/N infection experiments. Following overnight incubation at 37°C, spent virus-conditioned medium was removed and replaced with fresh virus-conditioned medium for 2 h. Donor cells for the two replicate experiments were then harvested, washed, and suspended in 160 and 291 µl DMEM/FBS, respectively.
For the 3-day periodic infection group, 6 x 106 donor testis cells were plated per 25-cm2 flask over mitomycin C-treated STO feeder cells with virus-conditioned medium as described for the O/N method. The next morning, the spent virus-conditioned medium was removed and replaced with new virus-conditioned medium for 2 h, followed by incubation with fresh DMEM/FBS for 1 h. This cycle (2 h in virus-conditioned medium followed by 1 h in fresh medium) was repeated three times each day for 2 days. On the third day, cultures were harvested for transplantation; 6 x 106 and 12 x 106 cells were cultured for two replicate experiments, and recovered cells were washed and resuspended in 115 and 132 µl DMEM/FBS, respectively.
Recipient Mice and Tranplantation Procedure
Approximately 10 µl of transgenic MT-lacZ or transduced SD donor rat testis cell populations (2.8 x 107–108 cells/ml) were transplanted by efferent duct injection [21] into NCr nude (nu/nu; Taconic, Germantown, NY) recipient mice, which lacked B-cells and thus made suitable hosts for rat testis cell transplantation [12, 16, 17]. Endogenous spermatogenesis in nude mouse recipient males was eliminated by i.p. injection of busulfan (44 mg/kg) 6–8 wk prior to the transplant. 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 (National Academy of Sciences, Assurance no. A3079-0).
Analysis of Recipient Testes
Recipient testes were collected 3 mo after transplantation and stained with X-gal to visualize donor-derived spermatogenesis [24]. Donor spermatogonial stem cells were defined by their ability to produce blue colonies of spermatogenesis in recipient testes. Other types of testis cells did not generate colonies of spermatogenesis, and endogenous recipient testis cells did not express the lacZ transgene. Spermatogonial stem cells from the SD rat donors, used in the viral transduction experiments, can only produce blue colonies of spermatogenesis if they have been infected by the Gen-pgkßgal virus. Colony number was counted manually using a dissecting microscope. Because the number of stem cells recovered and injected varied for each experiment, colony number was normalized to 106 injected cells per testis. Statistical analyses were performed using ANOVA, and significant differences between means were determined using a Tukey HSD multiple comparisons test (SPSS version 10.0; SPSS, Chicago, IL).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Analysis of nude mouse recipient testes 3 mo after transplantation revealed that freshly collected MT-lacZ rat pup (Day 12–15 postpartum) testis cells produced 168.9 ± 27.5 spermatogenic colonies per 106 cells injected (Fig. 1). Because spermatogonial stem cells are the only cell in the testis that can produce colonies of spermatogenesis and each colony is thought to arise from a single donor stem cell, the transplantation assay allows quantitative assessment of stem cell activity in fresh and cultured donor testis cell populations. The number of functional stem cells identified in the transplantation assay [9, 12, 24, 25] is 5%–10% of the number estimated by morphometric analyses of mouse and rat testes [26–28]. Total cell recovery after 1, 3, and 5 days of culture was 58%, 66%, and 46%, respectively. Day 0 cells were not cultured, so 100% of cells collected were injected. A significant (P < 0.001) and biphasic decrease in spermatogonial stem cell activity was observed during short-term culture, with only 30% of the stem cells originally plated surviving O/N culture (50.3/168.9 colonies/106 cells; Fig. 1). Stem cell activity plateaued between Days 1 and 3 of culture but was reduced to 7.2% of the original activity by Day 5. Similar losses in spermatogonial stem cell activity have been observed in mouse testis cell cultures [3].
|
Viral Transduction of Rat Spermatogonial Stem Cells
The purpose of the second set of experiments was to determine whether a foreign gene could be introduced into rat spermatogonial stem cells by retrovirus-mediated gene transfer using methods similar to those recently described for the mouse [3, 4]. Nagano and coworkers [3, 4] determined that the most effective method for introducing viral DNA into mouse spermatogonial stem cells is by periodic infection during several days of culture and that pup testis cells were more efficiently infected than adult cells. Therefore, we examined the efficiency of rat pup (Day 10–14 postpartum) spermatogonial stem cell transduction using a 3-day periodic infection protocol and high titer virus-conditioned medium (average virus titer for all treatments = 3.1 x 105 colony-forming units [cfu]/ml; Fig. 2). The results from two replicate experiments are summarized in Table 1. A total of 13.85 x 106 cells were cultured, exposed to Gen-pgkßgal virus-conditioned medium (multiplicity of infection = 1.31 virus particles/cell), and transplanted into nude mouse recipient testes (Table 1). These cells produced a total of 12 blue colonies of spermatogenesis. Histological examination revealed that many tubules contained spermatogenesis that did not stain blue in the presence of the X-gal substrate (data not shown). By definition, only a spermatogonial stem cell can produce a colony of spermatogenesis and because donor testis cells were from SD rats that do not express the lacZ transgene, only a virally transduced stem cell can produce a blue colony. Based on these observations, there were 0.87 virally transduced stem cells/106 cells cultured and injected (12 colonies/13.85 x 106 cells cultured; Table 1). Comparing these values to control values from 0- and 3-day culture of MT-lacZ rat testis cells revealed that 1 stem cell was infected and produced a transgenic colony of spermatogenesis for every 194 stem cells present in the fresh testis cell isolate at the beginning of the culture period (168.9/0.87 colonies per 106 cells cultured; Table 1). By similar calculation, there was 1 transduced stem cell for every 56 stem cells remaining at the time of injection after 3 days in culture (48.3/0.87 colonies per 106 cells cultured; Table 1). Transduced SD rat testis cells produced complete and normal spermatogenesis in nude mouse recipient testes, characterized by multiple germ cell layers and the presence of elongated spermatids in the lumina of the seminiferous tubule (Fig. 3).
|
|
|
The results from experiment 1 and previous studies [3] demonstrated a significant loss of spermatogonial stem cells during the culture period. Therefore, two short infection protocols were designed to maximize spermatogonial stem cell survival. An O/N viral infection protocol was evaluated in two replicate experiments. A total of 56.94 x 106 SD rat pup testis cells were cultured, exposed to Gen-pgkßgal virus-conditioned medium (multiplicity of infection = 0.29 virus particles/cell) and transplanted into nude mouse recipient testes. In contrast to the 3-day periodic infection, the O/N infection protocol failed to produce any blue colonies of spermatogenesis (Table 1). A 2.75-h virus infection protocol was similarly ineffective. A total of 54.13 x 106 SD rat pup testis cells were exposed to Gen-pgkßgal virus-conditioned medium (multiplicity of infection = 0.025 virus particles/cell) and transplanted into recipient testes but failed to produce any blue colonies of spermatogenesis (Table 1). The slight increase in stem cell number associated with the short infection protocols was apparently offset by decreases in multiplicity of infection and time of exposure to virus particles compared with the 3-day periodic infection protocol. In addition, the 3-day culture may allow or stimulate spermatogonial stem cell replication, which is estimated to occur every 56 h in vivo [29].
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The feasibility of using highly engineered, replication-defective retroviral vectors to introduce therapeutic or reporter transgenes into mammalian cells was first demonstrated about 20 yr ago [32–34]. In the rapidly developing field of somatic cell gene therapy, retroviruses remain the most widely used gene delivery vectors [35]. Retroviral vectors can shuttle approximately 8 kilobases of exogenous DNA into target cells [36], and the recombinant viral genome is randomly inserted into host chromosomes [37]. Retroviral vectors have a two-step mechanism of infection. First, host cell specificity and virus particle uptake is mediated by interaction of the viral envelope protein with cell surface molecules on the target cells [38]. Second, movement of the viral genome to the nucleus and integration into host cell chromosomes requires dissolution of the nuclear membrane during mitosis [39]. Therefore, the susceptibility of different cell types to retroviral transduction depends on cell surface characteristics and proliferative activity [40]. Stem cells are attractive targets for gene therapy because of their ability to generate and maintain self-renewing tissues. Hematopoietic and spermatogenic stem cells are the most productive and, upon transplantation, can functionally reconstitute the dependent systems of the body [4, 9, 10, 41–43].
Spermatogonial stem cells can only be identified retrospectively based on their capacity to produce spermatogenic colonies in a transplantation assay, because no distinguishing biochemical or morphological characteristics are known. The rat-to-nude mouse transplantation technique provides a valuable system to characterize spermatogonial stem cell activity in donor rat testis cell populations. Using this assay as a functional end point, we recently demonstrated that adult rat testes have a 9.5-fold higher concentration (169.2 vs. 17.9 colonies/106 cells) of spermatogonial stem cells than do adult mouse testes [12]. Results of the current study (Fig. 1) indicate that the spermatogonial stem cell concentration in rat pup testes (168.9 colonies/106 cells transplanted) is similar to that in the adult. These data, combined with the observation that mouse pup spermatogonial stem cells are about 5- to 10-fold more susceptible to retroviral transduction than are adult mouse stem cells [3], suggested that rat pup testis cells would be good candidate targets for retrovirus-mediated gene transfer.
The ability to maintain rat spermatogonial stem cells in short-term culture was critical to achieving retroviral transduction. Although some mouse spermatogonial stem cells can survive for >3 mo in culture [44], about 50% and 90% are lost after 3 and 7 days of culture, respectively [4]. A similar loss of stem cells was observed in the rat testis cell culture (Fig. 1). However, in this and previous studies retroviral transduction of the remaining stem cells in both species occurred [3, 4], providing indirect evidence that some stem cells divided in culture. The biological mechanisms that allow some stem cells to divide in the face of an overall decrease in stem cell numbers during the culture period are not known. These quantitative analyses provided the basis for calculating the efficiency of transducing rat pup spermatogonial stem cells using the 3-day periodic retroviral infection protocol. The stem cell transduction efficiency based on the number of functional stem cells remaining in culture after 3 days was 1.8% (0.87/48.3 colonies per 106 cells transplanted; Table 1). Compared with the number of functional stem cells present in the original uncultured control population at the beginning of the culture period, 0.5% were transduced and produced colonies of spermatogenesis in recipient testes (0.87/168.9 colonies per 106 cells transplanted; Table 1). This later efficiency reflects both stem cell transduction efficiency and stem cell survival during 3 days of culture. In the mouse, pup and cryptorchid adult donor testes contain 140 and 358 functional stem cells per 106 cells transplanted, respectively [4], and retroviral transduction of these donor cell populations resulted in 2.47 and 0.69 transduced colonies of spermatogenesis per 106 cells transplanted, respectively [3]. Based on these data, the efficiency of transducing mouse pup and cryptorchid adult spermatogonial stem cells was 1.8% (2.47/140) and 0.2% (0.69/358), respectively [3]. Using similar methods, retroviral transduction of mouse pup spermatogonial stem cells was 3.6-fold better than that in rat pups (1.8/0.5), and that in rat pups was 2.5-fold better than that in cryptorchid adult mice (0.5/0.2). The reason for the reduced transduction efficiency in rat pup spermatogonial stem cells compared to mouse pup cells is not known but may reflect differences in cell surface characteristics or cell cycle status in culture.
In addition to transduction efficiency and stem cell survival in culture, colonization efficiency after transplantation directly influences the quantity and quality of donor-derived spermatogenesis in mouse recipients. Infertile W mutant mouse recipient testes allow levels of donor-derived spermatogenesis sufficient to produce fertility in recipient animals [9], and the W pup is approximately 10-fold better at producing fertile recipients than is the W adult [10]. Thus, use of the W pup as a recipient [4] resulted in higher levels of transduced donor-derived spermatogenesis than in a previous study [3] or in the present study and enabled the production of transgenic progeny. A similar approach of optimizing the recipient testis model should result in the production of transgenic rats, using methods developed in this study.
The present investigation provides a framework for understanding the dynamics of spermatogonial stem cell activity in rat testes and in culture. The results indicate that rat pup spermatogonial stem cells can be maintained in short-term culture and are susceptible to retroviral transduction. The approach described here can be used to generate transgenic rats, facilitated by 1) enrichment of spermatogonial stem cells in the donor population, 2) development of culture conditions that promote stem cell survival and proliferation, 3) increasing virus titer and using alternative viral vectors, and 4) development of improved rat recipient models. Similar approaches are likely to be feasible in other species.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Financial support for the research was from the National Institutes of Health (NICHD 36504), the Commonwealth and General Assembly of Pennsylvania, and the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation. 
2 Correspondence: R.L. Brinster, School of Veterinary Medicine, University of Pennsylvania, 3850 Baltimore Ave., Philadelphia, PA 19104. FAX: 215 898 0667
Accepted: April 12, 2002.
Received: March 13, 2002.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  S. Goel, M. Fujihara, N. Minami, M. Yamada, and H. Imai Expression of NANOG, but not POU5F1, points to the stem cell potential of primitive germ cells in neonatal pig testis Reproduction, June 1, 2008; 135(6): 785 - 795. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. E. Orwig, B.-Y. Ryu, S. R. Master, B. T. Phillips, M. Mack, M. R. Avarbock, L. Chodosh, and R. L. Brinster Genes Involved in Post-Transcriptional Regulation Are Overrepresented in Stem/Progenitor Spermatogonia of Cryptorchid Mouse Testes Stem Cells, April 1, 2008; 26(4): 927 - 938. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Honaramooz, S. Megee, W. Zeng, M. M. Destrempes, S. A. Overton, J. Luo, H. Galantino-Homer, M. Modelski, F. Chen, S. Blash, et al. Adeno-associated virus (AAV)-mediated transduction of male germ line stem cells results in transgene transmission after germ cell transplantation FASEB J, February 1, 2008; 22(2): 374 - 382. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Goel, M. Sugimoto, N. Minami, M. Yamada, S. Kume, and H. Imai Identification, Isolation, and In Vitro Culture of Porcine Gonocytes Biol Reprod, July 1, 2007; 77(1): 127 - 137. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B.-Y. Ryu, K. E. Orwig, J. M. Oatley, C.-C. Lin, L.-J. Chang, M. R. Avarbock, and R. L. Brinster Efficient Generation of Transgenic Rats Through the Male Germline Using Lentiviral Transduction and Transplantation of Spermatogonial Stem Cells J Androl, March 1, 2007; 28(2): 353 - 360. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Fouchecourt, M. Godet, O. Sabido, and P. Durand Glial cell-line-derived neurotropic factor and its receptors are expressed by germinal and somatic cells of the rat testis. J. Endocrinol., July 1, 2006; 190(1): 59 - 71. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Kanatsu-Shinohara, K. Inoue, H. Miki, N. Ogonuki, M. Takehashi, T. Morimoto, A. Ogura, and T. Shinohara Clonal Origin of Germ Cell Colonies after Spermatogonial Transplantation in Mice Biol Reprod, July 1, 2006; 75(1): 68 - 74. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Ehmcke, J. Wistuba, and S. Schlatt Spermatogonial stem cells: questions, models and perspectives Hum. Reprod. Update, May 1, 2006; 12(3): 275 - 282. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Kanatsu-Shinohara, H. Miki, K. Inoue, N. Ogonuki, S. Toyokuni, A. Ogura, and T. Shinohara Long-Term Culture of Mouse Male Germline Stem Cells Under Serum-or Feeder-Free Conditions Biol Reprod, April 1, 2005; 72(4): 985 - 991. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Kanatsu-Shinohara, S. Toyokuni, and T. Shinohara Transgenic Mice Produced by Retroviral Transduction of Male Germ Line Stem Cells In Vivo Biol Reprod, October 1, 2004; 71(4): 1202 - 1207. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Oatley, D. M. de Avila, J. J. Reeves, and D. J. McLean Spermatogenesis and Germ Cell Transgene Expression in Xenografted Bovine Testicular Tissue Biol Reprod, August 1, 2004; 71(2): 494 - 501. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Barzon, M. Boscaro, and G. Palu Endocrine Aspects of Cancer Gene Therapy Endocr. Rev., February 1, 2004; 25(1): 1 - 44. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Honaramooz, E. Behboodi, S. O. Megee, S. A. Overton, H. Galantino-Homer, Y. Echelard, and I. Dobrinski Fertility and Germline Transmission of Donor Haplotype Following Germ Cell Transplantation in Immunocompetent Goats Biol Reprod, October 1, 2003; 69(4): 1260 - 1264. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. J. Brinster, B.-Y. Ryu, M. R. Avarbock, L. Karagenc, R. L. Brinster, and K. E. Orwig Restoration of Fertility by Germ Cell Transplantation Requires Effective Recipient Preparation Biol Reprod, August 1, 2003; 69(2): 412 - 420. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. G. Hawley and D. A. Sobieski Germline Stem Cells (The Origin of Teenage Mutant Ninja Turtles(R)?) Stem Cells, November 1, 2002; 20(6): 478 - 481. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
