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BOR - Papers in Press, published online ahead of print October 29, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.022483
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BIOLOGY OF REPRODUCTION 70, 625–631 (2004)
DOI: 10.1095/biolreprod.103.022483
© 2004 by the Society for the Study of Reproduction, Inc.

Testis

Testis Tissue Explant Culture Supports Survival and Proliferation of Bovine Spermatogonial Stem Cells1

Jon M. Oatley, David M. de Avila, Jerry J. Reeves, and Derek J. McLean2

Department of Animal Sciences and Center for Reproductive Biology, Washington State University, Pullman, Washington 99164


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study was designed to evaluate the survival and proliferation of bovine spermatogonial stem cells in an explant culture system over a 2-wk period. Explants of calf testicular parenchyma were placed on 0.45-µm pore membranes in culture and maintained for 1–2 wk. Histological examinations of fresh (t0) and cultured tissues revealed morphologically normal seminiferous tubules. Germ cell numbers/tubule increased (P <= 0.05) during culture when compared with t0, yet germ cell differentiation was not observed. Testosterone was present in medium throughout the culture period, indicating functional Leydig cells. Sertoli, spermatogonial, and spermatogonial stem cell viability was evaluated by reverse transcription-polymerase chain reaction for cell-specific gene expression of stem cell factor, protein gene product 9.5, and glial cell line-derived neurotrophic factor family receptor-{alpha}1, respectively. Results demonstrated the expression of all genes at t0, 1 wk, and 2 wk of culture. Single-cell suspensions were prepared from the testicular tissues at t0 and during culture and transplanted into nude mouse testes to investigate spermatogonial stem cell viability. One month after transplantation, colonies of round bovine cells were identified in all mouse testes analyzed, indicating survival of spermatogonial stem cells. The average number of resulting colonies in recipient testes was significantly (P <= 0.05) higher following 1 wk of culture compared with t0 and was numerically higher at 2 wk of culture compared with t0. This increase in colony numbers over time in culture indicates spermatogonial stem cell proliferation in vitro. This explant culture system appears to provide an environment that supports survival and proliferation of bovine spermatogonial stem cells.

gametogenesis, spermatogenesis, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sustained sperm production in the male relies on spermatogonial stem cells having the ability to undergo dual actions of 1) production of daughter progeny committed to the spermatogenic pathway and 2) self-replicating proliferation, which allows for continuous sperm production throughout the lifetime of a male. Theoretically, one cell has the ability to give rise to 4096 mature spermatozoa in the rat [1], although fewer than that reach maturity due to apoptosis. These cells have gained attention because of their demonstrated ability to restore fertility in infertile testes following transplantation in rodents [2, 3]. In mice, spermatogonial stem cells have been genetically modified in vitro and resulted in transgenic offspring being sired by transplanted recipients [4]. Thus, the genetic alteration of a single spermatogonial stem cell could give rise to thousands of modified spermatozoa after transplantation into a recipient testis.

In the mouse testis, gonocytes migrate to the basement membrane and undergo a conversion to stem cells from Day 0 to 5 postnatally, where spermatogonial stem cell niches are formed, from which all differentiating germ cells will arise [5]. In the bovine, this migration, conversion, and niche assembly occurs over a period of months; at 4 wk of age gonocyte migration occurs, and differentiated spermatogonia are not present until 8 wk of age [6]. Therefore, within this period of 4–8 wk of age, spermatogonial stem cell conversion and niche assembly must occur. Previously, we have demonstrated that transplantation of germ cells from bull calves older than 4 wk of age results in the best colonization in recipient mouse seminiferous tubules [7], further indicating that spermatogonial stem cell conversion is not complete until after 4 wk of age in the bovine.

Knowledge of spermatogonial stem cells and mechanisms regulating biological activity has been hindered due to their rarity in the testis and lack of methods to isolate them. Currently, spermatogonial stem cells can only be unequivocally identified on a functional basis by their ability to colonize recipient seminiferous tubules following transplantation. Recently, through the use of spermatogonial stem cell transplantation, glial cell line-derived neurotrophic factor (GDNF) has been demonstrated to play a role in regulating spermatogonial stem cell activity in mice [8, 9]. In several systems, including many types of neurons, GDNF has been shown to act on progenitor cells by binding to the receptor dimer of Ret tyrosine receptor kinase and GDNF family receptor-{alpha}1 (GFR{alpha}1) [10–12]. Therefore, it has been hypothesized that spermatogonial stem cells express the GFR{alpha}1 receptor.

Development of in vitro culture techniques that support the survival, proliferation, and differentiation of spermatogonial stem cells will aid in the ability to study them as well as modify their genome. In vitro culture of rodent spermatogonial stem cells has been demonstrated using feeder layers for periods extending up to 4 mo [13]. However, culture of bovine germ stem cells has been met with challenges [7, 14]. Maturation of bovine gonocytes through meiosis has been reported in vitro using calcium-alginate encapsulation [15], but stem cell activity using this method has not been investigated. The ability to stably modify bovine spermatogonial stem cells would be facilitated by use of an in vitro culture system that supports their survival and proliferation.

To date, spermatogonial stem cell transplantation between bulls is not possible; therefore, a bioassay model to evaluate bovine cells has been developed. Fresh bovine germ cells are capable of colonization and proliferation in recipient immunodeficient mouse seminiferous tubules [7, 14]. This bioassay can be used to evaluate culture and transfection techniques with bovine spermatogonial stem cells. The present study was designed to evaluate the survival and proliferation of bovine spermatogonial stem cells in an explant culture system over a 2-wk period. We hypothesized that bovine spermatogonial stem cells would proliferate in explant cultures as tested by cross-species transplantation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Testicular Tissue Collection and Culture

The Washington State University Institutional Animal Use and Care Committee approved all animal procedures. Testes removed from prepubertal bull calves (1–2 mo, n = 3) served as sources of donor testicular parenchyma. Pieces (10–20 mg) were removed from the testes and cultured on 0.45-µm pore membranes (Millipore, Bedford, MA) within six-well culture plates individualized by donor. Ten sections of tissue from each donor were placed on an individual membrane in a single well and 12 wells or two plates were used for each donor, equaling 120 sections per donor. Cultures were maintained for 1–2 wk in culture medium (Dulbecco modified Eagle medium [DMEM] containing 10% fetal bovine serum, 30 mg/ml penicillin, and 50 mg/ml streptomycin) at 32°C in an atmosphere of 5% CO2 in air. Media were collected every other day, stored at -20°C, and fresh media replaced. Fresh (t0) samples were used as a representation of the starting cell populations.

Histology and Testosterone RIA

Twenty tissue samples from each donor were fixed in Bouin solution overnight at 4°C at t0, 1, and 2 wk of culture. Tissues were subsequently dehydrated in 70% ethanol, embedded in paraffin, and sectioned at 6 µm. Sections were stained with hematoxylin to visualize cell nuclei. Slides were evaluated under inverted light microscopy and digital images were captured (Coolsnap Pro, MediaCybernetics, Carlsbad, CA). Average germ cell nuclei/tubule were quantified by calculating the average number of germ cell nuclei in 20 round tubules per sample.

Germ cell nuclei could be differentiated from somatic Sertoli cell nuclei by the distinct morphological differences between the two cell types. Round stained nuclei were considered to be germ cell, while cells without round nuclear morphology and a single distinctive nucleolus were considered Sertoli cells and not included in the overall germ cell counts. Average tubule diameter was measured using an eyepiece micrometer on 20 tubules per sample. Medium samples were assayed for testosterone concentration using a commercial kit (DSL-400, detection limit of 0.05 ng/ml; Diagnostic System Laboratories, Webster, TX). Control medium was culture medium before addition to the explant cultures.

Reverse Transcription-Polymerase Chain Reaction for Stem Cell Factor, Protein Gene Product 9.5, and GFR{alpha}1

At t0 and 1 and 2 wk of culture, total cellular RNA was collected from 20 tissue sections of each donor using the Trizol method (Invitrogen, Carlsbad, CA). Complimentary DNA for each sample was synthesized by oligo(dT)-primed reverse transcription using M-MLV reverse transcriptase (Invitrogen). Samples were then analyzed by polymerase chain reaction (PCR) for expression of the Sertoli cell-specific and spermatogonial-specific expression of stem cell factor (SCF) and protein gene product 9.5 (PGP 9.5), respectively. Samples were also assessed for possible spermatogonial stem cell survival by investigating the expression of GFR{alpha}1. All samples were also assayed for expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), to ensure the quality of the template cDNA. All primer sets were designed from available GenBank sequences for the bovine (SCF, GAPDH, GFR{alpha}1) and horse (PGP 9.5) using ABI Primer Express 3 (Applied Biosystems, Foster City, CA). Primer sequences were as follows: 5'-ATTGGTGGCAAATCTTCCCA-3', 5'-TGCACTCCACAAGGTCATCAA-3' (SCF), 5'-ACCCCGAGATGCTGAACAAAG-3', 5'-CCCAATGGTCTGCTTCATGAA-3' (PGP 9.5), 5'-ACGGCACAGTCAAGGCAGAG-3', 5'-GTGATGGCGTGGACAGTGGT-3' (GAPDH), and 5'-CCACCAGCATGTCCAATGAC-3', GAGCATCCCATAGCTGTGCTT-3' (GFR{alpha}1). Reactions of 25 µl were performed containing 2 mM MgCl2, 0.25 mM dNTPs, 1x PCR buffer, 5 pmol of each primer, and 1 U of Taq DNA polymerase. Reaction conditions were 94°C denaturation for 5 min followed by 35 cycles of 94°C for 30 sec, 58°C for 30 sec, and 72°C for 30 sec, with a final extension of 72°C for 10 min. Products were separated and visualized by 0.9% agarose gel electrophoresis with Gel Star staining (Molecular Probes, Eugene, OR).

Transplantation and Colonization Analysis

At t0 and 1 and 2 wk of culture, single cell suspensions were collected from 20 testicular tissue sections of each donor by two-step enzymatic digestion [7]. Cells were then diluted to 2 x 107 cells/ml, assessed for cell survival by trypan blue exclusion, and labeled with the fluorescent dye PKH26 (PKH26 Red Fluorescent Membrane Linker Dye; Sigma, St. Louis, MO). The labeling stably incorporates the fluorescent dye along with long aliphatic tails into regions of lipids of the cell membrane and has been used in germ cell transplantations between livestock species [16, 17]. Cells were then washed extensively in DMEM by centrifugation to ensure there was no residual dye in the suspension. Cells were again assessed for survival by trypan blue exclusion, diluted to 1 x 107 cells/ml, and visualized by ultraviolet light microscopy with a TRITC filter to ensure adequate staining. Approximately 10 µl of labeled cell suspension was microinjected into the seminiferous tubules of 8- to 10-wk-old recipient nude mice (n = three mice/donor/time point, totaling nine mice/time point) that had been treated with busulfan (33 mg/kg body weight) 4 wk prior in order to deplete endogenous germ cells [7].

Bovine germ cells were injected into the testes of recipient mice as described [7]. Briefly, recipient mice were anesthetized with a combination of ketamine (3.3 µg) and xylazine (150 µg) in sterile physiological saline. A midline incision was then made through the abdomen and the testes were exteriorized; a small hole was then made in the connective tissue surrounding the efferent bundle and a glass microneedle containing 10 µl of cell suspension was inserted into the rete testis. Cells were then infused through the rete testis into the seminiferous tubules, the testes were replaced, the incision was closed with suture, and the animal allowed to recover. In most recipients, one testis was injected while the contralateral one served as a negative control. One month after transplantation, recipient mice were killed by CO2 inhalation and the testes were removed. Seminiferous tubules were dispersed with collagenase (1 mg/ml) in Hank balanced salt solution (HBSS) at 37°C for 15 min with gentle agitation. Isolated tubules were then spread on glass slides and mounted with a cover slip under HBSS. Samples were visualized by ultraviolet light microscopy with a TRITC filter to identify donor bovine colonies. The numbers of colonies in the tubules from each recipient testis were counted and digital images were captured.

Statistical Analysis

The experiments were replicated using three different donor animals. All statistical analyses were conducted using the Proc GLM function of SAS (SAS Institute Inc., Cary NC). Differences were determined by analyzing the data using a Duncan test for significance between means. The main effects used in the model were time in culture, colony number, tubule diameter, average germ cell nuclei, and testosterone concentration. The level of significance was set at P <= 0.05. Data in text and figures are presented as the mean ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Histological Analysis and Testosterone Production

Histological assessment of t0 and 1- and 2-wk-cultured tissues was conducted to evaluate seminiferous epithelium morphology. Microscopic visualization revealed that seminiferous tubules were morphologically intact throughout the culture period and germ cells could be observed in tubules (Fig. 1). Quantification of the average number of germ cell nuclei/tubule revealed an increase of germ cells (P <= 0.05) in cultured tissue compared with t0 (Fig. 2). In contrast, seminiferous tubule diameter decreased in cultured tissues compared with t0 (Fig. 3). Although germ cell numbers increased during the culture period, no germ cell differentiation (i.e., meiotic cells) was observed.



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  		FIG. 1. Photomicrographs showing cross sections of (A and B) t0 testicular parenchyma and (C and D) 2-wk cultured testicular parenchyma. Cultured tissue had a significant increase in numbers of germ cells. Bar = 100 µm (A and C) and 30 µm (B and D)



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  		FIG. 2. Average germ cell numbers/tubule in t0 and cultured tissues. Data are presented as the mean ± SEM. Bars with different letters are different at P <= 0.05



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  		FIG. 3. Average seminiferous tubule diameter in t0 and cultured tissue. Data are presented as the mean ± SEM. Bars with different letters are different at P <= 0.05

To investigate Leydig cell viability during the culture period, medium collected on Days 7 and 14 from testis tissue explant cultures were assayed for production of testosterone. Testosterone was detected in the collected medium throughout the culture period (Fig. 4); concentrations peaked after Week 1 of culture (6.4 ± 2.7 ng/ml) and then declined during Week 2 (3.3 ± 1.3 ng/ml). Testosterone concentration in the control medium (culture medium before addition to testis tissue explant cultures) was below the detectable limit of the assay (0.05 ng/ml).



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  		FIG. 4. Testosterone concentration in tissue culture medium throughout a 2-wk period. Data are presented as the mean ± SEM

Sertoli Cell, Spermatogonial, and Spermatogonial Stem Cell-Specific Gene Expression

The function of Sertoli cells in the testis tissue explant cultures was assessed by evaluating the expression of SCF at each time point. The expression of SCF was detected by reverse transcription-polymerase chain reaction (RT-PCR) at t0 and 1 and 2 wk of culture (Fig. 5). Similarly, the presence of spermatogonia was assessed by RT-PCR for the expression of PGP 9.5. Like the expression of SCF, PGP 9.5 expression was detected at t0 and 1 and 2 wk of culture (Fig. 5). Spermatogonial stem cell survival was assessed by examination of the expression of GFR{alpha}1 in the fresh and cultured tissues. Expression of GFR{alpha}1 was detected in all tissues examined at t0 and 1 and 2 wk of culture (Fig. 5). Thus, Sertoli cells, spermatogonia, and spermatogonial stem cells were present and functional in the cultured tissues.



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  		FIG. 5. Expression of PGP 9.5, SCF, GFR{alpha}1, and GAPDH genes throughout the culture period; MW, 100-bp DNA ladder; 1, t0; 2, 1-wk culture; and 3, 2-wk culture

Transplantation

To investigate spermatogonial stem cell survival and proliferation during explant culture, fluorescently labeled single testicular cell suspensions (Fig. 6A) collected at t0 and 1 and 2 wk of culture were transplanted into busulfan-treated recipient immunodeficient mouse testes. Fluorescently labeled colonies of round donor bovine cells were identified in all recipient testes analyzed 1 mo after transplantation (Table 1). Microscopic visualization of donor testes revealed clusters or chains of donor cells in multiple areas throughout recipient seminiferous tubules (Fig. 6; Table 1). Negative control testes, those not injected, showed no fluorescent cell colonies. The number of resulting colonies from 1-wk cultured tissues was higher (P <= 0.05) than t0 colony numbers (Fig. 7). Colony numbers arising from 2-wk cultured tissues were numerically higher than those from t0; however, this difference was not statistically significant (Fig. 7).



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  		FIG. 6. Transplantation of bovine testis germ cells into mouse seminiferous tubules. Bovine germ stem cells that colonized recipient mouse seminiferous tubules formed chains or colonies of cells (arrows). A) Cultured single cell suspension after fluorescent staining before transplant (original magnification x100), (B) colonized recipient tubules 1 mo after transplant with 2-wk cultured bovine cells (original magnification x40), (C and D) colonized recipient tubule 1 mo after transplantation with 2-wk cultured bovine cells (original magnification x200). C) Bright light view, (D) corresponding fluorescent view, and (E and F) colonized recipient tubule 1 mo after transplant with 1-wk cultured bovine cells (original magnification x100). E) Bright light view, (F) corresponding fluorescent view). Bar = 100 µm (A, B, E, F) and 40 µm (C, D)


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  		TABLE 1. Colonization of recipient mouse testes by cultured bovine germ cells



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  		FIG. 7. Number of donor bovine colonies in recipient mouse seminiferous tubules 1 mo after transplantation with fresh and cultured bovine testicular germ cells. Data are presented as the mean ± SEM. Bars with different letters are different at P <= 0.05


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, a culture system was devised where Sertoli, Leydig, and spermatogonial stem cell interaction would be maintained. Results demonstrated that Sertoli cells remain viable at least 2 wk during tissue explant culture based on their ability to express SCF. This ligand secreted by Sertoli cells has been demonstrated to be essential in regulating spermatogonial proliferation in the testis through action on c-kit (SCF receptor) present on differentiated spermatogonia [18]. Interstitial Leydig cell testosterone production has been clearly demonstrated to be obligatory for spermatogenesis on both a qualitative and quantitative level [19–21]. The requirements of testosterone on spermatogonial stem cell actions are currently unknown, but undoubtedly play a role. Demonstration of testosterone production throughout the culture period in this study indicates survival of Leydig cells for at least 2 wk. Both stem cell factor and testosterone secretion in the testis are known to be under control of FSH and LH, respectively. The source of FSH and LH in the culture system used came from the serum added to the media (confirmed by RIA, LH 0.54 ng/ml and 38.13 ng/ml FSH). Immunohistochemical evaluation of the bovine testis has revealed the spermatogonial-specific expression of PGP 9.5 [22]; however, the exact spermatogonial cell type expressing it is unknown. Expression of this gene throughout the culture period indicates the survival of spermatogonia for at least 2 wk.

Development of in vitro culture systems for bovine spermatogonial stem cells has been a challenge due to the lack of a functional system to evaluate their presence in a cell suspension. Cross-species transplant of bovine cells into mouse testes is the only functional method for evaluating the survival and proliferation of bovine spermatogonial cells in culture [7, 14].

The demonstration in this study that explant cultured bovine cells could form round cell colonies in recipient mouse seminiferous tubules indicates the survival of spermatogonial stem cells. The observed round cell morphology of the colonized bovine cells is consistent with spermatogonial stem cell colonization as previously observed in other cross-species transplantations into recipient mouse testes [14, 23, 24]. Also, this observation is inconsistent with the appearance of somatic cell colonization in recipient testes, as has been previously demonstrated in rodent transplantations [25–27]. Donor bull germ cells used in this study were from animals at a developmental stage in which the testis germ cell population consists solely of undifferentiated gonocytes or spermatogonial stem cells. Therefore, it is unlikely that the colonizing donor cells in recipient mouse seminiferous tubules were differentiated germ cells or somatic cells.

The further demonstration that resulting colony numbers increased compared with t0 or fresh cells indicates that these cells were proliferating during the culture period. This observation in explant cultures of bovine testis is in contrast with feeder cell cocultures of mouse spermatogonial stem cells, in which the stem cells decreased in a time-dependent manner over a 7-day culture when compared with the fresh cell suspensions [8]. The observation that colony numbers were significantly higher in 1-wk cultured tissue compared with t0 but not statistically significant (P >= 0.05) at 2 wk of culture indicates that the spermatogonial stem cells are losing viability at 2 wk of explant culture. Another possible explanation of this observed effect is that the numerical and statistical difference between 1 and 2 wk of culture were minimal and could be a cause of a steady-state level of proliferation and apoptosis of the stem cells.

The exact mechanism that stimulated this proliferation remains unknown, but speculation could be made that Sertoli and interstitial cells are inducing proliferation through paracrine actions in response to lack of meiotic germ cells in the seminiferous epithelium. Recently, it has been demonstrated that glial cell line-derived neurotrophic factor (GDNF) induces proliferation of mouse spermatogonial stem cells [9]. Addition of GDNF to mouse testis germ cell cocultures has been demonstrated to enhance maintenance of spermatogonial stem cells [8]. It is possible that enhanced GDNF expression in the cultured bovine tissues induced spermatogonial stem cell proliferation, thus increasing the numbers of cells present compared with the fresh cell suspension. In neuronal progenitor cells, GDNF has been demonstrated to act on a receptor dimmer consisting of Ret tyrosine kinase and GFR{alpha}1. With the demonstration of GDNF actions on spermatogonial stem cells, it has been hypothesized that spermatogonial stem cells may express GFR{alpha}1. In this study, we were able to show the expression of the gene for GFR{alpha}1 throughout the culture period, thus supporting the conclusion of spermatogonial stem cell survival for at least 2 wk of explant testis tissue culture.

Another explanation of spermatogonial stem cell proliferation is related to the age of the donor animal; at 1–2 mo of age in the bovine, gonocytes have just migrated to the basement membrane and are likely undergoing conversion to stem cells and forming niches. Removal of the tissue at this age could trigger spontaneous stem cell conversion and proliferation in vitro. Culture of gonocytes from neonatal bull calves in a calcium alginate encapsulation system has been demonstrated to support the maturation of round spermatids by 10 wk of culture [15]. This maturation is at an accelerated rate compared with the in vivo situation of a normal bull, in which haploid germ cells are not present until 24–28 wk of age [6]. This accelerated rate of in vitro maturation in the bovine supports the theory that removal of the germ cells and somatic cells from a prepubertal animal stimulates spontaneous germ cell actions in vitro. Likewise, in vivo, the testis is maintained at a higher temperature than the culture temperature in this study. Many reports have demonstrated an inhibition of spermatogenesis at higher temperatures in bulls and rodents due to cryptorchidism [28–30]. The lowered temperature at which the tissue explants were cultured may have triggered stem cell proliferation, which would not normally be seen at 1–2 mo of age in the intact bull calf.

Histological examination of the cultured tissues revealed maintenance of seminiferous tubular structure and a significant increase in germ cell nuclei/tubule when compared with fresh tissue. The presence of meiotic cells in the cultured tissues was not observed, indicating spermatogonial proliferation and/or differentiation. Tubular diameter decreased and interstitial area appeared to increase in cultured tissues compared with t0. Similar to the apparent proliferation of spermatogonia, the exact reasons for these observations are elusive. Speculation could be made that the concentrations of FSH and testosterone in the medium were reduced compared with intertesticular concentrations in vivo. Sertoli and Leydig cells appeared to retain their functional abilities to produce growth factors and steroids, respectively. Taken together, the increase in germ cell nuclei/tubule and increased colonization of cultured cells supports the conclusion that spermatogonial stem cells proliferated during explant culture. In vivo, maturation of male germ cells in the testis is dependent on interaction with Sertoli cells. It has been well established that both testosterone and FSH are necessary for qualitative and quantitative spermatogenesis [31, 32]. However, the exact mechanisms that control spermatogonial stem cell activity in the testis are unknown.

The actions of the spermatogonial cell population govern the overall outcome of spermatogenesis, i.e., the number of spermatozoa produced daily in the testis. Spermatogonial stem cell proliferation is the determining factor of how many differentiated type A spermatogonia will be available to further mature, undergo meiosis, and transform into spermatozoa. Spermatogonial stem cells undergo both self-replicating proliferation and production of daughter progeny to become type A spermatogonia. These important aspects of spermatogonial stem cells have gained them much interest for use in reproductive technologies such as spermatogonial stem cell transplantation and transgenesis. Stable genetic modification of one male germ stem cell has the potential to provide a system by which thousands of genetically modified sperm could be produced. Moreover, in vitro culture of these cells has the ability to provide a model system to unravel the mechanisms controlling their actions in vivo. The ability to culture spermatogonial stem cells may give us a new avenue through male gametes to introduce transgenes into domestic animals.

The ultimate application of spermatogonial stem cell cultures and transplantation technology in livestock may be the ability to generate transgenic offspring. To accomplish this, several challenges must be overcome. One of these is the ability to culture spermatogonial stem cells from livestock animals. The current study demonstrates the survival and proliferation of bovine spermatogonial stem cells during explant testis tissue culture over a 2-wk period. This culture system could now be tested in conjunction with in vitro genetic modification techniques to alter bovine spermatogonial stem cells such as electroporation or retroviral transfection, with subsequent transfer into a recipient host's testis.


   FOOTNOTES
 
1 Supported by a Baxter Endowed Grant from Washington State University. J.M.O. is a recipient of a fellowship from Achievement Rewards for Collegiate Scientist. Back

2 Correspondence: FAX: 509 335 4246; dmclean{at}wsu.edu Back

Received: 19 August 2003.

First decision: 8 September 2003.

Accepted: 8 October 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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