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Testis |
Department of Animal Sciences and 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, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Another area of application for spermatogonial stem cell culture, cryopreservation, and transplantation is domestic livestock. Spermatogonial stem cell transplantation can potentially be utilized in domestic livestock as a means to preserve a male's germ cell line, increase genetic gain in domestic herds, and generate transgenic animals. In vitro culture and cryopreservation of spermatogonial stem cells are integral parts for the application of this technology. A system that supports the proliferation and maintenance of spermatogonial stem cells in vitro could be used to preserve and expand spermatogonial stem cell numbers as well as aid in genetic modification. Spermatogonial stem cell transplantation has been demonstrated in rodents [1, 2] and goats [7], and bovine spermatogonial stem cells have been demonstrated to be capable of colonizing recipient mouse seminiferous tubules, but not undergo differentiation into spermatozoa [8–10].
Over the past 10 years, all definitive reports of maintenance of spermatogonial stem cells in vitro have relied on coculture with a feeder cell monolayer [4, 6]. In initial studies with the mouse, coculture with an STO cell feeder line was demonstrated to support the survival of spermatogonial stem cells for several months [4]. Investigation of mouse spermatogonial stem cell maintenance during the first 7 days of coculture has shown a decline in stem cell number compared to the starting cell population [6]. Addition of exogenous glial cell line-derived neurotrophic factor (GDNF) was able to enhance the maintenance of spermatogonial stem cells within these cocultures [6]. Overexpression of GDNF in mice resulted in an accumulation of undifferentiated spermatogonia [11]. Both of these reports have implicated GDNF as a survival and proliferation factor for spermatogonial stem cells.
In recent years, maintenance of bovine germ cells in vitro without feeder cells has been demonstrated. Lee et al. [12] used a calcium alginate encapsulation method and demonstrated the differentiation of bovine gonocytes to haploid cells; however, spermatogonial stem cell maintenance was not evaluated. Izadyar et al. [13] used a gel matrix to support the proliferation and differentiation of bovine spermatogonia in vitro; however, analysis of spermatogonial stem cell survival was limited due to the lack of a labeling method for the donor cells prior to transplantation. We have previously demonstrated the survival and proliferation of bovine spermatogonial stem cells over a 1-wk period using a testis tissue explant culture system [10]. This system involves the culture of small pieces of testicular tissue and is limited because tissue infrastructure breakdown begins shortly after 1 wk of culture. Explant culture is a promising method for inducing short-term increases in spermatogonial stem cell numbers, but limited for long-term use and genetic modification of stem cells. A system that allows for the culture of a single cell suspension of bovine spermatogonial stem cells would be more beneficial for long-term survival and genetic modification.
Cryopreservation can be used to preserve a male spermatogonial stem cell line. Culture and cryopreservation in combination could be used to immortalize a male's genetic line through the germ cells due to the spermatogonial stem cells' ability to self-replicate. The efficacy of this approach with livestock spermatogonial stem cells has not been clearly demonstrated. The objective of the current study was to evaluate the maintenance of frozen-thawed bovine spermatogonial stem cells during in vitro culture with and without addition of exogenous GNDF. To accomplish this objective we developed a bovine embryonic fibroblast feeder cell line for coculture with bovine germ cells and hypothesized that bovine spermatogonial stem cell maintenance could be sustained in this system and enhanced by GDNF addition.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All animal procedures were approved by the Washington State University Institutional Animal Care and Use Committee. Testicular germ cells were collected from donor bull calves (1–2 mo of age, n = 3) by enzymatic digestion of testicular parenchyma as previously described [10]. Cells were diluted to 1 x 107 cells/ml and suspended in freeze medium (Dulbecco modified Eagle medium [DMEM] containing 10% fetal bovine serum [FBS], 30 mg/ml penicillin, 50 mg/ml streptomycin, and 10% dimethyl sulfoxide [DMSO]; Sigma, St. Louis, MO). The suspensions were placed into freeze vials at 1-ml volumes and kept at –80°C for 24 h. Vials were then transferred to liquid nitrogen for long-term storage. Approximately 10 mo after being cryopreserved the cell suspensions were thawed in a 37°C water bath, and the cells were diluted in DMEM. Cells were then collected by centrifugation and resuspended in DMEM containing 10% FBS. Cell concentration and survival was determined using a hemocytometer and trypan blue-exclusion, respectively. Collected cells were then used for transplantation into recipient testes immediately or for in vitro culture.
Bovine Embryonic Fibroblast Feeder Cell Coculture
A bovine embryonic fibroblast feeder cell line (BEF-JMO#5) was created for use in a coculture system with cryopreserved bovine germ cells. This cell line was created from a Day 35 bovine embryo following previously described procedures for creation of embryonic fibroblast feeder cell lines [14]. Briefly, a Day 35 bovine embryo was collected into physiological saline and chopped into small sections with a sterile razor blade. Chopped tissue was placed into a 10-ml solution of Hanks balanced salt solution (HBSS) containing 0.05% trypsin and 1 mM EDTA, and incubated at 37°C for 15 min. Tissue was then allowed to settle on ice for 2 min followed by passing the suspension through a 14-gauge needle several times. Another 10 ml of enzyme solution was added and incubated again at 37°C for 15 min. The suspension was again allowed to settle on ice for 2 min, and the supernatant was removed for cell collection by centrifugation at 300 x g for 5 min. Collected cells were resuspended in culture medium (DMEM with 10% FBS, 30 mg/ml penicillin, and 50 mg/ml streptomycin), grown in culture at 37°C, and frozen stocks were created.
After thawing, bovine germ cells were diluted to provide 1 x 105 live cells/cm2 and placed into 25 cm3 tissue culture flasks containing a BEF feeder cell monolayer that was mitotically arrested with mitomycin-C [4]. Bovine germ cells were cocultured for 1 or 2 wk at 32°C in an atmosphere of 5% CO2 in air. Culture medium consisted of DMEM containing 10% FBS, 1x Gibco vitamin solution (Invitrogen, Carlsbad, CA), 30 mg/ml penicillin, and 50 mg/ml streptomycin.
Xenogeneic Transplantation of Cryopreserved and Cocultured Bovine Germ Cells and Colonization Analysis
Bovine testis cells were collected just after thawing from cyropreservation or after 1 and 2 wk of coculture with BEF cells by trypsin-EDTA digestion, and diluted to 1 x 107 live cells/ml in HBSS. Cells were then labeled with the fluorescent PKH26 dye following the manufacturer's instructions (Sigma) and microinjected into the seminiferous tubules of recipient nude mice (n = 3 mice per donor per time point) that had been treated with busulfan (30 mg/kg body weight) to deplete endogenous germ cells as previously described [10]. Briefly, approximately 10 µl of cell suspension was microinjected through a beveled glass needle that was inserted into a small hole that was previously made in the connective tissue surrounding the efferent bundle. The cell suspension was then infused through the rete testis into the seminiferous tubules. With all recipient mice, one testis was transplanted with bovine germ cells and the contralateral testis used as a noninjected negative control.
One month after transplantation, recipient nude mice were killed by CO2 inhalation followed by cervical dislocation. The testes were removed, placed in HBSS, and detunicated. The seminiferous tubules were then manually dispersed, spread on a glass microscope slide, and mounted under a cover slip with HBSS. The slides were then analyzed using fluorescent microscopy for detection and quantification of donor bovine colonies at 100x magnification. The total number of fluorescent donor bovine colonies within the seminiferous tubules of each recipient mouse testis was manually counted, and digital images were captured with a CoolSnap cf digital camera (Media Cybernetics, Silver Spring, MD).
RNA Isolation and Reverse Transcription-Polymerase Chain Reaction
Total cellular RNA was collected from cocultures of bovine germ and BEF cells at 1 and 2 wk, and cultures of mitomycin-C treated BEF alone using the Trizol method (Invitrogen). Isolated RNA was reverse-transcribed to cDNA using oligo(d)T priming and Maloney murine leukemia virus (M-MLV) reverse transcriptase. Samples were then analyzed by polymerase chain reaction (PCR) for the expression of GDNF family receptor-
1 (Gfra1). A partial characterization of BEF feeder cells was conducted by analyzing samples of mitomycin-C treated BEF cells for the expression of the growth factors leukemia inhibitory factor (Lif); kit-ligand (Kitl), also known as stem cell factor; and Gdnf. All samples were also analyzed for the expression of glyceraldehyde 3-phosphate dehydrogenase (Gapdh), to ensure equivalency of template cDNAs. Primer sets were designed from available GenBank or TIGR (The Institute for Genomic Research) sequences of the bovine genes using ABI Primer Express 3 (Applied Biosystems, Foster City, CA). The primer sequences and GenBank accession numbers are as follows: Gfra1 (5'-CCACCAGCATGTCCAATGAC-3', 5'-GAGCATCCCATAGCTGTGCTT-3'; from TIGR gene index TC213602, expected product size of 120 base pairs [bp]), Lif (5'-CTGGTTCTCCACTGGAAACACG-3', 5'-TGCACAGCTTGTCCAGGTTGT-3'; from GenBank NM173931), Kitl (5'-ATTGGTGGCAAATCTTCCCA-3', 5'-TGCACTCCACAAGGTCATCAA-3'; from GenBank NM174375), Gdnf (5'-GCAGCCGAAACAATGTACGA-3', 5'-AAGGCGATGGGTCTGCAA-3'; from GenBank AY382559, expected product size of 112 bp), and Gapdh (5'-ACGGCACAGTCAAGGCAGAG-3', 5'-GTGATGGCGTGGACAGTGGT-3'; from GenBank U85042, expected product size of 300 bp). Reactions of 25 µl were performed containing 2 mM MgCl2, 0.25 mM deoxynucleotide triphosphates (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). Cultures of mitomycin-C treated BEF cells maintained for 2 wk in vitro were also used as negative control samples for the expression of the germ cell specific Gfra1.
Immunohistochemisty for GDNF Production by BEF Cells
Mitomycin-C treated BEF cells maintained in culture for 1 or 2 wk were fixed with acetone:methanol (1:1) for 10 min at 4°C followed by washing in PBS. Nonspecific antibody binding was then blocked by incubating samples with 10% nonimmune goat serum for 15 min at room temperature. Samples were then incubated at 4°C overnight with primary antibody (monoclonal mouse anti-human GDNF; Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:250 in PBS. The next day, samples were washed with three changes of PBS and incubated at 37°C for 1 h with horseradish peroxidase (HRP)-labeled secondary antibody (goat anti-mouse immunoglobulin G; Bio-Rad, Hercules, CA) diluted 1:100 in PBS. Samples were again washed with three changes of PBS, stained for HRP with an aminoethyl carbazole (AEC) kit following the manufacturer's instructions (AEC Substrate Kit; Santa Cruz Biotechnology). Counterstaining was performed with hematoxylin, samples were evaluated using light microscopy, and digital images were captured at 100x magnification. Controls consisted of cells incubated with the omission of primary antibody, secondary antibody, and AEC chromogen.
Exogenous GDNF Addition to Cultures
In order to evaluate the effect of exogenous recombinant GDNF on bovine spermatogonial stem cell survival and proliferation during BEF coculture, recombinant human GDNF was added to the cultures (PeproTech Inc., Rocky Hill, NJ). Bovine cells were maintained in BEF coculture as described above in DMEM with 10% FBS, 1x Gibco vitamin solution, 30 mg/ml penicillin, 50 mg/ml streptomycin, and 100 ng/ml GDNF added. Cultures were maintained for 1–2 wk followed by RNA isolation or trypsinization and transplant into recipient mouse seminiferous tubules as described.
Statistical Analyses
All data were analyzed using the SAS system software (SAS Institute, Cary, NC) with the Proc GLM function. Differences between means were determined using a Duncan test for significance for average donor bovine colonies arising from transplants of frozen-thawed germ cells, 1- and 2-wk BEF cocultured germ cells, and 1- and 2-wk BEF cocultured germ cells with exogenous GDNF. Data were considered significantly different at P 
0.05. In all figures, data are presented as the mean ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To investigate the ability of bovine spermatogonial stem cells to survive during cryopreservation, donor bovine germ cells were collected, cryopreserved for approximately 10 mo, then transplanted into recipient mouse seminiferous tubules. One month after transplantation, colonies of round donor bovine cells were identified within the seminiferous tubules of all recipient mouse testes (Fig. 1A). The average number of donor bovine colonies per testis was 25.9 ± 1.8 (Fig. 2).
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The maintenance of bovine spermatogonial stem cells during BEF coculture was also evaluated by cross-species testicular germ cell transplantation. Total cell populations from 1- and 2-wk-cryopreserved bovine germ cells cultured on BEF feeder cells were transplanted into the seminiferous tubules of recipient nude mice to investigate spermatogonial stem cell maintenance. One month after transplantation, colonies of round donor cells were detected in all recipient mouse testes analyzed (Fig. 1B). The average number of colonies from 1-wk cultures (49 ± 5.6) was significantly higher than the average number of colonies from cells immediately transplanted after thawing. The average number of colonies resulting from cells cultured for 2 wk (10.6 ± 3.6) was significantly lower than those arising from both frozen-thawed and 1-wk-cultured germ cells (Fig. 2). Negative control recipient testes showed no fluorescent colonies of cells.
Reverse Transcription-PCR for Germ Cell-Specific and Growth Factor Genes
Isolated RNA from testis cell cocultures at 1 and 2 wk were assayed by reverse transcription (RT)-PCR for expression of Gfra1, which has been shown to be expressed by undifferentiated spermatogonia and suggested as a spermatogonial stem cell marker [11, 15]. The presence of germ cells in both 1- and 2-wk cocultures of testis cells on BEF feeder cells was confirmed by expression of Gfra1 (Fig. 3), also suggesting the presence of spermatogonial stem cells. Control cultures of BEF cells alone showed no expression of Gfra1 (Fig. 3).
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GDNF Addition to Cultures
The factor GDNF has been shown to enhance the maintenance of murine spermatogonial stem cells in vitro [6]. The BEF feeder cells created in this study endogenously express GDNF (Fig. 4), suggesting this factor also enhances bovine spermatogonial stem cell maintenance in vitro. In order to investigate the effects of exogenous GNDF on maintenance of bovine spermatogonial stem cells during in vitro BEF coculture, a recombinant GDNF was added to the culture media at a concentration of 100 ng/ml. The average number of bovine spermatogonial stem cell colonies in recipient seminiferous tubules arising from cells cultured for 1 wk with GDNF was significantly lower than 1-wk cultures without exogenous GDNF (Fig. 2). In contrast, the average number of resulting colonies from 2-wk-cultured bovine germ cells with GNDF was significantly higher than 2-wk-cultured cells without the exogenous GDNF (Fig. 2).
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Characterization of BEF Feeder Cell Line
Microscopic evaluation of the BEF cell line created in this study revealed a fibroblast morphology (Fig. 4). In order to partially characterize the cell line, RT-PCR was used to investigate the expression of several growth factors suggested to act as mitogens on male germ cells (Lif, Kitl, and Gdnf). Of the three factors investigated by RT-PCR only Gdnf was found to be expressed by the BEF feeder cells (Fig. 4A). Immunohistochemical labeling of GDNF showed production of the protein by BEF cells as well, with intense cytoplasmic distribution (Fig. 4B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, germ cell suspensions from donor bulls were cryopreserved in liquid nitrogen for approximately 10 mo prior to thawing for culture and transplantation. Colonization of frozen-thawed cells in recipient mouse seminiferous tubules demonstrates the survival of these cells in a relatively simple cryopreservation system. The round cell morphology of colonized bovine cells in mouse seminiferous tubules was consistent with previous reports of other cross-species testicular transplantations [8–10, 16, 17]. This observation was in contrast to previous reports of somatic cell colonization in a recipient testis [18–20]. The donor bulls used in this study were 4–8 wk of age, a development stage in which gonocytes or spermatogonial stem cells are the only cell type present within the testis. Therefore, it is highly likely that any germ cell colonization in recipient mouse testes was undifferentiated spermatogonial stem cells rather than differentiated germ cell types. Long-term cryopreservation of mouse and human spermatogonial stem cells has also been reported using a similar system [3, 17]. Survival of bovine type A spermatogonia during cryopreservation has been reported using a very similar method to the one employed in this study; however, evaluation of spermatogonial stem cell survival was again limited due to lack of a means for identifying donor cells following transplantation [21]. Similarly, bovine gonocytes have been demonstrated to be capable of survival during cryopreservation and undergoing differentiation in vitro postthaw [12]. It appears that spermatogonial stem cells from many species are quite robust and capable of survival during cryopreservation in relatively simple conditions.
Culture of bovine germ cells on BEF feeder cells for 1 wk resulted in a significant increase in round cell colonies following transplantation compared to frozen-thawed germ cells transplanted prior to culture. This observation provides a strong indication of bovine spermatogonial stem cell survival and proliferation over a 1-wk period. Mouse spermatogonial stem cells have been shown to decline during the first 7 days of culture on feeder cells compared to the starting cell population [6]. However, the addition of GDNF to the medium enhanced spermatogonial stem cell numbers in these cultures, implicating GDNF as a factor for spermatogonial stem cell maintenance [6]. Other studies have also indicated the proliferative effects of GNDF on undifferentiated spermatogonia in the mouse [11]. Evaluation of the BEF feeder cell developed in this study demonstrated the expression of endogenous Gdnf mRNA and protein production, which may have contributed to the enhanced stem cell maintenance in 1-wk cultures.
A significant observation in this study was the decrease in bovine colony numbers in recipient mouse testes arising from 2-wk-cultured bovine germ cells. This would indicate that spermatogonial stem cell death or stem cell differentiation to committed spermatogonia occurred over the 2-wk period, therefore decreasing the percentage of stem cells within the overall germ cell population.
The number of bovine round cell colonies in the seminiferous tubules of recipient mice when germ cells were cocultured with BEF cells and exogenous GDNF was significantly lower compared to cultures without exogenous GDNF at 1 wk. This observation indicates that a higher concentration of GDNF above what was endogenously produced by the feeder cells had a negative impact on stem cell maintenance. Similar studies with porcine spermatogonia have also shown dose-dependent effects of exogenous growth factor addition on cell survival and proliferation in vitro, in which a negative effect on cell survival was seen past a threshold concentration [22]. The FBS added to the culture medium may have also contained GNDF; therefore, further addition of a recombinant GDNF along with endogenous BEF production may have again passed a threshold and had a negative impact on spermatogonial stem cell maintenance.
The addition of exogenous GDNF enhanced stem cell maintenance in 2-wk cultures of bovine germ cells in this study. A twofold increase of surviving stem cells was seen with exogenous GDNF compared to 2-wk cultures without exogenous GDNF. Currently, the means of action that GDNF has on spermatogonial stem cell maintenance is unknown. Whether it inhibits stem cell differentiation or promotes stem cell proliferation remains debatable. If stem cell differentiation was occurring during the 2-wk period, a higher stem cell pool may have been maintained compared to cultures without added exogenous GDNF. The maintenance of this pool may have been through inhibition of stem cell differentiation as proposed by Nagano et al. [6] or by inhibition of cell death.
If GDNF action on stem cells works through inhibiting differentiation, then stem cell numbers would increase due to autonomous self-renewal. This theory would support the results obtained in this study for 1-wk-cultured cells without exogenous GDNF and the loss of stem cells at 2 wk of culture could be attributed to cell death over that period. The enhanced stem cell maintenance at 1 wk of culture may have resulted in a subgroup of stem cells whose viability was short-lived and could not be maintained throughout the 2-wk period. The appearance of colonies from 2-wk-cultured cells may have resulted from another subgroup of stem cells that arose from enhanced maintenance at 1 wk that may not have been as short-lived and could be maintained for an extended period of time. Proliferative spermatogonial stem cells in vitro and in vivo may be a short-lived subpopulation in which cell death occurs if differentiation to committed spermatogonia is not supported or initiated. Another subpopulation of spermatogonial stem cells whose proliferation is not quite as robust, but have a more vigorous, long-term viability, may also be present.
Addition of exogenous GNDF above the endogenous BEF production and FBS concentration may have passed a threshold concentration that resulted in cell death at 1 wk of culture or resulted in a steady state level of proliferation and cell death. This would explain the decreased number of stem cells in 1-wk cultures with added GNDF. At 2 wk of culture, the enhanced maintenance of stem cells from cultures with added GDNF may have resulted from an increased percentage of long-term viable cells at 1 wk that were still present at 2 wk. It appears that GDNF may play an integral role in spermatogonial stem cell proliferation, inhibition of differentiation, and viability.
Based on expression of Gfra1, germ cell survival and viability was demonstrated throughout the 2-wk culture period. Also, Gfra1 expression suggested spermatogonial stem cell presence. In neuronal cells, GDNF has been shown to exert its effects on a target cell through the receptor dimer of Ret tyrosine kinase and GFRa1 [23–25]. Therefore, it has been hypothesized that spermatogonial stem cells may express the GFRa1 receptor. Both these receptors have also been shown to be expressed by a subpopulation of undifferentiated spermatogonia in the mouse testis [15].
Development of in vitro culture and cryopreservation techniques for livestock spermatogonial stem cells are necessary for the application of spermatogonial stem cell transplantation. This technology has potential for use as a reproductive tool to enhance genetic gain and generate transgenic animals through the male germ line. In this study a BEF feeder cell coculture system was developed that supports the maintenance of bovine spermatogonial stem cells. An effect of GDNF on spermatogonial stem cell maintenance was demonstrated using this culture system; however, it is important to note that these data are strictly correlative. Definitive identification of GDNF as a mitogen for spermatogonial stem cells cannot be made until functional studies are conducted. The BEF coculture system reported here has potential to be used as a means for accomplishing stable genetic modification and investigation of factors regulating the biological activity of spermatogonial stem cells.
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FOOTNOTES |
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2 Correspondence. FAX: 509 335 4246; dmclean{at}wsu.edu
Received: 23 February 2004.
First decision: 27 March 2004.
Accepted: 10 May 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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  P. M Aponte, T. Soda, K. J Teerds, S C. Mizrak, H. J G van de Kant, and D. G de Rooij Propagation of bovine spermatogonial stem cells in vitro Reproduction, November 1, 2008; 136(5): 543 - 557. [Abstract] [Full Text] [PDF]
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