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a Department of Cell Biology, Georgetown University Medical Center, Washington, District of Columbia 20007
b Gene Evaluation and Mapping Laboratory, U.S. Department of Agriculture, Beltsville, Maryland 20705
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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[9], transforming growth factor ß [10], seminiferous growth factor [11], and Sertoli cell-secreted growth factor [12]. The function of these growth factors in spermatogenesis remains an enigma. SCF has been shown to stimulate DNA synthesis in spermatogonial cells [13]. Apart from Sertoli cell-secreted factors, growth factors and cytokines elaborated by peritubular myoid cells, Leydig cells, and testicular macrophages may also have an effect on the regulation of spermatogonial proliferation and differentiation. Leukemia inhibitory factor, a cytokine produced by myoid cells of rat testis [14], has been shown to promote survival of proliferating gonocytes, and it stimulates proliferation of quiescent gonocytes in coculture with Sertoli cells [15]. Granulocyte macrophage-colony stimulating factor (GM-CSF), a glycoprotein hormone, is produced by testicular macrophages at very high concentrations [16] and can cross the blood-testis barrier in mice [17]. Since GM-CSF has been shown to stimulate the maturation and proliferation of granulocytes and macrophages [18], it could be hypothesized that GM-CSF alone or in combination with other factors has a role in the process of spermatogonial renewal and differentiation. Although both SCF and GM-CSF have been cloned and sequenced in the porcine model system [19, 20], and their effect on the stem cells of the hematopoietic system has been documented [21], very little is known about their effect on the spermatogonial stem cells in the testis. In the present study, we isolated type A spermatogonia from prepubertal pigs by sedimentation velocity at unit gravity and examined the effects of SCF and GM-CSF on their survival. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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BSA, hyaluronidase, and trypsin were purchased from Sigma Chemical Company (St. Louis, MO). Collagenase was obtained from Worthington Biochemicals (Freehold, NJ). Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 medium and KSOM medium were purchased from Irvine Scientific (Santa Ana, CA) and Molecular Biotechnologies Inc. (Lavallette, NJ), respectively. The MTT assay kit was obtained from Boehringer-Mannheim (Indianapolis, IN). Human recombinant SCF (rhSCF) and human GM-SCF (rhGM-CSF) were purchased from R & D Systems (Minneapolis, MN).
Isolation of Porcine Type A Spermatogonia
Eighty-day-old male Yorkshire pigs, provided by the Beltsville Agricultural Research Center (USDA, Beltsville, MD), were anesthetized with Telozol (Aveco, Fort Dodge, IA) and xylazine (Rompun; Haver-Lockhart, Bayvet Division, Miles Laboratories, Shawnee, KS). The testes were excised and decapsulated. The decapsulated testes were minced into small pieces and suspended in DMEM/F12 medium. Seminiferous epithelial cells were enzyme-dispersed and separated by the method of Bellvé and colleagues [22] with minor modification [23]. Briefly, the minced pieces of testes were suspended in DMEM/F12 containing collagenase (1.5 mg/ml) and DNase (1 µg/ml) and then incubated at 34°C for 15 min in a shaking water bath operated at 100 cycles/min. After three washes in DMEM/F12 medium, seminiferous cord fragments, mostly devoid of interstitial cells, were incubated in DMEM/F12 medium containing collagenase (1.5 mg/ml), hyaluronidase (1.5 mg/ml), trypsin (0.5 mg/ml), and DNase (1 µg /ml) for 30 min using the conditions described above. The dispersed cells were washed twice with medium and filtered through 80-µm and 40-µm nylon mesh (Tetco Inc., Briarcliff Manor, NY) successively. The cells of the dissociated epithelium were then separated by sedimentation velocity at unit gravity at 4°C with the use of a 2–4% BSA gradient in DMEM/F12 medium. The cells were bottom-loaded into an SP-120 chamber in a volume of 30 ml, and a BSA gradient was generated using 275 ml of 2% and 4% BSA. The cells were allowed to sediment for a standard period of 2.5 h, and then 35 fractions of 15-ml volume were collected at 90-sec intervals. The cells in each fraction were examined under a phase-contrast microscope; fractions containing cells of similar size and morphology were pooled and spun down by low-speed centrifugation and then resuspended in DMEM/F12 medium.
Purification of Spermatogonia by Differential Plating
The enriched spermatogonial fractions collected from the STAPUT apparatus were pooled and subjected to differential plating to eliminate the contaminating cells (myoid and Sertoli cells), which constitute about 10–15% of the cell population. The pooled cells were incubated in DMEM/F12 containing 5% horse serum for 4 h at 34°C. Sertoli and myoid cells attached to the culture plates. The spermatogonial cells, which remained in suspension, were collected and washed in DMEM/F12 before plating. The purity of the cell preparation was determined prior to and after differential plating using c-kit receptor immunostaining.
Characterization of Porcine Type A Spermatogonia
Phase-contrast microscopy The cells recovered after differential plating were examined by phase-contrast microscopy for an approximation of their size and identification of their morphological attributes.
Immunocytochemical localization of c-kit receptor The isolated spermatogonial cells were fixed and permeabilized with ice-cold methanol for 3 min. The immunostaining was carried out with Histostain-SP kits (Zymed Laboratories, San Francisco, CA) according to the manufacturer's instructions and as described by Dym et al. [23]. Briefly, endogenous peroxidase activity was blocked by a 45-sec treatment with Peroxi-Block (Zymed Laboratories, San Francisco, CA) solution, and endogenous biotin was blocked with the avidin solution (10 min), followed by blocking of nonspecific antibody binding with 10% nonimmune goat serum (10 min). The cells were then incubated at 37°C for 1 h in a moist chamber with normal rabbit serum (1:500) or with rabbit anti-mouse c-kit receptor antibody (1:500) raised against the whole protein (gift from Dr. Peter Besmer, Sloan-Kettering Memorial Cancer Institute, New York, NY). Biotinylated goat anti-rabbit antibody (10 min), streptavidin-peroxidase (10 min), and substrate-chromogen mixture (5–10 min) were then added, successively. The cells were counterstained for 3 min with hematoxylin and then examined with a Zeiss (Thornwood, NY) Axiophot light microscope fitted with Planapo objectives.
Cell Viability Assay
Cell viability was determined with the MTT colorimetric assay as described by Dirami et al. [24]. The spermatogonia were seeded at a density of 5 x 104 cells per well (n = 8) in 96-well plates. The cells were cultured in DMEM/F12 or KSOM medium at 34°C for up to 120 h, and the viability was assessed at 24-h intervals. The viability of spermatogonial cells cultured in KSOM in the presence of rhSCF (0.1, 1.0, 10, and 100 ng/ml), rhGM-CSF (0.01, 0.1, 1.0, and 10 ng/ml), or a combination of rhGM-CSF (1 ng/ml) and rhSCF (10 ng/ml) was determined after culture for 48 h. The results were expressed as a percentage of viable cells at time zero.
Statistical Analysis
All the experiments were performed with separate cell preparations of type A spermatogonia from different pigs at 80 days of age. Each experiment with isolated spermatogonia involved 5–13 replicate samples for each point. The results are presented as mean ± SEM of representative experiments. Significance between controls and experimental samples was established by the Student's t-test. The difference was considered significant when p was less than 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The morphological attributes of freshly isolated type A spermatogonia from 80-day-old pigs are revealed by a phase-contrast micrograph (Fig. 1). The spermatogonial cells appeared to be approximately 20–25 µm in diameter and had large spherical nuclei with a thin rim of cytoplasm.
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Using the c-kit receptor as a marker for the type A spermatogonia, the purity of the spermatogonial preparation before and after differential plating was established. The purity of the preparation before differential plating was in the range of 80–85%. After removal of myoid and Sertoli cell contaminants, the percentage of spermatogonia in the final preparation ranged between 95% and 98%.
Time Course of Survival of Porcine Type A Spermatogonia in DMEM/F12 and KSOM Media
Purified spermatogonia were cultured in either plain DMEM/F12 medium or KSOM medium for up to 120 h at 34°C, and the viability was monitored at 24-h intervals by the MTT colorimetric assay (Fig. 2). The spermatogonial cells cultured in DMEM/F12 medium showed a precipitous drop in viability by 48 h. Less than 20% of the cells were viable at this time, and the number of viable cells decreased further to undetectable levels by 120 h.
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In contrast, the percentage of viable type A spermatogonial cells cultured in KSOM medium was higher at all times studied than that of cells in DMEM/F12. Although a precipitous drop in the viability occurred by 48 h, 40–50% of cells still remained viable in comparison to less than 20% in DMEM/F12 (Fig. 2). Interestingly, 30–40% of cells were still viable at the end of 120 h.
Effect of rhSCF on the Survival of Porcine Type A Spermatogonia
The type A spermatogonia were cultured in KSOM medium supplemented with rhSCF at various concentrations (0.1, 1.0, 10, 100 ng/ml). In controls, where spermatogonia were cultured in KSOM medium alone, only 40–50% of spermatogonia remained viable by 48 h (Fig. 3). In cells cultured in KSOM medium with various concentrations of rhSCF, a significant increase in the percentage of viability of type A spermatogonia was observed at 100 ng/ml (p < 0.05), but not at the lower concentrations.
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Effect of rhGM-CSF on the Viability of Porcine Type A Spermatogonia
The type A spermatogonia were cultured in KSOM medium supplemented with rhGM-CSF at 0.01, 0.1, 1.0, and 10 ng/ml for 48 h. In controls, where spermatogonia were cultured in KSOM alone, viability decreased to the range of 40–50% (Fig. 4). A significant increase in the viability of type A spermatogonia was observed at 0.01, 0.1, and 1 ng/ml concentrations (*p < 0.05). At a much higher concentration (10 ng/ml), however, there was a significant drop in the viability of spermatogonial stem cells (p < 0.01).
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A Combination of rhSCF and rhGM-CSF Did Not Enhance the Viability of Porcine Type A Spermatogonia
The percentage of viability of type A spermatogonia cultured in KSOM medium supplemented with rhGM-CSF (1 ng/ml) was significantly higher than the percentage of viability of cells cultured in KSOM alone (p < 0.05). This effect was masked by addition of rhSCF (Fig. 5), which had no significant effect when added alone at the concentration of 10 ng/ml.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The maintenance and propagation of any mammalian species depend upon the production of male and female gametes and their union to form an embryo. Selection of male reproductive traits is based on testicular size, semen characteristics, serum testosterone levels, and breeding aggressiveness [25]. True selection at the level of germ cells is necessary for improving considerably the reproductive efficiency of pigs. Isolation of type A spermatogonia from pigs selected for a specific trait and identification of culture conditions for survival, renewal, and differentiation could prove highly useful for germ cell transplantation. Using the technique of sedimentation velocity at unit gravity (STAPUT technique) that has been standardized for the isolation of type A spermatogonia from prepubertal rats and mice [22, 23, 26], we isolated a nearly homogeneous population of type A spermatogonia from 80-day-old pigs. On the basis of our previous experience with the purification of rat type A spermatogonia [26], we further purified this population of spermatogonial cells from contaminating Sertoli and myoid cells by differential plating of the cells in DMEM/F12 medium containing 5% horse serum for 4 h. After differential plating, the population of cells in suspension was aspirated and characterized for morphology and expression of the c-kit receptor. Expression of the c-kit receptor as a marker for identification of type A spermatogonia in the mouse and rat has been demonstrated previously [23, 27]. In pigs, the c-kit gene has been identified and assigned to chromosome 8p12-p21 by FISH [28]. However, no information is available on the expression of c-kit receptor in the pig testis. Isolated type A spermatogonia from prepubertal pigs exhibited immunocytochemical staining for the c-kit receptor, and this property was used to establish the purity of the population in conjunction with the morphology.
This is the first report of the isolation of type A spermatogonia from pigs. The highly pure type A spermatogonia (95–98%) were cultured in DMEM/F12 medium in the absence of any growth factor supplements. The viability of the cells was monitored by the MTT colorimetric assay. We have previously used this assay for determining the viability of cultured Sertoli and spermatogonial cells isolated from rat testis [24, 26]. There was an acute drop in the viability of pig type A spermatogonia at 48 h; only less than 20% of cells were viable. The viability decreased further with time in culture to undetectable levels by 120 h. We have reported similar results for rat type A spermatogonial cells [26]. As an alternate to DMEM/F12 medium, we cultured the porcine type A spermatogonia in KSOM medium. This medium was developed by John Biggers and colleagues (Harvard University) and has been used for culture of preimplantation embryos [29, 30]. The percentage of viable cells in KSOM medium was approximately 50% by 48 h in comparison to less than 20% in DMEM/F12 medium. At the end of 120 h, 30–40% of cells were still alive in KSOM medium whereas all the cells cultured in DMEM/F12 were dead.
In the present study, we observed that type A spermatogonia of the pig expressed the c-kit receptor. The c-kit receptor is a tyrosine kinase receptor [31, 32]. The ligand for this receptor is SCF [33, 34]. Several groups have demonstrated the importance of SCF for the survival of primordial germ cells, mast cells, and intestinal stem cells [35–37]. Thus, we studied the effect of SCF in porcine type A spermatogonia cultured in KSOM medium. Since porcine SCF is not available, we used rhSCF. The proliferative effect of rhSCF on the stem cells of the porcine hematopoietic system has been documented [21]. Stimulation of DNA synthesis in spermatogonia by the soluble form of SCF has also been demonstrated in the mouse [13]. After 48 h of incubation with various concentrations of rhSCF (0.1–100 ng/ml), an increase in the percentage of survival of spermatogonia was observed at 100 ng/ml but not at lower concentrations. These results suggest that rhSCF is effective in promoting survival of porcine type A spermatogonia. We have reported a similar effect in rat type A spermatogonia [26].
Apart from Sertoli cell-produced growth factors, several other mitogenic factors produced in close proximity to spermatogonia may regulate type A spermatogonial survival and proliferation. One such factor is GM-CSF produced by the testicular macrophages [16]. Macrophages compose up to 25% of the interstitial cell population in the testis of mammals, including boars [38]. Rat testicular macrophages produce 8-fold greater levels of GM-CSF than peritoneal macrophages; the significance of this is not known [16]. The biological effects of GM-CSF are mediated through GM-CSF receptor, which belongs to the cytokine receptor superfamily [39]. The receptor comprises two subunits, and ß. The subunit is specific to GM-CSF whereas the ß subunit is common to the second subunit of interleukin-3 and interleukin-5 [40]. The expression of GM-CSF receptor in the female reproductive system has been demonstrated [41–43]. Although high concentrations of GM-CSF are produced by testicular macrophages, no information is available on the expression of the GM-CSF receptor in the testis. We have localized GM-CSF receptor in mouse type A spermatogonia (unpublished results). We hypothesized that GM-CSF may act upon type A spermatogonial stem cells and promote their survival and proliferation. Indeed, rhGM-CSF at very low concentrations, ranging from 0.01 to 1 ng/ml, enhanced the percentage of survival of spermatogonia. Similar or more pronounced effects of GM-CSF have been observed on the proliferation of hematopoietic precursor cells [44–46], alveolar type II cells [47], and osteoblastic cells [48]. The role of GM-CSF as a survival factor has also been demonstrated in hematopoietic cells [49]. At a much higher concentration (10 ng/ml) of rhGM-CSF, the percentage of viability of spermatogonia decreased below control levels. This could be attributed to the toxic effect of GM-CSF. A combination of rhSCF and rhGM-CSF did not show any synergistic effect on the viability of porcine type A spermatogonial cells but rather appeared to counteract the increase exhibited by rhGM-CSF (1 ng/ml) when given alone.
In conclusion, we have developed a long-term culture system for the maintenance of isolated porcine type A spermatogonia with KSOM as the basal medium. This system should prove useful in the study of the fate (renewal, differentiation, or apoptosis) of type A spermatogonial cells and in identifying the factors involved in regulation of the early events of spermatogenesis in vitro. Long-term culture of these stem cells is essential for the introduction of foreign genes to alter the reproductive performance of pigs, and such an approach may find parallel use in germ-line gene therapy for treating genetic diseases afflicting human beings. The present data suggest that both SCF and GM-CSF enhance the survival of porcine type A spermatogonial cells. From the present study, it is not clear whether these factors also promote proliferation of porcine type A spermatogonia. Detailed studies on the renewal and differentiation of type A spermatogonia under the influence of SCF and GM-CSF, and the mechanism(s) of action involved in these processes, are necessary.
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FOOTNOTES |
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2 Correspondence: Martin Dym, Department of Cell Biology, Georgetown University Medical Center, 3900 Reservoir Road, NW, Washington, DC 20007. FAX: 202 687 9864; dymm{at}gunet.georgetown.edu
Accepted: February 5, 1999.
Received: October 22, 1998.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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