- Email: [email protected]
The Effects of Growth Factor on Testicular Germ Cell Apoptosis in the Stallion
REFEREED
ORIGINAL RESEARCH
The Effects of Growth Factor on Testicular Germ Cell Apoptosis in the Stallion Casey L. Donnelly, MS,a Christophe Staub, PhD,b Dickson Varner, PhD, DVM,c Terry Blanchard, PhD, DVM,d Larry Johnson, PhD,b and David W. Forrest, PhDa ABSTRACT Apoptosis is necessary for both initiation and control of spermatogenesis; however, an increase in apoptosis can lead to subfertility/infertility in stallions, causing substantial financial loss in the equine industry. The ability of stem cell factor (SCF), leukemia-inhibiting factor (LIF), granulocyte–macrophage colony-stimulating factor (GM-CSF), and estradiol (E2), alone or in combination, to prevent apoptosis of germ cells in short-term equine testicular cultures was examined. Testicular tissue was sectioned into approximately 2-mm cubes and placed in media-filled culture chambers. Concentrations of SCF (100 ng/mL), LIF (10 ng/mL), GM-CSF (5 ng/mL), and E2 (109 mol/l) were added alone or in combination to each well. After 6 hours in culture, the tissue was fixed and immunohistochemically (terminal deoxynucleotidyl transferase-mediated nick-end labeling; TUNEL) stained for apoptosis detection. Apoptotic cells per 100 Sertoli cell nuclei within seminiferous tubules were counted until the 500th Sertoli cell nuclei was reached. This counting procedure was used for each slide. An analysis of variance (ANOVA) with a Tukey’s test was used to compare apoptotic rates. In comparison with the control, GM-CSF alone lowered apoptosis by 34.77%. GM-CSF–treated tissue combined with SCF and LIF as well as GM-CSF combined with SCF, LIF, and E2 reduced apoptosis when compared with the control (37.45% and 44.40%, respectively) or other treatment combinations. GM-CSF alone reduced apoptosis; results suggest possible synergy for the combinations of SCF and LIF with GM-CSF and for E2 with SCF, LIF, and GM-CSF. Keywords: Apoptosis; Stallion; Growth factors; Granulocyte–macrophage colony-stimulating factor (GM-CSF); Spermatogonia
From the Department of Animal Science,a the Department of Veterinary Integrative Biosciences,b and the Department of Large Animal Clinical Sciences,c Texas A&M University, College Station, TX; and Hill N Dale Farm, Lexington, KY.d Reprint requests to: David W. Forrest, PhD, Department of Animal Science, Texas A&M University, 2471, TAMU College Station, TX 77843-2471. 0737-0806/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jevs.2007.04.003
212
INTRODUCTION Stallion subfertility/infertility is common1 and may cause substantial financial loss in the equine industry.2-4 Among the possible abnormalities or causes of germ cell loss may be an increase in apoptosis at the germ-cell level. During normal spermatogenesis, up to one-half of all germ cells never differentiate into mature sperm because of apoptoticrelated mechanisms.5 Increased apoptosis can reduce sperm count,5,6 leading to subfertility. The growth factors used in this study have known roles in spermatogenesis and apoptosis. In the human, addition of 17b-estradiol (109 mol/l) inhibited germ cell apoptosis and reduced apoptosis in spermatogenic cells in vitro.7 In mice, reduced expression of stem cell factor (SCF) increased germ cell apoptosis,8 and leukemia-inhibiting factor (LIF) acted directly on primordial germ cells to suppress apoptosis.9,10 Both SCF and LIF act synergistically with colony-stimulating factors such as granulocyte–macrophage colony-stimulating factor (GM-CSF).10,11 In a test conducted on normal human hematopoietic cells, Leary et al10 suggest that LIF/DIA (differentiation-inhibiting activity) along with G-CSF (granulocyte colony-stimulating factor) may be involved in early hematopoietic stem cell regulation. Also, in a study conducted by Martin et al11 using genomic clones of human SCF, SCF showed strong synergistic effects with colony-stimulating factors. GM-CSF recently has been shown to cross the blood–testis barrier intact,12 and it is able to promote the survival of porcine type A spermatogonial cells.13 The objective of this study was to determine whether the growth factors LIF, SCF, E2, and GM-CSF alone or in combination, might lower the incidence of apoptosis in stallion testicular germ cells, thereby identifying a possible treatment for this condition.
MATERIALS AND METHODS Testes Collection, Culture, and Growth Factor Administration Two stallions aged 5 and 7 years and of known reproductive soundness (determined by daily sperm output, motility, and concentration over a 3-month period) were castrated under general anesthesia. A section of testes from each horse was wrapped in aluminum foil and put on ice (for no more than 10 minutes). The testicular Journal of Equine Veterinary Science
May 2007
Table 1. Mean number of apoptotic cells per 100 Sertoli cell nuclei and the percentage difference of each treatment versus the control Growth Factor Treatments Control E2 C1 mean 18.94 C2 mean 24.06 % Difference from control
SCF
LIF
SCFD SCFD LIFD SCFDLIFD E2DSCFD GMCSF* LIF GMCSF GMCSF GMCSF* LIFDGMCSF*
15.20 23.42 22.71 13.06 28.39 27.47 24.45 14.99 1.3 18.33 9.69 34.77
18.41 18.07 27.93 15.56 7.77 21.80
17.31 18.53 16.66
8.89 18.01 37.45
10.14 13.78 44.40
The first horse was designated as culture 1 (C1) and the second culture 2 (C2). Each treatment had two wells, with a piece of tissue in each well. The mean number of apoptotic cells per 100 Sertoli cell nuclei per treatment is shown. The last row is the overall percent difference of each treatment compared with the control. A negative number signifies an increase and a positive number a decrease. * P < .05.
Figure 1. Immunohistochemical localization of apoptotic cells within the seminiferous epithelium. Labeled apoptotic cells are shown in A, whereas few or no labeled apoptotic cells within the seminiferous epithelium are shown in B.
samples were sectioned into approximately 2-mm cubes and placed into culture chambers fitted with bicameral cell culture inserts (Becton Dickinson Labware, Franklin Lakes, NJ). They were filled with media (Hanks F12/ DMEM 1:1) supplemented with 4 mmol/l glutamine,14 4 mmol/l sodium lactate, 1 mmol/l sodium pyruvate,15 Volume 27, Number 5
and 50 mg/ml ascorbic acid as an antioxidant16 and incubated for 6 hours17 at 328C at 5% CO2. Dosages Media only was used as the control. SCF (100 ng/ml),13 LIF (10 ng/ml),18 GM-CSF (5 ng/ml),13 or E2 (109 M)7 213
Figure 2. Treatment effects on the average number of apoptotic cells per 100 Sertoli cells. Data are represented as mean standard error, n ¼ 4. Time ¼ 0 (T0) is the number of apoptotic cells per 100 Sertoli cell nuclei present when the tissue was fixed immediately and not cultured. E2, estradiol; SCF, stem cell factor; LIF, leukemia inhibiting factor; GM-CSF, granulocyte macrophage-colony stimulating factor.
(all Sigma Chemical Co., St. Louis, MO) were added to each well, either alone or in combination. Two sections from each of the two testes were placed in separate individual wells for each treatment. They were incubated for 6 hours as described previously. Immunohistochemistry After 6 hours in culture, the tissue was fixed in 4% paraformaldehyde embedded in paraffin and sectioned into 5-mm slices. The tissue was deparaffinized and analyzed for apoptotic cells using the ApopTag Peroxidase In Situ Apoptosis Detection Kit (terminal deoxynucleotidyl transferase-mediated nick-end labeling; TUNEL) (Intergen Company, Purchase, NY) and counterstained with Mayer’s hematoxylin to observe nonreactive cells.19 Data Collection and Statistical Analysis The numbers of apoptotic germ cells per 100 detected Sertoli cell nuclei were then counted using a Zeiss Axioplan 2 microscope system at 630 total magnification. These data are reported as the average number of apoptotic cells per 100 Sertoli cell nuclei of the two tissue samples in each of the two cultures. Starting with one corner of a tissue section (excluding the tubules on the edges because of an increase in artifactual apoptotic labeling caused by processing the tissue), the numbers of apoptotic and Sertoli cell nuclei in each seminiferous tubule were counted until 500 Sertoli cell nuclei had been enumerated. Data were analyzed by two people to test for evaluator effect, and no significant 214
Figure 3. Combined effect of GM-CSF treatments on the average number of apoptotic cells per 100 Sertoli cells. Data are represented as mean standard deviation. GM-CSF, granulocyte macrophage-colony stimulating factor.
difference was found between evaluator 1 and evaluator 2. ANOVA and Tukey’s test were used to compare apoptosis among treatment groups. Statistical significance was determined at P < .05. Percent differences for each treatment versus the control also were calculated.
RESULTS The number of apoptotic cells per 100 Sertoli cell nuclei for each treatment is shown in Table 1. Figure 1 shows immunohistochemical localization of apoptotic cells within the seminiferous epithelium. For example, the control tissue shown in Fig. 1A exhibits labeled apoptotic cells, whereas the GM-CSF–treated tissue in Fig. 1B shows few or no labeled apoptotic cells within the seminiferous epithelium. A summary of the number of apoptotic cells per 100 Sertoli cells (mean SD) for each of the growth factors and their combinations is shown in Fig. 2. ANOVA and Tukey’s test, comparing GM-CSF–treated tissue with non–GM-CSF–treated tissue, indicated that treatment with GM-CSF reduced the mean number of apoptotic cells when compared with controls (P < .05) and tissue treated without GM-CSF (P < .001) (Fig. 3). Time zero (T0) (tissue that was fixed immediately and not cultured) had a lower mean number of apoptotic cells per 100 Sertoli cell nuclei when compared with all other treatments including the control (Fig. 2).
DISCUSSION AND CONCLUSION This study shows that GM-CSF was able to reduce the mean number of apoptotic cells per 100 Sertoli cell nuclei. Dirami et al13 also showed that, at relatively low concentrations, GM-CSF promoted cell survival. Neither estradiol (E2), stem cell factor (SCF), nor LIF alone were able to alter the mean number of apoptotic testicular germ cells. It was expected that both E2 and SCF treatments would Journal of Equine Veterinary Science
May 2007
significantly reduce the incidence of apoptosis. SCF regulates the proliferation of spermatogonia in the adult human8,20 and acts directly on primordial germ cells (PGC) by suppressing apoptosis.9 LIF also acts to suppress apoptosis in PGCs9 while (dose dependently) stimulating PGC proliferation.21 In spite of GM-CSF’s and SCF’s ability to promote type A spermatogonial survival and cell proliferation, and block retinoic acid induced apoptosis in the human,13,22 no significant reduction in apoptosis was seen for this treatment. The individual cell survival capabilities of LIF and GMCSF; SCF, LIF, and GM-CSF; and E2, SCF, LIF, and GM-CSF suggest that these factors might synergistically reduce apoptosis. Tissue treated with GM-CSF alone or in combination with other growth factors reduced the mean number of apoptotic cells compared with control samples and the combination of treatments without GM-CSF. A significant difference in apoptotic rate also was seen with both SCF, LIF, and GM-CSF combined and with all four treatments (E2, SCF, LIF, and GM-CSF) combined. Data suggest there was a synergistic reduction in apoptosis when all three growth factors were combined. Furthermore, the addition of E2 to SCF, LIF, and GMCSF resulted in an even greater reduction in apoptosis than SCF, LIF, and GM-CSF combined. These results are also shown in the percent differences, in which GMCSF alone had a 34.77% decrease on the mean number of apoptotic cells per 100 Sertoli cell nuclei, GM-CSF, SCF, and LIF had a 37.45% decrease, and all four combined had a 44.40% reduction. The results from this study could in part be explained because of species specification. Human recombinant growth factors were used instead of equine. In addition, growth factor concentrations derived from literature were optimized for non-equine tissue. Also, a short-term culture (6 hours) was used for this experiment, but perhaps a culture of 24 to 48 hours would have given the growth factors more time to affect the incidence of apoptosis. Furthermore, incidence of apoptosis may be attributed to a lack of ability to maintain the necessary ratio of Sertoli cells needed or to chemical signals given off by round spermatids needed for germ cell survival.23 This study shows how growth factors can affect the pathways of apoptosis and supports the possibility that the addition of growth factors inhibits apoptotic activity. E2, SCF, LIF, and GM-CSF were hypothesized to reduce the incidence of apoptosis in testicular germ cells. However, E2, SCF, and LIF, when administered alone, did not significantly reduce apoptosis. Tissue treated with GM-CSF had reduced apoptosis when compared with controls. The combined treatment of SCF, LIF, and GM-CSF was also able to reduce apoptosis, and the addition of E2 to SCF, LIF, and GM-CSF enhanced the reduction in apoptosis. There may be a synergy between the combination of SCF, LIF, and GM-CSF and E2, SCF, LIF, and GMCSF. More research is necessary to determine the role of other growth factors in apoptosis. Volume 27, Number 5
ACKNOWLEDGMENT This study was funded in part by the Link Equine Endowment, Texas A&M University.
REFERENCES 1. Casey PJ, Robertson KR, Liu IK, Espinoza SB, Drobnis EZ. Column separation of motile sperm from stallion semen. J Androl 1993;14: 142–148. 2. Douglas R, Umphenour N. Endocrine abnormalities and hormone therapy. Vet Clin North Am 1992;8:237–248. 3. De Vries P. Diseases of the testes, penis, and related structures. In: McKinnon A, Voss J, eds. Equine reproduction. Philadelphia: Lea & Febiger; 1993:878–884. 4. Roser J. Subfertility and infertility in the stallion. World Equine Vet Rev 1999;4:32–37. 5. Lin W, Lamb D, Lipshultz L, Kim E. Absence of cyclic-AMP responsive element modulator expression at the spermatocyte arrest stage. Fertil Steril 1998;69:533–538. 6. Lin W, Lamb D, Wheeler T, Lipshultz L, Kim E. In-situ labeling of human testicular tissue demonstrates increased apoptosis in conditions of abnormal spermatogenesis. Fertil Steril 1997;68: 1065–1069. 7. Pentika¨inen V, Erkkila¨ K, Suomalainen L, Parvinen M, Dunkel L. Estradiol acts as a germ cell survival factor in the human testis in vitro. J Clin Endocrinol Metab 2000;85:2057–2067. 8. Mauduit C, Hamamah S, Benahmed M. Stem cell factor/c-kit system in spermatogenesis. Human Reprod Update 1999;5: 535–545. 9. Pesce M, Farrace MG, Piacentini M, Dolci S, De Felici M. Stem cell factor and leukemia inhibitory factor promote primordial germ cell survival by suppressing programmed cell death (apoptosis). Development 1993;118:1089–1094. 10. Leary AG, Wong GG, Clark SC, Smith AG, Ogawa M. Leukemia inhibitory factor differentiation-inhibiting activity/human interleukin for DA cells augments proliferation of human hematopoietic stem cells. Blood 1990;75:1960–1964. 11. Martin FH, Suggs SV, Langley KE, Lu HS, Ting J, Okino KH, et al. Primary structure and functional expression of rat and human stem cell factor DNAs. Cell 1990;63:203–211. 12. McLay RN, Banks WA, Kastin AJ. Granulocyte macrophage-colony stimulating factor crosses the blood-testis barrier in mice. Biol Reprod 1997;57:822–826. 13. Dirami G, Ravindranath N, Pursel V, Dym M. Effects of stem cell factor and granulocyte macrophage-colony stimulating factor on survival of porcine type A spermatogonia cultured in KSOM. Biol Reprod 1999;61:225–230. 14. Steinberger A, Steinberger E. Stimulatory effect of vitamins and glutamine on the differentiation of germ cells in rat testis organ culture grown in chemically defined media. Exp Cell Res 1966;44: 429–435. 15. Jutte NH, Jansen R, Grootegoed JA, Rommerts FF, van der Molen HJ. Protein synthesis by isolated pachytene spermatocytes in the absence of Sertoli cells. J Exp Zool 1985;233:285–290. 16. Erkkila K, Hirvonen V, Wuokko E, Parvinen M, Dunkel L. N-acetylL-cysteine inhibits apoptosis in human male germ cells in vitro. J Clin Endocrinol Metab 1983;83:2523–2531.
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17. Staub C. A century of research on mammalian male germ cell meiotic differentiation in vitro. J Androl 2001;22:911–926.
21. Nikolova D, Martinova Y, Seidensticker M, Bellve A. Leukemia inhibitory factor stimulates proliferation of prospermatogonial stem
18. Mauduit C, Goodard I, Besset V, Tabone E, Rey C, Gashier F, et al.
cells. Reprod Fertil Dev 1997;9:717–721.
Leukemia inhibiting factor antagonizes gonadotropin induced-
22. Horie M, Broxmeyer H. The combination of steel factor and
testosterone synthesis in cultured porcine leydig cells: sites of action. Endocrinology 2001;142:2509–2520.
GM-CSF blocks apoptosis induced by retinoic acid and upregulates AP-1 in a human growth factor dependent cell line. Exp Hematol
19. Intergen Company. The complete ApopTagÒ manual. New York: Intergen Company; 2001:14–16.
1995;23:168–173. 23. Zhao GQ, Deng K, Labosky PA, Liaw L, Hogan BL. The gene
20. Gnessi L, Fabbri A, Spera G. Gonadal peptides as mediators of develop-
encoding bone morphogenic protein 8B is required for the initiation
mental and functional control of the testis: an integrated system with
and maintenance of spermatogenesis in the mouse. Genes Dev
hormones and local environment. Endocr Rev 1997;18:541–609.
1996;10:1657–1669.
ERRATUM Cavinder CA, Vogelsang MM, Gibbs PG, Forrest DW, Schmitz DG. Endocrine Profile Comparisons of Fat Versus Moderately Conditioned Mares Following Parturition. J Equine Vet Sci 2007;27:72-79. In the aforementioned article, the authors would like to acknowledge Dr. Gary Williams at the Texas Agricultural Research Station in Beeville, Texas for the laboratory assistance provided for this research.
216
Journal of Equine Veterinary Science
May 2007
ORIGINAL RESEARCH
The Effects of Growth Factor on Testicular Germ Cell Apoptosis in the Stallion Casey L. Donnelly, MS,a Christophe Staub, PhD,b Dickson Varner, PhD, DVM,c Terry Blanchard, PhD, DVM,d Larry Johnson, PhD,b and David W. Forrest, PhDa ABSTRACT Apoptosis is necessary for both initiation and control of spermatogenesis; however, an increase in apoptosis can lead to subfertility/infertility in stallions, causing substantial financial loss in the equine industry. The ability of stem cell factor (SCF), leukemia-inhibiting factor (LIF), granulocyte–macrophage colony-stimulating factor (GM-CSF), and estradiol (E2), alone or in combination, to prevent apoptosis of germ cells in short-term equine testicular cultures was examined. Testicular tissue was sectioned into approximately 2-mm cubes and placed in media-filled culture chambers. Concentrations of SCF (100 ng/mL), LIF (10 ng/mL), GM-CSF (5 ng/mL), and E2 (109 mol/l) were added alone or in combination to each well. After 6 hours in culture, the tissue was fixed and immunohistochemically (terminal deoxynucleotidyl transferase-mediated nick-end labeling; TUNEL) stained for apoptosis detection. Apoptotic cells per 100 Sertoli cell nuclei within seminiferous tubules were counted until the 500th Sertoli cell nuclei was reached. This counting procedure was used for each slide. An analysis of variance (ANOVA) with a Tukey’s test was used to compare apoptotic rates. In comparison with the control, GM-CSF alone lowered apoptosis by 34.77%. GM-CSF–treated tissue combined with SCF and LIF as well as GM-CSF combined with SCF, LIF, and E2 reduced apoptosis when compared with the control (37.45% and 44.40%, respectively) or other treatment combinations. GM-CSF alone reduced apoptosis; results suggest possible synergy for the combinations of SCF and LIF with GM-CSF and for E2 with SCF, LIF, and GM-CSF. Keywords: Apoptosis; Stallion; Growth factors; Granulocyte–macrophage colony-stimulating factor (GM-CSF); Spermatogonia
From the Department of Animal Science,a the Department of Veterinary Integrative Biosciences,b and the Department of Large Animal Clinical Sciences,c Texas A&M University, College Station, TX; and Hill N Dale Farm, Lexington, KY.d Reprint requests to: David W. Forrest, PhD, Department of Animal Science, Texas A&M University, 2471, TAMU College Station, TX 77843-2471. 0737-0806/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jevs.2007.04.003
212
INTRODUCTION Stallion subfertility/infertility is common1 and may cause substantial financial loss in the equine industry.2-4 Among the possible abnormalities or causes of germ cell loss may be an increase in apoptosis at the germ-cell level. During normal spermatogenesis, up to one-half of all germ cells never differentiate into mature sperm because of apoptoticrelated mechanisms.5 Increased apoptosis can reduce sperm count,5,6 leading to subfertility. The growth factors used in this study have known roles in spermatogenesis and apoptosis. In the human, addition of 17b-estradiol (109 mol/l) inhibited germ cell apoptosis and reduced apoptosis in spermatogenic cells in vitro.7 In mice, reduced expression of stem cell factor (SCF) increased germ cell apoptosis,8 and leukemia-inhibiting factor (LIF) acted directly on primordial germ cells to suppress apoptosis.9,10 Both SCF and LIF act synergistically with colony-stimulating factors such as granulocyte–macrophage colony-stimulating factor (GM-CSF).10,11 In a test conducted on normal human hematopoietic cells, Leary et al10 suggest that LIF/DIA (differentiation-inhibiting activity) along with G-CSF (granulocyte colony-stimulating factor) may be involved in early hematopoietic stem cell regulation. Also, in a study conducted by Martin et al11 using genomic clones of human SCF, SCF showed strong synergistic effects with colony-stimulating factors. GM-CSF recently has been shown to cross the blood–testis barrier intact,12 and it is able to promote the survival of porcine type A spermatogonial cells.13 The objective of this study was to determine whether the growth factors LIF, SCF, E2, and GM-CSF alone or in combination, might lower the incidence of apoptosis in stallion testicular germ cells, thereby identifying a possible treatment for this condition.
MATERIALS AND METHODS Testes Collection, Culture, and Growth Factor Administration Two stallions aged 5 and 7 years and of known reproductive soundness (determined by daily sperm output, motility, and concentration over a 3-month period) were castrated under general anesthesia. A section of testes from each horse was wrapped in aluminum foil and put on ice (for no more than 10 minutes). The testicular Journal of Equine Veterinary Science
May 2007
Table 1. Mean number of apoptotic cells per 100 Sertoli cell nuclei and the percentage difference of each treatment versus the control Growth Factor Treatments Control E2 C1 mean 18.94 C2 mean 24.06 % Difference from control
SCF
LIF
SCFD SCFD LIFD SCFDLIFD E2DSCFD GMCSF* LIF GMCSF GMCSF GMCSF* LIFDGMCSF*
15.20 23.42 22.71 13.06 28.39 27.47 24.45 14.99 1.3 18.33 9.69 34.77
18.41 18.07 27.93 15.56 7.77 21.80
17.31 18.53 16.66
8.89 18.01 37.45
10.14 13.78 44.40
The first horse was designated as culture 1 (C1) and the second culture 2 (C2). Each treatment had two wells, with a piece of tissue in each well. The mean number of apoptotic cells per 100 Sertoli cell nuclei per treatment is shown. The last row is the overall percent difference of each treatment compared with the control. A negative number signifies an increase and a positive number a decrease. * P < .05.
Figure 1. Immunohistochemical localization of apoptotic cells within the seminiferous epithelium. Labeled apoptotic cells are shown in A, whereas few or no labeled apoptotic cells within the seminiferous epithelium are shown in B.
samples were sectioned into approximately 2-mm cubes and placed into culture chambers fitted with bicameral cell culture inserts (Becton Dickinson Labware, Franklin Lakes, NJ). They were filled with media (Hanks F12/ DMEM 1:1) supplemented with 4 mmol/l glutamine,14 4 mmol/l sodium lactate, 1 mmol/l sodium pyruvate,15 Volume 27, Number 5
and 50 mg/ml ascorbic acid as an antioxidant16 and incubated for 6 hours17 at 328C at 5% CO2. Dosages Media only was used as the control. SCF (100 ng/ml),13 LIF (10 ng/ml),18 GM-CSF (5 ng/ml),13 or E2 (109 M)7 213
Figure 2. Treatment effects on the average number of apoptotic cells per 100 Sertoli cells. Data are represented as mean standard error, n ¼ 4. Time ¼ 0 (T0) is the number of apoptotic cells per 100 Sertoli cell nuclei present when the tissue was fixed immediately and not cultured. E2, estradiol; SCF, stem cell factor; LIF, leukemia inhibiting factor; GM-CSF, granulocyte macrophage-colony stimulating factor.
(all Sigma Chemical Co., St. Louis, MO) were added to each well, either alone or in combination. Two sections from each of the two testes were placed in separate individual wells for each treatment. They were incubated for 6 hours as described previously. Immunohistochemistry After 6 hours in culture, the tissue was fixed in 4% paraformaldehyde embedded in paraffin and sectioned into 5-mm slices. The tissue was deparaffinized and analyzed for apoptotic cells using the ApopTag Peroxidase In Situ Apoptosis Detection Kit (terminal deoxynucleotidyl transferase-mediated nick-end labeling; TUNEL) (Intergen Company, Purchase, NY) and counterstained with Mayer’s hematoxylin to observe nonreactive cells.19 Data Collection and Statistical Analysis The numbers of apoptotic germ cells per 100 detected Sertoli cell nuclei were then counted using a Zeiss Axioplan 2 microscope system at 630 total magnification. These data are reported as the average number of apoptotic cells per 100 Sertoli cell nuclei of the two tissue samples in each of the two cultures. Starting with one corner of a tissue section (excluding the tubules on the edges because of an increase in artifactual apoptotic labeling caused by processing the tissue), the numbers of apoptotic and Sertoli cell nuclei in each seminiferous tubule were counted until 500 Sertoli cell nuclei had been enumerated. Data were analyzed by two people to test for evaluator effect, and no significant 214
Figure 3. Combined effect of GM-CSF treatments on the average number of apoptotic cells per 100 Sertoli cells. Data are represented as mean standard deviation. GM-CSF, granulocyte macrophage-colony stimulating factor.
difference was found between evaluator 1 and evaluator 2. ANOVA and Tukey’s test were used to compare apoptosis among treatment groups. Statistical significance was determined at P < .05. Percent differences for each treatment versus the control also were calculated.
RESULTS The number of apoptotic cells per 100 Sertoli cell nuclei for each treatment is shown in Table 1. Figure 1 shows immunohistochemical localization of apoptotic cells within the seminiferous epithelium. For example, the control tissue shown in Fig. 1A exhibits labeled apoptotic cells, whereas the GM-CSF–treated tissue in Fig. 1B shows few or no labeled apoptotic cells within the seminiferous epithelium. A summary of the number of apoptotic cells per 100 Sertoli cells (mean SD) for each of the growth factors and their combinations is shown in Fig. 2. ANOVA and Tukey’s test, comparing GM-CSF–treated tissue with non–GM-CSF–treated tissue, indicated that treatment with GM-CSF reduced the mean number of apoptotic cells when compared with controls (P < .05) and tissue treated without GM-CSF (P < .001) (Fig. 3). Time zero (T0) (tissue that was fixed immediately and not cultured) had a lower mean number of apoptotic cells per 100 Sertoli cell nuclei when compared with all other treatments including the control (Fig. 2).
DISCUSSION AND CONCLUSION This study shows that GM-CSF was able to reduce the mean number of apoptotic cells per 100 Sertoli cell nuclei. Dirami et al13 also showed that, at relatively low concentrations, GM-CSF promoted cell survival. Neither estradiol (E2), stem cell factor (SCF), nor LIF alone were able to alter the mean number of apoptotic testicular germ cells. It was expected that both E2 and SCF treatments would Journal of Equine Veterinary Science
May 2007
significantly reduce the incidence of apoptosis. SCF regulates the proliferation of spermatogonia in the adult human8,20 and acts directly on primordial germ cells (PGC) by suppressing apoptosis.9 LIF also acts to suppress apoptosis in PGCs9 while (dose dependently) stimulating PGC proliferation.21 In spite of GM-CSF’s and SCF’s ability to promote type A spermatogonial survival and cell proliferation, and block retinoic acid induced apoptosis in the human,13,22 no significant reduction in apoptosis was seen for this treatment. The individual cell survival capabilities of LIF and GMCSF; SCF, LIF, and GM-CSF; and E2, SCF, LIF, and GM-CSF suggest that these factors might synergistically reduce apoptosis. Tissue treated with GM-CSF alone or in combination with other growth factors reduced the mean number of apoptotic cells compared with control samples and the combination of treatments without GM-CSF. A significant difference in apoptotic rate also was seen with both SCF, LIF, and GM-CSF combined and with all four treatments (E2, SCF, LIF, and GM-CSF) combined. Data suggest there was a synergistic reduction in apoptosis when all three growth factors were combined. Furthermore, the addition of E2 to SCF, LIF, and GMCSF resulted in an even greater reduction in apoptosis than SCF, LIF, and GM-CSF combined. These results are also shown in the percent differences, in which GMCSF alone had a 34.77% decrease on the mean number of apoptotic cells per 100 Sertoli cell nuclei, GM-CSF, SCF, and LIF had a 37.45% decrease, and all four combined had a 44.40% reduction. The results from this study could in part be explained because of species specification. Human recombinant growth factors were used instead of equine. In addition, growth factor concentrations derived from literature were optimized for non-equine tissue. Also, a short-term culture (6 hours) was used for this experiment, but perhaps a culture of 24 to 48 hours would have given the growth factors more time to affect the incidence of apoptosis. Furthermore, incidence of apoptosis may be attributed to a lack of ability to maintain the necessary ratio of Sertoli cells needed or to chemical signals given off by round spermatids needed for germ cell survival.23 This study shows how growth factors can affect the pathways of apoptosis and supports the possibility that the addition of growth factors inhibits apoptotic activity. E2, SCF, LIF, and GM-CSF were hypothesized to reduce the incidence of apoptosis in testicular germ cells. However, E2, SCF, and LIF, when administered alone, did not significantly reduce apoptosis. Tissue treated with GM-CSF had reduced apoptosis when compared with controls. The combined treatment of SCF, LIF, and GM-CSF was also able to reduce apoptosis, and the addition of E2 to SCF, LIF, and GM-CSF enhanced the reduction in apoptosis. There may be a synergy between the combination of SCF, LIF, and GM-CSF and E2, SCF, LIF, and GMCSF. More research is necessary to determine the role of other growth factors in apoptosis. Volume 27, Number 5
ACKNOWLEDGMENT This study was funded in part by the Link Equine Endowment, Texas A&M University.
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