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Differential effects of prepubertal rat Sertoli cell secreted proteins on somatic testicular and nontesticular cells
Molecular and Cellular Endocrinology, 98 (1993) 75-79 0 1993 Elsevier Scientific Publishers Ireland, Ltd. 0303-7207/93/$06.00
75
MCE 03116
Differential
effects of prepubertal rat Sertoli cell secreted proteins on somatic testicular and nontesticular cells
’ Y.S. Martinova *, L.S. Kancheva, D.B. Nikolova, and V.D. Georgiev Institute of Cell Biology and Morphology, Bulgarian Academy of Sciences, Sofia II 13, Bulgaria
(Received 7 June 1993; accepted 6 September 1993)
Key words: Cultured prepubertal rat Sertoli cell; Secreted protein; Cell proliferation; Immunoregulation
Summary There is little information on the mitogenic and immunoregulatory activities of proteins, secreted by prepubertal Sertoli cells during the stage of meiosis initiation and before creation of the blood-testis barrier. We have previously demonstrated dose-dependent and age-related stimulation of BALB/c 3T3 fibroblasts and quiescent rat prespermatogonia (Kancheva et al., 1990) as well as inhibition of natural killer cell activity of mice, guinea pigs and human lymphocytes (Nikolova et al., 1992) by Sertoli cell-conditioned medium derived from lZday-old rats. In the current study, using splenic lymphocytes stimulated by PI-IA, LPS and Con A, we have shown a dose-dependent inhibition of T and B lymphocyte proliferation by prepubertal Sertoli cell-secreted proteins (pSCSP). These results suggest that by the time the blood-testis barrier had been formed, Sertoli cell in rat testis had already synthesized immunoregulatory proteins. In addition we have found that pSCSP stimulate the proliferation of TM, Leydig but not TM, Sertoli cells. The differential effect of pSCSP is an expression of the different balance between growth factors secreted by Sertoli cells, which in turn is dependent on the requirements of the cell types at each stage of testicular development.
Introduction A number of growth factors and peptides have recently been identified in the mammalian testis, including seminiferous growth factor (SGF), Sertoli cellsecreted growth factor (SCSGF), acidic and basic fibroblast growth factor (aFGF, bFGF), nerve growth factor (NGF), transforming growth factor (TGF-(Y and TGF-/3), activin, inhibin, etc. These factors are supposed to be complex and sensitive regulators of cellular growth and differentiation in the testicular tissue (Mullaney and Skinner, 1991). The majority of testicular growth factors are known to be produced by Sertoli cells and may act on cells within the basal and/or adlumenal compartment of the seminiferous tubules (Bell& and Zheng, 1991). The creation of the two compartments is due to structural maturation of Sertoli cell, which includes the formation of inter-Sertoli cell tight junctions - a major component of the functional blood-testis barrier (Dym and Fawcett, 1970). In the rat the blood-testis barrier develops between the 16th
* Corresponding
author.
and 19th day p.p., when junctional complexes first appear between Sertoli cells (Vitale et al., 1973). It is well known that Sertoli cells secrete part of the seminal plasma components, which in turn suppress numerous functions of the immune system, and lead to the suggestion that the testis belongs to the ‘immunologically privileged’ tissues (Head et al., 1983). The immunosuppressive effect of Sertoli cell proteins from 20-day-old rats as well as of inhibin, activin and TGF-P, on phytolectin-stimulated splenic rat lymphocytes and on thymocytes has been demonstrated (Wyatt et al., 1988; Hedger et al., 1989). Sertoli cell secretion activity is dependent on many factors including hormones, the cycle of seminiferous epithelium (Parvinen et al., 1986), the stage of Sertoli cell maturation (Ritzen et al., 1981) as well on the stage of germ cell maturation (Le Magueresse et al., 1988; Pineau et al., 1990). We have presented evidence about dose-dependent and age-related stimulation of BALB/c 3T3 fibroblasts and quiescent rat prespermatogonia by proteins secreted from cultured Sertoli cells derived from 6- and lZday-old rats (Kancheva et al., 1990). The testicular architecture of 12-day-old rat is rather rare. The germ cells have just entered the first meiotic prophase; Sertoli cells lose the ability to prolif-
76
erate, while Leydig cells actively divide and synthesize testosterone. It is of particular interest to study the autocrine and paracrine regulatory possibilities of pSCSP at this critical stage of testicular development. On the other hand, immunoregulatory functions of pSCSP before formation of the blood-testis barrier is not known. We have shown that pSCSP from 12-day-old rats inhibited natural killer cell activity of mice, guinea pigs and human lymphocytes (Nikolova et al., 1992). In the current study, using splenic lymphocytes stimulated by PHA, LPS and Con A, we have shown a dose-dependent inhibition of T and B lymphocyte proliferation by pSCSP from lZday-old rats. In addition we have found that pSCSP stimulates the proliferation of BALB/c 3T3 fibroblasts and TM, Leydig cells but not TM, Sertoli cells. Materials
and methods
Materials
Immature lZday-old and mature female inbred Wistar rats were purchased from the animal breeding farm of the Bulgarian Academy of Sciences, Sofia. Dulbecco’s modified Eagle’s medium (DMEM), Ham’s F12, fetal bovine serum (FBS), tissue culture dishes and 96-well multidishes were supplied by Flow Labs., UK, RPM1 1640, trypsin from bovine pancreas (3.1 U/mg) and 8000 molecular weight exclusion limit dialysis tubings were supplied by Serva, Germany; collagenase was obtained from Boehringer, Mannheim, Germany; methyl-3H-thymidine (specific activity 5 Ci/ mmol) was purchased from Amersham, UK, Polysep was supplied by Pharmachim, Bulgaria, concanavalin A (Con A> from Pharmacia, phytohaemagglutinin P (PHA) and E. coli lipopolysaccharide (LPS) from Difco, USA; TM, Leydig and TM, Sertoli cells were kindly provided by Prof. Anthony BellvC (Department of Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY). Methods
Primary culture from prepubertal lZday-old rat Sertoli cells was prepared as described previously (Martinova et al., 1988). Briefly, decapsulated testicular fragments were consecutively digested with collagenase, hyaluronidase and trypsin. Isolated Sertoli cells were plated in tissue culture dishes at density l-l.5 x lo6 cells/ml in DMEM/Ham’s F12 (1: 1, vol/vol) supplemented with 5% FBS. At 24 h after seeding the cells were washed twice and given DMEM/F12 without serum. The Sertoli cell-conditioned medium (SCCM) was collected on 4th and 7th day after seeding. SCCM was dialyzed against 6 X 20 volumes 5 mM ammonium bicarbonate buffer pH 7.4. The media were frozen, lyophilized and reconstituted in DMEM at different concentrations. Using two-dimensional gel
electrophoresis of SCCM after [35S]methionine labeling, the putative factor(s) was pre-characterized as a protein with a molecular weight over 8000, end-sensitive to heat and trypsin treatment (Martinova et al., 1988). The total amount of proteins measured according to the methods of Lowry et al. (1951) was 10 pg/ml. Lymphocyte separation
Spleens from adult female Wistar rats were aseptically removed. The spleen cells were pressed through a wire mesh screen and the resulting cell suspension pooled. The splenic mononuclear cells were obtained after centrifugation on Polysep (density gradient 1.077 g/cm3) at 300 x g for 30 min. The cells were washed twice and resuspended in RPM1 1640 medium supplemented with 10% FBS. Lymphocyte proliferation assay
Splenic lymphocytes were suspended at 1 x lo6 cells/ml in RPM1 1640 supplemented with 10% FBS and seeded in 96-well U-bottom polystyrene multidishes in 180 kl/well. Lymphocyte proliferation was induced by addition of optimal concentration of Con A (0.5 pg/ml), PHA (25 pg/ml) or LPS (50 pg/ml) to triplicates. The cells were grown at 37°C in a humidified atmosphere of 95% air and 5% CO, for 72 h and for an additional 18 h in the presence of 1 &i per well of 3H-thymidine. pSCSP was added in 20 ~1 per well at final concentrations of 20, 40 and 80 Fg/ml at 0, 24 and 48 h after seeding. The results are expressed as mitotic index k standard deviation (Chang et al., 1981).
Mitotic index =
cpm sample (Ly + mitogen + pSCSP) cpm control (Ly only)
Viability of lymphocytes treated with different doses of pSCSP was determined 48 h after seeding by the Trypan blue exclusion test and expressed as percentage of living cells. 3T3 cell proliferation assay
BALB/c 3T3 fibroblasts were grown to confluence in 96-well polystyrene multidishes at a density of 1 x lo6 cells/ml in 200 ~1 of DMEM supplemented with 10% FBS for 3 days. Subsequently the medium was changed with DMEM + 0.5% FBS for a further 7 days’ cultivation. The quiescent 3T3 cells were incubated 48 h in DMEM containing different concentrations of pSCSP and 5 ~1 of 3H-thymidine/per well. Controls were incubated in DMEM without pSCSP. The incorporation of the isotope into DNA of 3T3 cells was quantified by scintillation spectrometry (Klagsbrun et al., 1977).
77
TM, and TM, cells proliferation assay
TM, Leydig or TM, Sertoli cells were propagated in DMEM/Ham’s F12 (1: 1, vol/vol) containing 1.2 mg/ml sodium bicarbonate, 15 mM Hepes, 10 pg/ml gentamicin sulfate, and supplemented with 5% FCS. TM, or TM, cells were seeded at 25000 cells/ml in 24-well plates in DMEM/F12 supplemented with 1 ng/ml EGF, 10 pug/ml insulin and 5 pg/ml transferrin. At 48 h after seeding the cells were washed and given DMEM/F12, containing different concentrations of pSCSP. Controls were given DMEM/F12 supplemented with 5% FCS (positive control) or 5% PBS (negative control). The cells were cultured for a further 48 h, harvested by adding 1 ml of 0.05% trypsin, suspended by repeated aspiration with a Pasteur pipette and counted with a hemocytometer.
TABLE 1 VIABILITY IN VITRO
OF pSCSP-TREATED
SPLENIC LYMPHOCYTES
Additions
Viability * (%)
Ly + pSCSP (80 @g/ml) Ly + pSCSP (40 pg/ml) Ly + pSCSP (20 @g/ml) LY
96.1 98.0 97.3 97.0
* Average % viability from triplicate wells.
Results Effect of pSCSP on lymphocyte proliferation
Inhibition of lymphocyte proliferation is presented in Fig. 1. It shows that in the presence of pSCSP, the mitotic indices of cell proliferation are significantly lower in comparison with those of the controls. The results are similar after activation with T cell stimulatory lectins, PHA and Con A, as well as with polyclonal B cell mitogen, LPS. The lymphocyte proliferation was inhibited in a dose-dependent manner with the lowest mitotic indices when pSCSP was added at 0 h after seeding. When added at 24 and 48 h after seeding, pSCSP expressed a similar inhibitory effect (Fig. 1). Fig. 2. Effect of pSCSP on [‘Hlthymidine incorporation by BALB/c 3T3 fibroblasts in vitro. Means represent five experiments, each done in triplicate + SEM.
0
n
20
(4
(9 Contrds
Fig. 1. Effect of different concentration of pSCSP on mitotic index (MI) of rat splenic lymphocytes at 48 h after stimulation with LPS (50 pg/ml), Con A (0.5 pg/ml) and PI-IA (25 pg/mll. Means represent two experiments, each done in triplicate.
Fig. 3. Effect of pSCSP on TM, Leydig cell proliferation. Data were derived from two separate cultures, each done in triplicate and represented by means+ SEM.
20 -
(4
t-1 Cartfrds
Fig. 4. Effect of pSCSP on TM, Sertoli cell proliferation. Data were derived from two separate cultures, each done in triplicate and represented by means+SEM.
The inhibition was not due to the toxicity of pSCSP on lymphocytes at any of the tested concentrations (Table 1). pSCSP does not affect proliferation of mitogennon-stimulated lymphocytes. Induction of BALB/c 3T3 cell proliferation by pSCSP is shown in Fig. 2. Maximal mitogenic effect was attained between 10 and 30 pg/ml with considerably high 3H-thymidine incorporation: 7-fold over controls (p < 0.01). A similar protein concentration was maximally effective in the stimulation of TM, Leydig cells (Fig. 3). pSCSP was not mitogenic for TM, cells. In comparison with a relatively high value of negative control, the effect of 5 pg/ml could be accepted as an inhibition of TM, cell proliferation (Fig. 4). Discussion In this study we provide evidence on the effect of pSCSP derived from lZday-old rat testes on cell proliferation of different cell types from testicular and nontesticular origin. pSCSP inhibits phytolectin-stimulated lymphocyte proliferation in a dose-dependent manner when added at 0, 24 and 48 h after seeding. pSCSP induces 3T3 and TM, Leydig cells to proliferate, but is not mitogenic for TM, Sertoli cells. The inhibition of mitogen-stimulated lymphocyte proliferation is consistent with the effect of TGF-& and activin (Hedger et al., 1989) and with those of SCP from 20-day-old rats (Wyatt et al., 1988). Based upon the above-mentioned data and on the suppressive effect of pSCSP on natural killer cell activity (Nikolova et al., 1992), it could be assumed that by the time the blood-testis barrier had been formed, Sertoli cells in rat are already able to synthesize immunoregulatory peptides.
The response of 3T3 fibroblasts to pSCSP from lZday-old rats is similar with the effect of TGF-/3, (Hedger et al., 19891, SGF, aFGF, bFGF (Braunhut et al., 1990), as well as with the mitogenic effect of SCCM from 6-day-old rat testes (Kancheva et al., 1990), which stimulated 3H-thymidine incorporation. Comparable studies on two somatic cell lines from testicular origin, TM, Leydig and TM, Sertoli cells, have shown similarities between them and their presumed archetyped cell (Mather et al., 1982; Maggi et al., 1989), and so they are a successful tool in the study of local paracrine and/or autocrine regulation in the testis (BellvC and Zheng, 1991). pSCSP stimulated TM, Leydig cells to proliferate, but did not activate TM, Sertoli cells, which is proportionate to the effect of aFGF (Zheng et al., 1990). On the other hand, it is well known thatSertoli cell proliferation declines after birth and ceases completely on day 15 p.p. (Clermont and Perry, 1957). Therefore it is reasonable to expect a decrease in the level of mitogenic factor(s) synthesis and secretion by Sertoli cells on day 12 p.p. as an expression of their local autocrine regulatory properties. Mitogenic activity of Sertoli cell derived proteins was first detected during mouse testicular development on days 6-8 and 12-14 p.p. (Feig et al., 1980). We have shown before that Sertoli cell-conditioned medium from 6-day-old rats can stimulate DNA synthesis in quiescent prespermatogonia (Kancheva et al., 1990). SGF purified from 1-2-week old calf testis is shown to stimulate incorporation of 5-bromo-2’-deoxyuridine (BrdU) into prespermatogonial stem cells of 3-day-old rat testis as well (Bell& et al., 1993). Obviously, depending on the stage of testicular development, Sertoli cells secrete a great variety of growth factors and peptides. On day 6 p.p., when gonocytes reinitiate DNA synthesis in vivo, Sertoli cells secrete mitogenic factor(s) that stimulates quiescent prespermatogonia (Kancheva et al., 1990). On day 12 when the condition of the testis is rather different, pSCSP is mitogenic for quiescent prespermatogonia but in a lower degree than pSCSP from 6-day-old rats (Kancheva et al., 19901, does not stimulate TM, Sertoli cell but stimulates TM, Leydig cells. Bearing in mind the above-mentioned similarities between the effect of pSCSP and some purified growth factors, we may speculate that SGF, TGF-P,, aFGF, bFGF and activin are the most probable growth factors, secreted by rat Sertoli cells during the stage of meiosis initiation, i.e. about day 12 p.p. This is in agreement with the precise developmental expression of bFGF (Mullaney and Skinner, 19921, and of TGF-/3, (Teerds and Dorrington, 1993) during testicular development in the rat. It could be concluded that the balance between growth factors secreted by Sertoli cells is dependent on the requirements of different cell types at each stage of
79
testicular development. Thus, Sertoli cell-secreted growth factors in concert with pituitary ho~ones coordinate onset and maintenance of spermatogenesis. Acknowledgements We are greateful to Prof. Anthony BeIlv& for the possibility to undertake the experiments with TM, Leydig and TM, Sertoli cells in his laboratory in the College of Physicians and Surgeons, Columbia University, New York. This work was partly financed by grant K4 from the National Foundation “Scientific Researches”, Sofia. References Be&&, A.R. and Zheng, W. (1991) in Molecular Mechanisms in Cellular Growth and Differentiation (Bell&, A.R. and Vogel, H.J., eds), pp. 187-206,Academic Press, San Diego. BelM, A.R., Zheng, W., Martinova, Y.S. and Seidensticker, M.J. (1993) Methods Enzymol. (in press). Braunhut, S.J., Rupo, ,G.A., Ernisee, B.J., Zheng, W. and Bellvd, A.R. (1990) Biol. Reprod. 42, 639-648. Chang, T.W., Kung, P.C., Cingras, S.P. and Goldstein, G. (1981) Proc. Natl. Acad. Sci. USA 78, 18051808. Clermont, Y. and Perry, B. (1957) Am. J. Anat. 100,241-266. Dym, M. and Fawcett, D.W. (1970) Biol. Reprod. 3, 308-326. Feig, L.A., Bell& A.R., Horbach-Erickson, N. and Klagsbrun, M. (1980) Proc. Nat]. Acad. Sci. USA 77, 4774-4778. Head, J., Neaves, W. and Billingham, R.E. (1983) Transplantation 36,423-431.
Hedger, M.P., Drummond, A.E., Robertson, D.M., Risbridger, G.P. and De Kretser D.M. (1989) Mol. Cell. Endocrinol. 61, 133-138. Kancheva, L.S., Martinova, Y.S. and Georgiev, V.D. (1990) Mol. Cell. Eadocrinoi. 69, 121-127. Klagsbrun, M., Langer, R., Levenson, R., Smith, S. and Lellehei, C. (1977) Exp. Cell Res. 105, 99-108. Le Magueresse, B., Pineau, C., Guillou, F. and Jegou, B. (1988) J. Endocrinol. 188, R13-R16. Lowry, O., Rosenbrough, N., Farr, A. and Randall, R. (1951) 3. Biol. Chem. 193, 265-275. Maggy, M., Morris, P.L., Kassis, S. and Rodbard, D. (1989) Int. J. Androl. 12, 65-71. Martinova, Y.S., Kancheva, L.S. and Georgiev, V.D. (1988). C.R. Acad. Bulg. Sci. 41, 133-136. Mather, J.P., Zhuang, L.Z., Perez-lnfante, V. and Phillips, D.M. (1992) Ann. NY Acad. Sci. 383, 44-67. Mullaney, B.P. and Skinner, M.K. (1991) Baitli&re’s Clin. Endocrinoi. Metabol. 5, 771-790. Mullaney, B.P. and Skinner, M.K. (1992) Endocrinology 131, 292% 2934. Nikolova, D.B., Kancheva, L.S., Surneva, M.D. and Martinona, Y.S. (1992) Immunopharmacology 23, 15-20. Parvinen, M., Vihko, K.K. and Toppari, J. (19863 Int. Rev. Cytol. 104, 11.5-147. Pineau, C., Sharpe, R.M., Saunders, P.T.K., Gerard, N. and Jegou, B. (1990) Mol. Cell. Endocrinol. 72, 13-22. Ritzen, E.M., Hansson, V. and French, F.S. (1981) in The Testis (Burger, H. and De Kretser, D., eds.) pp. 171-194, Raven Press, New York. Teerds, K.J. and Do~ington, J.H. (1993) Biol. Reprod. 48, 40-45. Vitale, R., Fawcett, D.W. and Dym, M. (1973) Anat. Rec. 176, 333-334. Wyatt, C., Law, L., Magnuson, J., Griswold, M.D. and Magnuson, N.S. (1988) J. Reprod. Immunol. 14, 27-40. Zheng, W., Butwell, T.J., Heckert, L., Griswold, M.D. and Be% A.R. (1990) Growth Factors 3, 73-82.
75
MCE 03116
Differential
effects of prepubertal rat Sertoli cell secreted proteins on somatic testicular and nontesticular cells
’ Y.S. Martinova *, L.S. Kancheva, D.B. Nikolova, and V.D. Georgiev Institute of Cell Biology and Morphology, Bulgarian Academy of Sciences, Sofia II 13, Bulgaria
(Received 7 June 1993; accepted 6 September 1993)
Key words: Cultured prepubertal rat Sertoli cell; Secreted protein; Cell proliferation; Immunoregulation
Summary There is little information on the mitogenic and immunoregulatory activities of proteins, secreted by prepubertal Sertoli cells during the stage of meiosis initiation and before creation of the blood-testis barrier. We have previously demonstrated dose-dependent and age-related stimulation of BALB/c 3T3 fibroblasts and quiescent rat prespermatogonia (Kancheva et al., 1990) as well as inhibition of natural killer cell activity of mice, guinea pigs and human lymphocytes (Nikolova et al., 1992) by Sertoli cell-conditioned medium derived from lZday-old rats. In the current study, using splenic lymphocytes stimulated by PI-IA, LPS and Con A, we have shown a dose-dependent inhibition of T and B lymphocyte proliferation by prepubertal Sertoli cell-secreted proteins (pSCSP). These results suggest that by the time the blood-testis barrier had been formed, Sertoli cell in rat testis had already synthesized immunoregulatory proteins. In addition we have found that pSCSP stimulate the proliferation of TM, Leydig but not TM, Sertoli cells. The differential effect of pSCSP is an expression of the different balance between growth factors secreted by Sertoli cells, which in turn is dependent on the requirements of the cell types at each stage of testicular development.
Introduction A number of growth factors and peptides have recently been identified in the mammalian testis, including seminiferous growth factor (SGF), Sertoli cellsecreted growth factor (SCSGF), acidic and basic fibroblast growth factor (aFGF, bFGF), nerve growth factor (NGF), transforming growth factor (TGF-(Y and TGF-/3), activin, inhibin, etc. These factors are supposed to be complex and sensitive regulators of cellular growth and differentiation in the testicular tissue (Mullaney and Skinner, 1991). The majority of testicular growth factors are known to be produced by Sertoli cells and may act on cells within the basal and/or adlumenal compartment of the seminiferous tubules (Bell& and Zheng, 1991). The creation of the two compartments is due to structural maturation of Sertoli cell, which includes the formation of inter-Sertoli cell tight junctions - a major component of the functional blood-testis barrier (Dym and Fawcett, 1970). In the rat the blood-testis barrier develops between the 16th
* Corresponding
author.
and 19th day p.p., when junctional complexes first appear between Sertoli cells (Vitale et al., 1973). It is well known that Sertoli cells secrete part of the seminal plasma components, which in turn suppress numerous functions of the immune system, and lead to the suggestion that the testis belongs to the ‘immunologically privileged’ tissues (Head et al., 1983). The immunosuppressive effect of Sertoli cell proteins from 20-day-old rats as well as of inhibin, activin and TGF-P, on phytolectin-stimulated splenic rat lymphocytes and on thymocytes has been demonstrated (Wyatt et al., 1988; Hedger et al., 1989). Sertoli cell secretion activity is dependent on many factors including hormones, the cycle of seminiferous epithelium (Parvinen et al., 1986), the stage of Sertoli cell maturation (Ritzen et al., 1981) as well on the stage of germ cell maturation (Le Magueresse et al., 1988; Pineau et al., 1990). We have presented evidence about dose-dependent and age-related stimulation of BALB/c 3T3 fibroblasts and quiescent rat prespermatogonia by proteins secreted from cultured Sertoli cells derived from 6- and lZday-old rats (Kancheva et al., 1990). The testicular architecture of 12-day-old rat is rather rare. The germ cells have just entered the first meiotic prophase; Sertoli cells lose the ability to prolif-
76
erate, while Leydig cells actively divide and synthesize testosterone. It is of particular interest to study the autocrine and paracrine regulatory possibilities of pSCSP at this critical stage of testicular development. On the other hand, immunoregulatory functions of pSCSP before formation of the blood-testis barrier is not known. We have shown that pSCSP from 12-day-old rats inhibited natural killer cell activity of mice, guinea pigs and human lymphocytes (Nikolova et al., 1992). In the current study, using splenic lymphocytes stimulated by PHA, LPS and Con A, we have shown a dose-dependent inhibition of T and B lymphocyte proliferation by pSCSP from lZday-old rats. In addition we have found that pSCSP stimulates the proliferation of BALB/c 3T3 fibroblasts and TM, Leydig cells but not TM, Sertoli cells. Materials
and methods
Materials
Immature lZday-old and mature female inbred Wistar rats were purchased from the animal breeding farm of the Bulgarian Academy of Sciences, Sofia. Dulbecco’s modified Eagle’s medium (DMEM), Ham’s F12, fetal bovine serum (FBS), tissue culture dishes and 96-well multidishes were supplied by Flow Labs., UK, RPM1 1640, trypsin from bovine pancreas (3.1 U/mg) and 8000 molecular weight exclusion limit dialysis tubings were supplied by Serva, Germany; collagenase was obtained from Boehringer, Mannheim, Germany; methyl-3H-thymidine (specific activity 5 Ci/ mmol) was purchased from Amersham, UK, Polysep was supplied by Pharmachim, Bulgaria, concanavalin A (Con A> from Pharmacia, phytohaemagglutinin P (PHA) and E. coli lipopolysaccharide (LPS) from Difco, USA; TM, Leydig and TM, Sertoli cells were kindly provided by Prof. Anthony BellvC (Department of Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY). Methods
Primary culture from prepubertal lZday-old rat Sertoli cells was prepared as described previously (Martinova et al., 1988). Briefly, decapsulated testicular fragments were consecutively digested with collagenase, hyaluronidase and trypsin. Isolated Sertoli cells were plated in tissue culture dishes at density l-l.5 x lo6 cells/ml in DMEM/Ham’s F12 (1: 1, vol/vol) supplemented with 5% FBS. At 24 h after seeding the cells were washed twice and given DMEM/F12 without serum. The Sertoli cell-conditioned medium (SCCM) was collected on 4th and 7th day after seeding. SCCM was dialyzed against 6 X 20 volumes 5 mM ammonium bicarbonate buffer pH 7.4. The media were frozen, lyophilized and reconstituted in DMEM at different concentrations. Using two-dimensional gel
electrophoresis of SCCM after [35S]methionine labeling, the putative factor(s) was pre-characterized as a protein with a molecular weight over 8000, end-sensitive to heat and trypsin treatment (Martinova et al., 1988). The total amount of proteins measured according to the methods of Lowry et al. (1951) was 10 pg/ml. Lymphocyte separation
Spleens from adult female Wistar rats were aseptically removed. The spleen cells were pressed through a wire mesh screen and the resulting cell suspension pooled. The splenic mononuclear cells were obtained after centrifugation on Polysep (density gradient 1.077 g/cm3) at 300 x g for 30 min. The cells were washed twice and resuspended in RPM1 1640 medium supplemented with 10% FBS. Lymphocyte proliferation assay
Splenic lymphocytes were suspended at 1 x lo6 cells/ml in RPM1 1640 supplemented with 10% FBS and seeded in 96-well U-bottom polystyrene multidishes in 180 kl/well. Lymphocyte proliferation was induced by addition of optimal concentration of Con A (0.5 pg/ml), PHA (25 pg/ml) or LPS (50 pg/ml) to triplicates. The cells were grown at 37°C in a humidified atmosphere of 95% air and 5% CO, for 72 h and for an additional 18 h in the presence of 1 &i per well of 3H-thymidine. pSCSP was added in 20 ~1 per well at final concentrations of 20, 40 and 80 Fg/ml at 0, 24 and 48 h after seeding. The results are expressed as mitotic index k standard deviation (Chang et al., 1981).
Mitotic index =
cpm sample (Ly + mitogen + pSCSP) cpm control (Ly only)
Viability of lymphocytes treated with different doses of pSCSP was determined 48 h after seeding by the Trypan blue exclusion test and expressed as percentage of living cells. 3T3 cell proliferation assay
BALB/c 3T3 fibroblasts were grown to confluence in 96-well polystyrene multidishes at a density of 1 x lo6 cells/ml in 200 ~1 of DMEM supplemented with 10% FBS for 3 days. Subsequently the medium was changed with DMEM + 0.5% FBS for a further 7 days’ cultivation. The quiescent 3T3 cells were incubated 48 h in DMEM containing different concentrations of pSCSP and 5 ~1 of 3H-thymidine/per well. Controls were incubated in DMEM without pSCSP. The incorporation of the isotope into DNA of 3T3 cells was quantified by scintillation spectrometry (Klagsbrun et al., 1977).
77
TM, and TM, cells proliferation assay
TM, Leydig or TM, Sertoli cells were propagated in DMEM/Ham’s F12 (1: 1, vol/vol) containing 1.2 mg/ml sodium bicarbonate, 15 mM Hepes, 10 pg/ml gentamicin sulfate, and supplemented with 5% FCS. TM, or TM, cells were seeded at 25000 cells/ml in 24-well plates in DMEM/F12 supplemented with 1 ng/ml EGF, 10 pug/ml insulin and 5 pg/ml transferrin. At 48 h after seeding the cells were washed and given DMEM/F12, containing different concentrations of pSCSP. Controls were given DMEM/F12 supplemented with 5% FCS (positive control) or 5% PBS (negative control). The cells were cultured for a further 48 h, harvested by adding 1 ml of 0.05% trypsin, suspended by repeated aspiration with a Pasteur pipette and counted with a hemocytometer.
TABLE 1 VIABILITY IN VITRO
OF pSCSP-TREATED
SPLENIC LYMPHOCYTES
Additions
Viability * (%)
Ly + pSCSP (80 @g/ml) Ly + pSCSP (40 pg/ml) Ly + pSCSP (20 @g/ml) LY
96.1 98.0 97.3 97.0
* Average % viability from triplicate wells.
Results Effect of pSCSP on lymphocyte proliferation
Inhibition of lymphocyte proliferation is presented in Fig. 1. It shows that in the presence of pSCSP, the mitotic indices of cell proliferation are significantly lower in comparison with those of the controls. The results are similar after activation with T cell stimulatory lectins, PHA and Con A, as well as with polyclonal B cell mitogen, LPS. The lymphocyte proliferation was inhibited in a dose-dependent manner with the lowest mitotic indices when pSCSP was added at 0 h after seeding. When added at 24 and 48 h after seeding, pSCSP expressed a similar inhibitory effect (Fig. 1). Fig. 2. Effect of pSCSP on [‘Hlthymidine incorporation by BALB/c 3T3 fibroblasts in vitro. Means represent five experiments, each done in triplicate + SEM.
0
n
20
(4
(9 Contrds
Fig. 1. Effect of different concentration of pSCSP on mitotic index (MI) of rat splenic lymphocytes at 48 h after stimulation with LPS (50 pg/ml), Con A (0.5 pg/ml) and PI-IA (25 pg/mll. Means represent two experiments, each done in triplicate.
Fig. 3. Effect of pSCSP on TM, Leydig cell proliferation. Data were derived from two separate cultures, each done in triplicate and represented by means+ SEM.
20 -
(4
t-1 Cartfrds
Fig. 4. Effect of pSCSP on TM, Sertoli cell proliferation. Data were derived from two separate cultures, each done in triplicate and represented by means+SEM.
The inhibition was not due to the toxicity of pSCSP on lymphocytes at any of the tested concentrations (Table 1). pSCSP does not affect proliferation of mitogennon-stimulated lymphocytes. Induction of BALB/c 3T3 cell proliferation by pSCSP is shown in Fig. 2. Maximal mitogenic effect was attained between 10 and 30 pg/ml with considerably high 3H-thymidine incorporation: 7-fold over controls (p < 0.01). A similar protein concentration was maximally effective in the stimulation of TM, Leydig cells (Fig. 3). pSCSP was not mitogenic for TM, cells. In comparison with a relatively high value of negative control, the effect of 5 pg/ml could be accepted as an inhibition of TM, cell proliferation (Fig. 4). Discussion In this study we provide evidence on the effect of pSCSP derived from lZday-old rat testes on cell proliferation of different cell types from testicular and nontesticular origin. pSCSP inhibits phytolectin-stimulated lymphocyte proliferation in a dose-dependent manner when added at 0, 24 and 48 h after seeding. pSCSP induces 3T3 and TM, Leydig cells to proliferate, but is not mitogenic for TM, Sertoli cells. The inhibition of mitogen-stimulated lymphocyte proliferation is consistent with the effect of TGF-& and activin (Hedger et al., 1989) and with those of SCP from 20-day-old rats (Wyatt et al., 1988). Based upon the above-mentioned data and on the suppressive effect of pSCSP on natural killer cell activity (Nikolova et al., 1992), it could be assumed that by the time the blood-testis barrier had been formed, Sertoli cells in rat are already able to synthesize immunoregulatory peptides.
The response of 3T3 fibroblasts to pSCSP from lZday-old rats is similar with the effect of TGF-/3, (Hedger et al., 19891, SGF, aFGF, bFGF (Braunhut et al., 1990), as well as with the mitogenic effect of SCCM from 6-day-old rat testes (Kancheva et al., 1990), which stimulated 3H-thymidine incorporation. Comparable studies on two somatic cell lines from testicular origin, TM, Leydig and TM, Sertoli cells, have shown similarities between them and their presumed archetyped cell (Mather et al., 1982; Maggi et al., 1989), and so they are a successful tool in the study of local paracrine and/or autocrine regulation in the testis (BellvC and Zheng, 1991). pSCSP stimulated TM, Leydig cells to proliferate, but did not activate TM, Sertoli cells, which is proportionate to the effect of aFGF (Zheng et al., 1990). On the other hand, it is well known thatSertoli cell proliferation declines after birth and ceases completely on day 15 p.p. (Clermont and Perry, 1957). Therefore it is reasonable to expect a decrease in the level of mitogenic factor(s) synthesis and secretion by Sertoli cells on day 12 p.p. as an expression of their local autocrine regulatory properties. Mitogenic activity of Sertoli cell derived proteins was first detected during mouse testicular development on days 6-8 and 12-14 p.p. (Feig et al., 1980). We have shown before that Sertoli cell-conditioned medium from 6-day-old rats can stimulate DNA synthesis in quiescent prespermatogonia (Kancheva et al., 1990). SGF purified from 1-2-week old calf testis is shown to stimulate incorporation of 5-bromo-2’-deoxyuridine (BrdU) into prespermatogonial stem cells of 3-day-old rat testis as well (Bell& et al., 1993). Obviously, depending on the stage of testicular development, Sertoli cells secrete a great variety of growth factors and peptides. On day 6 p.p., when gonocytes reinitiate DNA synthesis in vivo, Sertoli cells secrete mitogenic factor(s) that stimulates quiescent prespermatogonia (Kancheva et al., 1990). On day 12 when the condition of the testis is rather different, pSCSP is mitogenic for quiescent prespermatogonia but in a lower degree than pSCSP from 6-day-old rats (Kancheva et al., 19901, does not stimulate TM, Sertoli cell but stimulates TM, Leydig cells. Bearing in mind the above-mentioned similarities between the effect of pSCSP and some purified growth factors, we may speculate that SGF, TGF-P,, aFGF, bFGF and activin are the most probable growth factors, secreted by rat Sertoli cells during the stage of meiosis initiation, i.e. about day 12 p.p. This is in agreement with the precise developmental expression of bFGF (Mullaney and Skinner, 19921, and of TGF-/3, (Teerds and Dorrington, 1993) during testicular development in the rat. It could be concluded that the balance between growth factors secreted by Sertoli cells is dependent on the requirements of different cell types at each stage of
79
testicular development. Thus, Sertoli cell-secreted growth factors in concert with pituitary ho~ones coordinate onset and maintenance of spermatogenesis. Acknowledgements We are greateful to Prof. Anthony BeIlv& for the possibility to undertake the experiments with TM, Leydig and TM, Sertoli cells in his laboratory in the College of Physicians and Surgeons, Columbia University, New York. This work was partly financed by grant K4 from the National Foundation “Scientific Researches”, Sofia. References Be&&, A.R. and Zheng, W. (1991) in Molecular Mechanisms in Cellular Growth and Differentiation (Bell&, A.R. and Vogel, H.J., eds), pp. 187-206,Academic Press, San Diego. BelM, A.R., Zheng, W., Martinova, Y.S. and Seidensticker, M.J. (1993) Methods Enzymol. (in press). Braunhut, S.J., Rupo, ,G.A., Ernisee, B.J., Zheng, W. and Bellvd, A.R. (1990) Biol. Reprod. 42, 639-648. Chang, T.W., Kung, P.C., Cingras, S.P. and Goldstein, G. (1981) Proc. Natl. Acad. Sci. USA 78, 18051808. Clermont, Y. and Perry, B. (1957) Am. J. Anat. 100,241-266. Dym, M. and Fawcett, D.W. (1970) Biol. Reprod. 3, 308-326. Feig, L.A., Bell& A.R., Horbach-Erickson, N. and Klagsbrun, M. (1980) Proc. Nat]. Acad. Sci. USA 77, 4774-4778. Head, J., Neaves, W. and Billingham, R.E. (1983) Transplantation 36,423-431.
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