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Effects of testosterone and oestradiol on [3H]-thymidine incorporation by porcine granulosa and theca cells
SCIENCE Animal Reproduction
Science 47 (1997) 229-236
Effects of testosterone and oestradiol on [ 3H]-thymidine incorporation by porcine granulosa and theta cells E.J. Ranson, H.M. Picton ‘, M.G. Hunter
*
Department of Physiology and Environmental Science, Universir): of Nottingham, Sutton Bonington Campus, Loughborough, LE12 SRD, UK Accepted 6 December
1996
Abstract Experiments were carried out to investigate the effects of varying physiological concentrations (0, 10, 100, and 1000 ng ml-‘) of oestradiol or testosterone on [3H]-tbymidine incorporation by porcine granulosa and theta cells in vitro. Granulosa cells only were recovered from small (l -3-mm) follicles and both granulosa and theta cells recovered from large (4-&mm) porcine follicles. Cells were cultured for 72 h in medium containing 10% foetal calf serum, 24 h in serum-free medium, and finally 40 h in serum-free medium containing [3H]-thymidine and appropriate steroid treatment. Although DNA per well was significantly higher (P < 0.05) at the end of culture in the theta cells than in the granulosa cells, neither steroid treatment had a significant (P > 0.1) effect on DNA concentration in either cell type. Overall, cells from small follicles incorporated significantly (P < 0.01) more [3H]-thymidine than those from medium follicles. Both oestradiol and testosterone significantly ( P < 0.01) inhibited thymidine incorporation by cells from both follicle size categories, with a significant (P < 0.05) hormone X dose interaction. Finally, there was a highly significant (P < 0.001) interaction between the response of cells to different hormone concentrations and the follicle size from which they were recovered. These results indicate that both oestradiol and testosterone may act in an autocrine/paracrine manner to inhibit proliferation and encourage differentiation in follicular cells and thus are likely regulators of the later stage of antral follicle development in the pig. 0 1997 Published by Elsevier Science B.V. Keywords: Pig-endocrinology;
Granulosa
cell; Theta cell; Proliferation;
Oestradiol;
Testosterone
* Corresponding author. ’Present address: Centre For Reproduction Growth and Development, Division of Obstetrics and Gynaecology, University of Leeds, Clarendon Wing, LGI, Belmont Grove, Leeds, LS2 9NS, UK. 037%4320/97/$17.00 0 1997 Published PII SO378-4320(97)00004-3
by Elsevier Science B.V. All rights reserved.
230
E.J. Ranson et al. /Animal Reproduction Science 47 (1997) 229-236
1. Introduction It is now well established that the pituitary gonadotrophins LH and FSH play a major role in controlling porcine follicle development, and that their actions are modulated locally by a variety of paracrine factors including growth factors (Hammond and English, 1987, May et al., 1988, Hammond et al., 19931, regulatory proteins (Tonetta and diZerega, 1989), relaxin (Zhang and Bagnell, 1993), and steroids, particularly oestrogens and androgens (Haney and Schomberg, 1978, Veldhuis et al., 1982, Hunter and Armstrong, 1987). As follicles undergo the latter stages of development, cells in both the theta and granulosa cell (GC) compartments proliferate and differentiate becoming increasingly steroidogenic as the follicle becomes competent to ovulate (Foxcroft and Hunter, 1985). This steroidogenic activity of porcine GC (Haney and Schomberg, 1978, Veldhuis et al., 1982) and theta cells (Hunter and Armstrong, 1987) can be modulated in vitro by the actions of androgens and oestrogens. The effects of steroids on cultured cells are, however, variable and depend on both the precise culture conditions, the concentrations of the steroids used (Hutz, 19891, and the origin of the tissue. Studies examining the effects of steroids on porcine ovarian cell division, for example, have concentrated on GC obtained from prepubertal gilts (Thanki and Channing, 1976, Hammond and English, 1987). The aim of the present study therefore, was to investigate the effects of oestradiol and testosterone on cell proliferation as assessed by the measurement of [3H]-thymidine incorporation by granulosa cells from small follicles from prepubertal animals and by both granulosa and theta cells from follicles recovered from mature, cyclic animals. Since the inn-a-ovarian concentrations of oestradiol and testosterone change markedly during follicle maturation (Grant et al., 1989), a range of concentrations of each steroid, within the physiological range, was studied. 2. Materials and methods 2.1. Tissue All ovarian tissue was of slaughterhouse origin. Follicles were divided into two categories-small actively growing follicles of l-3 mm diameter and larger fully differentiated follicles of 4-8 mm diameter. Small follicles were obtained from the ovaries of immature gilts and large follicles were taken from the ovaries of mature, cyclic animals which were estimated to be mid to late follicular phase (approximately Days 18-20 of the oestrous cycle). Follicles of > 8 mm diameter, although present in the ovary at this stage of the cycle, were not included in the study. The numbers of follicles of each size within the 4-8-mm range were standardised so that each repeat culture used the same number of follicles of each diameter. 2.2. Cell culture and L3H]-thymidine incorporation Granulosa cells were aspirated from small follicles using a 19-gauge hypodermic syringe. Large follicles were dissected into DMEM/Ham’s F12 medium. Follicles were hemisected and GC harvested by scraping the follicle shell with a sterile inoculating loop. The harvested cells were pelleted by centrifugation at 1200 r.p.m., and resus-
E.J. Ranson et al./Animal
Reproduction Science 47 (1997) 229-236
231
pended in fresh media. This washing procedure was repeated twice. Theta intema tissue was removed from the remaining follicle shell using fine forceps and the cells dissociated by incubation for approximately 30 min at 37°C in 10 ml Hank’s balanced salt solution containing 0.5% collagenase, 0.1% hyaluronidase, and 5% foetal calf serum. The tissue was agitated frequently during incubation using a Pasteur pipette and medium containing dissociated cells removed. Following collection theta cells were pelleted, as above, to remove the digestion enzymes, and washed in fresh media three times. Cell viability was assessed by trypan blue dye exclusion. Granulosa and theta cells were plated out at a density of 0.3 X lo6 viable cells ml-’ per well in 24-well culture plates (Costar UK Ltd., High Wycome, UK) and cultured for 72 h in DMEM/Ham’s F12 medium containing 10% foetal calf serum, followed by 24 h in serum-free medium (DMEM/RPMI1640). The cells were then treated for 40 h with 0, 10, 100, or 1000 ng ml-’ of testosterone or oestradiol in the presence of 3H-thymidine (Amersham International, plc., UK; 0.074 MBq 3H-thymidine in 10 l_~l ethanol per well). Each treatment was carried out in replicates of four and the cultures were repeated three times. At the end of the incubation time, the amount of labelled thymidine incorporated was measured as described by May et al. (1988). Briefly, culture media was removed, the cells washed and 1 ml ice-cold 5% trichloroacetic acid (TCA) and a 1% solution of sodium pyrophosphate was added slowly and left for 30 min. The TCA was then removed, and the fixed cell layer solubilised in 500 p,l 0.5 N sodium hydroxide. Duplicate 200-l.~l aliquots of this were then counted in a scintillation counter. All chemicals, culture media, and culture additives were obtained from Sigma Chemical Co. Poole, UK, unless otherwise stated. 2.3. DNA assay As a measure of comparative cell numbers at the point of thymidine assay, the DNA content of each well was estimated. Parallel cultures including all steroid treatments but substituting 10 pl ethanol for the tritiated thymidine were run and at the end of the culture period, the medium was removed from the wells and the cells washed. DNA was then measured according to the method of Labarca and Paigen (19801, using calf thymus DNA (Type 1, Sigma Chemical Co., Poole, UK) as the standard. 2.4. Statistical
analysis
Cell viabilities and DNA measurements were compared between cell types by Student’s t-test. Incorporation of [ 3H]-thymidine by different cell types, the effect of treatment and interactions were assessed by ANOVA. ANOVA was carried out using the GENSTAT statistical package (GENSTAT 5 Committee, 1989) to assess treatment effects. The pooled variance was used to calculate the standard error of the difference @ED).
3. Results At the start of culture the viability of the CC recovered from large follicles was significantly lower (P < 0.05) than the viability of GC from small follicles, or theta
232
E.J. Ranson et al. /Animal Reproduction Science 47 (1997) 229-236
Table 1 The effect of cell tvoe and follicle size on cell viabilitv and DNA at the start and end of culture Cell type
Granulosa (small follicles)
Granulosa (medium follicles)
Theta (medium follicles)
% Cell viability at start DNA at start (ng per well) DNA at end (ng per well) % Viable cell DNA at end
48.0 f 8.7 a 40145814 b 543*14a 28.2
25.9f 1.8 b 6957k514’ 429*29’ 23.8
64.8 f 2.7 a 2771f114b 614k29 b 34.2
“b P < 0.05 at least. kc a’c P < 0.01 at least. Values represent mean f SEM across treatments four.
for three repeat cultures, each with treatments
in replicates
of
cells (Table 1). Since 3 X lo6 viable cells were plated per well, irrespective of cell source, and since total DNA measurements made prior to culture include estimates of both live and dead cell numbers, the amount of DNA per well at the start of culture was highest (P < 0.01) in wells containing GC from large follicles which had the lowest viability. DNA measurements made at the end of culture more closely reflected viable cell number as dead cells had been removed with each successive media change. Taking these points into account an estimate of the % viable cell DNA content per well at the end of culture was calculated, as shown in Table 1. None of the steroid treatments had a significant effect on well DNA content at the end of culture compared with control wells treated with ethanol alone. There were however, significant differences in final DNA concentrations between the different cell types with theta cultures containing significantly more DNA than GC from small follicles (P < 0.05) which in turn contained more DNA than cells from large follicles (P < 0.01). In all three cell types, < 35% of the
i]
iSED:
0
10
,
,
100
1000
Steroid concentration (nglml) Fig. 1. The effects of cestradiol (a) and testosterone ( A ) on [‘HI-thymidine incorporation by granulosa cells recovered from l-3-mm (small) follicles. Values represent means of three repeat cultures, with each treatment in replicates of four.
E.J. Ranson et al/Animal
Reproduction
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Science 47 (1997) 229-236
SED (concentrations)
, 0
233
10
, 100
1000
Steroid concentration (nglml)
Fig. 2. The effects of oestradiol (0, ?? ) and testosterone (0, 0) on [‘HI-thymidine incorporation by granulosa (open symbols) and theta (solid symbols) cells, respectively, recovered from 4-8-mm (large) follicles. Values represent mean of three repeat cultures, with each treatment in replicates of four.
amount of viable cell DNA originally dispensed into the culture wells remained at the end of the culture period. The thymidine incorporation results are sumrnarised in Figs. 1 and 2, which show the means of three repeat cultures, in which treatments were replicated four times. Analysis of variance indicated that both oestradiol and testosterone significantly inhibited thymidine incorporation by cells from both size categories (P < 0.01) and there was a significant hormone X dose interaction (I’ = 0.011). Furthermore GC from small follicles incorporated significantly more thymidine than cells from large follicles (P < 0.01) and there was a highly significant difference in the response of cells from different-sized follicles to different doses of hormone (dose X size interaction, P < 0.001). There was no difference in tbymidine incorporation between theta and GC from large follicles (P > 0.1).
4. Discussion This study has shown that low concentrations (10 ng ml-‘) of oestradiol and testosterone significantly inhibit thymidine incorporation both by GC harvested from small undifferentiated follicles, and by GC and theta from large steroidogenically active follicles. This steroid concentration falls within the physiological range of oestradiol (7-10 ng ml-‘) and testosterone (lo-12 ng ml-‘) measured in the follicular fluid of small porcine follicles during the oestrous cycle (Babalola and Shapiro, 1988, Grant et al., 1989). The higher doses of steroid used in the study fall within the physiological range of concentrations measured in the follicular fluid (783 ng ml-’ for oestradiol and 213 ng ml-’ for testosterone) of large follicles of 4-8 mm diameter (Grant et al., 1989).
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E.J. Ranson et al. /Animal Reproduction Science 47 (1997) 229-236
In GC harvested from small follicles the inhibitory effect of testosterone on cell division was dependent on the dose of androgen used. In all cases the observed effects of treatment were specific to the addition of steroid as ethanol alone had no effect on thymidine incorporation by the cultured cells. Additionally, undifferentiated GC from small follicles incorporated approximately five times the amount of [ 3H]-thymidine than differentiated GC or theta from mature large follicles. Together these data suggest that actively dividing GC from small follicles in vivo are more sensitive to the inhibitory effects of steroids on subsequent cell division, and hence thymidine incorporation in vitro, than are tbeca and GC from mature steroidogenically active follicles in vivo. The data presented confirm previously published results which have shown testosterone to have a negative effect on ovarian cell growth in the immature rat (Payne and Runser, 1958, Louvet et al., 1975, Billig et al., 1993) and the ewe (Moor and Walters, 1979). The published effects of oestradiol are less consistent. Oestradiol has been reported to both stimulate follicle growth in vivo and in vitro (Thanki and Channing, 1976). In contrast, the in vitro studies of Hammond and English (1987) and Luciano et al. (1993) have reported that oestradiol alone had no effect on mitosis of granulosa cells from pigs and immature rats respectively, but inhibited the proliferative effects of agents such insulin, phorbol ester, gonadotrophins, or 8-bromo-cyclic AMP. Together all of these results suggest that the response of ovarian cells, cultured in the presence of serum, to physiological concentrations of oestradiol and testosterone is modulated by the presence/absence of culture media additives and may be influenced by the maturational state of the original tissue. Furthermore physiological interpretation of these data is hampered by the spontaneous luteinisation of follicular cells cultured in the presence of serum (May and Schomberg, 1981). Ultimately the direct effects of steroids on GC and theta cell function should be investigated in cells which retain their follicular phenotype when cultured in the absence of serum. To date, little is known about the proliferation of theta cells as most studies on ovarian cell growth have been carried on GC. Porcine theta cells have, however, been shown to proliferate in response to growth factors such as epidermal growth factor and platelet-derived growth factor (May et al., 1992). In addition, thymidine incorporation by porcine theta has been shown to be enhanced by relaxin (Zhang and Bagnell, 1993). The current data show that theta cells harvested from mature porcine follicles are sensitive to the inhibitory effects of low doses of both oestradiol and testosterone and that these cells incorporate thymidine at a similar rate to cells in the granulosa layer. The results presented support the hypothesis that in developing follicles in vivo, the local action of oestradiol and testosterone is to enhance the stimulation of GC and theta differentiation through the inhibition of cell proliferation. The results demonstrate that immature porcine GC from small actively growing follicles are more sensitive to the inhibitory effects of steroids, and specifically testosterone, than GC and theta from large fully differentiated follicles. This suggests that as small developing follicles pass through an androgen dominated growth phase, during which the aromatase enzyme system is not yet fully active, the androgens themselves may inhibit further cell division so providing the stimulus for follicular differentiation and the induction of aromatase activity which are both critical for the establishment of follicular ovulatory competence. In addition, during the follicular phase in the pig, high local concentrations of oestradiol and
E.J. Ranson et al./Animal
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Science 47 (1997) 229-236
235
testosterone produced by the more advanced follicles in the ovulatory hierarchy may feedback locally to inhibit proliferation and induce differentiation of less advanced follicles in the recruited pool (Foxcroft and Hunter, 1985, Hunter et al., 1992). Beyond 4 mm diameter when follicles are less sensitive to the inhibitory effects of steroids, further increases in the size of the ovulatory follicles will be largely due to increased antral fluid volume rather than continued cell division (Grant et al., 1989). The effect of steroids in inhibiting further cell division in larger follicles would not therefore prevent them from reaching preovulatory size. The significant effect of steroid treatment on thymidine incorporation in the absence of a significant effect on final DNA concentration per well confirms that thymidine incorporation is a more sensitive assay of mitogenesis than DNA measurement alone (Hammond and English, 1987). Since thymidine incorporation is associated with cell division and not cell death, the significant inhibitory effect of steroid treatment on thymidine incorporation by GC and theta in comparison with cells cultured under the same conditions, but receiving no treatment, indicates that the effects of the steroid treatments used are real and cannot be accounted for by the declining DNA concentration during culture. The difference in final DNA measurement between the cells in the present study suggests either different plating efficiencies between the cells types or different cell growth rates during culture. Initially cell viability was highest in the theta cells, probably because the tissue had undergone enzymatic digestion so removing non-viable cells. The precise reason for the variation in the initial viability of granulosa cells harvested from small and large follicles is unclear, but may relate to the less invasive collection method used for the small follicles. In conclusion, the results of this study have shown that both oestradiol and testosterone at physiological concentrations have a direct inhibitory effect on DNA synthesis by granulosa and theta cells from small and large porcine follicles. This effect of oestradiol and testosterone on cells from small actively growing follicles may play a significant role in the mechanism regulating the establishment of the ovulatory follicle population in the pig.
Acknowledgements We acknowledge
the BBSRC for financial
support.
References Babalola, GO., Shapiro, B.H., 1988. Correlation of follicular steroid hormone profiles with ovarian cyclicity in sows. J. Reprod. Fert. 84, 79-87. Billig, H., Furuta, I., Hsueh, A.J.. 1993. Estrogens inhibit and androgens enhance ovarian granulosa cell apoptosis. Endocrinology 133, 2204-2212. Foxcroft, G.R., Hunter, M.G., 1985. Basic physiology of follicular maturation in the pig. J. Reprod. Fert. Suppl. 33, 1-19. GENSTAT 5 Committee, 1989. GENSTAT 5 Reference Manual. Clarendon Press, Oxford. Grant, S.A., Hunter, M.G., Foxcroft, G.R., 1989. Morphological and biochemical characteristics during ovarian follicular development in the pig. J. Reprod. Fert. 86, 171-183.
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Hammond, J.M., English, H.F., 1987. Regulation of deoxyribonucleic acid synthesis in cultured porcine granulosa cells by growth factors and hormones. Endocrinology 120, 1039-1046. Hammond, J.M., Samaras, S.E., Grimes, R., Leighton, J., Barber, J., Canning, SF., Guthrie, H.D., 1993. The role of insulin-like growth factors and epidermal growth factor-related peptides in intraovarian regulation in the pig ovary. J. Reprod. Fert. Suppl. 48, 117-125. Haney, A.F., Schomberg, D.W., 1978. Steroidal modulation of progesterone secretion by granulosa cells from large porcine follicles: a role for androgens and oestrogens in controlling steroidogenesis. Biol. Reprod. 19, 242-248. Hunter, M.G., Armstrong, D.T., 1987. Oestrogens inhibit steroid production by dispersed porcine thecal cells. Mol. Cell. Endocr. 50, 165-170. Hunter, M.G., Biggs, C., Faillace, L.S., Picton, H.M., 1992. Current concepts of folliculogenesis in monovular and polyovular farm species. J. Reprod. Fert. Suppl. 45, 21-38. Hutz, R.J., 1989. Disparate effects of estrogens on in vitro steroidogenesis by mammalian and avian granulosa cells. Biol. Reprod. 40, 709-713. Labarca, C., Paigen, K., 1980. A simple, rapid, and sensitive DNA assay procedure. Anal. Biochem. 102, 344-351. Louvet, J., Harman, SM., Schreiber, J.R., Ross, G.T., 1975. Evidence for a role of androgens in folliculat maturation. Endocrinology 97, 366-372. Luciano, A.M., White, B., Peluso, J.J., 1993. Estradiol-17/3 (E2) as a negative regulator of immature rat granulosa cell (GC) mitosis. Biol. Reprod. 48 (Suppl. l), 67 (abstract). May, J.V., Schomberg, D.W., 1981. Granulosa cell differentiation in vitro: effect of insulin on growth and functional integrity. Biol. Reprod. 25, 421-431. May, J.V., Frost, J.P., Schomberg, D.W., 1988. Differential effects of epidermal growth factor, somatomedinC/insulin-like growth factor I, and transforming growth factor-p on porcine granulosa cell deoxyribonucleic acid synthesis and cell proliferation. Endocrinology 123, 168-179. May, J.V., Bridge, A.J., Gotcher, E.D., Gangrade, B.K., 1992. The regulation of porcine theta cell proliferation in vitro: synergistic actions of epidermal growth factor and platelet-derived growth factor. Endocrinology 131, 689-697. Moor, R.M., Walters, D.E., 1979. Interaction of ovarian tissues in the control of follicular steroidogenesis in culture. J. Endo. 80, 271-277. Payne, R.W., Runser, R.H., 1958. The influence of estrogen and androgen on the ovarian response of hypophysectomized immature rats to gonadotropins. Endocrinology 62, 313-321. Thanki, K.H., Channing, C.P., 1976. Influence of serum, estrogen, and gonadotropins upon growth and progesterone secretion by cultures of granulosa cells from small porcine follicles. Endocr. Res. Comm. 3, 319-333. Tonetta, S.A., diZerega, G.S., 1989. Intragonadal regulation of follicular maturation. Endocrine Rev. 10, 205-229. Veldhuis, J.D., Klase, P.A., Strauss, J.F., III, Hammond, J.M., 1982. The role of estradiol as a biological amplifier of the actions of follicle-stimulating hormone: in vitro studies in swine granulosa cells. Endocrinology 111, 144-151. Zhang, Q.. Bagnell, C.A., 1993. Relaxin stimulation of porcine granulosa in vitro: interactions with insulin-like growth factor 1. Endocrinology
cell deoxyribonucleic 132, 1643-1650.
acid synthesis
Science 47 (1997) 229-236
Effects of testosterone and oestradiol on [ 3H]-thymidine incorporation by porcine granulosa and theta cells E.J. Ranson, H.M. Picton ‘, M.G. Hunter
*
Department of Physiology and Environmental Science, Universir): of Nottingham, Sutton Bonington Campus, Loughborough, LE12 SRD, UK Accepted 6 December
1996
Abstract Experiments were carried out to investigate the effects of varying physiological concentrations (0, 10, 100, and 1000 ng ml-‘) of oestradiol or testosterone on [3H]-tbymidine incorporation by porcine granulosa and theta cells in vitro. Granulosa cells only were recovered from small (l -3-mm) follicles and both granulosa and theta cells recovered from large (4-&mm) porcine follicles. Cells were cultured for 72 h in medium containing 10% foetal calf serum, 24 h in serum-free medium, and finally 40 h in serum-free medium containing [3H]-thymidine and appropriate steroid treatment. Although DNA per well was significantly higher (P < 0.05) at the end of culture in the theta cells than in the granulosa cells, neither steroid treatment had a significant (P > 0.1) effect on DNA concentration in either cell type. Overall, cells from small follicles incorporated significantly (P < 0.01) more [3H]-thymidine than those from medium follicles. Both oestradiol and testosterone significantly ( P < 0.01) inhibited thymidine incorporation by cells from both follicle size categories, with a significant (P < 0.05) hormone X dose interaction. Finally, there was a highly significant (P < 0.001) interaction between the response of cells to different hormone concentrations and the follicle size from which they were recovered. These results indicate that both oestradiol and testosterone may act in an autocrine/paracrine manner to inhibit proliferation and encourage differentiation in follicular cells and thus are likely regulators of the later stage of antral follicle development in the pig. 0 1997 Published by Elsevier Science B.V. Keywords: Pig-endocrinology;
Granulosa
cell; Theta cell; Proliferation;
Oestradiol;
Testosterone
* Corresponding author. ’Present address: Centre For Reproduction Growth and Development, Division of Obstetrics and Gynaecology, University of Leeds, Clarendon Wing, LGI, Belmont Grove, Leeds, LS2 9NS, UK. 037%4320/97/$17.00 0 1997 Published PII SO378-4320(97)00004-3
by Elsevier Science B.V. All rights reserved.
230
E.J. Ranson et al. /Animal Reproduction Science 47 (1997) 229-236
1. Introduction It is now well established that the pituitary gonadotrophins LH and FSH play a major role in controlling porcine follicle development, and that their actions are modulated locally by a variety of paracrine factors including growth factors (Hammond and English, 1987, May et al., 1988, Hammond et al., 19931, regulatory proteins (Tonetta and diZerega, 1989), relaxin (Zhang and Bagnell, 1993), and steroids, particularly oestrogens and androgens (Haney and Schomberg, 1978, Veldhuis et al., 1982, Hunter and Armstrong, 1987). As follicles undergo the latter stages of development, cells in both the theta and granulosa cell (GC) compartments proliferate and differentiate becoming increasingly steroidogenic as the follicle becomes competent to ovulate (Foxcroft and Hunter, 1985). This steroidogenic activity of porcine GC (Haney and Schomberg, 1978, Veldhuis et al., 1982) and theta cells (Hunter and Armstrong, 1987) can be modulated in vitro by the actions of androgens and oestrogens. The effects of steroids on cultured cells are, however, variable and depend on both the precise culture conditions, the concentrations of the steroids used (Hutz, 19891, and the origin of the tissue. Studies examining the effects of steroids on porcine ovarian cell division, for example, have concentrated on GC obtained from prepubertal gilts (Thanki and Channing, 1976, Hammond and English, 1987). The aim of the present study therefore, was to investigate the effects of oestradiol and testosterone on cell proliferation as assessed by the measurement of [3H]-thymidine incorporation by granulosa cells from small follicles from prepubertal animals and by both granulosa and theta cells from follicles recovered from mature, cyclic animals. Since the inn-a-ovarian concentrations of oestradiol and testosterone change markedly during follicle maturation (Grant et al., 1989), a range of concentrations of each steroid, within the physiological range, was studied. 2. Materials and methods 2.1. Tissue All ovarian tissue was of slaughterhouse origin. Follicles were divided into two categories-small actively growing follicles of l-3 mm diameter and larger fully differentiated follicles of 4-8 mm diameter. Small follicles were obtained from the ovaries of immature gilts and large follicles were taken from the ovaries of mature, cyclic animals which were estimated to be mid to late follicular phase (approximately Days 18-20 of the oestrous cycle). Follicles of > 8 mm diameter, although present in the ovary at this stage of the cycle, were not included in the study. The numbers of follicles of each size within the 4-8-mm range were standardised so that each repeat culture used the same number of follicles of each diameter. 2.2. Cell culture and L3H]-thymidine incorporation Granulosa cells were aspirated from small follicles using a 19-gauge hypodermic syringe. Large follicles were dissected into DMEM/Ham’s F12 medium. Follicles were hemisected and GC harvested by scraping the follicle shell with a sterile inoculating loop. The harvested cells were pelleted by centrifugation at 1200 r.p.m., and resus-
E.J. Ranson et al./Animal
Reproduction Science 47 (1997) 229-236
231
pended in fresh media. This washing procedure was repeated twice. Theta intema tissue was removed from the remaining follicle shell using fine forceps and the cells dissociated by incubation for approximately 30 min at 37°C in 10 ml Hank’s balanced salt solution containing 0.5% collagenase, 0.1% hyaluronidase, and 5% foetal calf serum. The tissue was agitated frequently during incubation using a Pasteur pipette and medium containing dissociated cells removed. Following collection theta cells were pelleted, as above, to remove the digestion enzymes, and washed in fresh media three times. Cell viability was assessed by trypan blue dye exclusion. Granulosa and theta cells were plated out at a density of 0.3 X lo6 viable cells ml-’ per well in 24-well culture plates (Costar UK Ltd., High Wycome, UK) and cultured for 72 h in DMEM/Ham’s F12 medium containing 10% foetal calf serum, followed by 24 h in serum-free medium (DMEM/RPMI1640). The cells were then treated for 40 h with 0, 10, 100, or 1000 ng ml-’ of testosterone or oestradiol in the presence of 3H-thymidine (Amersham International, plc., UK; 0.074 MBq 3H-thymidine in 10 l_~l ethanol per well). Each treatment was carried out in replicates of four and the cultures were repeated three times. At the end of the incubation time, the amount of labelled thymidine incorporated was measured as described by May et al. (1988). Briefly, culture media was removed, the cells washed and 1 ml ice-cold 5% trichloroacetic acid (TCA) and a 1% solution of sodium pyrophosphate was added slowly and left for 30 min. The TCA was then removed, and the fixed cell layer solubilised in 500 p,l 0.5 N sodium hydroxide. Duplicate 200-l.~l aliquots of this were then counted in a scintillation counter. All chemicals, culture media, and culture additives were obtained from Sigma Chemical Co. Poole, UK, unless otherwise stated. 2.3. DNA assay As a measure of comparative cell numbers at the point of thymidine assay, the DNA content of each well was estimated. Parallel cultures including all steroid treatments but substituting 10 pl ethanol for the tritiated thymidine were run and at the end of the culture period, the medium was removed from the wells and the cells washed. DNA was then measured according to the method of Labarca and Paigen (19801, using calf thymus DNA (Type 1, Sigma Chemical Co., Poole, UK) as the standard. 2.4. Statistical
analysis
Cell viabilities and DNA measurements were compared between cell types by Student’s t-test. Incorporation of [ 3H]-thymidine by different cell types, the effect of treatment and interactions were assessed by ANOVA. ANOVA was carried out using the GENSTAT statistical package (GENSTAT 5 Committee, 1989) to assess treatment effects. The pooled variance was used to calculate the standard error of the difference @ED).
3. Results At the start of culture the viability of the CC recovered from large follicles was significantly lower (P < 0.05) than the viability of GC from small follicles, or theta
232
E.J. Ranson et al. /Animal Reproduction Science 47 (1997) 229-236
Table 1 The effect of cell tvoe and follicle size on cell viabilitv and DNA at the start and end of culture Cell type
Granulosa (small follicles)
Granulosa (medium follicles)
Theta (medium follicles)
% Cell viability at start DNA at start (ng per well) DNA at end (ng per well) % Viable cell DNA at end
48.0 f 8.7 a 40145814 b 543*14a 28.2
25.9f 1.8 b 6957k514’ 429*29’ 23.8
64.8 f 2.7 a 2771f114b 614k29 b 34.2
“b P < 0.05 at least. kc a’c P < 0.01 at least. Values represent mean f SEM across treatments four.
for three repeat cultures, each with treatments
in replicates
of
cells (Table 1). Since 3 X lo6 viable cells were plated per well, irrespective of cell source, and since total DNA measurements made prior to culture include estimates of both live and dead cell numbers, the amount of DNA per well at the start of culture was highest (P < 0.01) in wells containing GC from large follicles which had the lowest viability. DNA measurements made at the end of culture more closely reflected viable cell number as dead cells had been removed with each successive media change. Taking these points into account an estimate of the % viable cell DNA content per well at the end of culture was calculated, as shown in Table 1. None of the steroid treatments had a significant effect on well DNA content at the end of culture compared with control wells treated with ethanol alone. There were however, significant differences in final DNA concentrations between the different cell types with theta cultures containing significantly more DNA than GC from small follicles (P < 0.05) which in turn contained more DNA than cells from large follicles (P < 0.01). In all three cell types, < 35% of the
i]
iSED:
0
10
,
,
100
1000
Steroid concentration (nglml) Fig. 1. The effects of cestradiol (a) and testosterone ( A ) on [‘HI-thymidine incorporation by granulosa cells recovered from l-3-mm (small) follicles. Values represent means of three repeat cultures, with each treatment in replicates of four.
E.J. Ranson et al/Animal
Reproduction
I q
Science 47 (1997) 229-236
SED (concentrations)
, 0
233
10
, 100
1000
Steroid concentration (nglml)
Fig. 2. The effects of oestradiol (0, ?? ) and testosterone (0, 0) on [‘HI-thymidine incorporation by granulosa (open symbols) and theta (solid symbols) cells, respectively, recovered from 4-8-mm (large) follicles. Values represent mean of three repeat cultures, with each treatment in replicates of four.
amount of viable cell DNA originally dispensed into the culture wells remained at the end of the culture period. The thymidine incorporation results are sumrnarised in Figs. 1 and 2, which show the means of three repeat cultures, in which treatments were replicated four times. Analysis of variance indicated that both oestradiol and testosterone significantly inhibited thymidine incorporation by cells from both size categories (P < 0.01) and there was a significant hormone X dose interaction (I’ = 0.011). Furthermore GC from small follicles incorporated significantly more thymidine than cells from large follicles (P < 0.01) and there was a highly significant difference in the response of cells from different-sized follicles to different doses of hormone (dose X size interaction, P < 0.001). There was no difference in tbymidine incorporation between theta and GC from large follicles (P > 0.1).
4. Discussion This study has shown that low concentrations (10 ng ml-‘) of oestradiol and testosterone significantly inhibit thymidine incorporation both by GC harvested from small undifferentiated follicles, and by GC and theta from large steroidogenically active follicles. This steroid concentration falls within the physiological range of oestradiol (7-10 ng ml-‘) and testosterone (lo-12 ng ml-‘) measured in the follicular fluid of small porcine follicles during the oestrous cycle (Babalola and Shapiro, 1988, Grant et al., 1989). The higher doses of steroid used in the study fall within the physiological range of concentrations measured in the follicular fluid (783 ng ml-’ for oestradiol and 213 ng ml-’ for testosterone) of large follicles of 4-8 mm diameter (Grant et al., 1989).
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In GC harvested from small follicles the inhibitory effect of testosterone on cell division was dependent on the dose of androgen used. In all cases the observed effects of treatment were specific to the addition of steroid as ethanol alone had no effect on thymidine incorporation by the cultured cells. Additionally, undifferentiated GC from small follicles incorporated approximately five times the amount of [ 3H]-thymidine than differentiated GC or theta from mature large follicles. Together these data suggest that actively dividing GC from small follicles in vivo are more sensitive to the inhibitory effects of steroids on subsequent cell division, and hence thymidine incorporation in vitro, than are tbeca and GC from mature steroidogenically active follicles in vivo. The data presented confirm previously published results which have shown testosterone to have a negative effect on ovarian cell growth in the immature rat (Payne and Runser, 1958, Louvet et al., 1975, Billig et al., 1993) and the ewe (Moor and Walters, 1979). The published effects of oestradiol are less consistent. Oestradiol has been reported to both stimulate follicle growth in vivo and in vitro (Thanki and Channing, 1976). In contrast, the in vitro studies of Hammond and English (1987) and Luciano et al. (1993) have reported that oestradiol alone had no effect on mitosis of granulosa cells from pigs and immature rats respectively, but inhibited the proliferative effects of agents such insulin, phorbol ester, gonadotrophins, or 8-bromo-cyclic AMP. Together all of these results suggest that the response of ovarian cells, cultured in the presence of serum, to physiological concentrations of oestradiol and testosterone is modulated by the presence/absence of culture media additives and may be influenced by the maturational state of the original tissue. Furthermore physiological interpretation of these data is hampered by the spontaneous luteinisation of follicular cells cultured in the presence of serum (May and Schomberg, 1981). Ultimately the direct effects of steroids on GC and theta cell function should be investigated in cells which retain their follicular phenotype when cultured in the absence of serum. To date, little is known about the proliferation of theta cells as most studies on ovarian cell growth have been carried on GC. Porcine theta cells have, however, been shown to proliferate in response to growth factors such as epidermal growth factor and platelet-derived growth factor (May et al., 1992). In addition, thymidine incorporation by porcine theta has been shown to be enhanced by relaxin (Zhang and Bagnell, 1993). The current data show that theta cells harvested from mature porcine follicles are sensitive to the inhibitory effects of low doses of both oestradiol and testosterone and that these cells incorporate thymidine at a similar rate to cells in the granulosa layer. The results presented support the hypothesis that in developing follicles in vivo, the local action of oestradiol and testosterone is to enhance the stimulation of GC and theta differentiation through the inhibition of cell proliferation. The results demonstrate that immature porcine GC from small actively growing follicles are more sensitive to the inhibitory effects of steroids, and specifically testosterone, than GC and theta from large fully differentiated follicles. This suggests that as small developing follicles pass through an androgen dominated growth phase, during which the aromatase enzyme system is not yet fully active, the androgens themselves may inhibit further cell division so providing the stimulus for follicular differentiation and the induction of aromatase activity which are both critical for the establishment of follicular ovulatory competence. In addition, during the follicular phase in the pig, high local concentrations of oestradiol and
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testosterone produced by the more advanced follicles in the ovulatory hierarchy may feedback locally to inhibit proliferation and induce differentiation of less advanced follicles in the recruited pool (Foxcroft and Hunter, 1985, Hunter et al., 1992). Beyond 4 mm diameter when follicles are less sensitive to the inhibitory effects of steroids, further increases in the size of the ovulatory follicles will be largely due to increased antral fluid volume rather than continued cell division (Grant et al., 1989). The effect of steroids in inhibiting further cell division in larger follicles would not therefore prevent them from reaching preovulatory size. The significant effect of steroid treatment on thymidine incorporation in the absence of a significant effect on final DNA concentration per well confirms that thymidine incorporation is a more sensitive assay of mitogenesis than DNA measurement alone (Hammond and English, 1987). Since thymidine incorporation is associated with cell division and not cell death, the significant inhibitory effect of steroid treatment on thymidine incorporation by GC and theta in comparison with cells cultured under the same conditions, but receiving no treatment, indicates that the effects of the steroid treatments used are real and cannot be accounted for by the declining DNA concentration during culture. The difference in final DNA measurement between the cells in the present study suggests either different plating efficiencies between the cells types or different cell growth rates during culture. Initially cell viability was highest in the theta cells, probably because the tissue had undergone enzymatic digestion so removing non-viable cells. The precise reason for the variation in the initial viability of granulosa cells harvested from small and large follicles is unclear, but may relate to the less invasive collection method used for the small follicles. In conclusion, the results of this study have shown that both oestradiol and testosterone at physiological concentrations have a direct inhibitory effect on DNA synthesis by granulosa and theta cells from small and large porcine follicles. This effect of oestradiol and testosterone on cells from small actively growing follicles may play a significant role in the mechanism regulating the establishment of the ovulatory follicle population in the pig.
Acknowledgements We acknowledge
the BBSRC for financial
support.
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