0013-7227/91/1285-2663$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society

Vol. 12.8, No. 5 Printed in U.S.A.

ESTRADIOL-17B STIMULATES DNA SYNTHESIS IN RAT GRANULOSA CELLS: FACTOR-B

ACTION MEDIATED BY TRANSFORMING GROWTH

James J. Bendell and Jennifer Dorrington Banting and Best Department of Medical Research, and I n s t i t u t e of Medical Science, University of Toronto, Toronto, Ontario, M5G 1L6, CANADA ABSTRACT: Estradiol-176 i s a mitogen jhi vivo for the p r o l i f e r a t i o n of granulosa c e l l s in the r a t ovary. In t h i s study we have shown t h a t e s t r a d i o l - 1 7 6 a c t s on c u l t u r e s of immature r a t g r a n u l o s a c e l l s t o s t i m u l a t e DNA s y n t h e s i s . DNA synthesis stimulated by estradiol-176 was enhanced by FSH. We have shown previously that transforming growth factor-^ (TGF-B) s t i m u l a t e s DNA synthesis in r a t granulosa c e l l s i £ v i t r o and that t h i s effect i s augmented by FSH. The mitogenic action of estradiol-176 was m e d i a t e d , a t l e a s t in p a r t , by a TGF-g-like f a c t o r s i n c e in the presence of a n e u t r a l i z i n g antibody to TGF-6, DNA synthesis was a t t e n u a t e d . This conclusion was f u r t h e r supported by the novel finding that estradiol-17S stimulated the secretion of a TGF-g-like f a c t o r by r a t g r a n u l o s a c e l l s in c u l t u r e . In summary, t h e evidence suggests that the actions of estradiol-176 and TGF-6 on DNA synthesis are i n t e r r e l a t e d , and that a TGF-6-like factor mediates the mitogenic effects of estradiol-176 on r a t granulosa c e l l s . The a d m i n i s t r a t i o n of e s t r a d i o l - 1 7 6, or the synthetic estrogen d i e t h y l s t i l b e s t r o l , to female mammals stimulates the proliferation of granulosa c e l l s in ovarian f o l l i c l e s and rescues follicles from undergoing a t r e s i a ( 1 ) . The e s t r o g e n r e q u i r e d for the normal d e v e l o p m e n t of a preovulatory follicle is synthesized and secreted by the f o l l i c l e i t s e l f through a s e r i e s of gonadotrophin-dependent events. LH s t i m u l a t e s t h e c a l c e l l s to s y n t h e s i z e androgens (androstenedione and t e s t o s t e r o n e ) which are s u b s t r a t e s for FSH-induced a r o m a t i z a t i o n to e s t r o g e n s in the granulosa c e l l s ( 2 ) . Those follicles that undergo a t r e s i a do not acquire the ability to synthesize estrogen ( 3 ) . The l a t t e r finding pointed to the key role played by estrogen in promoting f o l l i c u l a r development to the preovulatory stage, thereby maintaining f e r t i l i t y . Even though the importance of estrogen in maintaining granulosa cell proliferation has been realized for many years (1), l i t t l e is known about the mechanisms by which estrogen exerts t h i s effect. In the meantime, however, knowledge has emerged t h a t growth f a c t o r s in g e n e r a l are required for the progression of the G. phase of the c e l l c y c l e ( 4 ) . The e a r l i e r work of Gospodarowicz and his colleagues (5-7) showing that granulosa cells from human, bovine and ovine ovaries respond to i n s u l i n , i n s u l i n - l i k e growth factor-I (IGF-I), fibroblast growth factor (FGF) and epidermal growth factor (EGF) are consistent with this hypothesis. Granulosa cells isolated from the rat ovary respond to TGF-B with an i n c r e a s e in DNA s y n t h e s i s , and t h i s a c t i v i t y is dramatically enhanced in the presence of FSH ( 8 ) . Since estrogen is known to be a potent mitogen for rat granulosa c e l l s ^n_ vivo, and TGF-6 is the only known growth factor to stimulate proliferaton of granulosa cells Jj^ vitro, the present experiments were designed to determine if the actions of estrogen and TGF-B were i n t e r r e l a t e d . The experiments showed for the f i r s t time t h a t estradiol-176 regulates the secretion of TGF-B by rat granulosa c e l l s , and identify TGF-B as the "missing-link" in the mitogenic action of estrogen in the rat ovary. MATERIALS

River Canada, Montreal, Canada) were k i l l e d at 25 days of age and the granulosa c e l l s were recovered from the ovaries as described previously ( 2 ) . Cell suspensions were plated as 0.5 ml aliquots in modified E a g l e ' s Minimum E s s e n t i a l Medium (MEM) into 48 multiwell Falcon t i s s u e c u l t u r e p l a t e s ( F a l c o n P l a s t i c s , Los Angeles, CA), and were cultured as described previously (2). After 14 h of treatment, the c e l l s were washed twice with c u l t u r e medium, fresh medium was added and the cultures were r e t r e a t e d . After a further 24 h of culture, the c e l l s were retreated, but the medium was n o t r e p l a c e d t o a l l o w t h e c o n t i n u e d accumulation of s e c r e t e d p r o d u c t s . At this time c u l t u r e s were t r e a t e d with NIDDK oFSH-17 (10 n g / m l ) or w i t h a polyclonal immunoglobulin G neutralizing antibody directed against TGF-B. The TGF-6 a n t i b o d y was p u r i f i e d by a f f i n i t y chromatography to remove serum components (R & D Systems, I n c . , Minneapolis, MN). Following 18 h of treatment the i n c o r p o r a t i o n of [ Hjthymldine i n t o DNA was then d e t e r m i n e d as d e s c r i b e d previously ( 9 ) . T h e c a l / i n t e r s t i t i a l c e l l s were isolated from the ovarian tissue that remained after the release of the granulosa cells ( 9 ) . For the generation of conditioned medium granulosa and t h e c a l / i n t e r s t i t i a l c e l l s were plat£d in 1% i e t a l calf serum at d e n s i t i e s of 7x10 and 3x10 cells/well respectively in 48 well p l a t e s . After 24 h with serum the cultures were washed three times with serum-free medium over a 24 h p e r i o d . Treatment with e s t r a d i o l - 1 7 6 (5x10 M) was i n i t i a t e d 4 h a f t e r p l a t i n g and repeated daily. Conditioned medium was collected over a further 48 h culture period and the l e v e l s of TGF-B were measured in a Mink Lung e p i t h e l i a l cell (MV 1 Lu) growth assay ( 1 0 ) . MV 1 Lu c e l l s were plated into 96 well plates as 200 ul aliquots containing 1.5x10 c e l l s in c u l t u r e medium plus 10% fetal calf serum. After 24 h at 37C the c e l l s were washed and the medium was replaced by 200 ul of culture medium c o n t a i n i n g 1% fetal calf serum. The c e l l s were treated with TGF-6 s t a n d a r d s , and c o n d i t i o n e d medium from g r a n u l o s a and thecal/interstitial cells. A f t e r 24 h of treatment [ H]thymidine (1 uCi/ml) .was added for an 8 h i n c u b a t i o n and t h e [ H j t h y m i d i n e incorporation into DNA was determined as described previously ( 9 ) . A standard dose-response curve for TGF-6 (0.001 to 3.3 ng/ml) was included on

AND METHODS

Female rats (Wistar CrL: (Wl)BR from Charles Received in Iowa City February 8, 1991. 2663

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each 96 well plate, and the conditioned medium was assayed at four dilutions. The specificity of the MV 1 Lu assay for TGF-3 was confirmed by the inability of 10 ng/ml EGF, TGF-a, IGF-1, basic and acidic FGF, FSH (50 ng/ml), estradiol-173 (10 _M), testosterone (10 M) and dihydrotesterone (10 M) to influence growth. Furthermore, dose-response curves for conditioned medium on MV 1 Lu c e l l s paralleled that for standard TGF-3. In a l l cases the results are presented as the standard error of the mean of triplicate points and are representative of three experiments, each of which showed similar s i g n i f i c a n t differences between treatment groups.

Endo • 1991 Voll28«No5

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RESULTS

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Pretreatment Granulosa cells isolated from normal immature r a t s were c u l t u r e d for 60 h in the presence and absence of estradiol-17g (5 x 10 M). Cells that were t r e a t e d with e s t r a d i o l - 1 7 g i n c o r p o r a t e d s i g n i f i c a n t l y elevated l e v e l s of [ Hjthymidine i n t o DNA d u r i n g a s u b s e q u e n t 4 h i n c u b a t i o n (D< 0.05) ( F i g . 1 ) . The i n c o r p o r a t i o n of [ H]thymidine into DNA was significantly enhanced in both c o n t r o l s and e s t r a d i o 1-1 7B t r e a t e d granulosa cells in the presence of FSH (p< 0.05) (Fig. 1).

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Figure 2: Effects of a neutralizing antibody to TGF-B ( 1 0 0 u g / m l ) on e s t r a d i o l - 1 7 3 (E 5xlO~ M)-induced DNA s y n t h e s i s in r a t granulosa cells. Cells were t r e a t e d with E. for 60 h and a n t i b o d y f o r 18 h p r i o r t o t n e a s s a y of [ H]thymidine incorporation into DNA. Secondly, the levels of TGF-3 in the c u l t u r e medium of c o n t r o l and e s t r a d i o l - 1 7 B - t r e a t e d granulosa c e l l s were measured in the MV Lu 1 c e l l a s s a y i n which TGF-£ h a s been shown t o s p e c i f i c a l l y i n h i b i t c e l l growth. The levels of active TGF-B in unheated c u l t u r e medium were low and t h e r e was no s i g n i f i c a n t e f f e c t of estradiol-173 treatment. Activation of the latent form of the TGF-6 by heating cau«6:d a significant increase in the amount of active TGF-3 in each lot of culture medium (p< 0 . 0 5 ) . Heat treatment of conditioned medium from e s t r a d i o l - 1 7 3 - t r e a t e d granulosa cells increased the TGF-3 a c t i v i t y to a level that was significantly higher (p.< 0.01) than that of heat activated medium from untreated c e l l s (Fig. 3 ) . The TGF-B n e u t r a l i z i n g antibody (100 ug/ml) blocked the actions of standard TGF-3 and 30% granulosa cell conditioned medium in the MV 1 Lu bioassay, confirming t h a t TGF-3 is indeed the factor secreted by granulosa c e l l s that i n h i b i t e d growth of the MV 1 Lu c e l l s .

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Figure 1: Effects of estradiol-173 (E., 5x10 and FSH (10 n g / m l ) on the i n c o r p o r a t i o n of [ H]thymidine into DNA of rat granulosa c e l l s j £ v i t r o . Granulosa c e l l s were treated with E~ for 60 h prior to a 4 h incubation with [ H]thymidine. Since we have previously shown that FSH and TGF-3 i n t e r a c t to s t i m u l a t e [ H ] t h y m i d i n e incorporation into the DNA of rat granulosa cells (8), the experiments shown in Figure 1 raised the p o s s i b i l i t y t h a t FSH may be i n t e r a c t i n g in the c u l t u r e s with endogenously generated TGF-B. To test this p o s s i b i l i t y two approaches were used: F i r s t l y , a specific antibody that n e u t r a l i z e s the actions of TGF-B on granulosa cell growth (11) was added to the granulosa cell cultures to determine if the binding of TGF-3-like factors in the medium would interfere with the r e s p o n s e . The addition of 100 ug/ml of antibody, a concentration that had previously been shown to be e f f e c t i v e ( 1 1 ) , s i g n i f i c a n t l y reduced t h e i n c o r p o r a t i o n of [ H] t h y m i d i n e into b o t h c o n t r o l and e s t r a d i o l - 1 7 $ - t r e a t e d rat granulosa cells (p