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Studies on the synergistic effect of androgen on the stimulation of progestin secretion by FSH in cultured rat granulosa cells: A search for the mechanism of action
Molecular and Cellular Endocrinology,
0 Elsevier/North-Holland
8 (1977) 20 l-2 11 Scientific Publishers, Ltd.
STUDIES ON THE SYNERGISTIC STIMULATION GRANULOSA
EFFECT OF ANDROGEN
ON THE
OF PROGESTIN SECRETION BY FSH IN CULTURED RAT CELLS: A SEARCH FOR THE MECHANISM OF ACTION
A. NIMROD Department
of Hormone Research,
Weizrnann Institute of Science, Rehovot, Israel
Received 2 February 1977; accepted 5 May 1977
Cultures of granulosa cells from immature hypophysectomized DES-treated rats were unable to maintain progestin production for more than 48 h in medium without hormone supplementation or in the presence of FSH only. Production of progestin (20adihydroprogesterone, as measured by radioimmunoassay) remained unimpaired in the presence of androstenedione (Ad) and was markedly increased in the presence of both Ad and FSH. The combined treatment with FSH and Ad during the first 48 h of culture brought about persistent changes in the cultured cells, since progestin accumulation did not decline upon subsequent removal of these hormones during days 3 and 4 of culture. Dibutyryl cyclic AMP (DBC) was able to mimic the changes in steroidogenic capability induced by the combined action of FSH and Ad. The extent of [ 12’1]FSH binding, FSH-stimulable CAMP accumulation and cyclic 3’,5’-nucleotide phosphodiesterase activity were not affected by addition of Ad to the culture medium. Ad synergized with DBC in the stimulation of progestin accumulation in granulosa cell cultures. It is suggested that androgen acts at a step in the regulation of progestin biosynthesis distal to CAMP production. Keywords:
follicular maturation; 20adihydroprogesterone; adenylate cyclase; phosphodiesterase.
cyclic AMP; FSH receptor;
Follicle stimulating hormone (FSH) is believed to act exclusively on the follicular granulosa cells of the ovary (Richards and Midgley, 1976) and to play an important role in follicular development (Hisaw, 1947; Eshkol and Lunenfeld, 1972). Androgens, known to be secreted by the thecal layer (Makris and Ryan, 197.5) have been shown to synergize with FSH in the stimulation of progestin accumulation (Nimrod and Lindner, 1976) and to enhance the sensitivity of the FSH-responsive mechanism in cultures of granulosa cells from immature hypophysectomized DEStreated rats (Nimrod, 1977). The mechanism by which androgen exerts its effect is not known. The possibility that the enhanced accumulation of 20a-dihydroprogesterone (~OCY-OH-P)and progesterone found in androgen-treated cultures results from competitive inhibition of Sa-reductase has been ruled out (Nimrod, 1977). A different mechanism of action has been proposed, involving cyclic AMP mediation (Nimrod and Lindner, 1976). In the present work this aspect of the mechanism of action of steroids on these cells was examined. 201
202
A. Niw-od
EXPERIMENTAL Granulosa cells were collected from immature hypophysectomized rats treated with diethylstilbestrol (0.5 mg/rat/day) for 5 days (Hx-DES cells) and cultured as described by Nimrod and Lindner (1976) except that 20% fetal calf serum, instead of rat serum, was included in the medium. Cells (3-10 X 10’ cells/ml) were introduced into culture dishes (Falcon Optilux tissue culture dishes, or Falcon No. 3008 Multiwell tissue culture plates), and human FSH (LER-1801-3; 0.1 I.cg/ml; generously made available by the National Institute of Arthritis, Metabolism and Digestive Diseases, Pituitary Hormone Distribution Program, through the courtesy of Dr. L.E. Reichert, Jr.), dibutyryl cyclic AMP (DBC; 0.5 mg/ml; Sigma Chemical Co., St. Louis, MO.), androst-4-ene-3,17-dione (Ad; 1 yg/ml; Ikapharm, Ramat Can, Israel) and 3-isobutyl-I-methylxanthine (IBMX; 100 pg/ml; Aldrich Chemical Co., Milwaukee, Wis.) were added in various combinations to the medium at the beginning of the culture period. When cultures were to be maintained for 4 days, the medium was removed after the first 2 days of culture, the attached cells were washed with fresh serum-free medium, and fresh medium was added. 20@-Dihydroprogesterone (20a-OH-P) concentration in the medium was measured by radioimmunoassay (Bauminger et al., 1974). FSH-stimulable cyclic AMP accumulation was determined in cell layers cultured in the presence or absence of Ad. After washing the layers with Krebs-Ringer bicarbonate buffer (KRB), containing glucose (1 mg/ml), they were incubated at 37°C for 20 min in 1 ml KRB-glucose containing IBMX (100 pg/ml). FSH (0.1 pg/ml) and Ad (1 pg/ml) were added to the samples at the beginning of incubation. The reaction was terminated by cooling the plates on ice and after separation of the incubation medium from the cells 1 ml of 0.05 M acetate buffer, pH 4.0, was added to the dishes. The cell layers were then detached with a rubber scraper, and the suspension homogenized in a Teflon-glass homogenizer. Cyclic AMP content was determined in aliquots of the incubation medium and homogenates by the method of Gilman (1970). For the determination of [‘*‘I]FSH binding to cultured granulosa cells, cell layers were washed once, harvested with a rubber scraper and suspended in assay buffer (Nimrod et al., 1976). The cell suspensions were centrifuged at 500g for 8 min, the pellet resuspended in assay buffer by repeated passage through a Pasteur pipette, and specific binding of [ ‘*’ I] FSH was assayed as described by Nimrod et al., (1976). Cyclic 3’S’-nucleotide degradation, presumably by phosphodiesterase (PDE) activity, was measured in homogenates of cultured Hx-DES cells essentially as described by Zor et al. (1976). Cells ( lo7 cells per sample) were scraped off the culture plate and homogenized in a Teflon-glass homogenizer, in 0.55 ml of 40 mM Tris-3.5 mM MgCl,, pH 7.0. 50 ~1 of cAMP solution were then added to give a final concentration of 0.2 PM CAMP, and the mixture incubated with shaking at 37°C. Aliquots of the incubation mixture were taken out at various times and the
Mechanism of action of androgen in granulosa cells
203
reaction stopped by addition of an equal volume of 0.05 M sodium acetate, pH 4.0, and boiling for 5 min. Cyclic AMP content was determined in duplicate as described by Gilman (1970). Statistical analysis of the difference between group means was performed using Student’s f-test. When group variances were significantly different (P< 0.05 by Ftest), a logarithmic transformation was performed before application of the test of significance.
RESULTS The effect of FSH and Ad and DBC on the attachment, survival and multiplication of granulosa cells in culture
The attachment of the granulosa cells was usually completed within 24-36 h of culture, as judged by microscopic examination. Plating efficiency, calculated from the number of attached cells after 48 h of culture, was 50-70% (cf. table 1). Addition of FSH (0.1 pg/ml), Ad (1 pg/ml), FSH + Ad or dibutyryl cyclic AMP (DBC; 1 .O mM) to the culture medium had no significant effect on plating efficiency or net growth rate of the cultures during a 48 h culture period (table 1). After 4 days of culture (data not shown) there was no significant net change in the number of surviving cells in the control cultures, and the values for cultures supplemented with FSH, Ad, or FSH + Ad were within 20% of the control value. Light microscopic examination of cell layers after 2 days or 4 days of culture did not reveal any differences between the various treatment groups with respect to general morphology, nucleus/cytoplasm ratios or distribution of Lipid droplets.
Table 1 The effect of FSH, Ad and dibutyryl Hx-DES rats in culture
cyclic AMP (DBC) on the survival of granulosa
Cells harvested/dish Culture
conditions
No additions Ad (1 pg/ml) FSH (0.1 pg/ml) FSH + Ad DBC (1.0 mM) ____ ..___
* __.__
No. x lo@
% of no. plated
1.61; 2.14; 2.24; 1.72; 1.65;
57 72 72 60 58
1.70 2.05 1.97 1.78 1.72
cells from
* 2.9 X lo6 cells were plated in 10 cm diameter culture dishes containing 8 ml medium. number of attached cells was determined after 48 h. The values of 2 determinations shown, each representing the combined cells from 2 dishes.
The are
A. Nimrod
204
The effect of FSH and Ad on progestin production by granulosa cells during 4 days of culture Granulosa cell cultures were divided into four groups maintained during the first 2 days (stage I) as follows: (i) without hormones, (ii) with FSH, (iii) with Ad and (iv) with FSH and Ad. During the following 2 days (stage 11) the groups were subdivided into four subgroups, each maintained with one of these hormone combinations. The accumulation of 2Oo-OH-P in the media during the two culture periods is shown in fig. 1. Progesterone concentrations were not measured, since they have been shown under similar conditions to contribute only about 10% of total progestin concentration (Nimrod and Lindner, 1976). Cells cultured without hormone supplementation during stage I showed a diminished production of 2Oo-O&P during stage II (P< 0.05), even after addition of FSH or Ad (P< 0.05), but failed to
First 2 days Without hormowr
First 2 days With FSH
Fint 2 days With Ad
First 2 days With FSH+Ad
rnd Hormones
C
C,EA,FA
F
C,F;A,FA
A
C.[A.FA
FA
C,F;A.FA
Days of l-2 3-4 I-2 3 -4 I -2 3 -4 I -2 3-4 culture Fig. 1. The effect of FSH and Ad on the con tinuation of ZOcu-OH-Paccumulation by granulosa cells during 4 days of culture. Hx-DES cells (8 X lo3 cells/ml) were plated in Multiwell plates containing 0.5 ml of medium alone (C), or with androstenedione (A; 1 pg/ml), FSH (F; 0.1 pg/ ml), or a combination of the two hormones (FA). After 2 days the medium was collected, the cell layers washed once with 1 ml of fresh medium and recultured for an additional 2 days with the same combinations of FSH and Ad. The vertical bars represent the mean +SE of 20a-OH-P content in the media of 3 replicate cultures, assayed by radioimmunoa~y.
Mechanism ofoction of androgen in granulosa cells
205
prevent the decline during stage II (P< 0.001). By contrast, when Ad was added during stage I, 20cu-OH-Paccumulation was maintained above the control level during stage II cultures without hormone supplementation, and was further augmented upon addition during stage II of FSH together with Ad (P< 0.05). Exposure of the cells during stage I to both FSH and Ad resulted in greater augmentation of 2Ocr-OH-Paccumulation during stage I than that attained with either hormone alone (P< 0.001). This stimulated rate was maintained during stage II when the cells were cultured without hormone supplementation (f > O.Ol), and greatly enhanced when the two hormones were present in the medium during stage II as well (P< 0.001; fig. 1).
In another experiment, cells were cultured for 2 days (stage I) in the presence of a high concentration of DBC (1 mM), and then washed and cultured for an additional 2 days (stage II) either without hormone supplementation or with a combination of FSH and Ad. Exposure of the cell cultures to DBC during stage I resulted in an even higher rate of 20cu-OH-Paccumulation (P < 0.001) during that stage than in the presence of FSH and Ad (table 2). The stimulated rate was maintained during stage II in a medium not supplemented with DBC (P > 0.1). Further enhancement of 2Oo-OH-P accumulation was obtained during stage II by supplementation of the medium with FSH + Ad (P< 0.05); the levels thus attained were similar to those seen upon 4-day continuous exposure to FSH and Ad (P> 0.1; table 2). Thus, DBC was able to mimic the effect of FSH and Ad in transforming the Hx-DES cells into active progestin-secreting cells. These results suggest that the synergistic effect of Ad on FSH-stimulated proges-
Table 2 The effectiveness of dibutyryl CAMP (DBC) in mimicking the action of FSH and Ad on granulosa cell cultures. Hx-DES cells (3.6 X lo5 cells/ml initially plated) were cultured for 2 periods of 2 days each, as described in the legend to fig. 1. .-----_-.--.-- ____ ___ ....__..L__-.. Concentration of 20~OH-P * (ng/ml) Culture conditions ~Days 3 -4 Days l-2 Days 1-2 Days 3 -4 ~-----~-_--_---1422 23t 1 No hormones No hormones FSH + Ad so+3 FSH + Ad
No hormones FSH + Ad
310 f 19
306 f 5 962 f 80
DBC
No hormones FSH + Ad
704 f 58
665 f 32 125Oi: 180
* Mean -+SE of 4 replicate cultures.
A. Nitnrod
206
Table 3 Specific binding of FSH to granulosa cells of Hx-DES rats after 3 days of culture, compared to freshly isolated cells ___----~--... -... .._ ..___._.__ _. _~__.._. -~~~ -.--_-. Culture conditions [ t *“I] FSH bound * (cpm/l O6 cells) (mean + SE) __--_ - .~~~ No additions 4530 c 193 Androstenedione (1 pg/ml) 4128 i_ 225 Uncultured (freshly isolated) cells 4404 2 180 -_
* The difference between the radioactivity bound to the cells after incubation in the presence or absence of an excess of unlabelled FSH, assayed in triplicate.
tin production may be mediated by cyclic AMP. Three possible mechanisms which Ad could enhance cyclic AMP accumulation were explored: 1. 7’he Granulosa [“%]FSH ture period
by
ejyect of Ad on the binding of ( ‘25/]FSH to cultured granulosa ceils. cells from Hx-DES rats retained their high capacity to speci~~ally bind during 3 days in culture. Treatment of the cells with Ad during this culhad no significant effect (I’> 0.1) on FSH binding (table 3).
2. The effect of Ad on FSH-stinzulable cyclic AMP accurnulatioil in cultured granulosa cells. Cyclic AMP accumulation in ceils cultured for 24 h or 48 h with or without Ad was measured during a 20 min challenge with FSH and Ad separately or in combination, in the presence of IBMX (table 4). FSH stimulated CAMP accumulation to the same degree (P> 0.1) whether or not Ad was present during the preceding 24 or 48 h culture periods (about 9-fold and 14-fold, respectively). However, the presence of Ad during culture caused a slight increase in extracellular CAMP accumulation; this increase was significant after 48 h (P < 0.05) but not after 24 h (P> 0.05) of culture. Ad added on its own to the culture medium, or during the 20 min challenge period, did not affect CAMP accumulation. Addition of Ad together with FSH during the challenge incubation did not significantly potentiate the FSH effect (P > 0.05). 3. The effect of Ad, FSH and DBC on ~~os~~odieste~ase activity in ~anulosa cell ~~~~es. Androstenedione could raise CAMP concentrations in the granulosa cell culture by inhibiting degradation of the cyclic nucleotide by phosphodiesterase (PDE), rather than by stimulating its biosynthesis. However, the rate of CAMP degradation by homogenates of cells cultured in the presence of Ad was found to be similar to that of control cell homogenates (fig. 2). FSH- and DBC-treated cells exhibited slightly higher rates of degradation. Additional evidence that PDE is not involved in the synergistic action of Ad was obtained by culturing cells with Ad in combination with a PDE inhibitor (IBMX).
Medium
Cell homogenate
24 24 48 48
No hormones Ad
48 48
No hormones Ad
No hormones Ad
24 24
No hormones Ad (1 ng/ml)
1.6 f 0.1 l.? f 0.1
1.8 f 0.1 1.7 f 0.1
0.9 f 0.1 1.1 f 0.1
1.0 f 0.1 1.1 + 0.1
1.9 i 0.1 1.9 c 0.1
1.8 + 0.1 1.6 + 0.1
1.2 f 0.1 1.1 + 0.1
1.0 f 0.1 1.1 +_0.1
2.5 _i 0.1 3.5 * 0.1
2.6 + 0.1 3.5 t 0.5
14.9 * 3.2 14.1 f 0.9
8.8 f 1.0 8.7 + 0.2
2.5 f 0.2 3.4 f 0.1
2.6 + 0.3 2.8 of 0.3
11.8 + 1.6 16.7 t 0.6
9.0 t 0.9 6.4 t 0.2
Stimulation of cyclic AMP accumulation by Ad and FSH in granulosa cells of Hx-DES rats after 24 h and 48 h of culture. Cell layers were washed free of culture medium and incubated for 20 min in KRB medium containing 100 pg/ml IBMX (see Methods), with or without the hormones indicated. The KRB medium was then separated, and the cells were scraped off and homogenized in 0.05 M acetate buffer, pH 4.0. Cyclic AMP content of both KRB medium and cell homogenates is shown as the mean f SE for 3 plates, each assayed in duplicate. ___.~ Fraction Culture Cyclic AMP formation during 20 min challenge (pmol/dish) Culture assayed conditions time (h) No hormones FSH (0.1 pg/ml) FSH + Ad Ad (1 erg/ml) ~_~. ______
Table 4
k? 2 3
:: 2. % 9 2 9 0 1 -. 3: 09 2 3 =.
A. Nimrod
208
Time,
min
Fig. 2. Cyclic 3’,5’-nucleotide phosphodiesterase activity in homogenates of granulosa cell cultures with various hormones. Hx-DES cells (8 X lo5 cells/ml) were plated and the cultures maintained for 3 days in dishes of 10 cm diameter containing 8 ml of medium supplemented as indicated: o, no hormones (control); A, FSH 0.1 pg/ml; 0, androstenedione (Ad) 1 pg/ml; n, FSH + Ad; o, dibutyryl cyclic AMP (DBC) 1.0 mM. The cells were washed with PBS, scraped
off and homogenized in Tris-Mg buffer (see Experimental section). The homogenates were incubated with 0.2 PM CAMP and CAMP concentration determined in duplicate at the times specified. Duplicate determinations agreed within 10%. Cultures with IBMX alone accumulated lo-fold more 20a-OH-P than cultures without the inhibitor (P< 0.001; table S), indicating that PDE activity was markedly inhibited. Nevertheless, addition of Ad in the presence of IBMX caused a further increase in 2Oo-OH-P production (P < 0.001). Interaction of Ad and DBC Cells were cultured for 2 days in the presence of varying amounts of DBC with or without the addition of Ad. The amounts of 20o-OH-P accumulated in the medium are presented in fig. 3. The lowest effective concentration of DBC to
Table 5 The effect of a phosphodiesterase inhibitor (IBMX, 100 pg/ml) on the stimulation of 20a-OH-P accumulation by androstenedione (1 rg/ml). Hx-DES cells (5.2 X 10’ cells/ml) were sown and the cultures maintained for 2 days in Multiwell plates containing 0.5 ml medium. Culture conditions No additions IBMX Ad Ad + IBMX
Concentration 30+
1
365 f 20 146 f 4 1212 f 132
* Mean + SE of 4 replicate cultures.
of 20&H-P
* (ng/ml)
Mechunism of action ofndrogen I
I
I
in gramdosa ceils 1
I
I
I
209 I
1
500 s ‘_o i ::=3 h 0s
400
300
Ad(lOng/ml
L$! $ tY= s
5
2oo
No Ad
100
O
~~~ 0 -4
IO Dibvfyryl-
25
64
160
400
1000
cyclic AMP@glml)
Fig. 3. Interaction
of Ad and dibutyryl cyclic AMP (DBC) in the stimulation of 20cu-OH-P accumulation by granulosa cells during 2 days of culture. Hx-DES cells (4 X i05 cells/ml) were plated into Multiwell plates containing 0.5 ml of medium with various concentrations of DBC, in the presence or absence of Ad (!O ng/ml). The vertical bars represent the mean GE of 20~ OH-P content in the media of 4 replicate cultures.
stimulate 2Oo-OH-P accumulation was 25 @g/ml (50 j&I); maximal stimulation was attained with 400 fig/ml (0.8 mM) DBC. Androstenedione (10 nglml) had a synergistic effect on this stimulation at DBC concentrations of 10-1000 pg/ml (P < O.Ol), with the exception of 400 pg/ml of DBC, where marked variation was observed (P > 0.1). An increase in Ad concentrations up to 0.36 pg/ml (data not shown) did not further augment the synergistic effect with DBC.
DISCUSSION The present study shows that granulosa cell cultures from Hx-DES rats are unable to maintain progestin production for more than 48 h without hormone supplementation, or in the presence of FSH only. Progestin production, however, remained unimpaired in the presence of androstenedione (fig. 1). Moreover, combined treatment with FSH and Ad brought about persistent changes in the cultured cells, since progestin accumulation was not affected by the subsequent removal of these hormones (fig. 1). A marked rise in progestin production by granulosa cells is frequently regarded as a marker for luteini~tion (~hanning, 1970). However, the FSH + Ad treated Hx-DES cells differ from ordinary luteal cells in that they are morphologically indistinguishable from untreated granulosa cells (see Results section), and do not specifically bind [ ‘*‘I]hCG (Nimrod et al., 1977). The increased
210
A. Nimrod
progestin production by these cultures in the presence of FSH and/or Ad or DBC was not attributable to an increase in cell numbers, since none of these treatments had a significant effect on plating efficiency and subsequent cell survival or rate of multiplication (table 1). Dibutyryl CAMP (DBC) was able to mimic the metabolic changes induced by the combined action of FSH and Ad (table 2). This finding suggested that CAMP was involved in the mediation of the synergistic effect of androgen. Since the content of CAMP in Hx-DES cells was below the limits of sensitivity of the assay, we chose to examine by indirect means three possible mechanisms by which CAMP accumulation could be enhanced by androgen, namely (i) an effect on the number of available FSH binding sites; (ii) activation of the coupling of the FSH receptor to adenylate cyclase, as reflected by increased CAMP accumulation following a 20 min challenge with FSH in the presence of a PDE inhibitor; and (iii) a change in CAMP degradation by cyclic 3’,5’-nucleotide phosphodiesterase. The present results provided no evidence that any of these mechanisms is operative: the extent of [12’I]FSH binding (table 3) and PDE activity (fig. 2) were not affected by culturing granulosa cells in the presence of Ad, and the effect of Ad on FSH-stimulable CAMP accumulation was small and inconsistent (table 4). Taken together, these findings do not support the contention that Ad directly influences FSH-stimulable CAMP accumulation in granulosa cells, and further suggest that Ad acts at a step in the regulation of progestin biosynthesis distal to CAMP production. This conclusion is supported by the observation that Ad exerted a synergistic effect on the stimulation of 20a-OH-P accumulation in granulosa cell cultures by DBC (fig. 3). This synergistic effect was most pronounced at 0.3 mM DBC concentration (160 /.rg/ml; P< 0.001 by Student’s t-test; fig. 3). At the maximally effective DBC concentration (0.8 mM) the androgen effect on the rise of 20aOH-P accumulation exhibited a wide variation and was statistically not significant (P> O.l), in accordance with earlier observations (Nimrod and Lindner, 1976). Cyclic AMP is believed to stimulate steroidogenesis through a multistage biochemical pathway (Marsh, 1975). The steps in this pathway that are influenced by androgens, and the mechanism of action of these steroids in granulosa cells, remain to be elucidated.
ACKNOWLEDGEMENTS
We thank Mrs. Genia Acevedo for her expert technical assistance, and Dr. Charles Faiman, Dr. H.R. Lindner and Sergio A. Lamprecht for their criticism and assistance in writing this paper. This work has been supported by a grant (to Dr. H.R. Lindner) from the Ford Foundation and the Population Council, New York.
Mechanism of action of androgrn irtgramlosa cells
211
REFERENCES Baulninger, S., Kohen, F. and Lindner, H.R. (1974) J. Steroid Biochem. 5,739-747. Bauminger, S., Lieberman, M.E. and Lindner, H.R. (1975) Prostag~and~s 9,753-764. Channing, C.P. (1970) Recent Prog. Horm. Res. 26,589-622. Eshkof, A. and Lunenfeid, B. (1972) in: Gonadotropins, Eds.: B.B. Saxena, G.C. BeBng and H.M. Gandy (Wiley, New York) pp. 335-346. Gilman, A.G. (1970) Proc. Nati. Acad. Sci, U.S.A. 67,305-312. Hisaw, F.L. (1947) Physiol. Rev. 27,95-146. Lamprecht, S.A., Zor, U., Tsafriri, A. and Lindner, H.R. (1973) J. Endocrinol. 57,217-233. Makris, A. and Ryan, K.J. (1975) Endocrinology 96,694-701. Marsh, JM, (1975) Adv. Cyclic Nucieotide Res. f&137-199. Nimrod, A. (1977) Mol. Cell. Endocrinol. 8,189-199. Nimrod, A. and Lindner, H.R. (1976) Mol. Cell. ~ndocr~noI.5,315-319. Nimrod, A., Erickson, G.F. and Ryan, K.J. (1976) Endocrinology 98,56-64. Nimrod, A., Tsafriri, A. and Lindner, H.R. (1977) Nature (London), in press. Richards, J.S. and Midgley, A.R., Jr. (1976) Biol. Reprod. 14, 82-94. Zor, U., Koch, R. and Naor, Z. (1976) Adv. Prostag~andin T~omboxane Res. 1,331-335.
0 Elsevier/North-Holland
8 (1977) 20 l-2 11 Scientific Publishers, Ltd.
STUDIES ON THE SYNERGISTIC STIMULATION GRANULOSA
EFFECT OF ANDROGEN
ON THE
OF PROGESTIN SECRETION BY FSH IN CULTURED RAT CELLS: A SEARCH FOR THE MECHANISM OF ACTION
A. NIMROD Department
of Hormone Research,
Weizrnann Institute of Science, Rehovot, Israel
Received 2 February 1977; accepted 5 May 1977
Cultures of granulosa cells from immature hypophysectomized DES-treated rats were unable to maintain progestin production for more than 48 h in medium without hormone supplementation or in the presence of FSH only. Production of progestin (20adihydroprogesterone, as measured by radioimmunoassay) remained unimpaired in the presence of androstenedione (Ad) and was markedly increased in the presence of both Ad and FSH. The combined treatment with FSH and Ad during the first 48 h of culture brought about persistent changes in the cultured cells, since progestin accumulation did not decline upon subsequent removal of these hormones during days 3 and 4 of culture. Dibutyryl cyclic AMP (DBC) was able to mimic the changes in steroidogenic capability induced by the combined action of FSH and Ad. The extent of [ 12’1]FSH binding, FSH-stimulable CAMP accumulation and cyclic 3’,5’-nucleotide phosphodiesterase activity were not affected by addition of Ad to the culture medium. Ad synergized with DBC in the stimulation of progestin accumulation in granulosa cell cultures. It is suggested that androgen acts at a step in the regulation of progestin biosynthesis distal to CAMP production. Keywords:
follicular maturation; 20adihydroprogesterone; adenylate cyclase; phosphodiesterase.
cyclic AMP; FSH receptor;
Follicle stimulating hormone (FSH) is believed to act exclusively on the follicular granulosa cells of the ovary (Richards and Midgley, 1976) and to play an important role in follicular development (Hisaw, 1947; Eshkol and Lunenfeld, 1972). Androgens, known to be secreted by the thecal layer (Makris and Ryan, 197.5) have been shown to synergize with FSH in the stimulation of progestin accumulation (Nimrod and Lindner, 1976) and to enhance the sensitivity of the FSH-responsive mechanism in cultures of granulosa cells from immature hypophysectomized DEStreated rats (Nimrod, 1977). The mechanism by which androgen exerts its effect is not known. The possibility that the enhanced accumulation of 20a-dihydroprogesterone (~OCY-OH-P)and progesterone found in androgen-treated cultures results from competitive inhibition of Sa-reductase has been ruled out (Nimrod, 1977). A different mechanism of action has been proposed, involving cyclic AMP mediation (Nimrod and Lindner, 1976). In the present work this aspect of the mechanism of action of steroids on these cells was examined. 201
202
A. Niw-od
EXPERIMENTAL Granulosa cells were collected from immature hypophysectomized rats treated with diethylstilbestrol (0.5 mg/rat/day) for 5 days (Hx-DES cells) and cultured as described by Nimrod and Lindner (1976) except that 20% fetal calf serum, instead of rat serum, was included in the medium. Cells (3-10 X 10’ cells/ml) were introduced into culture dishes (Falcon Optilux tissue culture dishes, or Falcon No. 3008 Multiwell tissue culture plates), and human FSH (LER-1801-3; 0.1 I.cg/ml; generously made available by the National Institute of Arthritis, Metabolism and Digestive Diseases, Pituitary Hormone Distribution Program, through the courtesy of Dr. L.E. Reichert, Jr.), dibutyryl cyclic AMP (DBC; 0.5 mg/ml; Sigma Chemical Co., St. Louis, MO.), androst-4-ene-3,17-dione (Ad; 1 yg/ml; Ikapharm, Ramat Can, Israel) and 3-isobutyl-I-methylxanthine (IBMX; 100 pg/ml; Aldrich Chemical Co., Milwaukee, Wis.) were added in various combinations to the medium at the beginning of the culture period. When cultures were to be maintained for 4 days, the medium was removed after the first 2 days of culture, the attached cells were washed with fresh serum-free medium, and fresh medium was added. 20@-Dihydroprogesterone (20a-OH-P) concentration in the medium was measured by radioimmunoassay (Bauminger et al., 1974). FSH-stimulable cyclic AMP accumulation was determined in cell layers cultured in the presence or absence of Ad. After washing the layers with Krebs-Ringer bicarbonate buffer (KRB), containing glucose (1 mg/ml), they were incubated at 37°C for 20 min in 1 ml KRB-glucose containing IBMX (100 pg/ml). FSH (0.1 pg/ml) and Ad (1 pg/ml) were added to the samples at the beginning of incubation. The reaction was terminated by cooling the plates on ice and after separation of the incubation medium from the cells 1 ml of 0.05 M acetate buffer, pH 4.0, was added to the dishes. The cell layers were then detached with a rubber scraper, and the suspension homogenized in a Teflon-glass homogenizer. Cyclic AMP content was determined in aliquots of the incubation medium and homogenates by the method of Gilman (1970). For the determination of [‘*‘I]FSH binding to cultured granulosa cells, cell layers were washed once, harvested with a rubber scraper and suspended in assay buffer (Nimrod et al., 1976). The cell suspensions were centrifuged at 500g for 8 min, the pellet resuspended in assay buffer by repeated passage through a Pasteur pipette, and specific binding of [ ‘*’ I] FSH was assayed as described by Nimrod et al., (1976). Cyclic 3’S’-nucleotide degradation, presumably by phosphodiesterase (PDE) activity, was measured in homogenates of cultured Hx-DES cells essentially as described by Zor et al. (1976). Cells ( lo7 cells per sample) were scraped off the culture plate and homogenized in a Teflon-glass homogenizer, in 0.55 ml of 40 mM Tris-3.5 mM MgCl,, pH 7.0. 50 ~1 of cAMP solution were then added to give a final concentration of 0.2 PM CAMP, and the mixture incubated with shaking at 37°C. Aliquots of the incubation mixture were taken out at various times and the
Mechanism of action of androgen in granulosa cells
203
reaction stopped by addition of an equal volume of 0.05 M sodium acetate, pH 4.0, and boiling for 5 min. Cyclic AMP content was determined in duplicate as described by Gilman (1970). Statistical analysis of the difference between group means was performed using Student’s f-test. When group variances were significantly different (P< 0.05 by Ftest), a logarithmic transformation was performed before application of the test of significance.
RESULTS The effect of FSH and Ad and DBC on the attachment, survival and multiplication of granulosa cells in culture
The attachment of the granulosa cells was usually completed within 24-36 h of culture, as judged by microscopic examination. Plating efficiency, calculated from the number of attached cells after 48 h of culture, was 50-70% (cf. table 1). Addition of FSH (0.1 pg/ml), Ad (1 pg/ml), FSH + Ad or dibutyryl cyclic AMP (DBC; 1 .O mM) to the culture medium had no significant effect on plating efficiency or net growth rate of the cultures during a 48 h culture period (table 1). After 4 days of culture (data not shown) there was no significant net change in the number of surviving cells in the control cultures, and the values for cultures supplemented with FSH, Ad, or FSH + Ad were within 20% of the control value. Light microscopic examination of cell layers after 2 days or 4 days of culture did not reveal any differences between the various treatment groups with respect to general morphology, nucleus/cytoplasm ratios or distribution of Lipid droplets.
Table 1 The effect of FSH, Ad and dibutyryl Hx-DES rats in culture
cyclic AMP (DBC) on the survival of granulosa
Cells harvested/dish Culture
conditions
No additions Ad (1 pg/ml) FSH (0.1 pg/ml) FSH + Ad DBC (1.0 mM) ____ ..___
* __.__
No. x lo@
% of no. plated
1.61; 2.14; 2.24; 1.72; 1.65;
57 72 72 60 58
1.70 2.05 1.97 1.78 1.72
cells from
* 2.9 X lo6 cells were plated in 10 cm diameter culture dishes containing 8 ml medium. number of attached cells was determined after 48 h. The values of 2 determinations shown, each representing the combined cells from 2 dishes.
The are
A. Nimrod
204
The effect of FSH and Ad on progestin production by granulosa cells during 4 days of culture Granulosa cell cultures were divided into four groups maintained during the first 2 days (stage I) as follows: (i) without hormones, (ii) with FSH, (iii) with Ad and (iv) with FSH and Ad. During the following 2 days (stage 11) the groups were subdivided into four subgroups, each maintained with one of these hormone combinations. The accumulation of 2Oo-OH-P in the media during the two culture periods is shown in fig. 1. Progesterone concentrations were not measured, since they have been shown under similar conditions to contribute only about 10% of total progestin concentration (Nimrod and Lindner, 1976). Cells cultured without hormone supplementation during stage I showed a diminished production of 2Oo-O&P during stage II (P< 0.05), even after addition of FSH or Ad (P< 0.05), but failed to
First 2 days Without hormowr
First 2 days With FSH
Fint 2 days With Ad
First 2 days With FSH+Ad
rnd Hormones
C
C,EA,FA
F
C,F;A,FA
A
C.[A.FA
FA
C,F;A.FA
Days of l-2 3-4 I-2 3 -4 I -2 3 -4 I -2 3-4 culture Fig. 1. The effect of FSH and Ad on the con tinuation of ZOcu-OH-Paccumulation by granulosa cells during 4 days of culture. Hx-DES cells (8 X lo3 cells/ml) were plated in Multiwell plates containing 0.5 ml of medium alone (C), or with androstenedione (A; 1 pg/ml), FSH (F; 0.1 pg/ ml), or a combination of the two hormones (FA). After 2 days the medium was collected, the cell layers washed once with 1 ml of fresh medium and recultured for an additional 2 days with the same combinations of FSH and Ad. The vertical bars represent the mean +SE of 20a-OH-P content in the media of 3 replicate cultures, assayed by radioimmunoa~y.
Mechanism ofoction of androgen in granulosa cells
205
prevent the decline during stage II (P< 0.001). By contrast, when Ad was added during stage I, 20cu-OH-Paccumulation was maintained above the control level during stage II cultures without hormone supplementation, and was further augmented upon addition during stage II of FSH together with Ad (P< 0.05). Exposure of the cells during stage I to both FSH and Ad resulted in greater augmentation of 2Ocr-OH-Paccumulation during stage I than that attained with either hormone alone (P< 0.001). This stimulated rate was maintained during stage II when the cells were cultured without hormone supplementation (f > O.Ol), and greatly enhanced when the two hormones were present in the medium during stage II as well (P< 0.001; fig. 1).
In another experiment, cells were cultured for 2 days (stage I) in the presence of a high concentration of DBC (1 mM), and then washed and cultured for an additional 2 days (stage II) either without hormone supplementation or with a combination of FSH and Ad. Exposure of the cell cultures to DBC during stage I resulted in an even higher rate of 20cu-OH-Paccumulation (P < 0.001) during that stage than in the presence of FSH and Ad (table 2). The stimulated rate was maintained during stage II in a medium not supplemented with DBC (P > 0.1). Further enhancement of 2Oo-OH-P accumulation was obtained during stage II by supplementation of the medium with FSH + Ad (P< 0.05); the levels thus attained were similar to those seen upon 4-day continuous exposure to FSH and Ad (P> 0.1; table 2). Thus, DBC was able to mimic the effect of FSH and Ad in transforming the Hx-DES cells into active progestin-secreting cells. These results suggest that the synergistic effect of Ad on FSH-stimulated proges-
Table 2 The effectiveness of dibutyryl CAMP (DBC) in mimicking the action of FSH and Ad on granulosa cell cultures. Hx-DES cells (3.6 X lo5 cells/ml initially plated) were cultured for 2 periods of 2 days each, as described in the legend to fig. 1. .-----_-.--.-- ____ ___ ....__..L__-.. Concentration of 20~OH-P * (ng/ml) Culture conditions ~Days 3 -4 Days l-2 Days 1-2 Days 3 -4 ~-----~-_--_---1422 23t 1 No hormones No hormones FSH + Ad so+3 FSH + Ad
No hormones FSH + Ad
310 f 19
306 f 5 962 f 80
DBC
No hormones FSH + Ad
704 f 58
665 f 32 125Oi: 180
* Mean -+SE of 4 replicate cultures.
A. Nitnrod
206
Table 3 Specific binding of FSH to granulosa cells of Hx-DES rats after 3 days of culture, compared to freshly isolated cells ___----~--... -... .._ ..___._.__ _. _~__.._. -~~~ -.--_-. Culture conditions [ t *“I] FSH bound * (cpm/l O6 cells) (mean + SE) __--_ - .~~~ No additions 4530 c 193 Androstenedione (1 pg/ml) 4128 i_ 225 Uncultured (freshly isolated) cells 4404 2 180 -_
* The difference between the radioactivity bound to the cells after incubation in the presence or absence of an excess of unlabelled FSH, assayed in triplicate.
tin production may be mediated by cyclic AMP. Three possible mechanisms which Ad could enhance cyclic AMP accumulation were explored: 1. 7’he Granulosa [“%]FSH ture period
by
ejyect of Ad on the binding of ( ‘25/]FSH to cultured granulosa ceils. cells from Hx-DES rats retained their high capacity to speci~~ally bind during 3 days in culture. Treatment of the cells with Ad during this culhad no significant effect (I’> 0.1) on FSH binding (table 3).
2. The effect of Ad on FSH-stinzulable cyclic AMP accurnulatioil in cultured granulosa cells. Cyclic AMP accumulation in ceils cultured for 24 h or 48 h with or without Ad was measured during a 20 min challenge with FSH and Ad separately or in combination, in the presence of IBMX (table 4). FSH stimulated CAMP accumulation to the same degree (P> 0.1) whether or not Ad was present during the preceding 24 or 48 h culture periods (about 9-fold and 14-fold, respectively). However, the presence of Ad during culture caused a slight increase in extracellular CAMP accumulation; this increase was significant after 48 h (P < 0.05) but not after 24 h (P> 0.05) of culture. Ad added on its own to the culture medium, or during the 20 min challenge period, did not affect CAMP accumulation. Addition of Ad together with FSH during the challenge incubation did not significantly potentiate the FSH effect (P > 0.05). 3. The effect of Ad, FSH and DBC on ~~os~~odieste~ase activity in ~anulosa cell ~~~~es. Androstenedione could raise CAMP concentrations in the granulosa cell culture by inhibiting degradation of the cyclic nucleotide by phosphodiesterase (PDE), rather than by stimulating its biosynthesis. However, the rate of CAMP degradation by homogenates of cells cultured in the presence of Ad was found to be similar to that of control cell homogenates (fig. 2). FSH- and DBC-treated cells exhibited slightly higher rates of degradation. Additional evidence that PDE is not involved in the synergistic action of Ad was obtained by culturing cells with Ad in combination with a PDE inhibitor (IBMX).
Medium
Cell homogenate
24 24 48 48
No hormones Ad
48 48
No hormones Ad
No hormones Ad
24 24
No hormones Ad (1 ng/ml)
1.6 f 0.1 l.? f 0.1
1.8 f 0.1 1.7 f 0.1
0.9 f 0.1 1.1 f 0.1
1.0 f 0.1 1.1 + 0.1
1.9 i 0.1 1.9 c 0.1
1.8 + 0.1 1.6 + 0.1
1.2 f 0.1 1.1 + 0.1
1.0 f 0.1 1.1 +_0.1
2.5 _i 0.1 3.5 * 0.1
2.6 + 0.1 3.5 t 0.5
14.9 * 3.2 14.1 f 0.9
8.8 f 1.0 8.7 + 0.2
2.5 f 0.2 3.4 f 0.1
2.6 + 0.3 2.8 of 0.3
11.8 + 1.6 16.7 t 0.6
9.0 t 0.9 6.4 t 0.2
Stimulation of cyclic AMP accumulation by Ad and FSH in granulosa cells of Hx-DES rats after 24 h and 48 h of culture. Cell layers were washed free of culture medium and incubated for 20 min in KRB medium containing 100 pg/ml IBMX (see Methods), with or without the hormones indicated. The KRB medium was then separated, and the cells were scraped off and homogenized in 0.05 M acetate buffer, pH 4.0. Cyclic AMP content of both KRB medium and cell homogenates is shown as the mean f SE for 3 plates, each assayed in duplicate. ___.~ Fraction Culture Cyclic AMP formation during 20 min challenge (pmol/dish) Culture assayed conditions time (h) No hormones FSH (0.1 pg/ml) FSH + Ad Ad (1 erg/ml) ~_~. ______
Table 4
k? 2 3
:: 2. % 9 2 9 0 1 -. 3: 09 2 3 =.
A. Nimrod
208
Time,
min
Fig. 2. Cyclic 3’,5’-nucleotide phosphodiesterase activity in homogenates of granulosa cell cultures with various hormones. Hx-DES cells (8 X lo5 cells/ml) were plated and the cultures maintained for 3 days in dishes of 10 cm diameter containing 8 ml of medium supplemented as indicated: o, no hormones (control); A, FSH 0.1 pg/ml; 0, androstenedione (Ad) 1 pg/ml; n, FSH + Ad; o, dibutyryl cyclic AMP (DBC) 1.0 mM. The cells were washed with PBS, scraped
off and homogenized in Tris-Mg buffer (see Experimental section). The homogenates were incubated with 0.2 PM CAMP and CAMP concentration determined in duplicate at the times specified. Duplicate determinations agreed within 10%. Cultures with IBMX alone accumulated lo-fold more 20a-OH-P than cultures without the inhibitor (P< 0.001; table S), indicating that PDE activity was markedly inhibited. Nevertheless, addition of Ad in the presence of IBMX caused a further increase in 2Oo-OH-P production (P < 0.001). Interaction of Ad and DBC Cells were cultured for 2 days in the presence of varying amounts of DBC with or without the addition of Ad. The amounts of 20o-OH-P accumulated in the medium are presented in fig. 3. The lowest effective concentration of DBC to
Table 5 The effect of a phosphodiesterase inhibitor (IBMX, 100 pg/ml) on the stimulation of 20a-OH-P accumulation by androstenedione (1 rg/ml). Hx-DES cells (5.2 X 10’ cells/ml) were sown and the cultures maintained for 2 days in Multiwell plates containing 0.5 ml medium. Culture conditions No additions IBMX Ad Ad + IBMX
Concentration 30+
1
365 f 20 146 f 4 1212 f 132
* Mean + SE of 4 replicate cultures.
of 20&H-P
* (ng/ml)
Mechunism of action ofndrogen I
I
I
in gramdosa ceils 1
I
I
I
209 I
1
500 s ‘_o i ::=3 h 0s
400
300
Ad(lOng/ml
L$! $ tY= s
5
2oo
No Ad
100
O
~~~ 0 -4
IO Dibvfyryl-
25
64
160
400
1000
cyclic AMP@glml)
Fig. 3. Interaction
of Ad and dibutyryl cyclic AMP (DBC) in the stimulation of 20cu-OH-P accumulation by granulosa cells during 2 days of culture. Hx-DES cells (4 X i05 cells/ml) were plated into Multiwell plates containing 0.5 ml of medium with various concentrations of DBC, in the presence or absence of Ad (!O ng/ml). The vertical bars represent the mean GE of 20~ OH-P content in the media of 4 replicate cultures.
stimulate 2Oo-OH-P accumulation was 25 @g/ml (50 j&I); maximal stimulation was attained with 400 fig/ml (0.8 mM) DBC. Androstenedione (10 nglml) had a synergistic effect on this stimulation at DBC concentrations of 10-1000 pg/ml (P < O.Ol), with the exception of 400 pg/ml of DBC, where marked variation was observed (P > 0.1). An increase in Ad concentrations up to 0.36 pg/ml (data not shown) did not further augment the synergistic effect with DBC.
DISCUSSION The present study shows that granulosa cell cultures from Hx-DES rats are unable to maintain progestin production for more than 48 h without hormone supplementation, or in the presence of FSH only. Progestin production, however, remained unimpaired in the presence of androstenedione (fig. 1). Moreover, combined treatment with FSH and Ad brought about persistent changes in the cultured cells, since progestin accumulation was not affected by the subsequent removal of these hormones (fig. 1). A marked rise in progestin production by granulosa cells is frequently regarded as a marker for luteini~tion (~hanning, 1970). However, the FSH + Ad treated Hx-DES cells differ from ordinary luteal cells in that they are morphologically indistinguishable from untreated granulosa cells (see Results section), and do not specifically bind [ ‘*‘I]hCG (Nimrod et al., 1977). The increased
210
A. Nimrod
progestin production by these cultures in the presence of FSH and/or Ad or DBC was not attributable to an increase in cell numbers, since none of these treatments had a significant effect on plating efficiency and subsequent cell survival or rate of multiplication (table 1). Dibutyryl CAMP (DBC) was able to mimic the metabolic changes induced by the combined action of FSH and Ad (table 2). This finding suggested that CAMP was involved in the mediation of the synergistic effect of androgen. Since the content of CAMP in Hx-DES cells was below the limits of sensitivity of the assay, we chose to examine by indirect means three possible mechanisms by which CAMP accumulation could be enhanced by androgen, namely (i) an effect on the number of available FSH binding sites; (ii) activation of the coupling of the FSH receptor to adenylate cyclase, as reflected by increased CAMP accumulation following a 20 min challenge with FSH in the presence of a PDE inhibitor; and (iii) a change in CAMP degradation by cyclic 3’,5’-nucleotide phosphodiesterase. The present results provided no evidence that any of these mechanisms is operative: the extent of [12’I]FSH binding (table 3) and PDE activity (fig. 2) were not affected by culturing granulosa cells in the presence of Ad, and the effect of Ad on FSH-stimulable CAMP accumulation was small and inconsistent (table 4). Taken together, these findings do not support the contention that Ad directly influences FSH-stimulable CAMP accumulation in granulosa cells, and further suggest that Ad acts at a step in the regulation of progestin biosynthesis distal to CAMP production. This conclusion is supported by the observation that Ad exerted a synergistic effect on the stimulation of 20a-OH-P accumulation in granulosa cell cultures by DBC (fig. 3). This synergistic effect was most pronounced at 0.3 mM DBC concentration (160 /.rg/ml; P< 0.001 by Student’s t-test; fig. 3). At the maximally effective DBC concentration (0.8 mM) the androgen effect on the rise of 20aOH-P accumulation exhibited a wide variation and was statistically not significant (P> O.l), in accordance with earlier observations (Nimrod and Lindner, 1976). Cyclic AMP is believed to stimulate steroidogenesis through a multistage biochemical pathway (Marsh, 1975). The steps in this pathway that are influenced by androgens, and the mechanism of action of these steroids in granulosa cells, remain to be elucidated.
ACKNOWLEDGEMENTS
We thank Mrs. Genia Acevedo for her expert technical assistance, and Dr. Charles Faiman, Dr. H.R. Lindner and Sergio A. Lamprecht for their criticism and assistance in writing this paper. This work has been supported by a grant (to Dr. H.R. Lindner) from the Ford Foundation and the Population Council, New York.
Mechanism of action of androgrn irtgramlosa cells
211
REFERENCES Baulninger, S., Kohen, F. and Lindner, H.R. (1974) J. Steroid Biochem. 5,739-747. Bauminger, S., Lieberman, M.E. and Lindner, H.R. (1975) Prostag~and~s 9,753-764. Channing, C.P. (1970) Recent Prog. Horm. Res. 26,589-622. Eshkof, A. and Lunenfeid, B. (1972) in: Gonadotropins, Eds.: B.B. Saxena, G.C. BeBng and H.M. Gandy (Wiley, New York) pp. 335-346. Gilman, A.G. (1970) Proc. Nati. Acad. Sci, U.S.A. 67,305-312. Hisaw, F.L. (1947) Physiol. Rev. 27,95-146. Lamprecht, S.A., Zor, U., Tsafriri, A. and Lindner, H.R. (1973) J. Endocrinol. 57,217-233. Makris, A. and Ryan, K.J. (1975) Endocrinology 96,694-701. Marsh, JM, (1975) Adv. Cyclic Nucieotide Res. f&137-199. Nimrod, A. (1977) Mol. Cell. Endocrinol. 8,189-199. Nimrod, A. and Lindner, H.R. (1976) Mol. Cell. ~ndocr~noI.5,315-319. Nimrod, A., Erickson, G.F. and Ryan, K.J. (1976) Endocrinology 98,56-64. Nimrod, A., Tsafriri, A. and Lindner, H.R. (1977) Nature (London), in press. Richards, J.S. and Midgley, A.R., Jr. (1976) Biol. Reprod. 14, 82-94. Zor, U., Koch, R. and Naor, Z. (1976) Adv. Prostag~andin T~omboxane Res. 1,331-335.