0021-972X/91/7206-1206$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1991 by The Endocrine Society
Vol. 72, No. 6 Printed in U.S.A.
Effect of Recombinant Activin on Androgen Synthesis in Cultured Human Thecal Cells* S. G. HILLIER, E. L. YONG, P. J. ILLINGWORTH, D. T. BAIRD, R. H. SCHWALL, AND A. J. MASON Department of Obstetrics and Gynecology (S.G.H., E.L. Y., P.J.I., D.T.B.), University of Edinburgh, Centre for Reproductive Biology, Edinburgh, EH3 9EW Scotland, United Kingdom; and Genentech, Inc. (R.H.S., A.J.M), South San Francisco, California 94080
ABSTRACT. The effect of activin-A on ovarian androgen synthesis was tested in vitro using serum-free monolayer cultures of human thecal cells. Maximal rates of androgen (androstenedione and dehydroepiandrosterone) production were induced by treating the cells for 4 days with LH (10 ng/mL) in the presence of insulin-like growth factor-I (>30 ng/mL). The additional presence of recombinant activin-A (1-100 ng/mL) in culture medium caused dose-dependent suppression of thecal cell androgen production, with 50% maximal inhibition occurring at an
activin-A concentration of about 10 ng/mL. Progesterone production was only suppressed by high dose (100 ng/mL) activinA, and inhibition of steroid production occurred without inhibition of DNA synthesis (tritiated thymidine uptake). These results reveal a potent and selective inhibitory action of activinA on thecal cell androgen synthesis, consistent with a paracrine function for activin(s) in modulating follicular androgen biosynthesis in the human ovary. (J Clin Endocrinol Metab 72: 12061211,1991)
A
CTIVIN is an approximately 24-kDa protein that has been isolated from ovarian follicular fluid as a homodimer of the inhibin/activin /SA-subunit (activinA) or a heterodimer of the inhibin/activin /3A- and /3Bsubunits (activin-AB) (1, 2). Both inhibin/activin subunits are encoded by separate mRNAs (3-6) expressed in granulosa cells under the control of FSH (7-9). Based on the pattern of /3B-subunit mRNA expression that occurs in cyclic monkey ovaries, it is likely that a 0B/ |8B-activin homodimer is also produced, particularly by granulosa cells in small antral follicles (10). An endocrine function for ovarian activin(s) has been proposed, since it stimulates FSH release by pituitary cells in vitro (11, 12). However, an intraovarian function(s) for the protein is also likely, supported by evidence that activin-A purified from porcine follicular fluid inhibits LH-stimulated androgen production by rat ovarian thecal/interstitial tissues in vitro (13). To determine whether activin might regulate thecal function in the adult human ovary, we tested the effect of recombinant human activin-A on steroid production by primary monolayer cultures of human thecal cells. Received November 1,1990. Address all correspondence and requests for reprints to: Dr. Stephen G. Hillier, Reproductive Endocrinology Laboratory, University of Edinburgh, Centre for Reproductive Biology, Edinburgh, EH3 9EW Scotland, United Kingdom. * This work was supported by MRC Programme Grant PG8929583 (to S.G.H. and D.T.B.).
Here, we report that when thecal cell steroid synthesis is induced by treatment with LH and (or) insulin-like growth factor-I (IGF-I), the presence of femtomolar concentrations of activin-A in serum-free culture medium causes a selective inhibition of androgen [androstenedione and dehydroepiandrosterone (DHA)] production. These data provide direct support for the concept of activin (s) as a paracrine modulator of androgen synthesis in the human ovary. Subjects and Methods Patients Ovarian tissue was obtained from six women who underwent surgery (hysterectomy with uni- or bilateral oophorectomy) to treat nonmalignant gynecological disease (severe uterine fibroids, endometriosis, and/or heavy and painful menstrual bleeding; Table 1). The study had the approval of the local ethics committee. Isolation of thecal cells
Resected ovarian tissue was placed in ice-cold physiological saline, transported to the laboratory, and transferred to culture medium, which was also used for follicular dissection: medium 199 containing Earle's salts, 25 mM HEPES buffer, extra (2 mM) L-glutamine, 50 IU/mL penicillin, 50 ng/mL streptomycin (all from Gibco Ltd., Paisley, Renfrewshire, United Kingdom), and 0.1% (wt/vol) BSA (from ICN Biomedicals, High Wy-
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ACTIVIN AND THECAL ANDROGEN SYNTHESIS combe, Bucks, United Kingdom). Dissection of follicles (Table 1) and isolation of thecae were carried out with visualization using zoom stereomicroscopic optics, as described previously (14). Only thecae from apparently healthy follicles were studied, i.e. yielding straw-colored follicular fluid, granulosa cells, and (usually) a cumulus-enclosed oocyte. After scraping all visible granulosa cells from the lamina basalis, hemisected thecae from individual or pooled follicles were rinsed in fresh medium and transferred to more of the same medium (2 mL) containing 0.1% (wt/vol) collagenase type II from Cl. histolyticum and 0.01% (wt/vol) deoxyribonuclease-I (both from Sigma Chemical Co., Poole, Dorset, United Kingdom). Complete dispersal into a cellular suspension was achieved by incubating the tissue in the enzyme solution for two successive 15-min periods at 37 C in a shaking water bath, with repeated pipetting after each incubation. The cells were sedimented by centrifugation (5 min at 800 x g), resuspended in fresh culture medium containing 5.0% (vol/vol) donor calf serum (Gibco), and counted in a hemocytometer. Cell viability, determined by staining with 0.4% (wt/vol) trypan blue, was consistently more than 90%. Thecal cell culture Twenty-four-well plastic dishes (Linbro Space Savers from Flow Laboratories, Rickmansworth, Herts, United Kingdom) were inoculated with replicate 500-/zL portions of thecal cell suspension (2.0-5.0 X 104 viable cells/well) in culture medium containing 5% donor calf serum. Preincubation in serum-containing medium to allow cell anchorage and recovery from the enzymatic dispersal procedure was carried out for 24 h at 37 C in a humidified tissue culture incubator gassed with 95% air5% CO2. After removal of serum-containing medium and washing with 1 mL prewarmed (37 C) Dulbecco's phosphate-buffered saline, the cultures received 500 nL serum-free medium containing human LH (LER-1972: donated by Dr. L. E. Reichert, Jr.), recombinant human IGF-I (CGP-35'-126, donated by Drs. H. H. Peter and K. Scheibli), and/or recombinant human activin-A (15). Incubation at 37 C was then performed for 4 days, with a medium change on day 2. Media collected on days TABLE 1. Details of patients and ovarian follicles studied Exp no.
Patient
Age (yr)
LMP (day)
No. and diameter (mm) of follicles studied
I II
A A
45 45
13 13
III IV V
B B C
31 31 44
°
VI VII
VIII
D D E
IX
F
1(14) 1(18)
1207
2 and 4 of culture were stored frozen at —20 C until assayed for steroid content, as described below. Assay of steroids in thecal cell culture medium Steroids in unextracted culture medium were measured individually by RIA using standard procedures, as previously described (14). The androstendione assay used a rabbit antiserum to androst-4-ene-3,17-dione-7a-carboxyethyl thioether conjugated to BSA. Major cross-reactions were: androstendione, 100%; androsterone, 46.3%; 5a-androstene-3,17-dione, 50%; testosterone, 37%; and less than 0.5% for all other steroids tested. The DHA assay used sheep anti-DHA-3-hemisuccinylovalbumin purchased from Steranti Research Ltd. (St. Albans, Herts, United Kingdom; product code A005). Major crossreactions were: DHA, 100%; DHA sulfate, 141%; 5a-androstane-3,17-dione, 7.9%; testosterone, 4.4%; androstenedione, 3.3%; and 0.5% or less for all other steroids tested. The progesterone antiserum was sheep antiprogesterone-lla-hemisuccinyl-BSA with major cross-reactants of: progesterone, 100%, lla-hydroxyprogestrone, 35.5%; deoxycorticosterone, 25.3%; 11-ketoprogesterone, 15.8%; and 20/3-hydroxyprogesterone, 12%. The sensitivity (90% B/B.) of each assay was approximately 0.05 pmol steroid/assay tube. The inter- and intraassay precisions for each steroid were less than 15%. Steroid production rates are expressed as picomoles of steroid per 1000 cells/ 48 h, related to the number of viable cells used to inoculate each culture well. Measurement of tritiated thymidine untake by cultured thecal cell monolayers Incorporation of tritiated thymidine into cultured thecal cell monolayers was determined using a modification of the method described by May et al. (17). Briefly, after the collection of spent culture medium on day 4, each monolayer was incubated for a further 18 h in 500 /xL serum-free culture medium containing 2 /^Ci [me£fry/-3H]thymidine (1.5 Ci/mmol; Amersham International, Aylesbury, Bucks. United Kingdom). The monolayer was then washed twice with 0.1 M phosphate-buffered saline and incubated for 20 min at 4 C in 1.0 mL 5% (wt/vol) trichloroacetic acid. The fixed monolayer was then washed once with 5% trichloroacetic acid and twice with methanol. The cells were dissolved by incubation for 5 min at room temperature in 500 tih 0.5 M NaOH. After neutralization by the addition of 500 nL 0.5 M HC1, the solution was taken for liquid scintillation counting. Statistics
8
20 (4-5)* 6 (6-7)* 3 (2-5)*
Analysis of variance with the Neuman-Keuls or paired Student's t test was used to analyze differences between experimental and control observations. Differences assigned a P value less than 0.05 were regarded as statistically significant.
36 36 57
8 8 27
1(7) 1(8) 16 (3-8)*
Results
38
27
17 (3-5)*
Stimulation of thecal cell androstenedione production by LH and IGF-I
LMP, Last menstrual period. " Acyclic. * Pooled.
a
Maximal rates of androgen production by thecal cell cultures occurred in medium containing LH plus IGF-I.
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HILLIER ET AL.
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If neither LH nor IGF-I was present, the average rate of androstenedione accumulation in the medium was 0.064 ± 0.020 pmol/1000 cells-48 h (mean ± SE; n = 7) on day 2, falling to 0.024 ± 0.008 pmol/1000 cells-48 h on day 4. The presence of LH (1-100 ng/mL) alone did not prevent this decline, whereas IGF-I (>30 ng/mL) reversed it completely (Fig. 1, left panel). IGF-I also increased thecal cell responsiveness to LH (Fig. 1, right panel). Thus, whereas LH (10 ng/mL) alone increased androstenedione accumulation on day 4 of culture by less than a third, the response was about 15 times greater if IGF-I (30 or 100 ng/mL) was also present (Fig. 2).
JCE & M • 1991 Vol 72 • No 6
Inhibition of thecal cell androstenedione production by activin-A Activin-A did not modify basal androgen production. However, the presence of activin-A in culture medium dose-dependently inhibited the production of androstenedione in response to treatment with LH and/or IGF-I. Fifty percent maximal inhibition occurred at an activin-A concentration approaching 10 ng/mL (Fig. 3). Inhibitory effects of activin-A (10 ng/mL) on IGF-I- and LH- plus IGF-I-stimulated androstenedione production from five individual experiments are summarized in Fig. 4.
IGF-I + LH
IGF-I alone 0.09 n
Selective action of activin-A on thecal cell steroid production
0
10
100
1000
0
10
100 1000
IGF-I concentration (ng/ml)
FIG. 1. Dose-related stimulatory effect of IGF-I on androstenedione production by human thecal cell cultures. Left panel, In the absence of LH; right panel, in the presence of LH (10 ng/mL). Thecal cell monolayers (5.0 x 104 viable wells/culture well) pooled from 20 follicles between 4-5 mm in diameter (Exp III) were incubated for 4 days in serum-free culture medium containing IGF-I with and without LH. Androstenedione accumulation in medium collected on days 2 and 4 of culture is shown. Data are the mean ± SE (n = 4). Asterisks denote significant stimulation by IGF-I relative to the corresponding treatment without IGF-I (*, P < 0.05; • * , P < 0.01).
The increased production of androstenedione due to combined treatment with LH and IGF-I was paralleled by increased production of DHA and progesterone. However, whereas only 1 ng/mL activin-A significantly reduced the production of androstenedione and DHA by at least 20%, 100 ng/mL of the protein were required to inhibit progesterone production by a similar degree (Fig. 5). Thus, in five of five experiments in which it caused an average 50% reduction in androstenedione production, activin-A (10 ng/mL) had no significant effect on LH- plus IGF-I-stimulated progesterone production (P = 0.098, by Student's paired t test). 0.41
0.30.4 n x: oo
il
0.3-
0.2-
2 o
0.2-
0.1"
0.1 0" 0 Control
LH
IGF-I
LH + IGF-I
Treatment
FIG. 2. Interaction between IGF-I and LH on androstenedione production by human thecal cell cultures: composite data from Exp IIIIX. Thecal cell monolayers were incubated for 4 days in serum-free culture medium without (control) and with LH (10 ng/mL), IGF-I (30 or 100 ng/mL), or IGF-I plus LH. Androstenedione accumulation is shown in medium collected on day 4. Data are the mean ± SE. Asterisks denote significant differences between the group means indicated (*, P < 0.05; • * , P < 0.001).
1
10
100
Activin-A concentration (ng/ml)
FIG. 3. Dose-dependent inhibitory effect of activin-A on androgen production by human thecal cell cultures. Thecal cell monolayers (2.0 X 10" viable wells/culture well) from a single follicle (7 mm in diameter; Exp VI) were incubated for 4 days in serum-free culture medium containing IGF-I (100 ng/mL) alone, LH (10 ng/mL) alone, or IGF plus LH, with and without activin-A at the concentrations indicated. Androstenedione accumulation is shown in medium collected on day 4. Data are the mean ± SE (n = 3). Asterisks denote statistically significant inhibition due to activin-A (*, P < 0.05; • * , P < 0.01).
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ACTIVIN AND THECAL ANDROGEN SYNTHESIS 20 000
IGF 1 n
'
1209 IGF-I + LH
alone
T 15 000 -
Ep
10 000 -
5 000 -
Control
LH
IGF-I
LH + IGF-I 300
Treatment
FIG. 4. Inhibition by activin-A of LH-, IGF-I-, and LH- plus IGF-Istimulated androstenedione production by human thecal cell cultures: composite data from Exp V-IX. Thecal cells were incubated for 4 days in serum-free culture medium in the absence and presence of activinA (10 ng/mL) without (control) and with LH (10 ng/mL), IGF-I (30 or 100 ng/mL), or IGF-I plus LH. Androstenedione accumulation is shown in medium collected on day 4. Data are the mean ± SE. Asterisks denote significant inhibition by activin-A (•, P < 0.05; **, P < 0.001). DHA
Androstenedione
0.4 -i
Progesterone
0.3"
Z.Z
A
0.2H
i| 2 o 0.1 •
0
1
10
100
0
1
10
100
0
1
10
100
Activin-A concentration (ng/ml)
FlG. 5. Relative inhibitory effect of activin-A on production of androstenedione (left panel), DHA {center panel), and progesterone (left panel) by human thecal cell cultures. Thecal cells (2.0 x 104 viable wells/culture well) from a single follicle (7 mm in diameter; Exp VI) were incubated for 4 days in serum-free culture medium containing IGF-I (100 ng/mL) plus LH (10 ng/mL), with and without activin-A at the concentrations indicated. Steroid accumulation is shown in medium collected on day 4. Corresponding basal rates of steroid accumulation (i.e. in the absence of LH and IGF-I) were 0.006 ± 0.002 (androstenedione), 0.012 ± 0.003 (DHA) and 0.006 ± 0.001 (progesterone) pmol steroid/1000 cells-48 h. Data are the mean ± SE (n = 3). Asterisks denote statistically significant inhibition due to activin-A (*, P < 0.05; • * , P < 0.01).
No effect of activin-A on IGF-I-stimulated uptake of tritiated thymidine by thecal cell monolayers Treatment with IGF-I (30-300 ng/mL) elicited an approximately 20-fold increase in tritiated thymidine uptake by cultured thecal cells, regardless of the presence of LH (10 ng/mL). This response was not significantly altered by the presence of activin-A at a dose (10 ng/ mL) that was sufficient to cause about 50% inhibition of androstenedione production (Fig. 6).
Discussion These results reveal a potent and selective inhibitory action of activin-A on androgen production by human
0
30
100
300
IGF-I concentration (ng/ml)
FIG. 6. Lack of effect of activin-A on IGF-I-stimulated uptake of tritiated thymidine by human thecal cells. Thecal cell monolayers (2.0 x 104 viable wells/culture well) pooled from 16 follicles between 3-8 mm in diameter (Exp VIII) were incubated for 4 days in serum-free culture medium containing IGF-I at the concentrations shown in the absence (left panel) or presence (right panel) of LH (10 ng/mL), with and without activin-A (10 ng/mL). Uptake of tritiated thymidine was determined as described in Subjects and Methods. Data are the mean ± SE (n = 3).
thecal cells in vitro. Synthesis of A4 (androstenedione) and A5 (DHA) androgens was suppressed by femtomolar concentrations of the protein, consistent with a physiologically significant paracrine mode of action. Additional experiments are needed to determine the mechanism of action involved. However, preferential inhibition of Ci9 steroid production relative to progesterone production points to an action at the level of 17-hydroxylase/Ci7_2o lyase, the rate-limiting enzyme in thecal androgen synthesis. Previous studies of the effect of activin on steroidogenesis in ovarian cells have been confined to experimental animal tissues. In rat granulosa cell cultures, activin-A purified from bovine follicular fluid enhanced FSH-stimulated aromatase activity, but inhibited progesterone production (18). Augmentation of aromatase activity by recombinant activin-A was also observed using cultured granulosa cells from a nonhuman primate (common marmoset, Callithrix jacchus) (19). The concept that activin produced by granulosa cells might function as a paracrine modulator of thecal androgen synthesis initially arose from in vitro experiments using prepubertal rat tissues (13). However, quantitative and qualitative changes in the pattern of ovarian androgen synthesis precede the onset of puberty in rats (20), raising the question of whether activin inhibition of androgen synthesis has any relevance to the situation in adult ovaries. The present data are novel in showing not only that activin inhibition of thecal androgen synthesis extends to humans, but also that it persists into adulthood, at least in women. It should be noted, however, that this study of activin-A action was confined to thecal cells from follicles 8 mm or less in diameter (Exp V-IX, Table 1). Further studies
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HILLIER ET AL.
on more mature follicles are necessary to determine how the protein affects thecal function at later stages of preovulatory development in the human ovary. The use of serum-free conditions of culture highlighted a striking ability of IGF-I to enhance basal and LHresponsive androgen synthesis in thecal cell monolayers. Previous studies have shown that treatment with insulin or IGF-I enhances basal and LH-stimulated synthesis of androgen in cultured thecal/interstitial cells from rat ovaries (21-23) as well as stromal tissue explants from human ovaries (24, 25). The present study complements and extends these earlier findings, showing IGF-I stimulation of thecal cell DNA synthesis as well as androgen synthesis. An intrafollicular paracrine function(s) for the growth factor is suggested by the evidence that IGF receptors are present on human thecal cells (26), and granulosa cells are intrafollicular sites of IGF-I mRNA expression (27) and IGF-I synthesis regulated by FSH (28, 29). Despite its marked stimulatory effect on thymidine uptake, IGF-I does not measurably increase thecal cell number under the conditions and duration of culture used here (Yong, E. L., and S. G. Hillier, unpublished observation). Nevertheless, we interpret these data to suggest that IGF-I of granulosa cell origin has the potential to exert positive paracrine control over the growth of the theca interna as well as modulate its steroidogenic function in vivo. The evidence that activinA overrides IGF-I-stimulated thecal androgen production without suppressing thecal DNA synthesis further indicates that a complex functional interaction is likely to exist between these two factors in the follicular paracrine system. Thecal androgens play fundamental roles in the ovary, serving as estrogen precursors and having local regulatory actions (20). Studies of follicular fluid levels of androgen and estrogen in relation to granulosa cell aromatase activity indicate that the capacity of the theca interna to generate aromatase substrate (androstenedione) increases hand in hand with granulosa cell aromatase activity in the human preovulatory follicle (14). It is of interest, therefore, that in primate ovaries, inhibin/ activin 0B-subunit mRNA is expressed in the greatest amounts by granulosa cells in small antral follicles, decreasing to undetectable levels during preovulatory follicular development (10). Activin-A and -B exhibit similar actions in pituitary and erythroid differentiation assays in vitro (30). Presumably, therefore, they also share the inhibitory action on thecal androgen synthesis that has been described here, in which case a development-related reduction in granulosa cell activin synthesis might constitute a paracrine mechanism for positively regulating thecal androgen synthesis in the human preovulatory follicle.
JCE & M • 1991 Vol 72 • No 6
Acknowledgments We are grateful to Prof. L. E. Reichert, Jr., of the Department of Biochemistry, Albany Medical College (Albany, NY), for the supply of LH, to Drs. H. H. Peter and K. Scheibli, Ciba-Geigy Ltd. (Basel, Switzerland) for the IGF-I, and to Mr. W. A. Ferguson for the steroid assays. Note added in proof: Subsequently, we have shown that recombinant inhibin-A stimulates basal and LH- plus IGF-Iresponsive androgen synthesis and overcomes the inhibitory action of activin-A in human thecal cell cultures (31).
References 1. Ying S-Y. Inhibins, activins, and follistatins: gonadal proteins modulating the secretion of follicle-stimulating hormone. Endocr Rev. 1988;9:267-93. 2. Vale W, Rivier C, Hsueh A, et al. Chemical and biological characterization of the inhibin family of protein hormones. Recent Prog Horm Res. 1988;44:l-30. 3. Mason AJ, Hayflick JS, Ling N, et al. Complementary DNA sequences of ovarian follicular fluid inhibin show precursor structure and homology with transforming growth factor-/?. Nature. 1985;318:659-63. 4. Mason AJ, Niall HD, Seeburg PH. Structure of two human ovarian inhibins. Biochem Biophys Res Commun. 1986;35:957-64. 5. Forage RG, Ring JW, Brown RW, et al. Cloning and sequence analysis of cDNA species coding for the two subunits of inhibin from bovine follicular fluid. Proc Natl Acad Sci USA. 1986;83:30915. 6. Esch FS, Shimasaki S, Cooksey K, et al. Complementary deoxyribonucleic acid (cDNA) cloning and DNA sequence analysis of rat ovarian inhibins. Mol Endocrinol. 1987;l:388-96. 7. Woodruff T, Meunier H, Jones PB, Hsueh AJW, Mayo KE. Rat inhibin: molecular cloning of a- and /3-subunit complementary deoxyribonucleic acids and expression in the ovary. Mol Endocrinol. 1987;l:561-9. 8. Woodruff TK, D'Agostino JB, Schwartz NB, Mayo KE. Dynamic changes in inhibin messenger RNAs in rat ovarian follicles during the reproductive cycle. Science 1988;239:1296-9. 9. Turner IM, Saunders PTK, Shimasaki S, Hillier SG. Regulation of inhibin subunit gene expression by FSH and estradiol in cultured rat granulosa cells. Endocrinology. 1989;125:2790-2. 10. Schwall RH, Mason AJ, Wilcox JN, Bassett SG, Zeleznik AJ. Localization of inhibin/activin subunit mRNAs within the primate ovary. Mol Endocrinol. 1990;4;75-9. 11. Vale W, Rivier J, Vaughan J, et al. Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid. Nature. 1986;321:776-9. 12. Ling N, Ying S-Y, Ueno N, et al. Pituitary FSH is released by a heterodimer of the 0-subunits from the two forms of inhibin. Nature. 1986;321:779-82. 13. Hsueh AJW, Dahl KD, Vaughan J, et al. Heterodimers and homodimers of inhibin subunits have different paracrine action in the modulation of luteinizing hormone-stimulated androgen biosynthesis. Proc Nat Acad Sci USA. 1987;84:5082-6. 14. Hillier SG, Reichert Jr LER, van Hall EV. Control of preovulatory follicular estrogen biosynthesis in the human ovary. J Clin Endocrinol Metab. 198l;52:847-56. 15. Schwall RH, Nikolocs K, Szonyi E, Mason AJ. Recombinant expression and characterization of human activin A. Mol Endocrinol. 1989;2:1237-42. 16. McNatty KP, Hillier SG, van den Boogard AMJ, Trimbos-Kemper TCM, Reichert Jr LE, van Hall EV. Follicular development during the luteal phase of the human menstrual cycle. J Clin Endocrinol Metab. 1981:56:1022-31. 17. May JV, Frost JP, Schomberg DW. Differential effects of epidermal growth factor, somatomedin-C/insulin-like growth factor 1, and
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ACTIVIN AND THECAL ANDROGEN SYNTHESIS
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24. 25.
transforming growth factor-/? on porcine granulosa cell deoxyribonucleic acid synthesis and cell proliferation. Endocrinology. 1988;123:168-79. Hutchison LA, Findlay JK, de Vos FL, Robertson DM. Effects of bovine inhibin, transforming growth factor-/? and bovine activinA on granulosa cell differentiation. Biochem Biophys Res Commun. 1987;146:1405-12. Hillier SG. Effects of activin on oestrogen synthesis in primate granulosa cells [Abstract]. J Endocrinol. 1990;127(Suppl):67. Hillier SG. Sex steroid metabolism and follicular development in the ovaries. Oxf Rev Reprod Biol. 1985;7:168-222. Erickson GF, Magoffin DA. Dyer CA, Hofeditz C. The ovarian androgen producing cells: a review of structure/function relationships. Endocr Rev. 1985;6:371-99. Cara JF, Rosenfield RL. Insulin-like growth factor I and insulin potentiate luteinizing hormone-induced androgen biosynthesis by rat ovarian theca-intersitial cells. Endocrinology. 1988;123:733-9. Hernandez ER, Resnick CE, Svoboda ME, Van Wyk JJ, Payne DW, Adashi EY. Somatomedin-C/insulin-like growth factor I as an enhancer of androgen biosynthesis by cultured rat ovarian cells. Endocrinology. 1988;122:1603-12. Barbieri RL, Makris A, Ryan KJ. Insulin stimulates androgen accumulation in incubations of human ovarian stroma and theca. Obstet Gynecol. 1984;64(Suppl):73S-80S. Barbieri RL, Makris A, Randall RW, Daniels G, Kistner RW, Ryan
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KJ. Insulin stimulates androgen accumulation in incubations of ovarian stroma obtained from women with hyperandrogenism. J Clin Endocrinol Metab. 1986;62:904-10. Poretsky L, Grigorescu F, Seibel M, Moses AC, Flier JS. Distribution and characterization of insulin and insulin-like growth factor receptors in normal human ovary. J Clin Endocrinol Metab. 1985;61:728-34. Oliver JE, Aitman TJ, Powell JF, Wilson CA, Clayton RN. Insulinlike growth factor I gene expression in the rat ovary is confined to the granulosa cells of developing follicles. Endocrinology. 1989;124:2671-79. Hammond JM, Baranao JLS, Skaleris D, Knight AB, Romanus JA, Rechler MM. Production of insulin-like growth factors by ovarian granulosa cells. Endocrinology. 1985;117:2553-5. Adashi EY, Resnick CE, Hernandez ER, Svoboda ME, Van Wyk JJ. Potential relevance of insulin-like growth factor I to ovarian physiology: from basic science to clinical application. Semin Reprod. 1989;7:794-9. Mason AJ, Schmelzer C, Schwall RH. Molecular and physiological studies on inhibin and activin. In: Josso N, ed. Development and function of the reproductive organs. Rome: Ares-Serono Symposia; 1989;3:153-7. Hillier SG, Yong EL, Illingworth PJ, Baird DT, Schwall RH, Mason AJ. Effect of recombinant inhibin on androgen synthesis in cultured human thecal cells. Mol Cell Endocrinol. 1991;75:R1R6.
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Vol. 72, No. 6 Printed in U.S.A.
Effect of Recombinant Activin on Androgen Synthesis in Cultured Human Thecal Cells* S. G. HILLIER, E. L. YONG, P. J. ILLINGWORTH, D. T. BAIRD, R. H. SCHWALL, AND A. J. MASON Department of Obstetrics and Gynecology (S.G.H., E.L. Y., P.J.I., D.T.B.), University of Edinburgh, Centre for Reproductive Biology, Edinburgh, EH3 9EW Scotland, United Kingdom; and Genentech, Inc. (R.H.S., A.J.M), South San Francisco, California 94080
ABSTRACT. The effect of activin-A on ovarian androgen synthesis was tested in vitro using serum-free monolayer cultures of human thecal cells. Maximal rates of androgen (androstenedione and dehydroepiandrosterone) production were induced by treating the cells for 4 days with LH (10 ng/mL) in the presence of insulin-like growth factor-I (>30 ng/mL). The additional presence of recombinant activin-A (1-100 ng/mL) in culture medium caused dose-dependent suppression of thecal cell androgen production, with 50% maximal inhibition occurring at an
activin-A concentration of about 10 ng/mL. Progesterone production was only suppressed by high dose (100 ng/mL) activinA, and inhibition of steroid production occurred without inhibition of DNA synthesis (tritiated thymidine uptake). These results reveal a potent and selective inhibitory action of activinA on thecal cell androgen synthesis, consistent with a paracrine function for activin(s) in modulating follicular androgen biosynthesis in the human ovary. (J Clin Endocrinol Metab 72: 12061211,1991)
A
CTIVIN is an approximately 24-kDa protein that has been isolated from ovarian follicular fluid as a homodimer of the inhibin/activin /SA-subunit (activinA) or a heterodimer of the inhibin/activin /3A- and /3Bsubunits (activin-AB) (1, 2). Both inhibin/activin subunits are encoded by separate mRNAs (3-6) expressed in granulosa cells under the control of FSH (7-9). Based on the pattern of /3B-subunit mRNA expression that occurs in cyclic monkey ovaries, it is likely that a 0B/ |8B-activin homodimer is also produced, particularly by granulosa cells in small antral follicles (10). An endocrine function for ovarian activin(s) has been proposed, since it stimulates FSH release by pituitary cells in vitro (11, 12). However, an intraovarian function(s) for the protein is also likely, supported by evidence that activin-A purified from porcine follicular fluid inhibits LH-stimulated androgen production by rat ovarian thecal/interstitial tissues in vitro (13). To determine whether activin might regulate thecal function in the adult human ovary, we tested the effect of recombinant human activin-A on steroid production by primary monolayer cultures of human thecal cells. Received November 1,1990. Address all correspondence and requests for reprints to: Dr. Stephen G. Hillier, Reproductive Endocrinology Laboratory, University of Edinburgh, Centre for Reproductive Biology, Edinburgh, EH3 9EW Scotland, United Kingdom. * This work was supported by MRC Programme Grant PG8929583 (to S.G.H. and D.T.B.).
Here, we report that when thecal cell steroid synthesis is induced by treatment with LH and (or) insulin-like growth factor-I (IGF-I), the presence of femtomolar concentrations of activin-A in serum-free culture medium causes a selective inhibition of androgen [androstenedione and dehydroepiandrosterone (DHA)] production. These data provide direct support for the concept of activin (s) as a paracrine modulator of androgen synthesis in the human ovary. Subjects and Methods Patients Ovarian tissue was obtained from six women who underwent surgery (hysterectomy with uni- or bilateral oophorectomy) to treat nonmalignant gynecological disease (severe uterine fibroids, endometriosis, and/or heavy and painful menstrual bleeding; Table 1). The study had the approval of the local ethics committee. Isolation of thecal cells
Resected ovarian tissue was placed in ice-cold physiological saline, transported to the laboratory, and transferred to culture medium, which was also used for follicular dissection: medium 199 containing Earle's salts, 25 mM HEPES buffer, extra (2 mM) L-glutamine, 50 IU/mL penicillin, 50 ng/mL streptomycin (all from Gibco Ltd., Paisley, Renfrewshire, United Kingdom), and 0.1% (wt/vol) BSA (from ICN Biomedicals, High Wy-
1206
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ACTIVIN AND THECAL ANDROGEN SYNTHESIS combe, Bucks, United Kingdom). Dissection of follicles (Table 1) and isolation of thecae were carried out with visualization using zoom stereomicroscopic optics, as described previously (14). Only thecae from apparently healthy follicles were studied, i.e. yielding straw-colored follicular fluid, granulosa cells, and (usually) a cumulus-enclosed oocyte. After scraping all visible granulosa cells from the lamina basalis, hemisected thecae from individual or pooled follicles were rinsed in fresh medium and transferred to more of the same medium (2 mL) containing 0.1% (wt/vol) collagenase type II from Cl. histolyticum and 0.01% (wt/vol) deoxyribonuclease-I (both from Sigma Chemical Co., Poole, Dorset, United Kingdom). Complete dispersal into a cellular suspension was achieved by incubating the tissue in the enzyme solution for two successive 15-min periods at 37 C in a shaking water bath, with repeated pipetting after each incubation. The cells were sedimented by centrifugation (5 min at 800 x g), resuspended in fresh culture medium containing 5.0% (vol/vol) donor calf serum (Gibco), and counted in a hemocytometer. Cell viability, determined by staining with 0.4% (wt/vol) trypan blue, was consistently more than 90%. Thecal cell culture Twenty-four-well plastic dishes (Linbro Space Savers from Flow Laboratories, Rickmansworth, Herts, United Kingdom) were inoculated with replicate 500-/zL portions of thecal cell suspension (2.0-5.0 X 104 viable cells/well) in culture medium containing 5% donor calf serum. Preincubation in serum-containing medium to allow cell anchorage and recovery from the enzymatic dispersal procedure was carried out for 24 h at 37 C in a humidified tissue culture incubator gassed with 95% air5% CO2. After removal of serum-containing medium and washing with 1 mL prewarmed (37 C) Dulbecco's phosphate-buffered saline, the cultures received 500 nL serum-free medium containing human LH (LER-1972: donated by Dr. L. E. Reichert, Jr.), recombinant human IGF-I (CGP-35'-126, donated by Drs. H. H. Peter and K. Scheibli), and/or recombinant human activin-A (15). Incubation at 37 C was then performed for 4 days, with a medium change on day 2. Media collected on days TABLE 1. Details of patients and ovarian follicles studied Exp no.
Patient
Age (yr)
LMP (day)
No. and diameter (mm) of follicles studied
I II
A A
45 45
13 13
III IV V
B B C
31 31 44
°
VI VII
VIII
D D E
IX
F
1(14) 1(18)
1207
2 and 4 of culture were stored frozen at —20 C until assayed for steroid content, as described below. Assay of steroids in thecal cell culture medium Steroids in unextracted culture medium were measured individually by RIA using standard procedures, as previously described (14). The androstendione assay used a rabbit antiserum to androst-4-ene-3,17-dione-7a-carboxyethyl thioether conjugated to BSA. Major cross-reactions were: androstendione, 100%; androsterone, 46.3%; 5a-androstene-3,17-dione, 50%; testosterone, 37%; and less than 0.5% for all other steroids tested. The DHA assay used sheep anti-DHA-3-hemisuccinylovalbumin purchased from Steranti Research Ltd. (St. Albans, Herts, United Kingdom; product code A005). Major crossreactions were: DHA, 100%; DHA sulfate, 141%; 5a-androstane-3,17-dione, 7.9%; testosterone, 4.4%; androstenedione, 3.3%; and 0.5% or less for all other steroids tested. The progesterone antiserum was sheep antiprogesterone-lla-hemisuccinyl-BSA with major cross-reactants of: progesterone, 100%, lla-hydroxyprogestrone, 35.5%; deoxycorticosterone, 25.3%; 11-ketoprogesterone, 15.8%; and 20/3-hydroxyprogesterone, 12%. The sensitivity (90% B/B.) of each assay was approximately 0.05 pmol steroid/assay tube. The inter- and intraassay precisions for each steroid were less than 15%. Steroid production rates are expressed as picomoles of steroid per 1000 cells/ 48 h, related to the number of viable cells used to inoculate each culture well. Measurement of tritiated thymidine untake by cultured thecal cell monolayers Incorporation of tritiated thymidine into cultured thecal cell monolayers was determined using a modification of the method described by May et al. (17). Briefly, after the collection of spent culture medium on day 4, each monolayer was incubated for a further 18 h in 500 /xL serum-free culture medium containing 2 /^Ci [me£fry/-3H]thymidine (1.5 Ci/mmol; Amersham International, Aylesbury, Bucks. United Kingdom). The monolayer was then washed twice with 0.1 M phosphate-buffered saline and incubated for 20 min at 4 C in 1.0 mL 5% (wt/vol) trichloroacetic acid. The fixed monolayer was then washed once with 5% trichloroacetic acid and twice with methanol. The cells were dissolved by incubation for 5 min at room temperature in 500 tih 0.5 M NaOH. After neutralization by the addition of 500 nL 0.5 M HC1, the solution was taken for liquid scintillation counting. Statistics
8
20 (4-5)* 6 (6-7)* 3 (2-5)*
Analysis of variance with the Neuman-Keuls or paired Student's t test was used to analyze differences between experimental and control observations. Differences assigned a P value less than 0.05 were regarded as statistically significant.
36 36 57
8 8 27
1(7) 1(8) 16 (3-8)*
Results
38
27
17 (3-5)*
Stimulation of thecal cell androstenedione production by LH and IGF-I
LMP, Last menstrual period. " Acyclic. * Pooled.
a
Maximal rates of androgen production by thecal cell cultures occurred in medium containing LH plus IGF-I.
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HILLIER ET AL.
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If neither LH nor IGF-I was present, the average rate of androstenedione accumulation in the medium was 0.064 ± 0.020 pmol/1000 cells-48 h (mean ± SE; n = 7) on day 2, falling to 0.024 ± 0.008 pmol/1000 cells-48 h on day 4. The presence of LH (1-100 ng/mL) alone did not prevent this decline, whereas IGF-I (>30 ng/mL) reversed it completely (Fig. 1, left panel). IGF-I also increased thecal cell responsiveness to LH (Fig. 1, right panel). Thus, whereas LH (10 ng/mL) alone increased androstenedione accumulation on day 4 of culture by less than a third, the response was about 15 times greater if IGF-I (30 or 100 ng/mL) was also present (Fig. 2).
JCE & M • 1991 Vol 72 • No 6
Inhibition of thecal cell androstenedione production by activin-A Activin-A did not modify basal androgen production. However, the presence of activin-A in culture medium dose-dependently inhibited the production of androstenedione in response to treatment with LH and/or IGF-I. Fifty percent maximal inhibition occurred at an activin-A concentration approaching 10 ng/mL (Fig. 3). Inhibitory effects of activin-A (10 ng/mL) on IGF-I- and LH- plus IGF-I-stimulated androstenedione production from five individual experiments are summarized in Fig. 4.
IGF-I + LH
IGF-I alone 0.09 n
Selective action of activin-A on thecal cell steroid production
0
10
100
1000
0
10
100 1000
IGF-I concentration (ng/ml)
FIG. 1. Dose-related stimulatory effect of IGF-I on androstenedione production by human thecal cell cultures. Left panel, In the absence of LH; right panel, in the presence of LH (10 ng/mL). Thecal cell monolayers (5.0 x 104 viable wells/culture well) pooled from 20 follicles between 4-5 mm in diameter (Exp III) were incubated for 4 days in serum-free culture medium containing IGF-I with and without LH. Androstenedione accumulation in medium collected on days 2 and 4 of culture is shown. Data are the mean ± SE (n = 4). Asterisks denote significant stimulation by IGF-I relative to the corresponding treatment without IGF-I (*, P < 0.05; • * , P < 0.01).
The increased production of androstenedione due to combined treatment with LH and IGF-I was paralleled by increased production of DHA and progesterone. However, whereas only 1 ng/mL activin-A significantly reduced the production of androstenedione and DHA by at least 20%, 100 ng/mL of the protein were required to inhibit progesterone production by a similar degree (Fig. 5). Thus, in five of five experiments in which it caused an average 50% reduction in androstenedione production, activin-A (10 ng/mL) had no significant effect on LH- plus IGF-I-stimulated progesterone production (P = 0.098, by Student's paired t test). 0.41
0.30.4 n x: oo
il
0.3-
0.2-
2 o
0.2-
0.1"
0.1 0" 0 Control
LH
IGF-I
LH + IGF-I
Treatment
FIG. 2. Interaction between IGF-I and LH on androstenedione production by human thecal cell cultures: composite data from Exp IIIIX. Thecal cell monolayers were incubated for 4 days in serum-free culture medium without (control) and with LH (10 ng/mL), IGF-I (30 or 100 ng/mL), or IGF-I plus LH. Androstenedione accumulation is shown in medium collected on day 4. Data are the mean ± SE. Asterisks denote significant differences between the group means indicated (*, P < 0.05; • * , P < 0.001).
1
10
100
Activin-A concentration (ng/ml)
FIG. 3. Dose-dependent inhibitory effect of activin-A on androgen production by human thecal cell cultures. Thecal cell monolayers (2.0 X 10" viable wells/culture well) from a single follicle (7 mm in diameter; Exp VI) were incubated for 4 days in serum-free culture medium containing IGF-I (100 ng/mL) alone, LH (10 ng/mL) alone, or IGF plus LH, with and without activin-A at the concentrations indicated. Androstenedione accumulation is shown in medium collected on day 4. Data are the mean ± SE (n = 3). Asterisks denote statistically significant inhibition due to activin-A (*, P < 0.05; • * , P < 0.01).
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ACTIVIN AND THECAL ANDROGEN SYNTHESIS 20 000
IGF 1 n
'
1209 IGF-I + LH
alone
T 15 000 -
Ep
10 000 -
5 000 -
Control
LH
IGF-I
LH + IGF-I 300
Treatment
FIG. 4. Inhibition by activin-A of LH-, IGF-I-, and LH- plus IGF-Istimulated androstenedione production by human thecal cell cultures: composite data from Exp V-IX. Thecal cells were incubated for 4 days in serum-free culture medium in the absence and presence of activinA (10 ng/mL) without (control) and with LH (10 ng/mL), IGF-I (30 or 100 ng/mL), or IGF-I plus LH. Androstenedione accumulation is shown in medium collected on day 4. Data are the mean ± SE. Asterisks denote significant inhibition by activin-A (•, P < 0.05; **, P < 0.001). DHA
Androstenedione
0.4 -i
Progesterone
0.3"
Z.Z
A
0.2H
i| 2 o 0.1 •
0
1
10
100
0
1
10
100
0
1
10
100
Activin-A concentration (ng/ml)
FlG. 5. Relative inhibitory effect of activin-A on production of androstenedione (left panel), DHA {center panel), and progesterone (left panel) by human thecal cell cultures. Thecal cells (2.0 x 104 viable wells/culture well) from a single follicle (7 mm in diameter; Exp VI) were incubated for 4 days in serum-free culture medium containing IGF-I (100 ng/mL) plus LH (10 ng/mL), with and without activin-A at the concentrations indicated. Steroid accumulation is shown in medium collected on day 4. Corresponding basal rates of steroid accumulation (i.e. in the absence of LH and IGF-I) were 0.006 ± 0.002 (androstenedione), 0.012 ± 0.003 (DHA) and 0.006 ± 0.001 (progesterone) pmol steroid/1000 cells-48 h. Data are the mean ± SE (n = 3). Asterisks denote statistically significant inhibition due to activin-A (*, P < 0.05; • * , P < 0.01).
No effect of activin-A on IGF-I-stimulated uptake of tritiated thymidine by thecal cell monolayers Treatment with IGF-I (30-300 ng/mL) elicited an approximately 20-fold increase in tritiated thymidine uptake by cultured thecal cells, regardless of the presence of LH (10 ng/mL). This response was not significantly altered by the presence of activin-A at a dose (10 ng/ mL) that was sufficient to cause about 50% inhibition of androstenedione production (Fig. 6).
Discussion These results reveal a potent and selective inhibitory action of activin-A on androgen production by human
0
30
100
300
IGF-I concentration (ng/ml)
FIG. 6. Lack of effect of activin-A on IGF-I-stimulated uptake of tritiated thymidine by human thecal cells. Thecal cell monolayers (2.0 x 104 viable wells/culture well) pooled from 16 follicles between 3-8 mm in diameter (Exp VIII) were incubated for 4 days in serum-free culture medium containing IGF-I at the concentrations shown in the absence (left panel) or presence (right panel) of LH (10 ng/mL), with and without activin-A (10 ng/mL). Uptake of tritiated thymidine was determined as described in Subjects and Methods. Data are the mean ± SE (n = 3).
thecal cells in vitro. Synthesis of A4 (androstenedione) and A5 (DHA) androgens was suppressed by femtomolar concentrations of the protein, consistent with a physiologically significant paracrine mode of action. Additional experiments are needed to determine the mechanism of action involved. However, preferential inhibition of Ci9 steroid production relative to progesterone production points to an action at the level of 17-hydroxylase/Ci7_2o lyase, the rate-limiting enzyme in thecal androgen synthesis. Previous studies of the effect of activin on steroidogenesis in ovarian cells have been confined to experimental animal tissues. In rat granulosa cell cultures, activin-A purified from bovine follicular fluid enhanced FSH-stimulated aromatase activity, but inhibited progesterone production (18). Augmentation of aromatase activity by recombinant activin-A was also observed using cultured granulosa cells from a nonhuman primate (common marmoset, Callithrix jacchus) (19). The concept that activin produced by granulosa cells might function as a paracrine modulator of thecal androgen synthesis initially arose from in vitro experiments using prepubertal rat tissues (13). However, quantitative and qualitative changes in the pattern of ovarian androgen synthesis precede the onset of puberty in rats (20), raising the question of whether activin inhibition of androgen synthesis has any relevance to the situation in adult ovaries. The present data are novel in showing not only that activin inhibition of thecal androgen synthesis extends to humans, but also that it persists into adulthood, at least in women. It should be noted, however, that this study of activin-A action was confined to thecal cells from follicles 8 mm or less in diameter (Exp V-IX, Table 1). Further studies
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HILLIER ET AL.
on more mature follicles are necessary to determine how the protein affects thecal function at later stages of preovulatory development in the human ovary. The use of serum-free conditions of culture highlighted a striking ability of IGF-I to enhance basal and LHresponsive androgen synthesis in thecal cell monolayers. Previous studies have shown that treatment with insulin or IGF-I enhances basal and LH-stimulated synthesis of androgen in cultured thecal/interstitial cells from rat ovaries (21-23) as well as stromal tissue explants from human ovaries (24, 25). The present study complements and extends these earlier findings, showing IGF-I stimulation of thecal cell DNA synthesis as well as androgen synthesis. An intrafollicular paracrine function(s) for the growth factor is suggested by the evidence that IGF receptors are present on human thecal cells (26), and granulosa cells are intrafollicular sites of IGF-I mRNA expression (27) and IGF-I synthesis regulated by FSH (28, 29). Despite its marked stimulatory effect on thymidine uptake, IGF-I does not measurably increase thecal cell number under the conditions and duration of culture used here (Yong, E. L., and S. G. Hillier, unpublished observation). Nevertheless, we interpret these data to suggest that IGF-I of granulosa cell origin has the potential to exert positive paracrine control over the growth of the theca interna as well as modulate its steroidogenic function in vivo. The evidence that activinA overrides IGF-I-stimulated thecal androgen production without suppressing thecal DNA synthesis further indicates that a complex functional interaction is likely to exist between these two factors in the follicular paracrine system. Thecal androgens play fundamental roles in the ovary, serving as estrogen precursors and having local regulatory actions (20). Studies of follicular fluid levels of androgen and estrogen in relation to granulosa cell aromatase activity indicate that the capacity of the theca interna to generate aromatase substrate (androstenedione) increases hand in hand with granulosa cell aromatase activity in the human preovulatory follicle (14). It is of interest, therefore, that in primate ovaries, inhibin/ activin 0B-subunit mRNA is expressed in the greatest amounts by granulosa cells in small antral follicles, decreasing to undetectable levels during preovulatory follicular development (10). Activin-A and -B exhibit similar actions in pituitary and erythroid differentiation assays in vitro (30). Presumably, therefore, they also share the inhibitory action on thecal androgen synthesis that has been described here, in which case a development-related reduction in granulosa cell activin synthesis might constitute a paracrine mechanism for positively regulating thecal androgen synthesis in the human preovulatory follicle.
JCE & M • 1991 Vol 72 • No 6
Acknowledgments We are grateful to Prof. L. E. Reichert, Jr., of the Department of Biochemistry, Albany Medical College (Albany, NY), for the supply of LH, to Drs. H. H. Peter and K. Scheibli, Ciba-Geigy Ltd. (Basel, Switzerland) for the IGF-I, and to Mr. W. A. Ferguson for the steroid assays. Note added in proof: Subsequently, we have shown that recombinant inhibin-A stimulates basal and LH- plus IGF-Iresponsive androgen synthesis and overcomes the inhibitory action of activin-A in human thecal cell cultures (31).
References 1. Ying S-Y. Inhibins, activins, and follistatins: gonadal proteins modulating the secretion of follicle-stimulating hormone. Endocr Rev. 1988;9:267-93. 2. Vale W, Rivier C, Hsueh A, et al. Chemical and biological characterization of the inhibin family of protein hormones. Recent Prog Horm Res. 1988;44:l-30. 3. Mason AJ, Hayflick JS, Ling N, et al. Complementary DNA sequences of ovarian follicular fluid inhibin show precursor structure and homology with transforming growth factor-/?. Nature. 1985;318:659-63. 4. Mason AJ, Niall HD, Seeburg PH. Structure of two human ovarian inhibins. Biochem Biophys Res Commun. 1986;35:957-64. 5. Forage RG, Ring JW, Brown RW, et al. Cloning and sequence analysis of cDNA species coding for the two subunits of inhibin from bovine follicular fluid. Proc Natl Acad Sci USA. 1986;83:30915. 6. Esch FS, Shimasaki S, Cooksey K, et al. Complementary deoxyribonucleic acid (cDNA) cloning and DNA sequence analysis of rat ovarian inhibins. Mol Endocrinol. 1987;l:388-96. 7. Woodruff T, Meunier H, Jones PB, Hsueh AJW, Mayo KE. Rat inhibin: molecular cloning of a- and /3-subunit complementary deoxyribonucleic acids and expression in the ovary. Mol Endocrinol. 1987;l:561-9. 8. Woodruff TK, D'Agostino JB, Schwartz NB, Mayo KE. Dynamic changes in inhibin messenger RNAs in rat ovarian follicles during the reproductive cycle. Science 1988;239:1296-9. 9. Turner IM, Saunders PTK, Shimasaki S, Hillier SG. Regulation of inhibin subunit gene expression by FSH and estradiol in cultured rat granulosa cells. Endocrinology. 1989;125:2790-2. 10. Schwall RH, Mason AJ, Wilcox JN, Bassett SG, Zeleznik AJ. Localization of inhibin/activin subunit mRNAs within the primate ovary. Mol Endocrinol. 1990;4;75-9. 11. Vale W, Rivier J, Vaughan J, et al. Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid. Nature. 1986;321:776-9. 12. Ling N, Ying S-Y, Ueno N, et al. Pituitary FSH is released by a heterodimer of the 0-subunits from the two forms of inhibin. Nature. 1986;321:779-82. 13. Hsueh AJW, Dahl KD, Vaughan J, et al. Heterodimers and homodimers of inhibin subunits have different paracrine action in the modulation of luteinizing hormone-stimulated androgen biosynthesis. Proc Nat Acad Sci USA. 1987;84:5082-6. 14. Hillier SG, Reichert Jr LER, van Hall EV. Control of preovulatory follicular estrogen biosynthesis in the human ovary. J Clin Endocrinol Metab. 198l;52:847-56. 15. Schwall RH, Nikolocs K, Szonyi E, Mason AJ. Recombinant expression and characterization of human activin A. Mol Endocrinol. 1989;2:1237-42. 16. McNatty KP, Hillier SG, van den Boogard AMJ, Trimbos-Kemper TCM, Reichert Jr LE, van Hall EV. Follicular development during the luteal phase of the human menstrual cycle. J Clin Endocrinol Metab. 1981:56:1022-31. 17. May JV, Frost JP, Schomberg DW. Differential effects of epidermal growth factor, somatomedin-C/insulin-like growth factor 1, and
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ACTIVIN AND THECAL ANDROGEN SYNTHESIS
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transforming growth factor-/? on porcine granulosa cell deoxyribonucleic acid synthesis and cell proliferation. Endocrinology. 1988;123:168-79. Hutchison LA, Findlay JK, de Vos FL, Robertson DM. Effects of bovine inhibin, transforming growth factor-/? and bovine activinA on granulosa cell differentiation. Biochem Biophys Res Commun. 1987;146:1405-12. Hillier SG. Effects of activin on oestrogen synthesis in primate granulosa cells [Abstract]. J Endocrinol. 1990;127(Suppl):67. Hillier SG. Sex steroid metabolism and follicular development in the ovaries. Oxf Rev Reprod Biol. 1985;7:168-222. Erickson GF, Magoffin DA. Dyer CA, Hofeditz C. The ovarian androgen producing cells: a review of structure/function relationships. Endocr Rev. 1985;6:371-99. Cara JF, Rosenfield RL. Insulin-like growth factor I and insulin potentiate luteinizing hormone-induced androgen biosynthesis by rat ovarian theca-intersitial cells. Endocrinology. 1988;123:733-9. Hernandez ER, Resnick CE, Svoboda ME, Van Wyk JJ, Payne DW, Adashi EY. Somatomedin-C/insulin-like growth factor I as an enhancer of androgen biosynthesis by cultured rat ovarian cells. Endocrinology. 1988;122:1603-12. Barbieri RL, Makris A, Ryan KJ. Insulin stimulates androgen accumulation in incubations of human ovarian stroma and theca. Obstet Gynecol. 1984;64(Suppl):73S-80S. Barbieri RL, Makris A, Randall RW, Daniels G, Kistner RW, Ryan
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KJ. Insulin stimulates androgen accumulation in incubations of ovarian stroma obtained from women with hyperandrogenism. J Clin Endocrinol Metab. 1986;62:904-10. Poretsky L, Grigorescu F, Seibel M, Moses AC, Flier JS. Distribution and characterization of insulin and insulin-like growth factor receptors in normal human ovary. J Clin Endocrinol Metab. 1985;61:728-34. Oliver JE, Aitman TJ, Powell JF, Wilson CA, Clayton RN. Insulinlike growth factor I gene expression in the rat ovary is confined to the granulosa cells of developing follicles. Endocrinology. 1989;124:2671-79. Hammond JM, Baranao JLS, Skaleris D, Knight AB, Romanus JA, Rechler MM. Production of insulin-like growth factors by ovarian granulosa cells. Endocrinology. 1985;117:2553-5. Adashi EY, Resnick CE, Hernandez ER, Svoboda ME, Van Wyk JJ. Potential relevance of insulin-like growth factor I to ovarian physiology: from basic science to clinical application. Semin Reprod. 1989;7:794-9. Mason AJ, Schmelzer C, Schwall RH. Molecular and physiological studies on inhibin and activin. In: Josso N, ed. Development and function of the reproductive organs. Rome: Ares-Serono Symposia; 1989;3:153-7. Hillier SG, Yong EL, Illingworth PJ, Baird DT, Schwall RH, Mason AJ. Effect of recombinant inhibin on androgen synthesis in cultured human thecal cells. Mol Cell Endocrinol. 1991;75:R1R6.
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