Effects Ects of Androgen on Progesterone Secretion From

Endocrinol. Japon. 1990, 37(1), 113-120

Effectsects of Androgen on Progesterone Secretion from Granulosa Cells Obtained from Antral Follicles of Rats

CHIKA KADOTA*, MITUAKI OHTA** AND MICHIO TAKAHASHI

Department of Veterinary Physiology, Veterinary Medical Science, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan

Abstract

The effects of testosterone (T) on the secretion of progesterone (P) by ovarian

granulosa cells obtained from immature rats pre-treated with pregnant mare's serum gonadotropin were examined in vitro. T (10 nM-10ƒÊM) enhanced both

basal and FSH- or cAMP-stimulated secretion of P in a dose-dependent manner.

Furthermore, T augmented FSH-stimulated cAMP production. The biphasic secretory pattern of P induced by continuous superfusion of granulosa cells with

FSH was much exaggerated in the cultures supplemented with T. A stimulatory effect of T on secretion of P was observed only in the medium that contained serum. T affected neither the basal nor the FSH-stimulated secretion on 20a-

dihydroprogesterone. Androsterone, a non-aromatizable and low-potency andro-

gen, at a similar concentration as T mimicked the effects of T on the secretion of progesterone. These results indicate that androgen stimulates mature

granulosa cells to enhance the secretion of P. This androgen action extends either up- or down-stream of cAMP in the process of steroidogenesis.

Androgen combined with FSH or by pig (Schomberg et al., 1976)]. In contrast, itself stimulated the production of progeste- an inhibitory effect of T on FSH-stimulated rone (P) by cultured granulosa cells [undif- production of P by cultured porcine granulosa ferentiated granulosa cells from immature, cells obtained from medium-size pre-pubertal hypophysectomized, estrogen-primed (H-E) follicles has been reported (Lishinsky et al., rats (Armstrong and Dorrington, 1976; Nim- 1983). The inhibitory effect of T was mim- rod and Lindner, 1976; Hillier et al., 1977), icked by androstenedione but not by either and differentiated granulosa cells from the dihydrotestosterone or androsterone (Evans et al., 1984). Using granulosa cells obtained Received October 12, 1989 from H-E rats the mechanism of the stimula- Present Address;* Institute for Developmental tory effect of androgens on the production Research, Aichi Prefectural Colony, Kasugai, of progestin has been extensively examined, Aichi 480-03. ** Department of Veterinary Physiology, College of Agriculture, University of and several lines of evidence indicate the Osaka Prefecture, Sakai, Osaka 591. possibility that androgens stimulate the ac- tivities of enzymes involved in the biosynthe- Requests for reprints: MICHIO TAKAHASHI, sis of P (Nimrod, 1989; Welsh et al., 1972). Department of Veterinary Physiology, Veteri- nary Medical Science, The University of Tokyo, Androgens are present in the follicular 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan. fiuid at high concentrations throughout the Endocrinol. Japon. 114 KADOTA et al. February 1990

estrous cycle (McNatty et al., 1975). But the Progesterone (P) and 20ƒ¿-dihydroptogeste- effect of the androgen on the granulosa cells rone (20ƒ¿-OH-P) were measured by radioimmu- does not seem to be uniform throughout noassay, using respective specific antibodies folliculogenesis. In the present study, we used raised in rabbits in our laboratory (cross-reac- tivity with testosterone and androsterone in each fully developed granulosa cells obtained from antibody was <0.1%). HCl-extracts of scraped antral follicles of immature rats pretreated cells were boiled and centrifuged. The super-

with pregnant mare's serum gonadotropin natant were acetylated (Kadota et al., 1988). (PMSG) and examined the effects of androgens either aromatizable or nonaromatizable, on the secretion of P from these granulosa cells.

Materials and Methods

Reagents NIADDK-rat-FSH-B-2 (3x NIH-FSH-S1 U/ mg) was a generous gift from the National Institute of Arthritis, Diabetes and Digestive Disease and Kidney, U.S.A. and 8-bromo- adenosine-3',5'-cyclic monophosphate (8Br- cAMP), testosterone and androsterone were pur- chased from Sigma (St. Louis, Mo).

Animals Immature (22-to 24-day-old) female rats of the Wistar-Imamichi strain (Imamichi Institute for Animal Reproduction, Ohmiya, Japan) were injected subcutaneously with 40 IU PMSG (Sankyo Co., LTD, Tokyo, Japan). The animals were killed by cervical dislocation 48 hr after the injection.

Granulosa cell culture

The granulosa cells were collected and cultured as described by Kadota et al.(1988). Briefly, 2.5•~105 viable cells (except in the case of the studies with cAMP where 1•~105 viable cells were used) were pre-cultured for 24 hr in plastic culture dishes (Falcon Plastics, Los Angeles, CA) or in siliconized glass dishes that Fig. 1. Effects of increasing concentrations of contained microcarrier beads (Pharmacia Fine testosterone (T) on the secretion of progeste- Chemicals, Uppsala, Sweden) in Dulbecco's rone (P). Granulosa cells which had been modified essential medium (Gibco, Grand Is- pre-cultured for 24 hr with or without T were land, NY), supplemented with 2.5 g/1 NaHCO3, incubated for 30 min in the presence of 100 100 U/ml penicillin, 100ƒÊg/ml streptomycin ng/ml FSH (•£) or 1mg/ml 8br-cAMP sulphate, and 10% v/v fetal calf serum (Gibco, (•œ), or in the absence of the stimulators

Grand Island, NY), in the presence or absence (•›). Data points represent means•}SE of of androgen. Secretion of progestin by pre- results from triplicate cultures. Similar results cultured granulosa cells was studied under static were obtained in 2 separate experiments. conditions or using the superfusion system Error bars not shown are contained within

(Kadota et al., 1988). the symbols. Vol.37, No.1 EFFECTS OF ANDROGEN ON GRANULOSA CELLS 115

The accumulation of intracellular cAMP was lation by FSH with 100 nM T. Thus, the assayed in acetylated samples using an[I125]- secretion of P stimulated by 8Br-cAMP (1 cAMP RIA kit (New England Nuclear, Boston, mg/ml) was not affected by increasing con- Ma). centrations of T up to 1,uM, but was aug- Statistical analysis mented by 10 pu,M T. The statistical analysis was performed by Student's t-test. Effects of testosterone on the patterns of progesterone secretion from superfused granulosa cells Results As shown in Fig. 2, P was secreted from superfused granulosa cells in a biphasic man- Effects of increasing concentrations ner with continuous presence of FSH (100 of testosterone on the secretion of ng/ml). The fluctuation in P concentrations progesterone without T was much smaller than that with T. Isolated granulosa cells were pre-cultured Two sequential 10 min pulses of FSH (100 for 24 hr without T or with various concen- ng/ml) induced discrete secretions of P (Fig. trations of T. The granulosa cells were then 3). A self-priming effect of FSH on the incubated with various additives for 30 min secretions of P was observed in the cultures in a fresh medium without T. As shown in with T, such that the second pulse of FSH Fig. 1, T stimulated both the basal and the induced a higher rate of secretion than the FSH (100 ng/ml)-stimulated secretion of P first pulse.However,in the cultures without in a dose-dependent manner. P levels after T, the self-priming effect of FSH on the secre- stimulation by 8Br-cAMP (1 mg/ml) without tion of P was not apparent. T were almost equivalent to those after stimu-

Fig. 2. Secretion of P(ng/ fraction, mean•}SE; n=3 columns) by superfused granulosa cells (2.5•~105 viable cells/column) plot- ted against time (min). The granulosa cells which had been pre-cultured for 24 hr with microcarrier beads in the presence (•£) or absence (•›) of 100 nM T were packed in a column. Two columns were superfused simultaneously. The flow rate was 0.1ml/min. The influent was pumped into the column with a peri- staltic pump. The efflu- ent was collected on a fraction collector in tubes changed at intervals of 10 min . Continuous superfusion with 100 ng/ml FSH was performed. Endocrinol. Japon. 116 KADOTA et al. February 1990

Fig. 3. Secretion of P

stimulated by two se-

quential 10-min pulses of 100 ng/ml FSH. Data

are means•} SE; n=3 columns. For further desig- nation see legend to Fig.

Presence of FCS for the expression of the effect by testosterone Isolated granulosa cells were cultured for 24 hr in the medium supplemented with or without FCS in combination with 100 nM T or without T. These pre-cultured granulosa cells were then incubated for 30 min in a fresh medium for P measurement. As shown in Fig. 4, the stimulatory effect of T on the basal secretion of P was observed only in the cultures grown in the medium that contained FCS (10% v/v).

Effect of testosterone on FSH-stimulated cAMP production Isolated granulosa cells were pre-cultured for 24 hr with or without T (1ƒÊM). Then, intracellular cAMP after incubation with FSH

(100 ng/ml) for 30 min in a fresh medium Fig. 4. Involvement of factor(s) in serum in was measured. As shown in Fig. 5, FSH- the effect of T on the secretion of P. Granu- stimulated cAMP production was significantly losa cells, which had been pre-cultured for more abundant in the cells pre-cultured with 24 hr with or without FCS (10% v/v) in T. combination with T (100 nM) or without T,

were incubated for 30 min in fresh medium. Values are means•}SE of results of triplicate Effects of androsterone, a non-aroma- cultures. tizable and low-potency androgen, on the secretion of progestin Isolated granulosa cells were pre-cultured Vol. 37, No.1 EFFECTS OF ANDROGEN ON GRANULOSA CELLS 117 for 24 hr with or without androgen (T or Fig.6a, stimulatory effects of androsterone androsterone). The granulosa cells were then (100nM) on both the basal and the FSH- incubated with or without FSH (100ng/ml) stimulated secretion of P were observed as for 30 min in a fresh medium. As shown in was the stimulatory effect of T (100nM).

base FSH

Fig.6. Effects of androsterone (A), a nonaromatizable and low-potency androgen, on

Fig.5. Effects of T on production of cAMP the secretion of P (a) and 20a-dihydropro-

stimulated by FSH. The granulosa cells which gesterone (20a-OH-P; b). Granulosa cells had been pre-cultured for 24 hr with (shadow which had been pre-cultured for 24 hr with bar) or without (open bar) T (1ƒÊM) were or without androgen (100nM) were incu-

incubated for 30 min in the presence of FSH bated for 30 min in the presence or absence (100ng/ml). Values are means•}SE of re- of FSH (100ng/ml). Values are means•}SE sults of triplicate cultures. Similar results were of results of triplicate cultures. Similar results obtained in 2 separate experiments. were obtained in 2 separate experiments. Endocrinol. Japon. 118 KADOTA et al. February 1990

Androgen, both T and androsterone had no priming effect of FSH on the secretion of P effect on either the basal or the FSH stimu- was observed in the cultures with andro- lated secretion of 20a-OH-P (Fig. 6b). sterone as well as in those with T. Granulosa cells pre-cultured without androgen, or with 100nM T or 100nM androsterone were given two sequential pulses of Discussion FSH (100ng/ml, each for 20 min) at an interval of 80 min. The incubation medium was exchanged every 20 min. The levels of The stimulatory effects of androgen on P induced by the first or the second exposure the production of progesterone (P) in cultured to FSH are shown in Fig. 7. The self- granulosa cells, in particular those obtained from H-E rats, has been demonstrated: androgen acts either synergistically with FSH or by itself to stimulate production of P (Arm- strong and Dorrington, 1976; Nimrod and Lindner, 1976; Lucky et al., 1977; Nimrod, 1981;. Welsh et al., 1982). In the present study, using fully matured granulosa cells obtained from immature, PMSG-treated rats, the effects of androgen in the secretion of P were re-evaluated. T stimulated the basal secretion of P in a dose-dependent manner (Fig. 1). This effect of T appears to depend factor(s) in the serum Fig.4). Stimulatory effects of androgen on the uptake of cholesterol (Nimrod, 1981) and utilization of lipoproteins (Schreiber et al., 1983) by granulosa cells have been reported. The serum factor required for the effect of T may be a lipoprotein that binds cholesterol which contribute to increasing the cholesterol pool in the granulosa cells. The stimulatory effect of T on the secretion of P was more apparent in terms of chronological fluctuations in the secretion of P after FSH stimulation. The biphasic pattern of secretion of P stimulated by continuous superfusion with FSH was much enlarged Fig.7. The self-priming effect of FSH on the secretion of P by granulosa cells cultured with at its first phase (Fig. 2). The self-priming androsterone (A). Granulosa cells which had effect of a pulsatile application of FSH on been pre-cultured without androgen (A) or the secretion of P was observable only if the with 100nM T (e) or with 100nM A (•›) cells were cultured with T (Fig. 3). were given two sequential pulses of FSH Although estrogen has a stimulatory effect (100ng/ml) for 20 min at an interval of on the secretion of P by differentiated porcine 80 min. The incubation medium was exchanged every 20 min. Data points are the granulosa cells (Veldhuis et al., 1981 and means•}SE of results of triplicate cultures. 1982), the stimulatory effect of T on the Error bars not shown are contained within secretion of P by matured rat granulosa cells the symbols. is not mediated by the formation of estrogen Vol. 37, No.1 EFFECTS OF ANDROGEN ON GRANULOSA CELLS 119 from T because androsterone, a non-aroma- 1976; Nimrod, 1977b). These results suggest tizable androgen, could reproduce the effect that when immature granulosa cells are used, of T at the same concentration, (Figs. 6 and the effect of androgen on the secretion of P 7). Androsterone, which is known as a low- is expressed distally to the effect on the pro- potency androgen cannot be converted to duction of cAMP. It is suggested that T 5a-dihydrotestosterone (DHT). Thus, the could stimulate P secretion from granulosa formation of DHT from T is not critical for cells independently of cAMP production and the action of T in the granulosa cells unlike irrespectively of cytodifferentiation of granu- that in the prostate and testis. In granulosa losa cells, and that an alternative pathway cells obtained from H-E rats, the A-ring seemed to be acquired with the maturation reduced C-19 steroids were much less active of granulosa cells, where T as well as andro- than •¢4-3 Keto androgens in terms of the sterone could act at a level proximal to the stimulation of production of progestin (Nim- production of cAMP. rod, 1977a). The metabolic route for this The present study demonstrated that T reduction may be operative in the regulation stimulates production of P by granulosa cells of steroidogenesis in undifferentiated granu- obtained from PMSG-treated rats. Androgens losa cells since the ovaries of immature rats are present at very high concentrations in contain a 5a-reductase (Eckstein and Nimrod, follicular fluid (Short, 1964; McNatty et al., 1977). As shown in Fig. 6, the presence of 1975), and are produced by the thecal and androgen increased the ratio of P to 20a- interstitial cells of the ovary (Louvet et al., OH-P during both basal secretion (ratios in 1975; Makris and Ryan, 1975). Thecal cell control cultures, 0.20•}0.01; in cultures with may generate T not only as a substrate for androsterone, 0.27•}0.02; in cultures with T, the aromatase enzyme in granulosa cells but 0.29•}0.02) and FSH-stimulated secretion also an effector of the biosynthesis of P which

(ratios in control cultures 0.30•}0.02; in in turn may serve as a substrate for produc- cultures with androsterone, 0.36•}0.00; in tion of androgen in thecal cells in large antral cultures with T, 0.37•}0.03). Thus androgen follicles. Such synergistic interactions between stimulation of P secretion may depend on the granulosa and thecal cells may be important suppression of activity of 20a-hydroxysteroid in effecting the maximal production of estro- dehydrogenase as well as the acceleration of gen by large antral follicles. the side-chain cleavage reaction. Increasing the concentration of T (10nM-1ƒÊM) had no effect on 8Br-cAMP-stimulated secretion Acknowledgment of P, and T, on the other hand, increased the intracellular level of cAMP (Fig. 5). The We are indebted to Dr. Kazutaka Honma enhancement of FSH-stimulated secretion of for his valuable suggestions. P by T, thus, appears to be due to the eleva- tion of not only the basal rate of production of P but also the responsiveness to FSH in References the cAMP production. The results of studies using immature granulosa cells, obtained from Armstrong, D. T. and J. H. Dorrington (1976). H-E rats, however, showed that androgen Androgens augment FSH-induced progeste- had no effect on the binding of FSH, produc- rone secretion by cultured rat granulosa cells. Endocrinology 99, 1411-1414. tion of cAMP or phospho-diesterase activity Eckstein, B. and A. Nimrod (1977). Properties

(Nimrod, 1977b), while androgen augmented of microsomal •¢4-3-ketosteroid 5a-reductase P production stimulated by the administration in immature rat ovary: inhibition by estradiol- of cAMP analogues (Nimrod and Lindner, 17ƒÀ. Biochim. Biophys. Acta. 499, 1-9. Endocrinol. Japon. 120 KADOTA et al. February 1990

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