0013-7227/90/1261-0159$02.00/0 Endocrinology Copyright© 1990 by The Endocrine Society

Vol. 126, No. 1 Printed in U.S.A.

Corticosteroid Regulation of Gonadotropin and Prolactin Secretion in the Rat* DARRELL W. BRANN, CARLA D. PUTNAM, AND VIRENDRA B. MAHESH Department of Physiology and Endocrinology, Medical College of Georgia, Augusta, Georgia 30912-3000

and inhibited LH at higher doses (0.5 and 1.0 mg/kg BW). A single low dose of dexamethasone (0.02 mg/kg BW) was found to significantly release LH. With respect to PRL secretion, a biphasic effect of dexamethasone was observed in that the lowest dose (0.01 mg/kg BW) stimulated PRL release while the highest dose (1.0 mg/kg BW) significantly inhibited PRL release. Triamicolone acetonide and deoxycorticosterone were found to require estrogen priming for their effects on gonadotropin secretion. The findings in this study raise the possibility that the beneficial effects seen with corticosteroids in inducing ovulation in polycystic ovarian syndrome may be due, in part, to their direct effects upon the release of gonadotropins. (Endocrinology 126: 159-166,1990)

ABSTRACT. The purpose of this study was to determine if glucocorticoids had any direct effects on the release of gonadotropin. In estrogen-primed ovariectomized immature rats, triamcinolone acetonide and deoxycorticosterone (1 mg/kg BW) caused a surge in both serum LH and FSH levels. Dexamethasone treatment (0.05 mg/kg BW) resulted in a highly significant selective release of FSH. Cortisol (1 mg/kg BW) suppressed serum FSH levels. A systematic dose-response study showed that triamcinolone acetonide significantly released LH and FSH and suppressed PRL at all doses tested (range, 0.25-4 mg/kg BW). Deoxycorticosterone was not as potent as triamcinolone acetonide and only doses greater than 0.8-1 mg/kg BW significantly released LH and FSH. Dexamethasone selectively released FSH at low doses (0.01, 0.02, 0.05, and 0.1 mg/kg BW)

W

(Brann, D. W., C. D. Putnam, and V. B. Mahesh, unpublished). These findings suggest that other mechanisms may be involved in some patients who respond to corticosteroid therapy. Therefore, the purpose of this study was to examine several natural and synthetic corticosteroids for their ability to regulate gonadotropin secretion in estrogen-primed and nonestrogen primed ovariectomized immature rats. The ability of these compounds to regulate PRL secretion was also assessed.

HILE the effects of estradiol and progesterone upon gonadotropin secretion and ovulation are well documented (1-3), the effects of corticosteroids have not been as extensively studied. Intriguingly, in human polycystic ovarian syndrome there have been numerous reports of improvement in menstrual rhythm and ovulatory activity after corticosteroid therapy (4-6). The suppression of serum androgen levels by corticosteroids resulting in increased gonadotropin secretion, particularly FSH, has been proposed to be a possible cause for the restoration of ovulatory cycles (7-10). However, corticosteroid therapy has also been reported effective in several normoandrogenic patients with anovulatory infertility (4, 11, 12) and no clear explanation has been found. Direct stimulatory effects of corticosteroids on gonadotropin secretion have been demonstrated in vitro in pituitary cell cultures (13, 14). Additionally, we have recently demonstrated that the corticosteroids, triamcinolone acetonide and deoxycorticosterone, can facilitate ovulation in PMSG-primed immature female rats, demonstrating that a hyperandrogenic state is not necessary for facilitation of ovulation by these corticosteroids

Materials and Methods Animal experiments Inhibition of estrogen-induced PRL release. The effects of corticosteroids on estrogen-induced PRL release were studied in a rat model described previously (15). Briefly, immature virus free female Holtzman rats (Madison, WI) were obtained at 26 days of age and bilaterally ovariectomized on the same day under ether anesthesia. They were maintained in air-conditioned rooms with a 14-h light; 10-h dark cycle (lights on at 0500 h; off at 1900 h) and were given water and rat chow ad libitum. At 28 days of age the rats were injected ip with 2 /xg estradiol in 0.2 ml 20% ethanol-saline at 0900 h. Thirteen hours later a second 2-/ng estradiol injection was administered (2200 h). One hour before the second estrogen injection either vehicle or corticosteroids were administered in 0.2 ml 50% ethanolsaline (2100 h). All animals were killed 12 h after the second estradiol injection (1000 h) by decapitation and trunk blood was collected. After clotting for 12 h at 4 C, the blood was

Received August 3, 1989. Address requests for reprints to: Dr. Virendra B. Mahesh, Department of Physiology/Endocrinology, Medical College of Georgia, Augusta, Georgia 30912-3000. * This investigation was supported by Research Grant HD-16688, NICHHD, NIH, U.S. Public Health Service.

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CORTICOSTEROID EFFECTS ON LH, FSH, AND PRL

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centrifuged at 2500 rpm for 30 min at 4 C and serum separated and stored at -20 C for subsequent RIA of PRL. Effect on gonadotropin secretion. The animal model used for the study of the action of corticosteroids on estrogen-induced PRL increase did not appear to be suitable for the study of steroid effect on gonadotropin release. In that model estradiol was injected at 0900 h and 2200 h and the animals were killed at 1000 h the next day. This time in the morning was unsuitable for measuring serum gonadotropin levels, since steroid-induced surges of gonadotropins have been shown to only occur in the afternoon hours. Therefore a new model was devised in which ovariectomized 26-day-old rats received a 2 ng injection of estradiol at 1700 h (day 27) and either vehicle or progesterone at 0900 h (day 28). Unfortunately, progesterone did not stimulate a surge of gonadotropins in this model when measured at 1500 h, 1600 h, and 1800 h (day 28). Therefore a second model was devised in which the length of estrogen priming was extended by 1 day. In this model, immature virus-free female Holtzman rats were obtained at 25 days of age and bilaterally ovariectomized at 26 days of age under ether anesthesia. They were maintained in air-conditioned rooms with a 14-h light, 10-h dark cycle (lights on at 0500 h; off at 1900 h) and were given water and rat chow ad libitum. At 1700 h on 27 and 28 days of age the animals received injections of 2 ng estradiol sc in 0.2 ml 20% ethanol-saline. At 29 days of age (0900 h) the animals were injected with either vehicle or corticosteroids in 0.2 ml 50% ethanol-saline. The animals were killed at 1500 h (29 days of age) and trunk blood collected. After clotting for 12 h at 4 C, the blood was centrifuged at 2500 rpm for 30 min at 4 C and serum separated and stored at -20 C for subsequent RIA of LH, FSH, and PRL. The validity of this animal model for the corticosteroid experiments was demonstrated by an absence of a gonadotropin surge on the afternoon of day 29 (see Fig. 2) unless progesterone was injected at 0900 h (see Fig. 3). In experiments in which estrogen priming was not carried out, the protocol was the same as above with the exception that vehicle (20% ethanol-saline) was administered at 27 and 28 days of age instead of estradiol. In all experiments at least six animals were used per group. Key experimental results were confirmed by repetition in two or three separate experiments. RIA ofLH, FSH, and PRL The concentrations of LH, FSH, and PRL in serum samples were analyzed by a double-antibody RIA method as described by Rao and Mahesh (3). The purified hormones and standards and the first antibody for LH [NIDDK-rLH-S-10 (rabbit)], FSH [NIDDK-rFSH-Sll (rabbit)], and PRL [NIDDK-r-PRLS-9 (rabbit)] were obtained from NIDDK. The purified hormone was iodinated with 125I (Amersham, Arlington Heights, IL) by the chloramine-T method (16). The second antibody was purchased from Arnell Inc. (Brooklyn, NY). A 25% binding was obtained at 1:46,825, and 1:25,000 dilutions for LH and FSH antisera, respectively; and at 1:2,500 dilution of the PRL antisera. The assay was linear at 4-128 ng/tube for LH, 32512 ng/tube for FSH, and 0.05-12.8 ng/tube for PRL. The intra- and interassay variabilities as determined by analysis of replicate serum pool samples were 9.6% and 12.4% for LH,

Endo • 1990 Vol 126 • No 1

4.1% and 9% for FSH, and 7% and 11% for PRL. Hormone levels are expressed in terms of NIDDK-RP-1 standard for LH and FSH and NIDDK-RP-3 standard for PRL. Statistical analysis The results given in the text are expressed as mean ± SEM. The difference between the experimental groups were analyzed using one-way analysis of variance, and P < 0.05 was considered significant.

Results Inhibition of estrogen-induced PRL release In order to examine the effect of natural and synthetic corticosteroids on estrogen-induced PRL release, the paradigm of two injections of 2 fig estradiol to immature ovariectomized rats was used. The test steroid was injected 1 h before the second estradiol injection. As shown in Fig. 1, cortisol, corticosterone, triamcinolone acetonide, and dexamethasone were all effective in antagonizing estrogen-induced PRL release, with dexamethasone being the most potent. Effect of corticosteroids on gonadotropin secretion A different model was used for the study of corticosteroids on gonadotropin secretion as described in Materials and Methods. Figure 2 illustrates the effect of the estrogen priming on LH, FSH, and PRL at various times in comparison to vehicle-treated controls. Serum LH levels in the estrogen treated group were significantly decreased (P < 0.05) from the vehicle control group at only 0900 h. FSH serum levels were decreased with estrogen treatment at 0900 h and 1200 h (P < 0.05) and were not different from controls at 1500 h and 1800 h. PRL appeared to be elevated at every time point excepting 1800 h in the estrogen-treated groups, but due to a large amount of variability the increase was only statistically significant at 1500 h (P < .05). Thus, the results Vehicle img/kgCortlwl Imgftg Cortlcosttront lmg/kg Trlimclnotont icctonlde lmg/kg Denmtthuont

FIG. 1. Effect of various glucocorticoids upon estrogen-induced PRL release. Two injections of 2 fig estradiol were administered to ovariectomized immature rats 0 h and 13 h. Controls received vehicle 1 h before the second estradiol injection, whereas the glucocorticoid-treated groups received 1 mg/kg BW of the test glucocorticoid 1 h before the second estradiol injection. *, P < 0.05; **, P < 0.01. n = at least six animals per group.

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CORTICOSTEROID EFFECTS ON LH, FSH, AND PRL LH

FSH

500-1

HOOk

I

3000-

0900t 300-

100-

400-

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1500h 400-

PRL

nooh

4000 i

161

isoot

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1

200-

1200h

1500h

i.

T

IL u, 1000

a a

?

13 Vch-Vch-Vch H

a

E2-E2-Vch

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FIG. 2. Effect of 2 days of estrogen-priming upon LH, FSH, and PRL serum levels in ovariectomized 29-day-old rats. Twenty six-day-old rats were ovariectomized and received an injection of estradiol (2 /xg/rat) or vehicle at 1700 h on days 27 and 28. On day 29 the animals were killed at 3-h intervals beginning at 0900 h up to 1800 h EST. *, P < 0.05. n = at least six animals per group. PRL EJ B D • H D

•J

VeHde N(ln«/k|BW) TA(ln«\|BW) &rUld(InVk|BW) Del(0.03n«*|BW) DOC(lin|/l|BW)

JOO-

FIG. 3. Effect of progesterone and various glucocorticoids upon LH, FSH, and PRL serum levels in estrogen-primed ovariectomized immature rats. The model is the same as described in Fig. 2. The test steroids were administered at 0900 h and the animals were killed at 1500 h of day 29. P4 (Progesterone), TA (Triamcinolone acetonide), Dex (Dexamethasone), DOC (Deoxycorticosterone). **, P < 0.01. n = at least six animals per group.

in Fig. 2 established that this regimen of estrogen treatment did not, of itself, cause an increase in serum LH and FSH levels at 1500 h and 1800 h. Progesterone treatment (1 mg/kg BW) at 0900 h caused a significant increase in serum LH and FSH levels at 1500 h (P < 0.01) (Fig. 3). Therefore this model was used for subsequent studies on the effects of corticosteroids on gonadotropin secretion. In preliminary experiments, a single dose of the various test corticosteroids was examined for its ability to modulate gonadotropin and PRL secretion (Fig. 3). The synthetic glucocorticoid, triamcinolone acetonide (1 mg/ kg BW) was equipotent to progesterone in increasing serum LH and FSH levels (P < 0.01). Triamcinolone acetonide also significantly inhibited serum PRL levels (P < 0.01). The natural glucocorticoid, cortisol (1 mg/kg BW), had no significant effect on serum LH or PRL levels, but did significantly inhibit serum FSH levels (P < 0.01). The natural mineralocorticoid, deoxycorticosterone, significantly elevated both serum LH and FSH levels (P < 0.01) but had no significant effect on serum PRL levels. Finally, the synthetic glucocorticoid, dexamethasone (0.05 mg/kg BW), had no effect on serum LH and PRL levels, but did cause a significant increase in serum FSH levels (P < 0.01). Since only a single dose of corticosteroids was used to examine their effect on gonadotropin secretion in preliminary studies, a dose-response relationship of the corticosteroid effects was examined next. As shown in Fig. 4,

cortisol significantly inhibited serum LH and FSH levels at every dose tested except for the 0.8 mg/kg BW dose. A release of PRL was not stimulated by 0.2 to 0.8 mg/kg BW of cortisol. However, 1.6 mg/kg BW of cortisol significantly stimulated serum PRL release (P < 0.05). The dose response for dexamethasone is shown in Fig. 5. A biphasic effect of dexamethasone was demonstrated for LH, in that a single low dose (0.02 mg/kg BW) caused a significant release of LH (P < 0.01), whereas the higher doses (0.5 mg/kg and 1 mg/kg BW) significantly inhibited serum LH levels. Serum PRL levels were not affected by intermediate doses of dexamethasone, however a similar biphasic effect was observed in that the lowest dexamethasone dose (0.01 mg/kg BW) stimulated PRL release (P < 0.05), while the highest dose (1 mg/kg BW) significantly inhibited PRL release (P < 0.01). Serum FSH levels were significantly elevated by each low dose of dexamethasone tested (0.01 mg/kg, 0.02 mg/kg, 0.05 mg/kg, and 0.1 mg/kg BW; P < 0.01, and P < 0.05, respectively). The higher doses (0.5 mg/kg and 1.0 mg/ kg BW) had no significant effect on serum FSH levels. Figure 6 illustrates the effect of different doses of the synthetic glucocorticoid, triamcinolone acetonide, on serum LH, FSH, and PRL levels. Triamcinolone acetonide significantly stimulated LH and FSH release at every dose tested (0.25 mg/kg BW-4.0 mg/kg BW). In contrast to its potent stimulatory effects on LH and FSH, triamcinolone acetonide inhibited serum PRL levels at each dose tested (P < 0.01).

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Endo • 1990 Vol 126 • N o 1

FSH

LH

PRL

E3 Vehicle

0 •

0.4m j/kj CortUol

•

OJoig/kg Cortbol

•

1.6oig/Vg Cortlsol

FIG. 4. Dose response of cortisol upon LH, FSH, and PRL serum levels in estrogen-primed ovariectomized immature rats. The model is the same as described in Figs. 2 and 3. *, P < 0.05. n = at least six animals per group. FSH

LH

PRL 600-

E3

Vehicle E3 Da(0J>lmgfli«BW) D a (0;0Imjflig BW) D D a (0.05m j/k»BW)

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• 400300200 100 -

I

B

Da(OJmgt|BW)

•S

De«(0.5nig/kjBW) Da(lJt>mg/kgBW)

FlG. 5. Dose response of the synthetic glucocorticoid, dexamethasone, upon LH, FSH, and PRL serum levels in estrogen-primed ovariectomized immature rats. The model is the same as described in Figs. 2 and 3. *, P < 0.05, **, P < 0.01. n = at least six animals per group. FSH

LH

PRL

1200 n 1000-

I FIG. 6. Dose response of the synthetic glucocorticoid, triamcinolone acetonide (TA), upon LH, FSH and PRL serum levels in estrogen-primed ovariectomized immature rats. The model is the same as described in Figs. 2 and 3. *, P < 0.05, **, P < 0.01. n = at least six animals per group. FSH 2000-1

4000 -i

PRL 200 n Vehicle

a DOC(0.2mg/kgBW) m DOC((Umifli«BW) (0Jmg/k| BW) o DOC a DOCtUmj^jBW) DOC(3.Jmg/kgBW) a 1000-

FIG. 7. Dose response of deoxycorticosterone (DOC) upon LH, FSH, and PRL serum levels in estrogen-primed ovariectomized immature rats. The model is same as described in Figs. 2 and 3. *, P < 0.05; **, P < 0.01. n = at least six animals per group.

The dose-response for the natural mineralocorticoid, deoxycorticosterone, is illustrated in Fig. 7. Deoxycorticosterone significantly increased serum LH levels at doses of 0.8,1.6, and 3.2 mg/kg BW (P < 0.05, P < 0.01, P < 0.01, respectively). The lower doses of deoxycorti-

costerone (0.2 and 0.4 mg/kg BW) had no significant effect on serum LH levels. Deoxycorticosterone also stimulated FSH secretion at doses of 1.6 and 3.2 mg/kg BW (P < 0.01). The lower doses, 0.2, 0.4, and 0.8 mg/kg BW, had no significant effect on serum FSH levels. Only

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CORTICOSTEROID EFFECTS ON LH, FSH, AND PRL one dose of deoxycorticosterone (1.6 mg/kg BW) significantly reduced (P < 0.05) serum PRL levels. Other doses did not influence serum PRL. Experiments were also conducted to determine if estrogen priming was necessary for triamcinolone acetonide, deoxycorticosterone and dexamethasone to exert their effects on gonadotropin secretion. As shown in Fig. 8, a 1 mg/kg BW dose of triamcinolone acetonide and deoxycorticosterone significantly stimulated LH secretion (P < 0.01) in the estrogen-primed animals, while dexamethasone significantly inhibited LH release (P < 0.01). In the nonestrogen-primed animals triamcinolone acetonide and deoxycorticosterone were no longer able to stimulate LH secretion; however, dexamethasone remained effective in inhibiting LH release (P < 0.01). In the estrogen-primed animals serum FSH levels were significantly elevated by triamcinolone acetonide and deoxycorticosterone administration (P < 0.01), whereas dexamethasone had no significant effect. In the nonestrogen-primed animals, triamcinolone acetonide and deoxycorticosterone were no longer effective in stimulating FSH release while dexamethasone was without effect. Serum PRL levels were significantly inhibited in estrogen-primed animals by triamcinolone acetonide and

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dexamethasone (P < 0.01), whereas deoxycorticosterone had no significant effect. In nonestrogen-primed animals both triamcinolone acetonide and dexamethasone remained effective in inhibiting basal serum PRL levels (P < 0.01 and P < 0.05, respectively). Deoxycorticosterone had no significant effect on basal PRL levels.

Discussion The interaction of steroid hormones in regulating the surge of gonadotropins leading to ovulation is complex (17). The effect of estradiol acting as the primary trigger for gonadotropin release has been well established (1, 17). The role of progesterone in the ovulatory surge has been more difficult to determine because its effects are dependent upon the time of its administration (1, 2, 17). Furthermore, it is often difficult in the cycling rat to determine the effect of progesterone from that of estradiol during the preovulatory gonadotropin surge. The understanding of the role of progesterone on regulating the gonadotropin surge has been enhanced by the use of ovariectomized rats primed with doses of estradiol sufficient to induce progesterone sensitivity but not large enough to induce a gonadotropin surge (2, 17). This type

ESTROflEN-PRIlylED

in

1500 n

NON-ESTROGEN-PRIMEn 1500 n

1000-

Ul

1000-

Veh TA DOC Dex

FSH

FiG. 8. Effect of triamcinolone acetonide (TA), dexamethasone (Dex) and deoxycorticosterone (DOC) with and without estrogen-priming in ovariectomized immature rats. The model is the same as described in Figs. 2 and 3, except that in the nonestrogen-primed study vehicle was administered instead of estradiol on days 27 and 28. **, P < 0.01. n = at least six animals per group.

FSH 6000-1

Veh TA DOC Dex

Veh TA DOC Dex

PRL

PRL 300 250 200 150 100 50

Veh

TA

DOC

Dex

Veh TA

DOC Dex

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CORTICOSTEROID EFFECTS ON LH, FSH, AND PRL

of model has been used in the present study to examine the effects of corticosteroids on gonadotropin secretion. In a previous paper, we had demonstrated that progesterone could antagonize estrogen-induced PRL secretion (15). In the present study, we extend this observation to corticosteroids. Cortisol, corticosterone, triamcinolone acetonide, and dexamethasone were all found to be effective in inhibiting estrogen-induced PRL secretion when administered 1 h before estrogen treatment (Fig. 1). The precise mechanism by which corticosteroids inhibit estrogen-induced PRL release is not clear. We have previously demonstrated that progesterone's mechanism of inhibiting estrogen-induced PRL release is via decreasing nuclear estradiol binding in the anterior pituitary (15, 18). A similar mechanism of action could be possible for corticosteroids. In support of this possibility, dexamethasone has been reported to decrease estradiol retention in the uterus, vagina, and anterior pituitary in the ovariectomized rat (19) and to decrease nuclear estradiol receptors in rat uteri (20). Additionally, corticosterone has been reported to suppress PRL synthesis and estrogen receptor levels in vitro in rat pituitary tumor cells (GH3) (21). From these findings, it is tempting to speculate that corticosteroid antagonism of estrogeninduced PRL release in our study could be due to a decrease in estradiol receptor binding in the anterior pituitary. However, one cannot rule out other effects such as direct gene regulation or modulation of opioid or dopaminergic systems which are known to be important in regulating PRL release. These systems were not investigated in this study. In the gonadotropin model, corticosteroids were administered 16 h after the second estrogen priming injection, and the time of killing was in the afternoon (1500 h). This model differs significantly from the model for studying corticosteroid effect on estrogen-induced PRL secretion in which corticosteroids were administered 1 h before estrogen administration and this interfered with the effect on the second estradiol injection. Whereas all corticosteroids were inhibitory in the estrogen-induced PRL model, only the synthetic corticosteroids, triamcinolone acetonide and dexamethasone, were inhibitory in the gonadotropin model (Figs. 5 and 7). Cortisol and corticosterone actually enhanced estrogen-induced PRL release at the highest doses (Fig. 4; corticosterone data not shown). Interestingly, dexamethasone also enhanced PRL release at a single low dose (0.01 mg/kg BW). Thus, dexamethasone exerted a biphasic effect on estrogeninduced PRL release, in that a single low dose enhanced release, whereas higher doses (0.5 and 1.0 mg/kg BW) inhibited release (Fig. 5). A similar biphasic effect of dexamethasone upon PRL secretion has been reported in vitro in pituitary cell cultures by Lamberts et al. (22). The mechanism behind such dose-dependent effects re-

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main unknown. It is also unclear why, in the same model, certain corticosteroids, stimulate PRL release whereas others are inhibitory. It is possible that some of these divergent effects may be due to a difference in potencies between the corticosteroids tested. With respect to gonadotropin secretion, divergent effects by the various test corticosteroids were also observed. Cortisol was found to suppress LH and FSH secretion (Fig. 4), whereas corticosterone had no significant effect on LH or FSH secretion (data not shown). Similar to its effect on PRL secretion, dexamethasone was found to exert a biphasic effect on LH secretion with a single low dose (0.02 mg/kg BW) stimulating LH release and higher doses (0.5 and 1.0 mg/kg BW) inhibiting LH release (Fig. 5). Such dose-dependent effects are intriguing and by no means unique for corticosteroids. Dose dependency for progesterone's effect on gonadotropin release (2), reduction of occupied nuclear estrogen receptors in the anterior pituitary (23), and antagonism of estrogen-induced PRL release (15, 24) have been reported earlier. Perhaps the most intriguing finding was the preferential release of serum FSH by all low doses of dexamethasone tested (Fig. 5). This finding is similar to in vitro results reported by Suter and Schwartz (14) and Kamel and Kubajak (13) in which the natural corticosteroids, cortisol and corticosterone, caused preferential release of FSH in pituitary cell cultures. Several studies have suggested that LH and FSH secretion operate under different control mechanisms (25-27). Selective suppression of FSH by inhibin is well documented (28-30). In the ovariectomized rat we have observed selective release of LH and FSH by the progesterone metabolites, 3ahydroxy-5a-pregnan-20-one and 5a-dihydroprogesterone, respectively (31, 32). We have also demonstrated differential suppression of FSH and LH by a variety of natural and synthetic estrogens in the ovariectomized rat (33). Since these studies were conducted in ovariectomized rats, gonadal peptides were not involved with the selective release or suppression. Recent work by Wood and Weibe (34) has demonstrated a selective suppression of FSH by the newly discovered steroid, 3ahydroxy-4-pregnen-20-one. To this growing list of selective modulators of gonadotropin release, we now are able to add the synthetic corticosteroid, dexamethasone. Triamcinolone acetonide and deoxycorticosterone were found to stimulate LH and FSH release, with triamcinolone acetonide being the most potent of the two corticosteroids. Additionally, triamcinolone acetonide and deoxycorticosterone, like progesterone, were able to facilitate ovulation in PMSG-primed immature female rats (Brann, D. W., C. D. Putnam, and V. B. Mahesh, unpublished). Competitive binding studies using the synthetic progestin, R5020 ([17a-methyl]17,21-dimethyl-19-

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CORTICOSTEROID EFFECTS ON LH, FSH, AND PRL nor-4, 9-pregnadiene-3,20-dione), have demonstrated that triamcinolone acetonide and deoxycorticosterone are effective competitors for the progesterone receptor (35, 36). Therefore, the stimulatory effects of these two steroids on gonadotropin secretion may be progesterone receptor mediated. In support of this possibility was our finding that triamcinolone acetonide and deoxycorticosterone were incapable of stimulating gonadotropin secretion when estrogen priming was omitted (Fig. 8). On the other hand, dexamethasone's effect on gonadotropin and PRL secretion appears to be corticosteroid receptor mediated since it was active in the absence of estrogen priming (Fig. 8). Triamcinolone acetonide also suppressed PRL secretion even when estrogen priming was omitted (Fig. 8). Further investigation is needed to answer the question of specific receptor mediation of these corticosteroid effects on gonadotropin and PRL secretion. The physiological significance of stimulatory effects of corticosteroids upon gonadotropin secretion remains to be determined. Adrenalectomy before day 25 in the rat delays puberty and corticosterone treatment reverses this effect of adrenalectomy (37). Adrenalectomized adult rats maintained on physiological saline exhibit normal cycles (38, 39). However, Mann et al. (39) have shown that the critical period for the LH release is prolonged in adrenalectomized animals maintained on physiological saline for 12-15 days and that their cycle length becomes less predictable. From their study it was suggested that the adrenal gland is involved in synchronizing the preovulatory release of gonadotropins (39). Other investigators have also suggested that adrenal steroid secretion on proestrus may play a role in regulating the preovulatory gonadotropin surge (40, 41). However, since the adrenal cortex secretes both progesterone and corticosterone it is difficult to determine which steroid is responsible for the reported potentiation and synchronization of preovulatory gonadotropin secretion. Corticosterone secretion has been shown to be greatly increased on the afternoon of proestrus (40, 41). It could be secreted in sufficient quantities to exert an effect on preovulatory gonadotropin secretion. Despite these suggestions in the literature, this study failed to demonstrate any effect by corticosterone on gonadotropin secretion and cortisol was found to actually suppress gonadotropin release. Corticosterone has been shown to potentiate the effect of GnRH in vivo and in vitro in the release of LH (42, 43). Therefore, corticosterone may still be capable of influencing preovulatory gonadotropin secretion through modulation of gonadotropin response to GnRH. Another product of the adrenal gland, deoxycorticosterone, was found to be an effective stimulator of LH and FSH release in our model, and to facilitate ovulation in PMSG-primed immature female rats (Brann, D. W., C.

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D. Putnam, and V. B. Mahesh, unpublished). At present, it is unknown whether deoxycorticosterone has any role in the physiological regulation of gonadotropin secretion or ovulation. In the past corticosteroid effects on gonadotropin secretion were usually examined in ovariectomized nonestrogen primed animals under a regime of chronic corticosteroid administration. The effects were always inhibitory to gonadotropin secretion and reproduction (44-46). In the ovariectomized estrogen-primed animal model treated acutely with natural corticosteroids, corticosterone had no significant effect on LH and FSH secretion, cortisol suppressed gonadotropin release, and deoxycorticosterone stimulated LH and FSH release. Furthermore, the synthetic corticosteroids, dexamethasone and triamcinolone acetonide, were found to exhibit specific effects on gonadotropin secretion with dexamethasone preferentially releasing FSH at low doses and triamcinolone acetonide stimulating both LH and FSH release at every dose tested. Several other studies have demonstrated that stress and adrenal corticosteroids can enhance gonadotropin secretion and ovulatory activity. Surgical stress, such as ovariectomy (but not adrenalectomy) on the morning of proestrus has been shown to actually advance the LH surge (47). Ramaley (48) has also demonstrated a close correlation between irregular estrous cycles and the absence of a corticosterone rhythm in female rats. Moreover, in aged mice that were not cycling, a week of intermittent stress increased fertility (49). These studies support the concept that adrenal corticosteroids may be important in the maintenance of a normal reproductive state in rats. This study also raises the possibility that the beneficial effects seen with glucocorticoids in inducing ovulation in polycystic ovarian syndrome may be, in part, due to their direct effects upon the release of gonadotropins. In the past cortisone, and more recently, dexamethasone, have been used in polycystic ovarian syndrome for the management of hirsutism. Triamcinolone acetonide might prove to be particularly intriguing as a candidate for management of polycystic ovarian syndrome by virtue of its dual ability as a glucocorticoid to suppress ACTH secretion, and thus reduce adrenal androgen production, and as a progestin to facilitate gonadotropin secretion and ovulation. References 1. Mann D, Barraclough CA 1973 Role of estrogen and progesterone in facilitating LH release in 4-day cyclic rats. Endocrinology 93:694 2. McPherson JC, Mahesh VB 1979 Dose-related effect of a single injection of progesterone on gonadotropin secretion and pituitary sensitivity to LHRH in estrogen-primed castrated female rats. Biol Reprod 20:763 3. Rao IM, Mahesh VB 1986 Role of progesterone in the modulation of the preovulatory surge of gonadotropins and ovulation in the PMSG primed immature rat and the adult rat. Biol Reprod 35:1154

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4. Greenblatt RB, Barfield WE, Lampros CO 1956 Cortisone in the treatment of infertility. Fertil Steril 7:203 5. Smith KD, Steinberger E, Perloff WH 1965 Polycystic ovarian disease. Am J Obstet Gynceol 93:994 6. Loy R, Seibel MM 1988 Evaluation and therapy of polycystic ovarian syndrome. In: Mahajan DK (ed) Endocrinology and Metabolism Clinics of North America: Polycystic Ovarian Disease. WB Saunders Co, Philadelphia, p 785 7. Toaff R, Toaff M, Gould S, Chayen R 1978 Role of androgenic hyperactivity in anovulation. Fertil Steril 29:407 8. Bardin C, Hembree W, Lipsett M 1968 Suppression of testosterone production rates with dexamethasone in women with idiopathic hirsutism and polycystic ovaries. J Clin Endocrinol Metab 28:1300 9. Mahesh VB, Greenblatt RB 1964 Steroid secretions of the normal and polycystic ovary. Recent Prog Horm Res 20:341 10. Mahesh VB, Mills TM, Bagnell CA, Conway BA 1987 Animal models for study of polycystic ovaries and ovarian atresia. In: Mahesh VB, Dhindsa D, Anderson E, Kalra S (eds) Regulation of Ovarian and Testicular Function. Plenum Press, New York p 237 11. Jefferies W, Weir W, Weir D, Prouty R 1958 The use of cortisone and related steroids in infertility. Fertil Steril 9:145 12. Steinberger E, Smith K, Tcholakian R, Rodrigues-Rigau L 1979 Testosterone levels in female partners of infertile couples. Relationship between androgen levels in the woman, the male factor, and the incidence of pregnancy. Am J Obstet Gynecol 133:133 13. Kamel F, Kubajak C 1987 Modulation of gonadotropin secretion by corticosterone: interaction with gonadal steroids and mechanism of action. Endocrinology 121:561 14. Suter D, Schwartz N 1985 Effects of glucocorticoids on secretion of luteinizing hormone and follicle-stimulating hormone by female rat pituitary cells in vitro. Endocrinology 117:849 15. Brann DW, Rao IM, Mahesh VB 1988 Antagonism of estrogeninduced prolactin release by progesterone. Biol Reprod 39:1067 16. Bolton AE 1977 Experimental protocols for the radioiodination of proteins and other compounds. In: Bolton AE (ed) Radioiodination Techniques, Review 18. Amersham/Searle Corp, Arlington Heights, p 45 17. Mahesh VB, Muldoon TG 1987 Integration of the effects of estradiol and progesterone in the modulation of gonadotropin secretion. J Steroid Biochem 27:665 18. Calderon J, Muldoon TM, Mahesh VB 1987 Receptor-mediated interrelationships between progesterone and estradiol action on the anterior pituitary-hypothalamic axis of the ovariectomized rat. Endocrinology 120:2428 19. Lisk RD, Reuter LA 1976 Dexamethasone: increased weights and decreased [3H] estradiol retention of uterus, vagina and pituitary in the ovariectomized rat. Endocrinology 99:1063 20. Campbell PS 1978 The mechanism of the inhibition of uterotrophic responses by acute dexamethasone pretreatment. Endocrinology 103:716 21. Haug E 1979 Progesterone suppression of estrogen-stimulated prolactin secretion and estrogen receptor levels in rat pituitary cells. Endocrinology 104:429 22. Lamberts SW, van Koetsveld P, Verleun T 1987 Prolactin releaseinhibitory effects of progesterone, megestrol acetate, and mifepristone (RU 38486) by cultured rat pituitary tumor cells. Cancer Res 47:3667 23. Fuentes M, Muldoon T, Mahesh VB 1988 The action of progesterone on occupied and total estrogen receptors in the adult ovariectomized rat primed with estradiol. Endocrinology [Suppl 122]:716 (Abstract) 24. Brann DW, Putnam CD, Mahesh VB 1989 Antagonism of estrogeninduced prolactin release by dihydrotestosterone. Biol Reprod 40:1201 25. Lloyd J, Childs GV 1988 Differential storage and release of luteinizing hormone and follicle releasing hormone from individual gonadotropes separated by centrifugal elutriation. Endocrinology 122:1282 26. Savoy-Moore RT, Schwartz NB 1980 Differential control of FSH

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and LH secretion. Int Rev Physiol 22:203 27. Mizunuma H, Samson WK, Lumpkin MO, Moltz J, Fawcett CP, McCann SM 1983 Purification of a bioactive FSH-releasing factor (FSHRF). Brain Res Bull 10:623 28. Franchimont P, Hagelstein M, Jasper J, Renard C, Demoulin A 1989 Inhibin and related peptides: mechanisms of action and regulation of secretion. J Steroid Biochem 32:193 29. Lumpkin M, Negro-Vilar A, Franchimont P, McCann S 1981 Evidence for a hypothalamic site of action of inhibin to suppress FSH release. Endocrinology 108:1101 30. Ying S, Czvik J, Becker A, Ling N, Ueno N, Guillemin R 1987 Secretion of follicle-stimulating hormone and production of inhibin are reciprocally related. Med Sci 84:4631 31. Murphy LL, Mahesh VB 1984 Selective release of luteinizing hormone by 3a-hydroxy-5a-pregnan-20-one in immature ovariectomized estrogen-primed rats. Biol Reprod 30:795 32. Murphy LL, Mahesh VB 1984 Selective release of follicle stimulating hormone by 5a-dihydroprogesterone in immature ovariectomized estrogen-primed rats. Biol Reprod 30:594 33. McPherson JC, Eldridge JC, Costoff A, Mahesh VB 1974 The pituitary-gonadal axis before puberty: effects of various estrogenic steroids in the ovariectomized rat. Steroids 24:41 34. Wood P, Weibe J 1989 Selective suppression of follicle-stimulating hormone secretion in anterior pituitary cells by the gonadal steroid 3a-hydroxy-4-pregnen-20-one. Endocrinology 125:41 35. McGuire WL, Horwitz KB, Pearson OH, Segaloff A 1977 Current status of estrogen and progesterone receptors in breast cancer. Cancer 39:2934 36. Booth BA, Colas AE 1975 Binding interactions of progesterone and other C2i steroids with rat uterine cytosol. Gynecol Invest 6:265 37. Gelato M, Meites J, Wuttke W 1978 Adrenal involvement in the timing of puberty in female rats: interaction with serum prolactin levels. Acta Endocrinol 89:590 38. Baldwin DM, Sawyer CH 1979 Light synchronization of the preovulatory LH surge in adrenalectomized rats. Proc Soc Exp Biol Med 161:295 39. Mann DR, Korowitz CD, Barrachough CA 1975 Adrenal gland involvement in synchronizing the preovulatory release of LH in rats. Proc Soc Exp Biol Med 150:115 40. Buckingham J, Dohlerk, Wilson C 1978 Activity of the pituitaryadrenocortical system and thyroid gland during the oestrus cycle of the rat. J Endocrinol 78:359 41. Raps S, Barthe PL, Desaulles PA 1970 Plasma and adrenal corticosterone levels during the different phases of the sexual cycle in normal female rats. Experientia 21:339 42. Cohen IR, Mann DR 1981 Influence of corticosterone on the response to gonadotropin-releasing hormone in rats. Neuroendocrinology 32:1 43. Fujihara N, Shiino M 1989 The participation of corticosterone in luteinizing hormone releasing hormone (LHRH) action on luteinizing hormone (LH) release from anterior pituitary cells in vitro. Life Sci 26:777 44. Smith ER, Johnson J, Weick RF, Levine S, Davidson JM 1971 Inhibition of the reproductive system in immature rats by intracerebral implantation of cortisol. Neuroendocrinology 8:94 45. Ringstrom SJ, Schwartz NB 1985 Cortisol suppresses the LH, but not the FSH, response to gonadotropin-releasing hormone after orchidectomy. Endocrinology 116:472 46. Li PS, Wagner WC 1983 In vivo and in vitro studies on the effect of adrenocorticotropic hormone or cortisol on the pituitary response to gonadotropin releasing hormone. Biol Reprod 29:25 47. Lawton IE 1972 Facilitatory feedback effects of adrenal and ovarian hormones on LH secretion. Endocrinology 90:575 48. Ramaley JA 1975 Differences in serum corticosterone patterns in individual rats: relationship to ovulatory cycles. J Endocrinol 66:241 49. Paris A, Kelly P, Ramaley JA 1973 Effects of short term stress upon fertility. II. After puberty. Fertil Steril 24:546

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