Effects of Progesterone on the Secondary Surge of Follicle ... - CiteSeerX

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BIOLOGY OF REPRODUCTION 57, 77-84 (1997)

Effects of Progesterone on the Secondary Surge of Follicle-Stimulating Hormone in the Rat' M. TMbar, ,2 3 J.Th.J. Uilenbroek, 4 P. Kramer, 4 R.H.N. van Schaik,4 C.D.J. Wierikx, 4 A. Ruiz, 3 F.H. de ong, 4 and .E. Sanchez-Criado 3

Department of Physiology, 3 Faculty of Medicine, University of C6rdoba, Spain Department of Endocrinology and Reproduction,4 Faculty of Medicine and Health Sciences, Erasmus University Rotterdam, The Netherlands preovulatory LH surge was blocked [10, 11]. These data indicate that the rise in serum concentrations of progesterone on proestrus, which is induced by the LH surge [12, 13], could be necessary to evoke the secondary surge of FSH. The stimulatory action of progesterone on FSH secretion might be exerted either directly at the pituitary level or indirectly through inhibition of ovarian inhibin production. The first possibility is supported by the finding that both basal FSH release and the maximal FSH response to LHRH in cultured anterior pituitary cells are stimulated by progesterone [14]. However, the fact that progesterone secretion on proestrus mediates the effects of the primary surge of LH on the ovary in follicular rupture [15-17], progesterone production [18, 19], and luteinization [20] supports an indirect effect of progesterone on FSH levels through the synergism with the LH surge in decreasing ovarian inhibin production. Therefore, the present study was undertaken to investigate whether, and by which mechanism, the LH surge-dependent secretion of progesterone on proestrus is involved in the regulation of the secondary surge of FSH.

ABSTRACT In the cyclic rat, the secondary surge of FSH on estrus appears to depend on the LH surge-induced fall in serum concentrations of inhibin. To investigate the involvement of progesterone in the regulation of the secondary surge of FSH, 4-day cyclic rats were treated on proestrus with an antagonist of LHRH (LHRHant) and with an ovulatory dose of ovine (o) LH, progesterone, the antiprogestin RU486, or the combination of RU486 and oLH. Serum concentrations of gonadotropins and inhibin at 1830 h on proestrus and at 0030 h on estrus were determined, and the expression of inhibin/activin subunit mRNAs in the ovary at 0030 h on estrus was analyzed by in situ hybridization. Rats receiving saline showed low expression of a-, A-, and IBsubunit mRNAs in the ovary and low serum levels of inhibin in conjunction with the elevated serum concentrations of FSH on estrus. Administration of LHRHant blocked the decrease in the synthesis and secretion of inhibin and abolished the FSH secondary surge, whereas the injection of oLH prevented these effects. Exogenous progesterone, compared with LHRHant injection, increased a-, A-, and PB-subunit mRNA hybridization intensity in the ovary and serum inhibin immunoreactivity, and also restored, in part, the surge of FSH on estrus. The antiprogestin RU486 did not modify the effect of oLH on either inhibin/ activin subunit mRNAs in the ovary or serum levels of inhibin, but blocked the FSH surge. These results indicate that, in the cyclic rat, 1) the secretion of progesterone on proestrous afternoon, induced by the LH surge, is not involved in the fall of ovarian inhibin synthesis and secretion; and 2) in combination with a drop in serum inhibin, a stimulatory action of progesterone on another factor, possibly pituitary activin, could be necessary to elicit a complete secondary surge of FSH.

MATERIALS AND METHODS Animals Adult female Wistar rats weighing 205 10 g were used. The rats were housed (4-5 per cage) under standard light (0500-1900 h) and temperature (20-23°C) conditions and had free access to rat chow and tap water. Vaginal smears were taken daily. Only those animals showing at least two consecutive 4-day estrous cycles were included in the experiments.

INTRODUCTION In the cyclic rat, after the preovulatory (primary) surge of gonadotropins, serum concentrations of FSH remain high during the first hours of estrus (secondary surge of FSH) [1]. The control of the secondary surge of FSH is not completely known although it is well established that it does not depend on LHRH [2, 3] but on the LH surge-induced drop in serum levels of inhibin [4-6]. Recently, it has been reported that administration of the antiprogestin RU486 on proestrus blunts the primary surge of LH and FSH and abolishes the secondary surge of FSH [7, 8]; the latter is not restored by injecting an ovulatory dose of LH [9]. Moreover, administration of progesterone increases serum concentrations of FSH at early estrus in rats in which the

Drugs and Treatments The antiprogestin RU486 (Mifepristone, 11 -(4-dimethylaminophenyl)- 173-hydroxy- 17ot(prop- 1-ynyl)-estra-9,9dien-3-one) was donated by Dr R. Deraedt (Roussel-Uclaf, Romainville, France). This compound also has a high affinity for glucocorticoid receptors [21]. Rats received a single s.c. injection of 4 mg RU486/200 1l oil at 0930 h on proestrus. The LHRH antagonist (LHRHant) used was ORG.30276 (Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Arg-LeuArg-Pro-D-Ala-NH2.CH 3.COOH) [22] (Organon International B.V., Oss, The Netherlands). Immediately before use, the peptide was dissolved in saline, and rats received s.c. injections of 1 mg/200 Il saline at 0900 h on proestrus. This dosage causes maximal suppression of endogenous LH secretion [23, 24]. Ovine LH (oLH; NIDDK oLH-26) was injected (i.v.) under ether anesthesia at 1700 h on proestrus at a dose of

Accepted February 24, 1997. Received December 23, 1996. 'This work has been subsidized by a grant from DGICYT (PB94-0449), Spain 2Correspondence: Marfa Tebar Castro, Department of Physiology, Faculty of Medicine, University of C6rdoba, Avda. Menendez Pidal s/n, 14004 C6rdoba, Spain. FAX: 957-218288; e-mail: [email protected]

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32 pLg/250 1l saline. This LH preparation contained less than 0.5% contamination of FSH. Progesterone (Sigma Chemical Company, St. Louis, MO) was suspended in oil and rats received s.c. injections of 10 mg/350 pld oil at 1500 h on proestrus. Experimental Groups To block the preovulatory surge of LH and in consequence the rise in serum concentrations of progesterone, five groups of 7-9 rats received injections of LHRHant and the appropriate vehicles (250p1 saline, 350t1 oil, and 200 ±l1oil), oLH, progesterone, RU486, or the combination of RU486 and oLH. Another group of rats received saline. Half of the animals in each group were killed by decapitation at 1830 h on proestrus and the other half at 0030 h on estrus. Trunk blood was collected and centrifuged, and the serum was stored frozen until the RIAs for LH, FSH, and inhibin. Ovaries from two to three rats per group were removed and dissected at 0030 h on estrus to measure follicular expression of a(-, A-, and B-inhibin/activin subunit mRNAs by in situ hybridization analysis. Six groups of 6 rats each were treated as described above and killed at 1000 h on estrus to assess ovulation by examining the ampullary region of the Fallopian tubes for the presence of eggs. Tissue Fixation and Preparation Within 5 min after the dissection of the ovaries, they were immersed in freshly prepared Bouin's fixative overnight at 4°C, then rinsed in ethanol 70% and embedded in paraffin. Sections (8 jpm thick) of the ovaries were cut and mounted on microscope slides coated with 3-aminopropyl ethoxysilane. Sections were dried at 37°C overnight before use. In Situ Hybridization Probes Complementary DNAs encoding part of the rat/human inhibin subunits were cloned after reverse transcriptase polymerase chain reaction (RT-PCR) with AmpliTaq (Roche Molecular Systems Inc., Branchburg, NJ). For the inhibin ot subunit probe, a rat cDNA Kpn IPst I fragment corresponding to nucleotides 759-1095 [25] was subcloned in pBluescript KS(-) (Stratagene, La Jolla, CA). For the inhibin PA and 3 B subunit probes, human cDNA clones were used corresponding to nucleotides 309-946 and 2631073, respectively [26], which were cloned after RT-PCR (unpublished results). In Situ Hybridization Digoxigenin (DIG)-uridine triphosphate (UTP)-labeled antisense mRNA probes specific for inhibin a, 3 A, and 3B subunits were generated from the appropriate linearized pBluescript subclones by in vitro transcription, using the DIG RNA Labeling Kit (Boehringer Mannheim Biochemica, Indianapolis, IN). Background levels were determined using the corresponding DIG-UTP-labeled sense mRNA probes. After deparaffination, sections were treated with 0.2 N HCI (20 min) and digested with proteinase-K (2 ipg/ml in 100 mM Tris and 50 mM ethylenediamine tetraacetic acid [EDTA]; incubation 40 min at 37C). After treatment with 0.2% glycine for 10 min at 4C and blocking of the nonspecific binding with 0.1 M triethanolamine, followed by 0.1 M triethanolamine with 0.25% acetic anhydride, the sections were incubated with prehybridization solution con-

taining 50% deionized formamide, 4-strength SSC (singlestrength SSC = 0.15 M sodium chloride, 0.015 M sodium citrate), single-strength Denhardt's solution, heat-denatured herring sperm DNA (125 [xg/ml), and yeast tRNA (125 pxg/ ml) for 2 h at 55°C in a humid chamber. Hybridization was carried out overnight at 55°C by incubating the sections with the DIG-UTP-labeled antisense or sense mRNA probes specific for inhibin oa, 3A, and PB subunits, diluted in the hybridization solution containing 50% deionized formamide, 4-strength SSC, single-strength Denhardt's solution, heat-denatured herring sperm DNA (125 Lg/ml), yeast tRNA (125 tpg/ml), and 10% dextran sulfate. The optimal concentration for each probe was determined experimentally (0.5 pl1, 0.9 pl. and 0.2 ,xl/150 ]l hybridization solution/slide for the inhibin -, PA-, and B-subunit mRNA probes, respectively). Slides were washed in 4-strength SSC at 55°C for 15 min, then treated with RNase A solution (20 pLg/ml in 10 mM Tris, 5 mM EDTA, and 0.3 M NaCl) for 30 min at 37°C, and washed again two times in doublestrength SSC (5 min at room temperature). The immunological detection was accomplished with the DIG Nucleic Acid Detection Kit (Boehringer Mannheim Biochemica). After incubation with the color solution in a humid chamber in the absence of light for 14-15 h, slides were dehydrated, placed in xylene, and mounted without counterstaining. Sections were analyzed under brightfield illumination and scored independently by four investigators. Histology Histological examinations of the follicles was based on sections stained with hematoxylin and eosin adjacent to those used for the in situ hybridization. Healthy follicles were grouped as large antral or preovulatory follicles (LA, > 450 pxm), small antral follicles (SA, 275-450 pLm), and preantral follicles (PA, < 274 rLm), according to Osman [27]. RIAs Serum concentrations of LH and FSH were measured using double-antibody RIA methods with the RIA kits supplied by NIH (Bethesda, MD), according to the microassay method described previously [24]. Rat LH-I-9 and FSH-I-8 were labeled with 1251 by the chloramine T method [28]. LH and FSH concentrations were expressed as nanograms per milliliter of serum of the reference preparation LH-ratRP-3 and FSH-rat-RP-2, respectively. All samples were run in the same assay. The intraassay coefficients of variation were 7% and 8% for LH and FSH, respectively. The sensitivities of the assays were 7.5 and 50 pg/tube for LH and FSH, respectively. Serum inhibin-like immunoreactivity was estimated according to the method described by Robertson et al. [29], using a bovine follicular fluid preparation with an arbitrary potency of 1 U/jpg protein as standard [30]. The antibody was raised against 32-kDa bovine follicular inhibin and cross-reacts with free at subunits. These materials were purchased from the Institute for Reproduction and Development, Monash University (Clayton, Victoria, Australia). All samples were run in the same assay, and the intraassay coefficient of variation was 12%. Data Evaluation and Statistical Analysis Results are given as the mean + SEM. Data were evaluated for statistically significant differences by one-way

PROGESTERONE AND FSH SECONDARY SURGE o Saline _ LHRHant+Vehicles ES LHRHant+oLH

ON LHRHant+P 022 LHRHant+RU486 EK LHRHant+RU486+oLH

a

TABLE 1. Ovulation rate at 1000 h on estrus in 4-day cyclic rats treated with LHRHant and injected with an ovulatory dose of either oLH, progesterone, the antiprogestin RU486, or RU486 + oLH.*

12 Groups, Saline LHRHant LHRHant LHRHant LHRHant LHRHant

8

4

NMNM I_a

N

Y7? b Nb NML-aN

NM _MNM

79

+ + + + +

Vehicles oLH P RU486 RU486 + oLH

No. of rats ovulating

No. of ova/ ovulating rat

6/6 0/6 6/6 0/6 0/6 6/6

14.3 _ 1.0

10.9 + 1.9

* Data are presented as mean - SEM (n = 6). ,Saline: 200 l saline s.c. at 0900 h; LHRHant: LHRH antagonist, 1 mg/200 I.l saline s.c. at 0900 h; Vehicles: 200 ,LI oil s.c. at 0930 h, 350 ,Il oil s.c. at 1500 h, and 250 Il saline i.v. at 1700 h; oLH: ovine LH, 32 Rig/250 LI saline i.v. at 1700 h; P: progesterone, 10 mg/350 1l oil s.c. at 1500 h; RU486: 4 mg/200 ,1 oil s.c. at 0930 h.

by LHRHant. Whereas administration of RU486 alone to LHRHant-injected rats increased serum concentrations of LH, progesterone did not modify the effect of LHRHant either on the afternoon of proestrus or the morning of estrus.

10

5

Serum Concentrations of FSH (Fig.

E

13.3 + 0.3

b)

Saline-injected rats showed the primary (proestrous) and secondary (estrous) surges of FSH. Administration of LHRHant prevented the FSH surges at 1830 h on proestrus and at 0030 h on estrus. Serum concentrations of FSH on proestrus afternoon in LHRHant-treated rats did not change after administration of oLH or progesterone. However, the ovulatory dose of oLH completely restored the secondary surge of FSH on estrus, whereas progesterone injection restored it partially. At both 1830 h on proestrus and 0030 h on estrus RU486 reduced serum concentrations of FSH in LHRHant-injected rats with or without the injection of oLH (Fig. lb).

50

Z 25

z

Serum Concentrations of Inhibin (Fig. 1c)

1830 h Proestrus

0030 h Estrus

FIG. 1. Serum concentrations of LH (a), FSH (b), and inhibin (c) at 1830 h on proestrus and at 0030 h on estrus in rats treated on proestrus with 1 saline (200 I saline s.c. at 0900 h) or with LHRHant (1 mg/200 RI saline 1 1 oil s.c. at s.c. at 0900 h) and vehicles (200 p1 oil s.c. at 0930 h, 350 1500 h, and 250 pI saline i.v. at 1700 h); ovine LH (oLH, 32 iRg/250 ,l saline i.v. at 1700 h); progesterone (P, 10 mg/350 pIl oil s.c. at 1500 h); RU486 (4 mg/200 1I oil s.c. at 0930 h); or a combination of RU486 and oLH. NM, not measurable. Values are mean _+SEM (n = 7-9). a, p < 0.01 vs. rats injected with saline; b, p < 0.01 and c, p < 0.05 vs. rats injected with LHRHant + vehicles (one-way ANOVA followed by Duncan's Multiple-Range test).

analysis of variance (ANOVA) and Duncan's MultipleRange test. A difference was considered to be statistically significant if p < 0.05. RESULTS Serum Concentrations of LH (Fig. 1a) Serum LH concentrations showed the expected rise at 1830 h on proestrus in rats injected with saline, which was completely absent in LHRHant-treated animals. Basal concentrations of LH at 0030 h on estrus also were reduced

There were significant differences at 1830 h on proestrus between serum concentrations of inhibin in rats that had received saline and in all groups of animals that had received injections of LHRHant. At 0030 h on estrus, salineinjected rats showed the expected drop in inhibin serum concentrations, whereas administration of LHRHant prevented the decrease in serum levels of inhibin. This effect was reversed by either the ovulatory dose of oLH or RU486, or the combination of oLH and RU486. In contrast, administration of progesterone significantly increased serum concentrations of inhibin in LHRHant-injected rats. Ovulation Data (Table 1) As expected, treatment with LHRHant completely blocked ovulation. The ovulatory dose of oLH restored the normal ovulation rate in LHRHant-injected rats, and this effect was not prevented by administration of RU486. Neither RU486 alone nor progesterone treatments induced ovulation in LHRHant-injected rats. Ovarian Expression of a-, 13-, and P13-lnhibin/Activin Subunit mRNAs (Table 2; Figs. 2 and 3) In saline-injected rats, granulosa cells of large antral or preovulatory follicles (LA) showed almost no expression of a- and PA-subunit mRNAs, whereas these mRNAs were

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TABLE 2. Follicular expression, measured by in situ hybridization, of inhibin/activin (1-, PA-and p,-subunit mRNAs at 0030 h on estrus in ovaries of 4-day cyclic rats treated with LHRHant and injected with an ovulatory dose of either oLH, progesterone, the antiprogestin RU486, or RU486 + oLH *

Groups, Saline LHRHant LHRHant LHRHant LHRHant LHRHant

+ Vehicles + oLH +P + RU486 + RU486 + oLH

P,-subunit mRNA

P,-Subunit mRNA

a-Subunit mRNA b

b

PAb

SAC

LAd

PA

SA'

LAd

PA

SAc

LAd,

+/0 +/0 +/O +/0 0 0

+ + ++ + +/0 +/0

+/0 + +++ +/0 ++++ ++ +/0

0 0 0 0 0 0

+ 0 + +/0 0 0

+/0 ++ +/0 +++ + +/0

0 0 0 0 0 0

+/0 0 +/0 +/0 0 0

0 + 0 +/+ + +/0 0

* The expression of mRNA was estimated on a scale of 1 to 4: 0, no blue grains above background; +, slight expression; + +, clear expression; + ++, high expression; ++++, very high expression. 1 1 a Saline: 200 pI saline s.c. at 0900 h; LHRHant: LHRH antagonist, 1 mg/200 I.L slaine s.c. at 0900 h; Vehicles: 200 pI oil s.c. at 0930 h, 350 pI oil s.c. at 1500 h, and 250 pI saline i.v. at 1700 h; oLH: ovine LH, 32 pLg/250 1LIsaline .v. at 1700 h; P: progesterone, 10 mg/350 I.l oil s.c. at 1500 h; RU486: 4 mg/200 pI oil s.c. at 0930 h. b PA: preantral follicles c SA: small antral follicles. d LA: large antral follicles or preovulatory follicles. Expression only in the antral layer of the granulosa cells.

moderately expressed by granulosa cells of small antral follicles (SA) (Fig. 2, a-c). 3B-subunit mRNA was detected in low levels only in SA (Fig. 2d). Administration of LHRHant enhanced the signal of all three subunit mRNAs in LA compared with vehicle-treated rats and completely abolished the expression of 3 subunit, but not that of a subunit, in SA (Fig. 2, e-h). The ovulatory dose of oLH reversed the changes induced by LHRHant on subunit mRNA expression in LA (Fig. 2, i-l), as it also did in combination with the RU486 injection (data not shown since no differences were found between rats receiving oLH alone and those receiving RU486 and oLH). Administration of progesterone increased the expression of inhibin/activin subunit mRNAs in LA of LHRHant-injected rats (Fig. 3, a-d). In contrast, the injection of RU486 alone to LHRHant-treated rats reduced the signal of all three subunit mRNAs in LA (Fig. 3, e-h). Although a-subunit mRNA was detected in SA regardless of RU486 treatment, only rats that had not received RU486 injection showed 3-subunit mRNA expression in these follicles. Granulosa cells of preantral follicles (PA) weakly expressed only a-subunit mRNA in all groups of animals except for those receiving RU486 injections. DISCUSSION This study confirms earlier reports regarding the blockade of the secondary surge of FSH [31-33] and the high ovarian mRNA expression and serum concentrations of inhibin at early estrus [5] in cyclic rats lacking the proestrous gonadotropin surge. Administration of an antagonist of LHRH (LHRHant) on the morning of proestrus completely abolished the preovulatory surge of LH, blocked ovulation, and slightly reduced serum concentrations of inhibin on the afternoon of proestrus. At early estrus, expression of inhibin/activin subunit mRNAs in the ovary and serum levels of immunoreactive inhibin were increased in relation to those in saline-injected rats, and the secondary surge of FSH was absent. LH replacement in LHRHant-injected rats induced full ovulation, lowered the expression of inhibin/ activin subunit mRNAs in preovulatory follicles below the detection limit, reduced inhibin serum concentrations, and restored the secondary FSH surge. Overall, our findings support others previously published which indicate that the proestrous LH surge modulates the release of FSH on estrus through the suppression of inhibin subunit mRNA expression in the ovary and inhibin levels in serum [4, 5, 34, 35].

Our previous data showing that exogenous progesterone increases the serum concentrations of FSH on estrus morning in a dose-dependent manner in LHRHant-injected rats [11] suggest that the rise in serum concentrations of progesterone on proestrus would elicit the secondary surge of FSH on estrus by synergizing with the LH surge in inducing the fall in inhibin synthesis and secretion. However, the present study showed that injection of progesterone into LHRHant-injected rats, in spite of partially restoring the secondary surge of FSH ([11], this study), did not reduce either inhibin/activin subunit mRNA expression in the ovary or inhibin serum levels at early estrus. This finding clearly indicates that the increased levels of FSH on estrus morning in LHRHant-injected rats after progesterone injection were not negatively correlated with the FSH-inhibiting signal, i.e., ovarian inhibin. Rather, since these animals had a greatly enhanced expression of 3-subunit mRNAs in the ovary at 0030 h on estrus, progesterone could stimulate the production of -subunit homo/hetero dimers, which are known to be FSH-releasing proteins [36]. In this respect, it has recently been suggested [37] that an ovarian factor, possibly progesterone, may up-regulate pituitary follistatin gene expression. The finding that activin stimulates pituitary follistatin mRNA levels [38, 39] could indicate that progesterone stimulates pituitary follistatin production by stimulating activin production in the pituitary. According to this hypothesis, the increased circulating levels of inhibin are not high enough to counteract the increased pituitary production of activin. Alternatively, the activin produced in the ovary might act as peripheral FSH-releasing hormone since the injection of activin in intact female rats causes an increase in serum FSH [40]. However, indirect evidence suggests that the major nonsteroidal signal from the ovary is inhibitory: 1) the administration of steroid-free follicular fluid reduces serum FSH concentrations in female rats [41]; 2) the amount of

FIG. 2. Micrographs showing the follicular expression, measured by in situ hybridization, of inhibin/activin a-, PA-, and PB-subunit mRNAs, at 0030 h on estrus in ovaries of 4-day cyclic rats treated on proestrus with saline (200 pI saline s.c. at 0900 h) or with LHRH antagonist (LHRHant, 1 1 mg/200 pIl saline s.c. at 0900 h) and vehicles (200 p oil s.c. at 0930 h, 350 pLIoil s.c. at 1500 h and 250 p.1saline i.v. at 1700 h) or ovine LH (oLH, 32 [pg/250 )pI saline i.v. at 1700 h). Horizontal bars represent 340 Ipm. H/E, sections stained with hematoxylin and eosin (all remaining sections shown here were coverslipped without staining).

PROGESTERONE AND FSH SECONDARY SURGE

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82 FIG. 3. Micrographs showing the follicular expression, measured by in situ hybridization, of inhibin/activin a-, A,-, and PBsubunit mRNAs, at 0030 h on estrus in ovaries of 4-day cyclic rats treated on proestrus with LHRH antagonist (LHRHant, 1 mg/200 Il] saline s.c. at 0900 h) and progesterone (P, 10 mg/350 Il oil s.c. at 1500 h) or RU486 (4 mg/200 tl oil s.c. at 0930 h). Horizontal bars represent 340 jIm. H/E, sections stained with hematoxylin and eosin (all remaining sections shown here were coverslipped without staining).

TEBAR ET AL.

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activin in bovine follicular fluid is very low compared with that of inhibin [42]; and 3) at any rate, the activin binding protein follistatin would neutralize its biological activity [42]. Furthermore, activin subunit mRNAs and proteins are detected in the pituitary [43, 44], and immunoneutralization of activin suppresses both expression and secretion levels of FSH in cultured rat pituitary cells [45], as well as the hypersecretion of FSH after ovariectomy of hypophysectomized/pituitary-grafted rats [46, 47]. These facts support the concept that pituitary rather than ovarian activin plays a role in FSH release. This does not exclude the possibility that ovarian factors can regulate the expression of inhibin/ activin mRNA in the pituitary [44, 48], since gonadotrophs contain progestin receptors in the afternoon and evening of proestrus [49], and progesterone has been found to stimulate basal FSH release by a direct action at the pituitary level [50, 51]. Hence, all these data together suggest that administration of progesterone into LHRHant-injected rats increased FSH secretion on estrus through a positive effect on pituitary activin production. Indeed, activin has been previously described to be involved in the regulation of the secondary FSH surge [47, 52]. Administration of the antiprogestin RU486 did not prevent the LH surge-dependent drop in either a-, 3A-, and PB-subunit mRNA expression in the ovary or serum concentrations of inhibin at early estrus ([8], this study). This demonstrates that the LH surge-dependent progesterone secretion in the afternoon of proestrus is not responsible for the drop in the synthesis and secretion of ovarian inhibin. However, despite the low levels of inhibin, the secondary surge of FSH was not present in RU486-injected animals. In consequence, the results presented in this study indicate that the action of progesterone in the afternoon of proestrus, together with the drop in ovarian inhibin, is necessary for the release of FSH on estrus. Nevertheless, the participation of other factor(s) blocked by this compound cannot be excluded. In fact, we have reported earlier that the rise in serum concentrations of corticosterone on proestrus afternoon mediates, in part, the secretion of FSH on estrus morning [33]. This action could be due to a direct effect of corticosterone on the anterior pituitary gland [53]. The finding that RU486 injection alone reduced a-, 3 A-, and 3B-subunit mRNA expression and inhibin immunoreactivity in LHRHant-injected rats can be explained on basis of the low FSH serum concentrations induced by RU486, since FSH is the primary stimulus for inhibin production [35, 48] and a-subunit mRNA levels change in concert with serum concentrations of FSH [54, 55]. The FSH secondary surge was prevented in this group of rats despite the reduced inhibin serum concentrations because progesterone and corticosterone actions were blocked by RU486. In summary, the data presented in this study demonstrate that the secretion of progesterone in the afternoon of proestrus does not synergize with the preovulatory surge of LH in reducing the synthesis and secretion of ovarian inhibin. Additionally, evidence is presented to suggest that proestrous progesterone might regulate the release of FSH on estrus by stimulating the production of activin at the pituitary level. ACKNOWLEDGMENTS The authors wish to thank Dr. R. DeRaedt (Roussel-Uclaf, Romainville, France) for the supply of the antiprogestin RU486. Their appreciation is also expressed to the NIDDK (Baltimore, MD) for the ovine LH and the materials used in the gonadotropin assays and to the Institute for Re-

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production and Development, Monash University (Clayton, VIC, Australia) for those used in the inhibin assay. They also thank M.A. Timmerman for her help with the RIA of inhibin.

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