Download Regulation of inhibin/activin subunits and follistatin mRNA

Life Sciences 67 (2000) 2549–2562
Regulation of inhibin/activin subunits and follistatin mRNA
expression in the rat pituitary at early estrus
M. Tébara,b*, F.H. de Jongb, J.E. Sánchez-Criadoa
a
Department of Physiology, Faculty of Medicine, University of Córdoba, 14004 Cordoba, Spain
b
Department of Endocrinology and Reproduction, Faculty of Medicine and Health Sciences,
Erasmus University Rotterdam, 3000 DR Rotterdam, The Netherlands
Received 27 October 1999; accepted 18 April 2000
Abstract
In the rat pituitary, activin stimulates whereas inhibin prevents FSH synthesis and secretion. Besides, the activin binding protein follistatin neutralizes the action of activin. The control of the FSH
secondary surge at early estrus is not completely understood. To investigate the regulation of the inhibin/
activin a-, bA- and bB-subunits and follistatin mRNA expression in the pituitary during the time of
the FSH secondary surge, cyclic rats treated with LHRH antagonist (ANT) and ovine LH (oLH),
progesterone (P), the anti-steroid RU486, adrenalectomy (ADX) or ADX plus corticosterone (B), were
killed at early estrus. The serum concentrations of FSH were measured and the mRNA levels of the
above mentioned transcripts were analysed and quantitated by using RNase protection assays. ANT
abolished the FSH secondary surge and increased mRNA for a- and bA-subunits and follistatin, but
reduced that for bB-subunit. Both oLH and P reversed these effects. RU486 blocked the effect of oLH
on FSH levels and prevented the reduction in the mRNA for follistatin. ADX in ANT1oLH-treated
rats reduced the serum FSH concentrations, enhanced mRNA for bA- and bB-subunits and, similar to
RU486, blocked the drop in follistatin mRNA. Finally, replacement of B in ADX animals reversed
these effects. These results demonstrate that, in the cyclic rat, the preovulatory secretion of LH and the
surges of P and B on proestrus regulate the synthesis of inhibin/activin subunits and follistatin mRNA
in the rat pituitary at early estrus, probably by reducing inhibin and follistatin and increasing activin.
Moreover, these effects of LH, P and B at the pituitary level, together with the decrease in the amount
of inhibin coming from the ovary, might be responsible for the occurrence of the FSH secondary surge.
© 2000 Elsevier Science Inc. All rights reserved.
Keywords: Inhibin/activin subunits and follistatin mRNA; Pituitary expression at early estrus in the cyclic rat
* Corresponding author. Respiratory Biology Group. P/A Anatomy–LUMC. P.O. Box 9602-2300 RC Leiden
(The Netherlands). Tel.: 31-71-5276692; fax: 31-71-5276511.
E-mail address: [email protected] (M. Tébar)
0024-3205/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved.
PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 8 3 9 -0
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Introduction
The structurally related proteins inhibin (dimer of one a-subunit and one bA- or bBsubunit) and activin (homo- or heterodimer of the two b-subunits) were originally isolated
from the ovary based on their ability to regulate pituitary FSH production. Inhibin decreases
while activin stimulates FSHb mRNA levels and FSH secretion [1–4]. On the other hand,
the structurally unrelated protein follistatin binds to and neutralises the action of activin [5].
Inhibin/activin subunits as well as follistatin have also been identified in the rat pituitary
[6,7].
In the cyclic rat, after the preovulatory surge of gonadotropins, FSH remains high during
the first hours of estrus (secondary surge of FSH) [8]. The secondary surge of FSH does not
depend on LHRH [9,10] but on the LH surge-dependent drop in ovarian mRNA expression
and serum levels of inhibin [11–14]. Administration of the anti-steroid RU486 on proestrus,
which blocks the actions of both the LH surge-induced rise in the secretion of progesterone
[15] and the LH-independent surge in serum levels of corticosterone [16], abolishes the secondary surge of FSH without preventing the drop in serum inhibin [14,17,18].
Exogenous progesterone increases serum concentrations of FSH at early estrus in rats in
which the proestrous LH surge was blocked [14,19,20]. Likewise, glucocorticoids tend to increase selectively the release of FSH in the female rat [21–23]. In addition, we have previously reported that adrenalectomy reduces whereas corticosterone restores the secondary
surge of FSH in rats treated with an LHRH antagonist and an ovulatory dose of oLH [24]. All
these data together suggest that progesterone and corticosterone surges on proestrous afternoon could be involved in the regulation of the secretion of FSH at early estrus by modifying
the production of inhibin/activin-related proteins in the pituitary. That is, the release of the
secondary surge of FSH in the cyclic rat would be the result of the reduction in the negative
signal coming from the ovary (inhibin), which is induced by the primary surge of LH, together with the increase in the positive signal (activin) and/or the decrease in the negative signal (inhibin and follistatin) at the pituitary level, which could be caused by the action of the
LH, progesterone and/or corticosterone surges.
Therefore, the purpose of this study was to investigate the regulation by LH, progesterone
and corticosterone of the mRNA expression of inhibin/activin a-, bA- and bB-subunits and
follistatin in the rat pituitary at the time of the secondary FSH surge.
Methods
Animals
Adult female Wistar rats weighing 180–220 g were used. The rats were housed (five per
cage) under controlled conditions of light (lights on from 0500 to 1900 h) and temperature
(20–238C), with free access to tap water and food available ad libitum. Vaginal smears were
examined daily. Only those animals exhibiting at least two consecutive 4-day estrous cycles
were used. All the experiments were conducted with the approval of the Animal Care and
Use Committee of the University of Cordoba, in accordance with the NIH Guide for Care
and Use of Laboratory Animals.
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Reagents and surgery
The LHRH antagonist (ANT) used was ORG.30276 (Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-TrpSer-Tyr-D-Arg-Leu-Arg-Pro-D-Ala-NH2.CH3.COOH) [25] (Organon International B.V., Oss,
The Netherlands). Immediately before use, the peptide was dissolved in saline and injected
subcutaneously.
Ovine LH (oLH, NIDDK oLH-26) was dissolved in saline and injected intravenously under ether anesthesia. This LH preparation contained less than 0.5% contamination of FSH.
Progesterone (P) (Sigma Chemical Company, St. Louis, MO, USA) was suspended in olive
oil and injected subcutaneously.
The antiprogesterone RU486 (Mifepristone, 11b-(4-dimethylaminophenyl)-17b-hydroxy17a(prop-1-ynyl)-estra-9,9-dien-3-one was donated by Dr Sitruk-Ware (Exelgyn, Paris, France).
This compound has a high affinity for progesterone and glucocorticoid receptors [27,28]. It
was suspended in olive oil and injected subcutaneously.
Corticosterone (B) (4-pregnene-11b,21-diol-3,20-dione; Sigma Chemical Company, St.
Louis, MO, USA) was suspended in olive oil and injected subcutaneously.
Adrenalectomy (ADX) was performed under ether anesthesia. Adrenals were removed
through a subcostal 1 cm incision on both sides.
Experiment 1
To investigate the role of the preovulatory surge of LH on proestrus in the regulation of the
expression of the mRNAs coding for inhibin/activin subunits and follistatin in the pituitary at
early estrus, one group of rats (n54) was injected with ANT (1 mg/0.2 ml saline at 0900 h on
proestrus). This dosage causes maximal suppression of endogenous LH secretion [26] and, in
consequence, also blocks the rise in serum concentrations of P. The control group (n54) received 0.2 ml saline.
Experiment 2
First, to study the participation of the LH-dependent surge of P on proestrus in the expression of inhibin/activin subunits and follistatin mRNA expression in the pituitary at early estrus, 12 animals were administered at 0900 h on proestrus with an injection of ANT (1 mg/
0.2 ml saline). Then they were divided in four groups (n53) and treated with RU486 (4 mg/
0.2 ml olive oil at 0930 h on proestrus), P (10 mg/0.35 ml olive oil at 1500 h on proestrus),
oLH (32 mg/0.25 ml saline at 1700 h on proestrus) or RU4861oLH, respectively. Second, to
investigate the role of the adrenal secretion of B on proestrus in the regulation of inhibin/
activin subunits and follistatin mRNA expression in the pituitary at early estrus, 6 animals were
ADX (between 1100 h and 1200 h on proestrus) and then divided in two groups (n53) which
received B (3 mg/0.2 ml olive oil at 1500 h and at 1700 h on proestrus) or not. All the ADX
animals were also treated with ANT and oLH (32 mg/0.25 ml saline at 1700 h on proestrus)
to overcome the possibility that the stress of the surgical operation would affect the preovulatory secretion of LH. Finally, the control group (n53) was treated with an injection of ANT
(1 mg/0.2 ml saline at 0900 h on proestrus) plus the corresponding vehicles (0.2 ml olive oil
at 0930 h on proestrus, 0.35 ml olive oil at 1500 h on proestrus, 0.25 ml saline at 1700 h
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on proestrus and 0.25 ml saline at 1700 h on proestrus) and the Sham-ADX operation was
performed.
All animals were sacrified by decapitation at 0030 h on estrus, like we did in a previous
study [14] and accordingly to the period of time when the rats in our laboratory display the
FSH secondary surge. Trunk blood as well as pituitaries were collected. Serum was stored
frozen until the radioimmunoassay for FSH was run, and pituitaries were snap frozen in
liquid nitrogen and stored at 2808C. Besides, another 4 animals per experimental group
were treated as described above and killed at 0030 h on estrus to measure FSH serum
concentrations.
RNA isolation
Frozen tissue was pulverized in liquid nitrogen and then total RNA was isolated using
TRIzol reagent (Gibco BRL, Gaithersburg, MD, USA), according to the manufacturer’s instructions. Total RNA was dissolved in RNase-free water and its concentration and purity
were determined by optical density measurements at 260 and 280 nm.
Radioimmunoassay (RIA) of FSH
Serum concentrations of FSH were measured in duplicate in 25 ml samples by doubleantibody RIA methods using the RIA kit supplied by NIADDK (Baltimore, MD, USA) and
following a microassay method described previously [26]. Rat FSH-I-8 was labelled with 125I
by the chloramine-T method [29]. Serum FSH concentrations are expressed as mg/l of the
reference preparation FSH-rat-RP-2. All samples were run in the same assay. The intra-assay
coefficient of variation was 7% and the sensitivity of the assay was 20 pg/tube.
cDNA for inhibin/activin subunits and follistatin
Complementary DNA (cDNA) encoding part of the rat inhibin/activin subunits were obtained from Dr. F.S. Esch (Salk Institute, La Jolla, CA, USA). For the inhibin a-subunit
probe, a rat cDNA PstI/KpnI fragment corresponding to nucleotides 759–1095 [30] was subcloned in pBluescript KS(2) (Stratagene, La Jolla, CA, USA). A cDNA fragment corresponding to nucleotides 40–1540 [29] was subcloned in the EcoRI cloning site of pBluescript
KS(2) for the inhibin/activin bA-subunit probe. For the inhibin/activin bB-subunit probe, a
rat cDNA fragment corresponding to nucleotides 322–632 [30] was subcloned in the SmaI
cloning site of pBluescript KS(1) (Stratagene, La Jolla, CA, USA). The cDNA encoding part
of the rat follistatin was cloned after reverse transcriptase-polymerase chain reaction (RTPCR) with AmpliTaq (Roche Molecular Systems, Branchburg, NJ, USA) from ovarian RNA,
using the following oligonucleotide primers (Eurogentec, Seraing, Belgium): GG CAGCTCCA
CTTGTGTGGT GG and CCGTAACAGC GACCGTGTCT GCC; for the follistatin probe, a
cDNA fragment corresponding to nucleotides 439–783 [31] was subcloned in the EcoRI
cloning site of pBluescript KS(2). Finally, for the rat glyceraldedyde 3-phosphate dehydrogenase (GAPDH) probe, used as an internal control, a 163 bp AccI/Sau3AI fragment of rat
GAPDH cDNA was subcloned in pBluescript KS(2). The GAPDH probe was obtained from
Dr. P. Elfferich (Leiden University, The Netherlands).
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RNase protection assay
RNase protection assays were performed as described previously [32]. In these specific assays, hybridization temperature was 458C for a-subunit and follistatin probes and 508C for
bA- and bB-subunits probes. Routinely, 10 mg of total RNA of each sample were analyzed in
two independent assays. Results were quantified using a PhosphorImager (Molecular Dynamics/B&L Systems, Maarsen, The Netherlands) and data are expressed relative to GAPDH
as relative units (ru). The RNase protection assays were conducted using two samples of total
RNA from rat ovary and from rat testis as positive and quality controls. The mRNA values of
these control samples were reproducible for all four probes in the present experiments as well
as in a previous study from our laboratory [33]. Besides, a sample of tRNA was included in
each assay to determine the background value, which was substracted from the original value
of each sample. Representative gel images are presented in Fig. 1.
Data evaluation and statistical analysis
Results are given as the mean 6 SEM. Data were evaluated for statistically significant differences by Student’s t test (experiment 1) or one-way ANOVA and Duncan Multiple Range
test (experiment 2). A difference was considered to be significant if P , 0.05.
Fig. 1. Representative images of RNase protection gels, showing the signals of protected fragments for a-, bAand bB-subunits and follistatin mRNAs, in pituitaries of cyclic rats treated on proestrus with LHRH antagonist
(ANT, 1 mg/0.2 ml saline s.c. at 0900 h). Control animals received 0.2 ml saline (Saline). tRNA: sample used as
background control. See Methods section for assay details.
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Table 1
Serum concentrations of FSH and quantitative analysis of RNase protection data for the expression
of pituitary inhibition/activin subunits and follistatin mRNA at 0030 h on estrus, in cyclic rats
treated on proesterus with LRHR antagonist
Groups
FSH (mg/l)
a-subunit (ru)
bA-subunit (ru)
bB-subunit (ru)
Follistatin (ru)
Saline
ANT
10.23 6 0.82
2.86 6 0.64**
5.00 6 0.75
8.39 6 1.14*
2.26 6 0.35
7.56 6 2.01*
10.90 6 1.98
6.14 6 0.7*
1.19 6 0.12
4.23 6 0.62**
Saline: 0.2 ml saline s.c. at 0900 h. ANT: 1 mg LHRH antagonist/0.2 ml saline s.c. at 0900 h. FSH serum concentrations: values are given as mean 6 S.E.M. (n58). RNase protection assays: signals of protected fragments
were quantified using a PhosphorImager; values are normalized for GAPDH and expressed in relative units (ru)
(mean 6 S.E.M., n54). * P , 0.05 and ** P , 0.01 vs Saline (Student’s t test).
Results
Effect of LHRH antagonist on proestrus on the serum concentrations of FSH and
on the pituitary expression of inhibin/activin a-, bA- and bB-subunits and
follistatin mRNA at 0030 h on estrus (Table 1)
The administration of ANT significantly decreased the serum concentrations of FSH at
early estrus when compared with saline-injected animals. The pituitary mRNA expression of
a- and bA-subunits was significantly increased by the injection of ANT in relation to rats receiving saline. In contrast, the expression of bB-subunit mRNA was decreased after administration of ANT. With regard to follistatin mRNA, the injection of ANT induced a strong increase in its expression.
Effect of oLH, P, RU486, ADX or ADX1B in LHRH antagonist-injected rats
on the serum concentrations of FSH (Table 2) and on the pituitary expression
of inhibin/activin a-, bA- and bB-subunits and follistatin mRNA
(Fig. 2) at 0030 h on estrus
The serum concentrations of FSH at early estrus in the control group (animals injected
with ANT plus the different vehicles and Sham-ADX) did not differ from those present in
ANT-treated rats (experiment 1, Table 1). The mean expression in relative units (ru) for a-,
bA- and bB-subunits and follistatin mRNA in the control group was 9.0962.10 ru,
10.5161.76 ru, 7.7961.34 ru and 3.9860.96 ru, respectively. Likewise, these values were
not significantly different from those found in animals injected only with ANT (experiment
1, Table 1). Therefore, it can be concluded that none of the procedures performed in the experiment 2, like anesthesia or Sham-ADX operation, had an effect on the serum concentrations of FSH or the pituitary mRNA expression of inhibin/activin subunits and follistatin at
early estrus.
Replacement of the secretion of LH in ANT-treated rats by administration of an ovulatory
dose of oLH on proestrous afternoon restored the release of the secondary FSH surge. Besides, oLH significantly decreased the pituitary expression of a- and bA-subunits and follistatin mRNA (Fig. 2a, b, d). In contrast, pituitary mRNA expression for bB-subunit was increased by oLH (Fig. 2c). The injection of P alone to ANT-treated rats significantly increased
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Table 2
Serum concentrations of FSH at 0030 h on esterus in rats treated on proestrus with LHRH antagonist (ANT) and:
vehicles (Veh) and sham-adrenalectomy (Sham), ovine LH (oLH), progesterone (2), RU486, the combination
of oLH and RU486, the combination of oLH and adrenalectomy (ADX) or the combination of oLH, ADX
and corticosterone (B)
Groups
ANT1Veh1Sham
ANT1oLH
ANT1P
ANT1RU486
ANT1oLH1RU486
ANT1oLH1ADX
ANT1oLH1ADX1B
FSH (mg/l)
2.25 6 0.40
9.72 6 1.05a
7.85 6 1.14a
2.09 6 0.30
1.97 6 0.26
5.20 6 0.60b,c
9.84 6 0.93a
ANT: 1 mg/0.2 ml saline s.c. at 0900 h. Veh1Sham: 0.2 ml oil s.c. at 0930 h, 0.35 ml oil s.c. at 1500 h, 0.25 ml
saline i.v. at 1700 h, 0.2 ml oil s.c. at 1500 h and 1700 h and Sham operation between 1100 h and 1200 h. oLH: 32
mg/0.25 ml saline i.v. at 1700 h. P: 10 mg/0.35 ml oil s.c. at 1500 h. RU486: 4 mg/0.2 ml oil s.c. at 0930 h. ADX:
operation between 1100 h and 1200 h. B: 3 mg/0.2 ml oil s.c. at 1500 h and 1700 h. Values are given as mean 6
S.E.M. (n57). a P , 0.01 and b P , 0.05 vs ANT1Veh1Sham; c P , 0.05 vs ANT1oLH (one-way ANOVA and
Duncan Multiple Range test).
the serum concentrations of FSH at early estrus, and further reduced the expression of a- and
bA-subunits and follistatin mRNA (Fig. 2a, b, d) while it increased that of bB-subunit to the
same extent as oLH administration did (Fig. 2c).
The antagonist RU486 blocked the effect of the ovulatory dose of oLH on the serum levels
of FSH. The expression of a-, bA- and bB-subunit mRNAs in ANT1oLH-treated rats was
unmodified by the injection of RU486 (Fig. 2a, b, c). However, it reversed the reduction in
the mRNA for follistatin induced by oLH in ANT-treated rats (Fig. 2d). Administration of
RU486 alone did not affect the serum concentrations of FSH at early estrus in ANT-treated
animals. However, the anti-steroid induced a strong reduction in the mRNA levels of a-subunit
(22% of value in ANT-treated rats) (Fig. 2a). bA- and bB-subunit mRNAs expression was decreased and increased, respectively (Fig. 2b, c), while follistatin mRNA expression was not
affected by RU486 alone in ANT-treated animals (Fig. 2d).
The removal of glucocorticoids by ADX was associated with a significant reduction of
FSH serum concentrations at early estrus compared with those found in ANT1oLH-treated
rats. While ADX did not change the levels of mRNA for a-subunit (Fig. 2a), it increased the
expression of both b-subunits and follistatin mRNA in comparison to ANT1oLH-treated
rats (Fig. 2b, c, d). When B was replaced in ADX-rats, the secondary surge of FSH was restored and the expression of all three subunits and follistatin was similar to that present in
ANT1oLH-treated rats (Fig. 2a, b, c, d).
Discussion
Inhibin, activin and the activin-binding protein follistatin were initially isolated from the
gonads and reported to affect the synthesis and secretion of FSH in the pituitary through an
endocrine action. However, the finding that these proteins are also extensively expressed in
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the pituitary [6,7] strongly indicates that they might also act as paracrine or autocrine factors
to regulate the secretion of FSH. In fact, all three inhibin/activin subunits as well as follistatin
are costored with LH and FSH in the gonadotropes [34,35] and it has been recently described
that, in the normal cyclic rat, activin increases while the pituitary content of follistatin drops
during the evening of proestrus, simultaneously with the FSH secondary surge [36]. On the
other hand, receptors for P and B are present in the rat pituitary [37,38] and the modulation
of the expression of inhibin-related proteins by steroids has been shown [39–41]. Therefore,
the possibility that surges of P and B in the afternoon of proestrus can affect the mRNA and
protein levels of inhibin/activin subunits and follistatin in the pituitary seems feasible.
This study proves that in vivo manipulations of cyclic rats during the day of proestrus have
an effect on the mRNA expression of inhibin/activin subunits and follistatin at the pituitary
level at early estrus. Besides, the present results confirm previous works from our laboratories demonstrating that these same treatments modulate the serum levels of FSH at that time
of the estrous cycle [14,24]. Abolition of the primary surge of LH through administration of
the ANT blocked the secondary surge of FSH and increased the pituitary mRNA expression
for a- and bA-subunits and follistatin. In contrast, the mRNA expression of bB-subunit was
diminished by the injection of the ANT. As expected, the ovulatory dose of oLH reversed all
the effects of the ANT on the FSH serum levels and on the pituitary mRNA expression of inhibin/activin subunits and follistatin. These results indicate that the preovulatory surge of
LH, besides reducing the production of a- and b-subunits at the ovarian level and hence the
serum concentrations of inhibin [11,12,14], also has an effect at the pituitary level. Since activin is preferentially produced when the ratio of b- to a-subunit is increased [41], it is likely
that the surge of LH reduces the production of inhibin, but stimulates that of activin B in the
pituitary. Moreover, by inducing the fall in pituitary follistatin, the LH surge would enable
activin B to act and increase the secretion of FSH [this study, 14]. In line with these data, a
role of activin B in regulating FSH secretion on estrus has been reported [42,43].
Administration of P to ANT-treated rats induced similar effects on FSH serum concentrations as well as on the pituitary expression of inhibin/activin subunits and follistatin mRNA
compared to the ovulatory dose of oLH. Besecke et al. [36] suggested that P, together with
estradiol, could act as potential mediator of follistatin gene expression in the pituitary. Actually, recent data of this group show a decrease of follistatin mRNA after administration of P
in vitro and in vivo [44]. Another gonadal steroid, testosterone, has also been reported to re-
Fig. 2. Quantitative analysis of RNase protection data for the expression of inhibin/activin a- (a), bA- (b) and
bB-subunit (c) and follistatin (d) mRNA at 0030 h on estrus in the pituitaries of cyclic rats treated on proestrus
with LHRH antagonist (1 mg/0.2 ml saline s.c. at 0900 h) and: vehicles and Sham-adrenalectomy (1Veh1Sham;
0.2 ml oil s.c. at 0930 h, 0.35 ml oil s.c. at 1500 h, 0.25 ml saline i.v. at 1700 h, 0.2 ml oil s.c. at 1500 h and 1700 h
and operation between 1100 h and 1200 h), ovine LH (1oLH, 32 mg/0.25 ml saline i.v. at 1700 h), progesterone
(1P, 10 mg/0.35 ml oil s.c. at 1500 h), RU486 (1RU486, 4 mg/0.2 ml oil s.c. at 0930 h), the combination of oLH
and RU486 (1oLH1RU486), the combination of oLH and adrenalectomy (1oLH1ADX) or the combination of
oLH, ADX and corticosterone (1oLH1ADX1B, 3 mg/0.2 ml oil s.c. at 1500 h and 1700 h). Signals of protected
fragments were quantified using a PhosphorImager. Values are normalized for GAPDH and expressed as percentage of 1Veh1Sham mean value in relative units (ru) (mean 6 S.E.M., n53). Columns with different letters differ significantly from each other (P , 0.05) (one-way ANOVA followed by Duncan Multiple Range test).
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duce follistatin mRNA levels in gonadectomized rats [41]. Taking into account that P participates in several LH-dependent processes in the ovary [45–48], it might be possible that the
secretion of P also mediates the action of the LH surge at the pituitary level on inhibin/activin
subunits and follistatin mRNA. To confirm this hypothesis, we examined the effect of antagonizing proestrous P action by injecting RU486. This anti-P completely abolished the secondary FSH surge while it only prevented the action of the ovulatory dose of oLH on the levels of follistatin mRNA. Our findings confirm those of Polak et al. [44] and demonstrate that
the secretion of P on proestrous afternoon might be involved in the FSH release at early estrus through the decrease in the production of pituitary follistatin. Moreover, they are in line
with recent data which suggest a role for the P receptor in the regulation of FSH secretion by
interference with the action of activin [49]. On the other hand, the surge of LH seems to have
an effect on the pituitary mRNA levels of a- and b-subunits which is not dependent on P action. This effect of LH might be exerted directly on the pituitary gland, since LH/hCG receptors are reported to be present in this brain area [50], or indirectly through the LH-induced
fall in serum concentrations of inhibin, which could influence the expression of a- and bsubunits mRNA at the pituitary level. In this respect, Bilezikjian et al. [41] have reported a
decrease in bB-subunit mRNA levels after addition of inhibin to cultured rat anterior pituitary cells.
In the present experiments, removal of adrenal steroids by ADX on proestrus in ANT1oLHtreated rats decreased the serum concentrations of FSH at early estrus, as we have already
demonstrated [24]. Furthermore, this study shows for the first time that ADX affected the
mRNA expression of bA- and bB-subunits in the pituitary at early estrus. Both b-subunit
mRNA levels were greatly enhanced in comparison with those found in ANT1oLH-treated
rats. All these effects were reversed by the injection of B. These findings indicate that adrenal
products, mainly B, are negatively regulating the synthesis of activin in the pituitary. The
present results seem to be in contradiction with several reports showing the stimulatory effect
of glucocorticoids on FSH release [22,23,51] and with our own results regarding the FSH
serum levels [this study, 24]. However, the effect of B probably is a reduction of inhibin production, caused by the decrease of the mRNA levels of b-subunits in the presence of an unchanged a-subunit mRNA expression. Moreover, these experiments also show that ADX increased the pituitary mRNA expression of follistatin in ANT1oLH-treated animals and that
B reversed this effect. Interestingly, this effect of glucocorticoids on follistatin has been previously demonstrated in porcine endothelial cells [52]. Therefore, all these data together suggest that glucocorticoids can influence FSH release through a negative action on pituitary inhibin and follistatin production, which would allow activin to increase the synthesis and
secretion of FSH.
Finally, our results show that administration of RU486 alone to ANT-treated rats affected
the mRNA expression of inhibin/activin subunits but not that of follistatin. Since ANTinjected animals exhibit normal serum levels of B on proestrous afternoon [24], we assume
that the injection of RU486 blocks the actions of B in these animals. This would explain the
increase in bB-subunit mRNA induced by the antagonist, as ADX also enhanced the mRNA
levels of this subunit in ANT1oLH-treated rats. As for follistatin mRNA, RU486 tended to
increase its expression like ADX did in ANT1oLH-treated animals, although the difference
was not significant in comparison with ANT-treated rats. It might be that blockade of B ac-
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tions in ANT-treated rats is not able to stimulate follistatin mRNA levels beyond those induced by the injection of ANT. Interestingly, RU486 reduced mRNA levels of both a- and
bA-subunits in ANT-injected animals. This effect can not be explained on basis of its antagonism of B actions, because ADX did not change a-subunit while raised bA-subunit mRNA
expression in ANT1oLH-treated rats. In these cases, a direct effect of RU486 on pituitary inhibin/activin subunits mRNA expression cannot be completely ruled out. In fact, actions of
RU486 on the gonadotropes in the absence of ligand have been described [53]. Moreover, recent data support the occurrence of ligand-independent activation of steroid receptors [54].
In summary, our results demonstrate that, in the cyclic rat, the actions of the preovulatory
secretion of LH and of the P and B surges on proestrous afternoon modify the mRNA patterns of inhibin/activin subunits and follistatin in the pituitary during the first hours of estrus.
Both LH and P secretions seem to induce a decrease in inhibin together with an increase in
activin B production, while only the secretion of P would decrease the synthesis of follistatin.
In its turn, the secretion of B appears to diminish the amount of both inhibin and follistatin. We
have shown previously that the decrease in the serum levels of ovarian inhibin at early estrus
is induced directly by the LH preovulatory surge [14]. As for ADX rats, we assume that serum
inhibin dropped in these animals because they received an ovulatory dose of oLH. Therefore,
taking all these data together, we propose a model in which the release of the secondary surge
of FSH—occurring during this period of the estrous cycle—would be caused by the combined
action of: 1) the LH preovulatory secretion, which reduces the inhibitory signal coming from
the ovary (inhibin) and within the pituitary (inhibin and, through the secretion of P, follistatin),
and increases the stimulatory signal in the pituitary (activin B); and 2) the secretion of B on
proestrus, which decreases the negative signal in the pituitary (inhibin and follistatin) (Fig. 3).
Fig. 3. Proposed model for the generation of the secondary surge of FSH at early estrus in the cyclic rat. LHRH:
gonadotropin-releasing hormone; CRH: corticotropin-releasing hormone; LH: luteinizing hormone; ACTH: corticotropin hormone; P: progesterone; B: corticosterone; FSH: follicle-stimulating hormone. (.): positive action;
(—): negative action. See explanation in the text of Discussion.
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Acknowledgments
The authors wish to thank Dr Sitruk-Ware (Exelgyn, Paris, France) for the supply of the
anti-steroid RU486. This work has been subsidized by a grant from DGESEIC (PM98-0167),
Spain.
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