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Endocrinology, doi:10.1210/en.2008-0604
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Endocrinology Vol. 149, No. 11 5783-5790
Copyright © 2008 by The Endocrine Society

Follicle-Stimulating Hormone Responses to Kisspeptin in the Female Rat at the Preovulatory Period: Modulation by Estrogen and Progesterone Receptors

J. Roa, E. Vigo, J. M. Castellano, F. Gaytan, D. García-Galiano, V. M. Navarro, E. Aguilar, F. A. Dijcks, A. G. H. Ederveen, L. Pinilla, P. I. van Noort and M. Tena-Sempere

Department of Cell Biology, Physiology, and Immunology (J.R., E.V., J.M.C., F.G., D.G.-G., V.M.N., E.A., L.P., M.T.-S.), University of Córdoba, and CIBER Fisiopatología de la Obesidad y Nutrición, 14004 Córdoba, Spain; and Organon NV (F.A.D., A.G.H.E., P.I.v.N.), 5340 BH Oss, The Netherlands

Address all correspondence and requests for reprints to: Manuel Tena-Sempere, Physiology Section, Department of Cell Biology, Physiology and Immunology. Faculty of Medicine, University of Córdoba, Avenida Menéndez Pidal s/n, 14004 Córdoba, Spain. E-mail: tufi1tesem{at}uco.es.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ovulation is triggered by the preovulatory surge of gonadotropins that, in rodents, is defined by the concomitant rise in circulating LH and FSH at the afternoon of proestrus (primary surge), followed by persistently elevated FSH levels at early estrus (secondary surge). In recent years, kisspeptins, products of the KiSS-1 gene that act via G protein-coupled receptor 54, have emerged as an essential hypothalamic conduit for the generation of the preovulatory LH surge by conveying positive feedback effects of estradiol onto GnRH neurons, an event that involves not only estradiol-induced transcription of the KiSS-1 gene at the anteroventral periventricular nucleus but also its ability to modulate GnRH/LH responses to kisspeptin. However, little is known about the potential modulation of FSH responsiveness to kisspeptin by sex steroids in the cyclic female. We report herein analyses on the consequences of selective blockade of estrogen receptors (ER)-{alpha} and -β, as well as progesterone receptor (PR), on the ovulatory surges of FSH and their modulation by kisspeptin. Antagonism of ER{alpha} or PR equally blunted the primary and secondary surges of FSH and nullified FSH responses to kisspeptin at the preovulatory period. Conversely, selective blockade of ERβ failed to induce major changes in terms of endogenous FSH surges, yet it decreased FSH responses to exogenous kisspeptin. In contrast, FSH responses to GnRH were fully conserved after ERβ blockade and partially preserved after inhibition of ER{alpha} and PR signaling. Finally, secondary FSH secretion was rescued by kisspeptin in females with selective blockade of ER{alpha} but not PR. In sum, our results substantiate a concurrent, indispensable role of ER{alpha} and PR in the generation of FSH surges and the stimulation of FSH responses to kisspeptin at the ovulatory period. In addition, our data suggest that ERβ might operate as a subtle, positive modulator of the preovulatory FSH responses to kisspeptin, a role that is opposite to its putative inhibitory action on kisspeptin-induced LH secretion and might contribute to the dissociation of gonadotropin secretion at the ovulatory phase in the cyclic female rat.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE NEUROENDOCRINE TRIGGER of ovulation is the so-called preovulatory surge of gonadotropins (1). In rodents, such a hormonal surge is defined by the concomitant rise in the circulating levels of pituitary LH and FSH, which takes place in the afternoon/evening of proestrus; i.e. the day preceding ovulation (1, 2, 3). This primary surge is followed by persistently elevated concentrations of FSH, but not of LH, during early estrus, which conform the so-called secondary surge of FSH (3, 4). The primary surge of gonadotropins is coupled to enhanced secretion of hypothalamic GnRH and GnRH self-priming at the pituitary, which drive the elevation of LH and FSH levels at proestrus (1, 2, 5). This phenomenon relies on the concerted action of estrogen and progesterone receptors (ER and PR, respectively), which mediate the positive feedback effects of sex steroids and appear crucial for both hypothalamic (increased GnRH) and pituitary (GnRH self-priming) components of the primary surge (1, 6, 7, 8). Yet, the neuroendocrine networks ultimately responsible for such positive feedback actions, which manifest only in the female, have remained elusive for decades (1, 2). Likewise, the mechanisms behind the selective elevation of FSH at the secondary surge have been the subject of extensive investigation and debate (3, 4, 9).

Kisspeptins, the peptide products of the KiSS-1 gene that operate through binding to GPR54 (10, 11), have recently emerged as essential gatekeepers of the gonadotropic axis in mammals (12, 13, 14). Indeed, discrete neuronal populations at the hypothalamus that express KiSS-1 have been suggested as pivotal integrators and transmitters for the effects of a diversity of regulators of gonadotropin secretion, including metabolic factors, environmental cues, and sex steroids (12, 15). On the latter, rodent data have led to the proposal that KiSS-1 neurons at the arcuate nucleus and the anteroventral periventricular nucleus (AVPV) play important roles in mediating the negative and positive feedback effects of estradiol, respectively (12, 15, 16, 17). In keeping with a pivotal role of KiSS-1 neurons at the AVPV in the induction of positive feedback effects of estrogen, these neurons are selectively activated at the time of the preovulatory surge (17) and are far more abundant in the female, where positive feedback is detected, (18), whereas the preovulatory surge of LH can be prevented by immunoneutralization of kisspeptins (19). In addition, we have recently reported that estrogen, acting at the hypothalamus via ER{alpha}, plays a critical role in enhancing LH responses to kisspeptin during the preovulatory phase, a phenomenon that is likely to contribute to the full expression of the LH surge that triggers ovulation (20). Yet, it was recently reported that mild induction of LH secretion could be detected in estrogen-primed ovariectomized mice with a congenital deficiency of G protein-coupled receptor 54 (GPR54) signaling, an observation whose physiological implications warrant further investigation (21).

Despite the complex nature of the preovulatory surge described above, most of the research efforts recently conducted to elucidate its molecular and neuroendocrine basis, which have included the assessment of ER subtypes involved and putative contribution of hypothalamic KiSS-1 (17, 21, 22), have focused in the analysis of LH secretory events, with little attention being paid to FSH surges. To provide a deeper insight into the neuroendocrine mechanisms of the latter, we report herein a series of analyses of FSH secretory patterns and responses to kisspeptin in the cyclic female rat at the periovulatory period, after selective blockade of ER{alpha}, ERβ, and PR in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and drugs
Adult Wistar female rats bred in the vivarium of the University of Córdoba were used. The animals were maintained under constant conditions of light (14 h light from 0700 hand temperature (22 C) and housed in individual cages with free access to pelleted food and tap water. Experimental procedures were approved by the Córdoba University Ethical Committee for animal experimentation and conducted in accordance with European Union normative for the use of experimental animals. Rat/mouse KiSS-1 (110–119)-NH2, namely kisspeptin-10, was obtained from Phoenix Pharmaceuticals Ltd. (Belmont, CA). Antagonists of ER{alpha} (ERA-90) and ERβ (ERB-88) as well as the antagonist of PR (ORG 31710) were selected and provided by Organon NV (Oss, The Netherlands). GnRH was purchased from Sigma Chemical Co. (St. Louis, MO).

Experimental designs
In experiment 1, the effects of an effective dose of kisspeptin-10 on FSH secretion were tested after selective blockade of ER{alpha}, ERβ,or PR in vivo, acutely at the preovulatory period. In detail, adult virgin female rats were monitored for estrous cyclicity by daily vaginal cytology. Only rats with at least two consecutive regular 4-d estrous cycles were used for the subsequent pharmacological studies. Groups of cyclic females (n = 20–24) were injected twice (at 2100 h of diestrus-2 and 0900 h of proestrus), via sc route, with one of the following compounds/dose regimens: ER{alpha} antagonist ERA-90 at 1.5 mg/kg, ERβ antagonist ERB-88 at 1.5 mg/kg, or PR antagonist ORG31710 at 8 mg/kg. Rats injected with vehicle (5% mannitol plus 0.5% gelatin in sterile deionized water) served as controls. Subsequently, each group was divided in two halves (n = 10–12) that received, at 1200 h of proestrus, a single intracerebroventricular (icv) injection of either 1 nmol kisspeptin-10 or sterile saline (10 µl). The experimental protocol and icv administration were as described in detail elsewhere (20). Similarly, the dose of 1 nmol kisspeptin per rat was selected on the basis of our recent data on its ability to elicit LH and FSH secretion in adult female rats (20, 23). Blood samples (250 µl) were obtained by jugular venipuncture before (0 min), and at 15, 60, 120, 210, 300, 390, and 480 min after icv injections. Additional blood samples were taken from each animal between 0900 and 1000 h of the following estrus.

In experiment 2, the effects of an effective dose of GnRH on FSH secretion were tested at proestrus, in adult female rats after selective blockade of ER{alpha}, ERβ, or PR in vivo, acutely at the preovulatory period. This experiment was conducted in parallel to experiment 1, thus allowing direct comparison between the FSH-releasing effects of kisspeptin-10 and GnRH in our models of ER/PR antagonism. Protocols of acute ER{alpha}, ERβ, and PR antagonism at the preovulatory period as well as of serial blood sampling (except at 300 and 480 min), were similar to those of experiment 1. An effective dose of GnRH (1 µg/rat in 100 µl sterile saline), delivered via the ip route, was selected based on previously published data (20). Independent control groups (injected sc with 5% mannitol/0.5% gelatin; injected ip with saline) were included in this experiment.

In experiment 3, the effects of an effective dose of kisspeptin-10 on FSH secretion were tested at proestrus, in adult female rats after a prolonged protocol of blockade of ER{alpha} or ERβ in vivo. Experimental procedures were similar to those of experiment 1, except that sc injections of the antagonists of ER{alpha} or ERβ were continued every 12 h for a complete cycle, starting at 2100 h of diestrus-2 and finishing at 0900 h of proestrus of the next cycle (n = 10 injections over 4.5 d). Intracerebral injections of kisspeptin-10 (1 nmol) were conducted at 1200 h on proestrus, as described for experiment 1. Blood samples (250 µl) were obtained by jugular venipuncture before (0 min) and at 15, 60, 120, 210, 390, and 480 min after icv injections. Additional blood samples were taken from each animal between 0900 and 1000 h of the following estrus.

Of note, the experimental designs described above are similar to those previously used by our group for the assessment of the endogenous LH surges and pharmacological LH responses to kisspeptin after manipulation of ER subtypes and PR in the adult female rat (20). The compounds used for selective blockade of ER{alpha}, ERβ, and PR were chosen on the basis of their biological profiles in different heterologous cell systems and in vivo tests, as described in detail in a recent publication from our group (20). In all experiments, selection of the protocols (doses/regimens) of administration of ER{alpha}, ERβ, and PR analogs was based on pharmacokinetic data provided by the supplier (Organon NV) (20).

FSH measurements by specific RIA
Serum FSH levels were determined in a volume of 25–50 µl using a double-antibody method and RIA kits supplied by the National Institutes of Health (Dr. A. F. Parlow, National Institute of Diabetes and Digestive and Kidney Diseases National Hormone and Peptide Program, Torrance, CA). Rat FSH-I-9 was labeled with 125I using iodogen tubes, following the instructions of the manufacturer (Pierce, Rockford, IL). Hormone concentrations were expressed using reference preparation FSH-RP-2 as standard. Intra- and interassay coefficients of variation were, respectively, less than 6 and 9%. The sensitivity of the assay was 20 pg/tube.

Presentation of data and statistics
Hormonal determinations were conducted in duplicate, with a minimal total number of 10 samples per group. When appropriate, in addition to individual time point determinations, integrated FSH secretory responses were expressed as the area under the curve AUC, calculated following the trapezoidal rule. Hormonal data are presented as mean ± SEM. Results were analyzed for statistically significant differences using single or repeated ANOVA followed by Student-Newman-Keuls multiple range test (SigmaStat 2.0, Jandel Corp., San Rafael, CA). P ≤ 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
FSH surge and responses to kisspeptin after acute blockade of ER{alpha}, ERβ, or PR at the preovulatory period
The effects of acute blockade of ER{alpha}, ERβ, or PR during the preovulatory phase on the magnitude of the endogenous primary surge of FSH, and of FSH responses to exogenous kisspeptin at proestrus, were analyzed in detail in cyclic female rats in experiment 1. As surrogate marker of the secondary surge of FSH, serum levels of this hormone were also determined at the morning of estrus in the above experimental groups.

In control cyclic female rats injected with vehicle, the expected preovulatory (primary) surge of FSH was detected, with low circulating levels of the hormone before 1400 h and a progressive increase thereafter that peaked at 2000 h. Also as expected for the secondary surge, FSH remained elevated at the morning of estrus. A similar profile was detected in females treated with the selective antagonist of ERβ, ERB-88, at late diestrus-2 (2100 h) and early proestrus (0900 h), which showed circulating FSH concentrations that raised form 1400 h proestrus onwards, peaked between 1700 h and 2000 h and remained elevated at the morning of estrus. In striking contrast, administration of two doses of the selective antagonist of ER{alpha}, ERA-90, at late diestrus-2 and early proestrus completely prevented the occurrence of the primary surge of FSH. Moreover, the secondary rise of this hormone was not detected at the morning of estrus. Likewise, blockade of PR, by means of administration of the selective PR antagonist ORG 31710 to cyclic females at late diestrus-2 and early proestrus abrogated the primary and secondary surges of FSH along the proestrus-to-estrus transition (Fig. 1AGo). Quantitative estimation of integrated secretory responses during the primary surge of FSH was obtained by calculation of AUC between 1200 and 2000 h of proestrus. As shown in Fig. 2Go, whereas antagonism of ERβ did not have any significant impact on the secretory mass of the FSH surge, this was clearly attenuated by the preovulatory blockade of either ER{alpha} or PR.


Figure 1
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  		FIG. 1. FSH surges and responses to kisspeptin after acute blockade of ER{alpha}, ERβ, or PR at the preovulatory period. A, Profiles of serum FSH levels at the ovulatory phase are shown from cyclic female rats acutely pretreated sc with vehicle (Veh), ER{alpha} antagonist (aER{alpha}), ERβ antagonist (aERβ), or PR antagonist (aPR), as described for experiment 1 and injected at 1200 h on proestrus with vehicle. B, Profiles of serum FSH levels at the ovulatory phase are presented from cyclic female rats subjected to similar protocols of ER or PR antagonism and injected at 1200 h on proestrus with kisspeptin-10 (Kp-10; 1 nmol icv). Numbers on the x-axis represent time (hours) at proestrus; 9-E, 0900 h on estrus.

 

Figure 2
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  		FIG. 2. Integrated FSH secretion at the primary surge and FSH responses to kisspeptin after acute blockade of ER{alpha}, ERβ, or PR at the preovulatory period. Integrated FSH secretion was calculated as the AUC for the period between 1200 and 2000 h on proestrus after icv injection of vehicle or kisspeptin-10 (Kp-10) to cyclic female rats pretreated sc with vehicle, ER{alpha} antagonist (aER{alpha}), ERβ antagonist (aERβ), or PR antagonist (aPR). Net fold increases over corresponding groups injected with vehicle are indicated in the insets of each histogram. **, P < 0.01 vs. groups pretreated sc with vehicle; a, P < 0.01 vs. corresponding groups icv injected with vehicle (ANOVA followed by Student-Newman-Keuls multiple-range test).

 
Dynamic profiles of FSH secretion and integrated hormonal responses were similarly evaluated in the above groups after a single icv injection of 1 nmol kisspeptin-10 at 1200 h on proestrus. In control female rats, kisspeptin administration evoked a durable FSH secretory response, which manifested as moderate elevation of FSH levels between 1230 and 1400 h, followed by a sharp and robust elevation of FSH concentrations that peaked at 2000 h and decreased thereafter to levels of the endogenous secondary surge (Fig. 1BGo). Indeed, when calculated as AUC, such a kisspeptin-induced FSH secretion corresponded to an approximately 3.2-fold increase over the secretory mass of the endogenous primary surge (Fig. 2Go). Notably, such robust responses were not detected in any of the groups treated with the antagonists of ER or PR, yet the magnitude of FSH secretion induced by kisspeptin significantly varied among the groups. Thus, animals treated with ERβ antagonist displayed a pattern of response to kisspeptin that was roughly similar to that of control animals, with modest elevation within the first 2 h after injection and progressive increase thereafter along the evening of proestrus (Fig. 1BGo). However, the magnitude of the latter was markedly lower than in control females. In fact, net kisspeptin responses represented only a 1.5-fold increase over the endogenous primary surge of FSH in females treated with the antagonist of ERβ and were only half of that detected in controls (Fig. 2Go). More markedly, females pretreated with ER{alpha} or PR antagonists from late diestrus-2 to proestrus showed virtually negligible FSH responses to kisspeptin along the afternoon of proestrus (Fig. 1BGo), with a secretory mass that was less than 20–25% of that of control animals and represented a nonsignificant 1.4- and 1- to 2-fold elevation over the endogenous primary surge of FSH in females treated with antagonists of either ER{alpha} or PR, respectively (Fig. 2Go). It’s worthy of note that despite severe suppression of the primary surge, icv injection of kisspeptin was able to restore FSH levels in the morning of estrus (to those of the secondary surge) in females pretreated with ER{alpha} antagonist but not with the antagonist of PR.

FSH responses to GnRH after acute blockade of ER{alpha}, ERβ, or PR at the preovulatory period
Using a similar setting, FSH responses to an effective dose of GnRH were evaluated in experiment 2 after acute blockade of ER{alpha}, ERβ, or PR during the preovulatory phase. In keeping with results of experiment 1, control cyclic females injected with vehicle and those treated with ERβ antagonist at late diestrus-2 and early proestrus exhibited the expected primary surge of FSH, with levels that peaked at 1830 h (the latest time point of proestrus assessed in this experiment). Likewise, both groups showed a persistent elevation of circulating FSH concentrations at the morning of estrus, indicative of the secondary surge. In clear contrast, treatment with either ER{alpha} or PR antagonists between late diestrus-2 and early proestrus fully blunted the expression of both primary and secondary surges of FSH (Fig. 3AGo). In line with the above secretory profiles, the integrated FSH secretion during the primary surge (calculated as AUC) was similar in controls and animals injected with ERβ antagonist, whereas it was significantly reduced by pretreatment with either ER{alpha} or PR antagonists (Fig. 4Go).


Figure 3
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  		FIG. 3. FSH surges and responses to GnRH after acute blockade of ER{alpha}, ERβ, or PR at the preovulatory period. A, Profiles of serum FSH levels at the ovulatory phase are shown from cyclic female rats acutely pretreated sc with vehicle (Veh), ER{alpha} antagonist (aER{alpha}), ERβ antagonist (aERβ), or PR antagonist (aPR), as described for experiment 2 and injected at 1200 h on proestrus with vehicle. B, Profiles of serum FSH levels at the ovulatory phase are presented from cyclic female rats subjected to the similar protocols of ER or PR antagonism and injected at 1200 h on proestrus with GnRH (1 µg ip). Numbers on the x-axis represent time (hours) at proestrus; 9-E, 0900 h on estrus.

 

Figure 4
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  		FIG. 4. Integrated FSH secretion at the primary surge and FSH responses to GnRH after acute blockade of ER{alpha}, ERβ, or PR at the preovulatory period. Integrated FSH secretion was calculated as the AUC for the period between 1200 and 1830 h on proestrus, after icv injection of vehicle or GnRH to cyclic female rats pretreated sc with vehicle, ER{alpha} antagonist (aER{alpha}), ERβ antagonist (aERβ), or PR antagonist (aPR). Net fold increases over corresponding groups injected with vehicle are indicated in the insets of each histogram. **, P < 0.01 vs. groups pretreated sc with vehicle; a, P < 0.01 vs. corresponding groups icv injected with vehicle (ANOVA followed by Student-Newman-Keuls multiple-range test).

 
In addition to endogenous surges, FSH secretion in response to GnRH stimulus was also evaluated in the above experimental groups. A single ip injection of 1 µg GnRH to control female rats at 1200 h on proestrus evoked robust FSH responses, whose secretory profile clearly differed from that induced by kisspeptin administration. Thus, although kisspeptin elicited a durable, progressive increase in FSH levels, with modest elevation within the first 2 h followed by a sharp increase thereafter, GnRH injection induced a rapid elevation of serum FSH concentrations that peaked at 2 h and partially declined thereafter, with FSH levels that remained elevated along the afternoon of proestrus and the morning of estrus (Fig. 3BGo). A strikingly similar profile of response to GnRH was detected in females pretreated with the antagonist of ERβ: rapid elevation of FSH levels followed by a partial decrease of FSH concentrations that, nonetheless, remained elevated along the proestrus-to-estrus transition (Fig. 3BGo). Again, this pattern clearly differed from that evoked by kisspeptin in the same experimental group. When calculated as AUC, FSH responses to GnRH along the afternoon of proestrus were similar between control and ERβ-antagonist groups and represented an approximately 2.4-fold increase over the corresponding values of the endogenous primary surge of FSH (Fig. 4Go).

Finally, FSH responses to GnRH were also monitored in female rats pretreated with antagonists of ER{alpha} or PR at the preovulatory period. In contrast to null responses to kisspeptin in these groups, GnRH administration elicited a transient, significant elevation in serum FSH levels, which remained increased for at least the first 3.5 h after GnRH injection (Fig. 3BGo). Yet, the net FSH secretory responses to GnRH (calculated as AUC) were about one half of those induced by GnRH in the control group (Fig. 4Go). Nonetheless, given the marked decrease in the magnitude of the endogenous primary FSH surges in animals pretreated with ER{alpha} or PR antagonists, the above responses to GnRH represented an increase of about 2.0-fold, i.e. roughly similar to relative responses to GnRH in control and ERβ-antagonist-treated groups. Finally, as was the case in animals injected with kisspeptin, administration of an effective dose of GnRH at 1200 h on proestrus was able to rescue the occurrence of the secondary surge of FSH in animals pretreated with ER{alpha} antagonist but not with an antagonist of PR (Fig. 3BGo).

Effect of chronic blockade of ER{alpha} or ERβ on FSH surges and preovulatory responses to kisspeptin
In addition to the effects of their acute blockade, we evaluated in experiment 3 the consequences of a prolonged protocol of antagonism of ER{alpha} or ERβ on the magnitude of the endogenous FSH surges and FSH responses to kisspeptin at proestrus. Control cyclic female rats displayed the expected rise in serum FSH concentrations at the afternoon of proestrus, with peak levels at 1830–2000 h (primary surge), and persistently elevated FSH concentrations at the morning of estrus (secondary surge). In good agreement with our results from experiment 1, icv injection of 1 nmol kisspeptin-10 at 1200 h on proestrus induced a consistent, progressive increase in FSH secretion along the afternoon of proestrus, with hormonal levels that were significantly higher than those of the endogenous primary surge (Fig. 5AGo). Conversely, chronic treatment with the antagonist of ER{alpha} fully prevented the occurrence of primary and secondary surges of FSH, in line with results from experiments 1 and 2. Moreover, as was the case in models of acute blockade, intracerebral administration of 1 nmol kisspeptin-10 failed to evoke any significant FSH responses during the afternoon of proestrus in females chronically pretreated with the antagonist of ER{alpha}. Yet, kisspeptin injection evoked the secondary surge of FSH in this group (Fig. 5BGo). Finally, female rats chronically treated with ERβ antagonist did show the primary and secondary surges of FSH and responded to kisspeptin administration with an elevation of FSH secretion during the afternoon of proestrus, yet the magnitude of such a response was significantly attenuated vs. control females (Fig. 5CGo).


Figure 5
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  		FIG. 5. FSH surges and responses to kisspeptin after chronic blockade of ER{alpha} or ERβ. Profiles of serum FSH levels at the ovulatory phase are shown from female rats pretreated sc for one complete cycle with vehicle (A), ER{alpha} antagonist (B), or ERβ antagonist (C), as described for experiment 3 and injected at 1200 h on proestrus of the last day of treatment with vehicle or kisspeptin-10 (Kp10). Numbers on the x-axis represent time (hours) at proestrus; 9-E, 0900 h on estrus.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although it is well known that the preovulatory surge of gonadotropins involves the elevation of circulating levels of both LH and FSH, most of the recent advancements in our knowledge about key neuroendocrine aspects of this phenomenon have considered only LH secretion, with less attention being paid to FSH secretory events (17, 21, 22). Although those analyses were obviously instrumental and fully valid, they might have also neglected some specific features and regulatory mechanisms of FSH secretion at this critical period of the ovarian cycle. In this context, our present study is the first to provide a detailed characterization of the functional consequences of selective blockade of ER{alpha}, ERβ, and PR on the endogenous ovulatory (primary and secondary) surges of FSH and their modulation by kisspeptin in the cyclic female rat, observations that might contribute to unravel the mechanisms responsible for FSH secretory dynamics during the ovulatory phase of the cycle.

Acute and chronic pharmacological blockade of ER{alpha} in cyclic female rats resulted in the complete elimination of the primary and secondary surges of FSH. Considering that, acting through ER{alpha}, estradiol has been proven to enhance KiSS-1 mRNA expression at the AVPV (16), it is presumable that, at least partially, the effects of ER{alpha} blockade on FSH secretion reported herein might derive from suppressed hypothalamic expression of KiSS-1 gene during the preovulatory period. Anyhow, our pharmacological data conclusively demonstrate that, regardless of potential transcriptional events, FSH secretory responses to kisspeptin are severely blunted in absence of ER{alpha} signaling. The molecular basis for such a phenomenon, which might involve decreased GPR54 expression and/or GnRH responsiveness, remains to be unfolded. Yet, it is notable that an analogous observation has been recently described by our group for the preovulatory LH responses to kisspeptin (20). These findings jointly point out that functional ER{alpha} is indispensable for attaining proper LH and FSH responsiveness to kisspeptin and, thereby, for the full expression of the preovulatory surge of both gonadotropins.

In contrast to ER{alpha}, pharmacological blockade of ERβ failed to induce significant changes in the profiles of the endogenous primary and secondary surges on FSH either after acute or chronic administration. This observation extends our recent reports on the lack of effects of antagonism of ERβ on the LH surge (20) and further confirms the dispensable role of this receptor subtype in the generation of the preovulatory rise of gonadotropins (20, 22). Notwithstanding, detailed analysis of the pattern of FSH responses to kisspeptin evidenced clear-cut effects of selective blockade of ERβ, effects that were diametrically opposite to those previously identified for LH (20). Thus, although acute or chronic treatment with the antagonist of ERβ resulted in the enhancement (~2-fold) of LH responses to kisspeptin at proestrus (20), similar protocols of antagonism induced a significant attenuation (about one half) of kisspeptin-induced FSH secretion (see Fig. 2Go). Taken together, these observations suggest that, acting via ERβ, estrogen might convey opposite regulatory signals to LH and FSH secretion at proestrus, because activation of ERβ would result in restraining LH responses but enhancing FSH responses to kisspeptin. The relative importance of these regulatory actions appears modest and subordinated to the dominant ER{alpha}-mediated effects. Yet, it is tempting to speculate that this dual mechanism might contribute to the dissociation of gonadotropin secretion observed along the proestrus-to-estrus transition. In this context, we have preliminarily observed that chronic treatment with a selective agonist of ERβ, in the presence of progesterone, elicits maximal FSH responses (our unpublished data) but attenuates LH secretion after kisspeptin administration (20).

Comparison of the profiles of FSH responses to GnRH and kisspeptin in female rats pretreated with ER{alpha} or ERβ antagonists revealed significant differences, which might help to define the putative sites of action of estrogen for its modulation of FSH secretion. Thus, considering that kisspeptin primarily operates via stimulation of GnRH release at the hypothalamus to induce gonadotropin secretion (12), it is worthy to note that FSH responses to kisspeptin were more severely affected by the blockade of ER{alpha} or ERβ than those elicited by GnRH. Indeed, although antagonism of ER{alpha} nullified FSH secretory responses to kisspeptin, GnRH stimulation in this model was able to evoke detectable elevations of FSH levels, whose relative magnitude was roughly similar to that observed in control females. Furthermore, blockade of ERβ, which significantly attenuated absolute and relative FSH responses to kisspeptin at proestrus, failed to alter the pattern of FSH secretion after GnRH stimulation. As a whole, these observations suggest that the effects of blockade of ER{alpha} and ERβ do not merely stem from changes in pituitary responsiveness to GnRH but rather point out that the modulatory actions of estrogen on FSH responsiveness to kisspeptin are taking place, at least partially, upstream of the pituitary level. Indeed, it has been recently demonstrated in mice that estradiol significantly potentiates hypothalamic GnRH neuronal activity in response to kisspeptin (24). In this context, our results suggest that ER{alpha}, and to a lesser extent ERβ, might be involved in such a phenomenon. Yet, the mechanism whereby activation of (hypothalamic) ERβ differentially affects LH and FSH responses to kisspeptin waits to be elucidated. Nonetheless, the fact that blockade of ER{alpha} significantly decreased absolute FSH responses to GnRH evidenced that part of the inhibition of FSH surges induced by antagonism of this receptor subtype is due to its effects at the pituitary level, where ER{alpha}-dependent GnRH self-priming has been proven essential for the generation of the preovulatory surge of gonadotropins (7, 8).

Administration of RU-486, a synthetic steroid with antiprogesterone and antiglucocorticoid activity, to cyclic female rats at proestrus has been previously shown to decrease basal FSH secretion and to blunt the primary and secondary surges of FSH (3, 4, 25), a phenomenon that is likely to involve the disruption of both ligand-dependent and -independent actions of the receptor. Our current data are in line with those observations, because the expected elevations of FSH levels at the afternoon of proestrus and the morning of estrus were not detected after administration of our selective PR antagonist. Moreover, FSH responses to kisspeptin at proestrus were abrogated after acute blockade of PR, suggesting a state of lower FSH responsiveness. Of note, however, whereas kisspeptin administration to females treated with PR antagonist failed to induce any detectable stimulation of FSH secretion, GnRH injection in this model did evoke a modest but significant elevation in FSH levels. This observation indirectly suggests that part of the effects of blockade of progesterone actions in terms of suppression of the FSH surge are due to putative actions of PR upstream of the pituitary, likely at the hypothalamus. Of note, treatment with RU-486 has been previously shown to induce a decrease in the secretion of GnRH during the afternoon of proestrus (25), an event that might be mechanistically related to the decrease in FSH responsiveness to kisspeptin reported herein but whose molecular nature is yet to be deciphered. Admittedly, however, the blockade of PR might also induce changes in KiSS-1 gene transcription at the hypothalamus, because progesterone has been recently shown to modulate KiSS-1 mRNA levels in sheep (26), a possibility that is yet to be confirmed in rodents. Finally, the fact that the absolute FSH responses to GnRH were significantly attenuated after anti-PR treatment further confirms the pivotal role of pituitary PR in the generation of the preovulatory surge of gonadotropins (8).

In the cyclic female rat, dissociation of gonadotropin secretion along the proestrus-to-estrus transition manifests in the lowering of circulating LH in the presence of persistently elevated FSH levels (3, 4), the latter being responsible for recruitment of the next cohort of follicles (27). The mechanisms for such a secondary surge of FSH have been the subject of extensive investigation and debate, but it is globally accepted that this surge is not GnRH driven (25) but rather dependent (among other factors) on the drop in inhibin-{alpha} levels evoked by the primary surge of LH (4, 28). In good agreement, in our experiments, acute or chronic antagonism of ER{alpha} (which abolished the preovulatory surge of LH), but not of ERβ, prevented also the elevation of FSH levels at the morning of estrus. Similarly, treatment with the antagonist of PR blunted the secondary rise in circulating FSH. However, the mechanisms behind these apparently similar effects might be different, because the rise in FSH concentrations at the morning of estrus could be rescued by treatment with kisspeptin or GnRH in females injected with the antagonist of ER{alpha}, but not with PR antagonist. On the latter, the persistent blockade of ligand-independent activation of PR might be responsible for the suppression of the secondary surge of FSH (4), despite exogenous stimulation with GnRH or kisspeptin. Conversely, the apparent recovery of the secondary surge of FSH after kisspeptin or GnRH injection to females treated with ER{alpha} antagonist is likely related to the detectable, albeit significantly suppressed, LH-secretory responses elicited by those secretagogues at the preovulatory period, even after blockade of ER{alpha} (20).

In summary, we provide herein compelling evidence for the indispensable roles of ER{alpha} and PR in the generation of the preovulatory surges of FSH. This function appears to involve their ability to positively modulate FSH responses to kisspeptin at the preovulatory period, a phenomenon that seems to be present also in humans, because maximal gonadotropin responses to kisspeptins have been detected in women at the preovulatory phase of the cycle (29). In addition, our data are the first to disclose the potential stimulatory effect of ERβ signaling in the control of FSH responsiveness to kisspeptin, an effect that is opposite to its (inhibitory) action on LH secretion at the preovulatory period (20). Given the so far conspicuous lack of evidence for a discernible role of ERβ in the feedback regulation of gonadotropin secretion (22), elucidation of the sites and mechanisms of action for those ERβ-mediated events, as well as their potential physiological relevance in terms of dissociation of gonadotropin secretion at the ovulatory phase, warrants further investigation.


   Acknowledgments
 


   Footnotes
 
This work was supported by a research contract between Organon NV and the University of Cordoba and grants from Instituto de Salud Carlos III (Red de Centros RCMN C03/08 and Project PI042082; Ministerio de Sanidad, Spain). CIBER is an initiative of Instituto de Salud Carlos III (Ministerio de Sanidad, Spain).

Disclosure Statement: F.A.D., A.G.H.E., and P.I.v.N. are employed by Organon NV; M.T.-S. is a recipient of grant support from Organon NV; other authors have nothing to declare.

First Published Online July 17, 2008

Abbreviations: AUC, Area under the curve; AVPV, anteroventral periventricular nucleus; ER, estrogen receptor; GPR54, G protein-coupled receptor 54; icv, intracerebroventricular; PR, progesterone receptor.

Received April 28, 2008.

Accepted for publication July 3, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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