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Pituitary Gonadotroph Estrogen Receptor- Is Necessary for Fertility in Females

Center of Excellence in Reproductive Sciences (M.C.G., H.J.K., C.K.) and Departments of Biology (M.C.G., C.K.) and Physiology (S.J.L.), University of Kentucky, Lexington, Kentucky 40536; Department of Anatomy and Neurobiology (H.J.K.), Institute of Health Sciences, School of Medicine, Gyeongsang National University, 660-751 Jinju, Korea; School of Biotechnology and Biomedical Sciences (Y.K.), Inje University, 621-749 Kimhae, South Korea; and Institut de Genetique et de Biologie Moleculaire et Cellulaire (A.K., P.C.) (Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, College de France), Institut Clinique de la Souris, 67404 Illkirch-Strasbourg, France

Address all correspondence and requests for reprints to: CheMyong Ko, Ph.D., Division of Clinical and Reproductive Sciences, University of Kentucky, Lexington, Kentucky 40536. E-mail: cko2{at}uky.edu.

	Abstract

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Estrogens play a central role in regulating female reproduction throughout the reproductive axis, and the pituitary is one of the major targets of estrogen action. We hypothesized that estrogen receptor (ER) mediates estrogen action in the pituitary gonadotroph. To test this hypothesis, we generated a mouse line with a selective ER deletion in the gonadotropin -subunit (GSU)-expressing pituitary cells (pituitary-specific ER knockout; ER^flox/flox GSU^cre). Although the ER^flox/flox GSU^cre female mice maintain a basal level of serum LH and FSH and their ovulatory capacity is comparable to that in controls, they do not display regular estrous cycles and are infertile, indicating a potential disorder in regulating LH and/or FSH secretion. The ER^flox/flox GSU^cre female mice express equivalent levels of LHβ and GSU mRNA compared with wild-type mice as determined by microarray analysis. Taken together, these findings indicate that pituitary gonadotroph ER carries out the effects of estrogens with regard to estrous cyclicity and ultimately fertility.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
ESTROGEN FEEDBACK to the hypothalamus and pituitary is the major factor regulating LH secretion, including the LH surge (1, 2, 3). Estrogens regulate LH secretion by controlling both the basal secretion of GnRH from the hypothalamus and the GnRH surge that stimulates tonic LH secretion and the LH surge, respectively (3). In addition, estrogens have also been shown to exert their feedback actions on LH secretion at the level of the pituitary (4, 5) and are believed, by a mechanism yet to be fully delineated, to prime the pituitary for the LH surge.

The nuclear receptor transcription factors, estrogen receptor (ER) and ERβ, mediate estrogen action by targeting transcription of genes whose products will ultimately alter the physiology of the cell. Both ER and ERβ are expressed in the pituitary gonadotroph (6), yet diverse lines of evidence indicate that ER is the major mediator of estrogen action in the pituitary. Agonists for ER but not ERβ are capable of inducing LH secretion in estrogen-primed pituitaries in vitro (7, 8). ER is suggested to be the primary mediator of estrogen-induced reversal of hypertrophied gonadotrophs after ovariectomy (9). In support of these findings, ER^–/– female mice are completely infertile, have elevated levels of LH, and do not ovulate, whereas ERβ^–/– mice can ovulate, although they are subfertile (10, 11). These findings provide strong evidence indicating that ER mediates estrogen action in the pituitary; however, the in vivo studies in particular cannot distinguish between the role of ER in the pituitary and hypothalamus in reproductive function. Therefore, it remains to be established that ER mediates estrogen action in the pituitary gonadotroph by isolating the estrogen-ER system in the pituitary gonadotroph in vivo.

In this study, we investigated whether or not ER is the mediator of estrogen action in the pituitary gonadotroph in vivo. To accomplish this, we used a genetic approach by deleting ER in the pituitary gonadotroph. The results demonstrate that ER in the gonadotroph is critical for estrous cyclicity and fertility in females.

	Materials and Methods

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Reagents
Antibody for ER was purchased from Novocastra (RTU-ER-6F11; Newcastle upon Tyne, UK). Polyclonal antiserum for LHβ and TSHβ was acquired from the National Hormone and Peptide Program (Harbor-UCLA Medical Center, Torrance, CA). Pregnant mare serum gonadotropin (PMSG), human chorionic gonadotropin (hCG), and 17β-estradiol were purchased from Sigma Chemical Co. (St. Louis, MO). Molecular reagents were purchased from Invitrogen Life Technologies, Inc. (Carlsbad, CA).

Generation of ER^flox/flox GSU^cre and ER^–/– mice
The ER^flox/flox GSU^cre mice were created using the cre/loxP approach (12). The ER^flox/flox mouse was created by a targeting strategy used to generate ER^–/– mice (10). As previously described, the floxed allele L2 was produced as a consequence of partial Cre-mediated deletion of the floxed cassette (10). Inbreeding of ER^L2/+ mice produced the conditional floxed ER^L2/L2 mice, referred to as ER^flox/flox mice in this paper. ER^flox/flox mice possess two loxP sites flanking exon 3 of the ER gene (10). To generate ER^flox/flox GSU^cre mice, an ER^flox/flox male was crossed with an GSU^cre female in which the 4.6-kb promoter of the glycoprotein hormone -subunit (GSU) gene drives the expression of Cre recombinase in the GSU-expressing cells (13). The F1 heterozygote (ER^flox/+ GSU^cre) mice were then bred with ER^flox/flox mice, which gave four genotypes: ER^flox/flox GSU^cre, ER^flox/+ GSU^cre, ER^flox/flox, and ER^flox/+. Genotyping was performed by PCR using ear-biopsy DNA. Genomic DNA was isolated from ear using the Easy-DNA Kit (Invitrogen). The primer combination of ERP2F (5'-gtg tca gaa aga gac aat-3') plus ERP3 (5'-ggc att acc act tct cct ggg agt ct-3') was used to determine the presence or absence of loxP sequences (flox or wild type) (Fig. 1A). The presence of GSU^cre recombinase was determined using the primers Cga (5'-aca ttg ttc ccc tca gat cg-3') and Cre (5'-ata gtt ttt act gcc aga cc-3'). Examples of banding patterns used to determine the genotypes for three of the possibilities are shown (Fig. 1B). The generation of ER^–/– resulted from a cross of male ER^flox/flox with female Zp3^cre, a line expressing cre recombinase in the oocyte specifically. The F1 heterozygote ER^flox/+ Zp3^cre was then bred with ER^flox/flox to produce ER^flox/flox Zp3^cre. Females that are ER^flox/flox Zp3^cre produce oocytes that are ER^–. Oocytes fertilized by sperm from ER^flox/flox males result in progeny that are ER^flox/–. The breeding of two ER^flox/– mice produces one fourth of progeny that are ER^–/–. The primer set ERP1 (5'-ttg ccc gat aac aat aac at-3') plus ERP3 was used to determine whether or not exon 3 had been deleted (ER^–). Presence of Zp3 Cre recombinase was determined using primers Cre-P1 (5'-gga cat gtt cag gga tcg cca ggc g-3') and Cre-P85 (5'-gtg aaa cag cat tgc tgt cac tt-3').

View larger version (78K): [in this window] [in a new window]   FIG. 1. Generation of ERflox/flox GSUcre mice. A, Schematic diagram showing targeted deletion of exon 3 of ER at the level of genome (left) and the resulting translated product (right). The resulting protein product lacks both the DNA-binding domain (DBD) and the hormone-binding domain (HBD). AF-1 and AF-2, Transactivation domains; H, hinge region; P1, P2, and P3, primer binding sites used for genotyping; WT, wild type. B, Representative gel of PCR banding patterns showing three possible genotypes for F2 progeny. Amplification using ER-P2F and ER-P3 primers were used to detect ER+ (543 bp) or ERflox (607 bp). Primers Cga and Cre were used to determine the presence or absence of Cre recombinase. C, ER protein expression in relation to the gonadotrophs and thyrotrophs was examined in the pituitary of ERflox/flox, ERflox/flox GSUcre, and ER–/– by double immunostaining with anti-ER and anti-LHβ or anti-ER and anti-TSHβ. In ERflox/flox, cells positive for both LHβ (red-brown, cytoplasm) and ER (brown, nuclear) or TSHβ (red-brown) and ER (brown) are present as well as cells positive for ER only. Note that cells staining positive for LHβ or TSHβ in the ERflox/flox GSUcre are devoid of ER (indicated by arrows). ER–/– shows no staining for ER.

Fertility assay
The 45- to 50-d-old mice were individually housed with control mates for either 3 continuous months or in three consecutive matings. In the control group, ER^flox/flox females were mated with ER^flox/flox males. In the experimental group, ER^flox/flox GSU^cre females were paired with ER^flox/flox males. Females were monitored for pregnancy, and after giving birth, the number of pups was counted.

Determination of estrous cyclicity
Using vaginal lavage techniques (15), the pattern of estrous cycles was determined in 45- to 50-d-old female mice for 15 d. Vaginal lavage was performed daily, at the same time each day, by flushing the vagina with 0.9% sodium chloride. The cell samples were then examined under the microscope and scored. Estrus was determined by the presence of cornified cells and a very dense number of cells overall. Metestrus was scored by the presence of large round cells with an irregular border. A high density of leukocytes indicated the stage of diestrus, whereas small nucleated cells indicated proestrus (15).

Tissue collection, histology, and immunohistochemistry
Before animals were killed, the stage of the estrous cycle was determined and recorded. Animals were deeply anesthetized and perfused intracardially with PBS, followed by 4% paraformaldehyde at 1500 h. The pituitary, ovaries with attached oviducts, and uterus were collected in 10% buffered formalin and then embedded in paraffin blocks. For histology, sections were cut at 7 µm, mounted on slides, and stained with hematoxylin and eosin. Ovaries were cut in serial sections. Immunohistochemistry was performed as described previously (16). Briefly, sections were deparaffinized by treatment with xylenes and rehydrated through a graded alcohol series. Antigen unmasking was performed by boiling the sections in sodium citrate buffer (10 mM sodium citrate; and 0.05% Tween 20, pH 6.0) for 10 min in a microwave oven. After the sections were allowed to cool to room temperature, they were rinsed briefly in PBS and treated with 0.3% hydrogen peroxide (Sigma) in water for 30 min to quench endogenous peroxidase activity. Next, staining was performed by the ABC method using the Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA). For double immunostaining, slides were first labeled for the nuclear antigen ER (prediluted), followed by the cytoplasmic LHβ (1:1000) or TSHβ (1:1000); the slides were incubated for 20 min with normal blocking serum at room temperature and then incubated for 1 h at room temperature with primary antibody. Biotinylated secondary antibody was applied for 30 min, and then the slides were incubated for 30 min with ABC reagent. Slides were developed with diaminobenzidine-Ni substrate (Vector) for the ER antigen and aminoethylcarbazole (Vector) for the LHβ or TSHβ antigen until the stain was evident.

Measurement of serum LH and FSH levels
Blood samples for hormone assay were obtained by cardiac puncture at 1500 h on the day of diestrus. Plasma LH concentrations were determined in 20- and 5-µl duplicate aliquots of serum using a modification of a previously described method (National Institutes of Health National Hormone and Peptide Program). Primary antibody (rat LH antiserum-rabbit; NIDDK anti-rLH-S-11) was diluted 1:200,000 and incubated for 48 h before adding 20,000 cpm/100 µl of trace (07-C65102; MP Biomedicals, Irvine, CA) to each tube. After an overnight incubation, secondary antibody at 1:50 (anti-RGG; B37I) was added, and the reaction was incubated for 6 h before washing, pouring off supernatant, and counting the precipitates. All incubations were performed at room temperature. The sensitivity (100% to 2 SD of maximum binding) averaged 0.03 ng/tube. RIA for FSH was performed by the University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core (17).

Measurement of gonadotropin mRNA expression
DNA microarray was performed to measure the gonadotropin subunit mRNA as previously described (14). Pituitaries were collected as described in Animals and treatments, and total RNA was extracted using Trizol reagent (Invitrogen) and then purified using an RNeasy kit (QIAGEN Inc., Valencia, CA). For the primary pituitary cell culture, anterior pituitary cells were isolated from 10-wk-old female C57BL/6 mice as previously described (18) with minor modification. Isolated cells were counted and plated on poly-L-lysine-coated culture dishes containing medium (20 mM HEPES and 0.3% BSA in DMEM) supplemented with 10% fetal bovine serum. Cells were incubated in a humidified incubator at 37 C with 5% CO₂. After 2 d of culture, the incubation medium was changed with medium supplemented with 10% charcoal-treated fetal bovine serum and cultured for an additional 2 d. The cells were then treated with charcoal-treated serum containing either 0.00001% ethanol or 1 nM 17β-estradiol in 0.00001% ethanol for 2 d. Two days after estrogen treatment, cells were harvested and total RNA was extracted. To verify RNA integrity, RNA was separated on a 1.5% agarose gel with ethidium bromide and the 28S rRNA and 18S rRNA bands visualized. The total RNA used for DNA microarray was pooled from at least five mice per group for the wild-type and at least two mice per group for the ER^–/– and ER^flox/flox GSU^cre, and the assay was done in duplicate. DNA microarray was performed at the DNA Microarray Core Facility of the University of Kentucky (Lexington, KY) using the Affymetrix Mouse 430 2.0 oligonucleotide array set.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Targeting of ER in GSU-expressing cells
In this study, the Cre recombinase expression was driven by the GSU promoter, which is the common subunit shared by LH and FSH of the gonadotrophs and TSH of the thyrotrophs, so that ER could be removed only in these cell types. The excision of exon 3 of ER results in a protein product lacking the DNA-binding and hormone-binding domains as well as the AF-2 transactivation domain (Fig. 1A). The successful removal of ER in the gonadotroph and thyrotroph was confirmed by the lack of ER protein expression in the gonadotrophs and thyrotrophs of the ER^flox/flox GSU^cre, in contrast to the strong ER immunostaining that was apparent in the wild-type counterparts (Fig. 1C).

Pituitary gonadotroph ER is critical for female fertility
To determine whether pituitary gonadotroph ER is necessary for female fertility, a mating assay was performed. Matings between female ER^flox/flox GSU^cre mice and proven males did not result in any pups over a 3-month period or three consecutive matings, indicating that female ER^flox/flox GSU^cre mice are infertile (Table 1). Vaginal plugs were observed in ER^flox/flox GSU^cre females, proving that the female ER^flox/flox GSU^cre are responsive to attempts to mate. In contrast, male ER^flox/flox GSU^cre mice are fertile (data not shown). To determine whether the ER^flox/flox GSU^cre maintain ovulatory capacity, immature mice were treated with exogenous gonadotropins PMSG and hCG, oocytes counted, and ovarian histology examined. Upon examination of the ovaries, it was evident that both ER^flox/flox and ER^flox/flox GSU^cre produced corpora lutea (Fig. 2A). The number of oocytes released after the superovulation regimen was not significantly different in ER^flox/flox GSU^cre compared with ER^flox/flox (Fig. 2B). Next, ovaries from mature 1.5- to 7-month-old ER^flox/flox GSU^cre mice were examined. Ovaries of the ER^flox/flox GSU^cre mice contained primary, secondary, and preovulatory follicles. The granulosa and theca cell layer of these follicles did not show any remarkable abnormality compared with controls. Furthermore, ovaries of the ER^flox/flox GSU^cre mice contained corpora lutea (Fig. 2C). Meanwhile, no hemorrhagic cysts were observed in ER^flox/flox GSU^cre ovaries in contrast to ER^–/– mouse ovaries (Fig. 2C, b and d). However, follicular cysts were occasionally observed in the ER^flox/flox GSU^cre ovaries (Fig. 2Cc).

View this table: [in this window] [in a new window]   TABLE 1. Fertility of ERflox/flox GSUcre in mating assay

View larger version (58K): [in this window] [in a new window]   FIG. 2. Ovaries of ERflox/flox GSUcre mice are capable of releasing oocytes and producing corpora lutea. A, Immature 24-d-old mice were primed with PMSG (5 IU) and hCG (5 IU) and oocytes counted at 20 h. Ovaries were formalin fixed, paraffin embedded, and stained with hematoxylin and eosin. Note the presence of corpora lutea in both genotypes. B, ERflox/flox (n = 2) produced an average of 55 oocytes per mouse, and ERflox/flox GSUcre (n = 3) produced 38 oocytes per animal on average. C, Ovaries from 1.5- to 7-month-old adult mice were examined in three genotypes: ERflox/flox, ERflox/flox GSUcre, and ER–/–. Corpora lutea are present in both ERflox/flox (a) and ERflox/flox GSUcre (d). Some ERflox/flox GSUcre display cysts (c, indicated by arrows), although not hemorrhagic bloody cysts like those of ER–/– (b). CL, Corpora lutea.

ER in the pituitary gonadotroph is required for estrous cyclicity but not basal LH and FSH secretion

View larger version (18K): [in this window] [in a new window]   FIG. 3. ERflox/flox GSUcre females have basal levels of LH and FSH secretion but irregular estrous cyclicity. A, Vaginal lavage was performed on ERflox/flox, ERflox/flox GSUcre, and ER–/– on a daily basis for 15 d. ERflox/flox females cycle every 4–5 d, whereas ERflox/flox GSUcre display a dichotomous pattern; some mice have many consecutive days of diestrus interspersed with cornified and nucleated cells, whereas other mice show long periods of cornified cells. ER–/– show constant diestrus. All profiles are representative. D, Diestrus; E, estrus; M, metestrus; P, proestrus. B and C, Serum LH and FSH were measured in mice during diestrus. Basal LH and FSH levels in the ERflox/flox GSUcre female are comparable to those in ERflox/flox mice. This is in contrast to elevated levels of LH in the ER–/–. Significance was determined by t tests comparing ERflox/flox and ER–/– or ERflox/flox GSUcre and ER–/–. Error bars represent SEM.

Expression of LHβ, FSHβ, or GSU mRNA is not regulated by pituitary gonadotroph ER

View larger version (37K): [in this window] [in a new window]   FIG. 4. 17β-Estradiol does not increase transcription of GSU, LHβ, FSHβ, and TSHβ. A, Immunostaining for LHβ was performed on pituitaries from regularly cycling mice. Wild-type cycling mice were monitored for estrous cyclicity for 2 wk and then killed on the afternoon of estrus, metestrus, diestrus 1, or proestrus. B, Vaginal lavage was performed on 45-d-old wild-type mice twice daily at 0900 and 1500 h for at least 10 d. Mice which showed a 4- to 5-d cycle were used and killed at 1700 h on the day of metestrus or proestrus. C, Mice were OVX at 1.5–4 months of age. Three weeks later, mice were injected with either 10 µg 17β-estradiol or oil (vehicle) at 0900 h of d 1 and 0900 h of d 2. Pituitaries were collected at 1500 h on d 2 from wild-type (WT) OVX plus vehicle, WT OVX plus 17β-estradiol, ER–/– OVX plus vehicle, ER–/– OVX plus 17β-estradiol, ERflox/flox GSUcre OVX plus vehicle, and ERflox/flox GSUcre OVXplus 17β-estradiol. D, Primary pituitary cells plus vehicle (n = 2), primary pituitary cells plus 17β-estradiol (n = 2). The total RNA used for DNA microarray was pooled from at least five mice for WT metestrus, WT proestrus, and WT OVX and at least two mice for ER–/– OVX and ERflox/flox GSUcre OVX. The array was done in duplicate. Data are represented by mean plus SEM from two independent array results.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Disruption of ER in the pituitary gonadotroph alone produces several notable differences compared with mice in which ER is globally deleted. ER^–/– females have elevated levels of serum LH as well as blood-filled hemorrhagic cysts, which form by 2 months of age, and absence of corpus lutea (10, 19, 20). Basal LH and FSH levels are not elevated in the ER^flox/flox GSU^cre. This finding in comparison with the ER^–/– phenotype provides support that in mice, the pituitary is not the primary target for the negative feedback of estrogens, but rather the hypothalamus, as has been concluded in other species (1). The normal level of serum FSH and LH secretion supports the observation of primary and secondary follicles in the ovaries of the ER^flox/flox GSU^cre mice. PMSG/hCG-induced ovulation resulted in release of a comparable number of oocytes from the ER^flox/flox GSU^cre ovary, demonstrating that the ovarian mechanisms necessary for ovulation are intact. Although a problem with the ovary cannot be ruled out, this result implies that the loss of the estrogen/ER pathway in the gonadotroph is responsible for the infertile phenotype of the ER^flox/flox GSU^cre female mice. Furthermore, corpora lutea are present in adult ER^flox/flox GSU^cre mouse ovaries, indicating that spontaneous ovulation may occur in the intact ER^flox/flox GSU^cre female mice. It is important to consider that corpora lutea can form in the absence of ovulation (21), and normal numbers of corpora lutea can form despite a reduced LH surge (22). However, the irregular estrous cycle of these mice implies that the LH surge is mistimed, attenuated, or absent, so it is probable that the ovulation could be caused by irregular elevations of LH in the ER^flox/flox GSU^cre mice.

ER is also absent in the thyrotrophs of ER^flox/flox GSU^cre mice; thus, it is possible that TSH could be affected in these mice. Changes in TSH and thyroid hormone can impact reproduction. Hypothyroid rats have irregular estrous cycles, specifically a prolonged diestrus (23). Although some ER^flox/flox GSU^cre mice display a prolonged diestrus, others display prolonged days of cornified cells. Additionally, hypothyroid female rats have a decreased basal serum LH (23). In contrast, the basal serum LH of ER^flox/flox GSU^cre mice is not decreased. Hypothyroid female rats that were given an hCG challenge on the day of diestrus showed a significant decrease in the number of oocytes released compared with controls (23). A challenge of PMSG and hCG to immature ER^flox/flox GSU^cre mice did not result in a significant decreased release of oocytes. This evidence suggests that the reproductive problems of the ER^flox/flox GSU^cre mice are not due to decreases in serum TSH.

Based on our evidence that ER is indeed the mediator of estrogen action in the gonadotroph, we examined the role of ER in gene expression of hormone subunits. First we tested whether 17β-estradiol induced the transcription of GSU, LHβ, FSHβ, and TSHβ. In neither regularly cycling wild-type mice nor the OVX model did 17β-estradiol increase the transcription of GSU, LHβ, or TSHβ. Thus, it is not surprising that absence of ER did not serve to reduce the amount of mRNA transcript for these three subunits (Fig. 4C). It was surprising to find that 17β-estradiol did not increase transcription of LHβ, given the fact that an estrogen-responsive element has been identified in the rat LHβ gene (24) and that estrogen directly increases transcription of the rat gene in vitro (25). However, it has been suggested that the positive-feedback action of estrogens that generate the LH surge regulates LH secretion, not synthesis (26), and our model was designed to mimic the long exposure of estrogens that occurs before the surge. It is possible that there may have been a small immediate increase in transcription after 17β-estradiol injection, which later returned to basal. There is also evidence that LHβ mRNA increases before the LH surge in cycling rats (27). This increase, however, does not point to a direct effect of estrogens and may be explained by increased secretion of GnRH. It has also been shown that the loss of ER in the total ER^–/– results in increases in transcript of gonadotropin subunits (20), suggesting that ER regulates this transcription. Once again, the total ER^–/– does not isolate effects in the pituitary because ER is absent in all tissues, and this increase may likely be due to some disruption of estrogen’s regulation of GnRH, and consequently GnRH’s control of gonadotrophin transcription, and may explain the difference in the ER^flox/flox GSU^cre that did not show an increase and are presumed to have normal GnRH pulsatility.

Estrogens have been shown to have positive feedback effect at the level of pituitary and pituitary gonadotroph in sheep and rats (1, 5). Recently, neuron-specific ER^–/– mice were found to be infertile and unable to respond to the positive-feedback action of estradiol, providing evidence that the brain is important for positive estrogen feedback as well (28). The lack of ER in the pituitary gonadotroph may affect positive feedback in the ER^flox/flox GSU^cre mice, which has yet to be tested. In fact, the ER agonist propylpyrazole-triol elicits LH secretion from propylpyrazole-triol-primed rat pituitaries in response to consecutive GnRH challenges in vitro, comparable to that induced by 17β-estradiol; the ERβ agonist diarylpropionitrile gave no such response (7). In addition, tamoxifen-treated rats induced ER expression in gonadotrophs, eliciting LH secretion, presumably through ER (8). Finally, estradiol treatment does not increase LH secretion in ER^–/– gonadotroph cells in vitro (29). Our results show that pituitary gonadotroph ER does not affect basal LH secretion; this result in combination with the above data leads us to speculate that gonadotroph ER does have a role in the positive feedback of estrogens. Interestingly, males do not have the unique positive-feedback system that governs the LH surge, and ER^flox/flox GSU^cre males are fertile. However, it has yet to be determined whether the cause of infertility in the ER^flox/flox GSU^cre female mice is due to a problem with ovarian function, oocyte health, maintenance of pregnancy, or disruption of positive feedback and the LH surge.

In conclusion, we have shown for the first time direct in vivo evidence that ER is the mediator of estrogen action in the pituitary gonadotroph. In particular, a direct action of estrogen/ER in the pituitary gonadotroph is required for fertility and estrous cyclicity, but not the regulation of basal LH and FSH secretion. We speculate that estrogen/ER is regulating positive feedback, the LH surge, and estrous cyclicity and that the genes under this control are involved in regulation of LH secretion itself, rather than expression of hormone subunits. Future studies will further investigate the role of ER and induced genes in LH secretion during the LH surge.

	Acknowledgments

 
We thank Dr. Phillip Bridges for his critical review and comments in the preparation of this manuscript and Dr. Dong-Wook Kang for consultation on immunostaining.

	Footnotes

 
This work was supported by Grants P20 RR15592 (C.K.) and 1RO1HD052694-01 (C.K.) from the National Institutes of Health. RIA for FSH was performed by the University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core [NICHD (SCCPRR) Grant U54-HD28934, University of Virginia, Charlottesville, VA].

Disclosure Summary: The authors have nothing to disclose.

First Published Online October 18, 2007

Abbreviations: ER, Estrogen receptor; GSU, glycoprotein hormone -subunit; hCG, human chorionic gonadotropin; PMSG, pregnant mare serum gonadotropin; OVX, ovariectomized.

Received August 6, 2007.

Accepted for publication October 10, 2007.

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