Pituitary gonadotroph estrogen receptor-alpha is necessary for fertility in females

Abstract

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 alpha (ERalpha) mediates estrogen action in the pituitary gonadotroph. To test this hypothesis, we generated a mouse line with a selective ERalpha deletion in the gonadotropin alpha-subunit (alphaGSU)-expressing pituitary cells (pituitary-specific ERalpha knockout; ERalpha(flox/flox) alphaGSU(cre)). Although the ERalpha(flox/flox) alphaGSU(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 ERalpha(flox/flox) alphaGSU(cre) female mice express equivalent levels of LHbeta and alphaGSU mRNA compared with wild-type mice as determined by microarray analysis. Taken together, these findings indicate that pituitary gonadotroph ERalpha carries out the effects of estrogens with regard to estrous cyclicity and ultimately fertility.

Figure 1

Generation of ERα^flox/flox αGSU^cre mice. A, Schematic diagram showing targeted deletion of exon 3 of ERα 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 ERα-P2F and ERα-P3 primers were used to detect ERα⁺ (543 bp) or ERα^flox (607 bp). Primers Cga and Cre were used to determine the presence or absence of Cre recombinase. C, ERα protein expression in relation to the gonadotrophs and thyrotrophs was examined in the pituitary of ERα^flox/flox, ERα^flox/flox αGSU^cre, and ERα^−/− by double immunostaining with anti-ERα and anti-LHβ or anti-ERα and anti-TSHβ. In ERα^flox/flox, cells positive for both LHβ (red-brown, cytoplasm) and ERα (brown, nuclear) or TSHβ (red-brown) and ERα (brown) are present as well as cells positive for ERα only. Note that cells staining positive for LHβ or TSHβ in the ERα^flox/flox αGSU^cre are devoid of ERα (indicated by arrows). ERα^−/− shows no staining for ERα.

Figure 2

Ovaries of ERα^flox/flox αGSU^cre 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, ERα^flox/flox (n = 2) produced an average of 55 oocytes per mouse, and ERα^flox/flox αGSU^cre (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: ERα^flox/flox, ERα^flox/flox αGSU^cre, and ERα^−/−. Corpora lutea are present in both ERα^flox/flox (a) and ERα^flox/flox αGSU^cre (d). Some ERα^flox/flox αGSU^cre display cysts (c, indicated by arrows), although not hemorrhagic bloody cysts like those of ERα^−/− (b). CL, Corpora lutea.

Figure 3

ERα^flox/flox αGSU^cre females have basal levels of LH and FSH secretion but irregular estrous cyclicity. A, Vaginal lavage was performed on ERα^flox/flox, ERα^flox/flox αGSU^cre, and ERα^−/− on a daily basis for 15 d. ERα^flox/flox females cycle every 4–5 d, whereas ERα^flox/flox αGSU^cre 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. ERα^−/− 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 ERα^flox/flox αGSU^cre female are comparable to those in ERα^flox/flox mice. This is in contrast to elevated levels of LH in the ERα^−/−. Significance was determined by t tests comparing ERα^flox/flox and ERα^−/− or ERα^flox/flox αGSU^cre and ERα^−/−. Error bars represent sem.

Figure 4

17β-Estradiol does not increase transcription of αGSU, 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, ERα^−/− OVX plus vehicle, ERα^−/− OVX plus 17β-estradiol, ERα^flox/flox αGSU^cre OVX plus vehicle, and ERα^flox/flox αGSU^cre 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 ERα^−/− OVX and ERα^flox/flox αGSU^cre OVX. The array was done in duplicate. Data are represented by mean plus sem from two independent array results.