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The estrogen receptor (ER) is expressed in two forms, ER␣ and ER␤. Here we show that ER␣ and ER␤, expressed both in vitro and in vivo, form heterodimers which bind to DNA with an affinity (K d of approximately 2 nM) similar to that of ER␣ and greater than that of ER␤ homodimers. Mutation analysis of the hormone binding domain of ER␣ suggests that the dimerization interface required to form heterodimers with ER␤ is very similar but not identical to that required for homodimer formation. The heterodimer, like the homodimers, are capable of binding the steroid receptor coactivator-1 when bound to DNA and stimulating transcription of a reporter gene in transfected cells. Given the relative expression of ER␣ and ER␤ in tissues and the difference in DNA binding activity between ER␣/ER␤ heterodimers and ER␤ it seems likely that the heterodimer is functionally active in a subset of target cells. Estrogen receptors (ER)1 were recently shown to be encoded by two distinct genes, ER␣ and ER␤ (1, 2). Reverse transcription-polymerase chain reaction (PCR) analysis indicates that ER␤ is highly expressed in prostate and ovary (1, 2), but moderate expression was detected in many other tissues including testis and uterus, some of which also seem to express ER␣ (3). The two receptors which share about 95% homology in the DNA binding domain and 55% homology in the ligand binding domain, both bind to a consensus estrogen response element (ERE) (4) and exhibit similar ligand binding properties (3). They are poorly conserved in the N-terminal domain but ER␤, like ER␣, appears to contain a similar activation domain, activation function 1 (AF-1) sensitive to a mitogen-activated protein kinase pathway (4 -6). In addition, both receptors contain a second activation domain, activation function 2 (AF-2) (7, 8), whose activity is enhanced by the coactivator SRC-1 (4, 9, 10). Thus, although the relative expression of ER␣ and ER␤ varies in cells, their ligand binding, DNA binding, and transactivation properties are rather similar to one another.Steroid hormone receptors usually bind to inverted DNA repeats as homodimers, although the glucocorticoid and mineralocorticoid receptors have been reported to form heterodimers, at least in vitro (11,12). In the classically accepted model of steroid hormone action, the estrogen receptor is sequestered in an inactive state in a multiprotein complex in the absence of hormone (13). Upon estrogen binding, the receptor forms homodimers which then interact with response elements in the vicinity of target genes and modulate rates of gene transcription. In view of the similarity of the ligand binding domain of ER␣ and ER␤ we investigated the possibility that the two receptors may form functional heterodimers in target cells. ER␣ and ER␤ were capable of forming heterodimers on DNA that could bind the coactivator, SRC-1, and appeared to stimulate transcription of a reporter gene. Moreover, we demonstrate that while the region of ER␣ required for homodimerization overlaps with that required for heterodimerization ...

Controlled ovarian hyperstimulation (COH) used in IVF produces lower implantation rates per embryo transferred compared to natural cycles utilized in ovum donation, suggesting a suboptimal endometrial development. Endometrial receptivity has recently been investigated in natural menstrual cycles with the aid of microarray technology. The aim of this study is to investigate the impact of COH using urinary gonadotrophins with a long protocol with GnRH agonists without progesterone supplementation (similar to the natural cycle) on endometrial gene expression profiles during the window of implantation by comparing the profiles at day hCG + 7 of COH versus LH + 7 of a previous natural cycle in the same women. For this purpose we have used microarray technology by Affymetrix (GeneChip HG_U133A), which allows more than 22,000 genes to be tested simultaneously. Results were validated by semi-quantitative PCR and quantitative PCR experiments. We found that more than 200 genes showed a differential expression of more than 3-fold when COH and normal cycles were compared at hCG + 7 versus LH + 7. We simultaneously re-analysed the LH + 2 versus LH + 7 endometrial gene expression profiles in previous natural cycles in the same subject using this specific GeneChip, the results obtained were consistent with our own published results. This is the first time that gene expression profiles of the endometrium during COH are reported. The large degree of gene expression disturbance is surprising and highlights the need for further efforts to optimize COH protocols.

LGR7 is a G-protein coupled receptor with structural homology to the gonadotrophin and thyrotrophin receptors. Recently, LGR7 was deorphanized, and it was shown that relaxin is the ligand for LGR7. To further study the function of this receptor, mice deficient for LGR7 were generated by replacing part of the transmembrane-encoding region with a LacZ reporter cassette. Here we show that LGR7 is expressed in various tissues, including the uterus, heart, brain, and testis. Fertility studies using female LGR7 ؊/؊ mice showed normal fertility and litter size. However, some females were incapable of delivering their pups, and several pups were found dead. Moreover, all offspring died within 24 to 48 h after delivery because female LGR7 ؊/؊ mice were unable to feed their offspring due to impaired nipple development. In some male LGR7 ؊/؊ mice, spermatogenesis was impaired, leading to azoospermia and a reduction in fertility. Interestingly, these phenomena were absent in mutant mice at older ages or in later generations. Taken together, results from LGR7 knockout mice indicate an essential role for the LGR7 receptor in nipple development during pregnancy. Moreover, a defect in parturition was observed, suggesting a role for LGR7 in the process of cervical ripening.LGR7 is a leucine-rich repeat-containing G-protein coupled receptor that was identified on the basis of its structural homology to the follicle-stimulating hormone, luteinizing hormone, and thyrotropin receptor family (4, 5). An interesting feature of these receptors are their large extracellular domains with leucine-rich repeats, which are commonly found in proteins involved in protein-protein interactions (8). Recently, LGR7 has been deorphanized, and it was shown that relaxin is the high-affinity ligand (6). The relaxin hormone has been studied in detail over the last few decades and has been implicated in many physiological processes related to the female reproductive system, including the induction of collagen remodeling and softening of the tissues of the birth canal prior to delivery. Also, the inhibition of uterine contractile activity and development of growth and differentiation of the mammary gland have been reported (2). In addition, relaxin has been implicated to trigger blood vessel dilatation in several organs and tissues, such as rat and mouse uterine endometrium and myometrium and mammary glands. Moreover, it has been shown that relaxin increases coronary flow, an effect which was paralleled by an increase of the production of nitric oxide, a potent vasodilatory agent (15).The observation that mice deficient for relaxin are unable to deliver milk to their pups due to impaired mammary gland development and the absence of nipple enlargement at the end of pregnancy has provided the first evidence for the function of relaxin. Moreover, it was reported that some of the mice were unable to deliver, most probably due to the absence of cervical softening or contractile activity (20). Phenotypic data related to the role of relaxin cardiac function indi...

Polycystic ovary syndrome (PCOS) affects 5% of reproductive aged women and is the leading cause of anovulatory infertility. A hallmark of PCOS is excessive theca cell androgen secretion, which is directly linked to the symptoms of PCOS. Our previous studies demonstrated that theca cells from PCOS ovaries maintained in long term culture persistently secrete significantly greater amounts of androgens than normal theca cells, suggesting an intrinsic abnormality. Furthermore, previous studies suggested that ovarian hyperandrogenemia is inherited as an autosomal dominant trait. However, the genes responsible for ovarian hyperandrogenemia of PCOS have not been identified. In this present study, we carried out microarray analysis to define the gene networks involved in excess androgen synthesis by the PCOS theca cells in order to identify candidate PCOS genes. Our analysis revealed that PCOS theca cells have a gene expression profile that is distinct from normal theca cells. Included in the cohort of genes with increased mRNA abundance in PCOS theca cells were aldehyde dehydrogenase 6 and retinol dehydrogenase 2, which play a role in all-trans-retinoic acid biosynthesis and the transcription factor GATA6. We demonstrated that retinoic acid and GATA6 increased the expression of 17␣-hydroxylase, providing a functional link between altered gene expression and intrinsic abnormalities in PCOS theca cells. Thus, our analyses have 1) defined a stable molecular phenotype of PCOS theca cells, 2) suggested new mechanisms for excess androgen synthesis by PCOS theca cells, and 3) identified new candidate genes that may be involved in the genetic etiology of PCOS.

Successful implantation absolutely depends on the reciprocal interaction between the implantation-competent blastocyst and the receptive uterus. Expression and gene targeting studies have shown that leukemia inhibitory factor (LIF), a cytokine of the IL-6 family, and Hoxa-10, an abdominalB-like homeobox gene, are crucial to implantation and decidualization in mice. Using these mutant mice, we sought to determine the importance of Msx-1 (another homeobox gene formerly known as Hox-7.1) and of Wnt4 (a ligand of the Wnt family) signaling in implantation because of their reported functions during development. We observed that Msx-1, Wnt4, and a Wnt antagonist sFRP4 are differentially expressed in the mouse uterus during the periimplantation period, suggesting their role in implantation. In addition, we observed an aberrant uterine expression of Msx-1 and sFRP4 in Lif mutant mice, and of Wnt4 and sFRP4 in Hoxa-10 mutant mice, further reinforcing the importance of these signaling pathways in implantation. Collectively, the present results provide evidence for a novel cytokine-homeotic-Wnt signaling network in implantation.

It recently has been recognized that men develop colonic adenomas and carcinomas at an earlier age and at a higher rate than women. In the Apc Pirc/+ (Pirc) rat model of early colonic cancer, this sex susceptibility was recapitulated, with male Pirc rats developing twice as many adenomas as females. Analysis of large datasets revealed that the Apc Min/+ mouse also shows enhanced male susceptibility to adenomagenesis, but only in the colon. In addition, WT mice treated with injections of the carcinogen azoxymethane (AOM) showed increased numbers of colonic adenomas in males. The mechanism underlying these observations was investigated by manipulation of hormonal status. The preponderance of colonic adenomas in the Pirc rat model allowed a statistically significant investigation in vivo of the mechanism of sex hormone action on the development of colonic adenomas. Females depleted of endogenous hormones by ovariectomy did not exhibit a change in prevalence of adenomas, nor was any effect observed with replacement of one or a combination of female hormones. In contrast, depletion of male hormones by orchidectomy (castration) markedly protected the Pirc rat from adenoma development, whereas supplementation with testosterone reversed that effect. These observations were recapitulated in the AOM mouse model. Androgen receptor was undetectable in the colon or adenomas, making it likely that testosterone acts indirectly on the tumor lineage. Our findings suggest that indirect tumor-promoting effects of testosterone likely explain the disparity between the sexes in the development of colonic adenomas.colon cancer | animal models | androgens | estrogens | intestinal regionality E pidemiologic studies have identified a number of factors that influence the risk of sporadic adenomas and colorectal cancer (CRC). Age, familial predisposition, racial background, diet, physical activity, obesity and the metabolic syndrome, smoking, and heavy alcohol use are all established risk factors for the development of CRC. In addition, the risk of CRC also shows sexual dimorphism, with a lower incidence and delayed onset in women (1, 2). Colonoscopic screening of asymptomatic individuals has corroborated male sex as a risk factor for the development of both adenomas and CRC in all age groups (3, 4); however, whether this disparity depends on protective factors in women, tumor-promoting factors in males, or both is unknown.A protective role of female hormones against the development of frank CRC is suggested by data from the Women's Health Initiative (WHI). In the WHI, two large randomized controlled trials examined the effects of hormonal replacement therapy on postmenopausal women over a 5-y period, using CRC development as one of the endpoints. The first study showed that combined treatment with both equine estrogen (E2) and medroxyprogesterone acetate (MPA) substantially reduced the risk of colorectal cancer compared with placebo (odds ratio, 0.63) after a 5-y follow-up (5). However, protection was not found in a second randomized cont...

Human thyrotropin (TSH), luteotropin (LH), follitropin (FSH), and chorionic gonadotropin are members of the heterodimeric glycoprotein hormone family. The common alpha subunit forms noncovalent heterodimers with different beta subunits. Two novel human glycoprotein hormonelike genes, alpha2 (A2) and beta5 (B5), recently have been identified. Using a yeast two-hybrid assay, the two subunits were found as potential heterodimerization partners. Immunological analyses confirmed the heterodimerization of A2 and B5 in transfected cells and their colocalization in the anterior pituitary. Recombinant A2/B5 heterodimeric glycoproteins, purified using cation exchange and size fractionation chromatography, activated human TSH receptors, but not LH and FSH receptors, and showed high affinity to TSH receptors in a radioligand receptor assay. The heterodimer also stimulated cAMP production and thymidine incorporation by cultured thyroid cells and increased serum thyroxine levels in TSH-suppressed rats in vivo. This new heterodimeric glycoprotein hormone was named as thyrostimulin based on its thyroid-stimulating activity. The expression of thyrostimulin in the anterior pituitary known to express TSH receptors suggested a paracrine mechanism. The present discovery of a new ligand based on genomic approaches could facilitate the understanding of the physiological roles of extra-thyroid TSH receptor systems and the structural-functional basis of receptor signaling by related glycoprotein hormones.