No abstract

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.