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Ovarian hyperstimulation syndrome (OHSS) is a severe complication arising from controlled stimulation treatment. Vascular endothelial growth factor (VEGF) has recently emerged as an important factor which may be responsible for the hyperpermeability seen in OHSS. The purpose of the present study was to investigate and compare the mechanisms by which ascites in patients with OHSS and ovarian carcinoma induce increases in vascular permeability in an in vitro assay and an in vivo animal experiment. We found 8-fold lower VEGF levels in ascites from patients with OHSS than in those from patients with ovarian carcinoma. Although VEGF is produced by the ovaries, it is not necessarily the factor responsible for hyperpermeability. We also demonstrated that the vascular hyperpermeability produced by OHSS ascites was not abolished by specific neutralizing anti-VEGF antibodies, and that not all of the VEGF found in the ascites fluid is biologically active. Moreover, our results strongly suggest that the vascular permeability produced by OHSS ascites may depend on activation of the kallikrein-kinin system. Possible evidence for this phenomenon was obtained by demonstrating that the hyperpermeability caused by the ascites could be blocked by Trasylol (known to inhibit bradykinin synthesis) and potentiated by captopril (a kininase II inhibitor). Taken together, the results suggest that, although VEGF is found in ascites fluid from patients with OHSS, it is unlikely that the cause of OHSS involves VEGF production by the ovaries. The kallikrein-kinin system may be more important in the hyperpermeability seen in OHSS.

Developmental exposure to a synthetic estrogen, diethylstilbestrol (DES), induces carcinogenesis in human and laboratory animals. In mice, neonatal DES treatment induces persistent proliferation and keratinization of the vaginal epithelium, even in the absence of the ovaries, resulting in cancerous lesions later in life. To understand the mechanisms underlying this persistent cell proliferation and differentiation, we characterized the gene expression patterns in the neonatally DES-exposed mouse vagina using DNA microarray and real-time quantitative RT-PCR. We found that genes related to cellular signaling, which are candidates for mediating the persistent proliferation and differentiation, were altered, and genes related to the immune system were decreased in the neonatally DES-exposed mouse vagina. We also noted high expression of interleukin-1 (IL-1)-related genes accompanied by phosphorylation of JNK1. In addition, expression IGF-I and its binding proteins was modulated and led to phosphorylation of IGF-I receptor and Akt, which is one of the downstream factors of IGF-I signaling. This led us to characterize the expression as well as the phosphorylation status of IL-1 and IGF-I signaling pathway components which may activate the phosphorylation cascade in the vagina of mice exposed neonatally to DES. These findings give insight into persistent activation in the vagina of mice exposed neonatally to DES.

In order to understand early events caused by estrogen in vivo, temporal uterine gene expression profiles at early stages were examined using DNA microarray analysis. Ovariectomized mice were exposed to 17beta-estradiol and the temporal mRNA expression changes of ten thousand various genes were analyzed. Clustering analysis revealed that there are at least two phases of gene activation during the period up to six hours. One involved immediate-early genes, which included certain transcription factors and growth factors as well as oncogenes. The other involved early-late genes, which included genes related to RNA and protein synthesis. In clusters of down-regulated genes, transcription factors, proteases, apoptosis and cell cycle genes were found. These hormone-inducible genes were not induced in estrogen receptor (ER) alpha knockout mice. Although expression of ERbeta is known in the uterus, these findings indicate the importance of ERalpha in the changes in gene expression in the uterus.

Administration of physiological and non-physiological estrogens during pregnancy or after birth is known to have adverse effects on the development of the reproductive tract and other organs. Although it is believed that both estrogens have similar effects on gene expression, this view has not been tested systematically. To compare the effects of physiological (estradiol; E2) and non-physiological (diethylstilbestrol; DES) estrogens, we used DNA microarray analysis to examine the uterine gene expression patterns induced by the two estrogens. Although E2 and DES induced many genes to respond in the same way, different groups of genes showed varying levels of maximal activities to each estrogen, resulting in different dose-response patterns. Thus, each estrogen has a distinct effect on uterine gene expression. The genes were classified into clusters according to their dose-responses to the two estrogens. Of the eight clusters, only two correlated well with the uterotropic effect of different doses of E2. One of these clusters contained genes that were upregulated by E2, which included genes encoding several stress proteins and transcription factors. The other cluster contained genes that were downregulated by E2, including genes related to metabolism, transcription and detoxification processes. The expression of these genes in estrogen receptor-deficient mice was not affected by E2 treatment, indicating that these genes are affected by the E2-bound estrogen receptor. Thus, of the many genes that are affected by estrogen, it was suggested that only a small number are directly involved in the uterotropic effects of estrogen treatment.

Prolactin (Prl) receptor (Prlr) gene is expressed in various brain regions, with the highest level present in the choroid plexus, a site for receptor-mediated PRL transport from the blood to cerebrospinal fluid. We investigated the regulatory mechanism of Prlr gene expression by PRL in the murine choroid plexus. We first examined the organization of the alternative first exons in murine Prlr gene. In addition to the three known first exons, mE1₁, mE1₂, and mE1₃, two first exons, mE1₄ and mE1₅, were newly identified by cDNA cloning. Each first exon variant of Prlr mRNA exhibited tissue-specific or generic expression. In the choroid plexus of mice, the expression levels of mE1 ₃ -, mE1 ₄ -, and mE1 ₅ -Prlr mRNAs were increased in the lactating mice compared with those in the diestrus mice. Furthermore, the expression level of mE1 ₄ -Prlr mRNA was decreased in the PRL-deficient (Prl ^−/− ) mice compared with the PRL-normal (Prl ^+/+ and Prl ^+/− ) mice. In the ovariectomized Prl ^−/− mice, the expression level of mE1 ₄ -Prlr mRNA was significantly increased by PRL administration but not by 17β-estradiol administration. The expression levels of the two last exon variants of Prlr mRNAs, encoding the long and short cytoplasmic regions of PRLR, were also increased in the lactating mice and decreased in the Prl ^−/− mice. These findings suggest that PRL stimulates the Prlr gene expression through the transcriptional activation of mE1₄ first exon, leading to increases in the long- and short-form variants of Prlr mRNA in the murine choroid plexus.

Recent studies have revealed that hundreds of genes in the uterus are activated by estrogen. Their expression profiles differ over time and doses and it is not clear whether all these genes are directly regulated by estrogen via the estrogen receptor. To select the genes that may be regulated by estrogen, we treated mice with several doses of estrogen and searched for those genes whose dose–response expression pattern mirrored the uterine growth pattern. Among those genes, we found that the dose-dependent expression of the adrenomedullin (ADM) gene correlated well with the uterotrophic effect of estrogen. ADM expression is induced early after estrogen administration and is restricted to the endometrial stroma. The spatiotemporal gene expression pattern of ADM was similar to that of receptor-modifying protein 3 (RAMP3). RAMP3 is known to modify calcitonin gene-related receptor (CRLR) so that it can then serve as an ADM receptor. Chromatin immunoprecipitation assays indicated that the estrogen receptor binds directly to the ADM promoter region and RAMP3 intron after estrogen administration. It was also shown that neither the ADM nor RAMP3 gene could be activated in estrogen receptor-α-null mouse. Although uterine ADM expression has been reported to occur in the myometrium, our observations indicate that estrogen-induced ADM is also expressed in the uterine stroma and that such variable, spatiotemporally regulated ADM expression contributes to a wider range of biological effects than previously expected.

Chicken ovalbumin upstream promoter-transcription factors (COUP-TFs) are orphan receptors involved in regulation of neurogenesis and organogenesis. COUP-TF family members are generally considered to be transcriptional repressors and several mechanisms have been proposed to underlie this activity. To explore novel transcriptional coregulators for COUP-TFs, we used the COUP-TFI as bait in a yeast two-hybrid screen of an adrenocortical adenoma cDNA library. We have identified Ubc9, a class E2 conjugating enzyme of small ubiquitin-related modifier (SUMO)-1 as a COUP-TFI corepressor. Ubc9 interacts with COUP-TFI in yeast and in glutathione S-transferase pulldown and coimmunoprecipitation assays. Fluorescence imaging studies show that both Ubc9 and COUP-TFI are colocalized in the nuclei of transfected COS-1 cells. The C-terminal region of Ubc9 encoding amino acids 59-158 interacts with the C-terminus of COUP-TFI encoding amino acids 383-403, in which transcriptional repression domains are located. Mammalian one-hybrid assays utilizing a variety of Ubc9 fragments fused to Gal4 DNA-binding domain show that a Ubc9 fragment encoding amino acids 1-89 contains autonomous transferrable repression domain. Transfection of Ubc9 into COS-1 cells markedly enhances transcriptional repression by Gal4 DNA-binding domain-fused to COUP-TFI(155-423), but not by Gal4-COUP-TFI(155-388) which lacks a repressor domain. Coexpression of a C-terminal deletion mutant of Ubc9(1-58), which fails to interact with COUP-TFI, but retains a transcriptional repression domain, has no effect on Gal4-COUP-TFI-mediated repression activity. These findings indicate that interaction of Ubc9 with COUP-TFI is crucial for the corepressor function of Ubc9. Overexpression of Ubc9 similarly enhances COUP-TFI-dependent repression of the promoter activity of the bovine CYP17 gene encoding steroid 17alpha-hydroxylase. In addition, the C93S mutant of Ubc9, which abrogates SUMO-1 conjugation activity, continues to function as a COUP-TFI corepressor. Our studies indicate that Ubc9 functions as a novel COUP-TFI corepressor, the function of which is distinct from its SUMO-1 conjugating enzyme activity.