During early pregnancy, steroid hormones estrogen (E) and progesterone (P) regulate a complex series of interactions between the implanting embryo and the uterus by controlling the proliferation and differentiation of uterine epithelium and stroma in a timely manner. To identify the steroid-regulated genes that control these functions, we performed messenger RNA profiling of mouse uterine tissues at the time of implantation. Our analysis revealed that the expression of the transcription factor CCAAT͞enhancer-binding protein ␤ (C͞EBP␤) is rapidly induced in the pregnant uterus at the time of blastocyst attachment. The expression of C͞EBP␤ increased further during the decidualization phase of pregnancy and was localized in the proliferating as well as the decidualized stromal cells surrounding the implanted embryo. Administration of E or P to ovariectomized females induced C͞EBP␤ expression in both uterine epithelium and stroma, showing a dual regulation of this gene by these hormones. The female C͞EBP␤-null mice are infertile. We, therefore, assessed steroid-hormone-dependent responses in the uteri of these mice. We observed that E-induced proliferation of uterine epithelial cells is markedly compromised in the absence of C͞EBP␤. Most strikingly, there was a complete lack of response of the C͞EBP␤-deficient uteri to an artificial deciduogenic stimulus, indicating a critical role of this transcription factor in regulating the decidualization program. Further analysis revealed defects in steroid-induced stromal cell proliferation and differentiation in C͞EBP␤-null uteri. Collectively, our studies established that C͞EBP␤ is a key mediator of steroid responsiveness of the epithelium and stroma in the mouse uterus.estrogen ͉ progesterone ͉ implantation ͉ decidualization T he physiological functions of the mammalian uterus are profoundly influenced by the concerted actions of steroid hormones estrogen (E) and progesterone (P). Waves of steroidhormone-induced cell proliferation and differentiation dictate the cyclical changes that occur in the uterine epithelium during the reproductive cycle (1-4). During pregnancy, these hormones orchestrate the changes in the uterine epithelium that make it competent to attach to the blastocyst to initiate the process of implantation (1-4). Subsequently, E and P regulate a series of complex interactions between the developing embryo and the cells in the stromal compartment leading to the formation of the decidua, which nourishes the embryo and maintains early pregnancy. Although the details of these events vary in different species, the central roles played by E and P in controlling various phases of early pregnancy are common to many mammals (5).The mouse has served as an important animal model to study the regulation of uterine functions by E and P (6-8). The development of mutant mouse models lacking the estrogen receptor (ER) ␣ and progesterone receptor (PR) has firmly established the requirement of these hormones and their downstream signaling pathways for successful establishmen...

The cellular actions of steroid hormone progesterone (P) are mediated via its nuclear receptors, which regulate the expression of specific target genes. The identity of gene networks that are regulated by the P receptors (PRs) in the uterus at various stages of the reproductive cycle and pregnancy, however, remain largely unknown. In this study, we have used oligonucleotide microarrays to identify mRNAs whose expression in the pregnant mouse uterus is modulated by RU486, a well-characterized PR antagonist, which is also an effective inhibitor of implantation. We found that, in response to RU486, expression of mRNAs corresponding to 78 known genes was down-regulated at least 2-fold in the preimplantation mouse uterus. The PR regulation of several of these genes was ascertained by administering P to ovariectomized wild-type and PR knockout (PRKO) mice. Detailed spatio-temporal analysis of these genes in the pregnant uterus indicated that their expression in the epithelium and stroma could be correlated with the expression of PR in those cell types. Furthermore, time-course studies suggested that many of these genes are likely primary targets of PR regulation. We also identified 70 known genes that were up-regulated at least 2-fold in the pregnant uterus in response to RU486. Interestingly, initial examination of a number of RU486-inducible genes reveals that their uterine expression is also regulated by estrogen. The identification of several novel PR-regulated gene pathways in the reproductive tract is an important step toward understanding how P regulates the physiological events leading to implantation.

The steroid hormone progesterone (P) plays a pivotal role during ovulation. Mice lacking P receptor (Pgr) gene fail to ovulate due to a defect in follicular rupture. The P receptor (PGR)-regulated pathways that modulate ovulation, however, remain poorly understood. To identify these pathways, we performed gene expression profiling using ovaries from mice subjected to gonadotropin-induced superovulation in the presence and in the absence of CDB-2914, a synthetic PGR antagonist. Prominent among the genes that were down-regulated in response to CDB-2914 was endothelin (ET)-2, a potent vasoactive molecule. ET-2 mRNA was transiently induced in mural granulosa cells of the preovulatory follicles immediately preceding ovulation. This induction was absent in the ovaries of PGR null mice, indicating a critical role of this receptor in ET-2 expression. To investigate the functional role of ET-2 during ovulation, we employed selective antagonists of endothelin receptors, ETR-A and ETR-B. Mice treated with an ETR-B antagonist exhibited a dramatic (>85%) decline in the number of released oocytes. Strong expression of ETR-B was observed in the mural and cumulus granulosa cells of the preovulatory follicles as well as in the capillaries lining the inner border of the theca interna. We also identified cGMP-dependent protein kinase II, a previously reported PGR-regulated gene, as a downstream target of ET-2 during ovulation. Collectively, our studies uncovered a unique pathway in which ET-2, produced by PGR in mural granulosa cells, acts in a paracrine or autocrine manner on multiple cell types within the preovulatory follicle to control the final events leading to its rupture.

Estrogen receptor-alpha (Esr1) mediates estrogen action in regulating at all levels of the hypothalamic-pituitary-ovarian axis. Whereas the importance of Esr1 in hypothalamus and pituitary has been demonstrated by loss of fertility in the neuron- and pituitary-specific Esr1 knockout mice, whether Esr1 plays a critical role in the ovary remains to be determined. In the ovary, Esr1 is mainly expressed in the theca/interstitial cells and germinal epithelium and thus is believed to mediate estrogen action in these cells. In this study, we assessed the importance of Esr1 in the ovarian theca cells in regulating female reproduction. The Cre-LoxP approach was used to selectively delete the Esr1 gene in the theca cells, and the reproductive consequence of the deletion was measured. Adolescent theca-specific Esr1 knockout (thEsr1KO) mice (<4 months of age) are fertile and cycling. However, they begin to display an erratic pattern of estrous cycles and become infertile before they reach the age of 6 months. The ovaries of thEsr1KOmice (>or=4 months) have fewer corpora lutea but more antral follicles than the age-matching wild-type mice. The numbers of 17-hydroxylase-expressing cells are largely increased in the interstitium of the thEsr1KO mouse ovary. Interestingly, whereas basal levels of serum testosterone and FSH were mildly elevated, LH level was either markedly lower or undetectable in the thEsr1KO mice. When superstimulated by exogenous gonadotropins, thEsr1KO mice released significantly fewer oocytes that wild-type littermates and developed multiple hemorrhagic cysts. Taken together, this study demonstrates that theca Esr1 plays a critical role in regulating female reproduction.

The 12/15-lipoxygenases (12/15-LOX) catalyze the stereo-specific oxygenation of arachidonic and linoleic acids into a complex series of signaling molecules, including the hydroxyeicosatetraenoic acids (HETEs) and hydroxyoctadecadienoic acids (HODEs). Our previous studies, using high density oligonucleotide microarrays, suggested a novel link between progesterone receptor (PR) signaling and 12/15-LOX-mediated fatty acid metabolism in preimplantation mouse uterus. In this paper, using PR knockout mice, we established that The steroid hormones progesterone (P) 1 and estrogen (E) play crucial roles during early pregnancy by coordinating a complex series of interactions between the implanting blastocyst and the uterus (1-5). In mice, implantation is initiated 4 days after fertilization when the blastocyst reaches the uterus (1, 6). It is thought that the action of the steroid hormones during the preimplantation phase prepares the endometrium for the attachment of the blastocyst and the subsequent events leading to the establishment of pregnancy. The cellular effects of P and E are mediated through intracellular progesterone receptor (PR) and estrogen receptor, which are hormone-inducible transcription factors (7,8). Hormone-occupied PR or estrogen receptor triggers the expression of specific gene networks in different cell types within the uterus, and the products of these genes mediate the hormonal effects during implantation. In order to understand the molecular basis of this complex physiological process, it is critical to identify the steroid-regulated pathways that are induced or suppressed at the time of implantation and analyze their functional roles.We recently used RU486, a well characterized antiprogestin, as a tool to uncover the PR signaling pathways in the pregnant uterus (9). RU486 counteracts the action of P by binding directly to PR and impairing its gene regulatory function (10, 11). Administration of RU486 to pregnant rodents during the preimplantation phase effectively blocks implantation by inhibiting PR-dependent gene expression (12). By using oligonucleotide microarrays, we identified several potential PR-regulated genes whose expression was markedly altered by RU486 in the uterus at the time of implantation (9). Interestingly, two of the mRNAs repressed in response to RU486 were leukocyte-12/15-lipoxygenase (L-12/15-LOX) and epidermal-12/15-lipoxygenase (E-12/15-LOX), which belong to a family of polyunsaturated fatty acid-metabolizing enzymes.The lipoxygenases metabolize arachidonic and linoleic acids, which are major cellular substrates in the synthesis of biological mediator substances known as eicosanoids (13, 14). There are three major types of mammalian lipoxygenase activity: 5-LOX, 12-LOX, and 15-LOX. The primary metabolites of arachidonic acid generated by the 5-LOX are the leukotrienes and lipoxines, whereas the 12-and 15-LOX enzymes produce hydroxyeicosatetraenoic acids (HETEs) (15,16). Metabolism of

PurposeTo investigate whether autologous platelet-rich plasma (PRP) treatment can improve regeneration of the endometrium in an experimental model of ethanol-induced damage.Materials and MethodsSixty female Sprague-Dawley rats were randomly assigned into three groups: control group, ethanol group, and PRP-treated group (administration of 0.25 mL of PRP into both uterine cavities 72 hours after ethanol injection). After 15 days of endometrial damage, all the animals were sacrificed during the estrous cycle, and samples were taken from the mid-uterine horn. Functional and structural recovery of the endometrium was analyzed by hematoxylin-eosin (H&E) and Masson trichrome (MT) staining, real-time polymerase chain reaction (PCR) assay, and immuno-histochemical (IHC) analyses.ResultsH&E and MT staining confirmed significantly decreased fibrosis and increased cellular proliferation in the PRP-treated group, compared to the ethanol group. The endometrial areas in the ethanol and PRP-treated groups were 212.83±15.84 µm2 and 262.34±12.33 µm2 (p=0.065). Significantly stronger IHC expression of cytokeratin, homeobox A10 (HOXA10), vascular endothelial growth factor (VEGF), and Ki-67 was found in the PRP-treated group, compared to the ethanol group. In real-time PCR analyses, interleukin-1β mRNA was down-regulated, while c-Kit mRNA was up-regulated, in the PRP-treated group, compared to the ethanol group.ConclusionIntrauterine administration of autologous PRP stimulated and accelerated regeneration of the endometrium and also decreased fibrosis in a murine model of damaged endometrium.

The steroid hormone progesterone (P) is a critical regulator of uterine receptivity during blastocyst implantation. The hormone acts through nuclear P receptors (PRs) to modulate the expression of specific gene networks in various uterine cell types. To identify the P-regulated pathways underlying uterine receptivity, we previously used oligonucleotide microarrays to analyze uterine mRNA profiles at the time of implantation in response to RU486, a PR antagonist. We reported that the mRNA corresponding to the immune-responsive gene 1 (Irg1), a previously described lipopolysaccharide-inducible gene, is one of the several mRNAs that are markedly down-regulated by RU486 in the preimplantation uterus. In the present study, we performed in situ hybridization to show that P stimulates Irg1 mRNA synthesis in the luminal epithelial cells of uteri of ovariectomized wild-type but not PR knockout mice. We also report that Irg1 mRNA was induced in the luminal epithelium of pregnant uterus between d 3 and 5, overlapping the window of implantation. To investigate the function of Irg1 during implantation, we administered sense or antisense oligodeoxynucleotides into preimplantation mouse uteri. Treatment with antisense oligodeoxynucleotides led to suppression in Irg1 mRNA expression without affecting unrelated mRNAs in the pregnant uterus. This intervention was also accompanied by impairment in embryo implantation, indicating that the phenotype is linked to the suppression of Irg1 mRNA. Collectively, our studies identified Irg1 as a novel target of PR in the pregnant uterus and also revealed that it is a critical regulator of the early events leading to implantation.

The antiobesity effect of wild ginseng (WG; Panax ginseng C.A. Meyer) in male obese leptin-deficient (B6.V-Lepob, 'ob/ob') mice was evaluated. WG was administered orally to mice at doses of 100 mg/kg and 200 mg/kg daily for 4 weeks. The WG-treated ob/ob mice showed a loss of body weight and a decrease in blood glucose levels compared with control mice. WG regulated the mRNA expression level especially, it increased peroxisome proliferators-activated receptors-gamma (PPAR-gamma) and lipoprotein lipase (LPL) in adipose tissue, as well as glucose transporter 4 (GLUT4) and insulin receptor (IR) in the skeletal muscle and liver. Taken together, these results suggest that WG may play a vital role in the antiobesity effect in ob/ob mice; this has importance in insulin sensitivity. This may prove to be of clinical importance in improving the management of obesity and related metabolic syndromes.