Sialomucin complex (Muc4) expression in the rat female reproductive tract

Previous studies in our laboratory demonstrated the presence of sialomucin complex (SMC)/Muc4 covering the rat uterine luminal epithelium. SMC/Muc4 expression in the uterus is regulated by estrogen and progesterone and lost at the time of receptivity. In contrast to this hormonal regulation at the uterine luminal surface, SMC/Muc4 in the uterine glandular epithelium, oviduct, cervix, and vagina was constitutively expressed at all stages of the estrous cycle. Furthermore, SMC was expressed in the cervix and vagina of the ovariectomized rat, even though it is not found in the uterine luminal epithelium. Both soluble and membrane-bound forms of SMC were present in these tissues. Immunohistochemical analyses showed distinctive localization patterns of SMC in the various tissues during the estrous cycle. Moreover, the previously unreported expression of SMC/Muc4 in the isthmus, ampulla, and infundibulum of the oviduct suggests potential functions in gamete development. These results indicate that SMC/Muc4 is expressed in most tissues of the female reproductive tract, in which it may have multiple functions. However, hormonal regulation appears to be restricted to the uterine luminal epithelium.     

Relaxin depresses small bowel motility through a nitric oxide-mediated mechanism. Studies in mice

Gastrointestinal motility is reduced and the incidence of functional gastrointestinal disorders is increased in pregnancy, possibly due to hormonal influences. This study aims to clarify whether the hormone relaxin, which attains high circulating levels during pregnancy and has a nitric oxide-mediated relaxant action on vascular and uterine smooth muscle, also reduces bowel motility and, if it does, whether nitric oxide is involved. Female mice in proestrous or estrous were treated for 18 h with relaxin (1 microg s.c.) or vehicle (controls). Isolated ileal preparations from both groups were used to record contractile activity, either basal or after acute administration of relaxin (5 x 10(-8) M). Drugs inhibiting nitric oxide biosynthesis or neurotransmission were used in combination with relaxin. Expression of nitric oxide synthase isoforms by the ileum was assessed by immunocytochemistry and Western blot analysis. Relaxin caused a clear-cut decay of muscle tension and a reduction in amplitude of spontaneous contractions upon either chronic administration to mice or acute addition to isolated ileal preparations. These effects were significantly blunted by N(G)-nitro-L-arginine, but not by the neural blockers we used. Moreover, relaxin increased the expression of nitric oxide synthases II and III, but not synthase I. Relaxin markedly inhibits ileal motility in mice by exerting a direct action on smooth muscle through the activation of intrinsic nitric oxide biosynthesis.     

Regulation of expression of the retinoic acid metabolizing enzyme CYP26A1 in uteri of ovariectomized mice after treatment with ovarian steroid hormones

The retinoic acid (RA) synthesizing enzymes, retinaldehyde dehydrogenases (RALDH), are expressed in specific spatial and temporal patterns in uterine tissues during estrous cycle and early pregnancy in mice. Expression of RALDH1 and 2 has been shown to be induced by estrogen treatment within the uterus. In this study, we determined the influence of progesterone and 17-ss-estradiol on the uterine expression of the RA-metabolizing enzyme CYP26A1 after specific time intervals (1, 4, 24, and 48 hr after treatment of ovariectomized mice). In a following experiment, we investigated the influence of gestagen (promegestone 0.3 mg/kg body weight), estrogen (estradiol 3 microg/kg), their combination, as well as the antagonizing anti-progesterone hormone (RU 486 10 mg/kg) on the uterine expression of CYP26A1. Expression of CYP26A1 was localized using in situ hybridization and quantified using RT-PCR. CYP26A1 mRNA expression was strongly--although transiently--induced in uterine endometrial epithelial and glandular cells after administration of gestagen or the combination of gestagen + estrogen, but not by estrogen alone. These observations were confirmed by semi-quantitative RT-PCR experiments on whole uteri. Thus, we show that the expression of CYP26A1 in endometrial epithelial cells is regulated by progesterone and not significantly influenced by co-administration of estrogen. These data indicate an additional level of hormonal control of endogenous RA levels in the mouse uterus, where its synthesis would rely on estrogen-dependent expression of RALDH enzymes, whereas its active metabolism would be triggered by progesterone-induced CYP26A1 expression.     

Mucin and proteoglycan functions in embryo implantation

Embryo implantation is a complex series of events that involves changes in pattern of expression of embryonic as well as uterine cell surface components. In the case of the embryo, these changes are driven by the developmental program. In the case of the uterus, these changes are triggered by both maternal hormonal influences as well as embryo-derived factors. Aspects of the implantation process vary among species; however, interaction between the external surface of the embryonic trophectoderm and the apical surface of the lumenal uterine epithelium is a common event. Progress is being made in defining the molecular players in these cell surface interactions. Large-molecular-weight mucin glycoproteins such as MUC1 are present at the apical surface of the uterine epithelium under most conditions. Under most circumstances, these mucins appear to protect the mucosal surface from infection and the action of degradative enzymes. These mucins are antiadhesive and also appear to represent a barrier to embryo attachment. Consistent with this model, reduction of mucin expression is observed in uterine lumenal epithelia in many species. Nonetheless, mucin expression persists in the human uterus during the proposed receptive phase. It is possible that mucin loss is localized to implantation sites in humans. Alternatively, mucins may function differently within the context of human implantation than in other species. Studies primarily performed in mice indicate that heparan sulfate proteoglycans, in particular, perlecan, appears on the exterior trophectodermal surface coincident with the acquisition of attachment competence. Various in vitro studies indicate that heparan sulfate proteoglycans support embryo attachment activity that may represent an early event in embryo–uterine interaction. Uterine epithelia cells express several complementary heparan sulfate-binding proteins that may participate in these attachment processes. Use of molecular genetic approaches in mouse models, as well as careful studies of the expression and function of these molecules in the context of implantation in various species are beginning to shed light on the key molecular events of implantation. BioEssays 20 :577–583, 1998. © 1998 John Wiley & Sons Inc.     

Effects of mifepristone (RU486) treatment on the development of uterine adenomyosis induced by pituitary grafting in mice

To evaluate the effects of mifepristone (RU486) on the development of uterine adenomyosis induced by pituitary grafting (PG), 3 groups of mice receiving pituitary grafts at 7 weeks of age were given RU486 in food (20 mg/kg chow) from 3-14 (RU486-3 group) or 10-14 (RU486-10 group) weeks of age, or were given no further treatment (PG control group), respectively. All the mice were killed at 14 weeks of age. The uterine weight was significantly decreased in both RU486-treated groups compared with the PG control group. The incidence of adenomyosis was also decreased significantly in both the RU486-3 group (0/10 mice) and RU486-10 group (2/10 mice) compared with the PG control group (7/9 mice). To look for vascular changes in the uterine tissues, which have been reported to be related to the development of adenomyosis, immunohistochemical staining of von Willebrand factor in the blood vessels was performed. The mean surface area and minor axis of blood vessels in the uterus were thereby found to be significantly decreased in the RU486-10 group compared to the PG control group. The results clearly indicated that RU486, a potent antiprogestin, could inhibit the genesis of uterine adenomyosis in mice, and at the same time caused shrinkage of the vascular system. As in humans, progesterone as well as the vascular system therefore appear to be important factors in the pathogenesis of uterine adenomyosis in this mouse model.     

Steroids modulate the expression of alpha4 integrin in mouse blastocysts and uterus during implantation

Blastocyst implantation and successful establishment of pregnancy require delicate interactions between the embryo and the maternal uterine milieu, which are controlled at the embryo-maternal interface by the coordinated interplay of a variety of growth factors, cytokines, hormones, and cell adhesion molecules expressed by both the decidualized endometrium and the trophoblast cells. Proper implantation of the embryo is solely dependent on the initial endometrial receptivity and the preparation of the blastocyst to glue itself to the uterine wall. Both these events are considered to be mediated by cell adhesion molecules and integrins expressed by the blastocyst as well by as the maternal endometrium. Integrin expression by the blastocyst and the uterus is a dynamic process. However, reports on the expression and the hormonal modulation of integrins and their role in blastocyst activation and uterine receptivity during implantation are meager. The present study investigates the expression and hormonal regulation of alpha4beta1 integrin by steroid hormones in the blastocyst and the receptive uterus using an in vivo, delayed-implantation mouse model system. The dormant and activated blastocysts as well as the uteri were recovered from ovariectomized mice after progesterone-alone and progesterone-plus-estrogen therapy, respectively. Immunolocalization of protein expression of alpha4 and beta1 integrin subunits indicate that steroids modulate the expression of alpha4beta1 integrin receptor in the mouse blastocyst as well as the uterus and that a differential expression is observed with exposure to progesterone and estrogen. Intrauterine blocking of alpha4 integrin by specific antibody resulted in implantation failure in normal as well as in delayed-implantation mice. Based on our data, we propose here, to our knowledge for the first time, that alpha4beta1 integrin, which is responsible for binding to fibronectin and vascular cell adhesion molecule-1, is induced by estradiol and is down-regulated by progesterone in mice during implantation. Furthermore, the results also indicate the direct role of alpha4 integrin in the process of implantation.     

LacZ expression in Fut2-LacZ reporter mice reveals estrogen-regulated endocervical glandular expression during estrous cycle, hormone replacement, and pregnancy

The secretor gene (FUT2) encodes an alpha(1,2)fucosyltransferase (E.C. 2.4.1.69) that elaborates alpha(1,2)fucose residues on mucosal epithelium and secreted mucins. Though uterine alpha(1,2)fucosylated glycans have been proposed to be involved in embryo adhesion, mice with a homozygous null mutation of Fut2 displayed normal fertility. To help develop alternative hypotheses for function, the cell type and regulation of Fut2 expression during the estrous cycle, hormone replacement, and pregnancy was examined in Fut2-LacZ reporter mice containing targeted replacement of Fut2 with bacterial lacZ. LacZ expression in the reproductive tract of Fut2-LacZ mice is most prominent in the glandular epithelium of the endocervix during estrus and pregnancy. Nuclear LacZ expression identifies cell-specific expression of Fut2 in mucus-secreting cells of the endocervix, uterine glands, foveolar pit and chief cells of the stomach, and goblet cells of the colon. In ovariectomized Fut2-LacZ mice, estradiol treatment stimulates X-gal staining in endocervix and uterus but does not affect expression in stomach and colon. Northern blot analysis in wild-type mice shows 15-fold elevations of Fut2 steady-state mRNA with estradiol treatment, whereas Fut1 varies little. Fut2 levels in the glandular stomach and distal colon remain constant, and uterine Fut2 levels vary eightfold during the estrous cycle. These data represent the first demonstration of a glycosyltransferase gene under tissue-specific hormonal regulation in a LacZ reporter mouse model. Endocervical expression of Fut2 in estrus and pregnancy may modify cervical mucus barrier properties from microbial infection analogous to the potential role of mucosal glycans in humans.     

Regulation of uterine 5 alpha-reductase type 1 in mice.

A null mutation in the murine gene encoding steroid 5 alpha-reductase type 1 (5 alpha R1) leads to failure of normal parturition at term. This observation, together with the finding that mRNA levels of uterine 5 alpha R1 increase significantly at term in normal pregnant animals, indicates that 5 alpha R1 plays an important role in murine parturition. The current studies were conducted to elucidate the regulation of 5 alpha R1 in uterine tissues of nonpregnant and pregnant animals. Nonpregnant, ovariectomized ICR mice were treated with vehicle (control), 17 beta-estradiol (E(2)), progesterone (P(4) ), or E(2)+P(4) for 3 days. Thereafter, uterine tissues were obtained for histology, quantification of 5 alpha R1 specific activity, and Northern blot analysis of 5 alpha R1 mRNA expression. The 5 alpha R1 enzyme activity was significantly increased in animals treated with E(2)+P(4). However, activity was much less in uterine tissues from E(2)+P(4)-treated animals than in uterine tissues from pregnant animals near term. To evaluate further the regulation of 5 alpha R1 during gestation, mice underwent unilateral tubal ligation before timed matings. The 5 alpha R1 activity increased eightfold in uterine tissues from the fetal horn from Gestational Days 12 to 18. This temporal pattern in 5 alpha R1 activity paralleled marked increases in uterine diameter. Taken together, these studies indicate that expression of 5 alpha R1 is regulated by E(2)+P(4) in uterine tissues. Whereas E(2) alone is insufficient to induce enzyme activity, E(2) may be required to increase P(4) receptors and, thereby, mediate the effects of P(4) on 5 alpha R1 gene expression. Further increases in enzyme activity during late gestation are mediated by fetal occupancy, possibly through stretch-induced increases in endometrial growth. Thus, like other genes involved in parturition, expression of 5 alpha R1 is regulated by both hormonal and fetal-derived signaling pathways.     

Expression of maspin in the early pregnant mouse endometrium and its role during embryonic implantation

Maspin (serpin B5), a tumor-suppressing member of the serine protease inhibitor family, participates in cell migration, adhesion, invasion, and apoptosis. These processes are also critical for embryo implantation, but the role of maspin in embryo implantation remains poorly understood. The aim of the present study was to investigate the spatiotemporal expression of maspin in early pregnant mouse endometrium and its role in embryo implantation. Real-time quantitative PCR analysis, immunohistochemistry, and Western blotting were used to detect mRNA and protein expression of maspin in the endometria of nonpregnant and early pregnant (days 0 to 7) mice. On day 3 of pregnancy, mice in the treated group (n = 20) were injected in the left uterine horn with antimaspin polyclonal antibody and in the right horn with purified rabbit IgG; control mice (n = 20) were injected only with purified rabbit IgG in the right uterine horn. Implanted embryos were counted on pregnant day 8. The mRNA and protein expressions of maspin were higher in the endometria of pregnant mice than nonpregnant mice; these levels gradually increased from day 1 of pregnancy, peaked on day 5, and then decreased on days 6 and 7. The mice treated with antimaspin polyclonal antibody group had far fewer implanted embryos than did the control group. Taken together, these results suggest that maspin, a tumor suppressor, may play an important role in embryo implantation.     

Mice-lacking LMP2, immuno-proteasome subunit,as an animal model of spontaneous uterine leiomyosarcoma

Uterine tumors are the most common type of gynecologic neoplasm. Uterine leiomyosarcoma (LMS) is rare, accounting for 2% to 5% of tumors of the uterine body. Uterine LMS develops more often in the muscle tissue layer of the uterine body than in the uterine cervix. The development of gynecologic tumors is often correlated with female hormone secretion; however, the development of uterine LMS is not substantially correlated with hormonal conditions, and the risk factors are not yet known. Radiographic evaluation combined with PET/CT can be useless in the diagnosis and surveillance of uterine LMS. Importantly, a diagnostic biomarker, which distinguishes malignant LMS and benign tumor leiomyoma (LMA) is yet to be established. Accordingly, it is necessary to analyze risk factors associated with uterine LMS in order to establish a method of treatment. LMP2-deficient mice spontaneously develop uterine LMS, with a disease prevalence of ~40% by 14 months of age. It is therefore of interest whether human uterine LMS shows a loss of LMP2 expression. We found LMP2 expression is absent in human LMS, but present in human LMA. Therefore, defective LMP2 expression may be one of the risk factors for LMS. LMP2 is potentially a diagnostic biomarker for uterine LMS, and gene therapy with LMP2-encording DNA may be a new therapeutic approach.     

Immunohistochemical detection of progesterone receptors in the canine uterus and their relation to sex steroid hormone levels

The aim of this immunohistochemical study is to describe the normal distribution of progesterone receptors in the various cell types of the canine uterine horns, body and cervix. The results can be used for research on uterine and endocrinological pathology, since the impact of progesterone on different uterine cell types is partly determined by the receptor availability. Nuclear staining for progesterone receptors was observed in epithelial cells of the surface epithelium, glandular ducts and basal glands of the endometrium, in endometrial stroma cells and in myometrial smooth muscle cells. This staining was positively correlated with the estradiol-17 beta:progesterone ratio, and reflects the positive effect of estradiol-17 beta and the negative influence of progesterone on the receptors. Staining scores were high during proestrus and decreased through estrus to early metestrus. In late metestrus, staining scores of the stromal and smooth muscle cells increased again. In anestrus, high scores of the surface-epithelial cells contrasted with minimal scores of the basal glands. This finding suggests a different hormonal regulation of the progesterone receptor expression in both epithelial cell groups. The higher staining intensities for progesterone receptors in stromal cells compared with epithelial cells might be explained by the fact that stromal cells mediate some effects of steroid hormones on the epithelial cells in the genital tract. Therefore, the role of stromal cells in regulation of the cyclic endometrial changes and in pathologic changes of uterine tissue should not be underestimated.     

Proteomic analysis of proteins differentially expressed in uterine lymphocytes obtained from wild‐type and NOD mice

Non‐obese diabetic (NOD) mice exhibit impaired fertility and decreased litter size when compared to wild type (WT) mice. However, it is unclear why allogeneic pregnant NOD mice are prone to spontaneous embryo loss. Herein, two‐dimensional gel electrophoresis (2‐DE) and mass spectrometry (MS) were used to detect differentially expressed proteins in the uterine lymphocytes isolated from these mice and WT BALB/c controls. We found 24 differentially expressed proteins. The differential expression of 10 of these proteins was further confirmed by Western blot analysis. Out of the 24 identified proteins, 20 were expressed in uterine lymphocytes of WT mice at a level at least 2 times higher than in NOD mice, whereas 4 were down‐regulated. Western blot analysis confirmed that 8 proteins were up‐regulated and 2 proteins were down‐regulated in WT mice compared with NOD mice, consistent with the results of 2‐DE and MS. Additionally, most of the highly expressed proteins in WT uterine lymphocytes were expressed at a significantly lower level in the corresponding splenic group (17/20). These results suggest that up‐regulated expression of these proteins may be specific to uterine lymphocytes. Reported functions of the highly expressed proteins affect key functions during pregnancy, including cell movement, cell cycle control, and metabolisms. Finally, we analyzed the constitutional ratio of CD3⁺ and CD49b⁺ cells in the isolated lymphocytes by flow cytometry. Our results suggest that the differentially expressed proteins may participate in the modulation of embryo implantation and early‐stage development of embryos, and subsequently influence pregnancy outcome. J. Cell. Biochem. 108: 447–457, 2009. © 2009 Wiley‐Liss, Inc.     

Role of early and late oestrogenic effects on implantation in the mouse

Oestrogen action in the uterus is expressed in an early phase (Phase I) and a late phase (Phase II). The role of this biphasic oestrogen action in implantation is not clear. To determine the relative importance of Phase I and II responses, triphenylethylene compounds (CI-628, LY-117018, nafoxidine, clomiphene citrate and tamoxifen) and oestrogens (oestriol and oestradiol-17 beta) were used in a physiologically relevant experimental system for studying implantation. All compounds elicited uterine water imbibition to various degrees in ovariectomized-progesterone-treated mice at 6 h (Phase I response) and their effectiveness in inducing implantation in delayed implanting mice correlated with their respective potency to increase uterine wet weight. This suggests that Phase I might be an essential component of oestrogen action in implantation and that the efficiency of a compound to elicit a Phase I response might serve as a predictive indicator of its potential action on implantation.     

Adult tissue angiogenesis: evidence for negative regulation by estrogen in the uterus.

Increased uterine vascular permeability and angiogenesis are two major events of embryo implantation and placentation during pregnancy. These latter processes require coordinated, uterine-specific interactions between progesterone (P4) and estrogen (E) signaling. Although roles of these steroids have long been suspected, definitive functions of E and/or P4 in uterine angiogenesis still remain elusive. We have therefore exploited the availability of reporter and mutant mice to explore the regulation of angiogenesis in response to steroid hormonal changes in vivo. We present here molecular, genetic, physiological, and pharmacological evidence that E and P4 have different effects in vivo: E promotes uterine vascular permeability but profoundly inhibits angiogenesis, whereas P4 stimulates angiogenesis with little effect on vascular permeability. These effects of E and P4 are mediated by differential spatiotemporal expression of proangiogenic factors in the uterus.     

Expression of hypoxia-inducible factors in the peri-implantation mouse uterus is regulated in a cell-specific and ovarian steroid hormone-dependent manner. Evidence for differential function of HIFs during early pregnancy

Increased uterine vascular permeability and angiogenesis are hallmarks of implantation and placentation. These events are profoundly influenced by vascular endothelial growth factor (VEGF). We previously showed that VEGF isoforms and VEGF receptors are expressed in the uterus, suggesting the role of VEGF in uterine vascular permeability and angiogenesis required for implantation and decidualization. We have recently shown that estrogen promotes uterine vascular permeability but inhibits angiogenesis, whereas progesterone stimulates angiogenesis with little effect on vascular permeability. However, the mechanism of differential steroid hormonal regulation of uterine angiogenesis remains unresolved. Oxygen homeostasis is essential for cell survival and is primarily mediated by hypoxia-inducible factors (HIFs). These factors are intimately associated with vascular events and induce VEGF expression by binding to the hypoxia response element in the VEGF promoter. HIFalpha isoforms function by forming heterodimers with the aryl hydrocarbon nuclear translocator (ARNT) (HIF-beta) family members. There is very limited information on the relationship among HIFs, ARNTs, and VEGF in the uterus during early pregnancy, although the role of HIFs in regulating VEGF and angiogenesis in cancers is well documented. Using molecular and physiological approaches, we here show that uterine expression of HIFs and ARNTs does not correlate with VEGF expression during the preimplantation period (days 1-4) in mice. In contrast, their expression follows the localization of uterine VEGF expression with increasing angiogenesis during the postimplantation period (days 5-8). This disparate pattern of uterine HIFs, ARNTs, and VEGF expression on days 1-4 of pregnancy suggests HIFs have multiple roles in addition to the regulation of angiogenesis during the peri-implantation period. Using pharmacological, molecular, and genetic approaches, we also observed that although progesterone primarily up-regulates uterine HIF-1alpha expression, estrogen transiently stimulates that of HIF-2alpha.     

Hormonal and nutritional regulation of muscle carnitine palmitoyltransferase I gene expression in vivo

Transgenic mice carrying the human heart muscle carnitine palmitoyltransferase I (M-CPTI) gene fused to a CAT reporter gene were generated to study the regulation of M-CPTI gene expression. When the mice were fasted for 48 h, CAT activity and mRNA levels increased by more than 2-fold in heart and skeletal muscle, but not liver or kidney. In the diabetic transgenic mice, there was a 2- to 3-fold increase in CAT activity and CAT mRNA levels in heart and skeletal muscle which upon insulin administration reverted to that observed with the control insulin sufficient transgenic mice. Feeding a high fat diet increased CAT activity and mRNA levels by 2- to 4-fold in heart and skeletal muscle of the transgenic mice compared to the control transgenic mice on regular diet. Overall, the M-CPTI promoter was found to be necessary for the tissue-specific hormonal and dietary regulation of the gene expression.     

Different binding to squamous and columnar epithelium of the uterine cervix as a marker of epithelial differentiation

Biotinylated lectins were used to investigate the expression of carbohydrate residues on columnar and squamous epithelium of the uterine cervix. Con A, WGA, RCA I, PNA, UEA I, DBA and SBA were used. In the native exocervical and in metaplastic squamous epithelium of the transformation zone, one group of lectins (Con A, WGA, RCA I and PNA) stained the cell periphery of all epithelial layers. A second group (UEA I, DBA and SBA) colored the cell periphery of the suprabasal cells. The basal layer was always negative. All lectins labeled the apical border and occasionally the cytoplasm of the endocervical columnar epithelium. Lectin-binding of metaplastic and native squamous epithelium could possibly be used as a marker of epithelial differentiation in normal and abnormal conditions.