Maternal exposure to bisphenol a during late pregnancy resulted in an increase of Calbindin-D9k mRNA and protein in maternal and postnatal rat uteri

It has been reported that Calbindin-D9k (CaBP-9k) is rapidly and strongly induced by environmental estrogenic compounds, possibly through estrogen receptors (ERalpha) in the uterus of mammals. CaBP-9k can be evaluated as an early gene marker for assaying estrogenic effects of putative environmental chemicals in the rat uterus. This study was undertaken to investigate CaBP-9k mRNA and protein expression in the postnatal rat uterus following maternal exposure to 17beta-estradiol (E2) and bisphenol A (BPA) during the neonatal period. Treatment with a high dose of BPA (600 mg/kg body weight (BW) per day) resulted in a 3-fold increase in CaBP-9k mRNA expression for 3 days, while a single dose of E2 (40 microg/kg BW per day) induced 2-fold increase of this gene in the maternal uterus. In an agreement with maternal CaBP-9k mRNA, postnatal CaBP-9k mRNA in the uterus increased 4-fold when treated with BPA (600 mg/kg BW per day). In addition, treatment with increasing concentrations of BPA resulted in significant increases in CaBP-9k protein in the maternal rat uterus. It is of interest that increasing doses of BPA induced a significant ERalpha mRNA increase in the postnatal uterus. Furthermore, immunohistochemistry revealed that treatment with BPA induced CaBP-9k protein in the maternal uterus. We demonstrated that maternal exposure to BPA during late pregnancy induced CaBP-9k mRNA and protein in maternal and postnatal rat uteri. These results suggest that rapid absorption and distribution of environmental estrogenic compounds occurs in maternal and neonatal rat uteri and these chemicals can easily pass though the placenta during pregnancy to affect postnatal reproductive functions.     

Expression of calbindin-D9k messenger ribonucleic acid in the gastrointestinal tract of dairy cattle

The calcium demands of pregnancy and lactation are known to up-regulate expression of Calbindin-D9k (CaBP-9k) mRNA in the intestines. The gastrointestinal CaBP-9k mRNA expressions has not been studied in dairy cows, which are bound to experience several pregnancies and lactation stages. In this study, the CaBP-9k mRNA expression were examined in the gastrointestinal tract of Holstein dairy cattle by Northern blot analysis. Detectable expression of CaBP-9k mRNA was localized in the proximal portion of the small intestines. These expressions were higher at the most proximal region of the duodenum and gradually decreased distally. The duodenal CaBP-9k mRNA was detected in all dairy cattle from 0.4 to 83.4 months old, but was not detectable in foetuses. There were no significant correlations between the age and the levels of CaBP-9k mRNA expression or between the plasma 1,25-(OH)2D3 concentrations and the levels of CaBP-9k mRNA expression.     

Calbindin-D9k mRNA expression in the rat uterus following exposure to methoxychlor: a comparison of oral and subcutaneous exposure

Calbindin-D(9k) (CaBP-9k) is a cytosolic calcium-binding protein that is induced by estrogenic compounds possibly through estrogen receptors. We compared CaBP-9k mRNA expression in the uterus with uterotrophic response in immature rats exposed to methoxychlor (MC), an environmental chemical with estrogenic activity. MC was orally or subcutaneously administered to 3-week-old female Sprague-Dawley rats for 3 days. The weights of the uterus and vagina significantly increased in the oral treatment group at a dose of 50, 100 and 200 mg/kg, but those of the subcutaneous (SC) treatment group only increased at 200 mg/kg. Northern blot analysis showed that CaBP-9k mRNA expression was significantly induced in a dose-dependent manner at doses of 50, 100 and 200 mg/kg/day in the oral treatment group. SC administration of MC induced significant expression at only a dose of 200 mg/kg/day; this was similar to the uterotrophic response. MC has an estrogenic effect on the uterus as shown by the increase in weight and induction of CaBP-9k mRNA expression, which were much greater following exposure via oral gavage than via the SC route. The strong correlation between the results of in vivo uterotrophic assay and CaBP-9k mRNA expression suggests that CaBP-9k mRNA expression in the rat uterus may be used as an early gene marker for detection of the estrogenic effects of putative environmental chemicals.     

Expression and Hormonal Regulation of E‐Cadherin in Canine Uterus During Early Pregnancy

E‐cadherin, a Ca^{2 +} ‐dependent cell adhesion molecule, is necessary for endometrial receptivity to blastocyst implantation. The aim of this study was to investigate the differential expression of E‐cadherin in canine uterus during early pregnancy and its regulation under different conditions by in situ hybridization. E‐cadherin mRNA expression was at a low level in the glandular epithelium on days 6, 12 and 17 of pregnancy. On days 20 and 23 of pregnancy, E‐cadherin mRNA was highly expressed in the glandular epithelium surrounding the embryo, but not in the luminal epithelium and declined in villi and placenta on day 28 of pregnancy. During oestrous cycle, a moderate level of E‐cadherin mRNA expression was found in the luminal and glandular epithelium of canine uteri at oestrus stage. The same expression was also found at anoestrus stage. Progesterone slightly induced the expression of E‐cadherin mRNA in the luminal and glandular epithelium of ovariectomized canine uterus. These results suggest that E‐cadherin expression is closely related to canine implantation and can be up‐regulated by progesterone.     

Conflict of estrogenic activity by various phthalates between in vitro and in vivo models related to the expression of Calbindin-D9k

Phthalates are suspected to disrupt the endocrine system, especially through estrogenic effects. In the present study, we investigated the effects of various phthalates and compared them with those of estrogenic compounds that disrupt the female reproductive system. To assess the effects of these phthalates, alteration of the Calbindin-D9k (CaBP-9k) gene was measured as a biomarker because rat CaBP-9k gene carries an estrogen response element (ERE) which is involved in estrogen responsiveness of the gene during the estrous cycle. In this study, phthalates were tested for estrogenic properties in in vitro and in vivo models. First, the E-Screen assay was used to measure the proliferation of MCF-7 cells, a human breast cancer cell line. Treatments with 17beta-estradiol (E2; 9-fold) and 17alpha-estradiol (EE; 9-fold) induced MCF-7 cell proliferation at concentrations of 10(-9) M. Phthalates induced an increase in MCF-7 proliferation at concentration of 10(-6) M up to 10(-4) M. Nbutyl benzyl phthalate (BBP; 6-fold vs. vehicle), dicyclohexyl phthalate (DCHP; 8-fold), 2-ethylhexyl phthalate (DEHP; 6-fold) and di-n-butyl phthalate (DBP; 7-fold) at the concentration of 10(-4) M induced in an increase in MCF-7 proliferation after 6 d of treatment compared to vehicle. However, significant increase in MCF-7 proliferation was induced by diethyl phthalate (DEP). Second, we investigated the expression of CaBP-9k in the uterus of immature rats after oral treatment with BBP, DCHP, DEHP, DBP or DBP (600 mg/kg per day) in this in vivo model, because the immature rat model is highly sensitive to exposure to estrogenic chemicals. None of the phthalates induced the expression of CaBP-9k mRNA and its protein in the neonatal uterus as analysed by Northern and Western blot analyses, respectively. Although phthalates induced an increase in MCF-7 cell proliferation by an estrogenic effect, they could not induce CaBP-9k expression in the in vivo system, suggesting that the assays of estrogenic effects of various phthalates conducted in vitro and in vivo expression of CaBP-9k may produce conflicting results.     

Epidermal growth factor and epidermal growth factor receptor immunoreactivity in the endothelial cells of the uterine artery and its branches during different stages of the estrous cycle in the pig

The uterine artery and its branches are the most important vessels that supply the uterus with blood, nutrients and active substances. Epidermal growth factor (EGF) and its receptor (EGFR) are expressed in many tissues, including reproductive organs, and is involved in angiogenesis, embryo implantation and development as well as in proliferation and differentiation of various cells. The aim of our study was to determine EGF and EGFR immunoexpression in the uterine artery and its branches during the estrous cycle in the pig. The experiment was performed on cryostat sections of the uterine artery and its branches stained immunohistochemically by ABC method. Light microscopic observations revealed the phase-related immunoreactivity of EGF and EGFR in the endothelial cells of the uterine artery and its branches. The highest intensity of EGF and EGFR immunoreaction in endothelial cells of the uterine artery was observed in the follicular phase. A significant decrease in the intensity of EGF and EGFR immunoreactivity was found in the middle luteal phase. Similar results of the immunostaining were found with regard to EGFR. In the endothelium of the uterine arterial branches, a significant increase in the intensity of EGF and EGFR-immunoreactivity was observed in the middle luteal phase. A decrease in the intensity of EGF immunostaining was observed in the late luteal phase. The phase-related expression of EGF and EGFR in the endothelium of the uterine artery and its branches suggest the modulatory effect of EGF and its receptor on the uterine artery and the region supplying these vessels.     

猪胚胎附植部分因子研究进展

产仔数性状是养猪业中一个重要的经济性状,而胚胎附植对猪产仔数的高低有重要的影响。胚胎附植的调控涉及到多个生物学过程,在其中发挥作用的物质（附植因子）很多。作者针对孕酮、孕酮受体、雌二醇、雌激素受体α（ESR1）和促红细胞生成素产生肝细胞受体配体（Eph-ephrin）系统这几种附植因子的相关研究进行了归纳总结,重点阐述了这些因子在母胎对话过程中的时空表达、功能、突变、调控等方面的研究进展。其中,孕酮及其受体在猪胚胎附植活动中全程发挥着重要作用,黄体分泌的孕酮与母猪子宫内膜腔上皮、腺上皮、基质和肌细胞中的孕酮受体结合发挥作用,使这些组织细胞分泌多种附植因子,这些附植因子进一步参与胚胎附植过程。雌二醇是雌激素中含量最多、活性最强的一种激素,主要由卵巢颗粒细胞分泌,雌激素受体α是子宫内雌二醇的主要受体,二者结合可使母猪发情;此外,附植中处于游离状态的胚泡也分泌雌二醇,其是一个让母猪子宫内膜得以识别的信号,允许胚泡附植。Eph-ephrin系统作用广泛,其在人、小鼠和猪的胚胎附植过程中发挥着重要作用。系统中的EphA1、ephrin A1、EphA4等基因在猪的胚胎附植期表达量显著高于空怀猪,它们在子宫内膜附植点的表达量显著高于附植点间的部位,且ephrin A1的抑制表达会降低子宫内膜上皮细胞的迁移和黏附能力。这些附植因子都在猪胚胎附植活动中发挥着重要作用,对它们的深入研究将有利于明确猪胚胎附植调控机理,进一步揭示猪高繁机理。     

Embryo gene expression in pig pregnancy

Pregnancy is a complex process in which significant changes occur continually in both the corpora lutea and in the endometrium of the females and varies depending on the embryonic, pre-implantation or foetal stages. In the embryonic stages, the majority of genes expressed in the pig embryo correspond to the loss of cellular pluripotency. In contrast, the implantation consists of three phases: elongation of the conceptus, adhesion and union of the embryo to the endometrial epithelium. During these phases, many factors are expressed, including growth factors, molecules that facilitate adhesion and cytokines. All these changes are ultimately regulated by different lipid and hormonal substances, specifically by progesterone, oestradiol and prostaglandins, which regulate the expression of many proteins necessary for the development of the embryo, endometrial remodelling and embryo–maternal communication. This paper is a review of primary gene regulatory mechanisms in pigs during different stages of implantation.     

The localization and expression of epidermal growth factor and epidermal growth factor receptor in bovine ovary during oestrous cycle

Epidermal growth factor (EGF) is one of the important regulatory factors of EGF family. EGF has been indicated to effectively inhibit the apoptosis of follicular cells, to promote the proliferation of granulosa cells and the maturation of oocytes, and to induce ovulation process via binding to epidermal growth factor receptor (EGFR). However, little is known about the distribution and expression of EGF and EGFR in cattle ovary especially during oestrous cycle. In this study, the localization and expression rule of EGF and EGFR in cattle ovaries of follicular phase and luteal phase at different time points in oestrous cycle were investigated by using IHC and real-time qPCR. The results showed that EGF and EGFR in cattle ovary were mainly expressed in granulosa cells, cumulus cells, oocytes, zona pellucida, follicular fluid and theca folliculi externa of follicles. The protein and mRNA expression of EGF/EGFR in follicles changed regularly with the follicular growth wave both in follicular and in luteal phase ovaries. In follicular phase ovaries, the protein expression of EGF and EGFR was higher in antral follicles than that of those in other follicles during follicular growth stage, and the mRNA expression of EGFR was also increased in stage of dominant follicle selection. However, in luteal phase ovaries, the growth of follicles was impeded during corpus luteum development under the action of progesterone secreted by granular lutein cell. The mRNA and protein expressions of EGF and EGFR in ovarian follicles during oestrous cycle indicate that they play a role in promoting follicular development in follicular growth waves and mediating the selection process of dominant follicles.     

Expression profiles of perforin,granzyme B and granulysin genes during the estrous cycle and gestation in the bovine endometrium

The conceptus is susceptible to destruction by maternal cytotoxic lymphocytes, which have cytotoxic potential. Therefore, it is expected that mechanisms for regulating cytotoxic lymphocytes exist, but little is known about the expression of cytotoxic genes in the endometrium. In the present study, we examined the spatial and temporal expression patterns of the cytotoxic genes perforin, granzyme B, and granulysin during the estrous cycle and gestation in the bovine endometrium. Endometrial tissues were collected from cows during the estrous cycle and gestation. The gene expression patterns of the three cytotoxic genes were examined using quantitative polymerase chain reaction and in situ hybridization, and cytotoxic lymphocyte subsets were characterized using immunohistochemistry. During mid‐ to late gestation in the intercaruncular (ICAR), granulysin expression was significantly increased, and a large number of granulysin‐expressing cells were localized in the luminal epithelium. Perforin and granzyme B displayed similar expression profiles and were highly expressed in the peri‐implantation endometrium, but few cells expressing these genes were found in the endometrial stroma. In conclusion, these findings suggest that in the ICAR epithelium granulysin may play important roles in the establishment and maintenance of gestation during normal pregnancy.     

Expression patterns of the implantation-associated genes in the uterus during the estrous cycle in mice

The mRNA expression patterns of EGF, HB-EGF, Amphiregulin, EGF receptor, IGF-1, CSF-1, IL-1 alpha, IL-1 beta, IL-1 receptor type 1, IL-1 receptor antagonist, LIF, COX-1, COX-2, Mucin-1, calcitonin, and rat USAG-1 mouse homologue, all of which are involved in the process of conceptus implantation to the endometrium, were examined during the estrous cycle by means of real-time quantitative PCR. COX-2, HB-EGF, LIF, Mucin-1, CSF-1, IL-1 alpha, IL-1 beta, and IL-1 receptor antagonist were temporally regulated during the estrous cycle and highly expressed during the estrous stage. In the case of COX-1, EGF, IGF-1, and EGF receptor, the highest mRNA expression was during the diestrous stage. In contrast, the rat USAG-1 mouse homologue mRNA expression did not change during the estrous cycle. These results indicate that rat USAG-1 mouse homologue expression at implantation might be specifically regulated by embryonic factors rather than the maternal environment.     

Expression Pattern Implicates a Potential Role for Luman Recruitment Factor in the Process of Implantation in Uteri and Development of Preimplantation Embryos in Mice

Luman/CREB3 recruitment factor (LRF or CREBRF) was identified as a regulator of Luman (or CREB3) that is involved in the unfolded protein response during endoplasmic reticulum stress. Luman is implicated in a multitude of functions ranging from viral infection and immunity to cancer. The biological function of LRF, however, is unknown. In this paper, we report that uteri of pregnant mice and embryos displayed enhanced LRF expression at all stages, and the expressed LRF was found to be localized specifically at implantation sites. On the other hand, uteri of mice induced for delayed implantation or pseudopregnant mice showed low levels of LRF expression, suggesting that LRF mediates uterine receptivity during implantation. Further, expression of LRF was found to be modulated by steroid hormones such as progesterone and estradiol. This study thereby identifies a potential role for LRF in the process of implantation in uteri and development of preimplantation embryos in mice.     

Pre-implantation exogenous progesterone and pregnancy in sheep: I. polyamines,nutrient transport,and progestamedins

Background: Administration of exogenous progesterone(P4) to ewes during the pre-implantation period advances conceptus development and implantation. This study determined effects of exogenous P4 on transport of select nutrients and pathways that enhance conceptus development. Pregnant ewes(n = 38) were treated with either 25 mg P4 in 1 mL corn oil(P4, n = 18) or 1 mL corn oil alone(CO, n = 20) from day 1.5 through day 8 of pregnancy and hysterectomized on either day 9 or day 12 of pregnancy. Endometrial expression of genes encoding enzymes for synthesis of polyamines,transporters of glucose, arginine, and glycine, as wel as progestamedins was determined by RT-qP CR.Results: On day 12 of pregnancy, conceptuses from P4-treated ewes had elongated while those from CO-treated ewes were spherical. The mR NA expression of AZIN2, an arginine decarboxylase, was lower in endometria of P4-treated than CO-treated ewes on day 9 of pregnancy. Expression of FGF10, a progestamedin, was greater in endometria of CO and P4-treated ewes on day 12 of gestation in addition to P4-treated ewes necropsied on day 9 of gestation. Treatment with P4 down-regulated endometrial expression of amino acid transporter SLC1 A4 on day 12 of pregnancy.Conclusions: Results indicated that administration of exogenous P4 during the pre-implantation period advanced the expression of FGF10, which may accelerate proliferation of trophectoderm cel s, but also was correlated with decreased expression of glycine and serine transporters and polyamine synthesis enzyme AZIN2. Further research with increased sample sizes may determine how differential expression affects endometrial functions and potentially embryonic loss.     

Endometrial expression of key genes related to fertility in repeat breeder and non-repeat breeder cows

The aim of the present study was to compare the endometrial gene expression of epidermal growth factor receptor (EGFR), nodal growth differentiation factor (NODAL), prostaglandin-endoperoxide synthase 2 (PTGS2), oestrogen receptor 1 (ESR1) and progesterone receptor (PGR) in repeat breeder cows (RBC) and non-RBC during diestrus. Endometrial samples were collected by cytobrush technique and stored in RNA stabilizing solution at −20°C until RT-qPCR analysis. Differences in endometrial mRNA expression of selected genes were assessed by ANOVA and simple (r) and the partial correlations (rp) among selected genes were performed. Results demonstrated that mRNA expression of EGFR and NODAL were higher in RBC than in non-RBC (3 and 25-fold change, p < .01 and p < .01, respectively), while the mRNA expression of PTGS2 was lower (1.56-fold change, p < .01). Although there were no differences detected in the mRNA expression of ESR1 and PGR, there was a positive correlation between the expression of ESR1 and EGFR (0.84, p < .05) and a negative correlation between PGR and PTGS2 (−0.49, p < .05). In conclusion, the difference on the endometrial mRNA expression of the genes included in the study between RBC and non-RBC indicates a deregulation of important mechanisms that are vital to establish a successful pregnancy. Thus, the present study provides useful insight as a base for future studies to elucidate the causes of RBC.