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Cigarette smoke (CS) is well known to be very harmful to human body functions such as fertility, reproduction, and development. CS is considered to more affect patients with hypertension (HT). To estimate the effect of CS associated with female rat's fertility, we examined the histopathological characteristics of the uterus and ovary which were obtained from the female rats exposed to smoke of the standard cigarette (3R4F) for 4 weeks (10h a week) according to the OECD guidelines. The female wild-type Wistar Kyoto (WK) rats (WTR) and spontaneously hypertensive WK rats (SHR) were used to compare the effect of CS on healthy and hypertensive rats. After CS exposure, we manufactured tissue slides from uterine and ovarian samples and evaluated the maturation of follicles of ovary and cell proliferation in the uterus by H&E staining and immunohistochemistry (IHC). In IHC analysis on ovarian tissues, the expression of proliferating cell nuclear antigen (PCNA) and the number of follicles were decreased by CS exposure. On the contrary, PCNA expression and cell proliferation in the uterine inner layers were increased by CS exposure. The protein expression of C/EBP homologous protein (CHOP), an endoplasmic reticulum (ER)-stress marker, and BAX, a pro-apoptotic protein, was decreased by CS exposure. This phenomenon was more exacerbated in SHR rats than in WTR rats. Taken together, acute exposure to CS induced the decreased maturation of ovarian follicles and abnormal over-growth of uterine inner wall, leading to a harmful effect on female rat's normal function. In addition, this harmful effect of CS may be displayed more seriously in rats with HT., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Plasma membrane sodium/calcium (Na(+)/Ca(2+)) exchangers are an important component of intracellular calcium [Ca(2+)](i) homeostasis and electrical conduction. Na(+)/Ca(2+) exchangers, NCX and NCKX, play a critical role in the transport of one [Ca(2+)](i) and potassium ion across the cell membrane in exchange for four extracellular sodium ions [Na(+)](e). Mammalian plasma membrane Na(+)/Ca(2+) exchange proteins are divided into two families: one in which Ca(2+) flux is dependent only on sodium (NCX1-3) and another in which Ca(2+) flux is also dependent on potassium (NCKX1-4). Both molecules are capable of forward- and reverse-mode exchange. In cells and tissues, Na(+)/Ca(2+) (and K(+)) gradients localize to the cell membrane; thus, the exchangers transport ions across a membrane potential. Uterine NCKX3 has been shown to be involved in the regulation of endometrial receptivity by [Ca(2+)](i). In the uterus and placenta, NCKX3 expression is regulated by the sex steroid hormone estrogen (E2) and hypoxia stress, respectively. In this chapter, we described the expression and regulation of these proteins for reproductive functions in various tissues including uterus, placenta, and kidney of humans and rodents. Evidence to date suggests that NCKX3 and NCX1 may be regulated in a tissue-specific manner. In addition, we focused on the molecular mechanism involved in the regulation of NCKX3 and NCX1 in mammals, based upon our recent results and those of others.

Triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol; TCS) is used as an antimicrobial agent in personal care, pharmaceutical, industrial, and household products. In this study, we established an in vivo model for screening estrogenic activity of TCS in the uteri of immature rats. In addition, we employed temporarily transfected cells with plasmids containing estrogen response element (ERE) and progesterone (P4) response element (PRE) sequences. We found that uterine weight was significantly increased by 17α-ethinylestradiol (EE) as a positive control and TCS at doses of 7.5, 37.4, and 187.5 mg/kg. In addition, the expressions of calbindin-D(9k) (CaBP-9k) and complement C3 (C3) were significantly induced by EE and TCS in the uteri of immature rats, indicating that TCS can induce their expression mediated by estrogenic activity. Co-treatment with steroid antagonists ICI 182,780 (ICI) and RU 486 in conjunction with TCS (37.5 mg/kg) reversed TCS-induced uterine weight and CaBP-9k mRNA and protein expression increases in immature rats. Moreover, ERE and PRE luciferase activity was evaluated in GH3 cells following treatment with TCS. Concentrations of TCS at increasing doses (10⁻⁹, 10⁻⁷, and 10⁻⁵ M) resulted in a significant increase in ERE luciferase activity compared to control; however, no difference was observed in PRE luciferase activity following TCS treatment. To confirm that ER signaling is involved in TCS-induced CaBP-9k expression, we treated GH3 cells with the anti-estrogen ICI, which can block TCS-induced up-regulation of CaBP-9k in these cells. Taken together, these results indicate that TCS has an estrogen-like property, which may be mediated through an ER-involved signaling pathway in both in vivo and in vitro models., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Ectonucleotide pyrophosphatase/phosphodiestrase 2 (Enpp2) isolated from the supernatant of human melanoma cells is a lysophospholipase D that transforms lysophosphatidylcholine into lysophospatidic acid. Although multiple analyses have investigated the function of Enpp2 in the hypothalamus, its role in the uterus during the estrous cycle is not well understood. In the present study, rat uterine Enpp2 was analyzed by RT-PCR, Western blotting, and immunohistochemistry. Quantitative PCR analysis demonstrated that uterine Enpp2 mRNA was decreased during estrus compared to proestrus and diestrus. To determine whether uterine Enpp2 expression is affected by sex steroid hormones, immature rats were treated with 17β-estradiol (E2), progesterone, or both on postnatal days 14 to 16. Interestingly, the expression of Enpp2 mRNA and protein were down-regulated by E2 in the uterus during estrus but not during proestrus or diestrus, suggesting that Enpp2 may play a role in uterine function during estrus. Enpp2 is primarily localized in the stromal cells of the endometrium during proestrus and estrus. During diestrus, Enpp2 was highly expressed in the epithelial cells of the endometrium. Taken together, these results suggest that uterine Enpp2 may be regulated by E2 and plays a role in reproductive functions during female rat development.

Four membrane calcium (Ca²⁺) channels are known to play an important role in calcium transport: transient receptor potential cation channel subfamily V members 5 and 6 (Trpv5/6), Na⁺/Ca²⁺ exchanger 1 (Ncx1) and plasma membrane Ca²⁺-transporting ATPase1 (Pmca1b). These channels have been shown to mediate the transport of Ca²⁺ between the cytosol and bloodstream in multiple tissues, including the duodenum, kidney, and uterus. Trpv5 and Trpv6 are known to selectively import Ca²⁺ into cells, while Ncx1 and Pmca1b function to extrude Ca²⁺. The present study examined and compared expression patterns of these genes in the canine duodenum, kidney and uterus. We confirmed expression of all four genes in this canine model. To collect tissues for analysis, two adult (2.5-year-old) female dogs were euthanized and tissue samples were collected from an abdominal incision. Expression of Trpv5/6, Ncx1 and Pmca1b was analyzed by reverse transcription polymerase chain reaction (RT-PCR), Western blot analysis, and immunohistochemistry. Trpv5, Trpv6 and Ncx1 mRNA were highly expressed in the kidney, while Pmca1b mRNA was primarily detected in the uterus. Protein expression was verified by Western blot analysis and localized by immunohistochemistry. Protein expression showed patterns similar to those of mRNA expression. Taken together, these results indicate that transcriptional and transitional expression of Tprv5, Trpv6, Ncx1 and Pmca1b may play a critical role in calcium transport in the canine duodenum, kidney and uterus.

Plasma membrane sodium/calcium exchangers are an important component of intracellular calcium homeostasis and electrical conduction. NCKX3 (gene SLC24A3), a potassium-dependent sodium-/calcium exchanger, plays a critical role in the transport of one intracellular calcium and potassium ion across the cell membrane in exchange for four extracellular sodium ions. NCKX3 transcripts are most abundant in the brain and smooth muscle, but many other tissues, in particular, the uterus, aorta and intestine, also express this gene at lower levels. However, the expression and physiological roles of NCKX3 in the uterus of rats during the estrous cycle are unknown. Thus, we examined the uterine expression of NCKX3 mRNA and protein at different stages of the estrous cycle in mature and immature female rats in the absence or presence of the sex-steroid hormones estrogen (E2) and progesterone (P4). During the estrous cycle, uterine expression of NCKX3 mRNA and protein was enhanced up to 4.0- and 2.5-fold, respectively, at proestrus compared to during estrus and diestrus. To examine the effect of sex steroids on NCKX3 regulation in the uterus, immature female rats were treated with E2 (40 µg/kg body weight; BW), P4 (4 mg/kg BW), or E2 plus P4 for 3 days. The expression of NCKX3 mRNA and protein was induced by E2, whereas P4 antagonized E2-induced NCKX3 expression. Subsequent immunohistochemical analysis revealed that uterine NCKX3 protein was abundantly localized in the cytoplasm of luminal and glandular epithelial cells throughout the estrous cycle. Taken together, these results indicate that uterine NCKX3 is abundantly expressed in the uterus and that its expression is regulated by the steroid hormones, E2 and P4. These findings suggest that NCKX3 may be involved in reproductive function during the estrous cycle in female rats., (Copyright © 2010 Wiley-Liss, Inc.)

Calbindin-D9k (CaBP-9k) is a cytosolic calcium-binding protein expressed in tissues in the intestine, uterus, placenta, kidney, pituitary gland and bone. Its exact function is unknown, but it is considered to regulate intracytoplasmic concentration and transport of free ions (Ca(2+)). CaBP-9k protein is involved in intestinal calcium absorption in the intestine and in the regulation of myometrial activity by intracellular calcium in the uterus. Renal CaBP-9k protein is expressed at the site of calcium re-absorption in the kidney and expressed in distal convoluted tubules, where it is thought to facilitate calcium re-absorption. Expression of the CaBP-9k gene has been explored in most mammalians except in a canine model. Presently, we elucidated the expression of CaBP-9k mRNA and protein in the duodenum, kidney and uterus in a canine model involving two adult (2.5-year-old) female beagles. To collect tissues, the dogs were euthanized and then the abdominal cavity was exposed by midline incision. The proximal duodenum, cortex of kidney and uterine horn were collected. Expression of CaBP-9k mRNA was confirmed by reverse transcriptionpolymerase chain reaction (RT-PCR) and real-time PCR. CaBP-9k protein expression and localization were ascertained by Western blot analysis and immunohistochemistry, respectively. CaBP-9k mRNA was detected in the duodenum, but not in the kidney and uterus. Its protein was expressed only in the enterocytes of the duodenum. Taken together, the results indicate that CaBP-9k mRNA and protein are highly expressed in the enterocytes of the duodenum of a canine model, consistent with findings in other mammalian species.

Plasma membrane sodium/calcium exchangers are an important component of intracellular calcium homeostasis and electrical conduction. A member of the family of potassium-dependent sodium/calcium exchangers, NCKX3 (SLC24A3) plays a critical role in the transport of an intracellular calcium and potassium ion in exchange for four extracellular sodium ions. NCKX3 mRNA transcripts are particularly abundant in brain and smooth muscle, but many other tissues, including uterus, aorta, and intestine, also express NCKX3, albeit at lower levels. In the present study, we examined uterine expression of NCKX3 at the mRNA and protein levels during the estrous cycle in mature female ICR mice in the absence or presence of the sex steroid hormones estrogen (E2) and progesterone (P4). During the estrous cycle in mice, uterine expression of NCKX3 mRNA and protein was elevated at estrus as compared to proestrus and diestrus. To determine the role of sex steroids in the regulation of NCKX3 expression in the uterus, mice were treated with E2 (40 microg/kg body weight), P4 (4 mg/kg body weight) or E2 plus P4 for 3 days. The levels of NCKX3 mRNA in the uterus of mice were significantly reduced by E2 or P4 treatment. Co-treatment with E2 and P4 caused a significant decrease in the transcription of NCKX3 as compared to E2 or P4 alone, but the effect was not synergistic. Using immunohistochemistry, we identified abundant levels of uterine NCKX3 in the cytoplasm of luminal and glandular epithelial cells at estrus. The results of the present study indicate that NCKX3 is abundantly expressed in the uterus and that its expression is regulated by the steroid hormones E2 and P4. Thus, uterine expression of NCKX3 might be involved in reproductive function during the estrous cycle in female mice.

Transient receptor potential cation channel, subfamily V, member 6 (TRPV6) is an epithelial Ca2+ channel protein expressed in calcium absorbing organs. In the present study, we investigated the expression and regulation of uterine and placental TRPV6 during gestation in rodents. Uterine TRPV6 peaked at pregnancy day (P) 0.5, P5.5 and, P13.5 and was detected in uterine epithelium and glands of rats, while placental TRPV6 mRNA levels increased in mid-gestation. Uterine and placental TRPV6 mRNA levels in rats appear to cyclically change during pregnancy, suggesting that TRPV6 may participate in the implantation process. In addition, uterine TRPV6 mRNA is only expressed in placenta-unattached areas of the uterus, and uterine TRPV6 immunoreactivity was observed in luminal and glandular epithelial cells. In the placenta, TRPV6 was detected in the labyrinth and spongy zone. These results may indicate that TRPV6 has at least two functions: implantation of the embryo and maintenance of pregnancy. To investigate the pathway(s) mediating TRPV6 expression in rodents, anti-steroid hormone antagonists were injected prior to maximal TRPV6 expression. In rats, TRPV6 expression was reduced by RU486 (an anti-progesterone) through progesterone receptors, and ICI 182,780 (an anti-estrogen) blocked TRPV6 expression via estrogen receptors in mice. The juxtaposition of uterine and placental TRPV6 expressed in these tissues supports the notion that TRPV6 participates in transferring calcium ions between the maternal and fetal compartments. Taken together, TRPV6 gene may function as a key element in controlling calcium transport in the uterus between the embryo and the placenta during pregnancy.

Calbindin-D(9k) (CaBP-9k) gene is expressed in the uterus of pregnant rats, which is regulated by steroid hormones during estrous cycle or gestation. We hypothesized that there is a positive correlation between altered CaBP-9k expression and change in one or more of the hormones to provide a clue to the mechanism responsible for the altered calcium levels in the uterus, placenta, and fetal membrane during late gestation. Thus, in the present study, we investigated the effects of the hormones including estradiol (E2), calcitonin (CT), and parathyroid hormone (PTH) on the regulation of CaBP-9k in these tissues. There was an increase in the level of CaBP-9k in the uterus, placenta, and extra-embryonic membrane at late gestation, as blood calcium level increased. The protein level of CaBP-9k remained lower in the uterus at two-thirds of pregnancy, and then it rebounded abruptly during late pregnancy. During late gestation, E2 is postulated to be a dominant factor in the regulation of uterine CaBP-9k gene expression. Furthermore, we assumed that there is a positive correlation between altered expression of CaBP-9k and blood calcium level during pregnancy. The present study demonstrated the regulation of CaBP-9k mRNA in the uterus, placenta, and fetal membrane of rats, implying a role for CaBP-9k gene in the control of blood calcium in placenta and the calcium passing from maternal blood to fetal circulation. Taken together, these results suggest that major alterations in calcium metabolism caused by maternal thyroparathyroidectomy (TPTX), are sufficient to affect the changes in reproductive tissues during late pregnancy. In addition, an increase of blood calcium level is one of the most significant factors in the regulation of CaBP-9k at the transcriptional and/or translational levels in the reproductive tissues during late pregnancy.

Polybrominated diphenyl ethers (PBDEs), a class of organic brominated flame retardants, have been increasing in the environment and in the tissues and milk of animals, including humans. To date, 209 PBDE congeners have been reported. Among these, 2,2',4,4'-tetrabromodiphenyl ether (BDE 47) is the dominant congener found in humans and animals. A number of studies have suggested that BDE 47 possesses the potential to disrupt the endocrine system, as well as reproductive functions. This suggests that BDE 47 may act as a developmental neurotoxin and endocrine disruptor. In this study, we employed immature rats as a developmental model to examine the potential involvement of BDE 47 in the induction of calbindin-D9k (CaBP-9k), which is a novel biomarker for screening estrogenic compounds. Beginning on postnatal day 16, BDE 47 was administered to immature rats in a dose- and time-dependent manner for 3 days. The biological effects of BDE 47 on the induction of CaBP-9k mRNA and protein were examined by semiquantitative RT-PCR and western blotting, respectively. In addition, the physiological role of the estrogen receptor (ER) in BDE 47-induced CaBP-9k expression was examined in vivo. Treatment with a high dose of BDE 47 (200 mg/kg body weight [BW]/day) resulted in a significant increase in CaBP-9k mRNA and protein 24 h after injection, whereas a modest increase was observed with low and medium doses (50 and 100 mg/kg BW/day). Additionally, treatment with the high dose of BDE 47 induced a clear uterotrophic response. Cotreatment with ICI 182,780, an ER antagonist, completely reversed the BDE 47-induced increases in uterine wet weight and CaBP-9k mRNA and protein. Taken together, these results demonstrate that BDE 47 exposure results in increases in CaBP-9k mRNA and protein in the uteri of immature rats. The biochemical pathway for BDE 47-induced activity may involve the ER-mediated signaling pathway. These results provide new insights into the estrogenic effects of BDE 47 at a critical developmental stage of the female reproductive system.

Various environmental chemicals, both natural and synthetic, are believed to act as endocrine disruptors (EDs) in mammals. In this study, a new in vivo model of immature rats was used to explore the induction of calbindin-D9k (CaBP-9k) following a single injection of EDs. In a time-dependent experiment, immature rats at postnatal day 16 were treated with high doses (600 mg/kg body weight [BW]) of 4-tert-octyphenol (OP), p-nonylphenol (NP), or bisphenol A (BPA), and euthanized at different time points (3, 6, 12, 24, or 48 h). For a dose-dependent study, immature rats were given different doses (200, 400, or 600 mg/kg BW) and euthanized at 24 h after injection. After treatment with these EDs, the effects on CaBP-9k mRNA and protein were examined by Northern and Western blot analyses, respectively. An anti-estrogen, ICI 182,780, was employed to examine the potential involvement of estrogen receptor (ER) in the induction of estrogen receptor-mediated physiologic responses in vivo. A single treatment with each of the chemicals, at 600 mg/kg BW, resulted in a significant increase in the expression of CaBP-9k mRNA and protein 24 h after injection. In addition, treatment with OP, NP, or BPA resulted in a positive uterotrophic response. Cotreatment with the ER antagonist ICI 182,780 completely prevented the ED-induced uterine weight gain. Taken together, these results demonstrate that a single injection of OP, NP, or BPA results in an increase of CaBP-9k mRNA and protein via an ER-dependent pathway in the uterus of immature rats. This new model may be important to elucidate the mechanism of action of xenoestrogens on estrogen-sensitive tissue.

A calcium binding protein, calbindin-D9k (CaBP-9k), is a cytosolic protein and regulated by steroid hormones in the reproductive tissues. Mouse CaBP-9k gene was predominantly regulated by progesterone (P4), whereas rat CaBP-9k was mainly regulated by 17beta-estradiol (E2) in the uterus. The induction of CaBP-9k can be employed as a biomarker for steroidal substrates as endocrine disruptors (EDs). Flutamide (FLU) is a non-steroidal anti-androgen or pro-drug that is rapidly metabolized to hydroxyflutamide, which may have both an anti-androgenic and anti-progestogenic activities. Thus, in the present study, we employed immature mice (14-day-old) subcutaneously injected with P4 (20 mg/kg/day) and/or FLU (5 mg/kg/day) for 3 consecutive days in the presence or absence of RU486, a pure PR antagonist (30 mg/kg/day), to analyze uterine CaBP-9k expression in this model. When immature mice were treated with P4, the expression levels of CaBP-9k mRNA and protein were significantly increased by P4. P4-induced expression levels of CaBP-9k mRNA and protein were abolished by FLU, in part, suggesting that FLU is a partial antagonist of P4 in the regulation of uterine CaBP-9k in immature mice. In addition, P4-induced CaBP-9k expression was completely reversed by RU486. Increased expression levels of CaBP-9k mRNA and protein were maintained for 24h after final injection with P4 in a time-dependent manner. However, CaBP-9k mRNA rapidly disappeared after 48 h and its protein level is similar with its mRNA. Treatment with FLU suppressed partially P4-induced CaBP-9k mRNA and protein until 24 h. Taken together, these results indicate that FLU has an anti-progestogenic activity and plays a role as a partial antagonist of P4 in the regulation of uterine CaBP-9k in immature mouse model.

Environmental estrogenic compounds which bind to the estrogen receptor (ER) can block or alter endogenous functions of estrogen in reproductive and developmental stages. A microarray technology is a very valuable method for the prediction of hormone-responsive activities in various gene expressions. Thus, we investigated the altered gene expression by estrogen and endocrine disruptors (EDs) using microarray technology in the uterus of immature rats. In this study, the expression levels of only 555 genes (7.42%) among the 7636 genes spotted on microarray chips were enhanced by more than two-fold following treatment with estradiol (E2), suggesting that direct or rapid response to E2 is widespread at the mRNA levels in these genes. In addition, elevated expression levels of the genes (over 2-fold) were observed by diethylstilbestrol (DES; 9.01%), octyl-phenol (OP; 8.81%), nonyl-phenol (NP; 9.51%), bisphenol-A (BPA; 8.26%) or genistein (9.97%) in the uterus of immature rats. The expression levels of representative genes, i.e., calbindin-D9k (CaBP-9k; vitamin D-dependent calcium-binding protein), oxytocin, adipocyte complement related protein (MW 30 kDa), lactate dehydrogenase A and calcium binding protein A6 (S100a6; calcyclin), were confirmed in these tissues by real-time PCR. In addition, the mRNA levels of these genes by real-time PCR were increased at follicular phase when E2 level was elevated during estrous cycle of adult female rats. In conclusion, these results indicate distinct altered expression of responsive genes following exposure to E2 and estrogenic compounds, and implicate distinct effects of endogenous E2 and environmental endocrine disrupting chemicals in the uterus of immature rats.

Calcium-related proteins include the calcium transporters 1 and 2 (CaT1 and CaT2), plasma membrane Ca2+-ATPase 1b (PMCA1b), and calbindin-D9k and -D28k. The expression of CaT1 and PMCA1b and their potential roles in the uterine tissue remain to be clarified. Thus, in the present study, the expression patterns of CaT1 and PMCA1b were examined to predict their roles in rat uterus during the estrous cycle. Both CaT1 and PMCA1b mRNAs were detected in rat uterus. Uterine CaT1 mRNA was highly expressed at diestrus compared with proestrus, whereas PMCA1b expression was not altered during the estrus cycle. To evaluate the sex steroids involved in uterine CaT1 mRNA regulation, 17beta-estradiol (E2) and/or progesterone (P4) were injected into immature rats. Treatment with P4 or E2 plus P4 resulted in an increase in CaT1 mRNA, but a synergetic effect of E2 plus P4 was not detected. Uterine CaT1 mRNA was induced by P4 in a time- and dose-dependent manner, with maximal transcript detected 12 h after the final P4 injection. Treatment with RU486, a progesterone receptor (PR) antagonist, completely blocked P4-induced CaT1 mRNA, indicating that P4 regulates CaT1 mRNA expression via a PR-mediated pathway. In addition, CaT1 mRNA was expressed in uterine endometrium and glandular endometrium at diestrus in P4-treated rats. Together, these results suggest that CaT1 is regulated by P4 at diestrus via a PR-dependent pathway.

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.

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.

It has been demonstrated in our previous studies that Calbindin-D9k (CaBP-9k) is a potent biomarker for screening estrogen-like chemicals in the rat model. Although treatments with 17beta-estradiol (E2) and endocrine disrupting compounds resulted in the up-regulation of uterine CaBP-9k, the mechanism of CaBP-9k induction by these compounds through two subtypes of estrogen receptors (ERalpha and ERbeta) is unclear. Thus, in the present study, immature rats were treated with propyl pyrazole triol (PPT, an ERalpha-selective ligand), diarylpropionitrile (DPN, an ERbeta-selective ligand), E2, or dimethyl sulfoxide (DMSO, a vehicle control) for three days in order to clarify which subtype of ER is involved in the uterine CaBP-9k induction. Following injection with these ER ligands, uterine CaBP-9k expression was analyzed by Northern blot and immunoblot assays. Uterine CaBP-9k expression is mainly mediated by PPT in a dose- and time-dependent manner in immature rats, whereas no significant alteration of the uterine CaBP-9k gene was observed after DPN treatment. In addition, an estrogenicity of PPT in inducing CaBP-9k expression was completely blocked by the anti-estrogen ICI 182,780, implying that uterine CaBP-9k is solely induced by ERalpha. A single treatment with PPT rapidly increased the protein levels of ERalpha and PR, an E2-mediated gene, in these tissues. Taken together, these results indicate that uterine CaBP-9k is induced by E2 and endocrine disrupting chemicals via the ERalpha pathway, but not ERbeta, in the uterus of immature rats.

Endocrine disruption is a major global health concern in the industrialized world. The induction of uterine calbindin-D9k (CaBP-9k), which belongs to a large family of intracellular calcium binding proteins, was used to assess the exposure of endocrine disruptors (EDs) in an immature mouse model. Sex steroid hormones have been demonstrated to regulate uterine CaBP-9k expression in the uterus of rats and mice. In particular, the mouse CaBP-9k gene was predominantly regulated by progesterone (P4), whereas rat CaBP-9k was mainly induced by 17-beta-estradiol (E2) in the uterus. In the present study, immature (14-day-old) female mice were injected with 4-tert-octylphenol (OP), nonylphenol (NP), bisphenol A (BPA), E2, or P4 to determine their effects on uterine CaBP-9k mRNA and protein expression. In addition, to specify estrogenic or progestogenic activity of EDs in the regulation of CaBP-9k, the mice were co-treated with ICI 182,780, an estrogen receptor (ER) antagonist, or RU486, a progesterone receptor (PR) antagonist,. Treatments with OP, NP, or BPA resulted in an increase in CaBP-9k mRNA and protein in the uterus of immature mice in a dose-dependent and time-dependent manner. The EDs-induced expression of CaBP-9k mRNA and protein was reversed or abolished by pretreatment with RU486 or ICI 182,780, suggesting that these synthetic chemicals may have both progestogenic and estrogenic properties by acting through PR or ER in the induction of uterine CaBP-9k mRNA and protein in this model. These results describe a novel in vivo model for detection of both estrogenic and progestogenic activities of EDs in the induction of CaBP-9k mRNA and protein in the uterus of immature mice.

Genistein, a phytoestrogen possessing a high affinity for estrogen receptor beta (ERbeta), is of increasing interest because of its possible influence on the physiology of mammalian reproductive tracts. Although estrogen has been demonstrated to regulate Calbindin-D9k (CaBP-9k) in the rat uterus as with other calcium binding proteins, the role of ERbeta on the modulation of CaBP-9k remains to be elucidated. To elucidate the effect of genistein as a selective ERbeta agonist on uterine expression of CaBP-9k mRNA and protein, immature female rats were injected with genistein daily for three consecutive days in a dose-dependent (0.4, 4, and 40 mg/kg/day) and time-dependent (40 mg/kg/day; 3, 6, 12, 24, 48, and 72 h) manner. Then, the expression of CaBP-9k mRNA and protein was analyzed by Northern hybridization and Western blot, respectively, in the absence or presence of ICI 182,780 (ICI), an estrogen antagonist. In addition, the protein levels of ERalpha and ERbeta and mRNA level of progesterone receptor (PR) were further measured following genistein treatment to elucidate which of ERs is involved in CaBP-9k modulation. In a dose-dependent experiment, the highest dose of genistein (40 mg/kg/day) for 3 days significantly induced uterine CaBP-9k protein as 17beta-estradiol (E2) did. In addition, its maximal mRNA expression was observed at 3 and 6 h, and it returned to control level at 24 h in a time-dependent experiment. In parallel with its mRNA level, the protein level of CaBP-9k was significantly induced by genistein at 3 h and sustained up to 48 h. The pretreatment with ICI, followed by genistein or E2, completely blocked genistein- and E2-induced CaBP-9k protein in the uterus of immature rats. Interestingly, genistein was demonstrated to induce ERalpha protein, but not ERbeta and PR mRNA, an E2-responsive gene, in this tissue. These results imply that genistein, an ERbeta ligand, may regulate CaBP-9k gene through ERalpha pathway. Taken together, the present study demonstrated that genistein enhanced CaBP-9k gene via ERalpha in the uterus of immature rats, suggesting that ERalpha may be a key mediator in uterine CaBP-9k gene induction in immature rats.

Environmental chemicals are proposed to possess hormone-like properties, such as mimicking natural hormones, inhibiting the action of hormones, and inducing abnormal gene expression. Among environmental chemicals, the alkylphenol products (APs), octylphenol (OP) and nonylphenol (NP), are derived from alkylphenol ethoxylates and have been reported to be environmentally persistent. Thus, in the present study, we examined the effect of two APs, OP and NP, on the expression of Calbindin-D(9k) (CaBP-9k) following maternal exposure during late pregnancy in maternal and fetal uteri. Treatment with a high dose (600 mg/kg body weight [BW]) of OP and NP resulted in an induction of CaBP-9k mRNA at Day 5 of lactation, as did a single treatment with diethylstilbestrol (DES) and 17beta-estradiol (E2) in maternal uteri. The expression of CaBP-9k mRNA was also induced following treatment with a high dose (600 mg/kg BW) of OP, transferred from the mother, exposed to fetuses during late pregnancy, and persisted through Day 5 of lactation. It is of interest that treatments with high doses of OP (400 and 600 mg/kg BW) reduced the expression of maternal estrogen receptor alpha (ERalpha) mRNA, as E2 did. However, all doses of NP resulted in an inhibition of neonatal ERalpha, while only the high does of OP (600 mg/kg BW) induced the reduction of neonatal ERalpha mRNA expression, as E2 did. Parallel to mRNA, the expression of CaBP-9k protein was significantly induced by treatment with a high dose of OP and NP. In conclusion, maternal exposure to APs, OP and NP, during late pregnancy increased the expressions of CaBP-9k mRNA and protein in maternal and neonatal uteri. These results suggest that the absorption and distribution of environmental estrogenic compounds in maternal and neonatal uteri are extremely rapid, and these chemicals can easily pass though the placenta during pregnancy to affect functions of neonatal reproductive tissues.

Calbindin-D(9k) (CaBP-9k) is a cytosolic calcium binding protein mainly expressed in the duodenum, placenta and uterus, and intestinal CaBP-9k is regulated by 1, 25-dyhydroxyvitamin D3. However, despite the presence of vitamin D receptors, uterine CaBP-9k is not under the control of vitamin D, but seems to be regulated by sex steroids. This steroids-dependent regulation of CaBP-9k is not only limited to a tissue-specific manner but also extends to a species-specific manner. In this study, we examined the regulation of CaBP-9k gene at the transcriptional and translational levels, and also localized CaBP-9k protein in the uterus of immature mice. Treatment with progesterone (P4) resulted in the induction of CaBP-9k mRNA, and a co-treatment with estrogen (E2) plus P4 evoked a synergic effect on its mRNA level in this tissue. Interestingly, the translation of CaBP-9k protein was enhanced by E2, while no difference was observed at the transcriptional level. Not only P4 but also E2 itself induced an increase of CaBP-9k protein, and co-treatment with E2 and P4 showed a similar effect on its protein level in the uterus of immature mice. The CaBP-9k protein was localized in the glandular epithelium of stroma in the uterus of immature mice at diestrus, indicating that the expression of CaBP-9k protein is differentially regulated by sex steroids. A potential mechanism of synergic effect of P4 and E2 may be E2 action in the increase of progesterone receptor (PR), with up-regulated PR increasing P4-induced CaBP-9k expression. This complicated relationship between CaBP-9k and steroid receptors suggests that P4 regulates CaBP-9k gene in the uterus of immature mice, in addition, E2 also can affect the expression of CaBP-9k through the regulation of PR. The expression levels of ERalpha and PR were further examined in this tissue. E2 stimulated the expression levels of ERalpha and PR mRNAs and P4 inhibited the expression of these transcripts at an early time point (12 h) and increased them at 24 and 48 h, while co-treatments with both steroids increased transcripts of ERalpha and PR at 24 h. In conclusion, P4 and PR may be dominant factors in the regulation of CaBP-9k. Also, E2 and ERalpha can influence the expression of the CaBP-9k gene via an indirect pathway in the uterus of immature mice.

Calbindin-D9k (CaBP-9k) is a member of intracellular calcium binding proteins, which have a high affinity to calcium. CaBP-9k is mainly expressed in the mammalian intestine, uterus and placenta, and is regulated in tissue- and species-specific manners. Previous studies have shown that CaBP-9k expression is mainly controlled by steroid hormones and their receptors. Thus, we further investigated the expression and regulation of CaBP-9k during an estrus cycle in the pig uterus by Northern blot and immunoblot analysis in this study. In addition, serum levels of estrogen (E2) and progesterone (P4) were measured using ELISA. The CaBP-9k mRNA is highly expressed in the porcine uterus during a luteal phase compared to a follicular phase, and its mRNA level in a luteal phase is increased up to 10-fold compared to a follicular phase. In parallel to the level of CaBP-9k mRNA, the CaBP-9k protein is also dominantly expressed in the porcine uterus, and strongly expressed in the epithelium and glands of the porcine uterus during a luteal phase. Although, the localization of the CaBP-9k protein is scarcely detected at follicular phase, it is dominantly expressed in the porcine uterus during a luteal phase. In addition, the serum P4 level was significantly increased during a luteal phase compared to a follicular phase, whereas no difference was observed in E2 levels between follicular and luteal phases, indicating that the ratio of P4/E2 is remarkably increased in porcine uterus during a luteal phase compared to a follicular phase. In conclusion, these results suggest that P4 may play an important role in the up-regulation of CaBP-9k gene in the porcine uterus in a luteal phase, which is unlike the condition in the rat uterus. In addition, the porcine CaBP-9k may be dominantly expressed in the epithelium and glandular structure of pig uterus during a luteal phase. It may also be differentially regulated during this cycle presumably by steroid hormones, especially up-regulated P4 levels in this tissue., (Copyright 2004 Wiley-Liss, Inc.)

The compounds that bind the estrogen receptors (ER) and induce or modulate an ER-mediated response can be defined as estrogenic endocrine disruptors (EDs). We demonstrated that environmental estrogens induced the uterine CaBP-9k mRNA in rats using Northern blot assay suggesting that CaBP-9k gene could be a biomarker for the estrogenicity of chemicals in the previous studies. In the present studies, we further collaborated this idea by investigating the regulation and localization of CaBP-9k protein in response to estrogenic compounds. Immature rats were injected with estrogenic chemicals, 4-tert-octylphenol (OP), nonylphenol (NP) and bisphenol A (BPA) or 17beta-estradiol (E2). After treatment with these estrogenic compounds, the effects on the accumulation of CaBP-9k protein and uterine localization were examined by Western blot analysis and immunohistochemical staining (IHC), respectively. A dose- and time-dependent increase in CaBP-9k protein was observed in the uterus of immature rats when treated with OP and NP. In addition, treatment with BPA resulted in a significant increase in CaBP-9k protein at dose of 500mg/kg BW/day. Taken together, CaBP-9k protein is strongly up-regulated by estrogenic compounds (OP, NP and BPA) and E2 itself in the uterus of immature rats. These results indicate that CaBP-9k protein can be a useful biomarker for detection of the estrogenicity of putative estrogenic compounds. Thus, regarding to risk assessment, we propose that CaBP-9k protein assay in the immature rat uterus can be a very sensitive and powerful tool to identify compounds with estrogenic activity when used in combination with classical assays.

Cigarette smoke (CS) has harmful effects on human fertility, reproduction, and development as well as on patients suffering from metabolic diseases such as diabetes than on healthy individuals. This study was conducted to investigate the relationship between CS exposure and histological alterations of reproductive organs in female diabetic rats. We evaluated the histology of uteruses and ovaries obtained from female rats exposed to smoke from standard cigarettes for 4 weeks (28 hours a week). After CS exposure, tissue slides were made from uterine and ovarian samples and examined after hematoxylin and eosin staining. Immunohistochemistry was used for detection of matrix metallopeptidase 9 (MMP9), C‐X‐C chemokine receptor type 4 (CXCR4), and estrogen receptor (ER)α in the uterus and ovary. MMP9 is an inflammatory biomarker that increases during progression to endometriosis. As a chemokine receptor, CXCR4 is involved in development of the inner wall of the uterus and cell adhesion. In the uterus, the occurrence of MMP9, CXCR4, and ERα and the number of endometrial glands were increased by CS exposure, while in the ovary, occurrence of MMP9, CXCR4, ERα, proliferating cell nuclear antigen and the number of corpus lutea or cyst follicles were increased by CS exposure. Collectively, this study indicates that CS induced abnormal development of the uterus and ovary under induced diabetes, leading to adverse effects on normal function of reproductive organs in female rats. Highlights: Cigarette smoke (CS) exposure adversely affected reproductive organs of diabetic female rats.In the uterus, expression of matrix metallopeptidase 9 (MMP9), C‐X‐C chemokine receptor type 4 (CXCR4), estrogen receptor (ER)α, and the number of endometrial glands were increased by CS exposure,In the ovary, the expression of MMP9, CXCR4, ERα, and proliferating cell nuclear antigen and the number of corpus lutea or cyst follicles were increased by CS exposure.Exposure to CS via the respiratory system exerted a harmful impact on the uterus and ovary in female rats with diabetes. [ABSTRACT FROM AUTHOR]

Abstract: Many environmental chemicals are known endocrine disruptors (EDs). These have the potential to alter endocrine systems via various mechanisms that include binding to hormone receptors, thereby either mimicking or blocking the hormone actions and causing abnormal gene expression. Here, to elucidate the molecular mechanism(s) underlying the detrimental effects associated with the estrogenicity of these chemicals, we determined whether gene profiles were altered in rats exposed to 4-tert-octyphenol (OP) and diethylstilbestrol (DES) in utero. Pregnant rats were treated with a high dose of OP (600mg/kgBW per day) or DES (500μg/kgBW per day) at gestational days (GD) 17, 18 and 19. Both dams and neonates were euthanized at lactation day (LD) 5. The transcript profiles of uterine tissue were compared in treated versus control in both maternal and neonatal sites using cDNA microarray to determine the expression levels of approximately 13,000 genes and expressed sequence tags (ESTs). The expression levels of some known estrogen-responsive genes, i.e., complement component 3, epidermal growth factor receptor or c-fos oncogene and calbindin 3, as well as some other randomly selected genes, including general transcription factor IIa, transcription factor 4 and lymphocyte specific 1, were increased by OP and/or DES treatment in the uteri of both maternal and neonate groups. However, the magnitude of these alterations in gene expression differed markedly between dams and neonates, most likely reflecting the temporal susceptibility of the reproductive tract to estrogenic chemicals. Importantly, the altered gene patterns identified by microarray analysis were confirmed by RT-PCR and real-time RT-PCR. Fifteen primers were designed to amplify specific altered genes. These genes were selected for validation because of their markedly increased expression levels and they were classified on the basis of gene ontology. Overall, a high correlation was observed between microarray and real-time PCR data. Taken together, these results indicate that placental exposure to OP or DES may cause temporal changes in gene expression in the uteri of dams and neonates. Moreover, these findings may provide useful indicators of the adverse effects of EDs and prove particularly important in elucidating the effects of xenoestrogens on estrogen-responsive tissues, such as the developing reproductive tract. [Copyright &y& Elsevier]