Biol Reprod Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print April 7, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.026146
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
71/2/669    most recent
biolreprod.103.026146v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hong, E.-J.
Right arrow Articles by Jeung, E.-B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hong, E.-J.
Right arrow Articles by Jeung, E.-B.
Agricola
Right arrow Articles by Hong, E.-J.
Right arrow Articles by Jeung, E.-B.
BIOLOGY OF REPRODUCTION 71, 669–675 (2004)
DOI: 10.1095/biolreprod.103.026146
© 2004 by the Society for the Study of Reproduction, Inc.

Female Reproductive Tract

Induction of Calbindin-D9k Messenger RNA and Protein by Maternal Exposure to Alkylphenols During Late Pregnancy in Maternal and Neonatal Uteri of Rats1

Eui-Ju Hong3, Kyung-Chul Choi4, and Eui-Bae Jeung2,3

Laboratory of Veterinary Biochemistry and Molecular Biology,3 College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, 361-763, Republic of Korea Department of Obstetrics and Gynecology,4 British Columbia Children's and Women's Hospital, British Columbia Research Institute for Children's and Women's Health, University of British Columbia, Vancouver, British Columbia, Canada V6H 3V5


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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-D9k (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 17ß-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} (ER{alpha}) mRNA, as E2 did. However, all doses of NP resulted in an inhibition of neonatal ER{alpha}, while only the high does of OP (600 mg/kg BW) induced the reduction of neonatal ER{alpha} 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.

environment, gene regulation, placental transport, pregnancy, steroid hormones


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Environmental chemicals are increasingly suspected of inducing estrogenic effects. It has been proposed that they 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. Alkyl groups are typically branched nonyl-, octyl-, or dodecyl-chains, forming a variety of isomers mostly in the para-position of the phenolic ring. Numerous APs have been identified that can bind to the estrogen receptor (ER) and initiate transcription of ER-regulated genes in vitro [1, 2], but their potency is about 1000- to 10 000-fold lower than 17ß-estradiol (E2) [3]. In addition, APs that are weakly estrogenic have been known to increase uterine weight in traditional uterotropic assays [4]. In vitro, E2 has been demonstrated to induce maximal proliferation of MCF-7 cells at 1 nM concentration, and OP and NP have been found to be considerably potent estrogenic factors at 1 and 10 µM, respectively. Treatment with OP and NP inhibited the binding affinity of E2 to ER in MCF-7 cells by a competitive ER binding assay [5]. In female rats, subcutaneous OP induced estrogen-like effects in the reproductive system, as evidenced by alterations in the estrous cycle and increased uterine weight [4, 6]. Sperm production and testes weight in offspring of dams exposed during pregnancy and lactation was reduced by administered OP in drinking water at approximately 0.1 ppm [7]. Although environmental exposure to these chemicals has the potential to disrupt or interfere with reproductive functions, the mechanism remains to be clarified.

E2 effects the temporal and cell type-specific expression of various genes, the products of which mediate cell growth, differentiation, and general homeostasis of reproductive and other systems [8, 9]. In rats, the concentration of E2 is consistently low throughout neonatal development and starts to increase after Day 28 of age [10]. The uterus and ovaries are two of the most sensitive tissues to estrogen, and both tissues express ER{alpha} and ERß. In particular, ER{alpha} is predominantly expressed in uteri, while ERß is expressed in ovaries [11, 12]. In the previous studies, we demonstrated that expressions of the Calbindin-D9k (CaBP-9k) gene is regulated through binding of the ER/estrogen complex to estrogen response element (ERE) in rats, and this binding enhanced expression of ER{alpha} mRNA in uteri treated with estrogenic compounds [6, 13]. The CaBP-9k that binds calcium with high affinity is expressed in the intestines, uterus, placenta, and kidneys [14, 15]. The CaBP-9k mRNA in the uterus is known to fluctuate during the estrous cycle of rats, depending on serum estrogen level. The CaBP-9k mRNA at diestrus was not detectable, but increased at proestrus, reached the highest level at estrus, then decreased as metestrus [16].

It was shown in a previous study that the rat CaBP-9k gene binds to ER because it contains an estrogen-response element in the encoding region [13]. The induction of CaBP-9k in rat placenta and endometrial epithelium at the time of maximal fetal bone growth and accumulation of calcium suggests its involvement in maternal-fetal calcium transport [17]. During late pregnancy, the expression of CaBP-9k mRNA in rat uteri increases from Day 16 of pregnancy, peaks at Day 21, and dramatically decreases after parturition from Day 1 to 2 of lactation [18]. In our previous studies, we demonstrated that maternal-fetal transfer of endocrine disruptors resulted in an increase in the induction of CaBP-9k mRNA and protein in the fetal uterus during late pregnancy. In particular, diethylstilbestrol (DES) and APs were transferred to fetuses, and the transplacental absorption of alkylphenols resulted in an induction of CaBP-9k in fetal uteri [19], which was not expressed in the uterus of mature ovariectomized and immature rats [20].

In the present study, we investigated the expression of CaBP-9k mRNA and protein in maternal and neonatal uteri following maternal exposure to OP and NP during late pregnancy. In addition, the effect of estrogenic compounds on the expression of ER{alpha} mRNA in these tissues was examined to elucidate correlative expressions of CaBP-9k and ER.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal Treatments

Pregnant Sprague-Dawley rats were obtained from Dae Han Biolink Co., Ltd. (Chungbuk, Korea). All animals were housed in polycarbonate cages, and used after acclimation to an environmentally controlled room (temperature, 23 ± 2°C; relative humidity, 50 ± 10%; frequent ventilation; and a 12L:12D cycle). They were fed with soy-free pellet food (Dyets Inc., Bethlehem, PA). In the experiment, six groups of five animals (total n = 30) were given subcutaneous (SC) injections with OP and NP at the doses of 200, 400, and 600 mg/kg body weight (BW) per day dissolved in corn oil (Sigma-Aldrich Corp., St. Louis, MO) during Days 17–19 of pregnancy. As control groups, three groups of maternal rats (total n = 15) were injected with a single dose of DES (500 µg/kg BW per day) and E2 (40 µg/kg BW per day) on Day 17 of pregnancy, or corn oil during Days 17–19 of pregnancy. On the fifth day after parturition, both dams and pups were killed (Fig. 1). The uteri were washed in cold sterile 0.9% NaCl solution (0.9% normal saline) and used for RNA and protein extraction. All experimental procedures and animal use were approved by the Ethics Committee of the Chungbuk National University.



View larger version (11K):
[in this window]
[in a new window]
  		FIG. 1. Experimental design to treat pregnant rats with OP and NP during pregnancy

Northern Blot Analysis

Total RNA was extracted with Trizol (Life Technologies, Rockville, MD) according to manufacturer's suggested procedure, and the concentration of RNA was determined by the absorbance at 260 nm. Northern blot analysis was performed to determine CaBP-9k mRNA, as described previously [19]. Briefly, 10–20 µm total RNA isolated from maternal uteri was electrophoresed and 28S rRNA was used as an indication of the quantity of total RNA. The total RNA was transferred overnight onto a nylon membrane (Schlicher and Schuell, Keene, NH), ultraviolet cross-linked, and prehybridized in buffer containing 50% formamide, 5x Denhardt (0.1% polyvinyl pyrrolidine, 0.1% BSA, 0.1% Ficoll), 5x SSPE (sodium chloride, sodium phosphate, EDTA), 0.1% SDS, and denatured salmon sperm DNA (100 µg/ml) for 2 h at 42°C before adding labeled probe. The cDNA for CaBP-9k was labeled with [32P]dCTP (3000 Ci/mmol; Amersham Biosciences, Little Chalfont, UK) using the Random-Primed DNA labeling kit (Takara, Shiga, Japan) and purified as indicated in the manufacturer's protocol. Hybridization was performed in the fresh buffer at 42°C for 18 h with 32P-labeled cDNA. After washing under stringent conditions, the membrane was exposed to Kodak X-AR film (Eastman Kodak, Rochester, NY) at –80°C.

Reverse Transcription-Polymerase Chain Reaction

Northern blot analysis was performed to analyze the expression of CaBP-9k mRNA in neonatal uteri, but CaBP-9k transcript was not detectable. Thus, reverse transcription-polymerase chain reaction (RT-PCR) was alternatively performed to detect CaBP-9k mRNA in this tissue. Total RNAs (5 µg) from the neonatal uterus were used for RT-PCR analysis. Total RNAs were subjected to first-strand cDNA synthesis using M-MLV reverse transcriptase (KOSCO Inc., Seoul, Korea) and random primer (9 mer). Aliquots of 1 µl were used for PCR at standard conditions. To determine the condition under which PCR amplification for CaBP-9k (314 base pair [bp]), ER{alpha} (325 bp), and Cytochrome c oxidase subunit 1 (1A, 968 bp) mRNA were in the logarithmic phase, the aliquots (1µl) were amplified using different numbers of cycles. The PCR product of the 1A gene was amplified to rule out the possibility of RNA degradation and was used to control the variation in mRNA concentrations in the RT reaction. A linear relationship between PCR products and amplification cycles was observed in CaBP-9k, ER{alpha} and 1A mRNAs. Twenty-five cycles for CaBP-9k and ER{alpha}, 18 cycles for 1A gene were employed for quantification. The cDNA was amplified in a 20-µl PCR reaction containing 1 unit Taq polymerase (Intron Co., Seoul, Korea) and its buffer, 1.5 mM MgCl2, 2 mM deoxy-NTP, and 100 pmol specific primers. PCR reactions were denatured at 95°C for 1 min, annealed at 55°C for 1 min, and extended at 72°C for 1 min 30 sec. The primers of CaBP-9k were 5'-AAG AGC ATT TTT CAA AAA TA-3' (sense, JK1) and 5'-GTC TCA GAA TTT GCT TTA TT-3' (antisense, JK2). The primers of ER{alpha} were 5'-ATG ATC AAC TGG GCA AAG A-3' (sense, ER1) and 5'-TGT ACA CTC CGG AAT TAA GC-' (antisense, ER2). The primers of the 1A gene were: 5'-GAT ATA GCA TTC CCA CGA ATA-3' (sense, A-1) and 5'-GGG CTT TTG CTC ATG TGT CAT-3' (antisense, A-2). Ten microliters of PCR products were fractionated on a 2% agarose gel, stained with ethidium bromide. The photograph was scanned and analyzed using molecular analysis program version 1.5 (Gel Doc 1000; Bio-Rad, Hercules, CA). The PCR product was then transferred from the agarose gel to the nylon membrane using Vacuum blotter (Bio-Rad) and ultraviolet cross-linked to the membrane using a Gene Cross-Linker (Bio-Rad). Membranes were prehybridized in 50% formamide, 5x SSPE, 5x Denhardt, 0.1% SDS, 0.1 mg/ml salmon sperm DNA for 3 h at 42°C. The [32P]dCTP-labeled probes were added to the hybridization solution and incubated for 16 h at 42°C. The membranes were washed three times at 42°C in 2x SSC, 0.1% SDS, at 54°C in 1x SSC, 0.1% SDS, and at 68°C in 0.1x SSC, 0.1% SDS. The membranes were then exposed to x-ray films (Eastman Kodak Co.). The film was scanned and analyzed by molecular analysis program version 1.5 (Gel Doc 1000; Bio-Rad).

Western Blot Analysis

The uteri were rapidly excised and washed in cold sterile 0.9% NaCl solution. Protein was extracted with Proprep (Intron Co.) according to the supplier's instruction. Fifty micrograms of cytosolic protein per lane were dissolved by SDS/PAGE (15% acrylamide) and transferred to polyvinylidene fluoride transfer membrane (Perkin Elmer Co., Wellesley, MA) by a TransBlot Cell (Bio-Rad) according to the manufacturer's protocol. The membranes were then blocked with phosphate-buffered saline containing 0.05% Tween-20 and 5% dry milk for overnight and were incubated sequentially with primary and secondary antibodies dissolved in 1% BSA for 1 h at room temperature. To detect CaBP-9k protein, a polyclonal antibody (1:2000; Swant, Bellinzona, Switzerland) to rat CaBP-9k was employed, and beta-actin antibody (42 kDa; Santa Cruz Biotechnology, Santa Cruz, CA) was used as an indication of the quantity of total protein [21]. Horseradish peroxidase-conjugated anti-rabbit IgG (Santa Cruz Biotechnology) was used as a second antibody and visualized using the ECL chemiluminescent system (Amersham Biosciences), followed by autoradiography. The level of CaBP-9k protein was quantitated by densitometry (NIH Image Data 3; http://rsb.info.nih.gov/nih-image/).

Immunohistochemical Staining

The localization of CaBP-9k protein was examined by immunohistochemistry. Small pieces of rat uteri were embedded in paraffin, and paraffin sections (5 µm) were deparaffinized in xylene and hydrated in descending grades of ethanol. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in methanol for 20 min. Incubation of the sections in 3% bovine serum albumin (BSA) blocked nonspecific reaction for 1 h at room temperature and sequentially incubated with a rabbit polyclonal antibody (1 µg/ml, 1:1000; Swant) specific to the rat CaBP-9k dissolved in 1% BSA at 4°C for overnight. After washing, the sections were incubated with the secondary biotinylated antibody (rabbit IgG; Vector Laboratories, Inc., Burlingame, CA) for 30 min, and further incubated with ABC-Elite for 30 min at room temperature. Diaminobenzidine (Sigma, St. Louis, MO) was used as chromogen, and the sections were counterstained with hematoxylin followed by mounting in Canada balsam.

Data Analysis

Data are presented as the mean ± SD. The data were analyzed by a nonparametric procedure of Kruskal-Wallis test, followed by the Dunnett test for two-pair comparisons. Each value of the Dunnett test was converted to rank for statistical analysis. All statistical analyses were performed with SAS (SAS Institute, Cary, NC). P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of CaBP-9k mRNA in Maternal and Neonatal Uteri

In maternal uteri, treatment with a high dose (600 mg/ kg BW) of OP and NP induced the expression of CaBP-9k mRNA on the fifth day after parturition, as did a single dose of DES or E2 (Fig. 2). The 600 mg/kg BW per day dose increased CaBP-9k 3-fold and 4-fold for OP and NP, respectively. CaBP-9k mRNA was induced by a single dose treatment with DES (5-fold, 50 µg/kg BW per day) and E2 (1.9-fold, 40 µg/kg BW) for 3 days. However, treatments of maternal rats with lower doses of OP and NP (200 and 400 mg/kg BW) for 3 days failed to change the expression level of CaBP-9k mRNA, as shown in Figure 2.



View larger version (48K):
[in this window]
[in a new window]
  		FIG. 2. Effect of OP and NP on the increase of CaBP-9k mRNA in maternal uteri. Northern blot analysis was performed, as described in the Materials and Methods section during the lactation period in maternal uteri. Data were analyzed by the nonparametric procedure of a Kruskal-Wallis test, followed by a Dunnett test for two-pair comparisons. The values represent means ± SD. a, P < 0.05 vs. vehicle

Using RT-PCR analysis, we examined the expression of CaBP-9k mRNA in neonatal uteri following maternal injection with APs, DES, and E2. The expression of neonatal CaBP-9k mRNA was significantly induced when treated with a high dose of OP (15-fold, 600 mg/kg BW per day) and DES (41-fold, 50 µg/kg BW per day), as seen in Figure 3. However, lower doses of OP and NP did not change the level of CaBP-9k mRNA following maternal exposure to these chemicals.



View larger version (41K):
[in this window]
[in a new window]
  		FIG. 3. Effect of OP and NP on the induction of CaBP-9k mRNA in neonatal uteri. RT-PCR/Southern blot assay was performed, as described in the Materials and Methods section during the lactation period in neonatal uteri. Data were analyzed by the nonparametric procedure of a Kruskal-Wallis test, followed by a Dunnett test for two-pair comparisons. The values represent means ± SD. a, P < 0.05 vs. vehicle

Expression of ER{alpha} mRNA in Maternal and Neonatal Uteri

The effect of estrogenic compounds on the expression of ER{alpha} mRNA in maternal and neonatal uteri was further examined to correlate CaBP-9k expression with ER{alpha} by RT-PCR. The expression of ER{alpha} mRNA was slightly reduced by OP (400 and 600 mg/kg BW), similar to what a single dose of E2 did in maternal uteri (Fig. 4). However, no significant difference was observed in treatments with all doses of NP and a low dose of OP (200 mg/kg BW) in this tissue.



View larger version (47K):
[in this window]
[in a new window]
  		FIG. 4. Effect of OP and NP on the induction of ER{alpha} mRNA in maternal uteri. A RT-PCR/Southern blot assay was performed as described in the Materials and Methods section during the lactation period in maternal uterus. Data were analyzed by the nonparametric procedure of a Kruskal-Wallis test, followed by a Dunnett test for two-pair comparisons. The values represent means ± SD. a, P < 0.05 vs. vehicle

Treatments with a high dose of OP (600 mg/kg BW) and all doses of NP resulted in a decrease of ER{alpha} in neonatal uteri, as demonstrated in Figure 5. A single treatment with E2 (0.29-fold, 40 µg/kg BW per day) decreased the level of ER{alpha} in this tissue as well (Fig. 5). Therefore, treatment with OP and NP resulted in a significant decrease in the expression level of ER{alpha} mRNA in neonatal uteri.



View larger version (49K):
[in this window]
[in a new window]
  		FIG. 5. Effect of OP and NP on the induction of ER{alpha} mRNA in neonatal uteri. RT-PCR/Southern blot assay was performed, as described in the Materials and Methods section during the lactation period in neonatal uteri. Data were analyzed by the nonparametric procedure of a Kruskal-Wallis test, followed by a Dunnett test for two-pair comparisons. The values represent means ± SD. a, P < 0.05 vs. vehicle

Expression of CaBP-9k Protein in Maternal Uteri

The level of CaBP-9k protein was examined in maternal uteri by immunoblot analysis. A significant amount of CaBP-9k protein was detected in maternal uterus. Treatments with a high dose (600 mg/kg BW per day) of OP and NP resulted in a significant increase of CaBP-9k protein less than 2-fold in maternal uteri, as demonstrated in Figure 6. In addition, a single dose of DES increased CaBP-9k protein expression (2-fold). However, we could not examine the expression level of CaBP-9k protein in neonatal uteri due to lack of sufficient protein from this tissue for immunoblot analysis.



View larger version (45K):
[in this window]
[in a new window]
  		FIG. 6. Effect of OP and NP on the increase of CaBP-9k protein in maternal uteri. An immunoblot assay was performed as described in the Materials and Methods section during the lactation period in maternal uteri. Data were analyzed by the nonparametric procedure of a Kruskal-Wallis test, followed by a Dunnett test for two-pair comparisons. The values represent means ± SD. a, P < 0.05 vs. vehicle

Localization of CaBP-9k Protein in Maternal Uteri

The CaBP-9k protein was localized in the endometrium and smooth myometrial fibers of maternal uteri by immunohistochemistry, as shown in Figure 7. In parallel with CaBP-9k protein level measured by immunoblot analysis, the expression of CaBP-9k protein was increased in a dose-dependent manner by immunohistochemistry following maternal exposure to APs in this tissue (Fig. 7). In particular, treatment with DES (50 µg/kg BW per day) resulted in an increase of CaBP-9k protein in this tissue. These results indicate that CaBP-9k protein, which is widely spread through stromal cells in the endometrium and myometrium of maternal uterus, may be induced by treatments with OP and NP.



View larger version (106K):
[in this window]
[in a new window]
  		FIG. 7. Localization of CaBP-9k protein by immunohistochemical staining in maternal uteri. Immunoreactivity of CaBP-9k protein expression following treatment with OP and NP was investigated in endometrium and smooth myometrial fibers, dose dependently. These proteins are widely spaced through the stromal cells in the endometrium; s, Stroma cells; le, luminal epithelial cell; ge, glandular epithelial cell. Magnification x100

Expression of CaBP-9k Protein in Neonatal Uterus

The CaBP-9k protein of neonatal uterus at Day 5 after delivery was further investigated following maternal injection with increasing doses of APs and a single treatment with DES and E2 by immunoblot analysis. Parallel to maternal CaBP-9k protein expression, treatment with DES resulted in upregulation of CaBP-9k protein in postnatal uteri. However, no significant difference was identified in the expression of CaBP-9k protein by APs or E2 in this tissue, as indicated in Figure 8.



View larger version (38K):
[in this window]
[in a new window]
  		FIG. 8. Effect of OP and NP on the induction of CaBP-9k protein in neonatal uteri. An immunoblot assay was performed as described in the Materials and Methods section during the lactation period in maternal uteri. Data were analyzed by the nonparametric procedure of a Kruskal-Wallis test, followed by a Dunnett test for two-pair comparisons. The values represent means ± SD. a, P < 0.05 vs. vehicle


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study suggests that estrogenic effects of APs and DES persist for 5 days after delivery following maternal exposure during late pregnancy. The treatment with a high dose of OP and NP significantly induced the expression of CaBP-9k mRNA at Day 5 of lactation (L5), and a single treatment with DES and E2 induced an increase of CaBP-9k mRNA in maternal uteri. As E2 is at a low level at L5, APs, OP and NP maternally injected, may function as estrogens during this period. It has been demonstrated that the CaBP-9k gene is regulated by estrogen and affected by estrogenic compounds in the uterus of rats [6, 20]. The expression of CaBP-9k mRNA in neonatal uteri was induced following treatments with APs that had transferred from the maternal side to fetus during late pregnancy and persisted at L5. Also, the treatment with OP and NP resulted in a significant decrease in the level of ER{alpha} mRNA in neonatal uteri to parallel the maternal uterus. We reasoned that up- or downregulation of these genes would be potentially important for detection of the estrogenic compounds from maternal transmission.

CaBP-9k gene expression is controlled by vitamin D in the intestines. However, this gene was induced by estradiol in the uterus, not by vitamin D, despite the presence of vitamin D receptor in the uterus [20]. E2 is one of the major factors controlling the expression of the CaBP-9k gene in rat uteri. During late pregnancy, the expression of CaBP-9k mRNA in rat uteri increases from Day 16 of pregnancy and peaks at Day 21 [18]. The induction of CaBP-9k in uterus at the time of maximal fetal bone growth and accumulation of calcium suggests its involvement in maternal-fetal calcium transport [17]. This gene starts to decrease during lactation and dramatically decreases at Day 5 of lactation. The lower level of CaBP-9k seems to be the result of the combination of high progesterone and low estrogen levels in the uterus of rats during lactation [22]. In the previous study, we demonstrated that CaBP-9k mRNA and protein were induced by environmental estrogenic compounds, which can be used as a marker gene for environmental estrogenic chemicals in the uterus of immature rats [6, 21]. The expression of CaBP-9k was induced by treatments with high doses of OP and NP in maternal uteri during late pregnancy, suggesting that the effects observed within the uterus of neonatal rats from maternal exposure reflect activation of APs. Parallel to CaBP-9k mRNA level, CaBP-9k protein was significantly increased by treatment with a high dose of OP and NP. Also, we performed immunohistochemistry to localize CaBP-9k protein in maternal uteri. The level of CaBP-9k protein in maternal uteri was dose-dependently increased by treatment with APs (OP and NP) and DES. The CaBP-9k protein is predominantly expressed in endometrial and myometrial stromal cells and smooth myometrial fibers in the estrous cycle of rats [23]. The present study demonstrated that CaBP-9k protein is induced by APs and DES in the subpopulation of stromal cells, but not in epithelial cells at L5.

A high dose of OP and DES significantly induced the expression of CaBP-9k mRNA in neonatal uteri, indicating that OP is a more potent estrogenic compound transferred from maternal exposure than NP. Maternally administered compounds can be transported to the fetus, and the capacity of compounds to cross the placenta is affected by hydrophilic, lipophilic, pH, partition coefficient, etc., properties [24, 25]. Placental transfer may require both lipophilic and hydrophilic properties [26]. In our previous study, we demonstrated that maternally injected estrogenic compounds resulted in an increase in the induction of CaBP-9k mRNA and protein in fetal uteri through the placenta [19]. Based on the dose-response data of molar equivalent doses in immature rats, the relative potencies of tested chemicals following subcutaneous injection are indicated as follows, 4-tert-octylphenol = 4-nonylphenol > bisphenol A [27]. Treatment with a high dose of OP and NP and a single dose of DES resulted in a significant increase of CaBP-9k protein in maternal uteri. Parallel to its mRNA level, a significant increase in CaBP-9k protein was demonstrated in neonatal uteri following treatment with DES maternally injected during pregnancy. However, there was no significant induction in the protein of CaBP-9k after treatment with APs (OP and NP) or E2 compared with DES. In the present study, the expression of CaBP-9k mRNA in neonatal uteri was induced following treatments with high doses of OP that had transferred from the maternal side to the fetus during late pregnancy and persisted through L5. OP has the greatest potential as an estrogenic compound among 4-alkylphenols, and it binds to ER, activates estrogen-responsive genes, and stimulates proliferation of MCF-7 cells [4, 28]. Exposure of pregnant rats to OP via drinking water has been reported to reduce testicular size and sperm production in the offspring [7]. Furthermore, NP was found to stimulate the growth of MCF-7 cells in vitro and enhance the uterotrophic response in mice and rats [27, 29, 30].

The actions of estrogen are primarily executed by its binding to nuclear estrogen receptors. Estrogens modulate the transcription of genes following binding as hormone receptor complexes to EREs in target promoters [31, 32]. APs have been reported to be weakly estrogenic. ER binding is primarily the result of interaction of the receptor with both a hydrophobic and a phenolic residue [33]. In the uteri of newborn mice after prenatal exposure, OP does not induce the expression of either ER{alpha} mRNA or protein in uterine epithelium. Furthermore, the protein level of ER{alpha} was decreased slightly in a dose-dependent manner [34]. In contrast with OP, methoxychlor has been shown to induce the expression of ER mRNA and protein in the uterine epithelium of mice [35]. In the present study, treatments with OP and E2 resulted in an inhibition of ER{alpha} mRNA in maternal uteri, whereas treatments with all doses of NP and a high dose of OP induced a decrease in ER{alpha} mRNA, as did E2. These results suggest that NP is more transferable from the maternal side to the fetus to affect the level of ER{alpha} mRNA in neonatal uterus than OP. The CaBP-9k gene carries an ERE, mediating expression in the rat uterus [36]. The CaBP-9k gene has an imperfect ERE that differs by only one nucleotide. Although this single nucleotide difference is important in the interaction between ER and ERE, the ERE in the promoter region of CaBP-9k gene binds to ER [37].

In conclusion, maternal exposure to the APs, OP and NP, during late pregnancy increased the expression levels 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.


   FOOTNOTES
 
1 Supported by Korea Research Foundation Grant (KRF 2003-041-E20238). Back

2 Correspondence: FAX: 82 43 267 3150; ebjeung{at}chungbuk.ac.kr Back

Received: 5 December 2003.

First decision: 29 December 2003.

Accepted: 30 March 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bolger R, Wiese TE, Ervin K, Nestich S, Checovich W. Rapid screening of environmental chemicals for estrogen receptor binding capacity. Environ Health Perspect 1998 106:551-557[Medline]
  2. Soto AM, Sonnenschein C, Chung KL, Fernandez MF, Olea N, Serrano FO. The E-SCREEN assay as a tool to identify estrogens: an update on estrogenic environmental pollutants. Environ Health Perspect 1995 103:suppl 7113-122
  3. Safe SH, Pallaroni L, Yoon K, Gaido K, Ross S, Saville B, McDonnell D. Toxicology of environmental estrogens. Reprod Fertil Dev 2001 13 307-315
  4. Bicknell RJ, Herbison AE, Sumpter JP. Oestrogenic activity of an environmentally persistent alkylphenol in the reproductive tract but not the brain of rodents. J Steroid Biochem Mol Biol 1995 54:7-9[CrossRef][Medline]
  5. Kwack SJ, Kwon O, Kim HS, Kim SS, Kim SH, Sohn KH, Lee RD, Park CH, Jeung EB, An BS, Park KL. Comparative evaluation of alkylphenolic compounds on estrogenic activity in vitro and in vivo. J Toxicol Environ Health A 2002 65:419-431[CrossRef][Medline]
  6. An BS, Kang SK, Shin JH, Jeung EB. Stimulation of calbindin-D(9k) mRNA expression in the rat uterus by octyl-phenol, nonylphenol and bisphenol. Mol Cell Endocrinol 2002 191:177-186[CrossRef][Medline]
  7. Sharpe RM, Fisher JS, Millar MM, Jobling S, Sumpter JP. Gestational and lactational exposure of rats to xenoestrogens results in reduced testicular size and sperm production. Environ Health Perspect 1995 103 1136-1143
  8. Hall JM, Couse JF, Korach KS. The multifaceted mechanisms of estradiol and estrogen receptor signaling. J Biol Chem 2001 276: : 36869-36872[Free Full Text]
  9. Nilsson S, Makela S, Treuter E, Tujague M, Thomsen J, Andersson G, Enmark E, Pettersson K, Warner M, Gustafsson JA. Mechanisms of estrogen action. Physiol Rev 2001 81:1535-1565[Abstract/Free Full Text]
  10. Noda S, Sawaki M, Shiraishi K, Yamasaki K, Yamaguchi R. Age-related changes of genital systems in the female Crj:CD (SD) IGS rats during sexual maturation. J Vet Med Sci 2002 64:315-319[CrossRef][Medline]
  11. Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson JA. Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta. Endocrinology 1997 138:863-870[Abstract/Free Full Text]
  12. Couse JF, Lindzey J, Grandien K, Gustafsson JA, Korach KS. Tissue distribution and quantitative analysis of estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta) messenger ribonucleic acid in the wild-type and ERalpha-knockout mouse. Endocrinology 1997 138 4613-4621
  13. Jeung EB, Leung PC, Krisinger J. The human calbindin-D9k gene. Complete structure and implications on steroid hormone regulation. J Mol Biol 1994 235:1231-1238[CrossRef][Medline]
  14. Christakos S, Gabrielides C, Rhoten WB. Vitamin D-dependent calcium binding proteins: chemistry, distribution, functional considerations, and molecular biology. Endocr Rev 1989 10:3-26[Medline]
  15. Wasserman RH, Fullmer CS. On the molecular mechanism of intestinal calcium transport. Adv Exp Med Biol 1989 249:45-65[Medline]
  16. Krisinger J, Dann JL, Currie WD, Jeung EB, Leung PC. Calbindin-D9k mRNA is tightly regulated during the estrous cycle in the rat uterus. Mol Cell Endocrinol 1992 86:119-123[CrossRef][Medline]
  17. Mathieu CL, Burnett SH, Mills SE, Overpeck JG, Bruns DE, Bruns ME. Gestational changes in calbindin-D9k in rat uterus, yolk sac, and placenta: implications for maternal-fetal calcium transport and uterine muscle function. Proc Natl Acad Sci USA 1989 86:3433-3437[Abstract/Free Full Text]
  18. Krisinger J, Dann JL, Applegarth O, Currie WD, Jeung EB, Staun M, Leung PC. Calbindin-D9k gene expression during the perinatal period in the rat: correlation to estrogen receptor expression in uterus. Mol Cell Endocrinol 1993 97:61-69[CrossRef][Medline]
  19. Hong E, Choi K-C, Jeung E-B. Maternal-fetal transfer of endocrine disruptors in the induction of Calbindin-D9k mRNA and protein during pregnancy in rat model. Mol Cell Endocrinol 2003 212:63-72[CrossRef][Medline]
  20. L'Horset F, Perret C, Brehier A, Thomasset M. 17 Beta-estradiol stimulates the calbindin-D9k (CaBP9k) gene expression at the transcriptional and posttranscriptional levels in the rat uterus. Endocrinology 1990 127:2891-2897[Abstract]
  21. An BS, Choi KC, Kang SK, Lee GS, Hong EJ, Hwang WS, Jeung EB. Mouse calbindin-D(9k) gene expression in the uterus during late pregnancy and lactation. Mol Cell Endocrinol 2003 205:79-88[CrossRef][Medline]
  22. Krisinger J, Dann JL, Jeung EB, Leung PC. Calbindin-D9k gene expression during pregnancy and lactation in the rat. Mol Cell Endocrinol 1992 88:119-128[CrossRef][Medline]
  23. Delorme AC, Danan JL, Acker MG, Ripoche MA, Mathieu H. In rat uterus 17 beta-estradiol stimulates a calcium-binding protein similar to the duodenal vitamin D-dependent calcium-binding protein. Endocrinology 1983 113:1340-1347[Abstract]
  24. Saillenfait AM, Payan JP, Fabry JP, Beydon D, Langonne I, Gallissot F, Sabate JP. Assessment of the developmental toxicity, metabolism, and placental transfer of Di-n-butyl phthalate administered to pregnant rats. Toxicol Sci 1998 45:212-224[Abstract/Free Full Text]
  25. Varma DR. Modification of transplacental distribution of salicylate in rats by acidosis and alkalosis. Br J Pharmacol 1988 93:978-984[Medline]
  26. Takahashi O, Oishi S. Disposition of orally administered 2,2-Bis(4-hydroxyphenyl)propane (bisphenol A) in pregnant rats and the placental transfer to fetuses. Environ Health Perspect 2000 108:931-935[Medline]
  27. Laws SC, Carey SA, Ferrell JM, Bodman GJ, Cooper RL. Estrogenic activity of octylphenol, nonylphenol, bisphenol A and methoxychlor in rats. Toxicol Sci 2000 54:154-167[Abstract/Free Full Text]
  28. Routledge EJ, Sumpter JP. Structural features of alkylphenolic chemicals associated with estrogenic activity. J Biol Chem 1997 272: : 3280-3288[Abstract/Free Full Text]
  29. Soto AM, Justicia H, Wray JW. Sonnenschein C. p-Nonyl-phenol: an estrogenic xenobiotic released from "modified" polystyrene. Environ Health Perspect 1991 92:167-173[Medline]
  30. Shelby MD, Newbold RR, Tully DB, Chae K, Davis VL. Assessing environmental chemicals for estrogenicity using a combination of in vitro and in vivo assays. Environ Health Perspect 1996 104:1296-1300[Medline]
  31. Jensen EV, DeSombre ER. Estrogen-receptor interaction. Science 1973 182:126-134[Abstract/Free Full Text]
  32. Evans RM. The steroid and thyroid hormone receptor superfamily. Science 1988 240:889-895[Abstract/Free Full Text]
  33. Yamakoshi Y, Otani Y, Fujii S, Endo Y. Dependence of estrogenic activity on the shape of the 4-alkyl substituent in simple phenols. Biol Pharm Bull 2000 23:259-261[Medline]
  34. Nielsen M, Hoyer PE, Lemmen JG, van der Burg B, Byskov AG. Octylphenol does not mimic diethylstilbestrol-induced oestrogen receptor-alpha expression in the newborn mouse uterine epithelium after prenatal exposure. J Endocrinol 2000 167:29-37[Abstract]
  35. Eroschenko VP, Rourke AW, Sims WF. Estradiol or methoxychlor stimulates estrogen receptor (ER) expression in uteri. Reprod Toxicol 1996 10:265-271[CrossRef][Medline]
  36. Darwish H, Krisinger J, Furlow JD, Smith C, Murdoch FE, DeLuca HF. An estrogen-responsive element mediates the transcriptional regulation of calbindin D-9K gene in rat uterus. J Biol Chem 1991 266: : 551-558[Abstract/Free Full Text]
  37. L'Horset F, Blin C, Colnot S, Lambert M, Thomasset M, Perret C. Calbindin-D9k gene expression in the uterus: study of the two messenger ribonucleic acid species and analysis of an imperfect estrogen-responsive element. Endocrinology 1994 134:11-18[Abstract]



This article has been cited by other articles:


Home page
 Toxicol SciHome page
V. H. Dang, T. H. Nguyen, K.-C. Choi, and E.-B. Jeung
A Calcium-Binding Protein, Calbindin-D9k, Is Regulated through an Estrogen-Receptor Mediated Mechanism following Xenoestrogen Exposure in the GH3 Cell Line
Toxicol. Sci., August 1, 2007; 98(2): 408 - 415.
[Abstract] [Full Text] [PDF]

Home page
 Toxicol SciHome page
N. Bechi, F. Ietta, R. Romagnoli, S. Focardi, I. Corsi, C. Buffi, and L. Paulesu
Estrogen-Like Response to p-Nonylphenol in Human First Trimester Placenta and BeWo Choriocarcinoma Cells
Toxicol. Sci., September 1, 2006; 93(1): 75 - 81.
[Abstract] [Full Text] [PDF]

Home page
 Toxicol SciHome page
G.-S. Lee, H.-J. Kim, Y.-W. Jung, K.-C. Choi, and E.-B. Jeung
Estrogen Receptor {alpha} Pathway Is Involved in the Regulation of Calbindin-D9k in the Uterus of Immature Rats
Toxicol. Sci., April 1, 2005; 84(2): 270 - 277.
[Abstract] [Full Text] [PDF]

Home page
 Toxicol SciHome page
Y.-W. Jung, E.-J. Hong, K.-C. Choi, and E.-B. Jeung
Novel Progestogenic Activity of Environmental Endocrine Disruptors in the Upregulation of Calbindin-D9k in an Immature Mouse Model
Toxicol. Sci., January 1, 2005; 83(1): 78 - 88.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
71/2/669    most recent
biolreprod.103.026146v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hong, E.-J.
Right arrow Articles by Jeung, E.-B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hong, E.-J.
Right arrow Articles by Jeung, E.-B.
Agricola
Right arrow Articles by Hong, E.-J.
Right arrow Articles by Jeung, E.-B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS