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Am J Physiol Endocrinol Metab 293: E132-E138, 2007. First published March 20, 2007; doi:10.1152/ajpendo.00666.2006
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Uterine TRPV6 expression during the estrous cycle and pregnancy in a mouse model

Geun-Shik Lee and Eui-Bae Jeung

Laboratory of Veterinary Biochemistry and Molecular Biology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea

Submitted 6 December 2006 ; accepted in final form 14 March 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Maintenance of uterine calcium balance is crucial for physiological functioning, including smooth muscle contraction and embryo implantation. Although calbindins were previously thought to act as important uterine calcium-processing genes for female reproductive function, they were not enough to attest the roles of calcium ions in the reproductive organs. Previously, we reported that rat transient receptor potential cation channel, subfamily V, member 6 (TRPV6) was expressed and regulated by hormones in the uterus. In the present study, we observed uterine TRPV6 expression in a mouse model to clarify the mutual roles of these two calcium-processing genes in female reproductive organs. We investigated uterine TRPV6 mRNA expression during the estrous cycle and pregnancy, as well as its regulation by the steroid hormones estrogen (E2) and progesterone (P4) in mice. Uterine TRPV6 mRNA levels increased at estrus and fluctuated in the uterus, placenta, and fetal membrane during pregnancy. Uterine TRPV6 mRNA increased in mid- and late pregnancy, and its expression was strongly induced in midpregnancy in the labyrinth and spongy zones of the placenta, and in the fetal membrane. E2 (17beta-estradiol) was found to regulate uterine TRPV6 expression in the luminal and glandular epitheliums. In addition, we determined that ER{alpha} tightly regulated uterine TRPV6 transcription. Together, these results suggest that for uterine function in normal pregnancy, TRPV6 is regulated by E2 via an ER{alpha}-dependent pathway.

estrogen receptor-{alpha}; uterus

IN MAMMALS, CALCIUM IONS may be involved in control of uterine muscle contraction and embryo implantation. The balance between contraction and relaxation is extremely important throughout pregnancy and during labor. However, regulation of uterine calcium is not yet fully understood. A model for calcium ions in intestinal cells suggests that calcium flows into the cytoplasm via channel proteins, i.e., calcium transporters. These transporters transfer calcium using calcium-binding proteins (Calbindin-D9k or -28k), which are extruded from the cell membrane by a plasma membrane Ca2+ ATPase (28, 31). Transient receptor potential vanilloid type 5 (TRPV5) and 6 (TRPV6) are found in the apical membranes of intestinal and renal epithelial cells and have been proposed as mediators for calcium uptake during trans-cellular transport (7). TRPV6 is also known as epithelial calcium channel 2 (EcaC2) and calcium transporter 1 (CaT1); TRPV5 is also called EcaC1 and CaT2. These closely related proteins are expressed primarily in cells of the duodenum and kidney that are involved in calcium absorption or reabsorption (25). TRPV6 was first cloned from rat duodenum and was detected subsequently in both human and mouse duodena (12, 22). The genes encoding these proteins are located closely together on the same chromosome and their genomic structures are similar (12, 19, 20). TRPV6 is expressed in the duodenum, jejunum, ileum, kidney, and exocrine tissues such as the pancreas, prostate, mammary, and sweat glands (7, 12, 29, 32). In contrast, TRPV5 is expressed in the kidney and human syncytiotrophoblast (4).

In the reproductive organs, TRPV6 is expressed in the placenta as well as in the uterus (14). Placental TRPV6 plays a role in calcium transport to the fetus (32). Duodenal and renal TRPV6 expression is regulated by vitamin D, estrogen, and dietary calcium. An active form of vitamin D increases duodenal calcium absorption and abnormal calcium absorption has been observed in vitamin D receptor-knockout mice (3, 28). Dietary calcium can also induce duodenal and renal TRPV6 mRNA expression (30, 31) and estrogen therapy in menopausal women induces duodenal TRPV6 mRNA, suggesting that this hormone modulates TRPV6 expression independently (32).

As mentioned above, maintenance of the uterine calcium balance is of crucial importance for many physiological functions, including smooth muscle contraction and embryo implantation. As certain calcium-processing proteins act systemically in the duodenum and kidney, we assumed that these proteins would also be functionally important in female reproductive organs. We therefore examined the expression of TRPV6 mRNA in the uterus, placenta, and fetal membrane during the estrous cycle and pregnancy in mature female mice. In addition, we investigated the effects of the steroid hormones estrogen (E2) and progesterone (P4), a spatial expression of reproductive tissues, and potential estrogen receptor (ER; ER{alpha} or ERbeta) pathways, using ER-specific antagonists and agonists, on the regulation of uterine TRPV6 expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and treatments. Mature (>14-wk-old) and immature (14-day-old) female ICR mice were obtained from Dae Han Biolink (Eumsung, Chungbuk, Korea). All animals were housed in polycarbonate cages and acclimatized to an environmentally controlled room (temperature: 23 ± 2°C, relative humidity: 50 ± 10%, frequent ventilation, and 12:12-h light-dark cycle) before use. Mature female mice (total n = 15) were divided into three groups (pro-, di-, and estrus) by vaginal cell morphology. Female mice were mated with adult males overnight (2) and then examined the following morning for the presence of a vaginal plug. This was designated as day 0 of pregnancy (P0). Mice were euthanized on each day of pregnancy (P5 to P18), the day of giving birth (birth), and lactation (L1 to L5).

Immature mice (14-day-old, total n = 20) were divided into four groups for the hormonal regulation experiments. Hormones were prepared in ethanol (Sigma, St. Louis, MO) and mice were treated daily for 3 days with subcutaneous injections of the following: 40 µg/kg body wt 17beta-estradiol (E2; Sigma), 4 mg/kg body wt progesterone (P4; Sigma), and E2 with P4 for 3 days and then euthanized 12 h following the final injection. In the time-dependent experiment, 30 mice were given a daily subcutaneous injection with E2 (40 µg/kg body wt) for 3 days and then euthanized (5 mice for each time) at 3, 6, 12, 24, 48, and 72 h following the final injection. In the dose-dependent experiment, five groups of five mice were treated daily with a subcutaneous injection of E2 (0.04, 0.4, 4, 40, and 80 µg/kg body wt, prepared in ethanol) for 3 days and then euthanized 12 h following the final injection.

To determine the effects of E2, 10 mice were separated into 2 groups, which were treated daily for 3 days with subcutaneous injections of ICI 182 780 (10 mg/kg body wt) 30 min before injection of either E2 (positive group, n = 5, 40 µg/kg body wt) or ethanol (negative control, n = 5). For the ER antagonist assay, mice (total n = 5 per group) were treated with daily subcutaneous injections of the following: ethanol, E2 (40 µg/kg body wt), propyl pyrazole triol (PPT; 1 mg/kg body wt, Tocris, Eslisvill, MO), and diarylpropionitrile (DPN; 1 mg/kg, Tocris) for 3 days (14). The Ethics Committee of the Chungbuk National University approved all experimental procedures and use of animals.

Preparation of total RNA and semiquantitative RT-PCR. Mice were euthanized, and the uteri were excised rapidly and then washed in cold sterile 0.9% NaCl. Total RNA was prepared from the uteri using TRIzol reagent (Invitrogen, Carlsbad, CA), and concentration was determined by absorbance at 260 nm. RT-PCR was performed as described previously (13). In brief, total RNA (1 µg) was reverse transcribed to first-stand complementary DNA (cDNA) using mMLV reverse transcriptase (iNtRON Bio; Sungnam, Kyungki-Do, Korea) and random primers (9 mer, TaKaRa Bio; Otsu, Shiga, Japan). TRPV6 cDNA was amplified in a 20-µl PCR reaction containing 1 U Taq polymerase (iNtRON), 1.5 mM MgCl2, 2 mM dNTP, and 50 pmol specific primers. The oligonucleotide sequences for TRPV6 were 5'-GTG CTG GGT GCC ATC TAC GT-3' (sense) and 5'-CAA TGA TGA CAT GGA ATG GCC-3' (antisense). PCR reactions were denatured at 95°C for 30 s, annealed at 60°C for 30 s, and extended at 72°C for 30 s. TRPV6 and 1A were quantified using 25 and 18 cycles, respectively. PCR products (10 µl) were separated on a 2% agarose gel, stained with ethidium bromide, and photographed under UV illumination. Photographs were taken using a Gel Doc EQ (Bio-Rad, Hercules, CA).

Real-time PCR. Real-time PCR was performed in 20-µl reactions containing 10 µl of TaqMan Universal PCR Master Mix (Applied Biosystems, Foster, CA), 1 µl of 20x Assays-on-Demand Gene Expression Assay Mix (Applied Biosystems, TRPV6: Mm00499069_m1, HPRT1: Mm00446968_m1), and 2 µl of cDNA. PCR amplification was conducted using a 7300 Real-Time PCR System (Applied Biosystems) starting with an initial denaturation at 50°C for 2 min, followed by 90°C for 10 min. Each of the 40 amplification cycles consisted of denaturation at 95°C for 15 s, followed by annealing and extension at 60°C for 1 min. Relative expression levels for each sample were determined using RQ software (Applied Biosystems) and TRPV6 expression was normalized relative to HPRT1.

Immunohistochemical staining. The localization of TRPV6 protein was examined by immunohistochemistry. Uteri were embedded in paraffin. Sections (7 µm) were deparaffinized in xylene and hydrated in descending grades of ethanol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in PBS-T for 20 min. Nonspecific reactions were blocked by incubating the sections in 10% normal goat serum (NGS) for 2 h at room temperature (RT). Sections were subsequently incubated at RT for 1 h with a polyclonal rabbit antibody directed against TRPV6 (diluted in 1:100, Almone Labs, Jerusalem, Israel) or GR (1:500, Santa Cruz Biotechnology) dissolved in 10% NGS. After being washed with PBS-T, the sections were incubated with the biotinylated secondary antibody (rabbit IgG, Vector Laboratories, Burlingame, CA) for 30 min at 37°C and further incubated with ABC-Elite for 30 min at 37°C. Diaminobenzidine (DAB; Sigma) was used as a chromogen, and the sections were counterstained with hematoxylin before being mounted with a coverslip.

Data analysis. Data were analyzed with a nonparametric one-way ANOVA, using the Kruskal-Wallis test, followed by Dunnett's test for multiple comparisons to the negative control. Data were ranked according to these tests. All statistical analyses were performed using SPSS for Windows (SPSS, Chicago, IL). P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Pattern of uterine TRPV6 mRNA expression during the estrous cycle and pregnancy. To investigate the expression of uterine TRPV6 during the estrous cycle, mature mice were divided into three groups (proestrus, estrus, and diestrus), according to vaginal cell morphology. Expression of uterine TRPV6 mRNA was detected using real-time PCR (Fig. 1A). Relative to HPRT1, TRPV6 mRNA was highly expressed at estrus compared with proestrus (10-fold) and diestrus (5-fold).


Figure 1
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  		Fig. 1. Uterine TRPV6 mRNA expression in adult female mice during estrous cycle and pregnancy. Mice were divided into 3 groups (Pro: proestrus, Es: estrus, and Di: diestrus) by vaginal smear. Uteri were sampled daily from pregnant mice (P5 to P18) and lactating mice (L1 to L5). Uterine TRPV6 mRNA during the estrous cycle (A) and during pregnancy and lactation (B) was examined by real-time PCR (bar graphs) and RT-PCR (gels). The bar graphs represent the analysis of real-time PCR data (means ± SE of duplicates from all samples, expressed as a percentage of TRPV6/HPRT1 mRNA). aSignificantly different (P < 0.05) level of expression compared with pro- and diestrus.

 
During pregnancy, uterine TRPV6 mRNA expression fluctuated (Fig. 1B). Two peaks of TRPV6 expression were observed in the middle of pregnancy (P10 and P13) and the maximum expression was detected at birth (Fig. 1B). However, TRPV6 transcripts in early (P5 to P7) and late (P14 to P16) pregnancy were scarcely detectable. The induction of TRPV6 mRNA expression at the time of birth was sustained through L1, after which it was not detected until L5 (Fig. 1B). In parallel with uterine TRPV6 expression, placental TRPV6 expression peaked at P10 in the middle of pregnancy, and moderate expression was observed at P14 and P17 (Fig. 2A). In the fetal membrane, TRPV6 transcript increased from P11, peaked at P13, then decreased until birth (Fig. 2B).


Figure 2
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  		Fig. 2. TRPV6 mRNA expression of placenta and fetal membrane in adult female mice during pregnancy. Placenta (A) and fetal membrane (B) were sampled daily from pregnant mice. TRPV6 mRNA was examined during pregnancy using real-time PCR (bar graphs) and RT-PCR (gels). The bar graphs represent the analysis of real-time PCR data (means ± SE of duplicates from all samples, expressed as a percentage of TRPV6/HPRT1 mRNA).

 
Thus, in the female reproductive organs, expression of TRPV6 was greatest at estrus and in the middle of pregnancy, implying that it may function in female reproduction during both the estrous cycle and during pregnancy.

Effects of sex steroid hormones on TRPV6 mRNA expression. To investigate the induction of TRPV6 mRNA, immature mice were treated with daily injections of two sex steroid hormones (E2 and/or P4) for 3 days. TRPV6 mRNA was induced significantly by E2 (10-fold vs. negative control) and E2 with P4 (13-fold vs. negative control) but was unaffected by P4 alone (Fig. 3A). This suggests that E2 secreted at estrous stage is responsible for the upregulation of the TRPV6 transcripts. Although the combined E2/P4 treatment (4 mg/kg) increased uterine TRPV6 mRNA significantly, no statistically synergistic effect was observed, compared with that of E2 alone (Fig. 3A).


Figure 3
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  		Fig. 3. Effect of steroid hormones on uterine TRPV6 mRNA expression. A: time-dependent experiment. Groups of immature mice were treated with ethanol (Ve, negative control), E2 (40 µg/kg body wt), P4 (4 mg/kg body wt), and E2 and P4 for 3 days and then euthanized 12 h following the final injection. B: dose-dependent experiment. Immature mice were treated daily with E2 (4 µg/kg body wt) for 3 days and euthanized at 3, 6, 12, 24, 48, and 72 h following final injection. C: six groups of immature mice were treated with E2 at the doses of 0, 0.04, 0.4, 4, 40, and 80 µg/kg body wt. Top: gel figures indicate RT-PCR of TRPV6 mRNA expression. Bottom: bar graphs represent the analysis of real-time PCR data (means ± SE of duplicates from all samples, expressed as a percentage of TRPV6/HPRT1 mRNA). aSignificantly different (P < 0.05) level of expression compared with the negative control.

 
To visualize a time course for TRPV6 mRNA regulation by E2, immature mice were injected daily with E2 (40 µg/kg) for 3 days, and their uteri were isolated at 3, 6, 12, 24, 48, and 72 h following the final injection. A maximum level of TRPV6 mRNA expression was observed at 12 h (3.5-fold vs. at 3 h) and although expression was still observed at 24 h, levels had reduced to background (as per the negative control) by 48 h (Fig. 3B). Dose-dependent E2 regulation of TRPV6 mRNA was investigated by daily treatment of immature mice with different doses of E2 (0.04, 0.4, 4, 40, and 80 µg/kg) for 3 days. TRPV6 mRNA exhibited a dose-dependent increase and significant induction was observed with 4, 40, and 80 µg/kg of E2 (Fig. 3C).

Effect of ER antagonist (ICI 182 780) and ER-specific ligands on uterine TRPV6 mRNA induction by E2. To investigate ER mediation of E2-induced uterine TRPV6 transcription, ICI 182 780 (an ER antagonist) was injected into immature mice 30 min before E2 (40 µg/kg) treatment for 3 days. Although a physiological dose of E2 induced uterine expression of TRPV6 mRNA significantly, pretreatment with the ER antagonist blocked E2-induced TRPV6 mRNA expression completely (Fig. 4A). This result suggests that ER, activated by E2, mediated the increased expression of TRPV6.


Figure 4
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  		Fig. 4. A: effect of estrogen receptor (ER) antagonist (ICI 182 780) and ER-specific ligands on uterine TRPV6 mRNA induction by E2. Three groups of mice were subcutaneously injected daily for 3 days with ICI 182 780 (an ER antagonist) 30 min before injection with 40 µg/kg body wt E2 (4 µg/kg body wt) and ethanol (n = 3, negative control). B: PPT (1 mg/kg, ER{alpha}-selective ligand), DPN (1 mg/kg, ERbeta-selective ligand), E2 (40 µg/kg body wt), and vehicle (ethanol) were injected daily into immature mice for 3 days. All mice were euthanized 12 h following the final injection. Top: gel indicates TRPV6 mRNA expression using RT-PCR. Bottom: bar graphs represent the analysis of real-time PCR data (means ± SE of duplicates from all samples, expressed as a percentage of TRPV6/HPRT1 mRNA). aSignificantly different to vehicle (P < 0.05). bSignificantly different to E2-treated group (P < 0.05).

 
To determine which subtype of ER is involved in the induction of uterine TRPV6 expression, immature mice were injected daily with PPT (1 mg/kg, ER{alpha}-specific ligand), DPN (1 mg/kg, ERbeta-specific ligand), E2, and the negative control "vehicle" for 3 days. PPT has a much greater affinity for ER{alpha} than ERbeta (11, 26), whereas DPN is 70 times more selective for ERbeta than ER{alpha} (18). TRPV6 mRNA was induced by PPT and E2, indicating that TRPV6 expression is mediated by ER{alpha} in uteri (Fig. 4B). TRPV6 expression was unaffected by the ERbeta-selective agonist (DPN).

Localization of TRPV6 expression in mouse uterus, placenta, and fetal membrane. To elucidate the spatial expression of TRPV6 protein expression in uterus, placenta, and fetal membranes, we performed immunohistochemistry on the tissue sections from E2-treated uteri, estrous cycling uteri (di- or estrus), and female reproductive organs at pregnancy day 10 or 13 to anti-TRPV6 antibody. After injecting OVX mice with E2 (Fig. 5A), the uteri were enlarged and TRPV6 was expressed in the apical luminal and glandular epitheliums. In addition, the uterus presented positive (brown color) signals in the same epitheliums during the estrous stage (Fig. 5B). Gestated uterus expressed TRPV6 protein in the epithelium; however, positive staining was not detected on the embryo attached to the uterus (Fig. 5C). In female reproductive organs, TRPV6 was highly expressed in the labyrinth and spongy zones of placenta (Fig. 5D) and in the fetal membrane (Fig. 5E).


Figure 5
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  		Fig. 5. Localization of TRPV6 expression in the mouse uterus, placenta, and fetal membrane. Immunohistochemistry was performed as described in MATERIALS AND METHODS. A: line represented OVX-treated uteri sections daily injected with vehicle (ethanol) or E2 (40 µg/kg body wt) for 3 days. B: uteri section during estrous cycles. C: gestated uterus between fetuses on P10 [the presence of a vaginal plug was designated as day 0 of pregnancy (P0)]. D: placenta in pregnancy day 10. E: fetal membrane on P13. The spotted rectangles represented the enlarged slides. Arrowhead, stained signals; f, fetus; gc, giant cells. Scale bar indicated 200 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
A number of calcium-regulating genes are involved in uterine function during estrous cycle and pregnancy (6). During the "window of receptivity" of the uterine endometrium, calcium-binding proteins (CaBP-9k and -28k), monoclonal nonspecific suppressor factor beta and splicing factor SC35, are involved in embryo implantation (15, 23, 24). In addition, there is evidence that CaBP-9k and -28k are required during the early phase of embryo implantation, suggesting that the regulation of calcium availability in the vicinity of the implanting embryo is critical for successful implantation (16, 17). In duodenum, CaBP-9k and TRPV6 were shown to have a similar expression pattern; however, uterine CaBP-9k is regulated by different hormones and expressed at different stages of the estrous cycle (5, 25). The expression of CaBP-9k is increased by E2 and at estrus in rats but its expression pattern is controlled by P4 and unaffected at diestrus in mice (1, 12). In our previous study, uterine TRPV6 expression in rats was found to be similar to that of CaBP-9k expression in mice (10). The present study examined the expression and regulation of TRPV6 in the uterus of mice during the estrous cycle and pregnancy to elucidate the functional relationships between these two uterine calcium-processing genes.

Expression of TRPV6 mRNA in the uterus varied during the estrous cycle (pro-, di-, and estrus). Expression of the less abundant TRPV5 mRNA was not determined in this study. However, Weber et al. (32) suggested that TRPV5 is expressed only in the kidney. The TRPV6 transcript was highly expressed at estrus, an E2-dominant phase in the reproductive cycle, implying that it might be involved in a specific uterine function such as calcium ion transport. In our previous study, rat TRPV6 transcripts in uterus were highly expressed at diestrus (10). The previous and present data indicated that uterine TRPV6 is regulated differently during the estrous cycle in rats and mice.

To further monitor the expression of TRPV6 gene in the mouse uterus, placenta, and fetal membrane, we investigated the expression level of TRPV6 during pregnancy and lactation. Our results indicated that mouse TRPV6 mRNA was actively expressed during pregnancy. Maximum expression was observed in the middle of gestation (P10 and P13), and this was followed by a decrease in the later stage of gestation. Immediately before birth, another peak in TRPV6 expression was observed, which disappeared during lactation. In particular, the regions of the uterine epithelium surrounded by the implanting embryo were negative for TRPV6 expression, whereas uniform expression was detected in regions of the entire luminal epithelium where no embryo was attached, as observed previously for CaBP-9k expression (2, 21). Placental TRPV6 expression followed the uterine expression pattern initially, with induction in the middle of gestation (P10 and P14) but not at the end. In the fetal membrane, TRPV6 mRNA was strongly induced in the middle of pregnancy and then decreased until birth. Although Bernucci et al. (4) studied the functional expression of calcium channels in human placental syncytiotrophoblast, the exact role of these channels remains to be determined. In a previous study on CaBP-9k, one of the uterine calcium-regulating genes, we observed a gradual increase in expression during late pregnancy (from day 12 to 18), followed by a decline at birth and during lactation (2). It has also been reported that serum E2 levels increase at the end of pregnancy and decline sharply following birth and during lactation, whereas serum P4 levels remain constant throughout late gestation and lactation (2). With regard to these studies, it is possible that the increased serum E2 induces uterine TRPV6 transcription at birth and during pregnancy and that it disappears during lactation when secreted E2 is no longer present. Our data may suggest that TRPV6 expression in the placenta and uterus during gestation takes part in fetal bone growth and maternal-fetal calcium transport (2, 17).

The sex steroids E2 and P4 are the main hormones that regulate uterine structural change and function, and they alternate periodically during the estrous cycle. To determine which of the hormones was involved in uterine TRPV6 transcription, immature mice were injected daily with E2 and/or P4. A physiological dose of E2, or E2 combined with P4, induced uterine TRPV6 mRNA, although a statistically significant synergistic effect between the two was not observed. These results imply that E2 may be the major regulator of uterine TRPV6 transcription of mice. In a time course experiment, mice were treated for 3 days with subcutaneous injections of E2. TRPV6 mRNA was elevated significantly 12 h following the final injection, although its expression disappeared by 48 h. In addition, TRPV6 mRNA expression was stimulated by E2 in a dose-dependent manner. These data suggest that in the mouse model uterine TRPV6 transcription may be completely dependent on E2.

To analyze the putative function of TRPV6 in female reproductive organs in this study, the expression of TRPV6 gene was compared with that of CaBP-9k gene, a well-documented calcium regulatory gene in the uterus. The pattern of expression of the former was the opposite to that of the latter. CaBP-9k was found to increase at diestrus and to be induced by P4 in the uterus of mice (10). The uterine regulating pattern of rat CaBP-9k is identical to that of uterine TRPV6 in mice, and E2 acts as an enhancer of both mouse TRPV6 and rat CaBP-9k expression (10). However, rat TRPV6 and mouse CaBP-9k expression are oppositely regulated (10). This distinct regulation of calcium-related genes in the uterus has not been fully explained, and uterine calcium ions might be regulated by complex mechanisms involving many proteins. This opposite regulation of TRPV6 and CaBP-9k in different rodent models could indicate that they compensate for each other in the uterus. Although CaBP-28k knockout (KO) mice demonstrate normal implantation, double KO mice (CaBP-9k and -28k) failed embryo implantation (16), indicating that either more than two calcium regulatory proteins participate in embryo implantation or that TRPV6 may provide uterine CaBPs at different stages of development. Based on this and previous studies, TRPV6 may be involved in a particular action for female reproduction related with other calcium-processing proteins (2, 10, 17).

Calcium ion transport occurs in calcium-absorbing tissues such as the intestine and kidney. TRPV6, CaBPs, and PMCA1 are thought to play major roles in this process and their function and regulation have been intensively studied in the gastrointestinal tract (30). Intestinal TRPV6 mRNA in vitamin D 1{alpha}-hydroxylase KO male mice and ovariectomized rats is upregulated following the administration of 17beta-estradiol, and its induction can be blocked by an ER antagonist (9, 30). CaBP transcripts were also increased by E2, although PMCA1 expression was unaffected. Although it was suggested that an estrogen response element (ERE) was not present in the putative TRPV6 promoter region, Arjmandi et al. (9, 30, 31) reported that TRPV6 possessed an ERE in the promoter sequence. In the intestinal model, Cromphaut et al. (32) used ER {alpha} and beta KO mice to demonstrate that ER{alpha} played a major role in the induction pathway of TRPV6 transcription. Thus estrogens are potent independent regulators of the expression of calcium influx genes and are involved in active intestinal calcium absorption (31). To determine the pathway of uterine TRPV6 induction by E2, an ER antagonist was injected into immature mice 30 min before E2 treatment. E2-induced uterine TRPV6 expression was blocked completely by the ER antagonist. These results suggest that E2 activates its receptor to induce uterine TRPV6 transcription, and this might be mediated by an ERE or via indirect stimulation of transcriptional factors (8, 9, 24, 27, 30, 31).

TRPV6 was thought to be a highly expressed gene in a number of exocrine organs including the pancreas, prostate, and mammary gland. However, its role in the uterus has not been established (32). Based on the results of previous studies and the work presented here, we have been able to compare the roles of two uterine calcium-regulating proteins, TRPV6 and CaBP-9k. In mice, TRPV6 was induced by E2 and localized to the luminal and glandular epithelium, whereas CaBP-9k was regulated by P4 and detected on the endometrium (1). Conversely, in rats, TRPV6 transcript levels were increased by P4 and expressed on the endometrium, whereas CaBP-9k was regulated by E2 and localized to the stroma and smooth muscle fibers (1, 10, 12, 14). This study suggests that uterine TRPV6 and CaBP-9k might play compensatory and/or cooperative roles in maintaining calcium balance in the uterus.

In summary, uterine TRPV6 expression in mice increased at estrus, and expression in the uterus, placenta, and fetal membrane fluctuated during pregnancy. Uterine TRPV6 mRNA increased in the middle and at the end of pregnancy, whereas placental and fetal membrane expression was elevated only in the middle of pregnancy. E2 was a major mediator in the regulation of TRPV6 in the uterus of immature mice. TRPV6 proteins were localized to the epithelium of the uterus, to the labyrinth and spongy zones of the placenta, and to the fetal membrane. E2 induced uterine TRPV6 transcription via an ER-dependent pathway that was inhibited by an ER-specific antagonist, and using ER{alpha} or -beta-specific ligands we identified ER{alpha} as a regulator of uterine TRPV6 transcription.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
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REFERENCES
 
This work was supported by the research grant of the Chungbuk National University in 2006.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Dr. E.-B. Jeung, Laboratory of Veterinary Biochemistry and Molecular Biology, College of Veterinary Medicine, Research Institute of Veterinary Medicine, Chungbuk National Univ., Cheongju, Chungbuk, 361-763, Republic of Korea (e-mail: ebjeung{at}chungbuk.ac.kr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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