{"paper_id":"e4713626-6a24-49e7-86ff-35ca356eacc5","body_text":"BioMed Central\nPage 1 of 11\n(page number not for citation purposes)\nReproductive Biology and \nEndocrinology\nOpen AccessResearch\nUterine and placental expression of TRPV6 gene is regulated via \nprogesterone receptor- or estrogen receptor-mediated pathways \nduring pregnancy in rodents\nBo-Mi Lee, Geun-Shik Lee, Eui-Man Jung, Kyung-Chul Choi and Eui-\nBae Jeung*\nAddress: Laboratory of Veterinary Biochemistry and Molecular Biology, College of Veterinary Medicine, Chungbuk National University, Cheongju, \nChungbuk, 361-763, Republic of Korea\nEmail: Bo-Mi Lee - bongmae@naver.com; Geun-Shik Lee - geunshiklee@paran.com; Eui-Man Jung - jemman@hanmail.net; Kyung-\nChul Choi - kchoi@cbu.ac.kr; Eui-Bae Jeung* - ebjeung@chungbuk.ac.kr\n* Corresponding author    \nAbstract\nTransient receptor potential cation channel, subf amily V, member 6 (TRPV6) is an epithelial Ca 2+\nchannel protein expressed in calcium absorbing organ s. In the present study, we investigated the\nexpression and regulation of uterine and placen tal TRPV6 during gestation in rodents. Uterine\nTRPV6 peaked at pregnancy day (P) 0.5, P5.5 and, P13.5 and was detected in uterine epithelium and\nglands of rats, while placental TRPV6 mRNA le vels increased in mid-gestation. Uterine and\nplacental TRPV6 mRNA levels in rats appear to cyclically change during pregnancy, suggesting that\nTRPV6 may participate in the implantation proc ess. In addition, uterine TRPV6 mRNA is only\nexpressed in placenta-unattached areas of the uterus, and uterine TRPV6 immunoreactivity was\nobserved in luminal and glandular epithelial ce lls. In the placenta, TRPV6 was detected in the\nlabyrinth and spongy zone. These results may in dicate that TRPV6 has at least two functions:\nimplantation of the embryo and maintenance of pregnancy. To investigate the pathway(s) mediating\nTRPV6 expression in rodents, an ti-steroid hormone antagonists were injected prior to maximal\nTRPV6 expression. In rats, TRP V6 expression was reduced by RU486 (an anti-progesterone)\nthrough progesterone receptors, and ICI 182,780 (an anti-estrogen) blocked TRPV6 expression via\nestrogen receptors in mice. The juxtaposition of  uterine and placental TRPV6 expressed in these\ntissues supports the notion that TRPV6 participat es in transferring calcium ions between the\nmaternal and fetal compartments. Taken together, TRPV6 gene ma y function as a key element in\ncontrolling calcium transport in the uterus between the embryo and the placenta during pregnancy.\nBackground\nUterine calcium ions are considered to be a critical factor\nfor smooth muscle contraction and embryo implantation.\nAt the implantation stage, these ions may help in secreting\nuterine extra-cellular matrix components at the site of\nimplantation, and during pregnancy and labor calcium\nions may regulate uterine tension [1,2]. Despite these crit-\nical roles of calcium, the regulation of uterine calcium lev-\nels is not yet fully understood. In calcium absorbing\norgans, i.e., intestine and kidney which express several cal-\ncium processing proteins, the regulation and function of\ncalcium ions and the associated genes are relatively well\nPublished: 21 May 2009\nReproductive Biology and Endocrinology 2009, 7:49 doi:10.1186/1477-7827-7-49\nReceived: 17 October 2008\nAccepted: 21 May 2009\nThis article is available from: http://www.rbej.com/content/7/1/49\n© 2009 Lee et al; licensee BioMed Central Ltd. \nThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), \nwhich permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\n\nReproductive Biology and Endocrinology 2009, 7:49 http://www.rbej.com/content/7/1/49\nPage 2 of 11\n(page number not for citation purposes)\nunderstood. Calcium ions are processed via entering,\ntransporting, and extruding proteins [3-5]. The transient\nreceptor potential cation channel, subfamily V, member\n(TRPV) 5 and 6 were found in apical membranes of intes-\ntinal and renal epithelial cells, and are proposed as medi-\nators for calcium uptake during trans-cellular transport\n[6]. Cytosolic calbindin-D9k and -28 k transport calcium\nions from apical to basolateral layers, and theses ions are\nfinally extruded from the cell membrane via plasma mem-\nbrane Ca\n2+ ATPase and sodium-calcium exchanger 1 [4,5].\nIn the past decade, uterine calcium-binding proteins have\nbeen studied to elucidate the function of uterine calcium\nions, however their regulation and mechanisms of action\nare not completely resolved [7-9].\nIn female reproductive organs, TRPV6 is expressed in the\nuterus as well as in the placenta [10-12]. In calcium\nabsorbing organs, TRPV6 expression is regulated by vita-\nmin D, estrogen and dietary calcium. An active form of\nvitamin D increases duodenal calcium absorption, and\nabnormally low calcium absorption has been observed in\nvitamin D receptor-knockout mice [4,13]. Dietary calcium\ncan also induce duodenal and renal TRPV6 mRNA expres-\nsion [5,14], and estrogen therapy in menopausal women\ninduces duodenal TRPV6 mRNA, suggesting that this hor-\nmone independently modulates TRPV6 expression [15].\nTRPV6 of the female reproductive organs is robustly\nexpressed; however its exact role is reproduction is not\nclearly understood. In the previous study, we reported\nthat rat TRPV6 is expressed in the uterine endometrium\nand glandular endometrium [11,12] and is up-regulated\nat diestrus in matured animals and by progesterone sup-\nplementation in immature or ovariectomized animals\n[11]. Recently, the physiological significance of TRPV6/\nCa\n2+ channel in maternal-fetal Ca2+ transport was investi-\ngated using TRPV6 knockout mice [16], demonstrating\nthat Ca\n2+ concentration in fetal blood and amniotic fluid\nwas significantly lower and the transport activity of radio-\nactive Ca 2+ from mother to fetuses was 40% lower in\nTRPV6 KO fetuses than in WTs. Progesterone appears to\nbe a dominant regulator of TRPV6 via progesterone recep-\ntor activation [11]. In a mouse model, uterine TRPV6\nexpression varied during estrous cycles, however it was\ndominantly observed at the estrous stage [12]. In addi-\ntion, estrogen seemed to be a major factor in the regula-\ntion of uterine TRPV6 transcription via estrogen receptor\nα pathway [12]. However, the role of TRPV6 in the\nimplantation process during pregnancy is not yet deter-\nmined.\nThus, in the present study, we employed a rat model to\nexamine the expression of TRPV6 mRNA in the uterus and\nplacenta during pregnancy. In addition, we investigated\nwhether sex hormones regulate uterine TRPV6 expression\nin rats and mice during pregnancy, using antagonists for\nestrogen and progesterone. Finally, we further determined\nthe localization of TRPV6 protein in the uterus and pla-\ncenta to elucidate the role of this protein during preg-\nnancy.\nMethods\nAnimals and treatments\nSprague-Dawley rats (male, 12-week-old; female, 10-\nweek-old) and pregnant ICR mice (12 to 15-week-old)\nwere obtained from KOATECH (Pyeongtaek, Gyeonggi,\nKorea). All animals were housed in polycarbonate cages,\nand acclimatized to an environmentally controlled room\nbefore experimentation (temperature, 23 ± 2°C; relative\nhumidity, 50 ± 10%; frequent ventilation and 12 h light\ncycle). Experiments were performed with the approval of\nthe Animal Ethics Committee at the College of Veterinary\nMedicine, Chungbuk National University. Adult female\nrats were mated with adult males overnight [17], then\nexamined the following morning for the presence of a vag-\ninal plug. This time point was designated as day 0.5 of\npregnancy (P0.5) when the morning a plug is found. The\nrats (n = 3 per group) were euthanized on each day of\npregnancy (P0.5 to P21.5).\nPregnant rats (P4.5 for uterine RNA preparation, P19.5 for\nplacental RNA preparation) received subcutaneous injec-\ntions of RU486 (RU, 2.5 mg per rat; Sigma-Aldrich Corp,\nSt. Louis, MO), ICI 182,780 (ICI, 0.5 mg per rat; Tocris,\nEslisvill, Missouri, USA) or 50% ethanol as a vehicle\n[11,18]. In addition, ICR mice at P9.5 were injected with\nRU (25 μg per mouse) or ICI (2 μg per mouse) [19]. The\nchemicals were dissolved in 0.2 ml ethanol and saline\nmixture (1:1), and 0.2 ml and 0.05 ml were administered\nto rats and mice, respectively. The pregnant rats were\ntreated with the antagonists one day before TRPV6 mRNA\nis maximally expressed depending on the tissues and spe-\ncies [12].\nTotal RNA extraction and reverse transcriptase (RT)-PCR\nAnimals were euthanized, and the uteri and placentas\nwere rapidly excised and washed in cold sterile saline\n(0.9% NaCl). Total RNA was prepared with TRIzol reagent\n(Invitrogen Life Technologies, Inc., Carlsbad, California,\nUSA), and the concentration was determined by measur-\ning light absorbance at 260 nm. Before RT-PCR, total RNA\nsamples were electrophoresed on 1% formaldehyde dena-\nturing agarose gels to validate the quality and purity. RT-\nPCR was performed, and the resulting products were visu-\nalized by agarose gel electrophoresis as described previ-\nously [20]. In brief, total RNA (1 μg) was reverse\ntranscribed to first strand complementary DNA (cDNA)\nusing mMLV reverse transcriptase (Invitrogen Life Tech-\nnologies, Inc.) and random primers (9 mers; TaKaRa Bio.\nInc., Otsu, Shiga, Japan). TRPV6 and 1A (cytochrome oxi-\ndase subunit I, a house keeping gene) were amplified in a\n\nReproductive Biology and Endocrinology 2009, 7:49 http://www.rbej.com/content/7/1/49\nPage 3 of 11\n(page number not for citation purposes)\n20 μl PCR reaction containing 1 U Taq polymerase\n(iNtRON Bio Inc, Sungnam, Kyungki-Do, Korea), 1.5 mM\nMgCl2, 2 mM dNTP, and 50 pmol TRPV6- or 1A specific\nprimers [12]. The oligonucleotide sequences for TRPV6\nwere 5'-GTG CTG GGT GCC ATC TAC GT-3' (sense) and\n5'-CAA TGA TGA CAT GGA ATG GCC-3' (antisense). The\nprimer sequences for 1A were 5'-CCA GGG TTT GGA ATT\nATT TC-3' (sense) and 5'-GAA GAT AAA CCC TAA GGC\nTC-3' (antisense). PCR reactions were denatured at 95°C\nfor 30 s, annealed at 60°C for 30 s and extended at 72°C\nfor 45 s. TRPV6 and 1A were quantified after 25 and 18\ncycles, respectively. PCR products (10 μl) were separated\non a 2% agarose gel, stained with ethidium bromide, and\nphotographed under UV illumination. Photographs were\ntaken using a Gel Doc EQ (Bio-Rad, Hercules, California,\nUSA).\nReal-time PCR using TaqMan™ probe\nReal-time PCR was performed in 20 μl reactions contain-\ning 10 μl TaqMan Universal PCR Master Mix (Applied\nBiosystems, Foster, CA, USA), and 1 μl of 20×Assays-on-\nDemand ™ Gene Expression Assay Mix (Applied Biosys-\ntems; rat TRPV6, Rn00586673_A1 [Assay ID in the\nApplied Biosystmes]; mouse TRPV6, Mm00499069_m1;\nrat HPRT1 (hypoxanthine guanine phosphoribosyl trans-\nferase 1, an internal control), Rn01527840_A1; mouse\nHPRT1, Mm00446968_m1) and 2 μl cDNA. PCR amplifi-\ncation was conducted using a 7300 Real-Time PCR System\n(Applied Biosystems), with initial enzyme activation at\n50°C for 2 min, followed by 90°C for 10 min. Each of 40\namplification cycles consisted of denaturation at 95°C for\n15 sec, followed by annealing and extension at 60°C for 1\nmin. Relative expression levels for each sample were deter-\nmined using RQ software (Applied Biosystems). The\nexpression of TRPV6 was normalized relative to that of\nHPRT1 [21,22]\nImmunohistochemistry\nThe tissue localization of TRPV6 protein was examined by\nimmunohistochemistry by using Vectastain Universal\nElite ABC Kit (Cat. No. PK-6200, Vacter Laboratories, Inc.,\nBurlingame, California, USA) as previously described\n[12]. The uterus from P5.5 to P 10.5 and placenta from\nP11.5 to P13.5 were embedded in paraffin. Sections (5\nμm) were deparaffinized in xylene and hydrated in\ndescending grades of ethanol. TRPV6 staining involved\nimmersion of the uterus and placenta sections in boiled\ncitrate target retrieval buffer (0.01 M sodium citrate and\n0.01 M citric acid, pH 6.0) at 100°C for 5 min. Endog-\nenous peroxidase activity was blocked with 3% hydrogen\nperoxide in PBS-T (PBS containing 0.05% tween20) for 30\nmin. Incubation of the section in 10% normal goat serum\n(NGS) for 2 h at room temperature blocked nonspecific\nreaction. Sections were subsequently incubated with a\npolyclonal rabbit antibody specific to TRPV6 (#ACC-036,\n0.8 mg/ml, diluted 1:100; Alomone Labs Ltd., Jerusalem,\nIsrael) dissolved in 10% NGS, at room temperature for 2\nh. A negative control was incubated with in 10% normal\nserum for 2 h at room temperature. After washing with\nPBS-T, the sections were incubated with biotinylated sec-\nondary antibody (anti-rabbit IgG; Vector Laboratories,\nInc.) for 30 min at 37°C and further incubated with\nFAST™ 3,3'-Diaminobenzidine Tablets (#D4293, Sigma-\nAldrich) for 30 min at 37°C. The sections were counter-\nstained with hematoxylin followed by mounting with a\ncoverslip.\nData analysis\nData were analyzed by nonparametric one-way analysis of\nvariance using the Kruskal-Wallis test, followed by Dun-\nnett's test for multiple comparisons to vehicle. All statisti-\ncal analyses were performed with SPSS for Windows\nEdition (SPSS, Chicago, Illinois, USA). p < 0.05 was con-\nsidered statistically significant.\nResults\nExpression of TRPV6 mRNA in the uterus and placenta \nduring pregnancy\nThe expression of rat TRPV6 mRNA during pregnancy was\nexamined by RT- and real-time PCR. The uteri were col-\nlected from rats at P0.5 to P21.5, and fetuses were\nremoved. TRPV6 mRNA was moderately expressed at\nP0.5, decreased through P4.5, and then suddenly\nincreased to the highest level at P5.5 (Figure 1). Following\nExpression of uterine TRPV6 mRNA during pregnancy in ratsFigure 1\nExpression of uterine TRPV6 mRNA during preg-\nnancy in rats. Uteri of gestating rats (n = 2) were collected \ndaily from P0.5 to P21.5, and expression of TRPV6 mRNA \nwas assayed by RT-PCR (Top panel, agarose gel image) and \nReal-Time PCR (Bottom panel, line graph). The unattached \nuteri containing uterine epithelial cells were used as a RNA \npreparation from P1.3.5. The line graph shows the analysis of \nReal-Time PCR data expressed as a percentage of TRPV6/\nHPRT1 (mean ± SEM of duplicates).\n\n\nReproductive Biology and Endocrinology 2009, 7:49 http://www.rbej.com/content/7/1/49\nPage 4 of 11\n(page number not for citation purposes)\nthis at mid-gestation, TRPV6 expression was moderate\nand then increased again at P13.5. From P12.5 to P21.5,\nthe expression of TRPV6 in the placenta steadily increased\nand expressed at the highest level at P20.5 in rats (Figure\n2).\nLocalization of TRPV6 protein in the uterus and placenta\nThe spatial distribution of TRPV6 protein in the rat uterus\nand placenta was investigated by using an anti-TRPV6\nantibody, respectively, when its transcripts were shown to\nbe expressed at relatively high levels. The rat uterus was\nlongitudinally sectioned. TRPV6 protein was detected on\nthe endometrial layer and glandular epithelium of the ges-\ntating uterus. TRPV6 was observed on the glandular epi-\nthelial apical layer but not on the basolateral membrane,\nimplying that this protein may control luminal calcium\nion transport (Figure 3). In addition, we observed that the\nsite of implantation was marked by swollen uterine tissue\n(Figure 3). The site was strongly stained by anti-TRPV6\nantibodies, supporting the notion that early embryo set-\ntlement might require the expression of TRPV6 protein\n(Figure 3). Between swollen areas in the rat uterus (Figure\n3), TRPV6 was observed in the endometrial apical layer,\nconsistent with our previous reports [11,12].\nTRPV6 protein was detected throughout the rat placenta\nas seen in Figure 4. The placenta was separated from fetus\nat P20.5 and the tissue was crossly sectioned. Three areas\nwere probed for TRPV6 expression: the inner and middle\nlabyrinth layers, and the spongy outer layer (Figure 4A, B\nand 4C). Although the tissue layer on the embryo-\nattached-side dominantly expressed TRPV6 protein, the\nmiddle labyrinth area was also shown to express this pro-\ntein. In the outer spongy layer containing giant cells,\nTRPV6 immuno-positive cells were also detected as dem-\nonstrated in Figure 4C.\nIn a time-dependent manner, uterine TRPV6 was mainly\ndetected in the epithelial and glandular cells of the non-\nattached uteri of rats (Figure 5A). The TRPV6 positive cells\nwere observed on the attached uterus, while modest\nTRPV6 signal was shown in the placental-like structure\nregarded as the implantation site (Figure 5A). In addition,\nTRPV6 positive cells were broadly observed through the\nplacenta containing the labyrinth and spongy zones in a\ntime-dependent manner (Figure 5B). The strong signals of\nTRPV6 protein were detected on the fetal membranes as\nshown.\nEffects of sex steroid receptor antagonists on uterine and \nplacental TRPV6 mRNA in a rat and mouse model\nRecently, uterine TRPV6 was shown to be regulated by\nprogesterone in immature and non-pregnant rats [11],\nwhereas its expression in a mouse model was shown to be\ncontrolled by estrogen [12]. To clarify which hormone\nmediates uterine TRPV6 expression, the rats and mice\nwere treated with a single treatment of RU or ICI to invoke\nprogesterone and estrogen receptor antagonism condi-\ntions, respectively. In the uterus at P5.5 (maximum\nexpression levels), RU treatment resulted in a significant\ninhibition of TRPV6 mRNA expression in the rat uterus. In\naddition, ICI significantly reduced TRPV6 transcription in\nthis tissue (Figure 6A). In the rat placenta, hormonal reg-\nulation was tested at P19.5 after a single treatment with\nRU or ICI. Treatment with RU significantly down-regu-\nlated the expression of placental TRPV6 at P20.5, while\nICI did not alter its expression in the rat placenta (Figure\n6B), suggesting that placental TRPV6 transcription in ges-\ntating rats may be controlled by progesterone and its\nreceptors.\nPreviously, we reported that uterine TRPV6 transcription\nin mice was regulated by estrogen via estrogen receptor α\n[12], but its placental regulation during pregnancy has not\nbeen investigated. In the previous study, steroid hor-\nmone-induced regulation of murine TRPV6 transcription\nwas shown by using ICI or RU at P10, a point in gestation\nwhen TRPV6 was previously reported to be highly\nexpressed [12]. As shown in Fig 6C, treatment with ICI sig-\nnificantly reduced uterine TRPV6 expression, whereas RU\ndid not affect uterine TRPV6 transcription levels in the\nmouse uterus. In addition, TRPV6 gene was significantly\ndown-regulated by ICI, while RU did not alter its mRNA\nlevels in the mouse placenta (Figure 6D). Taken together,\nPlacental TRPV6 mRNA expression during pregnancy in ratsFigure 2\nPlacental TRPV6 mRNA expression during pregnancy \nin rats. Placentas were collected daily from P13.5 to P21.5. \nPlacenta TRPV6 mRNA was examined by RT-PCR (Top panel, \nagarose gel image) and Real-Time PCR (Bottom panel, line \ngraph). The line graph represents the analysis of Real-Time \nPCR data expressed as a percentage of TRPV6/HPRT1 (mean \n± SEM of duplicates).\n\n\nReproductive Biology and Endocrinology 2009, 7:49 http://www.rbej.com/content/7/1/49\nPage 5 of 11\n(page number not for citation purposes)\nthese data indicate that uterine and placental TRPV6\nexpression may be primarily regulated by estrogen during\nnon-pregnancy and pregnancy in mice. This regulation of\nTRPV6 expression by estrogen in mice is in contrast to\nprogesterone-mediated regulation in rats.\nDiscussion\nSeveral uterine calcium-regulating genes are alternatively\nexpressed during estrous cycle and pregnancy [23]. In the\nimplantation period, calcium-binding proteins appear to\nplay a central role [8,12,24-33]. Calbindins are required\nduring the early phase of embryo implantation, implying\nthat the regulation of calcium availability in the vicinity of\nthe implanting embryo is critical for successful implanta-\ntion [8,9]. Although the importance of calbindins in\nimplantation has been established, the precise role of cal-\ncium ions during implantation remains unclear. Other\ncalcium processing genes have recently been identified in\nfemale reproductive organs, and TRPV6 is an interesting\nprotein in elucidating uterine process of calcium\n[11,12,34].\nUterine expression of TRPV6 varies during the estrous\ncycle in rodents. For instance, rat TRPV6 transcripts in the\nuterus were highly expressed at diestrus [11], while mouse\nTRPV6 mRNA was highly expressed at estrus [12]. Distinct\ntheir expression patterns indicate that uterine TRPV6 is\ndifferentially regulated during the estrous cycle in rats and\nmice. During pregnancy, mouse TRPV6 mRNA is actively\nexpressed with maximal expression observed in the mid-\ndle of gestation, followed by a reduction in the late\nperiod. Immediately prior to birth, another high level of\nTRPV6 expression is observed, which disappears during\nlactation [12]. In the present study, rat uterine TRPV6 was\nmodestly expressed at P0.5, which corroborates previous\nreports; and this gene was up-regulated by progesterone at\nLocalization of uterine TRPV6 at P6.5Figure 3\nLocalization of uterine TRPV6 at P6.5. Left panel, low power image of uterus showing representative TRPV6 immuno-\npositive regions restricted to three histological areas (A – C) as spotted rectangles. A, glandular epithelium; B, putative implan-\ntation site; C, epithelial layer. 'No first antibody' indicates immunoreactivity without anti-TRPV6 treatment, as a negative con-\ntrol. Arrows indicate immuno-positive staining.\n\n\nReproductive Biology and Endocrinology 2009, 7:49 http://www.rbej.com/content/7/1/49\nPage 6 of 11\n(page number not for citation purposes)\nthe diestrous stage in a rat model [11]. In days after P0.5,\nTRPV6 expression gradually decreased and was undetec-\nted at P4.5. However, at P5.5 a substantial increase and\npeak in TRPV6 expression was observed. This time point\ncoincides with the earliest sign of the implantation attach-\nment reaction which occurs around P4 or P5 in rodents\n[35]. Therefore, the maximal expression of TRPV6 gene at\nP5.5 may be related to the regulation of uterine calcium\nion concentration required for successful implantation.\nCalbindin-D9k, another uterine calcium associated pro-\ntein, gradually increases at pregnancy day 16.5, peaks at\nday 18.5, and declines at birth and the beginning of lacta-\ntion [17]. A comparison of uterine TRPV6 and calbindin-\nD9k time-dependent expressions in pregnant rats suggests\nthat these proteins may share in the function for main-\ntaining uterine calcium concentrations during pregnancy.\nIn mice, uterine calbindin-D9k showed a gradual increase\nin mRNA expression during late pregnancy (from day\n12.5 to 18.5), followed by a decline at birth and during\nlactation [17]. It has also been reported that serum estro-\ngen levels increase at the end of pregnancy and decline\nsharply following birth and during lactation, whereas\nserum progesterone levels remain constant throughout\nlate gestation and lactation [17]. With regard to these\nstudies, it is possible that increased serum estrogen levels\ninduce uterine TRPV6 transcription at birth and during\npregnancy, and that TRPV6 ceases during lactation when\nsecreted estrogen is no longer present. It was previously\nshown that mouse TRPV6 expression in the placenta ini-\ntially follows the pattern of uterine expression, with an\ninduction in the middle of gestation (P10.5 and P14.5)\nbut not at the end of gestation [12]. TRPV6 seems to have\na positive influence on transplacental calcium ion concen-\ntrations and to be a crucial factor in fetal development,\nbecause it is highly expressed in human placenta as com-\npared to levels in other organs [36]. In the present study,\nplacental expression of TRPV6 gene steadily increased\nfrom P12.5 to P20.5 before labor in rats. Taken together,\nthese results imply that constitutive expressions of TRPV6\nand/or other calcium processing genes may play a crucial\nrole in transporting calcium ions from maternal to fetal\nLocalization of placental TRPV6 at P20.5Figure 4\nLocalization of placental TRPV6 at P20.5. Left panel, overview of placenta divided into three distinct areas (A to C) \nshowing TRPV6 immuno-staining as spotted rectangles. A, inner labyrinth area; B, middle labyrinth; C, spongy zone. 'No first \nantibody' indicates immunoreactivity without anti-TRPV6 treatment, as a negative control. Arrows indicate immuno-positive \nstaining.\n\n\nReproductive Biology and Endocrinology 2009, 7:49 http://www.rbej.com/content/7/1/49\nPage 7 of 11\n(page number not for citation purposes)\nSpatial expression of uterine (A) and placental (B) TRPV6 expression in a time-dependent mannerFigure 5\nSpatial expression of uterine (A) and placental (B) TRPV6 expression in a time-dependent manner. A, Rat uteri \nfrom P5.5 to P10.5 were separately shown into the non-attached or attached uteri using anit-TRPV6 serum. u, attached uterus; \nf, placenta-like structure; arrows, epithelial and glandular cells. B, Rat placentas from P11.5 to P13.5 were presented in a time-\ndependent manner. la, labyrinth zone; sp, spongy zone; gc, giant cells; arrows, fetal membrane.\n\n\nReproductive Biology and Endocrinology 2009, 7:49 http://www.rbej.com/content/7/1/49\nPage 8 of 11\n(page number not for citation purposes)\nEffects of steroid receptor antagonists on uterine and placental TRPV6 mRNA expressionsFigure 6\nEffects of steroid receptor antagonists on uterine and placental TRPV6 mRNA expressions. Panel A (uteri at P5.5) \nand B (placenta at P20.5) presented the rat TRPV6 mRNA levels. Four groups of pregnant rats (n = 4 per group) were treated \nwith ethanol as a negative control (VE), progesterone receptor antagonist (RU, 2.5 mg per rat), or estrogen receptor antago-\nnist (ICI, 0.5 mg per mouse). Panel C (uteri at P10.5) and D (placenta at P10.5) showed the mouse TRPV6 mRNA expressions. \nFour groups of pregnant mice (n = 4 per group) were treated with ethanol as a negative control (VE), progesterone receptor \nantagonist (RU, 25 μg per mouse) or estrogen receptor antagonist (ICI, 2 μg per mouse). Murine TRPV6 mRNA levels were \nexamined by real-time PCR. The bar graph represents the analysis of real-time PCR data expressed as a percentage of TRPV6/\nHPRT1 (mean ± SEM of duplicates). a, statistically significant compared to a vehicle (P < 0.05).\n\n\nReproductive Biology and Endocrinology 2009, 7:49 http://www.rbej.com/content/7/1/49\nPage 9 of 11\n(page number not for citation purposes)\ncompartment during pregnancy. In addition, the changes\nin the uterine transcript levels of TRPV6 as a function of\npregnancy may be largely a reflection of myometrial\nchanges in its expression in both rats and mice.\nRat TRPV6 protein was detected in the endometrial layer,\nglandular epithelium and implantation site of the gestat-\ning uterus. This spatial pattern of TRPV6 expression\nbetween uterus and placenta was previously demon-\nstrated [12]. In parallel with TRPV6, rodent calbindin-D9k\nwas detected predominantly in luminal epithelial cells of\nthe uterus of pregnant rats [17,37]. The similar spatial dis-\ntribution of these gene products further supports the con-\ncept of a complementary function for TRPV6 and\ncalbindin-D9k in the control of calcium ions during preg-\nnancy. In rodent models, placental localization of TRPV6\nprotein in the labyrinth and spongy zone implies that this\nprotein may take part in transporting calcium ions\nbetween fetus and dam. In regard to its expression levels\nduring pregnancy, the expression of TRPV6 protein would\nbe highest in the myometrium as shown in this study.\nProgesterone has been implicated as a major regulator in\nthe expression of TRPV6 during the rat estrous cycle [11].\nThe progesterone receptor antagonist (RU) significantly\nblocked progesterone-induced TRPV6 gene transcription\n[11]. The expression of uterine TRPV6 mRNA at P5.5 was\ncompletely inhibited by RU treatment, and partially\ninhibited by treatments with ICI. Partial inhibition of\nTRPV6 expression by ICI may be attributed to the action\nof estrogen receptor at estrogen response elements (EREs)\nfound in the promoter region of the progesterone receptor\ngene. Although ICI partially down-regulated TRPV6 gene\nin the rat uterus, progesterone and its receptor appear to\nprimarily control uterine TRPV6 transcripts in this tissue\nduring pregnancy.\nA recent study provides the first in vivo evidence that\nTRPV6 is involved in maternal-fetal Ca\n2+ transport, pro-\nposing that TRPV6 functions as a Ca 2+ entry pathway,\nwhich is critical for fetal Ca 2+ homeostasis [16]. In addi-\ntion, a phenotype of mice with targeted disruption of\nTRPV6 gene was reported. TRPV6 knockout (KO) mice\nshowed disordered Ca\n2+ homeostasis and reduced fertil-\nity, and deficient maternal-fetal transfer of calcium ions\ncaused abnormal calcium ion deficiency during embry-\nonic development [38]. In this study, placental TRPV6\nexpression is expected to enhance the rate of maternal\ntransfer of calcium ions to the fetus during pregnancy\n[38]. In the present study, the expression of placental\nTRPV6 mRNA in rats was blocked by RU treatment, but no\nsignificant inhibitory effect was observed for ICI treat-\nments, suggesting that placental TRPV6 expression is\nunder the sole control of progesterone via a progesterone\nreceptor-mediated pathway. However we cannot rule out\nthe possibility that the difference in their doses may\naccount for the observed difference between rats and\nmice.\nIn contrast to rats, it was previously reported that murine\nuterine TRPV6 transcription at normal estrous cycles was\ntotally dependent on estrogen [12]. Uterine TRPV6 mRNA\nlevels increased at mid and late pregnancy, and were\nstrongly induced at mid-pregnancy in the labyrinth and\nspongy zones of the placenta, and in the fetal membrane\n[12]. To examine an expression pattern of TRPV6 tran-\nscripts by steroids compared to its level in rats during\npregnancy, we treated the mice with steroids, E2 and P4,\nand isolated the identical tissues from them to measure\nTRPV6 levels. In the present study, sex steroids controlled\nuterine and placental TRPV6 expression during preg-\nnancy. The expression of uterine TRPV6 mRNA at P10 was\nblocked by ICI, and placental TRPV6 transcription was\nblocked by ICI, suggesting that the expression of TRPV6\nwas primarily regulated by estrogen in gestating uteri and\nplacenta during pregnancy in mice. These results suggest\nthat TRPV6 may be distinctly regulated by different sex\nsteroids, progesterone in rats and estrogen in mice, in\nspite of shared similarities in their chromosomal make-up\n[12,20,39].\nThese results show that uterine and placental TRPV6\nmRNA levels in rats appear to cyclically change during\npregnancy, suggesting that TRPV6 protein may participate\nin the implantation process. It was of interest to note that\nuterine TRPV6 mRNA is only expressed in placenta-unat-\ntached areas of the uterus, indicating that TRPV6 has at\nleast two functions: implantation of the embryo and\nmaintenance of pregnancy. In rats, TRPV6 transcription\nappeared to be solely controlled by P4 through PRs, while\nits level was regulated by E2 via ERs in mice. In both\nrodents, uterine TRPV6 immunoreactivity was observed in\nluminal and glandular epithelial cells, and in the placenta\nTRPV6 was detected in the labyrinth and spongy zone.\nThe juxtaposition of uterine and placental TRPV6\nexpressed in these tissues can support a novel concept that\nTRPV6 participates in transporting calcium ions between\nthe maternal and fetal compartments. In conclusion,\nTRPV6 gene may function as a key element in controlling\ncalcium transport in the uterus between the embryo and\nthe placenta during pregnancy.\nCompeting interests\nThe authors declare that they have no competing interests.\nAuthors' contributions\nBL carried out the overall experiments including animal\ntreatments and molecular experiments. GL carried out the\nanimal treatments and molecular experiments with BL,\nand drafted the manuscript. EJ participated in the real-\n\nReproductive Biology and Endocrinology 2009, 7:49 http://www.rbej.com/content/7/1/49\nPage 10 of 11\n(page number not for citation purposes)\ntime PCR and immunohistochemical analysis. KC partic-\nipated in the design of the study, performed the statistical\nanalysis and helped to finalize the manuscript. EJ*\ndesigned and coordinated the overall study as a corre-\nsponding author and helped to draft the manuscript. All\nauthors read and approved the final manuscript.\nAcknowledgements\nThis work was supported by a grant (Code #20070401034011) from \nBioGreen 21 Program, Rural Development Administration, Republic of \nKorea.\nReferences\n1 . W a n g  J ,  M a y e r n i k  L ,  A r m a n t  D R :  Integrin signaling regulates\nblastocyst adhesion to fibronecti n at implantation: intracel-\nlular calcium transients and ve sicle trafficking in primary\ntrophoblast cells.  Developmental biology 2002, 245(2):270-279.\n2. 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An BS, Choi KC, Lee GS, Leung PC, Jeung EB: Complex regulation\nof Calbindin-D(9k) in the mouse placenta and extra-embry-\n\nPublish with BioMed Central   and  every \nscientist can read your work free of charge\n\"BioMed Central will be the most significant development for \ndisseminating the results of biomedical research in our lifetime.\"\nSir Paul Nurse, Cancer Research UK\nYour research papers will be:\navailable free of charge to the entire biomedical community\npeer reviewed and published immediately upon acceptance\ncited in PubMed and archived on PubMed Central \nyours — you keep the copyright\nSubmit your manuscript here:\nhttp://www.biomedcentral.com/info/publishing_adv.asp\nBioMedcentral\nReproductive Biology and Endocrinology 2009, 7:49 http://www.rbej.com/content/7/1/49\nPage 11 of 11\n(page number not for citation purposes)\nonic membrane during mid- and late pregnancy.  Mol Cell Endo-\ncrinol 2004, 214(1–2):39-52.\n38. 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