- Email: [email protected]
Expression profiling of vitamin D receptor in placenta, decidua and ovary of pregnant mice
Placenta 32 (2011) 657e664
Contents lists available at ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
Expression profiling of vitamin D receptor in placenta, decidua and ovary of pregnant mice M. Shahbazi a, M. Jeddi-Tehrani b, c, M. Zareie d, A. Salek-Moghaddam e, M.M. Akhondi f, M. Bahmanpoor g, M.R. Sadeghi f, A.H. Zarnani e, h, * a
Department of Immunology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran Immune and Gene Therapy Lab, CCK, Department of Oncology-Pathology, Karolinska University Hospital, Solna, Stockholm, Sweden d Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Canada e Immunology Research Center, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran f Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran g Department of obstetrics & Gynecology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran h Nanobiotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran b c
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 20 June 2011
Objectives: The presence of vitamin D receptor (VDR) and the identification of localized vitamin D3 synthesis in placenta and decidua implicate the importance of vitamin D3 in reproductive function. There is, however, no data on the expression profile of VDR in the mouse placenta and endometrium throughout the pregnancy period. Study design: In the present work expression of VDR in reproductive tissues of pregnant mice at different gestational phases has been addressed. Expression of VDR was determined by semi-quantitative RT-PCR, Western blotting and immunohistochemistry. Results: The results showed that VDR mRNA and protein were expressed in decidua, placenta and ovary throughout the pregnancy. VDR gene expression in placenta was significantly elevated in late pregnancy when compared to that of mid pregnancy. Additionally, VDR expression level in decidua rose significantly as pregnancy progressed from early to mid stages. VDR expression in decidua of pregnant mice was higher in comparison to endometrium of non-pregnant mice. Immunohistochemical analysis revealed that VDR protein is consistently expressed by luminal and glandular epithelial cells of decidua, giant cells, glycogen rich cells and labyrinth cells of placenta and by almost all follicular cell types of ovary. Surveying the expression of VDR at the protein level by Western blotting confirmed PCR results. Conclusion: It seems that expression of VDR in reproductive organs is finely tuned during pregnancy indicating its eminent role in reproductive biology. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Vitamin D receptor Placenta Decidua Ovary Pregnancy Mice
1. Introduction The active form of vitamin D3, 1,25 dihydroxy vitamin D3 (calcitriol), is a lipid-soluble secosteroid hormone which has wellestablished classic effects on bone metabolism and mineral homeostasis in the gastrointestinal tract and kidney. Vitamin D3 can be obtained from the diet or synthesized in the skin from 7-dehydrocholesterol during sunlight exposure. Upon intake, vitamin D3 is biologically inert and requires activation through a two-step enzymatic pathway involving 25-hydroxylase and 1a-hydroxylase * Corresponding author. Nanobiotechnology Research Center, Avicenna Research Institute, ACECR, P.O. Box: 1936773493, Tehran, Iran. Tel.: þ98 22432020; fax: þ98 22432021. E-mail addresses: [email protected], [email protected] (A.H. Zarnani). 0143-4004/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2011.06.013
for the conversion to the biologically active form [1]. Vitamin D3 exerts its effects upon a great variety of cell types by binding to the nuclear vitamin D receptor (VDR) which belongs to the nuclear receptor superfamily of ligand-dependent transcriptional factors. After binding to its ligand, VDR interacts with the nuclear receptor retinoic acid X receptor (RXR). In the presence of vitamin D3 the VDR/RXR complex binds small sequences of DNA known as vitamin D response elements (VDREs) and initiates a cascade of molecular interactions that modulate the transcription of multitude of genes in tissues throughout the body [1]. Regulation of VDR gene expression is one of the main mechanisms through which target cells respond to calcitriol. Therefore, biological action of this hormone is largely determined by the extent of VDR expression levels in a given cell type [2,3]. While a role for vitamin D3 in tissue growth and bone metabolism is well established, the presence of VDR and enzymes involved in the
658
M. Shahbazi et al. / Placenta 32 (2011) 657e664
hydroxylation of vitamin D3, and the identification of localized vitamin D3 synthesis in human placenta and decidua [4,5] implicates the importance of vitamin D3 in reproductive function. The elegant studies by Panda et al. clearly showed that targeted ablation of vitamin D3 1a-hydroxylase, the enzyme responsible for production of biologically active form of vitamin D3, is accompanied by several reproductive dysfunctions in female mice, including abnormal ovarian follicle development, uterine hyperplasia and infertility [6]. VDR knockout mice have a similar phenotype to that of 1a-hydroxylase knockout mice [7]. Female fertility and litter size is markedly reduced in vitamin D3 deficiency [8,9], but may involve calcium-dependent or independent mechanisms depending on the animal model used. In vitamin D3 deficient rats, decreased fertility and litter size seem to be solely due to diminished level of vitamin D3 and independent of calcium status [9]. In VDR knockout mice, however, female reproductive dysfunction seems to be under the direct influence of calcium levels [7,10]. VDR is widely expressed in most cells of the immune system, including antigen presenting cells such as macrophages and dendritic cells (DC), as well as activated CD4þ and CD8þ T lymphocytes. Dendritic cells are the main target of immunoregulatory effects exerted by vitamin D3. Vitamin D3 inhibits DC differentiation and maturation, decreasing their capability for stimulatory action, thus preventing T lymphocyte proliferation and activation [11], skewing cytokine profile toward T helper 2 (TH2) phenotype [12], and suppression of immunoactive NK cells [13]. Given the fact that DCs are crucial in establishment of maternal tolerance during pregnancy as our team and other groups have shown previously [14,15] and that increased activity of natural killer (NK) cells and T helper 1(TH1) cytokines, at least at midgestational period, are causative factors for pregnancy loss [16,17], it is conceivable to speculate that vitamin D3 could improve pregnancy outcome through its immunoregulatory function. Interestingly, vitamin D3 has been suggested as a new therapeutic agent for women with recurrent spontaneous abortion [18]. We have previously shown that VDR is present and differentially expressed in reproductive organs of cycling mice, including dendritic cells and macrophages of the endometrium, particularly during the estrus phase [19]. To further evaluate the functional role of the locally generated vitamin D3 in murine reproduction, the present work was designed to study the expression of the VDR at both gene and protein level in reproductive tissues of pregnant mice at different gestational phases. 2. Materials and methods 2.1. Animals
analysis. RNA purity was periodically assessed by agarose gel electrophoresis. Complementary DNA (cDNA) synthesis for the detection of mRNA for VDR and GAPDH (house keeping gene used as internal control) were performed according to the protocol we published recently [21]. In brief, RNA was denatured at 65 C and immediately placed on ice. A mixture containing 5 reaction buffer (Fermentase, Vilnius, Lithuania), 1 mM dNTP mix (Roche, Penzberg, Germany), 1 mM RandomHexamer (Cybergene, Stockholm, Sweden), and 20 IU/mL of Reverse Transcriptase M-MuLV (Fermentase) was added to the denatured RNA and mixed thoroughly. After incubation for 1 h at 42 C in a thermocycler, the resulting cDNA was aliquoted and stored at 20 C. Semi-quantitative RT-PCR was performed in a final volume of 25 mL containing one mL of cDNA, PCR buffer (10x), MgCl2 (2.5 mM for GAPDH and 2 mM for VDR), dNTP mix (0.4 U/mL), and Taq DNA Polymerase at 0.04 U/mL final concentration (Roche). One mL of each pair of primers, equivalent to final concentrations of 10 mM for GAPDH and 5 mM for VDR, were added. PCR was then performed as follows: GAPDH with the program 94 C 2 min (1 cycle), 94 C 30 s, 60 C 30 s, 72 C 30 s (30 cycles), and 72 C for 7 min (1 cycle); and VDR with the program 94 C 2 min (1 cycle), 94 C 30 s, 59 C 30 s, 72 C 30 s (35 cycles), and 72 C for 7 min (1 cycle). The cycle numbers used for VDR and GAPDH amplification were showed to be in the logarithmic phase of amplification as judged by the cycle gradients ran separately for each gene (Fig. 1). Oligonucleotide primers for GAPDH and VDR were as follows: VDR: F: 5-GAG-GTG-TCT-GAA-GCC-TGG-AG-30 ; R: 50 -ACC-TGC-TTT-CCT-GGG-TAGGT-30 , GAPDH: F: 50 -CAG-GAG-CGA-GAC-CCC-ACT-A-30 ; R: 50 -GGC-ATG-GAC-TGTGGT-CAT-GA-30 . The primers were designed to generate a fragment of 309 bp for GAPDH and a fragment of 155 bp for VDR. For VDR expression, the cDNA prepared from mouse kidney tissue served as positive control. A PCR system devoid of template cDNA and no amplification controls (no reverse transcriptase added) were included as negative controls. 2.4. Agarose gel electrophoresis and densitometry The PCR products for GADPH and VDR genes were electrophoresed on agarose gel. The gels were photographed with UV transilluminator Gel Doc (UVP, LLC, Upland, CA). For densitometry analysis, Alpha Ease software was used. Each photograph was first converted to 8 bit grayscale picture. For analysis of PCR bands, picture color was inverted so the bands appeared black. Afterward, contrast was adjusted so that black and white colors corresponded to 250 and 0, respectively. Using rectangle object selection tool, bands were then selected. After subtraction of background density of negative control, average densities of bands were quantified. Relative expression of VDR was presented as the percent of VDR/GADPH density ratio. 2.5. Immunohistochemical localization of VDR Immunolocalization of VDR in murine placenta, decidua and ovaries was performed as previously described [19]. Briefly, acetone-fixed cryostat sections were washed in Tris-buffered salineeBovine serum albumin 0.1% (TBS-BSA). After blocking with 5% normal goat serum for 15 min, endogenous biotin was blocked by Avidin/Biotin blocking solutions (Dako, Glostrop, Denmark) according to manufacturer’s instruction. Sections were then incubated with monoclonal anti-VDR primary antibody (NeoMakers, Fremont, CA, 2.5 mg/ml) for 90 min, washed and treated with 0.3% H2O2 in TBS for 10 min to block endogenous peroxidase, followed by incubation (45 min) with biotinylated goat anti-rat antibody (BD Biosciences Pharmingen, SanDiego, CA, 5 mg/ml). After rinsing, sections were processed further using streptavidin/peroxidase (Biosource, Camarillo, CA) (1:500 dilution, 30 min),
Female and male Balb/c mice aged 8e12 weeks were obtained from Pasteur Institute (Tehran, Iran). All studies carried out with the mice were approved by the ethical committee for experimental animals and in accordance with the institutional guidelines for care and use of laboratory animals at Avicenna Research Institute, Tehran, Iran. Female mice were mated with males and the onset of pregnancy was determined as we reported elsewhere [20]. 2.2. Tissue collection and preparation At each gestation phase [beginning (day 2), middle (day 10) and toward the end (day 18)], 4e5 mice were sacrificed and ovary, placenta and decidua samples were collected. For RNA extraction, specimens were immediately transferred into microtubes containing RNA-Bee solution (Nordic Biosite, Taby, Sweden). For immunohistochemistry, the samples were immersed in OCT medium (Sakura, Zoetenwoude, The Netherlands), flash frozen and stored at 70 C. Additional tissue samples were frozen in liquid nitrogen for Western blotting. Sections from the kidney of one mouse were treated accordingly and served as positive controls. 2.3. Isolation of total RNA and quantitative RT-PCR Total RNA was extracted from ovary, placenta and decidual samples by standard phenolechloroform method as described previously [19]. The resulting RNA pellet was dissolved in distilled water and samples were stored at 20 C for further
Fig. 1. Cycle gradient PCR. To ensure semi-quantitative amplification, cycle gradient PCR starring from 20 cycles was performed for VDR and reference gene (GAPDH). Numbers above the lanes indicate number of PCR cycles for each gene. Each PCR reaction was run in duplicate. NC: Negative control with no added cDNA; M: Size marker.
M. Shahbazi et al. / Placenta 32 (2011) 657e664
659
and reaction products were developed with 3,30 -diaminobenzide tetrahydrochloride (DAB) chromogen (Roche). In negative control sections primary antibody was omitted. All sections were counterstained with Harris hematoxylin for 5 s; dehydrated with increasing concentrations of ethanol and mounted using Enthelan glue (Merck, Darmstadt, Germany). 2.6. Western blot analysis of VDR expression Depending on the size of the samples, tissue were homogenized in 100e400 mL of lysis buffer (1% w/v Triton X-100, 50 mM Tris at pH ¼ 7.4, 0.1% sodium dodecyl sulfate, 1% v/v protease inhibitors, 150 mM NaCl, 1 mM sodium fluoride, 5 mM EDTA, and 1% v/v glycerol) using Pellet Pestle and left on ice for 1 h. Subsequently, the lysates were centrifuged (10,000 rpm, for 10 min at 4 C) and the supernatants were collected and the protein concentration was measured by Bradford assay. Western blotting of VDR and b actin were performed according to the protocol we published elsewhere with some modifications [22]. A total of 100 mg protein per lane were added to sample buffer (containing 5% 2-Mercapto Ethanol), boiled (2 min), and subjected to SDS-PAGE (3% stacking and 10% resolving) electrophoresis at 100 V for 90 min. The proteins were transferred onto polyvinylidene fluoride membrane (Roche). After blocking overnight with 5% nonfat dry milk in PBS containing 0.1% Tween (PBST), blots were incubated 2 h with agitation in monoclonal anti-VDR primary antibody (NeoMakers) at 1 mg/ml in PBST. After washing 5 10 min in PBST, blots were incubated 1 h in HRP-conjugated anti-rat IgG (BD Biosciences Pharmingen, 1:1000) and washed again. VDR was detected using an ECL Western blotting substrate kit (GE HealthCare, Buckinghamshire, UK) and digital images were obtained with a Gel Logic 2200 imaging system (Kodak, Tokyo, Japan). In negative control blots, primary antibody was omitted. Membranes were later stripped using Western Re-Probe (Calbiochem, SanDiego, CA) and re-incubated 2 h with agitation in anti-b actin antibody (Sigma, Kungsbacka, Sweden; 1:200), which was used as loading control. After washing 5 15 min in PBST, blots were incubated 45 min in HRP-conjugated Sheep anti-rabbit IgG (Avicenna Research Institute, Tehran, Iran, 1:500) and processed as above. Densities of VDR and b actin bands were quantified and the relative expression was calculated using Alpha Ease software as above.
Fig. 2. Expression of vitamin D3 receptor (VDR)-specific transcript in placenta, decidua and ovary of pregnant Balb/c mice at different periods of pregnancy. RT-PCR was run using specific primers for VDR and GAPDH amplification was used as the loading control. No amplification controls (NAC; no reverse transcriptase added were used to check for the amplification of genomic DNA). Results of 2 out of 4e5 experiments have been shown for each sample. EPD: Early pregnancy decidua; MPD: Mid pregnancy decidua; LPD: Late pregnancy decidua; EPO: Early pregnancy ovary; MPO: Mid pregnancy ovary; LPO: Late pregnancy ovary; MPP: Mid pregnancy placenta; LPP: Late pregnancy placenta; NC: Negative control with no added cDNA; PC: Kidney as positive control; M: Size marker.
2.7. Statistical analysis All data is presented as median (range). The density ratio (VDR/GAPDH and VDR/
b actin) data from each stage were analyzed using non-parametric ManneWhitney
test and statistically significant differences were accepted at P < 0.05.
3. Results 3.1. Detection of VDR mRNA expression in adult mice during pregnancy
luminal and glandular epithelial cells of decidua basalis staining the strongest (Fig. 4). In placenta, VDR expression was located on the Giant cells, glycogen rich cells, labyrinth cells, the chorionic membrane, and fetal neural tube cells (Fig. 5). Furthermore, immunohistochemical analysis of ovaries revealed VDR expression in almost all follicular cell types including granulosa cells, cumulus
Semi-quantitative RT-PCR was used to determine the presence of VDR gene transcripts in pregnant mice reproductive tissue. The 309 bp fragment of GAPDH enzyme mRNA was used as an endogenous control sequence. Fig. 2 illustrates that the VDR mRNA (155 bp) could be detected in every single sample of placenta, decidua and ovary regardless whether obtained from early, mid or late stages of pregnancy. Densitometry analysis revealed that, in relation to reference gene GAPDH, VDR gene expression in placenta was significantly elevated in late pregnancy when compared to that of mid pregnancy (P < 0.01). Additionally, VDR expression level in decidua rose significantly (P < 0.05) as pregnancy progressed from early to mid stages. However, there were no detectable differences in VDR expression in decidua samples from mid and late stages of pregnancy. More interestingly, significantly higher expression of VDR in decidual samples from pregnant mice was observed compared to endometrial samples of non-pregnant mice. (P < 0.001) (Fig. 3). 3.2. VDR protein expression: immunohistochemical localization VDR localization in ovaries, placenta and decidua of pregnant mice at early, mid and late stages of pregnancy was investigated by immunohistochemistry. VDR protein was detected throughout pregnancy in all tissue samples examined. In decidua, VDR staining was observed in both the cells of decidua capsularis and decidua basalis at early, mid and late stages of pregnancy, with both the
Fig. 3. Expression of VDR-specific message in endometrium of non-pregnant and decidua and placenta of pregnant Balb/c mice. Semi-quantitative RT-PCR was used to measure VDR expression in endometrium of non-pregnant mice at different stages of estrous cycle and decidua and placenta of pregnant mice at different periods of pregnancy. The density ratio was significantly higher in mice during estrous phase (E) compared to the other phases (P, M and D) (P < 0.01). During pregnancy, VDR expression was significantly increased. Each bar is representing median (range) of the data obtained from 4 (M, D, MPD, and MPP) or 5 mice (P, E, EPD, LPD, and LPP). P: Proestrous, E: Estrus, M: Metestrus, D: Diestrus, EPD: Early pregnancy decidua, MPD: Mid pregnancy decidua, LPD: Late pregnancy decidua, MPP: Mid pregnancy placenta, LPP: Late pregnancy placenta, AU: Arbitrary unit. *; P < 0.05, **; P < 0.01, ***; P < 0.001.
660
M. Shahbazi et al. / Placenta 32 (2011) 657e664
Fig. 4. Immunohistochemical analysis of vitamin D receptor (VDR) expression in decidua basalis of pregnant Balb/c mice at early (A), middle (B) and late stages (C) of pregnancy. Luminal and glandular epithelial cells were the prominent cell types that expressed VDR in all phases. There were also scattered positive cells in the decidua stroma. Each figure is a representative photograph from 4 to 5 animals. (D) Negative control. DB: Decidua basalis, EPC: Epithelial cells, Scale bar: A; 20mm, BeD; 40mm.
oophorus, corena radiata and cells in ovarian stroma at various stages of pregnancy (Fig. 6). There was minimal VDR staining in the cells of the theca externa and theca interna. 3.3. VDR protein expression: Western blotting As a complementary approach to immunohistochemistry and RT-PCR, Western blot analysis was carried out to assess the expression of VDR protein in ovaries, placenta and decidua of pregnant mice. Mouse kidney lysate was used as positive control. Western blot analysis detected the 52 kDa VDR protein in samples of placenta, decidua and ovary (Fig. 7) at early, mid and late stages of pregnancy. Quantification of VDR expression by densitometry revealed that the VDR protein level was elevated in late pregnancy placenta when compared to the placenta at mid pregnancy (P < 0.01). Compared to early pregnancy decidua, mid pregnancy decidua expressed higher levels of VDR protein (P < 0.05). 4. Discussion Recent findings show that calcitriol has several activities in regulation of immune system through antigen presenting cells and most of these actions are in accordance with a successful pregnancy [23,24]. Most researchers believe that, at least during midgestational period, successful pregnancy is associated with a shift of cytokine profile toward TH2 [25]. Considering the TH2 promoting activity of calcitriol, it seems that this hormone could have a supportive role in at least a period of conception. According to our recent report, dendritic cells and macrophages are among the main cells that express VDR in endometrial stroma [19].
Therefore it is conceivable that these cells have pivotal role in the regulation of local immunity toward the successful conception under the influence of vitamin D3. Interestingly, dendritic cells treated with vitamin D3 induce regulatory T cells [26,27]. These cells play a key role in creating conception tolerance [26]. The findings from this study showed that, VDR is expressed at all different stages of pregnancy in the placenta and decidua indicating the eminent role of this vitamin D3 in the process of reproduction. It was interesting that, the expression of this receptor in decidua was more prominent in the middle stages of pregnancy in comparison with early stage of pregnancy. In middle stages of murine pregnancy, the formation of placenta and its protrusion get to its highest level and this is expected to be accompanied by wellcontrolled local immune responses. Regarding the role of vitamin D3 in the control of immune responses, the increase in the expression of VDR in decidua would be imaginable in the middle stages of pregnancy. The level of VDR mRNA in human decidua is higher at the first compared to the second trimester [4]. The rationale behind the difference in time course expression of VDR in our finding and aforesaid report is the difference that exists between human and mouse implantation and placentation. In both human and mice, implantation takes place in a relatively same window period after fertilization (4e5 days for mice and 5e10 days for human), but considering the whole gestational period in these spices, it can be deduced that implantation begins very soon in human while it occurs in female mouse when she has passed one forth of her pregnancy period. Indeed, endometrial invasion by placental trophoblast occurs mostly during the mid-gestational period of mouse pregnancy, while in human it is started very soon after placental development at the first trimester. Based on this fact and also comparative developmental anatomy of murine
M. Shahbazi et al. / Placenta 32 (2011) 657e664
661
Fig. 5. Immunohistochemical analysis of vitamin D receptor (VDR) expression in placenta of pregnant Balb/c mice at middle, and late stages of pregnancy. VDR expression was located on the trophoblast giant cells (TGC), glycogen rich trophoblast cells (GlyT) and labyrinth trophoblast cells (LBT) at middle (A) and late stages of pregnancy (B). Fetal neural tube cells (C) at the mid pregnancy also expressed high levels of VDR. Different zones of placenta [Decidua zone (DZ), Junctional zone (JZ) and Labyrinth zone (LZ) have been specified in parts A and B. Each figure is a representative photograph from 4 to 5 animals. (D) Negative control. Scale bar: 40mm.
and human placenta, it is estimated that, temporally, the first trimester in the human may be equivalent to around mid-gestation in the mouse [28]. In addition to decidua, placenta expressed VDR in all stages of pregnancy, but the expression was significantly augmented in later stages of pregnancy. The rationale behind this is unclear, but it seems it has a role in the physiological processes such as absorption of calcium and its transition to the developing fetus before delivery. Several recent studies have examined VDR expression and function in human placental trophoblasts. The human placenta synthesizes 1,25(OH)2D3 and expresses VDR [29]. According to recent report, VDR is localized to syncytiotrophoblast, stromal and endothelial cells of human term placenta [5]. Vitamin D3 is involved in the regulation of placental lactogen expression, human chorionic gonadotropin (hCG) secretion and calcium transport in the placenta [30e32]. Surveying the expression of VDR at the protein level by Western blotting confirmed PCR results. In this experiment, the expression of VDR in decidua at the middle stage of pregnancy and in placenta at late gestation period were significantly more than those at early and mid pregnancy, respectively. The results of Immunohistochemical analysis were in line with those of PCR and Western blotting on this account that VDR was expressed at the feto-maternal interface in all duration of pregnancy. Immunohistochemistry revealed that, the glandular and luminal epithelial cells during all period of pregnancy are dominant cells expressing VDR in endometrium. It is notable that according to our recent report, these cells are the principal cells that express VDR in non-pregnant mice [19]. Our former findings indicate that, endometrial epithelial cells are the dominant cells, which express
mediators of immune regulators such as indoleamine 2,3,dioxigenase [22]. There is convincing evidence that mucosal epithelial cells, beyond their role in creating a physical barrier, are integral components of innate and adaptive immunity [33]. These cells have a substantial role in transition from innate immunity to adaptive immunity [34,35]. Based on the immunoregulatory activity of vitamin D3, our findings on the high expression of VDR in decidual epithelial cells is a further proof of the fundamental importance of these cells in shaping the local immune responses at the fetomaternal interface. Based on the wide distribution VDR in different cell types of decidua, placenta and ovary of the pregnant mice, it seems that vitamin D3 machinery is a basic requirement for most cells. In fact, VDR is expressed in over 30 different tissues, including the stomach, bone, brain, immune cells and other tissues [36]. It is also ubiquitous in proliferating cells [4]. The number of responding cells, availability of ligand and more importantly the density of VDR in the cells are key elements which determine the extent of vitamin D3 action on each specific organ. Our earlier studies showed that, VDR is expressed in the endometrium of non-pregnant mice in all stages of estrous cycle [19]. It was interesting that, in comparison with non-pregnant mice, expression of VDR increased significantly in the endometrium of pregnant mice. This finding emphasizes the role of vitamin D3 in conception process. In addition to immunologic roles, vitamin D3 has multiple physiologic activities during pregnancy. Low blood levels of vitamin D3 during pregnancy may be a risk factor forimpaired fetal development. Deficiency in vitamin D3 is accompanied with smaller litter size and number [8]. Mice having deficiency in VDR gene also have uterus hypoplasia, defect in
662
M. Shahbazi et al. / Placenta 32 (2011) 657e664
Fig. 6. Immunohistochemical analysis of vitamin D receptor (VDR) expression in ovary of pregnant Balb/c mice at early, middle and late stages of pregnancy. VDR was preferentially expressed by granulosa cells (GraC) of the ovary at early (A), middle (B) and late stages (C) of pregnancy. There are scattered immunoreactive stromal cells around the follicles. Each figure is a representative photograph from 4 to 5 animals. (D) Negative control. Scale bar: 40mm.
folliculogenesis and pregnancy [7,37]. According to Lappillone [38], vitamin D3 insufficiency during pregnancy is not only potentially associated with increased risk of preeclampsia, insulin resistance and gestational diabetes mellitus, but also may impair fetal brain and skeletal development.
Calcitriol has also axis role in blastocyst implantation through the increase in expression of such genes as HOXA10 [39]. According to recent findings, intraluminal injection of vitamin D3 in rat uterus causes pseudopregnancy and decidualization [40]. On the basis of the findings in this study, expression of VDR in decidua at the early
Fig. 7. Western blot analysis of vitamin D receptor (VDR) expression in placenta, decidua and ovary of pregnant mice at different periods of pregnancy. Expression of VDR in reproductive organs of pregnant Balb/c mice at different periods of pregnancy was analyzed by Western blotting. The results of the two samples out of 4e5 for each tissue are shown. b actin was used as internal standard (A). Relative expression of VDR in placenta and decidua is shown in part B. Each bar is representing the median (range) of the data obtained from 4 (MPD, and MPP) or 5 mice (EPD, LPD, and LPP). EPD: Early pregnancy decidua; MPD: Mid pregnancy decidua; LPD: Late pregnancy decidua; EPO: Early pregnancy ovary; MPO: Mid pregnancy ovary; LPO: Late pregnancy ovary; MPP: Mid pregnancy placenta; LPP: Late pregnancy placenta; NC: Negative control with no primary antibody; PC: Kidney as positive control, AU: Arbitrary unit. *; P < 0.05, **; P < 0.01.
M. Shahbazi et al. / Placenta 32 (2011) 657e664
stages of pregnancy increased substantially comparing to the nonpregnant mice and this finding can reveal the role of vitamin D3 in the processes of implantation. With relation to the expression of VDR at gene and protein level by both fetus and mother side of placenta, it seems that, vitamin D3 possesses a role in physiologic and immunologic regulation of mother and fetus activities. On the other hand, high expression of VDR in the fetus of mice during middle stages of pregnancy as judged by immunohistochemistry reveals that VDR expression is not solely confined to the mother and fetal cells also express this receptor. Data generated in this research showed that, VDR is expressed at gene and protein level by the ovary of pregnant mice in all stages of pregnancy. The role of VDR is not quite clear in the ovary but it may have a role in adjusting some of the physiological activities of the ovary. Vitamin D3 exists in human follicular fluid [41] and may have a role in regulation of granulosa cells activity through the interaction with VDR. Vitamin D3 in the fibroblasts of the human skin accelerates the conversion of androstenedione to estrogen through the augmentation of aromatase activity [42] and it is probable that, this vitamin D3 displays a similar role in the ovary. The granulosa cells of the ovary also have aromatase activity [19,43] and by means of producing estrogen have a crucial role in folliculogenesis and ovulation [44]. According to our recent study, VDR is expressed in epithelial cells of fallopian tube in all stages of estrous cycle [19]. Accordingly it seems that, this receptor is expressed in all pregnancy-related tissues in both pregnant and non-pregnant mice. These findings indicate the undeniable role of vitamin D3 in the process of reproduction. 5. Conclusion Collectively, the findings in this study showed that, VDR is expressed in the gene and protein level in endometrium, placenta, and ovary of pregnant mice during all stages of pregnancy and this phenomenon is probably in line with indispensible role of vitamin D3 in the adjustment of the physiologic and immunologic activities during pregnancy. Acknowledgment The authors wish to thank Mr. A. Moravej, Mrs. G.E. KazemiSefat and Miss. J. Ghasemi for their technical assistance. References [1] Jones G, Strugnell SA, DeLuca HF. Current understanding of the molecular actions of vitamin D. Physiol Rev 1998;78(4):1193e231. [2] Chen TL, Hauschka PV, Cabrales S, Feldman D. The effects of 1,25dihydroxyvitamin D3 and dexamethasone on rat osteoblast-like primary cell cultures: receptor occupancy and functional expression patterns for three different bioresponses. Endocrinology 1986;118(1):250e9. [3] Halloran BP, DeLuca HF. Appearance of the intestinal cytosolic receptor for 1,25-dihydroxyvitamin D3 during neonatal development in the rat. J Biol Chem 1981;256(14):7338e42. [4] Zehnder D, Evans KN, Kilby MD, Bulmer JN, Innes BA, Stewart PM, et al. The ontogeny of 25-hydroxyvitamin D(3) 1alpha-hydroxylase expression in human placenta and decidua. Am J Pathol 2002;161(1):105e14. [5] Pospechova K, Rozehnal V, Stejskalova L, Vrzal R, Pospisilova N, Jamborova G, et al. Expression and activity of vitamin D receptor in the human placenta and in choriocarcinoma BeWo and JEG-3 cell lines. Mol Cell Endocrinol 2009; 299(2):178e87. [6] Panda DK, Miao D, Tremblay ML, Sirois J, Farookhi R, Hendy GN, et al. Targeted ablation of the 25-hydroxyvitamin D 1alpha -hydroxylase enzyme: evidence for skeletal, reproductive, and immune dysfunction. Proc Natl Acad Sci U S A 2001;98(13):7498e503. [7] Yoshizawa T, Handa Y, Uematsu Y, Takeda S, Sekine K, Yoshihara Y, et al. Mice lacking the vitamin D receptor exhibit impaired bone formation, uterine hypoplasia and growth retardation after weaning. Nat Genet 1997;16(4): 391e6.
663
[8] Halloran BP, DeLuca HF. Effect of vitamin D deficiency on fertility and reproductive capacity in the female rat. J Nutr 1980;110(8):1573e80. [9] Kwiecinksi GG, Petrie GI, DeLuca HF. 1,25-Dihydroxyvitamin D3 restores fertility of vitamin D-deficient female rats. Am J Physiol 1989;256(4 Pt 1): E483e7. [10] Johnson LE, DeLuca HF. Vitamin D receptor null mutant mice fed high levels of calcium are fertile. J Nutr 2001;131(6):1787e91. [11] Adorini L, Penna G, Giarratana N, Roncari A, Amuchastegui S, Daniel KC, et al. Dendritic cells as key targets for immunomodulation by Vitamin D receptor ligands. J Steroid Biochem Mol Biol 2004;89e90(1e5):437e41. [12] Penna G, Adorini L. 1 Alpha,25-dihydroxyvitamin D3 inhibits differentiation, maturation, activation, and survival of dendritic cells leading to impaired alloreactive T cell activation. J Immunol 2000;164(5):2405e11. [13] Merino F, Alvarez-Mon M, de la Hera A, Ales JE, Bonilla F, Durantez A. Regulation of natural killer cytotoxicity by 1,25-dihydroxyvitamin D3. Cell Immunol 1989;118(2):328e36. [14] Zarnani AH, Moazzeni SM, Shokri F, Salehnia M, Dokouhaki P, Ghods R, et al. Microenvironment of the feto-maternal interface protects the semiallogenic fetus through its immunomodulatory activity on dendritic cells. Fertil Steril 2008;90(3):781e8. [15] Juretic K, Strbo N, Crncic TB, Laskarin G, Rukavina D. An insight into the dendritic cells at the maternal-fetal interface. Am J Reprod Immunol 2004; 52(6):350e5. [16] Raghupathy R. Pregnancy: success and failure within the Th1/Th2/Th3 paradigm. Semin Immunol 2001;13(4):219e27. [17] Choudhury SR, Knapp LA. Human reproductive failure I: immunological factors. Hum Reprod Update 2001;7(2):113e34. [18] Bubanovic I. 1alpha,25-dihydroxy-vitamin-D3 as new immunotherapy in treatment of recurrent spontaneous abortion. Med Hypotheses 2004;63(2):250e3. [19] Zarnani AH, Shahbazi M, Salek-Moghaddam A, Zareie M, Tavakoli M, Ghasemi J, et al. Vitamin D3 receptor is expressed in the endometrium of cycling mice throughout the estrous cycle. Fertil Steril 2010;93(8):2738e43. [20] Zarnani AH, Moazzeni SM, Shokri F, Salehnia M, Jeddi-Tehrani M. Kinetics of murine decidual dendritic cells. Reproduction 2007;133(1):275e83. [21] Moravej A, Jeddi-Tehrani M, Salek-Moghaddam AR, Dokouhaki P, Ghods R, Rabbani H, et al. Evaluation of thyroglobulin expression in murine reproductive organs during pregnancy. Am J Reprod Immunol; 2010. [22] Jeddi-Tehrani M, Abbasi N, Dokouhaki P, Ghasemi J, Rezania S, Ostadkarampour M, et al. Indoleamine 2,3-dioxygenase is expressed in the endometrium of cycling mice throughout the oestrous cycle. J Reprod Immunol 2009;80(1e2):41e8. [23] Hewison M, Freeman L, Hughes SV, Evans KN, Bland R, Eliopoulos AG, et al. Differential regulation of vitamin D receptor and its ligand in human monocyte-derived dendritic cells. J Immunol 2003;170(11):5382e90. [24] Kragballe K. Vitamin D analogues in the treatment of psoriasis. J Cell Biochem 1992;49(1):46e52. [25] Aagaard-Tillery KM, Silver R, Dalton J. Immunology of normal pregnancy. Semin Fetal Neonatal Med 2006;11(5):279e95. [26] Griffin MD, Xing N, Kumar R. Vitamin D and its analogs as regulators of immune activation and antigen presentation. Ann Rev Nutr 2003;23:117e45. [27] van Etten E, Mathieu C. Immunoregulation by 1,25-dihydroxyvitamin D3: basic concepts. J Steroid Biochem Mol Biol 2005;97(1e2):93e101. [28] Georgiades P, Ferguson-Smith AC, Burton GJ. Comparative developmental anatomy of the murine and human definitive placentae. Placenta 2002;23(1): 3e19. [29] Diaz L, Sanchez I, Avila E, Halhali A, Vilchis F, Larrea F. Identification of a 25hydroxyvitamin D3 1alpha-hydroxylase gene transcription product in cultures of human syncytiotrophoblast cells. J Clin Endocrinol Metab 2000; 85(7):2543e9. [30] Tuan RS, Moore CJ, Brittingham JW, Kirwin JJ, Akins RE, Wong M. In vitro study of placental trophoblast calcium uptake using JEG-3 human choriocarcinoma cells. J Cell Sci 1991;98(Pt 3):333e42. [31] Stephanou A, Ross R, Handwerger S. Regulation of human placental lactogen expression by 1,25-dihydroxyvitamin D3. Endocrinology 1994;135(6): 2651e6. [32] Barrera D, Avila E, Hernandez G, Mendez I, Gonzalez L, Halhali A, et al. Calcitriol affects hCG gene transcription in cultured human syncytiotrophoblasts. Reprod Biol Endocrinol 2008;6:3. [33] Saenz SA, Taylor BC, Artis D. Welcome to the neighborhood: epithelial cellderived cytokines license innate and adaptive immune responses at mucosal sites. Immunological Reviews 2008;226:172e90. [34] Schleimer RP, Kato A, Kern R, Kuperman D, Avila PC. Epithelium: at the interface of innate and adaptive immune responses. J Allergy Clin Immunol 2007;120(6):1279e84. [35] Hammad H, Lambrecht BN. Dendritic cells and epithelial cells: linking innate and adaptive immunity in asthma. Nat Reviews 2008;8(3):193e204. [36] Perez-Lopez FR. Vitamin D: the secosteroid hormone and human reproduction. Gynecol Endocrinol 2007;23(1):13e24. [37] Kinuta K, Tanaka H, Moriwake T, Aya K, Kato S, Seino Y. Vitamin D is an important factor in estrogen biosynthesis of both female and male gonads. Endocrinology 2000;141(4):1317e24. [38] Lapillonne A. Vitamin D deficiency during pregnancy may impair maternal and fetal outcomes. Med Hypotheses 2010;74(1):71e5. [39] Bagot CN, Troy PJ, Taylor HS. Alteration of maternal Hoxa10 expression by in vivo gene transfection affects implantation. Gene Ther 2000;7(16):1378e84.
664
M. Shahbazi et al. / Placenta 32 (2011) 657e664
[40] Halhali A, Acker GM, Garabedian M. 1,25-Dihydroxyvitamin D3 induces in vivo the decidualization of rat endometrial cells. J Reprod Fertil 1991;91(1): 59e64. [41] Potashnik G, Lunenfeld E, Levitas E, Itskovitz J, Albutiano S, Yankowitz N, et al. The relationship between endogenous oestradiol and vitamin D3 metabolites in serum and follicular fluid during ovarian stimulation for in-vitro fertilization and embryo transfer. Hum Reprod 1992;7(10): 1357e60.
[42] Hodgins MB, Murad S. 1, 25-Dihydroxycholecalciferol stimulates conversion of androstenedione into oestrone by human skin fibroblasts in culture. J Endocrinol 1986;110(1):R1e4. [43] Erickson GF, Magoffin DA, Cragun JR, Chang RJ. The effects of insulin and insulin-like growth factors-I and -II on estradiol production by granulosa cells of polycystic ovaries. J Clin Endocrinol Metab 1990;70(4):894e902. [44] Drummond AE. The role of steroids in follicular growth. Reprod Biol Endocrinol 2006;4:16.
Contents lists available at ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
Expression profiling of vitamin D receptor in placenta, decidua and ovary of pregnant mice M. Shahbazi a, M. Jeddi-Tehrani b, c, M. Zareie d, A. Salek-Moghaddam e, M.M. Akhondi f, M. Bahmanpoor g, M.R. Sadeghi f, A.H. Zarnani e, h, * a
Department of Immunology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran Immune and Gene Therapy Lab, CCK, Department of Oncology-Pathology, Karolinska University Hospital, Solna, Stockholm, Sweden d Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Canada e Immunology Research Center, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran f Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran g Department of obstetrics & Gynecology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran h Nanobiotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran b c
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 20 June 2011
Objectives: The presence of vitamin D receptor (VDR) and the identification of localized vitamin D3 synthesis in placenta and decidua implicate the importance of vitamin D3 in reproductive function. There is, however, no data on the expression profile of VDR in the mouse placenta and endometrium throughout the pregnancy period. Study design: In the present work expression of VDR in reproductive tissues of pregnant mice at different gestational phases has been addressed. Expression of VDR was determined by semi-quantitative RT-PCR, Western blotting and immunohistochemistry. Results: The results showed that VDR mRNA and protein were expressed in decidua, placenta and ovary throughout the pregnancy. VDR gene expression in placenta was significantly elevated in late pregnancy when compared to that of mid pregnancy. Additionally, VDR expression level in decidua rose significantly as pregnancy progressed from early to mid stages. VDR expression in decidua of pregnant mice was higher in comparison to endometrium of non-pregnant mice. Immunohistochemical analysis revealed that VDR protein is consistently expressed by luminal and glandular epithelial cells of decidua, giant cells, glycogen rich cells and labyrinth cells of placenta and by almost all follicular cell types of ovary. Surveying the expression of VDR at the protein level by Western blotting confirmed PCR results. Conclusion: It seems that expression of VDR in reproductive organs is finely tuned during pregnancy indicating its eminent role in reproductive biology. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Vitamin D receptor Placenta Decidua Ovary Pregnancy Mice
1. Introduction The active form of vitamin D3, 1,25 dihydroxy vitamin D3 (calcitriol), is a lipid-soluble secosteroid hormone which has wellestablished classic effects on bone metabolism and mineral homeostasis in the gastrointestinal tract and kidney. Vitamin D3 can be obtained from the diet or synthesized in the skin from 7-dehydrocholesterol during sunlight exposure. Upon intake, vitamin D3 is biologically inert and requires activation through a two-step enzymatic pathway involving 25-hydroxylase and 1a-hydroxylase * Corresponding author. Nanobiotechnology Research Center, Avicenna Research Institute, ACECR, P.O. Box: 1936773493, Tehran, Iran. Tel.: þ98 22432020; fax: þ98 22432021. E-mail addresses: [email protected], [email protected] (A.H. Zarnani). 0143-4004/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2011.06.013
for the conversion to the biologically active form [1]. Vitamin D3 exerts its effects upon a great variety of cell types by binding to the nuclear vitamin D receptor (VDR) which belongs to the nuclear receptor superfamily of ligand-dependent transcriptional factors. After binding to its ligand, VDR interacts with the nuclear receptor retinoic acid X receptor (RXR). In the presence of vitamin D3 the VDR/RXR complex binds small sequences of DNA known as vitamin D response elements (VDREs) and initiates a cascade of molecular interactions that modulate the transcription of multitude of genes in tissues throughout the body [1]. Regulation of VDR gene expression is one of the main mechanisms through which target cells respond to calcitriol. Therefore, biological action of this hormone is largely determined by the extent of VDR expression levels in a given cell type [2,3]. While a role for vitamin D3 in tissue growth and bone metabolism is well established, the presence of VDR and enzymes involved in the
658
M. Shahbazi et al. / Placenta 32 (2011) 657e664
hydroxylation of vitamin D3, and the identification of localized vitamin D3 synthesis in human placenta and decidua [4,5] implicates the importance of vitamin D3 in reproductive function. The elegant studies by Panda et al. clearly showed that targeted ablation of vitamin D3 1a-hydroxylase, the enzyme responsible for production of biologically active form of vitamin D3, is accompanied by several reproductive dysfunctions in female mice, including abnormal ovarian follicle development, uterine hyperplasia and infertility [6]. VDR knockout mice have a similar phenotype to that of 1a-hydroxylase knockout mice [7]. Female fertility and litter size is markedly reduced in vitamin D3 deficiency [8,9], but may involve calcium-dependent or independent mechanisms depending on the animal model used. In vitamin D3 deficient rats, decreased fertility and litter size seem to be solely due to diminished level of vitamin D3 and independent of calcium status [9]. In VDR knockout mice, however, female reproductive dysfunction seems to be under the direct influence of calcium levels [7,10]. VDR is widely expressed in most cells of the immune system, including antigen presenting cells such as macrophages and dendritic cells (DC), as well as activated CD4þ and CD8þ T lymphocytes. Dendritic cells are the main target of immunoregulatory effects exerted by vitamin D3. Vitamin D3 inhibits DC differentiation and maturation, decreasing their capability for stimulatory action, thus preventing T lymphocyte proliferation and activation [11], skewing cytokine profile toward T helper 2 (TH2) phenotype [12], and suppression of immunoactive NK cells [13]. Given the fact that DCs are crucial in establishment of maternal tolerance during pregnancy as our team and other groups have shown previously [14,15] and that increased activity of natural killer (NK) cells and T helper 1(TH1) cytokines, at least at midgestational period, are causative factors for pregnancy loss [16,17], it is conceivable to speculate that vitamin D3 could improve pregnancy outcome through its immunoregulatory function. Interestingly, vitamin D3 has been suggested as a new therapeutic agent for women with recurrent spontaneous abortion [18]. We have previously shown that VDR is present and differentially expressed in reproductive organs of cycling mice, including dendritic cells and macrophages of the endometrium, particularly during the estrus phase [19]. To further evaluate the functional role of the locally generated vitamin D3 in murine reproduction, the present work was designed to study the expression of the VDR at both gene and protein level in reproductive tissues of pregnant mice at different gestational phases. 2. Materials and methods 2.1. Animals
analysis. RNA purity was periodically assessed by agarose gel electrophoresis. Complementary DNA (cDNA) synthesis for the detection of mRNA for VDR and GAPDH (house keeping gene used as internal control) were performed according to the protocol we published recently [21]. In brief, RNA was denatured at 65 C and immediately placed on ice. A mixture containing 5 reaction buffer (Fermentase, Vilnius, Lithuania), 1 mM dNTP mix (Roche, Penzberg, Germany), 1 mM RandomHexamer (Cybergene, Stockholm, Sweden), and 20 IU/mL of Reverse Transcriptase M-MuLV (Fermentase) was added to the denatured RNA and mixed thoroughly. After incubation for 1 h at 42 C in a thermocycler, the resulting cDNA was aliquoted and stored at 20 C. Semi-quantitative RT-PCR was performed in a final volume of 25 mL containing one mL of cDNA, PCR buffer (10x), MgCl2 (2.5 mM for GAPDH and 2 mM for VDR), dNTP mix (0.4 U/mL), and Taq DNA Polymerase at 0.04 U/mL final concentration (Roche). One mL of each pair of primers, equivalent to final concentrations of 10 mM for GAPDH and 5 mM for VDR, were added. PCR was then performed as follows: GAPDH with the program 94 C 2 min (1 cycle), 94 C 30 s, 60 C 30 s, 72 C 30 s (30 cycles), and 72 C for 7 min (1 cycle); and VDR with the program 94 C 2 min (1 cycle), 94 C 30 s, 59 C 30 s, 72 C 30 s (35 cycles), and 72 C for 7 min (1 cycle). The cycle numbers used for VDR and GAPDH amplification were showed to be in the logarithmic phase of amplification as judged by the cycle gradients ran separately for each gene (Fig. 1). Oligonucleotide primers for GAPDH and VDR were as follows: VDR: F: 5-GAG-GTG-TCT-GAA-GCC-TGG-AG-30 ; R: 50 -ACC-TGC-TTT-CCT-GGG-TAGGT-30 , GAPDH: F: 50 -CAG-GAG-CGA-GAC-CCC-ACT-A-30 ; R: 50 -GGC-ATG-GAC-TGTGGT-CAT-GA-30 . The primers were designed to generate a fragment of 309 bp for GAPDH and a fragment of 155 bp for VDR. For VDR expression, the cDNA prepared from mouse kidney tissue served as positive control. A PCR system devoid of template cDNA and no amplification controls (no reverse transcriptase added) were included as negative controls. 2.4. Agarose gel electrophoresis and densitometry The PCR products for GADPH and VDR genes were electrophoresed on agarose gel. The gels were photographed with UV transilluminator Gel Doc (UVP, LLC, Upland, CA). For densitometry analysis, Alpha Ease software was used. Each photograph was first converted to 8 bit grayscale picture. For analysis of PCR bands, picture color was inverted so the bands appeared black. Afterward, contrast was adjusted so that black and white colors corresponded to 250 and 0, respectively. Using rectangle object selection tool, bands were then selected. After subtraction of background density of negative control, average densities of bands were quantified. Relative expression of VDR was presented as the percent of VDR/GADPH density ratio. 2.5. Immunohistochemical localization of VDR Immunolocalization of VDR in murine placenta, decidua and ovaries was performed as previously described [19]. Briefly, acetone-fixed cryostat sections were washed in Tris-buffered salineeBovine serum albumin 0.1% (TBS-BSA). After blocking with 5% normal goat serum for 15 min, endogenous biotin was blocked by Avidin/Biotin blocking solutions (Dako, Glostrop, Denmark) according to manufacturer’s instruction. Sections were then incubated with monoclonal anti-VDR primary antibody (NeoMakers, Fremont, CA, 2.5 mg/ml) for 90 min, washed and treated with 0.3% H2O2 in TBS for 10 min to block endogenous peroxidase, followed by incubation (45 min) with biotinylated goat anti-rat antibody (BD Biosciences Pharmingen, SanDiego, CA, 5 mg/ml). After rinsing, sections were processed further using streptavidin/peroxidase (Biosource, Camarillo, CA) (1:500 dilution, 30 min),
Female and male Balb/c mice aged 8e12 weeks were obtained from Pasteur Institute (Tehran, Iran). All studies carried out with the mice were approved by the ethical committee for experimental animals and in accordance with the institutional guidelines for care and use of laboratory animals at Avicenna Research Institute, Tehran, Iran. Female mice were mated with males and the onset of pregnancy was determined as we reported elsewhere [20]. 2.2. Tissue collection and preparation At each gestation phase [beginning (day 2), middle (day 10) and toward the end (day 18)], 4e5 mice were sacrificed and ovary, placenta and decidua samples were collected. For RNA extraction, specimens were immediately transferred into microtubes containing RNA-Bee solution (Nordic Biosite, Taby, Sweden). For immunohistochemistry, the samples were immersed in OCT medium (Sakura, Zoetenwoude, The Netherlands), flash frozen and stored at 70 C. Additional tissue samples were frozen in liquid nitrogen for Western blotting. Sections from the kidney of one mouse were treated accordingly and served as positive controls. 2.3. Isolation of total RNA and quantitative RT-PCR Total RNA was extracted from ovary, placenta and decidual samples by standard phenolechloroform method as described previously [19]. The resulting RNA pellet was dissolved in distilled water and samples were stored at 20 C for further
Fig. 1. Cycle gradient PCR. To ensure semi-quantitative amplification, cycle gradient PCR starring from 20 cycles was performed for VDR and reference gene (GAPDH). Numbers above the lanes indicate number of PCR cycles for each gene. Each PCR reaction was run in duplicate. NC: Negative control with no added cDNA; M: Size marker.
M. Shahbazi et al. / Placenta 32 (2011) 657e664
659
and reaction products were developed with 3,30 -diaminobenzide tetrahydrochloride (DAB) chromogen (Roche). In negative control sections primary antibody was omitted. All sections were counterstained with Harris hematoxylin for 5 s; dehydrated with increasing concentrations of ethanol and mounted using Enthelan glue (Merck, Darmstadt, Germany). 2.6. Western blot analysis of VDR expression Depending on the size of the samples, tissue were homogenized in 100e400 mL of lysis buffer (1% w/v Triton X-100, 50 mM Tris at pH ¼ 7.4, 0.1% sodium dodecyl sulfate, 1% v/v protease inhibitors, 150 mM NaCl, 1 mM sodium fluoride, 5 mM EDTA, and 1% v/v glycerol) using Pellet Pestle and left on ice for 1 h. Subsequently, the lysates were centrifuged (10,000 rpm, for 10 min at 4 C) and the supernatants were collected and the protein concentration was measured by Bradford assay. Western blotting of VDR and b actin were performed according to the protocol we published elsewhere with some modifications [22]. A total of 100 mg protein per lane were added to sample buffer (containing 5% 2-Mercapto Ethanol), boiled (2 min), and subjected to SDS-PAGE (3% stacking and 10% resolving) electrophoresis at 100 V for 90 min. The proteins were transferred onto polyvinylidene fluoride membrane (Roche). After blocking overnight with 5% nonfat dry milk in PBS containing 0.1% Tween (PBST), blots were incubated 2 h with agitation in monoclonal anti-VDR primary antibody (NeoMakers) at 1 mg/ml in PBST. After washing 5 10 min in PBST, blots were incubated 1 h in HRP-conjugated anti-rat IgG (BD Biosciences Pharmingen, 1:1000) and washed again. VDR was detected using an ECL Western blotting substrate kit (GE HealthCare, Buckinghamshire, UK) and digital images were obtained with a Gel Logic 2200 imaging system (Kodak, Tokyo, Japan). In negative control blots, primary antibody was omitted. Membranes were later stripped using Western Re-Probe (Calbiochem, SanDiego, CA) and re-incubated 2 h with agitation in anti-b actin antibody (Sigma, Kungsbacka, Sweden; 1:200), which was used as loading control. After washing 5 15 min in PBST, blots were incubated 45 min in HRP-conjugated Sheep anti-rabbit IgG (Avicenna Research Institute, Tehran, Iran, 1:500) and processed as above. Densities of VDR and b actin bands were quantified and the relative expression was calculated using Alpha Ease software as above.
Fig. 2. Expression of vitamin D3 receptor (VDR)-specific transcript in placenta, decidua and ovary of pregnant Balb/c mice at different periods of pregnancy. RT-PCR was run using specific primers for VDR and GAPDH amplification was used as the loading control. No amplification controls (NAC; no reverse transcriptase added were used to check for the amplification of genomic DNA). Results of 2 out of 4e5 experiments have been shown for each sample. EPD: Early pregnancy decidua; MPD: Mid pregnancy decidua; LPD: Late pregnancy decidua; EPO: Early pregnancy ovary; MPO: Mid pregnancy ovary; LPO: Late pregnancy ovary; MPP: Mid pregnancy placenta; LPP: Late pregnancy placenta; NC: Negative control with no added cDNA; PC: Kidney as positive control; M: Size marker.
2.7. Statistical analysis All data is presented as median (range). The density ratio (VDR/GAPDH and VDR/
b actin) data from each stage were analyzed using non-parametric ManneWhitney
test and statistically significant differences were accepted at P < 0.05.
3. Results 3.1. Detection of VDR mRNA expression in adult mice during pregnancy
luminal and glandular epithelial cells of decidua basalis staining the strongest (Fig. 4). In placenta, VDR expression was located on the Giant cells, glycogen rich cells, labyrinth cells, the chorionic membrane, and fetal neural tube cells (Fig. 5). Furthermore, immunohistochemical analysis of ovaries revealed VDR expression in almost all follicular cell types including granulosa cells, cumulus
Semi-quantitative RT-PCR was used to determine the presence of VDR gene transcripts in pregnant mice reproductive tissue. The 309 bp fragment of GAPDH enzyme mRNA was used as an endogenous control sequence. Fig. 2 illustrates that the VDR mRNA (155 bp) could be detected in every single sample of placenta, decidua and ovary regardless whether obtained from early, mid or late stages of pregnancy. Densitometry analysis revealed that, in relation to reference gene GAPDH, VDR gene expression in placenta was significantly elevated in late pregnancy when compared to that of mid pregnancy (P < 0.01). Additionally, VDR expression level in decidua rose significantly (P < 0.05) as pregnancy progressed from early to mid stages. However, there were no detectable differences in VDR expression in decidua samples from mid and late stages of pregnancy. More interestingly, significantly higher expression of VDR in decidual samples from pregnant mice was observed compared to endometrial samples of non-pregnant mice. (P < 0.001) (Fig. 3). 3.2. VDR protein expression: immunohistochemical localization VDR localization in ovaries, placenta and decidua of pregnant mice at early, mid and late stages of pregnancy was investigated by immunohistochemistry. VDR protein was detected throughout pregnancy in all tissue samples examined. In decidua, VDR staining was observed in both the cells of decidua capsularis and decidua basalis at early, mid and late stages of pregnancy, with both the
Fig. 3. Expression of VDR-specific message in endometrium of non-pregnant and decidua and placenta of pregnant Balb/c mice. Semi-quantitative RT-PCR was used to measure VDR expression in endometrium of non-pregnant mice at different stages of estrous cycle and decidua and placenta of pregnant mice at different periods of pregnancy. The density ratio was significantly higher in mice during estrous phase (E) compared to the other phases (P, M and D) (P < 0.01). During pregnancy, VDR expression was significantly increased. Each bar is representing median (range) of the data obtained from 4 (M, D, MPD, and MPP) or 5 mice (P, E, EPD, LPD, and LPP). P: Proestrous, E: Estrus, M: Metestrus, D: Diestrus, EPD: Early pregnancy decidua, MPD: Mid pregnancy decidua, LPD: Late pregnancy decidua, MPP: Mid pregnancy placenta, LPP: Late pregnancy placenta, AU: Arbitrary unit. *; P < 0.05, **; P < 0.01, ***; P < 0.001.
660
M. Shahbazi et al. / Placenta 32 (2011) 657e664
Fig. 4. Immunohistochemical analysis of vitamin D receptor (VDR) expression in decidua basalis of pregnant Balb/c mice at early (A), middle (B) and late stages (C) of pregnancy. Luminal and glandular epithelial cells were the prominent cell types that expressed VDR in all phases. There were also scattered positive cells in the decidua stroma. Each figure is a representative photograph from 4 to 5 animals. (D) Negative control. DB: Decidua basalis, EPC: Epithelial cells, Scale bar: A; 20mm, BeD; 40mm.
oophorus, corena radiata and cells in ovarian stroma at various stages of pregnancy (Fig. 6). There was minimal VDR staining in the cells of the theca externa and theca interna. 3.3. VDR protein expression: Western blotting As a complementary approach to immunohistochemistry and RT-PCR, Western blot analysis was carried out to assess the expression of VDR protein in ovaries, placenta and decidua of pregnant mice. Mouse kidney lysate was used as positive control. Western blot analysis detected the 52 kDa VDR protein in samples of placenta, decidua and ovary (Fig. 7) at early, mid and late stages of pregnancy. Quantification of VDR expression by densitometry revealed that the VDR protein level was elevated in late pregnancy placenta when compared to the placenta at mid pregnancy (P < 0.01). Compared to early pregnancy decidua, mid pregnancy decidua expressed higher levels of VDR protein (P < 0.05). 4. Discussion Recent findings show that calcitriol has several activities in regulation of immune system through antigen presenting cells and most of these actions are in accordance with a successful pregnancy [23,24]. Most researchers believe that, at least during midgestational period, successful pregnancy is associated with a shift of cytokine profile toward TH2 [25]. Considering the TH2 promoting activity of calcitriol, it seems that this hormone could have a supportive role in at least a period of conception. According to our recent report, dendritic cells and macrophages are among the main cells that express VDR in endometrial stroma [19].
Therefore it is conceivable that these cells have pivotal role in the regulation of local immunity toward the successful conception under the influence of vitamin D3. Interestingly, dendritic cells treated with vitamin D3 induce regulatory T cells [26,27]. These cells play a key role in creating conception tolerance [26]. The findings from this study showed that, VDR is expressed at all different stages of pregnancy in the placenta and decidua indicating the eminent role of this vitamin D3 in the process of reproduction. It was interesting that, the expression of this receptor in decidua was more prominent in the middle stages of pregnancy in comparison with early stage of pregnancy. In middle stages of murine pregnancy, the formation of placenta and its protrusion get to its highest level and this is expected to be accompanied by wellcontrolled local immune responses. Regarding the role of vitamin D3 in the control of immune responses, the increase in the expression of VDR in decidua would be imaginable in the middle stages of pregnancy. The level of VDR mRNA in human decidua is higher at the first compared to the second trimester [4]. The rationale behind the difference in time course expression of VDR in our finding and aforesaid report is the difference that exists between human and mouse implantation and placentation. In both human and mice, implantation takes place in a relatively same window period after fertilization (4e5 days for mice and 5e10 days for human), but considering the whole gestational period in these spices, it can be deduced that implantation begins very soon in human while it occurs in female mouse when she has passed one forth of her pregnancy period. Indeed, endometrial invasion by placental trophoblast occurs mostly during the mid-gestational period of mouse pregnancy, while in human it is started very soon after placental development at the first trimester. Based on this fact and also comparative developmental anatomy of murine
M. Shahbazi et al. / Placenta 32 (2011) 657e664
661
Fig. 5. Immunohistochemical analysis of vitamin D receptor (VDR) expression in placenta of pregnant Balb/c mice at middle, and late stages of pregnancy. VDR expression was located on the trophoblast giant cells (TGC), glycogen rich trophoblast cells (GlyT) and labyrinth trophoblast cells (LBT) at middle (A) and late stages of pregnancy (B). Fetal neural tube cells (C) at the mid pregnancy also expressed high levels of VDR. Different zones of placenta [Decidua zone (DZ), Junctional zone (JZ) and Labyrinth zone (LZ) have been specified in parts A and B. Each figure is a representative photograph from 4 to 5 animals. (D) Negative control. Scale bar: 40mm.
and human placenta, it is estimated that, temporally, the first trimester in the human may be equivalent to around mid-gestation in the mouse [28]. In addition to decidua, placenta expressed VDR in all stages of pregnancy, but the expression was significantly augmented in later stages of pregnancy. The rationale behind this is unclear, but it seems it has a role in the physiological processes such as absorption of calcium and its transition to the developing fetus before delivery. Several recent studies have examined VDR expression and function in human placental trophoblasts. The human placenta synthesizes 1,25(OH)2D3 and expresses VDR [29]. According to recent report, VDR is localized to syncytiotrophoblast, stromal and endothelial cells of human term placenta [5]. Vitamin D3 is involved in the regulation of placental lactogen expression, human chorionic gonadotropin (hCG) secretion and calcium transport in the placenta [30e32]. Surveying the expression of VDR at the protein level by Western blotting confirmed PCR results. In this experiment, the expression of VDR in decidua at the middle stage of pregnancy and in placenta at late gestation period were significantly more than those at early and mid pregnancy, respectively. The results of Immunohistochemical analysis were in line with those of PCR and Western blotting on this account that VDR was expressed at the feto-maternal interface in all duration of pregnancy. Immunohistochemistry revealed that, the glandular and luminal epithelial cells during all period of pregnancy are dominant cells expressing VDR in endometrium. It is notable that according to our recent report, these cells are the principal cells that express VDR in non-pregnant mice [19]. Our former findings indicate that, endometrial epithelial cells are the dominant cells, which express
mediators of immune regulators such as indoleamine 2,3,dioxigenase [22]. There is convincing evidence that mucosal epithelial cells, beyond their role in creating a physical barrier, are integral components of innate and adaptive immunity [33]. These cells have a substantial role in transition from innate immunity to adaptive immunity [34,35]. Based on the immunoregulatory activity of vitamin D3, our findings on the high expression of VDR in decidual epithelial cells is a further proof of the fundamental importance of these cells in shaping the local immune responses at the fetomaternal interface. Based on the wide distribution VDR in different cell types of decidua, placenta and ovary of the pregnant mice, it seems that vitamin D3 machinery is a basic requirement for most cells. In fact, VDR is expressed in over 30 different tissues, including the stomach, bone, brain, immune cells and other tissues [36]. It is also ubiquitous in proliferating cells [4]. The number of responding cells, availability of ligand and more importantly the density of VDR in the cells are key elements which determine the extent of vitamin D3 action on each specific organ. Our earlier studies showed that, VDR is expressed in the endometrium of non-pregnant mice in all stages of estrous cycle [19]. It was interesting that, in comparison with non-pregnant mice, expression of VDR increased significantly in the endometrium of pregnant mice. This finding emphasizes the role of vitamin D3 in conception process. In addition to immunologic roles, vitamin D3 has multiple physiologic activities during pregnancy. Low blood levels of vitamin D3 during pregnancy may be a risk factor forimpaired fetal development. Deficiency in vitamin D3 is accompanied with smaller litter size and number [8]. Mice having deficiency in VDR gene also have uterus hypoplasia, defect in
662
M. Shahbazi et al. / Placenta 32 (2011) 657e664
Fig. 6. Immunohistochemical analysis of vitamin D receptor (VDR) expression in ovary of pregnant Balb/c mice at early, middle and late stages of pregnancy. VDR was preferentially expressed by granulosa cells (GraC) of the ovary at early (A), middle (B) and late stages (C) of pregnancy. There are scattered immunoreactive stromal cells around the follicles. Each figure is a representative photograph from 4 to 5 animals. (D) Negative control. Scale bar: 40mm.
folliculogenesis and pregnancy [7,37]. According to Lappillone [38], vitamin D3 insufficiency during pregnancy is not only potentially associated with increased risk of preeclampsia, insulin resistance and gestational diabetes mellitus, but also may impair fetal brain and skeletal development.
Calcitriol has also axis role in blastocyst implantation through the increase in expression of such genes as HOXA10 [39]. According to recent findings, intraluminal injection of vitamin D3 in rat uterus causes pseudopregnancy and decidualization [40]. On the basis of the findings in this study, expression of VDR in decidua at the early
Fig. 7. Western blot analysis of vitamin D receptor (VDR) expression in placenta, decidua and ovary of pregnant mice at different periods of pregnancy. Expression of VDR in reproductive organs of pregnant Balb/c mice at different periods of pregnancy was analyzed by Western blotting. The results of the two samples out of 4e5 for each tissue are shown. b actin was used as internal standard (A). Relative expression of VDR in placenta and decidua is shown in part B. Each bar is representing the median (range) of the data obtained from 4 (MPD, and MPP) or 5 mice (EPD, LPD, and LPP). EPD: Early pregnancy decidua; MPD: Mid pregnancy decidua; LPD: Late pregnancy decidua; EPO: Early pregnancy ovary; MPO: Mid pregnancy ovary; LPO: Late pregnancy ovary; MPP: Mid pregnancy placenta; LPP: Late pregnancy placenta; NC: Negative control with no primary antibody; PC: Kidney as positive control, AU: Arbitrary unit. *; P < 0.05, **; P < 0.01.
M. Shahbazi et al. / Placenta 32 (2011) 657e664
stages of pregnancy increased substantially comparing to the nonpregnant mice and this finding can reveal the role of vitamin D3 in the processes of implantation. With relation to the expression of VDR at gene and protein level by both fetus and mother side of placenta, it seems that, vitamin D3 possesses a role in physiologic and immunologic regulation of mother and fetus activities. On the other hand, high expression of VDR in the fetus of mice during middle stages of pregnancy as judged by immunohistochemistry reveals that VDR expression is not solely confined to the mother and fetal cells also express this receptor. Data generated in this research showed that, VDR is expressed at gene and protein level by the ovary of pregnant mice in all stages of pregnancy. The role of VDR is not quite clear in the ovary but it may have a role in adjusting some of the physiological activities of the ovary. Vitamin D3 exists in human follicular fluid [41] and may have a role in regulation of granulosa cells activity through the interaction with VDR. Vitamin D3 in the fibroblasts of the human skin accelerates the conversion of androstenedione to estrogen through the augmentation of aromatase activity [42] and it is probable that, this vitamin D3 displays a similar role in the ovary. The granulosa cells of the ovary also have aromatase activity [19,43] and by means of producing estrogen have a crucial role in folliculogenesis and ovulation [44]. According to our recent study, VDR is expressed in epithelial cells of fallopian tube in all stages of estrous cycle [19]. Accordingly it seems that, this receptor is expressed in all pregnancy-related tissues in both pregnant and non-pregnant mice. These findings indicate the undeniable role of vitamin D3 in the process of reproduction. 5. Conclusion Collectively, the findings in this study showed that, VDR is expressed in the gene and protein level in endometrium, placenta, and ovary of pregnant mice during all stages of pregnancy and this phenomenon is probably in line with indispensible role of vitamin D3 in the adjustment of the physiologic and immunologic activities during pregnancy. Acknowledgment The authors wish to thank Mr. A. Moravej, Mrs. G.E. KazemiSefat and Miss. J. Ghasemi for their technical assistance. References [1] Jones G, Strugnell SA, DeLuca HF. Current understanding of the molecular actions of vitamin D. Physiol Rev 1998;78(4):1193e231. [2] Chen TL, Hauschka PV, Cabrales S, Feldman D. The effects of 1,25dihydroxyvitamin D3 and dexamethasone on rat osteoblast-like primary cell cultures: receptor occupancy and functional expression patterns for three different bioresponses. Endocrinology 1986;118(1):250e9. [3] Halloran BP, DeLuca HF. Appearance of the intestinal cytosolic receptor for 1,25-dihydroxyvitamin D3 during neonatal development in the rat. J Biol Chem 1981;256(14):7338e42. [4] Zehnder D, Evans KN, Kilby MD, Bulmer JN, Innes BA, Stewart PM, et al. The ontogeny of 25-hydroxyvitamin D(3) 1alpha-hydroxylase expression in human placenta and decidua. Am J Pathol 2002;161(1):105e14. [5] Pospechova K, Rozehnal V, Stejskalova L, Vrzal R, Pospisilova N, Jamborova G, et al. Expression and activity of vitamin D receptor in the human placenta and in choriocarcinoma BeWo and JEG-3 cell lines. Mol Cell Endocrinol 2009; 299(2):178e87. [6] Panda DK, Miao D, Tremblay ML, Sirois J, Farookhi R, Hendy GN, et al. Targeted ablation of the 25-hydroxyvitamin D 1alpha -hydroxylase enzyme: evidence for skeletal, reproductive, and immune dysfunction. Proc Natl Acad Sci U S A 2001;98(13):7498e503. [7] Yoshizawa T, Handa Y, Uematsu Y, Takeda S, Sekine K, Yoshihara Y, et al. Mice lacking the vitamin D receptor exhibit impaired bone formation, uterine hypoplasia and growth retardation after weaning. Nat Genet 1997;16(4): 391e6.
663
[8] Halloran BP, DeLuca HF. Effect of vitamin D deficiency on fertility and reproductive capacity in the female rat. J Nutr 1980;110(8):1573e80. [9] Kwiecinksi GG, Petrie GI, DeLuca HF. 1,25-Dihydroxyvitamin D3 restores fertility of vitamin D-deficient female rats. Am J Physiol 1989;256(4 Pt 1): E483e7. [10] Johnson LE, DeLuca HF. Vitamin D receptor null mutant mice fed high levels of calcium are fertile. J Nutr 2001;131(6):1787e91. [11] Adorini L, Penna G, Giarratana N, Roncari A, Amuchastegui S, Daniel KC, et al. Dendritic cells as key targets for immunomodulation by Vitamin D receptor ligands. J Steroid Biochem Mol Biol 2004;89e90(1e5):437e41. [12] Penna G, Adorini L. 1 Alpha,25-dihydroxyvitamin D3 inhibits differentiation, maturation, activation, and survival of dendritic cells leading to impaired alloreactive T cell activation. J Immunol 2000;164(5):2405e11. [13] Merino F, Alvarez-Mon M, de la Hera A, Ales JE, Bonilla F, Durantez A. Regulation of natural killer cytotoxicity by 1,25-dihydroxyvitamin D3. Cell Immunol 1989;118(2):328e36. [14] Zarnani AH, Moazzeni SM, Shokri F, Salehnia M, Dokouhaki P, Ghods R, et al. Microenvironment of the feto-maternal interface protects the semiallogenic fetus through its immunomodulatory activity on dendritic cells. Fertil Steril 2008;90(3):781e8. [15] Juretic K, Strbo N, Crncic TB, Laskarin G, Rukavina D. An insight into the dendritic cells at the maternal-fetal interface. Am J Reprod Immunol 2004; 52(6):350e5. [16] Raghupathy R. Pregnancy: success and failure within the Th1/Th2/Th3 paradigm. Semin Immunol 2001;13(4):219e27. [17] Choudhury SR, Knapp LA. Human reproductive failure I: immunological factors. Hum Reprod Update 2001;7(2):113e34. [18] Bubanovic I. 1alpha,25-dihydroxy-vitamin-D3 as new immunotherapy in treatment of recurrent spontaneous abortion. Med Hypotheses 2004;63(2):250e3. [19] Zarnani AH, Shahbazi M, Salek-Moghaddam A, Zareie M, Tavakoli M, Ghasemi J, et al. Vitamin D3 receptor is expressed in the endometrium of cycling mice throughout the estrous cycle. Fertil Steril 2010;93(8):2738e43. [20] Zarnani AH, Moazzeni SM, Shokri F, Salehnia M, Jeddi-Tehrani M. Kinetics of murine decidual dendritic cells. Reproduction 2007;133(1):275e83. [21] Moravej A, Jeddi-Tehrani M, Salek-Moghaddam AR, Dokouhaki P, Ghods R, Rabbani H, et al. Evaluation of thyroglobulin expression in murine reproductive organs during pregnancy. Am J Reprod Immunol; 2010. [22] Jeddi-Tehrani M, Abbasi N, Dokouhaki P, Ghasemi J, Rezania S, Ostadkarampour M, et al. Indoleamine 2,3-dioxygenase is expressed in the endometrium of cycling mice throughout the oestrous cycle. J Reprod Immunol 2009;80(1e2):41e8. [23] Hewison M, Freeman L, Hughes SV, Evans KN, Bland R, Eliopoulos AG, et al. Differential regulation of vitamin D receptor and its ligand in human monocyte-derived dendritic cells. J Immunol 2003;170(11):5382e90. [24] Kragballe K. Vitamin D analogues in the treatment of psoriasis. J Cell Biochem 1992;49(1):46e52. [25] Aagaard-Tillery KM, Silver R, Dalton J. Immunology of normal pregnancy. Semin Fetal Neonatal Med 2006;11(5):279e95. [26] Griffin MD, Xing N, Kumar R. Vitamin D and its analogs as regulators of immune activation and antigen presentation. Ann Rev Nutr 2003;23:117e45. [27] van Etten E, Mathieu C. Immunoregulation by 1,25-dihydroxyvitamin D3: basic concepts. J Steroid Biochem Mol Biol 2005;97(1e2):93e101. [28] Georgiades P, Ferguson-Smith AC, Burton GJ. Comparative developmental anatomy of the murine and human definitive placentae. Placenta 2002;23(1): 3e19. [29] Diaz L, Sanchez I, Avila E, Halhali A, Vilchis F, Larrea F. Identification of a 25hydroxyvitamin D3 1alpha-hydroxylase gene transcription product in cultures of human syncytiotrophoblast cells. J Clin Endocrinol Metab 2000; 85(7):2543e9. [30] Tuan RS, Moore CJ, Brittingham JW, Kirwin JJ, Akins RE, Wong M. In vitro study of placental trophoblast calcium uptake using JEG-3 human choriocarcinoma cells. J Cell Sci 1991;98(Pt 3):333e42. [31] Stephanou A, Ross R, Handwerger S. Regulation of human placental lactogen expression by 1,25-dihydroxyvitamin D3. Endocrinology 1994;135(6): 2651e6. [32] Barrera D, Avila E, Hernandez G, Mendez I, Gonzalez L, Halhali A, et al. Calcitriol affects hCG gene transcription in cultured human syncytiotrophoblasts. Reprod Biol Endocrinol 2008;6:3. [33] Saenz SA, Taylor BC, Artis D. Welcome to the neighborhood: epithelial cellderived cytokines license innate and adaptive immune responses at mucosal sites. Immunological Reviews 2008;226:172e90. [34] Schleimer RP, Kato A, Kern R, Kuperman D, Avila PC. Epithelium: at the interface of innate and adaptive immune responses. J Allergy Clin Immunol 2007;120(6):1279e84. [35] Hammad H, Lambrecht BN. Dendritic cells and epithelial cells: linking innate and adaptive immunity in asthma. Nat Reviews 2008;8(3):193e204. [36] Perez-Lopez FR. Vitamin D: the secosteroid hormone and human reproduction. Gynecol Endocrinol 2007;23(1):13e24. [37] Kinuta K, Tanaka H, Moriwake T, Aya K, Kato S, Seino Y. Vitamin D is an important factor in estrogen biosynthesis of both female and male gonads. Endocrinology 2000;141(4):1317e24. [38] Lapillonne A. Vitamin D deficiency during pregnancy may impair maternal and fetal outcomes. Med Hypotheses 2010;74(1):71e5. [39] Bagot CN, Troy PJ, Taylor HS. Alteration of maternal Hoxa10 expression by in vivo gene transfection affects implantation. Gene Ther 2000;7(16):1378e84.
664
M. Shahbazi et al. / Placenta 32 (2011) 657e664
[40] Halhali A, Acker GM, Garabedian M. 1,25-Dihydroxyvitamin D3 induces in vivo the decidualization of rat endometrial cells. J Reprod Fertil 1991;91(1): 59e64. [41] Potashnik G, Lunenfeld E, Levitas E, Itskovitz J, Albutiano S, Yankowitz N, et al. The relationship between endogenous oestradiol and vitamin D3 metabolites in serum and follicular fluid during ovarian stimulation for in-vitro fertilization and embryo transfer. Hum Reprod 1992;7(10): 1357e60.
[42] Hodgins MB, Murad S. 1, 25-Dihydroxycholecalciferol stimulates conversion of androstenedione into oestrone by human skin fibroblasts in culture. J Endocrinol 1986;110(1):R1e4. [43] Erickson GF, Magoffin DA, Cragun JR, Chang RJ. The effects of insulin and insulin-like growth factors-I and -II on estradiol production by granulosa cells of polycystic ovaries. J Clin Endocrinol Metab 1990;70(4):894e902. [44] Drummond AE. The role of steroids in follicular growth. Reprod Biol Endocrinol 2006;4:16.