ARTICLE |
Correspondence to: Kaname Saida, Nat. Inst. of Bioscience and Human-Technology, Agency of Industrial Science and Technology, 1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan. E-mail: ksaida@nibh.go.jp
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Summary |
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To understand the physiological roles of vasoactive intestinal contractor (VIC) and endothelin-2 (ET-2) in the uterus, we examined the expression levels of VIC mRNA by real-time quantitative reverse transcription-linked polymerase chain reaction (RT-PCR) and characterized the cellular distribution of VIC peptide and mRNA by immunostaining and in situ hybridization in mouse uterus. In pregnant mouse uterus, VIC mRNA expression changed considerably between Days 10.5 and 12.5 of pregnancy. The expression levels were significantly (p<0.05) higher (approximately fivefold) in the later stage of pregnancy (Days 12.517.5) than in the earlier stage (Days 7.510.5). In nonpregnant uterus, VIC mRNA expression was significantly (p <0.05) higher (approximately threefold) in proestrus and estrus than in diestrus. Immunohistochemical studies demonstrated the presence of VIC peptide in endometrial epithelial cells, myometrial cells, and vascular smooth muscle cells during the estrous cycle and pregnancy and after parturition. Notably, myometrial cells showed dominant immunostaining in proestrus and estrus, in the later pregnancy stage, and in the early postpartum period, analogous to the expression pattern of VIC mRNA. In situ hybridization confirmed localization of VIC mRNA in myometrial cells. These findings suggest that VIC may play an important role in the function of myometrial cells. (J Histochem Cytochem 48:699707, 2000)
Key Words: vasoactive intestinal contractor, (VIC), endothelin, mouse, uterus, estrous cycle, pregnancy, parturition, real-time quantitative PCR, in situ hybridization, immunohistochemistry
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Introduction |
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THE ENDOTHELIN (ET) FAMILY consists of the three isoforms: ET-1, vasoactive intestinal contractor (VIC)/ET-2, and ET-3. These isopeptides are composed of 21 amino acid residues and have a variety of biological roles, including involvement in vasoconstriction (
VIC is a murine-derived peptide characterized as a potent vasoactive and smooth muscle contracting compound (
Several studies concerning the presence of the ET system in the uterus have been reported. The presence of mRNA (
In this study, in an attempt to investigate the physiological roles of VIC/ET-2 in uterus in vivo, we characterized quantitative changes in VIC gene expression during the estrous cycle, pregnancy, and parturition in mouse uterus by real-time quantitative RT-PCR. The precise cellular distribution of VIC peptide and mRNA was examined using immunohistochemical and in situ hybridization techniques.
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Materials and Methods |
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Mice
Male and female ICR mice (10 weeks old) were obtained from Nippon Clea (Tokyo, Japan). Mice were mated, and noon on the day of the vaginal plug was designated Day 0.5 of pregnancy. Pregnant mice were divided into seven groups according to gestational age (Days 7.5, 8.5, 9.5, 10.5, 12.5, 14.5, and 17.5). Non-pregnant mice were divided into three groups dependent on the stage of the estrous cycle (diestrus, proestrus, and estrus) as determined by vaginal smear. Another group of mice were examined just after parturition. Each group contained three mice (n = 3). Our experimental procedures on animal subjects were in accordance with the Guidelines on Handling of Laboratory Animals for our institution.
Cloning of cDNAs
cDNA fragments of VIC and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified by PCR using primers designed in accordance with the published sequence in mouse (
Poly (A)+RNA Preparation.
The excised uteri were washed well with chilled PBS, pH 7.4, on ice and weighed. One hundred mg of uterus was immediately homogenized. Total RNAs were then prepared from the homogenate using a commercial phenol and guanidinethiocyanate solution (Isogen solution; Nippon Gene, Tokyo, Japan) according to the manufacturer's protocol. Poly(A)+ RNAs were obtained from the total RNAs by oligo(dT)-cellulose chromatography (Pharmacia; Uppsala, Sweden).
Gene Expression Analysis
We established a quantitative RT-PCR method for analysis of VIC gene expression using a real-time PCR system (PE Applied Biosystems) which is more accurate and less time-consuming than a conventional PCR system (
[Concentration of VIC mRNA in a sample/Concentration of GAPDH mRNA in a sample] x 100
Antibody for Immunohistochemistry
A rabbit antibody raised against the synthetic ET-2 peptide was used for immunohistochemistry. The IgG fraction was purified by affinity chromatography. After further purification on an ET-2-binding column followed by absorption with ET-1 and ET-3 peptide, IgG specific to ET-2 was obtained. Crossreactivity studies by ELISA showed that this antibody reacted at 100% with ET-2 and 90% with VIC but below 0.1% with ET-1 and ET-3. The absence of crossreactivity with tissue component proteins was also confirmed by Western blotting. This antibody (code no. 16152) was a gift from Immuno-Biological Laboratories (IBL; Fujioka, Japan). We used the antibody to detect VIC in mice.
Immunohistochemistry
The excised uteri were fixed with 4% (w/v) paraformaldehyde and embedded in paraffin by standard methods. Tissues sectioned at a thickness of 5 µm were deparaffined and rehydrated with graded alcohol. After washing with PBS, the sections were treated with methanol containing 0.3% hydrogen peroxide for 30 min to inactivate endogenous peroxidase activity. Immunostaining was carried out using a commercial kit (Vectastain ABC Kit; Vector Laboratories, Burlingame, CA). Briefly, the sections were washed and incubated in 10% normal goat serum in PBS for 30 min to block nonspecific immunoreactive sites. The sections were then reacted with 5 µg/ml of anti ET-2 rabbit antibody for 60 min at room temperature (RT). After washing with PBS, the sections were incubated with biotinylated goat anti-rabbit IgG for 30 min at RT, washed with PBS, and treated with streptavidinbiotinperoxidase complex (ABC reagent) for 30 min at RT. The complex was visualized by diaminobenzidine (DAB). Counterstaining for nuclei was done with Mayer's hematoxylin. Control studies included omission of the primary antibody, its replacement with preimmune rabbit IgG, and preabsorption tests using the antibody preincubated with an excess of synthetic VIC.
Riboprobes for In Situ Hybridization
Digoxygenin-labeled sense or antisense riboprobes for in situ hybridization were synthesized by T3 or T7 polymerases from the cloned cDNA plasmid described above in the presence of digoxygenindUTP using a commercial kit (DIG RNA Labeling Kit; Boehringer Mannheim, Mannheim, Germany).
In Situ Hybridization
The excised uteri were immersed in PBS containing 4% (w/v) paraformaldehyde overnight at 4C to fix and then serially placed into 10%, 20%, and 30% sucrosePBS at 4C. The tissues were frozen and embedded in a medium (OCT compound; Sakura Finetechnical, Tokyo, Japan) on dry ice and sectioned at a thickness of 10 µm using a cryostat (HM 500 OMV; Microm, Walldorf, Germany). The sections were immediately dried and stored at -80C until use. Before hybridization, the sectioned tissues were treated with 0.2 N HCl for 20 min to inactivate endogenous alkaline phosphatase, 5 µg/ml of proteinase K (Boehringer Mannheim) in PBS for 10 min at 37C, 2 mg/ml glycine in PBS for 15 min at RT for quenching, and 0.1 M triethanolamine hydrochloride, pH 8.0, containing 0.25% acetic anhydride for 15 min at RT for acetylation. After incubation in prehybridization buffer containing 50% formamide and 2 x SSC for 30 min at 50C, the sections were reacted with 1 ng/µl digoxygenin-labeled antisense riboprobe in hybridization buffer containing 50% formamide, 2 x SSC, 1 µg/µl tRNA, 1 µg/µl sonicated salmon sperm DNA, 1 µg/µl bovine serum albumin, and 10% dextran sulfate at 50C overnight. Sense riboprobe at the same concentration was used as a negative control. To lessen background signals, RNase A (20 µg/ml) treatment was carried out for 30 min at 37C. Bound digoxygenin-labeled riboprobe was detected with anti-digoxygenin antibody conjugated with alkaline-phosphatase (Boehringer Mannheim), and colored with nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphate (BCIP). Counterstaining for nuclei was carried out with methyl green.
Statistical Analysis
Data were analyzed for statistical significance by ANOVA using Fisher's Protected Least Significant Difference (Fisher's PLSD) test, requiring p<0.05 for significance.
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Results |
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VIC Gene Expression
Gene Expression During Pregnancy.
To examine the expression level of VIC mRNA in the uterus during its various physiological states, we established a real-time RT-PCR system for quantification of VIC mRNA. VIC expression levels in the pregnant uterus were quantified using this technique and are shown in Fig 1 as "gene expression rates"; the values are normalized to GAPDH expression (see Materials and Methods). The gene expression rates at Days 7.5, 8.5, 9.5, and 10.5 of pregnancy (the earlier stage of pregnancy) were 0.04, 0.05, 0.05, and 0.06, respectively. During the later stage of pregnancy, at Days 12.5, 14.5, and 17.5, and just after parturition (P-0), VIC gene expression rates were 0.36, 0.20, 0.15, and 0.19, respectively. The gene expression rates in the later stage of pregnancy and after parturition were significantly (p<0.05) higher (approximately fivefold) than those in the earlier stage.
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Gene Expression During the Estrous Cycle. The gene expression rates of VIC during the estrous cycle are shown in Fig 2. The gene expression rates in diestrus, proestrus, and estrus were 0.08, 0.22, and 0.35, respectively. The gene expression rates were significantly (p<0.05) higher (approximately threefold) in proestrus and estrus than in diestrus.
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Immunohistochemistry
To examine the cellular distribution of VIC peptide in the uterus, immunohistochemical studies were performed using a purified IgG antibody specific to ET-2, which detects VIC but not ET-1 and ET-3 in mouse (see Materials and Methods). Several cell types were positive for immunostaining for VIC in the pregnant and non-pregnant uterus. Fig 3 shows light micrographs of the pregnant uterus at Days 10.5 and 14.5. Strong immunostaining was observed in vascular smooth muscle cells (Fig 3A), weak immunostaining in endometrial epithelium cells (Fig 3C), and no apparent staining in endometrial stromal cells (Fig 3C) and vascular endothelial cells (Fig 3B) at Day 10.5. The same immunostaining patterns were also observed in the other pregnancy stages we examined (not shown). In myometrial cells, the positive patterns of immunostaining changed between the earlier (Days 7.510.5) and the later pregnancy stages (Days 12.517.5), with an increase in the degree of staining or immunopositivity occurring between Days 10.5 and 12.5, analogous to the increase observed in the gene expression rate. At Day 10.5, myometrial cells showed some positive staining (Fig 3D). However these cells showed more intense immunostaining at Day 14.5, with almost all myometrial cells staining positively for VIC (Fig 3E). The uterus after parturition showed the same pattern of immunostaining as in the later pregnancy stage.
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In non-pregnant mice in the diestrous, proestrous, and estrous uterus, specific immunostaining for VIC was dominant in vascular smooth muscle cells, faint in endometrial epithelium cells, and not apparent in stromal cells. Only in myometrial cells was a change in immunostaining observed during the estrous cycle. Myometrial cells in proestrus and estrus showed stronger positive immunostaining than those in diestrus (Fig 4).
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To examine the specificity of our antibody, control sections were immunostained with preimmune rabbit IgG and antibody preabsorbed with synthetic VIC. In these experiments we observed the elimination of specific signals observed in vascular smooth muscle cells, endometrial epithelial cells, and myometrial cells (Fig 3 and Fig 4).
In Situ Hybridization
We conducted in situ hybridization for VIC mRNA on the uterus in the proestrous and estrous stages, in the later pregnancy stage, and after parturition. In all of these stages, high expression of VIC mRNA was confirmed by real-time quantitative RT-PCR. Positive signal was located predominantly in myometrial cells (Fig 5A), was faint in endometrial epithelium cells, and was not observed in stromal cells (not shown). No signal was detected in the sections by a sense probe (Fig 5B). These findings, together with the results of the quantitative analysis for VIC mRNA and the immunohistochemical studies, demonstrate that myometrial cells in these stages strongly express VIC mRNA and produce peptide. It is also evident that cellular distribution of VIC mRNA is in accordance with that of VIC peptide.
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Discussion |
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The uterus is a physiologically active organ that undergoes various cellular changes in response to the estrous cycle, pregnancy, and parturition. Many factors, such as steroid hormones, growth factors, and cytokines, are involved in the regulation of uterine changes. We previously detected relatively high VIC expression in mouse uterus by semiquantitative RT-PCR (
Quantitative gene expression analysis by real-time RT-PCR demonstrated that the expression level of VIC mRNA in the uterus fluctuates during pregnancy (Fig 1). In particular, we observed a significant change in gene expression between the earlier (Days 7.510.5) and later (Days 12.517.5) stages of pregnancy. Gene expression rates in the later stage showed an approximately fivefold increase compared with those in the earlier stage (Fig 1). In accordance with this increase in the gene expression rate, myometrial cells showed a stronger positive pattern of immunostaining for VIC in the later stage of pregnancy (Fig 3). In the cycling non-pregnant uterus, the gene expression rate increased as the estrous cycle advanced from diestrus to proestrus and estrus (Fig 2), and concomitantly myometrial cells in proestrus and estrus showed a stronger positivity for VIC than in diestrus (Fig 4). On the basis of these results, we concluded that the expression of VIC mRNA and peptide increases in myometrial cells in the later stage of pregnancy and in proestrus and estrus. The results of in situ hybridization (Fig 5) support this conclusion.
The reason for the drastic changes in VIC gene expression during pregnancy and the estrous cycle is unclear. However, the most likely explanation for the change would be hormonal regulation by the ovarian steroids estrogen or progesterone, considering that the concentration of these agents in blood changes greatly during pregnancy ( (
(
For the pregnant uterus, there is another possible explanation for elevated VIC gene expression in the later stage of pregnancy. The mechanical stimulation of the uterus by the growing embryos, which drastically increase in size around this period, may play a role in the regulation of VIC expression. In vascular smooth muscle cells the expression, synthesis, and secretion of angiotensin II and transforming growth factor-ß are reported to be stimulated by mechanical stretching (
Cellular distribution of ET-1 in the uterus varies among species and among uterine physiological stages. In pregnant and parturient rabbit, a number of giant cells penetrating to the myometrium, which are proposed to be derived from trophoblastic knobs, showed intense ET-1 immunostaining, but myometrial cells did not (
The precise intracellular localization of VIC in myometrial cells is now unclear because we have not performed ultrastructural studies and there has been no previous report on the intracellular localization of VIC or ET-2. However, ET-1 has been demonstrated by an electron microscopic study to be in the endoplasmic reticulum (ER) and the Golgi apparatus of vascular endothelial cells and adrenal gland cells (
Both ETA and ETB receptors are expressed in myometrial cells (
In conclusion, we demonstrated that the expression level of uterine VIC changes during the estrous cycle and throughout pregnancy. We showed that myometrial cells in the proestrous and estrous stages, in late pregnancy, and in early postpartum dominantly express VIC mRNA and produce VIC peptide. Our present results, which reveal changes in the expression and production levels of VIC in response to various reproductive events in the uterus, suggest that VIC produced by myometrial cells may act in an autocrine/paracrine manner and play an important role in physiological functions including contraction, cell growth, and differentiation in the uterus.
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Acknowledgments |
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Supported by a project grant to K.S. of Research and Development for the Elucidation of Biological Functions from the Ministry of International Trade and Industry of Japan and by a project grant of the Cooperative System for Supporting Priority Research from the Science and Technology Agency of Japan.
K.S. gratefully acknowledges the encouragement and support of Drs Syuichi Oka and Noboru Tomizuka at NIBH. T.U. and K.S. thank Drs Norio Ishida, Tomoko Niki, Junji Magae, and Yasuo Tanaka at NIBH for helpful discussion, Ms Manami Nagano at PE Applied Biosystems for the design of and advice on TaqMan probes, and IBL for the gift of antibody.
Received for publication February 23, 1999; accepted December 22, 1999.
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