Molecular Human Reproduction, Vol. 7, No. 5, 437-446,
May 2001
© 2001 European Society of Human Reproduction and Embryology
Uterine physiology |
Changes in expression and subcellular localization of nuclear retinoic acid receptors in human endometrial epithelium during the menstrual cycle
Department of Obstetrics and Gynecology, Sapporo Medical University School of Medicine, South-1 West-16 Chuo-ku, Sapporo 060, Japan
Abstract
The endometrium is a uniquely dynamic tissue in that it undergoes monthly cycles of proliferation and secretory activity, and is regulated by ovarian steroid hormones. In this study, we focused on retinoic acid receptors (RAR and RXR) which are ligand-dependent transcription factors belonging to the large family of steroid hormones and are expected to affect to cell growth and differentiation in the endometrium. We analysed the expression and subcellular localization of the RA receptors in 57 samples of human endometrium by immunohistochemistry and Western blotting. In the nuclei of the endometrial epithelium, the RA receptors were expressed strongly in the proliferative phase. However, RAR were drastically reduced in the epithelial nuclei during the secretory phase in association with changes in serum oestradiol and in the expression of the oestrogen receptor. The expression of RXR was localized in the epithelial nuclei throughout the menstrual cycle. Confocal laser scanning microscopical observation clearly showed the difference in the localization between RAR and RXR in the secretory phase. Furthermore the findings of immuno-electron microscopy showed pooled RAR around the rough endoplasmic reticulum, suggesting that transport of these receptors to the nuclei is inhibited. These findings suggest that RAR and RXR work mainly in the proliferative phase and that in the endometrium RXR may play a different role to RAR during the secretory phase.
endometrium/menstrual cycle/nuclear localization/retinoic acid/retinoic acid receptor
Introduction
The endometrium is a uniquely dynamic tissue, consisting of epithelial glands and connective tissue stroma which undergo monthly cycles of proliferation, secretory activity, and breakdown in the absence of embryo implantation. Coordinated histological and cytological changes in both the endometrial epithelium and stroma occur during the reproductive cycle (Noyes et al., 1975
). These changes are mainly regulated by ovarian steroid hormones, oestrogen for cell proliferation and progesterone for cell differentiation. In the glands of cyclic endometria, proliferative activity, as revealed by expression of the Ki-67 antigen, is highest in the proliferative phase and early secretory phase. The proliferative activity decreases in the middle secretory phase (Pickartz et al., 1990). Throughout the menstrual cycle, it is expected that the stem cells of endometrial glands alternate between activity and inactivity under the control of ovarian steroid hormones (Saito et al., 1997c). In recent studies, we have reported the changes in the expression of molecules related to cell growth in association with the changes in cell growth and differentiation in the endometrium (Saito et al., 1997; Nei et al., 1999) and in this work we have focused on the receptors of retinoic acid (RA), which are known to be essential for growth, reproduction (conception and embryonic development), and resistance to and recovery from infection (Napoli, 1999). Retinoic acid also interacts with the oestrogen receptor, especially in breast cancer and ectocervical cells, both of which, like the endometrium, are target organs of the ovarian steroid hormones (Gorodeski et al., 1990; Dawson et al., 1995; Rosenauer et al., 1998; Zhu et al., 1999).
RA, one of the most potent of the naturally occurring retinoids (retinol and derivatives), is required in vivo for the maintenance of epithelial cell growth. The RA receptor (RAR) and retinoic X receptor (RXR) are members of the intercellular receptor superfamily that bind to their endogenous RA ligands with high affinity and specificity (Heyman et al., 1992; Levin et al., 1992; Allenby et al., 1993; Allegretto et al., 1995) and thereby regulate discrete sets of targets (Mangelsdorf et al., 1990). RAR bind to all-trans-RA and 9-cis-RA with approximately equal affinity, whereas RXR bind only to 9-cis-RA. There are three known subtypes of each RA receptor subfamily, classified as -
, -ß, and -
, which display specific patterns of expression throughout development and function in cell growth and differentiation processes (Fitzgerald et al., 1997; Lohnes, 1999). Though previous studies have demonstrated that RAR and RXR are expressed in the human endometrial epithelial and stromal cells, knowledge about their nature in the physiological condition is still limited (Prentice et al., 1992; Loughney et al., 1995; Kumarendran et al., 1996). Kumarendran et al. reported that the expression of RAR and RXR mRNA in endometrial epithelial and stromal cells varies in relation to the menstrual cycle (Kumarendran et al., 1996). They found that RAR and RXR were expressed at similar levels throughout the menstrual cycle both for stromal and epithelial cells, with the possible exception of RAR-ß, and they concluded that the result implied that any menstrual cycle-related function of RAR is controlled by the ligand availability rather than by changes in expression of the receptors. However, there is no report about the protein expression and the subcellular localization of the RA receptors, RAR and RXR, in human endometrial tissues. In this study, we have examined the protein expression and the cellular localization of RAR and RXR by immunohistochemistry and Western blotting for 57 endometrial tissue samples taken during various stages of the menstrual cycle.
Materials and methods
Patients and samples
Samples of endometrial tissues were obtained from 57 women who had undergone hysterectomy at the Sapporo Medical University Hospital according to institutional guidelines, and informed consent was obtained from patients. Normal endometrial tissue (n = 57) was taken from the unaffected endometrium of normally menstruating females with leiomyoma or adenomyosis of the uterus. All normal endometrial specimens were dated from haematoxylin and eosin sections, according to published criteria (Noyes et al., 1975). Of the 57 cases of normal endometrial epithelium, nine cases were of early proliferative (days 4–7), 11 of mid-proliferative (days 8–11), nine of late proliferative (days 12–14), nine of early secretory (days 15–19), 10 of mid-secretory (days 20–24) and nine of the late secretory phase (day 25 and later).
Immunohistochemistry
Tissues were fixed overnight in 10% buffered formalin, dehydrated, and embedded in paraffin. Serial sections (5 µm thick) of each sample were used in this study. Sections were cut, floated onto albumin-coated slides, dried at 56°C, deparaffinized in xylene, rehydrated, and washed with phosphate-buffered saline (PBS) for 15 min at room temperature. Specimens were treated in a microwave oven in 0.01 mol/l citrate buffer (pH 6.0) for 30 min at 100°C, slowly cooled to room temperature, and then washed with PBS for 5 min at room temperature. After quenching endogenous peroxidase with 3% hydrogen peroxide in PBS for 10 min at room temperature, the sections were incubated with a blocking solution (PBS containing 5% skimmed milk) for 60 min at room temperature. Then the slides were incubated overnight at 4°C with a 1:500 dilution of anti-RAR-, -ß and - (Santa Cruz Biotechnology, CA, USA), anti-RXR-, -ß and - (Santa Cruz Biotechnology) and with a 1:100 dilution of anti-ER, which recognizes the oestrogen receptor (ER- but not ER-ß) and anti-PR which recognizes the progesterone receptor (Novocastra Laboratories, UK). The manufacturer's data sheet reports that the antibodies to RAR and RXR have no cross-reactivity with other RAR or RXR. After several washes with PBS, the slides were incubated with a second antibody, a 1:200 dilution of peroxidase conjugated anti-rabbit immunogloblin (Dakopatts, Glostrup, Denmark) for RAR and RXR and a 1:200 dilution of peroxidase-conjugated anti-mouse immunogloblin (Dakopatts) for ER- and PR, for 2 h. The colour reaction was developed by the silver intensification procedure described previously (Saito et al., 1998). For the negative control, the same dilution of non-immunized mouse immunogloblin was used as the first antibody.
For confocal laser scanning microscopy (LSM) observation, after the first antibodies, a 1:100 dilution of fluorescein isothiocyanate-conjugated anti-mouse immunogloblin (Dakopatts) was employed as a second antibody, and then the slides were mounted using fluorescent mounting medium (Dakopatts). The observation by LSM was performed using MRC-1024ES (Bio-Rad, Hercules, CA, USA) at 0.2 µm thickness.
For immuno-electron microscopy, the endometrial tissues were fixed (4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 mol/l phosphate buffer, pH 7.2) and after dehydration, embedded in resin (Unicryl Resin Kit; British Bio Cell International, Cardiff, UK) according to the manufacturer's protocol and then ultrathin sections were prepared. After blocking by 5% bovine albumin, the sections were incubated in a 1:100 dilution of the first antibody (anti-RAR- or RXR-) for 30 min and then incubated in a 1:20 dilution of 10 nm gold-labelled goat anti-rabbit IgG (Auro ProbeTM EM GAR G10; Amersham, Little Chalfont, Bucks, UK). The specimens were examined under a Jeol 1200 EX electron microscope after uranyl acetate and lead citrate staining.
Staining evaluation
Staining evaluation was performed by two independent observers (K.F. and T.S.) without knowledge of clinical information. To quantify positivity for RAR, RXR, ER- and PR, immunoreacted cells whose nuclei were stained as intensively as observed in the mid-proliferative phase, were counted in the basal and functional layers of the normal endometrium, as were those whose cytoplasm was stained as intensively, as observed with the RAR of the mid-secretory phase. For each section, at least five fields were photographed under a microscope at x200. One hundred glandular cells in each field were selected and the positivity index (PI) was expressed as the number of positive cells per 100 cells. Statistical analyses were performed using Student's t-test and the Mann-Whitney test. One of the serial sections was stained with haematoxylin and eosin for histological evaluation.
Western blot analysis
For Western blotting, 10 endometrial samples were analysed, five from the proliferative phase and five from the secretory phase. Frozen tissues were disrupted for 30 s with a Brinkmann homogenizer in buffer solution containing 4 mmol/l NaHCO3, 2 mmol/l phenylmethylsulphonyl fluoride, 2 mg/ml aprotinin and 2 nmol/l sodium orthovanadate. After centrifugation at 10 000 g for 60 min, the pellets were solubilized with sodium dodecyl sulphate (SDS) electrophoresis sample buffer without mercaptoethanol (10 mmol/l Tris–HCl, pH 7.8, 1 mmol/l EDTA, 3% SDS, 5% glycerol) and sonicated in a Branson Sonifier. The extract was heated for 5 min at 95°C, and protein concentrations were determined using the Bio-Rad Detergent Compatible Protein Assay (Bio-Rad). Mercaptoethanol and Bromophenol Blue were added at 5% and 0.05% respectively, and 10 µg protein was run per lane on a 9% polyacrylamide electropheresis gel (Mini-Protein II, Bio-Rad), then blotted onto a polyvinylidene difluoride membrane (Bio-Rad). The filters were blocked in 5% (w/v) dry milk in PBS and incubated for 1 h at room temperature in anti-RAR-, -ß and - (Santa Cruz Biotechnology) and anti-RXR-, -ß and - (Santa Cruz Biotechnology) diluted 1:1000 in PBS. After four washes with 0.1% Tween in PBS, the blots were incubated for 1 h at room temperature with horseradish peroxydase anti-rabbit antibody diluted 1:2000 in PBS. The blots were then washed and treated with enhanced chemiluminescence Western blotting detection reagents (Amersham) and exposed to blue-light-sensitive autoradiographic film (Hyperfilm-ECL; Amersham). In the negative controls, normal rabbit serum was used as the first antibody.
Results
Immunohistochemistry of RAR and RXR in endometrium with the normal menstrual cycle
First, we analysed the subcellular localization and immunoreactivity of RA receptors, ER- and PR by immunohistochemistry in 57 cases of normal endometrial epithelium (nine cases of the early proliferative phase, 11 of the mid-proliferative phase, nine of the late proliferative phase, nine of the early secretory phase, 10 of the mid-secretory phase and nine of the late secretory phase) to determine the changes in the protein expression in the physiological condition. The intensity of staining in the functional layer of the endometrial epithelial cells was expressed as the PI of the nuclei and cytoplasm. Though RAR, RXR, ER- and PR were all expressed both in the endometrial epithelial and stromal cells (Figures 1 and 2), the immunoreactivity and subcellular localization were changed during the menstrual cycle. The mean of the evaluated PI in each phase of menstruation is shown in Figure 3a (functional layer) and 3b (basal layer) for the nuclei and in Figure 4a (functional layer) and 4b (basal layer) for the cytoplasm.
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As shown in Figure 3a
, there was a clear difference between the expression of RAR and the expression of RXR in the nuclei of the endometrial epithelium of the functional layer during the menstrual cycle. Though RAR were highly detected in the nuclei during the mid- and late proliferative phases (Figure 1b), they decreased in the early secretory phase and were rarely detected in the mid- and late secretory phases (Figure 2b). On the other hand, RXR were expressed throughout the menstrual cycle in the nuclei. Their expression increased during the proliferative phase and showed peak expression in the late proliferative phase (Figure 1c). Though they decreased in the secretory phase, they were still expressed in the nuclei in the late secretory phase; RXR- in particular was detected more intensively than RXR- and RXR-ß (Figure 3). The expression in the nuclei of the basal layer was almost identical to that in the functional layer, though it was weaker in the basal layer than in the functional layer (Figure 3b).
In the cytoplasm, both RAR and RXR were weakly detected in the proliferative phases, both functional (Figure 4a) and basal layers (Figure 4b). On the other hand, in the secretory phase, RXR were still weakly detected in the cytoplasm, but RAR showed stronger staining in cytoplasm during the secretory phase compared to the proliferative phase.
In contrast with the epithelium, subcellular localization in the stromal cells remained in the nuclei for all the RA receptors (data not shown). For RAR, the immunoreactivity was closely associated with that in the nuclei of the epithelial cells; that is, the intensity of staining was also strong in the proliferative phase (Figure 1b), but it decreased in the secretory phase (Figure 2b). On the other hand, the immunoreactivity to RXR was strong throughout the menstrual cycle, like that in the nuclei of the epithelial cells (Figure 1c and 2c).
Immunohistochemistry of ER- and PR in endometrium with the normal menstrual cycle
During the normal menstrual cycle, the concentrations and distribution of ER- and PR changed markedly both in functional and basal layers (Figure 5). The immunoreactivity of ER- in the endometrial epithelium was strong in the proliferative phase (Figure 1d) and weak in the secretory phase (Figure 2d). In the mid-proliferative phase, a small proportion of glandular cells stained positively for PR whereas staining for ER- was more intense and more frequent. During the late proliferative phase and early secretory phase, the staining for PR increased markedly in glandular cells. During the mid- and late secretory phase, ER- and PR were not detected in glandular cells (Figure 2d,e).
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Association between the serum sex steroid concentration and the immunoreactivity of RA receptors
To examine the association between the serum sex steroid concentration and the immunoreactivity of the RA receptors in endometrial nuclei, the serum oestradiol and progesterone concentrations were measured at the day of hysterectomy for 15 cases (Table I
). As shown in Table I, when oestradiol was high and progesterone low (samples 2–7), both RAR and RXR were highly expressed in nuclei. On the other hand, when progesterone was high (>10 ng/ml, samples 8–11), there was a decrease in nuclear RAR expression, while RXR were detected almost as strongly as in the high oestradiol (>100 pg/ml) and low progesterone (<10 ng/ml) condition. When both oestradiol and progesterone were low (samples 1, 12–15), the immunoreactivity of RAR was weak or absent; however, RXR were detected in the nuclei, albeit at lower levels then when oestradiol was high.
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Western blotting of the RA receptors in endometrium
To compare the expression of the RA receptors in the menstrual periods, Western blotting was performed (Figure 6
) for 10 samples, five from the proliferative phase and five from the secretory phase. All the RA receptors showed similar patterns of the expression, but a difference was found between RAR and RXR. In the proliferative phase, the expression of all the RA receptors was strong, but in the secretory phase, RAR were only weakly detected. RXR were detected in the secretory phase, though expression was lower than in the proliferative phase.
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Observation of subcellular localization by LSM and immuno-electron microscopy
To examine the subcellular localization of the RA receptors more precisely, we first observed the samples by LSM. Although both RAR-
and RXR- were localized in the nuclei in the proliferative phase (data not shown), RAR- was localized only in the cytoplasm (Figure 7a) in the secretory phase. In contrast, RXR- was localized in the nuclei (Figure 7b) in the secretory phase. Furthermore, to determine the organelle to which the cytoplasmic RAR were localized, we performed immuno-electron microscopy for RAR- and RXR- for the samples of the secretory phase. For RXR-, gold particles were observed on the chromatin of the nuclei (Figure 8b), but for RAR-, they were observed around the rough endoplasmic reticulum (rER) and not in the nuclei (Figure 8a).
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Discussion
The endometrium is a uniquely dynamic tissue. It consists of epithelial glands and connective tissue stroma, and undergoes monthly cycles of proliferation, secretory activity, and breakdown in the absence of embryo implantation. We have previously reported changes in expression of molecules related to the cell growth and differentiation (Saito et al., 1997b; Nei et al., 1999) and in this work we focused on RA and its receptors. These are known to affect cell differentiation in various organs (Napoli, 1999) and interact with the oestrogen receptor, especially in breast cancer and ectocervial cells, which are also targets of the ovarian steroid hormones, like endometrial tissue (Gorodeski et al., 1990; Dawson et al., 1995; Rosenauer et al., 1998). RA is also required for the normal differentiation of reproductive epithelia; however the exact role of RA in endometrial differentiation is poorly understood.
In this study, we analysed the expression and the subcellular localization of the nuclear RA receptors, RAR and RXR, in the endometrium in the physiological condition by immunohistochemistry. All the RA receptors were expressed in both endometrial epithelial cells and stromal cells. This was in accordance with previous studies showing that RAR and RXR mRNA were expressed in human endometrial epithelial and stromal cells (Siddiqui et al., 1994; Brar et al., 1996; Kumarendran et al., 1996). However, this is the first report to analyse the expression and the subcellular localization of the RA receptors in the endometrium by immunohistochemistry.
In the cytoplasm of the endometrial epithelial cells, both RAR and RXR were only weakly detected both in functional and basal layers. In the secretory phase, though RXR were hardly detected in the cytoplasm, RAR had a tendency to stain more strongly. In the nuclei they were changed drastically. In the proliferative phase, all the RA receptors were exposed strongly in epithelial nuclei, and after ovulation the intensity decreased. However, RAR and RXR showed different expressions in the secretory phase. Though RAR were rarely detected in the nuclei in the secretory phase, RXR were still detected. Western blotting supported the results of immunohistochemistry. Our results are not entirely in agreement with those of Kumarendran et al. on the expression of the RA receptors in the human endometrium during the menstrual cycle. They analysed mRNA of the nuclear RA receptors in endometrial epithelium and stroma by Northern blotting (Kumarendran et al., 1996) and concluded that RAR and RXR were expressed at similar levels throughout the menstrual cycle, with the possible exception of RAR-ß. This implied that any menstrual cycle-related function of RAR is controlled by ligand availability rather than by changes in expression of the receptors. We think that the difference between our results and those of Kumarendran et al. is related to the difference of the analysed material. We analysed the RA receptor expression at the protein level while Kumarendran analysed the mRNA level. The difference might be caused by the changes that have occurred after protein synthesis during the menstrual cycle, consistent with evidence that oestradiol enhancement of RAR- expression occurs in the presence of protein synthesis inhibitors and is not due to modulation of mRNA stability in mammary cancer cells (Sheikh et al., 1993). In the endometrial stromal cells, the intensity of staining was correlated with that in the nuclei of epithelial cells. These results suggested that the effects of RA on the endometrium were synchronized between the epithelial cells and the stromal cells.
In this study, the localization and expression of RAR and RXR were similar in the proliferative phase but different in the secretory phase. Though RAR were hardly detected in the nuclei during the secretory phase, RXR were still detected in the nuclei. Though RXR were still hardly detected in the cytoplasm, RAR tended to be stronger in the epithelial cytoplasm during secretory phase. The LSM observation clearly showed the difference. Furthermore, the observation of RAR- by immuno-electron microscopy showed that the protein was found mainly around the rough endoplasmic reticulum, suggesting that the synthesized protein was pooled around this organelle. The RA receptors are ligand-dependent transcription factors that belong to the large family of steroid and thyroid hormone receptors (Giguere and Evans, 1990; Mangelsdorf et al., 1990; Chambon, 1996). Activated by their ligand, the receptors interact with specific DNA sequences located in the 5'-flanking regions of target genes, resulting in the up- or down-regulation of the expression of these genes (Mangelsdorf et al., 1990; Rochette-Egly et al., 1997). Although the mechanism of cytoplasmic localization of nuclear receptors is still a point of discussion (Welshons et al., 1984; Jenster et al., 1993; Htun et al., 1999; Zhu et al., 1999) and the RA receptors are poorly characterized, it is possible that the capacity to transport the nuclear RA receptors, particularly the RAR, from the cytoplasm to nuclei declines in the secretory phase. Basically, the steroid hormone receptors are localized in the nuclei, regardless of hormonal status, and considerable amounts of unligated steroid hormone receptors may be present in the cytoplasm of target cells only in exceptional cases (Yamashita, 1998). Since there is no evidence that the nuclear receptors can work as ligand-dependent transcription factors when they exist in the cytoplasm, it may be reasonable to conclude that RAR work mainly in the proliferative phase, whereas RXR work throughout the menstrual cycle.
In this study, ER- showed strong nuclear staining in the proliferative phase, and PR showed positive staining in the mid-proliferative and early secretory phases. These results are in accordance with a previous report (Lessey et al., 1988). The expression of the RA receptors was associated with changes in the serum oestradiol level and the expression of oestrogen receptors. There is evidence that ER-positive cell lines express higher levels of the RA receptors than do ER-negative cells (Roman et al., 1993; Sheikh et al., 1993; Rishi et al., 1996) and oestradiol has been shown to cause a 2–3-fold up-regulation of RAR- gene expression in ER-positive MCF-7 and T47D HBC cells (Roman et al., 1993; van der Burg et al., 1993). Considering this evidence, it was suggested that the expression of the RA receptors in the nuclei was strongly affected by ER- and serum oestrogen in the endometrium. On the other hand, in this study, the expression of RA receptors decreased in the secretory phase and was lower in high serum progesterone conditions, even in the high serum oestradiol condition (<100 pg/ml). Thus it is possible that PR may have a down-regulatory effect on RA receptors in the endometrium, acting as an anti-oestrogenic molecule.
In previous studies, we reported on the molecules, connexin, telomerase and ß-catenin, all of which are associated with cell proliferation and differentiation in the endometrium (Saito et al., 1997c; Nei et al., 1999). We found that connexins 26 and 32, which are key proteins of intercellular communication through their roles as growth factors (Yamasaki and Naus, 1996; Saito et al., 1997a, 1998), were mainly expressed in the early and mid-secretory phases in the endometrial epithelium (Saito et al., 1997b). In recent studies, RA has been shown to enhance connexin expression in several tissues (Clairmont and Sies, 1997; Watanabe et al., 1999). Telomerase activity was detected in the late proliferative and early secretory phases in the endometrium, suggesting that its activity is associated with cell proliferation (Saito et al., 1997c). In a recent study, it was reported that the telomerase activity is suppressed during cell differentiation induced by RA (Albanell et al., 1996; Bednarek et al., 1999). ß-Catenin is known as the key molecule not only of the E-cadherin-associated cell adhesion molecules but also of the Wnt-1/TCF signal pathway (Miller and Moon, 1996), and the nuclear accumulation of ß-catenin results in cell proliferation (Behrens et al., 1996). In the endometrial endothelium, we have demonstrated that ß-catenin accumulates in the nuclei in the late proliferative and early secretory phases (Nei et al., 1999). In recent studies, it was revealed that RA increases cell–cell adhesion strength, as well as the stability of ß-catenin protein and its localization to the cell membrane (Vermeulen et al., 1995; Byers et al., 1996; Easwaran et al., 1999). Judging from this evidence, though there is no doubt that the endometrium is mainly under the influence of sex steroids, it could be suggested that RA and its receptors have effects on the changes in expression of various molecules in the late proliferative phase.
The difference in nuclear localization between RAR and RXR in the secretory phase suggests the possibility that all-trans-RA, which works as a ligand of RAR, and 9-cis-RA, which works as a ligand of both RAR and RXR, play different roles in the endometrium. In particular, 9-cis RA may have an effect on the endometrium throughout the menstrual cycle and we expect that understanding of these RA will help in hormone therapy for infertility and other diseases of the endometrium.
Acknowledgements
We thank Mr M.Kim Barrymore for editing the manuscript. Part of this work was supported by grants for Scientific Research from the Ministry of Education of Japan; contract grant numbers: 09470363, 09771289 and 11671638.
Notes
1 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Sapporo Medical University, S-1, W-16, Chuo-ku, Sapporo 060-0061, Japan. E-mail: tsaito{at}sapmed.ac.jp 
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Submitted on September 1, 2000; accepted on February 26, 2001.
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