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The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 3 1384-1389
Copyright © 2002 by The Endocrine Society

Other Original Articles

Existence of Placental Leucine Aminopeptidase/Oxytocinase/Insulin-Regulated Membrane Aminopeptidase in Human Endometrial Epithelial Cells

Shigeru Toda, Hisao Ando, Tetsuro Nagasaka, Shin-Ichiro Tsukahara, Masao Nomura, Yoshiaki Kotani, Seiji Nomura, Fumitaka Kikkawa, Masafumi Tsujimoto and Shigehiko Mizutani

Departments of Obstetrics and Gynecology (S.T., H.A., S.-I.T., M.N., Y.K, S.N., F.K., S.M), and Maternal and Perinatal Medicine (H.A., F.K., S.M.), Nagoya University School of Medicine, Nagoya 466-8550, Japan; Division of Pathology (T.N.), Clinical Laboratory, Nagoya University Hospital, Nagoya 466-8560, Japan; and Laboratory of Cellular Biochemistry (M.T.), The Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama 351-0198, Japan

Address all correspondence and requests for reprints to: Hisao Ando, M. D., Ph. D., Department of Obstetrics and Gynecology, Nagoya University School of Medicine, 65, Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail: . ando{at}med.nagoya-u.ac.jp

Abstract

Human endometrial epithelial cells are known to express oxytocin receptors around the time of ovulation. Moreover, oxytocin (OT) and OT-induced prostaglandins appear to play a pivotal role in the switching of endometrial glands from the proliferative to the secretory phase. However, there have been few studies of oxytocinase (OTase), which is identical to placental leucine aminopeptidase (P-LAP)/insulin-regulated membrane aminopeptidase (IRAP). We confirmed the expression of P-LAP/OTase in human endometrium and also observed the changes in the expression of P-LAP/OTase throughout the menstrual cycle. P-LAP/OTase and its mRNA were localized in endometrial epithelial cells but not in stromal cells. In the follicular phase, immunoreactive P-LAP/OTase was homogeneously distributed on the plasma membrane and in cytoplasmic granules. Immunoblot analysis demonstrated that the majority of P-LAP/OTase was produced around the time of ovulation. After ovulation, the immunostaining was restricted to the glycogen-rich subnuclear vacuoles, a glandular marker of progesterone release from the corpus luteum. Thereafter, the membrane-bound P-LAP/OTase was released by apocrine secretion during the period of blastocyst implantation and became depleted toward the time of menstruation. Further understanding of the function of P-LAP/OTase in the endometrium appears likely to yield insights into the cyclic changes during the normal menstrual cycle.

DURING A NORMAL menstrual cycle, the endometrium undergoes dynamic cyclic changes. While ovarian steroid hormones are thought to control the differentiation and function of the endometrium, several lines of evidence indicate that bioactive peptides such as oxytocin (OT) (1, 2) and angiotensin II (3, 4, 5) regulate reproductive phenomena such as ovulation (1, 4), oocyte maturation (5), and therefore also the cyclic changes in the endometrium.

OT, a neurophyseal hormone, is known to play roles in parturition and lactation and in the central nervous system as a neurotransmitter (reviewed in Ref. 6). Although the posterior lobe of the pituitary gland is the major source of OT in the peripheral blood in women during the menstrual cycle (7), Luck (8) has suggested that ovarian OT may have a crucial role in controlling the contractility of the fallopian tubes and uterus at the early to mid-luteal phase, and thereby influence the establishment of pregnancy. Because prostaglandins (PGs) are known to play a role in luteolysis, the existence of human endometrial oxytocin receptor (OTR), reported by Takemura et al. (9) and Dawood et al. (10), suggests that OT may also play a role in luteal regression by contributing to the regulation of the uterine synthesis of PGs in women (11). However, there have been few studies of the OT metabolizing enzyme oxytocinase (OTase) in the endometrium.

Placental leucine aminopeptidase (P-LAP, EC 3.4.11.3), which appears in the serum only during pregnancy (12), was previously found to be identical to cystine aminopeptidase and to function as OTase (13). It degrades AVP, angiotensin III (13), and somatostatin (14) in addition to OT, and appears to play important roles in both fetal development and the maintenance of homeostasis during pregnancy. We have cloned a cDNA for P-LAP/OTase from a human placental cDNA library, and analysis of this cDNA showed the enzyme to be a type-II membrane-spanning zinc metalloprotease (15). We also have shown that the tissue distribution of P-LAP/OTase mRNA was broad, rather than limited to the placenta (15). The results of a recent immunohistochemical study supported this widespread tissue distribution of P-LAP/OTase (16). P-LAP/OTase should be taken into consideration when attempting to understand the roles of OT in the nonpregnant human endometrium in relation to ovarian function. In this study, we have demonstrated the existence of P-LAP/OTase in the human endometrium in different phases of the menstrual cycle using RT-PCR, Western blot analysis, immunohistochemistry, in situ hybridization, and measurement of enzyme activity.

Materials and Methods

Tissues

We retrieved endometrial biopsy specimens from the pathology files at Nagoya University Hospital (Nagoya, Japan). Patient clinical charts were reviewed and cases were selected on the basis of a history of regular menstrual cycles, and no use of any intrauterine device or hormone therapy for at least 6 months before the biopsy. Histological slides of the endometrium were subsequently reviewed and cases were further selected on the basis of consistent histological findings. On the basis of these reviews, 44 cases, aged 32–43 yr, were selected for further study. Endometrial dating criteria were used to assess the phase of the menstrual cycle (17). Results of this histological categorization were as follows: 14 proliferative phase, 14 early secretory phase, 8 mid-secretory phase, 8 late secretory phase. All biopsy samples had been proved to be histologically benign. The use of these pathology slides for these studies was approved by our institutional review board. These samples were used for immunohistochemistry and in situ hybridization.

Fresh endometrial tissues were collected from women, aged 35–46 yr, who had undergone hysterectomy for leiomyoma at Nagoya University Hospital. Written informed consent was obtained from the women before the operation. Cases were further selected after careful review in a manner similar to that described for the biopsy specimens. Twenty-six samples (12 in the proliferative phase, 14 in the secretory phase) were used for RT-PCR, Western blotting, and measurement of enzymatic activity. Samples were also collected from patients, aged 34–44 yr, who had received oral administration of estrogen (one Premarin tablet daily, 0.625 mg of conjugated estrogens made from pregnant mare’s urine, Wyeth Lederle Japan, Tokyo, Japan; n = 3) or of estrogen-progestin combination (one Dolton tablet daily, 500 µg of norgestrel and 50 µg of ethinyl E2, Nihon Schering AG, Osaka, Japan; n = 4) for 21–28 d before hysterectomy. For all of these samples, informed consent was obtained from each patient before the study. Curetted samples were quickly washed to remove blood and secretions with cold PBS in the operating room. Then the tissue was placed in PBS, except for a small piece that was submerged in RNA stabilization solution (RNAlater; Ambion, Inc., Austin, TX). Thereafter, samples were immediately moved to our laboratory in an ice bucket. PBS was removed by brief centrifugation, and then the samples were stored at -80 C until use for protein extraction or RNA isolation.

RT-PCR

Total RNA was isolated from the frozen endometrial tissues using TRIZOL reagent (Life Technologies, Inc., Gaithersburg, MD) in accordance with the manufacturer’s protocol. RNA aliquots were stored at -80 C until use. RT-PCR was performed using specific primers for human P-LAP as described previously (18).

Western blotting

Western blotting was carried out according to the method reported previously (19) with minor modifications. Briefly, 10 µg of protein extract from each individual sample was separated by SDS/7.5% PAGE, transferred onto nitrocellulose membranes, and immunoblotted with rabbit anti-P-LAP polyclonal antibody at a 1:200 dilution. The primary antibody was raised against the N-terminal cytoplasmic domain (amino acids 55–82 of human P-LAP) (20). The biotinylated secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used at a 1:200 dilution. Immunoreactive proteins were stained using a chemiluminescence kit (ECL, Amersham Pharmacia Biotech, Buckinghamshire, UK). SDS-PAGE pre-stained standards (Bio-Rad Laboratories, Inc., Hercules, CA) served as molecular mass markers. In negative control experiments, the primary antibody was replaced with normal rabbit IgG.

Assay of enzyme activity

P-LAP/OTase activity in the detergent-solubilized tissue extracts was determined by measuring the rate of increase in absorbance at 405 nm using 1.6 mM L-leucyl-p-nitroanilide as a substrate in the presence of 20 mM L-methionine as described previously (13). Enzyme activity data were expressed as mean ± SEM. The data were evaluated by ANOVA.

In situ hybridization

An antisense and a sense RNA probe complementary to bases 959-1174 of the human P-LAP were synthesized and used to determine the localization of the mRNA that encodes the P-LAP protein. Tissue sections (3 µm thick) were warmed for 10 min in a 60 C oven just before melting the paraffinized sections. Then the sections were sequentially immersed in the following solutions at room temperature for the times indicated: three changes of xylene for 5 min, three changes of 100% ethanol for 1 min, and four changes of 95% ethanol for 1 min. Next the sections were rinsed in three changes of distilled water for 3 min. In situ hybridization was performed using the DAKO Corp. In situ Hybridization Detection System for Biotinylated Probes (DAKO Corp., Carpinteria, CA) according to the manufacturer’s protocol. In summary, the sections were heated at 95 C in Target Retrieval Solution, and hybridization was carried out in a moist chamber with 1 µg/ml of biotinylated RNA probe diluted in hybridization buffer containing 10% dextran sulfate, 1% SDS, 50% formamide, 6x SSC, sheared DNA, modified Denhardt’s solution, and 0.25 mg/ml denatured salmon sperm DNA overnight at 45 C. The sections were washed at high stringency in stringent wash solution at 56 C for 30 min. Streptavidin alkaline phosphatase was used to detect the biotin groups on the hybridized probe molecules. The sites of hybridization were visualized by the colorimetric reaction of the enzyme conjugate with its substrate, bromochloroindolyl phosphate/nitro blue tetrazolium. This reaction results in the deposition of an insoluble blue-purple product at the site of hybridization. Finally, the sections were counterstained using Nuclear Fast Red Counterstain (Biomeda, Foster City, CA).

Immunohistochemistry

Formalin-fixed, paraffin-embedded tissue sections were cut at a thickness of 3 µm. After melting, the paraffinized sections were treated three times for 5 min in 0.01 M citrate buffer at 90 C at 750 W in an H2500 microwave oven (M&M, Tokyo, Japan) for heat-induced epitope retrieval. Endogenous peroxidase activity was blocked by incubation with 0.5% (wt/vol) hydrogen peroxide in methanol for 10 min, and nonspecific Ig binding was blocked by incubation with 10% normal goat serum in PBS for 10 min. Immunohistochemical staining was carried out based on the labeled streptavidin-biotin method. A Ventana Basic DAB Detection Kit (Ventana Medical Systems, Tucson, AZ), yielding a brown product from diaminobenzidine/copper sulfate, was used to detect P-LAP. Staining procedures were done automatically using the Ventana’s BenchMark IHC Staining System (Ventana Medical Systems). Each primary antibody was diluted 1:100 in PBS. In negative control experiments, the primary antibody was replaced with rabbit IgG. The slides were counterstained with hematoxylin before mounting. The staining for P-LAP/OTase was carried out repeatedly for each sample. Stained sections were observed under an Olympus Corp. (Tokyo, Japan) BH2 microscope and photographed.

Results and Discussion

As shown in Fig. 1AGo, human endometrial tissues expressed P-LAP/OTase similar to that from human placental tissue (resulting in an 888-bp RT-PCR product). Nucleotide sequencing revealed that the cDNA fragment amplified with P-LAP/OTase primers was identical with the human placental P-LAP/OTase cDNA fragment (data not shown).



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  		Figure 1. Expression of P-LAP/OTase in endometrial tissue samples. A, RT-PCR for P-LAP/OTase mRNA. RNA samples from each individual were prepared from proliferative phase endometrium (lane 1), secretory phase endometrium (lane 2), and term placenta (lane 3). One microgram of total RNA was reverse transcribed and amplified with primers for P-LAP/OTase or with primers for GAPDH. Both primer pairs span introns. The molecular weight was estimated by coelectrophoresing molecular weight markers (lane M). B, Western blot for P-LAP/OTase in endometrial tissue samples. Protein samples from each individual were prepared from the early proliferative (lane 1), the mid-proliferative (lane 2), the late proliferative (lane 3), the early secretory (lane 4), the mid-secretory (lane 5), and the late secretory (lane 6) phase endometrium; from endometrium collected from women who had been taking estrogen tablets for 28 d (lane 7) and estrogen/progestin tablets for 21 d (lane 8); and from term placenta (lane 9). Samples containing 10 µg of protein were subjected to SDS/PAGE and Western blotting using anti-P-LAP/OTase antibody.

 
To confirm the expression of P-LAP/OTase in the human endometrium, Western blot analysis was conducted using an anti-P-LAP polyclonal antibody (20). Proteins similar in size (mol mass = 160 kDa) to the human placental membrane-bound P-LAP/OTase were detected using this antibody (Fig. 1BGo). The immunoreactive band was especially conspicuous in the samples in the late proliferative and early secretory phase, and also in the specimens from patients treated with estrogen tablets. However, the immunoreactive protein was less abundant in the samples from the mid- and the late secretory phases and from progestin-exposed endometrium than in the above samples. A negative control did not show any immunoreactive band (data not shown).

As shown in Table 1Go, P-LAP/OTase activity was higher in the samples from the proliferative and the early secretory phases. Samples from the mid- and the late secretory phase had significantly lower enzyme activity.


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  		Table 1. Enzyme activity of P-LAP/OTase in endometrial tissue samples

 
P-LAP/OTase mRNA was localized in endometrial epithelial cells (EECs) and, to a limited extent, in vascular endothelial cells, but was present only at negligible levels in stromal cells (ESCs) (Fig. 2Go).



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  		Figure 2. In situ hybridization detection of P-LAP/OTase mRNA in the proliferative phase endometrium. A, P-LAP/OTase mRNA is predominantly localized in glandular epithelial cells. Very weak specific hybridization is detectable in the stromal region may indicate vascular endothelial cells (indicated by arrows). B, This adjacent section is a negative control using a sense probe for P-LAP/OTase. The bar in B denotes 50 µm.

 
EECs showed positive immunoreactivity for P-LAP/OTase throughout the menstrual cycle, and the intracellular localization of the immunoreactivity changed according to the phase of the cycle (Fig. 3Go). P-LAP/OTase was localized mainly in some cytoplasmic granules as well as on the apical membrane in the proliferative phase (Fig. 3AGo). In the early secretory phase, P-LAP/OTase was clearly immunolocalized in the subnuclear vacuoles of the EECs (Fig. 3BGo). Thereafter, the staining for P-LAP/OTase shifted to the supranuclear region, and the cell-surface P-LAP/OTase was released into the glandular lumen by apocrine secretion (Fig. 3CGo). Our data suggest that P-LAP/OTase becomes depleted in the glandular epithelial cells in the late secretory phase (Fig. 3DGo).



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  		Figure 3. Immunohistochemical cellular localization changes of P-LAP/OTase in glandular epithelial cells during the ovulatory cycle, with high-magnification panels (within the low-magnification images) and also with negative control photographs (on the right side). For the negative control staining, rabbit IgG was used instead of anti-P-LAP. A, In the proliferative phase, P-LAP/OTase is localized mainly in some cytoplasmic granules in the epithelial cells, showing a homogeneous staining pattern. Apical membranes of the cells are also immunoreactive. B, In the early secretory phase epithelium, P-LAP/OTase is immunolocalized in glycogen-rich subnuclear vacuoles, the first endometrial marker of ovulation and luteal progesterone production. The apical side of the cytoplasm is only weakly stained for anti–P-LAP Ab. C, In the mid-secretory phase, the supranuclear region shows immunolocalization of P-LAP/OTase. Immunoreactive fragments of the apical membrane appear to be actively released into the lumen of the glands, indicating apocrine secretion. D, In the late secretory phase, weak staining restricted to the apical membrane indicates the depletion of P-LAP/OTase in the glandular epithelial cells. The bar in (d) denotes 25 µm for the low-magnification panels and 17 µm for the high-magnification panels.

 
This is the first report on the presence of P-LAP/OTase/IRAP in human EECs. The highest levels of OTR mRNA are detected around the time of ovulation in human EECs, but not in ESCs (9). Our data indicating the absence of P-LAP/OTase mRNA in ESCs are compatible with the findings about OTR. The serum concentration of OT has been reported to increase during the proliferative phase and reach its peak on the day following the LH surge in women with normal cycles (7). It has also been shown that the human ovary produces OT (21, 22). Therefore, the existence of P-LAP/OTase in the human endometrium supports the idea that OT has effects on the endometrium as well as on the subendometrial myometrium during the ovulatory cycle (10, 11).

Our results indicate that a large amount of P-LAP/OTase is produced in the endometrium around the time of ovulation, possibly due to the effect of estrogen. In addition to the cell-surface localization, P-LAP/OTase was homogeneously localized in some cytoplasmic granules of EECs in the proliferative phase. While the cell-surface P-LAP/OTase may degrade OT at the cell surface, the function of the homogeneously distributed P-LAP/OTase in the cytoplasm, on the other hand, is unknown at present. We speculate that progesterone may induce repeated fusion of the cytoplasmic granules on which P-LAP/OTase is localized, resulting in production of the subnuclear vacuoles. Consequently, the P-LAP/OTase on the apical side may decrease in the early secretory phase. This may result in increased OTR binding on EECs, which would then activate the secretory function, which peaks around the day of blastocyst implantation. Dawood et al. (10) proposed that OT-induced activation of phosphoinositidase C causes rapid mobilization of free calcium from intracellular stores and thus mediates cellular secretory activity. They also mentioned that OT-induced PG production may increase endometrial capillary permeability around the epithelium. The involvement of OT in the mechanism of uterine peristalsis has been proved, as has its involvement in the coordinated uterine contraction that directs sperm transport from the external cervical os of the uterine cervix to the isthmic part of the tube ipsilateral to the dominant follicle (23). It has been suggested that endometrial OT may pass the thin layer of stroma existing between the tip of the uterine glands and the myometrium, and interact directly with the subendometrial myometrial OTR (24).

We showed here that P-LAP/OTase was localized in the subnuclear vacuoles of EECs in the early secretory phase and then moved to the supranuclear region. It is known that, just after ovulation, glycogen begins to accumulate in the basal portion of EECs, and that some of their nuclei are displaced to the midposition. In formalin-fixed materials, the glycogen is solublized, leaving large vacuoles at the base of the cells. This widespread subnuclear vacuolation of the glandular epithelium is taken as evidence of the formation of the corpus luteum and the production of progesterone. Recently we isolated a P-LAP/OTase cDNA clone and showed that the enzyme encoded by this cDNA is a homologue of rat IRAP (15), which is colocalized with glucose transporter 4 (GLUT4) in specific secretory vesicles of rat adipocytes (25). Waters et al. (26) demonstrated that the amino terminus of IRAP may be critical in controlling intracellular trafficking of the GLUT4 vesicle. It is therefore interesting to speculate that the P-LAP/OTase localization in the subnuclear vacuoles, and later in the supranuclear region, might be involved in glucose transport in EECs.

It is well known that the EECs secrete a clear fluid after ovulation. The endometrial secretion is apocrine: the apical cell membrane of the endometrial cell ruptures, releasing secretory materials (27). Consequently, this phase of the cycle is called the secretory or luteal phase. Research on the components of this secretion has resulted in the identification of specific proteins produced by the epithelial cells, e.g. progesterone-associated endometrial protein and human {alpha}-uterine protein (28, 29). Although the physiological significance of these secretory products is not known, it is likely that they are important in supporting the embryo before implantation and during the early stage of placental development. It is probable that P-LAP/OTase is one of these secretory products. Moreover, P-LAP/OTase may be involved in the mechanism of apocrine secretion, since we have also observed P-LAP/OTase in the apocrine sweat gland (16).

Our present data showed that P-LAP/OTase tends to decrease in the glandular epithelial cells in the late secretory phase. This suggests that the endometrial OT concentration might be increased as a result of the decrease of its degradation by P-LAP/OTase. OTR has been reported to be expressed at the highest level at the end of the luteal phase and during menstruation in the human myometrium (30). Although it is not clear at present whether P-LAP/OTase functions as an oxytocinase in the endometrium, it is interesting to speculate that the predominance of OT within the endometrial-subendometrial unit due to the depletion of P-LAP/OTase causes uterine contraction and thus might contribute to the onset of menstruation.

In conclusion, our present study has provided evidence for the existence of P-LAP/OTase/IRAP in the human endometrium. Our results suggest that the receptor bindings for OT in the endometrium and myometrium may be modulated along with the changes of the cellular localization of P-LAP/OTase during the ovulatory cycle. Further understanding of the function of P-LAP as an oxytocinase in the endometrium appears likely to yield insights into the cyclic changes during the normal menstrual cycle, although extensive work will be required to prove the involvement of this enzyme in various physiological changes of the endometrium.

Acknowledgments

We are grateful for the technical assistance of Hiroko Sato and Yukiko Sugie in the immunohistochemistry analysis.

Footnotes

This work was supported in part by Grant-in-Aid for Scientific Research No. 12770910 (to H.A.) and No. 12470341 (to S.M.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by a research grant from the Ministry of Public Management, Home Affairs, Posts and Telecommunications of Japan (collaboration with Nagoya Teishin Hospital), and by a research grant (to H.A.) from Showa-kai, the Alumni of Department of Obstetrics and Gynecology, Nagoya University School of Medicine.

Abbreviations: EEC, Endometrial epithelial cell; ESC, endometrial stromal cell; IRAP, insulin-regulated membrane aminopeptidase; OT, oxytocin; OTase, oxytocinase; OTR, oxytocin receptor; PG, prostaglandin; P-LAP, placental leucine aminopeptidase.

Received June 11, 2001.

Accepted November 28, 2001.

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A. Iwase, H. Ando, T. Nagasaka, D. Shibata, T. Harata, Y. Shimomura, M. Goto, and F. Kikkawa
Neutral Endopeptidase Expressed by Decidualized Stromal Cells Suppresses Akt Phosphorylation and Deoxyribonucleic Acid Synthesis Induced by Endothelin-1 in Human Endometrium
Endocrinology, November 1, 2006; 147(11): 5153 - 5159.
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D. Shibata, H. Ando, A. Iwase, T. Nagasaka, A. Hattori, M. Tsujimoto, and S. Mizutani
Distribution of Adipocyte-derived Leucine Aminopeptidase (A-LAP)/ER-aminopeptidase (ERAP)-1 in Human Uterine Endometrium
J. Histochem. Cytochem., September 1, 2004; 52(9): 1169 - 1175.
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Y. Suzuki, K. Shibata, F. Kikkawa, H. Kajiyama, K. Ino, S. Nomura, M. Tsujimoto, and S. Mizutani
Possible Role of Placental Leucine Aminopeptidase in the Antiproliferative Effect of Oxytocin in Human Endometrial Adenocarcinoma
Clin. Cancer Res., April 1, 2003; 9(4): 1528 - 1534.
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