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Departments of Obstetrics and Gynecology (H.A., M.N., S.-I.T., Y.K., S.T., Y.M., A.I., S.M.) and Maternal and Perinatal Medicine (H.A., S.M.), Nagoya University School of Medicine, Nagoya 466-8550, Japan; and Division of Pathology, Clinical Laboratory, Nagoya University Hospital (T.N.), Nagoya 466-8560, Japan
Address all correspondence and requests for reprints to: Dr. Hisao Ando, 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
Aminopeptidase A (APA, BP-1) is a membrane-bound zinc metallopeptidase that converts angiotensin II (AngII) into AngIII by selectively hydrolyzing the N-terminal aspartyl residue. AngII has been proposed as a candidate for the initial vasoconstrictor of endometrial spiral arteries/arterioles in the preliminary step of menstruation. In the late secretory phase, endometrial stromal cells (ESC) around the blood vessels begin to differentiate into decidual cells, and AngII has been reported to accumulate around such vessels. However, whether there is a concurrent increase in renin or angiotensin-converting enzyme in this area has not been determined. We hypothesized that APA may be involved in the metabolism of AngII in the cycling endometrium.
Western blot analysis in the present study demonstrated that a considerable amount of APA was present in the secretory phase endometrium. ESC in the secretory phase showed strong expression of APA by immunohistochemical analysis and of APA mRNA by in situ hybridization. In contrast, both APA mRNA and protein were absent in decidual cells. The enzyme activity and the biosynthesis of [35S]methionine-labeled APA significantly decreased during the in vitro decidualization of cultured ESC.
These results suggest that the perivascular disappearance of APA is a differentiation-specific change that occurs along with the decidualization, and that the disappearance of APA might accelerate the accumulation of AngII around the vessels.
MENSTRUATION, THE PROCESS by which the superficial or functionalis layer of the endometrium disintegrates at the end of the nonpregnant cycle, is commenced by vasoconstriction of endometrial spiral arteries/arterioles, and cessation of menstrual bleeding is achieved by vasoconstriction of the remaining vessel fragments (1). The spiral arteries/arterioles are surrounded by endometrial stromal cells (ESC) that differentiate into decidual/predecidual cells (DC), which have a larger and rounder phenotype, in response to progesterone released by the corpus luteum. The decidualization of ESC is an essential step for menstruation. In women, it starts around the blood vessels on postovulation d 9 (POD+9), and the predecidua covers up to two thirds of the functionalis layer on POD+14, or the day preceding menstruation (2, 3).
The precise nature of the endometrial vasoconstrictor has not been established, although angiotensin II (AngII), endothelin-1, and PGF2
are among the possible candidates. PGF2 is regarded as a relatively weak pressor agent (4). Endothelin-1 has been found to cause a potent long-lasting contraction of human uterine arteries and veins (5), and thus it appears to be suitable as the vasoconstrictor in the cessation of menstruation. It has been known for some time that plasma AngII levels are higher in the luteal than in the follicular phase during the menstrual cycle. AngII-like immunoreactivity in human follicular fluid has been shown to be more than 10 times higher than that in plasma (6). As the whole renin-angiotensin system, consisting of renin, angiotensinogen, and angiotensin-converting enzyme, is present in both the ovary and endometrium (review in Refs. 7 and 8), it has been suggested that the sources of AngII in endometrial tissue include both the systemic circulation and the reproductive tract itself. Ahmed et al. (9) have determined the localization of AngII-like immunoreactivity throughout the menstrual cycle. Interestingly, during the premenstrual phase AngII-like immunoreactivity, which includes both AngII and AngIII, is enhanced in perivascular DC, with negligible staining in nondecidualized ESC. Therefore, we hypothesized that there may be a mechanism to control the degradation of tissue AngII.
Aminopeptidase A (APA; glutamyl aminopeptidase, EC 3.4.11.7) is a homodimeric membrane-bound zinc metallopeptidase that specifically hydrolyzes N-terminal acidic amino acid residues (glutamic or aspartic acid) from peptide substrates. Physiologically, APA is regarded as a representative angiotensinase in humans. We purified this enzyme from human placenta (10) and found that the purified APA cleaves the N-terminal aspartic acid of AngII, which converts AngII into AngIII (11). Li et al. (12) cloned the cDNA of human APA, which they call B lymphocyte differentiation antigen BP-1/6C3. Nanus et al. (13) also independently cloned the cDNA of human APA, and identified kidney differentiation antigen gp160 as APA. They also showed that the tissue distribution of APA mRNA is broad and not limited to the placenta. The fact that Naruse et al. (14) found high levels of AngIII in the human endometrium suggests that APA may also be present there. In the present study we evaluated the presence of APA in human endometrium in different phases of the menstrual cycle using Western blot analysis, immunohistochemistry, and in situ hybridization. As we observed strong localization of APA protein and mRNA in ESC and their absence in DC, we also examined the effect of decidualization in vitro on APA enzyme activity and synthesis of APA protein by metabolic labeling of cultured ESC with [35S]methionine, followed by immunoprecipitation and autoradiography.
Materials and Methods
Tissue collection
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. As a result of these reviews, the biopsy specimens of 39 patients, aged 34–43 yr, were available for examination. Endometrial dating criteria were used to assess the phase of the menstrual cycle (2). The results of this histological classification were as follows: 14 proliferative phase, 8 early secretory phase, 8 midsecretory phase, and 9 late secretory phase. Endometrial dating was strictly defined histologically by an expert in this technique (T.N.). All biopsy samples had been proven to be histologically benign. The use of pathology slides for this research was approved by the institutional review board. These samples, which had been fixed in 10% formalin and embedded in paraffin, were used for immunohistochemistry and in situ hybridization. For other experimental purposes, fresh endometrial tissues were collected by curettage from women, aged 34–46 yr, during hysterectomy operations for leiomyoma at Nagoya University Hospital. Cases were further selected after careful review in a manner similar to the biopsy specimens, with some exceptions for cases as described below. Accordingly, the fresh endometrial tissue samples were allocated to 1 of 4 groups: 14 proliferative phase, 7 early secretory phase, 6 midsecretory phase, and 8 late secretory phase. Samples were also collected from patients, aged 34–44 yr, who had received oral administration of estrogen (one Premarin tablet daily, 0.625 mg conjugated estrogens made from pregnant mare’s urine, Wyeth Lederle Japan, Tokyo, Japan; n = 3) or an estrogen-progestin combination (one Dolton tablet daily, 500 µg norgestrel and 50 µg ethinyl estradiol, Nihon Schering AG, Osaka, Japan; n = 4) for 21–28 d before hysterectomy. Endometrial tissues were also curetted from ectopic pregnancy patients, aged 26–35 yr, (n = 4; tubal pregnancy at 6–8 wk). For all of these samples informed consent was obtained from each patient before the study. Curetted samples were washed to remove blood and secretions with cold PBS in the operating room and 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 used for protein extraction. For the culture experiments to study in vitro decidualization, fresh sterile endometrial tissue samples collected by curettage from patients in the late proliferative phase were used.
Preparation of anti-APA IgG antibody
Rabbit polyclonal IgG antibody was raised against a synthetic multiple antigenic peptide (15) composed of the 18-amino acid sequence from Ser257 to Thr274 (SNMPVAKEESVDDKWTRT) of human APA, of which the complete amino acid sequence is described in Refs. 12 and 13 as described in detail in our previous report (16).
Western blot analysis
The frozen tissue samples were homogenized using a motor-driven Teflon pestle for 10 min on ice in PBS extraction buffer containing 1% Triton X-100 and protease inhibitors (1 mM phenylmethylsulfonylfluoride, 1 µg/ml aprotinin, and 10 µg/ml leupeptin). Tissue extracts were obtained as the supernatants after centrifugation at 15,000 x g for 30 min at 4 C and stored at -80 C. Protein concentrations were determined using a protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA). Immunoblot analysis was carried out according to the method reported previously (17) with some modification. Briefly, 10 µg protein extract were separated by SDS-7.5% PAGE, transferred onto a nitrocellulose membrane, and immunoblotted with the rabbit anti-APA polyclonal antibody at a dilution of 1:500. A biotinylated secondary antibody specific to rabbit IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used at a dilution of 1:1,000. Immunoreactive proteins were stained using a chemiluminescence kit (ECL, Pharmacia Biotech, Little Chalfont, UK). Densitometric analysis was applied for statistical comparison. Densitometric data from three independent experiments were expressed as the mean ± SEM.
Immunohistochemistry
Formalin-fixed, paraffin-embedded tissue sections were cut to a thickness of 3 µm. Deparaffinized sections in 0.01 M citrate buffer were treated three times for 5 min each time at 90 C at 750 watts 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 diaminobenzidine detection kit (Ventana Medical Systems, Tucson, AZ), yielding a brown product from diaminobenzidine/copper sulfate, was used to detect APA. Staining procedures were performed automatically using a Ventana’s BenchMark IHC staining system (Ventana Medical Systems) according to the manufacturer’s instructions; this system is a fully automatic computerized staining system that yields stable and reproducible data. The primary antibody against APA 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. Staining for APA was carried out repeatedly for each sample. Stained sections were observed under an Olympus Corp. (Tokyo, Japan) BH2 microscope and photographed using film (Fujicolor Superia 100, Fuji Photo Film Co., Ltd., Tokyo, Japan) and an Olympus Corp. camera.
Preparation of biotin-labeled RNA probes
An antisense and a sense RNA probe complementary to bases 480–657 of the human APA mRNA were synthesized and used to locate the mRNA that encodes the APA protein. The cDNA encoding this region was amplified from human placental mRNA by the RT-PCR method. Human placental total RNA was extracted using TRIzol reagent (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s protocol. The RT reaction using 1 µg total RNA was carried out using a Gene Amp RNA PCR kit (Perkin-Elmer Corp., Foster City, CA) following the manufacturer’s protocol. Thereafter, PCR was carried out with the sense primer (5'-CCCGGCCAGTGAGGATGAGAGC-3') and the antisense primer (5'-GAGCCGGGTGATCCTGGTCTCC-3'). The PCR product was subcloned in a pGEM-T plasmid according to the manufacturer’s protocol (pGEM-T and pGEM-T Easy Vector Systems, Promega Corp., Madison, WI). Sequence analysis confirmed that this PCR product contained the correct sequence of the human APA cDNA. The plasmid contained RNA polymerase promoter sites (T7 and SP6) and multiple restriction sites within the multiple cloning sites. The recombinant plasmid was linearized with appropriate restriction enzymes: NcoI to synthesize the antisense probe, and SalI to synthesize the sense probe. Transcription of biotin-labeled RNA probes was performed with SP6 RNA polymerase to synthesize the antisense probe and T7 RNA polymerase to synthesize the sense probe, using the Riboprobe In Vitro Transcription System (Promega Corp.) and biotin-14-CTP (Life Technologies, Inc.) according to the manufacturer’s protocol.
In situ hybridization
Tissue sections (3 µm thick) were warmed for 10 min in a 60 C oven just before deparaffinization. Then the sections were sequentially immersed in the following solutions at room temperature: 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 with the In Situ Hybridization Detection System for Biotinylated Probes (DAKO Corp., Carpinteria, CA) according to the manufacturer’s protocol. In brief, the sections were heated at 95 C in the target retrieval solution. Hybridization was carried out in a moist chamber with 1 µg/ml 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 site of hybridization was 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) before mounting. Stained sections were observed under an Olympus Corp. BH2 microscope and photographed.
Endometrial stromal cell (ESC) culture and in vitro decidualization
ESC were separated as described previously (18) with only slight modifications. In brief, endometrial tissue in the late proliferative phase was minced into small pieces (
1 mm3), and these pieces were filtered through a cell strainer consisting of 100-µm pore size nylon mesh (Becton Dickinson and Co., Franklin Lakes, NJ) to remove blood cells. Then the minced tissue was incubated with stirring at 37 C for 20 min in PBS, 0.5% collagenase (Wako, Osaka, Japan), and deoxyribonuclease (0.1 mg/ml; Sigma, St. Louis, MO). The dispersed ESC were separated from endometrial glands and undigested tissues by filtration through a cell strainer consisting of 70-µm pore size nylon mesh (Becton Dickinson and Co.). The separated ESC were washed and pelleted by centrifugation (400 x g, 10 min). The ESC were suspended (106 cells/ml) in RPMI 1640 (Sigma) containing FCS (10%, vol/vol; Life Technologies, Inc.), 100 IU/ml penicillin, and 100 µg/ml streptomycin and plated onto 6- or 10-cm Falcon dishes (Becton Dickinson and Co.) for 10–12 d at 37 C in a humidified atmosphere of 5% CO2 in air. Progesterone (10-6 M; Sigma), estrogen (17ß-E2; 10-8 M; Sigma), and (Bu)2cAMP (1 mM; Sigma) were added to the medium at the beginning of culture to induce decidualization. For inhibition of the in vitro decidualization, IL-1 (100 ng/ml; PeproTech, Rocky Hill, NJ), TNF (1 µg/ml; PeproTech), or TGFß1 (100 ng/ml; PeproTech) was also added as described previously (19, 20, 21). The culture media were replaced every 2 d to ensure the continuous presence of the reagents described above. In vitro decidualization was assessed by evaluating morphological changes and assaying the PRL in the conditioned medium (18, 19, 20).
Enzyme activity assay
APA enzyme activity in ESC cultures was measured spectrophotometrically as reported previously (13, 16, 22). In brief, intact cells (5x 105/ml) were prepared in 15-ml test tubes by trypsinization from subconfluent cultures, washed with PBS, and resuspended in 1 ml prewarmed 1.5 mM -L-glutamic acid-p-nitroanilide (Peptide Institute, Osaka, Japan) as a substrate in the presence of 2.5 mM CaCl2. After vortex-mixing, samples were incubated at 37 C with continuous agitation. At appropriate time points, the reaction was terminated by the addition of 2 vol ice-cold PBS, and after the solution was centrifuged for 5 min at 4 C, the OD of the supernatant at 405 nm was measured with a spectrophotometer (Multiskan Bichromatic Labsystems, Helsinki, Finland). Cell-free (BSA used instead of cells) and substrate-free blanks were run in parallel. All assays were conducted in triplicate and repeated three times. In some experiments cells were preincubated with amastatin (500 µg/ml), a potent APA inhibitor, followed by analysis as described above. Data were expressed as the mean ± SEM.
Metabolic labeling and immunoprecipitation
Cells were replated into 12-well dishes (250,000 cells/well) in 1 ml medium I [RPMI 1600 supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), and glutamine (2 mM)] containing 5% FCS and the indicated hormones and other substances after trypsinization by standard procedures at the indicated times during the culture. After incubation for 18 h, the cells were washed twice with cysteine- and methionine-free medium II (medium I supplemented with 5% dialyzed FCS) and labeled in 1 ml medium II containing 25 µCi/ml [35S]methionine and the indicated hormones and other substances for 7 h. Cell lysates were immunoprecipitated with polyclonal antibody against APA as described previously (23), and the precipitates were resolved on 5–15% gradient gels. Immunoprecipitated [35S]methionine-labeled APA was visualized by autoradiography. Quantification of APA was performed by densitometry. Densitometric data from three independent experiments were expressed as the mean ± SEM.
Statistical analysis
Statistical comparison was carried out by t test as appropriate. P less than 0.05 was considered statistically significant.
Results
APA expression in human endometrial tissue
Proteins similar to the human placental APA, 160 kDa in size (16), were detected in the endometrial tissue samples (Fig. 1
). The samples from the mid and the late secretory phases expressed a large amount of APA. However, the level of immunoreactive APA was significantly lower in the samples of cases treated with estrogen/progestin for 2–3 wk compared with those treated with estrogen only (18.4 ± 2.1% decrease; P < 0.05). Moreover, a barely detectable amount of APA was only expressed in the endometrial samples from ectopic pregnancy patients at 6–8 wk. These data indicate that extremely long exposure to progesterone may reduce the expression of APA. We speculate that the reduction might be caused by the decidual change, which is well underway in the samples of cases with extremely long exposure to progesterone.
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In the proliferative phase, endometrial epithelial cells (EEC) were stained for APA, whereas the immunoreactivity in ESC was rather weak (data not shown). After ovulation, intense staining for APA was observed in the entire stromal region, including the ESC near the blood vessels, and this tendency was more marked in the midsecretory phase, in contrast with negligible staining in EEC (Fig. 2A). In the late secretory phase, staining of immunoreactive APA was negligible or very weak in the well vascularized decidualized stroma, whereas staining in the other stromal regions remained very strong (Fig. 2B). The decidualized area, consisting of enlarged DC, was expanded around the spiral arteries/arterioles in the premenstrual phase endometrium, where the immunoreactivity for APA was negligible (Fig. 2C).
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The localization of APA mRNA was in agreement with the immunohistochemical data for each sample (Fig. 3). The DC, which surround the coiled spiral arteries/arterioles, consistently showed negligible staining for APA mRNA (Fig. 3, A and B), in contrast with the nondecidualized ESC, which showed intense staining (Fig. 3A).
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To verify the disappearance of APA during decidualization indicated by immunohistochemistry and in situ hybridization, we used the in vitro decidualization culture model described previously (18). The in vitro decidualization was confirmed by assessing the morphological changes in the cells from a fibroblast-like spindle shape to a large polygonal shape as well as by detection of the differentiation marker PRL in the conditioned medium (>3 ng/ml·48 h) on d 7 or 8 of the ESC culture (data not shown). The enzyme activity of APA was reduced about 50% in the decidualized stromal cells under the decidualization treatment for 10 d (Fig. 4). Both the appearance of PRL and disappearance of APA (50% decrease) were almost simultaneous. APA biosynthesis was also reduced with decidualization in the time-course experiment (Fig. 5A). The decrease in biosynthesis was 53.7 ± 4.3%. Treatment with IL-1, TNF, or TGFß1 prevented the decrease in APA synthesis (Fig. 5B).
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This is the first report of the presence of angiotensinase in human endometrium. Our data showed that in the proliferative phase APA was localized predominantly in EEC, but ESC were also stained in the late proliferative phase. According to a study of angiotensin receptors (9), both EEC and ESC express type 1 angiotensin (AT1) receptor in the proliferative phase. Therefore, it is suggested that APA might affect receptor binding for AngII on the surfaces of both endometrial cell types in the proliferative phase. Our data also showed that APA was strongly expressed in ESC during the luteal phase. The major role of perivascular stromal APA during the early and midsecretory phases is probably the degradation of AngII.
We further demonstrated that DC lack the expression of APA protein and its mRNA, as shown by immunohistochemistry and in situ hybridization, respectively. AngII-like immunoreactivity has been reported to increase around the spiral arteries/arterioles (9). This suggests that AngII might accumulate in the perivascular area due to the expanding decidualized area, which lacks APA. Although the withdrawal of progesterone is a prerequisite for the onset of menstruation, our present data suggest the possibility that AngII functions in the initiation of menstruation, as AngII accumulates in parallel with the decrease in APA in DC. A focal increase in AngII might be important in breakthrough bleeding, which occurs with continuous progestin treatment such as Norplant (levonorgestrel implants; Wyeth Pharmaceuticals, Collegeville, PA) or Mirena (levonorgestrel-releasing intrauterine system; Berlex Laboratories, Inc., Montville, NJ). Other mechanisms that prevent the arteries from constricting in the presence of continuous progestin should be further investigated. Another possible functional role of the AngII accumulation is the induction of vascular endothelial growth factor (VEGF). In addition to the direct effect of AngII on angiogenesis (24), recent reports (25, 26, 27, 28) have suggested the possibility that AngII induces VEGF production, which may increase vascular permeability and vessel elongation of spiral arteries/arterioles. The effect of AngII on DC and endometrial blood vessels should be evaluated in association with the effects of VEGF and other angiogenic factors. As decidualization of ESC is crucial for blastocyst implantation and the maintenance of pregnancy, the lack of APA in endometrial tissue might be associated with the mechanism of implantation.
The disappearance of APA observed in the DC appears to be a differentiation-specific change, as is true in the B cell developmental pathway (29, 30). Our in vitro data as well as histochemical observations have demonstrated that the disappearance of APA is dependent on the decidual change and not on the effect of progesterone. Therefore, an abundance of AngII might be inevitable in the DC. Interestingly, our previous data showed that in human placenta APA is predominantly expressed in the cytotrophoblast, whereas very low levels of APA are found in the syncytiotrophoblast, which is the more differentiated cell type (31). In contrast, increasing expression of aminopeptidase N (CD13, EC 3.4.11.2), which converts AngIII to AngIV, has been reported in human ESC in the secretory phase as an effect of progesterone, especially as decidualization proceeds (32). The same research group has reported that bestatin, an inhibitor of both aminopeptidase N and APA, inhibits in vitro decidualization of human ESC (33). The function of endometrial aminopeptidases in association with both decidualization and onset of menstruation seems likely, although extensive work will be required before we have a full understanding of both mechanisms.
In conclusion, our data indicate that APA may be involved in the metabolism of angiotensins in the human endometrium and thus in the abundance of AngII during decidualization of human endometrial stromal cells, including those around the spiral arteries/arterioles, and might thereby have some role in the initiation of menstruation.
Acknowledgments
We are grateful for the technical assistance of Hiroko Sato and Yukiko Sugie with the immunohistochemistry analysis.
Footnotes
This work was supported in part by a Grants-in-Aid for Scientific Research 12770910 (to H.A.) and 12470341 (to S.M.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, 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: AngII, Angiotensin II; APA, aminopeptidase A; DC, decidual/predecidual cells; EEC, endometrial epithelial cells; ESC, endometrial stromal cells; POD+9, postovulation d 9; VEGF, vascular endothelial growth factor.
Received August 8, 2001.
Accepted January 28, 2002.
References
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