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Original Studies |
Department of Gynecology and Obstetrics, Faculty of Medicine (S.Yo., H.F., S.Ya., K.T., T.N., T.H., T.I., S.F.), and Institute for Frontier Medical Science (M.M.), Kyoto University, Sakyo-ku, Kyoto, 606, Japan
Address all correspondence and requests for reprints to: Hiroshi Fujiwara, M.D., Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto, 606-8507, Japan. E mail: fuji@kuhp.kyoto-u.ac.jp.
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Abstract |
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In the preovulatory follicles, immunohistochemical study showed that ECE-1 was expressed, with moderate intensity, on the theca interna cells and weakly on the granulosa cells. In the menstrual and pregnant CL, ECE-1 was highly expressed on both large and small luteal cells, indicating that ECE-1 expression increases during luteinization. Western blotting analysis revealed that the molecular mass of the ECE-1 extracted from the menstrual CL was 130 kDa and that ECE-1 was more strongly expressed on the CL in early and midluteal phases than the CL in late luteal phases. In the isolated luteinizing granulosa cells obtained from patients undergoing in vitro fertilization, ECE-1 was immunohistochemically detected on their cell surface. The activity of ECE-1 was also detected on cultured luteinizing granulosa cells by measuring endothelin-1 production from its precursor. The activity of ECE-1 was significantly enhanced by the treatment of human CG (10 U/mL) and interleukin (IL)-1 (10 ng/mL) during 4-day culture, whereas no significant alteration was observed by IL-4 (10 ng/mL) and IL-10 (10 ng/mL) treatment.
These results indicate that ECE-1 is a cell surface differentiation-related molecule of human granulosa and of theca interna cells and suggest that the expression of ECE-1 is regulated by LH/human CG and cytokines.
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In addition to these aminopeptidases, we recently found that a membrane-bound carboxypeptidase, carboxypeptidase-M (CP-M, EC 3.4.17.12), was expressed on human theca interna cells and luteal cells (8). CP-M can degrade or activate several peptides (including bradykinin, enkephalin hexa- peptides, epidermal growth factor, and dynorphin A) by removing carboxyterminal basic amino acid, such as arginine or lysine (8, 22).
We previously reported that neutral endopeptidase (EC 3.4.24.11) is not expressed on human ovarian cells (1). Recently, endothelin-converting enzyme-1 (ECE-1, EC 3.4.23) was revealed to be a membrane-bound endopeptidase, which converts inactive intermediate-form big endothelin-1 to endothelin-1 by cleaving big endothelin-1 at the median portion (23). Endothelin-1 is a 21-amino acid vasoconstrictive peptide originally isolated from the supernatant of cultured porcine aortic endothelial cells (24). Endothelin-1 exerts a wide spectrum of biological activities other than vasoconstriction in different tissues, and this peptide was reported to be involved in ovarian functions (25, 26, 27). At present, there is no information on the expression of ECE-1 in the human ovary. In this study, we investigated the presence of ECE-1 in human follicles and corpora lutea (CL). Because the expression of DPP-IV on luteinizing granulosa cells is regulated by cytokines, but not by human CG (hCG) (28), we also examined the effect of cytokines and hCG on the ECE-1 activity of luteinizing granulosa cells.
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Materials and Methods |
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The murine antihuman ECE-1 monoclonal antibody (mAb, clone AEC32–136, IgG1 isotype) was a gift from Dr. K. Tanzawa (Biological Research Laboratories, Sankyo Co., Ltd., Tokyo, Japan) (29). The murine antihuman CP-M (clone 1C2, IgG1 isotype) was purchased from Novocastra Lab. Ltd., Newcastle, UK). The murine antitrinitrophenyl (TNP) mAb (IgG1) was used as a negative control (30).
Human ovaries
Ovarian follicles and CL of menstrual cycle were obtained from 22 women, 23–46 yr old. All had undergone an unilateral ovarian cystectomy or oophorectomy and a contralateral wedge resection to treat benign ovarian tumors. All the women had a history of regular menstrual cycles (28–30 days), and their ovulatory basal body temperature charts showed a normal luteal phase length. The CL of pregnancy were obtained from 5 pregnant patients, 28–46 yr old, who had undergone a hysterectomy at 6, 8, 9, 10, or 13 weeks of gestation, because of a uterine myoma and/or cervical cancer. In all 5 patients, fetal growth was normal, on ultrasonographic examination. The gestational weeks were determined from the last menstrual period of each patient. Macroscopically- and microscopically-normal regions of these tissues were used for this study. Informed consent was obtained from all patients before the study.
Immunohistochemical staining of frozen sections
Indirect immunofluorescence histochemistry was performed as previously described, with some modifications (6). Each specimen was embedded in an OCT compound (Tissue-Tec, Miles Scientific, Naperville, IL), snap-frozen in liquid nitrogen, and stored at -80 C. The frozen tissues were sliced into 7-µm thickness using a cryostat microtome (Cryocut 1800, Reichert-Jung, Heidelberg, Germany), immediately air-dried on Neoplene (Nisshin EM, Tokyo, Japan)-coated glass slides, and then fixed in acetone at -20 C for 5 min.
The following follicles and CL were studied: three growing follicles of 4 and 12 mm (n = 2) in diameter; two preovulatory follicles (18 and 21 mm in diameter); two CL on corpus luteum day 2 (the day after ovulation); two CL on day 3; two CL on day 6; two CL on day 8; three CL on day 9; one CL on day 11; one CL on day 12, one CL on day 14, and five CL of pregnancy at 6, 8, 9, 10, and 13 weeks of gestation. The growing follicles were morphologically evaluated by hematoxylin- and eosin-staining of the cryosections. Follicles obtained during the follicular phase, in which the granulosa cells had normally shaped nuclei, cytoplasm, stratified layers, and mitotic figures, were classified as growing or preovulatory follicles. Follicles that were irregularly shaped with blood cell invasion and that lacked mitotic figures were classified as atretic (31, 32). If this differentiation was difficult, hematoxylin- and eosin-stained sections from identical samples that had been fixed with 10% formalin and embedded with paraffin were used instead. The postovulatory date of the CL was evaluated according to the histological dating described by Corner, using hematoxylin- and eosin-stained sections of 10% formalin-fixed and paraffin-embedded samples (33). In this report, the term "CL day" was used, according to his definition. For example, CL day 2 is the day after ovulation.
The slides were steeped in 60-C phosphate-buffered saline (PBS) for 10 sec and then incubated with the anti-ECE-1 mAb (50 µg/mL) or with the anti-TNP mAb (50 µg/mL; a negative control) for 40 min at room temperature. After washing in PBS, they were incubated with the fluorescein isothiocyanate-conjugated rabbit antimouse Ig antibody (diluted 1:40, Dakopatts A/S, Glostrup, Denmark) for 40 min, at room temperature, in the dark. The slides were washed, mounted with Perma Fluor Aqueous Mounting Medium (Immunon, Pittsburgh, PA), and examined under a fluorescence microscope (Nikon, Tokyo, Japan). Serial cryosections were also stained with hematoxylin and eosin after the acetone fixation. The intensity of the antigen expression was graded from - to +++, based on its fluorescence intensity (-, absence of immunostaining; +, weak staining; ++, medium staining; +++, intense staining). The scores were judged by two independent examiners within 6 h.
Isolation of human luteinizing granulosa cells
Human granulosa cells were isolated from patients undergoing in vitro fertilization, as previously reported (28). Briefly, beginning on the first day of their cycle, the patients receiving a gonadotropin-releasing hormone analogue (buserelin acetate, Hoechst Japan Co. Ltd., Tokyo, Japan) were hyperstimulated with human menopausal gonadotropin (Organon Japan Co. Ltd., Tokyo, Japan) until the follicles reached maturity. The follicles were aspirated 36 h after the administration of hCG (Mochida Pharmaceutical Co. Ltd., Osaka, Japan). The follicular fluid was centrifuged, and the resuspended granulosa cells were overlayered onto Ficoll-Hypaque (Nacalai Tesque, Kyoto, Japan) and centrifuged at 400 x g for 30 min. The cells were then collected from the interphase.
Cell surface detection of ECE-1 on isolated human luteinizing granulosa cells, using indirect immunofluorescence staining
The isolated human granulosa cells were washed in HBSS containing 0.1% BSA and 0.1% NaN3, sedimented by centrifugation, and then incubated with 5 µL of the anti-ECE-1 mAb (1 mg/mL) or the anti-TNP mAb (1 mg/mL) for 30 min at 4 C. After washing in HBSS, the cell pellet was incubated with a fluorescein isothiocyanate-conjugated rabbit antimouse Ig for 30 min, at 4 C, in the dark. After washing in HBSS, the cells were resuspended in glycerin/PBS (1:1), mounted onto glass slides, and examined using a fluorescent microscope.
Culture of granulosa cells and enzyme assay of ECE-1
The isolated human granulosa cells were resuspended in culture
medium consisting of RPMI 1640 medium with 10% FCS (Flow
Laboratories, McLean, VA) and kanamycin sulfate (100 mg/L, Meiji Seika
Ltd., Tokyo, Japan). The cells collected after mild washing were
suspended in culture medium at a density of 3 x 105
cells/mL and were incubated in 96-well plates (Corning Inc.,
Corning, NY) at 100 µL/well. On the next day (day 2) and on
day 4, the medium was exchanged by fresh medium with or without hCG (10
IU/mL), recombinant human interleukin (IL)-1
(10 ng/mL, Dainippon
Pharmaceutical Co. Ltd., Osaka, Japan), recombinant human IL-4 (10
ng/mL), or recombinant human IL-10 (10 ng/mL) (Toyobo Co. Ltd., Osaka,
Japan).
On day 6, the medium was replaced by DMEM/F-12 (1:1 vol:vol,
Gibco BRL, Gaithersburg, MD) medium containing 10 mmol/L
HEPES (Nacalai Tesque) and 2 mg/mL BSA with or without big endothelin-1
(200 nmol/L, human, 1–39; Peptide Institute Inc., Osaka,
Japan) in the presence or absence of a potent inhibitor of ECE-1,
phosphoramidon (
0.1–100 µmol/L, Sigma Chemical Co.
Ltd., St. Louis, MO) (34). These culture media were harvested 3 h
later. The concentration of endothelin-1 in the harvested culture media
was measured using a commercial kit (endothelin-1 enzyme-linked
immunosorbent assay kit, Amersham Corp., Buckinghamshire, UK). The
minimum detectable concentration of endothelin-1 was 10 fmol/mL.
Enzymatic activity was expressed as endothelin-1 (fmol) produced per
105 cells in 60 min. Each experiment was performed in
triplicate. The culture was repeated five times. Data are shown as
means ± SEM.
RNA isolation
Human luteinizing granulosa cells, menstrual CL tissue on day 6, pregnant CL tissue at 9 weeks of gestation, and human term placenta tissue were obtained and immediately frozen in liquid nitrogen and stored at -80 C until RNA extraction. Total RNAs of these tissues were isolated using a commercial kit (TRIzol, Gibco BRL).
RT-PCR analysis of ECE-1 in the human CL
Five micrograms of total RNAs from luteinizing granulosa cells, CL, and placenta were reverse-transcribed, with random primers, by a commercial kit (First Strand complementary DNA (cDNA) Synthesis Kit; Pharmacia, Inc., Piscataway, NJ). The resulting cDNA mixtures were subjected to 30 cycles of PCR amplification with oligonucleotides from the human ECE-1 cDNA as primers (35) (sense primer 5'-CTACCGCAC CTCACCCTTCT-3': position 715–734; antisense primer 5'-TTCCTCATCCATCCACTTCA-3': position 1470–1489) or with human S26 primers (36) (sense primer 5'-GGTCCGTGCCTCCAAGATGA-3': position 8–27; antisense primer 5'-TAAATCGGGGTGGGGGTGTT-3': position 308–327). After PCR amplification, 10 µL from each PCR product was electrophoresed on a 1% agarose gel, and amplified bands were detected by ethidium-bromide staining.
Western blotting analysis of ECE-1 in the human CL
Fifty micrograms of placenta; CL of menstrual cycle on day 3 (n = 2), 4, 6, 9 (n = 2), 11, 13, 14; and CL of pregnancy at 9, 10, and 13 weeks of gestation were lysed in a sample buffer (2 mL vol containing 20 mmol/L Tris-HCl (pH 8.6), 1% SDS, 20% glycerol, bromophenol blue) containing protease inhibitors, 1 mmol/L phenylmethylsulfonyl fluoride hydrochloride (Wako Pure Chemicals, Osaka, Japan), 0.5 µg/mL leupeptin (Peptide Institute Inc.), 2 µg/mL aprotinin (Nacalai Tesque), and 1 µg/mL pepstatin (Peptide Institute Inc.). The lysed proteins were separated by 7.5% or 12% SDS-PAGE, under the reducing conditions, and were electrically transferred onto a polyvinilidene fluoride membrane (Millipore Corp., Bedford, MA) in a buffer containing 25 mmol/L Tris-HCl, 192 mmol/L glycine, and 20% methanol. The filter membranes were blocked with 5% nonfat dry milk in PBS. After the blocking, the membranes were washed in PBS three times and incubated with the anti-ECE-1 mAb (10 µg/mL), anti-CP-M mAb (diluted 1:100), or the anti-TNP mAb (10 µg/mL) as a control for 2 h at room temperature. After washing, the membranes were incubated with horseradish peroxidase-conjugated rabbit antimouse IgG (Dakopatts, diluted 1:1000) for 1 h at room temperature. The membranes were washed and then visualized with a chemiluminescence peroxidase substrate, according to the provider’s manual (ECL, Amersham Corp.). The image analyzing soft, NIH image 1.55 Plot Profile, was used to measure the amount of main antigenic protein of immunoreactive ECE-1.
Statistics
The production of endothelin-1, detected by EIA in five experiments, was analyzed by repeated-measures ANOVA, followed by the Scheffe F test. The data obtained from the Western blotting was classified into three groups of early, mid- and late luteal phases, and the amount of main antigenic protein detected by an image analyzer was analyzed by one-way ANOVA, followed by the Scheffe F test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the growing follicles of 4–12 mm in diameter, ECE-1 was moderately detected only on the theca interna cells, by immunohistochemistry (data not shown). In the preovulatory follicles, ECE-1 was moderately detected on the theca interna cells. Immunoreactive ECE-1 was also detected on the granulosa cells, but the intensity of expression was weak (Fig. 1).
In the menstrual CL, immunoreactive ECE-1 was strongly detected on both
large and small luteal cells in the early and midluteal phases (Fig. 1
). In the CL of late luteal phase, ECE-1 was moderately expressed on
both luteal cells (data not shown).
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These expression profiles in the follicles and CL are summarized in
Table 1.
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ECE-1 was detected on the cell surface of luteinizing granulosa
cells isolated from the patients undergoing the in vitro
fertilization treatment (Fig. 2).
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Immunoreactive ECE-1 was detected in the proteins extracted from
the placenta, the menstrual CL, and the pregnant CL. The molecular mass
of the main antigenic protein extracted from the menstrual CL and the
placenta was 130 kDa, whereas the additional specific 160 kDa-protein
band was also detected in the pregnant CL (Fig. 3A).
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The amount of ECE-1 protein (130 kDa) was analyzed by image analyzer.
The mean of ECE-1 protein detected from the CL in late luteal phase was
significantly lower than those from CL in the early and midluteal
phases (P < 0.05, Figs. 4 and 5).
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The expression of ECE-1 mRNA was observed in the placenta,
isolated granulosa cells, menstrual CL on day 6, and the pregnant CL at
9 weeks of gestation (Fig. 6). The
nucleotide sequence of the PCR product, 775 bp in length, in the CL on
day 6, was analyzed by DNA sequencer and confirmed to be identical to
that of ECE-1 cDNA, as previously reported (35). The expected PCR
product of S26 was also detected in placenta and CL.
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The production of endothelin-1 was not detected, by an enzyme assay, in the absence of big endothelin-1. On the other hand, the production of endothelin-1 was observed in the DMEM/F-12 medium of cultured luteinizing granulosa cells in the presence of big endothelin-1. Its production was suppressed by phosphoramidon in a dose-dependent manner (data not shown).
Luteinizing granulosa cells were cultured with or without hCG, IL-1,
TNF-, IL-4, or IL-10. After 4 days, there was no difference in the
number of viable cells among the groups. Endothelin-1 production by
granulosa cells cultured with hCG and IL-1 was significantly higher
[2.7-fold (P < 0.01) and 1.9-fold (P
< 0.05), respectively] than that of the control group. Endothelin-1
production by granulosa cells cultured with IL-4 or IL-10 was 0.6-fold
and 0.73-fold, as compared with the control group, but the differences
were not significant (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we detected immunoreactive ECE-1 on theca interna cells in the growing follicles and on luteal cells in the CL of both menstrual cycle and early pregnancy. Indirect immunofluorescence staining of isolated luteinizing granulosa cells showed the presence of ECE-1 on their cell surface. Western blotting analysis showed that the molecular mass of ECE-1 in the placenta and the menstrual CL was 130 kDa, which is compatible with previous reports (34). Not only the protein but also the mRNA of ECE-1 was detected in the CL and luteinizing granulosa cells, by RT-PCR. The activity of ECE-1 was detected on the cultured luteinizing granulosa cells. Thus, we concluded that ECE-1 is expressed on human granulosa, theca interna, and luteal cells.
Granulosa and theca interna cells in the preovulatory follicles express ECE-1 weakly or moderately, whereas ECE-1 was strongly expressed on the luteinizing granulosa and theca interna cells during CL formation. These expression profiles suggest the involvement of ECE-1 in the luteinization process of both cells, indicating that ECE-1 is a differentiation-related cell surface molecule of human granulosa and theca interna cells. ECE-1 is a membrane-bound endopeptidase, which has an extracellular catalytic site. We have previously proposed that the membrane-bound aminopeptidases and carboxypeptidase expressed on ovarian cells were involved in ovarian cell function and differentiation, by regulating the extracellular peptide concentrations (1, 2, 8, 19, 20). Similarly, ECE-1 may be a local regulator of granulosa, theca, and luteal cell function, which metabolize the biologically active peptides, including endothelin-1.
Not only big endothelin-1 but also bradykinin has been revealed to be a substrate for ECE-1 in 1997 (37). Bradykinin is also a substrate for membrane-bound carboxypeptidase, CP-M. CP-M is expressed on luteinizing granulosa and theca interna cells in the periovulatory follicles and on both luteal cells in CL of menstrual cycle and pregnancy, as similar to ECE-1 (8). Bradykinin is contained in follicular fluid and has been proposed to be involved in the process of follicle rupture as an inflammatory agent (38, 39). Therefore, it is suggested that ECE-1 is involved in ovulation and luteal function by regulating bradykinin metabolism in concert with CP-M.
The regulation of vasodilatation and vasoconstriction has been proposed to be related to the maintenance of the CL of pregnancy and the induction of luteolysis. Biologically active peptides, such as endothelin, are believed to participate in this regulatory pathway (40). Recently, in situ binding studies indicate that the majority of endothelin receptors are expressed in the blood vessels of the human ovary. In particular, endothelin A receptors are abundant in the ovulatory follicles and localized in the theca interna, suggesting that the endothelin system is primarily involved in the regulation of ovarian blood flow (27). Because ECE-1 and CP-M are expressed on the vascular-rich place in the follicles and CL, these membrane-bound peptidases may be related to ovarian function by regulating vasoconstrictor, endothelin-1, or vasodilator bradykinin.
The direct effect of endothelin on granulosa cells was also reported in various species (26, 41). The presence of endothelin receptor was reported on the human luteinizing granulosa cells, and progesterone production by them was shown to decrease in the presence of endothelin-1 in vitro, suggesting that endothelin serves as a suppressing factor against granulosa cell luteinization (41). In this study, the activity of ECE-1 in the cultured luteinizing granulosa cells was increased by hCG treatment. Furthermore, immunoreactive ECE-1 was more intensely detected in the CL in the early and midluteal phases than those in the late luteal phase, by immunohistochemistry and Western blotting analysis. These findings suggest that endothelin production via ECE-1 is induced and maintained when CL highly secretes progesterone. It is theoretically possible that LH/hCG promotes and maintains granulosa cell luteinization and the activity of ECE-1, and then endothelin, produced by ECE-1, may suppress the process of granulosa cell luteinization as a negative-feedback mechanism. If the function of ECE-1 is restricted only to the activation of endothelin precursor, ECE-1 (which is strongly expressed on CL of pregnancy) will produce a high amount of endothelin-1; and the activated endothelin-1 may inhibit progesterone production and blood supply within the CL of pregnancy, to induce luteolysis. However, this scenario is inconsistent with high activity of progesterone production in the CL of pregnancy. One possible explanation for the expression profiles of ECE-1 in the CL of pregnancy is that ECE-1 acts as a luteotropic factor, but not luteolytic, and that undefined substrates for ECE-1, other than big endothelin-1, may be implicated in CL function.
Previously, we proposed that CL of pregnancy is a further differentiated stage through menstrual CL, judging from expression profiles of cell surface molecules on luteal cells (3). In this study, the isotype of ECE-1 was suggested to be expressed in the CL of pregnancy, by Western blotting analysis. The isotype of ECE-1 at 160 kDa has not been reported. We cannot know whether this protein has the same enzyme activity as the 130-kDa ECE-1. It is also possible that this additional protein band is detected by cross-reaction of the anti-ECE-1 mAbs to the other unrelated 160-kDa protein, which is rich in CL of pregnancy. However, the similar additional protein band was also observed, by Western blotting, for CP-M, which is a 68-kDa protein and has not been reported in other organs. At present, there is no convincing evidence that the additional specific protein is the isotype of ECE-1. However, it can be considered that the induction of isotypes of cell surface molecules in the CL may occur during pregnancy, because the additional specific protein was also observed in CP-M, and these additional proteins were not detected in the menstrual CL.
Recently, the immune system has been proposed to play a role in ovarian
function (42, 43). We have previously reported that leukocyte
functional antigen (LFA)-3 and human leukocyte antigen-DR, which are
ligands for cell surface markers of T lymphocytes, CD-2 and CD-4, are
expressed on large luteal cells in the menstrual and pregnant CL (4, 5). Peripheral blood mononuclear cells (PBMC) stimulated
progesterone production by human luteinizing granulosa
cells (44). Furthermore, progesterone production of luteal
cells was also increased when luteal cells were cocultured with PBMC
derived from women in the luteal phase and early pregnancy (45).
According to these findings, we proposed that T lymphocytes communicate
with luteal cells during the formation of menstrual CL and pregnant CL
to facilitate their function and differentiation (5, 45). Because the
production of IL-4 and IL-10 was significantly elevated when PBMC were
cocultured with luteal cells, and progesterone production
of luteal cells was augmented by these cytokines (45), lymphocytes may
serve as a regulator of CL function through cytokine production.
Previously, we reported that the expressions of DPPIV and LFA-3 on
luteinizing granulosa cells were induced by IL-1 and TNF- in
vitro (5, 28), suggesting the regulatory role of these cytokines
in granulosa cell luteinization. In this study, the activity of ECE-1
in the cultured luteinizing granulosa cells was increased by IL-1, as
well as hCG. This also supports the stimulatory effects of IL-1 on
granulosa cell luteinization, because ECE-1 expression on granulosa
cells was immunohistochemically shown to increase during luteinization.
In addition to cytokines, lymphocytes are known to produce various
biologically active peptides, such as enkephalin (46, 47), and some are
the substrates of membrane-bound peptidases. Therefore, it is
speculated that the expression of membrane-bound peptidases is
regulated by lymphocytes that interact with luteal cells, and then the
enhanced membrane-bound peptidases are involved in luteal functions, in
concert with lymphocytes, by hydrolysis of soluble factors, which
lymphocyte may produce (although the identities of the substrates for
membrane-bound peptidases have not yet been clarified).
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Acknowledgments |
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Footnotes |
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Received June 16, 1998.
Revised August 3, 1998.
Accepted August 11, 1998.
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