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Department of Gynecology and Obstetrics, Faculty of Medicine (N.H., H.F., S.F.), Institute for Frontier Medical Science (M.M.), and Institute for Virus Research (M.U.), Kyoto University, Sakyo-ku, Kyoto, 606-8507, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Here, we show a new mAb, named HCL-3, raised against the luteal cells. An analysis of the partial amino acid sequence of purified antigen demonstrated that HCL-3 detected microsomal epoxide hydrolase (mEH, EC3.3.2.3). EH is an enzyme that converts epoxide to diol by adding H2O to epoxide (7). mEH is expressed in hepatocytes and is considered to play an important role in detoxification of several substances (7). It was reported that this enzyme was also expressed on human adrenal gland, but its physiological role remains unclear (8). In 1977, Oesch et al. (9) reported that rat gonads contained relatively high activity of mEH, but the precise localization or function of this activity has not yet been investigated. In the present study, we confirmed the enzyme activity of mEH in human corpora lutea (CL) in the ovary and examined the precise localization of mEH in human follicles and CL of various developmental stages.
Recently, anticonvulsant sodium valproate and its analogues were revealed to inhibit EH enzyme activity (10, 11). It also has been reported that endocrine disorders, including polycystic ovary syndrome, are associated with the sodium valproate administration (12, 13). In contrast to the so-called polycystic ovary syndrome, these drug-induced disorders were not associated with a high level of serum LH, although the morphological changes of polycystic ovary and hyperandrogenism were exhibited (12, 13). At present, no definite mechanism has been proposed for the induction of polycystic ovary syndrome by sodium valproate. In this paper, to examine the physiological role of mEH in ovarian function in relation to the etiology of anticonvulsant drug-induced endocrine disorders, we also investigated the effect of an inhibitor for EH on steroid hormone production by human luteinizing granulosa cells in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Production and selection of mAb
Luteal cells were isolated as described, with minor
modification (14). Briefly, the CL were separated from connective
tissue, then minced with scissors. They were incubated in RPMI 1640
medium (Flow Labs., Irvine, Scotland, UK) containing 0.2%
collagenase (Wako Pure Chemical Industries Ltd.,
Osaka, Japan) and 0.01% deoxyribonuclease I (Sigma, St.
Louis, MO), at 37 C for 20 min, in a test tube. Tissue fragments were
then incubated in RPMI 1640 containing 10% FCS (Flow Laboratories,
McLean, VA), 1000 U/ml dispase (Godo Shusei, Tokyo, Japan), and 0.01%
deoxyribonuclease I, at 37 C for 30 min. After the undissociated
materials were discarded, the cell suspension was collected and
centrifuged for 5 min at 160 x g. Some of the cells
were washed twice and suspended in PBS and immediately used for the
immunization, and the rest were stocked in liquid nitrogen for further
immunization. Eight-week-old BALB/c mice were injected ip with 2
x 106 luteal cells every 3 weeks, for 9 weeks.
The spleen cells of an immunized mouse were fused with X63Ag8.653 mouse
myeloma cells, using 50% polyethylene glycol 1500 (BDH Chemicals,
England, UK) on the third day after the last immunization (15).
The fused cells were cultured in RPMI medium supplemented with 15% FCS
and 10% BM-condimed H1 (Boehringer, Germany), including HAT
(Flow Co., Scotland, UK), in 96-well plates. An indirect
immunofluorescence method, using frozen sections of human CL as
described below, was used for screening the supernatants of growing
hybridomas. The positive hybridomas were cloned twice and were injected
ip into mice previously treated with pristane
(2,6,10,14-tetramethylpentadecane, Tokyo Kasei Co., Tokyo, Japan). IgG
was purified from ascitic fluids with Affi-Gel protein A (Bio-Rad Laboratories, Inc., Richmond, CA). Ig isotype was determined
using an isotype kit for mouse mAbs (Serotec Ltd., Oxford,
UK).
Immunohistochemical examination of the antigen expression
on various follicles, CL, and other tissues
The following follicles and CL were used for examination
(Tables 1
and 2): primordial and primary follicles
(n = 10), preantral follicles (n = 2), small follicle (less
than 1 mm in diameter, n = 1), growing follicles (4–16 mm in
diameter, n = 7), preovulatory follicles (18 and 20 mm in
diameter, n = 2), atretic follicles (2–16 mm in diameter, n
= 6), CL on CL day 2 (the day after ovulation, n = 2), CL on day 3
(n = 2), CL on day 4 (n = 4), CL on day 5 (n = 2), CL on
day 6 (n = 3), CL on day 7 (n = 4), CL on day 8 (n = 2),
CL on day 9 (n = 4), CL on day 10 (n = 1), CL on day 11
(n = 2), CL on day 12 (n = 3), CL on day 13 (n = 1), CL
on day 14 (n = 2), and CL of pregnancy at 6, 7, 8, 9, 10, 13, 14,
and 15 weeks of gestation (n = 9). Follicles obtained in the
follicular phase with granulosa cells having mitotic figures and
regularly shaped nuclei, cytoplasm, and stratified layers were
classified as growing follicles. Follicles that were irregularly shaped
with blood cell invasion and lacked mitotic figures were classified as
atretic (16). If the above judgment was difficult with cryosections of
follicles, the hematoxylin and eosin stained sections from the
identical samples that were fixed with 10% formalin and embedded with
paraffin were used. The postovulatory date of CL was evaluated
according to the histological dating described by Corner GW, using
hematoxylin and eosin-stained tissue sections of 10% formalin-fixed
and paraffin-embedded samples (17). In this work, the term "corpus
luteum (CL) day" was used according to his definition. For example,
CL day 2 is the day after ovulation.
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Purification of HCL-3 antigen from human CL and porcine
liver
The HCL-3 antigen was purified from human CL and porcine
liver as described previously, with slight modification (2). One gram
of tissue was homogenized with Polytron (Kinematica AG, Luzern,
Switzerland) in 10 ml of ice-cold 40 mM phosphate buffer
(pH 7.3) containing 5 mM EDTA, 150 mM NaCl, 1%
CHAPS (cholamidopropyl-dimethylammonio-propanesulfonic acid, Wako Pure Chemical Industries Ltd.) and the protease inhibitors, 1
mM (p-amidinophenyl) methanesulfonyl fluoride
hydrochloride (Wako Pure Chemical Industries Ltd.), 10
µg/ml leupeptin, and pepstatin (Peptide Institute,
Osaka, Japan). After centrifugation (10,000 x g, 20
min, 4 C), the concentration of CHAPS in the lysate was reduced to
0.3%, by dilution. The lysate was passed through the column of
Affi-gel 10 that was conjugated with anti-TNP mouse IgG1 mAb (2 mg
IgG/ml gel). The lysate was then incubated with 0.2 ml Affi-gel 10 that
was conjugated with HCL-3 or anti-TNP mAb (2 mg IgG/ml gel) at 4 C for
3 h. After sufficient washing with the buffer, the antigen was
eluted with 0.5 M NH4OH
containing 0.1% nonidet P-40 (Iwai Chemicals, Tokyo, Japan).
The eluate was dried in vacuo at room temperature. The samples were
dissolved in lysis buffer with 0.1 M
dithiothreitol (Wako Pure Chemical Industries Ltd.) and
were separated by SDS-PAGE (12% gel). Proteins in the gel were
visualized by the silver stain kit (Wako Pure Chemical Industries Ltd.).
Partial amino acid sequencing of the 46-kDa protein
purified from porcine liver
Porcine liver (45 g) was homogenized with Polytron in 500
ml of 40-mM phosphate buffer containing 5 mM
EDTA, 150 mM NaCl, 1% CHAPS, and 1 mM
(p-amidinophenyl) methanesulfonyl fluoride hydrochloride, as
described above. After absorbing the nonspecific binding, the lysate
was incubated with 1 ml affi-gel 10 conjugated with HCL-3 mAb at 4 C
for 4 h. The antigen was eluted with 0.5 M
NH4OH containing 0.1% nonidet P-40. This
procedure was repeated three times. The purified antigen was dissolved
in lysis buffer for SDS-PAGE. After the electrophoresis, the proteins
in the polyacrylamide gel were transblotted onto polyvinylidene
difluoride (PVDF) membrane (Millipore Corp.,
Bedford, MA) in Tris/Boric acid buffer. The protein on PVDF
membrane was stained with Coomassie blue, and the 46-kDa protein band
was cut and analyzed with an amino acid sequencer PSQ-1 (Shimazu Co.,
Kyoto, Japan). The SWISS-PROT and GenBank data bases were used in the
analysis of amino acid sequence homology.
Preparation of microsomal fraction of CL and porcine
liver
Tissue (1 g) was homogenized in 5 ml PBS with Dounce
homogenizer. The homogenate was centrifuged at 10,000 x
g for 20 min, and the supernatant was centrifuged at
100,000 x g for 60 min. The precipitate was suspended
in 1 ml PBS and was used as microsomal fraction.
Assay of epoxide hydrolase activity
Enzyme activity was assessed by the production rate
of 7-(2',3'-dihydroxy) propoxycoumarin (DHC) from 7-glycidoxycoumarin
(GOC), as described by Inoue et al. (19). Fifty microliters
of the microsomal fraction and 925 µl of 173-mM
Tris HCl (pH 8.6) were incubated at 37 C for 5 min. In some
experiments, HCL-3 antigen-binding gel was used instead of microsomal
fraction. Twenty-five microliters of GOC (2 mM)
in acetone were added to the mixture and incubated at 37 C for 10 min.
The reaction was stopped by cooling in ice water. Five milliliters of
benzene were immediately added, and the mixture was centrifuged at
2,000 rpm for 5 min. The upper benzene fraction was discarded, and the
lower water fraction was stored. This extraction procedure was repeated
once more. The fluorescence intensity of the water-soluble fraction was
monitored by fluorescence spectrometer F-2000 (Hitachi Scientific Instruments, Inc., Tokyo, Japan) at excitation 325 nm and
emission 391 nm. DHC in methanol was used as a standard. GOC and DHC
were synthesized by the method of Watabe et al. (20) and
were given to us by Dr. T. Aimoto (Faculty of Pharmaceutical Science,
Setsunan University, Osaka, Japan). The protein concentration was
determined by the method of Lowry, and the amount of HCL-3 antigen on
the Affi-gel was determined by comparing the intensities of visualized
bands, by the silver staining method, after SDS-PAGE. BSA
(crystallized, Sigma) was used as a standard.
Culture of human granulosa cells
Human granulosa cells were isolated from patients who had
undergone treatment for in vitro fertilization-embryo
transfer, as described (5). Briefly, patients receiving a GnRH agonist
(buserelin acetate, Hoechst Marion Roussel, Inc. Ltd.,
Tokyo, Japan), beginning on the first day of the cycle, were
hyperstimulated by im injection of human menopausal gonadotropin (HMG,
Organon Japan Co. Ltd., Tokyo, Japan). Follicles were
aspirated 36 h after the administration of hCG (5000 IU, im,
Mochida Pharmaceutical Co. Ltd., Osaka, Japan). The suspension of
granulosa cells was overlayered on Ficoll-Hypaque and centrifuged at
400 x g for 30 min. The cells collected from the
interface were suspended in culture medium consisting of RPMI 1640
medium supplemented with 10% FCS, and 100 mg/ml kanamycin sulfate
(Meiji Seika Ltd., Tokyo, Japan). To reduce contamination by adherent
peripheral mononuclear cells, the collected cells were incubated, for
60 min at 37 C, in humidified air containing 5% CO2, in 60-mm diameter
plastic tissue culture dishes (Falcon, Becton Dickinson and Co., Lincoln Park, NJ) pretreated with autologous serum (21).
The cells were then collected after a mild wash, and viable granulosa
cells suspended in culture medium (1 x
105/ml, 0.1 ml/well) were inoculated into each
well of 96-well plates (Falcon; Becton Dickinson and Co.). The next day (day 1), the medium was discarded, to
remove unattached cells, and replaced with fresh medium [F-12/DMEM 1:1
(Life Technologies, Tokyo, Japan) containing 2
mg/ml BSA (Nitta Gelatin, Inc., Tokyo, Japan)]. The
culture media were changed on day 3, and then cells were cultured in
the presence or absence of 10-7
M testosterone and various concentrations of
1,2-epoxy-3,3,3-trichloropropane (ETCP, 0–500
µM, Sigma), which is a competitive
inhibitor of EH. The cultured media were collected on day 4 for steroid
hormone assay.
Expression of HCL-3 antigen on cultured granulosa cells
detected by indirect immunofluorescence
On days 1 and 4, the cultured granulosa cells in 8-chamber
slides (Lab Tec, Chamber Slide, Nunc Inc., Naperville, IL) were washed
gently three times with PBS containing 1 mM
CaCl2 and 1 mM
MgCl2, thoroughly dried, and fixed with acetone
at -20 C. The slides were indirectly stained using HCL-3 and anti-TNP
mAbs, as described above. Three independent experiments were
performed.
Assay of steroid production by human luteinizing
granulosa cells
The concentrations of progesterone and estradiol were measured
using RIA kits (Daiichi Pharmaceutical Company Ltd. Radio
Isotope Research Inc., Tokyo, Japan). Inter and intraassay coefficients
of variation were 6.5% and 5.3% for the progesterone assay and 7.4%
and 6.3% for the estradiol assay, respectively.
Effect of ETCP on the epoxide hydrolase activity of
cultured granulosa cells
The granulosa cells (1 x 105/ml, 0.3
ml/well) were inoculated into each well of 48-well plates (Coster,
Corning, Inc., New York, NY). Cells were cultured
as above; and, on day 3, various concentrations of ETCP were added to
each well. Eight microliters of GOC (2 mM) in
dimethylsulfoxide were added to the culture mixture. After 10 min at 37
C, the 48-well plates were cooled on ice cold water and 1 ml of benzene
were immediately added, and DHC production in the water fraction was
monitored by fluorescence spectrometer, as described above.
Effect of ETCP on aromatase activity in the granulosa cell
and CL homogenate
The enzyme activity of aromatase in the granulosa cell and
CL homogenate was measured by the method described previously, with
slight modification (22, 23). Granulosa cells (3 x
106) or CL tissue (50 mg) were homogenized in 2
ml of 40-mM phosphate buffer containing 0.25 M
sucrose and 1 mM EDTA with sonicator. The supernatant was
collected after centrifugation at 160 x g for 5 min,
and was diluted with buffer to 3 mg protein/ml. The above
buffer, containing ßnicotinamide adenine dinucleotide phosphate
(reduced form, ß-NADPH, 2.2 mM,
Sigma), dithiothreitol (2.2 mM), and
testosterone (1.1 x 10-7
M) was used as a substrate mixture for the
aromatase reaction. For enzyme assay, 10 µl of each cell homogenate
was added to the prewarmed substrate mixture (90 µl) containing ETCP
(0–500 µM) in the tube and was incubated at 37
C for 10 min. The tube was dipped into ice-cold water to stop the
reaction. The enzyme in the sample was then inactivated by boiling for
3 min. The concentration of estradiol in the reaction mixture was
assayed by RIA, as described above. The boiling procedure did not
affect the estradiol concentration detected by RIA assay. The estradiol
production during the incubation period increased linearly for 15 min,
indicating that the initial rate of aromatase enzyme activity was
measured in this condition.
Statistical analysis
The concentrations of progesterone and estradiol are given as
means ± SEM. The difference of progesterone and
estradiol production was analyzed by the one-way ANOVA, followed by
Scheffé’s F test. The difference was considered to be
significant at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HCL-3 antigen expression in the follicles and CL
In the primordial and primary follicles, HCL-3 antigen was not
detected. In the preantral follicles that have two or three layers of
granulosa cells, HCL-3 antigen was not detected in granulosa or theca
interna cells. On the other hand, in a small follicle less than 1 mm in
diameter that contained several layers of granulosa cells, HCL-3
antigen was well detected in the theca interna cells but not in the
granulosa cells (Fig. 1 and Table 1
). In
the growing (4–16 mm in diameter) and preovulatory follicles, HCL-3
antigen was highly expressed in the theca interna cells and was also
detected in the granulosa cells with weak intensity (Fig. 2). In the atretic follicles (2–16 mm in
diameter), HCL-3 antigen was expressed weakly in granulosa cells and
intensely in theca interna cells (data not shown).
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and Table 2). In the midluteal and late
luteal phase of the menstrual cycle, HCL-3 antigen was expressed
intensely in small luteal cells and moderately in large luteal cells
(Figs. 3 and 4). Similarly to menstrual
CL, HCL-3 antigen was highly expressed in small luteal cells and
moderately expressed in large luteal cells in the CL of pregnancy (Fig. 4).
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). The purification was
independently repeated four times with identical results.
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, A and B).
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).
Assay of epoxide hydrolase activity
Antigenic molecules were affinity-purified from the microsomal
fraction of human CL of the midluteal phase on HCL-3 mAb-conjugated
Affi-gel 10. A part of the gel was used for the assay of EH enzyme
activity; and the rest, for the determination of the amount of the
46-kDa protein. The EH enzyme activity of the protein, purified from
human CL, was 84 µM DHC/10 min·µg 46-kDa protein
(Table 3). The activity of the protein
purified from the microsomal fraction of human liver was 65
µM DHC/10 min·µg 46-kDa protein. The enzyme activity
of EH in the nonpurified microsomal fraction from human CL of midluteal
phase and from human liver was 0.039 and 0.105 µM DHC/10
min·µg microsomal protein, respectively. The activity of the
purified protein from porcine liver was 76 µM DHC/10
min·µg 46-kDa protein, and that of the nonpurified microsomal
preparation of porcine liver was 0.053 µM DHC/10
min·µg microsomal protein. HCL-3 mAb (0.1 mg/ml) or CHAPS (0.3%)
did not affect the enzyme activity of EH in the microsomal fraction
from human CL. In the control incubation with the proteins which bound
to anti-TNP-mAb-conjugated gels, slight production of DHC was also
observed, but it did not differ from the DHC production in the control
buffer, indicating that the proteins associated with anti-TNP mAb had
no significant EH activity.
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The progesterone and estradiol production of granulosa cells
cultured with ETCP was assayed. Estradiol production was
significantly suppressed by ETCP at 100 µM or more (Fig. 7A). Progesterone production was also
reduced by ETCP, but only slightly (Fig. 7B).
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). The EH activity was
effectively inhibited by ETCP in a dose-dependent manner in these
homogenates (Fig. 8B).
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). Some cultured granulosa cells
were disrupted by sonication, and then GOC and ETCP was added
simultaneously. ETCP inhibited the enzyme activity in dose-dependent
manner similar to that of living cells. The enzyme activity was
monitored 5 or 24 h after the addition of ETCP. ETCP was not
effective at 20 µM or less in these conditions and
exhibited the inhibitory activity at higher concentrations (Fig. 9).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mEH is an enzyme that converts epoxide to diol by adding H2O to it, and is widely distributed not only in mammals, but also in other animals (7). mEH in the liver has been reported to be important for inactivation of most carcinogenic or cytotoxic electrophilic epoxide (7). Substrates for this enzyme include epoxide derivatives of certain pharmaceuticals, such as metabolites of phenytoin, carbamazepine, and other antiepileptic medications (30, 31). Epoxides of environmental toxins, such as the carcinogenic polyaromatic hydrocarbon, aromatic amines, and benzene, were also reported to be its substrates (32, 33, 34).
The study described here immunohistochemically demonstrated that
mEH was expressed on the steroid-hormone producing cells in the human
ovary. These findings imply that mEH may be involved in
steroidogenesis. mEH was reportedly expressed on cortical cells of
human adrenal gland (35). Papadopoulos et al. examined the
effect of mEH on steroid hormone synthesis using microsomal fractions
isolated from adrenal gland with 14C-labeled
progesterone. They found no differences in the steroid hormone
production by the treatment of inhibitors, concluding that the
physiological role of mEH in the adrenal gland remained unclear (8). We
could not examine the physiological role of mEH in theca interna cells
or small luteal cells in this experiment. A large amount of theca
interna cells or small luteal cells from human ovary sufficient for the
in vitro culture experiment cannot be obtained, and the
theca interna cells from porcine ovary cultured in vitro for
3 days did not maintain the expression of mEH in the cells (not shown).
The granulosa cells, on the other hand, are well known to have
aromatase enzyme activity like the hepatocytes. The present study
showed that mEH inhibitor, ETCP, reduced estradiol production from
testosterone in cultured granulosa cells (Fig. 7A). However, in the
preparation of granulosa cell homogenate, the inhibitor effectively
suppressed mEH activity (Fig. 8B) but not aromatase activity (Fig. 8A).
It shows that the inhibitor has no direct effect on the enzyme activity
of aromatase, and that granulosa cells indirectly require mEH activity
for the production of estradiol.
The concentration of ETCP required for the effective inhibition
of estradiol production by the cultured granulosa cells (Fig. 7A) was
very much higher than that for the inhibition of EH activity in the
granulosa homogenate (Fig. 8B). We then examined the change of
inhibitory effect of ETCP on EH activity during the granulosa cell
culture period (Fig. 9). ETCP, which was added to the cell culture
simultaneously with the enzyme substrate, GOC, effectively inhibited
the EH activity in the low concentrations. ETCP in the cell homogenate
disrupted by sonication also inhibited EH activity in the same
dose-dependent manner as in the living cells, which shows that GOC and
ETCP were cell membrane-permeable enough for the detection of enzyme
activities and inhibitory effects in the condition described here.
ETCP, added 5 or 24 h before the assay of enzyme activities, on
the other hand, inhibited the EH activity only in the high
concentrations, which was similar to the inhibitory effect shown in the
estradiol production during 24 h of incubation (Fig. 7A). In
conclusion, ETCP was shown to inhibit the EH activity and the estradiol
production of the granulosa cells in a similar dose-dependent
manner.
Anticonvulsant sodium valproate is reported to induce endocrine
disorders such as polycystic ovary syndrome. In the drug-induced
polycystic ovary syndrome, an elevated serum androgen level, irregular
menstruation, and polycystic morphological change of the ovary are
observed. Sodium valproate is known to interfere with the metabolism of
-aminobutyric acid (GABA) and is expected to increase the GABA
concentration in the central nervous system. It was therefore suspected
that sodium valproate activates LH secretion through GABA systems and
that the impaired LH secretion induces the hypersecretion of androgen
by theca interna cells concomitant with polycystic change of the ovary.
However, subsequent clinical studies did not support this explanation.
Elevation of the serum LH level was not observed in the
valproate-induced endocrine disorders (12, 13). The administration of
sodium valproate, in normal women, was shown to induce no alteration of
the basal secretion and the pulsatility of LH in both follicular and
luteal phases (36, 37). In patients with polycystic ovary syndrome,
sodium valproate administration also produced no significant change in
the frequency and amplitude of LH and FSH secretion (38). Furthermore,
it is reported that acute administration of sodium valproate suppresses
the LH secretion (39). In ovariectomized women, valproate did not
affect the pulsatile secretion of LH (40). Then, the effect of sodium
valproate on the induction of polycystic ovary syndrome would not be
caused by the alteration in the central nervous system through the GABA
metabolism. Some investigators suspected that sodium valproate may
inhibit conversion of testosterone to estradiol by an unknown mechanism
(38). Sodium valproate and its analogues were reported to inhibit EH
enzyme activity (10, 41). The present study showed that granulosa cells
in the follicles of more than 4 mm in diameter express mEH and that the
inhibition of mEH activity suppresses the conversion of testosterone to
estradiol by granulosa cells cultured in vitro. If sodium
valproate inhibits the mEH activity and then suppresses the aromatase
activity in granulosa cells, testosterone produced by theca interna
cells is not converted to estradiol in the follicles. This leads to the
androgen-dominant microenvironment in the ovary and then to polycystic
change of the ovary. This interpretation may explain the overall status
of valproate-induced polycystic ovary syndrome, where an elevated serum
testosterone concentration without an increase of peripheral LH and
estradiol levels are manifested. It is also possible that mEH in
theca interna cells regulates the steroidogenesis via the epoxide
degradation, and that it leads indirectly to the suppression of the
estradiol production by the granulosa cells.
In the small follicles, the protection of oocytes from adverse agents is important to preservation of healthy follicles. Although the basal lamina surrounding granulosa cells may serve as a barrier for macromolecules, it does not block low-molecular-weight reagents. In the initial developmental follicles, we observed the expression of LH receptor (42) and low-density lipoprotein receptor (43) on the theca interna cells. In addition, apolipoprotein-B was detected in the cytoplasm of theca interna cells in the small follicles less than 1 mm in diameter, suggesting the continuous uptake of low-density lipoprotein and steroid hormone synthesis by these cells (44). mEH in the liver is reported to inactivate the carcinogenic or cytotoxic electrophilic epoxides produced by the cytochrome P-450-dependent monooxygenase system (7). Interestingly, mEH was clearly detected on the theca interna cells in a small follicle with several granulosa cell layers. It is suggested that toxic substances would be inactivated by mEH, to maintain homeostasis of the microenvironment in the immature follicles, which protects oocytes from damage by some epoxides. It is also possible that a large amount of mEH found in theca interna cells in large follicles and in small luteal cells in CL inactivates the cytotoxic epoxides that were produced during the metabolic pathway of steroidogenesis.
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Acknowledgments |
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Footnotes |
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* This work was supported, in part, by grants-in-aid for scientific research (09671673, 09671674, and 09671676).
Received December 13, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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6 is a differentiation antigen of human granulosa cells.
J Clin Endocrinol Metab 80:2899–29056ß1 in ovarian functions. Horm Res [Suppl 2] 50:25–29
) function of murine B cells. J Immunol 144:4571–4578[Abstract]
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