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Original Studies |
Department of Obstetrics and Gynecology, Yamaguchi University School of Medicine, Ube 755-8505, Japan
Address correspondence and requests for reprints to: Norihiro Sugino, M.D., Department of Obstetrics and Gynecology, Yamaguchi University School of Medicine, Minamikogushi 1-1-1, Ube 755-8505, Japan. E-mail: obgyn{at}po.cc.yamaguchi-u.ac.jp
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials
RPMI 1640 was from Flow Laboratories Inc. (McLean, VA).
Streptomycin, penicillin, deoxynucleotide triphosphates, and Moloney
murine leukemia virus reverse transcriptase were from Life Technologies, Inc. (Grand Island, NY). hCG (catalog no. C1063)
was from Sigma (St. Louis, MO). Random hexamer and
Taq DNA polymerase were from Perkin-Elmer Corp.
(Foster City, CA). [
-32P]deoxycytidine
triphosphate was from Amersham Pharmacia Biotech
(Arlington Heights, IL). Isogen was from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
Tissue samples
Corpora lutea were obtained at hysterectomy from normally cycling women, 39–49 yr of age, who underwent surgery for myoma uteri or cervical cancer. The menstrual history and the endometrial dating diagnosed histologically according to the criteria of Noyes et al. (18) were used to determine the age of the CL. Corpora lutea of the cycle were classified into four different groups according to their age: early luteal phase (days 14–18), midluteal phase (days 19–24), late luteal phase (days 25–28), and regression phase (days 3–7), with day 1 being the day of the onset of menstruation. Corpora lutea of early pregnancy (6–8 weeks) were obtained from the patients, 24–30 yr of age, with ectopic pregnancy. Tissue samples were washed with saline to remove blood and immediately frozen in liquid nitrogen and stored at -80 C until RNA isolation and Western blot analysis. In some patients, blood samples were obtained at surgery for determination of serum progesterone concentrations.
Immunohistochemistry
The immunohistochemical staining was performed on two to four tissue samples from the midluteal phase, regression phase, and early pregnancy. Corpora lutea were fixed in Carnoy solution and embedded in paraffin and sectioned (8 µm thick). The tissue sections were deparaffinized in xylene and dehydrated in a graded series of ethanol. Immunohistochemistry for VEGF and flt-1 was performed with a peroxidase-antiperoxidase method (DAKO PAP kit; DAKO Corp., Tokyo, Japan) using rabbit antihuman VEGF polyclonal antibodies or rabbit antihuman flt-1 polyclonal antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After inhibition of endogenous peroxidase activity with 0.3% H2O2 for 50 min, the sections were incubated with 10% normal swine serum for 10 min at room temperature to avoid nonspecific binding. The sections were then incubated with the primary antibodies, at a dilution of 1:50 in PBS-BSA (1%) overnight at 4 C. After three washes with PBS for 5 min each, the sections were incubated with swine antirabbit immunoglobulin for 30 min at room temperature, washed three times with PBS for 5 min each, and reacted with rabbit-peroxidase-antiperoxidase for 40 min at room temperature. Immunohistochemistry for KDR was performed with a streptavidin-biotin-peroxidase complex method (SAB-PO kit; Nichirei Co. Ltd., Tokyo, Japan) using mouse anti-human KDR monoclonal antibodies (Santa Cruz Biotechnology, Inc.). After inhibition of endogenous peroxidase activity with 0.3% H2O2 for 50 min, the sections were incubated with 10% normal rabbit serum for 10 min at room temperature to avoid nonspecific binding. The sections were incubated with the primary antibody at a dilution of 1:50 in PBS-BSA (1%) overnight at 4 C. After three washes with PBS for 5 min each, the sections were incubated with biotinylated rabbit antimouse immunoglobulin for 10 min at room temperature, washed three times with PBS for 5 min each, and reacted with peroxidase conjugated streptavidin for 5 min at room temperature. Peroxidase activity was visualized by incubating the sections with 3, 3' diaminobenzidine•4HCl (Nacalai Tesque Co. Ltd., Tokyo, Japan) in 0.05 M Tris-HCl buffer (pH 7.6) containing 0.01% H2O2 for 2–3 min. Control sections were incubated with normal rabbit serum for VEGF and flt-1, normal mouse serum for KDR, or PBS. Counterstaining was performed with Meyer’s hematoxylin.
Incubation of corpora lutea
Corpora lutea obtained from the midluteal phase were sliced into small pieces and incubated in serum-free RPMI 1640 (35–60 mg wet weight/mL·tube) at 37 C for 1 h under an atmosphere of 95% O2:5% CO2 in a shaking water bath. The medium was then changed to the test medium containing hCG (1 IU/mL), and the incubation was continued for 6 h under the same atmosphere, as described above. After incubation, the CL tissue was immediately frozen in liquid nitrogen and stored at -80 C until RNA isolation and Western blot analysis. The incubation was run in triplicate.
RT-PCR
Total RNA was isolated from the corpora lutea with Isogen
by the method provided by the manufacturer. For mRNA analysis, RT-PCR
was performed as reported previously (16) with the oligonucleotide
primers for VEGF (5'-CACATAGGAGAGATGAGCTTC-3' and
5'-CCGCCTCGGCTTGTCACAT-3'), for flt-1
(5'-CTAGGATCCGTGACTTATTTTTTCTCAACAAGG-3' and
5'-CTCGAATTCAGATCTTCCATAGTGATGGGCTC-3'), and for KDR
(5'-CGTGGATCCACCAAAGGGGCACGATTCCGTC-3' and
5'-CTCGAATTCTGTAACAGATGAGATGCTCCAAGG -3'), designed by Athanassiades
et al. (19). Direct sequence analyses of the PCR products
were performed for sequence verification. Two oligonucleotide primers
(5'-CTGAAGGTCAAAGGGAATGTG-3' and 5'-GGACAGAGTCTTGATGATCTC-3') were also
used to amplify ribosomal protein L19 as an internal control (20). In
brief, 3 µg total RNA were reverse-transcribed at 42 C in a reaction
mixture (single-strength PCR buffer, 2.5 mM
deoxynucleotide triphosphate, 5 µM random
hexamer primer, 1.5 mM
MgCl2, and 200 U Moloney murine leukemia virus
reverse-transcriptase). The RT product was aliquoted equally into two
tubes for VEGF or flt-1 or KDR primers and L19 primers, and PCR was
performed. For PCR amplification, a mixture containing the
oligonucleotide primers (50 pmol),
[-32P]deoxycytidine triphosphate (2 µCi at
3000 Ci/mmol), and Taq DNA polymerase (2.5 U) was added to
each reaction. Amplification was carried out for 25 cycles consisting
of 94 C (1 min), 57 C (1 min) and 72 C (1 min) for VEGF, 28 cycles
consisting of 94 C (1 min), 60 C (1 min) and 72 C (1 min) for flt-1 and
25 cycles consisting of 94 C (1 min), 60 C (1 min) and 72 C (1 min) for
KDR, followed by 10 min of final extension at 72 C in a programmed
temperature control system PC-800 (ASTEC, Fukuoka, Japan). The
predicted sizes of the PCR-amplified products were 98 bp for VEGF121,
228 bp for VEGF165, 230 bp for flt-1, 209 bp for KDR, and 194 bp for
L19. A linear curve was plotted using number of cycles of amplification
vs. densitometric values of the PCR products, measured with
a BAS2000 (Fuji Photo Film Co., Ltd., Tokyo, Japan). The
optimal number of cycles for amplification that fit within the linear
range was chosen for each sets of primers of VEGF, flt-1, KDR, and L19
(data not shown). Reaction products were electrophoresed on an 8%
polyacrylamide nondenaturing gel.
Western blot analysis
Corpora lutea were homogenized with distilled water and centrifuged at 800 x g for 10 min at 4 C. The supernatant was used for Western blot analysis. Eighty micrograms of protein of the supernatant, determined by the Lowry’s method (21), was loaded in each sample and separated by SDS-PAGE in 15% gels for VEGF and in 7.5% gels for flt-1 and KDR under reduced conditions. The proteins on the gel were electrophoretically transferred to nitrocellulose membranes and reacted with antibodies used in the immunohistochemistry at a dilution of 1:50 with 0.5% skimmed milk in Tris-buffered saline (pH 7.5). The membranes were then immersed in the reaction buffer containing peroxidase-conjugated swine antirabbit immunoglobulin for VEGF and flt-1 or peroxidase-conjugated rabbit antimouse immunoglobulin for KDR. The reacted band was developed on a film with ECL kit (Amersham Pharmacia Biotech, Buckinghamshire, UK).
Progesterone assay
Progesterone concentrations in the serum and medium were determined by a specific RIA, as reported previously (22). The sensitivity of the assay was 100 pg/mL, and the intra- and interassay coefficients of variation were 7.0% and 14.4%, respectively.
Statistical analysis
Data were examined by ANOVA and Duncan’s new multiple range test. Where appropriate, Student’s t test was used. Differences were considered significant at P less than 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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VEGF immunostaining was localized in luteal cells, with strong
immunostaining at the midluteal phase and in early pregnancy (Fig. 1
, A and C), whereas luteal cells of the
regressing CL showed weak immunostaining (Fig. 1B). Negative control
sections for VEGF were consistently free from staining (Fig. 1D).
Immunostaining of flt-1 and KDR was localized in luteal cells and in
vascular endothelial cells, with strong immunostaining at the midluteal
phase and in early pregnancy (Fig. 1, E, G, I, and K), whereas the
luteal cells of the regressing CL showed weak immunostaining (Fig. 1, F
and J). Negative control sections for flt-1 and KDR were consistently
free from staining (Fig. 1, H and L). No difference was observed in the
intensity of those immunostaining in the luteal cells between granulosa
lutein cell layer and theca lutein cell layer.
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shows a representative
autoradiogram of RT-PCR for VEGF mRNA expression in corpora
lutea prepared from early luteal phase (n = 3), midluteal phase
(n = 4), late luteal phase (n = 3), regression phase (n
= 5), and early pregnancy (n = 5). To date, four VEGF splice
variants of 121, 165, 189, and 206 amino acids have been identified in
human tissues (4). Using RT-PCR, strong bands corresponding to VEGF121
and VEGF165 were detected in the human CL (Fig. 2). The mRNA expression
of both isoforms of VEGF in the CL remained constant from
the early luteal to late luteal phase and was lower in the regression
phase (Fig. 2). In the pregnant CL, VEGF mRNA expression was
higher compared with that in other luteal phases (Fig. 2).
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flt-1 mRNA expression was detected at constant levels from
the early luteal to late luteal phase and lower in the regressing CL
(Fig. 3A). In the pregnant CL,
flt-1 mRNA expression was the same level as that in the
midluteal phase (Fig. 3A). KDR mRNA expression tended to be
higher in the early luteal phase (Fig. 3B), but there was no
significant difference when the intensity of the KDR signals measured
by a bioimaging analyzer BAS2000 (Fuji Photo Film Co., Ltd.) was normalized to that of the internal control L19 and
analyzed semiquantitatively (data not shown). The regressing CL showed
lower KDR mRNA expression compared with other luteal phases
(Fig. 3B). In the pregnant CL, KDR mRNA expression was the
same level as that in the midluteal phase (Fig. 3B).
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shows representative immunoblots
for VEGF, flt-1, and KDR protein in corpora lutea obtained from
midluteal phase (n = 4), regression phase (n = 4), and early
pregnancy (n = 5). VEGF was highly expressed in the pregnant CL
compared with the midluteal phase CL (Fig. 4A). In contrast, VEGF
expression was remarkably low in the regressing CL (Fig. 4A). flt-1 and
KDR were barely detectable in the regressing CL, but they were clearly
expressed in the midluteal phase and early pregnancy (Fig. 4, B and C).
There was no significant difference in the expression of flt-1 and KDR
between midluteal phase and early pregnancy when the reacted bands of
flt-1 and KDR were scanned and their optical densities were measured by
NIH Image (data not shown).
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shows a representative autoradiogram
of RT-PCR and hCG increased the mRNA expression of both isoforms of
VEGF. Figure 5B shows a representative immunoblot and hCG increased
VEGF protein expression. Progesterone concentrations in the medium were
also significantly (P < 0.01) increased by hCG
(control, 3.0 ± 0.2 ng/mg wet wt; hCG, 5.7 ± 0.7 ng/mg wet
wt; mean ± SEM, n = 3).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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hCG has been reported to be involved in the rescue of the CL when pregnancy occurs (11, 14, 16). The present study showed that VEGF expression in the pregnant CL was higher than that in the midluteal phase CL and that VEGF expression was increased by hCG in vitro. This is consistent with the report showing that VEGF expression in human luteinized granulosa cells was enhanced by increasing amounts of hCG, with maximum enhancement at 1 IU/mL (7). These findings may suggest that hCG maintains luteal function via the increase in VEGF expression when pregnancy occurs. As for the mechanism that hCG prolongs the CL function, Christenson and Stouffer (24) showed that no significant increase in proliferation of endothelial cells was observed in the primate CL maintained by hCG, and they suggested that endothelial cell proliferation is not a critical factor in the rescue of the CL by hCG. It has also been reported that VEGF is expressed in some tissues that are not undergoing active angiogenesis (30). VEGF may function to increase vascular permeability, which in turn facilitates not only the supply of large amounts of cholesterol required for progesterone synthesis but also the delivery of progesterone to the circulation when pregnancy occurs.
In the present in vitro study, the effects of hCG were small, although there were significant differences when the band intensities of RT-PCR and Western blotting were analyzed. This may be because there was no protein included in the incubation medium to prevent nonspecific adsorption of hCG to the incubation vessel.
The present study also showed that two VEGF receptors were expressed in luteal cells and that the expression of those receptors was high in the CL during the luteal phase and in early pregnancy compared with the regression phase, suggesting that VEGF may exert a paracrine-autocrine role in regulating luteal function. Expression of VEGF receptors has been reported in a variety of nonendothelial cells (31, 32, 33, 34). It is of interest to note that VEGF can act as a survival factor and inhibit apoptosis (35, 36). Recent evidence has suggested that apoptosis plays important roles in the determination of luteal life span in humans (37, 38). The high expression of VEGF in the pregnant CL, the induction of VEGF expression by hCG, and the decline of VEGF, flt-1, and KDR expression in the regressing CL suggest an additional possibility that VEGF may be involved in the prolongation of the life span of the CL as a survival factor when pregnancy occurs.
In conclusion, the present study suggests that VEGF and its receptors play important roles in the development and function of the CL and that VEGF may exert a paracrine-autocrine role in regulating luteal function. hCG may act to prolong the life span of the CL by stimulating VEGF expression when pregnancy occurs. However, additional studies are needed regarding the differential roles of VEGF in the regulation of angiogenesis, vascular permeability, and apoptosis in the human CL.
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Footnotes |
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Received December 22, 1999.
Revised February 25, 2000.
Revised June 6, 2000.
Accepted June 20, 2000.
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: balance between growth and death
signals. J Mol Cell Cardiol. 29:1321–1330.[CrossRef][Medline]
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