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Original Studies |
Department of Obstetrics and Gynecology, Wakayama Medical College, 811–1 Kimiidera, Wakayama 641-0012, Japan
Address all correspondence and requests for reprints to: Dr. Ryosuke Nakano, Department of Obstetrics and Gynecology, Wakayama Medical College, 811–1 Kimiidera, Wakayama 641-0012, Japan. E-mail: ryosuken{at}wakayama-med.ac.jp
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Numerous candidates for potential regulators of angiogenesis have
been listed, including acidic fibroblast growth factor, basic
fibroblast growth factor, transforming growth factor-
, angiogenin,
and interleukin-8 (2). Although these molecules are able to promote
angiogenesis, it has been difficult to correlate such activity with the
physiological or pathological regulation of blood vessel growth.
Recently, many studies have been performed on vascular endothelial
growth factor (VEGF), a new potent regulator of angiogenesis. VEGF,
also known as vascular permeability factor, is a homodimeric
heparin-binding glycoprotein that has potent angiogenic activity (3).
In addition to stimulating endothelial cell growth in vitro,
VEGF is angiogenic in vivo and induces increased blood
vessel permeability (4). The sequence of the complementary DNA (cDNA)
and genomic clone of VEGF has revealed the existence of multiple
isoforms containing 206-, 189-, 165-, 145-, and 121-amino acid
residues. Human VEGF gene is composed of 8 exons, and these isoforms
arise from the same gene by alternative splicing (5). The VEGF isoforms
are distinguished by the presence or absence of the peptide encoded by
exons 6 and 7 of the VEGF gene. These isoforms appeared to have similar
biological activities in vitro.
VEGF works via two tyrosine kinase family receptors, fms-like tyrosine kinase (Flt-1) and kinase insert domain-containing receptor tyrosine kinase (3, 6). These receptors are structurally similar and have seven Ig-like domains in the extracellular region, a single transmembrane region, and a consensus tyrosine kinase sequence that is interrupted by the kinase insert domain. As assessed by Northern blot analysis, these receptors are expressed in vascular endothelial cells (6).
Several studies have indicated that VEGF is expressed in intact human ovaries (7) and in luteinized granulosa cells aspirated during in vitro fertilization oocyte retrieval (8). More recently, VEGF has been reported to be localized in human CL (9). However, little is known about the expression of VEGF and Flt-1 in human ovaries during the reproductive cycle. In this study, we examined the expression and localization of VEGF and Flt-1 in human ovaries and the concentrations of VEGF in lutein extracts to evaluate the potential role of VEGF in the ovarian physiology.
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Subjects and Methods |
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Subjects were chosen from patients who were admitted to Wakayama Medical College Hospital from June 1996 to December 1998. Human ovaries were obtained from 28 patients (aged 30–48 yr) with regular menstrual cycles who were undergoing abdominal hysterectomy for cervical cancer. The gestational CL were obtained from 14 patients with ectopic pregnancy and 3 normal pregnant women with ovarian tumor at 7–14 weeks of pregnancy. None of the patients received any hormone therapy before operation. The project was approved by the committee on investigations involving human subjects of Wakayama Medical College. Informed consent was obtained from each patient after the purpose and nature of the study had been fully explained. Six ovaries were obtained during the follicular phase, and 22 ovaries with CL were obtained during the luteal phase of the menstrual cycle. The histological features of the CL and the menstrual history of each subject were used to determine the phase of the menstrual cycle. Three luteal phases relative to an ideal 28-day cycle were used: 1) early luteal phase (ELP), cycle days 15–19, 7 cases; 2) midluteal phase (MLP), cycle days 20–23, 6 cases; and 3) late luteal phase (LLP), cycle days 24–28, 9 cases. The weeks of pregnancy were determined by menstrual history and ultrasound sonography.
Immunohistochemistry for VEGF and Flt-1
Ovarian tissues were rinsed in ice-cold saline and fixed in Bouin’s solution for 48 h at 4 C. The tissues were embedded in paraffin, cut into 5-µm sections, and mounted onto gelatin-precoated slides. The immunohistochemical procedure was performed by the avidin-biotin-peroxidase complex technique using a Vectastain ABC kit (Vector Laboratories, Inc., Burlingame, CA). After deparaffinization, sections were incubated with the antibody against VEGF (0.5 µg/mL) or Flt-1 (0.2 µg/mL). The primary antibody against VEGF was a polyclonal antibody directed against the amino-terminal epitope of human VEGF. The antibody against Flt-1 was a polyclonal antibody directed against the receptor molecules (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After the incubation with biotinylated goat antirabbit IgG and avidin-biotin-peroxidase complex, antigen-antibody complexes were visualized with diaminobenzidine as a chromogen. The sections were lightly counterstained with Mayer’s hematoxylin. The appearance of brown reaction products was observed under a light microscope. The endothelial cells in the ovarian tissue were stained with a monoclonal antibody against CD34 (Nichirei, Tokyo, Japan) at a concentration of 1 µg/mL. For negative controls, the primary antibodies were used after preabsorption with an excess of synthetic peptides of human VEGF or Flt-1. To analyze the intensity of the brown staining, images were imported into a personal computer (G3 Power Macintosh; Apple Computer Inc., CA) using a digital camera (Fujifilm, Tokyo, Japan) and a microscope (Olympus Corp., Tokyo, Japan). Images were analyzed using Photoshop (version 4, Adobe System, San Jose, CA) as previously described (10). The values of each phase were compared by ANOVA and Student’s unpaired t test, and P < 0.05 was considered statistically significant.
Measurement of VEGF concentrations in human CL
Luteal extracts were prepared by homogenizing of CL in 10 volume of chilled phosphate-buffered saline containing 1 mmol/L phenyl-methylsulfonylfluoride and 20 IU/ml aprotinin by glass homogenizer for 2 min to ensure efficient extraction. After centrifugation at 10,000 x g for 15 min at 4 C, supernatants were aspirated and stored at -20 C until assays for VEGF.
VEGF concentrations of lutein extracts were measured by a two-site enzyme-linked immunosorbent assay according to the protocol obtained from the manufacturer (Quantikine Human VEGF Immunoassay, R & D Systems, Inc., Minneapolis, MN). The assay employed the quantitative sandwich enzyme immunoassay technique with precoated monoclonal antibody and polyclonal antibody against recombinant human VEGF. A 96-well plate reader (Multiskan MS, Labsystems, Helsinki, Finland) set to read 450 nm emission was used to quantitate assay results. According to the manufacturer’s instruction, the assay limit of this kit was 5.0 pg/mL. The mean intra- and interassay variations were 4.7% and 6.7%, respectively. The protein concentrations of lutein extracts were measured by a Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Inc., Hercules, CA) at 595 nm.
RT-PCR
Human CL obtained in the MLP were dissected from surrounding tissues and rinsed in ice-cold saline. The tissues were immediately frozen in a dry ice-ethanol bath and stored at -80 C until further use. Total ribonucleic acid (RNA) was extracted from frozen human CL using STAT-60 (Tel-Test, Inc., Friendswood, TX). cDNA was synthesized using reverse transcriptase and random hexamer from 2 µg total RNA (First-Strand cDNA synthesis kit, Pharmacia Biotech, Bucks., UK). One third of an aliquot of cDNAs was amplified by 35 cycles (denaturing at 94 C for 45 s, annealing at 62 C for 45 s, and elongation at 72 C for 90 s), using the two VEGF-specific primers (5'-tcgggcctccgaaacctga-3' and 5'-cctggtgagagatctggttc-3') or the two Flt-1-specific primers (5'-ttgctgagcataaaacagtc-3' and 5'-tccgcagtaaaatccaagta-3'; GeneAmp PCR system 9700, Perkin Elmer Corp., Emeryville, CA). PCR products were separated on a 1.0% agarose gel containing ethidium bromide.
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Results |
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In primordial follicles, flattened granulosa cells were not
stained with antibody against VEGF, whereas oocytes were weakly stained
(Fig. 1A
). In small antral follicles, the
periantral granulosa cells (those that faced the follicular antrum) and
oocyte showed weak cytoplasmic staining for VEGF (Fig. 1B). We did not
detect the staining in thecal cells of small antral follicles (Fig. 1B). The immunostaining for VEGF was observed in the cytoplasm of
granulosa and thecal cells of medium-sized follicles. The intensity of
the staining in thecal cells was more intense than that in granulosa
cells (Fig. 1C). In preovulatory follicles, distinct staining for VEGF
was observed in granulosa and thecal cells (Fig. 1D). We observed clear
immunostaining for Flt-1 in the cytoplasm of thecal cells and to a
lesser degree in granulosa cells of preovulatory follicles (Fig. 1E).
However, endothelial cells, which were identified by the staining for
CD34 (Fig. 1F), were faintly stained with Flt-1 (Fig. 1E). We also
observed faint staining for Flt-1 in granulosa cells of small antral
follicles and in granulosa and thecal cells of medium-sized follicles
(data not shown).
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). Lutein cells
in the MLP exhibited distinct staining for VEGF (Fig. 2D). Staining for
Flt-1 was detected in the cytoplasm of lutein cells during the ELP
(Fig. 2B) and MLP (Fig. 2E), whereas it was weak in endothelial cells
of microvessels in the CL. The intensity of the staining for Flt-1 was
more intense in thecal lutein cells than in granulosa lutein cells
(Fig. 2, B and E). Endothelial cells were stained with CD34. A
developing capillary network was observed in the luteinized granulosa
lutein layer of the CL. (Fig. 2, C and F).
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) and in early pregnancy (Fig. 3D). The
intensity of the staining in gestational CL was stronger than that in
the LLP. The staining for Flt-1 was detected in the cytoplasm of
granulosa and thecal lutein cells during the LLP (Fig. 3B) and in early
pregnancy (Fig. 3E), whereas it was weak in the endothelial cells of
microvessels in the CL (Fig. 3, B and E). Endothelial cells were
identified by positive staining for CD34 (Fig. 3, C and F). When we
stained tissue sections with preabsorbed antibodies, we did not observe
any staining for VEGF (Fig. 3G) or Flt-1 (Fig. 3H) in the CL during the
MLP. We analyzed the intensity of staining for VEGF in CL using
Photoshop-based image analysis. The results of the mean ±
SD were 145.6 ± 39.6 in ELP, 199.8 ±
25.9 in MLP, 137.1 ± 39.1 in LLP, and 179.1 ± 24.0 in
gestational CL. The intensity of staining in MLP tended to be higher
than that in ELP, but not significant. On the other hand, it was
significantly high compared to that in LLP. The staining intensity in
gestational CL was significantly high compared to that in LLP.
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Serial dilution of representative lutein extracts generated
dose-response curves parallel to that of recombinant human VEGF165
(Fig. 4). The VEGF concentrations of
lutein extracts during the luteal phase and early pregnancy are shown
in Table 1. The VEGF concentrations of
lutein extracts were moderately high in the ELP and MLP, and they
tended to be decreased in the LLP. A measurable amount of VEGF was
detected in the gestational CL.
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We examined the expression of VEGF and Flt-1 in the CL obtained in
the MLP using specific primers for human VEGF and Flt-1 by RT-PCR. VEGF
primers were designed to amplify all splice variants that may be
expressed in human CL. PCR products of VEGF from the CL showed two
bands, migrating at 517 and 649 bp, which corresponded to VEGF121 and
VEGF165, respectively. The expression of Flt-1 messenger RNA (mRNA) was
demonstrated as RT-PCR products migrating at 565 bp (Fig. 5).
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Discussion |
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Developing ovarian follicles accumulate follicular fluid, at least a portion of which is derived from plasma (13). VEGF secreted by thecal cells may render the thecal microvascular hyperpermeable, leading to the increased extravasation of plasma and the accumulation of antral fluid in growing follicles (9). Several reports (14, 15) indicate the involvement of VEGF in the pathogenesis of ovarian hyperstimulation syndrome (OHSS). It has been reported that VEGF production by luteinized granulosa cells was enhanced by hCG in vitro (16, 17). Levin et al. (15) demonstrated that VEGF in the follicular fluid collected from patients with OHSS enhanced vascular permeability in vitro, and the effect was reversed by the VEGF antibody. Therefore, VEGF might contribute to the pathogenesis of OHSS.
It has been reported that VEGF stimulates the expression of urokinase and tissue-type plasminogen activator (PA) and plasminogen activator inhibitor in endothelial cells (18). For vascular formation, it is necessary to protrude endothelial cells beyond the vascular basement membrane. Collagenase, which is activated by plasmin, the product of PA action on plasminogen, contributes to the degradation of basement membrane (19). The same scenario has been reported when an ovarian follicle ovulates. PA converts plasminogen into plasmin, and the proteolytic cascade participates in ovulation (20). In humans, PA activity was demonstrated in granulosa cell lysates, and gonadotropin stimulation increased tissue-type PA expression in granulosa cells (21). In the present study, the immunolocalization of Flt-1 was observed in granulosa and thecal cells as well as in endothelial cells. It is possible that VEGF might have a role in the ovulatory process in humans.
We demonstrated that VEGF was produced by lutein cells in the ELP and MLP by immunohistochemical study, enzyme-linked immunosorbent assay, and RT-PCR. After ovulation and disintegration of the basal lamina, the thecal vessels invade the granulosa cell layer to form the luteal vascular network. During this period, massive proliferation of endothelial cells occurs, as indicated by the staining for Ki67 and the uptake of [3H]thymidine (22, 23, 24). Ferrara et al. (25) reported that the cyclical growth of blood vessels was essential for the development of the ovarian CL, and VEGF secreted during the periovulatory phase played a role in this physiological angiogenesis process. It is possible that VEGF secreted from lutein cells stimulates the proliferation of endothelial cells in the ELP. This is also confirmed by in vitro study in which VEGF treatment increased bromo-deoxyuridine incorporation in cultured endothelial cells isolated from the CL of rhesus monkey (26). Although the number of vessels in the CL increased from the ELP to MLP and reached a plateau in the LLP (23, 27, 28), the proliferation rate of endothelial cells was dramatically decreased in the MLP (22, 23). In the present study, we indicated that the intense staining for VEGF was supported by relatively high amounts of VEGF in lutein extracts in the MLP. These results could suggest different functions of VEGF other than angiogenesis during the MLP.
Furthermore, in the gestational CL, distinct cytoplasmic staining for VEGF was observed, and tissue concentrations of VEGF were measurable. The CL of early pregnancy is stimulated by hCG secreted from villous tissues. Some reports indicate that in vivo, treatment with hCG stimulated progesterone production by the CL, but did not increase the number of proliferating endothelial cells (22, 23). During early pregnancy, another role for VEGF may be proposed. The production pattern of VEGF in the CL might suggest a luteal function perhaps unrelated to angiogenesis. VEGF might increase the vascular permeability and the uptake of cholesterol to lutein cells that resulted in the enhancement of the luteal function, such as progesterone production during the MLP and pregnancy. It is also possible that VEGF might have an autocrine role in luteal function. In the present study, the expression of Flt-1 was detected in luteal tissues, and the immunolocalization of Flt-1 was observed in the cytoplasm of lutein cells as well as endothelial cells. These results might support an autocrine role of VEGF in the CL. Yan et al. (29) reported that Flt-1, kinase insert domain-containing receptor tyrosine kinase, and Flt-4 were not expressed in human cultured luteinized granulosa cells. The discrepancy may be due to the difference in the sensitivity of the experiments.
Luteal function is under the control of LH. Northern blot analysis revealed that the expression of LH receptor in human CL was increased from ELP to MLP and was dramatically decreased in the LLP (30). Human luteal regression mediated in part by apoptosis begins in the LLP, which is shown by DNA laddering and in situ analysis of DNA fragmentation (31). In the gestational CL, apoptotic DNA fragmentation did not occur, and the expression of LH receptor was maintained. It has been reported that VEGF production in cultured luteinized granulosa cells was enhanced by hCG (16, 17). Our study suggests that VEGF production may be enhanced by endogenous LH in the ELP and MLP and that the decrease in VEGF production in the LLP may be linked to the loss of LH receptors during luteolysis.
The VEGF gene consists of eight exons, and as a result of alternative splicing, different transcripts encoding VEGF forms have been identified (5). We showed that VEGF transcripts, VEGF121 and VEGF165, were produced by the human CL. This result fits with another report showing that VEGF121 and VEGF165 are the most common isoforms and have endothelial cell mitogenic activity (7). VEGF121 is a weakly acidic polypeptide and is secreted into the circulation. VEGF165 is slightly basic, and a significant fraction of the secreted protein remains bound to the cell surface and extracellular matrix (32). As VEGF121 does not have exon 7, which contains seven cystein residues, the crystal structure of VEGF121 and VEGF165 might be different (5). Although VEGF121 and VEGF165 are known to have different heparin binding abilities (32), the biological differences in these isoforms remain to be elucidated.
In summary, the immunolocalization of VEGF and Flt-1 was demonstrated in granulosa and thecal cells in human follicles. We found the expression of VEGF and Flt-1 in luteal tissues and the localization of Flt-1 in lutein cells as well as in endothelial cells during the menstrual cycle and early pregnancy. These data suggest that VEGF might have an autocrine role in ovulatory process and luteal function as well as a paracrine role in angiogenesis.
Received February 3, 1999.
Revised June 7, 1999.
Accepted June 22, 1999.
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