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Regular Article |
a Department of Obstetrics and Gynecology, Yamaguchi University School of Medicine, Ube 755-8505, Japan
ABSTRACT
The present study was undertaken to investigate the role of vascular endothelial growth factor (VEGF) in luteal angiogenesis and the regulation of VEGF in the corpus luteum (CL) during mid-pregnancy in rats. Protein concentrations and mRNA levels of VEGF in the CL significantly increased from Day 9 to Day 12 and remained at the same level as Day 12 until Day 15. To study whether estradiol is involved in VEGF expression between Day 12 and Day 15, rats undergoing hypophysectomy-hysterectomy on Day 12 were treated with estradiol until Day 15. Protein concentrations and mRNA levels of VEGF in the CL were significantly decreased by hypophysectomy-hysterectomy, and this inhibitory effect was completely reversed by estradiol treatment. Changes in vascular density in the CL were parallel to those in VEGF expression. To examine whether the effect of estradiol is mediated by VEGF, anti-VEGF antibody was administered to hypophysectomized-hysterectomized rats simultaneously with estradiol. The recovery in the vascular density, CL weight, and serum progesterone concentration caused by estradiol was significantly inhibited by the anti-VEGF antibody treatment. In conclusion, the present study has demonstrated that VEGF contributes to luteal angiogenesis, CL development, and progesterone production during mid-pregnancy in rats and that luteal VEGF expression is increased by estradiol.
corpus luteum, corpus luteum function, estradiol, growth factors, progesterone
INTRODUCTION
Angiogenesis has been recognized to play an important role in the development of the corpus luteum (CL) and maintenance of luteal function [1–4]. Especially, during mid-pregnancy, the CL rapidly increases in size, mainly due to hypertrophy of luteal cells, and the number of endothelial cells in the CL and luteal blood flow also increase during mid-pregnancy [1, 2, 5]. High vascularization seems to be necessary to provide luteal cells with the large amounts of cholesterol needed for progesterone synthesis and for the delivery of progesterone to the circulation. Angiogenic factors responsible for luteal development have been studied for many years. It is well known that vascular endothelial growth factor (VEGF) is a protein that has the potential to play a dynamic role in the regulation of vascular endothelial growth, angiogenesis, and vascular permeability [6]. Recent evidence has demonstrated that VEGF is expressed in the rat CL [7, 8] and is essential for luteal angiogenesis leading to CL development [4]. However, changes in VEGF expression in the CL during mid-pregnancy are unknown, and the regulation of VEGF is poorly understood although there seems to be some possible regulatory factors in the CL such as estrogen or relaxin [1, 2, 9].
It is well known that estradiol is necessary for the development of the CL and the maintenance of luteal function during mid-pregnancy [1, 2, 10]. It has also been reported that estradiol is responsible for the increase in angiogenesis in the CL during mid-pregnancy in rats [1]. However, it is unknown whether estrogen acts directly on the vascular endothelial cells in the CL during mid-pregnancy or is mediated via some growth factors. Recent evidence has shown that estradiol increases VEGF expression in bovine granulosa cells and in rat uteri [11–13]. Therefore, there is a possibility that the angiogenic effect by estrogen is mediated by VEGF. The present study was undertaken to investigate the change in VEGF expression in the CL during mid-pregnancy in rats and to clarify whether estradiol-induced angiogenesis is mediated by VEGF in the CL during mid-pregnancy.
MATERIALS AND METHODS
Animals
Sprague-Dawley rats (Japan SLC Inc., Hamamatsu, Japan), weighing 220–270 g, were housed at 24°C under controlled conditions (lights-on from 0500 to 1900 h) with free access to standard rat chow and water. Vaginal smears were obtained daily, and only those rats showing at least two consecutive 4-day estrous cycles were used. Proestrous rats were housed with males overnight, and Day 1 of pregnancy was defined as the day on which sperm were found through a vaginal smear. The experimental protocol was reviewed and approved by the Committee for the Ethics on Animal Experiments in Yamaguchi University School of Medicine under the Law (no. 105) and Notification (no. 6) of the Government.
Experiments
The first experiment was set up to determine the level of VEGF in the CL during mid-pregnancy in rats. Rats were laparotomized under ether anesthesia on Days 9, 12, and 15 of pregnancy, and blood samples were obtained from the portal vein. The ovaries were perfused with saline, via the portal vein during draining of the inferior vena cava, to remove the blood as reported previously [14], and removed. Corpora lutea were dissected and cleaned of adhering tissue in a watch glass. Corpora lutea were weighed to examine the change in size of the CL, immediately frozen in liquid nitrogen, and stored at -80°C until VEGF assay and RNA isolation. Serum samples were stored at -20°C until progesterone assay.
The second experiment was performed to study the effect of estradiol on VEGF expression in the CL during mid-pregnancy. Hypophysectomy and hysterectomy were performed on Day 12 of pregnancy under ether anesthesia. Hysterectomy was done through an abdominal midline incision. The uterine connections with the oviduct and cervix were ligated and severed, and then the abdominal midline incision was surgically closed. Hypophysectomy was done by the parapharyngeal approach as reported previously [15]. Completeness of removal of the pituitary gland was assessed by the absence of the pituitary gland in the fossa at autopsy. Rats were injected s.c. with either 100 µg of 17ß-estradiol (minimum 98%, catalog no. E-8875; Sigma Chemical Co., St. Louis, MO) dissolved in 0.2 ml sesame oil or sesame oil (control) daily until the morning of Day 15 of pregnancy. Serum estradiol concentrations were 59.3 ± 9.1 pg/ml (n = 5) in intact rats on Day 15 of pregnancy, 27.9 ± 5.2 pg/ml (n = 9) in hypophysectomized-hysterectomized rats treated with oil, and 2244 ± 489 pg/ml (n = 9) in hypophysectomized-hysterectomized rats treated with estradiol. These results were consistent with the report by Tamura and Greenwald [1].
The third experiment was performed to examine whether the effect of estradiol is mediated by VEGF. To neutralize the bioactivity of VEGF, goat anti-human VEGF polyclonal antibody (100 µg; R&D Systems, Minneapolis, MN) diluted with 5 ml of saline was daily injected i.p. simultaneously with estradiol in rats bearing hypophysectomy-hysterectomy on Day 12 of pregnancy. According to the manufacturer, this antibody can be used for neutralization of VEGF activity. The neutralization dose50 for this antibody defined as a concentration of antibody required to yield one-half maximal inhibition was determined to be approximately 3–6 µg/ml in the presence of 10 ng/ml VEGF, using the VEGF-dependent human umbilical vein endothelial cell proliferation assay. Also, rat VEGF has a high amino acid homology (90%) with human VEGF. A dose of antibody was determined by the studies reported previously [16–19] and the information provided by the manufacturer as described above. In those studies, a dose of 100 µg/body was effective in inhibiting VEGF action in the mouse [16–18], and the administration of 2 mg/body inhibited the angiogenesis in the CL of the marmoset [19]. Control rats received 5 ml saline because it has been reported that neutralizing activity of anti-VEGF antibody was not dependent on the nonspecific action of immunoglobulin, and PBS has been used as a control [16–18].
Immunohistochemistry
For immunohistochemistry, ovaries were fixed in Bouins solution and then embedded in paraffin. Paraffin-embedded ovaries were sectioned at 4 µm. The tissue sections were deparaffinized in xylene and dehydrated in a graded series of ethanol. Immunohistochemistry for VEGF was performed with a peroxidase-anti-peroxidase method (Dako PAP kit; Dako Japan, Co. Ltd., Tokyo, Japan) using rabbit anti-human VEGF polyclonal antibodies (NeoMarkers, Inc., Union City, CA), which is reactive with rat VEGF because of high amino acid homology (90%). 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 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 swine anti-rabbit immunoglobulin for 30 min at room temperature, washed three times with PBS for 5 min each, and reacted with rabbit-PAP for 40 min at room temperature. Immunohistochemistry for CD34, a marker of vascular endothelial cells, was performed by the streptavidin-biotin-peroxidase complex method (SAB-PO kit; Nichirei Co. Ltd., Tokyo, Japan) using mouse anti-CD34 monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). 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 anti-mouse 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 4 HCl (Nacalai Tesque Co. Ltd., Tokyo, Japan) in 0.05 M Tris HCl buffer (pH 7.6) containing 0.01% H2O2 for 2 min. Control sections were incubated with normal rabbit serum for VEGF or normal mouse serum for CD34. Counterstaining was performed with Myers hematoxylin.
Vascular Endothelial Growth Factor Assay
Corpora lutea were homogenized with Tris HCl buffer (pH 7.4) and centrifuged at 800 x g for 10 min at 4°C, and the supernatant was used for the determination of VEGF concentrations using a sandwich ELISA kit (mouse VEGF immunoassay; Quantikine M, R&D Systems), because rat VEGF has a high amino acid homology (98%) with mouse VEGF. The sensitivity of the assay was 3.0 pg/ml, and the intra- and interassay coefficients of variation were 8.2% and 8.4%, respectively. All data were expressed in pg per mg protein. Protein concentrations were determined by Lowry method [20].
Reverse Transcription-Polymerase Chain Reaction
Total RNA was isolated from CL with Isogen (Wako Pure Chemical Industries Ltd., Osaka, Japan) by the method provided by the manufacturer. For mRNA analysis, reverse transcription-polymerase chain reaction (RT-PCR) was performed as reported previously [21] with the oligonucleotide primers for VEGF (5'-CTGCTCTCTTGGGTGCACTGG-3' and 5'-GGTTTGATCCGCATAATCTGCAT-3') designed on the basis of the rat VEGF cDNA sequences [8]. This primer pair gives rise to a single 320-base pair (bp) PCR product generated from the mRNAs for all known forms of VEGF, and direct sequence analyses of the PCR products were performed for sequence verification [8]. Two oligonucleotide primers (5'-CTGAAGGTCAAAGGGAATGTG-3' and 5'-GGACAGAGTCTTGATGATCTC-3') were also used to amplify ribosomal protein L19 as an internal control [22]. In brief, 3 µg of total RNA were reverse-transcribed at 42°C in a reaction mixture (single-strength PCR buffer, 2.5 mM deoxynucleotide triphosphates, 5 µM random hexamer primer, 1.5 mM MgCl2, and 200 U Moloney murine leukemia virus reverse-transcriptase [Perkin-Elmer, Roche Molecular Systems Inc., Branchburg, NJ]). The RT product was divided into two equal aliquots (one tube was for L19 primers), and PCR was performed. For PCR amplification, a mixture containing the oligonucleotide primers (50 pmol), [
-32P]dCTP (2 µCi at 3000 Ci/mmol; Amersham, Arlington Heights, IL), and Taq DNA polymerase (2.5 U; Perkin Elmer) was added to each reaction. Amplification was carried out for 25 cycles using a 60°C annealing temperature in a program temperature control system PC-800 (Astec, Fukuoka, Japan). The predicted sizes of the PCR-amplified products were 320 bp for VEGF and 194 bp for L19. A linear curve was plotted using number of cycles of amplification versus densitometric values of the PCR products, measured with a BAS 2000 (Fuji Photo Film Co., Tokyo, Japan). The optimal number of cycles for amplification that fit within the linear range was chosen for each sets of primers of VEGF and L19 (data not shown). Reaction products were electrophoresed on an 8% polyacrylamide nondenaturing gel. After autoradiography, data were quantified using a bioimaging analyzer BAS2000.
Vascular Density and Luteal Cell Size in CL
Vascular density in the CL was determined by counting the number of CD34-positive vessels within a grid area of 0.0625 mm2 at 400x in the histological sections obtained from three animals in each experimental group. Counting was done on 10 randomly chosen areas independently by three observers. Luteal cell size was determined by length by width of the cell. The measurement was done on 10 randomly chosen microscopic fields independently by three observers. An observer-related mean was calculated for each slide, and the mean of the three observer-related means was used as a single observation.
Hormone Assay
Progesterone and estradiol concentrations in the serum were determined by the specific RIA as described previously [23, 24]. The sensitivities of the assay were 100 pg/tube for the progesterone assay and 2.7 pg/tube for the estradiol assay. The intra- and interassay coefficients of variation were, respectively, 7.0% and 14.4% for the progesterone assay, and 10.0% and 10.4% for the estradiol assay.
Statistical Analysis
Data were analyzed by analysis of variance and Duncan new multiple range test. Differences were considered to be significant if P < 0.05.
RESULTS
Figure 1 shows the immunostaining for VEGF in the CL on Day 15 of pregnancy. VEGF was localized in luteal cells and the pattern of staining was similar in the CL on Days 9, 12, and 15 of pregnancy (data not shown).
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To investigate the change in VEGF expression in the CL during mid-pregnancy, both protein concentrations and mRNA levels of VEGF were determined on Days 9, 12, and 15 of pregnancy. Protein concentrations and mRNA levels of VEGF in the CL significantly increased from Day 9 to Day 12 and remain same as the level of Day 12 until Day 15 (Fig. 2, A and B). Serum progesterone concentrations also increased from Day 9 to Day 12 and further increased until Day 15 (Fig. 2C).
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Estradiol has been reported to be responsible for the increase in luteal size and progesterone production during mid-pregnancy [1, 2, 10]. Recent evidence has also shown that VEGF expression is increased by estradiol in rat uteri and bovine granulosa cells [11–13]. To study whether estradiol is involved in high VEGF expression between Day 12 and Day 15 of pregnancy, VEGF expression was examined in the CL of hypophysectomized-hysterectomized rats treated with estradiol. In this rat model, hypophysectomy-hysterectomy remarkably decreased the weight of CL, luteal cell size, and serum progesterone concentration, while estradiol treatment significantly reversed the inhibitory effects of hypophysectomy-hysterectomy (Fig. 3). Protein concentrations and mRNA levels of VEGF in the CL were significantly decreased by hypophysectomy-hysterectomy, and this inhibitory effect was completely reversed by treatment with estradiol (Fig. 4). Figure 5 shows representative photomicrographs of CD34-positive cells, vascular endothelial cells, in the CL obtained from intact rats on Day 15 of pregnancy (Fig. 5A), hypophysectomized-hysterectomized rats (Fig. 5B), and hypophysectomized-hysterectomized rats treated with estradiol (Fig. 5C). As shown in Figure 5D, vascular density was estimated based on the number of the CD34-positive vessels and changes in vascular density in the CL were also parallel to those in VEGF expression shown in Figure 4.
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From these results, we decided to examine whether the effects of estradiol on CL weight, serum progesterone concentration, and vascular density are mediated by VEGF. For this purpose, anti-VEGF antibody, to neutralize the bioactivity of VEGF, was administered simultaneously to hypophysectomized-hysterectomized rats with estradiol. As shown in Figure 3A and Figure 5D, the recovery in CL weight and vascular density caused by estradiol was significantly, but in part, inhibited by treatment with anti-VEGF antibody. In addition, the recovery in serum progesterone concentrations by treatment with estradiol was completely inhibited by the anti-human VEGF antibody (Fig. 3C). Although the serum progesterone level of the anti-VEGF antibody group was the same as the oil group, the luteal cell size of the anti-VEGF antibody group was much larger than that of the oil group (Fig. 3B).
DISCUSSION
The present study has shown that VEGF is localized in the luteal cells of the CL during mid-pregnancy, which is in agreement with previous reports showing that VEGF is extensively expressed in the luteal cells forming the CL [25–28]. Corpora lutea of primates also express this growth factor, and changes in its levels at different stages indicate a correlation between the pattern of vasculature changes and VEGF expression [28, 29]. Tamura and Greenwald [1] reported that angiogenesis in the rat CL gradually increased from Day 8 to Day 12 of pregnancy and thereafter increased rapidly from Day 12 to Day 14 of pregnancy. It seems that the rapid change in luteal angiogenesis after Day 12 of pregnancy is due to the constant high levels of VEGF between Day 12 and Day 15 of pregnancy. It is difficult to explain clearly why changes in VEGF expression in the present study did not correlate with those in luteal angiogenesis reported by them.
The present study has demonstrated that estradiol activates angiogenesis via VEGF in the CL during mid-pregnancy because the treatment with immunoneutralizing anti-VEGF antibody significantly inhibited the increase in vascular density caused by estradiol. Our results are consistent with the recent reports that VEGF expression is increased by estradiol in the rat uterus and in the bovine granulosa cell [11–13]. Recently, Ferrara et al. [4] demonstrated that VEGF was essential for luteal angiogenesis leading to the development of the CL. The present study has also shown that VEGF induces angiogenesis in the CL and is involved in the increase in size of the CL during mid-pregnancy, suggesting that VEGF plays important roles in the development of the CL during mid-pregnancy. It is unclear why the inhibitory effect of the anti-VEGF antibody treatment on vascular density was incomplete. It may be possible that angiogenic factors other than VEGF are in part involved in angiogenesis in the CL during mid-pregnancy. Also, we cannot neglect the possibility that the dose of antibody was not enough to neutralize completely the VEGF action.
It is of interest to note that treatment with immunoneutralizing anti-VEGF antibody completely suppressed serum progesterone levels, whereas the inhibition of vascular density by anti-VEGF antibody was incomplete. In addition, luteal cell size in rats treated with anti-VEGF antibody was still large compared with the oil group showing luteal regression. These findings may suggest that VEGF contributes to the progesterone production as the potent enhancer of vascular permeability in addition to angiogenic function [30]. Thus, VEGF may work 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 during mid-pregnancy. Another possibility is that the decline in VEGF action causes inhibition of luteal function. For example, decreased VEGF action causes the reduction of luteal blood flow, which in turn generates superoxide radicals that inhibit progesterone production by luteal cells [14, 31]. It is well accepted that the superoxide radical and its scavenging system regulate luteal function [14, 32–40]. However, further studies are needed regarding the relation between VEGF and progesterone production.
In conclusion, the present study has demonstrated that VEGF contributes to luteal angiogenesis, development of the CL, and progesterone production during mid-pregnancy in rat and that luteal VEGF expression is increased by estradiol.
FOOTNOTES
1 This work was supported in part by a grant from the UBE Foundation and Grant-in-Aid 11671623 from the Ministry of Education, Science, and Culture, Japan. 
2 Correspondence: Norihiro Sugino, Department of Obstetrics and Gynecology, Yamaguchi University School of Medicine, Minamikogushi 1-1-1, Ube 755-8505, Japan. FAX: 81 836 22 2287; obgyn{at}po.cc.yamaguchi-u.ac.jp
Accepted: August 17, 2000.
Received: April 6, 2000.
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