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RESEARCH |
Division of Obstetrics and Gynecology, Department of Reproductive, Pediatric and Infectious Science, Yamaguchi University School of Medicine, Minamikogushi 1-1-1, Ube, 755-8505 Japan
Correspondence should be addressed to N Sugino; Email: sugino{at}yamaguchi-u.ac.jp
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Abstract |
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Introduction |
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The CL is essential for the maintenance of pregnancy throughout the entire pregnancy in rats. To maintain progesterone production for successful pregnancy, not only high vascularization but also stabilization of blood vessels in the CL is necessary to provide luteal cells with the large amounts of cholesterol needed for progesterone synthesis and to deliver progesterone to the circulation. Therefore, it seems that blood vessels in the CL need to stabilize or mature to serve as functional vessels (Jain & Booth 2003). However, little is known regarding the change in blood vessel stability and its regulation in the CL. Therefore, in the present study, we have evaluated blood vessel stability in the CL by quantifying vascular leakage, and furthermore the involvement of angiopoietins in the regulation of blood vessel stability has been examined in the CL during pregnancy in rats.
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Materials and Methods |
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The first experiment was planned to examine blood vessel stability in the CL during pregnancy. Blood vessel stability was evaluated on days 3, 7, 9, 12, 15 and 21 of pregnancy, based on vascular leakage, quantified by the Evans blue dye assay as described below.
The second experiment was set up to determine the levels of Ang-1, Ang-2 and Tie-2 in the CL during pregnancy. Rats were laparotomized under ether anesthesia on days 3, 7, 9, 12, 15 and 21 of pregnancy. The ovaries were perfused with saline via the portal vein during draining of the inferior vena cava to remove the blood, and then removed as reported previously (Sugino et al. 1993a). CL were dissected and cleaned of adhering tissue in a watch glass. Only newly formed CL were used in the present study. CL were immediately frozen in liquid nitrogen, and stored at –80 °C until RNA isolation for RT-PCR and protein isolation for Western blot analysis.
The third experiment was performed to study the involvement of estradiol in vascular leakage, and Ang-1 and Ang-2 expression in the CL during mid-pregnancy. For this purpose, a well-characterized estradiol-treated hypophysectomized and hysterectomized rat model was used. This model was reported by Gibori & Keyes (1978) and has been widely used to study the role of intraluteal estrogen in the regulation of rat CL function. Hypophysectomy and hysterectomy were performed on day 12 of pregnancy, and rats were injected s.c. with either 100 µg 17ß-estradiol (Sigma Chemical Co., St Louis, MO, USA) dissolved in 0.2 ml sesame oil or sesame oil (control) daily until the morning of day 15 of pregnancy, as reported previously (Kashida et al. 2001). Rats were used for the measurement of vascular leakage and Ang-1 and Ang-2 expression in the CL.
Autofluorescence imaging of Evans blue dye
Evans blue dye is widely used to measure vascular protein leakage (Udaka et al. 1970, Saria & Lundberg 1983, Murphy & Lever 2001, Hamer et al. 2002). Increased vascular leakage causes the leakage of albumin out of blood vessels. Since Evans blue dye binds to albumin in the circulation, vascular leakage can be evaluated by measuring the Evans blue dye that has exuded from blood vessels. First, we observed exudation of the Evans blue dye in the CL by autofluorescence imaging of Evans blue dye as reported previously (Murphy & Lever 2001). On days 3 and 15 of pregnancy, Evans blue dye (Sigma; 30 mg/kg) was injected via a femoral vein. The ovaries were perfused with saline to remove blood 30 min after injection and then taken out. The ovary was mounted in Tissue-Tek compound and cooled by liquid nitrogen. Frozen sections (6 µm) were cut at –21 °C on a Leica cryostat, dipped in cold acetone (–20 °C) for 1 min and then air-dried at room temperature. The sections were then dipped into xylene and mounted with a glass cover-slip. Alternate sections were stained with hematoxylin (Wako Pure Chemical Industries Ltd, Osaka, Japan) and eosin (Merck, Darmstadt, Germany) (HE), and mounted with Entellan neu (Merck). The HE-stained sections were viewed with bright-field light microscopy, while the unstained frozen sections were viewed by fluorescence microscopy with the use of a green wave-length filter set.
Quantification of vascular leakage
On days 3, 7, 9, 12, 15 and 21 of pregnancy, Evans blue dye (30 mg/kg) was injected via a femoral vein. The ovaries were perfused with saline to remove blood 30 min after injection and then taken out. CL were dissected, cleaned of adhering tissue and weighed. Evans blue dye exuded in the CL was extracted by incubating four to six CL in 0.15 ml formamide for 24 h at 65 °C, and dye concentrations were measured by absorption at 620 nm and determined by a standard curve as reported previously (Udaka et al. 1970, Saria & Lundberg 1983). The amount of Evans blue dye was expressed as ng of Evans blue dye per mg wet weight of the CL. The optimal number of CL for extraction was chosen since a linear Evans blue dye concentration was obtained between three and six CL in 0.15 ml formamide. The intra- and interassay coefficients of variation in this assay were 2.2 and 2.4% respectively.
To estimate the number of blood vessels in the CL, after Evans blue dye extraction they were embedded in paraffin and tissue sections were made. Blood vessels were identified with vascular endothelial cells in the HE-stained histological sections as shown in Fig. 2A
, and the number of blood vessels was counted within a microscopic field at x 400 (Fig. 2A). Counting was done on five randomly chosen fields from two or three CL of several sections. More than one field was evaluated per CL. The mean value was used as a number of blood vessels in a unit area. Since the size of luteal cells changes during pregnancy, the luteal cell size influences the proportion of the blood vessels per unit area of the histological section. In fact, the marked impact of luteal cell size on the proportion per unit area of blood vessels has been pointed out (Wulff et al. 2001, Sugino et al. 2005). For example, in early pregnancy, luteal cells are relatively small so that in a given unit area the number of blood vessels is relatively high. In contrast, in mid-pregnancy, the luteal cell size is relatively large so that in a given unit area the number of blood vessels is relatively low. Therefore, to adjust for this effect, the number of blood vessels in a unit area was multiplied by the CL wet weight in order to estimate the number of blood vessels in each CL. This value reflects the number of blood vessels in the whole CL and was used as a vascular index in the present study. Vascular leakage was determined by dividing the amount of Evans blue dye per CL by the vascular index, which indicates the leakage of Evans blue dye per blood vessel in the CL.
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RT-PCR
Total RNA was isolated from CL with Isogen (Wako) by the method provided by the manufacturer. For mRNA analysis, RT-PCR was performed as reported previously (Sugino et al. 1998) with the oligonucleotide primers for Ang-1 (5'-GTGGCTGGAAAAACTTGAGA-3' and 5'-TGGATTTCAAGACGGGATGT-3'), for Ang-2 (5'-GACCAGTGGGCATCGCTACG-3' and 5'-CTGGTTGGCTGATGCTACTG-3') and for Tie-2 (5'-TGCCACCATCACTCAATACC-3' and 5'-AAACGCCAATAGCACGGTGA-3') designed on the basis of the rat Ang-1, Ang-2 and Tie-2 cDNA sequences (Sato et al. 2001). Two oligonucleotide primers (5'-CTGAAGGTCAAAGGGAATGTG-3' and 5'-GGACAGAGTCTTGATGATCTC-3') were also used to amplify ribosomal protein L19 as an internal control (Chan et al. 1987). In brief, 3 µg 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, 1.5 mM MgCl2, and 200 U Moloney murine leukemia virus reverse-transcriptase (Perkin-Elmer, Roche Molecular Systems Inc., Branchburg, NJ, USA)). 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/mol; Amersham, Arlington Heights, IL, USA) and Taq DNA polymerase (2.5 U; Perkin-Elmer) was added to each reaction. Amplification was carried out for 30 cycles consisting of 94 °C (1 min), 60 °C (1 min) and 72 °C (1 min) for Ang-1, Ang-2 and Tie-2, 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 201 bp for Ang-1, 170 bp for Ang-2, 214 bp for Tie-2 and 194 bp for L19. A liner curve was plotted using number of cycles for amplification vs densitometric values of the PCR products, measured with an FLA2000 (Fuji Photo Film Co., Tokyo, Japan). The optimal number of cycles for amplification that fit within the linear ranges were chosen for each sets of primers for Ang-1, Ang-2, Tie-2 and L19 (data not shown). Reaction products were subjected to electrophoresis on an 8% (v/v) polyacrylamide non-denaturing gel. After autoradiography, band intensities were analyzed using a bioimaging Analyzer FLA2000. For quantification, the densities of Ang-1, Ang-2 and Tie-2 were normalized to that of the internal control L19.
Western blot analysis
CL were homogenized with PBS containing a protease inhibitor cocktail tablet (Complete Mini; Roche Diagnostics, Mannheim, Germany) and centrifuged at 800 g for 10 min at 4 °C. The supernatant was used for Western blot analysis as reported previously (Sugino et al. 2000b). In brief, 50 µg of protein of the supernatant, determined by the Lowry et al.(1951) method, were loaded in each sample and separated by SDS-PAGE in 7.5% (v/v) gels under reduced conditions. The proteins on the gel were electrophoretically transferred to nitrocellulose membranes and reacted with the rabbit polyclonal Ang-1 antibody (Santa Cruz Biotechnology) or rabbit anti-mouse Ang-2 antibody (Alpha Diagnostic International, San Antonio, TX, USA) at a dilution of 1:50 with 0.5% (w/v) skimmed milk in Tris-buffered saline (pH 7.5). The membranes were then immersed in the reaction buffer containing PAP conjugated swine anti-goat immunoglobulin (1:3000). The reacted band was developed on a film with an ECL kit (Amersham Pharmacia Biotech, Bucks, UK). To reuse of the blot, the membranes were stripped in Restore Western Blot Stripping Buffer (Pierce, Rockford, IL, USA) and reacted with mouse monoclonal ß-tubulin antibody (Sigma) at a dilution of 1:500 with 0.5% (w/v) skimmed milk in Tris-buffered saline (pH 7.5). ß-Tubulin is a ribosomal protein and was used as an internal control. The membranes were immersed in the reaction buffer containing PAP conjugated rabbit anti-mouse immunoglobulin (1:3000). The reacted band was developed on a film with the ECL kit.
Progesterone assay
Progesterone concentrations in the serum were determined by a specific RIA as reported previously (Kato et al. 1982). 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 analyzed by ANOVA and Duncan’s new multiple range test. Differences were considered to be significant if P < 0.05.
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Results |
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shows fluorescence images of Evans blue dye in the CL on day 3 (Fig. 1A) and on day 15 of pregnancy (Fig. 1C). The orange color indicates the Evans blue dye that has exuded from blood vessels, which was seen in the CL on day 3 of pregnancy, with intense staining in the center (Fig. 1A). In contrast, there was a faint staining in the CL on day 15 of pregnancy (Fig. 1C). This finding suggests that vascular leakage in the CL was more apparent on day 3 compared with on day 15 of pregnancy. Although there may be a fluorescence that biological tissues naturally have, so-called autofluorescence, there was a faint staining in the CL on day 15 of pregnancy, suggesting that autofluorescence in the CL tissue can be regarded as negligible.
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. As shown in Fig. 3, vascular leakage was highest on day 3 and thereafter decreased until days 12 and 15, which are lowest during pregnancy (Fig. 3). However, vascular leakage increased on day 21 of pregnancy again (Fig. 3). These findings suggest that the blood vessel is not yet stabilized on day 3 and stabilized during mid-pregnancy, but destabilized on day 21 of pregnancy.
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shows the immunostaining for Ang-1 (Fig. 4A) and Ang-2 (Fig. 4B) in the CL on day 15 of pregnancy. Both Ang-1 and Ang-2 were localized in luteal cells, with no visible difference between Ang-1 and Ang-2, and there was no difference in immunointensities between day 3, day 15 and day 21 of pregnancy (data not shown).
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). However, there was no significant change in Ang-2 mRNA levels (Fig. 5B) and Tie-2 mRNA levels (data not shown).
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shows Western blot analysis of Ang-1 and Ang-2 in the CL on days 3, 15 and 21 of pregnancy. Ang-1 protein expression was barely detectable on day 3 of pregnancy, but it was highly expressed on day 15 and also stronger compared with that on day 21 of pregnancy (Fig. 6A) There was no significant change in Ang-2 protein expression between days 3, 15 and 21 of pregnancy (Fig. 6B).
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). Vascular leakage was significantly increased by hypophysectomy–hysterectomy, and this effect was significantly reversed by estradiol treatment (Fig. 7C). Furthermore, Ang-1 protein expression was decreased by hypophysectomy–hysterectomy and this effect was reversed by estradiol treatment (Fig. 7D), whereas there was no significant change in Ang-2 protein expression by hypophysectomy–hysterectomy or estradiol treatment (Fig. 7E).
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Discussion |
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It is likely that the changes in vascular leakage and angiopoietins are related to angiogenesis in the CL. The number of blood vessels in the CL gradually increase until mid-pregnancy, as shown in the present result (Fig. 2B), which is consistent with the report by Tamura & Greenwald (1987). Since it is reported that Ang-2 is involved in destabilization of blood vessels and promotes vessel sprouting in the presence of VEGF, angiogenic changes seen in the early luteal phase may be induced by the Ang-2 action because of low Ang-1 expression, although blood vessels are still immature in the CL of the early luteal phase.
It has been reported that angiogenesis in the CL is activated and parallel to the rapid growth of the CL during mid-pregnancy (Tamura & Greenwald 1987, Kashida et al. 2001). The present data have also shown an increase in the vascular index in the CL during mid-pregnancy. On the other hand, the present study showed high Ang-1 expression and decreased vascular leakage between day 12 and day 15 of pregnancy, suggesting that blood vessel stability is parallel to the angiogenesis in the CL in this period. The present data, therefore, seem inconsistent with the story that angiogenesis is induced by VEGF when blood vessels are destabilized by Ang-2 action. However, we hypothesize that angiogenesis may occur accompanied by blood vessel stabilization in the CL during mid-pregnancy. Recently, Wulff et al.(2001) and our group (Sugino et al. 2005) reported the same hypothesis, in which they suggested increased angiogenesis together with blood vessel stabilization in the human CL of pregnancy because significant increases in the number of endothelial cells and perivascular cells (pericytes) were found in the CL rescued by human chorionic gonadotropin. In fact, overexpression of Ang-1 has been shown to produce highly branched and numerous leakage-resistant blood vessels in the skin of transgenic mice (Suri et al. 1998, Thurston et al. 1999). Mice lacking Tie-2 receptor or Ang-1 showed that endothelial cells are present in normal numbers and are assembled into tubes, but the blood vessels are immature, lacking branching networks and proper organization into large and small blood vessels (Sato et al. 1995, Suri et al. 1996). Furthermore, recent reports have shown that co-administration of Ang-1 and VEGF increases angiogenesis and reduces vascular leakage in the ischemic myocardium (Siddiqui et al. 2003), which is a rational approach for creating more stable vessels for functional improvement (Zhu et al. 2002, Yamauchi et al. 2003). These findings, overall, strongly suggest that both Ang-1 and VEGF are necessary for the formation of stabilized mature blood vessel networks. Thus, we presume that mature blood vessels are actively formed in the CL during mid-pregnancy in rats.
It is well known that estradiol is necessary for development of the CL and maintenance of CL function during mid-pregnancy in rats (Gibori et al. 1977, Bowen-Shauver & Gibori 2004). The present study has demonstrated that estradiol decreases vascular leakage with an increase in Ang-1 expression in the CL during mid-pregnancy. Since estradiol stimulates angiogenesis via VEGF in the rat CL during mid-pregnancy (Kashida et al. 2001), estradiol contributes to both blood vessel stabilization and angiogenesis in the CL during mid-pregnancy. This finding again supports our hypothesis that angiogenesis may occur accompanied by blood vessel stabilization in the CL during mid-pregnancy. The mechanism by which estradiol modulates Ang-1 expression is unclear at present. There is no estrogen response element in the rat Ang-1 promoter region (gene accession No. AB080023 [GenBank] ), and there is a report that estradiol decreased Ang-1 expression in non-reproductive tissues in rats, in contrast to the present result (Ye et al. 2002). These findings suggest the possibility that estradiol modulates Ang-1 expression through some mediators.
Also, the present data may seem to be inconsistent with the report that estrogen increases microvascular permeability via VEGF in the rat uterus, which is observed especially at implantation (Rockwell et al. 2002). Blood vessel stabilization is modulated by Ang-1 and it would be important to note that Ang-1 expression is still low in the mouse uterus at implantation (Matsumoto et al. 2002). As described above, it is likely that the regulation of Ang-1 expression by estrogen in the CL is different from that in other tissues. These findings may suggest that the estrogen-induced microvascular permeability is dependent on the tissue.
It is of interest to note that vascular leakage is lowest when progesterone production is most activated. This result suggests that maintenance of CL function may involve stabilization of blood vessels in the CL. Vascular networks in the CL need to not only supply nutrients or substrates to luteal cells for progesterone synthesis but also deliver progesterone into the circulation. It is presumed that blood vessels in the CL during mid-pregnancy are mature enough to serve as functional vessels for progesterone production. It has been reported that VEGF leads to immature, leaky and hemorrhagic blood vessels, which are non-functioning. However, Ang-1 leads to leakage-resistant blood vessels (Yancopoulos et al. 2000). Therefore, Ang-1 may contribute to CL function by stabilizing blood vessels in the presence of VEGF. In addition, it is of interest to note that vascular leakage reduced concomitantly with the prolongation of the life span of the CL; from day 3 of pregnancy, at which the CL is rescued by the pituitary prolactin, and from day 12 of pregnancy, at which the CL is rescued by placental lactogens. Further studies are needed regarding the correlation between CL function and blood vessel stability.
For blood vessel stabilization, Ang-1 has been reported to recruit pericytes (Hanahan 1997, Darland & D’Amore 1999, Yancopoulos et al. 2000). It is controversial at present whether pericytes exist in the rat CL (Tsukada et al. 1996, Arfuso & Meyer 2003, Pauli et al. 2005). There are some reports showing the presence of pericytes in the rat CL, although they are immature and few (Tsukada et al. 1996, Arfuso & Meyer 2003). However, Ang-1 can directly act on endothelial cells for vascular network stabilization (Papapetropoulos et al. 1999, Carlson et al. 2001). Ang-1 plays a crucial role in mediating interactions between endothelial cells, the surrounding matrix and pericytes (Suri et al. 1996, Hanahan 1997). Although it is unclear how much pericytes are involved in blood vessel stabilization in the rat CL, it is likely that direct action of Ang-1 on endothelial cells, at least in part, contributes to blood vessel stabilization.
Increased vascular leakage in the CL on day 21 of pregnancy suggests that blood vessels are destabilized in the CL during the regression phase. It has been reported that deletion of endothelial cells or detachment of endothelial cells from the basement membrane is involved in blood vessel regression during the CL regression (Azmi & O’Shea 1984, Modlich et al. 1996, Goede et al. 1998). In fact, the number of blood vessels decreased in the CL on day 21 of pregnancy in the present study. It has been reported that destabilization of blood vessels caused by Ang-2 in the absence of VEGF induces endothelial cell death, probably by apoptosis (Hanahan 1997). This may apply to the present result, because we reported that VEGF action is lacking due to the remarkably low expression of VEGF receptors in the rat CL on day 21 of pregnancy (Sugino et al. 2001). Although mechanisms of CL regression have been a matter of concern and still seem complex (Sugino et al. 1993b, 1996, 1997, 1999, 2000b, Kato et al. 1997, Takiguchi et al. 2000, 2004, Sugino 2005), destabilization of blood vessels and blood vessel regression may be the first event in the CL undergoing functional luteolysis in pregnant rats (Plendl 2000).
In conclusion, the present study, for the first time, showed changes in vascular leakage and angiopoietins in the rat CL throughout pregnancy. It is likely that angiopoietins are involved in the regulation of blood vessel stability in the CL. Especially, estradiol contributes to blood vessel stabilization with an increase in Ang-1 expression in addition to stimulating angiogenesis via VEGF in the CL during mid-pregnancy, which may play important roles in CL development and maintenance of CL function.
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Acknowledgements |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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