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Division of Obstetrics and Gynecology (N.S., A.S., I.M., H.A., T.T., Y.Y., H.T.), Department of Reproductive, Pediatric, and Infectious Science, Yamaguchi University School of Medicine, Ube 755-8505, Japan; and Department of Pathology (T.S.), Tohoku University School of Medicine, Sendai 980-8575, Japan
Address all correspondence and requests for reprints to: Norihiro Sugino, M.D., Ph.D., 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. E-mail: sugino{at}yamaguchi-u.ac.jp.
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Abstract |
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Objective: To study angiogenesis and blood vessel stabilization in the human corpus luteum, changes in expression of angiopoietin (Ang)-1, Ang-2, and their specific receptor, Tie-2, together with the number of blood vessels and pericytes were examined in the corpus luteum throughout the menstrual cycle and in early pregnancy.
Design: The number of blood vessels and pericytes was determined by immunohistochemistry for CD34 and 
-smooth muscle actin, respectively. Ang and Tie-2 expression were examined by immunohistochemistry or RT-PCR.
Results: The number of blood vessels increased during the early luteal phase, whereas the number of pericytes was small in the early luteal phase and increased in the midluteal phase, suggesting that angiogenesis is undergoing during the early luteal phase and blood vessels are stabilized in the midluteal phase. Blood vessels and pericytes decreased in number during the late luteal phase. The increased number of both blood vessels and pericytes seen in the corpus luteum of early pregnancy suggests that angiogenesis is undergoing accompanied by blood vessel stabilization. Ang-2 expression with low Ang-1 expression was found during the early luteal phase. Thereafter, increasing Ang-1 expression during the midluteal phase, declining Ang-1 expression with continued Ang-2 expression during the late luteal phase, and relatively high Ang-1 expression in early pregnancy were observed.
Conclusions: The change in Ang expression is closely associated with angiogenesis, blood vessel stabilization, and blood vessel regression during the divergent phases of luteal formation, luteal regression, and luteal rescue by pregnancy.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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To maintain progesterone production for successful pregnancy, especially when the corpus luteum is rescued by pregnancy, not only high vascularization but also stabilization of blood vessels in the corpus luteum is necessary to provide luteal cells with large amounts of cholesterol needed for progesterone synthesis and deliver progesterone to the circulation. Therefore, blood vessels in the corpus luteum need to stabilize and mature to serve as functional blood vessels (13). However, little is known regarding the blood vessel stability and its regulation in the human corpus luteum.
In addition to angiogenesis or blood vessel stabilization, blood vessel regression is also an important physiological phenomenon in the corpus luteum. Blood vessel regression is typically associated with tissue involution during structural luteolysis (14, 15). Deletion of endothelial cells or detachment of endothelial cells from the basement membrane has been reported to be found in the corpus luteum during structural luteolysis (14, 15, 16). However, it is not clarified whether angiopoietins regulate blood vessel regression in the human corpus luteum.
Recent studies have shown the expression of angiopoietins in the corpus luteum in cows, monkeys, and humans, suggesting a possibility that angiopoietins are involved in the regulation of corpus luteum development and luteal function via stabilization or destabilization of blood vessels in the corpus luteum (17, 18, 19). In the present study, to study angiogenesis and blood vessel stabilization in the human corpus luteum, changes in expression of angiopoietins and Tie-2 together with the number of blood vessels and pericytes were examined in the human corpus luteum throughout the menstrual cycle and in early pregnancy.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Materials
Deoxynucleotide triphosphate and Moloney murine leukemia virus reverse transcriptase were obtained from Life Technologies, Inc. (Grand Island, NY). Random hexamer and Taq DNA polymerase were obtained PerkinElmer Corp. (Foster City, CA). [-32P]deoxy-CPT was purchased from Amersham Pharmacia Biotech (Arlington Heights, IL). Isogen was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). 3,3'-Diaminobenzidine 4HCl was obtained from Nacalai Tesque Co. Ltd. (Tokyo, Japan).
Tissue samples
Corpora lutea were obtained from patients with normal menstrual cycles, aged 29–45 yr, undergoing hysterectomy for myoma uteri or cervical cancer. The determination of the age of the corpus luteum was based on the menstrual history and endometrial histological dating, which may be less accurate than measurement of the LH surge. For samples obtained in this study, the corpus luteum age determined from the menstrual history was consistent with the endometrial dating. Corpora lutea from the menstrual cycle were classified into five different groups according to age: the early stage of the early luteal phase (d 1–3 of luteal phase, with d 1 being the day of ovulation), the late stage of the early luteal phase (d 4–5), the midluteal phase (d 6–11), the late luteal phase (d 12–15), and the next follicular phase (after the onset of menstruation, d 3–7 of the follicular phase). Corpora lutea of early pregnancy (6–8 wk of pregnancy) were obtained from patients, aged 24–30 yr, with ectopic pregnancy or cervical cancer. Some tissue samples were washed with saline to remove blood, immediately frozen in liquid nitrogen, and stored at –80 C until RNA isolation.
Immunohistochemistry
The immunohistochemical staining was performed on tissue samples obtained from three different patients as reported previously (20). Corpora lutea were fixed in Carnoy solution, embedded in paraffin, and sectioned (3 µm thick). The tissue sections were deparaffinized in xylene and dehydrated in a graded series of ethanol. Then the slides were heated in an autoclave at 120 C for 5 min in a citric acid buffer [2 mM citric acid and 9 mM trisodium citrate dehydrate (pH 6.0)]. Immunohistochemistry for CD34, -smooth muscle actin, Ang-1, and Ang-2 was performed with the streptavidin-biotin amplification method using a Histofine kit (Nichirei Co. Ltd., Tokyo, Japan). The dilution of primary antibodies used in this study was 1:40 in PBS-BSA (1%) for Ang-1 and Ang-2 [monoclonal antibody; clone N-18 (sc 6319) and clone C-19 (sc 7015), respectively, Santa Cruz Biotechnology, Santa Cruz, CA] and 1:200 for CD-34 and -smooth muscle actin (polyclonal antibody; Dako Corp. Japan, Tokyo, Japan). The antigen-antibody complex was visualized by incubating the sections with 3,3'-diaminobenzidine 4HCl in 0.05 M Tris-HCl buffer (pH 7.6) containing 0.01% H2O2 for 2–3 min. For the negative control, normal mouse or rabbit serum was used instead of the primary antibodies, or the anti-Ang-1 and anti-Ang-2 antibodies were preabsorbed with an excess of Ang-1 and Ang-2 blocking peptides (sc-6319p and sc-7015p,; Santa Cruz), respectively, and no specific immunoreactivity was detected in these sections. Counterstaining was performed with Meyer’s hematoxylin.
Quantification of blood vessels and pericytes in the corpus luteum
Blood vessels were identified with CD34-positive vascular endothelial cells. A separated compartment of one or more CD34-positive endothelial cells was defined as one blood vessel. A string of CD34-positive endothelial cells with or without a lumen was counted as one blood vessel, and one CD34-positive endothelial cell around a luteal cell was also counted as one blood vessel. The number of blood vessels was counted within a unit area in the microscopic field at x200. Because the size of luteal cells changes throughout the menstrual cycle, the luteal cell size influences on the proportion of the vascular endothelial cells 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 (21). For example, in the early luteal phase or the late luteal phase, luteal cells are relatively small so that in a given unit area, the number of endothelial cells is relatively high. In contrast, in the midluteal phase or early pregnancy, the luteal cell size is relatively large so that in a given unit area, the number of endothelial cells is relatively low. Therefore, to adjust this effect, the number of luteal cells was counted in the same area together with the number of blood vessels, and the number of blood vessels was expressed per 100 luteal cells. The number of blood vessels per 100 luteal cells was used as vascular index in this study. This adjustment was made with modification of the method reported by Wulff et al. (21). Similarly, the number of -smooth muscle actin-positive pericytes was counted within a unit area in the microscopic field at x200, and the number of blood vessels per 100 luteal cells was used as pericyte index in this study. It is reported that -smooth muscle actin can be used to identify the pericyte in the human corpus luteum (19). However, because -smooth muscle actin also labels vascular smooth muscle cells in large luminal blood vessels, those large blood vessels with positive staining were excluded from the pericyte counting in this study. Histological sections were obtained from three individuals on each luteal phase. Counting was done on three randomly chosen areas. Quantification was performed independently by three observers. An observer-related mean was calculated for each histological section, and the mean of the three observer-related means was used as a single observation.
RT-PCR
Total RNA was isolated from the corpus luteum with Isogen using the method provided by the manufacturer. For mRNA analysis, RT-PCR was performed as reported previously (22) with the oligonucleotide primers for Ang-1 (5'-TCGTGAAGATGGAAGTCTAG-3' and 5'-TGCCACTTTATCCCATTCAG-3'), Ang-2 (5'-GGATCTGGGGAGAGAGGAAC-3' and 5'-CTCTGCACCGAGTCATCGTA-3'), and Tie-2 (5'-GGTTCCTTCATCCATT-3' and 5'-GTCCTTCCCATAAACC-3') designed by Hirchenhain et al. (23) and Currie et al. (24), respectively. 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 (22). 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, 1.5 mM MgCl2, and 200 U Moloney murine leukemia virus reverse transcriptase). The reverse transcription product was aliquoted equally into two tubes for Ang-1 or Ang-2 primers and L19 primers, and PCR was performed.
For PCR amplification, a mixture containing the oligonucleotide primers (50 pmol), [-32P]deoxy-CPT (2 µCi at 3000 Ci/mmol), and Taq DNA polymerase (2.5 U) was added to each reaction. Amplification was carried out for 28 cycles consisting of 94 C for 45 sec, 53 C for 45 sec, and 72 C for 45 sec for Ang-1, and 35 cycles consisting of 94 C for 30 sec, 60 C for 30 sec, and 72 C for 30 sec for 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 444 bp for Ang-1, 554 bp for Ang-2, 292 bp for Tie-2, and 194 bp for L19. A linear curve was plotted using the number of cycles of amplification vs. densitometric values of the PCR products, measured with a BAS 2000 (Fuji Photo Film C0., Ltd., Tokyo, Japan). The optimal number of cycles for amplification that fit within the linear range was chosen for each set of primers of Ang-1, Ang-2, Tie-2, and L19 (data not shown). To separate the band of Ang-1/Ang-2/Tie-2 and L19, reaction products were electrophoresed on an 8% polyacrylamide nondenaturing gel under 200 V for 2 h. After autoradiography, band intensities were analyzed using a bioimaging analyzer BAS2000. For quantification, the densities of Ang-1, Ang-2, and Tie-2 were normalized to that of L19.
Statistical analysis
Data were examined by ANOVA and Duncan’s new multiple range test. Differences were considered significant at P < 0.05.
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Results |
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shows the immunohistochemical staining for CD34, a marker of vascular endothelial cells. CD34-positive vascular endothelial cells were few in the early stage of the early luteal phase (Fig. 1A) and thereafter increased in number and appeared distributed throughout the corpus luteum in the late stage of the early luteal phase and in the midluteal phase (Fig. 1, B and C). In the late luteal phase, vascular endothelial cells appeared more sparsely distributed than the midluteal phase (Fig. 1D). In the regressed corpus luteum of the next follicular phase, the number in vascular endothelial cells was smaller, compared with the midluteal phase and late luteal phase (Fig. 1E). In the corpus luteum of early pregnancy, vascular endothelial cells increased in number, compared with the midluteal phase (Fig. 1F). The number of blood vessels in the corpus luteum was expressed as vascular index in Fig. 2. Vascular index significantly increased from the early stage to the late stage during the early luteal phase. Vascular index in the late stage of the early luteal phase was the same level as that in the midluteal phase and significantly decreased in the late luteal phase. In the corpus luteum of pregnancy, vascular index was significantly larger than that in the midluteal phase. In the corpus luteum of the next follicular phase, the vascular index could not be quantified because of cell death of the luteal cells.
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shows the immunohistochemical staining for -smooth muscle actin, a marker of pericytes. -Smooth muscle actin-positive vascular cells were very few in the early stage of the early luteal phase (Fig. 3A). Then the positive vascular cells increased in number and appeared sparsely distributed in the corpus luteum in the late stage of the early luteal phase (Fig. 3B), and the staining was observed in capillaries. In the midluteal phase, -smooth muscle actin staining was also found in the large luminal blood vessels in addition to small capillaries (Fig. 3C). In the late luteal phase, -smooth muscle actin-positive small capillaries decreased in number, although strong staining was still observed in large luminal blood vessels (Fig. 3D). In the regressed corpus luteum of the next follicular phase, there were few -smooth muscle actin-positive cells (Fig. 3E). In the corpus luteum of early pregnancy, there were a number of small capillaries and some luminal blood vessels with -smooth muscle actin staining (Fig. 3F). The number of pericytes in the corpus luteum was expressed as pericyte index in Fig. 4. Pericyte index was small in the early stage of the early luteal phase and then significantly increased from the early stage to the late stage during the early luteal phase and further increased during the midluteal phase. Thereafter pericyte index decreased in the late luteal phase. The number of them in early pregnancy was the same as that in the midluteal phase. In the corpus luteum of the next follicular phase, the pericyte index could not be quantified because of cell death of the luteal cells.
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. In the early stage of the early luteal phase, Ang-1 immunoreactivity was negligible (Fig. 5A) whereas Ang-2 expression was clearly observed in luteal cells throughout the corpus luteum (Fig. 5I). In the late stage of the early luteal phase, Ang-1 immunoreactivity was observed but still weak (Fig. 5B), whereas Ang-2 expression increased and strong immunoreactivity was observed (Fig. 5J). In the midluteal phase, Ang-1 immunoreactivity was strong in luteinized granulosa cells and luteinized theca cells (Fig. 5C), and Ang-2 was also moderately positive in luteinized granulosa cells and luteinized theca cells (Fig. 5K). In the late luteal phase, Ang-1 immunoreactivity was weak in luteinized granulosa cells and negative in luteinized theca cells (Fig. 5D), whereas Ang-2 was clearly expressed, compared with Ang-1 in both luteinized granulosa cells and luteinized theca cells (Fig. 5L). Both Ang-1 and Ang-2 immunoreactivities were still detectable in luteinized granulosa cells but not in luteinized theca cells in the corpus luteum of the next follicular phase (Fig. 5, E and M). In the corpus luteum of early pregnancy, Ang-1 immunoreactivity was strong (Fig. 5F), and Ang-2 was moderately positive in luteal cells throughout the corpus luteum (Fig. 5N). No Ang-1 and Ang-2 immunoreactivity was observed in the ovary after preabsorption of the antibodies with an excess of the blocking peptide (Fig. 5, G and O). Both Ang-1 and Ang-2 clearly showed the positive immunostaining in the glandular epithelium of the human endometrium used as a positive control (23) (Fig. 5, H and P).
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. During the menstrual cycle, Ang-1 mRNA expression was negligible in the corpus luteum in the early luteal phase, compared with the other phases, although there was no statistical significance throughout the menstrual cycle (Fig. 6A). However, Ang-1 mRNA expression in the corpus luteum of early pregnancy was significantly higher than that in any stages of the menstrual cycle (Fig. 6A). In contrast to Ang-1, Ang-2 mRNA expression was high in the early luteal phase, significantly decreased in the midluteal phase and thereafter increased in the corpus luteum of the late luteal phase and next follicular phase (Fig. 6B). Ang-2 mRNA expression in early pregnancy was the same level as the late luteal phase (Fig. 6B). Tie-2 mRNA expression tended to be higher in the late luteal phase, compared with the other luteal phases, but it was not statistically significant (Fig. 6C).
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Discussion |
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The number of blood vessels in the corpus luteum increased and reached the same level as the midluteal phase during the early luteal phase, suggesting that angiogenesis is undergoing during the early luteal phase and completed by the midluteal phase during the menstrual cycle in humans. On the other hand, the number of pericytes was smaller in the early luteal phase than that in the midluteal phase and further increased during the midluteal phase. These results suggest that blood vessels in the corpus luteum are not stabilized in the early luteal phase yet and stabilized in the midluteal phase. This finding may be explained by the change in angiopoietins shown in the present study. Angiogenesis is induced from the immature blood vessel under the environment of high Ang-2 expression in the presence of VEGF during the early luteal phase, and the increasing Ang-1 expression induces blood vessel stabilization via recruitment of pericytes during the midluteal phase. VEGF is certainly expressed in the human corpus luteum (4, 19, 25), and in fact, activated angiogenesis has been demonstrated in the early luteal phase corpus luteum by the high rate of endothelial cell proliferation in contrast with low proliferation rate of midluteal phase (26, 27, 28, 29). The present data on pericytes are also consistent with the report by Wulff et al. (21), and the increase in number of pericytes from the early luteal phase to the midluteal phase is likely due to the recruitment of pericytes because pericyte recruitment is observed during angiogenesis in the corpus luteum in bovines (16). However, the precise mechanism underlying the recruitment of pericytes is shrouded in mystery at present (13).
The present study, for the first time, showed the changes in the number of blood vessels and pericytes, Ang-1, Ang-2, and Tie-2 in the corpus luteum of pregnancy in humans. It is of special interest to note that the number of blood vessels in the corpus luteum in early pregnancy was significantly larger than that in the midluteal phase. This finding suggests that angiogenesis occurs in the corpus luteum in early pregnancy and is consistent with the report by Wulff et al. (21) in which the increase of endothelial cell proliferation is observed in the human corpus luteum rescued by human chorionic gonadotropin. However, in the present study, Ang-1 was expressed at high levels, compared with Ang-2 in early pregnancy. Therefore, the present data seem inconsistent with the story that angiogenesis is induced when blood vessels are destabilized by Ang-2 action.
In our previous report, VEGF expression in early pregnancy was remarkably high, compared with that in the midluteal phase (4). Therefore, we hypothesize that angiogenesis occurs, accompanied by blood vessel stabilization in the corpus luteum of pregnancy. In fact, we recently found low vascular leakage with high Ang-1 expression in the rat corpus luteum during midpregnancy in which angiogenesis is actively undergoing by the high VEGF expression (30). Wulff et al. (21) also suggested the increased angiogenesis with blood vessel stabilization in the corpus luteum of pregnancy from the finding that significant increases in number of endothelial cells and pericytes were found in the rescued corpus luteum. Furthermore, overexpression of Ang-1 has been shown to produce highly branched and numerous leakage-resistant blood vessels in the skin of transgenic mice (31, 32). Mice lacking Tie-2 receptor 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 (33). Ang-1 knockout mice also die with similar vascular defects to the Tie-2 knockout mice (8). Interestingly, recent reports have shown that coadministration of Ang-1 and VEGF increases angiogenesis and reduces vascular leakage in the ischemic organs (34, 35, 36), which is a rational approach for creating more stable vessels for functional improvement (37). These findings, overall, strongly suggest that both Ang-1 and VEGF are necessary for the formation of stabilized mature blood vessel networks.
Blood vessel stabilization may be involved in the maintenance of luteal function for successful pregnancy (13), especially, during the midluteal phase or when rescued by pregnancy. The present study showed high Ang-1 expression and the increased number of pericytes in the corpus luteum of the midluteal phase and early pregnancy, suggesting a possibility that blood vessels in the midluteal phase and in early pregnancy are mature and serve as functional blood vessels. The present study may, therefore, imply that blood vessel stabilization in the corpus luteum is a prerequisite factor for the rescue of the corpus luteum.
The present study showed that the decline in the number of blood vessels and pericytes was concomitant with declining Ang-1 expression and constant expression of Ang-2 in the corpus luteum during the late luteal phase. It is well known that loss of Ang-1 action in the absence of VEGF induces apoptosis of endothelial cells (11, 15). VEGF expression is demonstrated to be decreased in the primate corpus luteum toward the regression phase (4, 18). Taking relatively high expression of Tie-2 during the late luteal phase into consideration, declining Ang-1 expression and constant Ang-2 expression may induce endothelial cell death, likely by apoptosis, leading to blood vessel regression in the human corpus luteum. In fact, deletion of endothelial cells or detachment of endothelial cells from the basement membrane is observed in the corpus luteum during structural luteolysis (14, 15, 16, 29).
There seems to be some differences in mRNA expression of angiopoietins or Tie-2 between the published reports and the present result. Wulff et al. (19) reported in humans that Ang-2 mRNA expression remained unchanged from the early to late luteal phase and increased in the rescued corpus luteum, whereas Ang-1 uniformly expressed at a low level throughout the menstrual cycle and in the rescued corpus luteum by in situ hybridization. Hazzard et al. (18) reported in macaques that Ang-1 and Ang-2 mRNA expression was low in the early to midluteal phase but increased at the late luteal phase and decreased again in the late stage of the late luteal phase. The change in Ang-1 mRNA expression during the menstrual cycle reported by both research groups does not seem to largely differ from the present data, but there is a difference in Ang-2 mRNA expression of the early luteal phase. It is difficult to clearly explain the difference, but it may be due to some variation of individuals, the different methodology, or the difference in precise ages of the corpus luteum in the same luteal phase such as a difference between early stage and late stage of the early luteal phase. In addition, it is of interest to note that there is a difference in both Ang-1 and Ang-2 mRNA expression between the corpus luteum rescued by human chorionic gonadotropin administration and the corpus luteum of pregnancy, which may suggest the intrinsic difference between them, although it is not fully clarified yet. A similar difference has also been reported between the rescued corpus luteum and the corpus luteum of pregnancy (20). Regarding the Tie-2 expression, Wulff et al. (19) reported that Tie-2 mRNA expression was localized in endothelial cells in the human corpus luteum, and the area of expression was high in the early luteal phase and rescued corpus luteum by in situ hybridization, which is inconsistent with the present RT-PCR data showing no significant change in Tie-2 mRNA expression throughout the menstrual cycle and in early pregnancy. Although it may be due to the difference in methodology and quantification used, accumulation of the data would be required because little information is available about Tie-2 expression in the human corpus luteum. Also, there seems to be, in part, a discrepancy in this study between mRNA expression and protein expression of Ang-2, and this may be due to the different sensitivities of RT-PCR and immunohistochemistry.
Physiological angiogenesis and blood vessel regression are restricted to the female reproductive system, in which they occur cyclically in the ovary and uterus. In particular, the corpus luteum is a unique organ in the adult from this point of view. The present study strongly suggests that there appears to be collaboration in the human corpus luteum among Ang-1, Ang-2, and VEGF to not only form blood vessel networks for successful pregnancy but also induce blood vessel regression for the next reproductive cycle. Analysis of the process of angiogenesis and blood vessel regression in the corpus luteum would provide useful information to the field of not only reproductive biology but also regenerative medicine, therapeutic angiogenesis, and tissue engineering.
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Footnotes |
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First Published Online August 23, 2005
Abbreviations: Ang, Angiopoietin; VEGF, vascular endothelial growth factor.
Received March 23, 2005.
Accepted August 17, 2005.
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References |
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-induced luteolysis: possible mechanism for the initiation of luteal regression. J Reprod Dev 50:619–626[Medline]
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