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Original Studies |
Medical Research Council, Human Reproductive Sciences Unit and Department of Obstetrics and Gynecology (C.W., H.W., P.L., W.C.D., H.M.F.), Edinburgh, United Kingdom EH3 9ET; and Roslin Institute (D.A.), Roslin, United Kingdom EH25 9PS
Address all correspondence and requests for reprints to: Dr. C. Wulff, Medical Research Council, Human Reproductive Sciences Unit, 37 Chalmers Street, Edinburgh, United Kingdom EH3 9ET. E-mail: c.wulff{at}ed-rbu.mrc.ac.uk
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Abstract |
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Introduction |
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To begin to address the clinical relevance of these angiogenic factors in the human CL, we investigated the pattern of Ang-1, Ang-2, Tie-2, and VEGF messenger ribonucleic acid (mRNA) expression in CLs throughout the luteal phase and during simulated pregnancy by treatment with hCG.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Corpora lutea were enucleated at the time of hysterectomy as described previously (1, 20). All women were healthy and aged 32–45 yr. Only women with regular menstrual cycles who had not received any form of hormonal treatment during the previous 3 months took part in the study. The date of the preovulatory LH surge was determined by measuring LH concentration in serial early morning urine samples collected before operation (21). On this basis, six CL in each group were classified as early luteal (LH+1 to LH+5 days), midluteal (LH+6 to LH+10), and late luteal (LH+11 to LH+14 days). In addition, three women were given im injections of hCG (Profasi, Serono Laboratories, Inc., Welwyn Garden City, UK) from LH+7 in daily doubling doses, starting at 125 IU and continuing for 6–8 days until surgery (21). This regimen has been shown to rescue the CL and reproduce the hormonal changes of early pregnancy (22). There were no differences in average age among the groups.
CLs were enucleated from the ovary by blunt dissection. The tissue was immediately divided into radial blocks, and a portion was fixed in 4% paraformaldehyde for 24 h. These CLs were further used in studies to determine changes in other factors associated with control of luteal cell function described previously (1, 20, 21). An endometrial biopsy was also obtained to assist luteal staging by tissue morphology. In all cases, morphological dating of the luteal phase endometrium (23) was used to confirm the luteal phase classification. The study was approved by the Reproductive Medicine Branch of the South-East of Scotland medical ethics committee, and informed consent was obtained from all patients before tissue collection.
In situ hybridization
In situ hybridization was performed using complementary RNA probes for human Ang-1, Ang-2, Tie-2 [complementary DNA (cDNA) probes were provided by Dr. G. D. Yancopoulos, Regeneron Pharmaceuticals, Inc., Tarrytown, NY] and VEGF (cDNA probe was provided by Dr. S. Charnock-Jones, University of Cambridge, Cambridge, UK). Sense and antisense probes were prepared using a RNA transcription kit (Ambion, Inc., Austin, TX) and were labeled with [35S]UTP (NEN Life Science Products, Boston, MA). The synthesized probes were purified from free bases using Chroma Spin-100 columns (CLONTECH Laboratories, Inc., Palo Alto, CA).
Paraffin sections (5 µm) were mounted onto SuperFrost Plus glass slides (BDH, Dorset, UK). Sections were deparaffinized in xylene and hydrated through descending concentrations of ethanol. Sections were treated with 0.1 N HCl and then digested in proteinase K (5 mg/ml; Sigma, St. Louis, MO) for 30 min at 37 C. The digestion was stopped by treating the slides with 0.2% glycine for 10 min at 4 C, acetylated with 0.2% acetic anhydride in triethanolamine buffer (Sigma), and then washed in 4 x SSC (standard saline citrate). A prehybridization step was carried out by incubation in prehybridization buffer (50 mL/slide) containing 50% formamide, 4 x SSC, 1 x Denhardt’s, 125 mg/mL salmon testes DNA, 125 mg/mL yeast transfer RNA, and 10 mmol/L dithiothreitol at 55 C in a moist chamber for 2 h. Hybridization was performed in a moist chamber overnight at 55 C. The hybridization buffer was similar to the prehybridization buffer, but contained 10% dextran sulfate additionally. Two sections per slide were exposed to the antisense and the sense sequences. After hybridization, slides were rinsed in 4 x SSC and then treated with ribonuclease A (20 mg/mL; Sigma) for 30 min at 37 C to remove all excess probe, desalted in descending concentrations of SSC (2 x SSC for 30 min at room temperature, 2 x SSC at 45 C, and 0.5 x SSC at room temperature) and dehydrated for 2 min each in 50%, 70%, and 90% ethanol containing 0.3 mol/L ammonium acetate. Dry slides were dipped in Ilford G5 liquid emulsion (Ilford Imaging, Cheshire, UK), exposed for 5 weeks at 4 C, and subsequently developed (Kodak D19 developer, Eastman Kodak Co., Rochester, NY) and fixed (Kodak GBS). All slides were counterstained with hematoxylin and eosin, dehydrated, and mounted.
Analysis
The slides were analyzed under lightfield conditions to detect Ang-1, Ang-2, Tie-2, and VEGF mRNA expression qualitatively. Granulosa cells, theca luteal cell areas, and endothelial cells were identified by their localization and morphological appearance. Quantitative analyses were performed under darkfield conditions using an image-analyzing system linked to an Olympus Corp. camera (New Hyde Park, NY). The Image-Pro Plus version 3.0 for windows (Media Cybernetics, Silver Spring, MD) computer program was used. The slides were analyzed in two ways. Firstly, the grain density (number of grains per µm2) in 10 representative areas (3.2 x 105/µm2) was estimated. For a direct comparison of Ang-1 and Ang-2, overall grain density was measured in the CL and the surrounding stroma tissue, respectively. For Ang-2, Tie-2, and VEGF, the grain concentration per single cell was measured additionally. The tissue background density was calculated in all sense slides and subtracted from the antisense measurements to eliminate the signals of unspecific binding. Secondly, the area of mRNA expression in the same 10 fields (3.2 x 105/µm2) was measured for Ang-2, Tie-2, and VEGF. To obtain a figure per unit area, measurements were divided by the size of the field.
Results were compared by ANOVA; where a significant difference (P < 0.05) was found, pairwise comparisons were performed using Duncan’s multiple range tests. Values are given as the mean ± SEM.
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Results |
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Ang-1 was expressed uniformly in luteal and endothelial cells
throughout the CL (Fig. 1
), with no
visible differences between the granulosa and thecal compartments. A
uniform expression was also found in the stroma. Density measurements
revealed the highest average grain concentration in the CL during the
early luteal phase (0.026 ± 0.007
grains/µm2). Grain densities during mid
(0.005 ± 0.003 grains/µm2) and late
(0.008 ± 0.003 grains/µm2) luteal phases
and during luteal rescue (0.003 ± 0.001
grains/µm2) were lower than that during the
early luteal phase (P < 0.05; Fig. 2). The sense grain concentration was
significantly different from the antisense grain
concentration, but did not vary throughout the different cycle stages
(0.002 ± 0.0001 grains/µm2 during
the early luteal phase, 0.003 ± 0.0001
grains/µm2 during the midluteal phase,
0.003 ± 0.0001 grains/µm2 during the late
luteal phase, and 0.002 ± 0.0001
grains/µm2 during luteal rescue).
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Ang-2 mRNA was strongly expressed in a minority of individual
luteal and endothelial cells (0.11 ± 0.01
grains/µm2; Fig. 3), found either singly or in clusters.
Ang-2 mRNA expression was present at a lower level throughout the CL
(0.06 ± 0.04 grains/µm2) with an increase
in overall grain density in the rescued CL (P < 0.05;
Fig. 2
). No differences in cell distribution were found between the
thecal and the granulosa cell compartments. The grain density of single
cell areas did not vary significantly throughout the luteal cycle, but
an increase was found from 0.11 ± 0.01
grains/µm2 during the luteal phase to 0.18
± 0.01 grains/µm2 during luteal rescue
(P < 0.05; Fig. 4). The
background grain density of the sense probe did not vary within
different cycle stages (i.e. 0.015 ± 0.005
grains/µm2 during the early luteal phase,
0.01 ± 0.001 grains/µm2 during the
midluteal phase, 0.01 ± 0.002 grains/µm2
during the late luteal phase, and 0.015 ± 0.008
grains/µm2 during luteal rescue).
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). This was consistent with measurements
of the area of Ang-2 mRNA expression, which increased from 0.01 ±
0.005 µm2 during the early luteal phase to
0.16 ± 0.06 µm2 during luteal rescue
(Fig. 4). In the stroma, Ang-2 mRNA was only found in endothelial cells
(0.12 ± 0.01grains/µm2; Fig. 3). No
changes in grain concentration were found throughout the luteal phase
and rescue.
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As Ang-1 and Ang-2 have opposing effects, the arbitrary ratio of
the overall grain density of Ang-2/Ang-1 was calculated to demonstrate
the net effect of their changes in the luteal phase. The Ang-2/Ang-1
ratio increased from 2.02 during the early luteal phase to 11.25 during
the late luteal phase. The highest ratio of 47 was detected during
luteal rescue (Fig. 2).
Tie-2 mRNA expression
Tie-2 mRNA was exclusively expressed in endothelial cells of blood
vessels and individual capillary endothelial cells (Fig. 6), without any change in grain density
per cell (0.06 ± 0.004 grains/µm2) within
different cycle stages (Fig. 7). The
grain concentration in sense slides was low (0.001 ± 0.0001
grains/µm2), with no difference in the density
within different cycle stages. The grain concentration of the sense
slides was significantly lower than the grain concentration of the
antisense slides.
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). No
differences were detected between early and late luteal phases
(0.05 ± 0.003 µm2). The area of Tie-2
mRNA expression during luteal rescue (0.08 ± 0.003
µm2) was significantly higher
(P < 0.05) than those during the mid- and late luteal
phases (Fig. 7). VEGF mRNA expression
VEGF mRNA was intensely expressed in granulosa luteal cells (Fig. 8). Expression was apparently absent in
thecal and endothelial cells (Fig. 8). Grain density measurements
revealed a significant increase between the midluteal phase (0.05
± 0.003 grains/µm2) and luteal rescue
(0.07 ± 0.003 grains/µm2; Fig. 9). The sense grain density was
significantly lower (0.002 ± 0.0001
grains/µm2) than the antisense grain
concentration, and did not vary between different groups.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Interestingly, the mRNA of these angiogenic factors showed different cellular localizations. Thus, Ang-1 mRNA was expressed at a low level only in most luteal cells. An intense signal for Ang-2 was detected in individual luteal and endothelial cells. VEGF mRNA was highly expressed in the majority of granulosa luteal cells. The Tie-2 receptor was localized exclusively in endothelial cells. Collectively, these findings strongly indicate that luteal cells have an important paracrine role in regulation of vessel growth and development, regression, and rescue of the CL.
During the early luteal phase the development of new blood vessels required to nourish the CL takes place, and the high expression of VEGF is compatible with its established role in stimulating angiogenesis at this time (12). Ang-1 has been shown to be required for stabilization of newly formed, leakage-resistant capillaries (16, 17). Consistently, our results show a uniform expression of Ang-1 in the CL with the highest levels during the early luteal phase associated with high levels of its receptor Tie-2. During the midluteal phase, when the capillary network is established, Ang-1 and Tie-2 mRNA decrease. These results are consistent with a role for Ang-1 in stabilizing the newly formed capillary network during the early luteal phase.
The actions of Ang-2 are potentially more complex. Ang-2 is a competitive inhibitor of Ang-1 at the level of the Tie-2 receptor, opposing the effect of Ang-1-mediated vessel stabilization (14). The action of Ang-2 alone is believed to lead to disruption of newly formed blood vessels (14). However, in the presence of VEGF, Ang-2 enhances migration and neovascularization (13, 15, 18). In the rat CL (27) it has been shown that Ang-2 in an angiogenic environment is expressed by blood vessels at the same time as high VEGF expression. In contrast, in environments undergoing regression, Ang-2 is highly expressed in parenchymal cells, and VEGF is down-regulated (27). In the human CL, high levels of VEGF are found during the early luteal phase, whereas the late luteal phase is associated with a decrease in VEGF production (10). Our data show (although statistically not significant) a tendency toward decreasing VEGF mRNA expression as the life span of the CL progresses. We observed VEGF mRNA expression in numerous granulosa luteal cell and showed that Ang-2 mRNA is highly expressed in individual cells throughout the CL in addition to a low overall expression. It is reasonable to propose that during the early luteal phase these areas of high Ang-2 expression are regions of ongoing angiogenesis, as has been suggested for the rat CL (27). In the rat, punctuate Ang-2 mRNA was particularly apparent within a pericentral region of the developing CL, and expression was also found in occasional blood vessels at the periphery in the vicinity of the original thecal vasculature. Later in the cycle after the process of luteinization was complete, Ang-2 mRNA was no longer detectable (27). In contrast, the results of this study show no down-regulation of Ang-2 in the human CL. Although statistically not significant, the area of Ang-2 mRNA expression appears to increase from the early to the late luteal phase. The Ang-2/Ang-1 ratio (which is, however, dependent on the validity of the weak Ang-1 signal) increases from the early to the late luteal phase. This is clearly caused by the decreasing Ang-1 levels. Because of the competitive actions between Ang-1 and Ang-2, this leads potentially to increasing concentrations of Ang-2 acting on the Tie-2 receptor. A rise of the Ang-2/Ang-1 ratio has been shown in the bovine CL during luteolysis (28). Together with decreasing VEGF levels during the late luteal phase (10), the elevation of the Ang-2/Ang-1 ratio in the aging CL may promote vascular breakdown, which follows later during luteolysis.
A most interesting finding is that the area of Ang-2, VEGF, and Tie-2 mRNA expression increases during luteal rescue. Cell culture studies using human luteinized granulosa cells revealed that treatment with hCG leads to increased VEGF mRNA levels (26), and it was suggested that vascularization of the CL is induced by hCG-mediated effects of VEGF. In the current study after hCG treatment in vivo, mRNA levels for Ang-2 and Tie-2 were also increased. Tie-2 receptor expression followed the up-regulation of its ligands, Ang-1 or Ang-2, respectively. During the early luteal phase Tie-2 was up-regulated when Ang-1 expression was highest. After hCG treatment, Tie-2 increased together with Ang-2.
It might have been expected that during luteal rescue Ang-1 mRNA expression would increase, as Ang-1 is involved in blood vessel stabilization (16, 17). Previous studies revealed that luteal rescue is not associated with increased angiogenesis, implying that extended endothelial survival is the principal change involved (1, 4). It has also been shown that during luteal rescue a potential endothelial survival factor, insulin-like growth factor-binding protein-3, is elevated (29). In the current study we found increased VEGF and Ang-2 mRNA expression as well as an increased Ang-2/Ang-1 ratio. As Ang-2, Tie-2, and VEGF mRNA are up-regulated during simulated pregnancy, the elevated ratio suggests that after the initial angiogenic burst during the early luteal phase, the molecular environment would be conducive for further angiogenesis to take place in the rescued CL.
In conclusion, our results are consistent with the hypothesis that VEGF and angiopoietins play a major role in human CL regulation by paracrine actions and imply that angiopoietins are involved during the initial angiogenic phase and in luteal rescue. Future studies will be required to elucidate fully the molecular regulation and morphological changes in the vasculature within the CL of the cycle and especially during early pregnancy. Maintenance of luteal function is essential for continuation of pregnancy, and inhibition of VEGF-induced angiogenesis has been shown to have antifertility effects in experimental animals (11, 12). Thus, the normal processes of luteinization and luteolysis in the human could be profoundly influenced by manipulation of angiogenic factors. Our results indicate that angiopoietins have a role in the regulation of vascular development, especially during luteal rescue in women. This should add impetus to developing agents with angiogenic or antiangiogenic effects in the CL for therapeutic application.
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Acknowledgments |
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Footnotes |
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Received April 8, 2000.
Revised June 30, 2000.
Accepted July 24, 2000.
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) and Ang-2 (
)
overall grain density in the human CL throughout the luteal phase and
during luteal rescue. The highest Ang-1 grain density
(P < 0.05) was found during the early luteal
phase. During luteal rescue the highest grain concentration
(P < 0.05) was detected for Ang-2. Different
letters indicate significant differences. b, The Ang-2/Ang-1 ratio
increases during luteal rescue. Note the increase in the ratio from the
early to the late luteal phase. The highest ratio was calculated for
luteal rescue.
