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Inhibition of Early Luteal Angiogenesis by Gonadotropin-Releasing Hormone Antagonist Treatment in the Primate

Medical Research Council Human Reproductive Sciences Unit, Centre for Reproductive Biology, Edinburgh, EH3 9ET, United Kingdom

Address correspondence and requests for reprints to: Sarah E. Dickson, Medical Research Council Human Reproductive Sciences Unit, Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh, EH3 9ET, United Kingdom. E-mail: s.dickson{at}ed-rbu.mrc.ac.uk

	Abstract

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Angiogenesis during luteal development is essential for normal lutein cell function, but the control of this process and the relationships between the steroidogenic and endothelial cells have still to be elucidated. The aim of this study was to: 1) quantify endothelial cell proliferation throughout the luteal phase of the marmoset ovulatory cycle; 2) determine the effect of gonadotropin withdrawal using GnRH antagonist treatment on the early luteal phase angiogenesis peak; and 3) describe the resultant morphological changes in the corpus luteum (CL). Ovaries were collected during the early, mid-, and late luteal phase, and changes in angiogenic activity were determined by quantification of bromodeoxyuridine incorporation. Animals were treated with a GnRH antagonist, on luteal days 1 and 2, and ovaries were collected on day 3. A proliferation index was obtained by counting the number of bromodeoxyuridine immunopositive cells in luteal sections. Cell proliferation was maximal in the early luteal phase and fell significantly in the mid- and late CL. GnRH antagonist treatment reduced the early luteal phase proliferation peak by 90%, suppressed plasma progesterone, and severely disrupted lutein cell morphology. These results demonstrate that the intense angiogenesis in the early primate CL is dependent on gonadotropin stimulation of lutein cells.

	Introduction

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THE CORPUS luteum (CL) is formed subsequent to follicular development and rupture of the preovulatory follicle at ovulation (1). It is well established that LH is the major luteotrophic factor regulating the function of the primate CL. Withdrawal of LH by GnRH antagonist administration results in luteolysis of the CL in the Old World primate (2), New World primate (3), and women (4), as reflected by decreased plasma progesterone levels.

Luteinization is accompanied by prolific angiogenesis, which is essential for normal CL function. During the establishment of the CL, vascularization of the granulosa cell layer takes place as the basement membrane, which in the follicle separated the avascular granulosa from the highly vascularized theca, undergoes loss of integrity. The increased blood supply provides luteal steroidogenic (lutein) cells with oxygen, nutrients, and substrates for progesterone and growth factor biosynthesis, and it facilitates removal of end products of metabolism. The growth of new blood vessels can be monitored by measuring endothelial cell proliferation. Proliferation studies in the human (5, 6), nonhuman primate (7), ovine (8), and bovine (9) show peak levels at the early stages of luteal development, whereas CL regression is associated with a decreased proliferative rate.

Regulation of angiogenesis is controlled by endothelial growth factors, which regulate cell-cell interactions. Most attention has focused on vascular endothelial growth factor (VEGF). It has been shown that neutralization of VEGF in the rat (10) and marmoset monkey (11) blocks both luteal angiogenesis and progesterone production. In most tissues, VEGF is stimulated by hypoxia; but in the endocrine system, the tropic hormones are likely to play a role. A relationship between gonadotropin stimulation of cultured luteinized granulosa cells and VEGF production in vitro has been established (12, 13, 14, 15). In turn, addition of VEGF to isolated macaque luteal endothelial cells stimulates proliferation (16). However, the in vivo connection between gonadotropic stimulation of lutein cells and angiogenesis has not been demonstrated. In this study, endothelial cell proliferation in the CL throughout the luteal phase of the ovulatory cycle of the marmoset was evaluated. The most intense phase of angiogenesis was subsequently targeted for the effects of gonadotropin withdrawal by treatment with GnRH antagonist. Assessment of treatment effects was based on examination of luteal endothelial cell proliferation and morphology of the lutein cells.

	Materials and Methods

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Animals and treatments

Marmoset monkeys (Calithrix jacchus) were housed as described previously (17), and procedures were carried out in accordance with the Animals (Scientific Procedures) Act, 1986. Blood samples were collected by femoral venepuncture, three times per week, without anesthesia. Ovulatory cycles were monitored by RIA of plasma progesterone, as previously described (18). The day of ovulation (day 0 of the luteal phase) was taken as the day on which progesterone concentration rose above 30 nmol/L when followed by a sustained increase, characteristic of the luteal phase.

Ovaries were collected from animals in the early (luteal days 2–4), mid-(days 8–10), and late (days 16–20) luteal phase of the ovulatory cycle (n = 5 animals per group). Twenty milligrams of bromodeoxyuridine (BrdU; Roche Molecular Biochemicals, Sussex, UK), dissolved in 500 µl physiological saline, was administered iv, 1 h before tissue collection, to label proliferating cells in the S phase of the cell cycle, via incorporation into DNA. This allowed changes in endothelial cell proliferation throughout the luteal phase of the ovulatory cycle to be determined; and, because the early luteal phase exhibited most intense angiogenesis, this period was selected for targeting with GnRH antagonist to investigate the gonadotropin dependence of early luteal primate angiogenesis.

The animals to receive GnRH antagonist treatment and early luteal control animals were given 1 µg PG F₂ analog (Planate; Coopers Animal Health Ltd., Crewe, Cheshire, UK) im, in the mid- to late luteal phase of the pretreatment cycle, to induce luteolysis and synchronize subsequent ovulation, which was presumed to occur 10 days after PG treatment (luteal day 0) (19). Blood samples were collected three times per week, then daily from the day of presumed ovulation, and the animals were subsequently treated with either 1 mg/kg sc. GnRH antagonist, Antarelix (20) or vehicle on luteal days 1 and 2 (n = 4). Ovaries were collected on day 3, as previously described (21), 1 h after administration of BrdU to animals in both control and treatment groups. Ovaries were fixed immediately in 4% paraformaldehyde for paraffin-embedding, and a small piece of CL was fixed in 3% glutaraldehyde in 0.1 mol/L cacodylate buffer, pH 7.3, for araldite resin embedding.

Immunocytochemistry

Paraffin-embedded ovarian sections (5 µm) were mounted onto TESPA (Sigma, Dorset, UK) coated glass slides and dried at 50 C overnight. Fluorescent and colorimetric colocalization of BrdU (Roche Molecular Biochemicals) and the endothelial cell marker, von Willebrand Factor VIII-associated antigen (Factor VIII; DAKO Corp., Cambridge, UK) was carried out on early and mid-luteal ovarian tissue. Sections were dewaxed in Histoclear (National Diagnostics, Hull, UK), rehydrated in descending concentrations of industrial methylated spirits, and washed in distilled water. Antigen retrieval was performed by microwaving (750W) slides on full power in 0.01 mol/L citrate buffer, pH6, for 4 sessions of 5 min. A constant volume of buffer was kept by addition of warm distilled H₂O after each 5 min of microwaving. The slides remained in hot buffer for a further 20 min and were washed in PBS (0.01 mol/L PBS, pH 7.4, containing 2.7 mmol/L KCl, 0.137 mol/L NaCl). The following procedures, with the exception of immunostaining development, were carried out in Sequenza racks (Life Sciences International, Hampshire, UK). Sections were treated for 30 min, at room temperature, with normal goat serum, diluted 1:5 in PBS + 0.25 g BSA [normal goat serum (NGS) diluted 1:5 in PBS + 0.25 g BSA], followed by dual incubation in rabbit polyclonal anti-Factor VIII (22.8 µg/mL in NGS), and mouse monoclonal anti-BrdU (6.6 µg/mL in NGS) at 4 C, overnight. Immunolocalization was undertaken using anti-rabbit FITC conjugate (diluted 1:50 in NGS, Sigma F-6005) and anti-mouse TRITC conjugate (diluted 1:50 in NGS, Sigma T-5393) dual incubation for 45 min at room temperature. The U-MWIBA and U-MWG filters on the Olympus Corp. AX70 Provis fluorescent microscope were used to visualize FITC-stained Factor VIII positive cells, and TRITC-stained BrdU positive cells, respectively.

The occurrence of erythrocyte autofluorescence masked BrdU staining in most sections, and this prevented quantification of endothelial cell proliferation in the majority of CL. To circumvent this problem, a colorimetric approach was also used for analysis. Endogenous peroxidase activity was quenched with a 30-min incubation in 3% hydrogen peroxide in methanol, at room temperature. Microwaving and NGS [diluted in Tris-buffered saline (TBS) (pH 7.4), containing 50 mmol/L Tris-HCl, 150 mmol/L NaCl] blocking was performed as before. Sections were incubated with anti-Factor VIII (28.8 µg/mL in TBS) alone at 4 C, overnight. Immunolocalization was detected with the mouse EnVision system (DAKO Corp.), according to the guidelines of the manufacturer. For BrdU colocalization, nonspecific binding was blocked with normal rabbit serum in TBS (NRS). Sections were incubated in mouse monoclonal anti-BrdU (3.3 µg/mL, in TBS) at 4 C, overnight, or for 2 h at room temperature. Rabbit anti-mouse (DAKO Corp., 26.6 µg/mL in NRS), was applied for 30 min at room temperature. BrdU binding was visualized using mouse APAAP (DAKO Corp., 1 µg/mL in NRS) incubation for 30 min, and the Fast Blue detection system (1 mg Fast Blue BB salt, per 1 mL Fast Blue buffer, containing 20 mg naphthol AS-MX Phosphate, 2 mL dimethylformamide, and 98 mL 0.1 mol/L Tris (pH 8.2), all from Sigma). The reaction was stopped with distilled water, and the slides were very lightly counterstained with hematoxylin and mounted with Permafluor aqueous mounting medium (Coulter Electronics, Bedfordshire, UK).

The procedure for BrdU immunostaining alone to determine changes in cell proliferation throughout the cycle and after treatment was carried out as described previously (17). In this case the NBT (nitro-blue tetrazolium, Roche Molecular Biochemicals) detection method was used, and intense counterstaining was needed.

To examine the effect of GnRH antagonist treatment on CL morphology, treated and control sections were stained with toluidine blue (BDH, Poole, Dorset, UK). Araldite, resin-embedded, 1-µm sections were incubated in toluidine blue at 70 C for 8 min, dried, and examined under the light microscope.

Labeling index and statistical analysis

Manual counting procedures were used to calculate the percentage of proliferating endothelial cells for each CL. Quantification was performed blind on both fluorescent sections and those stained using the colorimetric approach. Both early and mid-luteal phase, left and right ovaries for each animal were examined for appropriately aged CL. Data from all CL fulfilling these criteria were collected for subsequent analysis. Six randomly chosen fields per CL, at x400 magnification, were analyzed using a grid overlay. The number of proliferating endothelial cells was expressed as a percentage of proliferating cells, i.e. (number of dual stained cells/total number of BrdU positive cells) x 100.

The proliferation index (PI) was calculated in a similar manner. Ovaries throughout the luteal phase of the cycle were stained with anti-BrdU only. The number of positive BrdU cells was expressed as a percentage of total number of cells per graticule field, i.e. (number of BrdU positive cells/total cells) x 100. Representative sections were also quantified by a second individual. The PIs obtained correlated highly (r = 0.97, P < 0.001).

PI and plasma progesterone data throughout the luteal phase of the cycle were analyzed using Fisher’s protected least-significant difference ANOVA test, at 5% significance. The effects of GnRH antagonist treatment on PI and plasma progesterone levels, as compared with controls, were determined using a two-tailed, unpaired t test, with a 95% confidence interval. Both tests were performed using Statview version 4.0.

	Results

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PI and plasma progesterone throughout the cycle

Figure 1 depicts the CL PI and plasma progesterone concentration throughout the luteal phase of the marmoset ovulatory cycle. Peak cell proliferation was observed in the early luteal phase CL and declined markedly (P < 0.001, as compared with early levels) in the mid- and late CL. The 2.5-fold decrease in PI from the mid- to late CL was also significant (P < 0.05). Plasma progesterone concentration increased (P < 0.01) from early luteal levels, to peak in the mid-luteal phase, subsequently decreasing (P < 0.01) from mid- to late luteal phase.

View larger version (14K): [in this window] [in a new window]   Figure 1. A, Cell proliferation in the marmoset CL; B, plasma progesterone concentration throughout the luteal phase of the ovulatory cycle. Values are means ± SEM. Different letters denote significant differences between categories, P < 0.05.

Figure 2 shows fluorescent colocalization of Factor VIII and BrdU. Fig. 2a demonstrates the green cytoplasmic immunostaining for Factor VIII, and Fig. 2b shows red nuclear fluorescent staining for BrdU in the same section. Fig. 2c illustrates colocalization of Factor VIII and BrdU by overlaying 2a and 2b. From quantification of this and the colorimetric method of dual staining, the mean percentage Factor VIII and BrdU colocalization in the early CL was 84.6 ± 0.9 (mean ± SEM); and in the mid-CL, 84.4 ± 1.3 (n = 4 per group).

View larger version (111K): [in this window] [in a new window]   Figure 2. Fluorescent colocalization of Factor VIII and BrdU, and effect of GnRH antagonist treatment on PI, cell morphology, and progesterone output. a–c, Fluorescent immunocytochemical localization of endothelial cell marker, Factor VIII(a); proliferation marker, BrdU (b); and colocalization of the two (c), in an early CL. Note that most BrdU positive cells are also Factor VIII positive. d–f, Immunocytochemical localization of BrdU in an early control CL (d) and the CL of a GnRH antagonist treated marmoset at the same stage of the cycle (e). Note the marked reduction in BrdU incorporation and cellular disruption after treatment. Note also, the presence of the blood vessel (BV), and possible apoptotic bodies indicated by the arrow. f, The effect of GnRH antagonist treatment on PI (% BrdU incorporation). Values are means ± SEM, P < 0.001. g, Toluidine blue-stained sections of early control CL; h, a CL after GnRH antagonist treatment. Note the healthy appearance of steroidogenic cells with abundant cytoplasm (C), clear nuclei with nucleoli (N), and probable endothelial cells (E) in the control CL. After treatment, the cellular integrity is severely disrupted; and dense bodies indicated by the arrowheads, lipid droplets (L), and some intact nuclei (N) are present. i, The effect of GnRH antagonist treatment on plasma progesterone concentration. Values are means ± SEM, P < 0.001. Scale bars, 50 µm.

An elevation of plasma progesterone was observed before treatment in all animals and confirmed by the identification of recently formed CL in control and GnRH antagonist-treated animals. Fig. 2, d–f, shows a comparison of BrdU immunolocalization and PI in an early luteal control CL and an Antarelix-treated CL. BrdU positive staining (dark nuclei) and hematoxylin staining (pale nuclei) are obvious. A significant decrease (P < 0.001) in PI is seen in CL from the Antarelix-treated group of animals, as compared with the early luteal controls. Clusters of densely stained nucleic fragments, characteristic of apoptotic cell death, were apparent in the Antarelix-treated CL (Fig. 2e).

Figure 2, g and h, shows toluidine blue-stained sections of an early control CL and an Antarelix-treated CL. There is a significant effect on lutein cell morphology in the treated CL, as compared with controls. The lutein cells of the early control CL appear normal for this species, with distinct cell margins, and well formed nuclei containing a single nucleolus contained in an abundant cytoplasm, typical of a steroidogenic lutein cell. This contrasts with the appearance of the CL after GnRH antagonist treatment. Here, the CL shows disorganization, and the lutein cells show much-less-clearly defined margins. In addition, there is evidence of lipid accumulation and the occurrence of dense bodies. Typical apoptotic bodies were not observed. Recognizable lutein cells are smaller than in the control tissue, even those in which the nucleus remains intact. Finally, GnRH antagonist treatment was associated with a marked reduction (P < 0.001) in plasma progesterone concentration, to follicular phase levels (Fig. 2i).

	Discussion

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This study has established, for the first time in vivo, that the intense angiogenesis in the primate CL associated with the early luteal phase is dependent on gonadotropin support. Over 80% of proliferating cells in the marmoset CL are endothelial cells; and thus, it is reasonable to consider cell proliferation in the CL, based on the PI from single BrdU immunostaining, as being reflective of endothelial cell proliferation. There is evidence that there is little or no proliferation of fully differentiated primate lutein cells (7), which implies that the remaining BrdU positive cells in the CL are most likely fibroblasts, infiltrating macrophages, or endothelial support cells, such as pericytes.

The quantification of endothelial cell proliferation throughout the luteal phase, using the so-called gold standard BrdU technique, shows peak endothelial cell proliferation in the early CL, which had significantly decreased by the mid- and late stages of luteal development. The results agree with studies evaluating cell proliferation in corpora lutea from other species (6, 7, 8, 9, 22). The intense proliferation in the early luteal phase leads to increased vascularization during luteinization and CL establishment. By the mid-luteal stage, it is thought that the majority of steroidogenic cells are in contact with at least one capillary (9, 22). Peak plasma progesterone concentrations in the mid-luteal phase correlate with the presence of an extensive capillary network in the CL, for optimal delivery of progesterone precursors to, and progesterone from, the cell. Decreased endothelial cell proliferation in the late luteal phase CL coincides with the observed fall in plasma progesterone concentration and luteal regression (22).

GnRH antagonist treatment was employed to examine the role of gonadotropin dependence of the intense angiogenesis associated with the early luteal phase. In the marmoset, direct effects of GnRH antagonist treatment on the CL are unlikely (23), so the treatment is presumed to be a consequence of pituitary withdrawal of LH, as described previously in the marmoset (3), macaque (2), and human (4) luteal phase. Deprivation of gonadotropin support to steroidogenic cells reduced endothelial cell proliferation by 90%, and plasma progesterone concentration by 95%, demonstrating the dependence of early luteal angiogenesis and function on gonadotropin support.

Light microscopy of toluidine blue-stained sections revealed a severe disruption of normal steroidogenic cell morphology in the GnRH antagonist-treated CL, when compared with early luteal controls. Accumulation of lipid, appearance of densely staining bodies, and the absence of distinct cell margins were obvious after treatment. There was evidence of fragmented nuclei, characteristic of apoptosis, in hematoxylin-stained CL from treated marmosets. However, analysis of semi-thin sections indicated widespread cell death by a process other than apoptosis, as also suggested previously (24, 25); and because ultrastructural studies of GnRH antagonist-induced luteolysis in the marmoset do not indicate widespread cell degeneration via apoptosis (26), it is difficult to ascertain the method of luteal cell death seen in this study. It is clear, however, that the extent of luteal cell disruption was such that steroidogenic cell function was significantly impaired, preventing secretion of progesterone and, most likely, other factors involved in normal CL function.

Early luteal phase administration of GnRH antagonist to macaques (27) and women results in suppression of progesterone secretion and a marked reduction, in response to exogenous hCG administered in the mid-luteal phase (4, 27). Here, we show that administration of GnRH antagonist in the early luteal phase causes a severe disruption of steroidogenic cell morphology and angiogenesis, thus providing an explanation, at the cellular level, for the effects of GnRH antagonist treatment seen previously.

It is tempting to suggest that the adverse effect of GnRH antagonist treatment on early luteal angiogenesis is predominantly a consequence of decreased VEGF production from the dysmorphic steroidogenic cells of the developing CL. VEGF is a secreted angiogenic mitogen (28), specific for endothelial cell receptors (29, 30). Both VEGF messenger RNA (mRNA) and its transcribed protein have been localized to the granulosa and theca lutein cells of the human CL (31, 32). Cultured human (12, 13) and macaque (14, 15) luteinized granulosa cell VEGF expression has been shown to be up-regulated by gonadotropins in vitro. In preliminary investigations, using immunocytochemistry to determine changes in VEGF in the CL after GnRH antagonist treatment, we did not observe a decrease in immunostaining (unpublished observation). Hypoxia-induced VEGF production; the presence of residual, nonactive, or metabolized VEGF; or nonspecific staining by lutein cell debris in the treated CL may explain the unexpected failure to demonstrate decreased VEGF. Angiogenesis is a multistep process involving a number of angiogenic growth factors, degradation of the extracellular matrix by matrix metalloproteinases, and cell-cell interactions. Neutralization of VEGF in the marmoset, over the same time period, reduces endothelial cell proliferation by 80%, as compared with control levels (11), a significantly greater suppression is demonstrated after GnRH antagonist treatment in this study. This suggests that the reduction in luteal angiogenesis seen here is most probably a consequence of a decrease in other angiogenic factors in addition to VEGF, and/or by a breakdown in endothelial-steroidogenic cell interactions after disruption of lutein cell morphology as caused by treatment. The reduction in local progesterone concentration may also act to reduce angiogenesis. Progesterone is thought to have a local action in the periovulatory follicle, to regulate expression of modulators of the angiogenic process, such as matrix metalloproteinase-1 and its tissue inhibitor (33), implying a role in angiogenesis in the early CL.

The potential of manipulation of angiogenesis in the human reproductive tract is currently generating considerable interest (10, 11, 17), and it will be important to elucidate fully the mechanisms regulating the physiological process in suitable primate models. The current results establish that the intense angiogenesis associated with the formation of the CL, and necessary for normal luteal function, is almost exclusively dependent on normal gonadotropin support.

	Acknowledgments

 
We are grateful to K. Morris and staff for animal management; Dr. R. Deghengi (Europeptides) for the gift of Antarelix; Dr. S. F. Lunn and Dr. C. Wulff for critical evaluation of the manuscript and assistance with photography and morphological analysis; M. Millar, S. MacPherson, and P. Largue for histological support; H. Wilson for assistance with PI quantification; and F. Pitt and I. Swanston for progesterone assay.

Received July 22, 1999.

Revised February 9, 2000.

Accepted February 21, 2000.

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				H. M. Fraser, H. Wilson, K. D. Morris, I. Swanston, and S. J. Wiegand Vascular Endothelial Growth Factor Trap Suppresses Ovarian Function at All Stages of the Luteal Phase in the Macaque J. Clin. Endocrinol. Metab., October 1, 2005; 90(10): 5811 - 5818. [Abstract] [Full Text] [PDF]

				P D Taylor, S G Hillier, and H M Fraser Effects of GnRH antagonist treatment on follicular development and angiogenesis in the primate ovary J. Endocrinol., October 1, 2004; 183(1): 1 - 17. [Abstract] [Full Text] [PDF]

				H. E. Turner, A. L. Harris, S. Melmed, and J. A. H. Wass Angiogenesis in Endocrine Tumors Endocr. Rev., October 1, 2003; 24(5): 600 - 632. [Abstract] [Full Text] [PDF]

				P. Kohen, O. Castro, A. Palomino, A. Munoz, L. K. Christenson, W. Sierralta, P. Carvallo, J. F. Strauss III, and L. Devoto The Steroidogenic Response and Corpus Luteum Expression of the Steroidogenic Acute Regulatory Protein after Human Chorionic Gonadotropin Administration at Different Times in the Human Luteal Phase J. Clin. Endocrinol. Metab., July 1, 2003; 88(7): 3421 - 3430. [Abstract] [Full Text] [PDF]

				A. J. Rowe, K. D. Morris, R. Bicknell, and H. M. Fraser Angiogenesis in the Corpus Luteum of Early Pregnancy in the Marmoset and the Effects of Vascular Endothelial Growth Factor Immunoneutralization on Establishment of Pregnancy Biol Reprod, October 1, 2002; 67(4): 1180 - 1188. [Abstract] [Full Text] [PDF]

				C. Wulff, S. E. Dickson, W. C. Duncan, and H. M. Fraser Angiogenesis in the human corpus luteum: simulated early pregnancy by HCG treatment is associated with both angiogenesis and vessel stabilization Hum. Reprod., December 1, 2001; 16(12): 2515 - 2524. [Abstract] [Full Text] [PDF]

				C. Wulff, H. Wilson, J. S. Rudge, S. J. Wiegand, S. F. Lunn, and H. M. Fraser Luteal Angiogenesis: Prevention and Intervention by Treatment with Vascular Endothelial Growth Factor TrapA40 J. Clin. Endocrinol. Metab., July 1, 2001; 86(7): 3377 - 3386. [Abstract] [Full Text] [PDF]

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