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a Division of Reproductive Sciences, Oregon National Primate Research Center/Oregon Health & Science University, Beaverton, Oregon 97006
b Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, Oregon 97201
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ovary, ovulation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is interesting that VEGFR1 is also produced by endothelial cells as a soluble receptor (sVEGFR1) by alternative splicing of the precursor mRNA [11]. Circulating levels of sVEGFR1 increase during pregnancy, when it may serve to neutralize the systemic or local actions of VEGF from the placenta [12, 13]. Therefore, sVEGFR1 could serve as a tool to pharmacologically block VEGF action; indeed, injection of a truncated form of sVEGFR1 into rats for 5 days during follicular stimulation protocols resulted in depressed circulating progesterone (P4) levels and corpora lutea nearly devoid of immunoreactive vascular endothelial cells [14]. Although treatment completely suppressed luteal angiogenesis, injections commenced during the follicular phase; therefore, it is unclear whether the effects of VEGF blockade are due to disruption of vascular events in the corpus luteum or its antecedent follicle. In other studies, nonhuman primates treated systemically with an anti-VEGF antibody [15] or a soluble truncated form of the VEGFR1 (VEGF TrapA40 [16]) displayed lower levels of serum P4 during the luteal phase. However, it is unclear whether the decline in serum P4 was due to direct effects on the ovary as opposed to indirect effects mediated by changes in the neuroendocrine reproductive axis. To study the local effects of antiangiogenic agents on follicular maturation or early luteal development, we recently developed a technique for injecting agents directly into the preovulatory follicle during the menstrual cycle of the rhesus monkey [17]. The current study was designed to determine the effects of neutralizing VEGF action by local administration of sVEGFR1 fusion protein into the preovulatory follicle of the rhesus monkey on periovulatory events and subsequent development and function of the corpus luteum.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The general care and housing of rhesus monkeys at the Oregon National Primate Research Center (ONPRC) was described previously [18]. Animal protocols and experiments were approved by the ONPRC Animal Care and Use Committee, and studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals [19].
In Vivo Studies Employing Intrafollicular Injection of sVEGFR1
Adult female rhesus monkeys exhibiting normal menstrual cycles of approximately 28 days were bled daily by saphenous venipuncture beginning 6–8 days after the onset of menses. Serum concentrations of estradiol (E2) and P4 were measured by radioimmunoassays that were validated for nonhuman primates [20, 21]. Estradiol values were used to estimate the day of the midcycle gonadotropin surge and time of ovulation as previously established in our laboratory [22]. Briefly, Day 1 of the luteal phase was designated as the day when preovulatory serum E2 levels (range, 250–600 pg/ml) decline to less than 100 pg/ml [22]. In order to administer the antiangiogenic compound prior to ovulation, intrafollicular injections were performed when E2 levels exceeded 150 pg/ml.
Intrafollicular injections (modified from the report by Ginther et al. [23]) were performed on anesthetized [24] animals during surgery to expose the ovary bearing the dominant follicle as previously described [17]. Briefly, an insulin syringe containing 50 µl of solution was inserted through the stroma of the ovary before penetrating the follicular wall. Then, 50 µl of follicular fluid was aspirated into the syringe, diluting the injectable by half, before injecting 50 µl of this mixed solution into the follicle. When the needle was removed, the follicle was observed to note whether any deflation had occurred. Ovaries were viewed by laparoscopy 3 days after injection for evidence of follicle rupture and luteinization. Blood samples were collected on a daily basis until the first day of menses and analyzed for serum P4 levels. The day of the LH surge was confirmed using a mouse Leydig cell bioassay for LH [25], and verified that all animals were injected on the day before (Day -1) or the day of (Day 0) the midcycle gonadotropin surge.
A sequential experimental design was applied in which animals (n = 14) received an intrafollicular injection of vehicle (PBS:0.1% BSA, control) during the first experimental cycle (protocol 1) and recombinant human Flt-1/Fc immunoglobulin (Ig) G chimera (sVEGFR1; R&D Systems, Minneapolis, MN) in the second experimental cycle (protocol 2). After menses in protocol 1, animals were permitted to recover for at least one menstrual cycle before blood samples were taken to start protocol 2, resulting in administration of either a low dose of 7.5 µg/ml (1.5 µg in a 200-µl follicle [follicular volume determined from aspiration studies]; n = 4) or a high dose of 37.5 µg/ml (7.5 µg in a 200-µl follicle; n = 4) of sVEGFR1.
In protocols 3 and 4, animals again acted as their own controls but received in random sequence an intrafollicular injection of either vehicle or VEGF antagonist (low or high dose, as described in protocol 2) on Day -1 or Day 0. Animals were laparotomized 3 or 6 days (n = 3 per treatment) after the injection, and the ovary bearing the injected follicle was removed and fixed in formaldehyde overnight before embedding in paraffin (Day 3) or it was fresh-frozen in Tissue-Tek II OCT (Miles, Inc., Elkhart, IN) and frozen in liquid propane (Day 6). After hemiovariectomy, the animals were again permitted to recover for at least one menstrual cycle before starting protocol 4 for injection of the agent that was not delivered in protocol 3.
Tissue Analysis
Paraffin-embedded tissues from ovaries collected on Day 3 after injection were serially sectioned by the ONPRC Imaging and Morphology Core Laboratory using an American Optical (Southbridge, MA) microtome and mounted on Superfrost plus slides (Fisher, Santa Clara, CA). Slides were stained with hematoxylin and eosin, and viewed to determine whether the oocyte was released from the follicle or trapped within the luteinized tissue.
Fresh-frozen ovaries collected on Day 6 after injection were cryosectioned at 5 µm at -18°C, mounted on Superfrost plus slides (Fisher), and stored at -70°C. Slides were analyzed for Ki67 (a cell proliferation marker [26]) and PECAM-1 (an endothelial cell marker [26]) using immunocytochemistry and for 3ß-hydroxysteroid dehydrogenase (3ß-HSD, a steroidogenic cell marker [26, 27]) by histochemistry.
Immunocytochemistry was performed as previously described [26, 28] with some modifications. Briefly, tissue sections were fixed in 4% paraformaldehyde/PBS pH 7.4, dipped in a quenching solution (3% hydrogen peroxide/60% methanal) to remove endoperoxidase activity, and then placed in 10% normal blocking serum (ABC kit, Vector Laboratories, Inc., Burlingame, CA) for 20 min before incubation overnight at 4°C with mouse anti-human antibody for Ki67 or PECAM-1 (DAKO Corporation, Carpenteria, CA), or with mouse IgG as the control. Thereafter, tissue sections were treated with biotinylated antibody (ABC kit) for 40–45 min at room temperature, followed by a 45-min incubation with the avidin-biotin-peroxidase complex (ABC kit). The antigen-antibody complex was visualized by incubation with freshly prepared 3,3'-diaminobenzidine (DAB kit, Vector Laboratories), and the tissue was counterstained with hematoxylin.
Histochemistry for 3ß-HSD was performed as reported previously [26, 27]. Frozen sections were fixed in 1% paraformaldehyde/0.1 M phosphate buffer (PB) pH 7.2 and incubated in staining buffer (0.1 M PB pH 7.2, 0.1% BSA, 0.02% nitroblue tetrazolium, 0.00058% 5ß-androstan-3ß ol-17 one [Sigma, St. Louis, MO], 0.1% NAD [Sigma] or without NAD as negative control) at 37°C for 3 h. After rinsing with water, the sections were counterstained with nuclear fast red.
Five fields were randomly selected from the parenchyma of each corpus luteum using a Zeiss Axioplan microscope (Carl Zeiss, Gottingen, Germany) at 400x magnification, and photographed using a Cool SNAP CDD camera (Roper Scientific, Inc., Tucson, AZ). Images were transferred to a laptop computer, and the number of Ki67-positive, PECAM-1-positive, and 3ß-HSD-positive cells were counted manually using Photoshop 5.5 software (Adobe Systems, Inc., San Jose, CA). The area constituting the vasculature (PECAM-1-positive cells) and extracellular space (open areas, see Fig. 3) was estimated using Image-Pro Plus 4.0 (Media Cybernetics, L.P., Silver Spring, MD). The investigator was blinded to the treatment group for each tissue.
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In Vitro Studies Using Macaque Follicle Cells
Granulosa cells were obtained from anesthetized monkeys by follicle aspiration as previously described [18]. Somatic cells were harvested from follicular aspirates, enriched for granulosa cells, and assessed for cell viability as described previously [4]. Cells were resuspended in Dulbecco modified Eagle medium F-12 with low density lipoprotein (2 µg/ml; Sigma), insulin/transferrin/sodium selenite (10 µl/ml; Sigma), and aprotinin (10 µg/ml; Sigma) and approximately 50 000 cells per 500 µl were plated in each well (48-well plate; Corning Costar Corp., Corning, NY). Lyopholized sVEGFR1 was resuspended in filter-sterilized PBS and 0.1% BSA, and added to the wells in final concentrations varying 100-fold from 0.03 to 37.5 µg/ml. Cells were incubated at 37°C in duplicate either alone (controls), with PBS/0.1% BSA, or one of nine doses of sVEGFR1 for 24 or 48 h. Incubation medium was removed and stored for P4 analysis. Cell number was determined by crystal violet staining [29] as previously described [4].
Statistics
Progesterone levels in serum and culture media, as well as cell numbers and tissue areas, were analyzed using a repeated measures ANOVA (STATPAK, Northwest Analytical, Portland, OR) to characterize differences between controls and the low and high doses of sVEGFR1. Differences were considered significant at P < 0.05, and values are presented as means ± SEM (n = 3–4 per treatment group).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Incubation with 0.03–37.5 µg/ml sVEGFR1 did not alter P4 levels or numbers of luteinized granulosa cells after 24 or 48 h of culture compared to controls (data not shown). Thus, two doses of VEGFR1 (7.5 µg and 37.5 µg/ml) were chosen to achieve intrafollicular concentrations, which are comparable to (7.5 µg/ml) or higher (37.5 µg/ml) than the systemic doses reported to suppress ovarian angiogenesis in rats [14].
In Vivo Studies: Intrafollicular Injections
The pattern and levels of serum E2 and P4 in vehicle-treated (control) animals were typically comparable to those observed during natural menstrual cycles in untreated animals in our colony [30]. Vehicle treatment resulted in 13 of 14 animals displaying the expected hormonal patterns. Estradiol levels peaked during the ovulatory surge of LH (not shown), which is designated as Day 0. Serum P4 levels (Fig. 1, shaded region) increased on Day 0 and continued to rise until peaking at midluteal phase (Days 6–8). Serum concentrations of P4 remain elevated until Days 11–12 and then declined through the late luteal phase until menstruation. One vehicle-treated animal did not have hormone patterns typical of untreated animals (not shown) and was dropped from further protocols.
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Serum P4 concentrations after injection of low-dose sVEGFR1 into the preovulatory follicle are depicted in Figure 1 (upper panel). Although this dose did not significantly alter P4 levels as a group (n = 4; not shown) compared with those of controls, when treated animals were examined individually, low-dose sVEGFR1 injection suppressed P4 concentrations during the early to midluteal phase in two of four animals. However, there was no change in the day of menstruation between controls and low-dose sVEGFR1-treated groups.
Administration of high-dose sVEGFR1 resulted in a consistent hormonal profile (Fig. 1, lower panel). In all four animals, serum P4 concentrations were significantly reduced compared with those in controls (P < 0.05), with suppressed levels by midluteal phase, which remained low until the late luteal phase. Again, however, there was no change in the day of onset of menstruation after sVEGFR1 treatment.
By 3 days after the intrafollicular injection, all vehicle-injected ovaries (Fig. 2A) displayed indices of follicle rupture (protruding stigmata) and luteinization (yellowish tissue with prominent capillaries). VEGFR1-treated (both low and high doses) ovaries exhibited signs of distension (torn surface epithelium/tunica albuginea) but not necessarily of follicle rupture (malformed or missing stigmata; e.g., Fig. 2B). Evaluation of serial sections of ovaries collected 3 days after the intrafollicular injection of vehicle revealed an ovulatory canal (Fig. 2C) that ran from within the luteinizing tissue (Fig. 2, C and D) to the surface of the ovary, as well as the absence of an oocyte. Conversely, serial sections from sVEGFR1-injected follicles revealed that ovulation occurred only 50% of the time (three of six animals). In three animals, ovulation was confirmed by the presence of an ovulatory canal as well as by the absence of an oocyte (not shown). In these sections, signs of luteinization (e.g., hypertrophy of cell layers) were apparent. In contrast, serial sections from three other animals were devoid of any ovulatory canal, and two of three animals had an intact oocyte remaining within the injected follicle (Fig. 2E). These follicles displayed few signs of luteinization, with small granulosa cells still lining the basement membrane, and they were occasionally found in the antral space (Fig. 2F). Blood cells were detectable within the antrum of two of three of these unruptured follicles.
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As reported previously by this group [26], appreciable numbers of cells stained positive for Ki67 in luteal tissue during the early (Day 3, Fig. 3) and mid (Day 6, not shown) luteal phase. Likewise, PECAM-positive and 3ß-HSD-positive cells were apparent within the corpus luteum at midluteal phase (Day 6, Fig. 3). There were no differences in the numbers of Ki67-positve (Fig. 3, A, D, and G), PECAM-positive (Fig. 3, B, E, and H) or 3ß-HSD-positive (Fig. 3, C, F, and I) cells between tissues from each treatment group (data not shown). Although the microvascular network formed by putative endothelial (PECAM-positive) cells appeared more developed in luteal tissue following vehicle injection (Fig. 3B) than after sVEGFR1 injection (Fig. 3, E and H), the percentage area of PECAM-1-positive cells in luteal tissue from sVEGFR1 animals was not significantly less than controls (13.2% ± 1.5% vs. 21.0% ±5.7%, respectively; P = 0.14). However, a general feature of corpora lutea from the sVEGFR-1 treatment groups was the pronounced areas of extracellular space, giving the luteal tissue a fragile, lattice (Fig. 3H) or porous (Fig. 3I) appearance. As such, the percentage area of extracellular space increased (P < 0.05) in a dose-dependent manner in luteal tissue following control and low- or high-dose sVEGFR1 treatment (4.6% ± 1.8%, 11.1% ± 2.3%, and 20.4% ± 2.8%, respectively).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This is the first report that local administration of a VEGF neutralizing compound can block ovulation in any species, including primates. Several physical differences, including the apparent clear surface of the protruding follicle, the distinct absence of a highly vascularized stigmata, and the presence of "trapped" oocytes, indicated that follicle rupture had not occurred by 3 days after sVEGFR1 treatment. Although we were able to block ovulation using sVEGFR1, a general antiangiogenic compound, TNP-470, did not prevent ovulation when locally administered in the preovulatory follicle [17]. This difference may be due to the additional role of VEGF as a vascular permeability factor (VPF [2]), which could alter local fluid fluxes and thus change hydrostatic pressure within the follicle. However, granulosa cell luteinization was also suppressed in unruptured follicles after VEGFR1 treatment, with no preovulatory rise in P4 at the time of the LH surge, which was characteristic of our controls and untreated cycles [30]. Previously, this laboratory demonstrated that blocking P4 production in the macaque follicle at midcycle will subsequently block ovulation [31]. Therefore, we cannot rule out that anovulation is secondary to sVEGFR1 suppression of P4 synthesis in the follicle. In any case, the presence of blood cells in the follicular antrum or luteal parenchyma suggests a disruption of vascular structure. Thus, these data suggest that intrafollicular administration of sVEGFR1 impairs or delays the ability of the follicle to rupture, possibly by interfering with vessel integrity, whereas TNP-470 may be less likely to disrupt existing vessels in the periovulatory follicle.
Intrafollicular administration of sVEGFR1 at midcycle also dose-dependently impairs subsequent luteal function. In this study, a low dose (7.5 µg/ml) inconsistently suppressed serum P4 concentrations and impaired only the early luteal phase rise in P4 levels, whereas a high dose (37.5 µg/ml) suppressed luteal function in all animals, and reduced serum P4 levels throughout the luteal phase. The data are consistent with the report [16] that daily s.c. injections of VEGF TrapA40 beginning on the day of ovulation suppresses P4 levels during the early luteal phase in marmosets. Further, neutralization of VEGF using antiserum during the luteal phase in marmosets suppressed P4 levels for the duration of antisera exposure [15]. In contrast, our data suggest that the targeted administration of a single dose of sVEGFR1 into the preovulatory follicle subsequently leads to reduced luteal function throughout the life span of the corpus luteum. It is interesting that unlike the current results, intrafollicular injection of a general antiangiogenic compound, TNP-470, did not cause a decline in circulating P4 levels until midluteal phase [17]. Although these compounds have different actions, VEGF reportedly is expressed in greater abundance in the developing versus fully formed corpus luteum [3, 32], which may explain the immediate action of low-dose and high-dose sVEGFR1 injection to suppress luteal function in the early luteal phase. In any case, sVEGFR1 is not merely delaying normal luteal function, because the duration of the luteal phase and timely menstruation occurred despite suppressed steroidogenic function.
To investigate cellular alterations in the corpus luteum following sVEGFR1 injection into the preovulatory follicle, ovaries were collected 3 or 6 days after treatment, and markers for cell proliferation and luteal cell types (nonsteroidogenic and steroidogenic) were analyzed, respectively. We previously reported [26] that proliferation, as judged by Ki67 nuclear staining, was highest in the macaque corpus luteum during the early luteal phase, and that 
95% of the proliferating cells costained for PECAM-1 (i.e., they were endothelial cells). Notably, acute exposure to sVEGFR1 in the periovulatory follicle did not alter the number of proliferating cells in the corpus luteum by Day 3 of the luteal phase. Also, the number of PECAM-1-positive endothelial cells in luteal tissue was not different between sVEGFR-1 and vehicle-treated monkeys by Day 6 of the luteal phase. Thus, administration of a VEGF antagonist did not prevent subsequent proliferation of endothelial cells in the developing corpus luteum, although other angiogenic factors from the pleiotropic basic fibroblast growth factor or the steroidogenic tissue-specific endocrine gland-VEGF [33] may compensate. It is interesting that Wulff et al. [16] demonstrated an up-regulation of the ligand angiopoietin 2 and its receptor, Tie-2, in the corpus luteum of marmosets treated with a VEGF TrapA40 beginning at ovulation. However, 3 days of treatment with VEGF TrapA40 also resulted in a marked reduction in endothelial cell area and less extensive microvessel development in the developing corpus luteum [16]. Although there was some morphologic evidence suggesting that the development of the luteal microvasculature was impaired by sVEGFR1 treatment in the current study (Fig. 3, B vs. H), despite typical endothelial cell proliferation, the effect appears less than that observed by Wulff et al. [16]. It remains unknown whether these differences are due to mode of administration, potency, duration of action of the VEGF antagonist, or to species differences. Finally, the enzyme 3ß-HSD, which is responsible for conversion of pregnenolone to P4 in steroid-producing cells, was present regardless of sVEGFR1 treatment. Further studies quantitating steroidogenic enzyme expression and activity are needed to determine whether changes in luteal cell activity are associated with the lower circulating P4 levels in VEGFR1-treated animals.
Alternatively, other tissue/cell defects may be responsible for suppression of corpus luteum function by sVEGFR1 injection into the antecedent follicle. Notably, histological sections revealed that treatment resulted in a "Swiss cheese" appearance in luteal tissue; specifically, a decrease in the density of cells with an increase in vacant extracellular space. The possibility exists that fewer luteal cells may exist after sVEGFR1 injection. However, a lack of luteal cell hypertrophy, which was apparent in follicles that contained "trapped" oocytes may be responsible for the change in tissue appearance, because steroidogenic luteal cells are known to increase in size during luteal development [34]. Conversely, the larger extracellular space may be a result of cell shrinkage, a characteristic of apoptosis [35]. It is interesting that Dickson and colleagues [36] recently reported that neutralization of VEGF during the midluteal phase increased apoptosis in the primate corpus luteum. Although it is unclear what accounts for the change in luteal density, it is likely that normal cell-cell interactions are disrupted after sVEGFR1 treatment. Increasing evidence exists for cell-cell interactions involving endothelial cells, which may be necessary for tissue differentiation [37], as well as proper transport of substrates, nutrients, and products to and from the luteal cells [38].
The results of this and other recent studies support the concept that alterations in the VEGF-receptor system may have clinical applications. Inadequate vascular dysfunction around ovulation could result in anovulatory cycles (e.g., luteinized unruptured follicles [39]) or luteal phase defects [40]. Treatment of these aberrant cases with VEGF may adequately restore function. Alternatively, treatment with VEGF antagonists could induce luteal or uterine dysfunction and thus possibly generate a contraceptive effect [41]. Finally, the pathologic vascular leakage occurring in ovarian hyperstimulation syndrome during assisted reproductive technology cycles in women may be due to excess ovarian VEGF production and may be prevented by VEGF antagonists [42]. However, the use of VEGF or its antagonists to control vessel growth and function in clinical situations is not without problems [43]. Further studies are required to determine the clinical relevance of VEGF antagonists in controlling ovarian and reproductive function.
In summary, this is the first report on local administration of a VEGF antagonist into the preovulatory follicle, with the goal of investigating the effects on ovulation and luteal development. We demonstrate that sVEGFR1 impairs ovulation and attenuates subsequent luteal function in primates possibly by altering normal steroidogenic-nonsteroidogenic cell interaction or differentiation without dramatically changing cell numbers. Thus, VEGF may play a critical role in periovulatory events that greatly affect the subsequent structure and function of the corpus luteum during the menstrual cycle. However, further studies are necessary to determine the molecular and cellular mechanisms through which VEGF and VEGF antagonists control ovarian function, cyclicity, and fertility.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Richard L. Stouffer, Oregon National Primate Research Center, 505 N.W. 185th Avenue, Beaverton, OR 97006. FAX: 503 690 5563; stouffri{at}ohsu.edu
Received: 30 November 2001.
First decision: 31 December 2001.
Accepted: 16 May 2002.
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and ß in the rhesus monkey corpus luteum during the menstrual cycle: regulation by luteinizing hormone and progesterone. Endocrinology 2000 141:1711-1717This article has been cited by other articles:
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