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Ovary |
a Division of Reproductive Sciences, Oregon National Primate Research Center,
b Department of Physiology and Pharmacology,
c Department of Obstetrics and Gynecology, Oregon Health & Science University, Beaverton, Oregon
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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corpus luteum, granulosa cells, growth factors, ovary, ovulation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The factors regulating VEGF production by the preovulatory follicle and developing corpus luteum have not been rigorously analyzed. Recent studies suggest that luteinizing granulosa cells isolated from monkeys and women synthesize VEGF [6, 7]. In vivo and in vitro studies by our group strongly suggest that one action of the midcycle gonadotropin surge is directly on granulosa cells to stimulate VEGF secretion [7]. Moreover, circulating LH may act to maintain VEGF secretion by luteinizing granulosa cells [7, 8] in the developing corpus luteum. However, it remains unclear whether major regulators of VEGF production in other cell types and tissues, such as hypoxia [9, 10] local cytokines, or growth factors [11], influence the ovarian VEGF system. Notably, the insulin-related regulatory system (including insulin-like growth factors -1 and -2 [IGF-1 and IGF-2]), which plays a key local role in ovarian biology [12], reportedly influences VEGF production in other tissues such as the retina [13, 14].
Therefore, studies were designed to investigate the influence of oxygen and IGFs on VEGF production by granulosa cells obtained from preovulatory follicles in rhesus monkeys. Experiments were performed to analyze effects of treatments in the presence and absence of gonadotropin. In addition, experiments were performed on cells collected prior to (i.e., nonluteinized cells; low VEGF secretion) and after (i.e., luteinizing cells; high VEGF secretion) in vivo exposure to an ovulatory gonadotropin bolus. Treatment effects on cell VEGF and progesterone secretion were compared.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult, female rhesus monkeys (Macaca mulatta) were maintained at the Oregon National Primate Research Center (ONPRC) as previously described [15]. Animal protocols 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 published by the National Academy of Sciences [16].
Beginning at menses, recombinant human gonadotropins (r-hFSH ± r-hLH, Ares Serono Group, Norwell, MA) were administered i.m. to promote development of multiple preovulatory follicles as reported previously [17]. A GnRH antagonist (Antide, Ares Serono Group) was concomitantly administered to prevent an endogenous LH surge [17]. Granulosa cells were obtained by follicle aspiration during laparoscopy of anesthetized animals [18] either the morning after the last FSH + LH treatment (nonluteinized granulosa cells, NLGCs) or 27 h after administration of an ovulatory bolus of r-hCG (Ares Serono Group [17]). Follicle growth was monitored from estradiol levels in daily serum samples and ultrasonography [17].
Cell Preparation and Culture Experiments
Follicular aspirates from an individual monkey were pooled. Following removal of oocytes, an enriched preparation of granulosa cells was obtained by Percoll (Sigma Chemical Co., St. Louis, MO) gradient centrifugation as previously described [19].
To test the effects of O2 concentration (study 1), granulosa cells were cultured on fibronectin (Sigma)-coated 96-well plates (Costar; Corning, Inc., Corning, NY) in chemically defined Dulbecco modified Eagle medium (DMEM)-Hams F12 medium containing ITSA (insulin, 2 µg/ml; transferrin, 5 µg/ml; H2SeO3, 5 ng/ml; aprotinin, 25 µg/ml; Sigma) and human low density lipoprotein (LDL, 25 µg/ml; Sigma). This media promotes and sustains basal and gonadotropin-stimulated progesterone (P4) and VEGF secretion by macaque LGCs for at least 6 days of culture [7]. Cells were plated at an initial density of 40 000 cells/well for 24 h in a 20% O2, 5% CO2, 75% N2 environment at 37°C, 95% humidity. Then the cultures were changed to media with or without a maximal stimulatory dose of hLH (100 ng/ml, AFP-4261A; National Pituitary Agency, Research Triangle Park, NC) or CoCl2 (100 mM) and transferred to atmospheres of 20%, 5%, and 0% O2 (deficits in O2 were replaced with N2). After 48 h, media were collected and cells were fixed in methanol for storage at -80°C until subsequent analyses. Four experiments were performed with both NLGCs and LGCs from different monkeys. Each treatment was tested in quadruplicate wells in each experiment.
To examine the effects of insulin-like factors (study 2), granulosa cells were cultured on fibronectin-coated 48-well Costar plates (Corning) in chemically defined culture media as described above, except that insulin was deleted (i.e., DMEM-F12 plus TSA and LDL). Cells were cultured for 72 h in a 20% O2, 5% CO2, 75% N2 environment at 37°C and 95% humidity, in the presence or absence of hCG (100 ng/ml CR123, National Pituitary Agency), or six concentrations of recombinant human IGF-1, IGF-2, or insulin (Sigma), or a combination of these. Media were changed daily and cultured cells were methanol-fixed after 3 days and then stored at -80°C until subsequent analyses. Three to five experiments were performed with both NLGCs and LGCs from different animals. Each treatment was tested in triplicate wells in each experiment.
Media samples were assayed for VEGF(-A) using an ELISA (Quantikine human VEGF ELISA, R&D Systems, Minneapolis, MN) validated for macaque VEGF [7]. Interassay and intraassay variation for VEGF assays were 9.1% and 14.9%, respectively. Progesterone levels were also measured by ELISA (Elecsys 2010, Roche Diagnostics, Indianapolis, IN). Interassay and intraassay variations for the P4 assay were 2.9% and 5.9%, respectively. Cellular DNA content per well was determined by the crystal violet assay, as reported by Brasaemle and Attie [20] and used by this laboratory [19].
Statistical Analyses
Statistical tests were performed using the Statpak (Northwest Analytical, Portland, OR) computer program. In study 1, differences in media VEGF and P4 levels and DNA content were determined by ANOVA using a complete randomized block design. In study 2, differences between treatments and time were determined by two-way ANOVA using a complete randomized block design. After a significant (P < 0.05) F test, a Duncan multiple range test was used to determine differences between means. If a Bartlett test indicated heterogeneity of variance, values were log transformed. However, all data are expressed as nontransformed values. Because a complete randomized block design was employed, the common estimate of variance (CEV; defined as the square root of MSE/n, where MSE is the mean square error from ANOVA of all data in a treatment group) is illustrated rather than SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nonluteinized granulosa cells secreted low but detectable levels of P4 (Fig. 1 upper panel) and VEGF (lower panel) during 48 h of culture in the various O2 environments. Whereas P4 levels were low in the absence of gonadotropin (i.e., controls) or cobalt alone, LH exposure increased P4 levels at all three O2 concentrations and in the presence of cobalt. Progesterone levels in the various treatment groups were comparable during culture in 20% and 5% O2. However, LH-stimulated P4 levels were significantly less at 0% O2. Likewise, LH-stimulated P4 levels were significantly less (0% O2, P < 0.05) in combination with CoCl2. In contrast, either LH or cobalt alone increased VEGF levels above controls in 20% and 5% O2 environments. However, the stimulation by cobalt was less than that by LH alone, and the combination of LH plus cobalt was not different from that of LH alone. At 0% O2, VEGF levels for controls, cobalt, and LH plus cobalt were significantly lower than these groups in 20% and 5% 02 environments.
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As reported previously [7], LGCs produce much higher levels of P4 (Fig. 2, upper panel) and VEGF (lower panel) than NLGCs during culture in chemically defined media. Note that media P4 and VEGF levels are 60-fold and 10-fold higher, respectively, compared with those in Figure 1. At 20% and 5% O2, LH alone significantly increased P4 levels above controls. In contrast, cobalt alone decreased P4 levels and prevented the stimulatory effect of LH exposure. At 0% O2, LH alone increased P4 levels, but control and LH-stimulated P4 levels were significantly less than in 20% or 5% O2. At all O2 tensions, P4 levels in the presence of cobalt alone or cobalt plus LH were not different from those of control. In contrast, O2 concentration had no effect on VEGF levels in any treatment (Fig. 2, lower panel). At 20% and 5% O2, LH alone and LH in the presence of cobalt increased VEGF levels. At 0% O2, LH alone increased VEGF levels. Cobalt alone did not alter VEGF levels. However, cobalt reduced LH-stimulated VEGF levels at 5% O2 (LH versus LH + Co, P < 0.05).
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There were no significant differences in DNA content between treatment groups for either NLGCs or LGC (data not shown). However, there was a 3-fold greater content in wells of LGCs versus NLGCs (0.178 versus 0.067 mean OD600 nm, P < 0.05).
Effects of Insulin-Like Factors
Addition of IGF-1, IGF-2, or insulin to the culture media resulted in a dose-dependent increase (P < 0.05) in VEGF levels during the first day of culture for NLGCs (not shown) and LGCs (Fig. 3). Because there was no significant difference in VEGF levels produced by 20 to 100 ng/ml of each factor, a concentration of 50 ng/ml was chosen as a maximal stimulatory dose in further studies.
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Figure 4 depicts the effects of IGF-1 (50 ng/ml) alone and in combination with gonadotropin (100 ng/ml hCG) on VEGF levels produced by NLGCs as a function of time in culture. On the initial day of culture, either IGF-1, hCG, or the combination of IGF-1 and hCG stimulated VEGF levels above those of control. However, by 3 days of culture, neither IGF-1 nor hCG alone stimulated VEGF levels compared with that of controls, plus levels declined significantly from Day 1 of culture. In contrast, IGF-1 plus hCG stimulated VEGF levels above controls and maintained levels at those observed on Day 1 of culture.
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Similar response patterns (not shown) were obtained with IGF-2 and insulin, and with LGCs. As summarized in Figure 5, only the combination of IGF-1 or IGF-2 with hCG stimulated VEGF levels by Day 3 of culture by NLGCs. Insulin tended to produce similar results, but the results were more variable. The apparent synergistic effect of IGFs and hCG was evident when analyzing the stimulatory effect on P4 as well as VEGF levels (e.g., IGF-2 and hCG; Fig. 6). This effect, however, was not evident when DNA content in treatment groups was analyzed (Fig. 6). Similar results were obtained with LGCs (not shown) except the fold stimulation was less (e.g., 35-fold versus 313-fold for P4 levels, LGCs versus NLGCs) because of the higher control levels.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The current evidence that reducing the O2 milieu or preventing O2 utilization with cobalt had minimal or no effect on VEGF-A secretion by nonluteinized and luteinized granulosa cells, respectively, was unexpected. Several groups report that hypoxic conditions increase VEGF mRNA or protein expression in a number of cell types or tissues in vivo and in vitro [22], including reproductive organs such as the uterus [10]. The discovery that the genes for VEGF-A as well as its receptor, VEGF-R1, contain a hypoxia-response element that binds a critical mediator of hypoxic signaling, the hypoxia-inducible transcription factor, HIF-1 [23], established a fundamental link between oxygen availability and VEGF expression/action. This research led to working models implicating hypoxic stress to VEGF-mediated angiogenesis in pathologic states such as solid tumor growth [22] and retinopathy [14], as well as normal events such as menstruation and endometrial repair during the menstrual cycle in primates [10].
Our data would not support a model for hypoxic stress promoting granulosa cell VEGF production for angiogenesis in the developing antral follicle, as proposed by Neeman and colleagues [22]. However, studies to date on the source and control of VEGF production in the follicle during development are very limited. Our data clearly indicate the granulosa cells from large antral follicles produce an order of magnitude less VEGF-A than LGCs collected from ovulatory follicles 27 h after exposure to a bolus of gonadotropin that stimulates follicle rupture, reinitiation of oocyte maturation, and corpus luteum development [24]. The failure of hypoxic treatments to enhance VEGF production cannot be attributed to an O2 "leak" or residue in the cultures because the O2-dependent steroidogenesis by these cells was markedly suppressed by 0% O2 or cobalt exposure. The low level of VEGF production and lack of hypoxic response by NLGCs correlates with evidence from immunocytochemical and in situ hybridization studies that VEGF is expressed mainly in granulosa cells around the oocyte (i.e., the cumulus oophorus) and theca cells in large antral follicles in several species [25, 26]. It is possible that hypoxic conditions develop in growing follicles, particularly in clinically controlled ovarian stimulation (COS) cycles [27], but Van Blerkom et al. [28] noted no significant difference between VEGF-A concentrations in follicular fluid of human follicles with O2 contents of 3.0%–5.5% (normoxic), 1.5%–2.5%, or <1.5% (severely hypoxic) after COS protocols. These data would suggest that in the developing antral follicle, 1) VEGF-A production and regulation by hypoxia is low in mural granulosa cells, and 2) granulosa expression of VEGF is primarily stimulated by the midcycle gonadotropin surge during luteinization [7, 29] presumably to promote ovulation or neovascularization of the developing corpus luteum.
The lack of response by granulosa cells does not preclude other ovarian cells, such as theca cells in the follicle or luteal cells in the corpus luteum, from responding to hypoxia with enhanced VEGF production. Indeed, this laboratory has preliminary evidence that dispersed luteal cells from the macaque corpus luteum respond to a 0% O2 environment or cobalt with a significant increase in VEGF secretion [30]. The latter may be relevant to the report by Freidman et al. [31], that human LGCs collected 35 h after the hCG bolus responded to hypoxic (1% O2) conditions or cobalt with a 2-fold to 5-fold increase in VEGF secretion during culture. Although we cannot rule out species or culture (e.g., presence of serum [32]) differences, it is possible that the hypoxic response observed in the study by Freidman et al. was due to retrieval of more luteinized cells at 35 h post-hCG, compared to our cells at 27 h post-hCG. Alternatively, the more rigorous oocyte retrieval methods used in clinical protocols may have permitted Freidman et al. [31] to sample the band of mural granulosa cells juxtaposed to the basement membrane, which exhibits greater VEGF expression in the late follicular phase [8], and possibly different regulation.
The ability of IGFs to promote VEGF secretion by ovarian follicle cells is consistent with recent reports that insulin [13] and IGF-1 [11] stimulate VEGF mRNA expression, protein expression, or both in normal and tumor cells from various tissues. Although there are reports that IGF-1 treatment can increase DNA synthesis or cell proliferation in granulosa cells [33], DNA content did not vary significantly between treatment groups in the current study. Thus, gonadotropin, insulin/IGFs, or the combination thereof appeared to increase cellular VEGF secretion independent of any potential changes in cell proliferation in granulosa cells from large antral follicles. Although the mechanism or mechanisms of action are unknown in follicle cells, both insulin and IGF-1 increase the cellular content of VEGF mRNA by promoting the rate of transcription and increasing mRNA stability [13].
Because of promiscuity in the binding of insulin-related factors to receptors, it is difficult to surmise from the current data which insulin/IGF pathways promote VEGF production by macaque granulosa cells. Insulin receptors and IGF type I and II receptors are expressed in human granulosa cells with greater IGF receptor expression in the dominant versus smaller antral follicles [12]. Moreover, a similar expression profile for IGFs and their receptors appears to occur in the ovary of rhesus monkeys [34]. Although it is believed that circulating levels of insulin are rarely high enough to bind to IGF receptors, this can occur in hyperinsulinemia [12]. It is possible that the higher levels of insulin used in the current study bound to IGF-1 receptors, although similar levels were shown to act via insulin receptors to stimulate steroid production by human granulosa cells [35]. Both IGF-1 and IGF-2 activate insulin and IGF receptors [36], but a recent report suggests that IGF-1 promotion of VEGF expression in colon carcinoma cells is mediated at least in part by IGF type I receptor. The IGF type II receptor may be primarily important for IGF-2 internalization and degradation, but there is evidence for a role in signaling associated with angiogenesis [37]. Further studies (e.g., employing anti-insulin [35] and anti-IGF receptor antibodies), are warranted to address the role of various insulin/IGF-receptor pathways in VEGF expression in the ovary.
Whether specific insulin-like growth factors promote VEGF expression in the ovarian follicle or corpus luteum in vivo is unknown. Unlike in rodents, IGF-2 rather than IGF-1 expression appears to predominate in the primate ovary [12]. Although appreciable levels of IGF-1 exist in follicular fluid, they are apparently generated via serum transudation [12]. The dynamics of IGF-2 expression and availability (as controlled by IGF binding proteins and their proteases) led Guidice [38] and others to propose a model wherein this parameter is vital for directing the growing antral follicle toward dominance (i.e., maturation) or atresia (i.e., degeneration). Because many of the earlier studies employed IGF-1, the current study provides novel evidence that IGF-2 not only promotes steroidogenesis, but also VEGF production, by granulosa cells from mature periovulatory follicles in primates.
The results also expand the hypothesis that locally produced IGFs synergize with gonadotropic hormones to promote granulosa cell function [12, 39], to now include production of the angiogenic/permeability factor, VEGF. Recent studies using methods to neutralize VEGF support a critical role for this factor in follicular development [40], ovulation [5], and corpus luteum formation [4, 5]. It is possible that one action for insulin/IGFs in the ovary is to promote the production of angiogenic factors that are essential for vascularization of the developing follicle and corpus luteum. Indeed, it is noteworthy that low IGF levels produced by genetic [14] methods prevent normal vascular growth in tissues such as the retina. Further studies are needed to examine the role of the insulin/IGF system, including the recently discovered insulin-like factor family (Insl3 [41]), in controlling the expression of angiogenic factors and their actions in the ovary.
In summary, the current data strongly suggest that unlike in many other normal and tumorigenic tissues, hypoxia is not a major stimulant for VEGF production by the avascular granulosa cell layer in the developing antral follicle in primates. Rather, the gonadotropin surge is the primary stimulant for increased VEGF production by granulosa cells in the ovulatory, luteinizing follicle, with insulin-like growth factors acting synergistically to promote and maintain VEGF secretion in developing luteal cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Richard L. Stouffer, Division of Reproductive Sciences, Oregon National Primate Research Center, 505 NW 185th Avenue, Beaverton, OR 97006. FAX: 503 690 5563; stouffri{at}ohsu.edu
Received: 10 September 2002.
First decision: 25 September 2002.
Accepted: 16 October 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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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  S. van den Driesche, M. Myers, E. Gay, K. J. Thong, and W. C. Duncan HCG up-regulates hypoxia inducible factor-1 alpha in luteinized granulosa cells: implications for the hormonal regulation of vascular endothelial growth factor A in the human corpus luteum Mol. Hum. Reprod., August 1, 2008; 14(8): 455 - 464. [Abstract] [Full Text] [PDF]
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 M. B. Stanek, S. M. Borman, T. A. Molskness, J. M. Larson, R. L. Stouffer, and P. E. Patton Insulin and Insulin-Like Growth Factor Stimulation of Vascular Endothelial Growth Factor Production by Luteinized Granulosa Cells: Comparison between Polycystic Ovarian Syndrome (PCOS) and Non-PCOS Women J. Clin. Endocrinol. Metab., July 1, 2007; 92(7): 2726 - 2733. [Abstract] [Full Text] [PDF]
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O J Ginther, E L Gastal, M O Gastal, and M A Beg In vivo effects of pregnancy-associated plasma protein-A, activin-A and vascular endothelial growth factor on other follicular-fluid factors during follicle deviation in mares Reproduction, April 1, 2005; 129(4): 489 - 496. [Abstract] [Full Text] [PDF]
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H. M. Fraser, J. Bell, H. Wilson, P. D. Taylor, K. Morgan, R. A. Anderson, and W. C. Duncan Localization and Quantification of Cyclic Changes in the Expression of Endocrine Gland Vascular Endothelial Growth Factor in the Human Corpus Luteum J. Clin. Endocrinol. Metab., January 1, 2005; 90(1): 427 - 434. [Abstract] [Full Text] [PDF]
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K. A. Young, B. Tumlinson, and R. L. Stouffer ADAMTS-1/METH-1 and TIMP-3 expression in the primate corpus luteum: divergent patterns and stage-dependent regulation during the natural menstrual cycle Mol. Hum. Reprod., August 1, 2004; 10(8): 559 - 565. [Abstract] [Full Text] [PDF]
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T.J. Acosta, E.L. Gastal, M.O. Gastal, M.A. Beg, and O.J. Ginther Differential Blood Flow Changes Between the Future Dominant and Subordinate Follicles Precede Diameter Changes During Follicle Selection in Mares Biol Reprod, August 1, 2004; 71(2): 502 - 507. [Abstract] [Full Text] [PDF]
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