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Regular article |
a Dipartimento di Scienze Veterinarie ed Agroalimentari, Fisiologia Veterinaria, Università di Teramo, 64020 Italy
b Dipartimento di Morfofisiologia Veterinaria e Produzioni Animali, Fisiologia Veterinaria, Università di Bologna, 44020 Italy
ABSTRACT
Angiogenesis is the process that drives blood vessel development in growing tissues in response to the local production of angiogenic factors. With the present research the authors have studied vascular endothelial growth factor (VEGF) production in ovarian follicles as a potential mechanism of ovarian activity regulation. Prepubertal gilts were treated with 1250 IU equine chorionic gonadotropin (eCG) followed 60 h later by 750 IU of human chorionic gonadotropin (hCG) in order to induce follicle growth and ovulation. Ovaries were collected at different times of the treatment and single follicles were isolated and classified according to their diameter as small (<4 mm), medium (4–5 mm), or large (>5 mm). VEGF levels were measured in follicular fluid by enzyme immunoassay, and VEGF mRNA content was evaluated in isolated theca and granulosa compartments. Equine chorionic gonadotropin stimulated a prompt follicular growth and induced a parallel evident rise in VEGF levels in follicular fluid of medium and large follicles. Analysis of VEGF mRNA levels confirmed the stimulatory effect of eCG, showing that it is confined to granulosa cells, whereas theca cells maintained their VEGF steady state mRNA. Administration of hCG 60 h after eCG caused a dramatic drop in follicular fluid VEGF that reached undetectable levels in 36 h. A parallel reduction in VEGF mRNA expression was recorded in granulosa cells. The stimulating effect of eCG was also confirmed by in vitro experiments, provided that follicles in toto were used, whereas isolated follicle cells did not respond to this hormonal stimulation. Consistent with the observation in vivo, granulosa cells in culture reacted to hCG with a clear block of VEGF production. These results demonstrate that while follicles of untreated animals produce stable and low levels of the angiogenic factor, VEGF markedly rose in medium and large follicles after eCG administration. The increasing levels, essentially attributable to granulosa cells, are likely to be involved in blood vessel development in the wall of growing follicles, and may play a local key role in gonadotropin-induced follicle development. When ovulation approaches, under the effect of hCG, the production of VEGF is switched off, probably creating the safest conditions for the rupture of the follicle wall while theca cells maintained unaltered angiogenic activity, which is probably required for corpus luteum development.
follicle, follicular development, granulosa cells, growth factors, hormone action, theca cells
INTRODUCTION
Ovarian activity is characterized by alternating phases of growth and regression that involve both follicular and luteal structures. This dynamic situation is paralleled by a continuous rearrangement of the blood vessel network that evolves in relation to the needs of the tissues and their different levels of activity [1]. This process, which adjusts local blood supply to the specific needs of a tissue, defined as angiogenesis, is triggered by the local production of specific angiogenic factors [2]. In response to these stimuli, endothelial cells from pre-existing vessels will proliferate and develop new capillaries toward the site of production of the factor [3, 4]. Although it is now well-recognized that follicular dynamics are strictly regulated by gonadotropins, the intimate, local mechanism that translates the systemic hormonal command into follicular growth and differentiation is still largely unknown. The regulation of blood vessel development, representing a crucial event in follicular maturation/regression, could be involved in this mechanism. It has long been known that follicle development is dependent on an increasing blood supply, whereas follicle atresia is related to a local reduction in blood flow [5, 6]. The study of the regulation of ovarian vascularization could provide a key to understanding the mechanisms by which endocrine as well as paracrine and autocrine factors operate in follicle selection, ovulation, or atresia [7, 8].
Previous investigations aimed to define the mechanisms that regulate the innervation of the wall of growing follicles. They revealed that the gonadotropin milieu that drives follicular growth simultaneously stimulates follicles to produce nerve growth factor [9, 10]. The same mechanism, which regulates innervation of growing follicles, equipped with specific gonadotropin receptors, could also coordinate the blood vessel network in the follicle wall. Recent investigations carried out in laboratory rodents and primates point to vascular endothelial growth factor (VEGF) as a key angiogenic factor [11–14] of the regulation of ovarian vascularization, and both in vivo and in vitro experiments suggest that the production of this angiogenic factor is influenced by gonadotropins. In women affected by ovarian hyperstimulation syndrome, which may result from superovulatory treatments, abnormal follicular development and extremely high blood vessel permeability are accompanied by ovarian production of abnormal quantities of VEGF [15]. Consistent with these data, gonadotropins have been shown to stimulate the production of VEGF in vitro [16–18].
In consideration of the potential role for angiogenesis in the control of folliculogenesis, this research has been designed to investigate the production of VEGF throughout follicle development in vivo using a model in which systemic endocrine changes were precisely timed and controlled. To this aim, the production of VEGF was monitored by measuring the amount of factor that accumulated in follicular fluid and by verifying the levels of expression of VEGF mRNA in the different types of follicular cells obtained from prepubertal gilts treated with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) to induce follicle growth and ovulation.
The investigation, supported by parallel experiments in vitro, reveals that granulosa cells actively secrete VEGF in follicular fluid during eCG-induced follicular maturation, whereas this activity is rapidly switched off by an ovulatory dose of hCG. Analysis of mRNA content confirms the prevalent involvement of granulosa cells in this local production and its positive connection with follicle growth, while the organization for VEGF production was completely reprogrammed after hCG when theca cells remain the only source of the angiogenic factor in preovulatory follicles.
MATERIALS AND METHODS
Experiments In Vivo: Animals and Hormonal Stimulation Protocols
Fifteen prepubertal Large White gilts with an average weight of 90 kg were injected i.m. with 1250 IU of eCG (Folligon, Intervet, Holland) to induce follicular growth and maturation. Sixty h later, they were injected with 750 IU of hCG (Corulon, Intervet) to induce ovulation within 40–44 h [19].
Groups of three animals each were ovariectomized at 0, 30, and 60 h after eCG injection and 18 and 36 h after hCG injection. Ovariectomy was carried out by laparotomy on animals that were preanesthetized by an injection of azaperone (6 ml/gilt; Stresnil, Janssen, Belgium) and atropine sodium salt (2 mg/gilt), and maintained under tiopenthal sodium (1.5 g/gilt; Pentothal Sodium, Gellini, Italy) anesthesia. All protocols had prior approval of the Ethical Committee of the University of Bologna.
Immediately after ovaries were removed, they were transported into the laboratory where single follicles were isolated in dissection medium (Dulbeccos phosphate buffer medium supplemented with 0.4% BSA) with the aid of a stereomicroscope. After measuring the diameter with a calibrated grid, each healthy follicle was dried on tissue paper to eliminate any trace of medium, and opened in a 35-mm Petri dish to collect follicular fluid. The samples of follicular fluid were then individually frozen until assayed for VEGF content.
The follicle wall obtained from each follicle was in parallel transferred in dissection medium to mechanically separate the granulosa layer from theca shells by gently scraping the follicle with a small spatula. The medium containing dispersed granulosa cells was collected and centrifuged, and the theca shell was then vigorously vortexed and carefully washed in order to remove any possible granulosa cell contamination. Preliminary histological analysis of the theca layer demonstrated that virtually no granulosa cells were present in this preparation. Theca and granulosa samples were then stored in liquid nitrogen for analysis of VEGF mRNA expression.
The results obtained were referred to as single follicles and were grouped on the basis of diameter: small, <4 mm; medium, 4–5 mm; or large, >5 mm.
In Vitro Experiments
In order to confirm the follicular synthesis of VEGF and to ascertain the regulatory role of gonadotropins in defined conditions, single follicles with a diameter of 5 mm, isolated from the ovaries of prepubertal gilts, were cultured as follicles in toto according to the method described by Moor and Trounson [8]. In brief, follicles that were judged healthy on the basis of their vascularization and translucent appearance were isolated and stripped of connective tissue with the aid of a stereomicroscope, and were then positioned on a stainless steel grid in a 35-mm Petri dish containing 2 ml of TCM 199 (Sigma Chemical Company, St. Louis, MO), supplemented with 10% fetal calf serum (FCS, Sigma). In this way, the follicles supported by the grid emerged from the bottom of the Petri dish by 
mm, thus facilitating gas diffusion. Dishes were then introduced to a hyperbaric chamber and cultured for 12 h in 5% CO2 and air at 1.2 atmosphere at 39°C with or without hormones (eCG, hCG, or both, 1 IU/ml each). At the end of the culture the follicles were removed, and single samples of follicular fluid were collected and stored until assayed for VEGF content.
In order to identify the compartment of the follicle wall involved in the production of this angiogenic factor, an in vitro study was carried out to compare VEGF production in purified preparations of theca and granulosa cells. To this aim, pig follicles were cut into two equal halves with a razor blade. One half, in which the theca and granulosa relationship was preserved, was cultured as a control, while the remaining half was used to isolate granulosa cells and theca layers by scraping the internal face of the follicle wall as previously described. Cultures consisting of 12 halves of theca fragments or granulosa cell preparations were then carried out for 12 h, with or without hormonal supplementation, and maintained under constant gentle agitation in order to avoid cell attachment to the bottom of the dishes. Previous investigations demonstrated that under these culture conditions, granulosa cells were prevented from undergoing luteinization in vitro [20]. At the end of the incubation, culture medium was collected, suspended cells were precipitated by centrifugation, and the supernatant was stored at -20°C until VEGF assay.
VEGF Assay
Samples of follicular fluid and culture medium were measured for their VEGF content by using a specific ELISA (Quantikine; R&D Systems, Minneapolis, MN). This highly specific sandwich assay recognizes VEGF 165 as well as VEGF 121, while it exhibits negligible cross-reactivity with all cytokines/growth factors tested. A 96-well plate reader (Biomek 1000; Beckman Instruments, Fullerton, CA) set to read at an emission of 450 nm was used to quantify the results. Because the VEGF kit was prepared to detect human VEGF, a test of parallelism was carried out to investigate whether the assay could be reliably used to measure pig VEGF. To this aim, the standard curve prepared with the points of human VEGF (0, 62.5, 125, 250, 500, and 1000 pg/ml) was compared with a curve that was obtained with a sample of follicular fluid diluted with the appropriate calibrator solution provided with the kit, in order to produce samples with values that fell within the dynamic range of the assay. The two curves were perfectly parallel, thus showing that the system does not reveal any major difference between human and pig VEGF. The sensitivity of the assay was equivalent to 5 pg/ml of follicular fluid. The levels of VEGF in samples of follicular fluid are expressed as ng/ml, while the levels produced in vitro are expressed on a per follicle basis (ng/follicle).
VEGF Messenger RNA Expression
Total RNA isolation Total cellular RNA was extracted from granulosa cells and theca tissues with the Tri-pure isolation Kit (Boehringer-Mannheim GmbH, Mannheim, Germany). RNA quantity and purity were determined spectrophotometrically. Integrity of the RNA samples was tested by the amplification of ß-actin.
Reverse transcription-polymerase chain reaction Oligonucleotide primers were selected on the basis of a bovine VEGF sequence (GenBank M32976) using Oligo software (Med Probe, Oslo, Norway) and synthesized by Pharmacia Biotech (Milan, Italy). Primers for VEGF were 5'-CCT GAT GCG GTG CGG GGG CT-3' (VEGF-1 nt 779–798) and 5'-TGG TGG TGG CGG CGG CTA TG-3' (VEGF-2 complementary to nt 1197–1216). Because these primers initiate before and terminate after the splicing site (nt 966) they enable amplification of all VEGF isoforms. VEGF 120 and VEGF 164 are the prevalent isoforms found within the ovary [21] and are the variants measured in follicular fluid, while larger isoforms predominantly occur as cell-associated isoforms for their strong heparin binding site [22]. Therefore, reverse transcription-polymerase chain reaction (RT-PCR) conditions were set in order to detect VEGF 120 and VEGF 164 mRNA splice variants in ethidium bromide staining. Primers for pig ß-actin were 5'-ATC GTG CGG GAC ATC AAG GA-3' (ActSS-1) and 5'-AGG AAG GAG GGC TGG AAG AG-3' (ActSS-2). All the chemicals were from Promega (Madison, WI). Equal amounts of total RNA from each sample (0.5 mg) were reverse transcripted at 42°C in a 20-µl reaction volume containing 1 U of RNasin, 2.5 U of avian mieloblastosis reverse transcriptase (AMV), 50 mM KCl, 50 mM Tris-HCl pH 8.3, 10 mM MgCl2, 1 mM deoxyribonucleoside triphosphate (dNTP), and 2.5 mM random primers. Then, 10 µl of this mixture was used in each PCR reaction, which contained 50 mM KCl, 10 mM Tris-HCl pH 9.0, 1% Triton X-100, 1.5 mM MgCl2, 200 mM dNTP, and 7 mM primers VEGF-1 and VEGF-2 or 0.5 mM of primers ActSS-1 and ActSS-2 in a final volume of 50 µl. The mixture was heated to 95°C for 10 min, cooled to 80°C, and Taq polymerase (Promega) was added to a final concentration of 10 U/ml. Samples were amplified using the following protocol: 24 cycles at 95°C for 1 min, 60°C for 1 min, 72°C for 1 min; then subjected to further extension at 72°C for 5 min, followed by incubation at 4°C. Products of the reaction were separated on 1.5% Amplisize agarose gel (BioRad, Hercules, CA) and visualized by ethidium bromide staining. Amplifications were also carried out on samples in which either RNA or RT were omitted from the RT mixture. Beta-actin mRNA have been found in pig follicle cells with levels that are independent of follicle status and size [23], and its expression is not affected by growth factors or gonadotropins [24, 25]. Therefore, VEGF mRNA levels were normalized on the basis of ß-actin mRNA content.
Southern blotting Southern blots for amplified VEGF complementary DNA fragments were performed to confirm the specificity of PCR by using a nonradioactive system (DIG Oligonucleotide 3'-End Labeling Kit; Boehringer) according to the manufacturer's instructions and revealed by chemiluminescent detection (CSPDâ; Boehringer). As a probe for hybridizations, we used a 40-mer oligo 5'-TGC TGG CTT TGG TGA GGT TTG ATC CGC ATA ATC TGC ATG G-3' (complementor to nt 850–890) selected by Oligo software. The relative densities of ethidium bromide-stained gels and exposed films were determined by densitometric scanning (GelDoc 1000; BioRad).
Statistical Analysis
VEGF results are presented as mean ± SD. Variations of VEGF production or mRNA levels during the gonadotropin treatment were analyzed by one-way ANOVA. Means were separated by Student's t-test. Differences were considered to be significant at P < 0.05 or less.
RESULTS
VEGF Production In Vivo
The population of follicles isolated from the ovaries of prepubertal untreated animals (24.33 ± 2.52 follicles/gilt) displayed wide variations in diameter: 40% of follicles were <4 mm (small), 50% had a diameter of 4–5 mm (medium), and only 5% of isolated follicles were >5 mm (large; Fig. 1A). Administration of eCG induced a prompt follicular growth and, after 30 h, 37% of the follicles recovered (23 ± 4.36 follicles/gilt) were larger than 5 mm. Sixty hours after eCG treatment, more than 85% of the follicles (27.33 ± 3.21 follicles/gilt) were larger than 5 mm, while the proportion of small follicles was reduced to 3%. Treatment with hCG further modified the population of isolated follicles: after 36 h no small follicles could be isolated, the percentage of medium-size ones were reduced, while the number of large follicles increased to 98% (27.67 ± 3.78 follicles/gilt).
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VEGF Levels in Follicular Fluid
As shown in Figure 1B, VEGF content in follicular fluid of control animals was similar in all the classes of follicle diameters analyzed, with values of 2.96 ± 1.49 ng/ml (mean ± SD, 20 replicates), 3.13 ± 2.65 ng/ml (n = 30), and 3.32 ± 1.32 ng/ml (n = 5) in small, medium, and large follicles, respectively.
At 30 h, eCG caused a rise in the levels of VEGF in follicular fluid of large follicles (18.43 ± 2.92 ng/ml; n = 27). The angiogenic factor only slightly increased in small follicles (4.36 ± 2.60 ng/ml; n = 11), while medium ones displayed wide variations in VEGF content, showing levels ranging from 2 to 15 ng/ml (n = 29; Fig. 1B). Constantly high levels of VEGF were recorded in the follicular fluid of large follicles (18.61 ± 3.48 ng/ml; n = 43) 60 h after eCG, while the factor ranged between average values of 3.17 ± 0.75 (n = 3) and 5.19 ± 4.81 ng/ml (n = 14) in small and medium follicles, respectively. Even at this time, medium follicles presented wide variations in VEGF content, despite their similar appearance.
Administration of hCG induced a marked inversion in VEGF levels that were progressively reduced in the follicular cavity (Fig. 1). The concentration of VEGF dropped to 0.77 ± 0.46 (n = 8) and 0.4 ± 0.38 ng/ml (n = 29) in medium and large follicles, respectively 18 h after hCG administration, to reach undetectable levels at 36 h (Fig. 1B).
VEGF Messenger RNA Content in Follicle Cells
PCR revealed that both theca and granulosa cells expressed VEGF 120 and VEGF 164 splice variants (Fig. 3). Densitometric values of the two variants were summed and considered as a cumulative mVEGF expression index.
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Analysis of these data revealed a different behavior in the two follicular compartments (theca and granulosa) throughout the experiment. Granulosa cells responded to eCG stimulation with a progressive increase in VEGF mRNA content. The values, expressed in arbitrary units, rose in large follicles (>5 mm) from 119.59 ± 81.69 before treatment to 484.65 ± 106.89 60 h after eCG treatment (P < 0.01; Fig. 1C). Consistent with VEGF content found in follicular fluid, the level of VEGF mRNA showed only a slight increase in granulosa cells of small follicles, while wide variations in mRNA levels were recorded in medium ones (Fig. 1C). Analysis of correlation revealed that VEGF levels recorded in follicular fluid are always strictly related to the expression of VEGF mRNA in granulosa compartments (r = 0.92, n = 48, P < 0.01; see Fig. 2a). By contrast, VEGF mRNA levels in theca shells remained unchanged following eCG treatment, with values ranging from 85.18 ± 20.30 to 138.21 ± 51.11, independently of the class of follicular diameter analyzed (Fig. 1D), which showed no correlation with the levels of VEGF recorded in follicular cavity (Fig. 2b).
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Administration of hCG caused a prompt reduction in VEGF mRNA levels in granulosa cells without affecting theca cell levels (Fig. 3). In detail, mRNA content dropped in large follicles from values of 484.66 ± 106.89, recorded 60 h after eCG treatment, to 87.12 ± 32.58 36 h after hCG stimulation (P < 0.01; Fig. 1C).
Theca shells were insensitive to hCG stimulation. The slight increase in mRNA expression recorded in medium and large follicles did not achieve statistical significance (109.93 ± 56.38 and 120.98 ± 24.22 ng VEGF/ml 60 h after eCG vs. 176.50 ± 66.77 and 208.57 ± 56.67 36 h after hCG in medium and large follicles, respectively; P > 0.05; Fig.1D).
Production of VEGF In Vitro
In order to confirm the data recorded in vivo, follicles isolated from ovaries of untreated prepubertal gilts with a diameter of about 5 mm were cultured as follicles in toto in the presence or absence of gonadotropin supplementation. The levels of VEGF in follicular fluid were then measured after 12 h of culture. In agreement with the previous observations in vivo, eCG produced a significant increase in the content of VEGF compared with the values recorded in unstimulated follicles (44.56 ± 11.63 ng/ml, n = 22 vs. 11.63 ± 4.22 ng/ml, respectively; n = 33; P < 0.01). In all the follicles tested, the presence of hCG during the culture caused a marked reduction in the levels of angiogenic factor assayed in follicular fluid (4.42 ± 1.46 ng/ml, n = 26, P < 0.05). The same marked inhibition was recorded when both the gonadotropins were added (5.09 ± 2.54 ng of VEGF/ml, n = 31; P < 0.05).
The second set of experiments was conducted to confirm the different contribution of the two compartments, theca or granulosa, in follicular VEGF production. To this aim, follicular walls and purified preparations of granulosa cells or theca shells were cultured with or without gonadotropins. In the absence of any hormonal stimulation, the granulosa compartment resulted in being the major producer of VEGF on a per follicle basis. As shown in Table 1, eCG supplementation was not able to stimulate VEGF production either in follicle halves, in theca layers, or in granulosa cells. The levels of VEGF accumulated in the media were, in fact, stable regardless of the presence or absence of eCG (Table 1). By contrast, hCG administration, alone or in combination with eCG, caused a marked inhibition in VEGF secretion both in follicle halves and in granulosa cells (Table 1), while hCG had no effect on theca shell VEGF production in the culture.
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DISCUSSION
The data reported here demonstrate that pig follicle cells produce substantial amounts of VEGF. The level of production is different according to the different phases of follicular dynamics. In prepubertal gilts with silent ovaries, low levels of the factor have generally been found in follicular fluid, independent of follicle size. When follicular activity has been switched on by the administration of eCG, the progressive increase in mean follicle diameter was paralleled by increasing levels of VEGF accumulating in the follicular fluid. Moreover, it appears that only follicles that enlarge beyond the limit of 4–5 mm in diameter can be engaged in a copious and constant production of the factor. The increased intrafollicular concentration of the angiogenic factor could depend on the increasing number of cells that occurs throughout follicle growth, but the results of VEGF mRNA levels, normalized with reference to actin mRNA, indicates that eCG does stimulate VEGF production rate on a per cell basis. The experiments in vitro, in the absence of any evident growth, clearly confirm the idea that follicles with the same diameter and with a similar background showed higher intrafollicular levels of VEGF when eCG was added to the culture medium. This suggests that the activation of VEGF production is more dependent on the dynamic status of the follicles and on their growth rate, rather than on their diameter per se.
As far as the meaning of this increased intrafollicular storage of VEGF is concerned, only hypotheses can be put forward at present. During follicular growth, the granulosa compartment seems to be the major producer of VEGF on a per follicle basis, as indicated by analysis of both mRNA expression and the in vitro production of the factor. In addition, we can suppose that while VEGF produced by the theca is likely cleared by blood circulation, the amounts produced by granulosa cells are more likely accumulated in follicular fluid due to the absence of blood vessels in this compartment. The analysis of VEGF mRNA after eCG stimulation demonstrates that only the granulosa compartments, probably for their follicle FSH receptor equipment, increased the production of the angiogenic factor, while the basal activity in theca compartments remained unchanged. Therefore, the production of VEGF becomes polarized throughout follicular growth, the major source of angiogenic factor being stored in the center of the follicle, in follicular fluid. The resulting scenario is therefore composed of a follicle wall with an external layer (theca compartment) in which there are blood vessels and a constant production rate of the factor, and an internal layer (granulosa-follicular fluid compartment) without blood vessels and with a VEGF synthesis that increases throughout follicular growth. The very high levels of VEGF that are reached in the antrum of preovulatory follicles are likely to diffuse toward the outer layers, thus creating an angiogenic gradient in the theca layer; the closer to the basal membrane, the higher the levels of VEGF, and the more intense angiogenesis should develop within the theca, the only vascularized compartment of follicle wall.
Early investigations by Hay and coworkers [26] demonstrated the importance of the blood vessel architecture, showing that follicle health depends on the presence of a rich network of capillaries localized in the innermost part of the wall, as close as possible to the basal membrane, so that oxygen and nutrients can be brought inside the follicle and particularly at the level of the cumulus-oocyte complex. Early signs of atresia include the selective disappearance of this inner vessel component without substantial modifications of outer theca vascularization [27]. The angiogenic gradient that results from the accumulation of VEGF in follicular fluid could represent the signal capable of developing and then maintaining the activity of these inner vessels. Consistent with these data, recent investigations by Van Blerkom and coworkers [28], demonstrated that the level of oxygen dissolved in follicular fluid is directly related to the follicular levels of VEGF and that adequate oxygen tension represents an important requisite for supporting normal developmental competence of the oocyte.
Equine chorionic gonadotropin, which is widely used to induce follicle growth in pigs, caused a progressive increase in mean follicle diameter and a parallel increase in the levels of VEGF accumulated in the follicular fluid of rapidly growing follicles. This response, which is consistent with experimental evidence obtained with different models, may represent a mechanism that guarantees fine coordination between angiogenesis and growth of the follicle wall, and leads to the maturity of follicle function. Thirty-sixty hours after eCG administration, follicles with a diameter larger than 5 mm invariably had high intrafollicular levels of VEGF, whereas those of 4–5 mm showed wide variations, with VEGF levels ranging from very low values (<1 ng/ml) to concentrations higher than 10 ng/ml, despite their similar morphological aspects. If the presence of VEGF is effectively correlated with follicle vascularization, these two kinds of follicles are likely to have different growth potentials. High VEGF levels could in fact guarantee the blood supply and vessel permeability that are required to deliver the amounts of gonadotropins that are adequate to sustain follicle development. On the contrary, a reduction in the rate of VEGF production could lead to follicular atresia. In this context, VEGF production could represent a key event in the process of follicle selection, and investigations are currently underway to assess this hypothesis.
The stimulatory effect of eCG treatment was confirmed in vitro only when follicles in toto were used. Follicle halves as well as granulosa and theca compartments cultured separately did not show any significant response to eCG. The different responses obtained by incubating follicle in toto or follicle halves, in which the structure of the follicle wall was retained intact, led us to hypothesize that the different culture conditions adopted for these two preparations could have been for this purpose.
Among the signals that regulate VEGF production, a low oxygen tension has always been indicated as a stimulating agent, while a high oxygen tension is likely to inhibit this production. Follicular environment is characterized by low oxygen tension [28], a condition particularly evident for large antral follicles, where gases must diffuse from the vessels present in the theca through the basal membrane to reach the granulosa layer, and accumulate into the follicular fluid. Follicle cells and the oocyte have become adapted to this special environment by developing an extreme sensitivity to oxidative stresses. Follicle cells removed from their natural, low-oxygen environment may have failed to produce VEGF in response to eCG for the excessively high oxygen tension experienced in CO2-air atmosphere, a condition capable of inhibiting VEGF production [10], while follicles cultured in toto, in which somatic cells live in reduced PO2, probably similar to the physiological environment, could respond to eCG in vitro, in agreement with the observations in vivo. Research is currently underway to investigate the effect of oxygen tension on the follicular production rate of angiogenic factors.
Apart from the intimate cell mechanism regulating the synthesis of angiogenic factors, the finding that gonadotropins stimulate VEGF production, possibly synchronizing growth and vascularization of the follicle wall, recalls our previous observations showing that gonadotropins induce follicle cells to secrete nerve growth factor [9], thus providing a mechanism that coordinates growth and innervation of the follicle wall. There seems to be a general system operating within the ovary, whereby structures growing under endocrine stimulation ensure that their development involves all the accessories that are essential for their function.
At the end of the growth phase, when the follicle becomes committed to ovulate in response to the gonadotropin surge, or to the corresponding administration of hCG in our experimental model, a progressive decrease in the production of VEGF was recorded and, after 36 h, intrafollicular levels of this factor were undetectable. The inhibitory effect of hCG on the production of the angiogenic factor was dependent on the progressive reduction of the expression of VEGF mRNA in the granulosa compartment, which has major responsibility for the secretion of VEGF. By contrast, the theca maintained its levels of VEGF mRNA throughout the entire periovulatory period. This follicular response to hCG treatment was further confirmed during the experiments carried out in vitro independently from the culturing conditions adopted. The inhibitory effect of hCG was already detectable after 12 h of culture and was not removed or mitigated by the simultaneous addition of eCG. This indicates that high levels of LH, probably identifiable with the ovulatory gonadotropin surge, are in any case capable of switching off VEGF production in granulosa cells. Theca cells have been an exception to this rule, and in all the experiments carried out, the production rate of the factor in this compartment tended to increase, although the rise did not achieve statistical significance.
Following hCG administration, it appears, therefore, that the polarization of VEGF production within the follicle is disrupted and the theca compartment remains, at least in the periovulatory period, the sole element maintaining what could be considered a basal production of the angiogenic factor.
The inhibitory effect of hCG reported here is in contrast with experimental evidence that showed a general stimulatory influence of LH or hCG [16–18]. The reasons for this discrepancy can, at present, only be hypothesized. In many works, the cells used for the experiments had been cultured for long times and, therefore, had probably undergone a nearly complete process of luteinization. When this occurs, the cells represent a suitable model for the study of luteal function, while they may not behave as somatic follicle cells. By contrast, our data have been obtained by analyzing the situation in vivo during a precisely controlled follicular development, and experiments in vitro have been conducted using the cells immediately after their isolation and, probably more important, maintaining, in most cases, the intact follicular structure (follicle in toto). Despite this discrepancy, which was confirmed in all our experiments in vivo and in vitro, the reduced production of VEGF caused by hCG is consistent with the reduction of VEGF mRNA expression and agrees with the clear reduction of blood vessel network that Cavender and coworkers [29] have observed in follicles just before ovulation. These changes in the immediate periovulatory period are particular interesting, warrant further investigation, and could provide precious insights in the mechanism of ovulation and, possibly, of final oocyte maturation.
In conclusion, our results indicate that follicular VEGF production is dependent on gonadotropin stimulation, particularly during the phase of follicular growth. The resulting vascularization is likely to provide the trophic support required by the actively dividing follicular somatic cells. In this context, VEGF production could be part of the mechanism of gonadotropin-induced follicular growth. Such a hypothesis may represent an important interpretation key for the understanding of the local events that regulate ovarian activity. Experiments are currently underway to test this hypothesis.
FOOTNOTES
First decision: 11 February 2000.
1 Supported by cofinanziamento Es fin. 1999. 
2 Correspondence: Barbara Barboni, Dipartimento di Scienze Veterinarie ed Agroalimentari, Fisiologia Veterinaria, Università degli Studi di Teramo, 64020 Nepezzano, Teramo, Italy. FAX: 39 861 558819; barboni{at}ifv.vet.unite.it
Accepted: April 25, 2000.
Received: January 18, 2000.
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 R.S. Robinson, L.T. Nicklin, A.J. Hammond, D. Schams, M.G. Hunter, and G.E. Mann Fibroblast Growth Factor 2 Is More Dynamic than Vascular Endothelial Growth Factor A During the Follicle-Luteal Transition in the Cow Biol Reprod, July 1, 2007; 77(1): 28 - 36. [Abstract] [Full Text] [PDF]
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A. E. Roberts, L. K. Arbogast, C. I. Friedman, D. E. Cohn, P. T. Kaumaya, and D. R. Danforth Neutralization of Endogenous Vascular Endothelial Growth Factor Depletes Primordial Follicles in the Mouse Ovary Biol Reprod, February 1, 2007; 76(2): 218 - 223. [Abstract] [Full Text] [PDF]
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M A Beg and O J Ginther Follicle selection in cattle and horses: role of intrafollicular factors. Reproduction, September 1, 2006; 132(3): 365 - 377. [Abstract] [Full Text] [PDF]
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D. Abramovich, F. Parborell, and M. Tesone Effect of a Vascular Endothelial Growth Factor (VEGF) Inhibitory Treatment on the Folliculogenesis and Ovarian Apoptosis in Gonadotropin-Treated Prepubertal Rats Biol Reprod, September 1, 2006; 75(3): 434 - 441. [Abstract] [Full Text] [PDF]
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 A Martelli, P Berardinelli, V Russo, A Mauro, N Bernabo, L Gioia, M Mattioli, and B Barboni Spatio-temporal analysis of vascular endothelial growth factor expression and blood vessel remodelling in pig ovarian follicles during the periovulatory period J. Mol. Endocrinol., February 1, 2006; 36(1): 107 - 119. [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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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]
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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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J. Greenaway, K. Connor, H. G. Pedersen, B. L. Coomber, J. LaMarre, and J. Petrik Vascular Endothelial Growth Factor and Its Receptor, Flk-1/KDR, Are Cytoprotective in the Extravascular Compartment of the Ovarian Follicle Endocrinology, June 1, 2004; 145(6): 2896 - 2905. [Abstract] [Full Text] [PDF]
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O.J. Ginther, E.L. Gastal, M.O. Gastal, C.M. Checura, and M.A. Beg Dose-Response Study of Intrafollicular Injection of Insulin-Like Growth Factor-I on Follicular Fluid Factors and Follicle Dominance in Mares Biol Reprod, April 1, 2004; 70(4): 1063 - 1069. [Abstract] [Full Text] [PDF]
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T. Shimizu, J.-Y. Jiang, K. Iijima, K. Miyabayashi, Y. Ogawa, H. Sasada, and E. Sato Induction of Follicular Development by Direct Single Injection of Vascular Endothelial Growth Factor Gene Fragments into the Ovary of Miniature Gilts Biol Reprod, October 1, 2003; 69(4): 1388 - 1393. [Abstract] [Full Text] [PDF]
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D. R. Danforth, L. K. Arbogast, S. Ghosh, A. Dickerman, R. Rofagha, and C. I. Friedman Vascular Endothelial Growth Factor Stimulates Preantral Follicle Growth in the Rat Ovary Biol Reprod, May 1, 2003; 68(5): 1736 - 1741. [Abstract] [Full Text] [PDF]
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T. Shimizu, J.-Y. Jiang, H. Sasada, and E. Sato Changes of Messenger RNA Expression of Angiogenic Factors and Related Receptors During Follicular Development in Gilts Biol Reprod, December 1, 2002; 67(6): 1846 - 1852. [Abstract] [Full Text] [PDF]
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M. Mattioli, B. Barboni, M. Turriani, G. Galeati, A. Zannoni, G. Castellani, P. Berardinelli, and P. A. Scapolo Follicle Activation Involves Vascular Endothelial Growth Factor Production and Increased Blood Vessel Extension Biol Reprod, October 1, 2001; 65(4): 1014 - 1019. [Abstract] [Full Text] [PDF]
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D. A. Redmer, V. Doraiswamy, B. J. Bortnem, K. Fisher, A. Jablonka-Shariff, A. T. Grazul-Bilska, and L. P. Reynolds Evidence for a Role of Capillary Pericytes in Vascular Growth of the Developing Ovine Corpus Luteum Biol Reprod, September 1, 2001; 65(3): 879 - 889. [Abstract] [Full Text] [PDF]
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C. Wulff, S. J. Wiegand, P. T. K. Saunders, G. A. Scobie, and H. M. Fraser Angiogenesis During Follicular Development in the Primate and its Inhibition by Treatment with Truncated Flt-1-Fc (Vascular Endothelial Growth Factor TrapA40) Endocrinology, July 1, 2001; 142(7): 3244 - 3254. [Abstract] [Full Text] [PDF]
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