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BOR - Papers in Press, published online ahead of print June 14, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.051052
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BIOLOGY OF REPRODUCTION 75, 434–441 (2006)
DOI: 10.1095/biolreprod.106.051052
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Effect of a Vascular Endothelial Growth Factor (VEGF) Inhibitory Treatment on the Folliculogenesis and Ovarian Apoptosis in Gonadotropin-Treated Prepubertal Rats1

Dalhia Abramovich 3, Fernanda Parborell 3, and Marta Tesone 2 34 

Instituto de Biología y Medicina Experimental (IByME)—CONICET,3 Departamento de Química Biológica,4 Facultad de Ciencias Exactas, Universidad de Buenos Aires, Buenos Aires 1428, Argentina

ABSTRACT

In the present study, we investigated whether vascular endothelial growth factor A (VEGFA) plays a critical intraovarian survival role in gonadotropin-dependent folliculogenesis. The effect of an intrabursal administration of a VEGFA antagonist on follicular development, apoptosis, and levels of pro- and antiapoptotic proteins of BCL2 family members (BAX, BCL2, and BCL2L1), as well as of TNFRSF6 (also known as FAS) and FAS ligand (FASLG), was examined. To inhibit VEGFA, a soluble FLT1/Fc Chimera (Trap) was administered to prepubertal eCG-treated rats. Injection of 0.5 µg of Trap per ovary did not change the number of preantral follicles (PFs) or early antral follicles (EAFs); however, it significantly decreased the number of periovulatory follicles 48 h after surgery and significantly increased the number of atretic follicles. No significant differences were found in any stage of the follicles either 12 or 24 h after injection. Cells undergoing DNA fragmentation were quantified by performing TUNEL on ovarian sections. Trap treatment caused a twofold increase in the number of apoptotic cells in EAFs. DNA isolated from antral follicles incubated for 24 h exhibited the typical apoptotic DNA pattern. Follicles obtained from Trap-treated ovaries showed a significant increase in the spontaneous onset of apoptotic DNA fragmentation. The injection of Trap significantly increased the levels of BAX and decreased the levels of BCL2 protein. The ratio of BCL2L1L:BCL2L1s was significantly diminished in follicles obtained from ovaries treated with Trap. No changes in the levels of TNFRSF6 or FASLG were observed after treatment. We concluded that the local inhibition of VEGFA activity appears to produce an increase in ovarian apoptosis through an imbalance among the BCL2 family members, thus leading a larger number of follicles to atresia.

angiogenesis, apoptosis, female reproductive tract, follicular development, growth factors, ovary, vascular endothelial growth factor A

INTRODUCTION

Angiogenesis is a process of vascular growth that is mainly limited to the reproductive system in healthy adult animals. The development of new blood vessels in the ovary is essential to guarantee the necessary supply of nutrients and hormones to promote follicular growth and corpus luteum formation. Preantral follicles (PFs) have no vascular supply of their own, but rather, they depend on vessels in the surrounding stroma [1]. However, during antrum development, the thecal layer acquires a vascular sheath consisting of two capillary networks located in the theca interna and externa. These newly formed ovarian blood vessels guarantee an increasing supply of gonadotropins, growth factors, oxygen, and steroid precursors, as well as other substances to the growing follicle. The acquisition of an adequate vascular supply is possibly a rate-limiting step in the selection and maturation of the dominant follicle destined to ovulate [1]. On the other hand, degeneration of the capillary bed in follicles that fail to develop is a relevant factor causing follicular atresia [2]. Both the ovarian follicle and corpus luteum have been shown to produce several angiogenic factors; however, vascular endothelial growth factor A (VEGFA) is thought to play a pivotal role in the regulation of angiogenesis in the ovary [3]. Expression of VEGFA in ovarian follicles depends on follicular size. In bovine and porcine follicles, VEGFA is weakly expressed during early ovarian follicular development and becomes more pronounced in granulosa and theca cells, along with dominant follicle development [4, 5]. Similar results were found in the rat ovary, where, in addition, some secondary follicles showed extremely strong VEGFA immunoreactivity in the zona pellucida [6].

Administration of a KDR (also known as FLK1 or VEGFR2) antibody inhibits gonadotropin-dependent follicular angiogenesis in mice, which, in turn, blocks development of mature antral follicles [7]. Furthermore, inhibition of VEGFA with a VEGFA Trap antagonist produces a decrease in follicular angiogenesis and development, as well as a decrease in FLT1 (also known as VEGFR1) and KDR expression in the marmoset monkey [8]. Hazzard et al. [9] have shown that intrafollicular injection of a VEGFA antagonist (soluble VEGF receptor 1 chimera) in rhesus monkeys can impair ovulation and the subsequent development and functional capacity of the corpus luteum.

However, the understanding of the distinct roles of VEGFA in ovarian angiogenesis and apoptosis is still limited. The molecular mechanism of apoptosis is a matter of an active debate. It is known that ovarian apoptosis takes place to eliminate follicular cells in atretic follicles [10–16]. FSH and LH are the primary survival factors for ovarian follicles; the antiapoptotic effects of these gonadotropins are probably mediated by the production of ovarian growth factors. It has been demonstrated that various growth factors and cytokines (IGF1, EGF, TGFA, FGF2, FGF7, and interleukin 1B) prevent apoptosis in antral follicles [12, 17–20]. Many of these reports use preovulatory follicles obtained from eCG-treated prepubertal rats and describe a significant degree of apoptosis within 24 h of incubation in serum-free medium and its prevention in the presence of FSH or growth factors. In addition, several molecules, including BCL2 [21, 22], BCL2L1 (also known as BCLX) [23], BAX [21], caspases [24, 25], TNFRSF6 (also known as FAS) and FAS ligand (FASLG) [26, 27], and inhibitor of apoptosis proteins (IAPs) [28], have been implicated to be directly involved in the regulation of ovarian apoptosis.

Particularly, the interaction between follicular angiogenesis and apoptotic cell death is poorly understood. In fact, the presence of VEGFA and its receptor in human and rodent ovaries has been reported at both protein and mRNA levels, in granulosa cells as well as in theca cells [29, 30]. We suggest that apoptotic cell death in granulosa cells of those follicles selected for ovulation can be prevented by the paracrine/autocrine actions of VEGFA. VEGFA is a cytoprotective agent for endothelial cells, which protects these cells from apoptosis [31] and induces the expression of antiapoptotic proteins in human endothelial and mouse ovarian cells [31, 32]. VEGFA exerts its cellular effects through interaction with its tyrosine kinase receptors FLT1 and KDR. At present, the precise role of VEGFA in nonvascular tissues is not yet known. Some reports have shown that VEGFA is cytoprotective for myocytes after ischemic injury [33] and that this factor exerts a direct neuroprotective effect through the inhibition of apoptosis and the stimulation of neurogenesis [34, 35]. Recently, Greenaway et al. [5] have shown that VEGFA also has a cytoprotective role in the bovine extravascular granulosa cell compartment and that this effect appears to occur via interaction with KDR and apoptotic cell death inhibition.

However, the relationship between angiogenesis and the molecular pathway involved in the regulation of apoptosis in the ovarian follicle growth is unknown. Therefore, in this study, we specifically investigated whether the VEGFA plays a critical intraovarian survival role for gonadotropin-dependent folliculogenesis. In particular, we examined the effect of the local administration of a VEGFA antagonist on follicular development, apoptosis, and the expression of BCL2 protein family members (BAX, BCL2, and BCL2L1), TNFRSF6, and FASLG in ovarian follicles from prepubertal eCG-treated rats.

MATERIALS AND METHODS

Materials and Reagents

Recombinant mouse-soluble VEGF receptor 1/Fc Chimera (Trap) (R&D Systems, Inc., Minneapolis, MN) was dissolved in PBS buffer with 0.1% BSA. The eCG (Novormon) was provided by Syntex S.A. (Buenos Aires, Argentina). Polyclonal primary antibodies for BAX (N-20), BCL2 (N-19), BCL2L1 (S-18), TNFRSF6 (FL-335), and FASLG (Q-20) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); actin B antibody was obtained from Novus Biologicals (ab-6276; Littleton, CO). Anti-rabbit and anti-goat secondary antibodies conjugated with horseradish peroxidase were purchased from Sigma-Aldrich. All other chemicals were of reagent grade and were obtained from standard commercial sources.

In Vivo Trap Treatment and Superovulation

General care and housing of rats was carried out at the Instituto de Biología y Medicina Experimental (IByME) in Buenos Aires. Prepubertal rats were from our colony. Immature female Sprague-Dawley rats (21–23 days) were allowed food and water ad libitum and were kept at room temperature (21–23°C) on a 12L:12D cycle. After being anesthetized with ketamine HCl (80 mg/kg; Holliday-Scott S.A., Buenos Aires, Argentina) and xylazine (4 mg/kg; König Laboratories, Buenos Aires, Argentina), the ovaries were exteriorized through an incision made in the dorsal lumbar region. Rats then received either 0.1 or 0.5 µg of Trap in 5 µl of PBS with 0.1% BSA under the bursa of one ovary (Trap ovary). The contralateral ovary was injected with 5 µl of vehicle (PBS with 0.1% BSA, control ovary). Taking into account that the VEGF Trap protein is a chimera that contains the Fc region of human immunoglobulin G (IgG), an additional control was designed in which one ovary was injected with 0.65 µg of human IgG, and the contralateral ovary was injected with vehicle. No significant differences were found in the number of follicles at any stage between the human IgG and the vehicle-injected ovary. Therefore, PBS with 0.1% BSA was chosen as the control. After injection, ovaries were replaced, and the incision was closed with skin adhesive. Subsequently, rats were injected s.c. with 0.1 ml of eCG (25 IU per rat). Rats were killed 12, 24, or 48 h after surgery by CO2 asphyxiation. The ovaries were removed and cleaned of adhering tissue in culture medium for subsequent assays.

The experimental protocols were approved by the Animal Experimentation Committee of the IByME.

Ovarian Morphology

To evaluate changes in general structure, the ovaries were removed and immediately fixed in 4% neutral-buffered formalin for 12 h and then embedded in paraffin. Four-micrometer step sections were mounted at 50-µm intervals onto microscope slides to prevent counting the same follicle twice, according to the method described by Woodruff et al. [36]. One set of slides was stained with hematoxylin-eosin in order to count the number of different stages of follicles per ovary section, and the others were immunostained by the TUNEL technique. Follicles were classified as preantral (PF) or early antral (EAF), according to the presence or absence of an antrum, or periovulatory follicles (POFs), which include the advanced preovulatory follicles plus the recently formed corpus luteum. Morphological characteristics of atretic follicles include the degeneration and detachment of the granulosa cell layer from the basement membrane, the presence of pyknotic nuclei in this cell type, and oocyte degeneration [37, 38]. The number of PFs, EAFs, POFs, and atretic follicles was determined in three ovarian sections from each ovary (three sections per ovary; six to eight ovaries per dose or time).

Follicle Isolation

The individual ovarian follicles were dissected from the ovary under a stereoscopic microscope as previously described [12, 39]. In brief, healthy antral follicles (300–450 µm in diameter) from eight ovaries per group were frozen and used for biochemical assays. From each ovary, a pool of isolated follicles was frozen, and the results obtained from each pool were considered a single datum.

TUNEL Technique

For immunohistochemical quantification of apoptosis, formalin-fixed tissue sections were processed for in situ localization of nuclei exhibiting DNA fragmentation by the TUNEL technique [40] with an apoptosis detection kit (Apoptag plus peroxidase in situ Apoptosis detection kit; Chemicon International, Inc., Temecula, CA) as previously described [37]. The 4-µm-thick tissue sections were deparaffinized and digested for 15 min at room temperature with proteinase K (20 µg/ml; Gibco, Grand Island, NY). Endogenous peroxidase was quenched with 3% hydrogen peroxide in PBS. The labeling reaction was carried out by incubating tissue sections with buffer containing digoxygenin-2'-deoxyuridine 5'-triphosphate prior to incubation with terminal deoxynucleotidyl transferase (TdT) for 1 h at room temperature. Tissues were then incubated for 30 min with a peroxidase-conjugated anti-digoxygenin monoclonal antibody, and apoptotic cells were visualized as positively immunostained structures after reaction with diaminobenzidine. Negative controls included TdT omission. Sections were counterstained with hematoxylin. The number of apoptotic cells was determined by counting labeled cells from follicles in 400x microscopic fields (three sections per ovary; five ovaries) and expressed as the apoptotic cell mean per field.

DNA Isolation and Fragmentation Analysis

Cellular DNA was extracted from 10 healthy antral follicles per ovary. Follicles were incubated for 24 h under serum-free conditions at 37°C in 500 µl of Dulbecco modified Eagle medium F12 (1:1) containing 10 mM Hepes, supplemented with fungizone (250 µg/ml), and gentamicin (10 mg/ml ) (five ovaries per group) and gassed with 95%O2:5% CO2 at the start of culture. This model has the advantage of keeping the integrity of the follicle. In addition, the incubation in serum-free conditions for 24 h allows an exhibition of the typical apoptotic DNA ladder: the presence of internucleosomal fragments of 180-bp multiples. The follicles from each culture were homogenized in a buffer containing 100 mM NaCl, 4 mM EDTA, 50 mM Tris-HCl, 0.5% SDS, pH 8, and proteinase K (100 µg/ml) at 55°C for 4 h to facilitate membrane and protein disruption. After incubation, samples were cooled for 30 min on ice in 1 M potassium acetate and 50% chloroform to initiate protein precipitation and then centrifuged at 9000 x g for 8 min at 4°C. Supernatants were then precipitated for 30 min in 2.5 volumes of ethanol at –70°C and centrifuged for 20 min at 5000 x g at 4°C. Finally, samples were extracted in 70% ethanol and resuspended in water. DNA content was measured by reading the absorbance at 260 nm and then incubated for 1 h with RNase (10 µg/ml) at 37°C. DNA samples (4 µg) were electrophoretically separated on 1.9% agarose gels containing ethidium bromide (0.4 µg/ml) in Tris-borate-EDTA buffer. Within each agarose gel, equal amounts of DNA were loaded into each well. DNA was visualized in an ultraviolet (302 nm) transilluminator and photographed with a Polaroid camera system. Densitometric analysis of low-molecular-weight (<15 kb) DNA was performed with an Image Scanner (Genius) by the software program Scion Image for Windows (Scion Corporation, Worman's Mill, CT). Quantitative results obtained by densitometric analysis of the low-molecular-weight DNA fragments represent the mean ± SEM of three independent gel runs.

Western Blots

One hundred follicles per ovary were resuspended in 5 volumes of lysis buffer (20 mM Tris-HCl [pH 8], 137 mM NaCl, 1% NP-40, and 10% glycerol) supplemented with protease inhibitors (0.5 mM PMSF, 0.025 mM N-CBZ-L-phenylalanine chloromethyl ketone, 0.025 mM N-p-tosyl-lysine chloromethyl ketone, and 0.025 mML-1-tosylamide-2-phenyl-ethylchloromethyl ketone) and homogenized with an Ultra-Turrax (IKA Werk, Breisgau, Germany) homogenizer. Samples were centrifuged at 4°C for 10 min at 10 000 x g, and the resulting pellets were discarded. Protein concentration in the supernatant was measured by the Bradford assay. After boiling for 5 min, 40 µg of protein was applied to a 12% SDS-polyacrylamide gel, and electrophoresis was performed at 25 mA for 1.5 h. The resolved proteins were transferred for 2 h onto nitrocellulose membranes. The blot was preincubated in blocking buffer (5% nonfat milk and 0.05% Tween-20 in 20 mM triethanolamine-buffered saline [TBS; pH 8.0]) for 1 h at room temperature and incubated with appropriate primary antibodies (TNFRSF6 1/500, FASLG 1/500, BAX 1/200, BCL2 1/500, and BCL2L1 1/200 in TBS) in blocking buffer overnight at 4°C. Then, it was incubated with anti-rabbit or anti-goat secondary antibodies conjugated with horseradish peroxidase (1:1000) and finally detected by chemiluminescence and autoradiography with x-ray film. Negative controls were obtained in the absence of the primary antibody. The density of each band was normalized to the density of the actin B band that was used as an internal control.

Quantification for Western Blot Assay

In each experiment, equal amounts of protein were loaded for all samples, and both groups in one experiment were loaded on the same gel. For quantification, a screening was performed on blots with x-ray film using different times of exposure to optimize the signal. The levels of protein were compared and analyzed with densitometric studies by Scion Image for Windows. Optical density data are expressed as arbitrary units ± SEM (n = 8).

Data Analysis

Data are expressed as the mean ± SEM. Representative gels are shown in the figures. Statistical analysis was performed by the paired or unpaired t-test. Values of P < 0.05 were considered significant.

RESULTS

Morphological and TUNEL Studies

The first objective was to analyze the effects of the inhibition of VEGFA on follicular development and apoptosis in the rat ovary. To inhibit VEGFA, a soluble truncated form of the fms-like tyrosine kinase (FLT) fused to IgG (VEGF Trap R1/Fc Chimera: Trap) was administered for different time periods. The time course effects of Trap on follicle growth are depicted in Figure 1. Rats were treated with intrabursal injections of 0.5 µg of Trap, and the ovaries were removed 12, 24, and 48 h later and then processed and analyzed as described above. Histological ovarian slides were stained with hematoxylin-eosin to determine the number of different follicle stages (Fig. 1A). Injection of 0.5 µg of Trap per ovary did not change the number of PFs or EAFs (Fig. 1B; a and b); however, it significantly decreased the number of POFs when compared to the control group 48 h after surgery (Fig. 1B; c) (control: 14.08% ± 1.49%; Trap: 8.54% ± 1.72%, P < 0.01, n = 6), and it significantly increased the number of atretic follicles (Fig. 1B; d) (control: 10.46% ± 0.54%; Trap: 16.38% ± 1.27%, P < 0.05, n = 6). No significant differences were found in any stage of follicles 12 or 24 h after injection. Additionally, there were no differences when 0.1 µg of Trap per ovary was injected (data not shown). Therefore, the 0.5-µg and 48-h treatments were used for the following assays. In addition, histological sections showed that the Trap treatment resulted in a decrease in the density of stromal cells with an increase in the extracellular space.


Figure 1
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  		FIG. 1. Time course of VEGFA inhibition (Trap treatment) on follicle growth in ovaries from gonadotropin-stimulated rats. A) Representative fields of x100 ovarian sections. Control ovary (a); Trap-treated ovary (b). EAFs, Early antral follicles; AtF, atretic follicles; POFs, periovulatory follicles; GC, granulosa cells; TC, theca cells. B) Preantral follicles (a); early antral follicles (b); periovulatory follicles (c) (control: 14.08% ± 1.49%; Trap: 8.54% ± 1.72%). d) Atretic follicles (control: 10.46% ± 0.54%; Trap: 16.38% ± 1.27%). Prepubertal rats were injected with 0.5 µg of Trap under the bursa of one ovary. The contralateral ovary served as a control and was injected with vehicle. Subsequently, 25 IU of eCG were administered. The ovaries were removed at 12, 24, or 48 h after injection and prepared for histology. *P < 0.05; **P < 0.01; n = 6

As granulosa cells of the follicle die by apoptosis during the process of atresia [11, 41], the TUNEL technique was performed on histological ovarian slides from ovaries obtained 48 h after the injection of Trap. Figure 2A shows representative fields of a control and a treated ovary. Specific staining was observed in granulosa cells, and Trap treatment caused a twofold increase in the number of apoptotic cells in EAFs (control: 3.17 ± 0.81 apoptotic cells per field; Trap: 6.81 ± 1.02 apoptotic cells per field, P < 0.05, n = 5) as shown in Figure 2B.


Figure 2
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  		FIG. 2. A) Representative fields of ovarian sections labeled by TUNEL technique. Control ovary (a); Trap-treated ovary (b). A portion of each section shows detail observed at high magnification. EAF, Early antral follicle; AtF, atretic follicle; GC, granulosa cell; TC, theca cell. B) In situ DNA fragmentation analysis (TUNEL technique). The number of apoptotic cells was determined by counting labeled cells in x400 randomly selected fields of antral follicles. Data were expressed as the apoptotic cell mean/field (control: 3.17 ± 0.82; Trap: 6.81 ± 1.02 apoptotic cells per field). The number of follicles analyzed is shown between parentheses. Three sections per ovary were analyzed (five ovaries/group). *P < 0.05 Paired Student t-test; n = 5

Agarose Gel Electrophoresis and Quantitation of DNA Fragmentation

The levels of DNA fragmentation were measured in antral follicles obtained from control and treated ovaries 48 h after surgery (Fig. 3A; lane 1, control; lane 2, Trap). Antral follicles cultured in serum-free medium showed a spontaneous onset of apoptotic DNA fragmentation. Follicles obtained from Trap-treated ovaries showed a significant increase (45%) in the spontaneous onset of apoptotic DNA fragmentation (Fig. 3B; control: 297.0 ± 4.7, Trap: 429.3 ± 36.6 arbitrary units; P < 0.05). DNA fragmentation was minimal in freshly isolated antral follicles (data not shown).


Figure 3
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  		FIG. 3. Effect of Trap injection on DNA fragmentation in cultured preovulatory follicles. A) Agarose gel showing DNA fragmentation. B) Quantitative estimation of DNA cleavage. Animals were injected under the bursa of one ovary with 0.5 µg of Trap. The other ovary was injected with vehicle and used as a control. Forty-eight hours after surgery, antral follicles were isolated and cultured for 24 h in serum-free medium. Four micrograms of follicular DNA extracted from each culture was analyzed by ethidium bromide staining. Low-molecular-weight DNA (<15 kb) from the gel was examined to calculate apoptotic DNA fragmentation. Control: 297.0 ± 4.7; Trap: 429.3 ± 36.6 arbitrary units. Data points represent the mean ± SEM of three independent experiments. *P < 0.05

Levels of Apoptosis-Related Proteins in Antral Follicles

Figure 4 shows the follicular contents of BCL2, BAX, BCL2L1s, BCL2L1L, TNFRSF6, and FASLG proteins measured by Western blotting from follicles isolated 48 h after Trap injection. The injection of Trap significantly increased the levels of BAX (control: 0.61 ± 0.05; Trap: 0.90 ± 0.01, P < 0.05, n = 8) (Fig. 4A) and decreased the levels of BCL2 protein (control: 2.08 ± 0.16; Trap: 1.35 ± 0.10, P < 0.01, n = 8) (Fig. 4B). The ratio of BCL2L1L:BCL2L1s was significantly diminished in follicles obtained from ovaries treated with Trap (control: 0.51 ± 0.02; Trap: 0.34 ± 0.04, P < 0.01, n = 8), with the reduction of BCL2L1L greater than that of BCL2L1S (Fig. 4C). No changes in the levels of TNFRSF6 or FASLG were observed after treatment (Fig. 4, D and E).


Figure 4
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  		FIG. 4. Effect of Trap treatment on proapoptotic and antiapoptotic protein content of antral follicles. A) Upper panel: Densitometric quantification of BAX content. Bars represent the mean ± SEM normalized to actin B. After homogenization, proteins were extracted, subjected to 12% SDS-PAGE, and transferred onto nitrocellulose membranes. BAX protein was visualized with an anti-BAX antibody. Lower panel: Representative immunoblot of BAX protein content in antral follicles from control and Trap-treated rats. Levels of BAX protein were significantly increased after Trap treatment (n = 8; P < 0.05). B) Upper panel: Densitometric quantification of BCL2 content. Bars represent the mean ± SEM normalized to actin B. Lower panel: Representative immunoblot of BCL2 protein content in antral follicles from control and Trap-treated rats. BCL2 protein was visualized with an anti-BCL2 antibody. Levels of BCL2 protein were significantly decreased after Trap treatment (n = 8; P < 0.01). C) Upper panel: Densitometric quantification of BCL2L1 content. Bars represent the mean ± SEM. Lower panel: Representative immunoblot of BCL2L1 protein content in antral follicles from control and Trap-treated rats. BCL2L1 protein was visualized with an anti-BCL2L1 antibody that recognizes both isoforms. The BCL2L1L:BCL2L1S ratio was significantly decreased after Trap treatment (n = 8; P < 0.01). D) Upper panel: Densitometric quantification of TNFRSF6 content. Bars represent the mean ± SEM normalized to actin B. Lower panel: Representative immunoblot of TNFRSF6 protein content in antral follicles from control and Trap-treated rats. TNFRSF6 protein was visualized with an anti-TNFRSF6 antibody. Levels of TNFRSF6 protein were not significantly different between control and Trap groups (n = 8). E) Upper panel: Densitometric quantification of FASLG content. Bars represent the mean ± SEM normalized to actin B. Lower panel: Representative immunoblot of FASLG protein content in antral follicles from control and Trap-treated rats. FASLG protein was visualized with an anti-FASLG antibody. Levels of FASLG protein were not significantly different between control and Trap groups (n = 8)

DISCUSSION

The data described in the present study reveal, for the first time, to our knowledge, that an in vivo intrabursal administration of a soluble form of VEGFR1 to inhibit the actions of VEGFA produces an increase in the apoptosis process in ovarian follicle cells from eCG-treated rats and that the changes observed in the expression of BCL2L1, BAX, and BCL2 are involved in this effect.

The inhibition of VEGFA by Trap produced an increase in the number of atretic follicles and a decrease in the number of POFs in gonadotropin-treated rat ovaries. Considering that follicular atresia is mediated by apoptosis, we analyzed the effect of Trap on programmed cell death. In fact, the injection of Trap caused an increase in the number of apoptotic granulosa cells in antral follicles and in the spontaneous DNA fragmentation of antral follicles cultured in serum-free medium. Ovarian follicles cultured in these conditions are currently being used to investigate the pathways that control apoptosis and follicular atresia [11, 23, 42]. Our results show that DNA isolated after incubation exhibited the typical apoptotic DNA fragmentation pattern and demonstrate that in vivo Trap treatment sensitizes granulosa cells to undergo apoptosis. These data suggest that VEGFA suppresses granulosa cell apoptosis and inhibits follicular atresia.

Taking into account all these results, we suggest that VEGFA plays an important role in follicular development and atresia mediated by apoptosis. Enhanced vascularity or vascular permeability near developing follicles could increase the delivery of endocrine or paracrine factors, such as growth factors and gonadotropins. Increased delivery of folliculotrophin-like substances could result in an enhancement in the follicular selection or a decrease of follicular atresia. In our model, we observed that Trap treatment resulted in a decrease in the density of stromal cells with an increase in the extracellular space. These results suggest a role of VEGF in the regulation of cell-cell interactions [43]. In vitro experiments are in progress in our laboratory to study the direct effect of VEGF inhibition on granulosa cell apoptosis.

The results presented in this study are consistent with previous data observed by other authors. Wulff et al. [44] have demonstrated that the blocking of VEGFA action by treatment with a soluble truncated form of the fms-like tyrosine kinase receptor resulted in a decrease in the proliferation of the theca cells of secondary and tertiary follicles and a marked decline in FLT1 mRNA expression in the primate ovary. Zimmermann et al. [7] have shown that the blockage of function of KDR (VEGF receptor 2) alters follicular development and hormone secretion, but the specific mechanism by which it occurs remains unclear. In addition, the impairment of oocyte release and steroid production and the subsequent development of the monkey corpus luteum after the injection of antiangiogenic factors into the preovulatory follicle was demonstrated [9, 45]. Moreover, a novel report has described the role of VEGFA in the molecular regulation of ovarian apoptosis, showing that VEGFA treatment reduces the incidence of apoptosis and active caspase-3 expression in culture endothelial and granulosa cells from rats and cattle [5].

Although several intracellular molecules, including BCL2 [21, 22], BCL2L1 [23], BAX [21], caspases [24, 25], and IAPs [28], have been implicated in the regulation of ovarian apoptosis, several studies have suggested that TNFRSF6 (FAS) and FASLG are central in the induction of follicular atresia [46, 47]. In the present study, the TNFRSF6/FASLG protein levels were not affected by Trap treatment. These results suggest that the other pathway, which is related to mitochondria protein regulation, is involved in the inhibition of activity. In this regard, members of the Bcl2 gene family have been described as main participants in the cascade of events that activate or inhibit apoptosis [48]. The BCL2-related proteins can be separated into anti- and proapoptotic members, and the balance between these counteracting proteins presumably determines cell fate [49]. In our experimental model, antral follicles obtained from Trap-treated rats showed a decrease in BCL2 and an increase in BAX protein levels. In addition, a reduction in the BCL2L1L:BCL2L1S ratio was observed in this group, with a reduction of BCL2L1L greater than that of BCL2L1S, showing that the expression of these antiapoptotic proteins is lower in follicles from intraovarian Trap-treated rats.

In conclusion, the inhibition of VEGFA activity appears to produce an increase in ovarian apoptosis through an imbalance in the ratio of antiapoptotic:proapoptotic proteins, leading a larger number of follicles to atresia. The mechanism could take place either through an increase in blood vessel extension or through a direct effect mediated by an ovarian VEGFA receptor in granulosa cells. Further information is needed to elucidate the relevance of the changes observed in the vasculature during folliculogenesis and to define the regulations and clarify the interactions between VEGFA and the hormones essential for normal ovarian function. Therefore, VEGFA would be a major limiting factor for follicular development and atresia.

The antiapoptotic actions of VEGFA in the ovary put this factor in a new position as one whose abnormal expression may lead to many diseases caused by dysregulations of programmed cell death. Moreover, the results described in the present study may provide insight into the mechanisms by which VEGFA has an effect on ovarian disorders (such as polycystic ovary syndrome and ovarian hyperstimulation syndrome). In addition, the use of antiangiogenic compounds may contribute to the development of new therapeutic strategies.

ACKNOWLEDGMENTS

We thank Dr. Richard L. Stouffer (Oregon National Primate Center/Oregon Health and Science University) for his helpful discussion. We also thank Pablo Do Campo and Diana Bas (IByME-CONICET) for their technical assistance.

FOOTNOTES

1 Supported by the Agencia Nacional de Promoción Científica y Tecnológica (BID 1201 OC-AR PICT 99:05–06384) and the Universidad de Buenos Aires (X237). Back

2 Correspondence. FAX: 54 011 4786 2564; mtesone{at}dna.uba.ar Back

Received: 27 January 2006.

First decision: 16 February 2006.

Accepted: 13 June 2006.

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