Effect of a Vascular Endothelial Growth Factor: DISCUSSION(4)

DISCUSSION(4)

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. The BCL2-related proteins can be separated into anti- and proapoptotic members, and the balance between these counteracting proteins presumably determines cell fate. 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.

Effect of a Vascular Endothelial Growth Factor: DISCUSSION(3)

Zimmermann et al. 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. 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.

Effect of a Vascular Endothelial Growth Factor: DISCUSSION(2)

DISCUSSION(2)

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 folliculotro-phin-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.

Effect of a Vascular Endothelial Growth Factor: DISCUSSION(1)

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.

Effect of a Vascular Endothelial Growth Factor: RESULTS(3)

RESULTS(3)

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 6 4.7, Trap: 429.3 6 36.6 arbitrary units; P < 0.05). DNA fragmentation was minimal in freshly isolated antral follicles (data not shown).

Effect of a Vascular Endothelial Growth Factor: RESULTS(2)

No significant differences were found in any stage of follicles 12 or 24 h after injection. Additionally, there were no differences when 0.1 ig of Trap per ovary was injected (data not shown). Therefore, the 0.5-ig 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.

Effect of a Vascular Endothelial Growth Factor: RESULTS(1)

RESULTS(1)

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 ig of Trap, and the ovaries were removed 12, 24, and 48 h later and then processed and analyzed as described above.

Effect of a Vascular Endothelial Growth Factor: MATERIALS AND METHODS(8)

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.

Effect of a Vascular Endothelial Growth Factor: MATERIALS AND METHODS(7)

METHODS(7)

Quantitative results obtained by densitometric analysis of the low-molecular-weight DNA fragments represent the mean 6 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) homog-enizer.

Effect of a Vascular Endothelial Growth Factor: MATERIALS AND METHODS(6)

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 ig/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.