99 EFFECT OF VASCULAR ENDOTHELIAL GROWTH FACTOR ON IN VITRO-PRODUCED PORCINE OOCYTES AND THEIR SUBSEQUENT DEVELOPMENT: A PARTHENOGENETIC STUDY

Abstract

The addition of vascular endothelial growth factor (VEGF) to maturation media has beneficial effects on oocyte maturation and blastocyst formation (Einspanier et al. 2002 Mol. Reprod. Dev. 62, 29–36). The present study was conducted to examine the effect of parthenogenesis on in vitro-matured porcine oocytes with VEGF along with porcine follicular fluid in the maturation media. Porcine ovaries were collected from a local slaughter house in physiological saline. After aspiration, COC were matured in vitro in TCM-199 supplemented with 10 ng mL^–1 of epidermal growth factor and (1) Group A: 10% pFF; (2) Group B: 10% pFF and 5 ng mL^–1 of VEGF; (3) Group C: 10% polyvinyl alcohol; or (4) Group D: 5 ng mL^–1 of VEGF plus 10% polyvinyl alcohol. Fifty COC were cultured for the first 22 h at 390°C in a humidified atmosphere of 5% CO₂ in 95% air with 4 IU mL^–1 of eCG and 4 IU mL^–1 of hCG. They were then transferred to hormone-free medium and cultured for an additional 20 h. After culture, COC were denuded with hyaluronidase, and a proportion were stained with Hoechst 33342 for evaluating the metaphase II stage. The remaining oocytes were subjected to electrical parthenogenesis by using a 1-mm fusion chamber and were activated by applying 2 direct current pulses of 110V for 60 µs. Cleavage and blastocyst formation rate were evaluated under a stereomicroscope at 48 and 168 h after activation, respectively. Blastocyst quality was assessed by differential staining of inner cell mass and trophectoderm cells according to a modified staining procedure (Thouas et al. 2001 Reprod. Biomed. Online 3, 25–29). All data are presented as mean ± SD and were analyzed by ANOVA followed by Duncan's multiple range test using SPSS 12.0 (SPSS Inc., Chicago, IL, USA). The maturation rate was significantly higher (P < 0.05) in Groups A and B than Groups C and D (76.1 ± 9.6, 78.9 ± 6.0 v. 60. ± 14.2 and 58.3 ± 14.3, respectively). The cleavage rate was significantly higher (P < 0.05) in Groups A and B (73.2 ± 1.8 and 64.6 ± 1.1, respectively) than Groups C and D (47.9 ± 1.8 and 48.3 ± 1.7, respectively). The blastocyst formation rate was significantly higher (P < 0.05) in Group B (32.6 ± 2.4) compared to other groups. There was no significant difference in blastocyst cell number (inner cell mass or trophectoderm) among these groups. These data indicate that the exogenous VEGF along with pFF in the maturation media helps to increase the blastocyst formation rate in vitro, and it might be due to presence of some ligand/protein kinase in the pFF that plays an important role during the cyclic growth of oocytes.