206 THE IMPORTANCE OF CUMULUS EXPANSION FOR MONOSPERMIC FERTILIZATION OF PORCINE CUMULUS–OOCYTE COMPLEXES

Abstract

In pigs, the exact role of the cumulus oöphurus during IVF still needs to be clarified. Indirect evidence exists that the rate of cumulus expansion is positively correlated with the defense against polyspermy. Epidermal growth factor (EGF) acts synergistically with FSH in the synthesis of hyaluronic acid, the deposition of which in the extracellular matrix is a prerequisite for cumulus expansion. Therefore, we aimed to evaluate the importance of cumulus expansion for fertilization results of porcine cumulus–oocyte complexes (COC) by using different EGF concentrations in the maturation medium. Cumulus–oocyte complexes were matured in vitro in NCSU23 medium supplemented with 10% follicular fluid (FF, obtained from 6- to 10-mm follicles) or 10% serum and 10, 20, or 50 ng mL^–1 of EGF (n = 480 per experiment). In vitro maturation (IVM) in the presence of 10% FF and 10 ng mL^–1 of EGF served as the control group. At 0, 22, 36, and 44 h of IVM, 20 COC of each group were selected for evaluation of cumulus expansion by measuring the maximum distance across the cumulus matrix (3 replications). Matured COC were co-incubated with frozen–thawed semen (6000 spermatozoa per oocyte) for 6 h. Subsequently, oocytes were cultured for 18 h. Zygotes were stained with 10 μg mL^–1 of bis-benzimide (Hoechst) to assess the fertilization rate, polyspermy, and sperm penetration index (sp index, mean number of penetrated spermatozoa per fertilized oocyte; 2 replications). Differences in cumulus diameter were analyzed by one-way ANOVA. Fertilization parameters were analyzed by applying a logistic regression model to the results. Cumulus–oocyte complexes selected for IVM had a mean diameter of 240 μm. After 22 h of IVM in 10% FF, the mean diameter of COC was 336, 313, and 300 μm for 10, 20, and 50 ng mL^–1 of EGF, respectively. After 44 h of IVM, these diameters had increased to 425, 388, and 397 μm. Twenty-two hours of IVM in 10% serum resulted in a COC diameter of 296, 305, and 276 μm for 10, 20, and 50 ng mL^–1 of EGF. After 44 h of IVM, these diameters reached 330, 325, and 275 μm, respectively. Only 10% serum with 50 ng mL^–1 of EGF proved unfavorable for cumulus expansion (P < 0.05). In vitro maturation for 44 h in 10% serum resulted in a smaller rate of cumulus expansion compared with IVM in 10% FF (P < 0.05), irrespective of EGF concentration. Penetration rate fluctuated between 84 and 100%, with no significant differences. Monospermic fertilization was lower in COC matured in the presence of 50 ng mL^–1 of EGF compared with the control group (P < 0.05). The sp index increased in parallel with EGF concentrations and was higher after IVM in 10% serum than in 10% FF. Oocytes were penetrated by 3.1, 4.3, and 6.0 spermatozoa after IVM in serum with 10, 20, and 50 ng mL^–1 of EGF, respectively. Results showed a tendency toward a lower rate of cumulus expansion concomitant with higher EGF concentrations. Follicular fluid was superior to serum in supporting cumulus expansion. Oocytes were penetrated by more spermatozoa when matured in 10% serum and 20 or 50 ng mL^–1 of EGF compared with 10 ng mL^–1 of EGF. Thus, the degree of cumulus expansion appears to be related to the sp index, confirming that the cumulus matrix may play a role in the polyspermy defense.