182 CHEMICAL ACTIVATION OF ZONA PELLUCIDA-FREE OOCYTES PROVOKES FULL CORTICAL REACTION: AN APPROACH TO STUDY CORTICAL GRANULE-DERIVED PROTEINS IN PIGS

Abstract

The cortical reaction is a mechanism that prevents polyspermy by cortical granule content being released into the periviteline space, modifying the zona pellucida (ZP). Knowledge about specific cortical granule-derived proteins has progressed slowly because these organelles contain only picogram quantities of proteins. An efficient method for collecting cortical granule content would help in its study; chemical activation of ZP-free oocytes has been successfully used in the murine model (Muñoz-Gotera et al. 2001 Mol. Reprod. Dev. 60, 405–413). Calcium ionophore A23187 is an effective chemical stimulator for provoking the cortical reaction in ZP-intact pig oocytes. However, the commonly used protocol (50 μM for 5min) cannot be employed with ZP-free oocytes because the oolemma is damaged, oocyte lysed and medium contaminated with ooplasm content, which is necessary to reduce the time and ionophore concentration (Romar et al. 2011 Reprod. Fertil. Dev. 23, 221 abst). The objective of this study was to evaluate the efficiency of this activation protocol for provoking the cortical reaction in ZP-free oocytes by assessment with confocal and electron microscopy. Immature cumulus–oocyte complexes from Landrace × Large White gilts were in vitro matured for 44 h in an NCSU-37 medium. After maturation, the oocytes were stripped of cumulus cells and their ZP were removed with pronase. Then, the ZP-free oocytes were incubated with calcium ionophore A23187 (6.5 μM for 2min), transferred to an exudate medium and incubated at 38.5°C, 5% CO2 and saturated humidity for 30 min. Control ZP-free oocytes were incubated without being activated. After incubation, ionophore-treated (n = 10) and control oocytes (n = 18) were used to assess the presence of a cortical granule monolayer. An aliquot was fixed, permeabilized (0.1% Triton), incubated with peanut agglutinin lectin conjugated to fluorescein isothiocyanate (10 μg mL–1 for 30 min) and examined under a confocal microscope. Presence or absence of a cortical granule monolayer at the equator level was recorded. Another aliquot was fixed and processed for electron microscopy observation. The cortical granules in the whole oocytes were counted and results are presented as the mean ± standard error of the mean. No cell lysis was observed in control or activated ZP-free oocytes after treatment and incubation time. The confocal study showed that the activation protocol provokes a full cortical reaction in 100% of A23187-treated oocytes, given that no peanut agglutinin labeling was observed in the cortical area. Presence of a cortical granule monolayer under the oolemma was observed in 100% of control oocytes. Cortical granule release was confirmed by electron microscopy. Control oocytes had 5.90 ± 1.78 cortical granules per 5 μm of oolemma, whereas activated oocytes exhibited a significant reduction (P < 0.05) of up to 0.71 ± 0.20. In conclusion, the presented activation protocol by using ZP-free oocytes is a valid method for provoking a complete cortical reaction and could be employed in the future as an efficient method to collect cortical granule-derived proteins in pig oocytes. Supported by CONACYT (0105961/I0110/194/09), MEC and FEDER (AGL2009-12512-C02-01).