Quantified analysis of cortical granule distribution and exocytosis of porcine oocytes during meiotic maturation and activation

Polyspermy is one of the unresolved problems that exist regarding pig oocytes matured and inseminated in vitro. Quantitative study of the changes in the cortical granule (CC) population in oocytes is essential for understanding the mechanism of how oocytes block polyspermic penetration and for developing the optimum conditions for in vitro maturation (IVM) and in vitro fertilization (IVF). The present study was conducted to quantify the CC distribution in pig oocytes during IVM and IVF by using fluorescein isothiocyanate-labeled peanut agglutinin with laser confocal microscopy. The results indicate that CCs are distributed in the cortex cytoplasm of oocytes at the germinal vesicle (CV) stage with a mean number of 33.8 +/- 7.3 CGs/100 mu m(2) of cortex. As nuclear maturation proceeded to metaphase I and metaphase II, CCs migrated to the cortex and formed a continuous monolayer under the oolemma. No distinct CC-free domain was observed in oocytes during maturation. The migration of CCs to the cortex continued during maturation, with an increased CC density after the CV stage. All oocytes penetrated by spermatozoa were activated and released CCs from ooplasm with an average residual number of 3.5 +/- 4.6 CGs/100 mu m(2) of cortex at 18 h after insemination. Complete CC exocytosis was observed in 45% of oocytes. Calcium ionophore did not induce oocyte nuclear activation, but CCs were released from oocytes with an average of 7.1 +/- 4.5 CGs/100 mu m(2) of cortex still present when examined 18 h after treatment. An electrical pulse induced 89% of nuclear activation in matured oocytes, and CC exocytosis was observed only in nuclear-activated oocytes with an average residual number of 6.4 +/- 9.4 CGs/100 mu m(2) of cortex. Complete CC exocytosis was induced by ionophore and electrical pulse in 10% and 25% of the oocytes, respectively. These results indicate that CCs migrate to the cortex in pig oocytes during IVM and that the matured oocytes obtained under these maturation conditions possess the ability to release CCs upon sperm penetration, ionophore treatment, and electrical pulse. However, a functional block to polyspermic penetration in oocytes after CC exocytosis was not fully established in these studies. The present methods and results provide the approach for further investigation of the reasons for polyspermy in pig oocytes matured and inseminated in vitro.