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 (CG) 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 CG distribution in pig oocytes during IVM and IVF by using fluorescein isothiocyanate-labeled peanut agglutinin with laser confocal microscopy. The results indicate that CGs are distributed in the cortex cytoplasm of oocytes at the germinal vesicle (GV) stage with a mean number of 33.8 +/- 7.3 CGs/100 microm2 of cortex. As nuclear maturation proceeded to metaphase I and metaphase II, CGs migrated to the cortex and formed a continuous monolayer under the oolemma. No distinct CG-free domain was observed in oocytes during maturation. The migration of CGs to the cortex continued during maturation, with an increased CG density after the GV stage. All oocytes penetrated by spermatozoa were activated and released CGs from ooplasm with an average residual number of 3.5 +/- 4.6 CGs/100 microm2 of cortex at 18 h after insemination. Complete CG exocytosis was observed in 45% of oocytes. Calcium ionophore did not induce oocyte nuclear activation, but CGs were released from oocytes with an average of 7.1 +/- 4.5 CGs/100 microm2 of cortex still present when examined 18 h after treatment. An electrical pulse induced 89% of nuclear activation in matured oocytes, and CG exocytosis was observed only in nuclear-activated oocytes with an average residual number of 6.4 +/- 9.4 CGs/100 microm2 of cortex. Complete CG exocytosis was induced by ionophore and electrical pulse in 10% and 25% of the oocytes, respectively. These results indicate that CGs migrate to the cortex in pig oocytes during IVM and that the matured oocytes obtained under these maturation conditions possess the ability to release CGs upon sperm penetration, ionophore treatment, and electrical pulse. However, a functional block to polyspermic penetration in oocytes after CG 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.
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