Wang W H, Sun Q Y, Hosoe M, Shioya Y, Day B N
Department of Reproduction, National Institute of Animal Industry, Tsukuba, Japan.
Biol Reprod. 1997 Jun;56(6):1376-82. doi: 10.1095/biolreprod56.6.1376.
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.
多精受精是猪卵母细胞体外成熟和体外受精过程中尚未解决的问题之一。对卵母细胞中皮质颗粒（CG）数量变化进行定量研究，对于理解卵母细胞阻止多精入卵的机制以及优化体外成熟（IVM）和体外受精（IVF）条件至关重要。本研究利用异硫氰酸荧光素标记的花生凝集素和激光共聚焦显微镜对猪卵母细胞体外成熟和体外受精过程中CG的分布进行定量。结果表明，在生发泡（GV）期，CG分布于卵母细胞的皮质细胞质中，皮质区每100μm²平均有33.8±7.3个CG。随着核成熟进展到中期I和中期II，CG迁移至皮质并在卵膜下形成连续单层。成熟过程中未观察到明显的无CG区域。成熟过程中CG持续向皮质迁移，GV期后CG密度增加。所有被精子穿透的卵母细胞均被激活并从卵质中释放CG，授精后18小时皮质区平均残留3.5±4.6个CG/100μm²。45%的卵母细胞观察到CG完全胞吐。钙离子载体未诱导卵母细胞核激活，但处理后18小时检查时，卵母细胞仍有平均7.1±4.5个CG/100μm²释放。电脉冲诱导89%的成熟卵母细胞核激活，仅在核激活的卵母细胞中观察到CG胞吐，皮质区平均残留6.4±9.4个CG/100μm²。分别有10%和25%的卵母细胞经离子载体和电脉冲诱导CG完全胞吐。这些结果表明，猪卵母细胞在体外成熟过程中CG迁移至皮质，在这些成熟条件下获得的成熟卵母细胞在精子穿透、离子载体处理和电脉冲作用下具有释放CG的能力。然而，这些研究中CG胞吐后卵母细胞对多精入卵的功能性阻断尚未完全建立。本方法和结果为进一步研究猪卵母细胞体外成熟和体外受精过程中多精受精的原因提供了途径。
