Kikuchi K, Naito K, Daen F P, Izaike Y, Toyoda Y
Department of Genetic Resources II, National Institute of Agrobiological Resources, Kannondai 2-1-2, Tsukuba, Ibaraki 305, Japan.
Theriogenology. 1995 Jan 15;43(2):523-32. doi: 10.1016/0093-691x(94)00044-u.
The present study was conducted to clarify the relationship between histone H1 kinase (H1K) activity and events associated with in vitro fertilization of pig follicular oocytes matured in vitro. Histone H1 kinase has been shown to be homologous with a maturation promoting factor (MPF). Cumulus-oocyte complexes obtained from prepubertal gilts were cultured for 46 h in a modified Waymouth's MB752/1 medium and were then inseminated in vitro with frozen-thawed and preincubated epididymal boar spermatozoa. At 4, 6, 8 and 10 h post insemination, the oocytes were stained with 10 microg/ml Hoechst-33342 and examined under a fluorescent microscope for the stage of fertilization, according to morphological changes of oocyte nuclear chromatin and the extent of sperm penetration. Sperm penetration was observed to occur within 4 h post insemination (20.5%), and the percentage of fertilized oocytes increased (P < 0.01) to 72.9% at 8 h post insemination. Pronuclear formation was observed from 6 h post insemination (3.3%) and the percentage increased (P < 0.01) to 46.8% at 10 h post insemination. In each examination period, H1K activities in unfertilized oocytes at metaphase-II remained unchanged (112.0 fmol/h/oocyte) and were higher (P < 0.01) than those in fertilized oocytes (30.1 fmol/h/oocyte). The H1K activity in fertilized oocytes such as oocytes emitting a second polar body, oocytes with an enlarging sperm head(s) and oocytes with multiple pronuclei did not differ significantly. These results suggest that MPF in pig oocytes is inactivated shortly after sperm penetration and is maintained at the basal level throughout pronuclear formation.
本研究旨在阐明组蛋白H1激酶（H1K）活性与体外成熟猪卵泡卵母细胞体外受精相关事件之间的关系。已证明组蛋白H1激酶与成熟促进因子（MPF）同源。从青春期前小母猪获取的卵丘-卵母细胞复合体在改良的Waymouth's MB752/1培养基中培养46小时，然后用冻融并预孵育的附睾公猪精子进行体外授精。在授精后4、6、8和10小时，用10微克/毫升的Hoechst-33342对卵母细胞进行染色，并在荧光显微镜下根据卵母细胞核染色质的形态变化和精子穿透程度检查受精阶段。观察到精子穿透在授精后4小时内发生（20.5%），授精后8小时受精卵母细胞的百分比增加（P<0.01）至72.9%。从授精后6小时观察到原核形成（3.3%），授精后10小时百分比增加（P<0.01）至46.8%。在每个检查期，处于中期II的未受精卵母细胞中的H1K活性保持不变（112.0飞摩尔/小时/卵母细胞），且高于受精卵母细胞中的活性（P<0.01）（30.1飞摩尔/小时/卵母细胞）。发出第二极体的卵母细胞、精子头部增大的卵母细胞和具有多个原核的卵母细胞等受精卵母细胞中的H1K活性无显著差异。这些结果表明，猪卵母细胞中的MPF在精子穿透后不久即失活，并在整个原核形成过程中维持在基础水平。
