Lechniak Dorota, Szczepankiewicz Dawid, Kauss Dominika, Szulc Joanna, Szydłowski Maciej
Department of Genetics and Animal Breeding, August Cieszkowski Agricultural University of Poznan, Wolynska 33, 60-637 Poznan, Poland.
Theriogenology. 2005 Jul 1;64(1):202-12. doi: 10.1016/j.theriogenology.2004.11.019. Epub 2005 Jan 9.
In the present study three factors were investigated that may affect the process of the first polar body extrusion in pig oocytes matured in vitro: IVM medium, oocyte diameter and donor genotype at the ryanodine receptor (RYR1) locus. In the first experiment, COCs were collected by the aspiration of slaughterhouse ovaries. Oocytes were matured in vitro at 39 degrees C, in humidified 5% CO(2) atmosphere for 44 h using the following media: (1) TCM199+hCG+eCG+follicular fluid (FF), (2) TCM199+hCG+17beta-estradiol and (3) NCSU23+hCG+eCG+FF. According to cytogenetic analysis, 98.1% of cells reached the second metaphase stage (MII). No significant differences were observed among IVM groups in terms of diploidy level. In the second experiment, oocytes collected by the aspiration or slicing of individual ovaries were matured in vitro in groups reflecting their origin. One ovary was considered a donor. IVM was carried out under conditions described in experiment I, with the use of TCM199+hCG+17beta-estradiol. A total of 68 ovaries/donors were included in this study. Granulosa cells collected from each ovary were used as DNA source in molecular (RFLP) analysis. Genotype frequencies at the RYR1 locus were as follows: CC, 0.46; CT, 0.48 and TT, 0.06. After maturation the diameter of each denuded oocyte was determined with the use of a computer aided system. Five size categories were distinguished: <90, 90-100, 100.1-110, 110.1-120 and >120 microm. The average diameter of haploid oocytes at MII stage was 111.7 microm, whereas that of diploid cells was 110.4 microm. According to statistical analysis, diploidy was not related to the oocyte diameter. That trait, however, was influenced by the donor genotype at the RYR1 locus. The TT genotype was associated with a higher rate of diploidy.
在本研究中，对可能影响体外成熟猪卵母细胞第一极体排出过程的三个因素进行了研究：体外成熟（IVM）培养基、卵母细胞直径以及位于兰尼碱受体（RYR1）位点的供体基因型。在第一个实验中，通过抽吸屠宰场的卵巢收集卵丘-卵母细胞复合体（COCs）。卵母细胞在39℃、湿度为5%二氧化碳的培养箱中，使用以下培养基进行44小时的体外成熟培养：（1）TCM199+人绒毛膜促性腺激素（hCG）+孕马血清促性腺激素（eCG）+卵泡液（FF）；（2）TCM199+hCG+17β-雌二醇；（3）NCSU23+hCG+eCG+FF。根据细胞遗传学分析，98.1%的细胞达到了第二次减数分裂中期（MII）。在二倍体水平方面，各IVM组之间未观察到显著差异。在第二个实验中，通过抽吸或切割单个卵巢收集的卵母细胞，根据其来源分组进行体外成熟培养。将一个卵巢视为一个供体。IVM在实验I所述的条件下进行，使用TCM199+hCG+17β-雌二醇。本研究共纳入68个卵巢/供体。从每个卵巢收集的颗粒细胞用作分子（限制性片段长度多态性，RFLP）分析的DNA来源。RYR1位点的基因型频率如下：CC为0.46；CT为0.48；TT为0.06。成熟后，使用计算机辅助系统测定每个去卵丘卵母细胞的直径。区分出五个大小类别：<90、90 - 100、100.1 - 110、110.1 - 120和>120微米。MII期单倍体卵母细胞的平均直径为111.7微米，而二倍体细胞的平均直径为110.4微米。根据统计分析，二倍体与卵母细胞直径无关。然而，该性状受RYR1位点的供体基因型影响。TT基因型与较高的二倍体率相关。
