King W A, Xu K P, Sirard M A, Greve T, Leclerc P, Lambert R D, Jacques P
Centre de Recherche en Reproduction Animale, Faculté de Médecine Vétérinaire, Université de Montréal St-Hyacinthe, Québec, Canada.
Gamete Res. 1988 Jul;20(3):265-74. doi: 10.1002/mrd.1120200303.
Three experiments were performed to evaluate the potential for parthenogenetic activation of bovine oocytes in in vitro fertilization systems and to determine the chromosome complement of the resulting parthenogenotes. In the first experiment, immature oocytes from slaughtered cattles were matured in vitro in Defined Medium (DM) for 24 h to simulate in vitro fertilization conditions. Subsequently, a portion was fixed, and the remainder were transferred to rabbit oviducts. Oocytes were then cultured for 6-8 h or for 24 h with Colcemid present during the last 6 to 8 h and fixed on slides and examined. In the second experiment, mature oocytes were collected from the preovulatory follicles, and the oocytes were subjected to the same culture as in experiment I. In the third experiment, oocytes were treated as in experiment II, except that instead of transfer to rabbit oviducts, they were cultured an additional 48 h in vitro. In experiment I, 131 oocytes were fixed after culture in DM. Of the 79 oocytes analyzed in the pre-rabbit group, 71 (90%) were at the second meiotic metaphase (MII), and 8 (10%) were at pre-MII stage; none were activated. After transfer to rabbits, 291 were fixed. Of these, 80 were analyzed; 37 (46.3%) were MII, 7 (8.6%) were pre-MII, and 36 (45%) were activated. Of the 36 activated oocytes, 26 (72.2%) were haploid, 4 (11.1%) were diploid, 1 (12.8%) was tetraploid, and 5 (13.8%) were in the process of endoreduplication. In experiment II, 51 oocytes were fixed after culture in DM of which 36 (70.6%) could be analyzed; 30 (83.3%) were MII, and 6 (16.7%) were pre-MII. After culture in the rabbit, 68 were fixed of which 27 (39.7%) could be analyzed. Of these 27, 20 (74.1%) were MII, and 7 (25.9%) were activated; 6 were haploid, and 1 was endoreduplicating. In experiment III, 30 oocytes were fixed at the end of the culture period; only 10 could be analyzed of which 8 (80%) were MII and 2 (20%) were pre-MII. In all, 46% of in vitro and 26% of in vivo matured oocytes were activated, based on chromosomal analysis. Of those activated, the majority (74.4%) were haploid, suggesting that activation occurs at or after completion of MII. Endoreduplication appears to be one of the mechanisms leading to the formation of diploid and polyploid parthenogenotes.
进行了三项实验，以评估体外受精系统中牛卵母细胞孤雌激活的可能性，并确定所得孤雌生殖体的染色体组成。在第一项实验中，将屠宰牛的未成熟卵母细胞在限定培养基（DM）中体外成熟24小时，以模拟体外受精条件。随后，一部分固定，其余转移到兔输卵管中。然后将卵母细胞培养6 - 8小时或在最后6至8小时添加秋水仙酰胺培养24小时，固定在载玻片上并进行检查。在第二项实验中，从排卵前卵泡中收集成熟卵母细胞，并对卵母细胞进行与实验I相同的培养。在第三项实验中，卵母细胞的处理与实验II相同，只是未转移到兔输卵管中，而是在体外再培养48小时。在实验I中，131个卵母细胞在DM中培养后固定。在兔前组分析的79个卵母细胞中，71个（90%）处于第二次减数分裂中期（MII），8个（10%）处于MII前期；无一被激活。转移到兔体内后，291个被固定。其中，80个被分析；37个（46.3%）为MII，7个（8.6%）为MII前期，36个（45%）被激活。在36个被激活的卵母细胞中，26个（72.2%）为单倍体，4个（11.1%）为二倍体，1个（12.8%）为四倍体，5个（13.8%）处于核内复制过程中。在实验II中，51个卵母细胞在DM中培养后固定，其中36个（70.6%）可以分析；30个（83.3%）为MII，6个（16.7%）为MII前期。在兔体内培养后，68个被固定，其中27个（39.7%）可以分析。在这27个中，20个（74.1%）为MII，7个（25.9%）被激活；6个为单倍体，1个处于核内复制。在实验III中，30个卵母细胞在培养期结束时固定；只有10个可以分析，其中8个（80%）为MII，2个（20%）为MII前期。基于染色体分析，总体而言，46%的体外成熟卵母细胞和26%的体内成熟卵母细胞被激活。在那些被激活的细胞中，大多数（74.4%）为单倍体，这表明激活发生在MII完成时或之后。核内复制似乎是导致二倍体和多倍体孤雌生殖体形成的机制之一。
