Morphologic changes in boar sperm nuclei with reduced disulfide bonds in electrostimulated porcine oocytes
Morphologic changes in boar sperm nuclei with reduced disulfide bonds in electrostimulated porcine oocytes
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DOI：
10.1530/rep.1.01001
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发表时间：
2006-03-01
期刊：
影响因子：
3.8
通讯作者：
Kikuchi, K
中科院分区：
文献类型：
--
作者：
Nakai, M;Kashiwazaki, N;Kikuchi, K
在猪中，注射到卵母细胞后精子核去浓缩失败已被报道。我们研究了用Triton x - 100 （tx - 100）和二硫苏糖醇（DTT）预处理精子头和注射精子头后电刺激卵母细胞对精子核的时间依赖性形态变化和囊胚期体外发育的影响。实验1，将精子用1% tx - 100和5 mM DTT （T + D）预处理或不处理，然后注射到体外成熟卵母细胞中。在注射后1 h对卵母细胞施加电刺激（11.5 kV/cm， 20 μ s直流脉冲）（刺激组）或不施加电刺激（未刺激组）。每隔一小时对每组的部分卵母细胞进行评估，直至注射后10小时，以观察精子核的形态变化。与注射T + d处理精子的卵母细胞相比，注射未经处理精子的未刺激卵母细胞的核去浓缩率峰值（分别为57.0%和52.6%，分别为1和2 h）延迟（39.4% - 44.1%，注射后3-6 h）。不论精子是否经过预处理，注射的卵母细胞受到电脉冲刺激后，雄性原核形成率在刺激后6小时达到峰值（40-60%）。在未受刺激的卵母细胞中，无论精子处理如何，整个培养期间雄性原核的形成率都没有增加，保持在基础水平（低于20%）。因此，对精子进行T + D处理不影响受精的完成。在实验2中，我们评估了电刺激和精子T + D处理对囊胚形成率和每个囊胚平均细胞数的影响。经T + d处理或未处理的精子注射后的卵母细胞囊胚形成率（分别为24.8%和27.1%）均显著高于未刺激的卵母细胞（经T + d处理和未处理的卵母细胞囊胚形成率分别为1.1%和4.1%;Duncan's多范围检验P < 0.01）。在T + d治疗组和未治疗组之间，囊胚形成率没有差异。每个囊胚的平均细胞数在任何组之间没有差异（14.0-29.4个细胞）。这些结果表明，用tx - 100和DTT预处理精子可以将精子核去浓缩的时间提前。然而，精子处理并没有促进原核的形成和囊胚期的发育。因此，电刺激注射的卵母细胞可以促进猪胚泡期的体外发育。
In pigs, failure of sperm nuclear decondensation has been reported after injection into oocytes. We examined the effects of pretreating sperm heads with Triton X-1 00 (TX-1 00) and dithiothreitol (DTT) and of electrical stimulation of oocytes after sperm head injection on time-dependent morphologic changes in sperm nuclei and in vitro development to the blastocyst stage. In experiment 1, spermatozoa were pretreated with 1 % TX-1 00 and 5 mM DTT (T + D) or not treated, and then injected into in vitro matured oocytes. Electrical stimulation (11.5 kV/cm, 20 mu s DC pulse) was applied to the oocytes 1 h after injection (stimulated group) or was not applied (unstimulated group). Some of the oocytes in each group were evaluated at hourly intervals until 10 h after injection for morphologic changes in the sperm nuclei. Unstimulated oocytes injected with untreated spermatozoa showed a delayed peak in the rate of nuclear decondensation (39.4-44.1 %, 3-6 h after injection) compared with oocytes injected with T + D-treated spermatozoa (57.0% and 52.6%, 1 and 2 h, respectively). The rate of male pronucleus formation peaked 6 h after stimulation (by 40-60%) after injected oocytes had been stimulated with an electrical pulse, irrespective of whether or not the spermatozoa had been pretreated. In unstimulated oocytes, the rate of male pronucleus formation did not increase and stayed at the basal level (less than 20%) throughout the culture period, regardless of the sperm treatment. Thus, T + D treatment of spermatozoa did not affect completion of fertilization. In experiment 2, we evaluated the effects of electrical stimulation and sperm treatment with T + D on the rate of blastocyst formation and the mean number of cells per blastocyst. Oocytes stimulated after injection with either T + D-treated or untreated spermatozoa showed significantly higher percentages of blastocyst formation (24.8% and 27.1 % respectively) than did unstimulated oocytes (1.1 % and 4.1 % for T + D-treated and untreated respectively; P < 0.01 by Duncan's multiple-range test). The rate of blastocyst formation did not differ between the T + D-treated and untreated groups. The mean number of cells per blastocyst did not differ among any of the groups (14.0-29.4 cells). These results suggest that pretreatment of sperm with TX-1 00 and DTT shifted the timing of sperm nuclear decondensation forward. However, pronucleus formation and development to the blastocyst stage in vitro were not improved by sperm treatment. Thus, electrical stimulation of injected oocytes enhances in vitro development to the blastocyst stage in pigs.