Oikawa Toshinori, Itahashi Tomoko, Yajima Risa, Numabe Takashi
Miyagi Prefectural Livestock Experiment Station, Miyagi 989-6445, Japan.
Miyagi Prefectural Sendai Livestock Hygiene Science Center, Miyagi 983-0832, Japan.
J Reprod Dev. 2018 Aug 20;64(4):303-309. doi: 10.1262/jrd.2018-023. Epub 2018 May 7.
Intracytoplasmic sperm injection (ICSI) was expected to enable more efficient use of sperm from sires with preferable genetic traits and result in a generation containing a larger number of offspring with superior genetic characteristics in livestock. However, the efficiency of the early development of embryos produced by ICSI is still far from satisfactory in cattle. The present study aimed to investigate the effects of the treatment of cryopreserved sperm with glutathione (GSH) on the early development of embryos produced by ICSI in Japanese Black cattle. Moreover, the disulfide bond state and mitochondrial function were investigated in the sperm treated with GSH to confirm the effectiveness of the abovementioned treatment. We also investigated the effect of 7% ethanol activation treatment on the developmental ability of ICSI embryos using GSH-treated sperm. There was no effect on the blastocyst rate from the activation treatment. When sperm-injected oocytes were cultured in vitro, the treatment with GSH significantly improved the early development of embryos. Specifically, the rates of embryos reaching the 4-8-cell stage and blastocyst stage were significantly higher in ICSI with GSH-treated sperm (71.4% and 31.0%, respectively) than that with the control sperm (36.6% and 7.0%, respectively). Moreover, the GSH-treated sperm treatment significantly decreased the number of disulfide bonds in the sperm head (as shown by monobromobimane staining) and enhanced the mitochondrial function in the sperm middle piece (as shown by Rhodamine 123 staining and the adenosine triphosphate-dependent bioluminescence assay). Based on these results, we suggest that the treatment of cryopreserved sperm with GSH might contribute to the improvement of ICSI techniques for the production of blastocysts in Japanese Black cattle.
胞浆内单精子注射（ICSI）有望更有效地利用具有优良遗传性状的种公牛的精子，并在牲畜中产生大量具有优良遗传特征的后代。然而，在牛中，通过ICSI产生的胚胎的早期发育效率仍远不能令人满意。本研究旨在探讨用谷胱甘肽（GSH）处理冷冻保存的精子对日本黑牛ICSI产生的胚胎早期发育的影响。此外，还对用GSH处理的精子的二硫键状态和线粒体功能进行了研究，以确认上述处理的有效性。我们还研究了7%乙醇激活处理对使用GSH处理的精子的ICSI胚胎发育能力的影响。激活处理对囊胚率没有影响。当将注射精子的卵母细胞进行体外培养时，GSH处理显著改善了胚胎的早期发育。具体而言，用GSH处理的精子进行ICSI时，达到4-8细胞期和囊胚期的胚胎率（分别为71.4%和31.0%）显著高于对照精子（分别为36.6%和7.0%）。此外，GSH处理的精子处理显著减少了精子头部的二硫键数量（如单溴代联苯胺染色所示），并增强了精子中段的线粒体功能（如罗丹明123染色和三磷酸腺苷依赖性生物发光测定所示）。基于这些结果，我们认为用GSH处理冷冻保存的精子可能有助于改进日本黑牛ICSI技术以生产囊胚。
