Shirazi Abolfazl, Golestanfar Arefeh, Bashiri Masomeh, Ahmadi Ebrahim, Shams-Esfandabadi Naser
Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran.
Department of Gametes and Cloning, Research Institute of Animal Embryo Technology, Shahrekord University, Shahrekord, Iran.
Avicenna J Med Biotechnol. 2018 Jan-Mar;10(1):41-48.
Failure of Male Pronucleus (MPN) formation is a major concern in the success of Intracytoplasmic Sperm Injection (ICSI) in some species. Despite the conducted unsuccessful efforts to improve ICSI efficiency in ovine, the present study was aimed to improve MPN formation and embryo development in ovine ICSI procedure through accompaniment of sperm pretreatment with co-injection of some compounds.
In experiment 1, sperm were treated with either 2-mercaptoethanol (2ME), glutathione (GSH), or DTT (dithiothreitol) in combination with Heparin (Hep). Following DNA integrity and fragmentation assessments, the best sperm pretreatment approach in induction of sperm head decondensation was applied for the second and third experiments. In experiment 2, matured oocytes were subjected to ICSI using pretreated sperm with or without GSH and Sperm Extract (SE) co-injection. In experiment 3, the procedure was followed as experiment 2 with acrosome reacted sperm.
The highest percentages of oocyte activation were observed in Hep+GSH and Hep+2ME groups. The greatest MPN formations were also observed in the same groups when ICSI procedure was accompanied with GSH co-injection. Despite the higher percentage of MPN formation and oocyte activation in Hep+GSH and Hep+2ME groups, none of the employed strategies could increase the cleavage and blastocyst rates compared to the control.
In our study condition, despite the lack of significant increase in embryo development in treated groups, the significant increase in MPN formation in Hep+ GSH+co.GSH and Hep+2ME+co.GSH groups indicates the lower chance of parthenote formation that means a higher chance of normal fertilization compared with control.
雄原核（MPN）形成失败是某些物种胞浆内单精子注射（ICSI）成功的一个主要问题。尽管在提高绵羊ICSI效率方面进行了一些未成功的尝试，但本研究旨在通过在精子预处理的同时共注射一些化合物来提高绵羊ICSI过程中MPN的形成和胚胎发育。
在实验1中，精子分别用2-巯基乙醇（2ME）、谷胱甘肽（GSH）或二硫苏糖醇（DTT）与肝素（Hep）联合处理。在进行DNA完整性和片段化评估后，将诱导精子头部去浓缩的最佳精子预处理方法应用于第二和第三个实验。在实验2中，使用经预处理的精子对成熟卵母细胞进行ICSI，同时共注射或不共注射GSH和精子提取物（SE）。在实验3中，对顶体反应的精子采用与实验2相同的程序。
在Hep+GSH和Hep+2ME组中观察到最高的卵母细胞激活率。当ICSI程序伴有GSH共注射时，在同一组中也观察到最大的MPN形成率。尽管Hep+GSH和Hep+2ME组中MPN形成和卵母细胞激活的百分比更高，但与对照组相比，所采用的任何策略都不能提高卵裂率和囊胚率。
在我们的研究条件下，尽管处理组的胚胎发育没有显著增加，但Hep+GSH+co.GSH和Hep+2ME+co.GSH组中MPN形成的显著增加表明孤雌胚形成的可能性较低，这意味着与对照组相比正常受精的可能性更高。
