Anzalone Debora A, Iuso Domenico, Czernik Marta, Ptak Grazyna, Loi Pasqualino
Faculty of Veterinary Medicine, University of Teramo, R. Balzarini Street 1, Campus Coste Sant'Agostino, 64100, Teramo, Italy.
National Research Institute of Animal Production, 1, Krakowska Street, 32-083, Balice n/Krakow, Poland.
J Assist Reprod Genet. 2016 Jun;33(6):757-63. doi: 10.1007/s10815-016-0709-1. Epub 2016 Apr 8.
This study aims to determine if the integrity of the sperm plasma membrane and acrosome vesicle could be limiting factors in sheep intracytoplasmic sperm injection (ICSI).
Prior to in vitro fertilization (IVF) or ICSI, the oocytes were subjected to in vitro maturation (IVM) for 24 h. First, to evaluate the need of artificial activation for ovine ICSI, 226 oocytes were injected with intact spermatozoa (IS), from which 125 were activated by incubation in ionomycin and 101 were cultured without activation. Next, spermatozoa were mechanically (by piezo-electrical pulses) and/or chemically (by ionomycin/Triton X-100) treated to break membranes and acrosomes and were injected into oocytes, grouped as follows: (i) piezo-pulsed spermatozoa (PPS), (ii) PPS pre-treated with ionomycin (PPS-I), (iii) PPS pre-treated with Triton X-100 (PPS-T), and (iv) intact and untreated spermatozoa as a control (CTR-IS).
No differences were observed in the zygote/cleavage/blastocyst rate between chemically activated and non-activated oocytes (50 vs. 45 %, 11.6 vs. 10.1 %; 1.8 vs. 1.1 %, respectively), after ICSI with CTR-IS. Injection of PPS compared to CTR-IS increased the proportion of zygotes and blastocysts (84.6 vs. 45 %, p < 0.01; 15.5 vs. 1.1 %, p < 0.0001, respectively). Moreover, the percentage of PPS-derived blastocysts was not significantly different from that obtained by conventional IVF (15.5 vs. 20.2 %). The ICSI blastocysts' development was also improved with PPS pre-treated with ionomycin (15.6 %), but was completely impeded with PPS pre-treated with Triton X-100 (0 %).
Our findings confirm that ICSI with spermatozoa whose plasma membrane and acrosome have been mechanically damaged substantially improves embryonic development until the blastocyst stage.
本研究旨在确定精子质膜和顶体囊泡的完整性是否可能是绵羊卵胞浆内单精子注射（ICSI）的限制因素。
在体外受精（IVF）或ICSI之前，将卵母细胞进行24小时的体外成熟（IVM）。首先，为了评估绵羊ICSI是否需要人工激活，向226个卵母细胞注射完整精子（IS），其中125个通过在离子霉素中孵育进行激活，101个未进行激活培养。接下来，对精子进行机械（通过压电脉冲）和/或化学（通过离子霉素/曲拉通X-100）处理以破坏膜和顶体，然后注射到卵母细胞中，分组如下：（i）压电脉冲精子（PPS），（ii）用离子霉素预处理的PPS（PPS-I），（iii）用曲拉通X-100预处理的PPS（PPS-T），以及（iv）完整且未处理的精子作为对照（CTR-IS）。
在使用CTR-IS进行ICSI后，化学激活和未激活的卵母细胞在受精卵/分裂/囊胚率方面未观察到差异（分别为50%对45%，11.6%对10.1%；1.8%对1.1%）。与CTR-IS相比，注射PPS增加了受精卵和囊胚的比例（分别为84.6%对45%，p<0.01；15.5%对1.1%，p<0.0001）。此外，PPS来源的囊胚百分比与传统IVF获得的百分比无显著差异（15.5%对20.2%）。用离子霉素预处理的PPS也改善了ICSI囊胚的发育（15.6%），但用曲拉通X-100预处理的PPS则完全阻碍了发育（0%）。
我们的研究结果证实，使用质膜和顶体已受到机械损伤的精子进行ICSI可显著改善胚胎发育直至囊胚阶段。
