Ressaissi Yosra, Anzalone Debora Agata, Palazzese Luca, Czernik Marta, Loi Pasqualino
Laboratory of Embryology, Faculty of Veterinary Medicine, University of Teramo, Via R. Balzarini, Teramo, 1 - 64100, Italy.
Laboratory of Embryology, Faculty of Veterinary Medicine, University of Teramo, Via R. Balzarini, Teramo, 1 - 64100, Italy.
Theriogenology. 2021 Jan 1;159:7-12. doi: 10.1016/j.theriogenology.2020.10.008. Epub 2020 Oct 10.
While intracytoplasmic sperm injection (ICSI) is an asset in human Assisted Reproduction Technologies (ART), its outcomes, in terms of blastocyst, is still unacceptably low in ruminants. The picture typically found in ICSI derived bovine and ovine embryos is an asymmetry between a high activation rate, marked by a pronuclear development, and a low first cleavage rate. Abnormal centriole function has been indicated as a possible factor which undermines embryonic development following ICSI, especially when Freeze Dried spermatozoa (FD) are used. In order to verify the hypothesis that centriole dysfunction might be responsible for low ICSI outcomes in sheep, we have investigated micro-tubular dynamics, markedly aster nucleation, in fertilized sheep zygotes by ICSI with frozen/thawed (FT) and FD spermatozoa; In Vitro Fertilized (IVF) sheep oocytes were used as control. The spermatozoa aster nucleation was assessed at different time points following ICSI and IVF by immune-detection of α-tubulin. Pronuclear stage, syngamy and embryo development were assessed. No difference was noticed in the timing of aster nucleation and microtubule elongation in ICSI-FT derived embryos with control IVF ones, while a delay was recorded in ICSI-FD ones. The proportion of 2-pronuclear stage zygotes was similar in ICSI-FT and ICSI-FD (47% and 53%, respectively), both much lower comparing the IVF ones (73%). Likewise, syngamy was observed in a minority of both ICSI groups (28.5% vs 12.5% in ICSI-FT/FD respectively) comparing to IVF controls (50%), with a high number of zygotes blocked at the 2-pronuclear stage (71.5% vs 87.5% respectively). While no significant differences were noticed in the cleavage rate between ICSI-FD, ICSI-FT and IVF groups (31%, 34% and 44%) respectively, development to blastocyst stage was markedly compromised in both ICSI groups, especially with FD spermatozoa (10% in ICIS-FD and 19% in ICSI-FT vs 33% in IVF (P < 0.005, ICSI-FD vs IVF and P < 0.05, IVF vs ICSI-FT, respectively). Hence, here we have demonstrated that the reduced cleavage, and the ensuing impaired development to blastocysts stage of ICSI derived sheep embryos is not related to centriole dysfunction, as suggested by other authors. The major recorded problem is the lack of syngamy in ICSI derived zygotes, an issue that should be addressed in further studies to improve ICSI procedure in sheep embryos.
虽然卵胞浆内单精子注射（ICSI）是人类辅助生殖技术（ART）中的一项有用技术，但其在反刍动物中产生囊胚的成功率仍然低得令人难以接受。在通过ICSI技术获得的牛和羊胚胎中，通常会出现一种情况：以原核发育为标志的高激活率与低首次卵裂率之间存在不对称性。有迹象表明，中心粒功能异常可能是导致ICSI后胚胎发育受损的一个因素，尤其是在使用冻干精子（FD）时。为了验证中心粒功能障碍可能是导致绵羊ICSI成功率低的原因这一假设，我们研究了通过ICSI技术，使用冷冻/解冻（FT）精子和FD精子使绵羊受精卵受精后的微管动力学，特别是星体形成；体外受精（IVF）的绵羊卵母细胞用作对照。通过α-微管蛋白的免疫检测，在ICSI和IVF后的不同时间点评估精子星体形成。评估原核期、配子融合和胚胎发育情况。ICSI-FT胚胎与对照IVF胚胎在星体形成时间和微管伸长方面没有差异，而ICSI-FD胚胎则出现延迟。ICSI-FT和ICSI-FD组中双原核期受精卵的比例相似（分别为47%和53%），与IVF组（73%）相比均低得多。同样，与IVF对照组（50%）相比，两个ICSI组中观察到配子融合的比例都较低（ICSI-FT/FD组分别为28.5%和12.5%），大量受精卵停滞在双原核期（分别为71.5%和87.5%）。虽然ICSI-FD、ICSI-FT和IVF组之间的卵裂率没有显著差异（分别为31%、34%和44%），但两个ICSI组发育到囊胚阶段的情况都明显受损，尤其是使用FD精子时（ICIS-FD组为10%，ICSI-FT组为19%，而IVF组为33%，P < 0.005，ICSI-FD与IVF相比；P < 0.05，IVF与ICSI-FT相比）。因此，我们在此证明，ICSI获得的绵羊胚胎卵裂减少以及随后发育到囊胚阶段受损与其他作者所认为的中心粒功能障碍无关。记录到的主要问题是ICSI获得的受精卵缺乏配子融合，这一问题应在进一步研究中加以解决，以改进绵羊胚胎的ICSI操作程序。
