Canel Natalia Gabriela, Suvá Mariana, Bevacqua Romina Jimena, Arias María Elena, Felmer Ricardo, Salamone Daniel Felipe
Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires/INPA-CONICET, Av. San Martín 4453, P.C. 1417, Buenos Aires, Argentina.
Laboratorio de Reproducción, Centro de Biotecnología de la Reproducción (CEBIOR-BIOREN), Universidad de La Frontera, Montevideo 0870, Temuco, Chile.
Theriogenology. 2018 Sep 1;117:26-33. doi: 10.1016/j.theriogenology.2018.05.017. Epub 2018 May 19.
In contrast to other species, intracytoplasmic sperm injection (ICSI) in bovine remains inefficient, resulting in low embryo developmental rates. It is unclear whether such inefficiency is due to the poor response of bovine ooplasms to the injection stimulus, or to the inability of bull sperm to induce oocyte activation. In order to facilitate these events, two strategies were assessed: the use of high concentration of cysteamine [Cys] during IVM; and the selection of sperm attached to cumulus cells after incubation with COCs for ICSI. First, COCs were IVM with increasing [Cys] and subjected to IVF. Zygotes from all groups were cultured under different O tensions and development to blastocyst was evaluated. In a second experiment, sperm were co-cultured for 3 h with COCs and acrosome reaction was studied. Afterwards, the best IVM and IVC conditions determined on Experiment 1 were used for ICSI assay. COCs were matured for 21 h with 1 (Cys 1) or 0.1 mM Cys (Cys 0.1 groups, standard condition). In addition, COCs were incubated for ≥3 h with 16 × 10 sperm/ml and only sperm attached to cumulus cells were selected for ICSI (ICSI + Co-cult groups). After chemical activation, embryos were cultured in SOF medium under low O tension. Cleavage and blastocyst rates were evaluated at days 2 and 7 of IVC, respectively. Finally, the relative expression of eight genes indicators of embryo quality was compared between ICSI and IVF control blastocysts by qPCR. Cleavage rates were higher for Cys 0.1 ICSI + Co-cult and Cys 1 ICSI + Co-cult groups (n = 117, 92% and n = 116, 79%, respectively) compared to their controls (n = 132, 60% for Cys 0.1 ICSI and n = 108, 52% for Cys 1 ICSI) (p ≤ 0.05). Interestingly, the combined treatment (Cys 1 ICSI + Co-cult) showed higher blastocyst rates than all other ICSI groups (23 vs. 11, 18 and 14% for Cys 0.1 ICSI + Co-cult, Cys 1 ICSI, and Cys 0.1 ICSI, respectively) (p ≤ 0.05). Moreover, incubation with COCs increased the rates of live acrosome reacted sperm (p ≤ 0.05). The relative abundance of mRNAs coding for INFτ, CAT, DNMT1, OCT4, and HDAC3 did not differ between treatments (p ≤ 0.05). SOD2, HADC1 and HADC2 expression was higher for Cys 0.1 ICSI than for IVF embryos (p ≤ 0.05). Group Cys 1 ICSI did not differ from IVF for those three genes, neither did Cys 1 ICSI + Co-cult, except for HDAC1 (p ≤ 0.05). In conclusion, the use of 1 mM Cys during IVM and of sperm incubated with mature COCs might be a good strategy to improve ICSI outcomes in cattle.
与其他物种不同，牛的胞浆内精子注射（ICSI）效率仍然较低，导致胚胎发育率低下。目前尚不清楚这种低效率是由于牛卵质对注射刺激反应不佳，还是由于公牛精子无法诱导卵母细胞激活。为了促进这些过程，评估了两种策略：在体外成熟（IVM）期间使用高浓度的半胱胺[Cys]；以及选择与卵丘细胞附着的精子用于ICSI，该精子是在与卵丘-卵母细胞复合体（COCs）孵育后获得的。首先，将COCs在不断增加[Cys]浓度的条件下进行IVM，然后进行体外受精（IVF）。将所有组的受精卵在不同的氧气张力下培养，并评估其发育至囊胚的情况。在第二个实验中，将精子与COCs共培养3小时，并研究顶体反应。之后，将在实验1中确定的最佳IVM和体外培养（IVC）条件用于ICSI试验。将COCs在1 mM（Cys 1组）或0.1 mM Cys（Cys 0.1组，标准条件）下成熟21小时。此外，将COCs与16×10个精子/ml孵育≥3小时，仅选择附着在卵丘细胞上的精子用于ICSI（ICSI + 共培养组）。化学激活后，将胚胎在低氧张力的输卵管液（SOF）培养基中培养。分别在IVC的第2天和第7天评估卵裂率和囊胚率。最后，通过定量聚合酶链反应（qPCR）比较ICSI和IVF对照囊胚之间八个胚胎质量基因指标的相对表达。与各自的对照组相比（Cys 0.1 ICSI组n = 132，卵裂率60%；Cys 1 ICSI组n = 108，卵裂率52%），Cys 0.1 ICSI + 共培养组和Cys 1 ICSI + 共培养组的卵裂率更高（n = 117，分别为92%和n = 116，79%）（p≤0.05）。有趣的是，联合处理（Cys 1 ICSI + 共培养）显示出比所有其他ICSI组更高的囊胚率（Cys 0.1 ICSI + 共培养组、Cys 1 ICSI组和Cys 0.1 ICSI组的囊胚率分别为23%、11%、18%和14%）（p≤0.05）。此外，与COCs孵育增加了活的发生顶体反应精子的比例（p≤0.05）。编码干扰素τ（INFτ）、过氧化氢酶（CAT）、DNA甲基转移酶1（DNMT1）、八聚体结合转录因子4（OCT4）和组蛋白去乙酰化酶3（HDAC3）的mRNA相对丰度在各处理之间没有差异（p≤0.05）。Cys 0.1 ICSI组超氧化物歧化酶2（SOD2）、组蛋白去乙酰化酶1（HADC1）和组蛋白去乙酰化酶2（HADC2）的表达高于IVF胚胎（p≤0.05）。对于这三个基因，Cys 1 ICSI组与IVF组没有差异，Cys 1 ICSI + 共培养组也没有差异，但组蛋白去乙酰化酶1（HDAC1）除外（p≤0.05）。总之，在IVM期间使用1 mM Cys以及使用与成熟COCs孵育后的精子可能是提高牛ICSI成功率的良好策略。
