Artificial oocyte activation is important for assisted reproductive technologies, such as fertilization with round spermatids (ROSI) or the production of cloned offspring by somatic cell nuclear transfer (SCNT). Recently, phospholipase Cζ (PLCζ)-cRNA was used to mimic the natural process of fertilization, but this method required the serial injection of PLCζ-cRNA and was found to cause damage to the manipulated oocytes. Here we tried to generate offspring derived from oocytes that were fertilized using round spermatid or somatic cell nuclear transfer with the co-injection of PLCζ-cRNA. After co-injecting round spermatids and 20 ng/µL of PLCζ-cRNA into the oocytes, most of them became activated, but the activation process was delayed by more than 1 h. With the co-injection method, the rate of blastocyst formation in ROSI embryos was higher (64%) compared with that of the serial injection method (55%). On another note, when SCNT was performed using the co-injection method, the cloned offspring were obtained with a higher success rate compared with the serial-injection method. However, in either ROSI or SCNT embryos, the birth rate of offspring via the co-injection method was similar to the Sr activation method. The epigenetic status of ROSI and SCNT zygotes that was examined showed no significant difference among all activation methods. The results indicated that although the PLCζ-cRNA co-injection method did not improve the production rate of offspring, this method simplified oocyte activation with less damage, and with accurate activation time in individual oocytes, it can be useful for the basic study of oocyte activation and development.

中文翻译：

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通过共注射 PLCζ-cRNA 从精细胞或体细胞产生后代

人工卵母细胞活化对于辅助生殖技术很重要，例如用圆形精子细胞 (ROSI) 受精或通过体细胞核移植 (SCNT) 生产克隆后代。最近，磷脂酶Cζ（PLCζ）-cRNA被用来模拟受精的自然过程，但这种方法需要连续注射PLCζ-cRNA，并被发现会对受精卵造成损害。在这里，我们尝试使用圆形精子细胞或体细胞核移植与 PLCζ-cRNA 的共同注射来产生源自卵母细胞的后代。将圆形精子细胞和 20 ng/µL PLCζ-cRNA 共同注入卵母细胞后，大部分被激活，但激活过程延迟了 1 小时以上。采用共注射法，与连续注射法（55%）相比，ROSI 胚胎的囊胚形成率更高（64%）。另一方面，当使用共注射方法进行 SCNT 时，与连续注射方法相比，克隆后代的成功率更高。然而，在 ROSI 或 SCNT 胚胎中，通过共注射方法的后代出生率与 Sr 激活方法相似。检查的 ROSI 和 SCNT 受精卵的表观遗传状态在所有激活方法中均无显着差异。结果表明，虽然PLCζ-cRNA共注射法没有提高子代产率，但该方法简化了卵母细胞的活化，损伤较小，单个卵母细胞活化时间准确，