Horiuchi Toshitaka
Faculty of Life and Environmental Sciences, Department of Life Sciences, Prefectural University of Hiroshima, Hiroshima, Japan.
J Reprod Dev. 2006 Feb;52(1):13-21. doi: 10.1262/jrd.17084.
This paper describes several technical improvements and our results in hamster intracytoplasmic sperm injection (ICSI), hamster round spermatid injection (ROSI) and bovine ICSI. The hamster is the mammalian species in which ICSI was first tried to produce fertilized oocytes. However, until recently, no live offspring following ICSI have ever been obtained. We reported the birth of live offspring following hamster ICSI. Improved points to success were 1) performing hamster ICSI in a dark room with a small incandescent lamp and manipulating both oocytes and fertilized eggs under microscope with a red light source and 2) injecting sperm heads without acrosomes. Under controlled illumination, the majority of the oocytes injected with acrosomeless sperm heads were fertilized normally, cleaved, and developed into morulae. Nine live offspring (19%) were born by transfer of hamster ICSI-derived embryos. Furthermore, we reported the birth of live offspring following hamster ROSI. About 70% of oocytes injected with round spermatids broken before injection were fertilized normally and about half of them developed to morulae and blastocysts. Three (5%) live young were born by transfer of hamster ROSI-derived embryos. On the other hand, in cattle, the main improvements were 1) injection of spermatozoa immobilized by scoring their tail just before injection into oocytes, and 2) additional ethanol activation 4 h after ICSI. About 70% of oocytes injected were activated 4 h after ICSI, and about 30% of them developed to blastocysts. Twenty-four live calves (39%) were born by non-surgical transfer of ICSI-derived embryos. Those results shows that, at present, live offspring are able to be obtained following hamster ICSI, ROSI and bovine ICSI, but further improvement is required due to higher production efficiency of offspring.
本文描述了仓鼠卵胞浆内单精子注射（ICSI）、仓鼠圆形精子细胞注射（ROSI）和牛ICSI的若干技术改进及我们取得的结果。仓鼠是首个尝试通过ICSI产生受精卵母细胞的哺乳动物物种。然而，直到最近，ICSI后从未获得过存活后代。我们报道了仓鼠ICSI后存活后代的诞生。成功的改进要点包括：1）在暗室中使用小白炽灯进行仓鼠ICSI，并在显微镜下用红色光源操作卵母细胞和受精卵；2）注射无顶体的精子头部。在可控照明条件下，大多数注射了无顶体精子头部的卵母细胞正常受精、分裂并发育成桑椹胚。通过移植仓鼠ICSI衍生的胚胎，诞生了9只存活后代（19%）。此外，我们报道了仓鼠ROSI后存活后代的诞生。约70%在注射前破裂的圆形精子细胞注射的卵母细胞正常受精，其中约一半发育成桑椹胚和囊胚。通过移植仓鼠ROSI衍生的胚胎，诞生了3只（5%）存活幼崽。另一方面，在牛中，主要改进包括：1）在即将注射前通过划破精子尾部使其固定后注射到卵母细胞中；2）ICSI后4小时额外进行乙醇激活。约70%注射的卵母细胞在ICSI后4小时被激活，其中约30%发育成囊胚。通过非手术移植ICSI衍生的胚胎，诞生了24头存活小牛（39%）。这些结果表明，目前，仓鼠ICSI、ROSI和牛ICSI后能够获得存活后代，但由于后代生产效率更高，仍需要进一步改进。
