273 REMOVAL OF ACROSOME FROM SPERM HEADS IMPROVES DEVELOPMENT OF RAT ZYGOTES THROUGH INTRACYTOPLASMIC SPERM INJECTION

Abstract

In intracytoplasmic sperm injection (ICSI), sperm chromatin is introduced into the oocyte together with acrosome, which does not enter cytoplasm of oocytes during normal fertilization. In mice, acrosome of sperm head has a detrimental effect on embryonic development of the ICSI oocytes (Morozumi et al. 2006 Proc. Natl. Acad. Sci. USA 103, 17 661–17 666). We examined the effect of acrosome removal of frozen/thawed (F/T) rat sperm on development to term of the ICSI oocytes in order to improve production efficiency of live offspring from F/T rat spermatozoa through ICSI. In experiment 1, epididymal spermatozoa of the Wistar rats were frozen as described previously (Seita et al. 2005 Reprod. Fertil. Dev. 18, 256 abst). The F/T spermatozoa were sonicated to separate sperm heads. The sperm heads were divided into 3 treatment groups; nontreated sperm head (control), sperm heads exposed in 0.02% of Triton X-100 solution for 1 min (TX), sperm heads exposed in 0.02% lysolectin solution for 1 min (LL). Acrosomal status of the sperm heads was then examined by FITC-peanut agglutinin stain. In experiment 2, sperm heads of the control, TX, and LL treatments were microinjected into denuded oocytes obtained from superovulated females (Hirabayashi et al. 2002 Transgenic Res. 11, 221–228). The ICSI oocytes were cultured and observed for the formation of pronuclei (2PN) for 6 h and blastocyst formation at 120 h of culture. In experiment 3, the ICSI oocytes cultured for 6 h were transferred to recipient females to examine development to term. Statistical analyses of the results were carried out by 1-way ANOVA. In experiment 1, the TX (76%) and LL (89%) treatments showed higher rates of acrosome removal than that of the control (24%) group (P < 0.05). In experiment 2, the percentage of 2PN formation at 6 h after ICSI was not significantly different among sperm-treated groups, although the TX (76%) and LL (70%) groups were higher than the control (24%) group at 4 h (P < 0.05). The percentages of blastocyst formation were not significantly different among sperm-treated groups (control: 10%; TX: 25%; and LL: 25%). In experiment 3, the efficiency of development to term of the TX treatment group (21%: 12/57) was significantly higher than the control (5%: 3/55; P < 0.05), although the LL treatment group (16%: 12/75) was not significantly different from the control and TX treatment groups. These results indicate that acrosome removal using TX of F/T rat sperm heads before ICSI improves development to term of the ICSI oocytes. This benefical effect of acrosome removal may be due to the quick release of sperm-activating factors from sperm heads after ICSI in the rat.