313 EFFECTS OF ETHANOL TREATMENT AFTER INTRACYTOPLASMIC SPERM INJECTION (ICSI) ON SPERM AFTER FORMATION AND THE MICROTUBULE ORGANIZATION OF BOVINE OOCYTES

Abstract

The cleavage rate of bovine embryos is very low without activation of oocytes after intracytoplasmic sperm injection (ICSI), although both male and female pronuclei are formed. We previously reported that the stimulus due to the injected sperm alone was sufficient to lower the MPF activity of bovine oocytes after ICSI, and the activation treatment of oocytes with ethanol at 4 h after ICSI served to maintain the low levels of MPF activity until the next cell cycle started (Fujinami et al. 2004 J. Reprod. Dev. 50, 171–178). These results suggested that activation treatment is necessary to improve the embryonic development after bovine ICSI. In bovine fertilization, the sperm introduces the centrosome into the oocyte. The centrosome acts as the microtubule-organizing center and microtubules are organized within the oocyte. It is reported that the sperm aster is important for the normal fertilization process. Therefore, failure of sperm aster formation possibly causes the failure of cleavage following fertilization. To investigate the reason of the low cleavage rate after bovine ICSI without artificial activation treatment, we examined sperm aster formation and the microtubule organization in bovine oocytes with or without activation treatment after ICSI. Bull spermatozoa immobilized by piezopulse was injected into bovine oocytes matured in vitro. At 4 h after ICSI, oocytes were treated with 7% ethanol in TCM199 for 5 min for activation. Oocytes were fixed at 6 and 12 h after ICSI, and the microtubule organization was examined by using specific antibodies and immunofluorescence microscopy. The cleavage rate (51% vs. 15%) and the developmental rate to the blastocyst stage (13% vs. 3%) were increased by ethanol treatment after ICSI (with or without ethanol treatment, respectively, P < 0.05). In oocytes activated with ethanol after ICSI, both the sperm aster formation rate at 6 h and the microtubule organization rate at 12 h after ICSI were significantly higher than in oocytes without activation treatment (58%, 80% vs. 12%, 26%, P < 0.05). It was reported that the sperm aster has an important role for the pronuclear movement to make the male and female pronuclei come into close apposition. From these results, it was concluded that oocyte activation after bovine ICSI promoted sperm aster formation and microtubule organization, and was effective to improve embryonic development.

This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan MEXT, and by a grant from the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence of the JST.