Activation with Ethanol Improves Embryo Development of ICSI-Derived Oocytes by Regulation of Kinetics of MPF Activity

Abstract

Abstract. Developmental potential of bovine embryos that are not artificially activated after intracytoplasmic sperm injection (ICSI) is generally very low. In this study, we investigated effects of artificial activation with ethanol on kinetics of maturation promoting factor (MPF) activity (p34 cdc2 kinase activity) and development of bovine oocytes following ICSI. Treatment of oocytes with ethanol at 4 h after ICSI improved their first cleavage and further preimplantation development (51% vs. 13%, 14% vs. 4%: treatment with vs. without ethanol, respectively). MPF activity of oocytes was lowered until at least 2 h after ICSI. In oocytes without activation after ICSI, MPF activity temporarily elevated at 6 h after ICSI, whereas this phenomena was not observed in the oocytes treated with ethanol. Furthermore, MPF activity was elevated 20 h after ICSI in oocytes activated with ethanol, whereas this elevation of MPF activity was not shown in oocytes without activation. These results indicate that the stimulus of sperm was sufficient to lower MPF activity of oocytes following ICSI, and moreover the activation treatment of bovine oocytes with ethanol after ICSI served to maintain the low levels of MPF activity until the next cell cycle started. Pronuclear formation in sperm-injected bovine oocytes was reported by Westhusin et al. [11]. Since that report, many investigators have tried to improve the efficiency of production of bovine ICSI e m b r y o s .H o w e v e r , t h e c l e a v a g e a n d developmental rate of ICSI-derived embryos to the blastocyst stage remain very low. Artificial activation of bovine oocytes after ICSI has been required for embryos to progress beyond the p r o n u c l e a r s t a g e a n d c o n t i n u e f u r t h e r development. While mechanical stimulus by an injection pipette can provide an activating stimulus for matured oocytes in some species, fewer than 5 % of bovine oocytes have been activated by shaminjection [12,13]. Parthenogenetic activation of bovine oocytes can be induced by various stimuli, such as exposure to ethanol [14,15], calcium

A combined treatment of ionomycin with ethanol improves blastocyst development of bovine oocytes harvested from stored ovaries and microinjected with spermatozoa

“…This blastocyst yield is comparable to or even higher than those in many reports where bovine oocytes harvested from freshly collected ovaries were used for ICSI [8,10,11,[13][14][15][16]. Simultaneously with the ICSI experiments in the present study, high blastocyst yield (40 ± 3%, 90/223) was obtained after routine 225 IVF protocol, suggesting that oocytes harvested from stored ovaries carry sufficient in vitro developmental potential after IVF and ICSI if they were subjected to appropriate activation regimen.…”

“…The blastocyst yield in non-treated group (12%) is higher than those (0-8%) reported previously by several laboratories [8,10,11,[14][15][16], but comparable to or even lower than those (15-23%) in other reports [12,13]. However, blastocysts/ 235 cleavage rate in our study (30%) is comparable to that reported by Wei and Fukui [12] (32%) which indicates that a failure in the early post-fertilization events -from fertilization until cleavage-is the main determiner to the successful development after bovine ICSI.…”

“…The ICSI procedure has resulted in birth of live offspring in many species [5]. Since the first attempt of bovine ICSI [6], the developmental potential of the ICSI 45 oocytes into blastocysts in vitro or live calves in vivo still low, unstable and far from satisfactory regardless of the numerous efforts [7][8][9][10][11][12][13][14][15][16]. Beside the technical difficulties caused by darkness of the ooplasm, large size of the sperm heads, and elasticity and toughness of the oolemma, the inability of the mechanical stimulation and/or the injected spermatozoon itself to induce proper oocyte activation after ICSI [17] may be responsible for this low blastocyst yields.…”

“…It has been reported that the pattern of calcium oscillations in bovine ICSI oocytes was abnormal [18] and the inactivation of the maturation promoting factors (MPF) occurred temporary [15]. This led to a hypothesis that the release and/or the activation of the sperm-borne oocyte activating factor (SOAF) are compromised after ICSI.…”

“…This hypothesis is supported by the low blastocyst yields after ICSI without any exogenous activation stimuli [8,10,11,[13][14][15][16]. To improve 55 the blastocyst yields after bovine ICSI, additional exogenous activation stimuli that induce intracellular calcium spike such as direct current [19], calcium ionophore [20], ionomycin [8,13,14,16,17] or ethanol [9][10][11]15,16] have been applied. However, the monotonic calcium increase triggered by these stimuli [21,22] was insufficient to completely inactivate the MPF due to re-accumulation of the cyclin B [23] and brought the oocytes to arrest again at the M-III stage 60 [17,23,24].…”