Activation regimens to prepare bovine oocytes for intracytoplasmic sperm injection

Abstract

Activation of bovine oocytes to produce a single haploid pronucleus in preparation for intracytoplasmic sperm injection (ICSI) has been investigated with various combinations of ionomycin and 6‐dimethylaminopurine (DMAP). Effects were evaluated by immunocytochemical staining, chromosomal analysis and assessment of development in vitro. Oocytes matured in vitro were exposed to: ionomycin alone (single or repeated treatments, Groups 1 and 2 respectively), ionomycin followed by DMAP (immediately or after a 3‐h delay, Groups 3 and 4), or no treatment (control, Group 5). They were then co‐cultured in M199 with bovine oviductal epithelial cells. Activation rates were not significantly different among groups but significantly fewer oocytes in Group 3 extruded a second polar body than in Groups 1, 2, and 4. Most parthenotes (60% to 80%) in Groups 1, 2, and 4 were haploid, whereas 82% in Group 3 were mixoploid or polyploid. Most of the parthenotes (88%) in Group 4 formed a single pronucleus besides extruding the second polar body and were therefore more suitable for ICSI than those of Groups 1 and 2 in which condensed chromosomes predominated. The respective rates of oocyte cleavage in Groups 1 to 4 were 24%, 36%, 70%, and 75%; corresponding blastocyst rates were 1%, 5%, 17%, and 8%. There were significantly fewer cells in the parthenotes of Groups 1, 2, and 4 than of Group 3, or of embryos produced by in vitro fertilization. Thus, delaying the addition of DMAP after ionomycin decreases chromosomal abnormalities and produces a high proportion of activated oocytes suitable for ICSI. Mol. Reprod. Dev. 50:485–492, 1998. © 1998 Wiley‐Liss, Inc.

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

“…Chemicals such as ethanol, ionomycin or calcium ionophore combined with or without CHX were not sufficient to induce parthenogenetic development in bovine oocytes [42,43]. Induction of diploidization by combining the calcium oscillation-inducing activators with cytochalasin or 6-DMAP was necessary to improve the 285 parthenogenetic development of bovine oocytes [24,44,45]. This may explain the complete failure or very low parthenogenetic developmental rate after different activation protocols in the present study.…”

“…Ionomycin + CHX [13]; Injected oocytes were first treated with ionomycin immediately after ICSI as described above, and followed by incubation in culture medium containing 10 µg/mL CHX for 5 h. (6) Ionomycin + 6-DMAP [24]; Injected oocytes were first treated with ionomycin immediately after ICSI as described above, and incubated in the TCM-199/FBS medium for 3 h. Next, the 150 oocytes were incubated in culture medium containing 1.9 mM 6-DMAP for an additional 3 h. The presence of the second polar body was recorded immediately before transferring the oocytes into culture medium (4 h after ICSI), except for the oocytes treated with ionomycin + CHX (5 h after ICSI when the oocytes were released from the CHX treatment) and ionomycin + 6-DMAP (3 h after ICSI when the oocytes were exposed to the 6-DMAP-containing medium). 155…”

“…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]. To avoid this phenomenon, these triggers have been accompanied with other chemicals that can directly or indirectly inactivate the MPF without changing the intracellular calcium profiles.…”

Parthenogenetic Development and Ploidy following Various Chemical Activation Regiments of Bovine Oocytes

“…During normal fertilization in mammalian species, sperm penetration into the oocyte triggers intracellular Ca 2+ oscillation followed by inactivation of the maturation promoting factor (MPF) composed of the catalytic subunit of cdc2p34 protein kinase and the regulatory subunit cyclin B1/B2 for about 2 hr [37]. Although artificial stimuli, such as Ca 2+ ionophore [19], ionomycin [23], ethanol [31], electric pulse [27] and strontium (SrCl 2 ) [21] can activate bovine oocytes by increasing intracellular Ca 2+ , the efficiency of artificial stimuli is limited [25,34]. Therefore, additional chemical agents, such as cycloheximide (CHX, protein phosphorylation inhibitor) [24] and 6-dimethylaminopurine (DMAP, protein synthesis inhibitor) [29,30], that prevent MPF re-accumulation by reducing cdc2 kinase activity [2] have also been applied.…”

The ability of freeze-dried bull spermatozoa to induce calcium oscillations and resumption of meiosis

“…Moreover, the adverse effect of freeze-drying was notable in the higher proportions of partially activated bovine oocytes after ICSI with FD versus control bull spermatozoa (Table 2). Such a re-arresting in the condensation stage has been reported to occur more frequently in bovine ICSI oocytes after weaker activation, single inonmycin treatment, compared to stronger activation as multiple ionomycin treatments or ionomycin plus 6-DMAP treatment [49,50]. Not only long-lasting but also highly frequent calcium oscillations may be required for the full activation of bovine ICSI oocytes.…”