Abstract:
Parthenogenetic activation of mammalian oocytes can be induced by several stimuli in vitro. In the mouse, ethanol and Sr2+ are commonly used artificial activating agents. The aim of this study was to investigate both the effects of these two activating agents and the genetic background of the oocyte on meiotic cell cycle progression following activation. Metaphase-II (MII) oocytes were collected from superovulated (5 IU eCG/5 IU hCG) 8-10-week-old female mice of hybrid B6D2F1, inbred C57BL/6, outbred CF-1 and nude (NU/+) strains at 16�h post-hCG. Oocytes were activated either by a 5-min exposure to 7% ethanol (E), followed by culture in KSOM, or by continuous culture in Ca2+ -free KSOM containing 10�mM SrCl2 (S). Starting 5�min after the activation stimulus, oocytes were fixed at 5-min intervals, until 20�min post-activation (pa), and then at 30-min intervals, until 6�h�pa. Fixed oocytes were processed for immunofluorescence analysis of chromatin (Hoechst), spindle (�/_-tubulin) and microfilaments (phalloidin) organization. The initial time course of activation (5-20�min pa), assessed by exit from MII arrest, was faster in E- than in S-activated oocytes, except in the C57BL/6 strain in which anaphase (AII) onset after ethanol exposure was significantly delayed in comparison to the other three strains. Progression from AII into telophase (TII), at 50 min pa, was faster in CF-1 and C57BL/6 E-activated oocytes (78.3-87.1%) and in B6D2F1 and CF-1 S-activated oocytes (93.1-94.2%) when compared to the other strains (60-68.8% and 41-62.5%, respectively). NU/+ S-activated oocytes showed the lowest activation rates (88.5% v. 100% at 6�h pa), and TII entry was also delayed both in comparison to the other strains (41% vs. 62.5-94.2% at 50�min pa) and in comparison to E-activated NU/+ oocytes (68.8%). Rotation of the meiotic spindle began at 15�min pa in both E- and S-activated C57BL/6 oocytes, at 20�min pa in B6D2F1 and CF-1, and at 50�min pa in NU/+ oocytes and, in general, spindle rotation was completed more rapidly following E activation. Second polar body (PB2) extrusion was also delayed in NU/+ oocytes (starting at 1�h 20�min pa) when compared to all other strains (starting at 50�min pa) irrespective of the activating agent used. In E-activated oocytes, TII exit commenced earlier in C57BL/6 oocytes (1�h 20�min pa) than in the other three strains (1�h 50�min-2�h 20�min pa), but a significant proportion of CF-1 and NU/+ oocytes (37-49% at 5�h 50�min pa) remained arrested at metaphase III. S-activation not only accelerated TII exit but also increased the rate of pronuclear formation in both CF-1 and NU/+ oocytes to 100%. In conclusion, the results demonstrate that both the activating stimulus and the genetic background of the oocyte have significant effects on key events of meiotic cell cycle progression following activation. Supported by USDA NRI 2001-35205-09966 and Charles River Labs.