Parthenogenetic Activation of Pig Oocytes with Calcium Ionophore and the Block to Sperm Penetration after Activation1

Abstract

Calcium ionophore A23187 can parthenogenetically activate oocytes in many animals. The present study was designed to analyze functionally the mechanism of A23187 activation of pig oocytes matured in vitro. In experiment 1, effects of the concentration of A23187 on intracellular calcium transients, cortical granule (CG) exocytosis, nuclear activation, and zona reaction, which was determined by zona hardening and sperm penetrability, were examined. Cumulus-free oocytes were exposed to 0-100 microM A23187 for 5 min. It was found that the amplitude of the intracellular calcium transients, percentage of CG exocytosis, and percentage of pronuclear formation were increased in a concentration-dependent manner. The time for dissolution of zona pellucida (ZP) was increased in the oocytes treated with 25-100 microM A23187. Penetration of ZP-intact oocytes by spermatozoa was decreased and only 3-4% of oocytes were penetrated by spermatozoa after 50-100 microM A23187 treatment. In experiment 2, oocytes were treated with 100 microM A23187 for 5 min and then cultured for 10 min or 3.5 h before insemination. No difference in penetration rates was observed between the two groups of oocytes (12.0% vs. 12.2%), but the penetration rates were significantly lower than those in controls (85.2% vs. 82.4%). In experiment 3, treatment of oocytes with 100 microM A23187 for 5 min was followed by removal of the ZP from a portion of the oocytes. ZP-intact and ZP-free oocytes were then inseminated for examination of sperm penetration. One of 65 (2%) oocytes with ZP and 48 of 52 (92%) oocytes without ZP were penetrated by spermatozoa. These results indicate that activation of pig oocytes by A23187 is the result of A23187-induced intracellular calcium increase and that A23187-induced cortical reaction can prevent sperm penetration of ZP-intact oocytes, but not ZP-free oocytes.

Calcium Release at Fertilization: Artificially Mimicking the Oocyte's Response to Sperm.

“…Intracellular-free calcium determinations using fura-2 or fluo-3 have d e m o n s t r a t e d t h a t i n c u b a t i o n o f m a t u r e mammalian oocytes with A23187 or ionomycin generates a single calcium transient increase [3,11,12,24,[139][140][141][142]. Mouse [140] and cattle [139] oocytes treated with 10 µM A23187 experience an intracellular calcium rise that is of greater amplitude and of longer duration than the first rise induced at fertilization.…”

“…Cortical granule exocytosis [126,127,[141][142][143][144] and zona pellucida modification, as assessed by the dissolution time of zonae in pronase and sperm penetrability [126,141], have been observed in mammalian oocytes following treatment with A23187. In bovine oocytes, treatment with A23187 alone induces a decrease in the levels of MPF activity within 30 min [118].…”

Effect of Oocyte Aging on Parthenogenetic Activation with Cycloheximide and A Iteration of the Activity of Maturation Promoting Factor during Aging of Bovine Oocytes.

“…Taken together, these results suggest that the agedoocytes are more sensitive to various activation stimuli than newly matured oocytes in all mammalian species. It is well known that when matured oocytes are treated with activation stimuli such as ethanol [21][22][23], Ca 2+ ionophore A23187 [24,25], IP 3 [26,27], thimerosal [26,28], strontium [24,29], and electric pulses [30,31], transient intracellular calcium [Ca 2+ ] i increases are triggered. Additionally, it is postulated that the sensitivity of the oocyte's activation mechanism to calcium increases with culture time following maturation.…”

Synchronization of In Vitro Maturation in Porcine Oocytes

“…The ability of IVM/IVF oocytes to develop to blastocyst stage was first confirmed by Mattioli et al (1989). Further, piglets were born after the transfer of IVM/IVF embryos that were cultured to the 2-4 cell stage Funahashi et al, 1996) or to morula stage (Day et al, 1998). However the first successful transfers of IVP embryos at blastocyst stage were reported only recently (Marchal et al, 2001;Kikuchi et al, 2002).…”

“…(which we used previously) regarding the cumulus expansion (Abeydeera et al, 1998;Abeydeera et al, 2000). Maturation culture was performed in Medium199 (prepared with 20mM HEPES; Gibco)…”