Functional analysis of activation of porcine oocytes by spermatozoa, calcium ionophore, and electrical pulse.

Abstract

The present study was conducted to examine differences in the activation of pig oocytes induced by sperm penetration or the artificial stimulators calcium ionophore A23187 and electrical pulse. Cumulus-oocyte complexes were cultured in NCSU 23 medium supplemented with 10% pig follicular fluid and 0.57 mM cysteine for 44 hr and then freed from cumulus and corona cells prior to activation with A23187 or an electrical pulse or inseminated with frozen-thawed ejaculated semen. Cortical granule (CG) exocytosis, zona reaction, nuclear activation, and developmental ability were examined after treatment. A23187 and electrical pulse induced 75.7% and 76.9% of CGs to be released from oocytes. Sperm penetration induced 86.3% of CGs to be released from the oocytes, which was significantly higher (P < 0.05) than those induced by artificial stimulators. Activation induced by A23187 and sperm penetration resulted in a zona reaction, which prevented sperm penetration after insemination or reinsemination, respectively. Activation induced by electrical pulse, however, did not cause a zona block since the penetration rate (80%) of oocytes was not different (P > 0.05) from that in control oocytes (87%). Electrical pulse induced 87% of the oocytes to form a pronucleus(ei), with 53% failing to release the second polar body. A23187 induced 62% of oocytes to form a pronucleus(ei), and 81% of these oocytes released the second polar body. Sperm penetration induced 98-100% of the oocytes to release the second polar body and form a female pronucleus, and 88-89% of sperm penetrated oocytes formed a male pronucleus(ei). Blastocyst formation of oocytes exposed to spermatozoa, electrical pulse, and A23187 was 27%, 10%, and 4% at Day 6 and 28%, 11%, and 5% at Day 7, respectively (P < 0.05). More nuclei were observed in the blastocysts derived from in vitro fertilization (32.3 +/- 12.9) than artificial stimulators. These results indicate that different and possibly overlapping mechanisms may be involved in the activation of pig oocytes by spermatozoa, electrical pulse, and A23187.