The optimal period of Ca-EDTA treatment for parthenogenetic activation of porcine oocytes during maturation culture

Abstract

The changes triggered by sperm-induced activation of oocytes, which are required for normal oocyte development, can be mediated by other agents, thereby inducing the parthenogenesis. In this study, we exposed porcine oocytes to 1 mM Ca-EDTA, a metal-ion chelator, at various intervals during 48 hr of in vitro maturation to determine the optimum period of Ca-EDTA treatment for parthenogenetic activation. When the oocytes were cultured with or without Ca-EDTA from 36 hr (post-12), 24 hr (post-24), 12 hr (post-36) and 0 hr (post-48) after the start of maturation culture, the blastocyst formation rates were significantly higher (P<0.05) in the post-24, post-36 and post-48 groups (3.3%, 4.0% and 2.6%, respectively) than those in the control group without treatment (0%). Furthermore, when the oocytes were cultured with Ca-EDTA for 0 hr (control), 12 hr (pre-12), 24 hr (pre-24), 36 hr (pre-36) and 48 hr (pre-48) from the start of maturation culture, the oocytes formed blastocysts only in the pre-36 and pre-48 groups (0.4% or 0.8%, respectively). Pronuclei (<66.7%) were observed only when the periods of Ca-EDTA treatment were more than 12 hr during maturation culture. In the control group, no pronuclei were detected. Our findings demonstrate that porcine immature oocytes can be parthenogenetically activated by Ca-EDTA treatment for at least 24 hr to 36 hr during maturation culture, leading to pronucleus formation followed by the formation of blastocysts.

Characterization of porcine oocytes stained with Lissamine Green B and their developmental potential in vitro

“…Nowadays, parthenogenetic activation is a modern tool, in which embryogenesis begins without previous sperm fertilization, which means that this method eliminates the influence of sperm and embryogenesis depends exclusively on the oocyte quality. The parthenogenetic activation of the oocyte can be initiated by various physical, mechanical, and chemical stimuli such as ethanol, ionomycin, ionophore, or direct electrical impulses (Alberio et al, 2001;Lane et al, 2002).…”

“…Electrochemical activation is based on pore formation in the oocyte membrane, which can increase levels of intracellular Ca 2+ due to the influx of extracellular Ca 2+ from the activation medium. Chemical activation is based on a combination of ionomycin and 6-DMAP (Alberio et al, 2001;Bing et al, 2001;Prochazka et al, 2011). Ionomycin can be characterized as a selective ionophore, which can bind intracellular Ca 2+ and 6-DMAP as an inhibitor of maturation promoting factors (MPF) and mitogen-activated proteinkinase (MAPK) activity, which leads to the successful formation of parthenogenetic embryos (Machaty and Prather, 1998;Alberio et al, 2001;Bing et al, 2001;Yin et al, 2002;Prochazka et al, 2011).…”