Artificial activation of porcine oocytes matured in vitro

Abstract

These studies were undertaken to understand the biological basis of artificially induced activation of meiotic metaphase II oocytes and to develop a source of oocytes as recipients for cloning by nuclear transfer. In vitro matured porcine oocytes were pulsed with various voltages of electricity and evaluated for pronuclear formation. The percentage of eggs that activated was significantly greater for the higher voltages. The effect on activation of the temperature of the ovaries returning from the abattoir was evaluated and it was found that oocytes derived from ovaries returning at 29 degrees C activated at lower rates (45.5%) than those returning at 36 degrees C (78.9%). An experiment was designed to evaluate the pH of electroporation medium (EM) and the duration of exposure to EM on activation. Oocytes were placed in EM at various pHs for 5 minutes, pulsed, and immediately removed to TL-Hepes or allowed an additional 2 minutes in EM prior to rinsing in TL-Hepes. The results indicate an optimum activation rate at a pH of 7.0 and allowing the additional 2 minutes in EM. Additional glucosamine (5 mM) had no affect on development of the oocyte to metaphase but reduced the percent pronuclear formation from 61% and 47%. A final experiment evaluated the developmental competence of oocytes subjected to a optimum combination of the above treatments and illustrated that a significant portion of the activated oocytes can show limited signs of cleavage. Thus in vitro matured pig oocytes can be induced to activate at high rates.

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

“…Application of one or more electric pulses has been s h o w n t o i n d u c e m e i o t i c r e s u m p t i o n a n d pronuclear formation in mouse [123,129], rabbit [8,124], sheep [130], cattle [6,131], pig [125,126,[132][133][134] and marmoset [135] oocytes. Furthermore, electrically activated rabbit [8], cattle [6] and pig [126,132,133,[136][137][138] oocytes undergo cleavage and parthenogenetic development to the blastocyst stage.…”

DNA methylation reprogramming and DNA repair in the mouse zygote

“…This ability allows different manipulations with the zygote such as the creation of parthenogenetic (monoparental) embryos, and nuclear/pronuclear transfer, including interspecies transfer (Chang et al, 2003;Hammer et al, 2001;Liu et al, 2004b;McGrath and Solter, 1983;McGrath and Solter, 1984;McGrath and Solter, 1986). The mature oocyte, awaiting the fertilization at metaphase II, gets activated by the penetrating sperm, but it also can be artificially activated in vitro by chemicals, or even by temperature and pH shifts (Meo et al, 2004;Nagai, 1987;Prather et al, 1991). After activation or fertilization, the oocyte completes meiosis II and one set of maternal chromosomes is extruded as second polar body.…”

Pronucleus formation in bovine oocytes activated by a single electric pulse

“…The sperm activation stimulus can be simulated artificially, ie parthenogenetically, via, for example by Ca 2+ ionophore A23187 (Ware et al, 1989), protein synthesis inhibitors (First et al, 1992) or ethanol (Nagai, 1987) and also by an electric pulse. It has been demonstrated that electrically activated oocytes exhibit similar characteristics to penetrated eggs: establishment of zona pellucida block prevents polyspermic penetration (Gwatkin et al, 1973;Behalova et al, 1992), completion of meiosis, chromatin decondensation and pronuclear formation (Landa and Hdjkovd, 1989;Prochazka et al, 1992), cleavage (Tarkowski et al, 1970;Kaufman et al, 1975; Prather et al, 1991;Prochazka et al, 1993) and in particular cases limited postimplantation development (Ozil, 1990). Although the kinetics of pronucleus formation in activated bovine oocytes has been monitored (Landa and Hajkova, 1989;Powell and Barnes, 1992) (Xu et al, 1986).…”