Development of Parthenogenetic and Cloned Ovine Embryos: Effect of Activation Protocols1

Abstract

Preliminary experiments carried out on ovine oocytes were designed to establish correlations between activation protocols and subsequent rates of embryonic development. The best activation protocols were thereafter used in studies on ovine parthenogenesis and cloning. The first study established that chemical activators induce pronuclear development at a slightly higher rate than physical activation (ionomycin, 96%; ethanol, 95%; electro activation, 80%). Inhibition of second polar body extrusion and one single pronucleus were observed in the majority of the oocytes (approximately 90%) treated for 3 h with 6-dimethylaminopurine (6-DMAP) following either ionomycin or ethanol activation. While over 80% of these oocytes cleaved after transfer to the oviducts of recipients, progression to the blastocyst stage was higher after ionomycin as compared with ethanol activation (58% vs. 19%). The ionomycin plus 6-DMAP activation protocol was used to produce parthenogenetic blastocysts whose subsequent development was monitored both by ultrasonography and by direct fetal examination. Over 70% of parthenogenotes were viable on Day 21 of pregnancy but dead by Day 25. The effects of 6-DMAP on nuclear remodeling and fetal development of cloned embryos was then investigated. Control cloned embryos underwent nuclear envelope breakdown (NEBD), premature chromatin condensation (PCC), and inhibition of DNA synthesis. By contrast, reconstructed embryos treated with 6-DMAP exhibited intact nuclear membranes, interphase chromatin, and no interference on DNA synthesis. Moreover, cloned embryos developed to blastocyst stage in higher percentage after 6-DMAP treatment (83% vs. 25%). We conclude that ionomycin followed by 6-DMAP incubation yields high percentages of diploid parthenogenetic embryos that develop to Day 25 before dying. Cloned embryos activated by the ionomycin-6-DMAP protocol develop readily to term.

Embryo transfer and related technologies in sheep reproduction

“…This procedure, however, is quite time-consuming because a two-step manipulation is required for embryo reconstruction. In subsequent work carried out in our laboratory, a high frequency of development to the blastocyst stage was obtained by transferring unsynchronized blastomeres from 16-32 cell embryos into MIT enucleated oocytes activated with ionomycin and a protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP) [58]. In previous work, activated mouse eggs treated with 6-DMAP showed an accelerated pronuclear organization [71], thereby indicating the inhibition of kinase(s) involved in the nuclear membrane assembly and chromatin structure.…”

The Effect of Activation Protocols on the Development of Cloned Goat Embryos

“…Activation is an important step in the NT procedure and ethanol and ionomycin have been widely used as activating agents in several species, such as cattle [15,20,21], mice [8], sheep [16], and goats [1,4,6,11,32]. There are a few reports on the development of goat oocytes after parthenogenetic activation by treatment with ionomycin and ethanol, both followed by exposure to 6-diethylaminopurine (6-DMAP) [17,18].…”

Factors inducing the activation of reconstructed oocytes of farm animals in somatic cloning procedures

“…A frequently used ionophore is ionomycin. Ionomycin has been successfully used in the activation ovine oocytes (Loi et al, 1998(Loi et al, , 2002 but also in activation of bovine (Wells et al, 1999) and goats (Keefer et al, 2001) oocytes.…”

“…Ethanol as an activating agent is particularly effective with regard to ovine oocytes (Loi et al, 1998), but less useful for activating cattle and porcine oocytes.…”