Development of parthenogenetic and cloned ovine embryos: effect of activation protocols

• Published in: Biology of reproduction 1998-05, Vol.58 (5), p.1177-1187
• Main Authors: Loi, P, Ledda, S, Fulka, J. Jr, Cappai, P, Moor, R.M
• Format: Article
• Language: English
• Subjects: 6-DIMETHYLAMINOPURINE; Adenine - analogs & derivatives; Adenine - pharmacology; Animals; Biological and medical sciences; Blastocyst - drug effects; Blastocyst - metabolism; Blastocyst - ultrastructure; Blastomeres - drug effects; Blastomeres - ultrastructure; Cell Nucleus - drug effects; Cell Nucleus - physiology; CLONACION; CLONAGE; Clone Cells - drug effects; Clone Cells - physiology; CLONING; CULTIVO DE EMBRIONES; CULTURE D'EMBRYON; DESARROLLO EMBRIONARIO; DEVELOPMENTAL COMPETENCE; DEVELOPPEMENT EMBRYONNAIRE; Diploidy; DNA REPLICATION; EMBRYO CLONING; EMBRYO CULTURE; EMBRYO TRANSFER; Embryo, Mammalian - cytology; Embryo, Mammalian - drug effects; Embryo, Mammalian - physiology; Embryology: invertebrates and vertebrates. Teratology; Embryonic and Fetal Development - drug effects; Embryonic and Fetal Development - physiology; EMBRYONIC DEVELOPMENT; ENZYME INHIBITORS; Enzyme Inhibitors - pharmacology; Female; FETAL DEVELOPMENT; Fundamental and applied biological sciences. Psychology; Germ Cells - drug effects; Germ Cells - physiology; Germ Cells - ultrastructure; INHIBIDORES DE ENZIMAS; INHIBITEUR D'ENZYME; Molecular embryology; NOYAU CELLULAIRE; NUCLEI; NUCLEO; NUCLEUS; OOCYTE ACTIVATION; OOCYTES; Oocytes - drug effects; Oocytes - physiology; Oocytes - ultrastructure; OVA; OVIN; OVINOS; OVULE; OVULO; PARTENOGENESIS; PARTHENOGENESE; PARTHENOGENESIS; Parthenogenesis - physiology; PARTHENOGENETIC EMBRYOS; Pregnancy; Protein Kinase Inhibitors; REPLICACION; REPLICATION; SHEEP; Stimulation, Chemical; TRANSFERENCIA DE EMBRIONES; TRANSFERT EMBRYONNAIRE
• ISSN: 0006-3363; 1529-7268
• DOI: 10.1095/biolreprod58.5.1177

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.