Developmental Consequences of Karyokinesis Without Cytokinesis during the First Mitotic Cell Cycle of Bovine Parthenotes1

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Abstract

Bovine parthenogenetic embryos and bovine embryos produced by in vitro fertilization were compared for chromosomal complement and developmental potential. Oocytes (n = 1885) were matured in vitro, fertilized (n = 1151) or activated (n = 734) by exposure to 5 μM ionomycin for 4 min, and then treated with 1.9 mM 6-dimethylaminopurine for 5 h to inhibit protein kinase functions and promote mitosis. Mean cleavage rates at 48 h were 76.3 ± 14.7% for fertilization and 60.1 ± 4.2% for activation (p < 0.05). A similar percentage of embryos had reached the blastocyst stage on Day 8 post fertilization/postactivation (16.4 ± 3.3%) and (15.8 ± 1.0%), respectively. Blastocysts (n = 53) produced by in vitro fertilization had higher total cell numbers (116.9 ± 5.5) than parthenotes (n = 71, 67.2 ± 3.5 cells, p < 0.05). Differential staining indicated a significant reduction in the number of blastomeres allocated to both the inner cell mass and trophectodermal lineages in parthenotes (p < 0.05). All parthenotes (n = 65) were polyploid or mixoploid, with observed karyotypes of 4n (61.53%), 2n/4n (30.76%), 2n/8n (4.61%), and 3n (3.07%). In contrast, only 9 control blastocysts (n = 53) revealed abnormal metaphases (16.9%). At 6 h postactivation (hpa), 70.7% of parthenotes (n = 65) demonstrated a fully formed pronucleus; and at 10 hpa (n = 86), 89% had completed pronuclear formation. Pronuclear DNA replication was observed by 6 hpa and resulted in the formation of a second pronucleus in 76.9% of activated oocytes (n = 104) by 24 hpa. These pronuclear kinetics lead to a high number of embryos with binucleate blastomeres upon cleavage. Thus, alterations in the DNA content (ploidy) of bovine parthenogenetic blastocysts reflect ongoing karyokinesis without cytokinesis during the first mitotic cell cycle after exposure to a protein kinase inhibitor.

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