Developmental and functional evidence of nuclear immaturity in prepubertal sheep oocytes.

ABSTRACT- Prepubertal sheep embryo transferred to foster mother for development to term is able to progress through the first part of gestation, while further fetal development is often interrupted. Disruption of maternal primary imprinting has been proposed as responsible for similar developmental arrest of neonatal mouse oocytes. On the other hand, in ovine and bovine species several structural and functional deficiencies of the cytoplasm are widely reported as causes of low developmental potential of prepubertal oocyte. In the light of these observations, developmental competence of each compartment of lamb oocyte, nucleus and cytoplasm, were studied first. To identify the compartment responsible for developmental failure, nucleus exchange between lamb and adult sheep oocyte followed by sperm injection into the reconstructed egg, were performed. Using this approach, we revealed that when lamb oocytes smaller than those from adult sheep were used for nuclear transfer, development of oocytes reconstructed with either sheep or lamb nucleus was arrested before blastocyst stage. This demonstrates that smaller oocytes lack both cytoplasmic and nuclear competence. Surprisingly, developmental progress of eggs reconstructed from nuclear exchange between adult and lamb oocytes similar in diameter, was significantly different for sheep- and lamb-nucleus derived embryos (18% vs. 0% blastocysts, respectively), indicating oocytes with diameters similar to adult still incompetent in terms of nuclear preparation. Secondly, to reveal if the epigenotype of prepubertal oocyte is appropriate for supporting normal development, global methylation level was assessed using anti-5-methylcytidine antibody. High levels of antibody incorporation were observed in nuclei of adult sheep oocytes (n=58) confirming fully established genome methylation while the intensity of nuclear immunofluorescence was invariably weak in similar size lamb oocytes (n=65) or even absent in more than half of smaller oocytes (34/62), indicative of epigenetic immaturity. In this study, we present developmental and functional outlines of nucleus immaturity reinforced by previous reports of late pregnancy loss following transfer of lamb embryos, leading to further insight on the causes of prepubertal oocytes developmental defects.[...]