Mammalian embryo development requires both paternal and maternal genomes which are imprinted differentially. The purpose of the present study is to contribute to better understanding of the processes and mechanisms of meiosis and of genomic imprinting in spermatogenic cells. The investigators will study how and when the nuclei of spermatogenic cells become competent to participate in normal embryo development. Nuclei of spermatogenic cells at various stages of differentiation (gonocyte through pachytene primary spermatocyte stages) will be individually injected into mature and immature oocytes to see if they are able to undergo normal meiotic divisions within oocytes. If they can, and if resultant zygotes develop into normal fertile offspring, genomic imprinting of male germ cell nuclei must either (a) be complete by the time of nuclear transfer or (b) proceed within the oocyte's cytoplasm in the intrinsic paternal fashion. Methylation patterns of three selected genes (Xist, H19 and Igf2r) in the nuclei of the following cells will be determined: (a) primordial germ cells, (b) oogonia, (c) maturing and mature oocytes, and (d) spermatogenic cells at various stages of development (gonocyte through mature spermatozoon). Methylation pattern of spermatogenic cells nuclei after transfer into maturing and mature oocytes will be compared with that of the nuclei within native cytoplasm. The investigators will culture early spermatogenic cells in vitro till they transform into round spermatids to see if their nuclei can be used to produce zygotes with full developmental potential. They will also investigate whether assisted fertilization by ICSI, which bypasses a number of normal physiological processes, has undesirable genetic consequences in immediate and later generations.
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