Marshall, Vivienne Shelley; (1995) Manipulation of oocytes and early embryos of the common marmoset monkey (Callithrix jacchus). Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Genomic imprinting is a phenomenon whereby some genes are expressed differently depending on whether they are maternally or paternally inherited. In mice, the most notable effects of genomic imprinting appear to be imposed during embryonic development. In humans, it is difficult to study the effects of genomic imprinting on development for obvious ethical reasons, however it is important to discover whether the effects of genomic imprinting on mouse embryonic development are paralleled in primates. The aim of this study was to determine the effects of genomic imprinting on the early embryonic development of a non-human primate, the common marmoset monkey. To facilitate the investigation, the fertilization rate of marmoset oocytes was increased from 53% to 76% (p<0.005) by altering the time between the administration of hCG and laparotomy, and duration of oocyte pre-incubation. The mean maximum cell number (MMCN) of in vitro fertilized (IVF) marmoset embryos was increased from 7.7 (± 0.7) when cultured in vitro to 15 (± 4.35) when cultured in the oviducts of live mice (p<0.003). The morphological determination of the parental origin of marmoset pronuclei was not possible because both pronuclei formed at the same time after insemination, they were both the same size and both first became visible near the centre of the zygote. Unlike similar studies using mouse zygotes, in marmoset zygotes it was not possible to visualize fluorescent paternal pronuclei after fertilization with marmoset sperm carrying DNA which was stained with a polyspecific fluorochrome. Pronuclear transfer and electrical fusion of marmoset one-cell embryos was successful in 7/15 (46%) embryos. Marmoset embryos which had undergone sham enucleation and were restored to a normal genetic constitution were able to develop to an average (± S.E.M) of 3.3 (± 2.3) cells and a maximum of 8 cells. Parthenogenetic activation of marmoset oocytes was achieved using ethanol (8/47; 17%) and electrical stimulation (68/74; 92%). Marmoset parthenogenones developed to a MMCN (± S.E.M.) of 4.0 ± 0.3 and reached a maximum of 16 cells in vitro. There was no significant difference between the percentage of parthenogenetic embryos and IVF embryos reaching each cell stage up to 16 cells. Three of four IVF embryos, and 2 of 3 marmoset parthenogenones transferred to synchronised recipient marmosets developed to post-implantation stages. To Day 33, when recipient animals were killed, progesterone and inhibin profiles of recipients carrying parthenogenetic embryos (RP) resembled those of recipients carrying normal embryos (RN). However, chorionic gonadotrophin of RP animals remained at non-pregnant levels. Histological analysis of RP animals showed syncytial invasion of the uterine stroma, but only remnants of embryonic membranes. The development of marmoset parthenogenones to the 16-cell stage is not significantly different from normal IVF embryos. Additionally, implantation of primate embryos can occur without the participation of the paternal genome. By developing the techniques of manipulation of primate embryos and oocytes, this study has provided the basis for further research to elucidate the role of genomic imprinting in primate embryonic development.

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