Production of Bovine Hand-Made Cloned Embryos by Zygote–Oocyte Cytoplasmic Hemi-complementation

Abstract

The aim of this study was to evaluate the effect of the cytoplast type and activation process on development of cloned embryos. Bovine oocytes (MII) or zygotes at the one-cell stage (IVF) were manually bisected and segregated in MII or IVF hemi-cytoplasts or hemi-karyoplasts. Adult skin cells from a bovine female were used as nucleus donors (SC). Experimental groups were composed of IVF embryos; parthenogenetic embryos; hand-made cloned (HMC) embryos; and reconstructed HMC embryos using IVF hemi-cytoplast + MII hemi-cytoplast + SC (G-I); IVF hemi-cytoplast + IVF hemi-cytoplast + SC (G-II); MII hemi-cytoplast + IVF hemi-karyoplast (G-III); and IVF hemi-cytoplast + IVF hemi-karyoplast (G-IV). Embryos from G-I to G-IV were allocated to subgroups as sperm-activated (SA) or were further chemically activated (SA + CA). Embryos from all groups and subgroups were in vitro cultured in the WOW system. Blastocyst development in subgroup G-I SA (28.2%) was similar to IVF (27.0%) and HMC (31.4%) controls, perhaps due to a to a more suitable activation process and/or better complementation of cytoplasmic reprogramming factors, with the other groups and subgroups having lower levels of development. No blastocyst development was observed when using IVF hemi-karyoplasts (G-III and G-IV), possibly due to the manipulation process during a sensitive biological period. In summary, the presence of cytoplasmic factors from MII hemi-oocytes and the sperm activation process from hemi-zygotes appear to be necessary for adequate in vitro development, as only the zygote-oocyte hemi-complementation was as efficient as controls for the generation of bovine cloned blastocysts.

Probability, odds and random chance: the difficult task of modulating the epigenetic profile of cloned embryos

“…In vivo-matured oocytes are routinely used for cloning in many animal species, as such oocytes are presumably more competent for nuclear reprogramming and to subsequent embryonic development (Campbell et al, 2005;Ribeiro et al, 2009;Wells, 2010;Mezzalira et al, 2011;Aguiar et al, 2016).…”

“…It is known that the exposure of a donor nucleus to an environment with active MPF, such as in the MII oocyte, will lead to a series of subcellular events promoting exposure of chromatin reprogramming factors also at high levels in the cytoplasm, which possibly favors epigenetic reprogramming (Mezzalira et al, 2011). Thus, in vitro embryo development is improved when cytoplasts at MII are used in association with post-fusion activation (Heyman et al, 2002;Mezzalira et al, 2011).…”

“…Thus, in vitro embryo development is improved when cytoplasts at MII are used in association with post-fusion activation (Heyman et al, 2002;Mezzalira et al, 2011). The time between the fusion of the cytoplast at MII with the karyoplast at G0/G1 and embryo activation is referred as reprogramming time, and such time appears to play an important role in chromatin exposure to oocyte cytoplasmic remodeling factors (Campbell et al, 2005).…”

“…As an additional oocyte source for cloning, also related to the cell cycle, the embryo reconstruction using oocytes at late telophase II (Bordignon and Smith, 1998) results in proper embryo development, but at slightly lower rates than MII oocytes, possibly due to MPF levels still being abundant to trigger events observed with MII oocytes, some associated with reprogramming and chromatin remodeling (Mezzalira et al, 2011). Such system is successfully in use in commercial cloning operations in Brazil and abroad.…”

“…Oocyte enucleation may result in loss of cytoplasm and, when excessive, can compromise subsequent embryo development (Ribeiro et al, 2009). On the other hand, the fusion of cell membranes leads to mosaicism and cytoplasmic heteroplasmy by mixing different cytoplasts, and it is still not clear which are the possible biological consequences on subsequent development (Ribeiro et al, 2009;Mezzalira et al, 2011). In addition, fusion aims to introduce a donor nucleus, typically at the G0/G1 phases of the cell cycle, into a MII oocyte.…”