Systematic analysis of the factors that adversely affect the rate of cell accumulation in mouse embryos during their culture in vitro

Abstract

BackgroundRetarded embryo growth is a pervasive effect of culture in vitro.MethodsA systematic analysis of the interactions between media design, embryo culture density, oxygen tension, amino acids, trophic ligands and the genetic background of the mouse on embryo growth rates in vitro was performed.ResultsGrowth retardation of mouse zygotes was greater in 20% O2 than 5%, a sequential media design was superior to static simple media designs, but the supplementation of simple media with mixed amino acids mitigated this difference. There was a beneficial effect of communal culture in small volumes, and supplementation with a trophic ligand (Paf) further enhanced growth rates. For hybrid strain zygotes (B6CBF1) communal culture in KSOM media supplemented with amino acids, albumin and Paf under 5% O2 resulted in complete rescue of their rate of accumulation of cells and blastocyst formation. Inbred strain (C57BL6/J) zygotes, however, still showed some retardation of development under these conditions. The additional supplementation of media with another trophic ligand (IGF1) showed a further additive beneficial effect on development of inbred strain embryos but they still showed a growth deficit of ~ 23% cell number. The results show that optimising the interactions between a range of culture conditions and media design can rescue hybrid strain embryos from a retarded rate of cell proliferation caused by culture in vitro, but this was incomplete for the B6 strain.ConclusionsThe results indicate that the growth requirement of embryos in vitro varies depending upon their genetic background and provide models for the further genetic analysis of embryo growth.

Combined effects of individual culture and atmospheric oxygen on preimplantation mouse embryos in vitro

“…Again, the blastocysts cultured individually in 20% oxygen had roughly half the number of cells compared with blastocysts cultured in groups in 5% oxygen. This reduction in cell numbers is in agreement with the majority of studies on single culture (Dai et al, 2012;Jin and O'Neill, 2014;Kato and Tsunoda, 1994;Lane and Gardner, 1992;O'Neill, 1998;Paria and Dey, 1990;Salahuddin et al, 1995;Stoddart et al, 1996;Vutyavanich et al, 2011) and 20% oxygen (Gardner and Lane, 1996;Karagenc et al, 2004;Quinn and Harlow, 1978;Rinaudo et al, 2006;Wale and Gardner, 2010), and may be caused by an increase in apoptosis that has been observed following single culture (Brison and Schultz, 1997;O'Neill, 1998) and culture in 20% oxygen (Van Soom et al, 2002;Yuan et al, 2003). Alternatively, 20% oxygen can also induce cellular senescence, which would slow the rate of cell divisions, resulting in fewer cells (Meuter et al, 2014).…”

Long term effects of ART: What do animals tell us?

“…The question of a direct transfer of conclusions from animals to humans becomes even more complex when considering the genetic effects observed in the response of mouse embryos to ART procedures. As mentioned above, genetic effects have been evidenced that may rely on differences in embryo metabolism and its interaction with lineage commitment (Schwarzer et al, ), resistance to stress of which oxydative stress, differences in the synthesis of autocrine embryotrophic factors (Jin & O'Neill, ), differences in modifiers genes involved in sensitivity of imprinting maintenance to ART conditions (Fauque et al, ). Such genetic differences very probably also exist between human embryos and may partly explain interindividual variability in the response to ART procedures.…”

“…It is well known that cell cleavage and preimplantation development kinetics is slower in in vitro cultured embryos than in their in vivo developed counterparts. This has been precisely evidenced using mouse BC57BL/6J X CBA/He (B6CBF1) hybrid strain embryos (Jin & O'Neill, 2014). Authors first counted the cells of embryos developed at 20% O 2 , in two different culture media (human tubal fluid medium supplemented with Glutamine and EDTA: GE-HTF and Sydney IVF medium) every 12 hr, and compared those to in vivo developed embryos.…”

A novel embryo culture media supplement that improves pregnancy rates in mice

“…We employed two different mouse strains to assess the effects of in vitro embryo treatment combinations on pregnancy rates: (1) C57BL/6 females that were mated with BalbC males to produce B6BcF1 embryos (B6 embryos). Embryos of C57BL/6 hybrid strains are sensitive to detrimental effects of in vitro culture in the absence of exogenous trophic ligands (Jin & O'Neill 2014) and so were used as a preliminary study to investigate the effects of treatment on embryo development and early pregnancy (to pregnancy day 8) in a susceptible model. (2) CBAB6F1 females (CBA X C57BL/6) that were mated with males of the same strain to produce CBAB6F2 embryos.…”

“…Ten embryos per 50 µL drop is within the published optimal mouse embryo density ranges summarized by Reed et al (2011) and a media volume per embryo intended to dilute the paracrine effects of endogenous IGF2 and waste products secreted by companion embryos. Five per cent oxygen was used as mouse embryos cultured in 21% oxygen (ambient) conditions show reduced development to the hatching stage (Jin & O'Neill 2014).…”