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

Abstract

Background: Retarded embryo growth is a pervasive effect of culture in vitro.

Methods: A 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.

Results: Growth 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.

Conclusions: The 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.

Figure 1

The effects of embryo culture on developmental viability. (A) Hybrid strain (B6CBF1) zygotes collected 20 h post-hCG or (B) 2-cell embryos 44 h post-hCG were cultured as 10 embryos in a drop of 10 μL GE-HTF medium or sIVF media under 5% CO₂ in air (20% O₂). The cell number of the cultured embryos and the embryos freshly collected from reproductive tracts at a 12 h interval from (A) 36 h post-hCG or (B) 60 h was recorded. Each time point is the record of least 80 embryos from 3 independent replicates. *p < 0.001, compared to the corresponding stage of cultured embryo. (C) Representative images of embryos stained with Hoechst 33342 and examples of fragmented nuclei shown with arrows.

Figure 2

The effect of O₂ tension on growth rate. Hybrid strain zygotes were cultured as 10 embryos in a drop of 10 μL GE-HTF medium, sIVF or KSOM media. The rates of blastocyst formation (A) 96 h and (B) 116 h post-HCG, (C) the rate of hatching blastocysts and (D) the cell number in the resultant blastocyst (116 h post-hCG) were analysed. Each treatment contained at least 80 embryos from two independent replicates. *p < 0.001, compared to the embryos cultured in 20% O₂. **p < 0.001, compared to modHTM or KSOM media in 5% O₂. (E) Representative images of embryos developed in different O2 tension stained with Hoechst 33342 and examples of fragmented nuclei shown with arrows.

Figure 3

The effect of duration and periods of changes in O₂ tension growth rates in vitro. Hybrid strain zygotes were cultured in groups of 10 in 10 μL of sIVF cleavage medium in 5% or 20% O₂ for 48 h. Each treatment was divided into two subgroups and cultured in sIVF blastocyst medium for a further 48 h in 5% or 20% O₂. The rate of blastocyst formation (A) 96 h post-hCG and (B) 116 h post-hCG, and the (C) the rate of blastocyst hatching, and (D) the total numbers of cells, (E) fragmented nuclei in the resultant 116 h post-hCG blastocyst was analysed. The results are representative of at least 100 embryos for each treatment from three independent replicates. *P < 0.001, compared to the corresponding treatment group of embryo that was cultured in different O₂ tension. **p < 0.001, compared to the embryos that were cultured in 5% O₂ for 48 or 96 h.

Figure 4

The effects of supplementation of media with mixed amino acids on growth rate of zygotes. Hybrid strain zygotes cultured in groups of 10 in 10 μL GE-HTF or sIVF media under the 5% oxygen tension. The developmental outcomes were assessed at 96 h for blastocyst formation and 116 h for the rates of blastocyst formation and hatching, and the number of the cells accumulated in the resultant blastocyst. At least 100 embryos for each medium from 3 independent replicates were tested. *P < 0.001, compared to embryos cultured in sIVF media.

Figure 5

The effects of O₂ tension and genetic background on growth rates in vitro. The effect of 5% or 20% O₂ on the proportion of (A) hybrid strain and (B) B6 mouse zygotes cultured as groups of 10 in 10 μL sIVF media achieving developmental landmarks at 48, 72, 96 and 116 h post-hCG. (C) the total number of cells in the resultant blastocysts, and (D) the number of these cells with fragmented nuclei in the resultant blastocyst 116 h post-hCG were assessed. At least 200 hybrid strain and 130 B6 embryos from 4 independent replicates were tested. *p < 0.01, compared to the embryos achieving the same landmarks in 20% O₂ condition.

Figure 6

The effects of embryo density in media on growth rates. Hybrid strain and B6 mouse zygotes were cultured individually or in groups of 10 embryos in 10 μL sIVF media under 5% O₂ condition. (A) the rates of blastocyst formation at 96 and 116 h post-hCG, and hatching blastocysts, (B) the number of the cells in each blastocyst and (C) the number of cells with fragmented nuclei were analysed. Each culture group had at least 60 hybrid strain embryos and 50 B6 embryos from 3 independent replicates. Statistically significant effects are shown on the graphs. DENSITY and STRAIN were the dependent variables, and INT (abbreviation of interaction) was the interaction effect between DENSITY and STRAIN.

Figure 7

The effects of exogenous Paf on growth rates. Hybrid strain and B6 mouse zygotes were cultured individually in a drop of 10 μL sIVF media with (Paf) or without (Control) supplementation with 37.2 nM Paf under 5% O₂. (A) the rates of the blastocyst formation at 96 and 116 h post-hCG, and the rate of blastocyst hatching, (B) the number of total cells in each blastocyst and (C) the number of the cells with fragmented nuclei were analysed. Each culture group had at least 60 hybrid strain embryos and 50 B6 embryos. Statistically significant effects are shown on the graphs. DENSITY and STRAIN were the dependent variables, and INT (abbreviation of interaction) was the interaction effect between DENSITY and STRAIN.

Figure 8

Testing the effects of optimised culture conditions on embryo growth rates of hybrid strain and inbred strain embryos. Embryos were collected directly from the reproductive tract during development or zygotes collected and cultured as groups of 10 in 10 μL KSOM media supplemented with amino acids and 37 nM Paf in 5% O₂ tension. The growth rate of each treatment was assessed by recording the number of cells in each embryo at 12 h interval up to 96 h post-hCG, compared to embryos collected directly from reproductive tract at the same times, in which method was designed as Figure 1. At least 60 embryos for each developmental time point were assessed. There was no differences between in vitro and in vivo hybrid strain embryos at any time point, *p < 0.001 shows the differences in growth rate of B6 embryos in vitro compared to those collected from the reproductive tract.

Figure 9

Effects of IGF1 on B6 embryo development in 5% O₂. B6 zygotes were cultured in groups of 10 μL KSOM media plus amino acid and Paf and supplemented with a range of doses of mouse recombinant IGF1. (A) The proportion of zygotes forming blastocysts were assessed 108 h post-hCG. There was an overall adverse dose-dependent effect IGF1 dose on the rate of blastocysts formation P < 0.001, and ** higher dose significant fewer cells (p < 0.01) compared to controls (B) total number of cells per blastocyst formed. There was an overall adverse dose-dependent effect of IGF1 (p < 0.001) and multiple comparison of means showed * 1 ng dose to have caused significantly more (p < 0.01) and ** higher dose significant fewer cells (p < 0.01) compared to controls. The results were representative of the three independent replicates with at least 80 embryos each dose. (C) The blastocyst rate of B6 zygotes cultured communally in KSOM media plus amino acid and Paf at 5% O₂ with or without 1 ng/mL IGF1 was shown, and the cell number was compared with blastocysts collected directly from the uterus 96 h post-hCG. *p < 0.001, compared to the fresh embryos. **p < 0.01, compared to the culture conditions without IGF1. The results were representative of three independent replicates and each treatment had at least 90 embryos.