CN110628704A

CN110628704A - In-vitro culture method of mouse early embryo

Info

Publication number: CN110628704A
Application number: CN201910997437.XA
Authority: CN
Inventors: 薛志刚; 曾桥
Original assignee: Tongji University
Current assignee: Tongji University
Priority date: 2019-10-17
Filing date: 2019-10-17
Publication date: 2019-12-31

Abstract

The invention discloses an in vitro culture method of mouse early embryos, which is characterized by comprising the following steps: the early-stage mouse embryo is treated by adopting 2000-5000 lux light source for 0.5-6 hours, and comprises single embryo cells in 2-cell stage, 4-cell stage, 8-cell stage, morula stage and blastocyst stage, so that the method has the advantage of effectively improving the cyst formation rate and the diameter of the blastocyst.

Description

In-vitro culture method of mouse early embryo

Technical Field

The invention relates to an in vitro culture method of mouse early embryos.

Background

Assisted reproduction techniques generally mean that the embryo is exposed to visible light during examination and transfer. Since the in vivo environment of an embryo is much darker than the in vitro environment, exposure to light is a non-natural stressor in the in vitro fertilization of embryos. Such high or prolonged light exposure is detrimental to the embryo during embryo handling, as they directly induce a number of stress-related metabolic processes or activate reactions leading to apoptosis, such as the production of reactive oxygen species in the cytoplasm, etc. However, the changes that occur throughout transcriptomics of embryos after light exposure are not clear. Because the preimplantation processes of different mammalian embryos share similarities in many respects, current research generally considers mouse embryos, as a substitute for human embryos, to be a better animal embryo model for improving assisted reproduction techniques.

Disclosure of Invention

The invention aims to provide an in vitro culture method for early mouse embryos, which can effectively improve the cyst formation rate and the diameter of blastocysts.

The technical scheme adopted by the invention for solving the technical problems is as follows: an in vitro culture method of mouse early embryos comprises the following steps: the early embryo of the mouse is treated by 2000-5000 lux light source for 0.5-6 hours.

The mouse early embryo comprises single embryo cells at 2 cell stage, 4 cell stage, 8 cell stage, morula stage and blastocyst stage.

Compared with the prior art, the invention has the advantages that: the invention discloses an in vitro culture method of mouse early embryo, which explains the damage of different illumination intensity and illumination time to the mouse embryo before implantation, and further clarifies transcriptomics change under high intensity and long-time illumination by single cell transcriptome sequencing.

Drawings

FIG. 1 is a graph showing the effect of different illumination times on the embryo encapsulation efficiency (percent encapsulation) of mice;

FIG. 2 is a graph showing the effect of different light intensities on the embryo encapsulation efficiency of mice;

FIG. 3 is a graph showing the effect of different light intensities on blastocyst morphology of a mouse embryo;

figure 4 is a graph of the effect of different light intensities on mouse blastocyst Diameter (Diameter), Bar 100 μm: pvalue is less than 0.05;

FIG. 5 is a graph of the effect of light on mouse early embryo transcriptome. The main component analysis of each stage comprises 2 cell stages (2-cell), 4 cell stages (4-cell), 8 cell stages (8-cell), mulberry blending stage (Morula) and Blastocyst stage (blast);

FIG. 6 shows the effect of light on mouse early embryo transcriptome (enrichment analysis of G0 signal pathway of gene difference between light group and non-light group at each stage);

FIG. 7 is a graph of the effect of light on mouse early embryo transcriptome: high expression gene wien map for 4 cell stage versus 2 cell stage.

Detailed Description

The invention is described in further detail below with reference to the accompanying examples.

In the following examples, five light treatment times and three light intensity treatments were selected, a mouse early embryo was selected as a study object, the effects of the treatment intensity and the treatment time on the mouse embryo cyst rate and the blastocyst morphology were analyzed, the treatment time and the light intensity affecting the development of the early embryo were determined, and further the effects of light on the development of the mouse embryo at each stage were determined by single-cell transcriptome high-throughput sequencing and analysis of the difference genes between the light group and the light-free group.

Example 1

1. We collected 2 cell stage mouse embryos for different light time treatments, specific numbers are as follows: no light treatment (0 hour) 227, 0.5 hour 102, 1 hour 98, 2 hours 103, 4 hours 104 and 6 hours 118.

2. And (3) treating the mouse embryos in the 2-cell period according to different illumination time, and continuously culturing in a dark state in an incubator at 37 ℃ after the treatment is finished.

3. The blastocyst rate of the mice treated at each illumination time was counted, and we found that the 6-hour treatment had a significantly decreased blastocyst rate, and the results are shown in fig. 1.

Example 2

1. We collected 2 cell stage mouse embryos for different light intensity treatments, specific numbers were as follows: no light treatment (0 lux) 332, 2000 lux 131, 3000 lux 152, 5000 lux 191.

2. And (3) treating the mouse embryos in the 2-cell period according to different illumination intensities, and continuously culturing in a dark state in an incubator at 37 ℃ after the treatment is finished.

3. The blastocyst rate of the mice treated by each light intensity was counted, and we found that the blastocyst rate of the mice treated by 6 hours was significantly reduced, and the results are shown in fig. 2.

4. The blastocyst morphology of the mice treated with each light intensity was counted and the results are shown in FIG. 3.

5. The diameter of the blastocyst of each light intensity treated mouse was counted, and the results are shown in FIG. 4,

Example 3

1. Cells of the light group (mouse embryo at the stage of 2 cells in 5000 lux light for 6 hours) and cells of the mouse embryo at each stage of the non-light group are collected, and single cell transcriptome expression data are obtained by a Smart-seq2 single cell transcriptome high-throughput sequencing method, sequence comparison and expression calculation.

4. Comparing the major components of the single-cell transcriptome in each period between the illuminated group and the non-illuminated group, the result shows that the transcriptome in each period has a large difference from the transcriptome in the non-illuminated group, and the transcriptomes are gathered at different positions, as shown in FIG. 5.

5. The differential genes of the unicellular transcriptome in each period of the illumination group and the non-illumination group are respectively compared, the differential genes are subjected to enrichment analysis of G0 signal paths (shown in figures 6A-E), the differential genes are enriched in transcription regulation (DNA template) signal paths in all embryo periods, the differential genes from 4 cell periods to 8 cell periods are highly enriched in the apoptosis process, and the differential genes are enriched in substrate adhesion-dependent cell diffusion in the period of the mulberry being matched to the blastocyst.

6. Further, by comparing the high-expression genes in the 4-cell stage and the 2-cell stage, 823 high-expression genes specific to the light group and 2853 high-expression genes without light group were found (FIG. 7).

Table 1 shows the enrichment analysis of the G0 signal path of 2853 differential genes with no illumination group specificity and high expression

As can be seen from the above Table 1, there is a difference in the mouse early embryo transcriptome in light, and genes with no difference in high expression in light are mainly enriched in oxidative phosphorylation, metabolic pathways, RNA transport, etc.

The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art should also realize that changes, modifications, additions and substitutions can be made without departing from the true spirit and scope of the invention.

Claims

1. An in vitro culture method of mouse early embryos is characterized by comprising the following steps: the early embryo of the mouse is treated by 2000-5000 lux light source for 0.5-6 hours.

2. The method of claim 1, wherein the method comprises the steps of: the mouse early embryo comprises single embryo cells at 2 cell stage, 4 cell stage, 8 cell stage, morula stage and blastocyst stage.