CN113430172B - Method for promoting in vitro embryo development by regulating metabolism - Google Patents

Abstract

The invention provides a method for promoting in vitro embryonic development by regulating metabolism, and relates to the technical field of biology. The inventor researches to find that the addition of L-2-hydroxyglutaric acid can block the erasure of histone methylation and the development of embryos during in vitro embryo culture, and the reduction of the content of L-2-hydroxyglutaric acid in the embryos can promote the erasure of histone methylation and the development of the embryos, so that the invention provides a method for regulating and controlling the in vitro embryo development of mammals, which comprises the steps of promoting the development of the embryos by reducing the content of the L-2-hydroxyglutaric acid in the embryos or inhibiting the development of the embryos by increasing the content of the L-2-hydroxyglutaric acid in the embryos. The method provided by the invention can effectively realize the regulation and control of in vitro embryo development and provide a new visual field for a molecular regulation and control mechanism in the early embryo development process.

Description

Method for promoting in vitro embryo development by regulating metabolism

Technical Field

The invention relates to the technical field of biology, in particular to a method for promoting in-vitro embryonic development by regulating metabolism.

Background

Pluripotent Stem Cells (PSCs) including Embryonic Stem Cells (ESCs) and Induced Pluripotent Stem Cells (iPSCs) are important tools for regenerative medicine because of their ability to differentiate into a variety of adult cells. The signal pathways, regulation of gene expression, and epigenetic regulation associated with pluripotent stem cells have been studied relatively intensively, and studies on the metabolism of pluripotent stem cells have just begun to reveal the important role of metabolism as a regulation of gene expression in the connection of the extracellular environment, the intracellular metabolic network, and the nucleus. Little is known about how these controls affect the mechanisms of acquisition, maintenance and withdrawal of the stem cell sternness. Meanwhile, recent studies have demonstrated in various systems that cellular metabolism can not only affect cell growth and proliferation by producing energy and synthesizing raw materials for each component of the cell, but also participate in epigenetic remodeling and expression of key genes, and affect cell fate determination, thereby affecting tumor occurrence, immune system activation, and early embryonic development and other physiological and pathological biological events. Early development of the embryo is especially likely to be a period of time when metabolic regulation and epigenetic regulation interact closely, because the process of the embryo from zygote to two-cell, four-cell, morula, pre-implantation blastocyst post-implantation is a process in which proliferation (cell proliferation) and large-scale epigenetic remodeling (epigenetic remodelling) of one cell occur simultaneously, and this process directly determines the decisions of the embryonic genome activation and cell fate. However, we are still not very clear about metabolic reprogramming and its effect on the regulation of appearance during early embryonic development.

The classical physiological analysis of embryos in the past mainly focuses on the uptake of nutrients, respiratory function and secretion of byproducts in the development process, for example, the embryo mainly utilizes pyruvate in the early development stage and converts to glucose in the later development stage, which also provides the best culture condition for the clinical practice of in vitro fertilization. In addition, blastocysts exhibit a rapid increase in respiration during development, but the underlying mechanisms of metabolic remodeling during this development are still not well elucidated. Recent omics analysis using low loading or single cell level RNA-seq technology and analysis of chromatin higher order structure provides us with a new field of view to understand the molecular regulatory mechanisms during early embryo development. On the other hand, however, metabolic genes are often considered housekeeping genes, and the metabolic state shift during early embryonic development is often ignored. Therefore, the changes of the expression condition, the epigenetic state and the kinetic characteristics of the metabolic genes in the development process of the early embryo of the mouse are not disclosed yet. The current understanding of the molecular mechanisms of embryonic metabolism is mainly based on the research on embryonic stem cells, the different pluripotent states of which can correspond to different embryonic stages, and the research on the cells can obtain some key information of synthesis and catabolism and how the metabolic network is integrated into the existing pluripotent network.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first purpose of the invention is to provide a method for regulating and controlling the in vitro embryonic development of mammals, which regulates and controls the embryonic development by reducing or increasing the content of L-2-hydroxy glutaric acid in the embryo.

The second purpose of the invention is to provide an application of L-2-hydroxyglutaric acid in preparing a product for inhibiting the in vitro embryonic development of mammals.

In a first aspect, the present invention provides a method of modulating mammalian in vitro embryonic development by reducing the level of L-2-hydroxyglutarate in an embryo;

alternatively, embryo development is inhibited by increasing the level of L-2-hydroxyglutarate in the embryo.

As a further technical scheme, the method for reducing the content of the L-2-hydroxyglutaric acid in the embryo comprises the following steps: increasing the content of L-2-hydroxyglutarate dehydrogenase in the fertilized egg;

preferably, the method for increasing the content of L-2-hydroxyglutarate dehydrogenase in the fertilized egg comprises: overexpresses L-2-hydroxyglutarate dehydrogenase in fertilized eggs.

As a further technical scheme, the method for over-expressing L-2-hydroxyglutarate dehydrogenase in fertilized eggs comprises the following steps: introducing L-2-hydroxyglutarate dehydrogenase mRNA into a fertilized egg;

preferably, the means of introduction comprises microinjection.

As a further technical scheme, the microinjection concentration of the L-2-hydroxyglutarate dehydrogenase mRNA is 200 ng/ul.

As a further technical scheme, the method for increasing the content of the L-2-hydroxyglutaric acid in the embryo comprises the following steps: the embryos are cultured in the presence of L-2-hydroxyglutarate.

As a further technical scheme, the method for increasing the content of the L-2-hydroxyglutaric acid in the embryo comprises the following steps: culturing the embryo in the presence of 0.1-1mM L-2-hydroxyglutaric acid;

as a further embodiment, the embryos are cultured in the presence of 0.3mM L-2-hydroxyglutarate.

As a further technical scheme, the embryo development is inhibited by increasing the content of L-2-hydroxy glutaric acid in the embryo from the 2-cell stage to the blastocyst stage.

As a further embodiment, the mammal includes a mouse, pig, rabbit, or human.

In a second aspect, the invention provides an application of L-2-hydroxyglutaric acid in preparing a product for inhibiting in vitro embryonic development of mammals.

Compared with the prior art, the invention has the following beneficial effects:

the inventor researches to find that the addition of L-2-hydroxyglutaric acid can block the erasure of histone methylation and the development of embryos during in vitro embryo culture, and the reduction of the content of L-2-hydroxyglutaric acid in the embryos can promote the erasure of histone methylation and the development of the embryos, so that the invention provides a method for regulating and controlling the in vitro embryo development of mammals, which comprises the steps of promoting the development of the embryos by reducing the content of the L-2-hydroxyglutaric acid in the embryos or inhibiting the development of the embryos by increasing the content of the L-2-hydroxyglutaric acid in the embryos. The method provided by the invention can effectively realize the regulation and control of in vitro embryo development and provide a new visual field for a molecular regulation and control mechanism in the early embryo development process.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a metabolomic analysis of 2 cell-like nuclear blastocysts; a: a workflow diagram for embryo and ES cell sample processing and targeted metabolomics analysis; b: mass spectrum quality control results; c: 2 main component analysis diagram of targeted metabonomics analysis of three biological repeated samples of cell and blastocyst; d: heat maps of the abundance of various metabolites in 2-cell embryos and blastocyst; e: the volcano plots represent fold differences of individual metabolites at the 2-cell and blastocyst stages and P-vaule values in the C plot, where blue dots (22 metabolites, including α -KG) and red dots (21 metabolites, including SAM and 2-HG) represent metabolites that are significantly reduced and upregulated in the 2-cell embryo and P-vaule values <0.05, respectively, compared to the blastocyst stage; f: the left side plate block: relative abundance of 2-HG in 2 cells and embryos at the blastocyst stage, middle plate: 2 relative abundance of α -KG in cells and embryos at the blastocyst stage, right panel: 2 ratio of alpha-KG/2-HG in the cell embryo and in the blastocyst stage embryo; G. h: 2 passage enrichment analysis of differential metabolites of the cell embryo and the blastocyst; i: relative methionine levels in oocytes and 2-cell embryos; j: 2 ratio of GSH/GSSH in cells and blastocysts;

FIG. 2 is metabolomics of 2 cell-like cells and ES cells; a: 2 flow results of cell-like cells and ES cells; b: 2 principal component analysis plot of targeted metabolomic analysis of two biological replicate samples of cell-like cells and ES cells; c: the volcano plots represent fold difference of the omics detected metabolites in 2-cell-like nuclear ES cells and P-vaule values, and blue dots (including 2-HG) and red dots (including SAM) in the plots represent metabolites that are significantly reduced and up-regulated, respectively, in ES cells and have P-vaule values <0.05, compared to ES cells; d: 2 higher levels of metabolites in each of the cell-like cells and the ES cells;

FIG. 3 is a comparison of embryonic metabolomics data with cellular metabolomics data; a: wien plots show higher levels of and overlapping metabolites in the blastocyst (compared to the 2c stage embryos) and ES cells (compared to the 2-cell like cells); b: wien plots show higher levels of overlapping metabolites in 2 cell embryos (compared to blastocyst stage embryos) and 2c-like cells (compared to ES cells); c: pathway enrichment analysis of metabolites in panel a; d: pathway enrichment analysis of metabolites in panel B;

FIG. 4 shows the content of L-2HG in early embryo at various developmental stages and the relationship between the content and alpha-KG; a: chromatograms of L-2-HG and D-2-HG detected in 2-cell embryos; b: L-2-HG and D-2-HG content in MII, zygote and 2 cell embryos, data shown as mean ± SEM of three biological replicates, # p <0.002, # p <0.001, (n ═ 3, unpaired t-test); c: L-2-HG and D-2-HG content in 2-cell embryos and blastocysts, data shown as mean ± SEM of three biological replicates, × p <0.002, × p <0.001, (n ═ 3, unpaired t-test); d: content of α -KG in MII, zygote and 2 cells, data shown as mean ± SEM of three biological replicates, × p <0.001, (n ═ 3, unpaired t-test); e: content of α -KG in 2 cells and blastocysts, data shown as mean ± SEM of three biological replicates, × p <0.001, (n ═ 3, unpaired t-test); f: ratios of α -KG/2-HG in MII, zygote and 2-cell embryos, data shown as mean ± SEM of three biological replicates, × p <0.033, (n ═ 3, unpaired t-test); g: ratios of α -KG/2-HG in 2-cell and blastocyst embryos, data shown as mean ± SEM of three biological replicates, { p ≦ 0.001 } (n ═ 3, unpaired t-test);

FIG. 5 is a schematic diagram of an embryo experimental method; a: schematic representation of Tunel assay, zygotes were collected 16 hours after hCG injection and cultured in KSOM containing varying concentrations of L-2-HG, embryos collected at the indicated times for further analysis; b: tunel test results of embryos cultured to different stages in A;

FIG. 6 is a graph showing that a reduction in 2-HG promotes global erasure of histone methylation; a: control and experimental embryos treated with 2-HG including zygote, early 2, late 2, and 4 cells were stained with H3K4me3 antibody, staining pattern in a: imaging embryos of a specific number (n) of embryos with FV3000 confocal equipment with maximum projection Z: female pronucleus and male pronucleus: male pronuclei, PB: pole body, 2 PN: zygote at 2PN stage, scale: 20um, histogram in A: quantification of fluorescence intensity of H3K4me3, data expressed as mean ± SEM, # p <0.002, # p < 0.001; b: control and experimental embryos treated with 2-HG included zygotes, early 2 cells, late 2 cells, 4 cells stained with H3K9me3 antibody, staining pattern in B: imaging embryos of a specific number (n) of embryos with FV3000 confocal equipment with maximum projection Z: female pronucleus and male pronucleus: male pronuclei, PB: polar body, 2 PN: zygote at 2PN stage, scale: 20um, B middle histogram: quantification of fluorescence intensity of H3K4me3, data expressed as mean ± SEM, # p <0.002, # p < 0.001;

FIG. 7 is a graph of the phenotypic effect of 2-HG on embryos; a: blastocyst formation, blastocyst collapse, hatchability, cell number and chamber area after 2-HG treatment (from 16h after hCG injection to the stage of blastocyst formation) versus normal embryos (Ctl, untreated), error bars indicate ± SEM, ± p <0.002, ± p < 0.001; b: imaged plots of blastocysts 3.5 days and 4.5 days after 2-HG treatment. This experiment was independently repeated three times, scale: 100 μm; c: schematic diagram of L-2-HG processing scheme at different stages of embryo development, wherein a red line segment is a 2-HG processing stage; d: bar graphs show the various embryogenesis rates in panel C after treatment with L-2-HG at different stages of embryo development, with the number of embryos from group a to group F being 22, 26, 28, 23, 25, 26, respectively;

FIG. 8 shows that 2-HG and α -KG have opposite effects during early embryo development, and that a reduction in 2-HG favors embryo development; a: qRT-PCR showed L2hgdh mRNA levels at different developmental stages, data shown as mean. + -. SEM for representative three biological replicates; b: proteomic data show L2hgdh protein levels at different developmental stages, data show mean ± SEM of representative three biological replicates; c: WB shows L2hgdh protein levels at different developmental stages; d: immunohistochemistry for L2hgdh in wild type mouse ovary; e: schematic representation of the zygotic stage after siL2hgdh mRNA injection and culture in KSOM containing 2-HG, immunofluorescence and qRT-PCR were performed on late 2 and 4 cell embryos, 5 embryos per time were collected for qRT-PCR; f: panel E after treatment, qRT-PCR showed L2hgdh mRNA levels after siL2hgdh mRNA injection from 2 and 4 cell embryos, rna (sinc) zygotes without specific targeting genes were injected as negative controls, data shown as mean ± SEM for representative three biological replicates,. p <0.01 from t-test; g: the same treatment method as that of the E picture is adopted, a control group (SiNC is injected) is set, SiNC, Sil2hgdh, SiNC +2-HG and Sil2hgdh +2-HG are injected respectively, the SiNC refers to a group injected with a common sequence without homology with a target gene sequence at the zygote stage, the SiNC +2-HG refers to a group cultured in a KSOM containing 2-HG after SiNC is injected at the zygote stage, the Sil2hgdh refers to a group injected with L2hgdh siRNA at the zygote stage, the Sil2hgdh +2-HG refers to a group cultured in a KSOM containing 2-HG after Sil2 hgh is injected at the zygote stage, immunofluorescence imaging of H3K4me3 is carried out on 2-cell embryos, the immunofluorescence imaging graph shows that under different conditions, a 3000 FV confocal device is used for carrying out Z-axis maximum projection on embryos, the Z-axis is represented by a scale, the average intensity of 3520 mu m is represented by a graph represented by a quantitative graph (3520 mu m), p <0.033, # p <0.002, # p <0.001 from the t-test; h: adopting the same processing mode as that of the E picture, setting a control group (injecting SiNC), and respectively carrying out immunofluorescence imaging of H3K4me3 on 4-cell embryos injected with SiNC, Sil2hgdh, SiNC +2-HG and Sil2hgdh +2-HG, wherein the immunofluorescence imaging in the H picture shows that under different conditions, Z-axis maximum projection imaging is carried out on a specific number (n) of embryos by using FV3000 confocal equipment, the scale is 20um, the columnar graph in the H picture shows that the average intensity of immunofluorescence of H3K4me3 is quantitative, p is less than 0.002, and p is less than 0.001 and comes from t test; i: injecting mRNA of L2hgdh into the oocyte of somatic cell nuclear transplantation, and counting the success rate of SCNT after over-expressing the L2hgdh, wherein the injection concentration is 200 ng/ul;

FIG. 9 is a graph of the effect of L-2-HG on TCA cycle and pluripotency during early embryonic development in mice; a: comparison between gene set enrichment analysis of TCA cycle-associated genes and pluripotency-associated genes of 2-HG treated blastocysts and untreated embryos, NES: a normalized enrichment score; b: comparison between gene set enrichment analysis of TCA cycle-related and pluripotency-related genes of α -KG treated blastocysts and untreated embryos, NES: a normalized enrichment score; c: qRT-QPCR showed expression of pluripotency gene Nanog in blastocysts after 2-HG and α -KG treatment, the number of embryos grown to blastocysts for qRT-QPCR was 5 under each condition, data is the average ± SEM of three biological replicates,. p < 0.033.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Those who do not specify the specific conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

In the present invention, the term "oxocyte: an oocyte; MII-Ocyte: MII stage oocytes; zygote: a zygote; BC: a blastocyst embryo; hCG: human chorionic gonadotropin; SCNT: somatic cell nuclear transfer technology; ctl: and (4) a control group.

In a first aspect, the present invention provides a method of modulating embryonic development in a mammal by reducing the level of L-2-hydroxyglutarate (L-2-HG) in the embryo to promote embryonic development;

alternatively, embryo development is inhibited by increasing the level of L-2-hydroxyglutarate in the embryo.

The inventor researches and discovers that the addition of L-2-hydroxyglutaric acid in the in vitro embryo culture process can block the erasure of histone methylation and the development of embryos, and the reduction of the content of L-2-hydroxyglutaric acid in the embryos can promote the erasure of histone methylation and the development of the embryos. The method provided by the invention can effectively realize the regulation and control of in vitro embryo development and provide a new visual field for a molecular regulation and control mechanism in the early embryo development process.

In some preferred embodiments, the method of reducing the level of L-2-hydroxyglutarate in an embryo comprises: increasing the content of L-2-hydroxyglutarate dehydrogenase in the fertilized egg;

preferably, the method for increasing the content of L-2-hydroxyglutarate dehydrogenase in the fertilized egg comprises: overexpresses L-2-hydroxyglutarate dehydrogenase in fertilized eggs.

L-2-hydroxyglutarate dehydrogenase (L2HGDH), which can convert L-2-hydroxyglutarate into alpha-ketoglutarate (alpha-KG), and overexpression of the L-2-hydroxyglutarate dehydrogenase in fertilized eggs can reduce the content of L-2-hydroxyglutarate.

In some preferred embodiments, the method of overexpressing L-2-hydroxyglutarate dehydrogenase in a fertilized egg comprises: introducing L-2-hydroxyglutarate dehydrogenase mRNA into a fertilized egg;

preferably, the means of introduction comprises microinjection.

In some preferred embodiments, the L-2-hydroxyglutarate dehydrogenase mRNA is microinjected at a concentration of 200 ng/ul.

In the invention, the overexpression of the intracellular L-2-hydroxyglutarate dehydrogenase is realized by injecting the mRNA of the L-2-hydroxyglutarate dehydrogenase with proper concentration on the premise of not influencing the metabolic growth of cells.

In some preferred embodiments, the method of increasing the level of L-2-hydroxyglutarate in an embryo comprises: the embryos are cultured in the presence of L-2-hydroxyglutarate.

In some preferred embodiments, the method of increasing the level of L-2-hydroxyglutarate in an embryo comprises: culturing the embryo in the presence of 0.1-1mM L-2-hydroxyglutarate; the concentration of L-2-hydroxyglutaric acid may be, for example, but not limited to, 0.1mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM, or 1 mM.

In some preferred embodiments, the embryo is cultured in the presence of 0.3mM L-2-hydroxyglutarate.

In some preferred embodiments, embryonic development is inhibited by increasing the level of L-2-hydroxyglutarate in the embryo from the 2-cell stage to the blastocyst stage.

The inhibition of the embryo development can be well realized by increasing the content of the L-2-hydroxy glutaric acid in the embryo from the 2-cell stage to the blastocyst stage.

In some preferred embodiments, the mammal includes, but is not limited to, a mouse, pig, rabbit, or human, or other mammals known to those of skill in the art.

In a second aspect, the invention provides an application of L-2-hydroxyglutaric acid in preparing a product for inhibiting in vitro embryonic development of mammals.

The inventor researches and discovers that the addition of L-2-hydroxyglutaric acid can block the erasure of histone methylation and the development of embryos in the in vitro embryo culture process, so that products containing the L-2-hydroxyglutaric acid, such as medicines, also have the effect of blocking the erasure of histone methylation and the development of embryos, and the L-2-hydroxyglutaric acid can be used for preparing products for inhibiting the development of in vitro embryos of mammals.

The present invention is further illustrated by the following specific examples and comparative examples, but it should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the invention in any way.

The experimental method comprises the following steps:

1. culturing feeder cells

Mouse fibroblast acquisition: a mouse with 13.5dpc of pregnancy was taken, and the mouse was dissected by a sterile method to obtain an embryo. The embryos were then placed in sterile 10mm petri dishes containing sterile and double-antibody-loaded PBS solution. The head and internal organs of the embryo were removed using ophthalmic forceps, the body was washed in PBS to remove blood, transferred to 0.5ml pancreatin, and embryonic tissue was minced with sterile ophthalmic scissors. Then 0.5ml of pancreatin was added and blown up uniformly using a 1ml pipette. After 5min, 5ml of DMEM was added for culture, the cells were again pipetted and mixed, centrifuged at 1100rpm for 3min at room temperature, resuspended in DMEM medium containing 10% FBS, and plated into 100mm dishes. The next day, passaging again according to cell number. After 80% of the cells have grown, they are passed on or used to make feeder layer cells. Mouse fibroblasts from generations F2-F4 were typically used to make the feeder layer.

Preparing a feeding layer: after the mouse fibroblasts were confluent, the culture medium was removed, washed with PBS, and washed with 10ug/ml mitomycin C in 5% CO₂The cells were treated in the 37 ℃ incubator for 2 hours, after which they were washed 5 more times with PBS. Cells were harvested using trypsinization, washed once with PBS and cryopreserved, thawed for use, and plated on 0.1% gelatin.

And (3) ES culture and passage: the Mouse embryonic stem cell line (Mouse embryonic stem cells, mESC) used was E14. E14 was cultured in LIF/2i medium and LIF/serum medium. Mouse embryonic stem cells E14 were cultured on mouse fibroblasts (MEF) that were not proliferated by mitomycin treatment after 0.1% gelatin coating. Passage at 37 deg.C and 5% CO₂In an incubator. The liquid was changed every day and passaged every other day.

2. ES cell freezing and resuscitation

When the ES cells grow to 90%, cells are digested with trypsin, and the ES cells are usually treated at (2-5). times.10 for 3-5min⁵And (5) freezing and storing each cell. The frozen stock solution was prepared in DMEM medium containing 40% FBS and 10% DMSO. ES cells are frozen as soon as possible during ES culture to reduce the number of culture generations. The time of cell passage, number of passage and location of the cryopreserved tubes were carefully recorded. When thawing, preparing a 37 deg.C water bath in advance, taking out cells from liquid nitrogen tank, unscrewing the freezing tube cover (preventing explosion), placing in 37 deg.C clean water bath, and shaking to only a little ice core within 1 min. Taking cells, wiping the frozen tube with alcohol, transferring to a biological safety cabinet, adding pre-warmed complete culture solution, transferring the liquid to a 15ml centrifuge tube, and centrifuging at 1100rpm for 3 min. The upper waste liquid was aspirated and the cell pellet was resuspended using complete medium. Cells were transferred and shaken well according to the size of the dish, transferred to 5% CO at 37 ℃₂Culturing in an incubator. Cell recovery was observed the next day.

3. Preparation of Dual reporter ES cell lines

E14 Stable reporter gene 2C: tdTomato was used to label two-cell like cells (2-cell like cells, 2 CLC). Transfection of Nanog: GFP-labeled naive cells. Cell transfection 2C was transfected with post lip 2000. tdTomato plasmid was screened for 48 hours 7 days later with 150. mu.g/ml hygromycin. E14 cells containing both the dual reporter 2C: tdTomato and GFP were cultured in LIF/Serum medium.

4. Flow sorting of small numbers of cells

Optimizing small-scale cellular metabolomics: ES cells (2C-like: tdTomato positive, ES: GFP positive) containing the double reporter system were trypsinized for 5min, the reaction was stopped with serum-containing medium, and then placed in an incubator at 37 ℃ for 30min to allow MEF to adhere to the wall, thereby separating from the ES cells. The supernatant suspended ES cells were collected, washed once with 0.2% BSA/PBS, and filtered through a 45 μm nylon mesh into a 5ml flow tube. The ES cells were then sorted in a FACS-Aria flow cytometer. The cell gradient was 2.5K, 5K, 10K, 20K, 40K, 80K, with 3 replicates per group.

2C-like cells and ES metabolomics: 10000 2-cell like and ES cells were collected for metabonomics studies. Sorting time for ES cells was 1min, and 2C-like ES sorting time was about 8 min.

All cells were kept on ice prior to sorting and immediately after sorting 500. mu.l of 80% methanol was precooled with dry ice. All samples were stored at-80 ℃. The mass spectrometric detection was carried out after vacuum drying in a centrifugal concentrator (SpeedVac).

5. Ovum retrieval

To obtain MII stage eggs, mature MII eggs were collected 12-14 hours after injection of human chorionic gonadotropin (hCG). M2 (commercial in vitro working fluid sigma (M7167)) which had been filtered was prepared as a microdrop, cervical dislocation was used to sacrifice the mice, and the oviduct was dissected and removed to place in the M2 drop. Using a sharp-pointed forceps to tear open the ampulla under a dissecting mirror to release the oocyte complex, using hyaluronidase of 0.3mg/ml to digest for 1min, transferring the oocyte complex into a new M2 microdroplet, observing the dropping condition of the granular cells, using a capillary with a larger caliber to transfer the embryo into a new M2 until the granular cells completely drop, and using a capillary with a small caliber to transfer the embryo.

6. Embryo acquisition and culture at different stages

After hCG injection, female mice and male mice are caged according to the ratio of 1: 1. Embryos were collected at different times as needed. The female mouse was removed for 16 hours to check for vaginal emboli. Collecting corresponding embryos according to the development time of the embryos after fertilization for experiment or culture. Fertilized eggs, early-2 cells, medium-2 cells, late-2 cells, 4-cells, 8-cells, morulae and blastocysts are obtained in vivo after hCG for 20-24 hours, 30-31 hours, 38-40 hours, 44-48 hours, 54-56 hours, 68-70 hours, 76-78 hours and 88-102 hours respectively. Blastocysts were flushed from the uterus using M2, and embryos were obtained from the fallopian tubes for the rest of the period. Embryos to be cultured, which need to be cleaned in M2, are transferred to KSOM droplets and cultured further in a 5% carbon dioxide incubator at 37 ℃.

9. 2-HG and alpha-KG processing of embryos

16 hours after hCG, fertilized eggs were collected from the oviduct, the embryos were treated with 0.3. mu.g/ml hyaluronidase to remove perivitelline granulosa cells, and polar-forming eggs were selected for culture in KSOM containing α -KG (0.15mM) or 2-HG (0.3 mM). Recording the research on embryos in different periods and the development condition of the embryos and collecting the embryos required by the experiment for the next experiment. When the embryo is transferred into a culture solution containing 2-HG, the embryo needs to be transferred in a culture dish for multiple times, so that the drug concentration in the culture solution is ensured.

10. Fetal metabolomics sample collection

Collecting early-stage mouse embryos at different stages according to the development rate of the mouse embryos. To reduce the in vitro time of embryos, no more than 3 mice were sacrificed at each time. Culture medium M2 from collected embryos was pre-warmed at 37 ℃. The collected embryos were rinsed 3-5 times in normal saline droplets, finally rinsed again in two wells with 0.9% NaCl, and finally transferred to 1.5ml EP tubes using capillaries. Then, the thin glass tube was blown in 0.9% NaCl to check the presence or absence of embryo residues, and it was confirmed that all of the embryos were transferred to the EP tube. Thereafter, a 1.5ml EP tube was placed in liquid nitrogen for condensation, and then 100. mu.l of 80% methanol pre-cooled in dry ice was added, and the EP tube was placed in dry ice or stored at-80 ℃. After collection of embryos, embryos were pooled by experimental group number and centrifuged in a 4 ℃ cryocentrifuge at 15,000rpm for 15 minutes. The supernatant was gently transferred to a new centrifuge tube and 80% methanol was pumped off at 30 ℃ on a vacuum pump for mass spectrometric detection. The centrifuged pellet was assayed for protein concentration using the BCA protein assay kit.

11. LC-MS mass spectrometry detection of ES or embryo

Dried metabolite experiments 30. mu.l of 0.03% deionized water were resuspended and shaken before centrifugation at 15,000rpm in a 4 ℃ cryocentrifuge for 15 minutes. The supernatant was subjected to Liquid Chromatography-Mass Spectrometry (LC-MS/MS). Wherein the liquid chromatography experiment is an ultra-high resolution liquid chromatography system, wherein the chromatographic column is ACQUITY UPLC HSS-T3 UPLC column (150 × 2.1mm,1.8 μm, Waters), and the mobile phase gradient is 0-3 min 1% mobile phase B in sequence; 1-99% of mobile phase B in 3-15 min; 15-17 min 99% mobile phase B; 17-17.1 min 99-1% mobile phase B; 17.1-20 min 1% mobile phase B.

Mobile phase a was a 0.03% formic acid/water solution and mobile phase B was a 0.03% formic acid/acetonitrile solution. The flow rate was 0.25ml/min, the column was maintained at 35 ℃, the sample in the autosampler was maintained at 4 ℃ and the injection volume was 20. mu.L. Mass spectrometry detection uses triple quadrupole rods and employs (QTRAP 6500+, SCIEX) in Mass Spectrometry multiple reaction monitoring mode (MRM). The chromatograms were examined and the peak areas calculated using MultiQuant software v.3.0 (SCIEX). The peak area for each detected metabolite was normalized to the total ion technique for that sample, and thus the resulting content was the ratio between the detected metabolite and the total metabolite. Since there was no difference in the total volume of 2 cell and blastocyst sizes, we normalized the two phases to total metabolite content, not to relative cell numbers. The peak area after normalization was used as a variable for multivariate and univariate data statistical analysis.

12. Quantification of 2-HG and alpha-KG by LC-MS mass spectrometry

Absolute quantification of L-2-HG and D-2-HG was performed using 20. mu.L (13c5-dl-2-HG, 100nM) as an internal standard added to a sample containing 80% methanol. After addition, vortexed and centrifuged at 4 ℃ for 15 minutes at 15,000rpm, the aspirated supernatant was evaporated to dryness using a SpeedVac concentrator. The quantification method for L-2-HG and D-2-HG is modified from the previously published literature (Sensitive Determination of on-methyl nucleotides of D-and L-2-hydroxy sulfate Enantiomers by Chiral differentiation Combined with Liquid Chromatography/Mass Spectrometry analysis. Z.Z.Y., J.X.Zhou, J.L., Shi, Z.Q.Liu.L.F., and Xin, G.Z (2019). A repair (16) O-/(18) O-isopropyl-related reagent analysis approach for the identification of 2-methyl nucleotides HG-2-hydroxy sulfate by chemical analysis of Taga. 174. A. 3. alpha. expression analysis. A. expression analysis of 2-methyl nucleotides).

50 μ L of TSPC (12.5mM ACN, 5% pyridine) was added to the dried metabolite for chiral derivatization. After 20 minutes of reaction at 25 ℃, the mixture was dried with SpeedVac and then redissolved in 50% ACN aqueous solution for LC-MS analysis. TSPC-labeled L-2-HG and D-2-HG were quantified on an LC-MS system using QTRAP 6500+ triple quadrupole mass spectrometer (SCIEX, Framingham, MA, USA) and electrospray ionization and Nexera 2 × LC-30A ultra high performance liquid chromatograph (Shimadzu Corporation, Kyoto, Japan). High performance liquid chromatography was performed at 45 ℃ using an ACQUITY UPLC HSS T3 column (2.1X 150mm,1.8 μm) with mobile phase A of 15mm ammonium acetate water + 0.1% formic acid (v/v) and mobile phase B of acetonitrile. The optimized gradient is 0-4 min in 10-31% B, 4-15.5 min in 31-32% B, 15.5-16 min in 32-95% B, 16-18 min in 95% B, and the balance is maintained in 10% B for 4 min. Mass spectrometry detection uses Multiple Reaction Monitoring (MRM) negative ion mode.

L-2-HG and D-2-HG are in 448.1>155.1, IS IS in 453.1>155.1 mode for MRM conversion.

13. Absolute quantification of alpha-KG

The alpha-KG content was determined using 13c 5-dl-2-hydroxyglutaric acid (100nM) as internal standard. The extraction and determination methods are the same as the targeted metabolomics methods. The MRM transition method 119 of 13C 5-dl-2-hydroxyglutaric acid is >74, and the MRM transition of 1,4-13C 2-succinic acid is 152>134 respectively.

14. Construction of expression vectors

Preparation of mRNA of the target gene by using the Gateway system in vitro transcription can be carried out by using a T7 in vitro transcription kit. The CDS fragment of the gene of interest was first loaded into the pDonor201 vector by BP reaction, at which time kana resistant culture plates were used for plating. And (4) sequencing after miniextraction, and performing LR reaction with pDEST-mCherry after sequencing of the insert without error. The gene of interest is inserted downstream of the mcherry. Ampicillin resistant plates were used at this time. The constructed plasmid was then extracted using a large-scale extraction kit.

15. In vitro transcription of mRNA

Since the efficiency of linearized plasmids is significantly higher in vitro than non-linearized plasmids, the expression plasmids are linearized by selecting a downstream restriction endonuclease (blunt end to prevent self-ligation) of the gene of interest before in vitro transcription, and the success of the linearization can be determined using agarose gel electrophoresis. After confirming that the linearization was complete, the linearized DNA was purified using a gel recovery kit. After purification the linearized plasmid was used to obtain mRNA using the T7 in vitro transcription kit.

The in vitro transcription system is as follows:

after gently mixing using a pipette, the PCR instrument was prevented from heating at 37 ℃ for 4 hours. Then, the DNA was removed, and 1. mu.l of Turbo DNase was added thereto and left at 37 ℃ for 15min to remove the DNA. Poly (a) was then ligated to the end of the mRNA using a tailing kit.

After the system was prepared, it was added to the previous 20. mu.l system. After mixing, the mixture was left at 37 ℃ for 1 hour to add Poly (A) to the end of the mRNA. Then, 50. mu.l of LiCl was added to the reaction system, and after mixing, the mixture was placed in a refrigerator at-20 ℃ overnight to precipitate RNA. The precipitated sample was centrifuged at 16000rpnm for 15min at 4 ℃. Washing with precooled 70% ethanol, centrifuging to remove ethanol, adding 20 μ l RNase water to dissolve RNA, measuring concentration, diluting to 0.5-1.0 μ g/ul, packaging, and storing at-80 deg.C for embryo microinjection.

16. Embryo RNA interference experiment

Exogenous mRNA can be injected into the embryo by using a micromanipulation system, thereby realizing high expression of the target gene. RNA interference typically uses the injection of siRNA against the gene of interest, thereby interfering with the mRNA transcribed from the gene of interest, reducing the protein level of the gene of interest. Both of these approaches are used to study the function of the gene of interest. Typically 3 siRNA lines were customized and used for embryo injection after confirmation of siRNA knockdown effect in cells using Lip2000 transfected ES.

The concentration of mRNA injected into the embryo is generally 0.5-1.0. mu.g/ul, and the concentration of siRNA injected is 20. mu.M. mRNA or siRNA was injected into the cytoplasm of the embryo using a syringe needle. Culturing embryo after microinjection at 37 deg.C and 5% CO₂An incubator. Embryo development was recorded daily and embryos were harvested for the required period for the experiment. Changes in the level of the target gene RNA are typically detected by qPCR, using immunofluorescence or western blot methods to detect changes in the level of the target gene protein.

17. Mouse embryo qRT-PCR

In vitro transcription of mouse embryos is described by Smart-seq2, and since mRNA levels are related to cell volume, typically 5-10 embryos are used (not much different from the stage of the embryo). The embryo in vitro reverse transcription-qPCR method is as follows:

preparing a lysate:

obtaining an embryo: embryos were washed 2-3 times with 0.2% BSA/PBS to remove medium.

The washed embryos were transferred to a 0.2ml PCR tube using a capillary tube, after which the capillary tube was purged with 0.2% BSA/PBS, confirming complete transfer of the embryos to the PCR tube. Without further experiments, the frozen product can be frozen in liquid nitrogen and stored in a refrigerator at-80 ℃. If the experiment is continued, the following lysis buffer is added.

Lysates were prepared as follows. After adding the lysis solution, the PCR tube was immediately placed in a PCR apparatus at 72 ℃ for 3min, and immediately after 3min, the PCR tube was placed on ice. This step enables Oligo-dT to bind to Poly (A) of mRNA.

Reverse transcription: the mRNA is reversely transcribed into cDNA, and the reverse transcription system is prepared as follows, the reaction conditions are that the temperature is 42 ℃ for 90min, and the temperature is 72 ℃ for 15 min. The reverse transcription product is stored at-20 ℃ or diluted to be subjected to the next qPCR reaction.

qPCR reaction: according to the enzyme preparation reaction system used by the fluorescence quantitative reagent of the experiment, Gapdh is taken as an internal reference. The first use of each primer requires the preparation of a melting curve. After the obtained CT values, the relative quantification of the detected genes was calculated using 2-DELTA. Ct.

18. Mouse embryonic RNA-seq

The concrete steps of mouse embryo RNA-seq are as follows:

and (3) embryo collection: 5-10 embryos were collected by the same method as the mouse embryo qPCR. Embryo experiments at this step 10U/ml Pronase removed the zona pellucida.

Mu.l lysis buffer (2. mu.l 0.2% Triton x-100, 1. mu.l 10mM dNTP, oligo-dT30) was added to each group of embryos. After centrifugation, the cells were incubated at 72 ℃ for 3 minutes and immediately placed on ice.

The reverse transcription system is as follows, the reverse transcription program is 42 ℃ for 90min, and 72 ℃ for 15 min.

Linear amplification: cDNA was amplified with KAPA Hifi HotStart for about 12 cycles.

Magnetic bead purification: the amplified DNA was purified using magnetic beads. After purification, the concentration of the Qubit is detected, and 1ng of DNA is taken according to the detection concentration for 2100 detection. Judging whether the reverse transcription cDNA is between 1.5Kb and 2 Kb according to the electrophoresis result. And if so, establishing a library.

Building a library: and selecting a corresponding library construction kit according to the content of the cDNA. The cDNA content in this experiment was low, so 1ng of the library construction kit was used.

The cDNA was first cleaved and ligated using the transposase of TTE Mix V1, and terminated by 5 XTS at room temperature immediately after 5 dishes at 55 ℃. Then, PCR amplification was performed by adding different indexes to each sample, and the amplification cycle was optimized. The constructed library was quality tested and sequenced on HiSeq-PE150, reading 150bp from each end.

19. Immunohistochemistry:

the mouse ovarian tissue is taken out according to the experimental purpose, washed by PBS, fixed in formalin and sent to a histochemical platform of Zhejiang medical college for immunohistochemistry. Immunohistochemistry was provided to the laboratory using antibodies in proportions according to the antibody instructions.

20. Immunoblotting

Immunoblotting (Western Blotting) is a commonly used method for detecting the molecular weight of proteins in biology today. The method uses sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to separate proteins with different molecular weights. Then, the protein is transferred to a nitrocellulose membrane (PVDF) by means of membrane transfer. Then, according to the size of the target strip, a corresponding antibody is hatched in the area, a secondary antibody with a label is used for recognizing the corresponding primary antibody, and the content of the target protein is detected through ECL chemiluminescence. The specific process is as follows:

protein sample preparation: a defined number of culture samples were added to a RIPA diluted 1 XSDS loading buffer and heated at 90-100 ℃ for 10 minutes to change the protein. The denatured protein can be directly electrophoresed or stored at-20 ℃.

Preparation of the glue: selecting concentrated glue and separation glue with different concentrations according to the molecular weight of the protein to be detected. After the separation gel is prepared, the gel surface is flattened by water and is placed at room temperature for more than 30min to be fully solidified. Then, a concentrated gel is prepared, the water for pressing the gel is poured off, and after the gel is sucked dry by using filter paper, the concentrated gel is added and immediately inserted into a sample comb. And waiting for half an hour again to fully solidify the concentrated gel, and then carrying out the next experiment.

Electrophoresis: and putting the plate-making clamp containing the glue into an electrophoresis device. After adding the electrophoresis buffer solution, the sample comb is pulled out, and an equal volume of protein sample with equal concentration is added into each sample space. In addition, protein marker is added, and the position of the target protein is laterally judged. If there are excess wells, fill in with 1 xSDS loading buffer. Electrophoresis uses a constant voltage mode where the sample is in the concentrated gel, using 90 volts, and after reaching the separation gel, 120 volts can be used.

Film transfer: and (5) performing film transfer by adopting a wet transfer mode. The membrane transfer time is determined by the molecular weight of the protein and the concentration of the separation gel. The transfer membrane uses a constant current mode. 120mM are typically transferred to the membrane for 2 h. Note that the front and back of the film, as well as the sample position, can be marked by cutting off the corners when the PVDF is removed.

And (3) sealing: 5% skim milk was prepared using TBST and the PVDF membrane was placed face up in blocking solution for 1 hour.

A first antibody: and (3) preparing a primary antibody by using the confining liquid, wherein the use ratio is determined according to the requirements of the antibody and the concentration of the protein in the sample. Incubate overnight in a refrigerator at 4 ℃.

First cleaning resistance: the primary antibody incubated PVDF membrane was washed off using TBST 3 times for 10min each.

Secondary antibody: PVDF membrane is incubated using blocking solution diluted to appropriate concentration according to antibody requirements. Incubation is typically carried out at room temperature for 1 hour.

Secondary antibody cleaning: PVDF secondary antibody was washed off 3 times for 10min using TBST buffer.

Exposure: preparing ECL exposure liquid on site, sucking the residual TBST on the film, adding ECL developing liquid, and placing in an exposure machine for development and exposure.

21. Immunofluorescence of mouse embryo

The embryo immunofluorescence can not only detect the position of the target protein of the mouse embryo, but also can quantify the target protein. Is beneficial to understanding the dynamic change of the target protein in the embryonic development. The method comprises the following specific steps:

removing the transparent tape: and determining whether the zona pellucida needs to be removed or not according to the period of the small embryo and the type of the target protein. For the embryo before fusion, the zona pellucida is not removed, and after the embryo blastomere fusion, the zona pellucida is removed, which is favorable for the detection of target protein in cell nucleus.

Embryo cleaning: embryos to be detected are washed 3 times in 3mg/ml PVP/PBS and the surface medium of the embryos is removed.

Fixing: the embryos are transferred into 4% Paraformaldehyde (PFA) to be fixed for 15-30 minutes, the fixed embryos can be permeabilized to continue the next experiment, and can also be stored in 3mg/ml PVP/PBS for one week at 4 ℃ in a refrigerator.

Permeabilization: embryos in the fixative were washed 3 times in 3mg/ml PVP/PBS and then permeabilized by transferring to 0.25% Triton X-100/PVP/PBS. The protein in cytoplasm is generally permeabilized for 20min, and the protein fixing time in nucleus can be prolonged to 1 hour.

And (3) sealing: after permeabilization, embryos are rinsed 1 time in the confining liquid, and confined after removing the permeabilization liquid, typically for 1 hour, 5mc and 5hmc are sealed overnight.

Primary antibody incubation and washing: antibody incubations were performed using blocking solution according to the primary antibody protocol. The incubation conditions were 4 ℃ overnight. The confining liquid was washed 4 times for 10 min/time.

And (3) secondary antibody incubation and washing: the secondary antibody was diluted with blocking solution. Secondary antibody selection was determined by primary antibody. The secondary antibody is diluted by half of the corresponding primary antibody, and the incubation time is generally 1 hour. Then the blocking solution is washed for 4 times and 10 min/time.

DAPI staining: after 10 minutes of staining with DAPI, wash 3 times with wash 3mg/ml PVP/PBS. The glass slide can be made into a confocal photographing detection.

Tabletting: 80% glycerol or shatter-resistant medium is added to the slide, and the cover glass is added after the embryo is transferred. Lanolin is coated on four corners of the cover glass, and the cover glass is placed on the embryo and squeezed to deform.

Confocal observation: pictures were taken using Olympus FV3000 confocal microscope. Photographs were taken with each layer having a z-axis of 0.5 μm. Acquisition parameters should avoid over-saturation of the signal. Such signal intensity accurately reflects the antigen level.

The average fluorescence intensity in each sample was calculated using the Fiji software.

5mc and 5hmc immunofluorescence: since both are modifications on the DNA, the use of 4N HCl is required to expose both modifications in the double stranded DNA. The immunofluorescence was improved as follows: after permeabilization, treatment with 4N hydrochloric acid for 15min, followed by transfer to 100mM Tris-HCl (pH8.0) for neutralization for 10 min. Then sealing and the like are carried out.

22. Mouse embryo OP-Puro

The experiment can detect the synthesis speed of the nascent protein in the cells. The main principle is to use a component of click-it Plus OPP to react with a group (pyridine azide and alkyne fragment) generated by a biological unique reaction and then with Alexa

The fluorescent dye binds to produce fluorescence, thereby fluorescently labeling the newly translated polypeptide.

To measure the content of nascent proteins at different stages, mouse embryos at different stages were cultured in EmbryoMax KSOM medium for 1 hour, using

Component A of the Plus OPP Protein Synthesis Assay Kit was used for a labeling experiment on embryos for 1 hour at a concentration of 50 μm.

After labeling, embryos were fixed in 4% paraformaldehyde-added PBS for 15 minutes at room temperature;

then 0.25% Triton X-100 in PBS was added for 15 minutes at room temperature.

Nuclei were stained with DAPI for 10min and embryos were imaged using an oil lens under a zeiss LSM880 fluorescence microscope.

23. TUNEL detection of apoptosis

TUNEL detection was performed on embryos cultured under different conditions using a one-step TUNEL apoptosis detection kit. And determining the concentration and the use time of the drug experiment.

Fixing: embryos were washed 3 times with PVP-PBS and fixed with 4% Paraformaldehyde (PFA) PBS for 30 min.

Permeabilization: then permeabilized with 0.25% Triton X-100PVP-PBS for 1 hour.

Dyeing: embryos were stained in TdT-FITC (fluorescein) solution at 37 ℃ for 60 minutes and washed with PVP-PBS.

DAPI staining: staining with DAPI for 10 min. And then cleaned.

Imaging: fluorescence of embryos was examined by slide scanning confocal laser microscopy (Olympus, FV 3000). FITC positive marker on nuclei was dead cells.

Counting: the total number of cells and the labeled dead cells were analyzed using the Fiji software.

24. Second generation sequencing (NGS) data analysis

The company, after sequencing, yielded transcriptome Data (Clean Data) provided by removing the linker and repeat sequences. Gene expression levels were calculated using the StringTie v1.3.4d (mihalea et al, 2015) and-e-B-G parameters using the Release M18 gene annotation downloaded from the gengene data portal. For each metabolic gene, the TPM (transcripts per million reads) was calculated from its genome position reads mapped to mm 10. We directly download gene expression numbers to obtain published single cell RNA-seq data of different mouse embryonic development stages. And FPKM was used as an expression abundance index. K-means clustering analysis was performed on the metabolic genes using the heatmap R software package. KEGG pathway enrichment analysis was performed on stage-specific metabolic gene clusters using clusterProfiler R package.

We used the same procedure as the RNA-seq read processing to customize and filter ATAC-seq and ChIP-seq reads. For each sample, the retained ATAC-seq and ChIP-seq data were first aligned to the mm10 genome using Bowtie2(version 2.3.4.1). The data read by ATAC-seq are aligned according to the following parameters, t-q-N1-L25-X2000, and no-mixed no-discordant is generated after alignment. ChIP-seq data were also aligned with mm10 with the option of-t-q-N1-L25. All reads and PCR repeats not aligned are deleted. The basetotal and bamcomp commands were included in the deepTools (version 2.5.3) (Ramirez et al, 2016) used for downstream analysis, a python tool for exploring deep sequencing data. Using the BamHCoverage command-normalized BPM-of bigwig-Binsize 100, we normalized the original read signal to Bins of the Per million map read (BPM) signal and converted the aligned bam file to a bigwig signal file. The "computeMatrix" and "plotProfile" commands of depTools are used to generate reads density profiles for the ATAC-seq and ChIP-seq signals of a given genomic region.

Construction of a regulatory network of developmental stage-specific TF-MG (transcription factor-metabolism related gene).

To reconstruct the phase-specific TF-MG regulatory network, we used a three-step "enrichment-search-selection" procedure. First, we used the HOMER software to find the corresponding TF binding motif for each identified Cluster. Next, we selected TFs with p-value <0.1 for subsequent analysis. Finally, we used MGs MouseNet V2(Kim et al, 2016) to find associations between transcription factors and metabolic genes in each Cluster. STRING V11(https:// STRING-db.org/cgi/input.pl) and gene co-expression networks were built by NetMiner (Yu et al, 2018). He used as input mouse embryonic total RNA-seq gene expression data at different developmental stages (Hua et al, 2018). Finally, we calculate the Person Correlation Coefficients (PCCs) between the transcription factors and the metabolic genes in each cluster, and choose the links contained in PCC >0.1 in MouseNet V2, STRING or NetMiner network, to get the TF-MG control network in different periods.

25. Statistics and analysis

And (4) carrying out statistical analysis on the SPSS (statistical testing system) in the experiment statistical experiment, wherein R software is adopted for the statistical analysis in the letter generation data. Detailed information for individual tests, including the number and type of experiments (n) and reported mean standard error (s.e.m), is listed in each legend. All difference statistics were calculated using p <0.05, p <0.01, p <0.001, using the two-tailed Wilcox signed rank test (paired samples) and the two-tailed Mann-Whitney U test (individual samples) or otherwise noted in the figure.

Example 1 metabolomic analysis shows that the TCA cycle of mitochondria is significantly enhanced during the 2-cell to blastocyst transition

To understand the dynamic metabolic remodeling during preimplantation embryo development, we used a mass spectrometry-based approach of small-scale cellular metabolomics to directly detect the abundance of various metabolites in embryos. We collected 100 embryos at 2-cell stage and 100 embryos at blastocyst stage, representing targeted metabolomic analysis when the zygotic genome was activated and when the pluripotent state was produced, i.e. ES cells, respectively, each condition consisting of three replicates of biological samples (a in fig. 1). A total of 113 metabolites were detected (Table 1), and compared to quality control, the signal intensity of the metabolites detected in the experimental group (including 2c: 2 cell stage embryos; BC: blastocyst stage embryos; GFP: ES cells; 2c-like 2 cell; QC: blank (group containing only 0.9% NaCl solution)) was significantly increased (B in FIG. 1). PCA analysis of targeted metabolite levels showed that 2 cells could be well distinguished from the blastocyst sample (C in fig. 1). Several of the most diverse metabolites include citrate, alpha-ketoglutarate (alpha-KG), succinate and glutamine, which are higher at the blastocyst stage, and 2-hydroxyglutarate (2-HG), S-adenosyl-methionine (S-adenosyl-methionine), Nicotinamide (NA), oxidized glutathione (GSSG), reduced Glutathione (GSH), spermidine and N-acetylputrescine (D in fig. 1), which are higher at the 2-cell stage. It is noteworthy that almost all metabolic intermediates in the TCA cycle are high in the blastocyst stage, including citrate, succinate and α -KG (P <0.05), but their competitive inhibitor 2-HG is higher in the 2-cell stage (P <0.001, E in fig. 1 and F in fig. 1). In fact, metabolite enrichment analysis indicated that the TCA cycle is the most highly enriched metabolome at the blastocyst stage, whereas "methionine metabolism", "spermidine and spermine biosynthesis" and "niacin and nicotinamide metabolism" are the most highly enriched metabolome at the 2-cell stage (G in fig. 1 and H in fig. 1). In addition, methionine was higher in oocytes than in 2 cells, indicating that some of the embryonic metabolites in 2 cells may be derived from oocytes (I in FIG. 1).

TABLE 1 detection of metabolites

To further validate these results, we tested ES (GFP +) cells and 2c-like (tdTomato +) cells cultured in vitro using the same metabolomics approach.

2C-like cells refer to ES cells cultured in vitro transfected with the reporter gene for 2C: tdTomato, thereby allowing a small population of 2C-like cells to be distinguished from ES cells. The ES cell line was also transfected with Nanog, a GFP reporter gene to indicate cells in a pluripotent state. Based on this cell line, we sorted 10000 tdTomato-positive 2C-like cells or GFP-positive ES cells, 2 biological replicates per cell for metabolomic analysis (a in fig. 2). PCA can also distinguish the two cells (B in fig. 2) consistent with metabolomics results from embryos, where higher levels of TCA cycle metabolites, such as succinic acid, fumaric acid, and malic acid, were also detected in ES cells, as well as higher methionine and polyamine metabolites, such as S-adenosyl-methionine, and N-acetylputrescine, in 2C-like cells (C in fig. 2 and D in fig. 2).

We compared the metabolomics data in embryos and ES, with 18 overlaps between the metabolites expressed higher in 2-cell embryos (compared to 2C-like cells) and the ones expressed higher in blastocyst stage (compared to ES cells) (a in figure 3), and 23 overlaps between the ones expressed higher in blastocyst stage (compared to 2-cell embryos) and the ones expressed higher in ES cells (compared to 2C-like cells) (B in figure 3). Metabolites enriched in both the 2-cell stage embryo and the 2-cell like cells include "spermidine and spermine biosynthesis" and "methionine metabolism". In addition, "purine metabolism" and "tricarboxylic acid cycle" were enriched in both blastocysts and tdTomato negative/GFP positive cells (C in FIG. 3 and D in FIG. 3). In summary, our fetal and in vitro cellular metabolomic data indicate that both TCA cycle and oxidative phosphorylation (OxPhos) levels are increased in mitochondria during the 2-cell to blastocyst stage transition. This is also evidenced by the relatively higher ratio of GSH/GSSG in the 2-cell embryos (J in FIG. 1), indicating that the embryos at the blastocyst stage are in a more oxidized state, while the embryos at the 2-cell stage are more reduced.

Example 2 correlation between alpha-KG and 2-HG

To see if metabolites are directly involved in gene regulation and embryonic development, we selected α -KG and 2-HG as subjects with established roles in epigenetic regulation. 2-HG has two enantiomers, D-2-HG and L-2-HG, respectively, and it has recently been found that L form of 2-HG is found under certain physiological conditions. The very high level of 2-HG at which whole genome epigenetic reprogramming occurs in early embryos has led us to the desire to further explore the subtype and absolute concentration of this metabolite in MII oocytes, 1-cell zygote stage and 2-cell embryos. We used isotopically labeled internal standards to distinguish the type of 2-HG (A in FIG. 4). Unexpectedly, we found that L-2-HG (B in FIG. 4) but not D-2-HG was present at very high concentrations in the zygotic stage of MII oocytes and in 2-cell embryos and gradually decreased as the development of the embryos proceeded (C in FIG. 4 and D in FIG. 4). The absolute concentration of α -KG in blastocysts was more than 10-fold higher than that of 2-cell embryos (E in fig. 4). Although α -KG decreased slightly from MII oocyte to 2-cell embryo, the α -KG/2-HG ratio increased significantly throughout early embryo development (F in fig. 4 and G in fig. 4).

Example 3 Effect of L-2-HG on embryonic development

The decrease in L-2-HG concentration after fertilization and the increase in the α -KG/2-HG ratio indicate that multiple methylated erasures may occur during this period. To test this hypothesis, we treated in vitro-developing embryos with L-2-HG concentrations (A in FIG. 5 and B in FIG. 5) that were non-toxic and did not affect the viability of 2-cell or 4-cell embryos.

When embryos were treated with 2-HG from the zygote stage, global erasure of both H3K4me3 and H3K9me3 from zygote to 4 cells was affected or abnormal hypermethylation occurred (A in FIG. 6 and B in FIG. 6) compared to control (no 2-HG treatment).

We have also found that the addition of L-2-HG not only alters the level of histone modification, but also causes abnormal embryonic development. Although the rate of blastocyst formation was not significantly reduced, the blastocysts were more likely to collapse after in vitro culture, and none of the blastocysts had hatched from the zona pellucida (a in fig. 7). It was found by DAPI labeling of nuclei that after addition of L-2-HG, the number of cells decreased and the area of blastocoel decreased (A in FIG. 7).

B in FIG. 7 shows the typical morphological changes of the embryos after addition of L-2-HG. To further study the period of action of L-2-HG, we treated L-2-HG at different times during in vitro embryo culture (C in FIG. 7), and it was found that addition of L-2-HG at early stage is more likely to hinder embryo development by counting the embryo development rate. In particular, when L-2-HG was added all the way, no blastocyst could be hatched, whereas in the 3.5dpc group (group E), 30.8% of blastocysts could be hatched from the zona pellucida at 6.5dpc in vitro (D in FIG. 7). Furthermore, we analyzed the transcriptome changes from the embryo development to the blastocyst stage after addition of L-2-HG and dm- α -KG. GSEA analysis found a decrease in embryonic TCA cycle and pluripotency gene expression after addition of L-2-HG (A in FIG. 9). In contrast, the embryonic TCA cycle and pluripotency gene expression increased after the embryo was treated with 0.15mM dm-alpha-KG that could be tolerated (B in FIG. 9). Fluorescent quantitative PCR also confirmed that α -KG promoted the expression of the pluripotent gene Nanog, while L-2-HG reduced the expression of Nanog (C in FIG. 9), suggesting that these two competing molecules have opposite effects on embryonic development.

Interestingly, the enzyme L2HGDH which converts L-2-HG to alpha-KG is highly expressed in embryos at the 2-cell stage. Both the published transcriptome data and early embryo qPCR demonstrated that L2HGDH expression started at 2 cells, reached a maximum after 4 cells and then gradually decreased (a in fig. 8 and B in fig. 8). In addition, the L2HGDH protein level and transcriptome were consistent, fertilized eggs were absent, 2 cells began to express and 4 cells were the highest, and 8 cells had significantly reduced blastocyst stage (C in FIG. 8). Immunohistochemistry detected L2HGDH in mouse ovaries and was found to be absent in ovarian primordial follicles as well as primary follicles (D in fig. 8). Therefore, the expression change of L2HGDH in ovum and embryo is consistent with the reduction of 2-HG in ovum height and after fertilization. Based on the fact that L2HGDH can reduce the content of L-2-HG, we knock out L2HGDH in fertilized eggs (E in FIG. 8), and detected that both H3K4me3 and H3K9me3 histone modifications are enhanced (G in FIG. 8 and H in FIG. 8). This effect was further amplified by treatment with L-2-HG based on this (G in FIG. 8 and H in FIG. 8), indicating that L-2-HG accumulation in the absence of L2HGDH prevented erasure of histone methylation. qPCR detection revealed a significant decrease in L2HGDH transcript levels in both 2 and 4 cells following siRNA injection into embryos (F in FIG. 8). These data indicate that a reduction in L-2-HG during post-fertilization and pre-implantation embryonic development is required for histone methylation erasure, and that accumulation of L-2-HG can hinder this epigenetic remodeling process and affect embryonic development. Since L2HGDH was able to convert 2-HG to α -KG, we overexpressed L2HGDH in fertilized eggs, so that the 2-HG content at the beginning of the fertilized egg stage was further decreased (I in fig. 8), and found that these fertilized eggs had a higher success rate of nuclear transfer in vitro (SCNT) compared to the control group. Indicating that a reduction in fertilized egg stage 2-HG levels can promote embryonic development.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and these modifications or substitutions do not depart from the spirit of the corresponding technical solutions of the embodiments of the present invention.

Claims (10)

1. A method of modulating embryo development in vitro in a mammal for non-therapeutic purposes, wherein embryo development is promoted by reducing the level of L-2-hydroxyglutarate in the embryo;

or inhibiting the development of the embryo by increasing the content of the L-2-hydroxyglutaric acid in the embryo;

the embryo is from 2 cell stage to blastocyst stage.

2. The method of modulating embryonic development in a mammal according to claim 1, wherein the method of reducing the level of L-2-hydroxyglutarate in an embryo comprises: increasing the content of L-2-hydroxyglutarate dehydrogenase in the fertilized egg;

the method for increasing the content of the L-2-hydroxyglutarate dehydrogenase in the fertilized egg comprises the following steps: overexpresses L-2-hydroxyglutarate dehydrogenase in fertilized eggs.

3. The method of regulating mammalian in vitro embryo development according to claim 2, wherein the method of overexpressing L-2-hydroxyglutarate dehydrogenase in fertilized eggs comprises: introducing L-2-hydroxyglutarate dehydrogenase mRNA into a fertilized egg.

4. The method of modulating embryonic development in mammals according to claim 3 wherein said means of introduction comprises microinjection.

5. The method of claim 3, wherein the microinjection concentration of the L-2-hydroxyglutarate dehydrogenase mRNA is 200 ng/ul.

6. The method of modulating mammalian embryonic development in vitro as claimed in claim 1, wherein the method of increasing the level of L-2-hydroxyglutarate in an embryo comprises: the embryos are cultured in the presence of L-2-hydroxyglutarate.

7. The method of modulating mammalian embryonic development in vitro according to claim 6, wherein the method of increasing the level of L-2-hydroxyglutarate in an embryo comprises: the embryos are cultured in the presence of 0.1-1mM L-2-hydroxyglutarate.

8. The method of claim 7, wherein the embryo is cultured in the presence of 0.3mM L-2-hydroxyglutarate.

9. The method of modulating mammalian in vitro embryonic development according to claim 1, wherein the mammal comprises a mouse, pig or rabbit.

The application of L-2-hydroxyglutaric acid in preparing products for inhibiting in vitro embryonic development of mammals.

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