Construction method and application of embryonic development retardation animal model

Abstract

The invention discloses a construction method and application of an animal model with delayed embryonic development, and relates to the technical field of animal models.The construction method of the animal model with delayed embryonic development comprises the following steps that S1, an early embryo is obtained; s2, performing contact culture on the early embryo and a development retardation inducer; s3, culturing to obtain a slowly developed embryo; the proteomics analysis method based on non-data-dependent acquisition is used for analyzing an animal model with slow embryonic development. The method comprises the following steps: providing an embryo sample; obtaining a protein expression profile by adopting a non-data-dependent acquisition proteomics method; according to a protein expression profile, identifying differential expression proteins related to the embryonic development retardation; the animal model with the delayed embryonic development is constructed, and an experimental model is provided for researching a molecular mechanism of the delayed embryonic development; a non-data-dependent collection proteomics analysis method is applied to the animal model, and compared with a traditional data-dependent collection method, the method has higher stability.

Description

Technical Field

[0001] This invention relates to the field of animal model technology, and in particular to a method for constructing an animal model of delayed embryonic development and its application. Background Technology

[0002] Embryonic development is a precisely regulated biological process, jointly controlled by spatiotemporally specific molecular networks and gene-encoded metabolic programs. Mice are a recognized model organism for mammalian developmental research, and models of delayed embryonic development can provide crucial mechanistic insights into infertility and pregnancy-related diseases. Fetal bovine serum (FBS) is widely used in embryonic development research to support in vitro co-culture systems of embryos and somatic cells, providing essential nutrients for cell proliferation. This application successfully constructed a mouse model of delayed embryonic development using FBS.

[0003] However, due to the scarcity of human embryos (typically only 10–15 per cycle), the complexity of FBS composition, and the potential for various molecular abnormalities to induce developmental abnormalities, traditional proteomics methods often struggle to achieve comprehensive analysis. To address these limitations, this application employs data-independent acquisition (DIA) proteomics technology, which offers greater stability and less data loss compared to data-dependent acquisition (DDA). More importantly, DIA can precisely quantify low-abundance proteins (including transcription factors and signaling molecules), thereby linking phenotypic changes with molecular characteristics and achieving a paradigm shift in embryo research.

[0004] This application combines DIA-based proteomics with multi-level functional validation to systematically elucidate the multidimensional regulatory mechanisms of delayed embryonic development. At the metabolic level, exogenous components disrupt lipid metabolism and amino acid homeostasis. Downregulation of fatty acid-binding protein 3 (FABP3) expression reduces fatty acid uptake efficiency, diverting metabolic flux to the less efficient glycolysis pathway. Simultaneously, disruption of the arginine-proline metabolic axis impairs nitric oxide (NO)-mediated signaling pathways, ultimately leading to insufficient embryonic energy.

[0005] In addition, abnormal expression of the nuclear transporter Kpna2 interferes with nucleoplasmic transport; the synergistic downregulation of small nucleonucleotide protein (snRNP) and ribosome assembly factors further exacerbates translation inefficiency and developmental asynchrony.

[0006] In summary, there is an urgent need to develop a method for stably constructing models of embryonic developmental delay and to combine this method with high-precision proteomics technology to comprehensively analyze its molecular mechanisms, so as to provide theoretical basis and experimental tools for research on embryonic developmental delay. Summary of the Invention

[0007] In view of this, the present invention addresses the deficiencies of existing technologies, and its main objective is to provide a method for constructing an animal model of embryonic developmental delay and its application. The method successfully constructs an animal model of embryonic developmental delay using fetal bovine serum or an inhibitor of fatty acid-binding protein 4 expression. The model is stable and reliable, with a clear phenotype, and the embryos exhibit developmental delay and a significant reduction in cell number, providing an ideal experimental model for studying the molecular mechanisms of embryonic developmental delay. The present invention applies a data-independent acquisition (DIA) proteomics analysis method to the constructed model. Compared with traditional data-independent acquisition methods, DIA technology has higher stability, less data loss, and can accurately quantify low-abundance proteins, enabling comprehensive analysis of protein expression profile changes related to embryonic developmental delay.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A method for constructing an animal model of delayed embryonic development includes the following steps: S1. Obtaining early embryos; S2. The early embryos obtained in step S1 are cultured in contact with a growth retardation inducer. S3. After culturing, embryos with delayed development are obtained, thus obtaining an animal model of embryonic developmental delay.

[0009] As a preferred embodiment: the developmental retardation inducer in step S2 is fetal bovine serum, and step S2 specifically involves: transferring early embryos into a culture medium containing fetal bovine serum for culture; wherein: the volume percentage concentration of fetal bovine serum in the culture medium is 10%, the early embryos are 2-cell embryos obtained 44-46 hours after hCG injection, the fetal bovine serum is inactivated fetal bovine serum, and the culture medium is G-1 culture medium.

[0010] As a preferred embodiment, the growth retardation inducer in step S2 is a fatty acid binding protein 4 expression inhibitor, i.e., a FABP4 expression inhibitor, wherein the FABP4 expression inhibitor is selected from at least one of a FABP4-specific chemical inhibitor or an anti-FABP4 antibody.

[0011] As a preferred embodiment: the developmental delay inducer in step S2 is a FABP4-specific chemical inhibitor, which is BMS-309403, and the concentration of BMS-309403 used is 200 nM.

[0012] As a preferred embodiment: the early embryo in step S1 is an early mammalian embryo, the mammal being a mouse, and the early embryo is in the 2-cell stage to the 8-cell stage.

[0013] Application of a data-independent acquisition-based proteomics analysis method in an embryonic developmental delay animal model constructed using the aforementioned method, the proteomics analysis method comprising the following steps: First, provide embryo samples for animal models of delayed embryonic development; Second, the embryonic samples were analyzed using a non-data-dependent acquisition proteomics method to obtain protein expression profiles; Third, based on the protein expression profile, identify differentially expressed proteins associated with delayed embryonic development and analyze animal models of delayed embryonic development.

[0014] As a preferred embodiment, the non-data-dependent proteomics acquisition method in step S2 includes: extracting proteins from embryonic samples and enzymatically digesting them to obtain peptide samples; performing mass spectrometry detection on the peptide samples using dia-PASEF mode; and analyzing the mass spectrometry data using DIA-NN software to obtain protein expression profiles.

[0015] As a preferred embodiment, the differentially expressed protein includes at least one of fatty acid-binding protein 4, fatty acid-binding protein 3, enoyl-CoAΔ isomerase, fumarate hydratase, monofunctional C1-tetrahydrofolate synthase, mitochondrial inner membrane transport subunit Tim9, and NADH dehydrogenase iron-sulfur protein 7.

[0016] The application of an animal model of embryonic developmental delay constructed by the method described above in screening candidate drugs for the prevention or treatment of embryonic developmental delay.

[0017] As a preferred approach, the screening process specifically involves: exposing early embryos to the candidate drug; detecting at least one of the following indicators: embryonic development rate, number of embryonic cells, reactive oxygen species (ROS) level, or FABP4 expression level; and determining the candidate drug as effective if, compared with the control group that has not been exposed to the candidate drug, the embryonic development rate is increased, the number of cells is increased, the ROS level is decreased, or the FABP4 expression level is restored.

[0018] Compared with the prior art, the present invention has obvious advantages and beneficial effects. Specifically, as can be seen from the above technical solution: First, this invention successfully constructed an animal model of embryonic developmental delay using fetal bovine serum or fatty acid-binding protein 4 (FABP4) expression inhibitors. The model is stable and reliable with a clear phenotype. The embryos exhibit developmental delay and a significant reduction in cell number, providing an ideal experimental model for studying the molecular mechanisms of embryonic developmental delay.

[0019] Secondly, this invention applies the data-independent acquisition (DIA) proteomics analysis method to the constructed model. Compared with traditional data-independent acquisition methods, DIA technology has higher stability, less data loss, and can accurately quantify low-abundance proteins. It can comprehensively analyze the protein expression profile changes related to embryonic developmental delay and successfully identified 165 differentially expressed proteins, including FABP4, FABP3, and Eci1, revealing multi-dimensional regulatory mechanisms such as lipid metabolism disorders, amino acid metabolism imbalances, and abnormal cytoskeleton assembly.

[0020] Third, the embryonic developmental delay animal model constructed by the method of the present invention can be used to screen candidate drugs for the prevention or treatment of embryonic developmental delay, and has good clinical application prospects and industrial value.

[0021] To more clearly illustrate the structural features and effects of the present invention, a detailed description is provided below in conjunction with the accompanying drawings and specific embodiments. Attached Figure Description

[0022] Figure 1 The development process of C-8C and M-8C group embryos of the present invention from day 0 (D0, i.e., immediately after the collection of 2-cell embryos) to days 1 to 4 (D1 to D4) is presented in microscopic images at a specified magnification. Figure 2 Volcano diagram of differentially expressed proteins between normally developing embryos and developmentally delayed embryos of the present invention; Figure 3 This is a box plot showing the differential protein expression levels between normally developing embryos and developmentally delayed embryos of the present invention. Figure 4 This is a schematic diagram of the subcellular localization of the normal development group and the developmental delay group of the present invention; Figure 5 This is a schematic diagram of the domain enrichment analysis of the normal development group and the developmental delay group in this invention; Figure 6 This is a statistical diagram of the gene ontology (GO) secondary annotation of differentially expressed proteins between the normal development group and the developmental delay group of the present invention. Figure 7 This is a schematic diagram of the KEGG pathway enrichment analysis (first 20 pathways) of differentially expressed proteins between the normal development group and the developmental delay group of the present invention. Figure 8 This is a schematic diagram of the upregulated KEGG pathway (nucleoplasmic transporter mmu03013) in the comparison between the M-8C group and the C-8C group of the present invention; Figure 9 This is a KEGG (splicing body mmu03040) pathway diagram comparing the M-8C group and the C-8C group of the present invention; Figure 10This is a KEGG pathway diagram (arginine biosynthesis mmu00220) comparing the M-8C group and the C-8C group of the present invention. Figure 11 This is a KEGG pathway diagram (arginine and proline metabolism mmu00330) comparing the M-8C group and the C-8C group of the present invention. Figure 12 This is a schematic diagram (confocal microscope) illustrating the effect of fetal bovine serum (FBS) of the present invention on the expression of the target protein in mouse embryos. Figure 13 This is a schematic diagram illustrating the effect of the interference treatment of the present invention on embryonic development at the D0, D1, and D2 stages. Figure 14 This is a schematic diagram illustrating the effect of fetal bovine serum (FBS) of the present invention on the level of reactive oxygen species (ROS) in mouse embryos; Figure 15 This is a schematic diagram illustrating the potential mechanism of the Fapp4-mediated signaling axis in early embryonic development according to the present invention. Detailed Implementation

[0023] The present invention is as follows Figure 1 As shown in Figure 15, a method for constructing an animal model of delayed embryonic development includes the following steps: S1. Obtaining early embryos; S2. The early embryos obtained in step S1 are cultured in contact with a growth retardation inducer. S3. After culturing, embryos with delayed development are obtained, thus obtaining an animal model of embryonic developmental delay.

[0024] The growth retardation inducer in step S2 is fetal bovine serum. Step S2 specifically involves: transferring early embryos into a culture medium containing fetal bovine serum for culture; wherein: the volume percentage concentration of fetal bovine serum in the culture medium is 10%, the early embryos are 2-cell embryos obtained 44-46 hours after hCG injection, the fetal bovine serum is inactivated fetal bovine serum, and the culture medium is G-1 culture medium.

[0025] The growth retardation inducer in step S2 is a fatty acid-binding protein 4 expression inhibitor, i.e., a FABP4 expression inhibitor, which is selected from at least one of FABP4-specific chemical inhibitors or anti-FABP4 antibodies.

[0026] The growth retardation inducer in step S2 is a FABP4-specific chemical inhibitor, namely BMS-309403, and the concentration of BMS-309403 used is 200 nM.

[0027] In step S1, the early embryo is a mammalian early embryo, the mammal being a mouse, and the early embryo is in the 2-cell to 8-cell stage.

[0028] An application of a data-independent acquisition-based proteomics analysis method in an embryonic developmental delay animal model constructed using this method is described. The proteomics analysis method includes the following steps: First, provide embryo samples for animal models of delayed embryonic development; Second, the embryonic sample was analyzed using a non-data-dependent acquisition proteomics method to obtain a protein expression profile; Third, based on this protein expression profile, differentially expressed proteins associated with delayed embryonic development were identified, and animal models of delayed embryonic development were analyzed.

[0029] The data-independent proteomics acquisition method in step S2 includes: extracting proteins from embryonic samples and enzymatically digesting them to obtain peptide samples; performing mass spectrometry detection on the peptide samples using dia-PASEF mode; and analyzing the mass spectrometry data using DIA-NN software to obtain protein expression profiles.

[0030] The differentially expressed proteins include at least one of fatty acid-binding protein 4, fatty acid-binding protein 3, enoyl-CoAΔ isomerase, fumarate hydratase, monofunctional C1-tetrahydrofolate synthase, mitochondrial inner membrane transport subunit Tim9, and NADH dehydrogenase iron-sulfur protein 7.

[0031] An application of an animal model of embryonic developmental delay constructed by this method in screening candidate drugs for the prevention or treatment of embryonic developmental delay.

[0032] The screening process involves: exposing early embryos to the candidate drug; detecting at least one of the following indicators: embryonic development rate, number of embryonic cells, reactive oxygen species (ROS) level, or FABP4 expression level; and determining the candidate drug as effective if, compared with the control group that has not been exposed to the candidate drug, the embryonic development rate is improved, the number of cells is increased, the ROS level is reduced, or the FABP4 expression level is restored.

[0033] Example: A method for constructing an animal model of embryonic developmental delay and its application. Animal model construction: Sexually mature female C57BL / 6 mice (Animal Experiment Center, Xinjiang Medical University), 6–8 weeks old, weighing 18–25 g, were housed in a specific pathogen-free (SPF) environment at a temperature of (22±1)℃ and humidity of (50±5)%, with a 12-hour light / 12-hour dark cycle. A total of 10 female mice were used in this application: 7 for model replication and validation (obtaining no fewer than 150 2-cell embryos and randomly dividing them into 2 groups), and 2 for experimental analysis (obtaining no fewer than 50 2-cell embryos and randomly dividing them into 2 groups). For DIA protein sequencing, each group had 3 biological replicates, with 8 embryos used in each replicate. This application was conducted in accordance with relevant guidelines for laboratory animals and was approved by the Laboratory Animal Ethics Committee of Urumqi Maternal and Child Health Hospital (Approval No.: XJFYLL2022025).

[0034] Estrus cycle synchronization: Vaginal smears were collected from mice daily from 09:00 to 10:00 for 5 consecutive days. Mice exhibiting estrus characteristics (predominantly leukocytes with a small number of epithelial cells) were selected for subsequent experiments.

[0035] Superovulation protocol: At 15:00 on day 1 of the estrus period, mice were intraperitoneally injected with 5–10 IU of pregnant mare serum gonadotropin (PMSG, dissolved in 0.1 mL of sterile saline) to mimic the effect of follicle-stimulating hormone (FSH). 48 h after PMSG injection (at 15:00 on day 3), mice were intraperitoneally injected with 5–10 IU of human chorionic gonadotropin (hCG) to mimic the luteinizing hormone (LH) surge and induce ovulation.

[0036] Cage combination and vaginal plug examination: Immediately after hCG injection, female and male mice were caged together at a ratio of 1:2 (male:female). The vaginal plug was examined at 09:00 the next day; the presence of a white keratinized mating plug indicated successful mating.

[0037] Ovulation site acquisition and treatment: Anesthesia and euthanasia: 42–44 h after hCG injection, mice were deeply anesthetized with sodium pentobarbital (50 mg / kg, intraperitoneal injection) and then euthanized by cervical dislocation.

[0038] Fallopian tube separation: The abdominal cavity is opened about 1.5 cm above the pubic symphysis, and the ampullae of both fallopian tubes are quickly separated and transferred to preheated G-MOPS culture medium (Vitrolife, catalog number: 510131).

[0039] Ovulation site counting: Under a stereomicroscope (20x magnification), the ampulla of the oviduct was mechanically torn open to release 2-cell embryos, and the number of 2-cell embryos was recorded (8-12 ovulations per mouse under normal ovulation, and 20-30 ovulations under superovulation).

[0040] Embryonic developmental arrest intervention experiment Experimental grouping and culture conditions Fetal bovine serum was inactivated in a 56°C water bath for 30 min and stored at 4°C for later use. Collected embryos were randomly divided into two groups: Treatment group: Early fertilized embryos (zygotic stage) were transferred into G-1 culture medium (Vitrolife, catalog number: 510112) containing 10% fetal bovine serum (FBS, Gibco, catalog number: 10099-141) and cultured to the target time point.

[0041] Control group: Embryos were cultured in basal G-1 medium without FBS as a synchronous control.

[0042] Sampling during embryonic development Normal embryonic development group (C-8C): After 24 h of culture in the control group, embryos that have developed to the 8-cell stage (E2.5) and have normal morphology (uniform cells, fragmentation rate <10%) were selected.

[0043] Delayed development embryo group (M-8C): After 48 h of culture, 8-cell stage embryos (homogeneous cells, fragmentation rate <10%) were collected from the treatment group.

[0044] After 24 h of culture, the embryonic developmental stages (4-cell stage, 8-cell stage) and the number of cells in each embryo were recorded. Each mouse was considered an independent biological replicate (n≥6), and paired t-tests were used for comparisons between two groups. Data are expressed as mean ± standard error (Mean ± SEM), and P < 0.05 was considered statistically significant.

[0045] Embryo sample processing and preservation Cryoprotectant loading: Target embryos were transferred to the bottom of 0.6 mL low-adsorption centrifuge tubes (Eppendorf, catalog number: 22431021) containing 3 μL of embryo cryopreservation solution (TMED, Kitazato, catalog number: CT-1123). Eight embryos were collected from each group. Cryopreservation: Embryos were rapidly frozen in liquid nitrogen and then stored long-term in an ultra-low temperature freezer (Thermo Scientific, model: 902-ULT) at -80°C.

[0046] DIA Inspection and Data Analysis Sample preparation For protein solution samples: Add SDT lysis buffer (4% sodium dodecyl sulfate SDS, 100 mM Tris-HCl, pH 7.6) directly to each sample.

[0047] For cell / embryo samples: use SDC lysis buffer instead (5% sodium deoxycholate SDC, 100 mM Tris-HCl, pH 8.5).

[0048] The lysate was sonicated (this step can be omitted for protein solutions), followed by boiling in a water bath for 15 minutes. After centrifugation at 14000×g for 40 minutes, the supernatant was used to determine the concentration using a BCA protein quantification kit. 15 μg of protein from each sample was added to 5 times the volume of loading buffer, boiled for 5 minutes, and separated by constant voltage electrophoresis in a 4–20% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) (180 V, 45 minutes). The gel was then stained with Coomassie Brilliant Blue R-250 for imaging.

[0049] For serum / plasma samples: Samples were centrifuged at 14000×g for 20 minutes, and the supernatant was used for BCA quantification. Similarly, 15 μg of protein was added, mixed with 5 times the volume of loading buffer, boiled for 5 minutes, and subjected to 4–20% SDS-PAGE electrophoresis (180 V, 45 minutes), followed by Coomassie Brilliant Blue R-250 staining. All samples were mixed in equal volumes to construct a quality control (QC) sample for subsequent analysis.

[0050] Sample enzymatic digestion Dithiothreitol (DTT) was added to each sample to reduce disulfide bonds, and the mixture was incubated at 37°C for 1.5 hours. Iodoacetamide (IAA) was then added and reacted at room temperature in the dark for 30 minutes to block the reduced cysteine ​​residues. Trypsin was added at a protein-to-trypsin ratio of 1:50, and the mixture was incubated overnight at 37°C (15–18 hours). The digests were desalted using an MCX solid-phase extraction column (Omicsolution, OS-MCX-1mL), concentrated under vacuum, and reconstituted in 20 μL of an aqueous solution containing 0.1% (v / v) formic acid. Peptide concentrations were estimated based on 280 nm UV absorbance. For data-independent acquisition (DIA) analysis, retention time calibration peptides (iRTs) were added to each sample before analysis.

[0051] Mass spectrometry analysis based on DIA strategy Peptides were detected in DIA mode using a timsTOF HT mass spectrometer (Bruker Daltonics) coupled with a nanoElate nanofluid chromatography system (Bruker Daltonics).

[0052] Full scan mass spectrometry (MS1): m / z scan range 300–1500.

[0053] Secondary mass spectrometry (MS2): dia-PASEF (parallel cumulative sequential fragmentation) acquisition mode was used. A total of 66 acquisition windows were set in DIA mode, with each window having an accumulation time of 50 ms. Under dia-PASEF conditions, the collision energy was linearly correlated with the inverse of ion mobility (1 / K0), ranging from 20–59 eV, corresponding to ion mobilities of 0.6–1.6 Vs / cm².

[0054] Mass spectrometry data analysis DIA data were processed using DIA-NN 2.0 software. The main software parameters were set as follows: enzyme specificity was set to trypsin, with a maximum allowed missed cleavage site of 1; cysteine ​​(C) carbamoyl methylation was a fixed modification, while methionine (M) oxidation and N-terminal acetylation were variable modifications. All results were based on a protein identification confidence level ≥99%, i.e., a false discovery rate (FDR) ≤1%. Label-free quantification (LFQ) was used, and median centering correction was applied to eliminate systematic bias. Differentially expressed proteins were screened using t-tests or analysis of variance (ANOVA), and multiple testing was performed using the Benjamini–Hochberg (BH) method. A |log2FC| ≥ 1.5 and a corrected P-value (FDR) < 0.05 were used as the screening criteria for differentially expressed proteins. Data have been uploaded to the Iprox database (https: / / www.iprox.cn / ), accession number: IPX0015024000.

[0055] Bioinformatics Analysis Cluster analysis: Hierarchical cluster analysis was performed using Cluster 3.0 and Java Treeview software. Euclidean distance was used as the similarity measure, and the average linkage method based on the observed centroids was employed for grouping.

[0056] Subcellular localization prediction: Protein subcellular localization prediction was performed using the CELLO classification system based on multi-class support vector machines.

[0057] Domain annotation: Using InterProScan software, the protein sequence was compared with the Pfam database in the InterPro member database to complete the identification of protein domains.

[0058] Gene Ontology (GO) annotation: Differentially expressed protein sequences were locally homologous aligned using the NCBI BLAST+ client and InterProScan, and then homologous sequences were matched and annotated into GO entries using Blast2GO software. A self-written R script was used to visualize the GO annotation results.

[0059] Kyoto Encyclopedia of Genetics and Genomes (KEGG) pathway analysis: After annotation, the target protein is compared with the KEGG database to obtain KEGG homology numbers, and then mapped to known KEGG pathways for functional enrichment analysis.

[0060] Early embryo FABP4 protein detection: Embryos were fixed in paraformaldehyde for 30 minutes, washed three times in wash buffer (WB) for 10 minutes each time, permeabilized in permeabilization buffer (PB) for 30 minutes, washed three times in wash buffer for 10 minutes each time, blocked in blocking buffer (BB) for 2 hours, and washed once. They were then incubated overnight at 4°C with Alexa Fluor® 647-labeled anti-FABP4 primary antibody (Abcam, Cambridge, UK). Imaging analysis was performed using a laser confocal microscope (Zeiss LSM880).

[0061] FABP4 protein inhibition assay: Embryos were divided into two groups. The experimental group was treated with G-1 culture medium containing 200 nM BMS-309403 (BMS, Princeton, USA), while the control group was treated with G-1 culture medium only. Two-cell embryos were cultured for one day, and embryonic development was observed. (The inhibition constant Ki of BMS-309403 for FABP4 is <2 nM, for FABP3 it is 250 nM, and for FABP5 it is 350 nM. At a concentration of 200 nM, FABP4 is completely inhibited while preserving the compensatory function of FABP3.) Reactive oxygen species (ROS) detection: Embryos were incubated in 10 µM DCFH-DA solution (Beyotime Biotechnology, Jiangsu) at 37°C for 30 minutes, then washed three times with PBS containing 0.1% PVP and placed on glass slides. Images were acquired using a fluorescence microscope (Nikon, Tokyo, Japan).

[0062] Results Discussion Embryonic development: All seven groups of embryos used for model validation exhibited a stable and consistent developmental delay phenotype. The normally developing embryo group (C-8C) developed according to the expected timeline; while the developmentally delayed group (M-8C) showed a 24-hour developmental lag starting from day 1 (D1). Notably, despite the developmental delay, the M-8C embryos still successfully developed to the blastocyst stage and completed hatching, confirming the stability and reliability of the model constructed in this application.

[0063] Table 1: Effects of fetal bovine serum (FBS) on the mean cell number of early mouse embryos in D1 stage.

[0064] Data are expressed as mean ± standard error (Mean ± SEM). Paired t-tests were used for statistical analysis. *P < 0.05 compared to the control group.

[0065] Differential protein analysis among different developmental groups: Univariate analysis was performed using fold change (FC) > 1.5 and P < 0.05 (t-test) as the criteria to screen differentially expressed proteins among the groups. Among the 2991 proteins detected, 165 differentially expressed proteins (DEPs) were identified, of which 62 were upregulated and 103 were downregulated.

[0066] Among the upregulated proteins, five are related to mitochondrial metabolism and energy homeostasis: enoyl-CoAΔ isomerase (Eci1), fumarate hydratase (Fh), monofunctional C1-tetrahydrofolate synthase (Mthfd1l), mitochondrial inner membrane transport subunit Tim9 (Timm9), and NADH dehydrogenase iron-sulfur protein 7 (Ndufs7).

[0067] Other differentially expressed proteins include: 3 cytoskeleton structural proteins, 2 proteins involved in protein synthesis and processing, 4 cell cycle, proliferation and signal transduction regulators, 4 intracellular transport and membrane transport-related components, and 3 transcription and epigenetic regulators.

[0068] Volcano plots visually demonstrate the differences in protein expression between the normal development group and the developmentally delayed group (Figure 2); box plots further illustrate the differences in protein expression levels among the groups. Figure 3 ).

[0069] Figure 2The volcano plot compared the protein expression profiles of the normal development group (C-8C) and the developmentally delayed group (M-8C). The horizontal axis represents the log2 fold change in protein expression, and the vertical axis represents the negative log10 (P-value), which indicates statistical significance. Red dots indicate significantly upregulated proteins, blue dots indicate significantly downregulated proteins, and gray dots indicate proteins with no significant difference in expression. The figure labels the top 10 differentially expressed proteins (including both upregulated and downregulated proteins) and their corresponding protein numbers.

[0070] Figure 3 The box plot shows the differential protein expression patterns between the normal development group (C-8C) and the developmental delay group (M-8C). The horizontal axis represents the experimental group, and the vertical axis represents the protein expression level after log2 transformation. Red and blue are used to distinguish the expression profiles of each differentially expressed protein in different samples. Statistical significance is indicated by an asterisk: * p < 0.05, ** p < 0.01, *** p < 0.001.

[0071] Bioinformatics analysis of differentially expressed proteins Bioinformatics analysis was performed on differentially expressed proteins (DEPs) between the normal development group (C-8C) and the developmental delay group (M-8C), including subcellular localization prediction and functional enrichment analysis based on the GO, KEGG and WikiPathways databases.

[0072] Subcellular localization analysis showed that differentially expressed proteins were mainly located in the nucleus (36.04%), cytoplasm (29.44%), mitochondria (12.69%), extracellular space (11.68%), and plasma membrane (6.6%). Figure 4 ).

[0073] Enrichment analysis of the top 20 enriched domains revealed that these domains are significantly involved in RNA processing and translation, lipid metabolism and signal transduction, cytoskeleton dynamics, calcium-mediated signaling pathways, and basal metabolic pathways. Figure 5 ).

[0074] Gene Ontology (GO) enrichment analysis categorizes differentially expressed proteins into three classes: cellular components, molecular functions, and biological processes.

[0075] The most significantly enriched cellular components included the cell membrane, extracellular regions, and organelles; in terms of molecular function, the differentially expressed proteins were mainly associated with RNA binding and calcium ion homeostasis; biological process analysis showed that they were enriched in metabolic pathways, cell proliferation, and signal transduction. Figure 6 ).

[0076] Pathway analysis by the Kyoto Encyclopedia of Genetics and Genomes (KEGG) showed that cytoskeleton regulatory pathways and spliceosome pathways were most significantly enriched. Figure 7 ).

[0077] For detailed localization of differentially expressed proteins in the spliceosome pathway, see [link to relevant documentation]. Figure 8 and Figure 9 The location of it in the amino acid metabolic pathway is shown in [reference needed]. Figure 10 and Figure 11 .

[0078] Figure 5 The table below shows the domain enrichment pattern, with the horizontal axis representing the enrichment factor (Rich Factor ≤ 1). The enrichment factor is defined as the proportion of differentially expressed proteins annotated to a specific domain to the total number of identified proteins annotated to that domain. The size of the bubbles corresponds to the number of differentially expressed proteins in each domain category. The color gradient reflects the statistical significance of domain enrichment obtained by Fisher's exact test; the intensity of the color represents the transformed p-value (−log10), with darker red indicating higher significance (smaller p-value). Domain prediction of differentially expressed proteins was performed using InterProScan software. Complete analysis results are shown in a supplementary table, with the protein counts for the top 20 domains displayed in a separate bar chart.

[0079] Figure 6 The results of gene ontology (GO Level 2) functional annotation are displayed, divided into biological processes (blue), molecular functions (red), and cellular components (orange). The vertical axis represents the functional category, and the horizontal axis represents the number of differentially expressed proteins annotated to each category.

[0080] Figure 7 This displays the pathway enrichment pattern. The horizontal axis represents the enrichment factor (Rich Factor ≤ 1), calculated as the proportion of differentially expressed proteins annotated to a specific KEGG pathway out of the total number of identified proteins annotated to that pathway. The size of the bubbles corresponds to the number of differentially expressed proteins within each pathway. The color intensity reflects the statistical significance of the enrichment (Fisher's exact test), and the gradient represents the transformed p-value (−log10), with darker red indicating higher significance (smaller p-value).

[0081] To investigate the role of FABP4 protein in embryonic development. FABP4 protein is expressed in early embryos and is mainly located in the cytoplasm (…). Figure 12 ) Figure 12 Compared with the control group, the fluorescence intensity of the FBS-treated group was significantly reduced, indicating that the expression of the target protein was inhibited. Scale bar = 100 μm. Inhibition of FABP4 significantly slowed down embryonic development. Figure 13 ), Figure 13 Representative images of embryos from the control and interference groups at stages D0, D1, and D2. Scale bar = 100 μm. Simultaneously, the level of reactive oxygen species in the embryos increased (…). Figure 14 ), Figure 14The ROS fluorescence intensity was significantly increased in the FBS-treated group, indicating an increase in oxidative stress levels. Scale bar = 100 μm.

[0082] This application employed data-independent acquisition (DIA)-based proteomics technology to successfully identify high-abundance proteins such as actin, GAPDH, heat shock protein 90 (HSP90), and proliferating cell nuclear antigen (PCNA) in mouse embryos. The detection of these proteins not only validated the sensitivity and reliability of our experimental method but also provided important evidence for elucidating the molecular mechanisms of embryonic development. Actin and GAPDH, encoded by housekeeping genes, are classic proteins involved in cytoskeleton maintenance and basal metabolic processes; their stable detection in various samples indicates that the experimental workflow, from tissue lysis and enzymatic digestion to mass spectrometry analysis, has been effectively optimized. PCNA was highly expressed during cleavage, a result confirmed by immunofluorescence and Western blot, further supporting the reliability of this dataset. Functional analysis showed that the identified proteins are associated with core developmental processes: high expression of ENO1 and LDHA suggests a shift in embryonic metabolism towards glycolysis, consistent with the early embryonic adaptation to hypoxic environments. Notably, complete knockout of histone H3.3 led to embryonic developmental arrest, highlighting its crucial role. Regarding the establishment of cell polarity, TPM3 participates in cell polarization by regulating actin filament assembly; while intermediate filament protein VIM maintains mechanical stability during embryonic development. In summary, DIA-based proteomics can effectively capture key molecular features related to metabolic reprogramming and morphogenesis during mouse embryonic development, and this dataset can provide valuable resources for subsequent mechanistic research and applications in reproductive medicine.

[0083] Among the metabolic regulation-related subtypes, Q5FW60 (major urinary protein 20, Mup20), P04117 (fatty acid-binding protein 3, FABP3), and P11404 (apolipoprotein A1, APOA1) (acetylated, N-terminus) were significantly downregulated. Notably, their corresponding peptides exhibited methylation modifications: DGETFQLMELYGREPDLSSDIKEK (M8), LVSSENFDDYMK (M11), DGDKLVVECVMK (C9), and ADAFVGTWK (acetylated). These modifications may affect lipid transport and energy metabolism. The downregulation of these proteins suggests that fetal bovine serum treatment may lead to impaired lipid-mediated energy supply. In contrast, homeostasis-related protein subtypes, including Q60590 (lipotransferrin 2, LCN2) and O08716 (retinol-binding protein 4, RBP4), maintained stable expression, supporting basic embryonic life activities by maintaining iron homeostasis and vitamin A metabolism. This application observed enrichment of the lipid transporter family, reflecting a relative increase in the abundance of some highly expressed subtypes, rather than an overall upregulation. Among them, P22935 and A2BIM8 were highly expressed in the treated group and met the differential expression criteria, significantly contributing to the domain enrichment signal. Downregulation of lipid metabolism-related subtypes signifies the embryonic developmental shift from dependence on maternal lipid energy supply to zygote-autonomous metabolic regulation.

[0084] GO enrichment analysis showed a significant enrichment of cytoskeleton-related proteins in the treatment group (P < 0.01, FDR = 0.0897). The proportion of these proteins in the interference group (14.55%) was significantly higher than that in the control group (5.82%), suggesting that cytoskeleton-related processes play a key role in fetal bovine serum-mediated developmental disturbances. Among the 24 enriched cytoskeleton-related proteins, several have been confirmed to be involved in neuronal differentiation and axonal guidance. Crmp1 mediates actin cytoskeleton rearrangement in response to extracellular signals; IQGAP1 regulates the dynamic assembly of the actin cytoskeleton; in addition, the filamentous actin-binding protein Afadin interacts with Nectin and is located in cadherin-dependent adhesion junctions. Embryonic sample data further suggest that the enrichment of cytoskeleton proteins reflects embryonic developmental delay or arrest.

[0085] Enrichment analysis of amino acid metabolism pathways showed that arginine biosynthesis (mmu00220) and arginine and proline metabolism (mmu00330) were moderately enriched in the treatment group. Within the arginine biosynthesis pathway, three related proteins were identified in the treatment group: P05202 (ornithine aminotransferase) and P15105 (arginine succinate synthase). This suggests certain biological characteristics: adaptive metabolic regulation: activation of arginine biosynthesis may be an adaptive response of the embryo to nitrogen metabolism requirements or specific cell type requirements, particularly related to rapid proliferation, antioxidant defense, or immune regulation.

[0086] Regarding the arginine and proline metabolic pathway (mmu00330), the enrichment of four proteins, including Q6P8J7 (proline dehydrogenase) and Q9D964 (arginase 2), showed critical significance (P = 0.047, FDR = 0.967), suggesting the following biological significance: Microenvironmental stress response: Proline metabolism may participate in cell osmolarity regulation or play a protective role against oxidative stress. In developmentally delayed embryos, significant downregulation of lipid transporter family members (including FABP3 and LCN2) and proteins related to the arginine-proline metabolic pathway (mmu00330) was observed. Reduced expression of FABP3 (fatty acid binding protein 3) may directly impair the embryo's utilization of maternal lipids for energy, forcing the embryo to compensate by shifting to glucose metabolism. This metabolic shift contradicts the characteristic of early embryos typically preferring lipid energy. Simultaneously, inhibition of arginine biosynthesis-related enzymes (P05202, P15105) disrupts urea cycle function and reduces NO signaling. These synergistic metabolic alterations collectively cause an imbalance in overall energy supply, which is the core mechanism leading to delayed embryonic development. Significant enrichment of cytoskeleton assembly-related proteins and S100 calcium-binding proteins suggests an abnormal differentiation pattern in delayed embryos. Specifically, impaired calcium-dependent function of S100A6 (O54774) disrupts cell polarity establishment, leading to defective blastocoel expansion. These observations are highly consistent with protein expression profiles reported in clinical cases of failed embryo compaction. Significant enrichment of negative mRNA splicing regulation (GO:0048025), particularly downregulation of SR protein family members, triggers aberrant alternative splicing, leading to an imbalance of key embryonic gene subtypes such as OCT4 and SOX2, ultimately disrupting the balance between pluripotency maintenance and differentiation initiation. These results suggest that exogenous components in fetal bovine serum (FBS) disrupt the fine regulatory network of zygotic genome activation.

[0087] Embryonic developmental rate has become a core focus of modern reproductive medicine research. Extensive evidence indicates that endogenous regulatory mechanisms have a profound impact on developmental processes: ovarian stimulation alters embryonic developmental dynamics; key mitochondrial metabolic factors determine developmental rate; and pre-ovulatory follicle diameter is significantly correlated with embryonic developmental potential. Regarding exogenous intervention mechanisms, studies have confirmed that serum oxidative stress metabolite levels during oocyte collection are associated with delayed blastocyst development after OPU-ICSI. Furthermore, the addition of IGF-1 can target and regulate embryonic growth rate, and temperature intervention can precisely control developmental timing. Clinical studies show that developmental dynamics are closely related to pregnancy outcomes: delayed blastocyst development may lead to recurrent implantation failure; increased oocyte cytoplasmic volume can accelerate cleavage; and in equines, in vitro developmental rate affects foal survival and sex ratio (p<0.05). Currently, quality control standards for human in vitro embryo culture systems mainly rely on mouse embryo testing, with blastocyst expansion rates typically exceeding 80%. Due to the practical difficulties in obtaining human embryos, most animal models employ single-gene knockout / knockdown strategies. The development of high-precision proteomics sequencing technology based on DIA in recent years has provided new tools for studying protein expression patterns in early embryos. This technology has been applied to the evaluation of disease-related embryonic models under multi-pathway regulation, laying an important foundation for the future development of human embryonic diagnostic technology.

[0088] Fatty acid-binding protein 4 (FABP4) is expressed in adipose tissue, macrophages, liver, limbs, whole brain, and placenta, playing multiple roles in reproduction, pregnancy, and offspring health. The most common physiological ligand for FABP4 is non-esterified fatty acids, which can be transported from the cell membrane to various organelles such as the nucleus and mitochondria. Inhibition or silencing FABP4 reduces intracellular free fatty acid levels, increases reactive oxygen species production, decreases mitochondrial membrane potential, and reduces ATP content. Fatty acids are not only crucial for energy substrate storage but also essential for maintaining cell membranes; the plasma membrane surface area increases significantly during embryonic division. The plasma membrane surface area increases by 74% from the 2-cell to 4-cell stage, suggesting a greater increase in the later stages of embryonic implantation. The fat content within the embryo decreases significantly during development. Inhibition of FABP4 leads to insufficient cellular energy supply and slowed cell membrane formation.

[0089] Fetal bovine serum-induced embryonic developmental delay disrupts normal embryogenesis by interfering with the dynamic coupling network that coordinates cytoskeleton assembly, nucleoplasmic transport, and metabolic processes, ultimately leading to a developmental delay phenotype. This developmental asynchrony may cause a temporal mismatch with the endometrial implantation window, potentially resulting in pregnancy failure.

[0090] This experiment also verified that the fatty acid transporter FABP4 exists in early mouse 8-cell embryos. A FABP4 inhibitor, BMS-309403, at a concentration of 200 nM, caused embryonic developmental delay and increased reactive oxygen species (ROS) levels.

[0091] The key design focus of this invention is: First, this invention successfully constructed an animal model of embryonic developmental delay using fetal bovine serum or fatty acid-binding protein 4 (FABP4) expression inhibitors. The model is stable and reliable with a clear phenotype. The embryos exhibit developmental delay and a significant reduction in cell number, providing an ideal experimental model for studying the molecular mechanisms of embryonic developmental delay.

[0092] Secondly, this invention applies the data-independent acquisition (DIA) proteomics analysis method to the constructed model. Compared with traditional data-independent acquisition methods, DIA technology has higher stability, less data loss, and can accurately quantify low-abundance proteins. It can comprehensively analyze the protein expression profile changes related to embryonic developmental delay and successfully identified 165 differentially expressed proteins, including FABP4, FABP3, and Eci1, revealing multi-dimensional regulatory mechanisms such as lipid metabolism disorders, amino acid metabolism imbalances, and abnormal cytoskeleton assembly.

[0093] Third, the embryonic developmental delay animal model constructed by the method of the present invention can be used to screen candidate drugs for the prevention or treatment of embryonic developmental delay, and has good clinical application prospects and industrial value.

[0094] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A method for constructing an animal model of delayed embryonic development, characterized in that: Includes the following steps: S1. Obtaining early embryos; S2. The early embryos obtained in step S1 are cultured in contact with a growth retardation inducer. S3. After culturing, embryos with delayed development are obtained, thus obtaining an animal model of embryonic developmental delay.

2. The construction method according to claim 1, characterized in that: The developmental delay inducer in step S2 is fetal bovine serum. Step S2 specifically involves: transferring early embryos into a culture medium containing fetal bovine serum for culture; wherein: the volume percentage concentration of fetal bovine serum in the culture medium is 10%, the early embryos are 2-cell embryos obtained 44-46 hours after hCG injection, the fetal bovine serum is inactivated fetal bovine serum, and the culture medium is G-1 culture medium.

3. The construction method according to claim 1, characterized in that: The growth retardation inducer in step S2 is a fatty acid binding protein 4 expression inhibitor, i.e., a FABP4 expression inhibitor, which is selected from at least one of FABP4-specific chemical inhibitors or anti-FABP4 antibodies.

4. The construction method according to claim 3, characterized in that: The growth retardation inducer in step S2 is a FABP4-specific chemical inhibitor, namely BMS-309403, and the concentration of BMS-309403 used is 200 nM.

5. The construction method according to claim 1, characterized in that: The early embryo in step S1 is an early mammalian embryo, and the mammal is a mouse. The early embryo is in the 2-cell stage to the 8-cell stage.

6. The application of a proteomics analysis method based on data-independent acquisition in an animal model of embryonic developmental delay constructed by the construction method according to any one of claims 1 to 5, characterized in that: The proteomics analysis method includes the following steps: First, provide embryo samples for animal models of delayed embryonic development; Second, the embryonic samples were analyzed using a non-data-dependent acquisition proteomics method to obtain protein expression profiles; Third, based on the protein expression profile, identify differentially expressed proteins associated with delayed embryonic development and analyze animal models of delayed embryonic development.

7. The application according to claim 6, characterized in that: The non-data-dependent proteomics acquisition method in step S2 includes: extracting proteins from embryonic samples and enzymatically digesting them to obtain peptide samples; performing mass spectrometry detection on the peptide samples using dia-PASEF mode; and analyzing the mass spectrometry data using DIA-NN software to obtain protein expression profiles.

8. The application according to claim 6, characterized in that: The differentially expressed proteins include at least one of fatty acid-binding protein 4, fatty acid-binding protein 3, enoyl-CoAΔ isomerase, fumarate hydratase, monofunctional C1-tetrahydrofolate synthase, mitochondrial inner membrane transport subunit Tim9, and NADH dehydrogenase iron-sulfur protein 7.

9. The use of an animal model of delayed embryonic development constructed by the method described in any one of claims 1 to 5 in screening candidate drugs for the prevention or treatment of delayed embryonic development.

10. The application according to claim 9, characterized in that: The screening process specifically involves: exposing early embryos to the candidate drug; detecting at least one of the following indicators: embryonic development rate, number of embryonic cells, reactive oxygen species (ROS) level, or FABP4 expression level; and determining the candidate drug as effective if, compared with the control group that has not been exposed to the candidate drug, the embryonic development rate is improved, the number of cells is increased, the ROS level is reduced, or the FABP4 expression level is restored.

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