Application of hypoxia inducible factor-1 alpha subunit activator in embryo telomere length recovery

Abstract

The invention discloses application of a hypoxia inducible factor-1 alpha subunit activator in embryo telomere length recovery, and belongs to the technical field of biological medicine. According to the present invention, embryos at different stages are subjected to single cell sequencing, a Tert gene single cell regulation network is constructed, key transcription factors affecting the specificity of the embryos at the early embryo development stage are screened, and a series of experiments verify that the Hif1alpha achieves the function through HRE in the region 2 of the Tert promoter region, has the transcription regulation effect at the upstream of the Tert, and can be used as a transcription regulation and control gene. Therefore, the telomere length of the embryo is influenced. Therefore, when the fertilized ovum is subjected to in-vitro culture, a hypoxia inducible factor-1alpha subunit activator is added in different stages of embryo culture, Hif1alpha expression is activated, Tert gene expression level is improved, telomere extension of the embryo is realized, and the telomere length of the embryo subjected to in-vitro culture is equivalent to that of an embryo obtained by natural pregnancy.

Description

Technical Field

[0001] The invention relates to application of a hypoxia inducible factor-1α subunit activator in restoring embryonic telomere length, and belongs to the technical field of biomedicine. Background Art

[0002] Assisted reproductive technology (ART) is a series of medical methods to help couples with fertility difficulties achieve fertility. These technologies include artificial insemination (AI) and in vitro fertilization-embryo transfer (IVF-ET) and their derivative technologies. IVF-ET refers to the process of removing eggs from female ovaries, fertilizing them with sperm in vitro and culturing them for 3-5 days, and then transplanting embryos that have developed to the blastomere stage or blastocyst stage into the uterine cavity to implant and develop into a fetus. It can be seen that ART can be divided into cleavage stage transplantation strategy and blastocyst stage transplantation strategy according to the different developmental stages of embryo transplantation. Compared with the cleavage stage transplantation strategy, extending the culture of fertilized eggs to the blastocyst stage before transplantation can screen and remove embryos with potential developmental disorders, which is more conducive to embryo implantation into the endometrium, and has a higher live birth rate, and is more widely used in clinical practice. However, the early embryonic development stage corresponding to the in vitro culture of embryos in ART is the critical time window for telomere extension. Studies have reported that the blastocyst stage transfer strategy will affect the telomerase-mediated telomere extension process during the development from morula to blastocyst, resulting in shortened telomere length in embryos and offspring.

[0003] Although the offspring of cleavage-stage transplants have longer telomeres, cleavage-stage transplants are not conducive to screening embryos with good developmental conditions, and the clinical live birth rate is lower, which will cause great pressure on patients. On the other hand, the cleavage-stage transplant strategy is not conducive to the synchronization of the physiological conditions of the embryo and the endometrium, and the risk of miscarriage and abnormal developmental patterns of offspring is greatly increased. Therefore, it is urgent to explore ART optimization strategies to restore the shortened embryo telomeres caused by the blastocyst-stage transplant strategy. Summary of the invention

[0004] In order to solve the above problems, the present invention adds a Hif1α small molecule activator to the embryo culture medium and then cultures the fertilized egg until the blastocyst stage, thereby achieving the effect of restoring the telomere length in the blastocyst.

[0005] One object of the present invention is to provide a product for restoring telomere length of embryos in vitro, wherein the product contains a hypoxia inducible factor-1α subunit activator.

[0006] Further, the product includes a medicine;

[0007] And / or, the embryo comprises a blastocyst.

[0008] Furthermore, when the product is used to restore embryonic telomere length, the step of adding the product during in vitro culture is included.

[0009] Further, the hypoxia-inducible factor-1α subunit activator includes a prolyl hydroxylase inhibitor;

[0010] And / or, the hypoxia inducible factor-1α subunit activator is selected from DMOG (Dimethyloxallyl Glycine), cobalt chloride (CoCl 2 ), deferoxamine (DFO), IOX2, DHB, GSNO (S-Nitroso-L-glutathione), hydralazine hydrochloride, mimosatin (L-Mimosine), Adaptaquin, PHD-1-IN-1, Enarodustat (JTZ-951), VH298, TP0463518, IOX4, Vadadustat (AKB-6548, B-506, PG-1016548), MK-8617, FG-2216, Molidustat (BAY 85-3934), Desidustat (ZYAN1, ZYAN1-1001), Daprodustat (GSK1278863) or more thereof;

[0011] And / or, the concentration of the hypoxia inducible factor-1α subunit activator in the in vitro culture medium is 0.001-500 μM.

[0012] Another object of the present invention is to provide a use of a hypoxia inducible factor-1α subunit activator in the preparation of a product for restoring the telomere length of an in vitro embryo or a product for restoring the telomere length of a newborn offspring obtained by embryo transplantation.

[0013] Further, the newborn offspring are human or non-human newborn offspring;

[0014] and / or, restoring embryonic telomere length comprises: adding a hypoxia-inducible factor-1α subunit activator during at least one stage of embryo culture to obtain embryos with extended telomeres;

[0015] And / or, restoring the telomere length of newborn offspring obtained by embryo transfer includes: adding a hypoxia-inducible factor-1α subunit activator during at least one stage of the embryo culture process until a blastocyst is obtained, and obtaining newborn offspring with extended telomeres through blastocyst transplantation.

[0016] Further, the hypoxia-inducible factor-1α subunit activator includes a prolyl hydroxylase inhibitor;

[0017] And / or, the hypoxia-inducible factor-1α subunit activator is selected from one or more of DMOG, cobalt chloride, deferiprone, IOX2, DHB, GSNO, hydralazine hydrochloride, mimosin, Adaptaquin, PHD-1-IN-1, enastat, VH298, TP0463518, IOX4, vadadustat, MK-8617, FG-2216, molistat, dedostat, and dapnostat;

[0018] And / or, the concentration of the hypoxia inducible factor-1α subunit activator in the in vitro culture medium is 0.001-500 μM.

[0019] Another object of the present invention is to provide an embryo in vitro culture method (for extending embryo telomere length), comprising the step of adding a hypoxia inducible factor-1α subunit activator at at least one stage during the embryo culture process.

[0020] Furthermore, the embryo in vitro culture method specifically comprises the following steps:

[0021] S1. Acquisition of sperm and eggs, and obtaining fertilized eggs through in vitro fertilization;

[0022] S2. Culturing the fertilized eggs in vitro until blastocysts are obtained, and adding a hypoxia-inducible factor-1α subunit activator in at least one stage of the in vitro culture.

[0023] Furthermore, the stage for adding the hypoxia-inducible factor-1α subunit activator includes the cleavage stage, the morula stage, or the blastocyst stage;

[0024] And / or, when the embryo is a human embryo, the morula stage is 72-120 hours after fertilization, and the blastocyst stage is 120-168 hours after fertilization.

[0025] Another object of the present invention is to provide a method for constructing a blastocyst model with restored telomere length, comprising the following steps:

[0026] The blastocyst model is constructed by adding a hypoxia-inducible factor-1α subunit activator during the culture process of at least one stage of the fertilized egg developing into the blastocyst.

[0027] A method for constructing an animal model for restoring telomere length by blastocyst stage transplantation comprises the following steps:

[0028] The animal model is constructed by adding a hypoxia-inducible factor-1α subunit activator during the culture process of at least one stage of the fertilized egg developing into a blastocyst, and producing newborn offspring through blastocyst embryo transplantation.

[0029] Further, the newborn offspring is a non-human animal;

[0030] and / or, the non-human animal comprises rats and / or mice;

[0031] and / or, the hypoxia inducible factor-1α subunit activator comprises a prolyl hydroxylase inhibitor;

[0032] And / or, the hypoxia-inducible factor-1α subunit activator is selected from one or more of DMOG, cobalt chloride, deferiprone, IOX2, DHB, GSNO, hydralazine hydrochloride, mimosin, Adaptaquin, PHD-1-IN-1, enastat, VH298, TP0463518, IOX4, vadadustat, MK-8617, FG-2216, molistat, dedostat, and dapnostat;

[0033] And / or, the concentration of the hypoxia inducible factor-1α subunit activator in the in vitro culture medium is 0.001-500 μM.

[0034] The present invention also provides an animal model constructed by the above construction method for restoring telomere length by blastocyst stage transplantation, and the application of the animal model in telomere function research, aging research or assisted reproductive technology research.

[0035] Beneficial effects of the present invention:

[0036] The present invention compares the Tert gene regulatory network in the 8-cell stage and the 32-cell stage, discovers the key transcription factor Hif1α that specifically regulates telomere extension in the embryonic stage, and explores its influence mechanism, confirming that Hif1α exerts its regulatory function by binding to HRE in region 2 of the Tert promoter region, thereby affecting the telomere length of the embryo, and through a series of experiments, proves that the regulatory effect of Hif1α on Tert occurs in the early embryonic development stage. Based on this innovative discovery, after adding Hif1α activator to the embryo culture medium, the embryo was cultured and it was found that it can activate Hif1α expression and upregulate the Tert gene level, thereby extending the telomere length, making it comparable to the telomere length of the embryo in the cleavage stage, i.e., the embryo transplanted in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The expression of TertmRNA in the blastocyst on the fourth day after fertilization obtained by the cleavage stage transfer strategy and the blastocyst stage transfer strategy.

[0038] Figure 2 Telomere length in day 4 post-fertilization blastocysts obtained for cleavage-stage transfer strategy and blastocyst-stage transfer strategy.

[0039] Figure 3This is the result of screening key transcription factors by constructing the Tert gene regulatory network.

[0040] Figure 4 The expression of Hif1α protein in comparative example 1 group and comparative example 2 group.

[0041] Figure 5 Schematic diagram of the Hif1α siRNA injection experimental process.

[0042] Figure 6 This is the expression level of Hif1α mRNA in the Hif1α siRNA injection experiment.

[0043] Figure 7 The expression level of Hif1α protein in the Hif1α siRNA injection experiment.

[0044] Figure 8 is the expression level of Tert mRNA in the cleavage-siRNA group, cleavage-control group and blastocyst-control group.

[0045] Fig. 9 Telomere length in the cleavage-siRNA group, cleavage-control group and blastocyst-control group.

[0046] Fig.10 Schematic diagram of the two HREs in the promoter region of the Tert gene.

[0047] Fig.11 The results show the enrichment of region 1 DNA fragments and region 2 DNA fragments by Hif1α antibody during normoxic and hypoxic culture.

[0048] Fig.12 Schematic diagram for the construction of luciferase reporter plasmids containing wild-type and mutant HRE Tert promoters.

[0049] Fig.13 The overexpression effect of Hif1α in MEFs cultured under hypoxia was measured with or without transfection of Hif1α overexpression plasmid.

[0050] Fig.14 To measure the expression of reporter genes in MEFs cultured in hypoxia after transfection with wild-type reporter gene plasmid or mutant reporter gene plasmid and transfection with Hif1α overexpression plasmid.

[0051] Fig.15 Schematic diagram of the action mechanism of DMOG.

[0052] Fig.16 Experimental groups set up for embryo culture with or without the addition of DMOG.

[0053] Fig.17The expression of Hif1α in Example 2, Comparative Example 1 and Comparative Example 2 is shown.

[0054] Fig.18 The Tert mRNA expression levels in Example 2, Comparative Example 1 and Comparative Example 2.

[0055] Fig.19 It is the telomere length of the blastocyst in Example 2, Comparative Example 1 and Comparative Example 2.

[0056] Fig. 20 The expression of Hif1α in Example 3, Comparative Example 1 and Comparative Example 2 is shown.

[0057] Fig.21 The expression levels of Tert mRNA in Example 3, Comparative Example 1 and Comparative Example 2.

[0058] Fig. 22 It is the telomere length of the blastocyst in Example 3, Comparative Example 1 and Comparative Example 2. DETAILED DESCRIPTION

[0059] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0060] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps described in the literature in the art can be used. If no detailed explanation is given for the reagents used in the examples, they are all conventional reagent products that can be obtained commercially.

[0061] Terminology explanation:

[0062] The "recovery" or "extension" of telomere length mentioned in the present invention have the same meaning, which means: during the in vitro culture of embryos, telomeres will shorten, which is reduced relative to the telomere length of embryos developing in vivo, thereby affecting the telomere length of newborn offspring produced by the embryo transplantation strategy, and the addition of hypoxia-inducible factor-1α subunit activator can overcome the telomere shortening caused by in vitro culture, so that the telomere length of embryos obtained in vitro is equivalent to that of natural pregnancy embryos, so that the telomere length of offspring obtained by embryo transplantation also reaches a level equivalent to that of natural pregnancy offspring. Specifically, the so-called "recovery" means that the telomere length of embryos cultured in vitro reaches a level equivalent to that of the reference, taking the mother's natural pregnancy embryo as a reference; the so-called "extension" means that the telomere length of embryos cultured in vitro without the addition of hypoxia-inducible factor-1α subunit activator is significantly extended compared with the reference (p<0.05).

[0063] About the technical solution of the present invention:

[0064] The present invention mainly provides a related product for in vitro fertilization-embryo transfer (IVF-ET) technology. IVF-ET is usually called test tube baby technology, which mainly includes the following steps:

[0065] (1) Sperm preparation: obtaining human or non-human semen samples, processing and screening out healthy sperm that meet the requirements;

[0066] (2) Egg retrieval: Hormonal drugs are administered to stimulate the ovaries to promote follicle development so that multiple eggs can be collected. When the follicles develop to an appropriate size, human chorionic gonadotropin (HCG) or similar drugs are injected to trigger ovulation, and mature eggs are retrieved before ovulation.

[0067] (3) In vitro fertilization: the obtained eggs and sperm are cultured together in the laboratory to achieve fertilization; or a single sperm is injected directly into the egg using intracytoplasmic sperm injection;

[0068] (4) Embryo culture: After fertilization, the fertilized egg is cultured in culture medium for several days until it develops into an early embryo;

[0069] (5) Embryo evaluation: Assess the quality of embryos before transplantation and select the embryos most likely to successfully implant;

[0070] (6) Embryo transfer: the selected embryo is placed into the mother's uterus;

[0071] (7) Progesterone support: After embryo transfer, progesterone and other hormone support are given to promote endometrial receptivity and embryo implantation;

[0072] (8) Subsequent testing and monitoring.

[0073] It can be seen from the above steps that the key step of this technology is embryo transplantation. As described in the background technology, embryos of different stages can be transplanted during embryo transplantation, mainly including the transplantation of cleavage-stage embryos and the transplantation of blastocyst-stage embryos. Compared with the cleavage-stage transplantation strategy, the blastocyst-stage transplantation strategy has the following advantages:

[0074] (1) Better selection of embryos with developmental potential: Blastocyst transfer can more effectively select embryos with developmental potential, because embryos with chromosomal abnormalities usually cannot develop to the blastocyst stage. By prolonging blastocyst culture, some embryos with chromosomal abnormalities and poor quality can be naturally eliminated, and blastocysts with strong survival ability and good quality can be selected.

[0075] (2) Temporal synchronization with endometrial development: The embryos transferred during the blastocyst stage are more physiologically synchronized with the development of the endometrium, which helps to improve the embryo implantation rate.

[0076] (3) Reduce the multiple pregnancy rate: Due to the high implantation rate of blastocyst transfer, the number of transfers can be appropriately reduced, thereby greatly reducing the risk of multiple pregnancy.

[0077] (4) Reduce the risk of ectopic pregnancy: Blastocysts are larger than cleavage-stage embryos and are more difficult to migrate into the fallopian tube. Therefore, the incidence of ectopic pregnancy is reduced after blastocyst transfer.

[0078] (5) Improve the live birth rate: Studies have shown that the live birth rate of blastocyst transfer is higher than that of cleavage stage embryo transfer.

[0079] (6) Helps in embryo genetic diagnosis: Blastocyst culture provides sufficient time for cleavage-stage embryo biopsy and preimplantation genetic diagnosis, which helps in embryo genetic diagnosis before implantation and reduces the incidence of genetic diseases.

[0080] Based on the advantages including but not limited to the above, the present invention improves and optimizes the blastocyst stage transplantation strategy. We also tested the telomere length of the blastocysts obtained by the cleavage stage transplantation strategy and the blastocyst stage transplantation strategy, and found that the telomere length of the blastocysts that have been cultured in vitro (blastocyst stage transplantation strategy) is significantly short. Therefore, the improvement and optimization of the present invention mainly focuses on how to solve the problem of hindered telomere extension in the blastocyst stage transplantation strategy, specifically including exploring what factors in the blastocyst stage transplantation strategy lead to shortened telomere length in the embryo, and whether there are corresponding optimization strategies for this factor.

[0081] The present invention first explores the factors that affect telomere extension during the development from morula to blastocyst. The details are as follows:

[0082] The embryonic development process starts from the fertilized egg and goes through multiple stages to form a blastocyst, including the zygote stage, 2-cell stage, 4-cell stage, 8-cell stage, 32-cell stage, and 64-cell stage.

[0083] 8-cell stage: After fertilization, the embryo begins to divide. About 3 days after fertilization, the embryo will develop into a mass of 8 cells. This stage is usually called the early embryo stage. At this stage, each cell of the embryo is totipotent, that is, each cell has the potential to develop into a complete embryo. The researchers also found that the activation of the zygotic genome in the 8-cell embryo is a key node, so studying the cell regulatory mechanism at this stage is of great significance to ensure good early embryonic development.

[0084] 32-cell stage: As the embryo continues to divide, the number of cells increases. Around the fourth day after fertilization, the embryo develops into a mass of about 32 cells. The embryo at this stage is called a morula. At the morula stage, the division of embryonic cells ends, and the next stage begins cell differentiation.

[0085] Therefore the present invention:

[0086] First, using the single-cell sequencing data of early mouse embryos, including the embryo data of 8-cell and 32-cell stages, the single-cell regulatory network of the Tert gene (encoding telomerase reverse transcriptase in the telomerase complex) was constructed using the SCENIC software to find the key transcription factors that regulate Tert expression during early embryonic development, in order to restore the telomere length during the development process from morula to blastocyst stage. SCENIC screened a variety of transcription factors and obtained positive transcription factors that regulate Tert expression. Among them, hypoxia-inducible factor-1α-Hif1α and sterol regulatory element binding transcription factor 1-Srebf1 were significant, and Hif1α had the highest enrichment coefficient, suggesting that it is a key transcription factor regulating Tert.

[0087] Subsequently, we injected Hif1α siRNA into fertilized eggs and cultured them in vitro until the blastocyst stage. We counted Tert mRNA and telomere length and found that as Hif1α expression was downregulated, Tert mRNA and telomere length were also downregulated, indicating that Hif1α is located upstream of Tert and has a transcriptional regulatory effect. Furthermore, we confirmed in mouse embryonic fibroblasts (MEFs) through chromatin immunoprecipitation combined with qPCR (ChIP) and luciferase reporter gene experiments that Hif1α can bind to HRE (hypoxia response element, Hif1α binding target) in the Tert promoter region to exert transcriptional regulatory functions;

[0088] At the same time, we compared the expression of Hif1α mRNA in embryonic cells in the cleavage transfer strategy and the blastocyst transfer strategy and found that there was no significant difference, indicating that the expression of Hif1α mRNA in the in vitro cultured embryos of the blastocyst transfer strategy was not affected, and the expression of Hif1α mRNA was not the main factor affecting telomere length. The study found that the lack of Hif1α mainly comes from the post-translational regulatory mechanism. Considering that the Hif1α small molecule activator does not directly stimulate the expression of Hif1α mRNA, but can increase the stability and activity of Hif1α protein, we tried to extend the telomere length of the embryo by adding the Hif1α small molecule activator to the culture medium.

[0089] In summary, we tried to add Hif1α small molecule activator during embryo culture in order to upregulate Tert and restore telomere length. The experimental process is as follows:

[0090] During the embryo in vitro culture process (including but not limited to the cleavage embryo culture process and the blastocyst culture process), Hif1α activator was added, and it was found that the expression of TertmRNA in the group with Hif1α activator was significantly increased compared with the group without addition, and the telomere length of the blastocyst stage was detected, and it was found that its length was successfully restored. Telomere recovery means that the telomere length of the blastocyst obtained by the method disclosed in the present invention is no different from or longer than that of the blastocyst obtained by the cleavage stage transplantation strategy, and is longer than that of the blastocyst obtained by the blastocyst stage transplantation strategy without adding Hif1α small molecule activator during conventional culture, and the above difference is statistically significant (P-value<0.05).

[0091] In summary, the present invention provides a product for restoring embryonic telomere length in vitro, wherein the product contains a hypoxia-inducible factor-1α subunit (Hif1α) activator.

[0092] Preferably, the embryo includes a blastocyst. In particular, the present invention finds that during the process of embryo in vitro culture, especially during the process of in vitro culture to blastocyst, the telomere length of the blastocyst will be shortened, and the specific factors related to this defect include the transcription factor Hif1α. When the expression of Hif1α in cells is reduced, the telomere length is reduced, and when Hif1α is overexpressed, the telomere length is extended.

[0093] The method adopted by the present invention also has the following advantages:

[0094] (1) A more natural regulation method: Adding small molecules to the culture medium to affect the expression of mRNA can simulate the biochemical processes that occur naturally in the body and may be more in line with the natural laws of embryonic development.

[0095] (2) Better spatiotemporal control: Adding small molecules to the culture medium can more precisely control the duration of the substance's action, thereby intervening at specific stages of embryonic development, which helps to precisely regulate gene expression during critical periods.

[0096] (3) Reduce immune response: Adding small molecules to the culture medium does not involve the direct introduction of exogenous genetic material, thereby reducing the risk of causing an immune response in the recipient.

[0097] (4) Reduce embryo damage: By adding small molecules to the culture medium, no destructive operations are performed on the embryo, thus avoiding physical damage to the embryo.

[0098] (5) Improve efficiency and repeatability: The method of adding small molecules to the culture medium is easier to standardize and automate, thereby improving efficiency in actual work.

[0099] (6) Reduce the risk of genetic manipulation: Compared with methods involving genetic manipulation such as directly injecting mRNA into the fertilized egg, adding small molecules to the culture medium is a milder method that can reduce direct genetic intervention on the embryo, thereby reducing the potential risk of causing unexpected gene mutations or non-target effects.

[0100] (7) Reduce ethical controversy and improve safety: Direct genetic manipulation of embryos may cause ethical controversy. Adding small molecules to the culture medium may reduce such controversy and avoid direct manipulation of the embryo's genetic material, which is better in terms of safety.

[0101] Moreover, it is well known that different cells, such as mature cells and embryonic cells, have significant differences in many aspects: mature cells are differentiated cells with specific functions and morphologies, while embryonic cells, especially cells before the blastocyst stage, are usually pluripotent or totipotent, and embryonic development is a dynamic process; for example, the gene expression characteristics of mature cells and embryonic cells are different. Mature cells express a specific set of genes, which are usually related to their functions and morphologies, while embryonic cells express a different set of genes, which are related to the pluripotency or totipotency of cells. Therefore, the results obtained when exploring the factors that affect their development may also be different. For example, in the present invention, the transcription factors that affect the telomere length of embryonic cells are sought, and mature cells are different from embryonic cells, and the same transcription factor may have completely different effects on the two. The reason is:

[0102] The cell state and function of mature cells are different from those of embryonic cells. In embryonic cells, transcription factors are usually in a state of activating pluripotency and differentiation, and these transcription factors may show different functional modes at different stages of embryonic development. Therefore, the effect of the same factor on cells is not clear, not to mention the difference between mature cells and embryonic cells. Moreover, in mature cells, transcription factors usually have more specific functions, mainly regulating the expression of genes related to their cell types, while in embryonic cells, the same transcription factor may simultaneously activate genes of multiple lineages to promote the determination of cell fate, so how it develops is unknown. In addition, in embryonic cells, transcription factors may form a complex interaction network with other transcription factors and regulatory factors, and may show the characteristics of "bidirectional activation", while in mature cells, the effect of transcription factors is usually unidirectional, focusing on specific cell functions. Therefore, the response mechanism of different cells to transcription factors under different physiological conditions is complex. It is necessary for the present invention to first screen and determine the factors affecting the telomere length of the blastocyst, and then verify the feasibility of restoring the telomere length.

[0103] Preferably, the product is a medicine.

[0104] Preferably, the hypoxia-inducible factor-1α subunit activator includes a prolyl hydroxylase (PHD) inhibitor; at two specific sites of the Hif1α protein, hydroxyl groups will be added (prolyl hydroxylation) under the action of prolyl hydroxylase (PHD), and this change enables Hif1α to be recognized and undergo ubiquitination and degradation. After adding a PHD inhibitor (i.e., an Hif1α activator), the unhydroxylated Hif1α will not be recognized and degraded, but directly enter the nucleus, thereby playing its role as a transcription factor and regulating the expression of a wide range of target genes involved in various important basic life activities, including the Tert gene among them. By upregulating Tert in this way, the telomere length can be further restored.

[0105] Preferably, the hypoxia-inducible factor-1α subunit activator is selected from one or more of DMOG (DimethyloxallylGlycine), cobalt chloride (CoCl 2 ), deferoxamine (DFO), IOX2, DHB, GSNO (S-Nitroso-L-glutathione), hydralazine hydrochloride, L-mimosine, Adaptaquin, PHD-1-IN-1, enarodustat (JTZ-951), VH298, TP0463518, IOX4, vadadustat (AKB-6548, B-506, PG-1016548), MK-8617, FG-2216, molidustat (BAY 85-3934), desidustat (ZYAN1, ZYAN1-1001), daprodustat (GSK1278863). In the examples of the present invention, IOX2 is most preferably used. It can be seen from the experimental results that under the same operating conditions, the concentration of IOX2 is only one-eighth of that of DMOG, but its effect on extending telomere length is better than that of DMOG.

[0106] Above, Adaptaquin has no definite Chinese name. The Chinese name of Enarodustat is "enasostat", the Chinese name of Vadadustat is "vadadustat", the Chinese name of Molidustat is "molidustat", the Chinese name of Desidustat is "desidustat", and the Chinese name of Daprodustat is "daprodustat". In the present invention, different names all refer to the same activator.

[0107] Preferably, the concentration of the hypoxia-inducible factor-1α subunit activator in the in vitro culture medium is 0.001-500 μM, for example, 0.1 μM, 1 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 45 μM, 55 μM, 60 μM, 80 μM, 100 μM, 150 μM, 200 μM, 250 μM, 300 μM, 350 μM, 360 μM, 370 μM, 380 μM, 390 μM, 450 μM, 480 μM, etc., including but not limited to the concentrations listed above.

[0108] The present invention provides the use of a hypoxia inducible factor-1α subunit activator in preparing a product for restoring the telomere length of embryos or the telomere length of newborn offspring obtained through embryo transplantation.

[0109] Preferably, the product is added during embryo culture to promote the recovery or extension of telomere length of in vitro embryos or human or non-human newborn offspring obtained by embryo transplantation. Restoring embryo telomere length helps restore telomere length of human or non-human newborn offspring transplanted at the blastocyst stage.

[0110] An embodiment of the present invention also provides an in vitro embryo culture method, comprising the step of adding a hypoxia inducible factor-1α subunit activator during at least one stage of the embryo culture process.

[0111] Preferably, the embryo culture process involving the addition of the hypoxia inducible factor-1α subunit activator includes cleavage stage culture and / or blastocyst stage culture.

[0112] The at least one "stage" mentioned in the present invention includes but is not limited to the cleavage stage, the morula stage, and the blastocyst stage.

[0113] Preferably, for human embryos, the cleavage period starts after fertilization, morulas are formed about 4 days after fertilization (such as 72-120 hours), and blastocysts are formed about 6 days after fertilization (such as 120-168 hours); for non-human embryos, there may be slight differences, such as the corresponding time of mouse morulas and blastocysts is about 3 days and 4 days after fertilization, respectively. Of course, those skilled in the art can make routine adjustments according to the species used.

[0114] Specifically, the method for culturing embryos in vitro or restoring embryo telomere length includes:

[0115] S1. Acquisition of sperm and eggs, and obtaining fertilized eggs through in vitro fertilization;

[0116] S2. Culture the fertilized eggs in vitro until blastocysts are obtained; and add a hypoxia-inducible factor-1α subunit activator during at least one stage of the in vitro culture.

[0117] Telomeres are DNA-protein complexes located at the ends of eukaryotic chromosomes, which protect the ends of chromosomes from being recognized as DNA breakage gaps and play a role in maintaining genome stability. However, as cells continue to divide, telomeres will continue to shorten with each cell division due to terminal replication problems. When telomeres are too short, the structure of the telomere complex is destroyed, and the chromosome protection function is lost, thereby activating the DNA damage response, further causing the downstream p53-p21 signaling pathway to activate, and the cells eventually enter a state of aging with cell cycle arrest. A large number of population studies have found that telomere length is related to the total mortality rate and life expectancy of the population. Telomere length is an important biological marker of population health. At the same time, a large number of animal experiments have also found that mice with telomere dysfunction will show a multi-system premature aging phenotype. Therefore, telomere shortening has been recognized as one of the most important signs of aging. The research on the above strategies also provides new clues and ideas for the study of telomere biology and the relationship between telomeres and aging.

[0118] The present invention also provides a method for constructing an animal model for restoring telomere length by blastocyst stage transplantation, comprising the following steps:

[0119] The animal model is obtained by adding a hypoxia-inducible factor-1α subunit activator during the culture process of at least one stage of the fertilized egg developing into the blastocyst, transplanting the obtained blastocyst stage embryo into a non-human surrogate mother, and producing newborn offspring.

[0120] Preferably, the method of obtaining the fertilized egg includes but is not limited to in vitro fertilization or in vivo fertilization.

[0121] Preferably, the concentration of the hypoxia-inducible factor-1α subunit activator in the in vitro culture medium is 0.001-500 μM, for example, 0.1 μM, 0.5 μM, 1 μM, 10 μM, 20 μM, 30 μM, 40 μM, 45 μM, 55 μM, 60 μM, 80 μM, 100 μM, 150 μM, 200 μM, 250 μM, 300 μM, 350 μM, 360 μM, 370 μM, 380 μM, 390 μM, 450 μM, 480 μM, etc., including but not limited to the concentrations listed above.

[0122] Specifically, the method for constructing an animal model includes:

[0123] S1. Acquisition of sperm and eggs, and obtaining fertilized eggs through in vitro fertilization;

[0124] S2, culturing the fertilized egg in vitro until a blastocyst is obtained; adding a hypoxia-inducible factor-1α subunit activator during at least one stage of the in vitro culture;

[0125] S3: Transplant the blastocyst obtained in S2 into a surrogate mother to produce newborn offspring.

[0126] The non-human animal is a rodent;

[0127] Optionally, the non-human animal is a mouse (Mus musculus);

[0128] Optionally, the mouse is selected from any of the following strains: ICR, A / He, A / J, A / SnSf, A / WySN, AKR, AKR / A, AKR / J, AKR / N, BALB / c, B6SJLF1, B6C3F1, B6D2F1, C3H, C3He, C3Hf, C57BR, C57L, C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57 BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, C58, CBA / Br, CBA / Ca, CBA / J, C BA / st, CBA / H, CB6F1, CD2F1, CFW, DBA / 1, DBA / 2, FACA, FVB, KM, NIH, NIH(S), RF, SJL, SWR, TA1, TA2, or 129;

[0129] Optionally, the male mice are 8 to 20 weeks old; preferably, 8 to 12 weeks old;

[0130] Optionally, the female mouse is an adolescent female mouse, which is 3 to 12 weeks old; preferably, 4 to 5 weeks old;

[0131] Optionally, the female surrogate mouse is 6 to 10 weeks old; preferably, 8 weeks old.

[0132] The present invention also provides an animal model obtained by the above construction method for restoring telomere length by blastocyst stage transplantation.

[0133] The animal model for restoring telomere length by blastocyst stage transplantation provided by the present invention can be used for telomere function research or aging research.

[0134] The present invention obtains a fertilized egg of a non-human animal by in vitro fertilization, and then by adding a Hif1α small molecule activator to the culture medium from the fertilized egg to the blastocyst stage, a blastocyst with restored telomere length can be obtained for subsequent embryo transplantation. The method disclosed in the present invention does not involve any steps for modifying the genetic material of mouse chromosomes or mitochondria, and the modification is for example but not limited to: gene mutation, gene editing, gene knockout, and mutagenesis. The method of the present invention does not require gene editing, has a short modeling cycle, and has reliable and stable effects; it only needs to add a Hif1α small molecule activator to the in vitro culture medium to increase the expression levels of Hif1α and TERT genes to achieve the effect of restoring the telomere length of the blastocyst. Therefore, the present invention can provide an animal model construction method and research direction for studying the mechanism of telomere shortening and exploring the optimization scheme of human clinical assisted reproductive technology.

[0135] Example 1: Exploration of the reasons why the blastocyst stage transfer strategy interferes with telomere extension

[0136] (1) Discovery of the relationship between the Tert gene and telomere elongation

[0137] We detected Tert mRNA and telomere length in blastocysts on the fourth day after fertilization obtained by cleavage stage transfer strategy and blastocyst stage transfer strategy, and found that Tert gene transcription was inhibited and telomere length was shortened in blastocysts that had been cultured in vitro in the blastocyst stage transfer strategy ( Figure 1 , 2). The results suggest that the in vitro culture process of the blastocyst transfer strategy may interfere with the transcription of the Tert gene and thus affect the telomere length.

[0138] (2) Construction of Tert gene regulatory network

[0139] Single-cell RNA expression data of mouse early embryos at different developmental stages were downloaded from the Gene Expression Omnibus (GEO) database with access number GSE136714. The split cell libraries were merged together using the DESeq2 R package. The transcript count / million sequencing fragment count (TPM) values ​​were converted to a mild logarithmic function log 2(1+TPM). A total of 28 8-cell stage embryonic cells and 53 32-cell stage embryonic cells were used for analysis. We used SCENIC software to construct a single-cell regulatory network of Tert during early embryonic development and inferred co-expression modules between transcription factors and candidate target genes. Subsequently, each co-expression module (including a transcription factor and its predicted target gene) was analyzed to identify enriched binding sites. Only modules enriched in transcription factor binding sites and their target genes were retained. Each transcription factor and its potential target gene constitute a regulator, thereby determining the transcription factors involved in regulating Tert. Hif1α was identified as having the highest enrichment coefficient, suggesting that it is a key transcription factor regulating Tert expression ( Figure 3 , where bits represents the specificity of the transcription factor binding at this position in the HUMAN.H11MO.0.C model of the hocomoco database).

[0140] (3) Effects of different treatments on Hif1α protein levels

[0141] Experimental groups:

[0142] Comparative Example 1 (positive control group, cleavage stage transplantation strategy, no DMOG): Cleavage medium without DMOG (manufacturer: Vitrolife, item number: 10128) was used during the culture process from fertilized eggs to 2-cell stage embryos. When the fertilized eggs developed to the 2-cell stage, they were transplanted into surrogate female mice. 15 2-cell stage embryos were transplanted into each female mouse, and they were cultured in vivo until they became blastocysts and then removed.

[0143] Comparative Example 2 (negative control group, blastocyst stage transfer strategy, no DMOG): During the development of fertilized eggs to the blastocyst stage, cleavage culture medium without DMOG and blastocyst culture medium without DMOG (manufacturer: Vitrolife, catalog number: 10132) were used for culture.

[0144] Experimental steps:

[0145] The blastocysts of Comparative Example 1 and Comparative Example 2 were collected about 96 hours after fertilization to extract proteins. SDS-PAGE gel electrophoresis was used to separate proteins of different molecular weights in the sample, and the electrophoresis conditions were 100V for 90min. After electrophoresis, the membrane was transferred to a 0.45μm PVDF membrane, and the transfer conditions were 90V for 120min. After the transfer, a 5% fetal bovine serum albumin solution was used for blocking at room temperature for 120min. After the blocking was completed, rabbit anti-Hif1α primary antibody (manufacturer: Cell Signaling Technology, catalog number: #36169) and rabbit anti-β-actin primary antibody (manufacturer: Cell Signaling Technology, catalog number: #3700) were incubated overnight at 4°C. After the primary antibody incubation was completed, TBST solution was used to wash 3 times, each time for 10min. After washing, rabbit secondary antibody (manufacturer: Cell Signaling Technology, catalog number: #7074) was incubated at room temperature for 60min. After the secondary antibody incubation was completed, TBST solution was used to wash 3 times, each time for 10min. After washing, the cells were exposed to the ECL method. The results showed that Hif1α was clearly expressed in Comparative Example 1, but was almost invisible in Comparative Example 2, indicating that the lack of Hif1α protein may indeed be the cause of the shortening of blastocyst telomeres in the blastocyst stage transfer strategy ( Figure 4 ).

[0146] (4) Hif1α siRNA injection experiment verifies the key transcription factors screened:

[0147] according to Figure 5 The experimental design was that the cleavage-siRNA group was injected with Hif1α siRNA during the fertilized egg period, and the rest of the operations were the same as those in the above comparative example 1. At the same time, the above comparative examples 1 and 2 were set as cleavage-control group and blastocyst-control group for comparison. The sequence of Hif1α siRNA was:

[0148] 5′-CUGAUGACCAGCAACUUGAdTdT-3′ (forward),

[0149] 5′-UCAAGUUGCUGGUCAUCAG dTdT-3′ (reverse). The blastocysts of each group were collected about 96 hours after fertilization for detection.

[0150] The results showed that the injection of Hif1α siRNA successfully downregulated the level of Hif1α mRNA ( Figure 6 ). Furthermore, the Hif1α protein level was also downregulated ( Figure 7 As Hif1α protein levels were downregulated, Tert mRNA and telomere length were also downregulated ( Figure 8 , Fig. 9). The above results indicate that Hif1α is indeed located upstream of Tert and telomere length in the early embryonic development stage, and has a regulatory effect on telomere length.

[0151] (5) Chromatin immunoprecipitation combined with qPCR experiment

[0152] To further elucidate the mechanism by which Hif1α regulates Tert expression in embryonic cells, we analyzed the Tert gene promoter region and found that (e.g. Fig.10 As shown in the figure, HRE is hypoxia response element, TSS is transcription start site, ATG is start codon, bold indicates classical HRE sequence, underline indicates non-classical HRE sequence), the Tert gene promoter region contains two regions containing HRE (Hif1α binding target). Magna ChIP was used to target these two regions TM ChIP-qPCR experiments were performed using A / G kit (manufacturer: Millipore) and Hif1α antibody (manufacturer: Cell Signaling Technology, catalog number: #36169) in mouse embryonic fibroblasts (MEFs) cultured under normoxic (Hif1α degradation, no Hif1α) and hypoxic (Hif1α activation, Hif1α). The results showed ( Fig.11 ), the use of Hif1α antibody could not enrich more region 1 DNA fragments, but could enrich more region 2 DNA fragments, indicating that Hif1α binds to region 2 of the Tert promoter region to exert a regulatory function.

[0153] (6) Luciferase reporter gene assay

[0154] To further demonstrate the binding of Hif1α to HRE in region 2 of the Tert promoter, luciferase reporter gene plasmids containing wild-type and mutant HRE Tert promoters were constructed for experiments ( Fig.12 , the bold part is the wild-type or mutant HRE sequence), the kit used is Dual- Reporter Assay System (manufacturer: Promega), the experiment was performed in hypoxic cultured MEFs. Wild-type reporter gene plasmid and mutant reporter gene plasmid were transfected, and Hif1α overexpression plasmid was transfected or not (to further upregulate Hif1α expression). The effect of Hif1α overexpression is shown in Fig.13 . The reporter gene results showed ( Fig.14), when the wild-type reporter gene plasmid was transfected, the luciferase signal increased significantly after overexpression of Hif1α, while when the mutant reporter gene plasmid was transfected, overexpression of Hif1α could not upregulate the luciferase signal, indicating that the HRE in region 2 is the functional site for Hif1α binding and plays a role in regulating Tert expression.

[0155] Example 2 Effect of adding Hif1α activator DMOG on telomere length in blastocyst stage transfer strategy

[0156] 1. Material Preparation

[0157] The sperm donor male mice (ICR strain, 8-12 weeks old) were housed in a single cage for one week before sperm collection. 30 minutes before sperm collection:

[0158] - In the sperm capacitation dish: make two 100 μL microdrops of conventional commercial sperm capacitation solution (e.g. TYH sperm capacitation solution, manufacturer: Aibei Biotechnology, item number: M2050) and cover with mineral oil;

[0159] - On the IVF dish: make a 100 μL droplet of conventional commercial IVF solution (e.g. HTF fertilization solution, manufacturer: Aibei Biotechnology, item number: M1150), cover with mineral oil, and place at 37°C, 5% CO 2 Equilibrate in incubator.

[0160] 2. Sperm Collection and Sperm Processing

[0161] Male mice aged 8-12 weeks were killed by cervical dislocation, the abdominal cavity was cut open, the tail of the epididymis was taken out and placed on sterile filter paper to remove blood, fat and other impurities, and the surface of the tail of the epididymis was blotted dry.

[0162] Place the cauda epididymis into the sperm capacitation solution droplet in the sperm capacitation dish and squeeze out the sperm paste. Pick the sperm paste into another sperm capacitation solution droplet. Place the sperm capacitation dish in an incubator for 1 hour to capamate the sperm. The culture conditions are 37°C, CO 2 Content 5%.

[0163] 3. Egg Preparation

[0164] Female mice (same strain) aged 4-5 weeks were superovulated and injected intraperitoneally with pregnant mare serum gonadotropin (PMSG) at a dose of 5 IU / mouse. 48 hours after PMSG injection, human chorionic gonadotropin (HCG) was injected at a dose of 5 IU / mouse. 15 hours after HCG injection, the female mice were killed by cervical dislocation, the abdominal cavity was cut open, and the fallopian tubes were taken out and placed in mineral oil in an in vitro fertilization dish. The dilatation of the fallopian tube was cut open, and both sides of the dilatation were squeezed to completely release the cumulus ovary-oocyte complexes (COCs) into the mineral oil. COCs were introduced into a 100 μL droplet of fertilization solution using ophthalmic forceps and placed in an incubator to wait for the sperm capacitation process. The culture conditions were a temperature of 37°C and CO 2 Content 5%.

[0165] 4. In vitro fertilization

[0166] 1-3 μL of sperm suspension was drawn from the outer edge of the sperm capacitation solution droplet and injected into the fertilization solution droplet containing COCs. The IVF dish was placed in an incubator for about 6 hours at a temperature of 37°C and CO 2 Content 5%.

[0167] 5. Embryo Culture

[0168] Prepare a cleavage embryo culture dish, add DMOG (manufacturer: Selleck, catalog number: S7483) to 100 μL of conventional commercial cleavage embryo culture medium (e.g. cleavage culture medium, manufacturer: Vitrolife, catalog number: 10128) to make the concentration of DMOG in the culture medium be 400 μM, cover with mineral oil, and place in an incubator in advance for 6 hours to equilibrate.

[0169] After 6 hours of in vitro fertilization, the fertilized eggs were washed three times with in vitro fertilization solution. After washing, the male and female pronuclei were observed, and the unfertilized eggs were removed. The fertilized eggs were transferred into the cleavage embryo culture medium and the cleavage embryo culture dish was placed in the incubator for culture at a temperature of 37°C and CO 2 After culturing for about 24 hours, observe the embryo status and remove the developmentally arrested embryos.

[0170] After the embryos that have developed normally to the 2-cell stage are retained and cultured for about 24 hours, a blastocyst culture dish is prepared. 100 μL of conventional commercial blastocyst culture medium (e.g., blastocyst culture medium, manufacturer: Vitrolife, catalog number: 10132) is added with 400 μM DMOG (manufacturer: Selleck, catalog number: S7483), covered with mineral oil, and placed in an incubator for 6 hours in advance for equilibrium. The development of the embryos is observed, developmentally arrested embryos are removed, and embryos that have developed normally to the 8-cell stage are selected and transferred into the blastocyst culture medium, which are then placed in an incubator for continued culture.

[0171] After about 48 hours of culture, the embryonic development was observed, developmentally arrested embryos were removed, and embryos that developed normally to the blastocyst stage were selected.

[0172] Combined with the experimental results of Example 1, in this example, we added Hif1α activator DMOG to the culture medium for embryo culture. The mechanism of action of DMOG is as follows: Fig.15 As shown, PHD activity is inhibited, Hif1α hydroxylation is prevented, and ubiquitination degradation after recognition is avoided, so that it can enter the nucleus to regulate target gene expression. As described above, 400 μM DMOG was added to the culture medium and then the embryos were cultured. Comparative Example 1 and Comparative Example 2 of Example 1 were also used as controls ( Fig.16 Here, since DMSO solvent is needed to prepare DMOG solution, it may interfere with the experimental results. Therefore, DMSO of the same concentration as that used in Example 2 was added to the culture medium of Comparative Examples 1 and 2 to ensure the comparability between the groups). Example 2, Comparative Example 1, and Comparative Example 2 were cultured for about 96 hours after fertilization, and the embryos were collected for detection. First, the Hif1α protein level was detected. It was found that the Hif1α protein level in Example 2 was significantly activated after DMOG treatment, which was equivalent to that in Comparative Example 1, while there was no Hif1α expression in Comparative Example 2 ( Fig.17 ). Further, the level of Tert mRNA was detected, and it was found that in Example 2, as Hif1α was activated, Tert mRNA was also upregulated accordingly ( Fig.18 Finally, the telomere length was detected and it was found that the telomere length of the blastocysts was successfully restored after DMOG treatment, which was equivalent to that of comparative example 1 and significantly longer than that of comparative example 2 ( Fig.19 ). This indicates that the addition of the Hif1α activator DMOG can restore the telomere length of the blastocysts that have been in the in vitro culture environment during the blastocyst stage transplantation strategy.

[0173] Example 3 Effect of adding Hif1α activator IOX2 on telomere length in blastocyst stage transplantation strategy

[0174] 400 μM Hif1α activator DMOG was replaced with 50 μM Hif1α activator IOX2, and the remaining steps were the same as in Example 2. The results were as follows:

[0175] After fertilization of Example 3, Comparative Example 1, and Comparative Example 2, the embryos were cultured for about 96 hours, and the Hif1α protein level was first detected. It was found that the Hif1α protein level was significantly activated after IOX2 treatment, which was equivalent to that of Comparative Example 1, while there was no Hif1α expression in Comparative Example 2 ( Fig. 20 ). Further, the level of Tert mRNA was detected, and it was found that in this embodiment, as Hif1α was activated, Tert mRNA was also upregulated accordingly ( Fig.21Finally, the telomere length was detected and it was found that the telomere length of the blastocysts was successfully restored after IOX2 treatment, which was equivalent to that of the positive control group and significantly longer than that of the negative control group ( Fig. 22 ). This indicates that the addition of another Hif1α activator, IOX2, also helps to restore the telomere length in the blastocysts that have been in in vitro culture during the blastocyst stage transfer strategy.

[0176] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A product for restoring telomere length of embryos in vitro, characterized in that: The product contains hypoxia-inducible factor-1α subunit activator.

2. The product according to claim 1, characterized in that Contain at least one of the following characteristics: (1) The product includes a drug; (2) The embryo includes a blastocyst.

3. The product according to claim 1, characterized in that Contain at least one of the following characteristics: (1) The hypoxia-inducible factor-1α subunit activator comprises a prolyl hydroxylase inhibitor; (2) the hypoxia-inducible factor-1α subunit activator is selected from one or more of DMOG, cobalt chloride, deferiprone, IOX2, DHB, GSNO, hydralazine hydrochloride, mimosacin, Adaptaquin, PHD-1-IN-1, enastat, VH298, TP0463518, IOX4, vadadustat, MK-8617, FG-2216, molistat, dedustat, and dapdustat; (3) The concentration of the hypoxia-inducible factor-1α subunit activator in the in vitro culture medium is 0.001-500 μM.

4. Use of a hypoxia-inducible factor-1α subunit activator in the preparation of a product for restoring the telomere length of an in vitro embryo or a product for restoring the telomere length of a newborn offspring obtained by embryo transplantation.

5. The use according to claim 4, characterized in that: Contains at least one of the following characteristics: (1) The newborn offspring are human or non-human newborn offspring; (2) Restoring embryonic telomere length includes: adding a hypoxia-inducible factor-1α subunit activator during at least one stage of embryo in vitro culture to obtain embryos with extended telomeres; (3) Restoring the telomere length of newborn offspring obtained by embryo transfer comprises: adding a hypoxia-inducible factor-1α subunit activator at at least one stage during the embryo culture process until a blastocyst is obtained, and obtaining newborn offspring with extended telomeres through blastocyst transfer.

6. A method for culturing embryos in vitro, characterized in that: The method comprises the step of adding a hypoxia inducible factor-1α subunit activator at least during at least one stage of the embryo in vitro culture process.

7. The method for culturing embryos in vitro according to claim 6, characterized in that: The embryo in vitro culture method specifically comprises the following steps: S1. Acquisition of sperm and eggs, and obtaining fertilized eggs through in vitro fertilization; S2, culturing the fertilized egg in vitro until a blastocyst is obtained, and adding a hypoxia-inducible factor-1α subunit activator during at least one stage of the in vitro culture; Among them, at least one of the following characteristics is included: (1) The stage for adding hypoxia-inducible factor-1α subunit activator includes cleavage stage, morula stage or blastocyst stage; (2) When the embryo is a human embryo, the morula stage is 72-120 hours after fertilization, and the blastocyst stage is 120-168 hours after fertilization.

8. The method for culturing embryos in vitro according to claim 7, characterized in that: Contain at least one of the following characteristics: (1) The hypoxia-inducible factor-1α subunit activator comprises a prolyl hydroxylase inhibitor; (2) the hypoxia-inducible factor-1α subunit activator is selected from one or more of DMOG, cobalt chloride, deferiprone, IOX2, DHB, GSNO, hydralazine hydrochloride, mimosacin, Adaptaquin, PHD-1-IN-1, enastat, VH298, TP0463518, IOX4, vadadustat, MK-8617, FG-2216, molistat, dedustat, and dapdustat; (3) The concentration of the hypoxia-inducible factor-1α subunit activator in the in vitro culture medium is 0.001-500 μM.

9. A method for constructing a blastocyst model with restored telomere length, characterized in that: The following steps are involved: The blastocyst model is constructed by adding a hypoxia-inducible factor-1α subunit activator during the culture process of at least one stage of the fertilized egg developing into the blastocyst.

10. A method for constructing an animal model for restoring telomere length by blastocyst stage transplantation, characterized in that: The following steps are involved: The animal model is constructed by adding a hypoxia-inducible factor-1α subunit activator during the culture process of at least one stage of the fertilized egg developing into a blastocyst, and producing newborn offspring through blastocyst embryo transplantation.

11. An animal model for restoring telomere length by blastocyst stage transplantation obtained by the construction method of claim 10.

12. Use of the blastocyst model obtained by the construction method of claim 9 or the animal model of claim 11 in telomere function research, aging research or assisted reproductive technology research.

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