Use of GSTO2 in improving the developmental potential of early mammalian embryos in vitro culture

Abstract

The application belongs to the field of reproductive biology and assisted reproductive technology, and discloses application of GSTO2 as an embryo development regulating factor in improving in vitro culture development potential of mammalian early embryos, promoting blastocyst formation and improving embryo implantation capacity. The application first discloses a key promoting role of GSTO2 in the process of mammalian early embryo development. It is shown that GSTO2 can not only be used as an important molecular marker for evaluating embryo development potential, but also be an important functional regulating factor for maintaining normal embryo development. The application can significantly promote early embryo development, improve blastocyst formation rate in the in vitro culture process, and enhance the implantation capacity after embryo transplantation by supplementing GSTO2 expression at the zygote stage, thereby providing a new technical means for improving the in vitro culture efficiency of mammalian embryos and improving the success rate of assisted reproduction.

Description

Technical Field

[0001] This invention belongs to the fields of reproductive biology and assisted reproductive technology, specifically involving the application of glutathione S-transferase Omega 2 (GSTO2) as an embryonic development regulator in improving the developmental potential of early mammalian embryos in vitro, promoting blastocyst formation, and enhancing embryo implantation ability. Background Technology

[0002] The glutathione S-transferase omega 2 (GSTO2) gene is located on human chromosome 10, region 10q25.1. It contains 7 transcripts (splicing variants), 542 orthologs, and 14 paralogs, and is widely expressed in various tissues, including skin and testes. The GSTO2 gene is highly conserved in various mammals, including chimpanzees, rhesus monkeys, dogs, cattle, mice, and rats. The protein encoded by GSTO2 belongs to the Omega-class glutathione S-transferase (GST) family and exhibits high levels of dehydroascorbic acid reductase activity among mammalian GST families. GSTO2 participates in the metabolism of exogenous compounds and potential carcinogens, and plays an important role in drug metabolism, redox homeostasis maintenance, and cellular antioxidant defense. It is a crucial protective factor against exogenous stimuli and oxidative stress damage. Previous studies have shown that GSTO2 is expressed in the sperm plasma membrane, and its redox regulatory function participates in sperm capacitation. Inhibition of GSTO2 activity leads to decreased tyrosine phosphorylation levels in the later stages of sperm capacitation, resulting in impaired acrosome reaction, exocytosis, and sperm motility. GSTO2 can utilize reduced glutathione as an electron donor, promoting sperm nucleus decondensation by regulating protein disulfide bond states, thereby affecting sperm function and fertilization capacity. Furthermore, genetic variations in GSTO2 may alter its biological function by affecting protein structural stability, functional activity, hydrophobic characteristics, and the formation of internal disordered regions, and are associated with the development of polycystic ovary syndrome (PCOS). However, the function of GSTO2 in early mammalian embryonic development and its role in regulating embryonic developmental potential remain insufficiently understood. Summary of the Invention

[0003] Based on the aforementioned deficiencies in existing technologies, this invention reveals for the first time the important role of GSTO2 in the regulation of early embryonic development, providing new molecular targets and technical strategies for improving the quality of mammalian embryo culture in vitro and optimizing assisted reproductive technologies.

[0004] The objective of this invention is achieved through the following technical solution:

[0005] This invention utilizes blastomere biopsy technology to obtain individual blastomeres cultured in vitro to the 4-cell and 8-cell stages. Bioinformatics analysis was performed on single-tube single-cell RNA sequencing (based on Smart-Seq2 amplification, obtaining full-length transcripts and then using Tn5 transposase for library construction and sequencing) to identify differentially expressed mRNAs between the groups. Specifically, blastomeres from cynomolgus embryos cultured in vitro at the 4-cell and 8-cell stages were mechanically biopsied. A single surviving blastomer was removed, rapidly frozen in 5 μL of lysis buffer, and used for transcriptome sequencing. The remaining embryos were cultured until day 7. Based on the developmental outcome of the remaining embryos, the biopsied blastomeres were divided into a high-potential group (the remaining embryos developed into expanded blastocysts) and a low-potential group (the remaining embryos developed arrested or degenerated). By comparing differentially expressed mRNAs between the two groups, we explored the molecular mechanisms of embryonic developmental arrest at the transcriptomic level and screened key genes that may ensure normal embryonic development.

[0006] Transcriptomic analysis revealed high transcriptional levels of GSTO2 mRNA in multiple blastomeres from the 4-cell and 8-cell high-development-potential groups, while expression was undetectable in both 4-cell and 8-cell blastomeres from the arrested group. GSTO2 expression persisted throughout the morula and blastocyst stages. Given the crucial antioxidant function of GSTO2 in maintaining reactive oxygen species (ROS) homeostasis, the inventors hypothesize that under in vitro culture conditions, both embryonic metabolic activity and external environmental factors can induce ROS production. Normal expression and translation of GSTO2 are essential for clearing excess ROS and maintaining redox balance. When GSTO2 expression is inhibited, or oxidative damage exceeds the embryo's clearance capacity, ROS levels continuously increase, ultimately leading to embryonic developmental arrest.

[0007] Based on the above findings, this invention designed a GSTO2 overexpression plasmid, which was introduced into the cytoplasm of fertilized eggs via microinjection. Experimental results showed that GSTO2 overexpression significantly increased the blastocyst formation rate of early cynomolgus monkey embryos (p < 0.01). Further embryo transfer experiments verified that the pregnancy rate of GSTO2-overexpressing blastocysts in cynomolgus monkey recipients increased from 39.13% (9 / 23) in the control group to 80.0% (4 / 5). This indicates that GSTO2 overexpression not only promotes in vitro embryonic development to blastocyst stage but also significantly enhances the implantation ability of blastocysts. Therefore, stable maintenance of GSTO2 mRNA expression is beneficial to the normal development and subsequent implantation of early cynomolgus monkey embryos and can serve as an effective target for improving the blastocyst rate and implantation rate in in vitro culture of early cynomolgus monkey embryos.

[0008] Therefore, the present invention first provides the application of GSTO2 as a target in the preparation, screening and / or development of functional products that promote early embryonic development in cynomolgus monkeys, wherein the functional products are used to increase the expression level of GSTO2.

[0009] Preferably, the application of promoting early embryonic development in cynomolgus monkeys refers to:

[0010] (1) Improve the blastocyst rate of cynomolgus monkey embryos in vitro, and / or

[0011] (2) Improve the implantation rate of cynomolgus monkey blastocysts.

[0012] To further elucidate the mechanism by which GSTO2 promotes embryonic development, this study performed single-embryo transcriptome sequencing analysis on 8-cell stage (GC8) and blastocyst (GBL) embryos overexpressing GSTO2. The results showed that GSTO2 expression was significantly upregulated in GC8 (p < 0.001), while there was no significant difference in GBL compared to the control group. GO / KEGG enrichment analysis of differentially expressed genes indicated that GSTO2 overexpression enhanced cell signaling, metabolic activity, and cell adhesion during the 8-cell stage, and promoted protein synthesis and transport during the blastocyst stage. Simultaneously, the embryos exhibited low levels of DNA damage response and ubiquitin-proteasome degradation system activity. These results suggest that GSTO2 promotes embryonic development and implantation preparation by coordinating multiple biological processes and remodeling the transcriptional program during the zygotic genome activation (ZGA) stage, providing important evidence for improving primate embryo quality.

[0013] Preferably, GSTO2 is a positive regulator of the developmental potential of early mammalian embryos in vitro.

[0014] In the specific process of early embryo in vitro culture, expression vectors containing the GSTO2 coding sequence or GSTO2 mRNA can be introduced into the cytoplasm of fertilized egg cytoplasm through microinjection at the fertilized egg stage to increase the expression level of GSTO2 in the early embryonic development stage and promote embryonic development.

[0015] Therefore, the present invention also provides a method for promoting in vitro embryonic development of cynomolgus monkeys, which involves introducing an expression vector containing the GSTO2 coding sequence or GSTO2 mRNA into the cytoplasm of the fertilized egg through microinjection during the cynomolgus monkey fertilized egg stage.

[0016] Preferably, in the method for promoting in vitro embryonic development of cynomolgus monkeys, the expression vector containing the GSTO2 coding sequence is a GSTO2-pEGFP-N1 overexpression vector, the sequence of which is shown in SEQ ID NO: 1.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention reveals for the first time the crucial promoting role of GSTO2 in early mammalian embryonic development. The study found that GSTO2 is specifically highly expressed in blastomeres with high developmental potential at the 4-cell and 8-cell stages, while its expression is absent in blastomeres with low developmental potential. Furthermore, it is persistently expressed in the morula and blastocyst stages, indicating that GSTO2 not only serves as an important molecular marker for evaluating embryonic developmental potential but also as a vital functional regulator for maintaining normal embryonic development.

[0019] This invention, by supplementing GSTO2 expression at the fertilized egg stage, can significantly promote early embryonic development, increase the blastocyst formation rate during in vitro culture, and enhance the implantation ability after embryo transfer, providing a new technical means to improve the efficiency of mammalian embryo in vitro culture and increase the success rate of assisted reproduction.

[0020] This invention establishes a research technology system for embryonic development regulation based on "blastomere biopsy - single-cell transcriptome analysis - candidate gene screening - functional verification". It can be used to systematically screen and verify key genes and functional molecules that regulate embryonic development potential, providing theoretical basis and technical support for the development of novel embryo culture additives, assisted reproductive optimization technologies and reproductive health-related products. Attached Figure Description

[0021] Figure 1 A schematic diagram of a single blastomere embryo biopsy and sampling.

[0022] Figure 2 Flowchart of single-tube, single-blastome transcriptome sequencing;

[0023] Figure 3 Volcano plot of C4LDP / C4HDP differentially expressed genes;

[0024] Figure 4 Analysis of GSTO2 expression patterns at different developmental stages in early cynomolgus monkey embryos;

[0025] Figure 5 Design a map for the GSTO2 overexpression plasmid (GSTO2-pEGFP-N1);

[0026] Figure 6 Electrophoresis image for GSTO2 overexpression plasmid digestion verification; Line 1 is GSTO2-pEGFP-N1 plasmid, Line 2 is GSTO2-pEGFP-N1 digested with KpnI-BamHI, Line M Marker is Plus (D2000 Plus), (1.0% agarose gel electrophoresis, EB staining);

[0027] Figure 7The peak diagram is a sequencing verification diagram of the GSTO2 overexpression plasmid;

[0028] Figure 8 Image of green fluorescent protein expression in embryos overexpressing GSTO2; Scale bar: 100 μm;

[0029] Figure 9 A statistical graph showing the effect of GSTO2 overexpression on the blastocyst rate of early cynomolgus monkey embryos;

[0030] Figure 10 A statistical chart showing the pregnancy rate after GSTO2-overexpressing blastocyst transfer;

[0031] Figure 11 To analyze the expression level of GSTO2 in 8-cell embryos (GC8) that overexpress GSTO2;

[0032] Figure 12 Analysis of GSTO2 expression levels in blastocysts (GBL) that overexpress GSTO2;

[0033] Figure 13 GO functional enrichment analysis for differentially regulated genes in GC8 / AC8;

[0034] Figure 14 GO functional enrichment analysis for differentially regulated genes in GBL / CBL;

[0035] Figure 15 GO functional enrichment analysis of differentially regulated genes in GBL / CBL. Detailed Implementation

[0036] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific drawings and embodiments. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0037] Example 1

[0038] I. Definition of High and Low Developmental Potential Groups in Embryonic Blastomere Biopsy

[0039] In mammalian embryonic development, the embryo begins with a fertilized egg. The fertilized egg undergoes cleavage to produce blastomeres, which are totipotent in early embryos (2-8 cells). Blastocyst biopsy is one of the important sampling methods for clinical preimplantation genetic testing (PGT). Embryos from different cynomolgus monkeys (ensuring samples from at least two individuals at each stage) are randomly selected, and blastomeres are biopsied at the 2-cell, 4-cell, and 8-cell stages. To ensure that the biopsy process does not affect embryonic development and the biological integrity of the retrieved blastomeres, this invention employs a mechanical method: a large "cross-shaped" incision is made in the zona pellucida, and a relatively thick biopsy needle is used to remove one blastomere. Figure 1 As shown, blastomeres of cynomolgus embryos cultured in vitro were mechanically biopsied at the 2-cell, 4-cell, and 8-cell stages. While obtaining a single surviving blastomere for transcriptome sequencing, the remaining blastomeres were cultured to the blastocyst stage. Furthermore, in this study, 2-cell embryos were mechanically divided in half, and one surviving hemimorula was extracted from the morula stage on day 5 for transcriptome sequencing. Based on the final developmental outcome on day 7, the sequencing samples from the early blastomeres and hemimorula were divided into two groups: high developmental potential (HDP) and low developmental potential (LDP), corresponding to samples that ultimately formed blastocysts and those that did not, respectively.

[0040] II. Transcriptomics Detection and Result Analysis

[0041] Samples were amplified within one week, and full-length transcripts were obtained using a Smart-Seq2-based single-tube, single-cell / single-embryo RNA sequencing method. Library construction and sequencing were then performed using Tn5 transposase. This amplification method can be used not only for quantitative transcript analysis but also for the interpretation of structural variations and functional information. The specific experimental and information analysis procedures are as follows: Figure 2 As shown.

[0042] This study performed a systematic transcriptome differential analysis on 2-cell, 4-cell, 8-cell, and morula embryos of HDP and LDP. The results are as follows: Figure 3 The results showed that GSTO2 was the gene with the greatest difference between the HDP and LDP groups at the 4-cell stage. Figure 4As shown, further analysis of the expression pattern of GSTO2 throughout the early embryonic developmental stages from MII oocytes to blastocysts revealed that GSTO2 was expressed at low levels in only a few samples during the MII oocyte to 2-cell embryo stage. During the zygote genome-activated 4-cell embryo stage, its expression level increased sharply in the high developmental potential group (C4HDP), showing a highly significant difference (p < 0.001) compared to the completely non-expressing low developmental potential group (C4LDP). During the 8-cell embryo stage, it continued to be expressed in the developmental group, while remaining unexpressed in the arrested group. The expression level of GSTO2 decreased during the development of morula and blastocyst stages.

[0043] III. GSTO2 plasmid microinjection

[0044] To verify the above analytical results, this invention designed the GSTO2-pEGFP-N1 overexpression vector ( Figure 5 ), and commissioned Tianyi Huiyuan Biotechnology Co., Ltd. to complete the whole gene synthesis, which was then digested with enzymes ( Figure 6 ) and sequencing ( Figure 7 The verification was correct. Thirteen cynomolgus monkeys with good superovulation were selected, and their embryos were collected and divided into experimental and control groups 8 hours after ICSI. In the experimental group, plasmid (concentration 30 ng / μL) was injected into the cytoplasm of fertilized egg cells via microinjection, while the control group received no treatment. Green fluorescent protein expression was observed in the embryos after injection. Figure 8 This indicates that the plasmid was successfully transfected and effectively expressed in the embryo.

[0045] IV. Statistical analysis of implantation outcomes after early embryo in vitro culture and embryo transfer

[0046] Embryos from both the control and experimental groups were cultured under the same conditions (37 ℃, 5% CO2, 5% O2, 90% N2). The medium was changed on days 3, 5, and 7, and developmental status was recorded. Blastocyst rate (%) = (number of blastocysts on day 7 / total number of dividing embryos) × 100%. Paired t-tests were performed on the blastocyst rates of the two groups using GraphPad Prism software. Figure 9 As shown, a p < 0.05 was considered statistically significant (marked with "*" in the figure). The analysis results showed that the experimental group was significantly different from the control group (p < 0.05). Therefore, it was concluded that during the in vitro culture of early cynomolgus monkey embryos, microinjection of the GSTO2 overexpression plasmid at the fertilized egg stage could increase the expression level of GSTO2 at the 4-cell and 8-cell stages, thereby significantly increasing the blastocyst rate. The blastocysts obtained from GSTO2 overexpression were transferred to the uterus of healthy recipient cynomolgus monkeys, with 3-4 blastocysts transferred to each recipient. The results are as follows... Figure 10As shown, the pregnancy rate increased significantly from 39.13% (9 / 23) in the control group to 80.0% (4 / 5), further verifying the promoting effect of GSTO2 overexpression on embryonic development and implantation.

[0047] V. Transcriptomic sequencing results of GSTO2 overexpressing single embryos

[0048] To further elucidate the mechanism by which GSTO2 promotes embryonic development, this study performed single-embryo transcriptome sequencing analysis on 8-cell stage (GC8) and blastocyst (GBL) embryos overexpressing GSTO2. The results are as follows: Figure 11 and Figure 12 The results showed that GSTO2 expression was significantly upregulated in GC8 (p < .001), while there was no significant difference between GBL and the control group, consistent with the expectation that GSTO2 is highly expressed in the ZGA stage and returns to baseline levels in the blastocyst stage. Figure 13 GO enrichment analysis of differentially regulated genes (GC8 / AC8) showed that GC8-upregulated genes were significantly enriched in pathways such as intracellular signal transduction, regulation of catalytic activity, calcium ion transmembrane transport, and homocellular adhesion mediated by plasma membrane adhesion molecules; for example... Figure 14 and Figure 15 GBL / CBL differential gene enrichment analysis (GO) revealed that GBL-upregulated genes were significantly enriched in translation and protein transport, while GBL-downregulated genes were significantly enriched in pathways involving cellular responses to DNA damage, protein transport, cell cycle, nuclear assembly, protein K48-linked ubiquitination, ubiquitin-dependent catabolism, endosome assembly, and proteasome-mediated ubiquitin-dependent catabolism. These results indicate that GSTO2 overexpression enhances cell signaling, metabolic activity, and cell adhesion at the 8-cell stage, while promoting protein synthesis and transport at the blastocyst stage, and simultaneously reducing the activity levels of DNA damage responses and the ubiquitin-proteasome degradation system. GSTO2, by coordinating multiple biological processes, remodels the transcriptional program at the ZGA stage, promoting embryonic development and implantation preparation, providing important evidence for improving primate embryo quality.

Claims

1. The application of GSTO2 as a target in the preparation, screening, and / or development of functional products that promote early embryonic development in mammals, characterized in that, The functional product is used to increase the expression level of GSTO2.

2. The application according to claim 1, characterized in that, The promotion of early embryonic development in mammals refers to: (1) Improve the blastocyst rate of mammalian somatic embryos in vitro, and / or (2) Improve the implantation rate of mammalian blastocysts.

3. A method for promoting in vitro embryonic development in mammals, characterized in that, In mammalian somatic zygotes, expression vectors containing the GSTO2 coding sequence or GSTO2 mRNA are introduced into the cytoplasm of zygote cytoplasm via microinjection.

4. The method for promoting in vitro embryonic development in mammals according to claim 3, characterized in that, The expression vector containing the GSTO2 coding sequence is a GSTO2-pEGFP-N1 overexpression vector, the sequence of which is shown in SEQ ID NO: 1.

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