Application of glycosylated phosphatidylinositol-specific phospholipase D1 (GPLD1) as sperm-egg recognition molecule

Abstract

The invention discloses an application of glycosylated phosphatidylinositol specific phospholipase D1 (GPLD1) as a sperm-egg recognition and / or binding regulation molecule.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the field of animal reproductive biology, and more specifically to the use of glycosylated phosphatidylinositol-specific phospholipase D1 (GPLD1) as a sperm-egg recognition molecule. Background Technology

[0002] With the development of in-vitro fertilization and in-vitro embryo production ( in vitro The widespread application of IVEP (in vitro embryo production) technology in livestock breeding, genetic improvement, and bio-breeding has made the recognition and fusion efficiency between sperm and oocytes a crucial limiting factor affecting fertilization outcomes and embryonic development quality. Pig sperm-oocyte recognition is a highly precisely regulated biological process involving multiple stages, including sperm acrosome reaction, sperm-oocyte membrane-specific recognition, membrane fusion, and post-fertilization regulation. The regulatory status of this process directly relates to the establishment of normal fertilization and subsequent embryonic developmental potential.

[0003] In porcine in vitro fertilization (IVF) systems, the sperm-egg microenvironment cannot fully mimic in vivo fertilization conditions, often resulting in unstable sperm-egg membrane recognition efficiency and a high rate of polysperm entry, which in turn affects the normal rate of double pronucleus formation and the ability of the embryo to continue developing. Current techniques mostly optimize the fertilization process by adjusting sperm concentration, fertilization time, or the composition of the culture system. However, these methods are primarily indirect, condition-level controls, making it difficult to precisely intervene in the sperm-egg recognition and membrane binding process at the molecular level. Furthermore, they are highly susceptible to experimental conditions, resulting in limited stability and reproducibility.

[0004] The sperm-egg recognition process relies on the specific interaction between sperm surface molecules and oocyte membrane receptors, and its regulatory mechanism is influenced by multiple factors, including the expression level, spatial distribution, and functional state of membrane-associated proteins. Although some molecules involved in sperm-egg recognition or fertilization regulation have been reported, research on the expression regulation of key molecules in animal sperm-egg recognition is still relatively insufficient, especially lacking a technical solution that can stabilize and regulate fertilization outcomes and improve embryonic development quality by modulating the expression status of endogenous sperm-egg recognition-related molecules.

[0005] Therefore, it is necessary to explore and establish a new sperm-egg recognition regulation strategy at the molecular level, in order to provide a more stable and controllable technical foundation for optimizing the porcine in vitro fertilization system, improving normal fertilization efficiency and embryo development quality. Summary of the Invention

[0006] In order to solve one of the above-mentioned technical problems in the prior art, the present invention elucidates the key role of glycosylphosphatidylinositol specific phospholipase D1 (GPLD1) in sperm-egg recognition and fertilization outcome regulation, and provides the use of GPLD1 active substances.

[0007] In some aspects of the invention, the use of glycosylated phosphatidylinositol-specific phospholipase D1 (GPLD1) as a sperm-egg recognition and / or binding regulatory molecule is provided.

[0008] In some aspects of the present invention, the use of GPLD1-related active substances in any one or more of the following is provided: a1) Regulating the efficiency of in vitro fertilization in animals; a2) Regulating the quality of embryonic development after in vitro fertilization; a3) Improve the selectivity of sperm and egg recognition; a4) Improve embryo cleavage rate; a5) Improves blastocyst formation rate; a6) Promotes continuous embryonic development; a7) Prepare products that regulate the efficiency of in vitro fertilization in animals; a8) Prepare products that regulate the quality of embryonic development after in vitro fertilization; a9) Prepare products that improve the selectivity of sperm-egg recognition; (a10) Prepare products that improve embryo cleavage rate; a11) Prepare products that improve blastocyst formation rate; (a12) Prepare products that promote continuous embryonic development.

[0009] In some embodiments, the active substance is capable of regulating GPLD1 levels and / or activity, wherein the GPLD1 levels and / or activity are the levels and / or activity of the GPLD1 protein or its encoded nucleic acid molecule.

[0010] In some embodiments, the active substance can regulate GPLD1 levels and / or activity in oocytes.

[0011] In some embodiments, the active substance can increase GPLD1 levels and / or activity.

[0012] In some embodiments, the active substance includes at least one of b1) to b6): b1) GPLD1 mRNA or its derivatives; b2) GPLD1 protein or its salts; b3) Biomaterials related to GPLD1 protein; b4) Substances that promote GPLD1 expression; b5) Substances that enhance GPLD1 activity; and b6) Substances that slow down the metabolism of GPLD1.

[0013] In some embodiments, the biomaterial associated with the GPLD1 protein is selected from at least one of the following: c1) Nucleic acid molecules encoding GPLD1; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A vector containing the nucleic acid molecule described in c1) or the expression cassette described in c2); c4) contains any one of c1) to c3).

[0014] In some embodiments, the GPLD1 protein has an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; or the nucleic acid molecule encoding GPLD1 has a nucleotide sequence as shown in SEQ ID NO: 2 or a nucleotide sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0015] In some embodiments, the GPLD1 active substance is selected from at least one of the following: a GPLD1 expression or overexpression vector; naked DNA and / or RNA containing a GPLD1 coding sequence; liposomes encapsulating DNA and / or RNA of a GPLD1 coding molecule; a GPLD1 precursor protein, conjugate, or complex capable of being converted into GPLD1 in vivo; a compound (e.g., a small molecule agonist) capable of increasing GPLD1 protein levels and / or function in vivo; an upregulator of GPLD1 gene promoter driving ability; or a downregulator of GPLD1 gene-specific microRNA.

[0016] In some embodiments, the GPLD1 active substance is a GPLD1 expression or overexpression vector.

[0017] In some embodiments, the GPLD1 expression or overexpression vector may include a eukaryotic expression vector and / or a prokaryotic expression vector. In some embodiments, the eukaryotic expression vector includes, for example, but not limited to, yeast expression vectors, mammalian expression vectors, and insect expression vectors. For example, the expression vector may include, but is not limited to, plasmids, retroviral vectors, lentiviral vectors, bacteriophage vectors, adenovirus vectors, adeno-associated vectors, or herpes simplex vectors.

[0018] In some embodiments, the carrier may be selected from nanoparticles, liposomes, exogenous bodies, microbubbles, or gene guns.

[0019] In some embodiments, regulating animal in vitro fertilization efficiency is to improve animal in vitro fertilization efficiency.

[0020] In some embodiments, the improvement of animal in vitro fertilization efficiency includes at least one of the following: Reduce the number of non-specific bindings between sperm and oocytes; Reduce the rate of polysperm entry into the egg; Increase the rate of normal dual pronucleus formation.

[0021] In some embodiments, regulating the quality of embryonic development after in vitro fertilization is to improve the quality of embryonic development after in vitro fertilization.

[0022] In some embodiments, the product is applied to mammals or mammalian cells.

[0023] In some embodiments, the mammal includes at least one of primates, rodents, livestock, and pets.

[0024] In some embodiments, the mammal includes at least one of the following: human, rat, mouse, pig, dog, horse, cow, rabbit, monkey, ape, and orangutan.

[0025] In some embodiments, the pig includes at least one of Duroc, Landrace, Yorkshire, and local breeds.

[0026] In some embodiments, the drug, pharmaceutical composition, reagent kit, or medical device.

[0027] In some embodiments, the product also includes other active ingredients that regulate the efficiency of in vitro fertilization in animals or the quality of embryonic development after in vitro fertilization.

[0028] In some embodiments, the product also includes a pharmaceutically acceptable carrier.

[0029] The beneficial effects of this invention are: This invention uses GPLD1 as a key molecule for sperm-egg recognition regulation. By regulating its expression level, it directly acts on the sperm-oocyte membrane recognition and binding process, thereby achieving molecular-level intervention in the sperm-egg recognition process and improving the stability and controllability of the fertilization process.

[0030] Overexpression of GPLD1 in oocytes or fertilization systems can significantly downregulate the protein expression level of the sperm-egg recognition protein JUNO, which can significantly increase the proportion of normal fertilization (2PN) during in vitro fertilization and simultaneously improve the embryo cleavage rate and blastocyst formation rate. This indicates that upregulation of GPLD1 is beneficial to the correct recognition of sperm and egg and the overall improvement of the subsequent embryonic development potential. Attached Figure Description

[0031] Figure 1 The expression and localization of GPLD1 in animal oocytes are shown. A represents GPLD1 expression (RPM) in human oocytes and early embryos; B represents GPLD1 expression (RPM) in mouse oocytes and early embryos; and C represents GPLD1 expression (TPM) in porcine oocytes and early embryos. In the diagram, NGO represents incompletely developing oocytes; GO represents quiescent phase; FGO represents fully developing oocytes; MI represents metaphase I of meiosis; MII represents metaphase II of meiosis; Zygote represents zygote stage; 2-cell represents 2-cell stage embryos; 4-cell represents 4-cell stage embryos; 8-cell represents 8-cell stage embryos; 16-cell represents 16-cell stage embryos; Morula represents morula; EM represents early morula; LM represents late morula; EB represents early blastocyst; LB represents late blastocyst; HB represents hatched blastocyst; ICM represents inner cell mass; and TE represents trophoblast cells. D shows representative immunofluorescence images of GPLD1 protein expression and localization in MII stage porcine oocytes and oocytes from different treatment groups 4 hours after in vitro fertilization.

[0032] Figure 2 This study describes the construction of the GPLD1 overexpression vector and its expression validation in porcine oocytes. Image A shows a schematic diagram of the GPLD1-EYFP overexpression plasmid vector, illustrating the fusion of the GPLD1 coding sequence with EYFP and its regulation by the CMV promoter. Image B shows a representative image of the microinjection of mRNA into porcine oocytes. Image C shows representative images of GPLD1-EYFP fluorescence signals in porcine oocytes from the control and GPLD1 overexpression groups. The control group showed almost no fluorescence signal, while significantly enhanced EYFP fluorescence was observed in the GPLD1 overexpression group, indicating successful expression of the overexpression vector in oocytes.

[0033] Figure 3The results of oocyte-sperm binding observation are shown. A is a schematic diagram showing the detection results of oocyte-sperm binding capacity in the control group (n=119) and the GPLD1 overexpression group (n=116) 4 hours after fertilization; B is a statistical analysis graph showing the number of oocytes bound to sperm in the control group (n=119) and the GPLD1 overexpression group (n=116) 4 hours after fertilization.

[0034] Figure 4 The results of observation on polyspermy in oocytes leading to the formation of multiple pronuclei are shown. Among them, A is a representative image of polyspermy in oocytes leading to the formation of multiple pronuclei in the control group (n=145) and the GPLD1 overexpression treatment group (n=149) 12 h after fertilization; B is a statistical analysis graph of the polyspermy in oocytes leading to the formation of multiple pronuclei in the control group (n=145) and the GPLD1 overexpression group (n=149) 12 h after fertilization.

[0035] Figure 5 The results of observation on double pronucleus formation in oocytes are shown. A represents the double pronucleus formation in oocytes from the control group (n=140) and the GPLD1 overexpression treatment group (n=145) 12 hours after fertilization; B is a statistical analysis graph of the double pronucleus formation rate in oocytes from the control group (n=140) and the GPLD1 overexpression treatment group (n=145) 12 hours after fertilization.

[0036] Figure 6 To show the effect of GPLD1 overexpression on JUNO expression in oocytes, representative immunofluorescence images of JUNO protein (red) in porcine MII stage oocytes and IVF fertilized eggs at 4 h and 12 h in the control and GPLD1 overexpression groups are presented, along with quantitative analysis of the immunofluorescence intensity of JUNO protein in the control and GPLD1 overexpression groups at the above stages.

[0037] Figure 7 The results show the embryo cleavage rate. In this image, A represents the embryo cleavage rate of the control group (n=159) and the GPLD1 overexpression group (n=170); B represents the embryo cleavage rate analysis of the control group (n=159) and the GPLD1 overexpression group (n=170).

[0038] Figure 8 The results show the blastocyst rate of embryos. A represents the blastocyst rate of the control group (n=238) and the GPLD1 overexpression group (n=235). B represents the blastocyst rate analysis of the control group (n=238) and the GPLD1 overexpression group (n=235).

[0039] Figure 9The results related to oocyte-sperm binding capacity are shown in Figure A, which is a schematic diagram of the detection results of oocyte-sperm binding capacity in the control group (n=119), GPLD1 interference treatment group (n=114), and 1,10-phenanthroline treatment group (n=120) 4 hours after fertilization; Figure B is a statistical analysis graph of the number of oocytes bound to sperm in the control group (n=119), GPLD1 interference treatment group (n=114), and 1,10-phenanthroline treatment group (n=120) 4 hours after fertilization.

[0040] Figure 10 To show the effect of GPLD1 knockdown on JUNO expression in oocytes, representative immunofluorescence images of JUNO protein (red) in porcine MII stage oocytes and IVF fertilized eggs at 4 h and 12 h in the control and GPLD1 knockdown groups, as well as quantitative analysis of the immunofluorescence intensity of GPLD1 and JUNO proteins in the control and GPLD1 knockdown groups at the above stages.

[0041] Figure 11 The results show the effect of 1,10-phenanthroline treatment on JUNO expression in oocytes. Representative immunofluorescence images of JUNO protein (red) in porcine MII stage oocytes and IVF fertilized eggs at 4 h and 12 h in the control and 1,10-phenanthroline treatment groups are shown. Quantitative analysis of the immunofluorescence intensity of GPLD1 and JUNO proteins in the control and 1,10-phenanthroline treatment groups at the above stages is also presented. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0043] definition Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0044] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0045] As used herein, "GPLD1" stands for glycosylphosphatidylinositol specific phospholipase D1. This gene encodes a specific enzyme, phospholipase D1, which is primarily responsible for hydrolyzing glycosylphosphatidylinositol (GPI) anchored proteins. The main function of GPLD1 is to release GPI-anchored proteins from the cell membrane through its encoded enzymatic activity. This release process is crucial for cell signaling, cell membrane component renewal, and intracellular substance transport. This application discloses for the first time the correlation between GPLD1 and the regulation of sperm-egg recognition and fertilization outcome, thereby providing a method to intervene at the molecular level in the sperm-egg recognition process by regulating GPLD1 levels in oocytes, thereby improving the stability and controllability of the fertilization process.

[0046] The GPLD1 protein may be encoded by the GPLD1 gene sequence or its CDS sequence, or by a homologous sequence having the same sperm-egg recognition function (e.g., a homologous sequence of GPLD1 can be obtained from databases or alignment software known in the art), variants, or modified forms. For example, the GPLD1 protein may be selected from, for example: (a) a protein encoded by the aforementioned porcine GPLD1 gene sequence or its CDS sequence or its homologous sequence; (b) a polypeptide having the amino acid sequence shown in SEQ ID NO:1 or its homologous sequence; or (c) a derived protein or polypeptide having a cell-depleting effect by substitution, deletion, or addition of one or more amino acids in the amino acid sequences defined in (a) and (b). In some embodiments, the GPLD1 protein may be the porcine GPLD1 protein or its homologous protein of NCBI Reference Sequence: XP_013832925.2. It should be understood that the GPLD1 protein involved in this article is preferably obtained from pigs, and other GPLD1 proteins obtained from other animals that are highly homologous to the pig GPLD1 protein (such as having more than 50%, preferably more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more preferably more than 85%, such as 85%, 90%, 95%, 98%, or even 99% or more of sequence identity) are also within the scope of the preferred consideration in this invention.

[0047] As used herein, the terms “GPLD1 / GPLD1 gene,” “GPLD1 encoding molecule / sequence,” or “GPLD1 protein encoding molecule / sequence” are used interchangeably and all refer to a sequence encoding the GPLD1 protein or polypeptide described herein. The terms “encoding molecule” and “polynucleotide encoding polypeptide” can include a polynucleotide encoding the polypeptide, or can include additional encoding and / or non-coding sequences. In some embodiments, the GPLD1 encoding molecule may have the nucleotide sequence shown in NCBI Reference Sequence: XM_013977471.2, or its homologous sequence, or may be a derived polynucleotide molecule that encodes a cell-killing effect by substitution, deletion, or addition of one or more nucleotides to the aforementioned sequence.

[0048] The GPLD1 / GPLD1 gene described herein may be selected from, for example, the porcine GPLD1 gene sequence or its CDS sequence, molecules hybridized to these sequences under stringent conditions, or family gene molecules highly homologous to the aforementioned molecules. It should be understood that the gene of this invention is preferably derived from pigs. Other genes from other animals that are highly homologous to porcine GPLD1, for example, having more than 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, more preferably more than 85%, such as 85%, 90%, 95%, 98%, or even 99% or more sequence identity, and having similar function and activity to the GPLD1 protein encoded by the porcine gene, are also within the preferred scope of this invention. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.

[0049] As used herein, the terms "vector" or "expression vector" and "expression construct" are used interchangeably for a DNA molecule that operatively links GPLD1 to an oocyte and directs its expression. The vector comprises a vector as a self-replicating nucleic acid structure and a vector incorporated into the genome of the oocyte to which it has been introduced. The expression vectors of this disclosure comprise expression cassettes. Expression vectors can perform transcription of large amounts of stable mRNA. Once the expression vector is within the oocyte, cellular transcription and / or translation mechanisms generate a ribonucleic acid molecule or protein encoded by the gene. In one embodiment, the expression vector of this disclosure comprises an expression cassette containing a polynucleotide sequence encoding GPLD1. The term "expression cassette" of this disclosure refers to a recombinant or synthetically produced polynucleotide having a series of nucleic acid elements that allow specific nucleic acids to be transcribed in target cells. Recombinant expression cassettes can be introduced into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, in addition to other sequences, the recombinant expression cassette portion of the expression vector includes the nucleic acid sequence to be transcribed and a promoter. In some embodiments, the expression cassette of this disclosure contains a polynucleotide sequence encoding GPLD1.

[0050] As used herein, the term "pharmaceutical composition" refers to a mixture containing one or more GPLD1 active substances and other chemical components, such as physiological / pharmaceutical-grade carriers or excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertment of its biological activity.

[0051] As used herein, the term "pharmaceuticalally acceptable excipient" refers to a component in a pharmaceutical composition that, apart from the active ingredient, is non-toxic to the subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizers, and / or preservatives.

[0052] The "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide or amino acid is present in both the target and reference sequences. Vacancies are not nucleotides or amino acids and are not counted in the target sequence. Similarly, vacancies in the reference sequence are not counted because nucleotides or amino acids from the target sequence are counted, but those from the reference sequence are not.

[0053] The percentage of sequence identity can be calculated as follows: determine the number of positions in both sequences where the same amino acid residue or nucleic acid base appears (the number of matching positions), divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available online and downloadable. Suitable software programs are available from various sources for protein and nucleotide sequence alignment. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information (NCBI) website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparing two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0054] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0055] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the invention. The reagents and / or kits used in the following embodiments are commercially available or can be synthesized by known methods.

[0056] It should be noted that, unless specific conditions are specified in the examples, experimental conditions should be performed according to standard conditions, manufacturer recommendations, or publicly reported experimental conditions. Reagents or instruments whose manufacturers are not specified are all commercially available, standard products. For reagents whose manufacturers are specified, similar products from other manufacturers are substitutes.

[0057] In this embodiment of the invention, the quantitative experimental results are expressed as mean ± standard error (SEM), and each experiment uses at least three independent biological replicates. All data were tested for normality using GraphPad software. One-way ANOVA was used to analyze significant differences among the three independent groups; the Student's t-test was used to compare significant differences between two independent groups, with P indicating statistical significance. Finally, graphs were generated using GraphPad software.

[0058] Sequence information The sequence information involved in this invention is shown in the table below: Table 1: Sequence Information

[0059] Example 1: Oocyte Acquisition and Sorting In this embodiment, the ovaries of healthy sows of reproductive age were used as the source of oocytes. The ovaries were collected immediately after slaughter, placed in sterile physiological saline containing an appropriate amount of antibiotics and preheated to 37°C, and transported to the laboratory within 1 hour for use.

[0060] Under aseptic conditions, follicular fluid with a diameter of 3-6 mm was aspirated using an 18G injection needle to collect cumulus-oocyte complexes (COCs). The obtained COCs were then morphologically screened under a stereomicroscope, selecting those with dense cumulus cell layers, intact cell containment, homogeneous oocyte cytoplasm, and no obvious vacuoles or fragments for subsequent in vitro fertilization and embryo development-related experiments.

[0061] Example 2: Expression and localization of GPLD1 in animal oocytes To verify the expression characteristics of GPLD1 in mammalian oocytes and during fertilization, this example analyzed the expression of GPLD1 in oocytes and early embryos of different species and its localization in porcine oocytes.

[0062] Analysis of GametesOmics and DevOmics databases and publicly available transcriptome data revealed that GPLD1 is expressed in oocytes and early embryos of humans, mice, and pigs. Figure 1 (A, B, and C) indicate that this molecule is somewhat conserved during mammalian reproduction. The expression dynamics of GPLD1 vary among different species, but all cover the stages of oocyte maturation and early post-fertilization.

[0063] Furthermore, ultramicro proteomics analysis confirmed (see Itze-Mayrhofer C, et al. J Proteomics. 2020 15;225:103884) that GPLD1 was continuously expressed in porcine MII stage oocytes and IVF-4h and IVF-24h fertilized eggs. Immunofluorescence and transmission electron microscopy (see Bianchi E, et al. Nature. 2014 24;508(7497):483-7) showed that ( Figure 1 In MII stage oocytes, GPLD1 is mainly distributed in the cytoplasmic region, and after fertilization, it is significantly enriched near the oocyte membrane, with some signals located in the perivitelline space. Co-staining with cortical granules indicates that GPLD1 is distributed in the cortical region before fertilization, and exhibits perimembranous relocation characteristics after fertilization.

[0064] The above results indicate that GPLD1 has clear expression and dynamic localization characteristics in porcine oocytes and fertilized eggs, providing experimental evidence for its role as a functional molecule regulating sperm-egg recognition.

[0065] Example 3: Effects of GPLD1 overexpression on the regulation of porcine sperm-egg recognition and fertilization outcome 1. Stable high expression of GPLD1 in porcine oocytes To achieve stable high expression of GPLD1 in porcine oocytes, this embodiment constructs a GPLD1 overexpression vector driven by the CMV promoter, as follows: Figure 2 As shown in Figure A.

[0066] The overexpression vector used in this embodiment uses Gs-CMV-100A-Bspal (Nanjing Genscript Biotech Co., Ltd.) as its backbone vector. Its main structure includes: CMV promoter, CMV enhancer, multiple cloning site, cYFP fusion tag, transcription terminator, and neomycin resistance selection marker (NeoR) and other functional elements. The coding nucleic acid sequence of GPLD1 was inserted into the multiple cloning site of the Gs-CMV-100A-Bspal vector to obtain the GPLD1 overexpression vector PIG-GPLD1. The GPLD1 mRNA was generated by in vitro transcription according to the method established by our team (see, for example, Zhang, et al. Biol Reprod. 2018 1;98(4):510-519).

[0067] Mature oocytes were randomly divided into the following two groups: ① Empty vector control group: microinjection (e.g., ... Figure 2(See Figure B) Empty vector mRNA; ② GPLD1 overexpression group: GPLD1 mRNA was microinjected. Mature oocytes were obtained by culturing the oocytes obtained in Example 1. The culture conditions are described in Zhang et al. Biol Reprod. 2018 1;98(4):510-519.

[0068] After injection, oocytes were cultured for another 12 hours (culture conditions are described in Zhang et al. Biol Reprod. 2018 1;98(4):510-519) to promote the expression of exogenous GPLD1 protein. CYFP signal was observed by fluorescence microscopy, and the expression level of GPLD1 protein was detected by immunofluorescence.

[0069] The results are as follows Figure 2 As shown in Figure C, the results show that GPLD1-CYFP fluorescence signal appeared in oocytes of the GPLD1 overexpression group, indicating that the overexpression vector can effectively drive high-level expression of GPLD1 in porcine oocytes.

[0070] 2. The promoting effect of GPLD1 overexpression on the normal fertilization rate in pigs. The GPLD1-overexpressing oocytes obtained in step 1 were used for in vitro fertilization experiments (see Zhang et al. Biol Reprod. 2018 1;98(4):510-519). Four hours after fertilization, the binding of sperm and oocytes was observed by treatment with dapi dye. Twelve hours after fertilization, the formation of multiple pronuclei due to polyspermy and the formation of double pronuclei in oocytes were observed by treatment with dapi dye.

[0071] The results are as follows Figures 3 to 5 As shown, the results indicated that compared with the control group (18.11 ± 0.32), the number of sperm-bound cells on the surface of oocytes in the GPLD1 overexpression group was significantly reduced (2.318 ± 0.13). P <0.05)( Figure 3 Compared with the control group (56.27 ± 1.21), the polyspermy rate in the overexpression group was significantly lower (13.30 ± 1.18%). Figure 4 Compared with the control group (38.72 ± 0.81%), the proportion of normal double pronuclear (2PN) formation in the overexpression group was significantly increased (80.33 ± 1.6%). Figure 5 The above results indicate that overexpression of GPLD1 can effectively increase the normal fertilization rate in porcine in vitro fertilization, which is an effective molecular regulatory strategy to improve the efficiency of porcine in vitro embryo production.

[0072] Meanwhile, quantitative analysis of immunofluorescence intensity (methods see Zhang et al. Biol Reprod. 2018 1;98(4):510-519) showed that, compared with the control group (53.15 ± 4.25), the expression level of JUNO in oocytes overexpressing GPLD1-EYFP was significantly decreased (4.211 ± 0.88) (P<0.05). Figure 6 This indicates that GPLD1 overexpression can significantly reduce the abundance of JUNO on the oocyte membrane, suggesting that GPLD1 may affect sperm-egg recognition by regulating JUNO activity.

[0073] 3. Effects of GPLD1 overexpression on blastocyst formation The fertilized eggs obtained from in vitro fertilization in step 2 were cultured under uniform in vitro culture conditions (see Cong et al., Theriogenology. 2004;61:1125–1135) until day 6. Blastocyst formation was observed, and the cleavage rate and blastocyst rate were calculated.

[0074] Cleavage rate was calculated using the following formula: Cleavage rate (%) = Number of cleaved embryos / Total number of fertilized eggs in culture × 100%. Culture continued until day 6, and embryo development to the blastocyst stage was observed under an inverted microscope. Blastocyst rate was calculated using the following formula: Blastocyst rate (%) = Number of blastocysts / Total number of fertilized eggs in culture × 100%. Each experiment was repeated at least three times, with consistent operating conditions between replicates. The data obtained were used for subsequent statistical analysis.

[0075] The results are as follows Figures 7 to 8 As shown in the figure. The results showed that compared with the control group (61.50 ± 1.10%), the cleavage rate of embryos in the GPLD1 overexpression group was significantly improved (72.33 ± 1.45%); compared with the control group (18.09 ± 0.09%), the blastocyst formation rate was significantly improved (27.50 ± 2.5%). P <0.05). The results showed that the blastocyst formation rate in the GPLD1 overexpression group was significantly higher than that in the control group, and the blastocysts had complete structures and clear cavities, further verifying the promoting effect of GPLD1 overexpression on the embryo's sustainable development potential.

[0076] Example 4: Effects of GPLD1 gene interference and GPLD1 functional inhibition on porcine sperm-egg recognition and JUNO protein Mature oocytes were randomly divided into the following three groups for pretreatment: Control group: injected with disordered RNA, the sequence of which is as follows: UUCUCCGAACGUGUCACGUTT. For the injection method, please refer to Zhang et al. Biol Reprod. 2018 1;98(4):510-519; GPLD1 gene interference group: GPLD1 expression in oocytes was interfered with using specific siRNA, the sequence of which is as follows: GCCAUUGUUGACUGCUCAUTT. The injection method is described in Zhang et al. Biol Reprod. 2018 1;98(4):510-519; GPLD1 function inhibition group (also known as 1,10-phenanthroline treatment group): 1000 μM 1,10-phenanthroline was added to the fertilization culture system (for the composition of the fertilization culture system, see Zhang et al. Biol Reprod. 2018 1;98(4):510-519), and oocytes were cultured for 1 h for subsequent experiments.

[0077] Pretreated oocytes were used for in vitro fertilization experiments (methods are described in Zhang et al. BiolReprod. 2018 1;98(4):510-519). Four hours after fertilization, the sperm-fertilization capacity of the oocytes was assessed, and the number of sperm-fertilized oocytes was counted. Results are as follows: Figure 9 As shown, compared with the control group, the GPLD1 interference group exhibited a significant increase in the number of sperm binding to the oocyte surface. At the 4h IVF stage, compared with the control group (13.85 ± 1.28), the immunofluorescence signal of JUNO in the fertilized eggs of the GPLD1 interference group was significantly enhanced (68.34 ± 1.66, P<0.05). Figure 10 ).

[0078] At the same time, a similar trend was observed in the GPLD1 function suppression group. Similarly, the results are as follows: Figure 9 As shown, compared with the control group, the GPLD1 function inhibition group exhibited a significant increase in the number of sperm binding to the oocyte surface. Immunofluorescence signal of JUNO in fertilized eggs at the 4-h stage of IVF was significantly enhanced after function inhibition treatment (43.89 ± 3.04). vs. The fluorescence intensity of GPLD1 was significantly reduced (P<0.05), while that of GPLD1 was 20.55±2.11. Figure 11 ).

[0079] The above results indicate that normal GPLD1 expression plays a crucial regulatory role in maintaining the selectivity of sperm-egg recognition, ensuring normal fertilization outcomes, and promoting continuous embryonic development. Compared with the control group, the number of sperm-bound cells on the oocyte surface was significantly increased under GPLD1 function inhibition conditions, suggesting that GPLD1 functional restriction weakens the selectivity of sperm-egg recognition, leading to enhanced non-specific sperm binding. This result serves to verify the important role of GPLD1 in the regulation of sperm-egg recognition from a reverse perspective.

[0080] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. Use of glycosylated phosphatidylinositol-specific phospholipase D1 (GPLD1) as a sperm-egg recognition and / or binding regulatory molecule.

2. Uses of GPLD1-related active substances in one or more of the following: a1) Regulating the efficiency of in vitro fertilization in animals; a2) Regulate the quality of embryonic development after in vitro fertilization; a3) Improve the selectivity of sperm and egg recognition; a4) Improve embryo cleavage rate; a5) Improves blastocyst formation rate; a6) Promotes continuous embryonic development; a7) Prepare products that regulate the efficiency of in vitro fertilization in animals; a8) Prepare products that regulate the quality of embryonic development after in vitro fertilization; a9) Prepare products that improve the selectivity of sperm-egg recognition; (a10) Prepare products that improve embryo cleavage rate; a11) Prepare products that improve blastocyst formation rate; (a12) Prepare products that promote continuous embryonic development.

3. The use according to claim 2, characterized in that, The active substance can regulate the level and / or activity of GPLD1, wherein the level and / or activity of GPLD1 is the level and / or activity of GPLD1 protein or its encoded nucleic acid molecule; Preferably, the active substance is capable of regulating the level and / or activity of GPLD1 in oocytes.

4. The use according to claim 3, characterized in that, The active substance can increase GPLD1 levels and / or activity; Preferably, the active substance comprises at least one of b1) to b6): b1) GPLD1 mRNA or its derivatives; b2) GPLD1 protein or its salts; b3) Biomaterials related to GPLD1 protein; b4) Substances that promote GPLD1 expression; b5) Substances that enhance GPLD1 activity; b6) Substances that slow down GPLD1 metabolism; Preferably, the biomaterial associated with the GPLD1 protein is selected from at least one of the following: c1) Nucleic acid molecules encoding GPLD1; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A vector containing the nucleic acid molecule described in c1) or the expression cassette described in c2); c4) contains cells that include any one of c1) to c3); Preferably, the GPLD1 protein has an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; or the nucleic acid molecule encoding GPLD1 has a nucleotide sequence as shown in SEQ ID NO: 2 or a nucleotide sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

5. The use according to claim 2, characterized in that, The regulation of animal in vitro fertilization efficiency is to improve the efficiency of animal in vitro fertilization. Preferably, the improvement of in vitro fertilization efficiency in animals includes at least one of the following: Reduce the number of non-specific bindings between sperm and oocytes; Reduce the rate of polysperm entry into the egg; Increase the rate of normal dual pronucleus formation.

6. The use according to claim 2, characterized in that, The regulation of embryo development quality after in vitro fertilization aims to improve the quality of embryo development after in vitro fertilization.

7. The use according to claim 2, characterized in that, The product is applied to mammals or mammalian cells; Preferably, the mammal includes at least one of primates, rodents, livestock, and pets; Preferably, the mammal includes at least one of the following: human, rat, mouse, pig, dog, horse, cow, rabbit, monkey, ape, and orangutan.

8. The use according to claim 2, characterized in that, The product is selected from at least one of the following: pharmaceuticals, pharmaceutical compositions, reagent kits, and medical devices.

9. The use according to claim 2, characterized in that, The product also includes other active ingredients that regulate the efficiency of in vitro fertilization in animals or the quality of embryonic development after in vitro fertilization.

10. The use according to claim 2, characterized in that, The product also includes a pharmaceutically acceptable carrier.

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