Biomarker for diagnosing male asthenospermia and infertility diseases and application thereof

Abstract

The invention provides a biomarker for male asthenospermia and infertility disease diagnosis and application thereof. The 541st base C of the nucleotide sequence of mRNA of a Tex44 mutant gene is mutated into T; preferably, the nucleotide sequence of the mRNA of the Tex44 mutant gene is as shown in SEQ ID NO. 4. The Tex44 gene can be used as a biomarker to be applied to preparation of a kit for diagnosing male asthenospermia and male infertility, and a new biomarker and a new target spot for treating male asthenospermia and / or male infertility are provided for an existing kit for diagnosing male infertility. Male asthenospermia and male infertility can be prevented and / or treated by improving or reducing the activity and / or expression quantity of the TEX44 protein.

Description

Technical Field

[0001] The present application relates to the field of biomedical technologies, and particularly to a biomarker for diagnosing male asthenospermia and infertility diseases and its application. Background Art

[0002] Asthenospermia is one of the most severe sperm quality defects. In most cases, it is due to gene mutations that regulate the encoding of sperm flagellum formation, resulting in changes in the ultrastructure of sperm flagella, which in turn affect sperm motility and fertilization, causing male infertility. Idiopathic asthenozoospermia (IAS) is a special clinical phenotype group among asthenospermic men, and its clinical manifestation is simply low sperm motility. However, the pathogenic mechanism of IAS men has not been elucidated yet, which is a major problem in the clinical diagnosis and treatment of male infertility, and this also leads to the lack of effective treatment means for IAS in clinical practice. Although some asthenospermic patients can obtain their own offspring through assisted reproductive technology (ART), for sperm abnormalities or male infertility caused by gene defects, assisted reproductive technology can cause their vertical transmission, thus affecting the reproductive health of the offspring.

[0003] Currently, the common method for judging male fertility is still based on the routine semen analysis (including sperm morphology, density, quantity, motility rate, viability, etc.) in the World Health Organization's Laboratory Manual for the Examination and Processing of Human Semen (6th Edition) combined with the analysis of serum hormone levels. However, the ability to predict male fertility status based on this is very limited. Routine semen analysis can only reflect the most basic sperm quality and cannot reflect other characteristics of sperm and sperm function defects. Therefore, it is necessary to identify new biomolecular markers for male infertility and / or asthenospermia to assist traditional routine semen analysis. Summary of the Invention

[0004] The purpose of the present application is to provide a biomarker for diagnosing male asthenospermia and infertility diseases and its application. The Tex44 gene and TEX44 protein can be used as biomarkers in the application of preparing kits for diagnosing male asthenospermia, teratospermia, abnormal fertilization, male infertility, etc., providing a new target for the treatment of male infertility. The specific technical solutions are as follows:

[0005] The first aspect of the present application provides a Tex44 mutant gene, wherein the 541st base of the nucleotide sequence of the mRNA of the Tex44 mutant gene is T; preferably, the nucleotide sequence of the mRNA of the Tex44 mutant gene is as shown in SEQ ID NO.4.

[0006] The second aspect of the present application provides a TEX44 mutant protein, wherein the 181st amino acid of the amino acid sequence of the TEX44 mutant protein is Q; preferably, the amino acid sequence of the TEX44 mutant protein is as shown in SEQ ID NO.5.

[0007] The third aspect of the present application provides any one of the following applications of a biomarker and / or a substance for detecting the biomarker:

[0008] A1) Application in the preparation of a product for studying the spermatogenesis process;

[0009] A2) Application in the preparation of a product for identifying the germ cell classification process;

[0010] A3) Application in the preparation of a product for screening male infertility diseases;

[0011] A4) Application in the preparation of a product for diagnosing male asthenospermia;

[0012] A5) Application in the preparation of a product for diagnosing abnormal sperm-egg fertilization;

[0013] A6) Application in the preparation of a product for diagnosing male teratozoospermia;

[0014] A7) Application in the preparation of a product for diagnosing male infertility;

[0015] Wherein, the biomarker is selected from the Tex44 gene, TEX44 protein, mRNA of the Tex44 gene, RNA of the Tex44 gene, miRNA targeting the TEX44 protein, the Tex44 mutant gene described in the first aspect of the present application, or the TEX44 mutant protein described in the second aspect of the present application.

[0016] In some embodiments of the present application, the substance is a reagent for detecting the presence or absence of the Tex44 gene, TEX44 protein, mRNA of the Tex44 gene, RNA of the Tex44 gene, miRNA targeting the TEX44 protein, the Tex44 mutant gene described in the first aspect of the present application, or the TEX44 mutant protein described in the second aspect of the present application; preferably, the detection sample for the detection is selected from at least one of blood, oral tissue, testicular tissue, and sperm.

[0017] In some embodiments of the present application, the nucleotide sequence of the Tex44 gene is as shown in SEQ ID NO.1; alternatively, the nucleotide sequence of the mRNA of the Tex44 gene is as shown in SEQ ID NO.2; alternatively, the amino acid sequence of the TEX44 protein is as shown in SEQ ID NO.3.

[0018] The fourth aspect of the present application provides a kit, wherein the kit includes reagents for detecting the presence of Tex44 gene, TEX44 protein, mRNA of Tex44 gene, RNA of Tex44 gene, the Tex44 mutant gene described in the first aspect of the present application, or the TEX44 mutant protein described in the second aspect of the present application, and the kit has at least one of the following uses:

[0019] A1) Studying the process of spermatogenesis;

[0020] A2) Identifying the process of germ cell classification;

[0021] A3) Screening male infertility diseases;

[0022] A4) Diagnosing male asthenospermia;

[0023] A5) Diagnosing abnormal sperm-egg fertilization;

[0024] A6) Diagnosing male teratozoospermia;

[0025] A7) Diagnosing male infertility.

[0026] In some embodiments of the present application, the kit includes at least one of primer pairs with nucleotide sequences shown in SEQ ID NO.6 and SEQ ID NO.7, primer pairs with nucleotide sequences shown in SEQ ID NO.8 and SEQ ID NO.9, and primer pairs with nucleotide sequences shown in SEQ ID NO.10 and SEQ ID NO.11.

[0027] In some embodiments of the present application, the nucleotide sequence of the Tex44 gene is as shown in SEQ ID NO.1; alternatively, the nucleotide sequence of the mRNA of the Tex44 gene is as shown in SEQ ID NO.2; alternatively, the amino acid sequence of the TEX44 protein is as shown in SEQ ID NO.3.

[0028] In some embodiments of the present application, the detection samples of the kit are selected from at least one of blood, oral tissue, testicular tissue, and sperm;

[0029] Preferably, the method for judging the detection result of the kit includes: if the Tex44 gene, TEX44 protein, mRNA of the Tex44 gene, RNA of the Tex44 gene, or miRNA targeting the TEX44 protein does not exist in the detection sample, the source of the detection sample is suspected of suffering from male asthenospermia, teratozoospermia, abnormal fertilization, or male infertility;

[0030] Alternatively, if the Tex44 mutant gene described in the first aspect of the present application or the TEX44 mutant protein described in the second aspect of the present application is present in the test sample, the source of the test sample is suspected of suffering from asthenozoospermia, teratozoospermia, abnormal fertilization, or male infertility.

[0031] The fifth aspect of the present application provides an application of a biomarker as a drug target in the preparation of a drug having any one of the following uses:

[0032] B1) Treating male asthenozoospermia;

[0033] B2) Treating male teratozoospermia;

[0034] B3) Treating male infertility;

[0035] B4) Improving male fertility;

[0036] B5) Treating sperm dysfunction;

[0037] B6) Promoting sperm fertilization;

[0038] B7) Improving the motility of sperm;

[0039] B8) Improving sperm quality;

[0040] B9) Repairing abnormal sperm morphology;

[0041] B10) Repairing abnormal sperm tail structure;

[0042] Wherein, the biomarker is selected from TEX44 protein or Tex44 gene.

[0043] Advantages of the present application:

[0044] The Tex44 gene and / or TEX44 protein described in the present application can be used as a biomarker in the application of preparing a kit for diagnosing male asthenozoospermia and male infertility, providing a new biomarker for the existing kit for diagnosing male infertility and a new target for treating male asthenozoospermia and / or male infertility. The presence of the Tex44 mutant gene or the TEX44 mutant protein will lead to a decrease in sperm motility, abnormal ultrastructure of the sperm tail, and a significant decrease in the fertilization rate. By detecting the Tex44 mutant gene or the TEX44 mutant protein, male asthenozoospermia can be diagnosed, which has great application value. Moreover, the knockout of the Tex44 gene can affect the expression level changes of 1542 genes related to spermatogenesis. By increasing or decreasing the activity and / or expression level of the TEX44 protein, male asthenozoospermia and male infertility can be prevented and / or treated.

[0045] Of course, when implementing any product or method of the present application, it is not necessarily required to achieve all the above-mentioned advantages simultaneously. Brief Description of the Drawings

[0046] To more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present application, and those of ordinary skill in the art can also obtain other embodiments based on these drawings.

[0047] Figure 1 It is a diagram showing the amino acid alignment results of TEX44 proteins of different mammals and humans in the present application;

[0048] Figure 2A It is a diagram showing the expression results (Q-PCR) of the Tex44 gene in different tissues of wild-type adult mice in Example 2 of the present application;

[0049] Figure 2B It is a diagram showing the expression results (Q-PCR) of the Tex44 gene in testes at different stages of wild-type mice in Example 2 of the present application;

[0050] Figure 2C It is a diagram showing the cellular localization of the TEX44 protein in the testes of wild-type mice in Example 2 of the present application;

[0051] Figure 2D It is a diagram showing the cellular localization of the TEX44 protein in human testes in Example 2 of the present application;

[0052] Figure 3A It is a diagram showing the gene knockout strategy design in Example 3 of the present application;

[0053] Figure 3B It is a diagram showing the results of PCR identification of the knockout mice in the F1 generation in Example 3 of the present application;

[0054] Figure 3C It is a diagram showing the results of PCR identification of the homozygous knockout mice in Example 3 of the present application;

[0055] Figure 3D It is a diagram showing the comparison results of the sizes of the testes and epididymides of wild-type mice (TEX44+ / + mice) and Tex44 gene knockout homozygous F2 mice (TEX44- / - mice) in Example 3 of the present application;

[0056] Figure 3E It is a diagram showing the comparison results of the volume sizes of the testes of wild-type mice (TEX44+ / + mice) and Tex44 gene knockout homozygous F2 mice (TEX44- / - mice) in Example 3 of the present application;

[0057] Figure 3FThis is the result graph of the weight change of testes of wild-type mice (TEX44+ / + mice) and Tex44 gene knockout homozygous F2 mice (TEX44- / - mice) at different weeks of age in Example 3 of this application;

[0058] Figure 4A This is the dynamic analysis graph of epididymal sperm of wild-type mice (WT mice) and Tex44 gene knockout homozygous mice (TEX44- / - mice) in Example 4 of this application;

[0059] Figure 4B This is the result graph of the comparison of sperm motility rates of wild-type mice (WT mice) and Tex44 gene knockout homozygous mice (TEX44- / - mice) in Example 4 of this application;

[0060] Figure 4C This is the result graph of the comparison of sperm densities of wild-type mice (WT mice) and Tex44 gene knockout homozygous mice (TEX44- / - mice) in Example 4 of this application;

[0061] Figure 4D This is the result graph of the comparison of forward motile sperm of wild-type mice (WT mice) and Tex44 gene knockout homozygous mice (TEX44- / - mice) in Example 4 of this application;

[0062] Figure 4E This is the result graph of the comparison of non-forward motile sperm of wild-type mice (WT mice) and Tex44 gene knockout homozygous mice (TEX44- / - mice) in Example 4 of this application;

[0063] Figure 4F This is the result graph of the comparison of immotile sperm of wild-type mice (WT mice) and Tex44 gene knockout homozygous mice (TEX44- / - mice) in Example 4 of this application;

[0064] Figure 5A This is the result graph of in vivo fertilization experiments in wild-type mice and TEX44- / - male mice in Example 5 of this application;

[0065] Figure 5B This is the result graph of in vivo fertilization experiments in TEX44- / - male mice of different weeks of age and wild-type female mice in Example 5 of this application;

[0066] Figure 5C This is the result graph of in vitro fertilization experiments of epididymal sperm of TEX44- / - male mice in Example 5 of this application;

[0067] Figure 6A This is the electron micrograph of the principal piece, midpiece and endpiece of the sperm flagellum of TEX44- / - mice in Example 6 of this application;

[0068] Figure 6B This is a scanning electron micrograph of different types of axoneme deletions in the sperm tails of TEX44 - / - mice in Example 6 of the present application;

[0069] Figure 6C This is a graph showing the percentage of different types of axoneme deletions in the sperm tails of TEX44 - / - mice in Example 6 of the present application;

[0070] Figure 7A This is a graph of the HE staining results of paraffin sections of testis and epididymis in Example 7 of the present application;

[0071] Figure 7B This is a graph of the HE staining results of paraffin sections of seminiferous tubules of testis in Example 7 of the present application;

[0072] Figure 8A This is a comparison graph of the testicular transcriptome sequencing results between TEX44 - / - mice and wild - type mice in Example 8 of the present application;

[0073] Figure 8B This is a graph of the GO analysis results in Example 8 of the present application;

[0074] Figure 8C This is a graph of the KEGG analysis results in Example 8 of the present application;

[0075] Figure 8D This is a graph of the structural angle analysis results in Example 8 of the present application;

[0076] Figure 8E This is a graph of the functional angle analysis results in Example 8 of the present application;

[0077] Figure 8F This is a graph of the further Reactome analysis results in Example 8 of the present application;

[0078] Figure 9 This is a scanning electron micrograph analysis of the sperm of a TEX44 mutant patient in Example 9 of the present application. Detailed implementation manners

[0079] Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all of the embodiments. All other embodiments obtained by those skilled in the art based on the present application belong to the scope of protection of the present application.

[0080] In the first aspect of the present application, a Tex44 mutant gene is provided, wherein the 541st base of the nucleotide sequence of the mRNA of the Tex44 mutant gene is T (mutated from C); preferably, the nucleotide sequence of the mRNA of the Tex44 mutant gene is as shown in SEQ ID NO.4.

[0081] SEQ ID NO.4 (Base C at position 541 mutated to T): actccctcca accgccacaa gcagcaaagggcatagatga ccacaggaga gccctagggg gaggccggct ccaccagcag ccccatacat ggcactcccagggtaccccc tgggcaacgtggatgacagc aggtctaagg acagcccagc aggagagccc caaggtcaggtcccactcac agcagatgtc ttggcagtgagcagctctgt cgcatccact gactggcagg atatagatcaggcctccttc aagacggcca cccccagggc catatcaacatctggagaca aagacaagag tgcagttgttccagaacacg gccagaagac acccagaaaa atcacacctc tgcttcccagccaaaatcca agtcccctgcaagtgtctat gagccttcag aatccagcgt gggacaggca ggttcaagac gcaaggacaagtcagagtctagtggttttc ccaagtcacc tcctgggtaa agacaagatg tcacaaatgg cgagtgttcctgagagagagccggagtcag ccccctctgc tccgagtgct gagctacagt ccacccagca catggaggctcagcccgtcg agagtgatgctgaccatgtc acagcaggtg ccaatggcca gcatggccct caggctgccagcaccaccaa gtctgctgag gagaaagctgagcatccaaa ggccccacac cctgaagctg aagctttaccatctgatgag tccccagtgg caatgggggc aaatgtggtggacagcttag gagacctgca gacttggttcttccctccac ctccagcagg cagtgtgtcc ccgtcgcctg gcccccacgaggtggccctg gggagaaggcccctggactc cagcctgtac acggccagtg aggagaacag ctacatgcgc tccatgaccagcctgctggacaggggcgag ggctccatca gctccctggc agacatcctg gtgtggtccg agaccaccatgggcatggccatagccacag gcttcctgga ttccggccac agcactgtgg cagacctgct gcacagctcggggcccagct tgcgctcggtccccagcctg gtgggaagcg tcagctcggc cttctcctct gggctggtgtcagggaccag ctcagccctg cgcaccatcacccgtgtgct ggagacagtg gagcagagga ccgtggagggcatccgctca gccatgcgct acctgaccag ccacctcaccccacgccagg cccaggctga ccccaactatgattagagcc ctgcacaggg acccccgaag ggctggttca gtggcctccagagatccctg ggaccctcacgccctgctcc cttgcccatt cttgctctgg acggttcccc cagcccatgc aataaatcaccagtgagcattta。

[0082] The second aspect of the present application provides a TEX44 mutant protein, wherein the 181st amino acid of the amino acid sequence of the TEX44 mutant protein is Q (glutamine); preferably, the amino acid sequence of the TEX44 mutant protein is as shown in SEQ ID NO.5.

[0083] SEQ ID NO.5 (the 181st amino acid is Q): malpgyplgn vddsrskdsp agepqgqvpltadvlavsss vastdwqdid qasfktatpr aistsgdkdk savvpehgqk tprkitpllp sqnpsplqvsmslqnpawdr qvqdartsqslvvfpshllg kdkmsqmasv perepesaps apsaelqstq hmeaqpvesdadhvtagang q hgpqaastt ksaeekaehpkaphpeaeal psdespvamg anvvdslgdl qtwffppppagsvspspgph evalgrrpld sslytaseen symrsmtslldrgegsissl adilvwsett mgmaiatgfldsghstvadl lhssgpslrs vpslvgsvss afssglvsgt ssalrtitrv letveqrtvegirsamryltshltprqaqa dpnyd。

[0084] The third aspect of the present application provides any one of the following applications of a biomarker and / or a substance for detecting the biomarker:

[0085] A1) Application in the preparation of a product for studying the process of spermatogenesis;

[0086] A2) Application in the preparation of a product for identifying the process of germ cell classification;

[0087] A3) Application in the preparation of a product for screening male infertility diseases;

[0088] A4) Application in the preparation of a product for diagnosing male asthenospermia;

[0089] A5) Application in the preparation of a product for diagnosing abnormal sperm-egg fertilization;

[0090] A6) Application in the preparation of a product for diagnosing teratozoospermia in males;

[0091] A7) Application in the preparation of a product for diagnosing male infertility;

[0092] Among them, the biomarker is selected from Tex44 gene, TEX44 protein, mRNA of Tex44 gene, RNA of Tex44 gene, miRNA targeting TEX44 protein, the Tex44 mutant gene described in the first aspect of the present application, or the TEX44 mutant protein described in the second aspect of the present application.

[0093] The Tex44 gene is conserved among multiple species such as cattle, sheep, mice, rhesus monkeys and humans, and is highly expressed in the testes of mammals. However, the spatio-temporal expression, subcellular localization, changes in expression level and function of the Tex44 gene in the testis have not been reported, and its biological function, molecular mechanism and related pathogenic mechanism are still unclear.

[0094] The inventors of the present application aligned the amino acids of TEX44 proteins from different mammals and humans. Figure 1 The result of the alignment is shown. The results show that the Tex44 gene is located on human chromosome 2 and consists of 395 amino acids. Through analysis and alignment using the BLAST software, it is found that the amino acid sequence of the TEX44 protein is highly homologous to that of mice, rhesus monkeys, cattle, sheep, etc.

[0095] The inventors of the present application found in the research that the presence of the Tex44 mutant gene or the TEX44 mutant protein can lead to a decrease in sperm motility, abnormal ultrastructure of sperm tails, and a significant decrease in fertilization rate. Detection of the Tex44 mutant gene or the TEX44 mutant protein can be used to diagnose male asthenospermia, which has great application value. Moreover, knockout of the Tex44 gene can affect the expression level changes of 1542 genes related to spermatogenesis. Preventing and / or treating male asthenospermia and male infertility can be achieved by increasing or decreasing the activity and / or expression level of the TEX44 protein.

[0096] In some embodiments of the present application, the substance is a reagent for detecting the presence or absence of Tex44 gene, TEX44 protein, mRNA of Tex44 gene, RNA of Tex44 gene, miRNA targeting TEX44 protein, the Tex44 mutant gene described in the first aspect of the present application, or the TEX44 mutant protein described in the second aspect of the present application.

[0097] In some embodiments of the present application, the test sample for the detection is selected from at least one of blood, oral tissue, testicular tissue and sperm.

[0098] In some embodiments of the present application, the nucleotide sequence of the Tex44 gene is as shown in SEQ ID NO.1; alternatively, the nucleotide sequence of the mRNA of the Tex44 gene is as shown in SEQ ID NO.2; alternatively, the amino acid sequence of the TEX44 protein is as shown in SEQ ID NO.3.

[0099]

[0100] SEQ ID NO.2 (Nucleotide sequence of the mRNA of human Tex44 gene): actccctcca accgccacaagcagcaaagg gcatagatga ccacaggaga gccctagggg gaggccggct ccaccagcag ccccatacatggcactccca gggtaccccctgggcaacgt ggatgacagc aggtctaagg acagcccagc aggagagccccaaggtcagg tcccactcac agcagatgtcttggcagtga gcagctctgt cgcatccact gactggcaggatatagatca ggcctccttc aagacggcca cccccagggccatatcaaca tctggagaca aagacaagagtgcagttgtt ccagaacacg gccagaagac acccagaaaa atcacacctctgcttcccag ccaaaatccaagtcccctgc aagtgtctat gagccttcag aatccagcgt gggacaggca ggttcaagacgcaaggacaagtcagagtct agtggttttc ccaagtcacc tcctgggtaa agacaagatg tcacaaatggcgagtgttcctgagagagag ccggagtcag ccccctctgc cccgagtgct gagctacagt ccacccagcacatggaggct cagcccgtcgagagtgatgc tgaccatgtc acagcaggtg ccaatggcca gcatggccctcaggctgcca gcaccaccaa gtctgctgaggagaaagctg agcatccaaa ggccccacac cctgaagctgaagctttacc atctgatgag tccccagtgg caatgggggcaaatgtggtg gacagcttag gagacctgcagacttggttc ttccctccac ctccagcagg cagtgtgtcc ccgtcgcctggcccccacga ggtggccctggggagaaggc ccctggactc cagcctgtac acggccagtg aggagaacagctacatgcgctccatgaccagcctgctgga caggggcgag ggctccatca gctccctggc agacatcctg gtgtggtccgagaccaccatgggcatggcc atagccacag gcttcctgga ttccggccac agcactgtgg cagacctgctgcacagctcg gggcccagcttgcgctcggt ccccagcctg gtgggaagcg tcagctcggc cttctcctctgggctggtgt cagggaccag ctcagccctgcgcaccatca cccgtgtgct ggagacagtg gagcagaggaccgtggaggg catccgctca gccatgcgct acctgaccagccacctcacc ccacgccagg cccaggctgaccccaactat gattagagcc ctgcacaggg acccccgaag ggctggttcagtggcctcca gagatccctgggaccctcac gccctgctcc cttgcccatt cttgctctgg acggttcccc cagcccatgcaataaatcaccagtgagcat tta。

[0101] SEQ ID NO.3 (Amino acid sequence of human TEX44 protein): malpgyplgn vddsrskdsp agepqgqvpltadvlavsss vastdwqdid qasfktatpr aistsgdkdk savvpehgqk tprkitpllp sqnpsplqvsmslqnpawdrqvqdartsqs lvvfpshllg kdkmsqmasv perepesaps apsaelqstq hmeaqpvesdadhvtagang qhgpqaasttksaeekaehp kaphpeaeal psdespvamg anvvdslgdl qtwffppppagsvspspgph evalgrrpld sslytaseensymrsmtsll drgegsissl adilvwsett mgmaiatgfldsghstvadl lhssgpslrs vpslvgsvss afssglvsgt ssalrtitrvletveqrtve girsamryltshltprqaqa dpnyd。

[0102] The fourth aspect of the present application provides a kit, wherein the kit includes reagents for detecting the presence of Tex44 gene, TEX44 protein, mRNA of Tex44 gene, RNA of Tex44 gene, the Tex44 mutant gene described in the first aspect of the present application, or the TEX44 mutant protein described in the second aspect of the present application. The kit has at least one of the following uses:

[0103] A1) Studying the process of spermatogenesis;

[0104] A2) Identifying the process of germ cell classification;

[0105] A3) Screening male infertility diseases;

[0106] A4) Diagnosing male asthenospermia;

[0107] A5) Diagnosing abnormal fertilization of sperm and egg;

[0108] A6) Diagnosing teratozoospermia in males; A7) Diagnosing male infertility.

[0109] The Tex44 gene and TEX44 protein described in the present application can be used as biomarkers in the preparation of a kit for diagnosing male asthenospermia and male infertility, providing a new biomarker for existing kits for diagnosing male infertility and a new target for treating male asthenospermia and / or male infertility.

[0110] In some embodiments of the present application, the kit includes at least one of primer pairs with nucleotide sequences shown in SEQ ID NO.6 and SEQ ID NO.7, primer pairs with nucleotide sequences shown in SEQ ID NO.8 and SEQ ID NO.9, and primer pairs with nucleotide sequences shown in SEQ ID NO.10 and SEQ ID NO.11.

[0111] SEQ ID NO.6 (upstream primer of human Tex44 gene DNA): TCCTCCTGTGTCCCTGTGGTTG;

[0112] SEQ ID NO.7 (downstream primer of human Tex44 gene DNA): TTGCTGCTTGTGGCGGTTGG;

[0113] SEQ ID NO.8 (upstream primer of human Tex44 gene mRNA): CTCCATAGGGCAAGATGTAGAG;

[0114] SEQ ID NO.9 (downstream primer of human Tex44 gene mRNA): CTCCGACTTGTACGGTTGTT;

[0115] SEQ ID NO.10 (upstream primer of human Tex44 gene mRNA): GTCCCACTCACAGCAGATGTC TTG;

[0116] SEQ ID NO.11 (downstream primer of human Tex44 gene mRNA): ACTTGTCCTTGCGTCTTGAACCTG.

[0117] In some embodiments of the present application, the nucleotide sequence of the Tex44 gene is as shown in SEQ ID NO.1; alternatively, the nucleotide sequence of the mRNA of the Tex44 gene is as shown in SEQ ID NO.2; alternatively, the amino acid sequence of the TEX44 protein is as shown in SEQ ID NO.3.

[0118] In some embodiments of the present application, the detection sample of the kit is selected from at least one of blood, oral tissue, testicular tissue, and sperm.

[0119] In some embodiments of the present application, the method for judging the detection result of the kit includes: if the Tex44 gene, TEX44 protein, mRNA of the Tex44 gene, RNA of the Tex44 gene, or miRNA targeting the TEX44 protein does not exist in the detection sample, the source of the detection sample is suspected of suffering from asthenospermia, teratospermia, abnormal fertilization, or male infertility;

[0120] Alternatively, if the Tex44 mutant gene described in the first aspect of the present application or the TEX44 mutant protein described in the second aspect of the present application is present in the test sample, the source of the test sample is suspected of suffering from asthenospermia, teratospermia, abnormal fertilization, or male infertility.

[0121] The fifth aspect of the present application provides the use of a biomarker as a drug target in the preparation of a drug having any one of the following uses:

[0122] B1) Treating asthenospermia;

[0123] B2) Treating teratospermia;

[0124] B3) Treating male infertility;

[0125] B4) Improving male fertility;

[0126] B5) Treating sperm dysfunction;

[0127] B6) Promoting sperm fertilization;

[0128] B7) Improving the motility of sperm;

[0129] B8) Improving sperm quality;

[0130] B9) Repairing abnormal sperm morphology;

[0131] B10) Repairing abnormal sperm tail structure;

[0132] Wherein, the biomarker is selected from TEX44 protein or Tex44 gene.

[0133] The present application provides the use of a novel TEX44 mutation site, namely a mutant gene / protein, as a molecular marker in the diagnosis of asthenospermia and male infertility diseases, and reveals the application of the TEX44 mutation site as a molecular marker in the preparation of a kit for diagnosing asthenospermia and male infertility, providing a new molecular marker and therapeutic target for existing kits for diagnosing male infertility and / or asthenospermia.

[0134] The present application also provides the use of Tex44 gene and / or TEX44 protein. Tex44 gene and / or TEX44 protein can be used as molecular markers to prepare a kit for diagnosing male infertility and / or asthenospermia, and at the same time can be used as a drug target to prepare drugs that can prevent and diagnose male infertility and / or asthenospermia; treat male infertility and / or asthenospermia; treat sperm dysfunction; promote sperm fertilization; improve the motility and fertilization ability of sperm; improve sperm quality.

[0135] Examples

[0136] Hereinafter, examples and comparative examples are given to more specifically illustrate the embodiments of the present application. Various tests and evaluations are carried out according to the following methods. In addition, unless otherwise specified, "parts" and "%" are based on mass.

[0137] The experimental methods in the following examples are all conventional methods unless otherwise specified; the experimental materials used in the following examples are all obtained from regular biochemical reagent stores unless otherwise specified.

[0138] All animal experiments in the following examples were carried out in the Animal Experiment Center of Peking University Health Science Center, and water and food were freely available during the experiment.

[0139] In the following examples, WT, TEX44+ / + and Tex44+ / + all represent wild-type mice, and TEX44- / -、TEX44-KO、KO and Tex44- / - all represent Tex44 gene knockout homozygous mice.

[0140] Example 1: Screening of patients with Tex44 mutant genes among asthenozoospermia patients and semen analysis

[0141] The research involved in this example was approved by the Ethics Committee of Peking University Third Hospital and informed consent was obtained from the patients. 106 discarded semen samples of male infertility and asthenozoospermia patients collected from Peking University Third Hospital were excluded from patients with organic lesions of the reproductive system, untreated endocrine disorders, drug or alcohol abuse within two years, chromosomal, AZF abnormalities, cryptorchidism, or semen abnormalities caused by known causes such as mumps.

[0142] Method: Use the TIANamp Micro DNA Kit to extract the patient's blood DNA. According to the operation method of the instruction manual, use the patient's blood genomic DNA as a template, amplify the exon sequence of the Tex44 gene through PCR reaction, and send it for sequencing. After the sequencing results are returned, sequence alignment is performed to screen patients containing the Tex44 mutant gene.

[0143] Result analysis: Two patients with Tex44 mutant defective genes were screened. Among them, the Tex44 gene mutation occurred in its exon. The nucleotide sequence was the 541st base C of the Tex44 gene sequence mutated to T (C.541C>T), and its mRNA nucleotide sequence was as shown in SEQ ID NO.4; or its amino acid sequence was the 181st amino acid of the TEX44 polypeptide mutated to Q (p.Q181), and the amino acid sequence of the TEX44 mutant protein was as shown in SEQ ID NO.5. The above results indicate that the TEX44-C.541C>T mutation site can be used as a molecular marker for the diagnosis of male asthenozoospermia.

[0144] Analysis of sperm conditions in patients with the above-mentioned Tex44 mutant defective gene; semen was collected after 5 days of abstinence. After liquefaction at 37°C for 30 min, CASA (computer-aided sperm analysis) was used to detect semen volume, concentration, sperm morphology, motility, and viability. The results are as follows: It was found that the sperm concentration, percentage of motile sperm, percentage of progressively motile sperm, and proportion of sperm with normal morphology in patients carrying the TEX44 mutant gene all decreased (the patient had undergone 2 times and the average value was taken).

[0145] Example 2: Fluorescent quantitative PCR detection and immunohistochemical detection

[0146] 1. Fluorescent quantitative PCR detection: Total RNA of mouse testes at different tissues and different stages was extracted using Trizol reagent (Thermo Fisher). Reverse transcription of 500 ng of total RNA was performed using the Prime-Script RT reagent kit (Takara RR047A). Using the cDNA products of different samples as templates, real-time fluorescent quantitative PCR detection was carried out. The expression level of Tex44 was normalized to Gapdh by the 2-ΔCt method and reported as fold change.

[0147] 2. Immunohistochemistry: Fresh testes and cauda epididymis of wild-type (WT) mice and male testicular biopsy tissue samples were fixed in 4% paraformaldehyde (Servicebio, G1101-500ML) and immunostained for 24 hours. Paraffin sections were boiled in 10 mM sodium citrate buffer (pH 6.0) for 15 min, gradually cooled to room temperature, and washed 3 times with PBS containing 0.1% Triton X-100. The sections were blocked with 3 w / v% bovine serum albumin (BSA) at room temperature for 1 hour and then incubated with the primary antibody overnight at 4°C. Next, the sections were incubated with the corresponding secondary antibody at room temperature for 1 hour. Then, these sections were washed, mounted, and analyzed according to the standard protocol. The antibodies used in this example included anti-human TEX44 (Humanatlas, HPA049917, 1:200) and anti-mouse TEX44 (Immunoway, YN5943, 1:200). Results: Figures 2A - 2D It is a diagram of the expression and testicular cell localization of the Tex44 gene in different tissues and different stages of mouse testes. Among them, from Figure 2A the Q-PCR results of different tissues of wild-type adult mice, it can be seen that Tex44 is a new testis-specific expression gene, which is specifically expressed only in adult mouse testes and not in tissues such as the heart, liver, kidney, lung, small intestine, spleen, and muscle of adult mice; the results show that Tex44 can be applied to the study of the process of spermatogenesis. By detecting mouse testicular cDNA at different time periods, the results are as Figure 2B shown, fromFigure 2B It can be seen that the Tex44 gene begins to be weakly expressed and transcribed initially at 18 days after the birth of mice, and then the transcription increases starting from 24 days (this stage is exactly the first wave of the transformation from round sperm to elongated sperm) and continues until adulthood. By immunohistochemical detection of the expression of TEX44 protein on mouse testicular tissue sections, the results are as Figure 2C shown. The TEX44 protein is only localized in the cytoplasm of mouse elongated sperm; the results indicate that the TEX44 antibody can be used as a biomarker for elongated sperm cells and applied to identify elongated sperm cells mixed in different types of germ cells (such as: elongated sperm cells mixed with Sertoli cells, interstitial cells, spermatogonial stem cells, spermatocytes, and round spermatids). By immunohistochemical detection of the expression of TEX44 protein on human testicular tissue sections, the results are as Figure 2D shown. The TEX44 protein is also localized in the cytoplasm of human elongated sperm; the above results show that Tex44 is a testis-specifically expressed gene and is only localized in the cytoplasm of elongated sperm; it can not only be applied to the study of the process of spermatogenesis, but also be used to distinguish different types of germ cells.

[0148] Example 3: Preparation and analysis of Tex44 gene knockout mice

[0149] 1. Using the CRISPR / Cas9 technology, two sgRNA sequences targeting the Tex44 gene (the number in the NCBI database is Gene ID: 71863) were designed through the CRISPR online website (http: / / crispr.mit.edu), and the design strategy is as Figure 3A shown. After synthesis and annealing, they were ligated to the pX458 vector expressing the Cas9 protein.

[0150] 2. The recombinant pX458 vector was transfected into embryonic stem cells. After flow cytometry screening, it was transferred into a culture dish and cultured for 24 hours. After the culture was completed, monoclonal picking and genotype identification were carried out.

[0151] 3. C57BL / 6N female mice superovulated by hormone treatment were mated with wild-type male mice in advance to obtain a large number of blastocyst cells (at the same time, ligated male mice were mated with C57BL / 6N female mice to produce pseudopregnant female mice). The gene-modified embryonic stem cells screened in the previous step were injected into the blastocysts obtained in this step. After short-term in vitro culture, the blastocysts were transplanted back into the uterus of pseudopregnant female mice to obtain F0 generation gene mutant mice.

[0152] 4. The genotype of F0 mice was identified by the PCR method, and the identification system was 25 μL; the primer sequences used were as follows:

[0153] Tex44-FP: GCTAAGACAGGAAGATCAGAGTG (SEQ ID NO.12);

[0154] Tex44-RP-KO: TGGAGAAGCCAGGAGAGTTAA (SEQ ID NO.13);

[0155] Tex44-RP-WT: ATGAAGGTATCAGTCCGAATGAAGTCC (SEQ ID NO.14).

[0156] 5. Crossbreed the heterozygous F1 mice with knocked-out Tex44 gene to obtain homozygous F2 mice with knocked-out Tex44 gene (hereinafter referred to as TEX44- / - mice) and wild-type mice (hereinafter referred to as TEX44+ / + mice).

[0157] 6. Identification and analysis of knocked-out mice

[0158] The results are as Figure 3B and Figure 3C shown, Figure 3B which is the PCR identification result diagram of the F1 generation of the knocked-out mice in Example 3 of this application; it can be seen from the figure that a 703bp-sized band was amplified in the F1 generation mice and wild-type with P1 as the primer, and a 500bp-sized band could be amplified for the allele containing the TEX44KO mutation in this system, indicating that the target band (exon sequence of Tex44 gene) has been knocked out.

[0159] Figure 3C This is the PCR identification result diagram of the homozygous knocked-out mice in Example 3 of this application; it can be seen from the figure that for the Tex44 gene of the F2 generation mice, it was found that the TEX44+ / + representing wild-type mice could amplify the Tex44 gene band, while the TEX44- / - knocked-out mice could not amplify the band, indicating that TEX44-KO mice have been prepared.

[0160] The above results show that the TEX44+ / + mice have a TEX44 protein-sized band; the TEX44- / - mice do not have a TEX44 protein-sized band, indicating that the Tex44 gene has been knocked out.

[0161] 7. Volume and weight of testes and epididymides of different mice

[0162] Take wild-type and Tex44 gene-knocked-out mice at 8 - 12 weeks old and sacrifice them by cervical dislocation; wipe the abdominal hair of the mice with an alcohol cotton ball, cut open the abdomen with a pair of dissecting scissors, pick up the gonads of the mice with forceps, and separate the testes, epididymides, and seminal vesicles, then measure and weigh them.

[0163] Figure 3DThis is a graph showing the comparison results of the sizes of the testes and epididymides of wild-type mice (TEX44+ / + mice) and homozygous F2 mice with Tex44 gene knockout (TEX44- / - mice) in Example 3 of this application. The results show no significant differences.

[0164] Figure 3E This is a graph showing the comparison results of the volumes of the testes of wild-type mice (TEX44+ / + mice) and homozygous F2 mice with Tex44 gene knockout (TEX44- / - mice) in Example 3 of this application; the results show no significant differences.

[0165] Figure 3F This is a graph showing the change results of the weights of the testes of wild-type mice (TEX44+ / + mice) and homozygous F2 mice with Tex44 gene knockout (TEX44- / - mice) at different weeks of age in Example 3 of this application; it can be seen from the graph that the weight of the testes of TEX44- / - mice becomes smaller as the weeks of age increase; the gap with the testes of wild-type mice is getting larger and larger, indicating that as the weeks of age of the mice increase, the volume of their testes becomes relatively smaller; it shows that fertility decreases significantly with age; among them: - / - represents homozygous mice with Tex44 gene knockout, and + / + represents wild-type mice.

[0166] The above results show that: there is no obvious difference in the gonad volume between adult male TEX44- / - mice and TEX44+ / + mice. There is no significant difference in the size and weight of the testes of TEX44- / - mice and wild-type mice; however, the size of the testes of older TEX44- / - mice is significantly reduced compared to that of wild-type mice.

[0167] Example 4: The sperm motility rate and the number of forward-moving sperm in Tex44 gene knockout male mice decrease significantly

[0168] 1. Observe and count the growth and development of TEX44- / - mice and TEX44+ / + mice obtained in Example 3. The results show that there is no significant difference in the survival rate, appearance, and overall behavior between TEX44- / - mice and TEX44+ / + mice; however, male TEX44- / - mice show asthenozoospermia, and the fertility of female TEX44- / - mice is not affected. The above results show that Tex44 gene knockout in mice leads to male asthenozoospermia.

[0169] 2. Take 3 adult male TEX44- / - mice and mate them with several female wild-type mice for 3 months. Observe whether there are sperm plugs in the reproductive tracts of female wild-type mice during the mating period. The results show that: there are sperm plugs in the reproductive tracts of female wild-type mice, but the rate of seeing plugs decreases. The above results show that Tex44 gene knockout affects the fertilization process of mice.

[0170] 3. Detect sperm motility and quantity through CASA experiments as follows: Place the mouse epididymis in CASA buffer, cut open the epididymis, and incubate it in an incubator at 37°C and 5% CO 2 for 5 minutes, 60 minutes, 90 minutes, and 120 minutes respectively, then collect sperm and analyze sperm motility. Sperm counting: Place the mouse epididymis in CASA buffer, cut open the epididymis (record the number of cuts and ensure that the number and size of each cut are uniform), maintain at 37°C, and after 30 minutes when sperm swim out, filter through a 40μm filter. After diluting 10 times, mix well, take 10μL and drop it onto a Biorad cell counting plate. Take a microscope photo and analyze the number of cells in each field of view using ImageJ. Based on the thickness of the counting plate, the conversion formula for the size of the photo field of view, and the sperm dilution factor and total volume, calculate the corresponding number of sperm. Among them, the CASA buffer formula: 120mM NaCl, 4.8mM KCl, 1.2mM MgSO 4 , 1mM CaCl 2 , 1.2mM KH 2 PO 4 , 21mM DL-sodium lactate (Na-dl-lactate), 5mM glucose, 25mM NaHCO 3 , 0.25mM sodium pyruvate (Napyruvate), 0.4μg / mL phenol red (Phenol red), 3mg / mL bovine serum albumin (BSAV).

[0171] The experimental results are shown in Table 1 and Figures 4A to 4F as follows.

[0172] Table 1 CASA analysis results of male sperm of Tex44 gene knockout homozygous mice and wild-type mice

[0173] WT(n = 2) Het(n = 1) TEX44 - / -(n = 3) <![CDATA[Sperm density (10 6 )]]> 88.75±32.08 66.81 72.53±8.64 Sperm motility rate (%) 58.70±10.26 54.89 19.85±7.12 Rapidly progressive sperm (%) 29.66±11.88 30 7.12±1.21 Slowly progressive sperm (%) 2.88±1.08 3 3.40±1.99 Non - progressive sperm (%) 26.15±2.70 21.89 9.33±4.01 Immotile sperm (%) 41.31±10.26 45.11 80.15±7.12 Abnormal sperm rate (%) 7.22±0.21 9.29 7.96±0.41

[0174] As can be seen from Table 1, the sperm motility rate of epididymal sperm in TEX44 - / - mice is significantly reduced, and the immotile sperm is significantly increased.

[0175] Figure 4A This is the dynamic analysis diagram of epididymal sperm of wild-type mice (WT mice) and Tex44 gene knockout homozygous mice (TEX44 - / - mice) in Example 4 of this application; where a) represents WT mice and b) represents TEX44 - / - mice. It can be seen that the motility of sperm in TEX44 - / - mice is significantly reduced.

[0176] Figure 4BThis is a graph showing the comparison results of sperm motility between wild-type mice (WT mice) and homozygous Tex44 gene knockout mice (TEX44- / - mice) in Example 4 of this application. It can be seen from the graph that the sperm motility of TEX44- / - mice has significantly decreased.

[0177] Figure 4C This is a graph showing the comparison results of sperm density between wild-type mice (WT mice) and homozygous Tex44 gene knockout mice (TEX44- / - mice) in Example 4 of this application. It can be seen from the graph that there is no significant difference in sperm density between TEX44- / - mice and wild-type mice.

[0178] Figure 4D This is a graph showing the comparison results of forward motile sperm between wild-type mice (WT mice) and homozygous Tex44 gene knockout mice (TEX44- / - mice) in Example 4 of this application. It can be seen from the graph that the motility rate of forward motile sperm in TEX44- / - mice has decreased.

[0179] Figure 4E This is a graph showing the comparison results of non-forward motile sperm between wild-type mice (WT mice) and homozygous Tex44 gene knockout mice (TEX44- / - mice) in Example 4 of this application. It can be seen from the graph that the non-forward motile sperm in TEX44- / - mice has decreased.

[0180] Figure 4F This is a graph showing the comparison results of immotile sperm between wild-type mice (WT mice) and homozygous Tex44 gene knockout mice (TEX44- / - mice) in Example 4 of this application. It can be seen from the graph that the immotile sperm in TEX44- / - mice has significantly increased.

[0181] The above results show that: compared with TEX44+ / +(WT) mice, the number of forward sperm in the cauda epididymis of TEX44- / - mice has decreased and the sperm motility has significantly decreased, showing asthenozoospermia.

[0182] 5. Observe the sperm morphology under an optical microscope as follows: Tear the epididymis into pieces, cut it several times with scissors, flick it gently, and place it at room temperature or 37°C for 5 minutes to allow the sperm to swim out. Aspirate the supernatant with a disposable pipette and filter it through a 100μm cell sieve. Take 5μL of the above suspension and drop it at one end of a glass slide coated with D-polylysine. Hold the spreader at a 30-degree angle to the glass slide, place it in front of the semen drop, and then move it slightly to touch the semen drop. It can be seen that the semen drop spreads along the lower edge of the spreader, driving the spread semen line. Pull the spreader evenly, without touching the glass slide, to form a semen smear, and observe the sperm morphology and the percentage of abnormal sperm under an optical microscope.

[0183] The results are as follows: Compared with TEX44+ / + mice, the morphology of sperm in TEX44- / - mice showed no obvious abnormalities in the appearance of the head and tail of sperm, but the sperm motility rate decreased significantly.

[0184] Example 5: In vivo and in vitro fertilization tests found that the fertilization rate of Tex44 gene knockout sperm decreased significantly

[0185] In vivo and in vitro fertilization techniques were used to study the role of TEX44 in the fertilization process, with the cleavage of 2-cell, 4-cell, and blastocysts as indicators of successful in vitro fertilization.

[0186] 1. Mouse superovulation procedure: Female wild-type mice aged 6-8 weeks were intraperitoneally injected with 5 IU of pregnant mare serum gonadotropin (Ningbo Second Hormone Factory, D-001), and 48 hours later, 5 IU of human chorionic gonadotropin was injected for superovulation (Ningbo Second Hormone Factory, X-001).

[0187] 2. In vivo fertilization experimental method: Superovulated female mice were mated overnight with wild-type and KO male mice, and the presence of vaginal plugs was detected. The fertilized eggs were removed 12-16 hours later and transferred to G1-plus culture drops (Vitrolife, 10128).

[0188] 3. In vitro fertilization experimental method: Superovulated female mice were sacrificed, and oocyte-corona radiata complex (COC) was dissected from the oviduct 12 hours later. The same number of COCs were transferred to 200 μL of HTF drops (Nanjing Aibei Biotechnology, M1150) containing 15 μL of sperm suspensions from wild-type mice and KO male mice respectively, and incubated at 37 °C, 5% CO 2 for 4 hours. After in vitro fertilization, the oocytes were washed and transferred to G1-plus culture drops (Vitrolife, 10128). The fertilization rate was recorded and calculated the next morning. The embryos were cultured for 3 days, and the embryonic development was recorded every day.

[0189] Results: Figure 5A This is the result graph of in vivo fertilization experiments of wild-type mice and TEX44- / - male mice. It can be seen from it that the fertilization rates of TEX44+ / + and TEX44- / - male mice were 68.91±14.53% and 21.64±18.58% respectively, P < 0.01; indicating that the fertilization rate and cleavage rate of TEX44- / - male mice were significantly lower than those of wild-type mice. Figure 5B This is the result graph of in vivo fertilization experiments of TEX44- / - male mice of different weeks of age and wild-type female mice. It can be seen from it that the fertilization rate of epididymal sperm of 24-week-old TEX44- / - mice decreased faster and dropped to zero. Figure 5CIt is a graph showing the results of in vitro fertilization experiments on epididymal sperm of TEX44 - / - male mice. It can be seen from the graph that the fertilization rates of TEX44 + / + and TEX44 - / - male mice are 66.66 ± 17.33% and 6.49 ± 5.28% respectively, with P < 0.001. For in vitro fertilization, the fertilization rate of eggs is very low, indicating that the fertilization rate of sperm from TEX44 - / - male mice is significantly reduced. The inventor believes that the deletion (knockout or mutation) of the Tex44 gene affects the fertilization process, causes abnormal fertilization, leads to a decrease in the fertilization rate or a low fertilization rate, and further results in infertility.

[0190] Example 6: The ultrastructure of the sperm tails of Tex44 gene knockout is abnormal, and some motile axonemes are missing

[0191] 1. Dissect the epididymis of adult mice. The mice to be tested are 8 - 12 - week - old male TEX44 + / + mice and male TEX44 - / - mice. Prepare paraffin sections (fixation - dehydration - clearing of tissues - infiltration with wax - embedding - sectioning - spreading - pasting), and analyze the epididymis of the mice to be tested by HE staining (dewaxing - rehydration - staining with hematoxylin solution - washing with water - differentiating with hydrochloric acid alcohol - rinsing - staining with eosin solution - dehydration - clearing - mounting).

[0192] The results are as follows: The amount of forward - moving sperm in the caudal epididymis of TEX44 knockout mice is significantly less than that of wild mice.

[0193] 2. Analyze the ultrastructure of sperm in the caudal epididymis of mice by transmission electron microscopy technology as follows: The samples are fixed overnight at 4°C in 2.5% glutaraldehyde solution (diluted with 0.1M PBS solution at pH 7.2). Aspirate the fixing solution, and rinse the samples three times with 0.1M PBS solution at pH 7.2, 15 minutes each time; fix the samples with 1% osmium tetroxide solution for 1 - 2 hours; remove the fixing solution, and rinse the samples three times with 0.1M PBS at pH 7.2, 15 minutes each time; dehydrate the samples with a gradient of ethanol solutions (including 30 vol%, 50 vol%, 70 vol%, 80 vol%, 90 vol% and 95 vol%), each concentration for 15 minutes, and then treat with 100% ethanol for 20 minutes; finally, treat with pure acetone for 20 minutes. Gradient infiltration with embedding agent: ① Treat the samples with a mixture of embedding agent and acetone (V / V = 1 / 1) for 1 hour; ② Treat the samples with a mixture of embedding agent and acetone (V / V = 3 / 1) for 3 hours; ③ Let the pure embedding agent infiltrate the samples overnight: In this step, transfer the samples to a new dry tube containing pure embedding agent. The infiltrated samples are aliquoted into 0.5 - mL eppendorf tubes for embedding and heated at 70°C overnight. Locate the sample direction. Cut the samples with an ultramicrotome, and place the samples on copper grids. Stain with uranyl acetate and observe under the electron microscope.

[0194] The results are as follows: The results are as Figures 6A - 6C shown in Figures 6A - 6B the scanning electron microscope of the sperm tails of TEX44 - / - mice in Example 6 of this application. Specifically, Figure 6A it is the electron micrograph of the principal piece, middle piece and end piece of the sperm flagella of TEX44 - / - mice in Example 6 of this application; it can be seen from it that the "9 + 2" axoneme assembly of the tails of TEX44 - / - mice is abnormally assembled, and some axonemes (DMT7) are missing. Figure 6B it is the scanning electron microscope of different types of axoneme deletions in the sperm tails of TEX44 - / - mice in Example 6 of this application; it can be seen from it that different types of "9 + 2" axonemes in the tails of TEX44 - / - mice are abnormal and axonemes are missing; in particular, axonemes lacking DMT7, DMTs4 - 7 and DMTs4 / 7 are more common. The axonemes lacking DMT7 and DMTs4 - 7 can be seen at the end of the sperm tail, indicating the absence of the axoneme movement component in the mouse tail, which causes the decline of sperm motility and further reduces the sperm viability. Figure 6C It is the percentage chart of different types of axoneme deletions in the sperm tails of Tex44 - / - mice. It can be seen from it that among the abnormal "9 + 2" axonemes in the tails of TEX44 - / - mice, the deletion rate of DMT7 axonemes is significantly higher than that of WT mice.

[0195] In summary, the "9 + 2" structure of the principal piece, middle piece and end piece of the sperm tails of TEX44 - / - mice is abnormal ( Figures 6A - 6C ); the ultrastructure of the sperm tails is significantly deformed, and the "9 + 2" axonemes of the sperm tails can be seen to be missing, resulting in the decline of sperm motility and viability, and further leading to the decline of fertilization rate and male sterility ( Figure 6C ).

[0196] Example 7: Tex44 gene knockout affects sperm differentiation and the ultrastructure of elongated sperm, and does not affect early sperm development

[0197] 1. Dissect the testes of adult mice to be tested, and the mice to be tested are 8 - 12 - week - old male TEX44 + / + mice and male TEX44 - / - mice. Analyze the testicular tissues of the mice by electron microscopy. The specific operation is the same as that in Example 2.

[0198] 2. Dissect the testes and epididymides of adult mice to be tested, and the mice to be tested are 8 - 12 - week - old male TEX44 + / + mice and male TEX44 - / - mice. Analyze the testicular tissues of the mice by HE staining. The specific operation is the same as that in Example 2.

[0199] The results are as follows: HE staining of paraffin sections of testes and epididymides. HE staining of paraffin sections shows that the testes of Tex44 - knockout male mice contain seminiferous tubules at different stages, and mature sperm can be seen in the epididymides ( Figure 7A), and it was found that there were 12 stages of seminiferous tubules in the testes of Tex44- / - male mice. Figure 7B ) The above results indicate that Tex44 gene knockout only affects sperm differentiation and the ultrastructure of elongated sperm, but does not affect early sperm development; TEX44 can be used as a biomarker for elongated spermatids; it can not only be applied to the study of the process of spermatogenesis, but also be used to identify different types of germ cells.

[0200] Example 8: Tex44 gene knockout affects the expression of 1542 sperm proteins and changes in multiple signaling pathways

[0201] Extract RNA from the testis tissues of knockout mice as follows: After washing the testis tissues with PBS, place them in RNase-free EP tubes. Add an appropriate amount of Triozol to each sample (1 ml of Trizol for 50 - 100 mg of sample) (Sigma), and homogenize the sample by breaking it at room temperature. Centrifuge at 12,000 rpm for 10 min at 4°C, transfer the supernatant to a new RNase-free EP tube, and discard the undissolved tissue. Incubate at room temperature for 5 min to ensure sufficient dissociation of nucleoprotein complexes. Add an appropriate amount of chloroform according to the initial amount of Trizol added (0.2 ml of chloroform for 1 mL of Trizol), shake vigorously by hand for 15 seconds, and let it stand at room temperature for 2 - 3 min. Centrifuge at 12,000 rpm for 15 min at 4°C, and the sample will be stratified. Carefully aspirate the upper aqueous phase to a new EP tube (roughly aspirate 80% of the upper aqueous phase), avoiding inhaling the middle phase and the lower red organic phase. Add an appropriate amount of 100% isopropanol according to the initial amount of Trizol (0.5 mL of isopropanol for 1 ml of Trizol), and let it stand at room temperature for 10 min. Centrifuge at 12,000 rpm for 10 min at 4°C. Remove the supernatant and retain the white RNA precipitate at the bottom of the tube. Add 75 vol% ethanol solution (prepared with DEPC-H 2 O, add 1 ml of 75 vol% ethanol solution for every 1 mL of Trizol), invert and shake several times, centrifuge at 7500 rpm for 5 min at 4°C, and discard the liquid. Repeat this step once. Open the lid and let it stand at room temperature to dry the RNA precipitate. Add an appropriate amount of RNase-free water, pipette several times to redissolve the RNA. Send the RNA samples to BGI for transcriptome sequencing.

[0202] The results are as follows: Figure 8A This is a comparison chart of the testis transcriptome sequencing results of TEX44- / - mice and wild-type mice in Example 8 of this application; it can be seen from it that GO and KEGG analyses show that the expression of 1542 genes / proteins is different (P < 0.05), and the expression of 28,940 genes is not significantly different; among them, the expression of 1352 genes is up-regulated and the expression of 190 genes is down-regulated. Figure 8BThis is the GO analysis result graph in Example 7 of this application; it can be seen from it that among the up-regulated differentially expressed genes, 84 genes closely related to spermatogenesis were found to be up-regulated, including 23 genes related to sperm motility rate; 50 genes related to flagellar movement were up-regulated; the above results indicate that the deletion of the TEX44 gene will affect spermatogenesis and lead to a decrease in sperm motility ability. Figure 8C This is the KEGG analysis result graph in Example 7 of this application; it can be seen from it that 22 genes related to sperm motility were up-regulated; the above results indicate that the deletion of the TEX44 gene will affect sperm motility ability, thereby leading to a decrease in sperm vitality. Figure 8D This is the structural angle analysis result graph in Example 7 of this application; it can be seen from it that 36 genes related to the "9+2" related to the sperm flagellar axis composition were up-regulated; the above results indicate that the deletion of the TEX44 gene will affect the abnormal assembly of the sperm flagellar axis, thereby leading to partial deletion of the flagellar axoneme and causing a decrease in vitality. Figure 8E This is the functional angle analysis result graph in Example 7 of this application; it can be seen from it that 25 genes related to oxidative phosphorylation were down-regulated; this may affect the metabolism of ATP energy, thereby leading to a decrease in the energy obtained by sperm and a decrease in motility rate. Figure 8F This is the further Reactome analysis result graph in Example 7 of this application; it can be seen from it that genes related to the electron transport chain and ATP synthesis were up-regulated, which may affect the mitochondrial electron transport chain and further affect the synthesis of ATP energy, resulting in a lack of energy supply for sperm and a decrease in vitality.

[0203] The above results indicate that the knockout of the Tex44 gene will directly affect the expression changes of 1542 proteins in sperm, especially proteins related to sperm motility, sperm motility metabolism-related pathways, and proteins related to the assembly of sperm tail motility structures. It shows that the decrease in the expression of the TEX44 gene / protein has an important impact on sperm motility ability and vitality, further leading to a decrease in fertilization rate and male infertility; increasing or restoring the activity and / or expression level of the TEX44 protein can improve the vitality and motility ability of sperm.

[0204] Example 9: Mutation screening and application of TEX44 gene in male infertility patients

[0205] The research involved in this example was approved by the Ethics Committee of Peking University Third Hospital and informed consent was obtained from the patients. Blood samples discarded after testing in the Reproductive Medicine Center of Peking University Third Hospital, testicular tissue samples discarded after testing, and / or semen samples of asthenospermia patients discarded after use in the assisted reproductive laboratory.

[0206] Method: Tissue samples of 106 patients were collected, and the specific operations were the same as those in Example 7 and Example 5. After sample processing, they were sent to BGI for whole-genome sequencing and transcriptome sequencing. Results: Through mutation screening and mutation site analysis in the TEX44 population, three mutations were found in two patients, and c.541C>T was the common mutation site. The details are shown in Table 2 below.

[0207] Table 2 Three mutation information of two mutant patients

[0208]

[0209] Scanning electron microscopy analysis was performed on the sperm of one TEX44 mutant patient (21Y03487), and it was found that the ultrastructure of the sperm flagella of patients with the c.541C>T mutation in the Tex44 gene was abnormal. The specific results are as Figure 9 shown, which is consistent with the phenotype of Tex44- / - male mice; where a) refers to human 21Y03487 and b) refers to Tex44- / - male mice; the results in the figure show that the axoneme of the patient is unstable and the structure of the main segment ("9+2" axoneme) is also damaged, which is consistent with the phenotype of Tex44- / - male mice.

[0210] Clinical data analysis of this patient found that the patient had been troubled by infertility for 4 years since marriage. After examination, his semen parameters were abnormal and he was diagnosed with male infertility. Semen examination showed that his sperm density was 1.09×10 6 / mL, only a few motile sperm were seen, and most sperm were stationary. This is consistent with the phenotype of Tex44- / - male mice. Due to the obvious decrease in his sperm motility, he was diagnosed with asthenozoospermia. This is consistent with the phenotype of Tex44- / - male mice. In vitro fertilization (IVF) treatment was performed on this patient (21Y03487), and it was found that he did not meet the conventional fertilization criteria and was also prone to low fertilization rate (abnormal fertilization) or fertilization failure.

[0211] Intracytoplasmic sperm injection (ICSI) fertilization was recommended. The specific results are shown in Table 3. 20 eggs were injected, 12 were fertilized, and the fertilization rate was 60%, which was lower than the average value of ICSI fertilization (>70%). It shows that the expression of TEX44 gene / protein has an important impact on male infertility; the decrease in the activity and / or expression level of TEX44 mutant protein can reduce the sperm motility rate and motility, thereby causing male asthenozoospermia and male infertility.

[0212] Table 3 Clinical outcomes of IVF treatment for the patient

[0213]

[0214] The present application provides the use of the Tex44 gene and its mutant genes as molecular markers in the diagnosis of male asthenospermia and male infertility diseases. Experiments have shown that the TEX44 mutant gene / TEX44 protein leads to sperm dysfunction, reduced sperm motility, abnormal sperm tail ultrastructure, abnormal fertilization, male asthenospermia and male infertility, etc. The Tex44 mutant gene / TEX44 protein of the present application can be used as a target gene for diagnosing male asthenospermia. At the same time, knocking out the Tex44 gene affects the up-regulation and down-regulation of 1542 testicular-expressed genes / proteins, affecting the motility of normal sperm and the assembly of sperm tail ultrastructure. A decrease in the activity and / or expression level of the TEX44 mutant protein can reduce the viability and motility of sperm, thereby causing abnormal fertilization, male asthenospermia and male infertility. Restoring or increasing the activity and / or expression level of the TEX44 protein can prevent and / or treat male asthenospermia.

[0215] The above are only the preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included in the scope of protection of the present application.

Claims

1. Tex44 mutant gene, among which, The 541st base of the nucleotide sequence of the mRNA of the Tex44 mutant gene is T; preferably, the nucleotide sequence of the mRNA of the Tex44 mutant gene is as shown in SEQ ID NO.

4.

2. TEX44 mutant protein, wherein The 181st amino acid in the amino acid sequence of the TEX44 mutant protein is Q; preferably, the amino acid sequence of the TEX44 mutant protein is as shown in SEQ ID NO.

5.

3. Any of the following uses of a biomarker and / or a substance for detecting the biomarker: A1) Use in the preparation of products for studying spermatogenesis; A2) Use in the preparation of a product for identifying a germ cell sorting process; A3) Use in the preparation of a product for screening male infertility diseases; A4) Use in the preparation of a product for diagnosing male asthenozoospermia; A5) Use in the preparation of products for diagnosing abnormal sperm and egg fertilization; A6) Use in the preparation of a product for diagnosing male teratozoospermia; A7) Use in the preparation of a product for diagnosing male infertility; in, The biomarker is selected from the group consisting of Tex44 gene, TEX44 protein, mRNA of Tex44 gene, RNA of Tex44 gene, miRNA targeting TEX44 protein, the Tex44 mutant gene according to claim 1 or the TEX44 mutant protein according to claim 2.

4. The use according to claim 3, wherein: The substance is a reagent for detecting whether the Tex44 gene, TEX44 protein, mRNA of the Tex44 gene, RNA of the Tex44 gene, miRNA targeting the TEX44 protein, the Tex44 mutant gene according to claim 1 or the TEX44 mutant protein according to claim 2 exists; preferably, the test sample for the test is selected from at least one of blood, oral tissue, testicular tissue and sperm.

5. The use according to claim 3 or 4, wherein: The nucleotide sequence of the Tex44 gene is shown as SEQ ID NO.1; or, the nucleotide sequence of the mRNA of the Tex44 gene is shown as SEQ ID NO.2; or, the amino acid sequence of the TEX44 protein is shown as SEQ ID NO.

3.

6. A kit, wherein: The kit comprises a reagent for detecting whether the Tex44 gene, TEX44 protein, Tex44 gene mRNA, Tex44 gene RNA, the Tex44 mutant gene according to claim 1 or the TEX44 mutant protein according to claim 2 exists, and the kit has at least one of the following uses: A1) Study the process of spermatogenesis; A2) Identification of the germ cell sorting process; A3) Screening for male infertility; A4) Diagnosis of male asthenozoospermia; A5) Diagnosis of abnormal sperm and egg fertilization; A6) Diagnosis of male teratozoospermia; A7) Diagnosis of male infertility.

7. The kit according to claim 6, wherein The kit comprises at least one of a primer pair having nucleotide sequences as shown in SEQ ID NO.6 and SEQ ID NO.7, a primer pair having nucleotide sequences as shown in SEQ ID NO.8 and SEQ ID NO.9, and a primer pair having nucleotide sequences as shown in SEQ ID NO.10 and SEQ ID NO.

11.

8. The kit according to claim 6, wherein The nucleotide sequence of the Tex44 gene is shown as SEQ ID NO.1; or, the nucleotide sequence of the mRNA of the Tex44 gene is shown as SEQ ID NO.2; or, the amino acid sequence of the TEX44 protein is shown as SEQ ID NO.

3.

9. The kit according to any one of claims 6 to 8, wherein The test sample of the kit is selected from at least one of blood, oral tissue, testicular tissue and sperm; Preferably, the method for judging the test result of the kit includes: if the Tex44 gene, TEX44 protein, Tex44 gene mRNA, Tex44 gene RNA or miRNA targeting TEX44 protein is not present in the test sample, then the source of the test sample is suspected of suffering from male asthenozoospermia, teratozoospermia, fertilization abnormality or male infertility; Alternatively, if the Tex44 mutant gene described in claim 1 or the TEX44 mutant protein described in claim 2 is present in the test sample, the source of the test sample is suspected of suffering from male asthenozoospermia, teratozoospermia, fertilization abnormality or male infertility.

10. Use of biomarkers as drug targets in the preparation of drugs for any of the following purposes: B1) Treatment of male asthenozoospermia; B2) Treatment of male teratozoospermia; B3) Treatment of male infertility; B4) Improve male fertility; B5) Treatment of sperm dysfunction; B6) Promote sperm fertilization; B7) Improve sperm motility; B8) Improve sperm quality; B9) Repair sperm morphological abnormalities; B10) Repair the abnormality of sperm tail structure; in, The biomarker is selected from TEX44 protein or Tex44 gene.

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