Application of HSFY2 protein in treatment of male infertility

Abstract

The invention discloses an application of an HSFY2 protein in treatment of male infertility. It is found through a large number of experiments that HSFY2 gene knockout can cause male sterility of mice, increase the whole sperm malformation rate and tail malformation rate of epididymis tails, inhibit normal formation of the tails of the mice sperms, reduce the motility of the mice sperms and reduce the protein tyrosine phosphorylation level related to mouse sperm capacitation; the percentage of sperms before growth is reduced, the expression of testis acetylation cytoskeleton protein is reduced, and the male fertility is reduced. The substance for improving the activity and / or expression quantity of the HSFY2 protein can be used for preventing and / or treating male infertility. Therefore, the HSFY2 protein has a remarkable application value in treatment of male infertility.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to application of HSFY2 protein in treating male infertility. Background Art

[0002] As people's living standards gradually improve, infertility has become an increasingly prominent social problem. Infertility caused by various environmental and genetic factors is a global health problem. The human male Y chromosome contains genes responsible for testicular development and the initiation and maintenance of spermatogenesis in adulthood. The long arm of the Y chromosome (Yq) contains many amplified and palindromic sequences, which make it easy for self-recombination to occur during spermatogenesis, and therefore susceptible to intrachromosomal deletions, which lead to copy number variations of Y chromosome genes and cause male infertility. There are three key regions on Yq, namely the azoospermia factor (AZF) region, including AZFa, AZFb and AZFc. The Y-linked heat shock transcription factor (HSFY2) gene is located in the AZFb region. Whether its deletion can cause male infertility is still unknown.

[0003] The HSFY2 gene (Gene ID: 71066 gene published by NCBI) is highly conserved and is expressed in humans, mice, cattle, chickens, dogs, etc. The most notable structure of its protein is the DNA binding region (DBD) containing a heat shock protein type. However, only 30% of the DBD of HSFY2 is homologous to the classical heat shock transcription factor. At the same time, studies have shown that the DBD of HSFY2 does not seem to be able to bind to heat shock elements, thereby controlling the expression of heat shock proteins. Therefore, the HSFY2 protein is likely to have a different function from the classical heat shock transcription factor. It is reported that in human testicular tissue, the HSFY2 protein is mainly expressed in the cytoplasm of supporting cells and the nucleus of spermatogenic cells, but the specific function of the HSFY2 protein in sperm has not yet been documented. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides the use of HSFY2 protein in treating male infertility. The present invention confirms through a large number of experiments that HSFY2 protein has significant application value in treating male infertility.

[0005] The present invention is achieved through the following technical solutions:

[0006] Use of a substance that increases the activity and / or expression of HSFY2 protein in the preparation of a product, wherein the function of the product is at least one of the following (1)-(9):

[0007] (1) Prevent male infertility; (2) Treat male infertility; (3) Promote sperm maturation; (4) Improve sperm motility; (5) Reduce sperm deformity rate; (6) Improve sperm motility; (7) Increase the level of protein tyrosine phosphorylation related to sperm capacitation; (8) Increase the percentage of sperm that mature; (9) Promote the expression of testicular acetylated tubulin.

[0008] Use of HSFY2 protein as a drug target in the preparation of a product, wherein the function of the product is at least one of the following (1)-(9):

[0009] (1) Prevent male infertility; (2) Treat male infertility; (3) Promote sperm maturation; (4) Improve sperm motility; (5) Reduce sperm deformity rate; (6) Improve sperm motility; (7) Increase the level of protein tyrosine phosphorylation related to sperm capacitation; (8) Increase the percentage of sperm that mature; (9) Promote the expression of testicular acetylated tubulin.

[0010] Use of HSFY2 protein in the preparation of a product, wherein the function of the product is at least one of the following (1)-(9):

[0011] (1) Prevent male infertility; (2) Treat male infertility; (3) Promote sperm maturation; (4) Improve sperm motility; (5) Reduce sperm deformity rate; (6) Improve sperm motility; (7) Increase the level of protein tyrosine phosphorylation related to sperm capacitation; (8) Increase the percentage of sperm that mature; (9) Promote the expression of testicular acetylated tubulin.

[0012] Preferably, the product is a medicine.

[0013] The application of HSFY2 protein is at least one of the following (1)-(9):

[0014] (1) Prevent male infertility; (2) Treat male infertility; (3) Promote sperm maturation; (4) Improve sperm motility; (5) Reduce sperm deformity rate; (6) Improve sperm motility; (7) Increase the level of protein tyrosine phosphorylation related to sperm capacitation; (8) Increase the percentage of sperm that mature; (9) Promote the expression of testicular acetylated tubulin.

[0015] A product comprising HSFY2 protein or a substance that increases the activity and / or expression of HSFY2 protein; the function of the product is at least one of the following (1)-(9):

[0016] (1) Prevent male infertility; (2) Treat male infertility; (3) Promote sperm maturation; (4) Improve sperm motility; (5) Reduce sperm deformity rate; (6) Improve sperm motility; (7) Increase the level of protein tyrosine phosphorylation related to sperm capacitation; (8) Increase the percentage of sperm that mature; (9) Promote the expression of testicular acetylated tubulin.

[0017] Use of a substance for detecting HSFY2 protein in preparing a product for diagnosing male infertility.

[0018] A kit for diagnosing male infertility, comprising a substance for detecting HSFY2 protein.

[0019] Preferably, it also includes a carrier recording the judgment criteria;

[0020] The judgment criteria are: if the expression level of HSFY2 protein in the sperm of the test subject is lower than the expression level of HSFY2 protein in the control sperm, the test subject is or is suspected to be a male infertility patient; if the expression level of HSFY2 protein in the sperm of the test subject is not lower than the expression level of HSFY2 protein in the control sperm, the test subject is not or is not suspected to be a male infertility patient; the control sperm is sperm with normal male fertility.

[0021] Application of HSFY2 protein as a biomarker in the development of diagnostic reagents for male infertility.

[0022] The beneficial effects of the present invention are as follows:

[0023] The present invention has found through a large number of experiments that HSFY2 gene knockout can lead to male infertility in mice, increase the overall sperm deformity rate and tail deformity rate of the epididymal tail, inhibit the normal formation of the tail of mouse sperm, reduce the motility of mouse sperm, reduce the protein tyrosine phosphorylation level related to mouse sperm capacitation, reduce the percentage of sperm prolapse, reduce the expression of testicular acetylated cytoskeleton protein, and reduce male fertility. Substances that increase the activity and / or expression of HSFY2 protein can prevent and / or treat male infertility. Therefore, HSFY2 protein has significant application value in treating male infertility. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The effects of HSFY2 gene knockout on mouse fertility in Example 1: A is the genotype identification of three groups of mice, namely HSFY2 gene knockout mice (HSFY2- / -), heterozygous (HSFY2+ / -) and normal (HSFY2+ / +); B is the fertility of the three groups of mice; C is the morphology of the testicles and epididymis of the three groups of mice; D is the ratio of testicle weight to body weight in the three groups of mice; E is the ratio of the tail of the epididymis of the three groups of mice;

[0025] Figure 2 The sperm concentration, motility and percentage of forward-moving sperm in the cauda epididymis of the HSFY2 gene knockout homozygous mice in Example 2 are detected: A is the sperm concentration and morphology of the cauda epididymis in the HSFY2+ / + and HSFY2- / - groups under the sperm analyzer, and the scale is 100 μm; B is the sperm concentration of the cauda epididymis in the HSFY2+ / + and HSFY2- / - groups; C is the percentage of sperm motility in the cauda epididymis in the HSFY2+ / + and HSFY2- / - groups; D is the percentage of forward-moving sperm in the cauda epididymis in the HSFY2+ / + and HSFY2- / - groups;

[0026] Figure 3 The abnormality rate of epididymal tail sperm in the HSFY2 gene knockout homozygous mice in Example 3 is detected: A is the representative epididymal tail sperm morphology of HSFY2+ / + and HSFY2- / - groups, and the scale is 5 μm; B is the overall abnormality rate and tail abnormality rate of epididymal tail sperm in HSFY2+ / + and HSFY2- / - groups; C is the representative epididymal tail sperm morphology of HSFY2+ / + and HSFY2- / - groups under scanning electron microscopy, and the scale is 1 μm;

[0027] Figure 4 The protein tyrosine phosphorylation level related to sperm capacitation in the cauda epididymis and testicular acetylated tubulin in the HSFY2 gene knockout homozygous mice in Example 4 is detected: A is the tyrosine (Tyr-P) phosphorylation level in sperm protein of the cauda epididymis sperm in the HSFY2+ / + and HSFY2- / - groups after incubation in sperm capacitation solution for 30 min and 90 min; B is the level of acetylated tubulin (Ac-tubulin) in the testis of the HSFY2+ / + and HSFY2- / - groups, wherein GAPDH protein is the internal reference protein. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0029] Unless otherwise specified, the technical means used in the following examples are all conventional means well known to those skilled in the art, and the experimental methods without specifying the specific conditions are all conventional methods in the art.

[0030] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0031] The experimental mice used in the following examples were purchased from Saiye Biotechnology Co., Ltd.

[0032] Example 1 Effect of HSFY2 gene knockout on mouse fertility

[0033] 1. Obtaining homozygous HSFY2 knockout mice

[0034] HSFY2 heterozygous (HSFY2+ / -, HET) mice were purchased from Saiye Biotechnology Co., Ltd. Adult (12 weeks) HSFY2 gene heterozygous male and female mice were mated to obtain HSFY2 gene knockout homozygous mice (HSFY2- / - mice, abbreviated as KO mice) and wild-type mice (HSFY2+ / + mice, abbreviated as WT mice). The identification of specific knockout mice was completed by PCR genotyping identification technology, and the operation was as follows:

[0035] (1) Cut a section of the mouse tail to be identified, add 200 μL of lysis buffer A (50 mM / L NaOH), cook at 100°C for 1 h, shake, mix, add 25 μL of lysis buffer B (1 M Tris-HCl, pH = 8.0) for neutralization, and centrifuge at 12000 g for 10 min.

[0036] (2) Polymerase chain reaction (PCR) reaction system: 5 μL of cleaved tail solution; add 1 μL of each forward and reverse primer for genotype identification; add 10 μL of 2×Premix Taq Polymerase, add ultrapure water to a total volume of 25 μL, use the conventional PCR temperature setting, 35 cycles, and agarose electrophoresis. The forward and reverse primers are any combination of the following:

[0037] ①F1 (SEQ ID NO.1): 5'-TGTACCTTACTCAGTACCCAGGAC-3';

[0038] R1 (SEQ ID NO. 2): 5'-CGACTCTTGTTGCACTTTTCAGAC-3'.

[0039] ②F2 (SEQ ID NO.3): 5'-CCCACTTAGCATTTTAACCATCCTT-3';

[0040] R1 (SEQ ID NO. 2): 5'-CGACTCTTGTTGCACTTTTCAGAC-3'.

[0041] The results are as follows Figure 1 As shown in A. The band of homozygous KO mice is 649 bp; the band of wild-type WT mice is 883 bp.

[0042] 2. Fertility test of homozygous HSFY2 knockout mice

[0043] WT, HET and KO male mice were bred with normal adult multiparous female mice for 3 months. Figure 1As shown in B, there was no significant difference in the fertility of heterozygous mice compared with the normal group, but the male KO mice were completely infertile, without any pups born, and the number of pups born was significantly lower than that of the normal group.

[0044] 3. Growth and development detection of WT mice and KO mice

[0045] The test was performed by observing the weight, survival rate and mating behavior of mice. Figure 1 As shown in center C, there was no difference in survival rate, morphology, and testis appearance between WT and KO male mice.

[0046] The testicles and epididymis tail are weighed. Figure 1 As shown in D, the ratio of testis weight to body weight did not differ significantly between the WT and KO groups. Figure 1 As shown in middle E, there was no significant difference in epididymal cauda epididymis weight between WT and KO.

[0047] The experimental results of this example show that HSFY2 gene knockout can lead to male infertility in mice, but this infertility is not reflected in testicular weight, testicular morphology, epididymal tail weight, mouse appearance and mouse body weight.

[0048] Example 2 Detection of sperm concentration, motility and percentage of premotile sperm in the cauda epididymis of HSFY2 gene knockout homozygous mice

[0049] The sperm of the cauda epididymis of the WT and KO mice in Example 1 were released into benchtop solution and incubated at 37° C. for 15 min. Then, the sperm concentration, sperm motility and percentage of progesterone in the two groups of mice were detected by sperm analyzer.

[0050] The results are as follows Figure 2 As shown in Figures AD, the sperm concentration, motility and percentage of promotor sperm in the KO group were significantly decreased compared with those in the normal group WT (P < 0.05).

[0051] The experimental results of this example show that knocking out the HSFY2 gene significantly affects sperm motility and concentration.

[0052] Example 3 Detection of sperm abnormality rate in the tail of epididymis of HSFY2 gene knockout homozygous mice

[0053] 1. Release the sperm of the cauda epididymis of WT and KO mice in Example 1 into a benchtop solution, then fix the cauda epididymis sperm in 4% paraformaldehyde for 30 minutes, and then evenly apply the sperm to an adhesive slide, dry naturally, stain with eosin and hematoxylin, and then seal the slide, observe the sperm morphology and count the sperm deformity rate under an upright microscope with a magnification of 40. The experimental results are as follows Figure 3As shown in A and B, the arrows indicate that the sperm in the tail of the HSFY2- / - epididymis is abnormal ( Figure 3 Middle (A) shows that the overall sperm deformity rate and sperm tail deformity rate of epididymal tail in KO group mice were significantly increased.

[0054] 2. The sperm of the WT and KO mice in Example 1 were fixed in 2.5% glutaraldehyde buffer, washed, fixed, dehydrated, dried, glued, and conductively treated, and then the sperm morphology was scanned. Figure 3 As shown in middle C, the sperm neck of the KO group epididymal tail was broken, the middle and main segments of the tail were broken, and the mitochondria were bent.

[0055] The experimental results of this example show that knockout of the HSFY2 gene significantly affects the morphology of mature sperm, especially the morphology of the sperm tail.

[0056] Example 4 Detection of protein tyrosine phosphorylation levels related to sperm capacitation in the cauda epididymis and testicular acetylated tubulin in HSFY2 gene knockout homozygous mice

[0057] 1. By immunoblotting (WB) method, the tyrosine phosphorylation level of spermatozoa of the cauda epididymis of WT and KO mice in Example 1 after incubation in in vitro capacitation solution for 30 min and 90 min was detected using Tyr-p antibody. Figure 4 As shown in middle A, the tyrosine phosphorylation level of 120kDa protein in sperm of the cauda epididymis of KO group mice was significantly reduced.

[0058] 2. The expression level of acetylated tubulin in the testis of WT and KO groups was detected using antibodies against acetylated tubulin. Figure 4 As shown in B, the acetylated tubulin in the testis of the KO group was significantly decreased.

[0059] The experimental results of this example show that knockout of the HSFY2 gene will lead to a decrease in the tyrosine phosphorylation level of sperm in the cauda epididymis and abnormal expression of testicular acetylated tubulin, which may be the reason for the increased sperm deformity rate and decreased sperm motility in the cauda epididymis of KO mice and sperm infertility.

[0060] The experimental results of the above examples show that HSFY2 protein is essential for male mice to maintain normal fertility, and substances that increase the activity and / or expression of HSFY2 protein can prevent and / or treat male infertility.

[0061] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. The scope of protection of the present invention shall be subject to the scope required by the claims. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work shall fall within the scope of protection of the present invention.

Claims

1. Use of a substance for increasing the activity and / or expression of HSFY2 protein in preparing a product, characterized in that: The function of the product is at least one of the following (1)-(9): (1) Prevent male infertility; (2) Treat male infertility; (3) Promote sperm maturation; (4) Improve sperm motility; (5) Reduce sperm deformity rate; (6) Improve sperm motility; (7) Increase the level of protein tyrosine phosphorylation related to sperm capacitation; (8) Increase the percentage of sperm that mature; (9) Promote the expression of testicular acetylated tubulin.

2. The use of HSFY2 protein as a drug target in the preparation of a product, characterized in that: The function of the product is at least one of the following (1)-(9): (1) Prevent male infertility; (2) Treat male infertility; (3) Promote sperm maturation; (4) Improve sperm motility; (5) Reduce sperm deformity rate; (6) Improve sperm motility; (7) Increase the level of protein tyrosine phosphorylation related to sperm capacitation; (8) Increase the percentage of sperm that mature; (9) Promote the expression of testicular acetylated tubulin.

3. The use of HSFY2 protein in preparing a product, characterized in that: The function of the product is at least one of the following (1)-(9): (1) Prevent male infertility; (2) Treat male infertility; (3) Promote sperm maturation; (4) Improve sperm motility; (5) Reduce sperm deformity rate; (6) Improve sperm motility; (7) Increase the level of protein tyrosine phosphorylation related to sperm capacitation; (8) Increase the percentage of sperm that mature; (9) Promote the expression of testicular acetylated tubulin.

4. The use according to any one of claims 1 to 3, characterized in that: The product described is a drug.

5. Application of HSFY2 protein, characterized in that: At least one of the following (1)-(9): (1) Prevent male infertility; (2) Treat male infertility; (3) Promote sperm maturation; (4) Improve sperm motility; (5) Reduce sperm deformity rate; (6) Improve sperm motility; (7) Increase the level of protein tyrosine phosphorylation related to sperm capacitation; (8) Increase the percentage of sperm that mature; (9) Promote the expression of testicular acetylated tubulin.

6. A product, characterized in that The product comprises HSFY2 protein or a substance that increases the activity and / or expression of HSFY2 protein; the function of the product is at least one of the following (1)-(9): (1) Prevent male infertility; (2) Treat male infertility; (3) Promote sperm maturation; (4) Improve sperm motility; (5) Reduce sperm deformity rate; (6) Improve sperm motility; (7) Increase the level of protein tyrosine phosphorylation related to sperm capacitation; (8) Increase the percentage of sperm that mature; (9) Promote the expression of testicular acetylated tubulin.

7. Use of a substance for detecting HSFY2 protein in the preparation of a product for diagnosing male infertility.

8. A kit for diagnosing male infertility, characterized in that: Included are substances for detecting HSFY2 protein.

9. The kit according to claim 8, characterized in that It also includes a carrier recording a judgment standard; the judgment standard is: if the expression level of HSFY2 protein in the sperm of the test subject is lower than the expression level of HSFY2 protein in the control sperm, the test subject is or is suspected to be a male infertility patient; if the expression level of HSFY2 protein in the sperm of the test subject is not lower than the expression level of HSFY2 protein in the control sperm, the test subject is not or is not suspected to be a male infertility patient; the control sperm is sperm with normal male fertility.

10. Application of HSFY2 protein as a biomarker in the development of reagents for diagnosing male infertility.

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