Application of quadruple peptide repetitive protein in diagnosis or treatment of male fertility defects

Abstract

The present invention provides methods for diagnosing or predicting a fertility defect, such as infertility, in a mammalian subject by detecting TTC6. The invention also provides kits and instruments for diagnosing or predicting fertility defects, such as infertility disorders, in mammalian individuals by detecting TTC6. The invention finds and proves for the first time that the knockout of the Ttc6 gene leads to sperm motility weakening, induces sperm to rotate, finally destroys the fertilization function and leads to male infertility, thereby providing a method for diagnosing infertility disorder of mammals including humans, and promoting development of a new treatment method for male infertility individuals influenced by gene mutation.

Description

Technical Field

[0001] The present invention relates to the field of diseases and diagnosis. Specifically, the present invention relates to methods and kits for diagnosing or treating fertility defects in mammals, especially humans. Background Art

[0002] Mammalian spermatozoa, like spermatozoa from most other vertebrates, carry a haploid genome in the head and use flagella for movement. The flagellum consists of a midsection, a principal segment, and a tail segment, including an axoneme that extends to all three segments. The midsection contains mitochondria, outer dense fibers, and a fiber sheath that extends further into the principal segment, while the tail segment lacks peri-axonemal structures. The axoneme is the main component of flagella and cilia, and is composed of microtubule doublets and various accessory structures, such as dynein motors, radial spokes, and nexin-dynein regulatory complexes. It has a "9+2" structure, consisting of two central single microtubules (C1 and C2) surrounded by a cylinder composed of nine microtubule doublets. The elaborate axoneme structure contributes to the bending of the cilia and the integrity of the axoneme, which is essential for the normal function of flagella and cilia.

[0003] The terminal annulus is a ring-shaped structure in the sperm flagellum that is closely associated with sperm health and fertility. A recent study of mammalian sperm revealed the proteins and processes involved in the annulus, finding that a group of proteins called septins are localized to the annulus. Studies in mice have shown that the annulus is a barrier to protein diffusion and organizes the mid-piece of the sperm tail, and that the absence of the annulus is associated with flagellar differentiation problems and azoospermia in humans. These studies highlight the important role of the annulus membrane in sperm health and motility, and suggest that the lack of septins or TAT1 on the annulus membrane could serve as a diagnostic marker for certain types of male infertility.

[0004] Sperm motility, a key factor in successful fertilization, is generated by the controlled sliding of the outer microtubule doublets facilitated by the inner and outer dynein arms. This interaction between dynein and microtubules causes the flagellum to bend and generate bending forces, and the activity of dynein is further regulated by the radial spokes and central microtubules. To ensure high-speed swimming and maintain its stability to its own beating motion, the flagellum relies on multiple intramicrotubule proteins and peri-axonal structures such as the outer dense fiber layer and the fibrous sheath. These structures, as well as accessory components such as the nexin-dynein regulatory complex and radial spokes, provide structural integrity and prevent flagella from deforming. The symmetry or asymmetry of flagellar beating determines the swimming path of sperm cells. When flagellar defects occur, resulting in straight, curved or even spiral paths, they hinder sperm motility and fertility. Although several genes have been implicated in such defects, including Tekt4 (20), Tecte1 (21), Cfap97d1 (22), Ttll3, and Ttll8 (23), the regulatory mechanisms of flagellar beating remain largely unexplored.

[0005] The tetratricopeptide repeat protein family, including TTC21a, TTC26, TTC29, TTC30a, and TTC30b, among others, plays an important role in the formation and operation of cilia and flagellar structures, which are key to cell motility and signal transduction. To date, only mutations in TTC21a and TTC29 have been reported to be associated with multiple morphological abnormalities of flagella. Tetratricopeptide repeat protein 6 (TTC6) is another member of the tetratricopeptide repeat protein family and has been considered a potential candidate gene for ciliopathies based on recent studies of the ciliopathy genome. For example, patients with Meckel-Gruber syndrome carrying TTC6 mutations show impaired cilia formation potential.

[0006] This field also needs to have a better understanding of the factors related to the male sperm structure of mammals, so as to detect and predict reproductive disorders such as infertility. Summary of the invention

[0007] The applicant first discovered that knockout of the Ttc6 gene leads to weakened sperm motility and induces sperm rotational movement, ultimately destroying fertilization function and leading to male infertility. Morphological analysis of epididymal sperm in Ttc6 knockout mice showed a hairpin phenotype. In addition, ultrastructural studies have shown that knockout of Ttc6 leads to decapitation of flagella at the junction of the midpiece and the main segment. These observations suggest that the role of TTC6 is to reinforce the terminal ring, thereby maintaining the integrity of the sperm flagellum and ensuring rapid directional movement of sperm.

[0008] The applicant thus provides a method for diagnosing fertility defects such as infertility in mammals, including humans, and further promotes the development of new treatments for male infertile individuals affected by gene mutations. Thus, the inventor provides a method for diagnosing or treating fertility defects such as infertility in mammals, especially humans.

[0009] Specifically, the present invention provides a method for diagnosing or predicting fertility defects such as infertility disorders in mammalian individuals of different sexes, which comprises detecting Ttc6 in the test individual.

[0010] TTC6, or tetratricopeptide repeat domain 6, is a highly conserved gene in mammals. The human Ttc6 gene has a Gene ID of 319089, while the mouse Ttc6 gene has a Gene ID of 70846.

[0011] In the application (including methods and related kits or instruments and devices) provided by the present invention for diagnosing fertility defects in mammals by detecting TTC6 of the test individual, whether the TTC6 of the test individual is abnormal is detected, which includes comparing the activity of the subject's Ttc6 gene or its regulatory factor, or the expression of TTC6 protein, or comparing with the situation of the gene or protein in normal people in the general or specific group. If there is a significant change, it is judged that an abnormality exists.

[0012] In the present invention, infertility disorders include but are not limited to teratozoospermia, azoospermia, oligozoospermia, asthenozoospermia, asthenozoospermia, etc. The present application found that knocking out the Ttc6 gene leads to weakened sperm motility and induces sperm rotation, destroying fertilization function and causing male infertility. Preferably, the infertility disorders include but are not limited to asthenozoospermia, asthenozoospermia, etc.

[0013] In the present invention, the mammal can be any mammal, including and not limited to rodents, canines, felines, equines, ovines, bovines, porcines and primates. The mammal typically includes humans.

[0014] The method and kit provided by the present invention are based on the discovery that the effect of TTC6 on the fertility of mammals is gender-dependent, so the diagnosis and prediction made by the method or kit provided by the present invention are different for mammals of different genders. Therefore, in the method provided by the present invention, it is also included to detect the gender of the subject. In the kit or instrument and device provided by the present invention, it is also included to detect the gender of the subject. Methods and reagents for detecting the gender of the subject are well known in the art, for example, including detecting the gender-specific proteins or genes of the subject, such as specific nucleic acid sequences on sex chromosomes, etc.

[0015] In one aspect of the present invention, the method for diagnosing fertility defects provided by the present invention comprises the step of detecting the Ttc6 gene.

[0016] The method for detecting genes used in the present invention is a method commonly used for detecting genes in the art and is not particularly limited. For example, mass spectrometry, microarray, sequencing, and detection methods using base sequence amplification methods such as PCR (polymerase chain reaction) can be cited.

[0017] In addition, the detection of PCR products obtained when PCR is performed using primers specific to each gene can be performed by any method commonly used to detect and quantify PCR products. For example, it can be detected by electrophoresis, by real-time PCR using fluorescent intercalators such as SYBR Green, or by single molecule fluorescence analysis.

[0018] The polynucleotide that can be used as the primer or probe for detecting gene is not particularly limited as long as it can be a polynucleotide that hybridizes with the partial region comprising the gene or its complementary strand. The design of polynucleotide can be carried out by any one of the well-known methods in the technical field. For example, it can be designed simply by utilizing known genome sequence data and universal primer design tool. As this primer design tool, for example, Primer3 that can be utilized on the network is arranged. In addition, known genome sequence data can be obtained in NCBI as an international base sequence database usually.

[0019] In one aspect of the present invention, the method for diagnosing fertility defects provided by the present invention includes the step of detecting TTC6 protein, such as detecting the expression of the protein (including whether it exists, and whether there is a phenomenon of reduced expression relative to normal or standard) or its activity.

[0020] In another aspect of the present invention, the method includes the step of detecting the expression of TTC6 protein by immunoassay. For example, the expression of TTC6 is detected by immunofluorescence or Western blotting using an antibody that specifically recognizes the protein. For another example, the expression of TTC6 is detected by detecting the presence or amount of mRNA of the protein, such as detecting the amount of mRNA encoding TTC6 in a sample by RT-PCR.

[0021] In yet another aspect of the present invention, the method comprises the step of determining the activity of TTC6 protein.

[0022] The present invention also provides a kit or instrument and device (including a gene chip) for diagnosing or predicting fertility defects such as infertility in a mammalian individual, which includes a reagent for detecting TTC6 in the subject.

[0023] In another aspect of the present invention, the kit or instrument includes a reagent for detecting the expression or activity of the TTC6 protein. For example, the kit or instrument includes a reagent for immunoassay, such as a reagent for detecting the expression of TTC6 by ELISA or Western blotting with an antibody that specifically recognizes the protein. For another example, the kit or instrument includes a reagent for detecting the expression of TTC6 by detecting the presence or amount of mRNA of the protein, such as a reagent for detecting the amount of mRNA encoding TTC6 in a sample by RT-PCR.

[0024] In yet another aspect of the present invention, the kit or apparatus comprises a reagent for detecting the Ttc6 gene.

[0025] In one aspect of the present invention, in the above-mentioned methods, kits or instruments of the present invention, the reagents for detecting genetic markers in samples include primers or probes for detecting Ttc6, or reagents for examining TTC6, such as specific antibodies or reagents for detecting the mRNA of the protein.

[0026] In the present invention, there is also provided the use of a reagent for detecting TTC6 in the preparation of a kit or instrument for diagnosing or predicting fertility defects such as infertility in a mammalian individual as described above. The reagent for detecting genetic markers includes a primer or probe for detecting the Ttc6 gene, or a reagent for checking the TTC6 protein, such as a specific antibody or a reagent for detecting the mRNA of the protein.

[0027] The present invention also provides a method for treating or avoiding fertility defects in a mammalian individual, which comprises treating an individual having a TTC6 defect, such as a Ttc6 mutation.

[0028] In this article, protein symbols are not italicized and are all capitalized; gene symbols are italicized. For example, TTC6 is a protein, and the gene encoding the protein is written as Ttc6. Sometimes in this article, protein symbols are not all capitalized. For example, sometimes "Ttc6" in this article means TTC6 protein. Sometimes in this article, gene symbols are not italicized. For example, sometimes "Ttc6" or "TTC6 gene" in this article means the gene Ttc6 encoding TTC6 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It was shown that Ttc6 gene knockout caused male infertility in mice.

[0030] (A) Tissue expression profile of Ttc6 mRNA in different adult mouse tissues determined by RT-PCR. GapdhmRNA was amplified as an internal control.

[0031] (B) RT-PCR was used to determine the expression profile of Ttc6 mRNA in testicular tissue at different postnatal ages. GapdhmRNA was amplified as an internal control.

[0032] (C) Production of Ttc6 - / - Schematic diagram of the gene knockout strategy for mice. gRNA3 and gRNA6 were designed to delete the region from exon 2 to exon 32.

[0033] (D) Genotyping of Ttc6-knockout mice.

[0034] (E) Ttc6 mRNA was detected in the testes of wild-type mice, whereas in Ttc6 - / - It is undetectable in the testes of mice.

[0035] (F)Ttc6 + / + and Ttc6 - / - Average litter size of male mice at 3 months (n=6 independent experiments). - / - Male mice were in subfertile state. Data are presented as mean ± SD. ***P < 0.001.

[0036] (G) All Ttc6 tested - / - All female mice are fertile. + / + and Ttc6 - / - Mean litter size of female mice at 3 months (n=4). ns, not statistically significant.

[0037] Figure 2 Display Ttc6 - / - Morphological and microscopic images of mouse testis.

[0038] (A)Ttc6 + / + and Ttc6 - / - There is no significant difference in testis size between mice. Scale bar is 2 mm.

[0039] (B)Ttc6 + / + and Ttc6 - / - Testis weight of mice. ns indicates no statistical significance.

[0040] (C)Ttc6 + / + and Ttc6 - / - Body weight of mice. ns indicates no statistical significance.

[0041] (D)Ttc6 + / +and Ttc6 - / - Ratio of testis weight to body weight of mice. ns indicates no statistical significance.

[0042] (E) Hematoxylin staining showed that Ttc6 - / - The histological morphology of mouse seminiferous tubules and Ttc6 + / + Mouse seminiferous tubules are similar. Scale bar, 50 μm.

[0043] (F)Ttc6 + / + and Ttc6 - / - PAS-hematoxylin staining analysis of mouse testicular seminiferous tubule cross sections. Arrows highlight germ cells at different stages of spermatogenesis. Pl, preleptotene; L, leptotene; Z, zygotene; P, pachytene; D, diplotene; RS, round spermatid; ES, elongating spermatid. Scale bar, 20 μm.

[0044] Figure 3 Display Ttc6 - / - The ability of sperm to fertilize in vivo and in vitro.

[0045] (A)Ttc6 + / + and Ttc6 - / - Hematoxylin staining of the mouse tail epididymis. Scale bar, 50 μm.

[0046] (B)Ttc6 - / - The number of sperm in the caudal epididymis of mice (n=3) and Ttc6 + / + There were no differences among mice (n=4). Data are presented as mean±SEM. Two-tailed Student's t-test; ns: not significant.

[0047] (C) 12-week-old Ttc6 + / + and Ttc6 - / - Immunofluorescence staining of mouse tubulin acetylation (green), glycosylation (red), and DAPI in mouse testis. Scale bar, 10 μm.

[0048] (D) From Ttc6 + / + and Ttc6 - / - Males inserted into fertilized oocytes collected from superovulated wild-type female mice. Morphology of two-cell and blastocyst-stage embryos. Scale bar, 100 μm.

[0049] (E) Using Ttc6 + / + and Ttc6 - / - Mouse spermatozoa were subjected to the sperm-zona binding assay. Scale bar, 50 μm.

[0050] (F)Ttc6 - / -Mouse sperm can fertilize normal mouse oocytes by in vitro fertilization. Morphology of binucleate, two-cell, and blastocyst stage embryos. Scale bar, 100 μm.

[0051] (G)Ttc6 - / - The ability of sperm and oocytes to fertilize in vitro is reduced, but the fertilized egg can still develop to the blastocyst stage, which is related to Ttc6 + / + There was no significant difference in sperm fertilization (n=3). Data are presented as mean ± SEM. Two-tailed Student's t-test. *ns: not significant.

[0052] Figure 4 Display Ttc6 - / - The sperm motility of mice was altered.

[0053] (A)Ttc6 - / - Mice (n=3) and Ttc6 + / + There were no differences in the frequency of motile sperm among mice (n=4). Data are presented as mean ± SEM. Two-tailed Student's t-test; ns: not significant.

[0054] (B) with Ttc6 + / + Compared with mice (n=4), Ttc6 - / - Progressive sperm frequency reduction in mice (n=3). Data are presented as mean ± SEM. Two-tailed Student's t-test; **P<0.01.

[0055] (C)Ttc6 + / + (n=3) and Ttc6 - / - Mean path velocity (VAP) of spermatozoa in mice (n=4). Data are presented as mean±SEM. Two-tailed Student's t-test; ***P<0.001.

[0056] (D)Ttc6 + / + Mice (n=3) and Ttc6 - / - Sperm progression velocity (VSL) of mice (n=4). Data are presented as mean±SEM. Two-tailed Student's t-test; ***P<0.001.

[0057] (E)Ttc6 + / + Mice (n=3) and Ttc6 - / - Sperm trajectory velocity (VCL) of mice (n=4). Data are presented as mean ± SEM. Two-tailed Student's t-test; **P<0.01.

[0058] (F)-(G) Ttc6 swimming freely near a glass surface + / + and Ttc6 - / - Color-coded time projection of a representative dark-field recording of sperm. Scale bar, 10 μm.

[0059] (H)-(I)Ttc6 + / + and Ttc6 - / - The swimming path of sperm. + / + Sperm swim along a twisting ribbon-like path, and Ttc6 - / - Spermatozoa swim in a circular path. Scale bars, 10 μm.

[0060] (J) Quantification of the swimming patterns observed in (H)-(I). Data are presented as mean ± SEM. Two-tailed Student's t-test; ***P < 0.001.

[0061] Figure 5 Display Ttc6 - / - The sperm flagella of mice are abnormal.

[0062] (A)Ttc6 + / + and Ttc6 - / - Morphology of mouse caudal epididymal sperm. Scale bars, 50 μm.

[0063] (B)Ttc6 + / + and Ttc6 - / - Representative TEM images of the mitochondrial sheath (MS) and fibrous sheath (FS) of mouse sperm. Scale bar, 500 μm.

[0064] (C) Representative TEM images showing Ttc6 + / + and Ttc6 - / - Cross-section of the mid-segment, principal segment, and terminal segment of a mouse sperm flagellum. Scale bar is 200 μm.

[0065] (D) Representative TEM image showing the unsheathed flagella region at the junction of the mid-lamella and the main lamella. Scale bar, 500 μm.

[0066] (E) Representative SEM images showing the unsheathed flagellar region at the junction of the mid-segment and main segment. Scale bar, 500 μm. DETAILED DESCRIPTION

[0067] The essential contents and beneficial effects of the present invention will be further described below in conjunction with embodiments, which are only used to illustrate the present invention rather than to limit the present invention.

[0068] Example 1 Ttc6 is a testis-enriched gene, and Ttc6 knockout male mice have severe fertility defects

[0069] To determine the specific expression profile of Ttc6, multiple tissue RT-PCR analysis was performed in adult mice. The results showed that Ttc6 was highly expressed in the testis, but its expression in other organs was negligible ( Figure 1A). RT-PCR analysis of testes at different postnatal ages showed that Ttc6 was expressed starting at postnatal day 21 and then continued to be expressed in adult mice ( Figure 1 B).

[0070] This timeline corresponds to the stages of sperm agglutination and flagella formation, implying that TTC6 plays an important role in the flagella formation process.

[0071] To elucidate the potential functional role of Ttc6 in spermatogenesis, Ttc6 knockout mice were generated using the CRISPR / Cas9 genome editing system provided by Cyagen Biosciences. The 173.79 kb testis-specific Ttc6 gene is located on chromosome 12 and contains an ATG start codon in exon 2 and a TGA stop codon in exon 32. Exons 2 to 32 were selected as target sites for this procedure ( Figure 1 C) Animals were genotyped by genomic DNA sequencing and further confirmed by polymerase chain reaction: Ttc6 + / + The size of the Ttc6 locus in male mice is 417 bp, while - / - The locus size for male mice is 600 bp ( Figure 1 D).

[0072] In the testes of Ttc6 knockout mice, no mRNA was detected by RT-PCR ( Figure 1 E).

[0073] No obvious abnormalities in development, behavior, or survival were observed in Ttc6 knockout mice. - / - To investigate the fertility of mice, adult male knockout mice were bred with wild-type mice for 3 months, and female mice were bred with wild-type mice for 3 months. + / + Male mice produce a normal number of offspring ( Figure 1 F) served as the control group for the entire study. + / + Males produced an average of 7.17±0.23 (SEM, n=6) offspring per litter, while Ttc6 - / - The average number of offspring per litter was 0.08 ± 0.05 (SEM, n = 6, Figure 1 F, S1A) offspring. Notably, two-thirds (66%) of Ttc6 - / - The males failed to produce any offspring. In contrast to the males, the fertility of the females appeared to be unaffected ( Figure 1 G).

[0074] In Ttc6 - / -The subfertility observed in the mice was not the result of hormonal changes, as testosterone levels remained constant, indicating that the Ttc6 gene is essential for maintaining normal male fertility in mice.

[0075] Example 2Ttc6 - / - Male spermatogenesis is normal but infertile

[0076] Sperm morphology and motility are important factors in predicting oocyte fertilization rate and overall pregnancy rate, and any abnormality in these factors can significantly lead to infertility. - / - The structure of the testis was observed macroscopically and microscopically. - / - and Ttc6 + / + Testis size in male mice ( Figure 2 A) Testicular weight ( Figure 2 B) Mouse weight ( Figure 2 C) and the ratio of testicular weight to total body weight ( Figure 2 D) There is no significant difference.

[0077] PAS staining showed that Ttc6 - / - The structure of the seminiferous tubules in the mouse testis remained normal ( Figure 2 E), Periodic acid-Schiff (PAS)-hemoglobin staining showed normal spermatogenesis, Ttc6 - / - Mouse sperm acrosome and nuclear morphology are intact ( Figure 2 F) These findings support the hypothesis that disruption of Ttc6 has little effect on acrosome biogenesis.

[0078] Example 3Ttc6 - / - Impaired fertility in male mice is associated with defects in sperm-oocyte interaction and zona pellucida binding

[0079] To further search for Ttc6 - / - Cause of male mouse infertility, from Ttc6 + / + or Ttc6 - / - Male mice were inserted into superovulated wild-type female mice to harvest fertilized eggs.

[0080] Tc6 + / + Both two-cell and blastocyst stage embryos appeared in large numbers in the Ttc6 group. - / - The group was significantly less common ( Figure 3 D), indicating that fertilization failure is the cause of Ttc6 - / - Causes of male mouse infertility.

[0081] Further attempts to identify Ttc6 - / -Whether the sperm-egg interaction or fertilization ability in vitro is affected. + / + Compared with sperm, Ttc6 - / - Spermatozoa incubated with granulosa-free eggs showed weaker zona pellucida binding ability ( Figure 3 E) Ttc6 - / - The ability of sperm and oocytes to fertilize in vitro is reduced, but the fertilized eggs can still develop to the blastocyst stage. + / + There is no significant difference between sperm and fertilized eggs ( Figure 3 F, 3G).

[0082] These observations suggest that the ability of sperm to bind to the zona pellucida may not be crucial for the fertilizing ability of sperm.

[0083] Example 4 Knockout of Ttc6 affects sperm motility

[0084] Spermatozoa were released from the cauda epididymis and their motility after capacitation was assessed by incubation in oviductal fluid. + / + and Ttc6 - / - There was no significant difference in the percentage of motile sperm between mice ( Figure 4 A) However, Ttc6 - / - The proportion of sperm with progressive motility in male mice was significantly reduced (26.41±2.14% vs. 13.64±0.79%; Figure 4 B).

[0085] Ttc6 - / - and Ttc6 + / + Mouse sperm motility parameters, which define sperm motility speed.

[0086] Decreases were detected in four kinematic parameters measuring sperm velocity, including mean path velocity (VAP, the average speed of the sperm head through its average trajectory) (107.98 ± 6.02 mm / s vs. 69.95 ± 1.83 mm / s; Figure 4 C), linear velocity (VAP, the average velocity of the sperm head through its average trajectory) (107.98 ± 6.02 mm / s vs. 69.95 ± 1.83 mm / s; Figure 4 D) and sperm velocity (VAP, the average velocity of the sperm head through its average trajectory). Figure 4 C), linear velocity (VSL, the average velocity of the sperm head through the straight line connecting the first position and the last trajectory) (73.13 ± 3.05 mm / s vs. 44.18 ± 1.46 mm / s; Figure 4D ) and curvilinear velocity (VCL, the average velocity of the sperm head through its actual path) (226.86 ± 9.61 mm / s vs. 173.51 ± 5.97 mm / s; Figure 4 E).

[0087] These results strongly suggest that Ttc6 - / - Reduced fertilization potential of sperm due to decreased sperm motility (asthenozoospermia) is a common factor leading to male infertility in mouse models.

[0088] Example 5 Knockout of Ttc6 changes the sperm flagellar beat pattern and causes abnormal sperm swimming path

[0089] After observing Ttc6 - / - After the sperm motility was reduced, the characteristics of the flagellar beat of these sperm were investigated. Single sperm cells with their heads tethered to a glass surface were analyzed in two dimensions. The analysis showed that Ttc6 + / + Sperm exhibit characteristic symmetrical flagellar beats ( Figure 4 F), while Ttc6 - / - Spermatozoa exhibit unusually stiff flagella that maintain a constant curvature from the mid-segment to the proximal region of the main lamella ( Figure 4 G). This indicates that knockout of Ttc6 alters the flagellar beating pattern of sperm.

[0090] Symmetrical flagellar beating is a prerequisite for progressive swimming and is expressed in Ttc6 - / - Next, we evaluated the expression of Ttc6 + / + and Ttc6 - / - Sperm swimming pattern. Head tracking of free-swimming sperm showed that Ttc6 + / + Sperm swim mainly in straight or curved paths ( Figure 4 H). In contrast, although Ttc6 - / - Sperm can swim, but their movements are mostly circular ( Figure 4 I, 4J). - / - The main defect of sperm cells is their nonlinear swimming pattern, which significantly hinders their ability to reach the site of fertilization.

[0091] Taken together, these results indicate that Ttc6 knockout leads to altered sperm flagellar beat patterns and atypical sperm swimming paths, thus providing new insights into the role of Ttc6 in the development of - / - The infertility of male sperm provides a reliable explanation.

[0092] Example 6Ttc6 - / - Mouse sperm exhibits abnormal morphology

[0093] Sperm motility has been shown to be related to its structural integrity and energy replenishment.

[0094] To test Ttc6 - / - To determine whether the reduced sperm motility could be attributed to defects in the sperm flagella structure, morphological analysis of sperm from the epididymis was performed.

[0095] The analysis showed that Ttc6 - / - Sperm have a hairpin phenotype, characterized by a bend at the junction of the mid-segment and main lamella ( Figure 5 A). The ultrastructure of sperm flagella in the cauda epididymis was studied using transmission electron microscopy (TEM). TEM images show that Ttc6 - / - The structural organization of the mitochondrial sheath and fibrous sheath of spermatozoa remains normal ( Figure 5 B).

[0096] Further investigation of the flagellar ultrastructure revealed that Ttc6 + / + Compared with sperm, Ttc6 - / - The "9+2" axoneme structure of spermatozoa showed no abnormality ( Figure 5 C) However, Ttc6 - / - TEM and SEM analysis of the longitudinal section of sperm flagella showed that there was an unsheathed flagella region at the junction of the middle piece and the main piece ( Figure 5 D, E), which is consistent with the hairpin phenotype found in sperm morphology analysis.

[0097] These results indicate that Ttc6 - / - The reduction in sperm motility in mice can be attributed to structural abnormalities in the flagellum.

[0098] The inventors of the present application discovered for the first time that TTC6 is essential for male mouse fertility. The inventors showed through a series of experimental analyses that knockout of the Ttc6 gene led to weakened sperm motility and induced sperm rotational movement, ultimately destroying fertilization function and leading to male infertility. Morphological analysis of epididymal sperm in Ttc6 knockout mice showed a hairpin phenotype. In addition, ultrastructural studies have shown that knockout of Ttc6 leads to desheathing of flagella at the junction of the mid-segment and the main segment. These observations indicate that the role of TTC6 is to reinforce the terminal ring, thereby maintaining the integrity of the sperm flagellum and ensuring rapid directional movement of sperm.

[0099] Applicants have thus provided methods for diagnosing fertility defects such as infertility disorders in mammals, including humans, and further facilitated the development of new treatments for male infertile individuals affected by gene mutations.

[0100] The above is an explanation of the present invention and cannot be regarded as a limitation of the present invention. Unless otherwise noted, the practice of the present invention will use the conventional techniques of organic chemistry, polymer chemistry, biotechnology, etc., and it is obvious that the present invention can also be realized in other ways except that it is particularly described in the above description and embodiments. Other aspects and improvements within the scope of the present invention will be apparent to those skilled in the art to which the present invention belongs. According to the teachings of the present invention, many changes and variations are feasible, and therefore they are within the scope of the present invention.

Claims

1. Use of a reagent for detecting TTC6 in a subject in the preparation of a kit or an apparatus for diagnosing and predicting the risk of fertility defects such as infertility in mammals.

2. The method of claim 1, wherein the infertility disorder is selected from teratozoospermia, azoospermia, oligozoospermia, asthenozoospermia, asthenozoospermia, in particular asthenozoospermia or asthenozoospermia.

3. The use according to claim 1, wherein the kit further comprises a reagent for detecting the gender of the subject.

4. The use according to any one of claims 1 to 3, wherein the reagent for detecting TTC6 in the subject is a reagent for detecting the Ttc6 gene.

5. The use according to any one of claims 1 to 3, wherein the reagent for detecting TTC6 in the subject is a reagent for detecting TTC6 protein, preferably, further comprising a reagent for determining the activity of TTC6.

6. The use according to claim 5, which comprises a reagent for detecting the expression of TTC6 by immunoassay, such as a reagent for detecting the expression of TTC6 by ELISA or Western blotting using an antibody that specifically recognizes the protein; or These include reagents for detecting TTC6 by detecting the presence or amount of TTC6 mRNA, such as reagents for detecting the amount of mRNA encoding TTC6 in a sample by RT-PCR.

7. A kit or device (eg, a gene chip) for diagnosing and predicting the risk of meiosis-related diseases, especially fertility defects or infertility, in mammals of different sexes, comprising a reagent for detecting TTC6 in a subject, and preferably, also comprising a reagent for detecting the sex of the subject.

8. The kit or device according to claim 7, wherein the reagent for detecting TTC6 in the subject is a reagent for detecting the Ttc6 gene.

9. The kit or device according to claim 7, wherein the reagent for detecting TTC6 in the subject is a reagent for detecting TTC6 protein, and preferably, further comprising a reagent for measuring the activity of TTC6.

10. The kit or device of claim 9, comprising a reagent for determining TTC6 by immunoassay, such as an antibody that specifically recognizes TTC6; or These include reagents for detecting the presence or amount of TTC6 mRNA to detect the expression of TTC6, such as reagents for detecting mRNA encoding TTC6 in a sample by RT-PCR.

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