阅读说明：本技术 Ak9作为靶标在检测和治疗原发性弱精子症中的应用 (Application of AK9 as target in detecting and treating primary asthenospermia ) 是由 沙艳伟 苏志英 于 2020-05-08 设计创作，主要内容包括：本发明公开了AK9作为靶标在检测和治疗原发性弱精子症中的应用。本发明中,AK9突变引起AK9功能蛋白缺失,可能破坏细胞内的核苷代谢,抑制糖酵解,引起弱精子症。(The invention discloses application of AK9 as a target in detecting and treating primary asthenospermia. In the invention, AK9 mutation causes AK9 functional protein loss, possibly destroys nucleoside metabolism in cells, inhibits glycolysis and causes asthenospermia.)

The application of AK9 as a detection target in preparing a kit for diagnosing primary asthenospermia.

The application of AK9 as a therapeutic target in preparing a kit for treating primary asthenospermia.

The application of AK9 mutated antagonistic substance in preparing medicine for preventing and treating primary asthenospermia.

4. A kit for diagnosing primary asthenospermia, characterized in that: reagents capable of detecting whether AK9 is mutated are included.

5. The kit of claim 4, wherein: the reagent capable of detecting whether AK9 is mutated or not includes a reagent for detecting whether AK9 is mutated or not by PCR.

6. A kit for treating primary asthenospermia, which is characterized in that: agents capable of treating AK9 mutations are included.

7. The kit of claim 6, wherein: the agent capable of treating AK9 mutation comprises an antagonist of AK9 mutation.

8. A medicine for preventing and treating primary asthenospermia is characterized in that: the effective component comprises AK9 mutated antagonistic substance.

9. The medicament of claim 8, wherein: the effective component is AK9 mutated antagonistic substance.

10. The medicament of claim 8 or 9, wherein: also comprises pharmaceutically acceptable auxiliary materials.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of AK9 as a target in detection and treatment of primary asthenospermia.

Background

In recent years, the incidence of male infertility has increased significantly, which seriously affects the family harmony and the sustainable development of the country. Clinical statistics show that: about 70% of the infertile men have varying degrees of manifestations of asthenospermia, an important factor in the induction of male infertility. The semen analysis standards promulgated by the world health organization indicate that: asthenospermia refers to a condition in which the proportion of forward motile sperm in semen is less than 32% or the proportion of forward motile sperm to non-forward motile sperm is less than 40%, and is mainly characterized by low forward motility of sperm. At present, besides secondary asthenospermia caused by age, malignant tumor, testicular injury, male accessory gland infection, cryptorchidism, varicocele, endocrine insufficiency or disorder, incomplete or blockage of vas deferens and ejaculatory ducts, seminal plasma abnormality, microbial infection and autoimmune deficiency, the pathogenesis of primary asthenospermia is still unknown in a certain proportion clinically. Therefore, the study on the pathogenesis of the primary asthenospermia in clinic is helpful for accurately solving the infertility caused by male asthenospermia in clinic.

A gene mutation is a change in the nucleotide sequence or number of DNA molecules. The gene mutation can cause the change of gene structure and function and participate in the occurrence of various diseases such as asthenospermia and the like. The research shows that: in the TEKT4 knockout mouse, the ultrastructure of a sperm electron microscope of a male mouse has no obvious abnormality, the advancing speed of the sperm is rapidly reduced, uncoordinated waveform motion appears along flagella, the marked asthenospermia is shown, and the fertility of the mouse is damaged. Wu et al demonstrated by clinical studies: the reduced expression of TEKT4 is closely associated with the occurrence of primary asthenospermia in humans. The mutation of polypeptide N-acetylgalactosamine aminotransferase 5(GALNTL5) can cause weak sperm disease and sterility in male and male mice in clinic. Furthermore, mutations in genes such as cyclooxygenase 1(COX1), Cyclooxygenase (COXII), cytochrome oxidase III (COIII) and the like have also been shown to be one of the causes of asthenospermia and infertility in men in clinical practice. Through previous researches, researchers find and disclose that SPAG17 and EIF4G1 gene mutation are closely related to the occurrence of clinical primary asthenospermia. Therefore, gene mutation may be an important cause of primary asthenospermia.

Disclosure of Invention

The invention aims to provide application of AK9 serving as a target in detecting and treating primary asthenospermia.

The technical scheme of the invention is as follows:

the AK9 is used as a detection target in the preparation of a kit for diagnosing primary asthenospermia.

The application of AK9 as a therapeutic target in preparing a kit for treating primary asthenospermia.

The application of AK9 mutated antagonistic substance in preparing medicine for preventing and treating primary asthenospermia is provided.

A kit for diagnosing primary asthenospermia comprising reagents capable of detecting whether AK9 is mutated.

In a preferred embodiment of the present invention, the reagent capable of detecting whether AK9 is mutated comprises a reagent for detecting whether AK9 is mutated by PCR.

A kit for treating primary asthenospermia comprising an agent capable of treating a mutation of AK 9.

In a preferred embodiment of the present invention, said agent capable of treating AK9 mutation comprises an antagonist of AK9 mutation.

A medicine for preventing and treating primary asthenospermia comprises AK9 mutated antagonistic substance as effective component.

In a preferred embodiment of the present invention, the effective ingredient is an antagonist substance of AK9 mutation.

Further preferably, the composition also comprises pharmaceutically acceptable auxiliary materials.

The invention has the beneficial effects that: in the invention, AK9 mutation causes AK9 functional protein loss, possibly destroys nucleoside metabolism in cells, inhibits glycolysis and causes asthenospermia.

Drawings

FIG. 1 is a diagram showing 4 cases of AK9 gene mutation primary asthenospermia patients and pedigrees in example 1 of the present invention. Among them, pedigree analysis of patients with the primary asthenospermia phenotype of example 4 carrying mutation of AK9 gene; black squares indicate infertile patients; the semi-black squares and semi-black circles indicate carriers of heterozygous mutations in the family.

FIG. 2 is a graph showing the expression abundance of AK9 gene in various tissues of human body in example 1 of the present invention. Wherein, (A) the expression abundance of AK9 gene in each tissue of human body in NCBI database; (B) the expression abundance of AK9 gene in various tissues of human body in HPA database.

FIG. 3 is a diagram showing the positions of mutation sites of AK9 gene in 4 patients in example 1 of the present invention on the genomic and protein domains. Wherein (a) shows the positions of 7 AK9 mutation sites in the inner genome. (B) The positions of 7 AK9 mutation sites on the protein domain of AK9 are shown. The orange box shows the ak (adenylate kinase) domain; blue boxes represent NMPbind domains; the green box shows the LID domain.

FIG. 4 is a graph showing the results of Sanger sequencing verification in example 1 of the present invention, wherein it was found that: patient R0008 was AK9 gene mutated to c.2005dupt: p.c669fs is a homozygous mutation, the parent is a carrier of the c.2005dupT heterozygous mutation, the site is not mutated and one child is bred. Patient R0022 is AK9 gene c.607 — 615 del: p.203-205 del and c.1266A > T: p.x422y compound heterozygous mutation, paternal c.607 — 615 del: the carrier of p.203-205 del heterozygous mutation, mother is the carrier of c.1266A > T heterozygous mutation. Patient R0038 is AK9 gene c.g 3391a: p.E1131K and c.C2444T: p.P815L complex heterozygous mutation, father c.G3391A: carriers of p.e1131 heterozygous mutations, maternal c.c 2444t: p.p815l heterozygous mutation carriers; patient R0052 is AK9 gene c.g 5074a: p.v16925 m and c.3257_3259 del: p.E1083del complex heterozygous mutation, father c.G5074A: carriers of p.v16925 m heterozygous mutations, maternal c.3257 — 3259 del: p.e1083del heterozygous mutation carriers; the results show that: gene mutations were co-isolated in these patient families, four patients had AK9 gene mutations inherited from their parents (see FIGS. 4A-D), the mutated genes were all located on the exon encoding the AK9 protein (see FIG. 4E), and the mutated genes affected the encoded proteins (see FIG. 4F).

FIG. 5 is a diagram of the results of four cases of flat-scan CT scans of paranasal sinuses, chest and chest of AK9 gene mutation asthenospermia patients in example 1 of the present invention. Wherein, the maxillary sinuses are positioned at two sides of the nasal cavity, the left and the right are basically consistent, the bone wall is clear, and the thickness of the mucosa along the sinus wall is not more than 1 mm. The upper frontal sinus is located at the inner upper part of the two eye sockets and is in a petal shape. The thoracic corridor is symmetrical, the veins of the two lungs are clear, the structure of the lung portal shadow is normal, the heart shadow is not large, the mediastinum is centered, the diaphragm surfaces of the two sides are smooth, the diaphragm angles of the ribs of the two sides are sharp, and the ribs on the diaphragm are not abnormal.

FIG. 6 is a graph showing the analysis of the motility of sperm cells of AK9 gene-mutated patients in example 1 of the present invention. Wherein (A) the forward motility of sperm of the AK9 gene mutant patient is significantly reduced compared with that of the control group; wherein: p is less than 0.001. (B) After ATP is added, the forward motility of the sperm of the AK9 gene mutation patient is obviously improved.

FIG. 7 is a graph showing the Papanicolaou staining of sperm of two cases of AK9 gene mutation asthenospermia patients in example 1 of the present invention. Wherein A is sperm of normal control, B is sperm of R0008 patient, and C is sperm of R0022 patient. Two cases of AK9 gene mutation patients had no obvious abnormality in sperm morphology compared with normal controls.

FIG. 8 is the structural diagram of two cases of the transmission electron microscope ultrastructures of the sperm of AK9 gene mutation asthenospermia patient in example 1 of the present invention. Wherein, MP is the cross section of the sperm in the middle section; both control and patient sperm had an intact "9 + 2" microtubule structure surrounded by an intact Mitochondrial Sheath (MS); PP, cross section of the sperm in the main section; both control and patient sperm had an intact "9 + 2" microtubule structure surrounded by an intact Fibrous Sheath (FS); compared with the normal control, the ultrastructure of the sperms of 2 patients with asthenospermia has no obvious abnormality.

FIG. 9 is a graph showing the expression of AK9mRNA in a patient and the expression and localization of AK9 protein in sperm in example 1 of the present invention. Wherein, (A) qRT-PCR assay detects the expression of AK9mRNA in patient blood cells; taking 2 patients as an example, the AK9mRNA of the R0008D patient is significantly reduced; however, the level of AK9mRNA expression of patient R0022 was not significantly affected; (B) western Blot experiment detects AK9 protein expression in sperm of patients. No expression of AK9 in the sperm sample of patient R0008; the level of AK9 expression in the sperm sample of patient R0022 was reduced but not significantly affected; (C) analyzing the expression and location of AK9 protein in sperm of a patient by an immunofluorescence staining experiment; AK9 was expressed along sperm flagella in normal controls; no expression of AK9 was observed on the sperm flagella of patient R0008; the expression of AK9 was observed in the sperm flagellum of patient R0022, but the expression amount was reduced. Blue color: DAPI; green: α -Tubulin; red: AK 9.

FIG. 10 is a graph showing the expression abundance of Ak9 gene in each tissue in the mouse of example 1 of the present invention. Among them, the expression abundance of Ak9 gene in each tissue of mouse in NCBI database was shown.

Fig. 11 is a design diagram of a gRNA of an Ak9 knockout mouse model constructed by the CRISPR-Cas9 technology in example 1 of the present invention. Wherein, the position and the sequence of the gRNA primer of the Ak9 knockout mouse model are shown.

FIG. 12 is a graph showing the PCR results of Ak9 knockout mice in example 1 of the present invention. Compared with a control group, the PCR strip position of the Ak9 gene knockout mouse is obviously different, and the strip size is obviously reduced.

FIG. 13 is a graph showing the body weight and reproductive development of AK9 knockout mice in example 1 of the present invention. Wherein, the weight (A) of the male mice with Ak9 gene knockout, the weight (B) of the testis, the weight (C) of the epididymis and the weight-to-body weight ratio (D) of the epididymis; compared with a control group, the Ak9 gene knockout adult mouse has no obvious abnormality in body weight, testis weight, epididymis weight and ratio of epididymis to body weight.

FIG. 14 is a graph showing the staining of Ak9 knockout mouse testis HE in example 1 of the present invention. Wherein, there are spermatogenic cells of each stage in the seminiferous tubule of adult mouse testis of control group, including: spermatogonia, spermatocytes at all levels, round spermatozoa and long spermatozoa; compared with a control group, the Ak9 gene knockout mouse has spermatogenic cells at various stages in testicular seminiferous tubules, and no obvious abnormality is seen.

FIG. 15 is a graph showing the epididymis morphology and the number of sperm of Ak9 knockout mice in example 1 of the present invention. Wherein, the (A) HE dyeing result shows that the form of the Ak9 gene knockout mouse epididymis is not obviously abnormal compared with that of a control group; (B) statistical results show that the number of sperms in the epididymis of Ak9 gene knockout mice is obviously reduced compared with that of a control group. Wherein: p is less than 0.05.

FIG. 16 is a sperm morphology map of AK9 knock-out mice in example 1 of the present invention. The Pasteur staining result shows that the sperm morphology of the Ak9 gene knockout mouse is not obviously abnormal compared with that of a control group.

FIG. 17 is a drawing showing the ultrastructure of sperm in Ak9 knockout mouse in example 1 of the present invention. Wherein, the results of MP section transmission electron microscope show that the sperm flagellum axis filaments of the contrast and gene knockout mice have complete '9 + 2' microtubule structures, and are surrounded by complete Mitochondrial Sheath (MS); the results of the PP section transmission electron microscope show that the sperms of the control mice and the Ak9 knockout mice have complete 9+2 microtubule structures, and are surrounded by complete Fibrous Sheaths (FS); compared with a control group, the Ak9 gene knockout mouse sperm ultrastructure has no obvious abnormality; MP, cross section of middle sperm. PP, cross section of main segment sperm.

FIG. 18 is a graph showing sperm motility analysis of Ak9 knockout mice in example 1 of the present invention. Wherein, the sperm motility in the epididymis of the adult mouse is analyzed; (A) compared with a control group, the sperm of the Ak9 gene knockout mouse has significantly reduced forward movement capability. Wherein: p is less than 0.001; (B) after ATP is added, the forward movement capacity of the sperm of the Ak9 gene knockout mouse is obviously improved.

Detailed Description

The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments.