Sperm treatment composition, sperm motility improving agent, sperm fertilization ability retaining agent, sperm motility improving method, and sperm fertilization ability retaining method

Abstract

The purpose of the present invention is to provide sperm having high motility. The present invention is a composition for sperm treatment containing exosomes. The composition for sperm treatment according to the present invention can be used as a sperm adjustment solution, a sperm dilution solution, a sperm preservation solution, an artificial insemination solution, an in vitro fertilization solution, a solution for improving sperm motility, or a solution for maintaining sperm fertilization ability.

Description

Technical Field

[0001] The present invention relates to a composition for sperm treatment, a sperm motility improver, a sperm fertilization ability retainer, a sperm motility improvement method, and a sperm fertilization ability retention method. Background Art

[0002] "Infertility" refers to the situation where healthy men and women with the intention of pregnancy have sexual intercourse without contraception but do not become pregnant within a certain period of time. The Japanese Obstetrics and Gynecology Society defines this "certain period" as "usually 1 year". It is said that about one in ten couples is infertile, and this proportion is actually higher because in recent years, the age at which people consider pregnancy has been increasing, and it is known that both men and women have difficulty getting pregnant as they age.

[0003] The causes of infertility include male factors, female factors, or cases where both factors are involved, as well as cases where the cause is unknown. In all cases, about half of the cases also involve male factors. As such male infertility causes, spermatogenic dysfunction, vas deferens obstruction, erectile dysfunction (ED), ejaculatory dysfunction such as intravaginal ejaculation disorder, and aging are known. It should be noted that generally, sperm quality gradually declines in men from around 35 years old. It has been reported that with the recent trend of late marriage, about one in ten men have semen problems before marriage.

[0004] In the treatment of infertility, the most suitable treatment method is selected according to the cause for treatment. The main treatment methods include timing selection method, ovulation induction method, artificial insemination, and further assisted reproductive therapies such as in vitro fertilization. Artificial insemination (AIH) is a treatment method in which sperm is injected into the uterine cavity in order to send sufficient sperm required for fertilization to the ampulla of the fallopian tube, which is the fertilization site. Oligospermia (sperm concentration below 15 million / ml), low sperm motility (motility rate below 40%), ejaculatory disorder, sperm-cervical mucus incompatibility (poor Huhner test), cases carrying anti-sperm antibodies, and cases of unexplained infertility are indications for AIH. However, it is said that the limit of AIH is that the total number of motile sperm after adjustment is 1 million to 5 million, and there are many cases where it is difficult to meet this condition. In addition, when cryopreserved semen is used for artificial insemination, sperm motility decreases due to freezing, and the pregnancy rate is not high. Therefore, a method for improving sperm motility is desired.

[0005] Mesenchymal stem cells are precursor cells with multipotential differentiation ability first isolated from bone marrow by Friedenstein (1982) (refer to Non-Patent Document 1). It has been clarified that mesenchymal stem cells exist in various tissues such as bone marrow, umbilical cord, and adipose tissue, and transplantation of mesenchymal stem cells is expected as a new treatment method for various intractable diseases (refer to Patent Documents 1-2). Recently, it has been found that cells with the same function exist in mesenchymal stromal cells of adipose tissue, placenta, umbilical cord, egg membrane, etc. Therefore, mesenchymal stem cells are sometimes also referred to as mesenchymal stromal cells.

[0006] Prior Art Documents

[0007] Patent Documents

[0008] Patent Document 1: Japanese Patent Application Laid-Open No. 2012-157263

[0009] Patent Document 2: Japanese Patent Application Laid-Open No. 2012-508733

[0010] Non-Patent Documents

[0011] Non-Patent Document 1: Pittenger F.M. et al., Science 284, pp. 143-147, 1999 Summary of the Invention

[0012] Problems to be Solved by the Invention

[0013] An object of the present invention is to provide spermatozoa with excellent motility under the above-mentioned circumstances.

[0014] Solutions to the Problems

[0015] In order to solve the above problems, intensive studies were conducted, and as a result, the present inventors found that exosomes can improve the motility of spermatozoa, thus completing the present invention. According to the present invention, spermatozoa with high motility and suppression of the acrosome reaction of spermatozoa can be provided. That is, the gist of the present invention is as follows.

[0016] [1] A composition for treating spermatozoa, which contains exosomes.

[0017] [2] The composition for treating spermatozoa according to [1], which is a spermatozoa adjusting solution, a spermatozoa diluting solution, a spermatozoa preserving solution, an artificial insemination solution, an in vitro fertilization solution, a solution for improving spermatozoa motility, or a solution for maintaining spermatozoa fertilization ability.

[0018] [3] The composition for treating spermatozoa according to [1], wherein the exosomes are derived from the culture supernatant of mesenchymal stem cells.

[0019] [4] The sperm processing composition according to [1], wherein the exosomes are isolated exosomes.

[0020] [5] A sperm motility improver comprising exosomes.

[0021] [6] A sperm fertilization ability retainer comprising exosomes.

[0022] [7] A method for improving sperm motility, characterized by culturing sperm in a solution containing exosomes.

[0023] [8] A method for retaining sperm fertilization ability, characterized by culturing sperm in a solution containing exosomes.

[0024] Effects of the Invention

[0025] According to the present invention, sperm with high motility and inhibited acrosome reaction of sperm can be provided. Compared with sperm adjusted by conventional methods, the sperm adjusted by the present invention has high motility and the acrosome reaction of sperm is inhibited, so the success rate of fertilization is significantly high. Description of the Drawings

[0026] Figure 1 A graph showing the motility (linear velocity) of mouse sperm under various conditions.

[0027] Figure 2 A graph showing the motility of mouse sperm under various conditions.

[0028] Figure 3 A graph showing the motility (linear movement) of mouse sperm under various conditions.

[0029] Figure 4 A graph showing the proportion of acrosome marker-positive sperm of mouse sperm under various conditions.

[0030] Figure 5 A graph showing the proportion of acrosome marker-positive sperm of bovine sperm under various conditions.

[0031] Figure 6 A graph showing the time-course change of oxygen consumption of mouse sperm under various conditions.

[0032] Figure 7 A graph showing the fluorescence staining image of mouse sperm treated with fluorescently labeled exosomes.

[0033] Figure 8 A graph showing the fluorescence staining image of mouse sperm treated with fluorescently labeled exosomes (after quenching treatment). Detailed Description of the Invention

[0034] The sperm treatment composition, sperm motility improver, sperm fertilization ability retainer, sperm motility improvement method, and sperm fertilization ability retention method of the present invention will be described in detail below.

[0035] [Composition for sperm treatment]

[0036] The sperm treatment composition of the present invention is characterized by containing exosomes. By containing exosomes, the sperm treatment composition of the present invention can provide sperm with high motility and suppressed acrosome reaction. For sperm whose motility is improved or enhanced and whose acrosome reaction is suppressed by treatment with the sperm treatment composition of the present invention, it is expected to improve the fertilization efficiency and pregnancy rate during artificial insemination and in vitro fertilization, and to promote early development, etc.

[0037] The motility of sperm can be represented by sperm motility parameters. As such motility parameters, for example, the amplitude of sperm head swing (amplitude of head swing; ALH), the number of sperm head swings (beat frequency; BCF), the speed relative to the total distance of sperm movement (curvilinear velocity; VCL), the speed relative to the straight-line distance of sperm movement (straight-line velocity; VSL), etc. can be cited. ALH / VCL (the value obtained by dividing ALH by VCL) represents the degree of motility in the characteristic zigzag movement of sperm. In addition, BCF / VSL (the value obtained by dividing BCF by VSL) represents the degree of motility of sperm in straight-line movement.

[0038] 〈Exosomes〉

[0039] The exosomes in the present invention are vesicles derived from the endosomes of cells and are microvesicles released by cells whose size can be confirmed by electron microscopy. As the specific size of the exosomes, the average particle diameter is 1 nm to 1000 nm, preferably 10 nm to 500 nm, and more preferably 30 nm to 200 nm. Here, the average particle diameter refers to the average value of the diameters of each particle measured by dynamic light scattering method or measurement using an electron microscope. The above exosomes may have a lipid bilayer surrounding biological molecules. In addition, the above exosomes may include, for example, substances called membrane particles, membrane vesicles, microvesicles, nanovesicles, microvesicles (average particle diameter 30 - 1000 nm), exosome-like microvesicles, ectosome-like microcapsules, ectosomes, and / or extracellular vesicles. Exosomes derived from different types of cells can also be distinguished based on their intracellular origin, exosome density in sucrose, shape, sedimentation velocity, lipid composition, protein markers, and secretion mode (i.e., after signal (inductive) or spontaneous (constitutive)). For example, in density gradient centrifugation, exosomes are fractionated into 1.0 - 1.5 g / mL, preferably 1.1 - 1.3 g / mL. In addition, exosomes contain any one of phosphatidylserine, cholesterol, sphingomyelin, and ceramide as their constituent lipids.

[0040] The exosomes in the present invention contain proteins derived from endosomes, proteins involved in intracellular trafficking, proteins derived from various secretory cells represented by proteins derived from cell membranes, RNA, and lipids derived from the cell membranes and endosomal membranes of secretory cells. Examples of proteins derived from endosomes include ESCRTs and TSG101, examples of proteins involved in intracellular trafficking include Rab and GTPase, examples of proteins derived from cell membranes include CD9, CD63, and CD81, and examples of lipids derived from endosomal membranes include cholesterol and sphingomyelin.

[0041] In addition, examples of other proteins and fatty acids contained in the exosomes in the present invention include IL-10, HGF (Hepatocyte Growth Factor), nonanoic acid, lauric acid, myristic acid, pentadecanoic acid, isopentadecanoic acid, palmitic acid, isopalmitic acid, margaric acid, isomargaric acid, stearic acid, isostearic acid, oleic acid, elaidic acid, linoleic acid, nonadecanoic acid, isononadecanoic acid, eicosanoic acid, 11Z-eicosenoic acid, dihomo-γ-linolenic acid, arachidonic acid, erucic acid, 13Z,16Z-docosadienoic acid, 13Z,16Z,19Z-docosatrienoic acid, docosatetraenoic acid, docosapentaenoic acid, etc. Preferably, IL-10, HGF, nonanoic acid, lauric acid, myristic acid, pentadecanoic acid, isopentadecanoic acid, palmitic acid, isopalmitic acid, margaric acid, isomargaric acid, stearic acid, isostearic acid, oleic acid, elaidic acid, linoleic acid, eicosanoic acid, 11Z-eicosenoic acid, arachidonic acid, erucic acid, 13Z,16Z-docosadienoic acid are listed. More preferably, IL-10, HGF, myristic acid, pentadecanoic acid, palmitic acid, margaric acid, isomargaric acid, stearic acid, oleic acid, linoleic acid, nonadecanoic acid, erucic acid, 13Z,16Z-docosadienoic acid are listed. Particularly preferably, IL-10, HGF, palmitic acid, margaric acid, stearic acid, oleic acid, 13Z,16Z-docosadienoic acid, etc. are listed, and the exosomes in the present invention preferably contain these.

[0042] In the present invention, palmitic acid refers to a saturated fatty acid having a carbon number and double bond number of 16:0 as represented by C 16 H 32 O2. In the present invention, stearic acid refers to a saturated fatty acid having a carbon number and double bond number of 18:0 as represented by C 18 H 36 O2. In the present invention, oleic acid is one of the unsaturated fatty acids represented by C 18 H 34 O2 and is a cis-monounsaturated fatty acid.

[0043] The exosomes in the present invention can be those contained in cells, those contained in cell culture supernatants, or exosomes isolated from cells or cell culture supernatants. Examples of the method for isolating exosomes include ultracentrifugation, microfiltration, capture using antibodies, and use of microfluidic systems. The isolated exosomes can contain cells such as mesenchymal stromal cells (mesenchymal stem cells), and can also contain culture media such as media for mesenchymal stromal cells (mesenchymal stem cells).

[0044] There is no particular limitation on the source of the exosomes in the present invention. They can be cells of mammals such as humans, horses, cows, goats, sheep, pigs, dogs, cats, rabbits, mice, rats, etc., or they can be microorganisms or plants such as rice. They can be recovered from the culture supernatant obtained by culturing mesenchymal stem cells, or from plants or microorganisms such as rice containing exosomes. In addition, the exosomes in the present invention are not limited to exosomes produced by organisms and can be artificial vesicles such as liposomes.

[0045] When the source of the exosomes in the present invention is the above-mentioned mammals, examples of the source cells include, for example, mesenchymal stem cells, liver-derived cells, fibroblasts, epithelial cells, myoblasts, pluripotent stem cells, and plant stem cells. Among them, mesenchymal stem cells and pluripotent stem cells are preferred, and mesenchymal stem cells are more preferred.

[0046] The case where the source of the exosomes in the present invention is mesenchymal stem cells will be described below.

[0047] When the above cells are mesenchymal stem cells, the exosomes contained in the sperm treatment composition of the present invention can be recovered from the culture supernatant obtained by culturing mesenchymal stem cells. For example, mesenchymal stem cells are allowed to form a sub-confluent or confluent state in a culture vessel, and after changing to a new culture medium, they are further cultured for 1 to 5 days, and the culture supernatant is recovered. Exosomes can be obtained by separating the culture supernatant of mesenchymal stem cells using ultracentrifugation separation, density gradient centrifugation, various exosome isolation kits, etc.

[0048] In the sperm treatment composition of the present invention, in addition to containing the above exosomes, it can also contain mesenchymal stem cells themselves that contain the above exosomes or have the ability to secrete the above exosomes. It should be noted that when the sperm treatment composition contains mesenchymal stem cells that contain the above exosomes or have the ability to secrete the above exosomes, it can also be interpreted that by containing the above mesenchymal stem cells, the requirement of containing exosomes as described above is of course satisfied at the same time. As the exosomes contained in the sperm treatment composition of the present invention, exosomes isolated from the culture supernatant of mesenchymal stem cells and the like are preferred.

[0049] (mesenchymal stem cells)

[0050] In the present invention, mesenchymal stem cells refer to cells that have the ability to differentiate into one or more cells belonging to mesenchyme (osteocytes, cardiomyocytes, chondrocytes, tendon cells, adipocytes, etc.) and can proliferate while maintaining this ability. The term "mesenchymal stem cells" used in the present invention refers to the same cells as mesenchymal stromal cells, and no special distinction is made between the two. Additionally, it is sometimes abbreviated as "mesenchymal cells". Examples of tissues containing mesenchymal stem cells include, for example: adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amnion, chorion, decidua, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, dental pulp, tooth germ, etc. Examples of mesenchymal stem cells in the present invention include mesenchymal stem cells derived from adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amnion, chorion, decidua, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, dental pulp, tooth germ, etc. Among them, mesenchymal stem cells derived from adipose tissue, umbilical cord, and bone marrow are preferred, and mesenchymal stem cells derived from adipose tissue and umbilical cord are more preferred.

[0051] Examples of the species of mesenchymal stem cells in the present invention include humans, horses, cows, goats, sheep, pigs, dogs, cats, rabbits, mice, and rats. The mesenchymal stem cells in the present invention and the treatment object (subject) can be derived from the same species or from different species.

[0052] Mesenchymal stem cells can be, for example, cells provided by companies such as PromoCell, Lonza, Biological Industries, Veritas, R&D Systems, and Corning, or cells prepared by those skilled in the art through well-known methods. Additionally, mesenchymal stem cells can be primary cells isolated from donor tissues or cell line-derived cells.

[0053] In the present invention, the medium for culturing mesenchymal stem cells is not particularly limited as long as it can culture mesenchymal stem cells while maintaining a good state. For example, a medium that can enable human mesenchymal stem cells to proliferate while maintaining the ability to differentiate into osteocytes, chondrocytes, and adipocytes is preferred.

[0054] The culture medium used in the present invention can be prepared by adding one or more serum substitutes such as serum, and / or albumin, transferrin, fatty acids, insulin, sodium selenite, cholesterol, collagen precursors, trace elements, 2-mercaptoethanol, 3'-mercapto glycerol, etc. to a basal medium. In addition, in these culture media, amino acids such as glutamine, sugars such as glucose, metal salts such as sodium chloride and magnesium sulfate, trace metals such as selenium, lipids such as cholesterol and unsaturated fatty acids, vitamins such as pantothenic acid, albumin, insulin, transferrin, growth factors, proliferation factors, cytokines and other proteins, polysaccharides, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts and other substances can be further added as needed.

[0055] Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's modified basal medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, MCDB201 medium, and mixed media thereof.

[0056] From the perspective of sperm processing, the medium used for culturing mesenchymal stem cells in the present invention is preferably a medium (free of heterologous substances) that does not contain heterologous components such as serum. As such a medium, for example, Mesenchymal Stem Cell Growth Medium 2 (Ready-to-use, manufactured by PromoCell), Mesenchymal Stem Cell Growth Medium XF (Ready-to-use, manufactured by PromoCell), MSCGM BulletKit tm, MSCGM tm Mesenchymal Stem Cell Growth Medium BulletKit tm (manufactured by Lonza), Serum-Free Medium for Human Mesenchymal Stem Cells (MSC NutriStem (registered trademark) XF, manufactured by Biological Industries), MesenCult-ACF Plus (manufactured by Veritas), StemXVivo tm Serum-Free Human MSC Expansion Media (manufactured by R&D Systems, Corning), Serum-Free Medium for Adipose-Derived Stem Cells (KBM ADSC-4, manufactured by Kohjin-bio), and Serum-Free Medium for Mesenchymal Stem Cells (R: STEM Medium for hMSC High Growth, manufactured by Rohto) can be cited as media provided in advance for the use of mesenchymal stem cells (mesenchymal stromal cells).

[0057] Examples of the above-mentioned serum include, but are not limited to, human serum, fetal bovine serum (FBS), bovine serum, calf serum, goat serum, horse serum, pig serum, sheep serum, rabbit serum, rat serum, etc. When using serum, it can be added at 5 v / v% to 15 v / v%, preferably 10 v / v%, relative to the basal medium.

[0058] (Preparation of mesenchymal stem cell culture supernatant)

[0059] The supernatant of mesenchymal stem cells obtained by the following method can be used as the mesenchymal stem cell culture supernatant in the present invention. In addition, substances obtained by removing unnecessary components from the supernatant by means such as dialysis and ultrafiltration, fractions obtained by fractionating the supernatant using a column, etc., fractions selected using antibodies against specific molecules, fractions obtained by centrifugation, etc. can also be used as the mesenchymal stem cell culture supernatant in the present invention.

[0060] As the medium used for obtaining the culture supernatant, the same medium as that used for culturing mesenchymal stem cells can be used. The method for obtaining the culture supernatant is not particularly limited as long as it is suitable for culturing each mesenchymal stem cell. For example, it is a method of culturing mesenchymal stem cells at a temperature of 20°C to 37°C, in an environment of 2% to 7% CO2 and 5% to 21% O2, preferably at room temperature to 37°C and in an environment of 5% CO2, and obtaining the culture supernatant thereof.

[0061] For the culture supernatant of the mesenchymal stem cells of the present invention, as long as the mesenchymal stem cells are in contact with the medium, the washing solution obtained by washing the mesenchymal stem cells with the medium can also be used as the culture supernatant in the present invention. The contact time between the mesenchymal stem cells and the medium is, for example, within 14 days, preferably within 10 days, more preferably within 7 days, and even more preferably within 5 days. Specifically, it is preferred to, for example, form a sub-confluent or confluent state of the mesenchymal stem cells in a culture container, replace it with a new medium, and further culture for 1 to 5 days, and recover the culture supernatant thereof. The culture for obtaining the culture supernatant can be monolayer culture attached to a flask or suspension and agitation culture attached to microbeads or the like.

[0062] (Isolation of exosomes)

[0063] When isolating exosomes, the culture supernatant of the mesenchymal stem cells recovered by the above method can be obtained by known methods such as ultracentrifugation separation method, density gradient centrifugation method, affinity purification method, size exclusion chromatography method, and various exosome isolation kits. The method for isolating exosomes is preferably the ultracentrifugation separation method, affinity purification method, or size exclusion chromatography method.

[0064] The amount of exosomes contained in the sperm treatment composition of the present invention, based on the exosome concentration in the sperm treatment composition, is 0.001 pg / mL or more and 100 μg / mL, preferably 0.005 pg / mL or more and 100 μg / mL or less, more preferably 0.01 pg / mL or more and 10 μg / mL or less, and particularly preferably 0.1 pg / mL or more and 100 pg / mL or less. By setting the amount of exosomes contained in the sperm treatment composition of the present invention within the above numerical range, the sperm treatment composition can inhibit damage to sperm and exhibit excellent effects of improving sperm motility and inhibiting the acrosome reaction of sperm.

[0065] In addition to containing exosomes, the sperm treatment composition of the present invention may contain other components within the scope that does not hinder the effects of the present invention. Examples of other components include protective agents such as dimethyl sulfoxide (DMSO) and albumin, antibiotics, sugars, amino acids, vitamins, carriers, excipients, disintegrants, buffers, emulsifiers, stabilizers, preservatives, antiseptics, physiological saline, etc. In addition, buffers sold as buffers for eggs and sperm (washing solutions) etc. can also be used. Examples of such buffers include HTF medium (manufactured by Fujifilm Wako Pure Chemical Corporation) containing NaCl, KCl, KH2PO4, MgSO4·7H2O, CaCl2·2H2O, NaHCO3, glucose, sodium pyruvate, sodium lactate, phenol red, Hepes, gentamicin sulfate, etc.

[0066] The species of sperm that is the subject of the sperm treatment composition of the present invention may be any mammalian species, and examples include humans, horses, cows, goats, sheep, pigs, dogs, cats, rabbits, mice, rats, rare animals, etc.

[0067] The sperm treatment composition of the present invention can be used as a sperm conditioning solution, sperm diluent, sperm preservation solution, artificial insemination solution, in vitro fertilization solution, sperm motility improvement solution, sperm fertilization ability maintenance solution, etc.

[0068] A sperm conditioning solution refers to a solution used to wash sperm and then suspend it in order to recover sperm showing good motility when preparing sperm for purposes such as artificial insemination and in vitro fertilization. A sperm diluent is a solution used to dilute sperm to an appropriate concentration and inject it into the uterus during artificial insemination. A sperm preservation solution is a solution that can be used for refrigerated or frozen preservation of sperm. An artificial insemination solution is a solution used to increase the fertilization rate during artificial insemination. An in vitro fertilization solution is a solution used to increase the fertilization rate during in vitro fertilization. A sperm motility improvement solution is a solution used to improve the motility of sperm. A sperm fertilization ability maintenance solution is a solution used to maintain the fertilization ability of sperm.

[0069] The sperm treatment composition of the present invention can be prepared by mixing the necessary other components into the above-mentioned exosomes by conventional methods.

[0070] [Sperm motility improver, sperm fertilization ability maintainer]

[0071] The sperm motility improver and sperm fertilization ability maintainer of the present invention are characterized by containing exosomes. By containing exosomes, the sperm motility improver and fertilization ability maintainer of the present invention can provide sperm with improved motility and inhibited acrosome reaction. Sperm treated with the sperm motility improver or sperm fertilization ability maintainer of the present invention is expected to improve the fertilization efficiency in, for example, artificial insemination and in vitro fertilization, increase the conception rate, and promote early development, etc.

[0072] The sperm motility improver and sperm fertilization ability maintainer of the present invention are substantially the same as the above-mentioned sperm treatment composition. Therefore, for specific descriptions, the sperm treatment composition can also be replaced with the sperm motility improver or sperm fertilization ability maintainer and the descriptions in the sperm treatment composition item can be directly applied.

[0073] As a method for treating sperm with the sperm motility improver or fertilization ability maintainer of the present invention, a state in which the sperm motility improver or sperm fertilization ability maintainer comes into contact with sperm is formed in vivo or in vitro. In the case of in vivo, it is considered that the sperm motility improver or fertilization ability maintainer can be administered to the seminal vesicle, which is the sperm storage organ. As long as the state in which the sperm motility improver or sperm fertilization ability maintainer of the present invention comes into contact with sperm is formed after administration, the administration site and administration method are not particularly limited. On the other hand, in order to form a state in which the sperm motility improver or sperm fertilization ability maintainer of the present invention comes into contact with sperm in vitro, for example, the pre-prepared sperm can be mixed with the sperm motility improver or sperm fertilization ability maintainer of the present invention in an appropriate container in vitro and treated for a certain period of time. The treatment temperature is 4°C to 40°C, preferably 15°C to 40°C, more preferably 25°C to 39°C, and further preferably a temperature close to body temperature, that is, 36°C to 38°C. The treatment time is 10 minutes to 48 hours, preferably 20 minutes to 24 hours, more preferably 30 minutes to 4 hours, and further preferably 1 hour to 2 hours.

[0074] Sperm treated with the sperm motility improver or sperm fertilization ability maintainer of the present invention can be used for artificial insemination and in vitro fertilization (to improve success rate and efficiency), livestock breeding (to improve the success rate and efficiency of artificial insemination), breeding and species maintenance (such as the maintenance of endangered species, the maintenance of pet strains or hybridization), the treatment and improvement of various diseases accompanied by sperm disorders or with sperm disorders as the main or secondary cause (such as varicocele, male infertility, cryptorchidism, sperm disorders after X-ray irradiation, after malignant tumor surgery or chemotherapy), sperm preservation, improvement of motility, maintenance of fertilization ability and preservation of immature sperm, etc.

[0075] When artificial insemination is carried out using sperm treated with the sperm motility improver or fertilization ability maintainer of the present invention (including artificial insemination for livestock breeding, breeding, and species maintenance), a state is formed in which sperm with improved motility and / or maintained fertilization ability coexist with eggs (egg cells, ova) in vivo or in vitro. To form a coexistence state in vivo, sperm with improved motility and / or maintained fertilization ability are given (transplanted) to the biological site where the eggs are located. For example, in the case of mammals, sperm treated with this agent are injected into the uterus. On the other hand, if a coexistence state is formed in vitro, eggs prepared by separating from a living organism, receiving from a cell bank, etc. and sperm treated with this agent are placed in the same container and incubated, or sperm with improved motility and / or maintained fertilization ability are inserted into the eggs, etc. using a micromanipulator, etc. Except for the characteristic conditions of the present invention, that is, using sperm treated with the sperm motility improver or sperm fertilization ability maintainer of the present invention, it can be carried out according to conventional methods.

[0076] [Method for improving sperm motility, method for maintaining sperm fertilization ability]

[0077] The present invention also includes: a method for improving sperm motility and a method for maintaining sperm fertilization ability, characterized in that sperm are cultured in a solution containing exosomes. The method for improving sperm motility and the method for maintaining sperm fertilization ability of the present invention can improve and / or enhance the motility of sperm or maintain the fertilization ability of sperm while maintaining the state of the acrosome by culturing sperm in a solution containing exosomes. Sperm with improved motility and inhibited acrosome reaction by the method for improving sperm motility or the method for maintaining sperm fertilization ability of the present invention can be expected to improve the fertilization efficiency and pregnancy rate during artificial insemination and in vitro fertilization, and promote early development, etc.

[0078] The method for improving sperm motility and the method for maintaining sperm fertilization ability of the present invention are methods for treating sperm using the above-mentioned sperm motility improver or sperm fertilization ability maintainer of the present invention, so specific descriptions can refer to the items of sperm motility improver and sperm fertilization ability maintainer.

[0079] Examples

[0080] The following examples and test examples are listed to illustrate the present invention in detail, but the present invention is not limited by these examples, etc.

[0081] [Example 1: Preparation 1 of exosomes]

[0082] Human umbilical cord mesenchymal stem cells (C-12971 Human Mesenchymal Stem Cells from Umbilical Cord Matrix (hMSC-UC), manufactured by Promocell) were cultured in RIM medium (serum-free medium for mesenchymal stem cells, containing EGF, bFGF, albumin, transferrin, and insulin, manufactured by Rohto) for 3 days to obtain a culture supernatant (hereinafter referred to as "3D RIM culture supernatant"). In addition, the medium was changed later, and the cells were cultured in RIM medium for 1 more day to obtain a culture supernatant (hereinafter referred to as "1D RIM culture supernatant"). Exosomes (hereinafter referred to as "1D exosomes" and "3D exosomes") were obtained from the 1D RIM culture supernatant and the 3D RIM culture supernatant, respectively, using ExoTrap Exosome Isolation Spin Column.

[0083] [Example 2: Preparation of Exosomes 2]

[0084] Human adipose-derived stem cells (PT-5006 HADSC-Human Adipose-Derived Stem Cells, manufactured by LONZA) were cultured in RIM medium (serum-free medium for mesenchymal stem cells, containing EGF, bFGF, albumin, transferrin, and insulin, manufactured by Rohto) for 3 days to obtain a culture supernatant (hereinafter referred to as "ADSC culture supernatant"). Exosomes (hereinafter referred to as "ADSC exosomes") were obtained using ExoTrap Exosome Isolation Spin Column.

[0085] [Example 3: Lipidome Analysis]

[0086] In the same manner as in Example 1, human umbilical cord mesenchymal stem cells (C-12971 Human Mesenchymal Stem Cells from Umbilical Cord Matrix (hMSC-UC), manufactured by Promocell) were cultured in RIM medium (serum-free medium for mesenchymal stem cells, containing EGF, bFGF, albumin, transferrin, and insulin, manufactured by Rohto) for 1 day or 3 days to obtain culture supernatants (hereinafter referred to as "1D RIM culture supernatant" and "3D RIM culture supernatant", respectively). Exosomes (hereinafter referred to as "1D exosomes" and "3D exosomes") were obtained from the 1D RIM culture supernatant and the 3D RIM culture supernatant, respectively, using ExoTrap Exosome Isolation Spin Column. In addition, the fatty acids contained in these exosomes were measured by GC-MS. The results are shown in Table 1.

[0087] [Table 1]

[0088] 1D exosome 3D exosome caproic acid 1.29 0.48 palmitic acid 6.15 4.08 stearic acid 4.13 1.84

[0089] [Example 4: Sperm motility]

[0090] In a 37 °C water bath, mouse sperm (C57 / BL6, 12 weeks old) was suspended in HTF medium (manufactured by Fujifilm Wako Pure Chemical Corporation) supplemented with 3D exosomes at various concentrations (3D exosome concentrations: 0.6 pg / mL, 6.0 pg / mL, 60 pg / mL), HTF medium without exosomes as a negative control (control), and ADSC culture supernatant (containing 60 pg / mL exosomes) as samples. Then, the samples were cultured at 37 °C for 60 minutes. Next, 3 μL of each sample was analyzed using a sperm motility analysis device (Computer Assisted Sperm Analysis (CASA) system). The straight-line velocity (VSL), linearity (LIN), and motility (sperm motility) of all observed sperm were analyzed. The median value at 60 minutes of incubation for the control was set to 100, and the median value of VSL of the medium supplemented with 3D exosomes at 60 minutes of incubation ( Figure 1 ), the average values of sperm motility and LIN are shown in Figure 2 and Figure 3 .

[0091] As Figures 1 to 3 shown, it was confirmed that by adding exosomes, the median value increased and sperm motility increased. It was also confirmed that the separated exosomes had an excellent effect of improving sperm motility compared to the ADSC culture supernatant containing an equal amount of exosomes.

[0092] [Example 5: Inhibition of sperm acrosome reaction]

[0093] Sperm were recovered from the epididymis of male mice (C57 / BL6, 12 weeks old) and suspended in HTF medium (manufactured by Fujifilm Wako Pure Chemical Corporation) supplemented with 60 pg / mL of 3D exosomes and HTF medium without exosomes as a negative control. After culturing for 60 minutes, the proportion of sperm maintaining the acrosome was analyzed by counting the number of positive sperm visually. The proportion of positive sperm maintaining the acrosome is shown in Figure 4 .

[0094] As Figure 4 shown, it was confirmed that the addition of exosomes inhibited the sperm acrosome reaction.

[0095] [Example 6: Motility of bovine spermatozoa]

[0096] Frozen bovine semen (purchased from the Livestock Improvement Corporation, a general incorporated association) was thawed by heating in a water bath at 37°C for 15 seconds and then washed twice with HTF medium (manufactured by Fujifilm Wako Pure Chemical Corporation). Spermatozoa were suspended in HTF medium (manufactured by Fujifilm Wako Pure Chemical Corporation) supplemented with 60 pg / mL of 3D exosomes and HTF medium without exosomes as a negative control. After culturing for 60 minutes, the proportion of spermatozoa that retained the acrosome was analyzed by counting the number of positive spermatozoa visually. The proportion of positive spermatozoa that retained the acrosome is shown in Figure 5 .

[0097] As Figure 5 shown, it was confirmed that the acrosome reaction of bovine spermatozoa was also inhibited by adding exosomes.

[0098] [Example 7: Aerobic respiration]

[0099] Spermatozoa were recovered from the epididymides of male mice (C57 / BL6, 12 weeks old) and suspended in HTF medium (manufactured by Fujifilm Wako Pure Chemical Corporation) for analysis using a flux analyzer for real-time measurement of cellular oxygen consumption. Six minutes after the start of culture, 20 μL of HTF medium containing 60 pg / mL of 3D exosomes or HTF medium containing stearic acid (8 ng) / palmitic acid (36 ng) as saturated fatty acids was added, and then the change in oxygen consumption of spermatozoa was studied every 6 minutes for a total of 60 minutes. The first measured value of the control was set to 100%, and the time-course change in its oxygen consumption is shown in Figure 6 .

[0100] As Figure 6 shown, it was confirmed that the aerobic respiration of spermatozoa was continuously promoted by adding exosomes.

[0101] [Example 8: Sperm staining]

[0102] Sperm were recovered from the epididymis of male mice (C57 / BL6, 12 weeks old), and the sperm were suspended in HTF medium (manufactured by Fujifilm Wako Pure Chemical Corporation) supplemented with D3 exosomes fluorescently labeled using the Exosparkler Exosome Membrane Labeling Kit (Dojindo), and cultured at 37°C for 30 minutes (Exosome). In addition, in order to identify the fluorescent label taken up into the cells, a quenching treatment to inactivate the fluorescent label outside the cells was performed (cultured with 0.4% trypan blue solution for 5 minutes) (Exosome after quenching). The above-stained sperm were smeared onto a glass slide by Cytospin and observed using a fluorescence microscope. The fluorescent staining images are shown in Figure 7 and 8 .

[0103] As Figure 7 and 8 shown, it was confirmed that the stained exosomes were taken up by sperm and membrane fusion occurred.

[0104] Industrial Applicability

[0105] According to the present invention, sperm with high motility and suppressed acrosome reaction of sperm can be provided. Compared with sperm adjusted by other methods, the sperm adjusted by the present invention have high motility and the acrosome reaction of sperm is suppressed, so the success rate of fertilization is significantly high.

Claims

1. A composition for sperm treatment, which comprises exosomes.

2. The composition for sperm treatment according to claim 1, which is a sperm conditioning solution, a sperm diluent, a sperm preservation solution, an artificial insemination solution, an in vitro fertilization solution, a solution for improving sperm motility or a solution for maintaining sperm fertilization ability.

3. The composition for sperm treatment according to claim 1, wherein, Exosomes are derived from the culture supernatant of mesenchymal stem cells.

4. The composition for sperm treatment according to claim 1, wherein, The exosomes are isolated exosomes.

5. A sperm motility improver, which comprises exosomes.

6. A sperm fertilization ability maintainer, which comprises exosomes.

7. A method for improving sperm motility, which is characterized in that, Sperm are cultured in a solution containing exosomes.

8. A method for maintaining sperm fertilization ability, which is characterized in that, Sperm are cultured in a solution containing exosomes.

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