Sperm motility improver and sperm motility improvement method

Abstract

[Problem] To provide a sperm motility improving agent that is highly safe and that can effectively improve sperm motility. [Solution] A sperm motility improving agent containing a nicotinamide mononucleotide as an active ingredient.

Description

[Technical Field]

[0001] The present invention relates to a sperm motility improver and a method for improving sperm motility. [Background technology]

[0002] Fertilization is the process in which a sperm, a male germ cell, fuses with an egg, a female germ cell, and the nuclei of both cells combine to form a single cell (zygote). In many mammals, fertilization occurs in the ampulla of the fallopian tube. When an egg is released from the ovary, it is drawn into the fimbria of the fallopian tube and moves to the ampulla of the fallopian tube. On the other hand, after being ejaculated, sperm travel through the female reproductive tract, including the uterus and fallopian tubes, before reaching the ampulla of the fallopian tube.

[0003] For sperm to fertilize an egg, they must move within the reproductive tract in search of the egg, so sperm motility is one of the important factors in determining whether or not fertilization occurs.

[0004] Sperm are generally divided into three parts: the head, neck, and tail. The tail is an extremely long and developed single flagellum (cilia), within which microtubules are accumulated in a unique arrangement. Mitochondria are wrapped around the midsection, where the tail begins, and supply ATP, the energy for sperm motility. Sperm motility is generated by consuming ATP produced by the mitochondria and shaking the tail. Specifically, this movement is thought to be caused by a structural change in the axoneme within the flagellum, where dynein, a molecular motor, consumes ATP as energy, causing microtubule sliding motion, which is then converted into flagellar beating motion. During fertilization, sperm receive various external factors from their surroundings, and dynein activity is regulated based on these factors. The flagellum is composed of peripheral microtubules, dynein, radial spokes, and the nexin-dynein regulatory complex (N-DRC). Improper formation of these components can result in abnormal flagellum formation and impaired motility.

[0005] As can be seen from the above explanation, sperm motility is an important factor in determining whether fertilization will occur. Semen contains a mixture of sperm of various qualitative levels, and the condition of these sperm is evaluated by motility in addition to sperm concentration and morphology. A condition in which sperm motility is reduced is called asthenozoospermia, and it is one of the major causes of infertility or reduced fertility due to male factors. In humans, semen analysis is performed according to the WHO standard manual, and asthenozoospermia is generally diagnosed when fewer than 50% of sperm are progressively motile and fewer than 25% are rapidly moving sperm.

[0006] In Japan, the number of couples struggling with infertility is increasing; it is said that one in every 5.5 couples suffers from infertility, and it is estimated that approximately 500,000 people are undergoing some form of infertility treatment. It has also become clear that approximately 80% of infertility caused by men is due to asthenozoospermia. The cause of asthenozoospermia has been pointed out as being due to the influence of harmful substances such as environmental hormones, but the exact cause is not yet clear.

[0007] Infertility treatments include drug therapy such as ovulation inducers, fallopian tube ventilation for tubal canalization disorders, salpingoplasty, vasectomy for vas deferens dysfunction, artificial insemination, and in vitro fertilization. Infertility treatments vary depending on the patient's age and illness, and patients undergoing infertility treatment suffer from physical pain, mental depression, and financial burdens. Despite the availability of these infertility treatments, the birth rate resulting from infertility treatment in Japan remains very low.

[0008] On the other hand, in livestock, asthenozoospermia is generally diagnosed when 50% or fewer of the sperm show active forward movement at the time of semen collection. When asthenozoospermia is diagnosed, causes such as testicular atrophy, inflammation of the testes, accessory sex glands, or urethra, or urine contamination are suspected, and treatment involves procedures to eliminate the cause, but the prognosis is generally poor. Cattle breeding is carried out using artificial insemination using frozen preserved sperm from superior breeding bulls (elite bulls), but it has been known that the conception rate (the percentage of cattle that become pregnant among those that have been artificially inseminated) has been decreasing year by year. As such, infertility is a major problem in the livestock world as well.

[0009] In view of this situation, various agents and methods have been developed to improve sperm motility, which is one of the causes of male infertility. For example, one example has been reported in which a method for improving sperm motility and / or sperm production capacity in a mammal other than human is administered to the mammal other than human with reduced sperm motility and / or sperm production capacity by enzymatically treating subcutaneous adipose tissue collected from the mammal other than human (Patent Document 1). A method for improving sperm motility and / or sperm production capacity in a mammal other than human is administered to the mammal other than human with reduced sperm motility and / or sperm production capacity by enzymatically treating subcutaneous adipose tissue collected from the mammal other than human (A). Method for improving sperm motility and / or sperm production in mammals other than humans

[0010] Other reported methods include a pharmaceutical composition for increasing sperm motility, which contains extracts of oysters and ginseng as active ingredients (Patent Document 2), a sperm motility improver consisting of a gas containing 1 (v / v)% or more hydrogen molecules (Patent Document 3), a sperm activator containing protamine (Patent Document 4), and a method for promoting sperm motility ex vivo, which involves adding a pharmaceutical composition containing a glycoprotein-containing high molecular weight protein with a molecular mass of 66 kDa that activates sperm motility and a pharmaceutically acceptable excipient to semen ex vivo, thereby improving sperm motility related to the treatment of human infertility and livestock breeding (Patent Document 5). [Prior art documents] [Patent documents]

[0011] [Patent Document 1] Japanese Patent Application Laid-Open No. 2017-25038 [Patent Document 2] Japanese Patent Application Laid-Open No. 2004-83517 [Patent Document 3] Re-tabled publication No. 2015-64109 [Patent Document 4] Japanese Patent Application Laid-Open No. 2010-6785 [Patent Document 5] Japanese Patent Application Publication No. 11-279075 Summary of the Invention [Problem to be solved by the invention]

[0012] An object of the present invention is to provide a sperm motility improver and a sperm motility improving method that are highly safe and can effectively improve sperm motility. [Means for solving the problem]

[0013] As a result of intensive research to solve the above problems, the present inventors discovered that nicotinamide mononucleotide, an intermediate metabolite involved in the biosynthesis of the coenzyme NAD (nicotinamide adenine dinucleotide), has an excellent effect of improving sperm motility, and thus completed the present invention.

[0014] The present invention is as follows. [1] A sperm motility improver containing nicotinamide mononucleotide as an active ingredient. [2] The sperm motility improver according to [1] above, further comprising pyrroloquinoline quinone. [3] The sperm motility improver according to [1] or [2], wherein the sperm are mammalian sperm. [4] Sperm concentration is 5×10 6 ~50×10 6The sperm motility improver according to any one of [1] to [3] above, wherein the nicotinamide mononucleotide is added to a semen concentration of 0.05 to 150 μM per 1 ml of semen. [5] Sperm concentration is 5×10 6 ~50×10 6 The sperm motility improver according to any one of [2] to [4] above, wherein the pyrroloquinoline quinone nicotinamide is added to a concentration of 20 to 200 nM per ml of semen. [6] The sperm motility improver according to any one of [2] to [5] above, wherein the molar ratio of the nicotinamide mononucleotide to the pyrroloquinoline quinone is 1:1 to 1500:1. [7] The sperm motility improver according to any one of [1] to [6] above, which is a pharmaceutical product for improving sperm motility. [8] The sperm motility improver according to any one of [1] to [6] above, which is a food or drink for improving sperm motility. [9] A method for improving sperm motility, comprising contacting nicotinamide mononucleotide with sperm in vitro or in vivo.

[10] The method according to [9], further comprising contacting pyrroloquinoline quinone with sperm in vitro or in vivo.

[11] The method according to [9] or

[10] above, wherein the sperm are mammalian sperm.

[12] Sperm concentration is 5×10 6 ~50×10 6 The method according to any one of [9] to

[11] above, comprising adding the nicotinamide mononucleotide to 1 / ml of semen so that the concentration of the nicotinamide mononucleotide is 0.05 to 150 μM.

[13] Sperm concentration is 5×10 6 ~50×10 6 The method according to any one of

[10] to

[12] above, comprising adding the pyrroloquinoline quinone nicotinamide to 1 / ml of semen so that the concentration of the pyrroloquinoline quinone nicotinamide is 20 to 200 nM.

[14] Sperm treated by the method described in any one of [9] to

[13] above. [Effects of the Invention]

[0015] The present invention can effectively improve sperm motility and increase NAD in vivo. + Since the active ingredient is nicotinamide mononucleotide, an intermediate metabolite involved in the biosynthesis of nicotinamide, it is highly safe and can be used with confidence. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 is an explanatory diagram showing the metabolic pathway involved in niacin (a collective term for nicotinamide and nicotinic acid). [Figure 2a] 1 is a graph showing the forward motility rate (%) of pig sperm when cultured with NMN alone or with the addition of NMN+PQQ. [Figure 2b] 1 is a graph showing the total motility rate (%) of pig sperm when cultured with NMN alone or with the addition of NMN+PQQ. [Figure 2c] 1 is a graph showing the rectilinear velocity (μM / s) of boar sperm when cultured with NMN alone or with the addition of NMN+PQQ. [Figure 3] 10 is a photograph showing the trajectory of sperm movement during porcine sperm culture in the presence of NMN alone or NMN+PQQ. DETAILED DESCRIPTION OF THE INVENTION

[0017] The sperm motility improver of the present invention contains nicotinamide mononucleotide as an active ingredient and exhibits the effect of improving sperm motility. As shown in the examples described below, the present invention is particularly effective in increasing the linear motility rate and linear velocity of sperm, which are essential for sperm to move upward in the reproductive tract. In addition, in the present invention, improving sperm motility does not only mean improving sperm motility in the narrow sense, but also includes preventing, stopping, or delaying the progression of decreased sperm motility.

[0018] The detailed mechanism by which the use of nicotinamide mononucleotide as an active ingredient improves sperm motility is currently under investigation, but is presumed to be as follows: Adding nicotinamide mononucleotide to semen increases the amount of NAD / NADH in sperm, activates the electron transport chain, and increases the ATP concentration in mitochondria. ATP serves as an energy source for sperm motility, causing microtubule sliding movement due to structural changes in dynein within the axoneme, which is then converted into flagellar movement, enhancing sperm motility. The present invention will be described in detail below.

[0019] Nicotinamide mononucleotide (chemical formula: C 11 H 15 N2O8P) is a compound represented by the following structural formula [Chemical Formula 1], which is produced in the bodies of many living organisms, including humans. It is commonly called NMN (nicotinamide mononucleotide) and is a coenzyme NAD + It is known as an intermediate metabolite involved in the biosynthesis of

[0020] [ka]

[0021] Nicotinamide mononucleotide, the active ingredient of the sperm motility improver, is produced in vivo through the NAD metabolic pathway in liver tissue, i.e., the pathway involved in the synthesis of nicotinamide adenine dinucleotide (NAD) from quinolinic acid via the kynurenine pathway. This point will be explained in detail with reference to Figure 1. Figure 1 is an explanatory diagram showing the metabolic pathway involved in niacin (a collective term for nicotinamide and nicotinic acid), also known as vitamin B3. Nicotinic acid ingested through diet is taken up by the liver and converted to nicotinamide, which is then distributed throughout the body via the bloodstream. Each cell takes up nicotinamide from the blood and converts it to NAD and NADP for use. Nicotinamide can also be biosynthesized from tryptophan.

[0022] As shown in Figure 1, in vivo, when tryptophan is used as a starting material, tryptophan is converted to quinolinic acid (QA) via the kynurenine pathway, which is the tryptophan metabolic pathway, and then to nicotinic acid mononucleotide (NaMN). On the other hand, when nicotinic acid (Na) is used as a starting material, nicotinic acid is directly converted to NaMN. NaMN then passes through nicotinic acid adenine dinucleotide (NaAD) and is interconverted to NAD, nicotinamide (NaM), and nicotinamide mononucleotide via the NAD cycle. Nicotinamide (NaM) is converted to nicotinamide mononucleotide by nicotinamide phosphoribosyltransferase (NAMPT), and nicotinamide mononucleotide is then converted to NAD by nicotinamide mononucleotide adenyltransferase (NMNAT). Nicotinamide mononucleotide can also be produced from nicotinamide riboside (NR), an NAD intermediate.

[0023] Nicotinamide mononucleotide exists as two optical isomers, α- and β-, and the β-isomer is used in the present invention. Nicotinamide mononucleotide can be obtained, for example, by synthesizing nicotinamide riboside from nicotinamide and ribose (see Bioorg. Med. Chem. Lett., 12, 1135-1137 (2002)), followed by phosphorylating the 5-hydroxyl group of the ribose moiety (see Chem. Comm., 1999, 729-730). Specifically, for example, nicotinamide and L-ribose tetraacetate are first dissolved in anhydrous acetonitrile, and an excess amount of trimethylsilyltrifluorosulfonic acid is added under a nitrogen stream. The mixture is stirred at room temperature, and the reaction is terminated by adding methanol. The reaction solution is then loaded onto a column packed with activated carbon, washed with distilled water, and eluted with methanol to recover the product. Next, to phosphorylate the 5-hydroxyl group of the L-ribose moiety of this product, the product is dissolved in trimethoxyphosphate, and phosphorus oxychloride is added dropwise under ice cooling. The mixture is stirred under a nitrogen stream, and aqueous sodium hydroxide is added to neutralize the reaction. To the reaction solution, which has been stopped, a cold acetonitrile-ether solution is added. The lower layer (aqueous phase) is then passed through an anion exchange resin to recover the reaction product, which is then further purified with a cation exchange resin to recover highly pure nicotinamide mononucleotide. Nicotinamide mononucleotide is also commercially available, and these commercially available products can be purchased and used.

[0024] The nicotinamide mononucleotide is a purified product with a low content of impurities, and its purity is preferably 90% or more, and more preferably 95% or more. If the purity is less than 90%, an unpleasant odor may be generated or the effect of nicotinamide mononucleotide may be weakened, making it difficult to fully achieve the effects of the present invention.

[0025] In the sperm motility improver of the present invention, the content of the active ingredient nicotinamide mononucleotide is not particularly limited. The amount of the sperm motility improver to be used may be appropriately determined while observing the effect. In one embodiment, the amount of the sperm motility improver to be used may be determined appropriately while observing the effect of the sperm motility improver when the sperm concentration (total sperm count) is 5 × 10 6 ~50×10 6 When a semen concentration of 100 ml is used as a standard, the sperm motility improver is added to the semen so that the concentration of the nicotinamide mononucleotide, which is the active ingredient contained in the sperm motility improver, is 0.05 to 150 μM, preferably 0.05 to 120 μM, more preferably 1 to 100 μM. If the concentration is less than 0.05 μM, the effects of the present invention may not be obtained, while if the concentration exceeds 150 μM, the obtained effects will not change much and it will be economically disadvantageous. Furthermore, when pyrroloquinoline quinone, which will be described later, is added, in one embodiment, the sperm concentration (total sperm count) is 5×10 6 ~50×10 6 / ml of semen as a standard, it is preferable to add the sperm motility improver to the semen so that the concentration of the nicotinamide mononucleotide, which is an active ingredient contained in the sperm motility improver, is 1 to 10 μM.

[0026] The sperm motility improver of the present invention can be easily produced by using nicotinamide mononucleotide alone or by mixing it with other components. The other components are not particularly limited as long as they can achieve the effects of the present invention.

[0027] In the present invention, another ingredient that is particularly effective in enhancing the sperm motility-improving effect is pyrroloquinoline quinone (PQQ) (including its salts). According to the presumed mechanism of the present invention described above, adding nicotinamide mononucleotide to semen (contacting it with sperm) increases the amount of NAD / NADH in sperm, activates the electron transport chain, and increases the ATP concentration in mitochondria, thereby enhancing sperm motility. However, activation of the electron transport chain also increases the production of reactive oxygen species (ROS), which is expected to have a negative effect on the durability of the effects of nicotinamide mononucleotide. Based on this prediction, the present inventors conducted research and found for the first time that the durability of the sperm motility-improving effect of nicotinamide mononucleotide can be enhanced by selecting and adding pyrroloquinoline quinone, in particular, from among many antioxidants.

[0028] Pyrroloquinoline quinone functions as an essential redox coenzyme for the energy acquisition system of living organisms, and has been shown to have many physiological activities, including function as a B vitamin, radical scavenger, brain function improvement, neuroprotective effect, nerve growth factor enhancement, antioxidant effect, mitochondrial biogenesis, memory improvement, and discrimination ability improvement, making it a compound with promising applications. Pyrroloquinoline quinone is widely distributed throughout the living world, found in high concentrations in breast milk and in trace amounts in various vegetables and meat. It is particularly abundant in tea, natto, and fruit.

[0029] In the present invention, pyrroloquinoline quinone (or a salt thereof) may be a hydrate or solvate thereof. Examples of the salt of pyrroloquinoline quinone include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, organic amine salts such as ammonium salt, triethanolamine salt and trimethylamine salt, and basic amino acid salts such as lysine salt and arginine salt. The salt of pyrroloquinoline quinone may be used alone or in combination.

[0030] In one embodiment of the present invention, pyrroloquinoline quinone (reduced) represented by the following structural formula [Chemical Formula 2] is used. Reduced pyrroloquinoline quinone is a water-soluble substance obtained by reducing the quinone moiety of oxidized pyrroloquinoline quinone. Reduced pyrroloquinoline quinone is generally more effective than oxidized pyrroloquinoline quinone. It is believed that oxidized pyrroloquinoline quinone is reduced to reduced pyrroloquinoline quinone in vivo. Reduced pyrroloquinoline can be obtained, for example, by reducing oxidized pyrroloquinoline quinone using various reducing agents, such as sodium borohydride and sodium hyposulfite.

[0031] [ka]

[0032] Pyrroloquinoline quinone may be prepared by extraction and purification from any source material, or may be synthesized. Examples of methods for producing pyrroloquinoline quinone include organic chemical methods and fermentation methods. For example, pyrroloquinoline quinone can be produced by culturing a bacterium capable of utilizing methanol and producing pyrroloquinoline quinone using methanol as a carbon source. Commercially available products are also available. For example, pyrroloquinoline quinone disodium salt is commercially available from Mitsubishi Gas Chemical Company, Inc.

[0033] In the sperm motility improver of the present invention, the content of pyrroloquinoline quinone is not particularly limited. The amount of the sperm motility improver to be used may be appropriately determined depending on the effect. In one embodiment, the amount of the sperm motility improver to be used may be determined based on the effect of the sperm motility improver. 6 ~50×10 6 When the concentration of semen is based on 1 / ml, the sperm motility improver is added to the semen so that the concentration of the pyrroloquinoline quinone nicotinamide becomes 20 to 200 nM, preferably 50 to 150 nM, more preferably 100 to 150 nM. If the concentration is less than 20 nM, the effect of the present invention may not be obtained, whereas if the concentration exceeds 200 nM, the effect obtained is not significantly different and it is economically disadvantageous.

[0034] In one embodiment, the sperm motility improver of the present invention has a molar ratio of the nicotinamide mononucleotide to the pyrroloquinoline quinone of 1:1 to 1500:1, preferably 1:1 to 1200:1, and more preferably 1:1 to 1000:1. If the molar ratio is outside the range of 1:1 to 1500:1, the effects obtained by the present invention may be reduced.

[0035] The method for producing the sperm motility improver is not particularly limited, and a general production method used for producing the agent may be appropriately selected depending on its form. For example, if the agent is in the form of a powder, it can be produced by uniformly kneading nicotinamide mononucleotide and other ingredients, such as pyrroloquinoline quinone, which are added as needed. Note that the active ingredient, nicotinamide mononucleotide, is distributed on the market and can be commercially obtained. In particular, with regard to nicotinamide mononucleotide, a quality control system and mass production system have been established in recent years, making it possible to supply nicotinamide mononucleotide.

[0036] The sperm motility improver of the present invention can be used as a pharmaceutical (including quasi-drugs) or a food or drink. The dosage form of the pharmaceutical is not particularly limited, and examples thereof include oral administration preparations such as powders, tablets, sustained-release tablets, chewable tablets, effervescent tablets, troches, buccal tablets, sublingual tablets, capsules, fine granules, granules, pills, dry syrups, liquids, suspensions, syrups, and elixirs, as well as injections, infusions, suppositories, and topical preparations. The intake amount of the pharmaceutical can be appropriately determined depending on the type of food, the age, sex, weight, expected effects, symptoms, etc. of the subject who will take it.

[0037] The pharmaceutical may be appropriately blended with known pharmaceutically acceptable additives suitable for the dosage form, taking into consideration physicochemical properties, biological properties, etc. Examples of such additives include excipients (lactose, starch, crystalline cellulose, sodium phosphate, etc.), solvents (water, soybean oil, saline, non-aqueous solvents for injection, etc.), binders (starch, gelatin, gum arabic, sodium alginate, carmellose sodium, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, etc.), disintegrants (starch, carmellose sodium, etc.), lubricants (talc, magnesium stearate, calcium stearate, macrogol, sucrose fatty acid esters, etc.), coating agents (sucrose, HPC, shellac, gelatin, glycerin, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate, cellulose acetate phthalate, etc.), stabilizers (sodium bisulfite, sodium thiosulfate, sodium edetate, sodium citrate, ascorbic acid, dibutylhydroxybenzoate, etc.), and the like. Preservatives (methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, benzyl alcohol, phenol, chlorobutanol, benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, thimerosal, etc.), thickeners (methylcellulose, carmellose sodium, chondroitin sulfate, sodium alginate, etc.), suspending agents (various nonionic surfactants, methylcellulose, carmellose sodium, etc.), Examples of such additives include emulsifiers (gum arabic, cholesterol, sorbitan sesquioleate, polysorbate 80, sodium lauryl sulfate, etc.), buffers (citric acid, acetic acid, sodium phosphate, boric acid), surfactants (hydrogenated castor oil, polysorbate 80, etc.), colorants (water-soluble food dyes, lake dyes, etc.), flavoring agents (lactose, sucrose, glucose, mannitol, etc.), flavoring agents (aromatic essential oils, etc.), plasticizers (phthalate esters, vegetable oils, polyethylene glycol, etc.), etc.

[0038] The sperm motility improver of the present invention can be used as a food or drink (including supplements). When taken daily in the form of a food, the effects of the present invention are continuously exerted, which is particularly effective in enjoying the effects at a higher level. The types of foods that can be used in the present invention are not particularly limited, and can include general foods, functional foods, foods for specified health uses, nutritional supplements, food additives, feed, nursing care foods, dietary foods, therapeutic foods, and diet foods. Specific examples include confectioneries (gum, candy, cookies, gummies, biscuits, cakes, chocolates, Japanese sweets, jellies, etc.), bread, noodles, processed rice and grain products (cereals, etc.), processed meat products, processed seafood products, processed vegetables, prepared dishes, fermented foods, seasonings (sauces, dressings, ketchup, etc.), spices, dairy products (yogurt, cheese, milk, etc.), ice cream, frozen foods, retort pouch foods, beverages (carbonated drinks, soft drinks, dairy drinks, alcoholic drinks, sports drinks, fruit juice drinks, teas, nutritional drinks, concentrated drinks, etc.), powdered beverages (powdered juice, powdered soup, etc.), etc. The form of the food is not limited, and in the case of functional foods and foods for specified health uses, for example, the food can be processed and provided as a powder, tablet, pill, granule, hard capsule, soft capsule, jelly, liquid, paste, etc. The intake amount of the food can be determined appropriately depending on the type of food, the age, sex, weight, expected effects, symptoms, etc. of the subject taking the food.

[0039] The food product is safe and has no particular side effects, so it can be taken over a long period of time and is applicable to both the elderly and young people.

[0040] Another aspect of the present invention is a method for improving sperm motility, which comprises contacting sperm with nicotinamide mononucleotide in vitro or in vivo. The species from which the sperm are derived is not limited, but in one embodiment, may be a mammal, specifically, a human, a pig, a cow, a horse, a sheep, a goat, a monkey, a rat, a mouse, a rabbit, a dog, a cat, etc.

[0041] Furthermore, as described in the section on the sperm motility improver of the present invention, contacting nicotinamide mononucleotide with pyrroloquinoline quinone in addition to nicotinamide mononucleotide with sperm can enhance the duration of the sperm motility improving effect of nicotinamide mononucleotide. The application amounts of nicotinamide mononucleotide and pyrroloquinoline quinone are as described above.

[0042] In the method for improving sperm motility of the present invention, the method for contacting nicotinamide mononucleotide and, if necessary, pyrroloquinoline quinone with sperm in vitro is not particularly limited. For example, the method can be carried out by placing the target semen in a container, adding nicotinamide mononucleotide and pyrroloquinoline quinone thereto, and culturing the mixture for a predetermined period of time. The contact of nicotinamide mononucleotide, pyrroloquinoline quinone with sperm can also be carried out multiple times by adding them again at predetermined time intervals. Alternatively, for example, the two can be contacted by using an aqueous solution containing nicotinamide mononucleotide and pyrroloquinoline quinone as a diluent for semen.

[0043] In the method for improving sperm motility of the present invention, the method for contacting nicotinamide mononucleotide and, if necessary, pyrroloquinoline quinone with sperm in vivo is not particularly limited, for example, by injecting nicotinamide mononucleotide and pyrroloquinoline quinone together with sperm into the uterus of a mammal.

[0044] The sperm treated by the sperm motility improving agent and sperm motility improving method of the present invention have good sperm motility, particularly forward motility, and are safe. The present invention can be used to improve asthenozoospermia and ultimately to treat infertility, and is extremely useful in the fields of assisted reproductive medicine and livestock farming. [Example]

[0045] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0046] [Example] (Effect of nicotinamide mononucleotide (NMN) on sperm motility) (1) Method Packs containing diluted boar semen (sperm concentration (total sperm count) 30,000,000 / ml, diluted with Hiroswein Solution B (Hiroshima Cryopreservation Services)) provided by the Oita Prefectural Agriculture, Forestry and Fisheries Research and Guidance Center were immediately stored in an incubator at 15°C upon arrival. The next day, 5 ml of the semen from each pack was transferred to a 15 ml centrifuge tube and cultured at 37°C for up to 6 hours using Hiroswein Solution B as the culture medium, with NMN added to concentrations ranging from 0.1 to 100 μM (0.1 μM, 1 μM, 10 μM, 100 μM). Some diluted semen was cultured in the same manner, with or without NMN, but with pyrroloquinoline quinone (PQQ) (Mitsubishi Gas Co.) added to a concentration of 100 nM. Sperm were collected at 0, 2, 4, and 6 hours after the start of culture, and their sperm motility was analyzed using a CASA (HT CASA-Ceros II (product name), Hamilton Thorne). Sperm motility was analyzed using the following: total motility rate (%), which is the ratio of motile sperm to the total sperm count; forward motility rate (%), which is the ratio of forward-moving sperm to the total number of motile sperm; and forward velocity (μm / s) of forward-moving sperm. The results are shown in Figures 2a, 2b, and 2c, respectively. The error bars in Figure 2 represent standard error. Sperm motility trajectories were photographed using a phase-contrast microscope 2, 4, and 6 hours after the start of culture, and are shown in Figure 3. (2) Results The total motility rate was not affected by differences in the concentration of NMN added (Figure 2a). However, the sperm motility rate and rectilinear velocity, which are essential for sperm to move upward through the reproductive tract, significantly increased in the NMN-only group (PQQ-), and this effect was observed up to 4 hours later (Figures 2b, 2c, and 3). However, in the NMN-only group, the improvement in motility rate and rectilinear velocity due to the addition of NMN alone disappeared after 6 hours (Figures 2b, 2c, and 3). On the other hand, in the NMN-PQQ+ group, the effect of adding NMN and PQQ was observed at lower concentrations (0.1 μM and 1 μM) (after 4 hours), and this effect continued for up to 6 hours (Figures 2b, 2c, and 3). (3) Consideration From the above experimental results, it was confirmed that NMN is utilized by sperm, and that NMN has the effect of enhancing the rate and speed of straight-line motility of sperm.Furthermore, by further adding PQQ, the duration of the effect of NMN is increased, so it was confirmed that combined treatment with PQQ is effective.In the group where NMN alone was added, the straight-line motility of sperm was enhanced, but the effect did not last for a long time, but the addition of PQQ provided a sustained effect.According to the above experimental results, the mechanism by which the effect of the present invention is brought about is as mentioned above: by contacting NMN with sperm, the amount of NAD / NADH in sperm increases, the electron transport chain is activated, the ATP concentration in mitochondria increases, and sperm motility is enhanced.At this time, reactive oxygen is also produced, which inhibits the duration of the effect of NMN, and it is speculated that by adding PQQ, PQQ blocks the action of reactive oxygen, thereby prolonging the effect of NMN.

Claims

1. A sperm motility improving agent comprising nicotinamide mononucleotide and pyrroloquinoline quinone as active ingredients, wherein the molar ratio of nicotinamide mononucleotide to pyrroloquinoline quinone is 1:1 to 1000:

1.

2. The sperm motility improver according to claim 1, wherein the sperm are mammalian sperm.

3. Sperm concentration is 5 x 10 ６ ~50 x 10 ６ A sperm motility improving agent according to claim 1 or 2, wherein the nicotinamide mononucleotide is added to semen at a concentration of 0.05 to 150 μM per ml.

4. Sperm concentration is 5 x 10 ６ ~50 x 10 ６ A sperm motility improving agent according to any one of claims 1 to 3, wherein the pyrroloquinoline quinone is added to ml of semen at a concentration of 20 to 200 nM.

5. The sperm motility improver according to any one of claims 1 to 4, wherein the sperm motility improver is a pharmaceutical product for improving sperm motility.

6. The sperm motility improver according to any one of claims 1 to 4, wherein the sperm motility improver is a food or drink for improving sperm motility.

7. A method for improving sperm motility (excluding medical procedures for humans), comprising contacting nicotinamide mononucleotide and pyrroloquinoline quinone with sperm in vitro or in vivo, wherein the molar ratio of the nicotinamide mononucleotide to the pyrroloquinoline quinone is 1:1 to 1000:

1.

8. 8. The method of claim 7, wherein the sperm are mammalian sperm.

9. Sperm concentration is 5 x 10 ６ ~50 x 10 ６ The method according to claim 7 or 8, comprising adding the nicotinamide mononucleotide to semen at a concentration of 0.05 to 150 μM.

10. Sperm concentration is 5 x 10 ６ ~50 x 10 ６ 10. The method according to claim 7, further comprising adding the pyrroloquinoline quinone to a semen concentration of 20 to 200 nM.

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