Sperm motility improver and method for improving sperm motility

Abstract

[Problem] To provide a sperm motility improver that is highly safe and can effectively improve sperm motility. [Solution] A sperm motility improver comprising nicotinamide mononucleotide as an effective ingredient.

Description

Technical Field

[0001] This invention relates to sperm motility improvers and methods for improving sperm motility. Background Technology

[0002] Fertilization is the process by which a sperm cell from a male reproductive cell fuses with an egg cell from a female reproductive cell until the zygote formed from the nuclei of both cells becomes 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 attracted by the fimbriae of the fallopian tube and transferred 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, and eventually reach the ampulla of the fallopian tube.

[0003] In order to fertilize an egg, sperm need to pursue the egg and move within the reproductive tract. Therefore, sperm motility is one of the important factors that determines whether fertilization is successful.

[0004] Sperm are generally divided into three parts: head, neck, and tail. The tail is formed by a single, extremely long flagellum (cilia), within which microtubules are clustered in a specific configuration. Mitochondria are coiled together at the middle of the tail, supplying ATP, the energy source for sperm motility. Sperm motility is then generated by consuming ATP produced by the mitochondria and shaking the tail. Specifically, it is believed that within the axoneme of the flagellum, dynein, acting as a molecular motor, consumes ATP for energy, causing structural changes that convert the sliding motion of the microtubules into the undulating motion of the flagellum. During fertilization, the sperm receives various external factors from its surroundings and regulates the activity of dynein based on these factors. The flagellum is composed of peripheral microtubules, dynein, radial rays, and the neuron-dynein regulatory complex (N-DRC). If these components do not form properly, it can lead to abnormal flagellum formation and reduced motility.

[0005] As explained above, sperm motility is a crucial factor in determining whether fertilization occurs. Semen contains sperm of varying levels of motility; in addition to sperm concentration and morphology, motility is also used to evaluate the state of these sperm. Reduced sperm motility is called asthenospermia, a major cause of male infertility or low fertilization rates. In humans, semen analysis is performed according to the WHO guidelines. Generally, asthenospermia is diagnosed when less than 50% of sperm are progressively motile and less than 25% are rapidly progressing.

[0006] In Japan, the number of couples suffering from infertility is increasing, reportedly one in 5.5 couples. It is estimated that approximately 500,000 people are undergoing some form of infertility treatment. Furthermore, it is known that about 80% of male-caused infertility is due to asthenospermia (low sperm motility). While the effects of harmful substances such as environmental hormones have been identified as contributing factors to asthenospermia, the exact causes remain unclear.

[0007] Infertility treatments include drug therapy such as ovulation induction agents, tubal insufflation to address fallopian tube blockages, tubal reconstruction surgery, vasectomy to address vas deferens dysfunction, artificial insemination, and in-vitro fertilization. Treatment methods vary depending on the patient's age and the specific condition. Patients undergoing infertility treatment experience physical discomfort, emotional distress, and financial burden. Furthermore, despite these treatments, Japan faces a very low birth rate due to infertility treatment.

[0008] On the other hand, in livestock, asthenospermia is generally diagnosed when less than 50% of sperm exhibit active forward motility during semen collection. In cases diagnosed asthenospermia, suspected causes include testicular atrophy, inflammation of the testes, accessory sex glands, urethra, and urinary contamination. Treatment aims to eliminate the underlying cause, but the prognosis is generally poor. Cattle reproduction has been achieved through artificial insemination using cryopreserved sperm from superior bull breeds (Sperm Hero bulls), but the conception rate (the proportion of pregnant cattle after artificial insemination) is known to be decreasing year by year. Thus, infertility has become a significant problem in the livestock world.

[0009] In view of this situation, various reagents and methods have been developed to improve sperm motility, which is one of the causes of male infertility. For example, one method for improving sperm motility and / or sperm production capacity in mammals other than humans is reported. This method involves administering cells containing mesenchymal cells derived from adipose tissue obtained by enzymatic treatment of subcutaneous adipose tissue collected from mammals other than humans to mammals other than humans with reduced sperm motility and / or sperm production capacity (Patent Document 1). Another method for improving sperm motility and / or sperm production capacity in mammals other than humans involves administering cells containing mesenchymal cells derived from adipose tissue obtained by enzymatic treatment of subcutaneous adipose tissue obtained from mammals other than humans (A) to mammals other than humans (B) with reduced sperm motility and / or sperm production capacity.

[0010] In addition, the following have been reported: a pharmaceutical composition for increasing sperm motility, containing extracts of oyster and ginseng as active ingredients (Patent Document 2); a sperm motility improver composed of a gas containing more than 1 (v / v)% hydrogen molecules (Patent Document 3); a sperm activator containing protamine (Patent Document 4); and a method for promoting sperm motility in vitro, comprising adding a pharmaceutical composition containing a high molecular weight protein and pharmaceutically permissible excipients to semen in vitro, thereby improving sperm motility related to the treatment of human infertility and the reproduction of livestock, wherein the high molecular weight protein has a molecular weight of 66 kDa, can activate sperm motility, and contains glycoproteins (Patent Document 5), etc.

[0011] Existing technical documents

[0012] Patent documents

[0013] Patent Document 1: Japanese Patent Application Publication No. 2017-25038

[0014] Patent Document 2: Japanese Patent Application Publication No. 2004-83517

[0015] Patent Document 3: Japanese Re-evaluation No. 2015-64109

[0016] Patent Document 4: Japanese Patent Application Publication No. 2010-6785

[0017] Patent Document 5: Japanese Patent Application Publication No. 11-279075 Summary of the Invention

[0018] The problem that the invention aims to solve

[0019] The present invention aims to provide a sperm motility improver that is highly safe and effective in improving sperm motility, as well as a method for improving sperm motility.

[0020] means for solving problems

[0021] The inventors of this invention conducted in-depth research to solve the above-mentioned problems and discovered that nicotinamide mononucleotide, an intermediate metabolite related to the biosynthesis of coenzyme NAD (nicotinamide adenine dinucleotide), has an excellent effect on improving sperm motility, thus completing this invention.

[0022] The present invention is as follows.

[0023] [1] A sperm motility improver, wherein the sperm motility improver contains nicotinamide mononucleotide as an active ingredient.

[0024] [2] According to the sperm motility improver described in [1], wherein the sperm motility improver further comprises pyrroloquinoline quinone.

[0025] [3] The sperm motility improver according to [1] or [2], wherein the sperm is mammalian sperm.

[0026] [4] The sperm motility improver according to any one of [1] to [3], wherein the concentration is 5 × 10⁻⁶ relative to the sperm concentration. 6 ~50×10 6 The nicotinamide mononucleotide was added to a volume of semen at a concentration of 0.05–150 μM.

[0027] [5] The sperm motility improver according to any one of [2] to [4], wherein the concentration is 5 × 10⁻⁶ relative to the sperm concentration. 6 ~50×10 6 / ml of semen was added at a concentration of 20-200 nM for pyrroloquinoline quinone.

[0028] [6] The sperm motility improver according to any one of [2] to [5], wherein the molar ratio of the nicotinamide mononucleotide to the pyrroloquinoline quinone is 1:1 to 1500:1.

[0029] [7] The sperm motility improver according to any one of [1] to [6], wherein the sperm motility improver is a pharmaceutical product for improving sperm motility.

[0030] [8] The sperm motility improver according to any one of [1] to [6], wherein the sperm motility improver is a food or beverage for improving sperm motility.

[0031] [9] A method for improving sperm motility, wherein the method comprises contacting sperm with nicotinamide mononucleotide in vitro or in vivo.

[0032]

[10] According to the method of [9], wherein the method further comprises contacting sperm with pyrroloquinoline quinone in vitro or in vivo.

[0033]

[11] According to the method described in [9] or

[10] , wherein the sperm is mammalian sperm.

[0034]

[12] The method according to any one of [9] to

[11] , wherein the method comprises a sperm concentration of 5 × 10⁻⁶. 6 ~50×10 6Nicotinamide mononucleotide was added to the semen at a concentration of 0.05–150 μM per ml.

[0035]

[13] The method according to any one of

[10] to

[12] , wherein the method comprises a sperm concentration of 5 × 10⁻⁶. 6 ~50×10 6 The pyrroloquinoline quinone is added to the semen at a concentration of 20–200 nM per ml.

[0036]

[14] A sperm, wherein the sperm is processed by any one of the methods described in [9] to

[13] .

[0037] Invention Effects

[0038] This invention can effectively improve sperm motility and serves as a catalyst for NAD+ interaction within the body. + The active ingredient is nicotinamide mononucleotide, an intermediate metabolite involved in the biosynthesis of nicotinamide, which is therefore highly safe and can be used with confidence. Attached Figure Description

[0039] Figure 1 This is an illustration of the metabolic pathways related to niacin (the collective name for nicotinamide and niacin).

[0040] Figure 2a This is a chart showing the forward motility (%) of porcine sperm during culture under conditions of NMN alone or NMN+PQQ addition.

[0041] Figure 2b This is a graph showing the total motility (%) of porcine sperm during culture under conditions of adding NMN alone or adding NMN+PQQ.

[0042] Figure 2c This is a graph showing the straight-line velocity (μM / s) of porcine sperm during culture under conditions of adding NMN alone or adding NMN+PQQ.

[0043] Figure 3 These are photographs showing the sperm motility trajectories during porcine sperm culture under conditions of adding NMN alone or adding NMN+PQQ. Detailed Implementation

[0044] The sperm motility improver of this invention uses nicotinamide mononucleotide as its active ingredient to improve sperm motility. As shown in the examples described later, this invention is particularly effective in increasing the straight-line motility rate and speed of sperm, which are essential for upward movement of sperm within the reproductive tract. Furthermore, in this invention, sperm motility improvement includes not only improvement of sperm motility in the narrow sense, but also prevention, cessation, and delay of decreased sperm motility.

[0045] The detailed mechanism of action of nicotinamide mononucleotide (NMN) in improving sperm motility is currently under investigation, but the following is a hypothesis: It is believed that adding NMN to semen increases the NAD / NADH ratio in sperm, activates the electron transport system, and raises the ATP concentration in mitochondria. ATP is the energy source for sperm motility, and the sliding motion of microtubules within the axoneme, caused by structural changes in dynein, is converted into flagellar movement, thus enhancing sperm motility. The present invention will now be described in detail.

[0046] Nicotinamide mononucleotide (Chemical formula: C 11 H 15 N2O8P is a compound produced in the bodies of many organisms, including humans, and is represented by the following structural formula [Chemical 1]. It is generally known as NMN (Nicotinamide mononucleotide) and is used in conjunction with the coenzyme NAD+. + It is known for its intermediate metabolites involved in biosynthesis.

[0047] [Chemistry 1]

[0048]

[0049] Nicotinamide mononucleotide, the active ingredient in the aforementioned sperm motility improver, is produced in vivo in the liver tissue via the NAD metabolic pathway, specifically the pathway involved in the synthesis of nicotinamide adenine dinucleotide (NAD) from quinolinic acid via the kynurenine pathway. For more information, see [link to relevant documentation]. Figure 1 Please provide a detailed explanation. Figure 1 This is a diagram illustrating the metabolic pathways associated with niacin (a collective term for niacin and nicotinamide), known as vitamin B3. Niacin obtained from the diet is ingested by the liver and converted into nicotinamide, which is then supplied throughout the body via the bloodstream. Cells take up nicotinamide from the blood and convert it into NAD and NADP for utilization. Nicotinamide can also be biosynthesized from tryptophan.

[0050] like Figure 1As shown, in living organisms, when tryptophan is used as the starting material, it is converted to quinolinic acid (QA) via the kynurenine pathway, which is part of the tryptophan metabolic pathway, and subsequently forms nicotinic acid mononucleotide (NaMN). Conversely, when nicotinic acid (Na) is used as the starting material, it is directly converted to NaMN. NaMN then undergoes interconversion with NAD, nicotinamide (NaM), and nicotinamide mononucleotide via the NAD cycle, through nicotinic acid adenine dinucleotide (NaAD). Nicotinamide (NaM) is converted to nicotinamide mononucleotide by nicotinamide phosphoribosyltransferase (NAMPT), and then nicotinamide mononucleotide is converted to NAD by nicotinamide mononucleotide adenosyltransferase (NMNAT). Furthermore, nicotinamide mononucleotide can also be produced from nicotinamide nucleoside (NR), an intermediate metabolite of NAD.

[0051] Nicotinamide mononucleotides exist as optical isomers, namely the α-form and the β-form, but the β-form is used in this invention. Nicotinamide mononucleotides can be obtained, for example, by synthesizing nicotinamide nucleosides from nicotinamide and ribose (see Bioorg. Med. Chem. Lett., 12, 1135-1137 (2002)), followed by phosphorylation of the 5-hydroxyl group of the ribose moiety (see Chem. Comm., 1999, 729-730). Specifically, for example, firstly, nicotinamide and L-ribose tetraacetate are dissolved in anhydrous acetonitrile. Under a nitrogen stream, excess trimethylsilyl trifluorosulfonic acid is added, and the mixture is stirred at room temperature. Methanol is added to stop the reaction, and the reaction solution is then attached to a column packed with activated carbon. After washing with distilled water, the product is eluted with methanol and recovered. Next, to perform phosphorylation of the 5-hydroxyl group of the L-ribose moiety of the product, the product was dissolved in trimethoxyphosphate, phosphoryl chloride was added dropwise under ice-cold conditions, and the mixture was stirred under a nitrogen stream. Sodium hydroxide aqueous solution was added for neutralization to stop the reaction, and a cold acetonitrile-ether solution was added to the reaction solution. The lower layer (aqueous phase) was then passed through an anion exchange resin to recover the reactants, and further purified using a cation exchange resin, thereby recovering nicotinamide mononucleotide with high purity. Nicotinamide mononucleotide is commercially available and can be purchased for use.

[0052] The aforementioned nicotinamide mononucleotide is a purified product with low impurity content, preferably with a purity of 90% or higher, and more preferably 95% or higher. If the purity is less than 90%, there is a risk that off-odors may develop, or the effect of nicotinamide mononucleotide may be weakened, thus failing to fully obtain the effects of the present invention.

[0053] In the sperm motility improver of the present invention, the content of nicotinamide mononucleotide, as an active ingredient, is not particularly limited. Furthermore, the dosage of this sperm motility improver can be appropriately determined while observing the effects. In one embodiment, the dosage is set at a sperm concentration (total sperm count) of 5 × 10⁻⁶. 6 ~50×10 6 Based on a semen volume of / ml, the sperm motility improver is added at a concentration of 0.05–150 μM, preferably 0.05–120 μM, and more preferably 1–100 μM, relative to the semen volume. If the concentration is less than 0.05 μM, there is a risk that the effects of the present invention will not be obtained; on the other hand, if the concentration exceeds 150 μM, the obtained effect is not correspondingly changed, which becomes economically disadvantageous. Furthermore, in the case of including pyrroloquinoline quinone (described later), in one embodiment, the sperm concentration (total sperm count) is 5 × 10⁻⁶. 6 ~50×10 6 Based on a volume of semen of 1 / ml, it is preferable to add the sperm motility improver at a concentration of 1 to 10 μM relative to the semen volume of the sperm motility improver as the active ingredient.

[0054] The sperm motility improver of the present invention can be readily manufactured by using nicotinamide mononucleotide alone or in combination with other ingredients. Other ingredients are not particularly limited as long as they achieve the effects of the present invention.

[0055] In this invention, pyrroloquinoline quinone (PQQ) (including its salts) is listed as another component particularly effective in enhancing sperm motility. According to the proposed mechanism of the invention described above, by adding nicotinamide mononucleotide to semen (in contact with sperm), the NAD / NADH ratio in sperm increases, the electron transport system is activated, the ATP concentration in mitochondria rises, and sperm motility is enhanced. However, if the electron transport system is activated at this time, the production of reactive oxygen species (ROS) also increases, and it is predicted that these ROS will negatively affect the persistence of the effect of nicotinamide mononucleotide. Therefore, based on this prediction, the inventors of this invention have studied countermeasures and, for the first time, discovered that by specifically selecting and adding pyrroloquinoline quinone from a large number of antioxidants, the persistence of the sperm motility-enhancing effect of nicotinamide mononucleotide can be improved.

[0056] Pyrroloquinoline quinone functions as an essential redox coenzyme for the energy acquisition system of organisms. Its functions as a B vitamin and free radical scavenger are now well-established, including improving brain function, neuroprotection, enhancing nerve growth factor, antioxidation, mitochondrial regeneration, improving memory, and enhancing recognition abilities. It is a compound with promising applications. Pyrroloquinoline quinone is widely distributed in the biological world, present in high concentrations in breast milk, and also found in trace amounts in various vegetables and meats. It is particularly abundant in tea, natto, and fruits.

[0057] In this invention, pyrroloquinoline quinone (or its salts) can be its hydrates or solvates. Examples of salts of pyrroloquinoline quinone include alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as magnesium and calcium salts, ammonium salts, organic amine salts such as triethanolamine and trimethylamine salts, and basic amino acid salts such as lysine and arginine salts. Salts of pyrroloquinoline quinone can be used alone or in combination.

[0058] In one embodiment of this invention, pyrroloquinoline quinone (reduced form) as shown in the following structural formula [Chemical 2] is used. Reduced pyrroloquinoline quinone is a water-soluble substance obtained by reducing the quinone portion of oxidized pyrroloquinoline quinone. Reduced pyrroloquinoline quinone is generally more potent than oxidized pyrroloquinoline quinone. It is believed that oxidized pyrroloquinoline quinone is reduced in vivo to form reduced pyrroloquinoline quinone. Reduced pyrroloquinoline quinone can be obtained, for example, by reducing oxidized pyrroloquinoline quinone using various reducing agents such as sodium borohydride, sodium hyposulfite, etc.

[0059] [Chemistry 2]

[0060]

[0061] Pyrroloquinoline quinone can be prepared by extraction and purification from various sources, or it can be synthesized. Methods for manufacturing pyrroloquinoline quinone include organic chemical methods and fermentation methods. For example, it can be manufactured by culturing bacteria that are methanol-assimilating and capable of 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 Co., Ltd.

[0062] In the sperm motility improver of the present invention, the content of pyrroloquinoline quinone is not particularly limited. Furthermore, the dosage of this sperm motility improver can be appropriately determined while observing the effects; in one embodiment, the dosage is 5 × 10⁻⁶ for a sperm concentration (total sperm count). 6 ~50×10 6Based on a semen volume of / ml, the sperm motility improver is added relative to this semen at a concentration of 20-200 nM, preferably 50-150 nM, and more preferably 100-150 nM. If the concentration is less than 20 nM, there is a risk that the effects of this invention will not be achieved; on the other hand, if the concentration exceeds 200 nM, the obtained effect does not change accordingly, which becomes economically disadvantageous.

[0063] In one embodiment of the sperm motility improver of the present invention, the molar ratio of the above-mentioned nicotinamide mononucleotide to the above-mentioned pyrroloquinoline quinone is 1:1 to 1500:1, preferably 1:1 to 1200:1, and more preferably 1:1 to 1000:1. If the ratio is outside the range of 1:1 to 1500:1, there is a risk of reduced efficacy obtained according to the present invention.

[0064] There are no particular limitations on the manufacturing method of the aforementioned sperm motility improver. The general manufacturing method used to manufacture the sperm motility improver can be selected appropriately according to its form. For example, if it is in powder form, it can be manufactured by uniformly mixing nicotinamide mononucleotide and other ingredients such as pyrroloquinoline quinone, as needed. Furthermore, nicotinamide mononucleotide, as the active ingredient, is commercially available and readily available. In particular, for nicotinamide mononucleotide, a quality management system and mass production system have been established in recent years, ensuring its supply.

[0065] The sperm motility improver of this invention can be used as a pharmaceutical (including quasi-pharmaceuticals) and a food product. There are no particular limitations on the dosage form of this pharmaceutical product; examples include, but are not limited to, powders, tablets, sustained-release tablets, chewable tablets, effervescent tablets, lozenges, buccal lozenges, sublingual tablets, capsules, granules, pellets, pills, dry syrups, liquids, suspensions, syrups, elixirs, and other oral administration preparations, as well as injections, infusions, suppositories, and topical preparations. The dosage of the above-mentioned pharmaceutical product can be appropriately determined based on the type of food, the age, sex, weight of the person consuming it, the desired effect, and the symptoms.

[0066] Considering the physicochemical and biological properties, pharmaceutically acceptable and well-known formulation additives suitable for their dosage forms can be appropriately added to the aforementioned pharmaceutical products. Examples of such formulation additives include, for instance, excipients (lactose, starch, crystalline cellulose, sodium phosphate, etc.), solvents (water, soybean oil, saline solution, non-aqueous solvents for injection, etc.), binders (starch, gelatin, gum arabic, sodium alginate, sodium carboxymethyl cellulose, methylcellulose, ethylcellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose, polyvinylpyrrolidone, etc.), disintegrants (starch, sodium carboxymethyl cellulose, etc.), lubricants (talc, magnesium stearate, calcium stearate, polyethylene glycol, sucrose fatty acid esters, etc.), coating agents (white sugar, HPC, shellac, gelatin, glycerin, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, cellulose acetate phthalate, etc.), stabilizers (sodium bisulfite, sodium thiosulfate, sodium edetate, sodium citrate, ascorbic acid, butylated hydroxytoluene, etc.), and preservatives. Agents include: (methylparaben, ethylparaben, propylparaben, benzyl alcohol, phenol, chlorobutanol, benzalkonium chloride, benzyl chloride, sodium dehydroacetate, thimerosal, etc.), thickeners (methylcellulose, sodium carboxymethylcellulose, chondroitin sulfate, sodium alginate, etc.), suspending agents (various nonionic surfactants, methylcellulose, sodium carboxymethylcellulose, etc.), 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 colorings, lake pigments, etc.), flavoring agents (lactose, white sugar, glucose, mannitol, etc.), odorants (aromatic essential oils, etc.), and plasticizers (phthalates, vegetable oils, polyethylene glycol, etc.).

[0067] The sperm motility improver of the present invention can be used as a food product (including supplements). When ingested daily in the form of food, the above-mentioned effects of the present invention are continuously exerted, thus being particularly effective in enjoying even greater effects. There are no particular limitations on the types of food to which the present invention is applicable; in addition to general foods, functional foods, specific health foods, nutritional supplements, food additives, animal feed, nursing foods, therapeutic foods, and weight-loss foods are also applicable. Specifically, examples include snacks (chewing gum, candy, cookies, gummy candies, biscuits, cakes, chocolates, Japanese sweets, jellies, etc.), bread, noodles, processed rice / grain products (oatmeal, etc.), processed meat products, processed fish and shellfish products, processed vegetable products, side dishes, fermented foods, seasonings (sauces, condiments, 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, tea, nutritional drinks, concentrated drinks, etc.), and powdered beverages (powdered fruit juice, powdered soup, etc.). Furthermore, the form of these foods is not limited, especially in the case of functional foods and foods for specific health purposes, for example, they can be processed into powders, tablets, pills, granules, hard capsules, soft capsules, jellies, liquids, pastes, etc. The intake of these foods can be appropriately determined based on the type of food, the age, gender, weight of the recipient, the desired effect, and symptoms.

[0068] The above-mentioned foods are safe and no side effects have been specifically identified, so they can be consumed long-term. They are suitable not only for the elderly but also for young people.

[0069] Another aspect of the present invention is a method for improving sperm motility, comprising contacting sperm with nicotinamide mononucleotide (NMN) in vitro or in vivo. The biological species from which the sperm is derived are not limited, but in one embodiment, mammals may be cited as examples, specifically including humans, pigs, cattle, horses, sheep, goats, monkeys, rats, mice, rabbits, dogs, cats, etc.

[0070] Furthermore, as described in the section on sperm motility improvers of the present invention, by contacting sperm together with pyrroloquinoline quinone and nicotinamide mononucleotide, the duration of the sperm motility-improving effect of nicotinamide mononucleotide can be enhanced. The appropriate amounts of nicotinamide mononucleotide and pyrroloquinoline quinone are as described above.

[0071] In the sperm motility improvement method of the present invention, the method of contacting nicotinamide mononucleotide and, if necessary, pyrroloquinoline quinone with sperm in vitro is not particularly limited. For example, this can be carried out by adding the target semen to a container, adding nicotinamide mononucleotide and pyrroloquinoline quinone thereto, and culturing for a predetermined time. Contact between nicotinamide mononucleotide, pyrroloquinoline quinone, and sperm can also be performed multiple times by adding them again at predetermined time intervals. Alternatively, for example, contact can be achieved by using an aqueous solution containing nicotinamide mononucleotide and pyrroloquinoline quinone as a diluent for the semen.

[0072] In the method for improving sperm motility of the present invention, there is no particular limitation on the method of contacting nicotinamide mononucleotide and, if necessary, pyrroloquinoline quinone with sperm in vivo. For example, this can be done by injecting nicotinamide mononucleotide and pyrroloquinoline quinone together with sperm into the uterus of a mammal.

[0073] Sperm treated with the sperm motility improver and method of the present invention exhibit good and safe sperm motility, particularly forward motility. This invention relates to the improvement of asthenospermia, and further to its application in infertility treatment, finding significant usefulness in assisted reproductive technology, animal husbandry, and other fields.

[0074] Example

[0075] The present invention will now be described in detail based on embodiments, but the present invention is not limited to these embodiments.

[0076] [Example]

[0077] Effects of nicotinamide mononucleotide (NMN) on sperm motility

[0078] (1) Method

[0079] Upon arrival of packages containing diluted bovine semen (sperm concentration (total sperm count) 30,000,000 / ml, diluted with HIRO-SWINE B solution (manufactured by Hiroshima Sperm Refrigeration Technology Service Co., Ltd.)) supplied by the Oita Prefectural Agricultural, Forestry and Fisheries Research and Guidance Center, they were immediately stored in an incubator at 15°C. The following day, 5 ml of the above semen was transferred from each package to 15 ml centrifuge tubes, and NMN was added at a concentration of 0.1–100 μM (0.1 μM, 1 μM, 10 μM, 100 μM) using HIRO-SWINE B solution as the culture medium, and incubated at 37°C for 6 hours. In a portion of the diluted semen, pyrroloquinoline quinone (PQQ) (manufactured by Mitsubishi Gas Co., Ltd.) was added at a concentration of 100 nM, with or without NMN, and the same incubation process was performed. Sperm were collected at 0, 2, 4, and 6 hours after the start of culture, and sperm motility was analyzed using CASA (HT CASA-Ceros II, manufactured by Hamilton Thorne). Regarding sperm motility, the following parameters were analyzed: the percentage of motile sperm relative to the total sperm count (total motility rate, %); the percentage of linearly motile sperm relative to the total number of motile sperm (linear motility rate, %); and the linearly motile sperm velocity (μm / s). The results are presented separately. Figure 2a , Figure 2b , Figure 2c Furthermore, the error bars in Figure 2 represent standard errors. Additionally, sperm motility trajectories were captured using a phase-contrast microscope at 2 hours, 4 hours, and 6 hours after the start of culture, as shown below. Figure 3 .

[0080] (2) Results

[0081] Depending on the concentration of NMN added, it has no effect on the overall exercise rate. Figure 2a However, the straight-line motility rate and speed of sperm, which are essential for upward movement of sperm within the reproductive tract, were significantly increased in the NMN-only addition zone (PQQ-), and this effect was confirmed up to 4 hours later. Figure 2b , Figure 2c , Figure 3 However, in the NMN-only addition area, the improvement in straight-line rate and speed brought about by NMN-only addition disappeared after 6 hours. Figure 2b , Figure 2c , Figure 3 On the other hand, in the compound addition zone with PQQ (PQQ+), the additive effects of NMN and PQQ were confirmed from lower concentrations (0.1 μM, 1 μM) (after 4 hours), and this effect lasted until 6 hours. Figure 2b , Figure 2c , Figure 3 ).

[0082] (3) Investigation

[0083] Based on the above experimental results, it was confirmed that NMN is utilized by sperm, and that NMN enhances the straight-line motility and speed of sperm. Furthermore, by adding PQQ, the duration of the above-mentioned effects brought about by NMN was improved, thus confirming the effectiveness of the combined treatment with PQQ. In the NMN-only addition area, sperm straight-line motility was enhanced, but the effect did not last long. However, based on the above experimental results showing that the effect was prolonged by adding PQQ, it is speculated that the mechanism of action of this invention is as described above: by bringing NMN into contact with sperm, the amount of NAD / NADH in sperm increases, the electron transport system is activated, the ATP concentration in mitochondria rises, and sperm motility is enhanced. However, at this time, reactive oxygen species are also generated, which hinders the duration of the NMN effect. If PQQ is added, PQQ inhibits the action of reactive oxygen species, thus prolonging the effect of NMN.

Claims

1. A sperm motility improver, wherein, The sperm motility improver contains nicotinamide mononucleotide and pyrroloquinoline quinone as active ingredients, and 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 in question is from mammals.

3. The sperm motility improver according to claim 1 or 2, wherein, Relative to a sperm concentration of 5×10 6 ~50×10 6 The nicotinamide mononucleotide was added to a volume of semen at a concentration of 0.05–150 μM.

4. The sperm motility improver according to claim 1 or 2, wherein, Relative to a sperm concentration of 5×10 6 ~50×10 6 / ml of semen was added at a concentration of 20~200nM for pyrroloquinoline quinone.

5. The sperm motility improver according to claim 1 or 2, wherein, The sperm motility improver is a pharmaceutical product used to improve sperm motility.

6. The use of a nicotinamide mononucleotide in the manufacture of a sperm motility enhancer, wherein, The application involves contacting nicotinamide mononucleotide and pyrroloquinoline quinone with sperm in vitro or in vivo, wherein the molar ratio of nicotinamide mononucleotide to pyrroloquinoline quinone is 1:1 to 1000:

1.

7. The application according to claim 6, wherein, The sperm in question is from mammals.

8. The application according to claim 6 or 7, wherein, The application includes sperm concentrations of 5 × 10⁻⁶. 6 ~50×10 6 Nicotinamide mononucleotide was added to the semen at a concentration of 0.05-150 μM per ml.

9. The application according to claim 6 or 7, wherein, The application includes sperm concentrations of 5 × 10⁻⁶. 6 ~50×10 6 The pyrroloquinoline quinone is added to the semen at a concentration of 20-200 nM per ml.

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