Fragmentation inhibitor for fertilized eggs

Abstract

Provided is a novel means of inhibiting fertilized egg fragmentation. The present invention provides an inhibitor of fertilized egg fragmentation, said inhibitor comprising selenoneine or ergothioneine, a tautomer or dimer thereof, or a pharmaceutically acceptable salt of the same as an active ingredient.

Description

[Technical field]

[0001] The present invention relates to an agent for suppressing the fragmentation of a fertilized egg, and a method for utilizing said fragmentation suppressor. [Background technology]

[0002] In recent years, the number of patients undergoing infertility treatment has been increasing year by year due to social factors such as late marriage and childbirth at an older age. There are various causes of infertility, such as a decrease in sperm count and motility rate with age, and a decrease in egg count and fertilization rate.

[0003] As types of infertility treatment, techniques such as artificial insemination, in vitro fertilization, and intracytoplasmic sperm injection are known. In these techniques, sperm may be used after collection or stored. In order to suppress the motility and metabolism of sperm during storage, sperm are diluted with a diluent and then refrigerated or frozen for low-temperature storage. The freezing and thawing processes can have adverse effects on sperm, causing a decrease in fertilization rate, development rate after fertilization, and implantation rate. In order to reduce the adverse effects of the freezing and thawing processes, cryoprotectants are used, and medium components such as buffers and sugars have been improved. In addition, water-soluble polymeric compounds of high molecular weight organic polymers and amino acids as auxiliary components thereof are known as protective agents for low-temperature storage (see, for example, Patent Document 1: International Publication No. 2008 / 007463).

[0004] Although artificial insemination and in vitro fertilization techniques are improving year by year, even if fertilization is successfully achieved using these techniques, the probability that the fertilized egg will divide smoothly and become a good embryo is not high. Fertilized eggs obtained through in vitro fertilization or intracytoplasmic sperm injection may be blastomerized unevenly during the process of culturing them to the 4-8 cell stage, causing fragmentation. The cause of fragmentation is not fully understood.

[0005] On the other hand, it has been revealed that selenoneine, a selenium-containing compound contained in fish such as tuna, mackerel, and yellowtail, has a high antioxidant effect and has physiological effects such as cell protection or cell proliferation promotion in cultured cells (Patent Document 2: JP 2019-76039 A). In addition, ergothioneine, in which the selenium in selenoneine is replaced with sulfur, is also known to have a high antioxidant effect. Ergothioneine has been reported to have elastase inhibitory effects and tyrosinase inhibitory effects, and is particularly attracting attention in the beauty and food fields, such as whitening and wrinkle prevention. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2008 / 007463 [Patent Document 2] JP 2019-76039 A Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a novel means for suppressing fragmentation of fertilized eggs.

[0008] The present inventors focused on selenoneine, an organic selenium compound discovered in fish, among various natural substances, and investigated its usefulness in regenerative medicine and reproductive medicine. They found that selenoneine and ergothioneine, which has a similar structure, significantly suppress the fragmentation of fertilized eggs, thus completing the present invention.

[0009] That is, the present invention includes the following inventions. [1] An agent for suppressing the fragmentation of fertilized eggs, comprising selenoneine or ergothioneine, or a tautomer or dimer thereof, or a pharma- ceutical acceptable salt thereof. [2] A sperm or egg treatment agent comprising the fragmentation inhibitor according to item 1, or selenoneine or ergothioneine, or a tautomer or dimer thereof, or a medicamentously acceptable salt thereof. [3] An embryo development promoter comprising the fragmentation inhibitor according to item 1, or selenoneine or ergothioneine, or a tautomer or dimer thereof, or a pharma- ceutical acceptable salt thereof. [4] A medium for sperm culture, oocyte culture, fertilized egg culture, or fertilization, comprising the fragmentation inhibitor according to item 1, or selenoneine or ergothioneine, or a tautomer or dimer thereof, or a medicamentously acceptable salt thereof. [5] A medium for freezing sperm, eggs, or fertilized eggs, comprising the fragmentation inhibitor described in item 1, or selenoneine or ergothioneine, or a tautomer or dimer thereof, or a pharma- ceutical acceptable salt thereof. [6] A sperm culture method comprising culturing sperm in a medium containing the fragmentation inhibitor according to item 1, or selenoneine or ergothioneine, or a tautomer or dimer thereof, or a pharma- ceutical acceptable salt thereof. [7] A method for culturing an egg or a fertilized egg, comprising culturing the egg or the fertilized egg in a medium containing the fragmentation inhibitor described in item 1, or selenoneine or ergothioneine, or a tautomer or dimer thereof, or a pharma- ceutical acceptable salt thereof. [8] Culturing an ovum in a medium containing the fragmentation inhibitor according to item 1, or selenoneine or ergothioneine, or a tautomer or dimer thereof, or a pharma- ceutical acceptable salt thereof; fertilizing the cultured eggs with sperm; A step of culturing the fertilized eggs in a fertilized egg culture medium. A method for producing a cleavage embryo, a morula or a blastocyst, comprising: [9] Culturing sperm in a medium containing the fragmentation inhibitor according to item 1, or selenoneine or ergothioneine, or a tautomer or dimer thereof, or a medicamentously acceptable salt thereof; fertilizing the cultured sperm with an egg; A step of culturing the fertilized eggs in a fertilized egg culture medium. A method for producing a cleavage embryo, a morula, or a blastocyst, comprising:

[10] The method according to item 8 or 9, further comprising a step of freezing the cleavage embryo, morula or blastocyst by liquid nitrogen.

[11] The method according to any one of items 8 to 10, wherein the fertilization step is carried out by in vitro fertilization or intracytoplasmic sperm injection.

[12] The method according to any one of items 8 to 11, wherein the fertilized egg culture medium further contains the fragmentation inhibitor.

[13] An infertility improving agent comprising selenoneine or ergothioneine, or a tautomer or dimer thereof, or a pharma- ceutical acceptable salt thereof.

[14] Selenoneine or ergothioneine, or a tautomer or dimer thereof, or a pharma- ceutically acceptable salt thereof, for use in the treatment of infertility.

[15] A method for treating infertility, comprising administering selenoneine or ergothioneine, or a tautomer or dimer thereof, or a medicamentously acceptable salt thereof.

[16] Use of selenoneine or ergothioneine, or a tautomer or dimer thereof, or a medicament acceptable salt thereof for the manufacture of an agent for improving or treating infertility. Effect of the Invention

[0010] According to the present invention, it is possible to suppress fragmentation in eggs and / or fertilized eggs. Furthermore, according to the present invention, a protective effect for sperm is also exhibited. [Brief description of the drawings]

[0011] [Figure 1]FIG. 1 shows the results of analytical HPLC of the 80% ethanol supernatant fraction obtained by purifying crude selenoneine by preparative HPLC. [Diagram 2] FIG. 2 shows the percentage of motile sperm (A) and well-motile sperm (B) among sperm cultured for 3 hours in the presence (SeN) or absence (Control) of purified selenoneine, and the percentage of motile sperm (C) and well-motile sperm (D) among sperm cultured for 6 hours. [Diagram 3] Fig. 3A shows micrographs of fertilized eggs cultured in the presence (SeN) or absence (Control) of purified selenoneine, Fig. 3B shows the rate of two-cell formation in fertilized eggs cultured in the presence (SeN) or absence (Control) of purified selenoneine, and Fig. 3C shows the rate of fragmented fertilized eggs cultured in the presence (SeN) or absence (Control) of purified selenoneine. [Figure 4] FIG. 4A shows the percentage of two-cell formation in fertilized eggs fertilized with sperm cultured for 6 hours in the presence (SeN) or absence (Control) of purified selenoneine, and FIG. 4B shows the percentage of fragmented fertilized eggs in fertilized with sperm cultured for 6 hours in the presence (SeN) or absence (Control) of purified selenoneine. [Diagram 5] FIG. 5A shows the percentage of two-cell formation in fertilized eggs fertilized with sperm cultured for 6 hours in the presence of purified selenoneine (SeN) or purified ergothioneine (Erg), and FIG. 5B shows the percentage of fragmented fertilized eggs in fertilized with sperm cultured for 6 hours in the presence of purified selenoneine (SeN) or purified ergothioneine (Erg). [Figure 6] FIG. 6 shows the percentage of oocytes that exhibited fragmentation 12 hours, 15 hours, 18 hours, 21 hours, and 24 hours after culturing the oocytes in TYH medium containing 10 nM, 100 nM, 1 μM, 10 μM, and 100 μM selenoneine. [Figure 7]FIG. 7 shows the percentage of oocytes that exhibited fragmentation 12 hours, 15 hours, 18 hours, 21 hours, and 24 hours after culturing the oocytes in TYH medium containing 10 nM, 100 nM, 1 μM, 10 μM, and 100 μM ergothioneine. [Figure 8] Figure 8A shows the fluorescence microscopic images of reactive oxygen species (ROS), reactive nitrogen species (RNS), and superoxide after culturing oocytes in a control (Vehicle) containing only DMSO, a selenoneine-containing medium (SeN), an ergothioneine-containing medium (Erg), and an ergothioneine and ebselen-containing medium (Erg+Eb). Figures 8B-D show the relative amounts of reactive oxygen species (ROS), reactive nitrogen species (RNS), and superoxide in the cells, respectively. [Figure 9] FIG. 9A shows fluorescence micrographs of sperm nuclei stained with Hoechst and propidium iodide after incubation in a selenoneine-containing medium. Circled sperm represent sperm stained with Hoechst but not with propidium iodide. FIG. 9B shows the ratio of the number of sperm stained with Hoechst but not with propidium iodide divided by the number of Hoechst-stained sperm for sperm cultured for 3, 6, and 9 hours in TYH medium with selenoneine concentrations of 10 nM, 1 μM, and 100 μM. FIG. 9C shows the ratio of the number of sperm stained with Hoechst but not with propidium iodide divided by the number of Hoechst-stained sperm for sperm cultured for 3, 6, and 9 hours in TYH medium with ergothioneine concentrations of 10 nM, 1 μM, and 100 μM. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Below, we will explain in detail the embodiment of the present invention (hereinafter referred to as the "present embodiment"); however, the present invention is not limited to this embodiment, and various modifications are possible without departing from the gist of the present invention.

[0013] (Fragmentation inhibitor) The fertilized egg fragmentation inhibitor of this embodiment has the following formula (I): [ka] (wherein X is selenium (Se) or sulfur (S). or a medicamentously acceptable salt thereof, as an active ingredient. The OH group, XH group, NH group, etc. in the molecule may be in a state without a hydrogen atom, i.e., in an ionized state. The compound may exist as any optical isomer, geometric isomer, tautomer, or dimer, or a mixture thereof. As a result of tautomerization and dimerization, the compound of the present invention may be in any form of the following formulae (I) to (III): [ka] (wherein X is selenium (Se) or sulfur (S). The fragmentation inhibitor may contain compounds of the forms of formulae (I) to (III) in any ratio. The dimerized compound may be reduced to a monomer depending on the medium environment. In addition, in order to maintain the monomer, the composition containing the compound represented by formula (I) or (II) may further contain a reducing agent. As such a reducing agent, any reducing agent may be used, and examples thereof include glutathione (GSH), dithiothreitol (DTT), and mercaptoethanol. In this specification, the "fragmentation inhibitor" may mean the active ingredient selenoneine or ergothioneine, or a tautomer or dimer thereof, or a medicamentously acceptable salt thereof.

[0014] In one embodiment, the compound represented by formula (I) or (II) is a compound in which X is a selenium atom, and hereinafter, this compound is also referred to as selenoneine. In yet another embodiment, the compound represented by formula (I) or (II) is a compound in which X is a sulfur atom, and this compound is also referred to as ergothioneine. As long as selenoneine or ergothioneine has the basic structure of formula (I) or (II), the OH group, SeH group, NH group, etc. in the molecule may be in a state without a hydrogen atom, i.e., in an ionized state.

[0015] Selenoneine can be produced appropriately by those skilled in the art. Selenoneine can be produced by chemical synthesis (Angew. Chem. Int. Ed. 2019, 58, 1-6), extraction from biological tissues containing selenoneine, or fermentation by microorganisms. For example, as described in Japanese Patent No. 5669056, it can be extracted from tissues of squids, fish, birds, mammals, etc.; as described in Pluskal T et al. (PLoS One 2014 May 14;9(5):e97774), it can be extracted from Schizosaccharomyces pombe ( Schizosaccharomyces pombe ) which is described in WO 2017 / 026173, and which utilizes histidine and selenium compounds in Aspergillus sojae ( Aspergillus sojae ), Aspergillus oryzae ( Aspergillus oryzae ), Aspergillus niger ( Aspergillus niger ) and Escherichia coli ( Escherichia coli Among these, when the fragmentation inhibitor of one embodiment of the present invention is produced on an industrial scale, the method described in WO 2017 / 026173 is preferred from the viewpoint of producing selenoneine in high yield.

[0016] In the method using a transformant overexpressing selenoneine synthase described in WO 2017 / 026173, ergothioneine is produced simultaneously with selenoneine and it is difficult to separate them, so that the resulting selenoneine-containing transformant extract may contain ergothioneine in addition to selenoneine. If available, the selenoneine is preferably purified selenoneine. Selenoneine can be purified by a method known to those skilled in the art, such as HPLC.

[0017] Ergothioneine may be commercially available, or may be produced by a person skilled in the art. As a method for producing ergothioneine, a chemical synthesis method (JP Patent Publication No. 2006-160748) and a method for extracting ergothioneine from organisms known to contain ergothioneine (e.g., basidiomycetes such as Pleurotus cornucopiae) (JP Patent Publication No. 4865083 and Patent Publication No. 5231025) as well as a method for extracting ergothioneine from microorganisms capable of producing ergothioneine (WO 2016 / 104437) are known.

[0018] As used herein, the term "fertilized egg" refers to a fertilized egg, and includes an early embryo in which cleavage has occurred. The term "early embryo" refers to an embryo from the 1-cell stage to the blastocyst stage after fertilization, and an embryo in the blastocyst stage may be hatched. Among early embryos, an embryo in the 2-cell stage to the 8-cell stage is particularly called a cleaved embryo. The early embryo forms a morula after the 8-cell stage, and then a blastocyst. The fertilized egg may be derived from any mammal, including a human. As a mammal, a human is preferable, but in another embodiment, the fertilized egg may be derived from a non-human mammal, such as a livestock animal, a laboratory animal, a zoo animal, or a pet animal. As a livestock animal, cows and pigs, which are subjected to artificial insemination or in vitro fertilization, are particularly mentioned.

[0019] Fertilized eggs grow into embryos by repeated division, and "inhibition of fragmentation" as used herein means inhibition of the formation of fragments during the growth process, particularly in early embryos at the 2-cell to 8-cell stages. Fragmentation usually occurs when only the cytoplasm divides, without nuclear division. The fragmentation of cytoplasm without cell nuclei can be confirmed by visual inspection in a bright field using a stereomicroscope. The degree of fragmentation is usually judged by visual inspection, and can be judged by classifying into grades 1 to 5 according to the Veeck classification according to the amount of fragmentation (Takahiro Nakayama: Clinical Gynecology and Obstetrics 2013:67(10), 1030-1037). The presence or absence of cell nuclei can be confirmed by visual inspection, as well as by observation with a fluorescent microscope, by adding a DNA-binding fluorescent reagent (Hoechst 33342) to the medium and leaving it in an incubator at 37°C for 10 minutes.

[0020] The fragmentation inhibitor may be added to a medium used for in vitro fertilization, artificial insemination, and intracytoplasmic sperm injection, or may be administered intravaginally. The fragmentation inhibitor may be added to a medium for gametes or fertilized eggs. In one embodiment, the fragmentation inhibitor may be added to a medium for sperm. The medium for sperm may be a medium for culturing sperm until they acquire fertilization capacity, or may be a medium for refrigerated or frozen storage. A fertilized egg fertilized or inseminated using sperm cultured in a culture medium containing a fragmentation inhibitor may suppress fragmentation in the subsequent development process. By culturing or storing sperm in a sperm medium to which a fragmentation inhibitor has been added, and washing the sperm with the medium before fertilization, it is possible to suppress the introduction of an excess amount of selenium into the egg. In another embodiment, the fragmentation inhibitor may be added to a medium for eggs and / or fertilized eggs. The medium for eggs may be a medium for refrigerated or frozen storage, or may be a medium for fertilization. Fragmentation can be inhibited during early embryo development by including a fragmentation inhibitor in one or more of the egg storage medium, fertilization medium, and fertilized egg culture medium.

[0021] Therefore, the fragmentation inhibitor of the present invention can be called a sperm or egg treatment agent, and fragmentation after fertilization is suppressed by treating sperm or eggs with a medium containing the fragmentation inhibitor. In yet another embodiment, the fragmentation inhibitor of the present invention can also be called an embryo development promoter. By suppressing fragmentation while promoting embryo development, it is possible to produce cleaved embryos, morulae, or blastocysts with a high implantation rate.

[0022] In yet another embodiment, the present invention relates to a medium for sperm, ovum, fertilized egg, or early embryo, comprising the fragmentation inhibitor of the present invention. These media may be medium for storage or culture. The storage medium may be a medium for refrigerated storage or frozen storage. The medium for sperm, medium for ovum, medium for fertilized egg, and medium for early embryo may be the same or different. When a medium exchange is performed after fertilization, a different medium may be used. Selenoneine or ergothioneine may be added at a final concentration of 1 nm to 10 μM in each medium. From the viewpoint of exerting the fragmentation inhibitory effect, the final concentration is usually preferably 5 nM or more, more preferably 10 nM or more, and even more preferably 50 nM or more. From the viewpoint of not exerting toxicity, the final concentration is usually preferably 100 μM or less, more preferably 50 μM or less, and even more preferably 20 μM or less.

[0023] The medium for sperm, eggs, fertilized eggs, or early embryos may be any medium available or commonly used in the art, to which the fragmentation inhibitor of the present invention may be added. Such media include, but are not limited to, TYH medium, HTF medium, CZB medium, and the like. Such media contain any components commonly used in the art. Components contained in these media include, for example, electrolytes, polymers, substances that serve as energy sources, pH adjusting substances, and serum components, and may be appropriately selected according to the intended medium. In general, freezing media may not contain electrolytes in order to prevent salt damage, and a cryoprotectant such as glycerol may be added in order to prevent freezing damage.

[0024] Examples of electrolytes include sodium chloride, potassium chloride, calcium chloride, magnesium chloride, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium bicarbonate, sodium acetate, and magnesium sulfate. Electrolytes can be added to adjust pH and osmotic pressure. Examples of polymers include polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), and Ficoll. Furthermore, examples of energy sources include glucose, lactic acid, pyruvic acid, and salts thereof (such as sodium lactate and sodium pyruvate). Examples of pH adjusting substances include buffers such as HEPES, HES, TRIS, and MES.

[0025] Furthermore, the fertilization medium may contain antibiotics such as penicillin, streptomycin, and amphotericin B, if necessary. In addition, the medium may contain a pH indicator such as phenol red so that the change in pH of the medium can be visually observed.

[0026] The fragmentation inhibitor may contain one or both of selenoneine and ergothioneine as active ingredients. The fragmentation inhibitor may further contain a known substance that is known to have an effect of suppressing the fragmentation of fertilized eggs.

[0027] (Sperm culture method) In another embodiment of the present invention, the present invention also relates to a method for culturing sperm using a medium containing the fragmentation inhibitor of the present invention. Specifically, the method for culturing sperm includes a step of culturing collected sperm (unfrozen sperm) or frozen-thawed sperm in a medium containing a fragmentation inhibitor. When frozen-thawed sperm are used, selenoneine is particularly preferable as the fragmentation inhibitor. In one example of a method for culturing sperm, a droplet of culture medium is formed on a dish, mineral oil is layered, and collected sperm is added to the medium placed under a 37°C 5% carbon dioxide atmosphere and cultured. By culturing for 30 minutes to several hours, the sperm usually undergo hyperactivation. The cultured sperm can be used for artificial insemination or in vitro fertilization. When cryopreserving, the collected sperm can be transferred to a freezing medium, dispensed into containers such as straws for cryopreservation, and frozen in liquid nitrogen. Before cryopreservation, the sperm may be cultured in a medium containing a fragmentation inhibitor, or the freezing medium may contain a fragmentation inhibitor and then frozen, or the sperm may be washed or cultured after freezing and thawing by adding a fragmentation inhibitor to the washing solution, thawing solution or culture medium.

[0028] (Oocyte culture method) In another embodiment of the present invention, the present invention also relates to a method for culturing eggs using a medium containing the fragmentation inhibitor of the present invention. Specifically, the method for culturing eggs includes a step of culturing collected eggs (unfrozen eggs) or frozen-thawed eggs in a medium containing a fragmentation inhibitor. As an example of the method for culturing eggs, droplets of culture medium are formed on a dish, mineral oil is overlaid, and collected eggs are cultured in the medium placed under a 37°C, 5% carbon dioxide atmosphere. In vitro fertilization (IVF) can be performed by adding a culture medium containing sperm to the culture medium containing eggs. In the case of cryopreservation, collected eggs can be transferred to a freezing medium, placed in a straw or wire mesh for cryopreservation, or frozen in liquid nitrogen. Before cryopreservation, eggs may be cultured in a medium containing a fragmentation inhibitor, or a fragmentation inhibitor may be included in the freezing medium and frozen, or a fragmentation inhibitor may be included in the washing solution, melting solution, or culture medium after freezing and thawing to wash or culture.

[0029] (fertilization method) Another embodiment of the present invention may relate to a fertilization method using a medium containing the fragmentation inhibitor according to the present invention. In the fertilization method of the present invention, fertilization is performed using a medium containing the fragmentation inhibitor. More specifically, the medium in which the egg is placed may contain the fragmentation inhibitor, or the medium in which the sperm is cultured may contain the fragmentation inhibitor. It is preferable that the medium in which the egg is placed and the medium in which the sperm is cultured both contain the fragmentation inhibitor. The fertilization method includes a step of mixing the medium in which the egg is placed with the medium containing the sperm. In yet another method, fertilization can be performed by selecting and obtaining sperm in good condition from the medium containing the sperm, and injecting the sperm into the egg by intracytoplasmic sperm injection. The fertilized egg after fertilization may be cultured in the same medium, or may be cultured by replacing the medium as the embryo develops. The replaced medium may also contain the fragmentation inhibitor. The fertilized egg after fertilization can be transferred to a freezing medium, placed in a straw or wire mesh for cryopreservation, or the like, and frozen in liquid nitrogen. Before cryopreservation, the eggs may be cultured in a medium containing a fragmentation inhibitor, or the freezing medium may contain a fragmentation inhibitor and then frozen, or the eggs may be washed or cultured after freezing and thawing by adding a fragmentation inhibitor to the washing solution, thawing solution or culture medium.

[0030] (Method of culturing fertilized eggs or embryos) After fertilization, the fertilized egg can be cultured as it is, or the medium can be changed. It is preferable to culture in a medium containing a fragmentation inhibitor. The fertilized egg or embryo may be unfrozen or may be frozen and thawed. In the method for culturing the fertilized egg, the fertilized egg may be used immediately after fertilization, or the fertilized egg that has been cryopreserved may be used. After fertilization, the fertilized egg passes through the 2-cell stage, the 4-cell stage, and the 8-cell stage over several days to form a morula, and then forms a blastocyst. Therefore, the method for culturing the fertilized egg or embryo, together with the fertilization method, can be said to be a method for producing a cleaved embryo, a morula, or a blastocyst. The cleaved embryo, the morula, or the blastocyst thus produced can be returned to the uterus.

[0031] (Infertility improver) The present invention also relates to an infertility improving agent comprising selenoneine or ergothioneine, or a tautomer or dimer thereof, or a medicamentously acceptable salt thereof. It has been reported that ergothioneine is not produced in the mammalian body, and accumulates in a specific organ when orally ingested. It is known that ergothioneine is distributed in the seminal vesicles together with its transporter, the ergothioneine transporter, and is contained in the seminal plasma secreted from the seminal vesicles (J. Physiology and Pharmacology (2011), 62, 4, 411-419). It is also known that ergothioneine accumulates in eggs of birds when they are fed a diet containing ergothioneine (International Publication No. WO 2015 / 098380). Selenoneine is a serine analog of ergothioneine, and is therefore thought to be distributed in the same manner in the living body. Selenoneine and ergothioneine can be accumulated in the seminal vesicles or ovaries by oral ingestion, are contained in the seminal plasma secreted from the seminal vesicles, and have a fragmentation inhibitory effect by acting on sperm and eggs. Therefore, selenoneine or ergothioneine, or its tautomer or dimer, or its medicamentously acceptable salt, can be used as an infertility improver and can be formulated for oral ingestion. The infertility improver of the present invention can be particularly used as an infertility improver for men or women. The infertility improver of the present invention can be sold as a medicine, a quasi-drug, or a functional food. Examples of functional food include supplements, nutritionally enhanced foods, and nutritionally enhanced drinks. When used as an infertility improver for oral ingestion, for example, 0.1 to 100 mg of selenoneine or ergothioneine, or its tautomer or dimer, or its medicamentously acceptable salt can be administered as a daily dose.

[0032] (Infertility treatment methods) Infertility treatment methods typically include artificial insemination, in vitro fertilization, and microinsemination. These treatment methods include culturing collected sperm or eggs, or fertilized eggs, in a medium containing a fragmentation inhibitor. Specific steps of the infertility treatment method are described, for example, in (Fertility and Sterility (2017) vol. 107, No.4 pp.882-896). In yet another embodiment, a fragmentation inhibitor, or selenoneine or ergothioneine, or a tautomer or dimer thereof, or a medicamentously acceptable salt thereof, can also be administered for infertility treatment. Any route of administration may be used, but oral administration may be used as an example. The subjects of administration are women who wish to become pregnant and their partners. In particular, the agent may be administered to male subjects suffering from male infertility, such as spermatogenesis failure.

[0033] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples and can take various forms as long as the object of the present invention can be achieved. All documents referred to in this specification are incorporated herein by reference in their entirety. EXAMPLES

[0034] Example 1. Preparation of selenoneine Preparation of selenoneine-containing fungal extract and ergothioneine-containing fungal extract A selenoneine-containing fungal extract was obtained by the method described in Example 7 using transformed Aspergillus sojae transformed with the gene AsEgtA described in WO 2017 / 026173.

[0035] Purification of selenoneine (1) Preparation of test samples and reference samples A portion of the selenoneine-containing fungal extract obtained in Example 1 (selenoneine concentration: 136.8 μg / mL) was subjected to freeze-drying and then concentrated by dissolving in a small amount of water. The concentrated sample was fractionated under the following HPLC conditions. Equipment: Pump LC-8A, UV / VIS detector SPD-20A, fraction collector FRC-10A (Shimadzu Corporation) Column: Develosil RPAQUEOUS-AR-5 20 x 250 mm (Nomura Chemical) Eluent: 5mM ammonium acetate Flow rate: 5mL / min Detection: 220nm Fractions: 1.25 mL each

[0036] A portion of the selenoneine obtained through the steps of fractionation by preparative HPLC, freeze-drying, and dissolution in 80% ethanol was dried in a centrifugal evaporator and then dissolved in water. The concentration was confirmed by LC-ICP-MS, and then adjusted to 10 mM (2.77 mg / mL) with water. The results of analytical HPLC of the 80% ethanol supernatant fraction after preparative HPLC purification are shown in FIG.

[0037] Example 2. Sperm motility test A TYH medium having the following composition was prepared. [Table 1]

[0038] A 100μL drop of TYH medium was prepared on a plastic petri dish, and liquid paraffin was layered over the drop until it was completely covered. The plastic dish was then left to stand for 16 hours in an incubator set to 5% CO2 and 37℃. The TYH medium drop on the left plastic dish was used for sperm motility and fertilization tests.

[0039] A mass of sperm collected from the epididymis of 8-12 week-old sexually mature male mice (C57BL / 6J strain) was transferred to a drop made from TYH medium. The plastic petri dish was then placed back in the incubator and left to stand for 1 hour. After leaving it for 1 hour, 1 μL of selenoneine (10 μM) prepared in Example 1 was added to the drop of fertilization medium containing sperm (no addition as a control), and left to stand in the incubator. After 3 hours of incubation, the motility of sperm was measured using CASA, and the sperm were classified into motile sperm and progressive sperm, and the percentage of sperm was measured (Figures 2A and B). After 6 hours of incubation, the sperm were classified based on their motility (VCL) in the same way, and the percentage of sperm was measured (Figures 2C and D). No effect on sperm motility was observed between the control group and the selenoneine-added group.

[0040] Example 3: Inhibitory effect of selenoneine treatment on fertilized egg fragmentation Female mice (C57BL / 6J strain: 8 to 12 weeks old) were subjected to superovulation treatment according to the following procedure to obtain oocytes. 1. Serotonin for animals (PMSG, Aska Pharmaceutical) and gonadotropin for animals (hCG, Aska Pharmaceutical) were dissolved in physiological saline to a concentration of 37.5 units / mL. 2. Mice were intraperitoneally administered PMSG at 0.1 mL (5 units) / mouse, and 48 hours later, intraperitoneally administered hCG at 0.1 mL (5 units) / mouse. 3. After administration of hCG, cumulus cell-oocyte masses were extracted from the mouse oviducts 14 to 16 hours later.

[0041] Cumulus cell-egg masses collected from the oviducts of superovulated mice were transferred to 100 μL drops of TYH medium that had been incubated in advance at 5% CO 2 and 37° C., and then left to stand in an incubator.

[0042] Selenoneine treatment of eggs The obtained eggs were cultured in TYH medium for 2 hours, and then 1 μL of the selenoneine (10 μM) solution prepared in Example 1 was added to the drop of TYH medium (no addition as a control), and the final concentration of selenoneine in the medium was set to 100 nM, and the eggs were cultured for another 2 hours. After the culture, 10 μL of the sperm incubated in the control TYH medium without selenoneine in Example 2 for 6 hours was taken together with the medium and added to the TYH medium containing the cumulus cell-egg mass. After 24 hours in the presence of selenoneine, the fertilization rate was measured, and the number of normal 2-cell stage embryos and fragmented embryos was examined. The fertilization rate was determined by visually observing the release of the second polar body and the presence or absence of cell nuclei present in each blastomere of the 2-cell stage embryo in the bright field of a stereomicroscope, and the normal 2-cell stage embryos and fragmented embryos were also visually observed in the bright field of a stereomicroscope. A photograph in the bright field is shown in FIG. 3A. Although there was no difference in the rate of formation of 2-cell stage embryos (Fig. 3A), no embryos underwent fragmentation when eggs were treated with selenoneine, and selenoneine suppressed fragmentation (Fig. 3B).

[0043] Selenoneine treatment of sperm As in Example 2, spermatozoa incubated in TYH medium containing selenoneine (final concentration 100 nM) for 6 hours were separated together with the medium, washed in TYH medium previously incubated at 37°C under 5% CO2 atmosphere, and the washed spermatozoa were separated and added to the TYH medium containing the cumulus cell-egg mass. After 24 hours, the fertilization rate was measured, and the number of normal 2-cell stage embryos and fragmented embryos was examined. The fertilization rate was determined by visually observing the release of the second polar body and the presence or absence of cell nuclei present in each blastomere of the 2-cell stage embryo in a bright field of a stereomicroscope, and the number of normal 2-cell stage embryos and fragmented embryos was also determined by visually observing the bright field of a stereomicroscope. The results are shown in Figure 4. There was no difference in the formation rate of 2-cell stage embryos (Figure 4A), but when the eggs were treated with selenoneine, there were no fragmented embryos, and selenoneine suppressed fragmentation (Figure 4B).

[0044] Example 4: Fragmentation suppression effect by ergothioneine treatment Ergothioneine and selenoneine treatment of sperm As in the selenoneine treatment of sperm in Example 3, the inhibitory effect of ergothioneine treatment on the fragmentation of fertilized eggs was examined using commercially available ergothioneine (final concentration 100 nM) instead of selenoneine (Figure 5). There was no difference in the rate of formation of 2-cell stage embryos between ergothioneine treatment and selenoneine treatment (Figure 5A). On the other hand, no embryos underwent fragmentation in the case of selenoneine treatment, whereas the rate of embryos underwent fragmentation in the case of ergothioneine treatment was reduced compared to the untreated control (Figure 5B).

[0045] Example 5: Inhibitory effect of selenoneine and ergothioneine on stress-induced fragmentation Selenoneine treatment of eggs The mouse cumulus cell-egg mass obtained by the method of Example 3 was stored in TYH medium, and cumulus cells were removed by treatment with 300 μg / mL hyaluronidase to recover only the eggs. TYH medium containing 10 nM, 100 nM, 1 μM, 10 μM, and 100 μM selenoneine was prepared and incubated on a hot plate set at 37° C. THY medium without selenoneine was used as a control. The recovered eggs were transferred to the incubated TYH medium containing selenoneine and cultured in the dark. After 12, 15, 18, 21, and 24 hours, the eggs were observed under a stereomicroscope to determine the proportion of fragmented eggs (FIG. 6). The numbers of oocytes cultured in each medium containing selenoneine were as follows: 10 nM: 107, 100 nM: 161, 1 μM: 155, 10 μM: 118, 100 μM: 85, and control: 83.

[0046] By culturing eggs on a hot plate rather than in a CO2 incubator, stress is applied to the eggs. After 18 hours of culturing on the hot plate, the percentage of eggs that showed fragmentation increased. On the other hand, when the eggs were cultured in TYH medium containing selenoneine at concentrations of 100nM, 1μM, or 10μM, fragmentation of the eggs was significantly suppressed (p<0.05). The number of eggs cultured in each medium was 10nM: 63, 100nM: 66, 1μM: 63, 10μM: 71, 100μM: 54, and control: 83.

[0047] Ergothioneine treatment of eggs The mouse cumulus cell-egg mass obtained by the method of Example 3 was stored in TYH medium, and the cumulus cells were removed by treatment with 300 μg / mL hyaluronidase to recover only the eggs. TYH medium containing 10 nM, 100 nM, 1 μM, 10 μM, and 100 μM ergothioneine was prepared and incubated on a hot plate set at 37°C. As a control, THY medium without ergothioneine was used. The recovered eggs were transferred to the incubated TYH medium containing selenoneine and cultured in the dark, and observed under a stereomicroscope after 12, 15, 18, 21, and 24 hours to examine the proportion of fragmented eggs (Figure 7).

[0048] By culturing eggs on a hot plate rather than in a CO2 incubator, stress is applied to the eggs. After 18 hours of culturing on the hot plate, the percentage of eggs that showed fragmentation increased. After 21 and 24 hours of culturing, when the eggs were cultured in TYH medium containing ergothioneine at a concentration of 1 μM, fragmentation of eggs was significantly suppressed (p<0.05), and a tendency for suppression was observed at concentrations of 10 nM to 10 μM after 21 and 24 hours of culturing.

[0049] Example 6: Oxidative stress test The mouse cumulus cell-egg mass obtained by the method of Example 3 was stored in TYH medium, and the cumulus cells were removed by treatment with 300 μg / mL hyaluronidase to recover only the oocytes. TYH medium containing 1 μM selenoneine (SeN), TYH medium containing 1 μM ergothioneine (Erg), and TYH medium containing 1 μM ergothioneine and 1 μM ebselen (Tokyo Chemical Industry Co., Ltd.) (Erg+Eb) were prepared and incubated on a hot plate set at 37°C. As a control, TYH medium containing DMSO (0.1%) used to dissolve ebselen (Vehicle) was used. The recovered oocytes were transferred to the incubated selenoneine-containing TYH medium and cultured for 15 hours in the dark. The reagents of the ReOIS-ID ROS / RNS Detection Kit (Enzo) were added to the medium containing the oocytes after the culture according to the product instructions. In addition, DAPI (Wako Pure Chemical Industries, Ltd.) was added separately in advance to stain the nuclei. Nuclei (DAPI), reactive oxygen species (ROS), reactive nitrogen species (RNS), and superoxide in the oocytes were observed under a fluorescent microscope (Figure 8A). Fluorescence intensity was calculated and graphed (Figures 8B-D).

[0050] Reactive oxygen species increased significantly in the medium supplemented with selenoneine and ergothioneine, whereas they decreased in the medium supplemented with the antioxidant ebselen (Erg+Eb). Reactive nitrogen species decreased significantly with the addition of selenoneine, but no change was observed with the addition of ergothioneine, and they increased in the medium supplemented with the antioxidant ebselen (Erg+Eb). Superoxide did not change with the addition of selenoneine or ergothioneine, but they increased significantly in the medium supplemented with ebselen (Erg+Eb). In summary, the amounts of reactive oxygen species, reactive nitrogen species, and superoxide were not consistent, and no association was found between selenoneine and ergothioneine and oxidative stress.

[0051] Example 7: Protective effect of selenoneine and ergothioneine treatment on sperm Selenoneine treatment of sperm A mass of sperm collected from the epididymis of a sexually mature male mouse (C57BL / 6J strain) aged 8 to 12 weeks was transferred to a drop made from TYH medium. The plastic petri dish was then returned to the incubator and left to stand for 1 hour. After standing for 1 hour, 1-selenoneine prepared in Example 1 was added to the drop of fertilization medium containing sperm so as to have a final concentration of 10 nM, 1 μM, and 100 μM (no addition as a control), and the mixture was cultured in the incubator for 3 hours, 6 hours, and 9 hours to obtain sperm. In addition, sperm before culture (0 h) was obtained. 2 μg / mL propidium iodide (PI: Sigma-Aldrich) and 16.2 μM Hoechst 3342 (Hoechst 3342: Invitrogen) were added to the obtained sperm to stain the sperm nuclei, and the sperm nuclei were observed under a fluorescent microscope (FIG. 9A). The percentage of sperm with intact membranes was calculated by dividing the number of sperm whose nuclei were not stained with propidium iodide by the number of sperm whose nuclei were stained with Hoechst (Figure 9B).

[0052] Hoechst is cell membrane permeable and can stain the nuclei of all sperm, whereas propidium iodide is membrane impermeable and can stain only the nuclei of dead sperm or sperm with damaged membranes. Therefore, sperm that are not stained with propidium iodide and are stained only with Hoechst represent normal sperm with membranes maintained. The percentage of sperm with membranes maintained decreased with the passage of culture time. Selenoneine exerted a sperm membrane protective effect when added at concentrations of 10 nM to 100 μM, and after 9 hours of culture, it significantly protected the sperm membrane at a concentration of 100 μM (p<0.05).

[0053] Ergothioneine processing of sperm A mass of sperm collected from the epididymis of a sexually mature male mouse (C57BL / 6J strain) aged 8 to 12 weeks was transferred to a drop made from TYH medium. The plastic petri dish was then returned to the incubator and left to stand for 1 hour. After leaving it for 1 hour, 1-selenoneine prepared in Example 1 was added to the drop of fertilization medium containing sperm so as to have a final concentration of 10 nM, 1 μM, and 100 μM (no addition as a control), and the mixture was cultured in the incubator for 3 hours, 6 hours, and 9 hours to obtain sperm. In addition, sperm before culture (0 h) was obtained. 2 μg / mL propidium iodide (PI: Sigma-Aldrich) and 16.2 μM Hoechst 3342 (Hoechst3342: sold by Invitrogen) were added to the obtained sperm to stain the sperm nuclei, and the sperm nuclei were observed under a fluorescent microscope. The percentage of sperm with intact membranes was calculated by dividing the number of sperm whose nuclei were not stained with propidium iodide by the number of sperm whose nuclei were stained with Hoechst (Figure 9C). Hoechst is cell membrane permeable and can stain the nuclei of all sperm, whereas propidium iodide is membrane impermeable and can stain only the nuclei of dead sperm or sperm with damaged membranes. Therefore, sperm that are not stained with propidium iodide and are stained only with Hoechst represent normal sperm with membranes maintained. The percentage of sperm with membranes maintained decreased with the passage of culture time. Ergothioneine exerted a sperm membrane protective effect when added at concentrations of 10 nM to 100 μM, and after 9 hours of culture, it significantly protected the sperm membrane at a concentration of 100 μM (p<0.05). [Industrial Applicability]

[0054] The fragmentation inhibitor according to one embodiment of the present invention is capable of suppressing fragmentation and destruction of eggs and fertilized eggs by the active ingredient selenoneine or ergothioneine, and also capable of protecting the cell membrane of sperm, and is therefore considered to contribute to the improvement of infertility treatment. More specifically, it is expected to improve the implantation rate and pregnancy rate in infertility treatment such as artificial insemination and in vitro fertilization. It is also expected to improve the implantation rate and pregnancy rate in fields such as livestock farming, where artificial insemination and in vitro fertilization are often performed.

Claims

[Claim 1] An agent for suppressing the fragmentation of fertilized eggs, comprising selenoneine or ergothioneine, or a tautomer or dimer thereof, or a pharma- ceutical acceptable salt thereof.

Images