Embryo preservation solution containing trehalose and diarylheptanoid compounds

Abstract

To provide means for obtaining a high-quality embryo that develops well and is fit for embryo transfer to an animal.SOLUTION: A liquid comprising trehalose or a derivative thereof or salts thereof and a diarylheptanoid compound or a derivative thereof or salts thereof is used to preserve an embryo.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a preservation solution for embryos containing trehalose or its derivatives or their salts, and diarylheptanoid compounds or their derivatives or their salts, a powder preparation for preparing the preservation solution, and a method for preserving embryos using the preservation solution.

Background Art

[0002] Technologies such as in vitro fertilization, embryo culture, and embryo transfer are used in the medical field for regenerative medicine, assisted reproductive medicine, etc., and in the livestock field for increasing the production of excellent livestock and livestock improvement, etc., and have now become indispensable technologies. Here, after fertilization, the blastomeres of mammalian embryos increase into 2-cell stage embryos, 4-cell stage embryos, and 8-cell stage embryos by cleavage. Then, through the morula stage, it develops into a blastocyst (early blastocyst - hatched blastocyst). However, even when mammalian embryos cultured in vitro, for example, hatched blastocysts are transplanted into the uterus, the success rate (pregnancy rate) is low. For example, in cows, it remains at about 40%, and in humans, it remains at about 30%. Generally, the quality of embryos produced by in vitro culture is lower compared to embryos fertilized in vivo, so there is a strong demand for technologies to obtain high-quality embryos suitable for embryo transfer.

[0003] Here, referring to Patent Document 1 as a technology for obtaining high-quality mammalian embryos, for example, a method of immersing mammalian embryos in a medium containing 20 - 80% (v / v) serum and 10 - 100 mM Good buffer and preserving the embryos at a non-freezing low temperature is disclosed. Also, referring to Non-Patent Document 1, a method of treating oocytes with trehalose during in vitro maturation is disclosed. Furthermore, referring to Patent Document 2, a method of irradiating mammalian embryos with near-infrared light is disclosed.

[0004] On the other hand, referring to Non-Patent Document 2, it has been reported that when curcumin, a yellow polyphenol compound found in turmeric (Curcuma longa), is administered to mouse blastocysts, apoptosis significantly increases, the total number of cells decreases, and furthermore, a decrease in implantation rate in the mouse uterus and a decrease in fetal weight in embryo transfer assays are observed. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2014 / 030211 Pamphlet [Patent Document 2] Japanese Patent Publication No. 2016-146823 [Non-patent literature]

[0006] [Non-Patent Document 1] Theriogenology 2020, 141, 91-97 [Non-Patent Document 2] Int.J.Mol.Sci. 2010, 11, 2839-2855 [Overview of the project] [Problems that the invention aims to solve]

[0007] The object of the present invention is to provide a means for obtaining high-quality embryos that develop well and are suitable for embryo transfer to animals. [Means for solving the problem]

[0008] Referring to Non-Patent Document 2 mentioned above, curcumin is thought to inhibit embryonic development. However, the inventors of this invention have surprisingly found that when curcumin is added to an embryo preservation solution containing trehalose, embryonic development is actually better compared to when curcumin is not added. This invention was completed based on this finding.

[0009] In other words, the present invention is defined as follows: [1] An embryo preservation solution comprising trehalose or its derivatives or salts thereof, and diarylheptanoid compounds or their derivatives or salts thereof. [2] The preservative solution described in [1] above, wherein the diarylheptanoid compound is a curcuminoid. [3] The preservative solution described in [2] above, wherein the curcuminoid is curcumin. [4] A preservative solution according to any one of [1] to [3] above, comprising trehalose or its derivatives or salts thereof, and curcumin or its derivatives or salts thereof in an isotonic solution. [5] The storage method described in [4] above, where the isotonic solution is Ringer's solution. use liquid. [6] A preservative solution according to any of [1] to [5] above, further comprising polysaccharides or their derivatives or salts thereof. [7] The preservative solution described in [6] above, wherein the polysaccharide is dextran. [8] A preservative solution according to any of [1] to [7] above, wherein the concentration of trehalose or its derivative or salt thereof is 2.0 to 6.0 (w / v)%. [9] The concentration of diarylheptanoid compounds or their derivatives or salts thereof is 0. 000 2-0. 000 A preservative solution according to any of the above [1] to [8], which is 6(w / v)%.

[10] A preservative solution according to any of [1] to [9] above, wherein the concentration of polysaccharides or their derivatives or salts thereof is 4.0 to 7.0 (w / v)%.

[11] A preservation solution described in any of [1] to

[10] above for preserving embryos that have been 2 to 7 days after fertilization.

[12] A preservation solution described in any of [1] to

[11] above for preserving embryos for 12 to 36 hours.

[13] A preservation solution described in any of [1] to

[12] above for preserving embryos at 0 to 40°C.

[14] A preservation solution according to any of [1] to

[13] above, used to suppress a decrease in the rate of embryonic morphological recovery, a decrease in the rate of development, or an increase in the rate of apoptosis.

[15] A preservation solution according to any of [1] to

[14] above, wherein the embryo is a blastocyst-stage embryo.

[16] A preservation solution according to any of [1] to

[15] above, wherein the embryo is a mammalian embryo.

[17] A preservation solution according to any of [1] to

[16] above, wherein the embryo is a human, pig, cattle, or horse embryo.

[18] A powder formulation containing trehalose or a derivative thereof or a salt thereof, and diarylheptanoid compounds or derivatives thereof or salts thereof, for preparing the preservative solution described in any of [1] to

[17] above.

[19] A method for preserving embryos, comprising the step of preserving the embryos in a preservation solution containing trehalose or its derivatives or salts thereof, and diarylheptanoid compounds or their derivatives or salts thereof.

[0010] Other embodiments of the present invention include, for example, the following:

[20] A method for embryo transfer, comprising the step of administering a solution containing an embryo, trehaloses, and diarylheptanoid compounds to a subject requiring embryo transfer (for example, a mammal such as a human, pig, cattle, or horse).

[21] A method for embryo transfer comprising the steps of: preparing a preservation solution by adding an embryo to a solution containing trehaloses and diarylheptanoid compounds (preferably an isotonic solution), or by adding trehaloses and diarylheptanoid compounds to a solution containing an embryo (preferably an isotonic solution); preserving the embryo in the prepared preservation solution; and administering the preservation solution containing the preserved embryo to a subject requiring embryo transfer (for example, a mammal such as a human, pig, cattle, or horse).

[22] A solution containing embryos, trehaloses, and diarylheptanoid compounds for use in the treatment of diseases or conditions requiring embryo transfer therapy (e.g., infertility). Use of trehaloses and diarylheptanoid compounds in the production of a preservation solution for embryos. Use of trehaloses and diarylheptanoid compounds for preserving a solution containing embryos. Use of trehaloses and diarylheptanoid compounds for suppressing a decrease in the morphological recovery rate, a decrease in the growth rate, or an increase in the apoptosis rate of embryos. Trehaloses and diarylheptanoid compounds for use in a preservation solution for embryos. A solution containing embryos and comprising trehaloses and diarylheptanoid compounds. A method for preparing a preservation solution containing embryos, the method comprising a step of preparing the preservation solution by adding embryos to a solution containing trehaloses and diarylheptanoid compounds (preferably an isotonic solution), or by adding trehaloses and diarylheptanoid compounds to a solution containing embryos (preferably an isotonic solution).

Advantages of the Invention

[0011] According to the present invention, by using a solution containing trehalose or its derivative or their salts, and a diarylheptanoid compound or its derivative or their salts for embryo preservation, high-quality embryos with good growth and suitable for embryo transfer to animals can be obtained.

Embodiments for Carrying Out the Invention

[0012] The preservation solution for embryos of the present invention is a solution (sometimes referred to as "the preservation solution of the present case" in the present application) containing trehalose or its derivative or their salts (sometimes referred to as "trehaloses" in the present application), and a diarylheptanoid compound or its derivative or their salts (sometimes referred to as "diarylheptanoid compounds" in the present application), which is specified for the use of "preserving embryos".

[0013] Among the trehaloses mentioned above, examples of trehaloses include α,α-trehalose, a disaccharide in which two α-glucose molecules are linked by a 1,1-glycosidic bond; α,β-trehalose, a disaccharide in which α-glucose and β-glucose molecules are linked by a 1,1-glycosidic bond; and β,β-trehalose, a disaccharide in which two β-glucose molecules are linked by a 1,1-glycosidic bond. Of these, α,α-trehalose is preferred. These trehaloses can be produced by any known method, such as chemical synthesis, microbial production, or enzymatic production, but commercially available products can also be used. For example, commercially available products such as α,α-trehalose (manufactured by Hayashibara Corporation or Fujifilm Wako Pure Chemical Industries, Ltd.) can be used.

[0014] The trehalose derivatives in the above-mentioned trehaloses are not particularly limited as long as they are glycosyltrehaloses in which one or more sugar units are bonded to the disaccharide trehalose, and glycosyltrehaloses include glucosyltrehalose, maltosyltrehalose, maltotriosyltrehalose, etc.

[0015] Examples of salts of trehalose or its derivatives in the above-mentioned trehalose compounds include acid addition salts such as hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate, sulfate, acetate, propionate, toluenesulfonate, succinate, oxalate, lactate, tartrate, glycolate, methanesulfonate, butyrate, valerate, citrate, fumarate, maleate, and malate; metal salts such as sodium salt, potassium salt, and calcium salt; and ammonium salts and alkylammonium salts. These salts are used as solutions, and it is preferable that their effects are the same as those of trehalose. These salts may form hydrates or solvates, and either one or two or more can be used individually or in appropriate combinations.

[0016] The concentration of trehaloses in the preservation solution should be such that it suppresses the reduction in embryo morphological recovery rate, the decrease in developmental rate, or the increase in apoptosis rate. For example, it should be 0.1(w / v)% or more, preferably 0.3(w / v)% or more, more preferably 0.6(w / v)% or more, even more preferably 1.0(w / v)% or more, and most preferably 2.0(w / v)% or more in terms of trehalose. Furthermore, from the viewpoint of avoiding adverse effects on the embryo, for example, it should be 40(w / v)% or less, preferably 20(w / v)% or less, more preferably 15(w / v)% or less, even more preferably 10%(w / v)% or less, and most preferably 6.0(w / v)% or less in terms of trehalose. Therefore, the concentration of trehaloses in the preservation solution is, for example, in the range of 0.1 to 40 (w / v)% in terms of trehalose, preferably 0.3 to 20 (w / v)%, more preferably 0.6 to 15 (w / v)%, even more preferably 1.0 to 10% (w / v)%, and most preferably 2.0 to 6.0 (w / v)%.

[0017] The diarylheptanoid compounds in the above-mentioned group of diarylheptanoid compounds may be linear or cyclic diarylheptanoid compounds, but linear diarylheptanoid compounds are preferred. Curcuminoids are examples of linear diarylheptanoid compounds. Examples of curcuminoids include curcumin (enol and keto forms), demethoxycurcumin, bisdemethoxycurcumin, and tetrahydrocurcumin. Further analogues include dihydrocurcumin, dihydrodemethoxycurcumin, dihydrobisdemethoxycurcumin, tetrahydrodemethoxycurcumin, tetrahydrobisdemethoxycurcumin, dihydroxytetrahydrocurcumin, di-O-demethylcurcumin, and O-demethyldemethoxycurcumin. Curcumin is particularly preferred as its effects have been demonstrated in the embodiment described later. In the present invention, diarylheptanoid compounds can be produced by known methods such as chemical synthesis and extraction from plants, but commercially available products can also be used. For example, curcumin can be obtained by extraction from plants belonging to the genus Curcuma of the family Zingiberaceae. There are no particular restrictions on the plants belonging to the genus Curcuma of the family Zingiberaceae, but turmeric (Curcuma longa) is preferred. In addition to curcumin, turmeric also contains demethoxycurcumin, bisdemethoxycurcumin, and tetrahydrocurcumin.

[0018] The diarylheptanoid compound derivatives in the above-mentioned diarylheptanoid compounds are not particularly limited, but examples include curcumin derivatives, demethoxycurcumin derivatives, bisdemethoxycurcumin derivatives, and tetrahydrocurcumin derivatives, which have substituents on the phenyl group of curcumin, demethoxycurcumin, bisdemethoxycurcumin, and tetrahydrocurcumin.

[0019] Examples of salts of diarylheptanoid compounds or their derivatives in the above-mentioned diarylheptanoid compounds include acid addition salts such as hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate, sulfate, acetate, propionate, toluenesulfonate, succinate, oxalate, lactate, tartrate, glycolate, methanesulfonate, butyrate, valerate, citrate, fumarate, maleate, and malate; metal salts such as sodium salt, potassium salt, and calcium salt; and ammonium salts and alkylammonium salts. These salts are used as a solution, and it is preferable that their action is the same as that of the diarylheptanoid compound or its derivative. These salts may form hydrates or solvates, and either one or two or more can be used individually or in appropriate combinations.

[0020] The concentration of diarylheptanoid compounds in the preservation solution should be such that it suppresses the reduction in embryo morphological recovery rate, the decrease in developmental rate, or the increase in apoptosis rate. For example, a concentration equivalent to 0. 000 0.1(w / v)% or higher, preferably 0. 000 0.5% (w / v) or higher, more preferably 0%. 000 1% (w / v) or more, more preferably 0%. 000 2% (w / v) or more, most preferably 0%. 000 The content is 3(w / v)% or higher. Furthermore, from the perspective of avoiding adverse effects on the embryo, for example, in terms of diarylheptanoid compounds... 0.00 4(w / v)% or less, preferably 0.00 2% (w / v) or less, more preferably. 0.00 1% (w / v) or less, more preferably 0%. 000 8(w / v)% or less, more preferably 0%. 000 6(w / v)% or less, most preferably 0. 000 The concentration is 5(w / v)% or less. Therefore, the concentration of diarylheptanoid compounds in the preservation solution is, for example, 0. 000 01~ 0.00Within the range of 4(w / v)%, preferably 0. 000 1~ 0.00 2% (w / v), more preferably 0%. 000 2~ 0.00 1% (w / v), more preferably 0%. 000 2-0. 000 8% (w / v), more preferably 0%. 000 2-0. 000 6% (w / v), most preferably 0%. 000 3-0. 000 The concentration is 5(w / v)%. That is, the concentration of diarylheptanoid compounds in the storage solution is, for example, in the range of 0.27 to 108 μM in terms of diarylheptanoid compounds, preferably 2.7 to 54 μM, more preferably 5.4 to 27 μM, even more preferably 5.4 to 21.6 μM, even more preferably 5.4 to 16.2 μM, and most preferably 8.1 to 13.5 μM.

[0021] The preservation solution in this specification can be any solution capable of preserving embryos (e.g., isotonic, hypotonic, or hypertonic solution) containing trehaloses and diarylheptanoid compounds, with isotonic solutions being a particularly suitable example. In this specification, "isotonic solution" means a solution having an osmotic pressure approximately the same as that of body fluids or cell fluid, specifically a solution having an osmotic pressure in the range of 250 to 380 mOsm / L. In this specification, "hypotonic solution" means a solution having an osmotic pressure lower than that of body fluids or cell fluid, specifically a solution having an osmotic pressure of less than 250 mOsm / L. As such a hypotonic solution, a hypotonic solution that does not cause cells to rupture (specifically, a solution having an osmotic pressure in the range of 100 to less than 250 mOsm / L) is preferred. Furthermore, in this specification, "hypertonic solution" means a solution having an osmotic pressure higher than that of body fluids or cell fluids, and specifically means an osmotic pressure greater than 380 mOsm / L (preferably in the range of greater than 380 mOsm / L to 1000 mOsm / L).

[0022] The above-mentioned isotonic solution is not particularly limited as long as it is an isotonic solution in which the salt concentration, sugar concentration, etc., is adjusted with sodium ions, potassium ions, calcium ions, etc., so that it is approximately the same as the osmotic pressure of body fluids or cell fluid. Specifically, examples include physiological saline, buffered physiological saline (e.g., PBS, Tris-buffered saline (TBS), HEPES-buffered saline), Ringer's solution, lactated Ringer's solution, acetate Ringer's solution, bicarbonate Ringer's solution, 5% glucose aqueous solution, basal culture media for animal cells (e.g., DMEM, EMEM, RPMI-1640, α-MEM, F-12, F-10, M-199), isotonic agents (e.g., glucose, D-sorbitol, D-mannitol, lactose, sodium chloride), etc., and among these, lactated Ringer's solution is preferred. The isotonic solution may be commercially available or prepared by the user. Examples of commercially available products include Otsuka Saline Injection (manufactured by Otsuka Pharmaceutical Factory Co., Ltd.) (physiological saline solution), Ringer's Solution "Otsuka" (manufactured by Otsuka Pharmaceutical Factory Co., Ltd.) (Ringer's solution), Lactec® Injection (manufactured by Otsuka Pharmaceutical Factory Co., Ltd.) (lactated Ringer's solution), Veen® F Infusion (manufactured by Fuso Pharmaceutical Industries Co., Ltd.) (acetate Ringer's solution), Otsuka Sugar Solution 5% (manufactured by Otsuka Pharmaceutical Factory Co., Ltd.) (5% glucose aqueous solution), Bicarbonate® Infusion (manufactured by Otsuka Pharmaceutical Factory Co., Ltd.) (bicarbonate Ringer's solution), Cellstore S (manufactured by Otsuka Pharmaceutical Factory Co., Ltd.), and Cellstore W (manufactured by Otsuka Pharmaceutical Factory Co., Ltd.).

[0023] The preservation solution in question may contain trehaloses and diarylheptanoid compounds individually, or it may contain two or more compounds selected from trehaloses and diarylheptanoid compounds. Furthermore, the solution may also contain any additional components besides trehaloses and diarylheptanoid compounds.

[0024] In this specification, "optional components" include, for example, isotonic agents (e.g., glucose, sorbitol, mannitol, lactose, sodium chloride), chelating agents (e.g., EDTA, EGTA, citric acid, salicylate), solubilizers, preservatives, antioxidants, amino acids (e.g., proline, glutamine), polymers (e.g., polyethers), and phospholipids (e.g., lysophosphatidic acid [LPA]). Examples of ingredients include acids, pH adjusters (e.g., alkalis such as hydroxides, acetates, carbonates, and bicarbonates; acids such as citric acid, succinic acid, acetic acid, lactic acid, glacial acetic acid, and hydrochloric acid), vitamins (vitamin A, vitamin B [B1, B2, B3 i.e., niacin, B5, B6, B7, B9, B12, etc.], vitamin C i.e., ascorbic acid, vitamin D, vitamin E, etc.), and polyphenols (catechin, epicatechin, gallocatechin, epigallocatechin, catechin gallate, epicatechin gallate, gallocatechin gallate, epigallocatechin gallate, rutin, quercetin, anthocyanins, chlorogenic acid, flavonoids, etc.). In this specification, "optional ingredients" means ingredients that may or may not be included. Furthermore, the present invention includes a powder formulation for preparing the preservation solution, comprising trehaloses and diarylheptanoid compounds. The powder formulation may also contain the above-mentioned optional components.

[0025] As the preservation solution in this application, trehaloses and diarylheptanoid compounds alone exert an inhibitory effect on reducing the rate of embryo morphological recovery, the rate of development, or the rate of apoptosis. Therefore, the solution may not contain any other components that have an inhibitory effect on reducing the rate of embryo morphological recovery, the rate of development, or the rate of apoptosis (for example, polysaccharides or derivatives thereof such as acarbose, stachyose, dextran, hydroxyethyl starch [HES], or salts thereof; monosaccharides or derivatives thereof such as glucose, or salts thereof). However, in order to further enhance the inhibitory effect on reducing the rate of embryo morphological recovery, the rate of development, or the rate of apoptosis, or to enhance the effect on increasing the total number of cells in the blastocyst, it is preferable to further contain the above components, specifically polysaccharides or derivatives thereof, or salts thereof. Since the effect of this has been demonstrated in the embodiment described later, a solution further containing dextran or its derivatives or salts thereof (sometimes referred to as "dextrans" in this application) can be suitably exemplified.

[0026] In the above-mentioned dextrans, the dextran is a polysaccharide consisting of D-glucose (C6H 10 O5) n The dextran is not particularly limited as long as it has an α1→6 linkage as its main chain, and examples of weight-average molecular weight (Mw) dextrans include dextran 40 (Mw=40000) and dextran 70 (Mw=70000). These dextrans can be produced by any known method, such as chemical synthesis, microbial production, or enzymatic production, but commercially available products can also be used. Examples of commercially available products include dextran 40 (manufactured by Tokyo Chemical Industry Co., Ltd.), dextran 70 (manufactured by Tokyo Chemical Industry Co., Ltd.), and low molecular weight dextran L injection (10[w / v]% dextran-containing Ringer's lactate solution) (manufactured by Otsuka Pharmaceutical Co., Ltd.).

[0027] Examples of dextran derivatives in the above-mentioned dextrans include dextran sulfate, carboxylated dextran, and diethylaminoethyl (DEAE)-dextran.

[0028] Examples of salts of dextran or its derivatives in the above-mentioned dextran compounds include acid addition salts such as hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate, sulfate, acetate, propionate, toluenesulfonate, succinate, oxalate, lactate, tartrate, glycolate, methanesulfonate, butyrate, valerate, citrate, fumarate, maleate, and malate; metal salts such as sodium salt, potassium salt, and calcium salt; and ammonium salts and alkylammonium salts. These salts are used as solutions, and it is preferable that their action is the same as that of dextran. These salts may form hydrates or solvates, and either one or two or more can be used individually or in appropriate combinations.

[0029] The concentration of dextran in the preservation solution should be such that it suppresses the reduction in embryo morphological recovery rate, the decrease in developmental rate, or the increase in apoptosis rate, or increases the total number of cells in the blastocyst. For example, the concentration should be 0.1(w / v)% or more, preferably 0.3(w / v)% or more, more preferably 0.6(w / v)% or more, even more preferably 1.0(w / v)% or more, even more preferably 2.0(w / v)% or more, and most preferably 4.0(w / v)% or more in terms of dextran. Furthermore, from the viewpoint of avoiding adverse effects on the embryo, the concentration should be such that it is 50(w / v)% or less, preferably 20(w / v)% or less, more preferably 15(w / v)% or less, even more preferably 12%(w / v)% or less, even more preferably 9.0%(w / v)% or less, and most preferably 7.0(w / v)% or less in terms of dextran. Therefore, the concentration of dextran in the preservation solution is, for example, in the range of 0.1 to 50 (w / v)% in terms of dextran, preferably 0.3 to 20 (w / v)%, more preferably 0.6 to 15 (w / v)%, even more preferably 1.0 to 12% (w / v)%, even more preferably 2.0 to 9.0% (w / v)%, and most preferably 4.0 to 7.0% (w / v)%.

[0030] The present invention relates to an embryo preservation method which includes the step of preserving an embryo that has been fertilized for a specified period of time at a specified temperature in a liquid containing trehaloses and diarylheptanoid compounds for a specified period of time (this may be referred to as "the present preservation method" in this application).

[0031] In the present invention, the preferred period of time after fertilization of the embryo to be preserved is a period during which, when the embryo is preserved in the preservation solution, a decrease in the morphological recovery rate of the embryo in the preservation solution, a decrease in the developmental rate, or an increase in the apoptosis rate can be suppressed. For example, 2 to 7 days after fertilization; 2 to 6 days after fertilization; 2 to 5 days after fertilization; 2 to 4 days after fertilization; 2 to 3 days after fertilization; 3 to 7 days after fertilization; 3 to 6 days after fertilization; 3 to 5 days after fertilization; 3 to 4 days after fertilization; 4 to 7 days after fertilization; after fertilization Examples of embryos that have progressed for 4 to 6 days; 4 to 5 days after fertilization; 5 to 7 days after fertilization; 5 to 6 days after fertilization; 6 to 7 days after fertilization; etc. It is preferable to preserve embryos that have progressed for 2 to 7 days after fertilization, more preferable to preserve embryos that have progressed for 3 to 6 days after fertilization, and even more preferable to preserve embryos that have progressed for 4 to 6 days after fertilization. As the effect of this has been demonstrated in the embodiment described later, it is particularly preferable to preserve embryos that have progressed for 5 days after fertilization.

[0032] In the present invention, the preferred period for storing the embryo is a period during which, when the embryo is stored in the storage solution, a decrease in the morphological recovery rate of the embryo, a decrease in the developmental rate, or an increase in the apoptosis rate of the embryo in the storage solution can be suppressed. For example, this period may be 6 hours or more, 12 hours or more, 18 hours or more, or 24 hours or more. Furthermore, since storing the embryo for too long may have adverse effects on the embryo, from the viewpoint of avoiding such adverse effects, the preferred period may be 72 hours or less, 60 hours or less, 48 ​​hours or less, 42 hours or less, 36 hours or less, 30 hours or less, or 24 hours or less. Therefore, the above storage periods are, for example, 6 to 72 hours; 6 to 60 hours; 6 to 48 hours; 6 to 42 hours; 6 to 36 hours; 6 to 30 hours; 6 to 24 hours; 12 to 72 hours; 12 to 60 hours; 12 to 48 hours; 12 to 42 hours; 12 to 36 hours; 12 to 30 hours; 12 to 24 hours; 18 to 72 hours; 18 to 60 hours; 18 to 48 hours; 18 to 42 hours; 18 Examples of storage times include 12 to 36 hours; 18 to 30 hours; 18 to 24 hours; 24 to 72 hours; 24 to 60 hours; 24 to 48 hours; 24 to 42 hours; 24 to 36 hours; 24 to 30 hours; and so on. Storage for 12 to 36 hours is preferred, and storage for 18 to 30 hours is more preferred. As the effect of this has been demonstrated in the embodiment described later, storage for 24 hours can be particularly favorably exemplified.

[0033] In the present invention, the preferred temperature for preserving embryos is one that, when the embryos are preserved in the preservation solution, suppresses a decrease in the morphological recovery rate, a decrease in the developmental rate, or an increase in the apoptosis rate of the embryos in the preservation solution, and is usually within the range of 0 to 40°C, preferably within the range of 0 to 30°C (room temperature). The preservation method preferably involves preserving the preservation solution containing the embryos under temperature conditions in which the preservation solution exists in a liquid state. Furthermore, it is preferable that the preservation method does not include a step of preserving the solution under temperature conditions in which the preservation solution exists in a solid state (for example, a step of freezing).

[0034] The preservation solution in question is a liquid that effectively suppresses the decrease in morphological recovery rate, decrease in developmental rate, or increase in apoptosis rate that occurs when embryos are preserved in liquid. For this reason, it is preferable that the preservation solution in question be specifically designated for the following purposes: "to suppress the decrease in the morphological recovery rate of embryos"; "to suppress the decrease in the developmental rate of embryos"; and / or "to suppress the increase in the apoptosis rate of embryos". The suppression of the decrease in morphological recovery rate or decrease in developmental rate of embryos preserved in the preservation solution in question can be confirmed by morphological observation under a microscope. In this case, morphological recovery is exemplified by determining whether or not the embryo re-expands after 24 hours from the start of culture, or it may be determined by determining whether or not the embryo re-expands between 24 and 48 hours from the start of culture. Embryo development is exemplified by determining whether or not the embryo emerges from the zona pellucida within 48 hours from the start of culture, or embryos in the process of emerging from the zona pellucida may be considered as embryos that have emerged from the zona pellucida. The suppression of the apoptosis rate in embryos stored in this preservation solution can be confirmed using known methods capable of detecting cell death, such as the Trypan Blue staining method, the TUNEL method, the Nexin method, and the FLICA method. Furthermore, since trehaloses and diarylheptanoid compounds are considered unlikely to have adverse effects on the animal's body, embryos stored in this preservation solution may be administered directly into the animal's body without replacing them with a new transplantation solution. For this reason, this preservation solution may be further specified for the purpose of "embryo transplantation."

[0035] When the preservation solution containing the embryo is used directly for transplantation, a solution suitable for embryo transplantation is preferred. Such a solution suitable for embryo transplantation preferably does not contain substances unsuitable for embryo transplantation, such as biological components (e.g., serum or serum-derived components [e.g., albumin]), or components that suppress the decrease in embryo viability when embryos are cryopreserved or freeze-dried, such as cryoprotective agents or freeze-drying protective agents, such as dimethyl sulfoxide [DMSO], glycerin, ethylene glycol, trimethylene glycol, dimethylacetamide, polyethylene glycol [PEG], polyvinylpyrrolidone, serum or serum-derived components (e.g., albumin).

[0036] In the present invention, the embryo includes any embryo before the start of cleavage (fertilized egg of the 1-cell stage) and any embryo after the start of cleavage (2-cell stage embryo, 4-cell stage embryo, 8-cell stage embryo, morula, blastocyst stage embryo (early blastocyst to hatched blastocyst)), but it is preferable that the embryo is after the start of cleavage and preferably does not include embryos before the start of cleavage (fertilized egg of the 1-cell stage), that is, it is preferable that the embryo is from the 2-cell stage to the blastocyst stage, and particularly preferably a blastocyst stage embryo. The stage at which an embryo is present can be easily determined, for example, by comparing a microscopic image of the embryo with an image published in the International Embryo Transfer Society (IETS) manual. In the present invention, the embryo can be prepared by known methods. For example, it can be prepared by in vitro fertilization, in vitro fertilization, nuclear transfer, etc. Furthermore, in the present invention, the embryo may be a transgenic embryo in which genes have been introduced by genetic engineering techniques using various vectors, or an embryo whose genetic information has been processed by techniques such as gene knockout or conditional knockout. Furthermore, in the present invention, the embryo may include embryonic stem cells, induced pluripotent stem cells, or stem cells artificially generated or selected by external stimuli. Also, in the present invention, the embryo may not be merely an embryo, but may be an embryoid body that does not necessarily develop into an individual or fetus, but differentiates only into organs of each lineage. In this case, it may be an embryo from which a portion has been removed for analysis, i.e., a biopsied embryo.

[0037] In the present invention, the embryo can be an animal embryo, and examples of animals include mammals, amphibians, reptiles, birds, fish, etc., with mammalian embryos being particularly preferred. Examples of mammals in the present invention include humans, pigs, cows, horses, goats, sheep, wild boars, rabbits, mice, rats, hamsters, guinea pigs, monkeys, rhesus macaques, crab-eating macaques, marmosets, orangutans, chimpanzees, dogs, cats, etc., with humans, pigs, cows, and horses being particularly preferred. Furthermore, in the present invention, the embryo may be a chimeric embryo in which cells of multiple types of animals are mixed. In this case, cells of animals other than mammals may be combined with the chimeric embryo.

[0038] In the present invention, examples of mammalian embryos include embryos for use in medical fields such as assisted reproductive technology for diseases or conditions requiring mammalian embryo transfer therapy (e.g., infertility), embryos for use in regenerative medicine, etc., or embryos for use in livestock farming fields such as embryos for use in livestock production, embryos for use in livestock improvement, etc. [Examples]

[0039] The present invention will be described more specifically below with reference to examples, but the technical scope of the present invention is not limited to these examples.

[0040] 1. Preparation of in vitro fertilized pig eggs For the preservation test, in vitro fertilized eggs prepared using the following method were used. (1) Collection of oocytes Ovaries of pigs (three-way crossbreed) collected at a slaughterhouse were immersed in sterile saline solution kept at 30°C and brought back to the laboratory within 3 hours. After washing the ovaries 2-3 times with sterile saline solution, follicles measuring 2-6 mm in diameter were incised on a 90 x 20 mm petri dish using a sterile scalpel. The follicular fluid containing the collected oocytes was collected in a 15 mL tube and allowed to stand for 15 minutes. After removing the supernatant, it was washed with Embryotec solution (manufactured by Nippon Zenyaku Kogyo Co., Ltd.) containing 100 IU / mL penicillin (manufactured by Meiji Seika Co., Ltd.) and 100 μg / mL streptomycin (manufactured by Meiji Seika Co., Ltd.), and then oocytes were collected under a stereomicroscope. Only oocytes with two or more layers of cumulus cells attached and morphologically uniform cytoplasm were selected and collected.

[0041] (2) In vitro maturation The mature culture medium used was Tissue Culture Medium (TCM) 199 (Thermo Fisher Scientific). To this, 10% (v / v) porcine follicular fluid, 0.6 mM L-cysteine ​​(Sigma-Aldrich), 50 μM sodium pyruvate (Sigma-Aldrich), 2 mg / mL D-sorbitol (Wako Pure Chemical Industries), 50 μM β-mercaptoethanol (Wako Pure Chemical Industries), 10 IU / mL pregnant mare serum gonadotropin (eCG; Kyoritsu Pharmaceutical Co., Ltd.), 10 IU / mL human chorionic gonadotropin (hCG; Kyoritsu Pharmaceutical Co., Ltd.), and 50 μg / mL gentamicin (Sigma-Aldrich) were added. After washing the selected oocytes with culture medium, approximately 50 cells were dispensed into 4-well dishes (Thermo Fisher Scientific) and matured in 500 μL of mature culture medium at 39.0°C under 5% CO2 gas phase conditions for 22 hours. Subsequently, they were transferred to the aforementioned hormone-free mature culture medium and matured for another 22 hours.

[0042] (3) In vitro fertilization Oocytes that had matured in vitro were washed with porcine-fertilized semen (PFM; manufactured by Functional Peptide Research Institute Co., Ltd.). 250 μL of PFM was dispensed into 4-well dishes, approximately 50 oocytes were transferred to each dish, and the mixture was incubated for approximately 30 minutes until sperm was added. Next, the frozen semen was thawed at 37°C, diluted with porcine-fertilized semen, and centrifuged at 500 × g for 5 minutes. After centrifugation, the supernatant was removed, and the mixture was diluted again with porcine-fertilized semen to a concentration of 2 × 10⁶. 6 The sperm was diluted to a concentration of sperm / mL. 250 μL of diluted semen was added to 250 μL of porcine semen containing the oocytes mentioned above, resulting in a final concentration of 1 × 10⁻⁶. 6 After adjusting to sperm / mL, the embryos were co-cultured for 5 hours under gas phase conditions of 39.0°C, 5% CO2, 5% O2, and 90% N2. These in vitro fertilized embryos were cultured for 3 days in porcine embryo development medium (PZM-5; Functional Peptide Research Institute Co., Ltd.), and thereafter in porcine blastocyst medium (PBM; Functional Peptide Research Institute Co., Ltd.) in an incubator under conditions of 5% CO2, 5% O2, 90% N2, and 39°C, and used in the experiments described later. Hereafter, "culture" refers to culture in an incubator under conditions of 5% CO2, 5% O2, 90% N2, and 39°C.

[0043] 2. Examination of short-term preservation solutions and additives for the preservation of pig embryos. (1) Comparative study of short-term storage solutions using porcine blastocyst stage embryos To compare the effectiveness of preservation solutions, blastocyst-stage embryos 5 days after in vitro fertilization were used. (i) Group that continued culturing without using preservation solution: No preservation We then compared this with the group that was preserved using each of the following preservation solutions (ii) to (iv). (ii) Pig blastocyst medium used for pig embryo culture: PBM (porcine blastocyst medium) solution (PBM, manufactured by Functional Peptide Research Institute Co., Ltd.) (iii) A commercially available cell preservation solution containing trehalose and dextran: Solution S (Cellstore S, manufactured by Otsuka Pharmaceutical Co., Ltd., composition is shown in Table 1.) (iv) Commercial cell preservation solution containing trehalose: Solution W (Cellstore W, manufactured by Otsuka Pharmaceutical Co., Ltd., composition is shown in Table 1.) Embryos were placed in 0.6 mL tubes containing each preservation solution, sealed, and stored at 25°C for 24 hours (hereafter, "preservation" refers to storage at 25°C under sealed conditions). After preservation, the embryos were cultured in PBM solution for 2 days, and morphological observation under a microscope was used to determine subsequent morphological recovery (survival; defined as embryo re-expansion observed between 24 and 48 hours after the start of culture) and embryonic development (defined as embryos in the process of emerging from the zona pellucida or those that have emerged within 48 hours after the start of culture). In addition, blastocysts were fixed and double staining with DAPI and TUNEL was performed to evaluate the total cell number of blastocysts and DNA fragmentation. The apoptotic rate (% of apoptotic cells), which is the ratio of apoptotic cells to the total number of cells in the blastocyst, was calculated by dividing the number of TUNEL-positive cells by the total number of cells. Statistical analysis was performed using STATVIEW (Abacus Concepts, Inc., Berkeley, CA, USA), following analysis of variance (ANOVA), and Fisher's protected least significant difference (PLSD) test was conducted. The significance level was set at p<0.05 (Table 2).

[0044] [Table 1]

[0045] As shown in Table 2, the morphological recovery rate (the percentage of blastocysts that recovered morphology out of the total number of blastocysts tested) and embryo development rate (the percentage of blastocysts that developed out of the total number of blastocysts tested) were significantly improved when stored in S solution or W solution compared to when stored in PMB solution. There were no significant differences in the morphological recovery rate, embryo development rate, and apoptosis rate between storage in S solution and storage in W solution, but the total number of cells in blastocysts stored in S solution was significantly higher than in those stored in W solution.

[0046] [Table 2]

[0047] (2) Comparative study of additives to solution S using porcine blastocyst embryos To investigate the effect of additives in the preservation solution, blastocyst-stage embryos 5 days after in vitro fertilization were treated with curcumin (10 μM; i.e., approximately 0.0 μM). 000 The cells were placed in 0.6 mL tubes containing solution S (equivalent to 37% w / v) or β-mercaptoethanol (50 μM), stored at 25°C for 24 hours, and then cultured in PBM solution for 2 days as in (1) above. Morphological recovery, embryonic development, total cell number of blastocysts, and apoptosis rate were then compared (Table 3).

[0048] As shown in Table 3, the morphological recovery rate was significantly higher when the embryos were stored in S solution with curcumin added compared to when they were stored in S solution with β-mercaptoethanol added. Furthermore, compared to storage in S solution alone (Control), the embryos stored in S solution with curcumin tended to have a higher morphological recovery rate and a lower apoptosis rate. The embryo development rate also tended to be higher in S solution with curcumin added, and was significantly higher, especially compared to storage in S solution alone (Control). In conclusion, it was shown that adding curcumin to S solution and using it for the preservation of porcine blastocyst-stage embryos can effectively suppress the decrease in morphological recovery rate, decrease in embryo development rate, and increase in apoptosis rate of porcine blastocyst-stage embryos.

[0049] [Table 3]

[0050] (3) Comparative study of additives to W solution using pig one-cell stage embryos To investigate the effects of additives to the preservation solution, one-cell stage embryos 10 hours after in vitro fertilization were placed in 0.6 mL tubes containing solution W, which was supplemented with chlorogenic acid (50 μM), curcumin (10 μM), and β-mercaptoethanol (50 μM), and stored at 25°C for 48 hours. They were then cultured for 3 days in PZM5 solution (Pig culture embryo development medium, PZM-5; manufactured by Functional Peptide Research Institute Co., Ltd.) and for 4 days in PBM solution. Cleavage (defined as cleavage observed during the culture period), embryonic development, total cell number of blastocysts, and apoptosis rate were examined using microscopic observation and double staining with DAPI and TUNEL as described in (1) above (Table 4).

[0051] As shown in Table 4, in the preservation of one-cell stage embryos, the cleavage rate (the ratio of embryos that underwent cleavage to the total number of eggs tested) was significantly reduced in the W solution preservation group with curcumin added, and a downward trend in embryonic development was also observed. Therefore, it was shown that adding curcumin to the W solution for preserving porcine one-cell stage embryos inhibits the development of porcine one-cell stage embryos. This result was surprising as it contradicted the previously mentioned finding that the addition of curcumin to the preservation solution resulted in good embryonic development for porcine blastocyst stage embryos. In summary, it was suggested that in this invention, the effect of adding curcumin to the preservation solution containing trehalose differs depending on the developmental stage of the target embryo, and that it improves embryonic development only for embryos that have progressed through cleavage after fertilization.

[0052] [Table 4] [Industrial applicability]

[0053] This invention can be used in the medical field for assisted reproductive technology, in vitro fertilization, in vitro culture, embryo transfer, etc., and in the livestock field for livestock production, etc., and is therefore applicable to industrial use.

Claims

1. A preservation solution for mammalian blastocyst-stage embryos, comprising trehalose or a salt thereof, and curcumin or a salt thereof, used to suppress a decrease in the developmental rate of the blastocyst-stage embryos.

2. The preservation solution according to claim 1, comprising trehalose or a salt thereof, and curcumin or a salt thereof in an isotonic solution.

3. The preservation solution according to claim 2, wherein the isotonic solution is Ringer's solution.

4. Furthermore, the preservative liquid according to any one of claims 1 to 3, comprising a polysaccharide or a salt thereof.

5. The preservative solution according to claim 4, wherein the polysaccharide is dextran.

6. The preservative solution according to any one of claims 1 to 5, wherein the concentration of trehalose or its salt is 2.0 to 6.0 (w / v)%.

7. The preservative solution according to any one of claims 1 to 6, wherein the concentration of curcumin or its salt is 0.0002 to 0.0006 (w / v)%.

8. The preservative solution according to any one of claims 4 to 7, wherein the concentration of polysaccharides or their salts is 4.0 to 7.0 (w / v)%.

9. A preservation solution according to any one of claims 1 to 8 for preserving embryos for 12 to 36 hours.

10. A preservation solution according to any one of claims 1 to 9 for preserving embryos at 0 to 40°C.

11. A preservation solution according to any one of claims 1 to 10, used to suppress a decrease in the rate of embryonic morphological recovery or an increase in the rate of apoptosis.

12. The preservation solution according to any one of claims 1 to 11, wherein the embryo is a human, pig, bovine, or horse embryo.

13. A powder formulation containing trehalose or a salt thereof, and curcumin or a salt thereof, for preparing a preservative solution according to any one of claims 1 to 12.

14. A method for preserving mammalian blastocyst-stage embryos, comprising the step of preserving the mammalian blastocyst-stage embryo in the preservation solution described in any one of Claims 1 to 12.

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