WO2018101270A1 - Cold storage solution and storage method for mouse sperm - Google Patents

Cold storage solution and storage method for mouse sperm Download PDF

Info

Publication number
WO2018101270A1
WO2018101270A1 PCT/JP2017/042653 JP2017042653W WO2018101270A1 WO 2018101270 A1 WO2018101270 A1 WO 2018101270A1 JP 2017042653 W JP2017042653 W JP 2017042653W WO 2018101270 A1 WO2018101270 A1 WO 2018101270A1
Authority
WO
WIPO (PCT)
Prior art keywords
sperm
dimethyl sulfoxide
refrigerated
storage
concentration
Prior art date
Application number
PCT/JP2017/042653
Other languages
French (fr)
Japanese (ja)
Inventor
直己 中潟
透 竹尾
英高 吉本
Original Assignee
国立大学法人熊本大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人熊本大学 filed Critical 国立大学法人熊本大学
Publication of WO2018101270A1 publication Critical patent/WO2018101270A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms

Definitions

  • the present invention relates to a preservation solution and a preservation method for refrigerated preservation of mouse sperm.
  • a mouse bank has been established all over the world as an institution that supports research using genetically modified mice, and genetically modified mice are widely used for medical research as a model animal for gene function analysis and human disease. Most mouse banks are responsible for the collection, storage and supply of genetically modified mice, and researchers can easily obtain them as needed.
  • transport by living organisms frozen sperm or frozen embryos is used as a method for transporting genetically modified mice.
  • Transport by living body has many problems from the viewpoints of the spread of microbiological contamination, the risk of escape and death during transport, the regulations on the use of genetically modified organisms, and the welfare of laboratory animals.
  • a dedicated transport container such as a dry shipper is required during transport, and there is a drawback that specialized techniques must be acquired to produce frozen sperm and frozen embryo. Therefore, the development of new transportation technology that can replace these methods is required.
  • the development of a method for refrigerated storage of the mouse epididymis tail has been promoted as a simple transportation technique that replaces the above method.
  • the epididymis tail which is a male reproductive organ that stores mature sperm
  • a refrigerated preservation solution a refrigerated preservation solution
  • sperm is transported under low temperature conditions.
  • Lifor registered trademark
  • preservation solution (Lifeblood® Medical, Inc.) is a refrigerated preservation solution for human organs compared to liquid paraffin, M2 medium and CPS-1. It was found and reported to show a high low-temperature protection effect (Non-patent Document 1).
  • Lifor is an artificial stock solution containing nutrients, growth factors, and non-protein oxygen and nutrient carriers.
  • the present inventors have improved fertilization rate for mouse frozen sperm by using methyl- ⁇ -cyclodextrin (MBCD) and reduced glutathione (GSH) for in vitro fertilization, and the technology is refrigerated sperm. It was also reported that it is effective for improving the fertilization rate (Non-patent document 2, Non-patent document 3, Patent document 1, Patent document 2). With these technical improvements, the refrigerated storage period of mouse sperm could be extended to 3 days.
  • MBCD methyl- ⁇ -cyclodextrin
  • GSH reduced glutathione
  • JP 2006-204180 A International Publication No. WO2012 / 036107 International Publication WO2013 / 047665 International Publication WO2014 / 162910
  • An object of the present invention relates to refrigerated storage of sperm, and is to provide a storage solution and a storage method that can be refrigerated for a longer period of time than conventional techniques.
  • the present inventors have conducted intensive research. As a result, in refrigerated preservation of sperm, particularly the epididymis tail containing sperm, dimethyl sulfoxide (DMSO), quercetin and dimethyl sulfite It has been found that by using a combination of phosphooxide soot (DMSO), it is possible to maintain a high fertilization rate and motility even in sperm that has been refrigerated for a longer period of time, thereby completing the present invention.
  • DMSO dimethyl sulfoxide
  • the present invention includes the following. (1) A preservation solution for refrigerated preservation of mouse sperm, which contains dimethyl sulfoxide at a concentration of 1% or more. (2) The preservation solution according to the above (1), wherein the concentration of dimethyl sulfoxide is 5% or more. (3) A preservation solution for refrigerated preservation of mouse sperm, which contains quercetin and dimethyl sulfoxide. (4) The preservation solution according to (3), wherein the mouse sperm is mouse sperm retained in the mouse epididymis tail. (5) The preservation solution according to (4), wherein the quercetin concentration is 10 ⁇ g / ml to 200 ⁇ g / ml, and the dimethyl sulfoxide concentration is 1% to 20%.
  • the preservation solution is a Lifor (registered trademark) preservation solution or an M2 preservation solution containing dimethyl sulfoxide or quercetin.
  • Refrigerated storage of mouse sperm (16) The refrigerated storage method according to (15), wherein the concentration of dimethyl sulfoxide is 5% or more.
  • Refrigeration of mouse sperm including the step of immersing a mouse testis upper body tail excised from male mice in a preservation solution containing quercetin and dimethyl sulfoxide and refrigerated at a temperature of 4 ° C to 10 ° C. Preservation method.
  • One embodiment of the present invention relates to refrigerated storage of mouse sperm, and provides a storage solution and storage method that can be refrigerated for a longer period of time than conventional techniques.
  • Another embodiment of the present invention is a refrigerated storage solution and refrigeration that can provide sperm that can achieve motility, fertility, and fecund development without any practical problems even after refrigerated storage of mouse sperm, for example, for 7 days.
  • a storage method is provided.
  • mouse sperm can be refrigerated for a longer period of time than the prior art by the preservation solution and preservation method of the present invention.
  • mouse spermatozoa stored using the present invention have motility, fertility and offspring development ability that are not problematic in practice.
  • FIG. A shows the proportion of sperm with motor ability (Motile sperm) in the total number of sperm.
  • FIG. A shows the proportion of sperm with motor ability (Motile sperm) in the total number of sperm.
  • FIG. B shows the proportion of sperm (Progressive motile sperm) having forward motility in the total number of sperm.
  • FIG. A shows the proportion of sperm with motor ability (Motile sperm) in the total number of sperm.
  • Quercetin glucoside which is a flavonoid glycoside compound
  • Quercetin glucoside has been proposed as a preservative for cryopreservation of biomaterials, and it has been reported that the survival rate of cells is increased and a protective effect against cold injury is obtained (Patent Document 3).
  • the same inventor has also proposed a preservative for cryopreservation of a biomaterial containing a flavonoid compound (eg, quercetin) in addition to a flavonoid glycoside compound (eg, quercetin glucoside) (Patent Document 4).
  • the flavonoid glycoside compound alone has almost no low-temperature damage protection effect, or the combination of the flavonoid glycoside compound and the flavonoid compound in a concentration range where sufficient effects cannot be obtained, and both are synergistic. It is described that a low-temperature damage protection effect can be obtained by acting on
  • dimethyl sulfoxide has a sperm protective effect, and confirmed that dimethyl sulfoxide can extend the refrigerated storage period of sperm, particularly the refrigerated storage period of the epididymis tail. And by using dimethyl sulfoxide alone or in combination with quercetin and dimethyl sulfoxide, it was confirmed that sperm could be refrigerated for a longer period of time than conventional methods. It was confirmed to have no motor ability, fertility and offspring development ability. The effect is particularly remarkable when used in combination.
  • Quercetin (also referred to as quercetin) used in the present invention is a kind of flavonoid and is a compound represented by the following formula. In the present invention, either quercetin, hydrates or salts thereof can be used. . In the present specification, the term “quercetin” is used to mean quercetin and its hydrates and salts.
  • Quercetin is commercially available. Commercial quercetin is sold, for example, as a hydrate, and can be used without limitation in the present invention. A commercially available dimethyl sulfoxide can be used without limitation in the present invention.
  • refrigerated storage means storage at 0 ° C. to 10 ° C., preferably 4 ° C. to 8 ° C.
  • refrigerated storage it is preferable to remove the epididymis tail from the mouse and then immerse it in the preservation solution of the present invention to make it 10 ° C. or less, preferably 8 ° C. or less as soon as possible.
  • the suspension is mixed in the preservation solution of the present invention, and 10 ° C or less, preferably as soon as possible. Is preferably 8 ° C. or lower.
  • Mouse spermatozoa stored refrigerated in the preservation solution of the present invention or refrigerated by the preservation method of the present invention include both sperm of the mouse sperm suspension and sperm contained in the epididymis tail, but the testis It is preferable to preserve
  • operations such as extraction of the epididymis tail from male mice and immersion in a preservation solution can be performed according to known methods. There are no particular limitations on the type of mouse from which mouse spermatozoa that are refrigerated and stored in the present invention are derived, and any type or strain of mouse may be used.
  • BALB / c, C3H / He, C57BL / 6J, C57BL / 6N, DBA / 2N, ICR, FVB / N, BDF1, B6CF3F1, 129T2 / SvEmsJ, NOD, CBA / J, MSM / Ms, JF1 / Ms, A / J, CD1, etc. can be mentioned.
  • the concentration of quercetin in the preservation solution of the present invention is usually 1-1000 ⁇ g / ml, preferably 10-200 ⁇ g / ml, more preferably 50-200 ⁇ g / ml, and even more preferably 50 in the case of preservation of the epididymis tail.
  • it is usually 1 to 1000 ⁇ g / ml, preferably 10 to 200 ⁇ g / ml, more preferably 10 to 100 ⁇ g / ml, and further preferably 10 to 50 ⁇ g / ml. It is.
  • the concentration of dimethyl sulfoxide in the preservation solution of the present invention is usually 1 to 20%, preferably 5 to 20%, more preferably 5 to 10% in the case of preservation of the epididymis tail. In the case of a turbid liquid, it is usually 1 to 20%, preferably 1 to 10%, more preferably 1 to 5%.
  • the preservation solution of the present invention is prepared by adding dimethyl sulfoxide alone or a combination of quercetin and dimethyl sulfoxide to any known preservation solution used as a frozen or refrigerated preservation solution of cells or tissues, preferably embryos or sperm. Can be prepared at a concentration as described above.
  • known preservation solutions include, but are not limited to, Lifor (registered trademark) preservation solution (sold by Lifeblood Medical, Inc.) and M2 preservation solution (commercially available from multiple manufacturers). be able to.
  • quercetin is poorly water-soluble, so after dissolving quercetin in dimethyl sulfoxide to the final concentration of interest, add the solution to the stock solution. It is preferable to do.
  • the preservation solution of the present invention includes a kit that can be prepared at the time of use, and specifically, a mouse sperm preservation solution kit comprising quercetin, dimethyl sulfoxide, and a preservation solution kit. Is included.
  • a mouse sperm preservation solution kit comprising quercetin, dimethyl sulfoxide, and a preservation solution kit.
  • the preservation solution kit of the present invention when quercetin is dissolved in dimethyl sulfoxide, and when the total amount or a constant amount thereof is mixed with the preservation solution, the final concentration of quercetin and the final concentration of dimethyl sulfoxide are predetermined concentrations. It is combined to become.
  • the final concentrations of quercetin and dimethyl sulfoxide are each as described above.
  • any known preservation solution used as a frozen or refrigerated preservation solution for cells or tissues, preferably embryo or sperm can be used, but is not limited thereto, for example, Lifor preservation solution, M2 preservation solution You can give liquid.
  • the method of the present invention is a method for refrigerated storage of sperm comprising the following steps (a) and (b).
  • a mouse will be described as an example.
  • Step (a) A step of extracting the mouse epididymis tail from the male mouse. Extraction of the epididymis of the mouse testis can be performed based on a conventional method. For example, the literature of the present inventors (Non-patent Document 1) can be referred to.
  • the immersion operation in the preservation solution and the refrigerated preservation method can be performed according to conventional methods.
  • spermatozoa are separated from the excised mouse epididymis tail, sperm is suspended in the preservation solution of the present invention, and refrigerated at a temperature of 4 ° C. to 10 ° C. as a mouse sperm suspension.
  • concentrations described for the preservation solution of the present invention can be applied as they are in the method of the present invention.
  • the epididymal tail refrigerated storage solution is Lifor (registered trademark) storage solution (Lifeblood Medical, Inc.) (hereinafter referred to as “Lifor”). Abbreviated) or M2 stock solution (Sigma-Aldrich) was used.
  • DMSO dimethyl sulfoxide
  • Quercetin hereinafter abbreviated as “Que” was dissolved in DMSO so as to be 1 mg / mL, and added to Lifo so that the final concentrations of Que were 1, 5, and 100 ⁇ g / mL.
  • sperm pre-culture medium and in vitro fertilization medium The sperm pre-culture medium was free-TYH from which BSA was removed from modified Krebs-Ringer bicarbonate solution (TYH), 0.75 mM methyl- ⁇ -cyclodextrin (MBCD) and CTYH added with 100 mg / 100 mL of polyvinyl alcohol was used. Moreover, modified human tubal fluid (mHTF) containing 1.0 mM reduced glutathione (GSH) was used as the in vitro fertilization medium. The prepared medium was sterilized by filter filtration (0.22 ⁇ m) and stored at 4 ° C.
  • TYH modified Krebs-Ringer bicarbonate solution
  • MCD methyl- ⁇ -cyclodextrin
  • GSH reduced glutathione
  • Embryo culture medium Embryos obtained by in vitro fertilization were subjected to in vitro culture to the blastocyst stage using potassium simplex optimized medium (KSOM). The prepared medium was sterilized by filter filtration (0.22 ⁇ m) and stored at 4 ° C.
  • KSOM potassium simplex optimized medium
  • sperm suspension For refrigerated storage of the sperm suspension, a CARD refrigerated transport kit (Kudo Co., Ltd.) that had been refrigerated in advance was used.sperm were collected from the epididymis tail of adult male mice euthanized. The collected sperm was introduced into a refrigerated stock solution in which DMSO and Que of various concentrations were added to an M2 stock solution that is a refrigerated stock solution to form a sperm suspension. The sperm suspension was then transferred to a 0.2 mL tube.
  • the tube was put in a paper box together with a button thermometer, and the paper box was sealed in a thermos bottle, and then packed in a polystyrene foam box together with a cooling agent.
  • the storage container was stored in the refrigerator (4 ° C.) until use (3 days).
  • the sperm suspension was recovered and the refrigerated storage solution was removed by centrifugation (600 g, 4 ° C., 5 minutes).
  • sperm was introduced into 100 ⁇ L of sperm preculture medium. The introduced sperm was pre-cultured at 37 ° C. and 5% CO 2 for 60 minutes in an incubator and used for each study.
  • the tube was put in a paper box together with a button thermometer, and the paper box was sealed in a thermos bottle, and then packed in a polystyrene foam box together with a cooling agent.
  • Storage containers were stored in a refrigerator (4 ° C.) until use ( ⁇ 7 days).
  • the epididymis tail was collected, and sperm was collected in 100 ⁇ L of sperm preculture medium.
  • pre-culture was performed in an incubator at 37 ° C. and 5% CO 2 for 60 minutes and used for each study. On day 0 of storage, spermatozoa were collected and used in the sperm preculture medium from the epididymis tail collected from adult male mice.
  • the percentage (%) of sperm with a progressive motility is that the average value of the velocities in the direction of sperm (Path Velocity) is 50 ⁇ m / sec or more out of the total number of sperm, and the straightness (Straightness ) Indicates the percentage of sperm with 50% or more.
  • Experimental Example 1 Effect of DMSO or Que on sperm motility The effect of DMSO or Que on sperm motility in refrigerated storage of the epididymis tail was examined. Epididymal tails taken from euthanized male mice were transformed into Lifor, Lifo containing DMSO with various concentrations (5, 10%), or Que with various concentrations (50, 100 ⁇ g / mL) and various concentrations (5, 10%). ) For 4 days or 7 days in DMSO-containing Lifor.sperm were then pre-cultured for 60 minutes in free-TYH (cTYH) containing 0.75 mM methyl- ⁇ -cyclodextrin (MBCD).
  • cTYH free-TYH
  • MCD methyl- ⁇ -cyclodextrin
  • sperm was introduced into 1.0 mM GSH-containing mHTF from which the ovum was collected, and in vitro fertilization was performed.
  • sperm stored refrigerated for up to 5 days maintained the same fertilization rate as sperm stored for 0 days (fresh sperm).
  • the fertilization rate equivalent to fresh sperm was maintained in the sperm which was refrigerated for 7 days by adding DMSO + Que.
  • spermatozoa were collected from the epididymis tail transported refrigerated and pre-cultured with cTYH for 60 minutes.
  • the storage time from the start of refrigerated storage to sperm collection was 7 days.
  • pre-cultured sperm was diluted with mHTF, and sperm motility was analyzed with HTM-IVOS.
  • FIG. In the case of refrigerated storage using Que-containing Lifor, the ratio of sperm with motility and the ratio of sperm with forward motility were significantly improved as compared with the case of Lifor alone. Subsequently, fertilization ability was confirmed using spermatozoa recovered from the epididymis tail transported in a refrigerator.
  • Example 7 Refrigerated Storage Using M2 Stock Solution
  • motility of sperm was confirmed when stored refrigerated with M2 stock solution containing DMSO or DMSO + Que. Refrigerate for 4 days using M2 stock solution, M2 stock solution containing 10% DMSO (M2-DMSO), or M2 stock solution containing 10% DMSO and 100 ⁇ g / mL Que (M2-DMSO + Q), respectively. After that, the sperm motility was measured. The results are shown in FIG. Even when the M2 preservation solution was used, the ratio of fertilized spermatozoa was significantly improved by the addition of DMSO or DMSO + Que.
  • the method of the present invention can provide a sperm that can achieve motility, fertility and fecundity without any practical problems even after refrigerated storage of mouse sperm, for example, for 7 days, and is useful as a refrigerated storage technique for mouse sperm. It is.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Dentistry (AREA)
  • Environmental Sciences (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The purpose of the present invention is to provide, with respect to the cold storage of mouse sperm, a cold storage solution and a storage method that enable cold storage for a longer period of time than the prior art. The present invention provides a cold storage solution for the cold storage of the mouse sperm held in cauda epididymis, wherein the storage solution contains dimethyl sulfoxide or dimethyl sulfoxide and quercitin. The present invention also provides a method for the cold storage of mouse sperm using this storage solution. Mouse sperm that has been cold-stored in accordance with the present invention can maintain a high fertility and motility.

Description

マウス精子の冷蔵保存液及び保存方法Mouse sperm refrigerated storage solution and storage method
　本発明は、マウスの精子を冷蔵保存するための保存液および保存方法に関する。 The present invention relates to a preservation solution and a preservation method for refrigerated preservation of mouse sperm.
　遺伝子改変マウスを用いた研究を支援する機関として、マウスバンクが世界中に設立されており、遺伝子改変マウスは、遺伝子機能の解析およびヒト疾患のモデル動物として、医学研究に広く利用されている。ほとんどのマウスバンクは、遺伝子改変マウスを収集、保管および供給する機能を担っており、研究者は必要に応じてそれらマウスを容易に入手することが可能である。 A mouse bank has been established all over the world as an institution that supports research using genetically modified mice, and genetically modified mice are widely used for medical research as a model animal for gene function analysis and human disease. Most mouse banks are responsible for the collection, storage and supply of genetically modified mice, and researchers can easily obtain them as needed.
　現在、遺伝子改変マウスの輸送方法として、生体、凍結精子あるいは凍結胚による輸送が利用されている。生体による輸送は、微生物学的汚染の拡大、輸送中の逃亡や死亡のリスク、遺伝子組み換え生物の使用に関する法規制および実験動物の福祉の観点から多くの課題がある。一方、凍結精子または凍結胚による輸送では、輸送中にドライシッパーなど専用の輸送容器が必要であり、凍結精子や凍結胚を作製するために専門の技術を習得しなければならない等の欠点があることから、これらの方法に代替する新規輸送技術の開発が求められている。 Currently, transport by living organisms, frozen sperm or frozen embryos is used as a method for transporting genetically modified mice. Transport by living body has many problems from the viewpoints of the spread of microbiological contamination, the risk of escape and death during transport, the regulations on the use of genetically modified organisms, and the welfare of laboratory animals. On the other hand, when transporting by frozen sperm or frozen embryo, a dedicated transport container such as a dry shipper is required during transport, and there is a drawback that specialized techniques must be acquired to produce frozen sperm and frozen embryo. Therefore, the development of new transportation technology that can replace these methods is required.
　そこで、上記方法に替わる簡便な輸送技術として、マウス精巣上体尾部の冷蔵保存法の開発が進められてきた。この方法は、成熟した精子を貯蔵する雄性生殖器官である精巣上体尾部を冷蔵保存液に浸漬し、低温条件下で精子を輸送する技術である。輸送された精巣上体尾部から精子を回収し、体外受精および胚移植を行うことで、特定の病原体を持たないマウス個体を作製することが可能である。 Therefore, the development of a method for refrigerated storage of the mouse epididymis tail has been promoted as a simple transportation technique that replaces the above method. In this method, the epididymis tail, which is a male reproductive organ that stores mature sperm, is immersed in a refrigerated preservation solution, and sperm is transported under low temperature conditions. By collecting sperm from the transported epididymal tail, performing in vitro fertilization and embryo transfer, it is possible to produce a mouse individual that does not have a specific pathogen.
　これまで、マウス精巣上体尾部の冷蔵保存において、流動パラフィンやミネラルオイルを冷蔵保存液として用いた場合、冷蔵保存精巣上体尾部から採取した精子の受精能保持時間はせいぜい１日であった。そこで、本発明者らは、透明帯をレーザーで穿孔した卵子を用いて体外受精を行うことにより、受精率を向上さることに成功した。しかしながら、レーザー穿孔卵の作製には、高価な装置や専門の技術が必要となること、また、レーザー穿孔により胚の発生能が低下するため、冷蔵保存液そのものの改良が望まれた。 So far, when liquid paraffin or mineral oil is used as a refrigerated storage solution in the refrigerated storage of the mouse upper body of the testis, the retention time of spermatozoa collected from the refrigerated storage of the upper body of the testis has been at most one day. Accordingly, the present inventors have succeeded in improving the fertilization rate by performing in vitro fertilization using an egg in which a zona pellucida has been perforated with a laser. However, the production of laser perforated eggs requires expensive equipment and specialized techniques, and the ability to develop embryos is reduced by laser perforation.
　本発明者らは様々な保存液を検討した結果、ヒト臓器の冷蔵保存液であるＬｉｆｏｒ（登録商標）保存液(Lifeblood Medical, Inc.) が、流動パラフィン、Ｍ２培地およびＣＰＳ－１に比べて、高い低温保護効果を示すことを見いだし報告した（非特許文献１）。Ｌｉｆｏｒは、栄養素、成長因子、および非タンパク性の酸素および栄養素のキャリアを含む人工の保存液である。 As a result of studying various preservation solutions, the present inventors have found that Lifor (registered trademark) preservation solution (Lifeblood® Medical, Inc.) is a refrigerated preservation solution for human organs compared to liquid paraffin, M2 medium and CPS-1. It was found and reported to show a high low-temperature protection effect (Non-patent Document 1). Lifor is an artificial stock solution containing nutrients, growth factors, and non-protein oxygen and nutrient carriers.
　また本発明者らは、メチル－β－シクロデキストリン（ＭＢＣＤ）と還元グルタチオン（ＧＳＨ）を体外受精に用いることにより、マウス凍結精子に対して受精率を改善したこと、および、その技術は冷蔵精子に対しても受精率改善に有効であることを報告した（非特許文献２、非特許文献３、特許文献１、特許文献２）。これらの技術改良により、マウス精子の冷蔵保存期間は、３日間まで延長することができた。 In addition, the present inventors have improved fertilization rate for mouse frozen sperm by using methyl-β-cyclodextrin (MBCD) and reduced glutathione (GSH) for in vitro fertilization, and the technology is refrigerated sperm. It was also reported that it is effective for improving the fertilization rate (Non-patent document 2, Non-patent document 3, Patent document 1, Patent document 2). With these technical improvements, the refrigerated storage period of mouse sperm could be extended to 3 days.
　現在、日本国内における精巣上体尾部の冷蔵輸送は、３日間の輸送時間があれば可能である。しかしながら、国際輸送では、輸送時のトラブルや通関の遅延等の理由で、輸送時間が延長することがあり、高い受精能を維持した精子を輸送するには、更なる冷蔵保存期間の延長が必要である。 Currently, refrigerated transport of the epididymis in Japan is possible if transport time is 3 days. However, in international transportation, the transportation time may be extended due to problems such as transportation troubles and delays in customs clearance. To transport sperm with high fertility, it is necessary to further extend the refrigerated storage period. It is.
特開２００６－２０４１８０号公報JP 2006-204180 A 国際公開ＷＯ２０１２／０３６１０７号公報International Publication No. WO2012 / 036107 国際公開ＷＯ２０１３／０４７６６５号公報International Publication WO2013 / 047665 国際公開ＷＯ２０１４／１６２９１０号公報International Publication WO2014 / 162910
　本発明の目的は、精子の冷蔵保存に関し、従来技術より長期間の冷蔵保存が可能な保存液および保存方法を提供することである。 An object of the present invention relates to refrigerated storage of sperm, and is to provide a storage solution and a storage method that can be refrigerated for a longer period of time than conventional techniques.
　前記課題を解決するために本発明者らは鋭意研究をした結果、精子、特には精子を含む精巣上体尾部の冷蔵保存において、ジメチルスルフォオキサイド（ＤＭＳＯ）、またはケルセチン (quercetin)およびジメチルスルフォオキサイド (ＤＭＳＯ)の組合せを用いることにより、より長い期間、冷蔵保存した精子においても高い受精率及び運動能を保持できることを見出し、本発明を完成した。 In order to solve the above-mentioned problems, the present inventors have conducted intensive research. As a result, in refrigerated preservation of sperm, particularly the epididymis tail containing sperm, dimethyl sulfoxide (DMSO), quercetin and dimethyl sulfite It has been found that by using a combination of phosphooxide soot (DMSO), it is possible to maintain a high fertilization rate and motility even in sperm that has been refrigerated for a longer period of time, thereby completing the present invention.
　本発明は以下のものを含む。
（１）マウス精子を冷蔵保存するための保存液であって、ジメチルスルフォオキサイドを１％以上の濃度にて含有する保存液。
（２）ジメチルスルフォオキサイドの濃度が５％以上である前記（１）に記載の保存液。
（３）マウス精子を冷蔵保存するための保存液であって、ケルセチンおよびジメチルスルフォオキサイドを含有する保存液。
（４）前記マウス精子がマウス精巣上体尾部中に保持されたマウス精子である前記（３）に記載の保存液。
（５）ケルセチン濃度が１０μｇ／ｍｌ～２００μｇ／ｍｌであり、ジメチルスルフォオキサイドの濃度が１％～２０％である、前記（４）に記載の保存液。
（６）ケルセチン濃度が５０μｇ／ｍｌ以上であり、ジメチルスルフォオキサイドの濃度が５％以上である、前記（５）に記載の保存液。
（７）前記マウス精子がマウス精子懸濁液である前記（３）に記載の保存液。
（８）ケルセチン濃度が１０μｇ／ｍｌ～１００μｇ／ｍｌであり、ジメチルスルフォオキサイドの濃度が１％～１０％である、前記（７）に記載の保存液。
（９）ケルセチン濃度が１０μｇ／ｍｌ～５０μｇ／ｍｌであり、ジメチルスルフォオキサイドの濃度が１％～５％である、前記（８）に記載の保存液。
（１０）前記保存液が、ジメチルスルフォオキサイドまたはケルセチンを含有するＬｉｆｏｒ（登録商標）保存液またはＭ２保存液である前記（１）～（９）のいずれか一つに記載の保存液。
（１１）前記（１）～（１０）のいずれか一つに記載の保存液を用いてマウス精子を保存する方法。
（１２）マウス精子を冷蔵保存するための保存液のキットであって、ケルセチン、ジメチルスルフォオキサイド、および保存液からなるキット。
（１３）ケルセチンをジメチルスルフォオキサイドに溶解した後、保存液と混合した場合、ケルセチンの最終濃度が１０μｇ／ｍｌ～２００μｇ／ｍｌ、ジメチルスルフォオキサイドの最終濃度が１％～２０％となるように組み合わされている、前記（１２）に記載のキット。
（１４）前記保存液が、Ｌｉｆｏｒ（登録商標）保存液またはＭ２保存液である前記（１２）または（１３）に記載のキット。
（１５）ジメチルスルフォオキサイドを１％以上の濃度にて含有する保存液中に雄マウスから摘出したマウス精巣上体尾部を浸漬し、４℃～１０℃の温度にて冷蔵保存する工程を含む、マウス精子の冷蔵保存方法。
（１６）ジメチルスルフォオキサイドの濃度が５％以上である前記（１５）に記載の冷蔵保存方法。
（１７）ケルセチンおよびジメチルスルフォオキサイドを含有する保存液中に雄マウスから摘出したマウス精巣上体尾部を浸漬し、４℃～１０℃の温度にて冷蔵保存する工程を含む、マウス精子の冷蔵保存方法。
（１８）ケルセチン濃度が１０μｇ／ｍｌ～２００μｇ／ｍｌであり、ジメチルスルフォオキサイドの濃度が１％～２０％の濃度である、前記（１７）に記載の冷蔵保存方法。
（１９）ケルセチン濃度が５０μｇ／ｍｌ～１００μｇ／ｍｌであり、ジメチルスルフォオキサイドの濃度が５％～１０％の濃度である、前記（１８）に記載の冷蔵保存方法。
（２０）ケルセチンおよびジメチルスルフォオキサイドを含有する保存液中に雄マウスから得られたマウス精子を懸濁し、該精子懸濁液を、４℃～１０℃の温度にて冷蔵保存する工程を含む、マウス精子の冷蔵保存方法。
（２１）前記保存液が、ケルセチン濃度が１０μｇ／ｍｌ～５０μｇ／ｍｌであり、ジメチルスルフォオキサイドの濃度が１％～５％である前記（２０）に記載の冷蔵保存方法。
（２２）前記保存液が、ジメチルスルフォオキサイドまたはケルセチンを含有するＬｉｆｏｒ（登録商標）保存液またはＭ２保存液である前記（１５）～（２１）のいずれか一つに記載の冷蔵保存方法。 The present invention includes the following.
(1) A preservation solution for refrigerated preservation of mouse sperm, which contains dimethyl sulfoxide at a concentration of 1% or more.
(2) The preservation solution according to the above (1), wherein the concentration of dimethyl sulfoxide is 5% or more.
(3) A preservation solution for refrigerated preservation of mouse sperm, which contains quercetin and dimethyl sulfoxide.
(4) The preservation solution according to (3), wherein the mouse sperm is mouse sperm retained in the mouse epididymis tail.
(5) The preservation solution according to (4), wherein the quercetin concentration is 10 μg / ml to 200 μg / ml, and the dimethyl sulfoxide concentration is 1% to 20%.
(6) The preservation solution according to (5), wherein the quercetin concentration is 50 μg / ml or more and the dimethyl sulfoxide concentration is 5% or more.
(7) The preservation solution according to (3), wherein the mouse sperm is a mouse sperm suspension.
(8) The preservation solution according to (7), wherein the quercetin concentration is 10 μg / ml to 100 μg / ml, and the dimethylsulfoxide concentration is 1% to 10%.
(9) The preservation solution according to (8), wherein the quercetin concentration is 10 μg / ml to 50 μg / ml, and the dimethyl sulfoxide concentration is 1% to 5%.
(10) The preservation solution according to any one of (1) to (9), wherein the preservation solution is a Lifor (registered trademark) preservation solution or an M2 preservation solution containing dimethyl sulfoxide or quercetin.
(11) A method for preserving mouse sperm using the preservative solution according to any one of (1) to (10).
(12) A kit of a preservation solution for refrigerated storage of mouse sperm, the kit comprising quercetin, dimethyl sulfoxide, and a preservation solution.
(13) When quercetin is dissolved in dimethyl sulfoxide and then mixed with a stock solution, the final concentration of quercetin is 10 μg / ml to 200 μg / ml, and the final concentration of dimethyl sulfoxide is 1% to 20%. The kit according to (12), wherein the kit is combined.
(14) The kit according to (12) or (13), wherein the storage solution is a Lifor (registered trademark) storage solution or an M2 storage solution.
(15) A step of immersing a mouse testis upper body tail extracted from a male mouse in a preservation solution containing dimethyl sulfoxide at a concentration of 1% or more and refrigerated at a temperature of 4 ° C. to 10 ° C. is included. , Refrigerated storage of mouse sperm.
(16) The refrigerated storage method according to (15), wherein the concentration of dimethyl sulfoxide is 5% or more.
(17) Refrigeration of mouse sperm, including the step of immersing a mouse testis upper body tail excised from male mice in a preservation solution containing quercetin and dimethyl sulfoxide and refrigerated at a temperature of 4 ° C to 10 ° C. Preservation method.
(18) The refrigerated storage method according to (17) above, wherein the quercetin concentration is 10 μg / ml to 200 μg / ml, and the dimethyl sulfoxide concentration is 1% to 20%.
(19) The refrigerated storage method according to the above (18), wherein the quercetin concentration is 50 μg / ml to 100 μg / ml, and the dimethyl sulfoxide concentration is 5% to 10%.
(20) suspending mouse sperm obtained from male mice in a preservation solution containing quercetin and dimethyl sulfoxide, and storing the sperm suspension in a refrigerator at a temperature of 4 ° C. to 10 ° C. , Refrigerated storage of mouse sperm.
(21) The refrigerated storage method according to (20), wherein the storage solution has a quercetin concentration of 10 μg / ml to 50 μg / ml and a dimethylsulfoxide concentration of 1% to 5%.
(22) The refrigerated storage method according to any one of (15) to (21), wherein the storage solution is a Lifor (registered trademark) storage solution or an M2 storage solution containing dimethyl sulfoxide or quercetin.
　本発明の一つの態様は、マウス精子の冷蔵保存に関し、従来技術より長期間の冷蔵保存が可能な保存液および保存方法を提供するものである。
　本発明の別の一つの態様は、マウスの精子を例えば７日間冷蔵保存した後でも、実用上問題のない運動能、受精能および産子発生能を達成できる精子を提供できる冷蔵保存液および冷蔵保存方法を提供するものである。 One embodiment of the present invention relates to refrigerated storage of mouse sperm, and provides a storage solution and storage method that can be refrigerated for a longer period of time than conventional techniques.
Another embodiment of the present invention is a refrigerated storage solution and refrigeration that can provide sperm that can achieve motility, fertility, and fecund development without any practical problems even after refrigerated storage of mouse sperm, for example, for 7 days. A storage method is provided.
　本発明の保存液及び保存方法により、従来技術より長期間にわたり、マウス精子を冷蔵保存できる。また、本発明を用いて保存されたマウス精子は、実用上問題のない運動能、受精能および産子発生能を有する。 The mouse sperm can be refrigerated for a longer period of time than the prior art by the preservation solution and preservation method of the present invention. In addition, mouse spermatozoa stored using the present invention have motility, fertility and offspring development ability that are not problematic in practice.
精子の運動能に関し、精巣上体尾部の冷蔵保存におけるジメチルスルフォオキサイド（ＤＭＳＯ）またはＤＭＳＯ＋ケルセチン（Ｑｕｅ）の影響を検討した結果である。図ＡおよびＢは４日間冷蔵保存精子の結果、図ＣおよびＤは７日間冷蔵保存精子の結果を示した。図ＡおよびＣは、精子全体数のうち、運動能を持つ精子（Motile sperm）の割合を示した。図ＢおよびＤは、精子全体数のうち、前進運動能を持つ精子（Progressive motile sperm）の割合を示した。測定値は平均値±標準偏差で示した（ｎ＝３）。＊Ｐ＜０．０５は、各実験区におけるＬｉｆｏｒの値と比較した。It is the result of examining the influence of dimethyl sulfoxide (DMSO) or DMSO + quercetin (Que) in refrigerated storage of the epididymis tail in relation to motility of sperm. Figures A and B show the results of sperm stored for 4 days, and Figures C and D show the results of sperm stored for 7 days. Figures A and C show the proportion of sperm with motor ability (Motile sperm) in the total number of sperm. FIGS. B and D show the proportion of sperm with a progressive motility (Progressive motile sperm) out of the total number of sperm. The measured value was expressed as an average value ± standard deviation (n = 3). * P <0.05 was compared with the value of Lifor in each experimental group. 精子の受精能に関し、精巣上体尾部の冷蔵保存におけるＤＭＳＯまたはＤＭＳＯ＋Ｑｕｅの影響を検討した結果である。測定値は平均値±標準偏差で示した（ｎ＝４）。＊Ｐ＜０．０５は、Ｌｉｆｏｒの値と比較した。It is the result of investigating the influence of DMSO or DMSO + Que in refrigerated storage of the epididymis tail with respect to fertilizing ability of sperm. The measured value was expressed as an average value ± standard deviation (n = 4). * P <0.05 compared to the value of Lifor. 精子の運動能に関し、精巣上体尾部の冷蔵保存におけるＤＭＳＯの影響を検討した結果である。図Ａは、精子全体数のうち、運動能を持つ精子（Motile sperm）の割合を示した。図Ｂは、精子全体数のうち、前進運動能を持つ精子（Progressive motile sperm）の割合を示した。測定値は平均値±標準偏差で示した（ｎ＝３～５）。＊Ｐ＜０．０５は、各実験区における新鮮精子（０日）の値と比較した。It is the result of examining the influence of DMSO in the refrigerated storage of the epididymis tail in relation to the motility of sperm. FIG. A shows the proportion of sperm with motor ability (Motile sperm) in the total number of sperm. FIG. B shows the proportion of sperm (Progressive motile sperm) having forward motility in the total number of sperm. The measured value was expressed as an average value ± standard deviation (n = 3 to 5). * P <0.05 compared with the value of fresh sperm (day 0) in each experimental section. 精子の運動能に関し、精巣上体尾部の冷蔵保存におけるＤＭＳＯ＋Ｑｕｅの影響を検討した結果である。図Ａは、精子全体数のうち、運動能を持つ精子（Motile sperm）の割合を示した。図Ｂは、精子全体数のうち、前進運動能を持つ精子（Progressive motile sperm）の割合を示した。測定値は平均値±標準偏差で示した（ｎ＝３～５）。＊Ｐ＜０．０５は、各実験区における新鮮精子（０日）の値と比較した。It is the result of examining the influence of DMSO + Que in refrigerated storage of the epididymis tail in relation to sperm motility. FIG. A shows the proportion of sperm with motor ability (Motile sperm) in the total number of sperm. FIG. B shows the proportion of sperm (Progressive motile sperm) having forward motility in the total number of sperm. The measured value was expressed as an average value ± standard deviation (n = 3 to 5). * P <0.05 compared with the value of fresh sperm (day 0) in each experimental section. 精子の受精能に関し、精巣上体尾部の冷蔵保存におけるＤＭＳＯまたはＤＭＳＯ＋Ｑｕｅの影響を検討した結果である。測定値は平均値±標準偏差で示した（ｎ＝３～５）。＊Ｐ＜０．０５は、各実験区における新鮮精子（０日）の値と比較した。It is the result of investigating the influence of DMSO or DMSO + Que in refrigerated storage of the epididymis tail with respect to fertilizing ability of sperm. The measured value was expressed as an average value ± standard deviation (n = 3 to 5). * P <0.05 compared with the value of fresh sperm (day 0) in each experimental section. 国内冷蔵輸送試験における冷蔵輸送キット内部の温度変化を示した結果である。It is the result which showed the temperature change inside a refrigerator transport kit in a domestic refrigerator transport test. 国内冷蔵輸送における、精子の運動能に対するＱｕｅの効果を検討した結果である。図Ａは、精子全体数のうち、運動能を持つ精子（Motile sperm）の割合を示した。図Ｂは、精子全体数のうち、前進運動能を持つ精子（Progressive motile sperm）の割合を示した。測定値は平均値±標準偏差で示した（ｎ＝３）。＊Ｐ＜０．０５は、Ｌｉｆｏｒの値と比較した。It is the result of examining the effect of Que on motility of sperm in domestic refrigerated transport. FIG. A shows the proportion of sperm with motor ability (Motile sperm) in the total number of sperm. FIG. B shows the proportion of sperm (Progressive motile sperm) having forward motility in the total number of sperm. The measured value was expressed as an average value ± standard deviation (n = 3). * P <0.05 compared to the value of Lifor. 国内冷蔵輸送における、精子の受精能に対するＱｕｅの効果を検討した結果である。＊Ｐ＜０．０５は、Ｌｉｆｏｒの値と比較して算出した。It is the result of examining the effect of Que on fertilizing ability of sperm in domestic refrigerated transport. * P <0.05 was calculated by comparing with the value of Lifor. Ｍ２保存液を用いて冷蔵保存を行った結果である。精子全体数のうち、運動能を持つ精子（motile）の割合および前進運動能を持つ精子（progressive）の割合を示した。測定値は平均値±標準偏差で示した（ｎ＝４）。異文字間に有意差あり（大文字：Ｐ＜０．０１，小文字：Ｐ＜０．０５）。It is the result of having carried out refrigeration storage using M2 preservation solution. Of the total number of sperm, the percentage of sperm with motile and the percentage of progressive sperm (progressive) are shown. The measured value was expressed as an average value ± standard deviation (n = 4). There is a significant difference between different characters (upper case: P <0.01, lower case: P <0.05). 精子の運動能に関し、精子懸濁液の冷蔵保存におけるＤＭＳＯまたはＤＭＳＯ＋Ｑｕｅの影響を検討した結果である。精子全体数のうち、運動能を持つ精子（motile）の割合および前進運動能を持つ精子（progressive）の割合を示した。測定値は平均値±標準偏差で示した（ｎ＝３）。It is the result of examining the influence of DMSO or DMSO + Que in refrigerated storage of the sperm suspension with respect to the motility of sperm. Of the total number of sperm, the percentage of sperm with motile and the percentage of progressive sperm (progressive) are shown. The measured value was expressed as an average value ± standard deviation (n = 3).
　以下、本発明を、例示的な実施態様を例として詳細に説明するが、本発明は以下に記載の実施態様に限定されるものではない。
　なお、文中で特に断らない限り、本明細書で用いるすべての技術用語及び科学用語は、本発明が属する技術分野の当業者に一般に理解されるのと同じ意味をもつ。また、本明細書に記載されたものと同等又は同様の任意の材料および方法は、本発明の実施において同様に使用することができる。
　また、本明細書に記載された発明に関連して本明細書中で引用されるすべての刊行物および特許は、例えば、本発明で使用できる方法や材料その他を示すものとして、本明細書の一部を構成するものである。 Hereinafter, the present invention will be described in detail by way of exemplary embodiments, but the present invention is not limited to the embodiments described below.
Unless otherwise noted in the text, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, any materials and methods equivalent or similar to those described herein can be used as well in the practice of the invention.
In addition, all publications and patents cited in this specification in relation to the invention described herein are hereby incorporated by reference, as examples of methods, materials and the like that can be used in the present invention. Part of it.
　本明細書中で、「Ｘ～Ｙ」という表現を用いた場合は、下限としてＸを、上限としてＹを含む意味で用いる。 In this specification, when the expression “X to Y” is used, X is used as the lower limit and Y is used as the upper limit.
　本発明者らは、ケルセチンに着目し、マウス精子の冷蔵保存、特に精巣上体尾部の冷蔵保存に対するケルセチンの効果を確認した。
　フラボノイド配糖体化合物であるケルセチングルコシドは、生体材料の低温保存用の保存剤として提案されており、細胞の生存率が高められ低温障害保護効果が得られると報告されている（特許文献３）。また、同じ発明者により、フラボノイド配糖体化合物（例えば、ケルセチングルコシド）に加えてフラボノイド化合物（例えば、ケルセチン）を含む生体材料の低温保存用の保存剤も提案されている（特許文献４）。ここでは、フラボノイド配糖体化合物単独では低温障害保護効果がほとんど認められないか、十分な効果が得られない濃度範囲で、フラボノイド配糖体化合物とフラボノイド化合物を併用することにより、両者が相乗的に作用して低温障害保護効果が得られると記載されている。 The present inventors focused on quercetin and confirmed the effect of quercetin on refrigerated storage of mouse sperm, particularly on the chilled storage of the epididymis tail.
Quercetin glucoside, which is a flavonoid glycoside compound, has been proposed as a preservative for cryopreservation of biomaterials, and it has been reported that the survival rate of cells is increased and a protective effect against cold injury is obtained (Patent Document 3). . The same inventor has also proposed a preservative for cryopreservation of a biomaterial containing a flavonoid compound (eg, quercetin) in addition to a flavonoid glycoside compound (eg, quercetin glucoside) (Patent Document 4). Here, the flavonoid glycoside compound alone has almost no low-temperature damage protection effect, or the combination of the flavonoid glycoside compound and the flavonoid compound in a concentration range where sufficient effects cannot be obtained, and both are synergistic. It is described that a low-temperature damage protection effect can be obtained by acting on
　本発明者らはまた、ジメチルスルフォオキサイドに精子保護効果があることを見いだし、ジメチルスルフォオキサイドにより、精子の冷蔵保存期間、特には精巣上体尾部の冷蔵保存期間を延長できることを確認した。
　そして、ジメチルスルフォオキサイド単独、またはケルセチンとジメチルスルフォオキサイドを組み合わせて用いることにより、従来の方法より長い期間、精子を冷蔵保存できることを確認し、冷蔵保存されたマウス精子は、実用上問題のない運動能、受精能および産子発生能を有することを確認した。組み合わせて用いた場合、効果は特に顕著である。 The inventors have also found that dimethyl sulfoxide has a sperm protective effect, and confirmed that dimethyl sulfoxide can extend the refrigerated storage period of sperm, particularly the refrigerated storage period of the epididymis tail.
And by using dimethyl sulfoxide alone or in combination with quercetin and dimethyl sulfoxide, it was confirmed that sperm could be refrigerated for a longer period of time than conventional methods. It was confirmed to have no motor ability, fertility and offspring development ability. The effect is particularly remarkable when used in combination.
　本発明において用いるケルセチン（クエルセチンともよばれる）とは、フラボノイドの一種で、以下の式で表される化合物であり、本発明においては、ケルセチン、およびその水和物や塩のいずれも用いることができる。本明細書中で、ケルセチンという場合は、ケルセチンおよびその水和物や塩を含む意味で用いられる。 Quercetin (also referred to as quercetin) used in the present invention is a kind of flavonoid and is a compound represented by the following formula. In the present invention, either quercetin, hydrates or salts thereof can be used. . In the present specification, the term “quercetin” is used to mean quercetin and its hydrates and salts.
Figure JPOXMLDOC01-appb-C000001
　ケルセチンは市販されている。市販のケルセチンは、例えば、水和物として販売されているが、それを本発明において制限なく用いることができる。
　ジメチルスルフォオキサイドは、市販されているものを、本発明において制限なく用いることができる。
Figure JPOXMLDOC01-appb-C000001
Quercetin is commercially available. Commercial quercetin is sold, for example, as a hydrate, and can be used without limitation in the present invention.
A commercially available dimethyl sulfoxide can be used without limitation in the present invention.
　本発明において冷蔵保存とは、０℃～１０℃、好ましくは４℃～８℃での保存をいう。冷蔵保存では、精巣上体尾部をマウスから摘出した後、本発明の保存液中に浸漬し、できる限り早くに１０℃以下、好ましくは８℃以下にすることが好ましい。懸濁した精子の冷蔵保存の際は、マウスから摘出した精巣上体尾部から精子を回収した後、その懸濁液を本発明の保存液中に混合し、できる限り早くに１０℃以下、好ましくは８℃以下にすることが好ましい。 In the present invention, refrigerated storage means storage at 0 ° C. to 10 ° C., preferably 4 ° C. to 8 ° C. In refrigerated storage, it is preferable to remove the epididymis tail from the mouse and then immerse it in the preservation solution of the present invention to make it 10 ° C. or less, preferably 8 ° C. or less as soon as possible. In the case of refrigerated storage of suspended sperm, after collecting sperm from the epididymis tail excised from the mouse, the suspension is mixed in the preservation solution of the present invention, and 10 ° C or less, preferably as soon as possible. Is preferably 8 ° C. or lower.
　本発明の保存液中で冷蔵保存されるまたは本発明の保存方法で冷蔵保存されるマウス精子は、マウス精子懸濁液の精子および精巣上体尾部中に含まれる精子のいずれも含むが、精巣上体尾部中に保持された状態で保存されるのが好ましい。また、雄マウスからの精巣上体尾部の摘出および保存液中への浸漬等の操作は、公知の方法に従って行うことができる。
　本発明で冷蔵保存されるマウス精子が由来するマウスの種類は特に制限なく、いずれの種類のまたは系統のマウスでもよい。これに限定されないが、例えば、ＢＡＬＢ／ｃ，Ｃ３Ｈ／Ｈｅ，Ｃ５７ＢＬ／６Ｊ，Ｃ５７ＢＬ／６Ｎ，ＤＢＡ／２Ｎ，ＩＣＲ，ＦＶＢ／Ｎ，ＢＤＦ１，Ｂ６ＣＦ３Ｆ１，１２９Ｔ２／ＳｖＥｍｓＪ，ＮＯＤ，ＣＢＡ／Ｊ，ＭＳＭ／Ｍｓ，ＪＦ１／Ｍｓ，Ａ／Ｊ，ＣＤ１等をあげることができる。 Mouse spermatozoa stored refrigerated in the preservation solution of the present invention or refrigerated by the preservation method of the present invention include both sperm of the mouse sperm suspension and sperm contained in the epididymis tail, but the testis It is preferable to preserve | save in the state hold | maintained in the upper body tail part. In addition, operations such as extraction of the epididymis tail from male mice and immersion in a preservation solution can be performed according to known methods.
There are no particular limitations on the type of mouse from which mouse spermatozoa that are refrigerated and stored in the present invention are derived, and any type or strain of mouse may be used. Although not limited to this, for example, BALB / c, C3H / He, C57BL / 6J, C57BL / 6N, DBA / 2N, ICR, FVB / N, BDF1, B6CF3F1, 129T2 / SvEmsJ, NOD, CBA / J, MSM / Ms, JF1 / Ms, A / J, CD1, etc. can be mentioned.
　本発明の保存液におけるケルセチンの濃度は、精巣上体尾部の保存の場合は、通常、１～１０００μｇ／ｍｌ、好ましくは１０～２００μｇ／ｍｌ、より好ましくは５０～２００μｇ／ｍｌ、さらに好ましくは５０～１００μｇ／ｍｌであり、精子懸濁液の保存の場合は、通常、１～１０００μｇ／ｍｌ、好ましくは１０～２００μｇ／ｍｌ、より好ましくは１０～１００μｇ／ｍｌ、さらに好ましくは１０～５０μｇ／ｍｌである。 The concentration of quercetin in the preservation solution of the present invention is usually 1-1000 μg / ml, preferably 10-200 μg / ml, more preferably 50-200 μg / ml, and even more preferably 50 in the case of preservation of the epididymis tail. In the case of storing a sperm suspension, it is usually 1 to 1000 μg / ml, preferably 10 to 200 μg / ml, more preferably 10 to 100 μg / ml, and further preferably 10 to 50 μg / ml. It is.
　本発明の保存液におけるジメチルスルフォオキサイドの濃度は、精巣上体尾部の保存の場合は、通常、１～２０％、好ましくは５～２０％、より好ましくは５～１０％であり、精子懸濁液の場合は、通常、１～２０％、好ましくは１～１０％、より好ましくは１～５％である。 The concentration of dimethyl sulfoxide in the preservation solution of the present invention is usually 1 to 20%, preferably 5 to 20%, more preferably 5 to 10% in the case of preservation of the epididymis tail. In the case of a turbid liquid, it is usually 1 to 20%, preferably 1 to 10%, more preferably 1 to 5%.
　本発明の保存液は、細胞または組織、好ましくは胚または精子の凍結または冷蔵保存液として用いられている任意の公知の保存液に、ジメチルスルフォオキサイド単独、またはケルセチンおよびジメチルスルフォオキサイドの組合せを上記の濃度で添加することにより調製できる。公知の保存液としては、これに限定されないが、例えば、Ｌｉｆｏｒ（登録商標）保存液（Lifeblood Medical, Inc.により販売されている）、Ｍ２保存液（複数のメーカーから市販されている）をあげることができる。
　ケルセチンおよびジメチルスルフォオキサイドの組合せを添加する場合、ケルセチンが難水溶性であるので、目的の最終濃度となるようにしてケルセチンをジメチルスルフォオキサイドに溶解した後、その溶解液を保存液に添加することが好ましい。 The preservation solution of the present invention is prepared by adding dimethyl sulfoxide alone or a combination of quercetin and dimethyl sulfoxide to any known preservation solution used as a frozen or refrigerated preservation solution of cells or tissues, preferably embryos or sperm. Can be prepared at a concentration as described above. Examples of known preservation solutions include, but are not limited to, Lifor (registered trademark) preservation solution (sold by Lifeblood Medical, Inc.) and M2 preservation solution (commercially available from multiple manufacturers). be able to.
When adding a combination of quercetin and dimethyl sulfoxide, quercetin is poorly water-soluble, so after dissolving quercetin in dimethyl sulfoxide to the final concentration of interest, add the solution to the stock solution. It is preferable to do.
　本発明の保存液は、用時調製可能なキットを含むものであり、具体的には、これに限定されないが、ケルセチン、ジメチルスルフォオキサイド、および保存液のキットからなるマウス精子保存液のキットを含むものである。
　本発明の保存液のキットにおいては、ケルセチンをジメチルスルフォオキサイドに溶解した後、その全量または一定量を保存液と混合した場合、ケルセチンの最終濃度およびジメチルスルフォオキサイドの最終濃度が所定の濃度となるように組み合わされている。ケルセチンおよびジメチルスルフォオキサイドの最終濃度は、それぞれ、上記した濃度である。保存液は、細胞または組織、好ましくは胚または精子の凍結または冷蔵保存液として用いられている任意の公知の保存液を用いることができ、これに限定されないが、例えば、Ｌｉｆｏｒ保存液、Ｍ２保存液をあげることができる。 The preservation solution of the present invention includes a kit that can be prepared at the time of use, and specifically, a mouse sperm preservation solution kit comprising quercetin, dimethyl sulfoxide, and a preservation solution kit. Is included.
In the preservation solution kit of the present invention, when quercetin is dissolved in dimethyl sulfoxide, and when the total amount or a constant amount thereof is mixed with the preservation solution, the final concentration of quercetin and the final concentration of dimethyl sulfoxide are predetermined concentrations. It is combined to become. The final concentrations of quercetin and dimethyl sulfoxide are each as described above. As the preservation solution, any known preservation solution used as a frozen or refrigerated preservation solution for cells or tissues, preferably embryo or sperm, can be used, but is not limited thereto, for example, Lifor preservation solution, M2 preservation solution You can give liquid.
　本発明の方法は、以下の工程（ａ）、（ｂ）を含む精子の冷蔵保存方法である。以下、マウスを例に説明する。
工程（ａ）：雄マウスからマウス精巣上体尾部を摘出する工程。マウス精巣上体尾部の摘出は常法に基づいて行うことができるが、たとえば、本発明者らの文献（非特許文献１）を参照することができる。
工程（ｂ）：摘出したマウス精巣上体尾部を、ジメチルスルフォオキサイド、または、ジメチルスルフォオキサイドおよびケルセチンを含有する保存液中へ浸漬し、４℃～１０℃の温度にて冷蔵保存する工程。保存液中への浸漬操作および冷蔵保存方法は、常法に従い行うことができる。
　或いは、摘出したマウス精巣上体尾部から精子を分離し、本発明の保存液に精子を懸濁し、マウス精子懸濁液として、４℃～１０℃の温度にて冷蔵保存する。
　ジメチルスルフォオキサイドおよびケルセチンの含有量、および保存液については、本発明の保存液に関して記載した濃度を本発明の方法においてもそのまま適用できる。 The method of the present invention is a method for refrigerated storage of sperm comprising the following steps (a) and (b). Hereinafter, a mouse will be described as an example.
Step (a): A step of extracting the mouse epididymis tail from the male mouse. Extraction of the epididymis of the mouse testis can be performed based on a conventional method. For example, the literature of the present inventors (Non-patent Document 1) can be referred to.
Step (b): A step of immersing the excised mouse epididymis tail in a storage solution containing dimethyl sulfoxide or dimethyl sulfoxide and quercetin and refrigerated at a temperature of 4 ° C. to 10 ° C. . The immersion operation in the preservation solution and the refrigerated preservation method can be performed according to conventional methods.
Alternatively, spermatozoa are separated from the excised mouse epididymis tail, sperm is suspended in the preservation solution of the present invention, and refrigerated at a temperature of 4 ° C. to 10 ° C. as a mouse sperm suspension.
Concerning the contents of dimethyl sulfoxide and quercetin and the preservation solution, the concentrations described for the preservation solution of the present invention can be applied as they are in the method of the present invention.
　以下、実施例により、本発明を具体的に説明するが、本発明は以下の実施例に限定されるものではない。 Hereinafter, the present invention will be specifically described by way of examples. However, the present invention is not limited to the following examples.
１．材料と方法
（１－１）動物
　精子および卵子の採取には、性成熟期に達した雄（１０～１５週齢）および雌（８～１２週齢）のＣ５７ＢＬ／６Ｊマウス（日本クレア）を使用した。胚移植用のレシピエントマウスおよび精管結紮雄には、成熟雌（８～１２週齢）および雄（１２～２０週齢）のＩＣＲマウス（日本クレア）を用いた。飼育環境は、７時から１９時までの明期、１９時から７時までの暗期、室温は２２±１℃、固形飼料および水は不断給与した。なお、全ての動物実験は、熊本大学動物実験指針に準じて行った。 1. Materials and Methods (1-1) Animals For the collection of sperm and eggs, male (10-15 weeks old) and female (8-12 weeks old) C57BL / 6J mice (Claire Japan) that have reached the sexual maturity stage are collected. used. Recipient mice for embryo transfer and vas deferens males were mature female (8-12 weeks old) and male (12-20 weeks old) ICR mice (CLEA Japan). The breeding environment was a light period from 7 o'clock to 19 o'clock, a dark period from 19 o'clock to 7 o'clock, room temperature was 22 ± 1 ° C, and solid feed and water were constantly fed. All animal experiments were conducted in accordance with the Kumamoto University animal experiment guidelines.
（１－２）保存液および培地
（ｉ）精巣上体尾部冷蔵保存液
　精巣上体尾部の冷蔵保存液は、Ｌｉｆｏｒ（登録商標）保存液（Lifeblood Medical,Inc.）（以下、”Ｌｉｆｏｒ”と略す）またはＭ２保存液（Sigma-Aldrich）を使用した。以下の実験例において、ジメチルスルフォオキサイド（ＤＭＳＯ）は、最終濃度が１、５、１０％（ｖ／ｖ）になるようＬｉｆｏｒに添加した。また、ケルセチン（以下、”Ｑｕｅ”と略す）は、１ ｍｇ／ｍＬとなるようにＤＭＳＯに溶解し、Ｑｕｅの最終濃度が１、５、１００ μｇ／ｍＬになるようＬｉｆｏｒに添加した。 (1-2) Preservative solution and medium (i) Epididymal chilled storage solution The epididymal tail refrigerated storage solution is Lifor (registered trademark) storage solution (Lifeblood Medical, Inc.) (hereinafter referred to as “Lifor”). Abbreviated) or M2 stock solution (Sigma-Aldrich) was used. In the following experimental examples, dimethyl sulfoxide (DMSO) was added to Lifor to a final concentration of 1, 5, 10% (v / v). Quercetin (hereinafter abbreviated as “Que”) was dissolved in DMSO so as to be 1 mg / mL, and added to Lifo so that the final concentrations of Que were 1, 5, and 100 μg / mL.
（ｉｉ）精子前培養培地および体外受精培地
　精子前培養培地はmodified Krebs-Ringer bicarbonate solution（ＴＹＨ）よりＢＳＡを除去したｆｒｅｅ－ＴＹＨに、０．７５ ｍＭのメチル－β－シクロデキストリン（ＭＢＣＤ）および１００ ｍｇ／１００ｍＬのポリビニルアルコールを添加したｃＴＹＨを使用した。また、体外受精培地には、１．０ ｍＭの還元グルタチオン（ＧＳＨ）を含有したmodified human tubal fluid（ｍＨＴＦ）を使用した。なお、調製した培地は、フィルター濾過（０．２２ μｍ）により滅菌した後、４℃で保存した。 (Ii) Sperm pre-culture medium and in vitro fertilization medium The sperm pre-culture medium was free-TYH from which BSA was removed from modified Krebs-Ringer bicarbonate solution (TYH), 0.75 mM methyl-β-cyclodextrin (MBCD) and CTYH added with 100 mg / 100 mL of polyvinyl alcohol was used. Moreover, modified human tubal fluid (mHTF) containing 1.0 mM reduced glutathione (GSH) was used as the in vitro fertilization medium. The prepared medium was sterilized by filter filtration (0.22 μm) and stored at 4 ° C.
（ｉｉｉ）胚培養培地
　体外受精により得られた胚は、potassium simplex optimized medium（ＫＳＯＭ）により、胚盤胞期まで体外培養を行った。なお、調製した培地は、フィルター濾過（０．２２ μｍ）により滅菌した後、４℃で保存した。 (Iii) Embryo culture medium Embryos obtained by in vitro fertilization were subjected to in vitro culture to the blastocyst stage using potassium simplex optimized medium (KSOM). The prepared medium was sterilized by filter filtration (0.22 μm) and stored at 4 ° C.
（１－３）精子の回収
（ｉ）新鮮精子
　精子は、安楽死させた成熟雄マウスの精巣上体尾部から採取した。採取した精子は、１００ μＬのｃＴＹＨに導入し、３７℃、５％ ＣＯ2のインキュベーター中で、６０分間の前培養を行い、各検討に用いた。 (1-3) Recovery of sperm (i) Fresh sperm Sperm was collected from the epididymis tail of a mature male mouse euthanized. The collected sperm was introduced into 100 μL of cTYH, pre-cultured for 60 minutes in an incubator at 37 ° C. and 5% CO 2 and used for each study.
（ｉｉ）冷蔵保存精子（精子懸濁液）
　精子懸濁液の冷蔵保存には、予め冷蔵したＣＡＲＤ冷蔵輸送キット（九動株式会社）を使用した。精子は、安楽死させた成熟雄マウスの精巣上体尾部から採取した。採取した精子は、冷蔵保存液であるＭ２保存液に各種濃度のＤＭＳＯおよびＱｕｅを添加した冷蔵保存液に導入して精子懸濁液にした。その後、精子懸濁液を０．２ ｍＬチューブに移した。続いて、ボタン温度計とともにチューブを紙箱に入れ、紙箱を魔法瓶に封入した後に、保冷剤と共に発泡スチロールの箱に梱包した。保存容器は、使用するまで冷蔵庫内（４℃）で保存した（３日間）。保存後、精子懸濁液を回収し、遠心処理（６００ｇ、４℃、５分）によって冷蔵保存液を除去した。その後、１００ μＬの精子前培養培地中に精子を導入した。導入した精子は、インキュベーター中で、３７℃、５％ ＣＯ2にて、６０分間の前培養を行い、各検討に用いた。 (Ii) refrigerated sperm (sperm suspension)
For refrigerated storage of the sperm suspension, a CARD refrigerated transport kit (Kudo Co., Ltd.) that had been refrigerated in advance was used. Sperm were collected from the epididymis tail of adult male mice euthanized. The collected sperm was introduced into a refrigerated stock solution in which DMSO and Que of various concentrations were added to an M2 stock solution that is a refrigerated stock solution to form a sperm suspension. The sperm suspension was then transferred to a 0.2 mL tube. Subsequently, the tube was put in a paper box together with a button thermometer, and the paper box was sealed in a thermos bottle, and then packed in a polystyrene foam box together with a cooling agent. The storage container was stored in the refrigerator (4 ° C.) until use (3 days). After storage, the sperm suspension was recovered and the refrigerated storage solution was removed by centrifugation (600 g, 4 ° C., 5 minutes). Thereafter, sperm was introduced into 100 μL of sperm preculture medium. The introduced sperm was pre-cultured at 37 ° C. and 5% CO 2 for 60 minutes in an incubator and used for each study.
（ｉｉｉ）冷蔵保存精子（精巣上体尾部）
　精巣上体尾部の保存は、竹尾らの方法に従って行った（非特許文献１）。精巣上体尾部の冷蔵保存には、予め冷蔵したＣＡＲＤ冷蔵輸送キット（九動株式会社）を使用した。精巣上体尾部の保存は、まず、安楽死させた成熟雄マウスから精巣上体尾部を採取し、冷蔵保存液であるＬｉｆｏｒまたはＭ２保存液に各種濃度のＤＭＳＯおよびＱｕｅを添加した冷蔵保存液で満たした０．２ ｍＬチューブに精巣上体尾部を移した。続いて、ボタン温度計とともにチューブを紙箱に入れ、紙箱を魔法瓶に封入した後に、保冷剤と共に発泡スチロールの箱に梱包した。保存容器は、使用するまで冷蔵庫内（４℃）で保存した（～７日間）。保存後、精巣上体尾部を回収し、１００ μＬの精子前培養培地中に精子を採取した。採精後、インキュベーター中で、３７℃、５％ ＣＯ2にて、６０分間の前培養を行い、各検討に用いた。保存０日は、成熟雄マウスから採取した精巣上体尾部から、精子前培養培地中に精子を採取して用いた。 (Iii) refrigerated spermatozoa (epididymis tail)
The preservation of the epididymis tail was performed according to the method of Takeo et al. (Non-patent Document 1). CARD refrigerated transport kit (Kudo Co., Ltd.) refrigerated in advance was used for refrigerated storage of the testis upper body tail. The epididymis tail is preserved by first collecting the epididymis tail from an euthanized adult male mouse, and using a refrigerated preservation solution in which various concentrations of DMSO and Que are added to Lifor or M2 preservation solution. The epididymis tail was transferred to a filled 0.2 mL tube. Subsequently, the tube was put in a paper box together with a button thermometer, and the paper box was sealed in a thermos bottle, and then packed in a polystyrene foam box together with a cooling agent. Storage containers were stored in a refrigerator (4 ° C.) until use (˜7 days). After storage, the epididymis tail was collected, and sperm was collected in 100 μL of sperm preculture medium. After collection, pre-culture was performed in an incubator at 37 ° C. and 5% CO 2 for 60 minutes and used for each study. On day 0 of storage, spermatozoa were collected and used in the sperm preculture medium from the epididymis tail collected from adult male mice.
（１－４）精子運動能の評価
　前培養した精子をｍＨＴＦにより希釈し、専用チャンバー（Hamilton Thorne, Inc.）に精子を導入した。その後、ＨＴＭ－ＩＶＯＳ（Hamilton Thorne, Inc.）にセットし、精子運動能を解析した。運動能を持つ精子（Motile sperm）の割合（％）は、精子全体数のうち、１秒間に５ μｍ 以上動いた精子の割合を示した。前進運動能を持つ精子（Progressive motile sperm）の割合（％）は、精子全体数のうち、精子進行方向性速度の平均値（Path Velocity）が５０ μｍ／秒 以上であり、かつ直線性（Straightness）が５０ ％ 以上である精子の割合を示した。 (1-4) Evaluation of sperm motility Pre-cultured sperm was diluted with mHTF, and sperm was introduced into a dedicated chamber (Hamilton Thorne, Inc.). Then, it set to HTM-IVOS (Hamilton Thorne, Inc.), and analyzed the sperm motility. The ratio (%) of sperm with motor ability (%) showed the ratio of sperm that moved more than 5 μm per second out of the total number of sperm. The percentage (%) of sperm with a progressive motility (%) is that the average value of the velocities in the direction of sperm (Path Velocity) is 50 μm / sec or more out of the total number of sperm, and the straightness (Straightness ) Indicates the percentage of sperm with 50% or more.
（１－５）卵子の回収
　過排卵処理のため、成熟雌マウスに、７．５ ＩＵ pregnant mare serum gonadotropin（ＰＭＳＧ）を腹腔内投与し、４８時間後に７．５ ＩＵ human chorionic gonadotropin（ｈＣＧ）を腹腔内投与した。ｈＣＧ投与後１４～１７時間後に、過排卵処理した雌マウスを安楽死させ、採取した卵管膨大部から、卵子卵丘細胞複合体を、パラフィンオイルで被覆した２００ μＬの１．０ ｍＭ 還元グルタチオン（ＧＳＨ）含有ｍＨＴＦ（体外受精培地）に導入した。導入後、インキュベーター中で、３７℃、５％ ＣＯ2にて、３０～６０分間の前培養を行った。 (1-5) Ovum recovery For treatment of superovulation, 7.5 IU pregnant mare serum gonadotropin (PMSG) was intraperitoneally administered to mature female mice, and after 48 hours, 7.5 IU human chorionic gonadotropin (hCG) was administered. It was administered intraperitoneally. From 14 to 17 hours after hCG administration, superovulated female mice were euthanized, and 200 μL of 1.0 mM reduced glutathione coated with paraffin oil from the enormous portion of the collected oviduct was collected. (GSH) -containing mHTF (in vitro fertilization medium) was introduced. After the introduction, preculture was performed in an incubator at 37 ° C. and 5% CO 2 for 30 to 60 minutes.
（１－６）体外受精、胚培養および胚移植
　体外受精、胚培養および胚移植は、常法に従い行った。 (1-6) In vitro fertilization, embryo culture and embryo transfer In vitro fertilization, embryo culture and embryo transfer were performed according to conventional methods.
（１－７）統計解析
　実験結果は、Ｓｔａｔｃｅｌ３を用いて分散分析を行い、有意差が認められた場合に限り、ＴｕｋｅｙまたはＤｕｎｎｅｔｔの方法により各群間の有意差検定を行った。有意水準 Ｐ＜０．０５のとき、その結果に有意に差があると判定した。 (1-7) Statistical analysis As for the experimental results, analysis of variance was performed using Statcel3, and only when a significant difference was observed, a significant difference test between groups was performed by the method of Tukey or Dunnett. When the significance level was P <0.05, the results were determined to be significantly different.
２．実験例
（２－１）実験例１：精子の運動能に対するＤＭＳＯまたはＱｕｅの影響
　精巣上体尾部の冷蔵保存におけるＤＭＳＯまたはＱｕｅの精子の運動能に及ぼす影響を検討した。安楽死させた雄マウスから採取した精巣上体尾部を、Ｌｉｆｏｒ、各種濃度（５、１０％）のＤＭＳＯ含有Ｌｉｆｏｒ、あるいは各種濃度（５０、１００μｇ／ｍＬ）のＱｕｅおよび各種濃度（５、１０％）のＤＭＳＯ含有Ｌｉｆｏｒ中において４日間あるいは７日間冷蔵保存した。その後、精子を、０．７５ ｍＭ メチル－β－シクロデキストリン（ＭＢＣＤ）含有ｆｒｅｅ－ＴＹＨ（ｃＴＹＨ）で６０分間前培養した。続いて、前培養した精子をｍＨＴＦにより希釈し、ＨＴＭ－ＩＶＯＳにて精子運動能を解析した。結果を図１に示す。
　運動能を持つ精子の割合および前進運動能を持つ精子の割合が、ＤＭＳＯの添加により顕著に向上した。また、ＤＭＳＯに加えてＱｕｅを添加することにより、それらの向上がより顕著になった。 2. Experimental Example (2-1) Experimental Example 1: Effect of DMSO or Que on sperm motility The effect of DMSO or Que on sperm motility in refrigerated storage of the epididymis tail was examined. Epididymal tails taken from euthanized male mice were transformed into Lifor, Lifo containing DMSO with various concentrations (5, 10%), or Que with various concentrations (50, 100 μg / mL) and various concentrations (5, 10%). ) For 4 days or 7 days in DMSO-containing Lifor. Sperm were then pre-cultured for 60 minutes in free-TYH (cTYH) containing 0.75 mM methyl-β-cyclodextrin (MBCD). Subsequently, pre-cultured sperm was diluted with mHTF, and sperm motility was analyzed with HTM-IVOS. The results are shown in FIG.
The percentage of sperm with motor ability and the percentage of sperm with forward movement ability were significantly improved by the addition of DMSO. Moreover, those improvements became more remarkable by adding Que in addition to DMSO.
（２－２）実験例２：精子の受精能に対するＤＭＳＯまたはＱｕｅの影響
　精巣上体尾部の冷蔵保存におけるＤＭＳＯまたはＱｕｅの精子の受精能に及ぼす影響を検討した。安楽死させた雄マウスから採取した精巣上体尾部を、Ｌｉｆｏｒ、各種濃度（１、５、１０％）のＤＭＳＯ含有Ｌｉｆｏｒ、あるいは各種濃度（１０、５０、１００μｇ／ｍＬ）のＱｕｅおよび各種濃度（１、５、１０％）のＤＭＳＯ含有Ｌｉｆｏｒ中において７日間冷蔵保存した。その後、精子を、ｃＴＹＨで６０分間前培養した。続いて、卵子を回収した１．０ ｍＭ ＧＳＨ含有ｍＨＴＦに精子を導入し、体外受精を行った。受精率（Fertilization rate）は次式にて算出した。Fertilization rate （％）＝二細胞期胚数／供試卵子数× １００。結果を図２に示す。
　受精能を持つ精子の割合がＤＭＳＯの添加により顕著に向上した。また、ＤＭＳＯに加えてさらにＱｕｅを添加することにより、向上がより顕著になった。
　また、ＤＭＳＯ含有Ｌｉｆｏｒ、またはＤＭＳＯ＋Ｑｕｅ含有Ｌｉｆｏｒにて冷蔵保存を行った精巣上体尾部から採取した精子を用いた体外受精を行ったところ、いずれの場合も体外受精により得られた二細胞期胚は、正常な産子へ発生した。 (2-2) Experimental Example 2: Effect of DMSO or Que on Fertilization Capacity of Sperm The effect of DMSO or Que on fertilization capacity of sperm in the refrigerated storage of the epididymis tail was examined. Epididymal tails collected from euthanized male mice were transformed into Lifor, DMSO-containing Lifor with various concentrations (1, 5, 10%), or Que with various concentrations (10, 50, 100 μg / mL) and various concentrations ( (1, 5, 10%) in a DMSO-containing Lifor for 7 days. Sperm were then pre-cultured with cTYH for 60 minutes. Subsequently, sperm was introduced into 1.0 mM GSH-containing mHTF from which the ovum was collected, and in vitro fertilization was performed. Fertilization rate was calculated by the following formula. Fertilization rate (%) = number of two-cell stage embryos / number of test eggs × 100. The results are shown in FIG.
The proportion of fertile sperm was significantly improved by the addition of DMSO. Moreover, the improvement became more remarkable by adding Que in addition to DMSO.
Moreover, when in vitro fertilization was performed using sperm collected from the epididymis tail that had been refrigerated and stored in DMSO-containing Lifor or DMSO + Que-containing Lifor, in either case, the two-cell stage embryo obtained by in vitro fertilization was Occurred to normal offspring.
（２－３）実験例３：精子の運動能に対するＤＭＳＯの影響
　ＤＭＳＯが冷蔵保存精子の運動能に及ぼす影響を検討した。安楽死させた雄マウスから採取した精巣上体尾部を、Ｌｉｆｏｒ、１０％のＤＭＳＯ含有Ｌｉｆｏｒ中において０、１、２、３、４、５、６、７日間冷蔵保存しした。その後、精子を、ｃＴＹＨで６０分間前培養した。続いて、前培養した精子をｍＨＴＦにより希釈し、ＨＴＭ－ＩＶＯＳにて精子運動能を解析した。結果を図３に示す。
　ＤＭＳＯの添加により、７日間まで、冷蔵保存した精子において、運動能を持つ精子の割合および前進運動能を持つ精子の割合が、０日間保存精子（新鮮精子）と同等に維持された。 (2-3) Experimental Example 3: Effect of DMSO on sperm motility The effect of DMSO on the motility of refrigerated sperm was examined. Epididymis tails collected from euthanized male mice were refrigerated for 0, 1, 2, 3, 4, 5, 6, 7 days in Lifor containing 10% DMSO. Sperm were then pre-cultured with cTYH for 60 minutes. Subsequently, pre-cultured sperm was diluted with mHTF, and sperm motility was analyzed with HTM-IVOS. The results are shown in FIG.
By the addition of DMSO, the proportion of sperm with motor ability and the proportion of sperm with forward motility in sperm stored refrigerated for up to 7 days was maintained at the same level as sperm with 0 day preservation (fresh sperm).
（２－４）実験例４：精子の運動能に対するＱｕｅの影響
　Ｑｕｅが冷蔵保存精子の運動能に及ぼす影響を検討した。安楽死させた雄マウスから採取した精巣上体尾部を、Ｌｉｆｏｒ、１０％のＤＭＳＯおよび１００μｇ／ｍＬのＱｕｅ含有Ｌｉｆｏｒ中において０、１、２、３、４、５、６、７日間冷蔵保存しした。その後、精子を、ｃＴＹＨで６０分間前培養した。続いて、前培養した精子をｍＨＴＦにより希釈し、ＨＴＭ－ＩＶＯＳにて精子運動能を解析した。結果を図４に示す。
　ＤＭＳＯ＋Ｑｕｅの添加により、７日間まで、冷蔵保存した精子において、運動能を持つ精子の割合および前進運動能を持つ精子の割合が、０日間保存精子（新鮮精子）と同等に維持された。 (2-4) Experimental Example 4: Effect of Que on Sperm Motility The effect of Que on the motility of refrigerated sperm was examined. Epididymis tails collected from euthanized male mice were refrigerated for 0, 1, 2, 3, 4, 5, 6, 7 days in Lifor containing 10% DMSO and 100 μg / mL Que. did. Sperm were then pre-cultured with cTYH for 60 minutes. Subsequently, pre-cultured sperm was diluted with mHTF, and sperm motility was analyzed with HTM-IVOS. The results are shown in FIG.
By the addition of DMSO + Que, the proportion of sperm with motility and the proportion of sperm with forward motility in sperm stored refrigerated for up to 7 days was maintained at the same level as sperm with 0 day storage (fresh sperm).
（２－５）実験例５：精子の受精能に対するＤＭＳＯまたはＱｕｅの影響
　ＤＭＳＯ、またはＤＭＳＯおよびＱｕｅを含有するＬｉｆｏｒで０～７日間冷蔵保存した精巣上体尾部から採取した精子の受精能を検討した。安楽死させた雄マウスから採取した精巣上体尾部を、Ｌｉｆｏｒ、１０％のＤＭＳＯを含有するＬｉｆｏｒ、１０％のＤＭＳＯ＋１００μｇ／ｍＬのＱｕｅを含有するＬｉｆｏｒ中において０、１、２、３、４、５、６、７日間冷蔵保存した。その後、精子をｃＴＹＨで６０分間前培養した。続いて、卵子を回収した１．０ ｍＭ ＧＳＨ含有ｍＨＴＦに精子を導入し、体外受精を行った。なお、受精率（Fertilization rate）は次式にて算出した。Fertilization rate （％）＝二細胞期胚数／供試卵子数 × １００（ｎ＝３～５）。結果を図５に示す。
　ＤＭＳＯの添加により、５日間まで冷蔵保存した精子において、０日間保存精子（新鮮精子）と同等の受精率を維持した。また、ＤＭＳＯ＋Ｑｕｅの添加により、７日間まで冷蔵保存した精子において、新鮮精子と同等の受精率を維持した。 (2-5) Experimental Example 5: Effect of DMSO or Que on Sperm Fertility Examination of fertilizing ability of spermatozoa collected from the epididymis tail refrigerated with DMSO or Lifor containing DMSO and Que for 0-7 days did. Epididymal tails taken from euthanized male mice were transferred to Lifor containing Lifor, 10% DMSO, Lifo containing 10% DMSO + 100 μg / mL Qu, 0, 1, 2, 3, 4, Refrigerated for 5, 6 and 7 days. Subsequently, sperm were pre-cultured with cTYH for 60 minutes. Subsequently, sperm was introduced into 1.0 mM GSH-containing mHTF from which the ovum was collected, and in vitro fertilization was performed. The fertilization rate was calculated by the following formula. Fertilization rate (%) = number of embryos at the two-cell stage / number of test eggs × 100 (n = 3 to 5). The results are shown in FIG.
By adding DMSO, sperm stored refrigerated for up to 5 days maintained the same fertilization rate as sperm stored for 0 days (fresh sperm). Moreover, the fertilization rate equivalent to fresh sperm was maintained in the sperm which was refrigerated for 7 days by adding DMSO + Que.
（２－６）実験例６：本発明の冷蔵保存液を用いた輸送
　本発明の冷蔵保存液および方法を用い、旭川医科大学より熊本大学へ国内冷蔵輸送試験を行った。
　安楽死させた雄マウスから採取した精巣上体尾部を、Ｌｉｆｏｒ、１０％のＤＭＳＯおよび１００μｇ／ｍＬのＱｕｅ含有Ｌｉｆｏｒ中においてＣＡＲＤ冷蔵輸送キットを用いて冷蔵保存し、旭川医科大学より熊本大学へ冷蔵輸送を行った。輸送キットの内部に設置した温度計の測定結果を図６に示す。輸送を開始した時間を０とし、精子を回収した時間まで温度を測定した。保存温度は１４℃から漸次低下し、５．５℃で一定となった。 (2-6) Experimental Example 6: Transport using the refrigerated preservation solution of the present invention A domestic refrigerated transportation test was conducted from Asahikawa Medical University to Kumamoto University using the refrigerated preservation solution and method of the present invention.
The epididymis tail collected from euthanized male mice was refrigerated using a CARD refrigerated transport kit in Lifor containing 10% DMSO and 100 μg / mL Que, and refrigerated from Asahikawa Medical University to Kumamoto University. Transported. The measurement result of the thermometer installed inside the transportation kit is shown in FIG. The time when the transportation was started was set to 0, and the temperature was measured until the time when the sperm was collected. The storage temperature gradually decreased from 14 ° C. and became constant at 5.5 ° C.
　次いで、冷蔵輸送された精巣上体尾部から精子を回収し、ｃＴＹＨで６０分間前培養した。なお、冷蔵保存開始から精子回収までの保存時間は７日間であった。続いて、前培養した精子をｍＨＴＦにより希釈し、ＨＴＭ－ＩＶＯＳにて精子運動能を解析した。結果を図７に示す。
　Ｑｕｅ含有Ｌｉｆｏｒを用いて冷蔵保存した場合は、Ｌｉｆｏｒのみの場合に比べて、運動能を持つ精子の割合および前進運動能を持つ精子の割合が著しく向上した。
　続いて、冷蔵輸送した精巣上体尾部から回収した精子を用いて受精能を確認した。卵子を回収した１．０ ｍＭ ＧＳＨ含有ｍＨＴＦに精子を導入し、体外受精を行った。受精率（Fertilization rate）は、以下により算出した。Fertilization rate（％）＝二細胞期胚数／供試卵子数×１００（ｎ＝３）。結果を図８に示す。
　Ｑｕｅ含有Ｌｉｆｏｒを用いて冷蔵保存した場合は、Ｌｉｆｏｒのみの場合に比べて、精子の受精率が著しく向上した。
　また、Ｑｕｅ含有Ｌｉｆｏｒにて冷蔵保存を行った精巣上体尾部から採取した精子を用いた体外受精を行ったところ、体外受精により得られた二細胞期胚は、正常な産子へ発生した。 Next, spermatozoa were collected from the epididymis tail transported refrigerated and pre-cultured with cTYH for 60 minutes. The storage time from the start of refrigerated storage to sperm collection was 7 days. Subsequently, pre-cultured sperm was diluted with mHTF, and sperm motility was analyzed with HTM-IVOS. The results are shown in FIG.
In the case of refrigerated storage using Que-containing Lifor, the ratio of sperm with motility and the ratio of sperm with forward motility were significantly improved as compared with the case of Lifor alone.
Subsequently, fertilization ability was confirmed using spermatozoa recovered from the epididymis tail transported in a refrigerator. Sperm was introduced into 1.0 mM GSH-containing mHTF from which the egg was collected, and in vitro fertilization was performed. Fertilization rate was calculated as follows. Fertilization rate (%) = number of embryos at the two-cell stage / number of test eggs × 100 (n = 3). The results are shown in FIG.
When refrigerated storage was performed using Que-containing Lifor, the fertilization rate of sperm was significantly improved compared to the case of Lifor alone.
Moreover, when in vitro fertilization was performed using sperm collected from the epididymis tail that had been refrigerated with Que-containing Lifor, the two-cell embryo obtained by in vitro fertilization developed into a normal offspring.
（２－７）実験例７：Ｍ２保存液を用いた冷蔵保存
　実施例１と同様にして、ＤＭＳＯまたはＤＭＳＯ＋Ｑｕｅを含有するＭ２保存液で冷蔵保存した場合の精子の運動能を確認した。Ｍ２保存液、１０％のＤＭＳＯ含有Ｍ２保存液（Ｍ２－ＤＭＳＯ）、あるいは１０％のＤＭＳＯおよび１００μｇ／ｍＬのＱｕｅを含有するＭ２保存液（Ｍ２－ＤＭＳＯ＋Ｑ）、のそれぞれを用いて４日間冷蔵保存を行ったのち、精子の運動能を測定した。結果を図９に示す。
　Ｍ２保存液を用いた場合でも、受精能を持つ精子の割合が、ＤＭＳＯの添加あるいはＤＭＳＯ＋Ｑｕｅの添加により顕著に向上した。 (2-7) Experimental Example 7: Refrigerated Storage Using M2 Stock Solution In the same manner as in Example 1, motility of sperm was confirmed when stored refrigerated with M2 stock solution containing DMSO or DMSO + Que. Refrigerate for 4 days using M2 stock solution, M2 stock solution containing 10% DMSO (M2-DMSO), or M2 stock solution containing 10% DMSO and 100 μg / mL Que (M2-DMSO + Q), respectively. After that, the sperm motility was measured. The results are shown in FIG.
Even when the M2 preservation solution was used, the ratio of fertilized spermatozoa was significantly improved by the addition of DMSO or DMSO + Que.
（２－８）実験例８：精子懸濁液の冷蔵保存
　精子懸濁液の冷蔵保存におけるＤＭＳＯまたはＱｕｅの精子の運動能に及ぼす影響を検討した。安楽死させた雄マウスから採取した精巣上体尾部から回収した精子の懸濁液を、Ｍ２保存液、各種濃度（１、５、１０％）のＤＭＳＯ含有Ｍ２保存液、あるいは各種濃度（１０、５０、１００μｇ／ｍＬ）のＱｕｅおよび各種濃度（１、５、１０％）のＤＭＳＯ含有Ｍ２保存液中において３日間冷蔵保存した。その後、精子を、ｃＴＹＨで６０分間前培養した。続いて、前培養した精子をｍＨＴＦにより希釈し、ＨＴＭ－ＩＶＯＳにて精子運動能を解析した。結果を図１０に示す。
　運動能を持つ精子の割合および前進運動能を持つ精子の割合が、５％のＤＭＳＯの添加により顕著に向上した。また、１％のＤＭＳＯ＋１０μｇ／ｍＬのＱｕｅを組み合わせて添加することにより、同様に運動能を持つ精子の割合が顕著に向上した。 (2-8) Experimental Example 8: Refrigerated Storage of Sperm Suspension The effect of DMSO or Que on sperm motility in refrigerated storage of sperm suspension was examined. A suspension of sperm collected from the epididymis tail collected from euthanized male mice was prepared using M2 stock solution, DMSO-containing M2 stock solution with various concentrations (1, 5, 10%), or various concentrations (10, Refrigerated for 3 days in M2 stock solutions containing DMSO containing 50, 100 μg / mL) Que and various concentrations (1, 5, 10%). Sperm were then pre-cultured with cTYH for 60 minutes. Subsequently, pre-cultured sperm was diluted with mHTF, and sperm motility was analyzed with HTM-IVOS. The results are shown in FIG.
The proportion of sperm with motility and the proportion of sperm with forward motility increased significantly with the addition of 5% DMSO. Also, the combination of 1% DMSO + 10 μg / mL Que significantly increased the proportion of sperm with similar motility.
　上記の記載は、本発明の目的及び対象を単に説明するものであり、添付の特許請求の範囲を限定するものではない。添付の特許請求の範囲から離れることなしに、記載された実施態様に対しての、種々の変更及び置換は、本明細書に記載された教示より当業者にとって明らかである。 The above description merely explains the objects and objects of the present invention, and does not limit the scope of the appended claims. Various changes and substitutions to the described embodiments will be apparent to those skilled in the art from the teachings described herein without departing from the scope of the appended claims.
　本発明の方法により、マウスの精子を例えば７日間冷蔵保存した後でも、実用上問題のない運動能、受精能および産子発生能を達成できる精子を提供でき、マウス精子の冷蔵保存技術として有用である。 The method of the present invention can provide a sperm that can achieve motility, fertility and fecundity without any practical problems even after refrigerated storage of mouse sperm, for example, for 7 days, and is useful as a refrigerated storage technique for mouse sperm. It is.

Claims (22)

  1. 　マウス精子を冷蔵保存するための保存液であって、ジメチルスルフォオキサイドを１％以上の濃度にて含有する保存液。 A storage solution for refrigerated storage of mouse sperm, containing dimethyl sulfoxide at a concentration of 1% or more.
  2. 　ジメチルスルフォオキサイドの濃度が５％以上である請求項１に記載の保存液。 The preservation solution according to claim 1, wherein the concentration of dimethyl sulfoxide is 5% or more.
  3. 　マウス精子を冷蔵保存するための保存液であって、ケルセチンおよびジメチルスルフォオキサイドを含有する保存液。 A preservation solution for refrigerated preservation of mouse sperm, which contains quercetin and dimethyl sulfoxide.
  4. 　前記マウス精子がマウス精巣上体尾部中に保持されたマウス精子である請求項３に記載の保存液。 The preservation solution according to claim 3, wherein the mouse sperm is a mouse sperm retained in a mouse testis upper body tail.
  5. 　ケルセチン濃度が１０μｇ／ｍｌ～２００μｇ／ｍｌであり、ジメチルスルフォオキサイドの濃度が１％～２０％である、請求項４に記載の保存液。 The preservation solution according to claim 4, wherein the concentration of quercetin is 10 μg / ml to 200 μg / ml, and the concentration of dimethyl sulfoxide is 1% to 20%.
  6. 　ケルセチン濃度が５０μｇ／ｍｌ以上であり、ジメチルスルフォオキサイドの濃度が５％以上である、請求項５に記載の保存液。 The preservation solution according to claim 5, wherein the concentration of quercetin is 50 µg / ml or more and the concentration of dimethyl sulfoxide is 5% or more.
  7. 　前記マウス精子がマウス精子懸濁液である請求項３に記載の保存液。 The preservation solution according to claim 3, wherein the mouse sperm is a mouse sperm suspension.
  8. 　ケルセチン濃度が１０μｇ／ｍｌ～１００μｇ／ｍｌであり、ジメチルスルフォオキサイドの濃度が１％～１０％である、請求項７に記載の保存液。 The preservation solution according to claim 7, wherein the concentration of quercetin is 10 μg / ml to 100 μg / ml, and the concentration of dimethyl sulfoxide is 1% to 10%.
  9. 　ケルセチン濃度が１０μｇ／ｍｌ～５０μｇ／ｍｌであり、ジメチルスルフォオキサイドの濃度が１％～５％である、請求項８に記載の保存液。 The preservation solution according to claim 8, wherein the concentration of quercetin is 10 μg / ml to 50 μg / ml, and the concentration of dimethyl sulfoxide is 1% to 5%.
  10. 　前記保存液が、ジメチルスルフォオキサイドまたはケルセチンを含有するＬｉｆｏｒ（登録商標）保存液またはＭ２保存液である請求項１～９のいずれか一つに記載の保存液。 The preservation solution according to any one of claims 1 to 9, wherein the preservation solution is a Lifor (registered trademark) preservation solution or an M2 preservation solution containing dimethyl sulfoxide or quercetin.
  11. 　請求項１～１０のいずれか一つに記載の保存液を用いてマウス精子を保存する方法。 A method of storing mouse sperm using the storage solution according to any one of claims 1 to 10.
  12. 　マウス精子を冷蔵保存するための保存液のキットであって、ケルセチン、ジメチルスルフォオキサイド、および保存液からなるキット。 A kit of a storage solution for refrigerated storage of mouse sperm, comprising quercetin, dimethyl sulfoxide, and a storage solution.
  13. 　ケルセチンをジメチルスルフォオキサイドに溶解した後、保存液と混合した場合、ケルセチンの最終濃度が１０μｇ／ｍｌ～２００μｇ／ｍｌ、ジメチルスルフォオキサイドの最終濃度が１％～２０％となるように組み合わされている、請求項１２に記載のキット。 When quercetin is dissolved in dimethyl sulfoxide and then mixed with a stock solution, it is combined so that the final concentration of quercetin is 10 μg / ml to 200 μg / ml and the final concentration of dimethyl sulfoxide is 1% to 20%. The kit according to claim 12.
  14. 　前記保存液が、Ｌｉｆｏｒ（登録商標）保存液またはＭ２保存液である請求項１２または１３に記載のキット。 The kit according to claim 12 or 13, wherein the storage solution is a Lifor (registered trademark) storage solution or an M2 storage solution.
  15. 　ジメチルスルフォオキサイドを１％以上の濃度にて含有する保存液中に雄マウスから摘出したマウス精巣上体尾部を浸漬し、４℃～１０℃の温度にて冷蔵保存する工程を含む、マウス精子の冷蔵保存方法。 Mouse sperm comprising the step of immersing the mouse testis upper body tail excised from male mice in a preservation solution containing dimethyl sulfoxide at a concentration of 1% or more and refrigerated at a temperature of 4 ° C. to 10 ° C. Refrigerated storage method.
  16. 　ジメチルスルフォオキサイドの濃度が５％以上である請求項１５に記載の冷蔵保存方法。 The refrigerated storage method according to claim 15, wherein the concentration of dimethyl sulfoxide is 5% or more.
  17. 　ケルセチンおよびジメチルスルフォオキサイドを含有する保存液中に雄マウスから摘出したマウス精巣上体尾部を浸漬し、４℃～１０℃の温度にて冷蔵保存する工程を含む、マウス精子の冷蔵保存方法。 A method for refrigerated storage of mouse sperm, comprising a step of immersing a mouse testis upper body tail excised from a male mouse in a storage solution containing quercetin and dimethyl sulfoxide and refrigerated at a temperature of 4 ° C to 10 ° C.
  18. 　ケルセチン濃度が１０μｇ／ｍｌ～２００μｇ／ｍｌであり、ジメチルスルフォオキサイドの濃度が１％～２０％の濃度である、請求項１７に記載の冷蔵保存方法。 The refrigerated storage method according to claim 17, wherein the quercetin concentration is 10 μg / ml to 200 μg / ml, and the dimethyl sulfoxide concentration is 1% to 20%.
  19. 　ケルセチン濃度が５０μｇ／ｍｌ～１００μｇ／ｍｌであり、ジメチルスルフォオキサイドの濃度が５％～１０％の濃度である、請求項１８に記載の冷蔵保存方法。 The refrigerated storage method according to claim 18, wherein the concentration of quercetin is 50 μg / ml to 100 μg / ml, and the concentration of dimethyl sulfoxide is 5% to 10%.
  20. 　ケルセチンおよびジメチルスルフォオキサイドを含有する保存液中に雄マウスから得られたマウス精子を懸濁し、該精子懸濁液を、４℃～１０℃の温度にて冷蔵保存する工程を含む、マウス精子の冷蔵保存方法。 Mouse sperm comprising the steps of suspending mouse sperm obtained from a male mouse in a preservation solution containing quercetin and dimethyl sulfoxide and refrigeratedly storing the sperm suspension at a temperature of 4 ° C. to 10 ° C. Refrigerated storage method.
  21. 　前記保存液が、ケルセチン濃度が１０μｇ／ｍｌ～５０μｇ／ｍｌであり、ジメチルスルフォオキサイドの濃度が１％～５％である請求項２０に記載の冷蔵保存方法。 21. The refrigerated storage method according to claim 20, wherein the storage solution has a quercetin concentration of 10 μg / ml to 50 μg / ml and a dimethyl sulfoxide concentration of 1% to 5%.
  22. 　前記保存液が、ジメチルスルフォオキサイドまたはケルセチンを含有するＬｉｆｏｒ（登録商標）保存液またはＭ２保存液である請求項１５～２１のいずれか一つに記載の冷蔵保存方法。 The refrigerated storage method according to any one of claims 15 to 21, wherein the storage solution is a Lifor (registered trademark) storage solution or an M2 storage solution containing dimethyl sulfoxide or quercetin.
PCT/JP2017/042653 2016-11-29 2017-11-28 Cold storage solution and storage method for mouse sperm WO2018101270A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2016230757A JP6857874B2 (en) 2016-11-29 2016-11-29 Refrigerated storage solution and storage method for mouse sperm
JP2016-230757 2016-11-29

Publications (1)

Publication Number Publication Date
WO2018101270A1 true WO2018101270A1 (en) 2018-06-07

Family

ID=62242890

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2017/042653 WO2018101270A1 (en) 2016-11-29 2017-11-28 Cold storage solution and storage method for mouse sperm

Country Status (2)

Country Link
JP (1) JP6857874B2 (en)
WO (1) WO2018101270A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115251042A (en) * 2022-09-08 2022-11-01 安徽科技学院 Poultry semen dilution buffer solution, preparation method thereof and poultry semen cold storage method

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998037182A1 (en) * 1997-02-19 1998-08-27 Universite Laval Oviductal catalase binding to the membranes of spermatozoa and uses thereof
WO2014162910A1 (en) * 2013-04-03 2014-10-09 石原産業株式会社 Preservative for cryopreservation of biological materials and method for preserving biological materials at low temperature

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998037182A1 (en) * 1997-02-19 1998-08-27 Universite Laval Oviductal catalase binding to the membranes of spermatozoa and uses thereof
WO2014162910A1 (en) * 2013-04-03 2014-10-09 石原産業株式会社 Preservative for cryopreservation of biological materials and method for preserving biological materials at low temperature

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
JOHINKE, D. ET AL.: "Quercetin reduces the in vitro production of H2O2 during chilled storage of rabbit spermatozoa", ANIMAL REPRODUCTION SCIENCE, vol. 151, no. 3-4, 2014, pages 208 - 219, XP055489444 *
JONES, R. C.: "THE USE OF DIMETHYL SULPHOXIDE, GLYCEROL, AND RECONSTITUTED SKIM MILK FOR THEPRESERVATION OF RAM SPERMATOZOA", AUSTRALIAN JOURNAL OF BIOLOGICAL SCIENCES, vol. 18, no. 4, 1965, pages 877 - 885, XP055489430 *
KHALIFA, T. A. A. ET AL.: "Testing Usability of Butylated Hydroxytoluene in Conservation of Goat Semen", REPRODUCTION IN DOMESTIC ANIMALS, vol. 43, no. 5, 2008, pages 525 - 530, XP055489437 *
TAKEO, TORU ET AL.: "Establishment of a transport system for mouse epididymal sperm at refrigerated temperatures", CRYOBIOLOGY, vol. 65, no. 3, 2 June 2012 (2012-06-02), pages 163 - 168, XP055489424 *
YOSHIMOTO, HIDETAKA ET AL., BIOLOGY OF REPRODUCTION, vol. 97, no. 6, 2017, pages 883 - 891 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115251042A (en) * 2022-09-08 2022-11-01 安徽科技学院 Poultry semen dilution buffer solution, preparation method thereof and poultry semen cold storage method
CN115251042B (en) * 2022-09-08 2023-06-02 安徽科技学院 Poultry semen dilution buffer solution and preparation method thereof and poultry semen cooling method

Also Published As

Publication number Publication date
JP2018087163A (en) 2018-06-07
JP6857874B2 (en) 2021-04-14

Similar Documents

Publication Publication Date Title
ES2672630T3 (en) Sperm cryoprotectants
Comizzoli et al. Comparative cryobiological traits and requirements for gametes and gonadal tissues collected from wildlife species
Pérez-Cerezales et al. Evaluation of oxidative DNA damage promoted by storage in sperm from sex-reversed rainbow trout
Butts et al. Cryopreservation of Atlantic cod Gadus morhua L. spermatozoa: effects of extender composition and freezing rate on sperm motility, velocity and morphology
Men et al. Effect of trehalose on DNA integrity of freeze-dried boar sperm, fertilization, and embryo development after intracytoplasmic sperm injection
Long et al. Cryopreservation of turkey semen: Effect of breeding line and freezing method on post-thaw sperm quality, fertilization, and hatching
CN104663649B (en) Human oocytes cryoprotective agent
Yoshikawa et al. Production of tiger puffer Takifugu rubripes from cryopreserved testicular germ cells using surrogate broodstock technology
Nynca et al. Effect of postthaw storage time and sperm-to-egg ratio on fertility of cryopreserved brook trout sperm
JP2021500927A (en) Compositions and Methods for Improving Gamete Viability and Function
Buarpung et al. Feline spermatozoa from fresh and cryopreserved testicular tissues have comparable ability to fertilize matured oocytes and sustain the embryo development after intracytoplasmic sperm injection
Takeo et al. Establishment of a transport system for mouse epididymal sperm at refrigerated temperatures
Thiangtum et al. Assessment of basic seminal characteristics, sperm cryopreservation and heterologous in vitro fertilisation in the fishing cat (Prionailurus viverrinus)
Liu et al. Sperm cryopreservation in different grouper subspecies and application in interspecific hybridization
US20200267969A1 (en) Use of tree sap to preserve sperm cell lines
Kim et al. Cryopreservation of sperm from farmed Pacific abalone, Haliotis discus hannai
Barfield et al. Effect of dehydration prior to cryopreservation of large equine embryos
WO2018101270A1 (en) Cold storage solution and storage method for mouse sperm
Namula et al. Effects of Tris (hydroxymethyl) aminomethane on the quality of frozen-thawed boar spermatozoa
Silva et al. Description of ultrastructural damages in frozen-thawed canine spermatozoa
EP2639298B1 (en) The method for preparing human semen for in vitro fertilization or for artificial insemination
Keeley et al. Cryopreservation of epididymal sperm collected postmortem in the Tasmanian devil (Sarcophilus harrisii)
Cejko et al. Application of sodium alginate solution for short-term storage of different volumes of sex-reversed rainbow trout (Oncorhynchus mykiss) testicular sperm
Albal et al. Successful recovery of motile and viable boar sperm after vitrification with different methods (pearls and mini straws) using sucrose as a cryoprotectant
Hu et al. Current status of Male fertility preservation in humans

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17876813

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17876813

Country of ref document: EP

Kind code of ref document: A1