JP6857874B2 - Refrigerated storage solution and storage method for mouse sperm - Google Patents
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- 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
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- 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
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Description
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 storage solution and a storage method for refrigerating 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。 Mouse banks have been established all over the world as an institution to support research using genetically modified mice, and genetically modified mice are widely used in medical research as an analysis of gene function and as a model animal for human diseases. Most mouse banks are responsible for collecting, storing and supplying 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, as a method for transporting genetically modified mice, transport by living organisms, frozen sperms or frozen embryos is used. Biological transport presents many challenges in terms of increased microbiological contamination, risk of escape and death during transport, legislation on the use of genetically modified organisms and the welfare of laboratory animals. On the other hand, transportation by frozen sperm or frozen embryo requires a dedicated transportation container such as a dry shipper during transportation, and has drawbacks such as having to acquire specialized techniques for producing frozen sperm and frozen embryo. Therefore, the development of new transportation technology to 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, as a simple transportation technique to replace the above method, the development of a refrigerated storage method for the epididymis of mice has been promoted. This method is a technique in which the epididymis tail, which is a male reproductive organ that stores mature sperm, is immersed in a refrigerated storage solution to transport sperm under low temperature conditions. By collecting sperm from the transported epididymal tail and performing in vitro fertilization and embryo transfer, it is possible to produce mouse individuals that do 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。 Until now, when liquid paraffin or mineral oil was used as a refrigerated storage solution in the refrigerated storage of the epididymis of mice, the fertility retention time of sperms collected from the tail of the epididymis in the refrigerated storage was at most one day. Therefore, the present inventors have succeeded in improving the fertilization rate by performing in vitro fertilization using an egg in which the zona pellucida is perforated with a laser. However, since the production of laser-perforated eggs requires expensive equipment and specialized techniques, and the developmental ability of embryos is reduced by laser perforation, improvement of the refrigerated storage solution itself has been desired.
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 examining various preservation solutions, the present inventors have found that Lifeblood Medical, Inc., which is a refrigerated preservation solution for human organs, is compared with liquid paraffin, M2 medium and CPS-1. , And reported that it shows a high low temperature protection effect (Non-Patent Document 1). Life is an artificial preservation solution containing nutrients, growth factors, and non-proteinaceous 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 the fertilization rate for frozen mouse sperm by using methyl-β-cyclodextrin (MBCD) and reduced glutathione (GSH) for in vitro fertilization, and the technique is refrigerated sperm. It was also reported that it is effective in improving the fertilization rate (
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 transportation of the epididymis and tail in Japan is possible if the transportation time is 3 days. However, in international transportation, the transportation time may be extended due to troubles during transportation, delays in customs clearance, etc., and it is necessary to further extend the refrigerated storage period in order to transport sperms that maintain high fertility. Is.
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 is to provide a storage solution and a storage method capable of refrigerating and storing sperms for a longer period of time than in the prior art.
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。 As a result of diligent research to solve the above problems, the present inventors have conducted diligent studies and found that dimethyl sulfoxide (DMSO), or quercetin and dimethylsul, in the cold storage of sperm, especially the epididymal tail containing sperm. The present invention has been completed by finding that a high fertilization rate and motility can be maintained even in sperms stored in a refrigerator for a longer period of time by using a combination of fluoride (DMSO).
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 refrigerating mouse sperm, which contains dimethylsulfoxide at a concentration of 1% or more.
(2) The preservation solution according to (1) above, wherein the concentration of dimethylsulfoxide is 5% or more.
(3) A preservation solution for refrigerating mouse sperm, which contains quercetin and dimethylsulfoxide.
(4) The preservation solution according to (3) above, wherein the mouse sperm is a mouse sperm retained in the epididymis of the mouse.
(5) The preservation solution according to (4) above, wherein the quercetin concentration is 10 μg / ml to 200 μg / ml, and the dimethylsulfoxide concentration is 1% to 20%.
(6) The storage solution according to (5) above, wherein the quercetin concentration is 50 μg / ml or more, and the dimethylsulfoxide concentration is 5% or more.
(7) The preservation solution according to (3) above, wherein the mouse sperm is a mouse sperm suspension.
(8) The preservation solution according to (7) above, wherein the quercetin concentration is 10 μg / ml to 100 μg / ml, and the concentration of dimethylsulfoxide is 1% to 10%.
(9) The preservation solution according to (8) above, wherein the quercetin concentration is 10 μg / ml to 50 μg / ml, and the dimethylsulfoxide concentration is 1% to 5%.
(10) The preservation solution according to any one of (1) to (9) above, wherein the preservation solution is a Lifor (registered trademark) preservation solution or an M2 preservation solution containing dimethylsulfoxide or quercetin.
(11) A method for preserving mouse sperm using the preservative solution according to any one of (1) to (10) above.
(12) A kit of a preservation solution for refrigerating mouse sperm, which comprises quercetin, dimethylsulfoxide, and a preservation solution.
(13) When quercetin is dissolved in dimethylsulfoxide and then mixed with a preservation solution, the final concentration of quercetin is 10 μg / ml to 200 μg / ml, and the final concentration of dimethylsulfoxide is 1% to 20%. The kit according to (12) above, which is combined with.
(14) The kit according to (12) or (13) above, wherein the preservation solution is a Life® preservation solution or an M2 preservation solution.
(15) The step of immersing the epididymis tail of a mouse removed from a male mouse in a storage solution containing dimethylsulfoxide at a concentration of 1% or more and refrigerating at a temperature of 4 ° C. to 10 ° C. is included. , How to refrigerate mouse sperm.
(16) The refrigerated storage method according to (15) above, wherein the concentration of dimethylsulfoxide is 5% or more.
(17) Refrigeration of mouse sperm, which comprises a step of immersing the epididymis tail of a mouse removed from a male mouse in a preservation solution containing quercetin and dimethylsulfoxide and refrigerating 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 dimethylsulfoxide concentration is 1% to 20%.
(19) The refrigerated storage method according to (18) above, wherein the quercetin concentration is 50 μg / ml to 100 μg / ml, and the concentration of dimethylsulfoxide is 5% to 10%.
(20) Including a step of suspending mouse sperm obtained from a male mouse in a storage solution containing quercetin and dimethylsulfoxide, and refrigerating the sperm suspension at a temperature of 4 ° C. to 10 ° C. , How to refrigerate mouse sperm.
(21) The refrigerated storage method according to (20) above, 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) above, wherein the storage solution is a Lifer (registered trademark) storage solution or an M2 storage solution containing dimethylsulfoxide 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 aspect of the present invention is to provide a storage solution and a storage method capable of refrigerating and storing mouse sperm for a longer period of time than in the prior art.
Another aspect of the present invention is a refrigerated storage solution and refrigeration capable of providing sperm capable of achieving practically acceptable motility, fertilization ability and sperm development ability even after refrigerating mouse sperm for, for example, 7 days. It provides a storage method.
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。 According to the preservation solution and preservation method of the present invention, mouse sperm can be refrigerated for a longer period of time than the prior art. In addition, mouse sperms stored using the present invention have motility, fertilization ability, and sperm development ability that are not practically problematic.
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 exemplifying an exemplary embodiment, but the present invention is not limited to the embodiments described below.
Unless otherwise specified in the text, all technical terms and scientific terms used in the present specification have the same meanings as generally understood by those skilled in the art to which the present invention belongs. Also, any material and method equivalent to or similar to that described herein can be used in the practice of the present invention as well.
In addition, all publications and patents cited herein in connection with the invention described herein are described herein, for example, as indicating methods, materials, etc. that can be used in the present invention. It constitutes a part.
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 the present specification, when the expression "XY" is used, it is used in the sense that X is included as the lower limit and Y is included 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。
Focusing on quercetin, the present inventors confirmed the effect of quercetin on the refrigerated storage of mouse sperm, especially the epididymal 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 cell viability is enhanced and a cryopreservation effect can be obtained (Patent Document 3). .. The same inventor has also proposed a preservative for low-temperature storage of biomaterials containing a flavonoid compound (eg, quercetin) in addition to a flavonoid glycoside compound (eg, quercetin glucoside) (Patent Document 4). Here, by using the flavonoid glycoside compound and the flavonoid compound in combination in a concentration range in which the flavonoid glycoside compound alone has almost no chilling injury protection effect or a sufficient effect cannot be obtained, both are synergistic. It is stated that it acts on chilling injury to protect against chilling injury.
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 present inventors also found that dimethylsulfoxide has a sperm-protecting effect, and confirmed that dimethylsulfoxide can extend the refrigerated storage period of sperm, particularly the epididymal tail.
Then, it was confirmed that sperm can be refrigerated for a longer period than the conventional method by using dimethylsulfoxide alone or in combination with quercetin and dimethylsulfoxide, and refrigerated mouse sperm are practically problematic. It was confirmed that it had no motility, fertility and sperm development ability. When used in combination, the effect is particularly remarkable.
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 called quercetin) used in the present invention is a kind of flavonoid and is a compound represented by the following formula. In the present invention, quercetin and any hydrate or salt thereof can be used. .. In the present specification, the term quercetin is used to include quercetin and its hydrates and salts.
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。
Quercetin is commercially available. Commercially available quercetin is, for example, sold as a hydrate, which can be used without limitation in the present invention.
As the dimethylsulfoxide, commercially available ones 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 that the epididymis tail is removed from the mouse and then immersed in the preservation solution of the present invention to bring the temperature to 10 ° C. or lower, preferably 8 ° C. or lower as soon as possible. In the case of refrigerated storage of suspended sperm, after collecting sperm from the epididymal tail removed from the mouse, the suspension is mixed with the preservation solution of the present invention, and the temperature is preferably 10 ° C. or lower 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。
Mouse sperms stored refrigerated in the preservative solution of the present invention or stored refrigerated by the storage method of the present invention include both spermatozoa in mouse sperm suspensions and spermatozoa contained in the epididymis tail, but the testis. It is preferably stored while being held in the tail of the upper body. In addition, operations such as removal of the epididymis tail from a male mouse and immersion in a preservation solution can be performed according to a known method.
The type of mouse from which the mouse sperm stored refrigerated in the present invention is derived is not particularly limited, and any type or strain of mouse may be used.
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 to 1000 μg / ml, preferably 10 to 200 μg / ml, more preferably 50 to 200 μg / ml, still more preferably 50 in the case of preservation of the tail of the testis. It is ~ 100 μg / ml, and in the case of storage of the sperm suspension, it is usually 1 to 1000 μg / ml, preferably 10 to 200 μg / ml, more preferably 10 to 100 μg / ml, still more preferably 10 to 50 μg / ml. 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 dimethylsulfoxide 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 and tail, and sperm suspension. In the case of a turbid liquid, it is usually 1 to 20%, preferably 1 to 10%, and 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 any known preservation solution used as a frozen or refrigerated preservation solution for cells or tissues, preferably embryos or sperms, with dimethylsulfoxide alone or in combination with quercetin and dimethylsulfoxide. Can be prepared by adding the above concentration. Known preservatives include, but are not limited to, Lifor® preservatives (sold by Lifeblood Medical, Inc.) and M2 preservatives (commercially available from multiple manufacturers). be able to.
When a combination of quercetin and dimethylsulfoxide is added, quercetin is poorly water-soluble, so after dissolving quercetin in dimethylsulfoxide to the desired final concentration, add the solution to the preservation solution. It is preferable to do so.
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, but is not limited to a kit of mouse sperm preservation solution, which comprises a kit of quercetin, dimethylsulfoxide, and a preservation solution. Is included.
In the preservation solution kit of the present invention, when quercetin is dissolved in dimethylsulfoxide and then the whole amount or a certain amount is mixed with the preservation solution, the final concentration of quercetin and the final concentration of dimethylsulfoxide are predetermined concentrations. It is combined so as to be. The final concentrations of quercetin and dimethylsulfoxide are the above-mentioned concentrations, respectively. As the preservation solution, any known preservation solution used as a frozen or refrigerated preservation solution for cells or tissues, preferably embryos or sperms can be used, and the preservation solution is not limited to, for example, Life preservation solution, M2 preservation solution. You can give the 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 refrigerating sperm, which comprises the following steps (a) and (b). Hereinafter, a mouse will be described as an example.
Step (a): A step of removing the epididymis tail of a mouse from a male mouse. The epididymis of the mouse can be removed according to a conventional method, and for example, the documents of the present inventors (Non-Patent Document 1) can be referred to.
Step (b): The removed mouse epididymal tail is immersed in dimethylsulfoxide or a preservation solution containing dimethylsulfoxide and quercetin, and refrigerated at a temperature of 4 ° C. to 10 ° C. .. The operation of immersing in the storage liquid and the method of refrigerating storage can be performed according to a conventional method.
Alternatively, sperm are separated from the removed epididymis of the mouse, the sperm are suspended in the preservation solution of the present invention, and stored in a cold storage at a temperature of 4 ° C. to 10 ° C. as a mouse sperm suspension.
Regarding the contents of dimethylsulfoxide and quercetin, and the preservative solution, the concentrations described for the preservative 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 with reference to Examples, but 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、成熟雄（１２〜２０週齢）および雌（８〜１２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. 1. Materials and methods (1-1) Animals For collection of sperms and eggs, C57BL / 6J mice (Claire Japan) of males (10 to 15 weeks old) and females (8 to 12 weeks old) who have reached sexual maturity were used. used. As recipient mice for embryo transfer and vas deferens ligated males, mature male (12 to 20 weeks old) and female (8 to 12 weeks old) ICR mice (Claire Japan) were used. The breeding environment was a light period from 7:00 to 19:00, a dark period from 19:00 to 7:00, a room temperature of 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) Epididymis Tail Refrigerated Preservation Solution The epididymis tail refrigerated storage solution is referred to as Lifeblood Medical, Inc. (hereinafter referred to as "Lifor"). (Omitted) or M2 preservative (Sigma-Aldrich) was used. In the following experimental examples, dimethyl sulfoxide (DMSO) was added to Life to a final concentration of 1, 5, 10% (v / v). In addition, quercetin (hereinafter abbreviated as "Que") was dissolved in DMSO so as to be 1 mg / mL, and added to Lift so that the final concentration of Que was 1, 5, 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 preculture medium and in vitro fertilization medium The sperm preculture medium was prepared by removing BSA from modified Krebs-Ringer bicarbonate solution (TYH), and 0.75 mM methyl-β-cyclodextrin (MBCD) and 0.75 mM methyl-β-cyclodextrin (MBCD). CTYH supplemented with 100 mg / 100 mL of polyvinyl alcohol was used. In addition, a 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 then 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 cultured in vitro by potassium simplex optimized medium (KSOM) until the blastocyst stage. The prepared medium was sterilized by filter filtration (0.22 μm) and then 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。
（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。
(1-3) Recovery of sperm (i) Fresh sperm Sperm were collected from the epididymal tail of euthanized mature male mice. The collected spermatozoa were introduced into 100 μL of cTYH, precultured for 60 minutes in an incubator at 37 ° C. and 5% CO 2, and used for each study.
(Ii) Refrigerated sperm (sperm suspension)
A pre-refrigerated CARD refrigerated transportation kit (Kyudo Co., Ltd.) was used for refrigerated storage of the sperm suspension. Sperm were collected from the epididymal tail of euthanized mature male mice. The collected sperms were introduced into a refrigerated storage solution in which various concentrations of DMSO and Que were added to the M2 storage solution, which is a refrigerated storage solution, to prepare a sperm suspension. The sperm suspension was then transferred to a 0.2 mL tube. Subsequently, the tube was placed in a paper box together with a button thermometer, the paper box was sealed in a thermos bottle, and then packed in a styrofoam box together with a cold insulator. The storage container was stored in a refrigerator (4 ° C.) until it was used (3 days). After storage, the sperm suspension was collected and the refrigerated storage solution was removed by centrifugation (600 g, 4 ° C., 5 minutes). Then, sperm were introduced into 100 μL of pre-culture medium for sperm. The introduced sperm were pre-cultured in an incubator at 37 ° C. and 5% CO 2 for 60 minutes and used for each study.
（ｉｉ）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。
(Ii) Refrigerated sperm (epididymis tail)
The epididymal tail was preserved according to the method of Takeo et al. (Non-Patent Document 1). A pre-refrigerated CARD refrigerated transport kit (Kyudo Co., Ltd.) was used for refrigerated storage of the epididymis tail. To preserve the epididymal tail, first, the epididymal tail is collected from an euthanized mature male mouse, and a refrigerated storage solution in which various concentrations of DMSO and Que are added to a refrigerated storage solution, Lifor or M2 preservative solution, is used. The epididymal tail was transferred to a filled 0.2 mL tube. Subsequently, the tube was placed in a paper box together with a button thermometer, the paper box was sealed in a thermos bottle, and then packed in a styrofoam box together with a cold insulator. The storage container was stored in the refrigerator (4 ° C.) until it was used (~ 7 days). After storage, the epididymal tail was collected and sperm were collected in 100 μL of pre-sperm culture medium. After semen collection, pre-culture was performed in an incubator at 37 ° C. and 5% CO 2 for 60 minutes, and used for each study. On the 0th day of storage, sperms were collected from the epididymal tail collected from mature male mice into a sperm preculture medium and used.
（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 The pre-cultured sperm was diluted with mHTF and sperm was introduced into a dedicated chamber (Hamilton Thorne, Inc.). Then, it was set in HTM-IVOS (Hamilton Thorne, Inc.) and sperm motility was analyzed. The percentage of sperms with motility (Motile sperm) showed the percentage of sperms that moved 5 μm or more per second in the total number of sperms. The percentage (%) of sperm with progressive motile sperm is such that the average value (Path Velocity) of the sperm traveling direction velocity is 50 μm / sec or more and the linearity (Straightness) is obtained. ) Is 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) Egg collection For overovulation treatment, 7.5 IU pregnant mare serum gonadotropin (PMSG) was intraperitoneally administered to mature female mice, and 48 hours later, 7.5 IU human chorionic gonadotropin (hCG) was administered. It was administered intraperitoneally. 14 to 17 hours after administration of hCG, hyperovulatory female mice were euthanized, and 200 μL of 1.0 mM reduced glutathione coated with paraffin oil was used to coat the ovum cumulus cell complex from the ampulla of uterine tube collected. It was introduced into mHTF (in vitro fertilization medium) containing (GSH). 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 a conventional method.
（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 The experimental results were analyzed by analysis of variance using Statcel3, and only when a significant difference was observed, a significant difference test between each group was performed by Tukey's or Dunnett's method. When the significance level P <0.05, it was judged that there was a significant difference in the results.
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 investigated. Epididymal tail collected from euthanized male mice is subjected to Lifor, various concentrations (5, 10%) of DMSO-containing Life, or various concentrations (50, 100 μg / mL) of Que and various concentrations (5, 10%). ) In the DMSO-containing Life, it was stored refrigerated for 4 days or 7 days. The sperm were then precultured in free-TYH (cTYH) containing 0.75 mM methyl-β-cyclodextrin (MBCD) for 60 minutes. Subsequently, the pre-cultured sperm were diluted with mHTF and the 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 were significantly improved by the addition of DMSO. Moreover, by adding Que in addition to DMSO, those improvements became more remarkable.
（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 sperm fertility The effect of DMSO or Que on sperm fertility in refrigerated storage of the epididymis tail was investigated. Epididymal tails collected from euthanized male mice are subjected to Lifor, various concentrations (1, 5, 10%) of DMSO-containing Life, or various concentrations (10, 50, 100 μg / mL) of Que and various concentrations (10, 50, 100 μg / mL). It was stored refrigerated for 7 days in a DMSO-containing Life (1, 5, 10%). The sperm were then precultured in cTYH for 60 minutes. Subsequently, sperm were introduced into mHTF containing 1.0 mM GSH from which eggs were collected, and in vitro fertilization was performed. The fertilization rate was calculated by the following formula. Fertilization rate (%) = number of embryos in the two-cell stage / number of test eggs x 100. The results are shown in FIG.
The proportion of fertile sperm was significantly improved by the addition of DMSO. Further, by adding Que in addition to DMSO, the improvement became more remarkable.
In addition, in vitro fertilization was performed using sperm collected from the epididymis tail that had been refrigerated in DMSO-containing Life or DMSO + Que-containing Life, and in each case, the two-cell stage embryos obtained by in vitro fertilization were obtained. , 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. Epididymal tails collected from euthanized male mice were refrigerated in Life, 10% DMSO-containing Life, for 0, 1, 2, 3, 4, 5, 6, and 7 days. The sperm were then precultured in cTYH for 60 minutes. Subsequently, the pre-cultured sperm were diluted with mHTF and the sperm motility was analyzed with HTM-IVOS. The results are shown in FIG.
With the addition of DMSO, the proportion of sperm with motility and the proportion of sperm with forward motility were maintained in the sperm refrigerated for up to 7 days at the same level as the sperm stored for 0 days (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. Epididymal tails collected from euthanized male mice were refrigerated in Life, 10% DMSO and 100 μg / mL Que-containing Life for 0, 1, 2, 3, 4, 5, 6 and 7 days. did. The sperm were then precultured in cTYH for 60 minutes. Subsequently, the pre-cultured sperm were diluted with mHTF and the sperm motility was analyzed with HTM-IVOS. The results are shown in FIG.
With the addition of DMSO + Que, the proportion of sperm with motility and the proportion of sperm with forward motility were maintained in the sperm refrigerated for up to 7 days at the same level as the sperm stored for 0 days (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 Examining the fertilizing ability of sperm collected from the epididymal tail that was refrigerated for 0 to 7 days in a Refor containing DMSO or DMSO and Que. did. Epididymal tails collected from euthanized male mice were placed in Life containing 10% DMSO, 10% DMSO + 100 μg / mL Que in 0, 1, 2, 3, 4, Refrigerated for 5, 6 and 7 days. Then, sperm were pre-cultured in cTYH for 60 minutes. Subsequently, sperm were introduced into mHTF containing 1.0 mM GSH from which eggs were collected, and in vitro fertilization was performed. The fertilization rate was calculated by the following formula. Fertilization rate (%) = number of two-cell stage embryos / number of test eggs x 100 (n = 3 to 5). The results are shown in FIG.
By adding DMSO, sperms refrigerated for up to 5 days maintained a fertilization rate equivalent to that of sperms stored for 0 days (fresh sperms). In addition, by adding DMSO + Que, the fertilization rate of spermatozoa refrigerated for up to 7 days was maintained at the same level as that of fresh spermatozoa.
（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 storage solution of the present invention Using the refrigerated storage solution and method of the present invention, a domestic refrigerated transportation test was conducted from Asahikawa Medical University to Kumamoto University.
The epididymal tail collected from euthanized male mice was refrigerated in Lift, 10% DMSO and 100 μg / mL Que-containing Life using a CARD refrigerated transport kit, and refrigerated from Asahikawa Medical University to Kumamoto University. Transported. FIG. 6 shows the measurement results of the thermometer installed inside the transportation kit. 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）。
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, sperm were collected from the epididymal tail that had been refrigerated and transported, and pre-cultured in cTYH for 60 minutes. The storage time from the start of refrigerated storage to the collection of sperm was 7 days. Subsequently, the pre-cultured sperm were diluted with mHTF and the sperm motility was analyzed with HTM-IVOS. The results are shown in FIG.
When refrigerated storage using Que-containing Life, the proportion of sperm having motility and the proportion of sperm having forward motility were significantly improved as compared with the case of only Lift.
Subsequently, fertility was confirmed using sperms collected from the epididymis tail that had been refrigerated and transported. Sperm was introduced into mHTF containing 1.0 mM GSH from which eggs were collected, and in vitro fertilization was performed. The fertilization rate was calculated as follows. Fertilization rate (%) = number of two-cell stage embryos / number of test eggs x 100 (n = 3).
When refrigerated storage was carried out using Que-containing Life, the fertilization rate of sperm was significantly improved as compared with the case where only Life was used.
Further, when in vitro fertilization was performed using sperms collected from the epididymis tail that had been refrigerated with Que-containing Life, the two-cell stage embryos obtained by in vitro fertilization developed into 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 preservation solution In the same manner as in Example 1, sperm motility was confirmed when refrigerated storage in M2 preservation solution containing DMSO or DMSO + Que. Refrigerate for 4 days using M2 preservation solution, M2 preservation solution containing 10% DMSO (M2-DMSO), or M2 preservation solution containing 10% DMSO and 100 μg / mL Que (M2-DMSO + Q). After that, the sperm motility was measured. The results are shown in FIG.
Even when the M2 preservative solution was used, the proportion of sperms having fertilizing ability 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 investigated. Suspensions of sperm collected from the epididymal tail collected from euthanized male mice are subjected to M2 preservation solution, various concentrations (1, 5, 10%) of DMSO-containing M2 preservation solution, or various concentrations (10, It was stored refrigerated for 3 days in M2 preservatives containing 50, 100 μg / mL) of Que and various concentrations (1, 5, 10%) of DMSO. The sperm were then precultured in cTYH for 60 minutes. Subsequently, the pre-cultured sperm were diluted with mHTF and the 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 were significantly improved by the addition of 5% DMSO. Further, by adding 1% DMSO + 10 μg / mL Que in combination, the proportion of sperms having the same motility was significantly improved.
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 describes the purpose and subject matter 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 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。 According to the method of the present invention, it is possible to provide sperm capable of achieving practically no problem in motility, fertilization ability and sperm development ability even after refrigerating mouse sperm for, for example, 7 days, which is useful as a refrigerating storage technique for mouse sperm. Is.
Claims (20)
１１または１２に記載のキット。 Claim that the preservation solution is a Life® preservation solution or an M2 preservation solution.
The kit according to 11 or 12.
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