WO2018101270A1 - Solution de stockage à froid et procédé de stockage de sperme de souris - Google Patents

Solution de stockage à froid et procédé de stockage de sperme de souris Download PDF

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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
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sperm
dimethyl sulfoxide
refrigerated
storage
concentration
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PCT/JP2017/042653
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English (en)
Japanese (ja)
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直己 中潟
透 竹尾
英高 吉本
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国立大学法人熊本大学
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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
    • 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.

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Abstract

La présente invention concerne le stockage à froid de sperme de souris, une solution de stockage à froid et un procédé de stockage qui permettent le stockage à froid sur une durée plus longue que celle de l'état de la technique. La présente invention concerne une solution de stockage à froid pour le stockage à froid de sperme de souris contenu dans la queue de l'épididyme, la solution de stockage contenant du sulfoxyde de diméthyle ou du sulfoxyde de diméthyle et de la quercitine. La présente invention concerne également un procédé de stockage à froid de sperme de souris utilisant cette solution de stockage. Le sperme de souris qui a été stocké à froid selon la présente invention peut conserver une fertilité et une motilité élevées.
PCT/JP2017/042653 2016-11-29 2017-11-28 Solution de stockage à froid et procédé de stockage de sperme de souris WO2018101270A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115251042A (zh) * 2022-09-08 2022-11-01 安徽科技学院 一种家禽精液稀释缓冲液及其制备和家禽精液冷存方法

Citations (2)

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WO1998037182A1 (fr) * 1997-02-19 1998-08-27 Universite Laval Fixation de la catalase de l'oviducte aux membranes de spermatozoides et ses applications
WO2014162910A1 (fr) * 2013-04-03 2014-10-09 石原産業株式会社 Conservateur pour la cryoconservation de matériels biologiques et procédé de conservation de matériels biologiques à basses températures

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
WO1998037182A1 (fr) * 1997-02-19 1998-08-27 Universite Laval Fixation de la catalase de l'oviducte aux membranes de spermatozoides et ses applications
WO2014162910A1 (fr) * 2013-04-03 2014-10-09 石原産業株式会社 Conservateur pour la cryoconservation de matériels biologiques et procédé de conservation de matériels biologiques à basses températures

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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 *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115251042A (zh) * 2022-09-08 2022-11-01 安徽科技学院 一种家禽精液稀释缓冲液及其制备和家禽精液冷存方法
CN115251042B (zh) * 2022-09-08 2023-06-02 安徽科技学院 一种家禽精液稀释缓冲液及其制备和家禽精液冷存方法

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