JPS63217269A - Assay of y-spermatozoon of mammal - Google Patents
Assay of y-spermatozoon of mammalInfo
- Publication number
- JPS63217269A JPS63217269A JP62050350A JP5035087A JPS63217269A JP S63217269 A JPS63217269 A JP S63217269A JP 62050350 A JP62050350 A JP 62050350A JP 5035087 A JP5035087 A JP 5035087A JP S63217269 A JPS63217269 A JP S63217269A
- Authority
- JP
- Japan
- Prior art keywords
- sperm
- spermatozoa
- phosphate buffer
- quinacrine
- quinacrine mustard
- Prior art date
- Legal status (The legal status 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 status listed.)
- Granted
Links
- 238000003556 assay Methods 0.000 title claims abstract description 4
- 241000124008 Mammalia Species 0.000 title abstract description 7
- 239000008363 phosphate buffer Substances 0.000 claims abstract description 19
- UKOBAUFLOGFCMV-UHFFFAOYSA-N quinacrine mustard Chemical compound C1=C(Cl)C=CC2=C(NC(C)CCCN(CCCl)CCCl)C3=CC(OC)=CC=C3N=C21 UKOBAUFLOGFCMV-UHFFFAOYSA-N 0.000 claims abstract description 16
- 108091005804 Peptidases Proteins 0.000 claims abstract description 14
- 239000004365 Protease Substances 0.000 claims abstract description 14
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract description 11
- 238000000034 method Methods 0.000 claims description 18
- 210000002593 Y chromosome Anatomy 0.000 abstract description 5
- 238000004140 cleaning Methods 0.000 abstract description 2
- 229960000901 mepacrine Drugs 0.000 abstract description 2
- GPKJTRJOBQGKQK-UHFFFAOYSA-N quinacrine Chemical compound C1=C(OC)C=C2C(NC(C)CCCN(CC)CC)=C(C=CC(Cl)=C3)C3=NC2=C1 GPKJTRJOBQGKQK-UHFFFAOYSA-N 0.000 abstract description 2
- 238000004043 dyeing Methods 0.000 abstract 2
- 108010007093 dispase Proteins 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 18
- 210000000582 semen Anatomy 0.000 description 13
- 238000000926 separation method Methods 0.000 description 10
- 238000005119 centrifugation Methods 0.000 description 8
- 238000012360 testing method Methods 0.000 description 7
- 241001465754 Metazoa Species 0.000 description 5
- 210000001766 X chromosome Anatomy 0.000 description 5
- 210000003765 sex chromosome Anatomy 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- 241000283690 Bos taurus Species 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000007447 staining method Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 241000282472 Canis lupus familiaris Species 0.000 description 3
- 102000035195 Peptidases Human genes 0.000 description 3
- 241000700159 Rattus Species 0.000 description 3
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 238000000432 density-gradient centrifugation Methods 0.000 description 3
- 239000000975 dye Substances 0.000 description 3
- 230000004720 fertilization Effects 0.000 description 3
- 239000007850 fluorescent dye Substances 0.000 description 3
- 244000144972 livestock Species 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 229920001917 Ficoll Polymers 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 241000282887 Suidae Species 0.000 description 2
- 244000309464 bull Species 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 239000006059 cover glass Substances 0.000 description 2
- 239000012997 ficoll-paque Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 240000006432 Carica papaya Species 0.000 description 1
- 235000009467 Carica papaya Nutrition 0.000 description 1
- 108090000317 Chymotrypsin Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- BRLQWZUYTZBJKN-UHFFFAOYSA-N Epichlorohydrin Chemical compound ClCC1CO1 BRLQWZUYTZBJKN-UHFFFAOYSA-N 0.000 description 1
- 241000282575 Gorilla Species 0.000 description 1
- 239000012981 Hank's balanced salt solution Substances 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 241001416505 Mastomys Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 108010059712 Pronase Proteins 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- -1 actinase Proteins 0.000 description 1
- 239000002390 adhesive tape Substances 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229960002376 chymotrypsin Drugs 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 238000003977 dairy farming Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000000918 epididymis Anatomy 0.000 description 1
- 201000010063 epididymitis Diseases 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 238000010030 laminating Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 230000020509 sex determination Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 210000000538 tail Anatomy 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Sampling And Sample Adjustment (AREA)
- Surgical Instruments (AREA)
- Media Introduction/Drainage Providing Device (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は哺乳動物の精液からＹ染色体を有する精子（以
下Ｙ−精子と呼ぶ）を選択的に染色するための方法、殊
に精液からＹ−精子とＸ染色体を有する精子（以下Ｘ−
精子と呼ぶ）とを分離する技術を実施する場合、その分
離程度を検定するための方法に関する。[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for selectively staining spermatozoa having a Y chromosome (hereinafter referred to as Y-sperm) from mammalian semen. - Sperm and sperm with X chromosome (hereinafter referred to as X-
This invention relates to a method for testing the degree of separation when implementing a technique for separating spermatozoa (referred to as spermatozoa).
更に具体的には、本発明は蛍光色素のキナクリンマスタ
ードを用いてＹ−精子を特異的に染色することによりＸ
−精子とＹ−精子とを区別し、両者の分離程度を検定す
るための方法に関する。本発明による蛍光染色法によっ
て識別したＸ−精子またはＹ−精子を人工受精や体外受
精などの繁殖手段に利用することにより所望の性の産仔
を得ることかで−きる。More specifically, the present invention utilizes the fluorescent dye quinacrine mustard to specifically stain Y-sperm.
-Relates to a method for distinguishing between sperm and Y-sperm and testing the degree of separation between the two. By utilizing the X-sperm or Y-sperm identified by the fluorescent staining method of the present invention in reproductive methods such as artificial fertilization and in vitro fertilization, offspring of the desired sex can be obtained.
家畜繁殖の分野で雌雄の生み分けが出来ればその効果は
きわめて大であって、例えば酪農においては雄は不要で
あり、種牝豚を取り扱う業者でも雄は必要がない。また
純粋な種牡豚や優良種牡牛を取り扱う業者は雌を必要と
しない。If it were possible to separate males and females in the field of livestock breeding, the effect would be extremely large; for example, in dairy farming, males would not be necessary, and even in businesses that handle breeding sows, males would not be necessary. Also, businesses that deal in purebred bulls and high-quality bulls do not need females.
しかしながら自然の状態の家畜の繁殖では雄と雌の出産
の確率はそれぞれ２で上記のような要求には出産した個
体数の半分しか合致しない。However, in breeding livestock under natural conditions, the probabilities of male and female births are each 2, and only half of the number of birthing individuals meets the above requirements.
そこで人工的に雌雄を生み分ける方法が求められるので
ある。Therefore, there is a need for a method to artificially separate males and females.
ところで性を決定する因子は性染色体であることが知ら
れている。Ｙ−精子とＸ染色体を有する卵子との結合に
より性染色体がＸＹである子が、またＸ−精子とＸ染色
体を有する卵子との結合により性染色体がＸＸである子
が生まれる。このように雌雄の性決定は精子がＹ染色体
を有するか、Ｘ染色体を有するかにかかることになる。By the way, it is known that the factors that determine sex are sex chromosomes. The combination of a Y-sperm and an egg with an X chromosome produces a child with XY sex chromosomes, and the combination of an X-sperm with an egg with an X chromosome produces a child with XX sex chromosomes. In this way, sex determination depends on whether the sperm has a Y chromosome or an X chromosome.
そこで精子中の性染色体がＹまたはＸのみのものを分別
する手法によって雌雄を生み分けることが原理的には可
能である。Therefore, in principle, it is possible to differentiate between males and females by separating sperm containing only Y or X sex chromosomes.
そしてこの精子中の性染色体がＹまたはＸのものを分別
するためにはＹまたはＸ染色体の質量差に基づく比重の
差による分別手段が提案されている。これまでに、ヒト
、牛では精液に生理食塩水を加え希釈、混和、遠心分離
を繰返して洗浄した精子にパーコール（Ｐｅｒｃａｌｌ
）密度勾配遠心法を適用してＸ−精子とＹ−精子との分
離に成功している。In order to separate whether the sex chromosomes in the sperm are Y or X, a method of separation based on the difference in specific gravity based on the difference in mass of the Y or X chromosomes has been proposed. Until now, in humans and cows, semen has been washed with physiological saline, diluted, mixed, and centrifuged repeatedly.
) Successfully separated X-sperm and Y-sperm using density gradient centrifugation.
また豚については本願と同日に出願した本出願人の発明
のように、精液を予め特定の手段で洗浄し、これを上記
と同様にパーコール密度勾配遠心法により分別してＸ−
精子とＹ−精子とに分離することができる。Regarding pigs, as in the present applicant's invention filed on the same day as the present application, semen is washed in advance by a specific means, and this is separated by Percoll density gradient centrifugation in the same manner as above.
It can be separated into sperm and Y-sperm.
しかしながら、これらのＸ−精子とＹ−精子との分離方
法において、その分離程度を確実にかつ簡単に検定する
方法が必要となる。However, in these methods of separating X-sperm and Y-sperm, a method is required to reliably and easily verify the degree of separation.
このためにはＹ−精子のみを特異的に染色する染料の使
用が考慮される。For this purpose, the use of a dye that specifically stains only Y-sperm may be considered.
ところでＹ染色体の長腕部の中心からはなれた領域にお
いてキナクリン（キナタリン塩酸塩）で染まる部分すな
わちＦ一体（Ｆ−ｂｏｄｙ）が存在しこれはヒトおよび
ゴリラのリンパ球の染色体標本では知られているが、他
の動物の染色体標本には適用できない（Ｎａｔｕｒｅ
Ｍｏｌ ２３１．、 Ｊｕｎｅ ４゜（１９７１）３２
６〜３２９頁）。またヒト精子ではキナクリンマスター
ドで染色されたものがＦ一体と視認されている（Ｎａｔ
ｕｒｅ Ｍｏｌ ２２６．、 Ｊｕｎｅ ６．（１９７
０）９６１〜９６２頁）がこの染色方法は他の哺乳動物
のＹ−精子の染色にはそのままで用いることができなか
った。By the way, there is a region far from the center of the long arm of the Y chromosome that is stained with quinacrine (quinataline hydrochloride), the F-body, and this is known from human and gorilla lymphocyte chromosome specimens. However, it cannot be applied to chromosome specimens of other animals (Nature
Mol 231. , June 4゜ (1971) 32
6-329). Furthermore, human spermatozoa stained with quinacrine mustard are visually recognized as F-integrated (Nat
ure Mol 226. , June 6. (197
0) pp. 961-962), but this staining method could not be used as is for staining Y-sperm of other mammals.
上記したように、哺乳動物の精子を分離してＸ−精子と
Ｙ−精子を得る場合、その分離の程度の検定のためにＹ
−精子の蛍光染料による染色を行うことが考慮されるが
、これ迄には特定の動物種に適用しうる染料と方法しか
なく、この染色手段を総ての哺乳動物に適用可能とする
ことが求められてきた。従って本発明はＹ−精子を特異
的に染色する染料および染色方法を提供し、かくして総
ての哺乳動物についてＹ−精子を染色しもってＹ−精子
を定量しようとするものである。As mentioned above, when separating mammalian sperm to obtain X-sperm and Y-sperm, Y-sperm is used to test the degree of separation.
- Staining of spermatozoa with fluorescent dyes has been considered, but until now there are only dyes and methods that can be applied to specific animal species, and it is not possible to make this staining method applicable to all mammals. I've been asked for it. Therefore, the present invention provides a dye and staining method that specifically stains Y-sperm, and thus attempts to stain Y-sperm and quantify Y-sperm in all mammals.
本−発明者らは、家畜、実験動物など哺乳動物の精子を
予めリン酸塩緩衝液で洗浄し、次いでプロテアーゼで処
理することによってＹ−精子をキナクリンマスタードに
よって染色しうろことを見出して本発明を完成した。The present invention - The inventors have discovered that scales of Y-sperm can be stained with quinacrine mustard by washing the sperm of mammals such as livestock and laboratory animals in advance with a phosphate buffer and then treating them with protease. completed.
すなわち、本発明者らは、マウス、ラット、マストミス
、犬、牛などの動物種にあってはその精液を採取し、遠
心分離に付するが付することなく（この遠心分離処理は
粗大異物の除去を目的とし１５００〜２０００ｒｐｍで
行うもの）リン酸塩緩衝液に加えて遠心分離処理に付し
、上澄部を廃棄し、そして必要によってこのリン酸塩緩
衝液による処理を２〜５回程度繰返し、このよう°にし
て得られるリン酸塩緩衝液で処理した精子についてプロ
テアーゼを含有する溶液を加えて３７℃程度の温度に１
０〜２０分間保持した場合には、この精子は容易にキナ
クリンマスタードにより染色されうろことを見出した。In other words, the present inventors collected semen from animal species such as mice, rats, mastomyces, dogs, and cows, and subjected it to centrifugation without contamination (this centrifugation process removes coarse foreign substances). (Carry out at 1,500 to 2,000 rpm for the purpose of removal) Add to phosphate buffer, centrifuge, discard the supernatant, and if necessary, treat with this phosphate buffer about 2 to 5 times. Repeatedly, a solution containing protease was added to the phosphate buffer-treated spermatozoa obtained in this manner, and the mixture was heated to a temperature of about 37°C for 1 hour.
When held for 0-20 minutes, the spermatozoa were found to be easily stained with quinacrine mustard scales.
また豚のようにその精液中に夾雑物が大量に含まれその
ために予備処理を必要とするものについてはその精液を
例えばフィコール４００の商品名で知られたファルマシ
ア社製の製品であるショ糖とエピクロルヒドリンとの水
溶性の共重合物質とジアドリゾエートナトリウムとの混
合物（溶液１００岐中にフィコール４００を５．７ｇと
、ジアドリゾエートナトリウムを９ｇ含有する）すなわ
ちフィコール−パーク（Ｆ １ｃｏｌｌ−Ｐａｑｕｅ）
のような浸透圧が制御されていて生体膜を通過すること
のない媒体と共に遠心分＃ｌ器中で遠心分離操作処理に
付し、精液中の夾雑物を取り除き、かくして得られた精
子を含む上層部についてこれを上記と同様のリン酸塩緩
衝液に加えて遠心処理に付し、上澄部を廃棄し、そして
必要に応じてこのリン酸塩緩衝液による処理を２〜５回
程度繰り返し、このようにして得られるリン酸塩緩衝液
で処理した精子についてプロテアーゼを含有する溶液を
加えて３７℃程度の温度に１０〜２０分間保持した場合
については同様にこの精子は容易にキナクリンマスター
ドにより染色されうろことを見出した。In addition, for pigs whose semen contains a large amount of impurities and therefore requires pretreatment, the semen may be treated with sucrose, a product manufactured by Pharmacia, known under the trade name Ficoll 400. A mixture of a water-soluble copolymer with epichlorohydrin and sodium diadrizoate (containing 5.7 g of Ficoll 400 and 9 g of sodium diadrizoate in 100 g of solution), namely Ficoll-Paque (F 1coll- Paque)
Contaminants in the semen are removed by centrifugation in a centrifuge with a medium that has a controlled osmotic pressure and does not pass through biological membranes, such as For the upper layer, add it to the same phosphate buffer as above, centrifuge it, discard the supernatant, and repeat the treatment with this phosphate buffer 2 to 5 times as necessary. Similarly, when a solution containing protease is added to the sperm treated with the phosphate buffer solution obtained in this way and the sperm is kept at a temperature of about 37°C for 10 to 20 minutes, the spermatozoa are easily treated with quinacrine mustard. I found dyed scales.
このように染色された精子については、蛍光顕微鏡で検
鏡することにより染色されたＦ一体を観察することがで
き、そしてこの染色されたＦ一体の数をカウントするこ
とによってＹ−精子の数を知ることができるのである。For spermatozoa stained in this way, the stained F-sperms can be observed by examining them with a fluorescence microscope, and the number of Y-sperms can be determined by counting the number of stained F-sperms. It is possible to know.
本発明の方法において精液の洗浄に用いられるリン酸塩
緩衝液としては、リンゲル液、ロック液、タイロード液
、ハンクス液、ダルベツコ液などの生理的リン酸塩類溶
液が挙げられる。Phosphate buffers used for washing semen in the method of the present invention include physiological phosphate solutions such as Ringer's solution, Locke's solution, Tyrode's solution, Hank's solution, and Dulbecco's solution.
これらのリン酸塩類溶液の使用量は洗浄目的が達成され
る限りにおいていかなる量で用いても良いが、通常は１
回の遠心分離処理当り精子を含む分画の１容量当り１〜
５倍容量、通常は２〜３倍容量で用いられる。These phosphate solutions may be used in any amount as long as the cleaning purpose is achieved, but usually 1.
1 to 1 volume of sperm-containing fraction per centrifugation process
It is used at 5 times the capacity, usually 2 to 3 times the capacity.
本発明の方法で使用されるプロテアーゼとしては、至適
ＰＨが中性域にある動物由来、植物由来および微生物由
来のプロテアーゼのいずれもが用いえられ、そしてこれ
らプロテアーゼの具体例としてはディスパーザ、パパイ
ア、プロテアーゼ、トリプシン、キモトリプシン、アク
チナーゼ、プロネースなどが挙げられる。そしてこれら
のプロテアーゼ中でディスパーザが殊に好ましく用いら
れる。As the protease used in the method of the present invention, any of animal-derived, plant-derived, and microbial-derived proteases whose optimal pH is in the neutral range can be used, and specific examples of these proteases include Dispaza, Papaya , protease, trypsin, chymotrypsin, actinase, pronase, etc. Among these proteases, disperser is particularly preferably used.
キナクリンマスタードによる染色はリン酸塩緩衝液中に
０．００２５〜０．００５０％程度の量で溶解させたキ
ナクリンマスタード溶液を用いて行われる。この溶液の
少屯を染色しようとする精子に加え、２０〜３５℃で１
００〜１８０分程度振盪し程度ら保持することによって
行われる。Staining with quinacrine mustard is performed using a quinacrine mustard solution dissolved in a phosphate buffer in an amount of about 0.0025 to 0.0050%. Add a small amount of this solution to the spermatozoa to be stained and
This is done by shaking and holding for about 0.00 to 180 minutes.
このようにして本方法によってはＹ−精子とＸ−精子を
分離する前の精子混合物についてその中に存在するＹ−
精子を検定することができるのは勿論であるが、Ｙ−精
子とＸ−精子とを分離する技術における分離程度を検定
するための方法として本方法は殊に有用である。すなわ
ち、種々の分離手段によって分離したＹ−精子とＸ−精
子とについて、その分離程度の検定は人工受精や体外受
精による繁殖手段における圧子の好ましい性のコントロ
ールのために必須であって、この方法はその−ための有
効な手段を提供するものである。In this way, the present method allows the sperm mixture, prior to separation of Y-sperm and X-sperm, to
Of course, sperm can be assayed, but this method is particularly useful as a method for assaying the degree of separation in a technique for separating Y-sperm and X-sperm. In other words, testing the degree of separation of Y-sperm and provides an effective means for this purpose.
以下に実施例によってこの発明を更に詳細に説明する。The present invention will be explained in more detail with reference to Examples below.
実施例 ｌ
牛の凍結精液（市販品）を室温で解凍し、１５００〜２
００Ｏｒｐｍで１０分間遠心分離処理を行って粗大異物
を除去し、精子塊を取り出した。これにダルベツコのリ
ン酸塩緩衝液を加え、混和後再び遠心分離して上清を除
いた。残った精子塊に同じダルベツコのリン酸塩緩衝液
を加えて遠心分離する操作をさらに２回行った。Example 1 Frozen cow semen (commercially available) is thawed at room temperature and
Centrifugation was performed at 00 rpm for 10 minutes to remove coarse foreign matter and the sperm mass was taken out. Dulbecco's phosphate buffer was added to this, mixed, and then centrifuged again to remove the supernatant. The same Dulbecco's phosphate buffer was added to the remaining sperm mass and centrifugation was performed two more times.
このようにリン酸塩緩衝液で洗浄処理した牛の精液のリ
ン酸塩緩衝液中における懸濁液（約５ＸｌＯ’精子／顧
）の０ 、１ ＊（ｌにプロテアーゼ液（Ｄｉｓｐａｓ
ｅ三光純薬製、４０００ Ｐ、ｕ、／ｄ） ０．１ｘＱ
を加え、３７℃で１０〜２０分間保持した。A suspension of bovine semen washed in phosphate buffer (approximately 5XlO' sperm/sperm) was added with protease solution (Dispas) to 0,1*(l).
e Sanko Pure Chemical Industries, 4000 P, u, /d) 0.1xQ
was added and held at 37°C for 10-20 minutes.
このようにプロテアーゼ処理した精液にダルベツコのリ
ン酸塩緩衝液中に０．００２５〜ｏ、ｏｏｓ％の濃度で
溶解したキナクリンマスタード（Ｓｉｇｍａ社製）溶液
を０ ； ２ ｘ（ｌ加え、２０〜２５℃で１００〜１
８０分間振盪しながら保持した。To the semen treated with protease in this way, a solution of quinacrine mustard (manufactured by Sigma) dissolved in Dulbecco's phosphate buffer at a concentration of 0.0025 to 0.000% was added at a concentration of 0; 2.times. 100-1 at °C
Hold with shaking for 80 minutes.
このようにキナクリンマスタードで染色した精子液の１
滴をスライドグラス上に取りカバーグラスで覆いカバー
グラス周辺をパラフィンで封じた。One of the sperm fluids stained with quinacrine mustard like this.
The drop was placed on a slide glass, covered with a cover glass, and the area around the cover glass was sealed with paraffin.
この染色標本について蛍光顕微鏡（オリンパスＢＨ３−
ＲＦＫ、励起フィルター：４４０ｒ＋ｌｌ、バリアフィ
ルター＋ ９１０ ｒｖ、光源：超高圧水銀灯ＵＳＨ−
２ＧＯＭｂ）で観察し１００〜２００個の精子のうちで
その頭部にＦ一体（蛍光を発する小体）が認められるも
のの数をカウントした。かくして１ｏｏｏｏ精子以上を
観察した結果、４３％にＦ一体が認められた。This stained specimen was examined using a fluorescence microscope (Olympus BH3-
RFK, excitation filter: 440r+ll, barrier filter + 910rv, light source: ultra-high pressure mercury lamp USH-
2GOMb), and out of 100 to 200 spermatozoa, the number of spermatozoa in which F-units (fluorescent bodies) were observed in their heads was counted. As a result of observing more than 100 sperm, 43% were found to contain F.
同様の操作を犬の射出精子、ウサギの射出精子およびマ
ウス、ラット、マストミスの精子（精巣上体尾部から夫
々採取）について夫々行ったところ、犬：２０％、ウサ
ギ：４６％、ラット、マウス、マストミス：５〜１３％
の精子にＦ一体が認められた。Similar operations were performed on dog ejaculated sperm, rabbit ejaculated sperm, and mouse, rat, and mastomys sperm (collected from the cauda epididymis, respectively), and the results were as follows: dog: 20%, rabbit: 46%, rat, mouse, Must miss: 5-13%
F-1 was detected in the sperm of the patient.
実施例 ２
フィコール−パーク３ ｘＱを遠沈管に取り、その上に
４ｊ１１２の豚の精液を重層した。２００Ｇで５分間遠
心分離後精子を含む上層をパスツールピペットで採取し
、これにダルベツコのリン酸塩緩衝液を２〜３倍量（容
量）加え、混和後８０Ｇで１０分間遠心分離し上清を除
いた。Example 2 Ficoll-Paque 3 xQ was placed in a centrifuge tube, and 4j112 pig semen was layered on top of it. After centrifugation at 200G for 5 minutes, collect the upper layer containing sperm with a Pasteur pipette, add 2 to 3 volumes of Dulbecco's phosphate buffer, mix, and centrifuge at 80G for 10 minutes to remove the supernatant. was excluded.
残った精子塊に３〜４ ｘＱのダルベツコのリン酸塩緩
衝液を加え同様の遠心分離処理をさらに２回繰返した。3 to 4 x Q of Dulbecco's phosphate buffer was added to the remaining sperm mass, and the same centrifugation process was repeated two more times.
このように洗浄した精子塊についてパーコール（Ｐｅｒ
ｃｏｌｌ）密度勾配遠心法によりＸ−精子とＹ−精子の
分離を行いその分離程度を検定した。The thus washed sperm mass was treated with Percoll (Percoll).
(coll) X-sperm and Y-sperm were separated by density gradient centrifugation, and the degree of separation was examined.
その手順は次の通りである。The procedure is as follows.
１）パーコール液の調製
１１ａｍＦ＋を液を溶媒として、３２．３８，５０．６
Ｇ、６５．７０゜７５％のパーコール液を作り、ＨＥＰ
ＥＳ ｂｕｆｆｅｒでＰＨ７，４に調整後、ミリポアフ
ィルタ−（０，４５μ）で濾過滅菌した。1) Preparation of Percoll solution 11amF+ as a solvent, 32.38, 50.6
G, 65.70° Make a 75% Percoll solution and apply HEP
After adjusting the pH to 7.4 with ES buffer, it was sterilized by filtration using a Millipore filter (0.45μ).
２）パーコール液、精液の重層
プラスチック製試験管に熱した注射針（１８Ｇ）を用い
て各パーコール層の界面の位置に穴をあけ、粘着テープ
で封じる。2) A hole is made at the interface of each Percoll layer using a heated injection needle (18G) in a multilayer plastic test tube containing Percoll liquid and semen, and the hole is sealed with adhesive tape.
この試験管にパーコール液を７５．７０，６５．６０，
５０゜３８．３２％の順に０，７顧ずつ積層した後、洗
浄した精子懸濁液１．４顧を重層した。Add Percoll solution to this test tube at 75.70, 65.60,
After laminating 0.7 layers in the order of 50° and 38.32%, 1.4 layers of the washed sperm suspension were layered.
３）分画の採取
上記プラスチック製試験管を２５０Ｇで５〜２０分間遠
心分離した後、各密度界面にあらかじめ設けた小孔から
注射針を挿入して各分画に存在する精子を採取した。3) Collection of fractions After centrifuging the above plastic test tubes at 250G for 5 to 20 minutes, a syringe needle was inserted through a small hole previously provided at each density interface to collect sperm present in each fraction.
４）精子の酵素処理
各々の分画に存在する精子の懸濁液（約５×１０′精子
／ ｘ（１） ０．１ｘ（ｌにプロテアーゼ液（Ｄｉｓ
ｐａｓｅ三光純薬製、４０００Ｆ’、ｕ、／ｌＱ ）
０．１ｘｆ２を加え３７°Ｃで１０分間保持した。4) Enzymatic treatment of sperm The suspension of sperm present in each fraction (approximately 5 x 10' sperm/x (1)
pase Sanko Pure Chemical Industries, 4000F', u, /lQ)
0.1xf2 was added and kept at 37°C for 10 minutes.
５） 染 色
プロテアーゼ液で処理した夫々の分画の精子懸濁液にダ
ルベツコのリン酸塩緩衝液中に０．００２５％の濃度で
溶解したキナクリンマスタード溶液をＱ、２岐加え、２
０〜２５℃で１００分間振盪しながら保持した。5) A quinacrine mustard solution dissolved in Dulbecco's phosphate buffer at a concentration of 0.0025% was added to the sperm suspension of each fraction treated with the staining protease solution.
It was held at 0-25°C for 100 minutes with shaking.
６） Ｘ−精子、Ｙ−精子の分離の検定上記のように
各分画の精子をキナクリンマスタートで染色したものに
ついて、Ｘ−精子（Ｆ−ｂｏｄｙ陰性）とＹ−精子（Ｆ
−ｂｏｄｙ陽性）の割合を調べた。結果を次の表で示す
。6) Assay of separation of X-sperm and Y-sperm The sperm of each fraction was stained with quinacrine mustard as described above, and the X-sperm (F-body negative) and Y-sperm (F-body negative) were separated.
-body positive) was examined. The results are shown in the table below.
３２％と３８％の界面 −＊
３８％と５０％の界面 −＊＊５０％と６０
％の界面 １５０／ ２０９ ７１．８６０％
と６５％の界面 ８４／１ｇ３ ４５．９６
５％と７０％の界面 ２９／ １６８ １７
．３７０％と７５％の界面 ２１／１７４
１２．１沈降精子 １５／ １８９ ７
．９＊ 精子ナシ
＊木精子極小
次いで夫々の分画の精子を用いて人工受精を行った結果
、Ｆ−ｂｏｄｙ７２％のＹ−精子分画を用いた群では雄
２１頭、雌９頭の子豚が得られ、Ｆ−ｂｏｄｙ ７．９
％のＸ〜精子分画を用いた群では雄３頭、酸２０頭が得
られた。Interface between 32% and 38% -* Interface between 38% and 50% -**50% and 60
% interface 150/209 71.860%
and 65% interface 84/1g3 45.96
Interface between 5% and 70% 29/ 168 17
．． Interface between 370% and 75% 21/174
12.1 Sedimented sperm 15/ 189 7
．． 9 * No sperm * Minimal tree sperm Next, as a result of artificial fertilization using the sperm of each fraction, 21 male piglets and 9 female piglets were obtained in the group using the Y-sperm fraction with 72% F-body. was obtained, F-body 7.9
In the group using the %X~ sperm fraction, 3 males and 20 males were obtained.
Claims (1)
プロテアーゼで処理したものについて、キナクリンマス
タードでＹ−精子を染色し、Ｙ−精子のＦ−体を視認で
きるようにしたことを特徴とするＹ−精子の検定方法。The method is characterized in that the Y-sperm of mammalian sperm was washed in advance with a phosphate buffer and then treated with protease, and the Y-sperm was stained with quinacrine mustard to make the F-form of the Y-sperm visible. Y-Sperm assay method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62050350A JP2511445B2 (en) | 1987-03-06 | 1987-03-06 | Pig Y-sperm assay method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62050350A JP2511445B2 (en) | 1987-03-06 | 1987-03-06 | Pig Y-sperm assay method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS63217269A true JPS63217269A (en) | 1988-09-09 |
| JP2511445B2 JP2511445B2 (en) | 1996-06-26 |
Family
ID=12856462
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62050350A Expired - Fee Related JP2511445B2 (en) | 1987-03-06 | 1987-03-06 | Pig Y-sperm assay method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2511445B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103822816A (en) * | 2013-12-16 | 2014-05-28 | 内蒙古赛科星繁育生物技术股份有限公司 | Method for identifying mixed sperms by using fluorescent dyes FITC (fluorescein isothiocyanate) and MITO (Mitotracker) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100949923B1 (en) | 2008-04-21 | 2010-03-30 | 중앙대학교 산학협력단 | Selection method for poor fertility boars |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5356381A (en) * | 1976-10-20 | 1978-05-22 | Bhattacharya Chandra Bhairab | Method and apparatus for separating sperm cell from seminal fluid |
-
1987
- 1987-03-06 JP JP62050350A patent/JP2511445B2/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5356381A (en) * | 1976-10-20 | 1978-05-22 | Bhattacharya Chandra Bhairab | Method and apparatus for separating sperm cell from seminal fluid |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103822816A (en) * | 2013-12-16 | 2014-05-28 | 内蒙古赛科星繁育生物技术股份有限公司 | Method for identifying mixed sperms by using fluorescent dyes FITC (fluorescein isothiocyanate) and MITO (Mitotracker) |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2511445B2 (en) | 1996-06-26 |
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