CN101865844B - Acrosome reaction kit, method for inducing acrosome reaction and , and method for evaluating acrosome reaction condition - Google Patents
Acrosome reaction kit, method for inducing acrosome reaction and , and method for evaluating acrosome reaction condition Download PDFInfo
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Abstract
The invention discloses an acrosome reaction kit, which comprises tissue slide preserving fluid, a fluorescent conjugate and a fluorescent sealing solid liquid, wherein each 100 ml of the tissue slide preserving fluid contains 0.5ml of glutaraldehyde, 5ml of methanol, 2ml of ethanol, 0.2g of polyvinylpyrrolidone (PVP), 1ml of formalin and the balance of purified water. The kit can carry out a sperm-induced acrosome reaction and detection; and if calcium ion induction is not carried out, the kit can carry out the acrosome reaction condition evaluation and the acrosome integrity determination. By the tissue slide preserving fluid, the test process for the sperm-induced acrosome reaction and detection is carried out by two parts, samples and test results which are obtained in the first part can be preserved and the final test result cannot be influenced if the operations of the second part are carried out in the subsequent 72 hours, so that the work intensity can be relieved. The fluorescence of a fluorescent staining system is strong and cannot be attenuated after being stabilized for 15 days, so that the detection result is more stable and is easy to interprete and the repeatability of the result can be ensured. The kit and a determination method can be clinically applied and popularized.
Description
Technical field
The present invention relates to the examination of perforatorium function; Particularly simulate the environment of being fertilized in the human body external; At external startup capacitation, bring out acrosome reaction; Estimate the ability of capacitation spermatogenesis acrosome reaction through fluorescent dye, thus the ability that the assessment sperm penetrates egg mother cell and makes it to be fertilized, for the selection of sterility etiological analysis, supplementary reproduction therapeutic scheme provides theoretical foundation.
Technical background
The male sex and correlative factor thereof account for 50% of infertile Mr. and Mrs' cause of disease at least.Main in the clinical practice by detecting the seminal fluid conventional analysis of indexs such as sperm concentration, vigor and form; Estimate male fecundity; But the index that conventional semen analysis provides has only reflected the quality of sperm; And the function of having only sperm could reflect the insemination ability (fecundity) of sperm inside and outside, therefore only relies on the seminal fluid routine to explain that the reason of male sterility and judgement male fecundity are far from being enough.Yet in the clinical practice, the thoroughly evaluating sperm function is extremely difficult.
In recent decades; Around smart ovum meet, combine, a series of physiology courses such as insemination and embryo growth growth; Researchist and clinical position person have explored the multiple sperm function test that can reflect these physiology courses, wherein the most important thing is the perforatorium function test.Acrosome is the distinctive eucaryotic cell structure of sperm, and the most essential steps of fertilization process such as participate in comprising that smart ovum is discerned, combined, penetrates plays a decisive role to sperm insemination ability.After acrosome reaction occurs in sperm and the egg mother cell oolemma combines, sperm penetrates oocyte membrane and makes before the oocyte fertilization, is one of necessary link of egg mother cell natural fertilization.As do not have the round end sperm disease of acrosome, and sperm can not combine with ovum fully, treatment can not be inseminated even such patient uses inseminatio externalis-embryo transfer technology (IVF-ET).
The perforatorium function test comprises that sperm morphology and integrality determination test, oolemma bring out sperm acrosome reaction determination test etc. at present; Although these tests have positive role to the success ratio of assessment sperm fertilizing ability, selection and prediction IVF (inseminatio externalis) or ICSI (injection in the monosperm ovarian follicle slurry); But because of these detection method many places in conceptual phase; The methodology of world health organisation recommendations is too loaded down with trivial details and complicated; Methodological stability is relatively poor with repeatability, is difficult to be able to popularize as conventional method clinically, lacks the product of industrialization.The current experiments method requires entire test to accomplish without any letup, and brings out smear fluorescent dye and interpretation as a result from the pre-service of semen sample, external capacitation, calcium ion, time-consuming 6～7 hours of whole test, and laboratory strength is big, is difficult to clinical practice and universal.And the fluorescent dye system strength of existing test method is not high, can in half an hour, decay rapidly usually, has influenced the accuracy of interpretation as a result, can cause the instability of testing result.
Summary of the invention
The technical matters that the present invention will solve is; Provide a kind of can with sperm bring out the acrosome reaction test experience be divided into two parts carry out with reduce laboratory strength, simultaneously make test findings more accurately, the kit of good reproducibility; Provide simultaneously and utilize this kit to carry out sperm to bring out the method that acrosome reaction detects, and provide and utilize this kit to carry out the method for sperm acrosome reaction condition evaluation.
For solving the problems of the technologies described above, the present invention provides a kind of sperm acrosome reaction kit, it is characterized in that, comprises tissue preservation liquid, fluorescence bond, fluorescence sealing liquid.
Said tissue is preserved the every 100ml of liquid and contained: glutaraldehyde 0.5ml, methyl alcohol 5ml, ethanol 2ml, polyvinyl pyrrolidone PVP0.2g, formalin 1ml, surplus is a pure water.
Said fluorescence bond is preserved liquid by fluorescence bond PSA-FITC and fluorescence bond and is formed pH7.5～8.0, adding 1mg fluorescence bond PSA-FITC in every 100ml fluorescence bond preservation liquid;
It is formulated with the following material of dissolved in purified water that wherein said fluorescence bond is preserved liquid, and every 100ml contains:
Fluorescein-labelled thing: 0.5～10ng, wherein, luciferin is the huge isothiocyanic acid rhodamine of fluorescein isothiocynate or tetramethyl; Being labeled thing is pisum sativum agglutinin, peanut agglutinin or concanavalin A;
Mol ratio is 1: 2 methacrylic acid and methylmethacrylate copolymer: 0.4～2.4g;
Tris damping fluid: 0.7～3.5g;
NaCl：0.3～1.5g；
Casein: 0.1～0.5g;
Sodium salicylate: 0.2～2.0mg;
Antiseptic Proclin300:20～100ul;
Surplus is a pure water.
Said fluorescence sealing liquid is formulated with the following material of dissolved in purified water, pH8.5-9.0, and every 100ml contains:
N-(1, the 3-dimethylbutyl)-N '-diphenyl-para-phenylene diamine: 0.05～0.5g;
PVAC polyvinylalcohol: 0.2～1.6g;
Polyglycol 8000:0.1～1.5g;
Tris damping fluid: 1.0～5.0g;
NaCl：0.4～1.6g；
BSA BSA:0.5～5g;
NaN
3Or thimerosal: 0.01～0.05g;
Glycerine: 6%～60%;
Surplus is a pure water.
Said sperm acrosome reaction kit also comprises Calcium ionophore.
Said Calcium ionophore is a calcium ion carrier A 23187.
Said sperm acrosome reaction kit also comprises 80% density gradient solution, 40% density gradient solution, sperm cleansing solution A, sperm nutrient solution, 5 * sperm washing lotion B, contrast liquid, immobile liquid, organizes slide glass, microscler cover glass.
Simultaneously, the present invention also provides a kind of and utilizes said kit to carry out the method that sperm brings out the acrosome reaction detection, it is characterized in that, comprises the steps:
Step 1: fresh semen sample is liquefied fully, and again with density gradient method or go up the swimming method and extract motile sperm, counting extracts the density of back motile sperm then, is 1 * 10 with sperm nutrient solution preparation density
6The sperm suspension of motile sperm/ml;
Step 2: the clean Eppendorf pipe that with the capacity is 1.5ml is as reaction tube, and every example detects sample and all establishes simultaneously and measure pipe and control tube, and every pipe adds the sperm suspension 0.5ml for preparing in the step 2, at CO
2Account in the CO2gas incubator that volume content 5%, air account for volume content 95% and under 37 ℃, hatched 3 hours, to induce capacitation;
Step 3: lucifuge adds Calcium ionophore 2.5ul in measuring pipe, in control tube, adds contrast liquid 2.5ul, respectively mixing; Manage at CO two
2Account in the CO2gas incubator that volume content 5%, air account for volume content 95% and under 37 ℃, hatched 15 minutes;
Step 4: take out reaction tube, add sperm washing lotion B application liquid 1ml in control tube respectively with measuring in the pipe rapidly; To remove supernatant behind the centrifugal 5min of 600g RCF, add sperm washing lotion B application liquid 1.5ml, once more to remove supernatant behind the centrifugal 5min of 600g RCF, adjust the sperm suspension volume to 100ul with sperm washing lotion B application liquid then;
Step 5: get 10 μ l sperm suspensions that step 4 obtains and evenly coat and organize being coated with in the gate on the slide glass, air dry in the air;
Step 6: will organize slide glass to be soaked in fully in the reaction tank that fills immobile liquid, the rearmounted room temperature of the tight lid of lid is 30min fixedly; 2min is soaked in physiological saline washing 3 times at every turn;
Step 7: remove and to organize the slide glass surface and to be coated with the moisture in the gate, in being coated with gate, add said fluorescence bond 40 μ l rapidly after organizing the slide glass drying, be placed horizontally in the wet box, 37 ℃ of reaction 60min with distilled water washing 3 times, soak 2min at every turn;
Step 8: after the tissue air dry that step 7 obtains;, each adds 1 fluorescence sealing liquid in being coated with gate, covered, observations under 100 times of object lens of fluorescent microscope of exciting light 450-490nm, blue color filter; Wherein, Each is coated with gate and observes 400 sperms of counting at least, and the sperm quantity of acrosome reaction takes place record, and carries out result's calculating and judgement.
In the method that the acrosome reaction of bringing out described sperm detects; When can not disposable continuous completion testing; Then can be after said step 5, before the step 6, carry out following steps: with dried organize slide glass to be soaked in fully to fill tissue preserve in the reaction tank of liquid, the tight lid of lid is placed on 2-8 ℃ of preservation; In 72 hours, take out, continue to carry out said step 6～eight then.
The present invention also provides a kind of method of utilizing said kit to carry out the sperm acrosome reaction condition evaluation, it is characterized in that, comprises the steps:
Step 1: simply wash sperm sample or above swimming method and extract high motile sperm or extract high motile sperm, with physiological saline sperm concentration is adjusted to 5 * 10 then with density gradient method
6/ ml;
Step 2: get 10 μ l sperm suspensions and evenly coat and organize being coated with in the gate on the slide glass, air dry in the air;
Step 3: will organize slide glass to be soaked in fully in the reaction tank that fills immobile liquid, the rearmounted room temperature of the tight lid of lid is 30min fixedly; 2min is soaked in physiological saline washing 3 times at every turn;
Step 4: remove and organize the slide glass surface and be coated with the moisture in the gate, in being coated with gate, add fluorescence bond 50 μ l rapidly, be placed horizontally in the wet box, 37 ℃ of reaction 60min;
Step 5: with distilled water washing 3 times, at every turn soak 2min then;
Step 6: after the tissue air dry; Each is coated with and adds 1 fluorescence sealing liquid in the gate, covered, observations under 100 times of object lens of fluorescent microscope of exciting light 450-490nm, blue color filter; Wherein, Each is coated with gate and observes 400 sperms of counting at least, writes down all kinds of perforatorium amount of state: it is that acrosome is complete that sperm head 1/2 has bright uniform fluorescence with the upper part, counts AI; Only having a fluorescent belt or all acrosome districts all not to have fluorescence at the sperm head equatorial plate is acrosome reaction, counts AR; Other sperm is unusual acrosome; Calculating AI, AR account for the percent of observed sperm.
Beneficial effect of the present invention:
The method that the acrosome reaction of bringing out kit of the present invention and sperm thereof detects and the experimental technique of sperm acrosome reaction condition evaluation; Through system optimization; The traditional experiment method of comparing operation is easier; Process of the test is compact more, has guaranteed accuracy of experimental results and repeatability, can promote the clinical application.Contain tissue in the sperm acrosome reaction detection kit of the present invention and preserve liquid, can let whole sperm bring out acrosome reaction detection process of the test separated into two parts and carry out: first is that the pre-service of semen sample, external capacitation and calcium ion bring out; Second portion is smear fluorescent dye and result's interpretation.This tissue is preserved liquid can effectively stop and preserve sample and the test findings that first's experiment obtains; The operation of carrying out second portion in the random time in 72 hours subsequently can not influence final test findings, the working strength when having alleviated clinical extensive detection.Experiment can separated into two parts after, make capacitation and the calcium ion sample after bringing out temporarily to store, can concentrate in second day and to carry out fluorescent dye, so that the clinical mass that carries out detects.As shown in Figure 1; Three experimental specimens are after first's experiment is accomplished; Divide 4 different times to carry out the second portion experiment respectively; Be respectively not to be stored in tissue fluid and directly to carry out second portion experiment and be stored in the said tissue fluid to take out respectively carrying out the second portion experiment in 24 hours, 48 hours and 72 hours, visible from figure, as far as same sample; It is very little that the acrosome that 4 different times obtain brings out the rate fluctuation, and prove to preserve to preserve in the liquid in tissue did not influence the experimental result of bringing out the acrosome reaction detection in 72 hours.
Further in the technical scheme; Kit of the present invention provides a kind of new fluorescent dye system, comprises fluorescence bond and fluorescence sealing liquid, makes fluorescence intensity strengthened greatly; Reaction time shortens greatly; Fluorescence intensity can stablize 15 days unattenuated, thereby make testing result stable more, be prone to interpretation, also guaranteed result's repeatability.As shown in Figure 2; Be to adopt the kit of invention to carry out the microphoto that fluorescent dye obtains; Can see very clearly that the whole sperm head acrosome of the sperm that acrosome reaction does not take place district fluoresces, and several sperm heads that acrosome reaction takes place only equatorial plate fluoresce.Like Fig. 3 is fluorescence sealing liquid of the present invention and existing other similar reagent performance comparison diagram; Fig. 3 A～Fig. 3 C is anti-fluorescence decay coefficient (histogram) and EM1 brightness value (broken line graph) comparison diagram that under 1%ND, 3%ND, 10%ND, carries out respectively; Can see among the figure; SlowFade light commercially available reagent in fluorescence sealing liquid of the present invention and traditional glycerine scheme, conventional fluorescent sealing liquid (PPD) scheme and the state of the art and ProLong commercially available reagent are learnt after relatively; The anti-fluorescence decay coefficient of fluorescence sealing liquid of the present invention is near 100%, and anti-fluorescence decay coefficient and brightness value all are higher than existing other fluorescence sealing liquid schemes far away.Fig. 4 is 4-8 ℃ of preservation after the solid tissue of fluorescence sealing fluid-tight of the present invention; Within 4 months, carry out tissue fluorescent stability testing result weekly; The histogram that different time is corresponding among the figure can see that the total trend of anti-fluorescence decay coefficient is that decay is arranged, but decay seldom; Can see EM in the broken line graph
1The total trend of brightness value is to reduce, but the amount that reduces seldom.This explains all that fluorescence sealing of the present invention system fluorescence is strong and stablize, is difficult for decaying.
Do not bring out if reagent of the present invention does not carry out calcium ion, then can be used for the sperm acrosome reaction condition evaluation, as the reagent that the perforatorium integrality is measured, promptly same kit can satisfy the needs of two Clinical detection projects simultaneously.
Description of drawings
Fig. 1 is that sperm of the present invention brings out method that acrosome reaction detects and after first's experiment is accomplished, sample is stored in tissue and preserves in the liquid and the different time in 72 hours takes out and carries out the acrosome that the second portion experiment obtains and bring out rate column diagram relatively.
Fig. 2 adopts the kit of invention to carry out the microphoto that fluorescent dye obtains, and Fig. 2 A is the Sperm characteristics that acrosome reaction does not take place; Fig. 2 B is the Sperm characteristics that acrosome reaction takes place.
Fig. 3 is the similar reagent performance with other of a fluorescence sealing liquid of the present invention comparison diagram; Fig. 3 A is anti-fluorescence decay coefficient (histogram) and the EM that under 1%ND, carries out
1Brightness value (broken line graph) comparison diagram;
Fig. 3 B is anti-fluorescence decay coefficient (histogram) and the EM that under 3%ND, carries out
1Brightness value (broken line graph) comparison diagram; Fig. 3 C is anti-fluorescence decay coefficient (histogram) and the EM that under 10%ND, carries out
1Brightness value (broken line graph) comparison diagram.
Fig. 4 is 4-8 ℃ of preservation after the solid tissue of fluorescence sealing fluid-tight of the present invention, and the different time within 4 months takes out and carries out tissue fluorescent stability testing result, and wherein histogram is represented anti-fluorescence decay coefficient, and broken line graph is represented EM
1Brightness value.
Concrete embodiment
Inspection principle of the present invention is that Calcium ionophore brings out, fluorescence colour.Calcium ion flows in the sperm body can start the spermatogenesis acrosome reaction.The use calcium ion carrier A 23187 is handled, and it is inner to induce calcium ion to flow into sperm, makes it that acrosome reaction take place; Because the glycoprotein that can combine with the agglutinin specificity is contained on perforatorium film surface, observe the variation in perforatorium district through the agglutinin fluorescent dye, thereby judge the situation occurred of acrosome reaction.
The kit of present embodiment is made up of (1)～(13) as follows:
1 bottle of 25ml of (1) 80% density gradient solution
1 bottle of 25ml of (2) 40% density gradient solutions
(3) 1 bottle of 80ml of sperm cleansing solution A
(4) 1 bottle of 80ml of sperm nutrient solution
1 bottle of 50ml of (5) 5 * sperm washing lotion B is diluted to 250ml
(6) 1 bottle of 150ul of Calcium ionophore
(7) 1 bottle of 300ul of contrast liquid
(8) 1 bottle of 100ml of immobile liquid
(9) tissue is preserved 1 bottle of 100ml of liquid
(10) 1 bottle of freeze-dried powder of fluorescence bond (FITC) redissolves to 5ml
(11) 1 bottle of 5ml of fluorescence sealing liquid
(12) 10/box of tissue slide glass 2 boxes, 8 holes/piece
(13) microscler cover glass is 20.
The present invention is suitable for instrument:
1. this kit is suitable for instrument: fluorescent microscope
2. other equipment of using in the operating process:
1) 2-8 ℃ of medicine preserved case 5) optical microscope
2) CO2gas incubator ℃ refrigerator 6-20)
3) superclean bench 37 ℃ of water baths 7)
4) low speed centrifuge
3. other materials that need:
1) 50ml glass cylinder seminal fluid sampling equipment 8)
2) purified water 9) the 50ml glass beaker
3) the 1-10 μ l adjustable liquid-transfering gun of numeral and a suction nozzle 10) 10ml taper centrifuge tube
4) the 20-100 μ l adjustable liquid-transfering gun of numeral and a suction nozzle 11) the Eppendorf pipe
5) the 100-1000 μ l adjustable liquid-transfering gun of numeral and a suction nozzle 12) 20ml dress plastics reagent bottle
6) disposable plastic tube 13) wet box
7) 5ml, 10ml glass pipette 14) disposable use gloves
One, kit preparation
1. select the universal product of the prior art for use, be ready to 80% density gradient solution, 40% density gradient solution, sperm cleansing solution A (potential of hydrogen is 7.2～7.6), sperm nutrient solution, 5 * sperm washing lotion B, Calcium ionophore, contrast liquid, immobile liquid, organize slide glass, microscler cover glass by above-mentioned specification and consumption.The A23187 Calcium ionophore that provides for Sigma company of Calcium ionophore wherein.Also can not comprise the material of these existing products in the kit, and before experiment, prepare these materials voluntarily by the user.
2. the preparation tissue is preserved liquid.Get glutaraldehyde 0.5ml, methyl alcohol 5ml, ethanol 2ml, PVP (polyvinyl pyrrolidone) 0.2g and formalin 1ml and mix, obtain the 100ml tissue and preserve liquid with pure water.
3. prepare the fluorescence bond.At first prepare the fluorescence bond and preserve liquid: get fluorescein-labelled thing: 0.5ng, methacrylic acid, methylmethacrylate copolymer (1: 2): 0.4g, Tris damping fluid: 0.7g; NaCl:0.3g; Casein: 0.1g, sodium salicylate: 0.2mg, the antiseptic Proclin300:20ul of Sigma company; In the pure water of surplus, the fluorescence bond that is mixed with pH7.5～8.0 is preserved liquid 100ml with these substance dissolves.Again fluorescence bond PSA-FITC 1mg is added the 100ml fluorescence bond that obtains and preserve in the liquid, process 1 bottle of freeze-dried powder then, be the fluorescence bond in this kit.
Wherein, this fluorescein-labelled thing is the PSA-FITC that Sigma company produces, and luciferin is the huge isothiocyanic acid rhodamine of fluorescein isothiocynate, tetramethyl; Being labeled thing is pisum sativum agglutinin, peanut agglutinin or concanavalin A.
Wherein, Tris buffer formulation: Tris (trishydroxymethylaminomethane) 60.57g, the about 420ml of 1N HCL, distilled water; Tris damping fluid compound method: (300～500ml) dissolving Tris behind the adding HCl, transfer to 7.6 with HCl (1N) or NaOH (1N) with pH, and last distilled water adds to 1000ml with a small amount of distilled water earlier.This liquid is storing solution, preserves in 4 ℃ of refrigerators.
4. prepare fluorescence sealing liquid.Get bSA BSA:0.5g, NaN that N-(1, the 3-dimethylbutyl)-N '-diphenyl-para-phenylene diamine: 0.05g, PVAC polyvinylalcohol: 0.2g, polyglycol 8000:0.1g, Tris damping fluid: 1.0g, NaCl:0.4g, Sigma company provide
3: 0.01g; Glycerine: 6%, these substance dissolves in the pure water of surplus, are mixed with the fluorescence sealing liquid of 100ml pH8.5-9.0.Only need 5ml in the kit.
Two, bringing out sperm acrosome reaction detects
(1) reagent is prepared
1. the density gradient solution of two kinds of concentration, sperm washing lotion A and sperm nutrient solution all do not contain antiseptic, when drawing reagent the reagent bottle after uncapping, should note avoiding polluting.Must not use the liquid reagent that has produced flocky precipitate.Recommend: the back disposable packing-20 ℃ preservation of mentioned reagent Kaifeng, thaw with preceding taking-up, do not use up reagent and put 2-8 ℃ of preservation (no longer frozen), avoid repeatedly using repeatedly as far as possible.
2. sperm washing lotion B application liquid preparation: the purified water that 50ml 5 * sperm washing lotion B adds 4 parts of volumes is diluted to 250ml, and mixing obtains sperm washing lotion B application liquid.Preserve available 2-3 week for 4 ℃.
3. before using, need the kit balance, and density gradient solution, sperm washing lotion A, the sperm nutrient solution of two kinds of concentration are put 37 ℃ hatched 0.5-1 hour to room temperature.
4. in fluorescence bond freeze-drying bottle, add the 5ml pure water, mixing, the fluorescence bond that obtains redissolving is subsequent use.
(2) carry out trace routine
1. fresh semen sample is put 37 ℃ or room temperature 30-60min, treat that it liquefies fully.If liquefaction is slow, then can in seminal fluid, add a small amount of sperm washing lotion A after, it liquefies fully to blow and beat seminal fluid promotion repeatedly with suction pipe.
2. the above-mentioned seminal fluid of liquefaction fully that obtains is extracted motile sperm with density gradient method, method is following:
1) draw 80% density gradient solution 0.5ml in cleaning awl end test tube bottom, above the liquid layer, add 40% density gradient solution 0.5ml at it gently again, careful operation is noted the interface of not upsetting two kinds of liquid.
2) add the 0.4ml seminal fluid gently on 40% density gradient solution.Centrifugal 20 minutes of 400g.
3) remove seminal fluid and 40% density gradient solution (noting: do not blow the bottom sperm) with careful suction of suction pipe; Change a new suction pipe, pipette is pinched into negative pressure state in advance after, be inserted into awl bottom tube bottommost then gently, slowly draw the spermatium suspension 0.2-0.4ml that is deposited in the bottom, this liquid draw is transferred to another new tapered tube.
4) add sperm washing lotion A 1.5ml in the new tapered tube, after mixing gently, centrifugal 5 minutes of 400g.
5) the careful suction gone the sperm washing lotion, stays bottom sperm deposition group; Add sperm nutrient solution 0.2ml, mixing.
6) counting extracts the density of back motile sperm, is 1 * 10 with sperm nutrient solution preparation density
6(for example: the density of extracting the back motile sperm is 50 * 10 to the sperm suspension 1.2ml of motile sperm/ml
6/ ml then gets this sperm suspension 25ul, adds sperm nutrient solution 1.2ml, and mixing gets final product).
3. the clean Eppendorf pipe that with the capacity is 1.5ml is as reaction tube.Every example detects sample and all establishes mensuration pipe and control tube simultaneously.Every pipe adds the above-mentioned sperm suspension that has prepared, and (density is 1 * 10
6The 0.5ml of motile sperm/ml), (5%CO in CO2gas incubator
2, 95% air, 37 ℃) hatched 3 hours, to induce capacitation.
4. lucifuge adds Calcium ionophore 2.5ul in measuring pipe, in control tube, adds contrast liquid 2.5ul, mixing; With two pipe (5%CO in CO2gas incubator
2, 95% air, 37 ℃) hatched 15 minutes.
5. the taking-up reaction tube adds sperm washing lotion B application liquid 1ml in control tube respectively with measuring in the pipe rapidly; Remove supernatant behind the centrifugal 5min of 600g, add sperm washing lotion B application liquid 1.5ml, remove supernatant behind the centrifugal 5min of 600g once more, adjust the sperm suspension volume to 100ul with sperm washing lotion B application liquid at last.
6. getting 10 μ l sperm suspensions evenly coats and organizes being coated with in the gate on the slide glass, air dry in the air.
7. organize slide glass to be soaked in fully to fill in the reaction tank that tissue preserves liquid with dried, the tight lid of lid is placed on 2～8 ℃ of preservations.(note: the tissue in the reaction tank is preserved liquid and can be used at least repeatedly 3～5 times, should avoid preserving liquid in the use as far as possible and directly be exposed in the air, in order to avoid reagent volatilizees and lost efficacy.)
8. when depositing 36 hours, take out and organize slide glass, with organizing slide glass to be soaked in fully in the reaction tank that fills immobile liquid, the rearmounted room temperature of the tight lid of lid is 30min fixedly; 2min is soaked in physiological saline washing 3 times at every turn.(note: the immobile liquid in the reaction tank can use 3～5 times at least repeatedly, should avoid immobile liquid directly to be exposed in the air in the use as far as possible, in order to avoid reagent volatilizees and lost efficacy.)
9. remove and organize the slide glass surface and to be coated with moisture in the gate (can ear washing bulb blow gently several down), in being coated with gate, add fluorescence bond (FITC) the 40 μ l after redissolving rapidly after organizing the slide glass drying, be placed horizontally in the wet box 37 ℃ of reaction 60min.With distilled water washing 3 times, soak 2min at every turn.
10. after the tissue air dry, each is coated with and adds 1 fluorescence sealing liquid in the gate, covered, observations under exciting light 450-490nm (blue color filter) fluorescent microscope 100 * object lens.Each is coated with gate and observes 400 sperms of counting at least, and the sperm quantity of acrosome reaction takes place record.The sperm sum of counting can not be less than 400, in order to avoid produce bigger counting error.The quality of fluorescent microscope and fluorescence intensity directly influence the accuracy that the result observes, and recommend to use power to be not less than the fluorescent mercury lamps of 100W.
(3) result judges and calculates
The Sperm characteristics of acrosome reaction does not take place: head acrosome district fluoresce (consulting Fig. 2 A).
The Sperm characteristics of acrosome reaction takes place: head only equatorial plate fluoresces (consulting Fig. 2 B), and perhaps all acrosome districts all do not have fluorescence.
The result calculates:
Measure sperm sum * 100% that is observed in the sperm quantity ÷ control tube of generation acrosome reaction in pipe acrosome reaction rate (%)=mensuration pipe)=200 ÷, 400 * 100%=50%;
Sperm sum * 100% that is observed in the sperm quantity ÷ control tube of generation acrosome reaction in control tube acrosome reaction rate (%)=control tube)=20 ÷, 400 * 100%=5%;
The acrosome reaction rate (%) that Calcium ionophore brings out=mensuration pipe acrosome reaction rate-control tube acrosome reaction rate=45%.
Conclusion: judge that according to the world health organisation recommendations reference value tested seminal fluid is the normal seminal fluid of acrosome reaction.
The world health organisation recommendations reference value is:
Normally: acrosome reaction rate >=15% that Calcium ionophore brings out;
Unusually: acrosome reaction rate＜10% that Calcium ionophore brings out;
Maybe be unusual: between the acrosome reaction rate 10～15% that Calcium ionophore brings out;
Too early acrosome reaction: after hatching 3h, control tube acrosome reaction rate＞20%.
Three, carrying out the sperm acrosome reaction condition evaluation detects
1. the sperm sample is prepared
Simple washing sperm:
1. get fresh semen 0.2ml in awl end centrifuge tube, add 10ml physiological saline, mix centrifugal 10 minutes of 500g.
2. topple over abandoning supernatant, leave and take bottom sperm deposition group.
3. add 10ml physiological saline, mix sperm suspension gently, centrifugal 10 minutes of 500g with suction pipe.
4. topple over abandoning supernatant, leave and take bottom sperm deposition group.
5. add a small amount of physiological saline, mixing, the counting sperm concentration is regulated sperm concentration to 5 * 10
6/ ml.
2. getting 10 μ l sperm suspensions evenly coats and organizes being coated with in the gate on the slide glass, air dry in the air.
3. will organize slide glass to be soaked in fully in the reaction tank that fills immobile liquid, the rearmounted room temperature of the tight lid of lid is 30min fixedly; 2min is soaked in physiological saline washing 3 times at every turn.(note: the immobile liquid in the reaction tank can use 3-5 time at least repeatedly, should avoid immobile liquid directly to be exposed in the air in the use as far as possible, in order to avoid reagent volatilizees and lost efficacy.)
4. remove and organize the slide glass surface and be coated with the moisture (available ear washing bulb dries up gently) in the gate, in being coated with gate, add fluorescence bond 50 μ l rapidly, be placed horizontally in the wet box, 37 ℃ of reaction 60min.
5. with distilled water washing 3 times, soak 2min at every turn.
6. after the tissue air dry, each is coated with and adds 1 fluorescence sealing liquid in the gate, covered, observations under exciting light 450-490nm (blue color filter) fluorescent microscope 100 * object lens.Each is coated with gate and observes 400 sperms of counting at least, writes down all kinds of perforatorium amount of state.
7. the result observes and calculates.
Acrosome complete (AI): sperm head 1/2 has bright uniform fluorescence (consulting figure A) with the upper part.
Acrosome reaction (AR): only at the sperm head equatorial plate fluorescent belt (consulting figure B) is arranged, perhaps all acrosome districts all do not have fluorescence.
Unusual acrosome: except other sperm.
Calculate the percent of AI, AR.400 sperms have been observed in the present embodiment, 44 of AR sperms, then AR (%)=44 ÷ 400 * 100%=11%.
One, the preparation of kit
The kit of embodiment 2 is prepared roughly with embodiment 1, and different is, when preparation fluorescence bond; Preparation fluorescence bond is preserved in the liquid process, gets fluorescein-labelled thing: 5ng, methacrylic acid, methylmethacrylate copolymer (1: 2): 1.5g; Tris damping fluid: 2.5g, NaCl:0.9g, casein: 0.3g; Sodium salicylate: 1.4mg, antiseptic Proclin300:60ul, the preparation method is with embodiment 1.When preparation fluorescence sealing liquid, get N-(1, the 3-dimethylbutyl)-N '-diphenyl-para-phenylene diamine: 0.2g, PVAC polyvinylalcohol: 0.9g; Polyglycol 8000:0.8g, Tris damping fluid: 2.5g, NaCl:1.0g; The bSA BSA:3g that Sigma company provides; The thimerosal 0.03g that Sigma company provides, glycerine: 30%, the preparation method is with embodiment 1.
Two, bringing out sperm acrosome reaction detects
(1) reagent is prepared
With embodiment 1.
(2) carry out trace routine
1. fresh semen sample is put 37 ℃ or room temperature 30-60min, treat that it liquefies fully.If liquefaction is slow, then can in seminal fluid, add a small amount of sperm washing lotion A after, it liquefies fully to blow and beat seminal fluid promotion repeatedly with suction pipe.
2. with the above swimming method extraction of the above-mentioned seminal fluid that liquefies fully that obtains motile sperm, and with sperm nutrient solution preparation density is 1 * 10
6The sperm suspension 1.2ml of motile sperm/ml.
Then carry out embodiment 1 described step 3-6, evenly coat and organize being coated with in the gate on the slide glass, air dry in the air up to getting 10 μ l sperm suspensions.Then the dried slide glass of organizing is not stored in the tissue preservation liquid, but the step 8 described in the embodiment 1 of and then directly consulting and carrying out, with organizing slide glass to be soaked in fully in the reaction tank that fills immobile liquid, the rearmounted room temperature of the tight lid of lid is 30min fixedly; 2min is soaked in physiological saline washing 3 times at every turn.Carry out the step 9-10 described in the embodiment 1 then.
(3) result judges and calculates
Measure sperm sum * 100% that is observed in the sperm quantity ÷ control tube of generation acrosome reaction in pipe acrosome reaction rate (%)=mensuration pipe)=120 ÷, 400 * 100%=30%;
Sperm sum * 100% that is observed in the sperm quantity ÷ control tube of generation acrosome reaction in control tube acrosome reaction rate (%)=control tube)=80 ÷, 400 * 100%=20%;
The acrosome reaction rate (%) that Calcium ionophore brings out=mensuration pipe acrosome reaction rate-control tube acrosome reaction rate=30%-20%=10%.
Conclusion: the tested seminal fluid of judging present embodiment 2 according to the world health organisation recommendations reference value is the unusual seminal fluid of acrosome reaction.
Three, carrying out the sperm acrosome reaction condition evaluation detects
1. the sperm sample is prepared
Directly detect the sperm sample after simple washing among the embodiment 1; But the preferred high motile sperm sample completion detection of using after extracting; Because through density gradient method or last swimming method sperm is separated from chaff interferences such as fragment, leucocyte, androgone and Necrospermia, can be obtained testing result more accurately.Therefore extract high motile sperm with the swimming method in the present embodiment 2, step is following:
1. draw the bottom of seminal fluid to the clean round bottom small test tube that 0.5-1ml liquefied.
2. change a new pipette, add 1.0ml sperm nutrient solution (or other type sperm nutrient solution) gently on the seminal fluid upper strata.
3. do not upset the liquid level layering, keep the test tube 45 to tilt, hatch 1h for 37 ℃, motile sperm will move about in nutrient solution.
4. carefully draw the nutrient solution 0.6-0.8ml that motile sperm is contained in the superiors.
5. the superiors' liquid of drawing is transferred at the bottom of another awl that 10ml physiological saline is housed in the centrifuge tube;
6. 300-500g is centrifugal 5 minutes, does not use damper brake;
7. discard the physiological saline of sperm deposition group top, make the remaining liquid measure of sperm deposition group top be no more than 2mm;
8. add a small amount of physiological saline, mixing, the counting sperm concentration is regulated sperm concentration to 5 * 10
6/ ml.
Carry out corresponding step 2-6 among the embodiment 1 then, then carry out the result and observe and calculate.481 sperms have been observed in the present embodiment, 50 of AR sperms, then AR (%)=50 ÷ 481 * 100%=10.4%.
One, kit preparation
The preparation of the kit of embodiment 2 is roughly with embodiment 1, and different is, when preparation fluorescence bond; Preparation fluorescence bond is preserved in the liquid process, gets fluorescein-labelled thing: 10ng, methacrylic acid, methylmethacrylate copolymer (1: 2): 2.4g; Tris damping fluid: 3.5g, NaCl:1.5g, casein: 0.5g; Sodium salicylate: 2.0mg, antiseptic Proclin300:100ul, the preparation method is with embodiment 1.When preparation fluorescence sealing liquid, get N-(1, the 3-dimethylbutyl) N '-diphenyl-para-phenylene diamine: 0.5g, PVAC polyvinylalcohol: 1.6g, polyglycol 8000:1.5g, Tris damping fluid: 5.0g, NaCl:1.6g, bSA BSA:5g, NaN
30.05g, glycerine: 60%, the preparation method is with embodiment 1.
Two, bringing out sperm acrosome reaction detects
(1) reagent is prepared
With embodiment 1.
(2) carry out trace routine
Step 1-7 carries out with reference to the step 8 of embodiment 1 with the step 1-7 of embodiment 1 then: take out in when depositing 72 hours and organize slide glass, with organizing slide glass to be soaked in fully in the reaction tank that fills immobile liquid, the rearmounted room temperature of the tight lid of lid is 30min fixedly; 2min is soaked in physiological saline washing 3 times at every turn.Then carry out with embodiment 1 described step 9-10.
(3) result judges and calculates
Measure the sperm sum * 100%=200 ÷ 400 * 100%=50% that is observed in the sperm quantity ÷ control tube of generation acrosome reaction in pipe acrosome reaction rate (%)=mensuration pipe;
Sperm sum * 100% that is observed in the sperm quantity ÷ control tube of generation acrosome reaction in control tube acrosome reaction rate (%)=control tube)=40 ÷, 400 * 100%=10%;
The acrosome reaction rate (%) that Calcium ionophore brings out=mensuration pipe acrosome reaction rate-control tube acrosome reaction rate=50%-10%=40%.
Conclusion: judge that according to the world health organisation recommendations reference value tested seminal fluid is the acrosome reaction normal semen.
Three, carrying out the sperm acrosome reaction condition evaluation detects
Method is wherein extracted high motile sperm with density gradient method roughly with embodiment 1, with density gradient method operation steps in " bringing out sperm acrosome reaction detects " program.Sperm concentration after will extracting with physiological saline then is adjusted to 5 * 10
6/ ml.Carry out corresponding step 2-6 among the embodiment 1 then, then carry out the result and observe and calculate.
435 sperms have been observed in the present embodiment, 65 of AR sperms, then AR (%)=65 ÷ 435 * 100%=15%.
Claims (7)
1. a sperm acrosome reaction kit is characterized in that, comprises tissue preservation liquid, fluorescence bond, fluorescence sealing liquid;
Said tissue is preserved the every 100ml of liquid and is contained: glutaraldehyde 0.5ml, methyl alcohol 5ml, ethanol 2ml, polyvinyl pyrrolidone PVP0.2g, formalin 1ml, and surplus is a pure water;
Said fluorescence bond is preserved liquid by fluorescence bond PSA-FITC and fluorescence bond and is formed pH7.5～8.0, adding 1mg fluorescence bond PSA-FITC in every 100ml fluorescence bond preservation liquid;
It is formulated with the following material of dissolved in purified water that wherein said fluorescence bond is preserved liquid, and every 100ml contains:
Fluorescein-labelled thing: 0.5～10ng, wherein, luciferin is the huge isothiocyanic acid rhodamine of fluorescein isothiocynate or tetramethyl; Being labeled thing is pisum sativum agglutinin, peanut agglutinin or concanavalin A;
Mol ratio is 1: 2 methacrylic acid and methylmethacrylate copolymer: 0.4～2.4g;
Tris damping fluid: 0.7～3.5g;
NaCl：0.3～1.5g；
Casein: 0.1～0.5g;
Sodium salicylate: 0.2～2.0mg;
Antiseptic Proclin300:20～100ul;
Surplus is a pure water;
Said fluorescence sealing liquid is formulated with the following material of dissolved in purified water, pH8.5-9.0, and every 100ml contains:
N-(1, the 3-dimethylbutyl)-N '-diphenyl-para-phenylene diamine: 0.05～0.5g;
PVAC polyvinylalcohol: 0.2～1.6g;
Polyglycol 8000:0.1～1.5g;
Tris damping fluid: 1.0～5.0g;
NaCl：0.4～1.6g；
BSA BSA:0.5～5g;
NaN
3Or thimerosal: 0.01～0.05g;
Glycerine: 6%～60%;
Surplus is a pure water.
2. sperm acrosome reaction kit according to claim 1 is characterized in that, said sperm acrosome reaction kit also comprises Calcium ionophore.
3. sperm acrosome reaction kit according to claim 5 is characterized in that, said Calcium ionophore is a calcium ion carrier A 23187.
4. sperm acrosome reaction kit according to claim 1; It is characterized in that said sperm acrosome reaction kit also comprises 80% density gradient solution, 40% density gradient solution, sperm cleansing solution A, sperm nutrient solution, 5 * sperm washing lotion B, contrast liquid, immobile liquid, organizes slide glass, microscler cover glass.
5. one kind is utilized each described kit of claim 1-4 to carry out the method that sperm brings out the acrosome reaction detection, it is characterized in that, comprises the steps:
Step 1: fresh semen sample is liquefied fully, and again with density gradient method or go up the swimming method and extract motile sperm, counting extracts the density of back motile sperm then, is 1 * 10 with sperm nutrient solution preparation density
6The sperm suspension of motile sperm/ml;
Step 2: the clean Eppendorf pipe that with the capacity is 1.5ml is as reaction tube, and every example detects sample and all establishes simultaneously and measure pipe and control tube, and every pipe adds the sperm suspension 0.5ml for preparing in the step 2, at CO
2Account in the CO2gas incubator that volume content 5%, air account for volume content 95% and under 37 ℃, hatched 3 hours, to induce capacitation;
Step 3: lucifuge adds Calcium ionophore 2.5ul in measuring pipe, in control tube, adds contrast liquid 2.5ul, respectively mixing; Manage at CO two
2Account in the CO2gas incubator that volume content 5%, air account for volume content 95% and under 37 ℃, hatched 15 minutes;
Step 4: take out reaction tube, add sperm washing lotion B application liquid 1ml in control tube respectively with measuring in the pipe rapidly; To remove supernatant behind the centrifugal 5min of 600g RCF, add sperm washing lotion B application liquid 1.5ml, once more to remove supernatant behind the centrifugal 5min of 600g RCF, adjust the sperm suspension volume to 100ul with sperm washing lotion B application liquid then;
Step 5: get 10 μ l sperm suspensions that step 4 obtains and evenly coat and organize being coated with in the gate on the slide glass, air dry in the air;
Step 6: will organize slide glass to be soaked in fully in the reaction tank that fills immobile liquid, the rearmounted room temperature of the tight lid of lid is 30min fixedly; 2min is soaked in physiological saline washing 3 times at every turn;
Step 7: remove and to organize the slide glass surface and to be coated with the moisture in the gate, in being coated with gate, add said fluorescence bond 40 μ l rapidly after organizing the slide glass drying, be placed horizontally in the wet box, 37 ℃ of reaction 60min with distilled water washing 3 times, soak 2min at every turn;
Step 8: after the tissue air dry that step 7 obtains;, each adds 1 fluorescence sealing liquid in being coated with gate, covered, observations under 100 times of object lens of fluorescent microscope of exciting light 450-490nm, blue color filter; Wherein, Each is coated with gate and observes 400 sperms of counting at least, and the sperm quantity of acrosome reaction takes place record, and carries out result's calculating and judgement.
6. sperm according to claim 5 brings out the method that acrosome reaction detects; It is characterized in that, when can not disposable continuous completion testing, then can be after said step 5, before the step 6; Carry out following steps: with dried organize slide glass to be soaked in fully to fill tissue preserve in the reaction tank of liquid; The tight lid of lid is placed on 2-8 ℃ of preservation, in 72 hours, takes out, and continues to carry out said step 6～eight then.
7. a method of utilizing each described kit of claim 1-4 to carry out the sperm acrosome reaction condition evaluation is characterized in that, comprises the steps:
Step 1: simply wash sperm sample or above swimming method and extract high motile sperm or extract high motile sperm, with physiological saline sperm concentration is adjusted to 5 * 10 then with density gradient method
6/ ml;
Step 2: get 10 μ l sperm suspensions and evenly coat and organize being coated with in the gate on the slide glass, air dry in the air;
Step 3: will organize slide glass to be soaked in fully in the reaction tank that fills immobile liquid, the rearmounted room temperature of the tight lid of lid is 30min fixedly; 2min is soaked in physiological saline washing 3 times at every turn;
Step 4: remove and organize the slide glass surface and be coated with the moisture in the gate, in being coated with gate, add fluorescence bond 50 μ l rapidly, be placed horizontally in the wet box, 37 ℃ of reaction 60min;
Step 5: with distilled water washing 3 times, at every turn soak 2min then;
Step 6: after the tissue air dry; Each is coated with and adds 1 fluorescence sealing liquid in the gate, covered, observations under 100 times of object lens of fluorescent microscope of exciting light 450-490nm, blue color filter; Wherein, Each is coated with gate and observes 400 sperms of counting at least, writes down all kinds of perforatorium amount of state: it is that acrosome is complete that sperm head 1/2 has bright uniform fluorescence with the upper part, counts AI; Only having a fluorescent belt or all acrosome districts all not to have fluorescence at the sperm head equatorial plate is acrosome reaction, counts AR; Other sperm is unusual acrosome; Calculating AI, AR account for the percent of observed sperm.
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| CN103335933A (en) * | 2013-06-28 | 2013-10-02 | 浙江星博生物科技有限公司 | Flow cytometry-based sperm acrosome reaction detection reagent |
| EP3452805A4 (en) * | 2016-05-03 | 2020-04-01 | Rarecyte, Inc. | Solutions for sample processing |
| CN108593393A (en) * | 2018-03-20 | 2018-09-28 | 安徽安科生物工程（集团）股份有限公司 | Spermatozoon activity oxygen staining kit and its application method |
| CN110463687B (en) * | 2019-07-10 | 2021-07-27 | 安徽农业大学 | Preservation solution and preservation method for sheep semen solution |
| CN114231590B (en) * | 2021-12-16 | 2023-03-24 | 浙江省人民医院 | Sperm quality evaluation method |
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