CN102002515A - Sperm treating method - Google Patents

Sperm treating method Download PDF

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Publication number
CN102002515A
CN102002515A CN 200910194916 CN200910194916A CN102002515A CN 102002515 A CN102002515 A CN 102002515A CN 200910194916 CN200910194916 CN 200910194916 CN 200910194916 A CN200910194916 A CN 200910194916A CN 102002515 A CN102002515 A CN 102002515A
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sperm
suspension
naoh
icsi
concentration
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施惠娟
李翀
宋丽伟
林菊芳
贾晓峰
史庭燕
袁瑶
王会
徐彦
牛志宏
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Shanghai Institute of Planned Parenthood Research
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Shanghai Institute of Planned Parenthood Research
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Abstract

The invention provides a sperm treating method. The method comprises the following step of: (1) hatching sperm suspension at the temperature of between 0 and 4DEG C for 0.5 to 3 hours to obtain intracytoplasmic sperm injection (ICSI) sperms, wherein the sperm suspension contains sperms and sodium hydroxide (NaOH); and according to the total volume of the sperm suspension, the concentration of the sodium hydroxide is 5 to 30nM.

Description

A kind of treatment process of sperm
Technical field
The present invention relates to artificial auxiliary procreation technology, relate in particular to a kind of treatment process of sperm.
Background technology
(intracytoplasmic sperm injection, ICSI) technology is an artificial auxiliary procreation technology that grows up in the later stage eighties 20th century in the monosperm microinjection.It is by the micrurgy instrument, and a sperm or androgone are directly injected in the ovum kytoplasm, makes smart ovum in conjunction with the process of finishing fertilization.Utilization ICSI technology prepares the efficiency ratio male pronucleus microinjection of transgenic animal and wants high, is carrier with the sperm therefore, has opened up a simple technological line in conjunction with the ICSI technology, for the research of transgenic animal.
The mouse sperm tail is long, needs to use physics or chemical process that the sperm tail is removed before ICSI and just can successfully carry out the ICSI operation.Therefore, can remove sperm tail effectively, and don't the method for damage sperm DNA seems extremely important.The method that currently used sperm truncates has sperm freeze thawing, ultrasonic, enzymic digestion etc., wherein sperm freeze thawing and to use non-ionic type washing agent TritonX-100 be the most frequently used method.Report is also arranged with low concentration alkali solution-treated mouse sperm, sperm is separated end to end, but isolating end to end effect is undesirable, generally docking rate has only about 10%.
Therefore, this area presses for provides a kind of new isolating end to end method of sperm that makes, and described method can keep the integrity of DNA under the high situation of docking rate.
Summary of the invention
The present invention aims to provide a kind of sperm treatment process.
Another object of the present invention provides a kind of isolating end to end sperm that obtains of handling through aforesaid method.
In a first aspect of the present invention, a kind of treatment process of sperm is provided, described method comprises step:
(1) sperm suspension was hatched 0.5-3 hour at 0-4 ℃, obtain monosperm microinjection (intracytoplasmic sperm injection, ICSI) preceding sperm;
Contain sperm and sodium hydroxide (NaOH) in the described sperm suspension, in the cumulative volume of described sperm suspension, wherein concentration sodium hydroxide is 5-30mM.
In another preference, incubation time 1-3 hour, in the described sperm suspension, naoh concentration was 5-20mM.
In another preference, described sperm suspension was hatched 1.5-2.5 hour at 0-2 ℃, and in the described sperm suspension, naoh concentration is 8-12mM.
In another preference, in the cumulative volume of sperm suspension, wherein sperm concentration is 5-10 * 10 6/ mL.
In another preference, contain the sperm nutrient solution in the described sperm suspension.
In another preference, also comprise step after the described step (1):
(2) sperm suspension and mineral acid are mixed.
In another preference, described sperm is selected from mouse sperm, rat sperm, Niu Jingzi, sheep sperm, pig sperm or people's sperm.
In a second aspect of the present invention, provide a kind of monosperm microinjection (intracytoplasmic sperminjection, ICSI) preceding sperm, before the described ICSI sperm by as the method for following step obtain:
(1) sperm suspension was hatched 0.5-3 hour at 0-4 ℃, obtain monosperm microinjection (intracytoplasmic sperm injection, ICSI) preceding sperm;
Contain sperm and sodium hydroxide (NaOH) in the described sperm suspension, in the cumulative volume of described sperm suspension, wherein concentration sodium hydroxide is 5-30mM.
In another preference, described sperm is selected from mouse sperm, rat sperm, Niu Jingzi, sheep sperm, pig sperm or people's sperm.
In view of the above, the invention provides a kind of new isolating end to end method of sperm that makes, and described method can keep the integrity of DNA under the high situation of docking rate.
Description of drawings
Fig. 1 has shown that NaOH concentration and operative temperature are to the mouse sperm influence of separating effect end to end.
Fig. 2 has shown the influence to the mouse sperm membrane integrity of NaOH concentration and operative temperature; Wherein
A is a blank;
B is that 50mM NaOH is at 4 ℃ of mouse sperms of hatching 30min;
C is the mouse sperm before NaOH handles;
D is the mouse sperm after 0mM NaOH is hatched 30min;
E is 5mM NaOH at 4 ℃ of mouse sperms after hatching 30min;
F is 50mM NaOH at 4 ℃ of mouse sperms after hatching 30min;
A, B are the cell state of sperm before and after Na0H handles of seeing under the fluorescent microscope, C, and D, E and F are the quantitative datas that draws through cells were tested by flow cytometry.
Fig. 3 has shown the variation of mouse sperm plasma membrane percentage of head rice with NaOH concentration and incubation temperature.
Fig. 4 has shown acridine orange dyeing back mirror observation mouse sperm cell DNA damage situations down, and the streaming of different treatment sample detects figure; Wherein
A is a mouse sperm of observing the different DNA states in acridine orange dyeing back under the fluorescent microscope:
A, DNA are not impaired; B, the impaired chain DNA of partly separating; C, the DNA that is badly damaged and unwinds fully;
B is a normal control;
C is 0 ℃, 50mM NaOH;
D is 37 ℃, 50mM NaOH.
Fig. 5 has shown the influence to mouse sperm docking rate of 0 ℃ of following NaOH concentration of treatment and treatment time.
Fig. 6 has shown the influence to people's plasmalemmae of sperms integrity of NaOH concentration and operative temperature; Wherein
A is that 10mM NaOH was hatched 30 minutes after the people's sperm after the acridine orange dyeing at 37 ℃; B is the sperm (being the stand-by people's sperm of prepared fresh) before NaOH handles.
Fig. 7 has shown the influence to people's sperm DNA integrity of NaOH concentration and operative temperature; Wherein
A is 0 ℃, and 0mM NaOH is hatched people's sperm of 2 hours; B is 0 ℃, and 10mM NaOH is hatched the people's sperm after 2 hours.
Embodiment
The contriver is surprised to find that at low temperatures that through extensive and deep research (0-4 ℃) mixes lower concentration NaOH (5-30mM) and sperm, hatches reasonable time (0.5-3 hour) sperm is effectively truncated, and can keep the integrity of DNA simultaneously.
The present invention uses the sperm chromatin structure assay technology on flow cytometer (sperm chromatinstructure assay SCSA) studies NaOH concentration, operative temperature and time respectively, explores the optimum condition of the preceding sperm of NaOH processing IC SI.
Treatment process
Sperm treatment process provided by the invention is that sperm suspension was hatched 0.5-3 hour at 0-4 ℃, obtains the preceding sperm of ICSI; Contain sperm and sodium hydroxide (NaOH) in the described sperm suspension, in the cumulative volume of described sperm suspension, wherein concentration sodium hydroxide is 5-30mM.
Contain sperm and sperm nutrient solution in the described sperm suspension.The vigor of sperm needs to be preferably greater than 90% greater than 80%; The density of sperm is counted 5-10 * 10 with the cumulative volume of sperm suspension 6/ mL, preferred 7-8 * 10 6/ mL; Can use sperm nutrient solution well known in the art, such as but not limited to, L 108 nutrient solutions, HTF, BWW etc.
The present invention can use the mineral acid stopped reaction, and described mineral acid is selected from hydrochloric acid, sulfuric acid, phosphoric acid or its mixing, preferred hydrochloric acid (HCl).The mineral acid that is used for stopped reaction is preferably with the NaOH equivalent.
Sperm before the ICSI
As used herein, " sperm before the ICSI " is meant process processing before carrying out ICSI, helps carrying out the sperm of ICSI, and described processing comprises that sperm separates end to end, the sperm braking, preferably makes the isolating end to end sperm of sperm.
ICSI is except simple making sperm-ovum insemination, and the ICSI method is still set up a kind of method of transgenic animal model.
Sperm docking rate reaches more than 20% before the ICSI provided by the invention, preferably is more than 30%, more preferably to reach more than 40%.Not significantly damage of the DNA of sperm before the ICSI provided by the invention.
The above-mentioned feature that the present invention mentions, or the feature that embodiment mentions can arbitrary combination.All features that this case specification sheets is disclosed can with any composition forms and usefulness, each feature that is disclosed in the specification sheets can anyly provide the alternative characteristics of identical, impartial or similar purpose to replace.Therefore removing has special instruction, and the feature that is disclosed only is the general example of equalization or similar features.
Major advantage of the present invention is:
1, sperm treatment process provided by the invention is simple to operation.
2, sperm treatment process provided by the invention can reach isolating end to end ideal effect, and not remarkable to the integrity influence of DNA.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (NewYork:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise all percentage ratio, ratio, ratio or umber by weight.
Unit in the percent weight in volume among the present invention is well-known to those skilled in the art, for example is meant the weight of solute in 100 milliliters solution.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
The material that uses among the embodiment provided by the invention is as follows:
Laboratory animal
The male mouse of ripe (3-6 monthly age) ICR is available from Shanghai Xi Pu-Bi Kai laboratory animal company limited, and raises in the SPF of Shanghai Family Planning Science and Research Institute. level laboratory rodent room (22 ℃, illumination 14 hours every days).
Main agents and instrument
Transgreen/PI fluorescence is redyed liquid: Transgreen is available from Shanghai Gan Feng Bioisystech Co., Ltd.Propidium iodide PI (Propidium lodide), Sumitomo Acridine Orange RK conc AO (Acri dine Orange), NaOH (1mol/L), HCL (1mol/L) and Hepes-CZB preparation agents useful for same are available from U.S. Sigma company, flow cytometer is that U.S. Beckman company product, inverted fluorescence microscope are Japanese Nikon eclipse 50i, and thermostatted is a German Eppendorff company product.
The method of using among the embodiment provided by the invention is as follows:
The processing of mouse sperm:
Get 1-2 male ICR mouse, the neck dislocation method is put to death, and gets its cauda epididymis, removes fat and bloodstain, puts in the Hepes-CZB drop of people's preparation in advance, shreds tissue, after the vibration sperm is swum out of a little, 37 ℃, 5%CO 2Incubator leaves standstill 10min, obtains the sperm of motility＞90%, adjusts density to 5-10 * 10 6/ mL.Divide to be filled in the EP pipe, add respectively 1M NaOH to final concentration be 0mM, 5mM, 10mM, 20mM, 30mM, 50mM, flick the tube wall mixing, in the thermostatted of preset temp, hatched 0.5-3 hour.After reaction finishes, add the HCl stopped reaction of equivalent in every pipe respectively, obtain the preceding sperm of ICSI, and place to be measured on ice.
The processing of people's sperm:
1, gets the people's of the normal fertility of the fresh liquefaction of 1ml seminal fluid (coming from the infertile outpatient service of Shanghai hospital of family planning institute), put into the aseptic taper centrifuge tube of a 15ml, adding the Earle sperm nutrient solution of augmenting then up gently (forms: Earle balanced salt solution 46ml, for serum 4ml, burnt Sodium.alpha.-ketopropionate 1.5mg, Sodium.alpha.-hydroxypropionate (60% syrup) 0.18ml, sodium bicarbonate 100mg) 1-2ml;
2, centrifuge tube 45 degree that tilt place 37 ℃, 5%CO2 incubator to hatch 1 hour;
3, again that test tube is vertical gently, take out the 1ml liquid of the superiors, augment the dilution of Earle sperm nutrient solution, the centrifugal 5min of 500g with 8 times of amounts then;
4, at last in being suspended from the Earle sperm nutrient solution that 0.5ml augments, shake up gently as far as possible, stand-by.
Sperm is the detection of separation efficiency end to end
Sperm suspension takes out 10 μ l in Macro sperm count plate with the every pipe among the HCl and back, microscopically direct viewing statistics spherospermium and total sperm quantity, at least 200 sperms of each sample counting.
Figure B2009101949164D0000061
Transgreen/PI detects the plasmalemmae of sperms integrity
With Transgreen/PI redye sperm method (see Zhao Hongxin, Yuan Yao, Hua Minmin, Zhang Huiqin, it is beautiful to oblige. detect the survival rate of sperm with Transgreen/PI fluorescence redyeing process. Chinese andrology magazine .2008:22 (4): 1-4).In the sperm suspension (sperm before being ICSI) of 40 μ l, adding five times of Hepes-CZB nutrient solution dilutions to density is 1-2 * 106/mL, respectively adding 2 μ l concentration is that 0.5 μ M Transgreen solution (being dissolved in DMSO) and 2 μ l concentration are PI solution (the being dissolved in ddH2O) mixing of 1.2mM, after room temperature leaves standstill 20min, with flow cytometer (Beckman, USA) membrane integrity of detection sperm.Flow cytometer argon ion excitation wavelength is 488nm, and light source is 200mW.In wavelength is the fluorescence (FL1) that detects Transgreen under the 520nm condition, detects the fluorescence (FL3) of PI in the 610nm condition.
The SCSA method detects the sperm DNA integrity
In the 40 μ l sperm suspensions (be ICSI before sperm), add 160 μ l TNE buffer (1mM disodium EDTA, pH 7.4 for 0.01MTris-Cl, 0.15M NaCl), dilution sperm-suspension to volume is 200 μ 1, and density is 1-2 * 10 6/ mL.Add 400 μ l pickling agents (0.1% (w/v) Triton X-100, pH 1.2 for 0.08M HCl, 0.15MNaCl) vibration mixing at once.Behind the 30s, (6 μ g/ml AO are dissolved in 0.037M citric-acid, 0.12M Na to the A0 dye liquor of adding 1.2mL 2HPO 4, 1.1mM disodiumEDTA, 0.15M NaCl, pH 6.0), in 3 minutes, sample upflowing cell instrument is surveyed sperm DNA fragmentation index (DFI).The control flow velocity is less than 300 sperm per seconds.
Statistical study
Each experimental group independence triplicate carries out statistical procedures with the SPSS13.0 statistical software, relatively adopts variance analysis between group, and there is statistical significance P＜0.05 for difference.
Embodiment 1
NaOH is to the mouse sperm influence of separating effect end to end
The contriver is under 0-37 ℃, and with the NaOH solution-treated mouse epididymis tail sperm of 5-50mM, microscopically is observed, and finds sperm after 5mM NaOH concentration is handled 30 minutes, and its motility rate has reduced to 0, only has the only a few sperm still moving about.See Fig. 1.
The result shows, along with the raising of NaOH concentration, isolating end to end efficient is progressively improving, but the different treatment temperature to sperm end to end separating effect have remarkably influenced, under 0-4 ℃, sperm separating effect end to end significantly is lower than other temperature condition.
Embodiment 2
NaOH is to the influence of mouse sperm membrane integrity
Sperm after the contriver redyes NaOH and handles with Transgreen/PI is observed down and is detected its membrane integrity (Fig. 2) with flow cytometer respectively at fluorescent microscope, and experimental data shows (Fig. 3).
The result shows that 5mM NaOH handled sperm 30 minutes, and the sperm of injury of plasmalemmae has all reached 90%, and the injury of plasmalemmae of sperms degree that the above concentration NaOH of 10mM handles is near 100%.
Embodiment 3
NaOH is to the influence of mouse sperm DNA integrity
Along with the increase of NaOH concentration, the degree of sperm DNA damage is big more.The results are shown in Figure 4 and table 1.
Table 1NaOH concentration and incubation temperature are to mouse sperm DNA
Figure B2009101949164D0000081
Influence
Figure B2009101949164D0000082
*P＜0.05 is compared with negative control group
The result shows that under 37 ℃ of temperature, even 10mM NaOH also has remarkable damage to sperm DNA, low temperature then can reduce the influence of NaOH to sperm DNA, and under 0 ℃ of condition, 50mM NaOH was hatched sperm 30 minutes, and significant damage does not take place sperm DNA.
Embodiment 4
NaOH and sperm incubation time altogether separate end to end to sperm and the influence of DNA integrity
Lower concentration NaOH is to the damage minimum of sperm DNA under the low temperature, but this condition is lower to the isolating end to end effect of sperm.The contriver is hatched the NaOH of sperm and different concns under 0 ℃ of condition altogether, and incubation time is extended to 3h respectively, the results are shown in Figure 5 and table 2.
0 ℃ of following different N aOH concentration of treatment of table 2SCSA analysis and time are to mouse sperm DNA loss percentage
Figure B2009101949164D0000091
Influence
Figure B2009101949164D0000092
The result shows, 0 ℃ of condition following time, sperm and 10mM NaOH solution were hatched 2 hours altogether, and sperm separation efficiency end to end will be increased to 55%, and remarkable damage does not take place sperm DNA simultaneously.
Embodiment 5
10mM NaOH handles the sperm 2h of the people behind the upstream under 0 ℃ of condition, control group (0 ℃, 0mM NaOH is hatched people's sperm of 2 hours) compares with the data of experimental group (0 ℃, 10mM NaOH is hatched the people's sperm after 2 hours), the results are shown in Figure 6 and 7, and following data:
(1) docking rate: 1.81% (control group) and 26.0% (experimental group)
(2) membrane integrity: 60.84% (control group) and 27.06% (experimental group)
(3) DNA integrity: loss percentage: 0.19% (control group) and 0.28% (experimental group)
The result shows that 0 ℃, 10mM NaOH is to not influence of people's sperm DNA damage.Same condition, the mouse sperm film is more responsive than people sperm membrane, shows the short-tail rate, and people's sperm will hang down.
Discuss
(DNA fragmentation index DFI) is the quantizating index of sperm DNA integrity to the dna fragmentation index, and sperm chromatin structure assay test (SCSA) is one of at present the most frequently used sperm DNA integrity detection technology.SCSA is tested in 1980 and is at first proposed by Evenson, it is existing that oneself is widely used in the detection of sperm DNA integrity, it detects principle is that sperm dyes with acridine orange (AO) after low pH damping fluid is handled, detect ratio red, green sperm by flow cytometer then, thus the DNA integrity of judgement sample.Eupyrene sperm DNA is in conjunction with closely having resistance to acid, and is double-stranded stable, and immature staining of sperm matter is loosely organized, and its DNA sex change under the effect of acid forms strand.AO combines with double-stranded DNA and is monomeric form and sends green fluorescence, is polymer form and sends red fluorescence and combine with single stranded DNA, and the sperm of rubescent look fluorescence shared ratio in total sperm is DFI, just is positioned at the cell beyond the main group on streaming figure.This method is highly sensitive, and detection speed is fast, and the contriver dyes with acridine orange to the sperm sample of handling well (being the preceding sperm of ICSI).Utilization SCSA method, the contriver can detect the DNA damage situations of sperm fast and accurately.
The above only is preferred embodiment of the present invention, be not in order to limit essence technology contents scope of the present invention, essence technology contents of the present invention is broadly to be defined in the claim scope of application, any technology entity or method that other people finish, if it is defined identical with the claim scope of application, also or a kind of change of equivalence, all will be regarded as being covered by among this claim scope.

Claims (9)

1. the treatment process of a sperm is characterized in that, described method comprises step:
(1) sperm suspension was hatched 0.5-3 hour at 0-4 ℃, obtain monosperm microinjection (intracytoplasmic sperm injection, ICSI) preceding sperm;
Contain sperm and sodium hydroxide (NaOH) in the described sperm suspension, in the cumulative volume of described sperm suspension, wherein concentration sodium hydroxide is 5-30mM.
2. treatment process as claimed in claim 1 is characterized in that, incubation time 1-3 hour, in the described sperm suspension, naoh concentration was 5-20mM.
3. treatment process as claimed in claim 1 is characterized in that, described sperm suspension was hatched 1.5-2.5 hour at 0-2 ℃, and in the described sperm suspension, naoh concentration is 8-12mM.
4. treatment process as claimed in claim 1 is characterized in that, in the cumulative volume of sperm suspension, wherein sperm concentration is 5-10 * 10 6/ mL.
5. treatment process as claimed in claim 1 is characterized in that, contains the sperm nutrient solution in the described sperm suspension.
6. treatment process as claimed in claim 1 is characterized in that, also comprises step after the described step (1):
(2) sperm suspension and mineral acid are mixed.
7. as the arbitrary described treatment process of claim 1-6, it is characterized in that described sperm is selected from mouse sperm, rat sperm, Niu Jingzi, sheep sperm, pig sperm or people's sperm.
8. a monosperm microinjection (intracytoplasmic sperm injection, ICSI) preceding sperm is characterized in that, by as the method for following step obtain:
(1) sperm suspension was hatched 0.5-3 hour at 0-4 ℃, obtain monosperm microinjection (intracytoplasmic sperm injection, ICSI) preceding sperm;
Contain sperm and sodium hydroxide (NaOH) in the described sperm suspension, in the cumulative volume of described sperm suspension, wherein concentration sodium hydroxide is 5-30mM.
Monosperm microinjection 9. as claimed in claim 8 (intracytoplasmic sperm injection, ICSI) preceding sperm is characterized in that, described sperm is selected from mouse sperm, rat sperm, Niu Jingzi, sheep sperm, pig sperm or people's sperm.
CN 200910194916 2009-09-01 2009-09-01 Sperm treating method Pending CN102002515A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103271778A (en) * 2013-04-09 2013-09-04 华东师范大学 Mouse microorganism purifying method utilizing egg cell cytoplasm sperm injection method
CN109843217A (en) * 2016-10-18 2019-06-04 伺马般有限公司 Device for mammalian artificial's insemination

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103271778A (en) * 2013-04-09 2013-09-04 华东师范大学 Mouse microorganism purifying method utilizing egg cell cytoplasm sperm injection method
CN109843217A (en) * 2016-10-18 2019-06-04 伺马般有限公司 Device for mammalian artificial's insemination
CN109843217B (en) * 2016-10-18 2021-03-12 伺马般有限公司 Device for artificial insemination of mammals
US11529222B2 (en) 2016-10-18 2022-12-20 Smartbow Gmbh Device for artificially inseminating a mammal

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Application publication date: 20110406