CN111289424A - Method for detecting sperm mitochondrial membrane potential and active oxygen by double-standard method - Google Patents
Method for detecting sperm mitochondrial membrane potential and active oxygen by double-standard method Download PDFInfo
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- G01N15/10—Investigating individual particles
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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Abstract
The invention discloses a method for detecting sperm mitochondrial membrane potential and active oxygen by a double-labeling method, which is characterized by comprising the steps of adding 5-10 ten thousand sperms into 500 mu L sperm culture buffer solution preheated at 37 ℃ after the semen is naturally liquefied, uniformly mixing, placing in a water bath tank at 37 ℃, adding 10uL sperm mitochondrial membrane potential and active oxygen combined dye solution, uniformly mixing, incubating at 37 ℃ in the dark, and detecting by an up-flow cytometer within 5-30 minutes; and finally, respectively collecting three color fluorescence signals: green, orange and red; analyzing results by using flow cytometry self-contained software, and determining the level of mitochondrial membrane potential according to green and orange fluorescence conditions; the method determines the content of the active oxygen according to the red fluorescence condition, has the advantages of realizing the detection of the sperm mitochondrial membrane potential and the active oxygen at one time, along with simple and quick operation and good repeatability.
Description
Technical Field
The invention relates to the field of in-vitro diagnostic reagents, in particular to a method for detecting sperm mitochondrial membrane potential and active oxygen by a double-label method.
Background
The primary function of mitochondria is to produce energy, providing ATP for sperm motility. The functional state of mitochondria plays an important role in sperm survival because sperm motility is closely related to mitochondrial activity, and energy required for whole sperm metabolism is mainly provided by mitochondria. Changes in mitochondrial activity can affect sperm energy supply. Studies have shown that mitochondria are also the regulatory center of apoptosis. The mitochondrial membrane potential is the earliest factor for starting the apoptosis process, and the measurement of the mitochondrial membrane potential is particularly important for the measurement of the sperm quality and function. Thus, the functional status of mitochondria is a critical indicator of the quality of sperm function. Normally, the assessment of mitochondrial activity is mainly analyzed by detecting changes in the transmembrane potential of mitochondria.
Transmembrane potentials are present on the mitochondria of sperm, and therefore mitochondrial activity is measured primarily by detecting changes in mitochondrial transmembrane potentials. Currently, the fluorescent probes for detecting the activity of sperm mitochondria mainly include Rhodamine123 (Rhodamine123, R123), MitoTracker Green FM (MITO), and 5, 5 ', 6, 6' -tetrachloro-1, 1 ', 3, 3' -tetraethylbenzimidazolyl-carbonyl-iodonium (5, 5 ', 6, 6' -tetrachloro-1, 1 ', 3, 3' -tetraethylene benzazolyl-carbocyanineiodide, JC-1). Although JC-1 was widely used in many experiments, its poor water solubility characteristics also made it difficult to use in some experiments. JC-1 precipitates in an aqueous buffer even at low concentrations. JC-10(5, 6-dichoro-1, 1 ', 3, 3' -tetra-acetyl-aminocyanine iodide; Enhanced JC-1) is a possible alternative to JC-1 in experiments where high concentrations of dye are required. JC-10 has better water solubility than JC-1, JC-10 can selectively enter mitochondria, and JC-10 is considered to be the most suitable fluorescent probe for detecting the mitochondrial function of sperms and has better staining specificity than JC-1. JC-10 molecular formula of C25H27Cl2IN4The molecular weight is 583.34, when the potential of the sperm mitochondrial membrane is low in the process of detecting the sperm mitochondrial function, the sperm mitochondrial membrane exists in a monomer form, the excitation wavelength Ex is 490nm, and the green fluorescence wavelength Em is 525 nm; when the mitochondrial membrane potential is high, a dimer is formed and emits orange fluorescence (emission wavelength Em of 590 nm). Although JC-10 staining alone can determine the level of mitochondrial membrane potential in sperm, it cannot accurately determine the state of oxidative stress active oxygen in sperm.
Reactive Oxygen Species (ROS) refers to a class of oxygen-containing substances with active chemical properties and strong oxidizing ability in the organism or in the natural environment, and is the normal organism tissueMainly comprising hydrogen peroxide (H) is provided2O2) The compound has a nuclear mismatching pair electron, a peroxide anion (O2-), a hydroxyl (-OH), active nitrogen (NO and the like), and the like, and can generate oxidation reaction with any molecule so as to change the structure and the function of the latter. Studies have shown that ROS are required at physiological levels and play an important role in maintaining the physiological functions of male reproduction, such as cell signaling, hormone production, sperm capacitation, acrosome reaction, and sperm motility. Sperm capacitation is closely related to the tyrosine phosphorylation level of the tail part of the sperm, particularly the middle section of the tail part, and promotes the combination of the sperm and the zona pellucida of the oocyte by influencing the intracellular cyclic adenosine monophosphate (cAMP) level and lipid peroxidation, thereby promoting the tyrosine phosphorylation-dephosphorylation of sperm protein. If the production of ROS is blocked, the sperm will not be able to capacitate. ROS may serve as second messengers in many different cell types, and they are also important regulators of sperm function. However, when the ROS exceeds the reducing capacity of the body's antioxidant system, the body is under oxidative stress, which destroys cellular DNA, proteins and lipids, causing cellular gene, structure and function to be damaged, and even cell death. ROS can cause damage to sperm mitochondria, oxidative stress can cause the potential of sperm mitochondrial membranes to be reduced in an early process, so that the sperm mitochondria can fall off, the quantity of the sperm mitochondria is reduced, energy synthesis is reduced, lipid in the sperm mitochondrial membranes is oxidized, and DNA of nuclear genome is damaged, which not only influences the exertion of respiratory chains on the mitochondrial membranes and the normal functions of adenosine triphosphatase generation, but also influences the synthesis of DNA coding protein necessary for the mitochondrial respiratory function, and even causes poor embryo pre-implantation development, high early abortion rate, offspring gene defect, sterility, tumor and the like.
Fluorescent probes for detecting singlet oxygen in active oxygen include DPAX, DMAX, and fluorin analog (FCLA); a fluorescent probe for detecting hydrogen peroxide of active oxygen is DCFH-DA (2 ', 7' -D ich fluorosceitin), which is a non-fluorescent substance and can be converted into DCF (2 ', 7' -D ich fluoroscein), i.e., dichlorofluorescein, which has strong fluorescence. Can also be used based on red light emitting naphthalene fluorescein probeNeedle N aph tho-Peroxyfluob1(N PF1), and the like. The fluorescent probe Hydroeth idine (HE) for detecting the superoxide anion in the active oxygen is a fluorescent probe for detecting the superoxide anion with good selectivity and sensitivity, and the Mito-HE synthesized on the basis is a fluorescent probe for detecting the superoxide anion based on ethidium bromide and has longer excitation wavelength than the HE; another substance of the class of Dihydroethidium (Dihydroethidium) has a molecular formula of C21H21N3Molecular weight is 315.41, and the fluorescent probe can be taken as superoxide anion, and has an excitation wavelength of 480-535nm and an emission wavelength of 610nm (red fluorescence). The fluorescent probes MitoAR and MitoHR for detecting hydroxyl radicals in active oxygen almost do not have fluorescence, but generate a strongly fluorescent substance HM TMR after reacting with the active oxygen, and the two substances are used for measuring the active oxygen in living mitochondria. A chloromethyl functional compound (MTCM) with weak mercapto reactivity is in non-fluorescent reduction state, and can emit fluorescence when oxidized, the dye can also dye the mitochondria of living cells, the accumulation of the dye depends on membrane potential, and the dye can be stably stored after the acetaldehyde is fixed, and the molecular formula of the dye is C34H36Cl2N2Molecular weight 543.58, a far-red fluorescent dye (absorption/emission wavelength approximately 640/662nm) that stains living cell mitochondria and accumulates depending on membrane potential. After fixation with acetaldehyde, the dye is stable for storage. The detection of the mitochondrial membrane potential and the active oxygen of the sperm is based on a single fluorescent probe staining method (single-standard method), and if two targets are detected, two different experimental operations are required. Therefore, the above conventional detection methods all have a disadvantage that the mitochondrial membrane potential and the active oxygen of the sperm cannot be detected simultaneously.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for detecting the mitochondrial membrane potential and the active oxygen of the sperm by a double-standard method, which is simple and rapid to operate and has good repeatability.
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for detecting mitochondrial membrane potential and active oxygen of sperm by a double-standard method comprises the following steps:
(1) after the semen is naturally liquefied, adding 5-10 ten thousand sperms into 500 microliter sperms culture buffer solution preheated at 37 ℃, uniformly mixing, and placing in a water bath cabinet at 37 ℃;
(2) adding 10 mu L of sperm mitochondrial membrane potential and active oxygen combined dye solution into the sperm culture buffer solution obtained in the step (1), uniformly mixing, incubating at 37 ℃ in the dark, and detecting by an up-flow cytometer within 5-30 minutes;
(3) three color fluorescence signals were collected separately: green, orange and red; analyzing results by adopting flow cytometry with software, and determining that the mitochondrial membrane potential is high or low, the orange fluorescent cell is a cell with high membrane potential, and the green fluorescent cell is a cell with low membrane potential according to green and orange fluorescent conditions; and determining the content of active oxygen according to the red fluorescence, wherein the cells with high red fluorescence are the cells with high active oxygen content, and the cells with weak red fluorescence are the cells with low active oxygen content.
The sperm culture buffer solution formula in the step (1) is as follows: 7.4g/L sodium chloride (NaCl), 0.366g/L sodium bicarbonate (NaHCO)3) 0.285g/L potassium chloride (KCl), 0.154g/L disodium hydrogen phosphate (Na)2HPO4) 0.153g/L magnesium sulfate (MgSO)4·7H2O), 0.083g/L potassium dihydrogen phosphate (KH)2PO4) 0.044g/L calcium chloride (CaCl)2·2H2O), 5.21g/LHepes sodium salt, 0.55-1.1g/L D-glucose and 10-50g/L human serum albumin, and the pH value is 7.0-7.8. Saline solutions to aid in sperm motility and staining.
The sperm mitochondrial membrane potential and active oxygen combined staining solution in the step (2) comprises two double-label staining solutions based on single laser and double lasers.
The double-label staining solution suitable for the flow cytometer of the single laser emitter is JC-10 and dihydroethidium double-label staining solution prepared by dissolving JC-10 and dihydroethidium in dimethyl sulfoxide (DMSO), wherein the concentration of JC-10 in the JC-10 and dihydroethidium double-label staining solution is 1-50 mu mol/L, and the concentration of dihydroethidium is 1-100 mu mol/L. Lipophilic cationic dye can enter mitochondria through transmembrane under the action of mitochondrial membrane potential, and a laser (488nm) with the same wavelength is adopted to respectively detect the fluorescence of three colors: green, orange and red.
The double-label dyeing solution suitable for the flow cytometer of the double laser emitter is JC-10 and MTCM double-label dyeing prepared by dissolving JC-10 and a chloromethyl functional group compound (MTCM) with weak sulfydryl reactivity in dimethyl sulfoxide (DMSO), wherein the concentration of JC-10 in the JC-10 and MTCM double-label dyeing solution is 1-50 mu mol/L, and the concentration of MTCM is 1-100 mu mol/L. With lasers of different wavelengths (488nm and 635nm), the fluorescence of three colors: green, orange and red.
Compared with the prior art, the invention has the advantages that: the invention discloses a method for detecting the mitochondrial membrane potential and active oxygen of sperms by a double-label method for the first time. The method adopts a double-standard method, namely, the detection is carried out by adopting a single laser flow cytometer after JC-10 and ethidium dihydrogen are jointly dyed, or the detection is carried out by adopting a double laser flow cytometer after JC-10 and MTCM are dyed, and the conditions of mitochondrial membrane potential and active oxygen of sperms are simultaneously detected, so that the purposes of one-time detection of two target objects are realized, and the method is simple and efficient.
Drawings
FIG. 1 shows the results of detecting mitochondrial membrane potential and active oxygen in JC-10 and ethidium dihydride staining sperm, wherein the five borders in the figure are sperm with high mitochondrial membrane potential, the four borders in the figure are sperm with low mitochondrial membrane potential, the short bar in the right figure is sperm with low active oxygen, and the long bar in the right figure is sperm with high active oxygen;
FIG. 2 shows the results of detection of mitochondrial membrane potential and active oxygen in sperm by JC-10 and MTCM double-label staining, wherein the five borders in the figure are sperm with high mitochondrial membrane potential, the four borders in the figure are sperm with low mitochondrial membrane potential, the short bar in the right figure is sperm with low active oxygen, and the long bar in the right figure is sperm with high active oxygen.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
Detailed description of the preferred embodiment
A method for detecting mitochondrial membrane potential and active oxygen of sperm by a double-standard method comprises the following steps:
1. after the semen is naturally liquefied, adding 5-10 ten thousand sperms into 500 microliter sperms culture buffer solution preheated at 37 ℃, uniformly mixing, and placing in a water bath cabinet at 37 ℃; the preparation method of the sperm culture buffer solution comprises the following steps:
1) weighing 7.4g NaCl, 0.366g NaHCO3，0.285g KCl，0.154g Na2HPO4，0.153g MgSO4·7H2O，0.083g KH2PO4，0.044g CaCl2·2H2O, 5.21g Hepes sodium salt, 0.55-1.1g D-glucose and 10-50g human serum albumin, and adding into 750ml purified water;
2) adjusting the pH to 7.0-7.8 (preferably 7.4) with 1mol/L sodium hydroxide (NaOH);
3) adding purified water to a constant volume of 1000 ml;
4) filtering the dissolved staining buffer solution with a 0.22 mu m filter membrane, subpackaging 100mL, and storing at 2-8 ℃;
2. adding 10 mu L of sperm mitochondrial membrane potential and active oxygen combined dye solution into the sperm culture buffer solution obtained in the step (1), uniformly mixing, incubating at 37 ℃ in the dark, and detecting by an up-flow cytometer within 5-30 minutes; the specific process is as follows:
1) adding 10 mu L of JC-10 and ethidium dihydrogen double-label staining solution into the sperm culture buffer solution obtained in the step (1), uniformly mixing, and incubating at 37 ℃ in a dark place; the preparation method of the JC-10 and ethidium dihydrochloride double-label staining solution comprises the following steps:
A. carefully weighing 1mg JC-10 and 2mg of dihydro ethidium dye in a dark place and placing the dye in a brown reagent bottle;
B. adding 7.1mL DMSO into a reagent bottle, shaking while adding DMSO to avoid JC-10 and ethidium dihydrogen agglomeration and precipitation, and vigorously shaking the prepared reagent for 5-10min by using a vortex shaker to ensure that JC-10 and ethidium dihydrogen are fully dissolved (after dissolution, the concentration of JC-10 is 16.7 mu mol/L, and the concentration of ethidium dihydrogen is 63.5 mu mol/L);
filtering the dissolved dye solution with a C.0.22 μm filter membrane, subpackaging 500 μ L, and freezing at-20 deg.C and keeping away from light;
2) starting a stabilized voltage supply and a Calibur flow cytometer, checking a waste liquid barrel and a sheath liquid barrel, and starting a single laser transmitter;
3) setting flow cytometry signal collecting conditions, including adopting the same wavelength laser (488nm), detecting the fluorescence of three colors respectively: green, orange and red, corresponding to FL1, FL2, FL3 channels, respectively; setting the detection flow rate, 100-;
3. three color fluorescence signals were collected separately: green, orange and red; results were analyzed using CellQuestPro data analysis software in Calibur flow cytometer: according to the green and orange fluorescence conditions, the mitochondrial membrane potential is determined (the membrane potential is high and emits orange fluorescence, and conversely, the cells emitting green fluorescence, which are high and low membrane potentials on two fluorescence two-dimensional scatter diagrams are presented as two groups, and the proportion of each group of cells can be further quantitatively analyzed); according to the red fluorescence condition, determining the content condition of active oxygen (the content of the red fluorescence is high, and the content of the red fluorescence is low otherwise). JC-10 and ethidium dihydride double-label staining detection of sperm mitochondrial membrane potential and active oxygen detection results are shown in figure 1, wherein the five borders in the figure are sperms with high mitochondrial membrane potential, the four borders in the figure are sperms with low mitochondrial membrane potential, the short bar in the right figure is sperm with low active oxygen, and the long bar in the right figure is sperm with high active oxygen.
Detailed description of the invention
A method for detecting mitochondrial membrane potential and active oxygen of sperm by a double-standard method comprises the following steps:
1. after the semen is naturally liquefied, adding 5-10 ten thousand sperms into 500 microliter sperms culture buffer solution preheated at 37 ℃, uniformly mixing, and placing in a water bath cabinet at 37 ℃; wherein the preparation method of the sperm culture buffer solution is the same as that of the first embodiment;
2. adding 10 mu L of sperm mitochondrial membrane potential and active oxygen combined dye solution into the sperm culture buffer solution obtained in the step (1), uniformly mixing, incubating at 37 ℃ in the dark, and detecting by an up-flow cytometer within 5-30 minutes; the specific process is as follows:
1) adding 10 mu L of JC-10 and MTCM double-label staining solution into the sperm culture buffer solution obtained in the step (1), uniformly mixing, and incubating at 37 ℃ in a dark place; the preparation method of the JC-10 and MTCM double-label staining solution comprises the following steps:
A. carefully weighing 1mg JC-10 and 2mg MTCM dyes in a dark place and placing the dyes in a brown reagent bottle;
B. adding 7.1mL of DMSO into a reagent bottle, shaking while adding to avoid JC-10 and MTCM aggregation and precipitation, and violently shaking the prepared reagent for 5-10min by using a vortex oscillator to ensure that JC-10 and MTCM are fully dissolved (after dissolution, the concentration of JC-10 is 16.7 mu mol/L, and the concentration of MTCM is 36.8 mu mol/L);
filtering the dissolved dye solution with a C.0.22 μm filter membrane, subpackaging 500 μ L, and freezing at-20 deg.C and keeping away from light;
2) starting a stabilized voltage supply and a Calibur flow cytometer, checking a waste liquid barrel and a sheath liquid barrel, and starting a double-laser transmitter;
3) setting flow cytometry signal collecting conditions, including adopting lasers (488nm and 635nm) with different wavelengths to respectively detect red fluorescence, orange fluorescence and red fluorescence values, which respectively correspond to FL1, FL2 and FL3 channels; setting the detection flow rate, 100-;
3. three color fluorescence signals were collected separately: green, orange and red; results were analyzed using CellQuestPro data analysis software in Calibur flow cytometer: according to the green and orange fluorescence conditions, the level of the mitochondrial membrane potential is determined (the membrane potential is high and emits orange fluorescence, and conversely emits green fluorescence); according to the red fluorescence condition, determining the content condition of active oxygen (the content of the red fluorescence is high, and the content of the red fluorescence is low otherwise). JC-10 and ethidium dihydride double-label staining detection of sperm mitochondrial membrane potential and active oxygen detection results are shown in figure 1, wherein the five borders in the figure are sperms with high mitochondrial membrane potential, the four borders in the figure are sperms with low mitochondrial membrane potential, the short bar in the right figure is sperm with low active oxygen, and the long bar in the right figure is sperm with high active oxygen.
The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art should also realize that changes, modifications, additions and substitutions can be made without departing from the true spirit and scope of the invention.
Claims (5)
1. A method for detecting the mitochondrial membrane potential and active oxygen of sperm by a double-standard method is characterized by comprising the following steps:
(1) after the semen is naturally liquefied, adding 5-10 ten thousand sperms into 500 microliter sperms culture buffer solution preheated at 37 ℃, uniformly mixing, and placing in a water bath cabinet at 37 ℃;
(2) adding 10 mu L of sperm mitochondrial membrane potential and active oxygen combined dye solution into the sperm culture buffer solution obtained in the step (1), uniformly mixing, incubating at 37 ℃ in the dark, and detecting by an up-flow cytometer within 5-30 minutes;
(3) three color fluorescence signals were collected separately: green, orange and red; analyzing results by adopting flow cytometry with software, and determining that the mitochondrial membrane potential is high or low, the orange fluorescent cell is a cell with high membrane potential, and the green fluorescent cell is a cell with low membrane potential according to green and orange fluorescent conditions; and determining the content of active oxygen according to the red fluorescence, wherein the cells with high red fluorescence are the cells with high active oxygen content, and the cells with weak red fluorescence are the cells with low active oxygen content.
2. The method for detecting mitochondrial membrane potential and active oxygen in sperm according to claim 1, wherein the sperm culture buffer of step (1) has the following formula: 7.4g/L sodium chloride, 0.366g/L sodium bicarbonate, 0.285g/L potassium chloride, 0.154g/L disodium hydrogen phosphate, 0.153g/L magnesium sulfate, 0.083g/L potassium dihydrogen phosphate, 0.044g/L calcium chloride, 5.21g/L Hepes sodium salt, 0.55-1.1g/L D-glucose and 10-50g/L human serum albumin, and the pH value is 7.0-7.8.
3. A method of dual-label detection of mitochondrial membrane potential and reactive oxygen species in sperm according to claim 1, wherein: the sperm mitochondrial membrane potential and active oxygen combined staining solution in the step (2) comprises two double-label staining solutions based on single laser and double lasers.
4. A method according to claim 3, wherein the method comprises the steps of: the double-label staining solution suitable for the flow cytometer of the single laser emitter is JC-10 and dihydroethidium double-label staining solution prepared by dissolving JC-10 and dihydroethidium in dimethyl sulfoxide, wherein the concentration of JC-10 in the JC-10 and dihydroethidium double-label staining solution is 1-50 mu mol/L, and the concentration of dihydroethidium is 1-100 mu mol/L.
5. A method according to claim 3, wherein the method comprises the steps of: the double-label dyeing solution suitable for the flow cytometer of the double laser emitter is JC-10 and MTCM double-label dyeing prepared by dissolving JC-10 and a chloromethyl functional group compound with weak mercapto reactivity in dimethyl sulfoxide, wherein the concentration of JC-10 in the JC-10 and MTCM double-label dyeing solution is 1-50 mu mol/L, and the concentration of MTCM in the MTCM double-label dyeing solution is 1-100 mu mol/L.
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