CN111323361B - Method for quickly separating sperm head, sperm tail and normal viable sperm - Google Patents
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- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1429—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its signal processing
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
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Abstract
The invention discloses a method for quickly separating sperm head, sperm tail and normal viable sperm, belonging to the technical field of reproductive medicine. Sperm, sperm head and sperm tail are sorted by using a flow cytometer based on differences in lateral scattered light-area (SSC-A) and forward scattered light-area (FSC-A), lateral scattered light-width (SSC-W) and lateral scattered light-height (SSC-H) of cells in a sperm sample. The method can realize the rapid separation of the sperm head, the sperm tail and the normal viable sperm, and has high purity and simplicity.
Description
Technical Field
The invention belongs to the technical field of reproductive medicine, and relates to a method for quickly separating sperm heads, sperm tails and normal viable sperms.
Background
Abnormal spermatogenesis is the leading cause of male sterility, and teratospermia is a common consequence of abnormal spermatogenesis. The kinds of teratospermia are also varied, such as broken head, broken tail, round head, etc., and the pathological mechanism of such diseases is not yet elucidated. Clinically, a single normal sperm is selected and matured, and in vitro fertilization is performed using ICSI (Intracytoplasmic sperm injection), and then offspring are obtained from such patients by tube infants. However, recent researches show that the offspring which is not generated by the natural conception mode has great health risk, so that the research on the cause and the pathological mechanism of the teratospermia is clear, and the method has great significance for treating the diseases and obtaining the offspring by the natural conception mode. In basic research, the separation of normal sperm by micromanipulation, or the separation of different components of sperm is extremely inefficient, which is not conducive to the development of relevant basic research, since the development of many scientific experiments requires a large number of samples. The same difficulties are encountered in studying the pathogenesis of teratospermia using model animals such as mice. The method has the advantages that components such as the head and the tail of the sperms are efficiently and highly obtained, or the teratospermia and the normal sperms are separated, the regulation and control functions of key proteins in the components are respectively researched, interference is eliminated, the fine regulation and control mechanism of key molecules in specific components of the sperms is researched, and the method has important significance for disclosing the regulation and control mechanism of spermatogenesis and researching the regulation and control of functions of mature sperms. At present, researchers separate sperm heads and sperm tails by using a density gradient centrifugation method, but the method is time-consuming and complex in operation on one hand, and cannot separate viable sperm simultaneously on the other hand. While viable sperm can be obtained by low-speed centrifugation such as the upstream method or percoll, sperm head and sperm tail with high purity cannot be obtained by splitting. Therefore, the invention of a method for rapidly separating the viable sperms, the sperm heads and the sperm tails is urgently needed to provide technical support for the development of related basic research.
Disclosure of Invention
In view of the above-mentioned deficiencies of the prior art, the present invention provides a method for rapidly separating sperm fractions from normal viable sperm, which is primarily suitable for rodents such as mice.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for rapidly sorting sperm, sperm head and sperm tail comprises the following steps:
step 1, processing a sperm sample: placing the epididymis tail and the vas deferens of a mouse into HS solution, cutting, incubating for 15 min at constant temperature of 35 ℃, filtering to remove cell clusters and other impurities, washing for 1 time by using the HS solution, then resuspending by using the HS solution to obtain a sperm sample, and carrying out ultrasonic treatment on the sperm sample, wherein the ultrasonic conditions are as follows: 500W, 5 s of over-treatment and 5 s of stop, and taking 5 min in total to obtain a mixed sperm sample;
step 2, preparing sperm head samples: taking the sperm sample in the step 1, adding 10% SDS and 20 mg/mL proteinase K, treating for 5 min at 37 ℃, washing for 2 times by using an HS solution, and then re-suspending by using the HS solution to obtain a sperm head sample;
step 3, respectively detecting the sperm head sample and the sperm sample by using a flow cytometer, determining the grouping range of the sperm head and the normal sperm, and referring to the preset voltage parameter and the accurate picture of the normal sperm cell and the sperm head;
step 4, sorting the sperms, the sperm heads and the sperm tails: detecting the mixed sperm sample in the step 1 by using a flow cytometer, adjusting voltage by taking the voltage parameter in the step 3 as reference, dividing the cells into three groups according to the parameter difference of the side scattering light-area and the forward scattering light-area, and respectively drawing the three groups as P1, P2 and P5 gates, further dividing the cells in the P1 gate into two gates of P3 and P4 according to the difference of the side scattering light-width and the side scattering light-height, and similarly, dividing the cells in the P2 gate into two gates of P6 and P7 and dividing the cells in the P5 gate into two gates of P8 and P9, and respectively collecting the cell groups, namely finishing the sorting of the sperms, the sperm heads and the sperm tails.
A method of rapidly sorting viable sperm cells comprising the steps of:
step 1, processing a sperm sample: taking epididymis and spermaduct of a mouse, shearing, incubating for 15 min at constant temperature of 35 ℃, filtering to remove cell mass and other impurities, washing for 1 time by using HS solution, then re-suspending by using the HS solution to obtain a sperm sample, adding 10 mg/mL of propidium iodide solution into the sperm sample suspension, and incubating for 5 min at 25 ℃ to obtain a sample;
step 2, preparing sperm head samples: taking the sperm sample in the step 1, adding 10% SDS and 20 mg/mL proteinase K, treating for 5 min at 37 ℃, washing for 2 times by using an HS solution, and then re-suspending by using the HS solution to obtain a sperm head sample;
step 3, respectively detecting the sperm head sample and the sperm sample by using a flow cytometer, determining the grouping range of the sperm head and the normal sperm, and referring to the preset voltage parameter and the accurate picture of the normal sperm cell and the sperm head;
and 4, sorting the viable sperm cells: detecting the sample in the step 1 by using a flow cytometer, adjusting voltage by taking the voltage parameter in the step 3 as reference, finding out cells, firstly, dividing the cells into three groups according to the parameters of side scattering light-area and forward scattering light-area, respectively drawing the three groups as P1, P2 and P5 gates, dividing the cells in the P1 gate into two gates of P3 and P4 according to the difference of side scattering light-width and side scattering light-height, similarly, dividing the cells in the P2 gate into two gates of P6 and P7, dividing the cells in the P5 gate into two gates of P8 and P9, selecting the group of normal sperms from the two gates, drawing the group as G1 gate, and then excluding dead cells with positive staining through a PI histogram under the G1 gate to obtain the group of cells representing the living sperms with negative PI staining, namely the G1 gate.
Has the advantages that: compared with a method for separating sperm heads and sperm tails by an ultradense gradient centrifugation method, the separation method is simpler, more convenient, quicker and more efficient; the difficulty that the low-speed gradient centrifugation such as percoll is used to separate the rodent sperms such as the mouse with viability is also overcome, and the purity and the vitality of the separated sperms are obviously improved.
Drawings
FIG. 1 is a graphical depiction of the gating strategy for sperm head, sperm tail and whole sperm sorting in example 1.
FIG. 2 shows the results of sperm heads, sperm tails and intact sperm cells isolated in example 1.
FIG. 3 is a graphical depiction of the viable sperm sorting strategy of example 1.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. The experimental methods and reagents of the formulations not specified in the examples are in accordance with the conventional conditions in the art.
The formula of the HS solution (HS high-saline solution) adopted in the invention is as follows: 135 mM NaCl, 5 mM KCl, 1 mM MgSO4，2 mM CaCl2，20 mM HEPES，5 mM glucose，10 mM lactic acid and 1 mM Na-pyruvate at pH 7.4 with NaOH。
Example 1
1. Treatment of mouse sperm samples
Dissecting the mouse, separating clean epididymis tail and vas deferens, placing in a 2 cm culture dish containing 2 mL HS solution, shearing, incubating at 35 deg.C for 15 min, vibrating or beating 3-5 times to make sperm fully free. Filtering with 70 μm cell sieve to remove cell mass and other impurities. HS washing is carried out once at 800 g for 5 min. Resuspending the sperm sediment by 2 mL HS, reserving 400 μ L of sperm sample, and carrying out ultrasonic treatment on the remaining 1.5 mL of sperm sample under the conditions of 500W, 5 seconds exceeding and 5 seconds stopping for 5 min in total.
2. Preparation of sperm head positive control sample
mu.L of the sperm sample was collected from the remaining 400. mu.L, 10. mu.L of 10% SDS and 5. mu.L of 20 mg/mL proteinase K were added thereto, and the mixture was treated at 37 ℃ for 5 min. 10 mu.L of the sperm is sucked and placed on a glass slide, the sperm lysis condition is observed under a microscope after a cover glass is covered, and generally 99.9 percent of the sperm only remain sperm heads. HS washing is carried out for 5 min at 1000 g for 2 times. 200 μ L HS was resuspended for use.
3. Sorting sperm, sperm head and sperm tail by flow cytometry
a. Detecting a reserved sperm sample (normal sperm cells) of 200 mu L on a machine, adjusting voltage parameters, excluding cell fragments with small values of FSC-A (side scattering light-area) and SSC-A (forward scattering light-area), finding out a main population of complete sperm cells in an FSC-A/SSC-A scatter diagram, drawing a P1 gate which represents most of the complete sperm and making reference for pre-adjusting voltage parameters and accurately drawing the gate of the normal sperm cells.
b. Detecting 200 μ L sperm head sample on a computer, adjusting voltage parameters, finding out main population of sperm head in SSC-A/FSC-A scatter diagram, drawing two gates P2 and P5, both representing sperm head, and making reference for pre-adjusting voltage parameters and accurately drawing gates of sperm head.
c. Detecting the mixed sperm sample after ultrasonic treatment, wherein the mixed sperm sample contains complete sperms, sperm heads and sperm tails. And (c) adjusting the voltage according to the parameters of the step a and the step b to find cells, and dividing the samples into three groups according to the difference of SSC-A and FSC-A parameters of sperms, sperm heads and sperm tails, wherein the three groups are respectively drawn as gates P1, P2 and P5. The P1 gates were selected individually and the cells were divided into two groups, P3 and P4, based on the difference between SSC-W (side scatter-width) and SSC-H (side scatter-height). Similarly, cells within the P2 and P5 gates were divided into two populations, P6, P7 and P8, P9, respectively. The size of each gate was fine-tuned under the same collection parameters to differentiate the populations of each cell as much as possible.
Cell groups in the gates of P3, P4, P5, P6 and P7 are collected respectively, and the forms and the statistical purities of the components collected in the gates of P3, P4, P5, P6 and P7 are analyzed under a 40-fold microscope.
The gate drawing strategy and experimental results of each component in the above flow are shown in fig. 1.
Through parameter optimization and later verification and analysis, due to large difference of sperm heterogeneity, it is known that the whole sperm is selected in the gates P3 and P4, the sperm head is selected in the gates P5, P7, P8 and P9, and the sperm tail is selected in the gate P6. The results of the separation of the intact sperm, sperm head and sperm tail are shown in FIG. 2.
In the future practical application, the same components selected from different departments can be combined and researched according to different practical requirements or respectively researched according to the practical requirements of purity difference and heterogeneity research.
4. Flow cytometer sorting viable sperm cells
Dissecting the mouse, separating clean epididymis tail and vas deferens, placing in a 2 cm culture dish containing 2 mL HS solution, shearing, incubating at 35 deg.C for 15 min, vibrating or beating 3-5 times to make sperm fully free. The cell is sieved by a 50 μm cell sieve to remove cell aggregates and other impurities. HS washing is carried out once at 800 g for 5 min. The sperm pellet was resuspended in 2 mL HS, sperm motility was measured (70% assay), and 2. mu.L of 10 mg/mL Propidium Iodide (PI) solution was added to the sperm suspension and incubated for 5 min at room temperature. And (3) performing machine detection, selecting a normal sperm subgroup according to the parameters of sorting sperm heads, sperm tails and intact sperm, drawing a G1 gate, and then excluding dead cells with positive staining through a PI histogram under a G1 gate, namely drawing a PI staining negative cell subgroup G1 gate representing live sperm. Cells in the G1, G2 gates were collected by sorting. The gating strategy for each component in the above flow is shown in FIG. 3. The computer aided sperm analysis system analyzes the shape, purity and vitality of the sperm.
The purity of the sperm head separated by the rapid separation technology of the invention reaches more than 90 percent, the purity of the sperm tail reaches 98 percent, and the purity of the complete dead sperm reaches more than 90 percent. By adopting the technical scheme of the invention, the sperms with the motility of more than 90% and the purity of more than 98% can be separated from the roughly separated sperms with the motility of about 70%, as shown in figure 2. Compared with a method for separating sperm heads and sperm tails by an ultradense gradient centrifugation method, the separation method is simpler, more convenient, quicker and more efficient; also overcomes the difficulty that the low-speed gradient centrifugation such as percoll is difficult to separate the viable mouse sperms, and the purity and the vitality of the separated sperms are obviously improved.
Claims (2)
1. A method for rapidly sorting sperms, sperm heads and sperm tails is characterized in that: the method comprises the following steps:
step 1, processing a sperm sample: placing the epididymis tail and the vas deferens of a mouse into HS solution, cutting, incubating at constant temperature of 35 ℃ for 15 min, filtering by a 70-micron cell filter to remove cell clusters and other impurities, washing by the HS solution, re-suspending by the HS solution to obtain a sperm sample, and carrying out ultrasonic treatment on the sperm sample under the ultrasonic conditions: 500W, 5 s of over-treatment and 5 s of stop, and taking 5 min in total to obtain a mixed sperm sample;
step 2, preparing sperm head samples: taking the sperm sample in the step 1, adding 10% SDS and 20 mg/mL proteinase K, treating for 5 min at 37 ℃, washing with HS solution, and then resuspending with HS solution to obtain a sperm head sample;
step 3, respectively detecting the sperm head sample in the step 2 and the sperm sample in the step 1 by using a flow cytometer, determining the grouping range of the sperm heads and the normal sperms, and taking the preset voltage parameters and the accurate drawing of the sperm heads as reference for the normal sperms and the sperm heads;
step 4, sorting the sperms, the sperm heads and the sperm tails: detecting the mixed sperm sample in the step 1 by using a flow cytometer, adjusting voltage by taking the voltage parameter in the step 3 as a reference, dividing the cells into three groups according to the parameter difference of lateral scattering light-area and forward scattering light-area, respectively drawing as gates P1, P2 and P5, dividing the cells in the gate P1 into two gates P3 and P4 according to the difference of lateral scattering light-width and lateral scattering light-height, similarly, dividing the cells in the gate P2 into two gates P6 and P7, dividing the cells in the gate P5 into two gates P8 and P9, respectively collecting cell groups, and knowing that the cells in the gates P3 and P4 are complete sperms, the cells in the gates P5, P7, P8 and P9 are sperm heads, and the cells in the gate P6 are sperm tails, thereby completing the sorting of the sperms, the sperm heads and the sperm tails.
2. A method for rapidly sorting viable sperm cells, comprising: the method comprises the following steps:
step 1, processing a sperm sample: taking epididymis and spermaduct of a mouse, shearing, incubating at constant temperature of 35 ℃ for 15 min, filtering by a 70-micron cell filter to remove cell clusters and other impurities, washing for 1 time by using HS (high speed) solution, then re-suspending by using the HS solution to obtain a sperm sample, adding 10 mg/mL of propidium iodide solution into the sperm sample suspension, and incubating at 25 ℃ for 5 min to obtain a sample;
step 2, preparing sperm head samples: taking the sperm sample in the step 1, adding 10% SDS and 20 mg/mL proteinase K, treating for 5 min at 37 ℃, washing for 2 times by using an HS solution, and then re-suspending by using the HS solution to obtain a sperm head sample;
step 3, respectively detecting the sperm head sample and the sperm sample by using a flow cytometer, determining the grouping range of the sperm head and the normal sperm, and pre-adjusting voltage parameters and accurately drawing a gate for the normal sperm cell and the sperm head to be used as reference;
and 4, sorting the viable sperm cells: detecting the sample in the step 1 by using a flow cytometer, adjusting voltage by taking the voltage parameter in the step 3 as reference, finding out cells, firstly, dividing the cells into three groups according to the parameters of side scattering light-area and forward scattering light-area, respectively drawing the three groups as P1, P2 and P5 gates, dividing the cells in the P1 gate into two gates of P3 and P4 according to the difference of side scattering light-width and side scattering light-height, similarly, dividing the cells in the P2 gate into two gates of P6 and P7, dividing the cells in the P5 gate into two gates of P8 and P9, selecting the group of normal sperms from the two gates, drawing the group as G1 gate, and then excluding dead cells with positive staining through a PI histogram under the G1 gate to obtain the group of cells representing the living sperms with negative PI staining, namely the G1 gate.
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Application publication date: 20200623 Assignee: AFFILIATED HOSPITAL OF NANTONG University Assignor: NANTONG University Contract record no.: X2023980052952 Denomination of invention: A Rapid Method for Separating Sperm Head, Sperm Tail, and Normal Vigorous Sperm Granted publication date: 20220510 License type: Common License Record date: 20231219 |