A method for preparing a slide for evaluating chromosome ploidy and structure of mammalian eggs

Abstract

The present invention belongs to the technical field of chromosome detection, and specifically relates to a method for preparing a slide for evaluating the chromosome ploidy and structure of mammalian eggs. The present invention sequentially treats eggs with a first embryo culture medium containing a kinesin Eg5 inhibitor and an acidic buffer solution to obtain eggs with zona pellucida removed and corresponding first polar bodies. By respectively spreading and immunofluorescence staining the chromosomes in the eggs and the corresponding first polar bodies on a porous slide, not only can the chromosome ploidy of the eggs be determined, but also the presence of sister chromosomes in the cells can be accurately detected, so as to achieve simultaneous detection of the chromosome ploidy and chromosome structure of the eggs, expand the detection range, and effectively eliminate the errors of the detection technology source that occur in the detection process of the prior art; each cell is detected independently to reduce cross contamination and improve the detection accuracy; in addition, the present invention adopts a porous slide, which can achieve multiple groups of simultaneous detection, effectively improving the detection and analysis efficiency.

Description

Technical Field

[0001] The invention belongs to the technical field of chromosome detection, and in particular relates to a method for preparing a glass slide for evaluating the chromosome ploidy and structure of mammalian eggs. Background Art

[0002] Chromosomal abnormalities are very common in mammalian eggs, especially in humans, and increase significantly with age. It is now clear that aneuploid embryos caused by chromosomal errors in eggs are the main cause of clinical problems such as early miscarriage, infertility, and congenital malformations. Therefore, it is crucial to study the regulation of chromosome segregation in mammalian oocytes and its use in detecting and evaluating the ploidy and structure of mammalian egg chromosomes.

[0003] At present, the technologies used to detect the chromosome ploidy of mammalian eggs mainly include traditional chromosome spread staining methods and NGS (Next generation sequencing) technology developed in recent years. Among them, the chromosome spread staining methods mainly include the following two schemes: 1) By using a mixture of formaldehyde and acetic acid as a fixative to fix the egg chromosomes, then stain and mark the chromosomes with dyes such as Giemsa or DAPI, and finally observe and analyze under a microscope. The advantages of this method are simple operation and low reagent cost; the disadvantage is that it is impossible to stain and analyze the chromosomes at the protein level on the spread. 2) By using paraformaldehyde as a fixative to fix the egg chromosomes, then using immunofluorescence staining technology to mark the chromosome centromere and other structures, and then using DAPI and other dyes to counterstain and mark the chromosomes, and finally observe and analyze under a fluorescence microscope. The main advantage of this method is that the reagent cost is relatively low, and the chromosome structure in the egg can be more clearly marked. However, the disadvantages of this scheme are as follows: if chromosome spreading analysis is performed on a single or a few eggs in a fixed area of ​​the slide, not only will it lead to a waste of reagents and consumables, but it will also be difficult or even impossible to find the chromosome structure of the egg on the slide when analyzed under a microscope, seriously affecting the detection efficiency and there is a high risk of losing the target sample; if chromosome spreading analysis is performed on a larger number of eggs in a smaller fixed area of ​​the slide, the possibility of cross-contamination of chromosomes between different cells during the spreading process cannot be avoided, thereby affecting the accuracy of the test results; the chromosomes on the slide may be lost during the entire chromosome spreading and staining detection process, and it is therefore impossible to determine whether the test result of too few chromosomes in the egg is a loss caused by the technology or a problem with the egg itself.

[0004] In addition, NGS genome sequencing technology used to detect egg chromosome ploidy is currently mainly used in clinical practice. It can detect the number of chromosomes in cells, such as too many or too few paired sister chromosomes. However, its application in basic research, especially in animal model eggs, is very limited. The most fundamental reason is that the detection and analysis is expensive, and it is impossible to distinguish whether the sister chromosomes in the egg are paired or prematurely separated. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a slide for evaluating the ploidy and structure of mammalian egg chromosomes. The method of the present invention can simultaneously detect chromosomes in the egg and its first polar body cell, which can effectively improve the detection accuracy, expand the detection range, and improve the detection and analysis efficiency.

[0006] The present invention provides a method for preparing a glass slide for evaluating the chromosome ploidy and structure of mammalian eggs, comprising the following steps:

[0007] placing the eggs in a first embryo culture medium containing a kinesin Eg5 inhibitor to obtain treated eggs;

[0008] Transferring the treated eggs to an acidic buffer solution to obtain eggs with zona pellucida removed and the corresponding first polar body; the pH of the acidic buffer solution is 2.2-2.8;

[0009] The oocytes with the zona pellucida removed and the corresponding first polar bodies are respectively transferred to the fixatives in different wells of a multi-porous glass slide for spreading and air-drying to obtain air-dried multi-porous glass slides;

[0010] The air-dried multi-well glass slide is subjected to immunofluorescence staining to obtain a glass slide for evaluating the chromosome ploidy and structure of mammalian eggs.

[0011] Preferably, the kinesin Eg5 inhibitor comprises Monastrol; and the concentration of the kinesin Eg5 inhibitor in the first embryo culture medium is 100-200 μmol / L.

[0012] Preferably, the time for treating the oocytes in the first embryo culture medium containing the kinesin Eg5 inhibitor is 1 to 2 hours.

[0013] Preferably, the diameter of the pores of the porous glass slide is 4 to 6 mm; the volume of the fixing solution in the porous glass slide is 5 to 10 μL / well.

[0014] Preferably, the fixing solution comprises components with the following concentrations: PFA 10 g / L, Triton X-100 0.15 Vol % and DTT 3 mM; the pH of the fixing solution is 9.2.

[0015] Preferably, before transferring the treated eggs to the acidic buffer solution, the step further includes: transferring the treated eggs to a second embryo culture solution for a first static treatment.

[0016] Preferably, before transferring the egg and the corresponding first polar body, the method further includes: transferring the egg with the zona pellucida removed and the corresponding first polar body to a third embryo culture medium for a second static treatment.

[0017] Preferably, the volume ratio of the acidic buffer solution to the treated eggs is 20-50 μL: 1-6 eggs.

[0018] Preferably, the immunofluorescence staining method comprises sequentially performing a first washing, blocking, adding a primary antibody that marks the chromosome centromere structure, a first incubation, a second washing, adding a secondary antibody that marks the chromosome centromere structure, a second incubation, a third washing, staining and sealing on the air-dried porous slide.

[0019] Preferably, the total amount of the primary antibody for labeling the chromosome centromere structure added to the air-dried porous glass slide is 100-200 μL; the first incubation is an overnight incubation at room temperature;

[0020] The total amount of the secondary antibody added to the air-dried porous glass slide for marking the chromosome centromere structure is 100-200 μL; the second incubation is incubated at room temperature in the dark for 1-2 hours.

[0021] The invention provides a method for preparing a slide for evaluating the chromosome ploidy and structure of mammalian eggs, comprising the following steps: placing an egg in a first embryo culture medium containing a kinesin Eg5 inhibitor for treatment to obtain a treated egg; transferring the treated egg to an acidic buffer solution to obtain an egg with a zona pellucida removed and a corresponding first polar body; the pH of the acidic buffer solution is 2.2-2.8; transferring the egg with a zona pellucida removed and the corresponding first polar body to a fixing solution in a porous slide for spreading, air-drying, and obtaining an air-dried porous slide; and performing immunofluorescence staining on the air-dried porous slide to obtain a slide for evaluating the chromosome ploidy and structure of mammalian eggs. The present invention can not only determine the chromosome ploidy of the egg, but also accurately detect the existence of sister chromosomes in the cell by spreading and immunofluorescence staining the chromosomes in the egg and its corresponding first polar body on a porous glass slide, thereby achieving simultaneous detection of the chromosome ploidy and chromosome structure of the egg, expanding the detection range, and effectively eliminating errors from the detection technology that occur in the detection process of the prior art; each cell is detected independently to reduce cross contamination and improve the detection accuracy; in addition, the present invention adopts a porous glass slide to achieve multiple groups of simultaneous detection, effectively improving the detection and analysis efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0023] Figure 1 It is a schematic diagram of a multi-well glass slide;

[0024] Figure 2 This is a schematic diagram of chromosome and centromere staining in an egg and its corresponding first polar body;

[0025] Figure 3 This is a schematic diagram of normal chromosome ploidy and early separation staining of sister chromosomes;

[0026] Figure 4 This is a schematic diagram of chromosomes not being stained;

[0027] Figure 5 This is a schematic diagram of the incomplete removal of staining of the zona pellucida of the egg;

[0028] Figure 6 This is a schematic diagram of over-dispersed staining during chromosome spread;

[0029] Figure 7 This is a schematic diagram of staining with bubbles during antibody incubation;

[0030] Figure 8 Schematic diagram of traditional dyeing method. DETAILED DESCRIPTION

[0031] The invention provides a method for preparing a slide for evaluating the chromosome ploidy and structure of mammalian eggs, comprising the following steps: placing an egg in a first embryo culture medium containing a kinesin Eg5 inhibitor for treatment to obtain a treated egg; transferring the treated egg to an acidic buffer solution to obtain an egg with a zona pellucida removed and a corresponding first polar body; the pH of the acidic buffer solution is 2.2-2.8; transferring the egg with a zona pellucida removed and the corresponding first polar body to a fixing solution in a porous slide for spreading, air-drying, and obtaining an air-dried porous slide; and performing immunofluorescence staining on the air-dried porous slide to obtain a slide for evaluating the chromosome ploidy and structure of mammalian eggs.

[0032] The present invention preferably places the cumulus-oocyte complex in a pre-treatment embryo culture medium and gently blows with a pipette to obtain an egg with somatic cells removed. In the present invention, the pre-treatment embryo culture medium preferably includes M2 culture medium; the pre-treatment embryo culture medium preferably also contains 300μg / mL hyaluronidase. In the present invention, the surface of the pre-treatment embryo culture medium is preferably covered with mineral oil, and it is preferably preheated at 37°C for 0.5-2h before use, more preferably preheated at 37°C for 0.5-1h, and more preferably preheated at 37°C for 0.5h. Covering the surface of the pre-treatment embryo culture medium of the present invention with mineral oil can avoid evaporation of the pre-treatment embryo culture medium, and preheating at 37°C before use can improve experimental stability. The present invention places the egg in the pre-treatment embryo culture medium and gently blows with a pipette to remove somatic cells such as granulosa cells around the egg, avoiding the influence of the chromosomes of somatic cells such as granulosa cells on subsequent exhibition and staining.

[0033] After obtaining the egg with somatic cells removed, the present invention places the egg with somatic cells removed in a first embryo culture medium containing a kinesin Eg5 inhibitor to obtain the treated egg. In the present invention, the first embryo culture medium preferably includes M2 culture medium; preferably, the surface of the first embryo culture medium is covered with mineral oil, and it is preheated at 37°C for 0.5 to 2 hours before use, more preferably 1 to 2 hours at 37°C, and more preferably 1 hour at 37°C. Covering the surface of the first embryo culture medium with mineral oil can avoid evaporation of the first embryo culture medium, and preheating at 37°C before use can improve experimental stability. The kinesin Eg5 inhibitor of the present invention preferably includes Monastrol; the concentration of the kinesin Eg5 inhibitor in the first embryo culture medium is preferably 100 to 200 μmol / L, more preferably 120 to 180 μmol / L, and more preferably 160 μmol / L. The time for placing the egg in the first embryo culture medium containing the kinesin Eg5 inhibitor for treatment is preferably 1 to 2 hours, more preferably 1 to 1.5 hours, and more preferably 1.5 hours. In the present invention, the inhibitor of kinesin Eg5 can induce the formation of a single stellate spindle in the egg, which is beneficial to the subsequent unfolding of chromosomes.

[0034] After obtaining the treated eggs, the present invention preferably transfers the treated eggs to a second embryo culture medium for a first static treatment to obtain a second treated egg. The second embryo culture medium of the present invention preferably includes M2 culture medium; preferably, the surface of the second embryo culture medium is covered with mineral oil, and is preheated at 37°C for 0.5 to 2 hours before use, more preferably preheated at 37°C for 0.5 to 1 hour, and more preferably preheated at 37°C for 0.5 hours. Covering the surface of the second embryo culture medium of the present invention with mineral oil can avoid evaporation of the second embryo culture medium, and preheating at 37°C before use can improve experimental stability. The present invention can remove the kinesin Eg5 inhibitor by transferring the treated eggs to the second embryo culture medium.

[0035] After obtaining the second treated egg, the present invention preferably transfers the second treated egg to an acidic buffer solution to obtain an egg with the zona pellucida removed and the corresponding first polar body. The pH of the acidic buffer solution of the present invention is preferably 2.2-2.8, and more preferably 2.3-2.6; the volume ratio of the acidic buffer solution to the treated eggs is preferably 20-50 μL: 1-6; more preferably 20-50 μL: 1-5; preferably 1-6 second treated eggs are transferred into the acidic buffer solution of the present invention. Preferably, the surface of the acidic buffer solution of the present invention is covered with mineral oil, and is preheated at 37°C for 0.5-2h before use, more preferably preheated at 37°C for 0.5-1h, and more preferably preheated at 37°C for 0.5h. Covering the surface of the acidic buffer solution of the present invention with mineral oil can prevent the evaporation of the acidic buffer solution, and preheating at 37°C before use can improve the experimental stability. In a specific embodiment of the present invention, the acidic buffer solution is preferably Tyrode's acidic buffer solution; the acidic buffer solution in the present invention can dissolve the zona pellucida of the egg, and the zona pellucida can be effectively removed by using the acidic buffer solution to avoid the influence of the zona pellucida on subsequent spreading and staining.

[0036] After obtaining the egg with the zona pellucida removed and the corresponding first polar body, the present invention preferably transfers the egg with the zona pellucida removed and the corresponding first polar body to the third embryo culture medium for a second static treatment. The third embryo culture medium of the present invention preferably includes M2 culture medium; preferably, the surface of the third embryo culture medium is covered with mineral oil, and is preheated at 37°C for 0.5 to 2h before use, more preferably at 37°C for 0.5 to 1h, and more preferably at 37°C for 0.5h. Covering the surface of the third embryo culture medium of the present invention with mineral oil can avoid evaporation of the third embryo culture medium, and preheating at 37°C before use can improve experimental stability. In the present invention, transferring the egg with the zona pellucida removed and the corresponding first polar body to the third embryo culture medium can remove the acidic buffer solution attached to the surface of the egg and the corresponding first polar body, and avoid the acidic buffer solution from continuously digesting the egg and the first polar body cells, resulting in the lysis and fragmentation of the egg and the first polar body cells.

[0037] In a specific embodiment of the present invention, the acidic buffer solution, the second embryo culture solution, and the third embryo culture solution are preferably in the same culture dish.

[0038] The present invention transfers the zona pellucida-removed ovum and the corresponding first polar body to a fixing solution in a porous glass slide for spreading, air-drying, and obtaining an air-dried porous glass slide. The porous glass slide in the present invention is preferably 6-well, 8-well, 10-well or 12-well. The present invention spreads on a porous glass slide, and can simultaneously detect multiple groups of ovum and their corresponding first polar bodies, effectively improving the efficiency of detection and analysis.

[0039] In the present invention, the diameter of the pores of the porous glass slide is preferably 4 to 6 mm, more preferably 5 mm; the volume of the fixative in the porous glass slide is preferably 5 to 10 μL / pore, more preferably 7 μL / pore. Adding an appropriate volume of fixative to the pores of the porous glass slide of the present invention can promote the unfolding and fixation of chromosomes.

[0040] In the present invention, the fixing solution preferably includes the following components in the following concentrations: PFA 10g / L, TritonX-100 0.15Vol% and DTT 3mM; the pH of the fixing solution is preferably 9.2. In the present invention, the PFA can fix the chromosome structure in the egg; the TritonX-100 can lyse the egg after the zona pellucida is removed to release the chromosomes therein; and the DTT makes the proteins on the chromosomes easier to be identified and combined in the subsequent immunofluorescence staining process.

[0041] The present invention preferably performs a first cleaning on the air-dried porous glass slide; the first cleaning is preferably performed with a TBST solution. The present invention preferably performs a sealing treatment on the porous glass slide after the first cleaning; the sealing is preferably performed with a PBS solution at room temperature for 1 to 2 hours; the PBS solution preferably contains 30 g / L of BSA.

[0042] The present invention preferably adds a primary antibody that marks the chromosome centromere structure to the sealed porous glass slide; the total amount of the primary antibody that marks the chromosome centromere structure on the sealed porous glass slide is preferably 100 to 200 μL, and more preferably 100 μL. After adding the primary antibody that marks the chromosome centromere structure to the porous glass slide, the present invention preferably covers the porous glass slide with a sealing film to obtain a porous glass slide with the primary antibody added and covered with a sealing film. The present invention uses a sealing film to cover the porous glass slide, which can ensure that each hole in the porous glass slide is fully in contact with the primary antibody to prevent the primary antibody from evaporating.

[0043] The present invention preferably performs a first incubation on the porous glass slide with the primary antibody added and the sealing film covered; the first incubation is preferably an overnight incubation at room temperature. The present invention preferably performs a second wash on the porous glass slide after the first incubation, and the second wash is preferably washed with a TBST solution.

[0044] The present invention preferably adds a secondary antibody that marks the chromosome centromere structure to the porous glass slide after the second cleaning; the total amount of the secondary antibody that marks the chromosome centromere structure on the porous glass slide after the second cleaning is preferably 100 to 200 μL, and more preferably 100 μL. After adding the secondary antibody that marks the chromosome centromere structure to the porous glass slide, the present invention preferably covers the porous glass slide with a sealing film to obtain a porous glass slide with the secondary antibody added and covered with a sealing film. The present invention uses a sealing film to cover the porous glass slide, which can ensure that each hole in the porous glass slide is fully in contact with the secondary antibody and prevent the evaporation of the secondary antibody.

[0045] The present invention preferably performs a second incubation on the porous glass slide with the secondary antibody added and the sealing film covered; the second incubation is preferably incubated at room temperature in the dark for 1 to 2 hours, more preferably 1 to 1.5 hours. The present invention preferably performs a third cleaning on the porous glass slide after the second incubation, and the third cleaning is preferably cleaned with a TBST solution, and attention should be paid to the light-proof operation during the cleaning.

[0046] In the present invention, the TBST solution preferably contains 0.05 Vol% Triton X-100. The primary antibody and secondary antibody of the present invention are preferably specific primary antibodies capable of labeling proteins on centromeres and corresponding fluorescent secondary antibodies.

[0047] In the present invention, the staining is preferably performed by staining the chromosomes on the porous glass slide using a DAPI solution. The present invention preferably performs a cover glass and sealing treatment on the stained porous glass slide to obtain a glass slide for evaluating the ploidy and structure of the chromosomes of mammalian eggs; the sealing is preferably performed by sealing with transparent nail polish.

[0048] To further illustrate the present invention, the method for preparing a glass slide for evaluating the chromosome ploidy and structure of mammalian eggs provided by the present invention is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0049] In a specific embodiment of the present invention, the acidic buffer solution is Tyrode's acid solution, and the embryo culture medium is M2 culture medium.

[0050] Example 1

[0051] A method for preparing a slide for evaluating the ploidy and structure of chromosomes of mammalian eggs, comprising the following steps:

[0052] 1. Preparation of acidic buffer solution and embryo culture medium droplets

[0053] Add hyaluronidase (Sigma, H4272) at a final concentration of 300 μg / mL to M2 culture medium to prepare pre-treated embryo culture medium. Drop 200 μL of pre-treated embryo culture medium into the first 35 mm culture dish, cover it completely with mineral oil (Sigma, M5310) to prevent evaporation, and preheat it at 37°C for 0.5 h before use.

[0054] Add Monastrol (Sigma, M8515) at a final concentration of 100 μmol / L to the M2 culture medium to prepare the first embryo culture medium. Drop 30 μL of the first embryo culture medium into the second 35 mm culture dish, cover it completely with mineral oil (Sigma, M5310) to prevent evaporation, and preheat it at 37°C for 1 h before use.

[0055] In the third 35mm culture dish, add 30μL M2 culture medium, 30μL Tyrode's acid solution and 30μL M2 culture medium in sequence at equal distances to prepare the second embryo culture medium, Tyrode's acid solution and the third embryo culture medium. Completely cover the second embryo culture medium, Tyrode's acid solution and the third embryo culture medium with mineral oil (Sigma, M5310) to prevent evaporation, and preheat at 37°C for 0.5h before use.

[0056] 2. Collect mouse cumulus oocyte complexes and place them in the pre-treated embryo culture medium for 1 to 2 minutes. Use a pipette to gently blow several times to remove the granulosa cells around the oocytes to obtain oocytes without granulosa cells and other somatic cells.

[0057] 3. Transfer the eggs with somatic cells such as granulosa cells removed from the pre-treatment embryo culture medium to the first embryo culture medium for 1 hour to ensure that there are no somatic cells such as granulosa cells.

[0058] 4. Transfer the eggs from the first embryo culture medium to the second embryo culture medium and wash to remove Monastrol.

[0059] 5. Transfer the egg from the second embryo culture medium to the Tyrode's acid solution for 15 to 60 seconds, and continue to observe the zona pellucida under a microscope. When the zona pellucida disappears from around the egg, transfer the egg and its corresponding first polar body to the third embryo culture medium.

[0060] 6. Prepare a 12-well glass slide with a pore diameter of 5 mm (Electron Microscopy Sciences, 63425-05), and add 7 μL of fixative solution to each well; the components of the fixative solution are PFA 10 g / L, Triton X-100 0.15 Vol% and DTT 3 mM; the pH value of the fixative solution is 9.2.

[0061] 7. Under a microscope, transfer the eggs and first polar bodies with the zona pellucida removed from the third embryo culture medium to the middle of the corresponding upper and lower holes on the multi-porous slide, with the eggs in the upper row and the first polar bodies in the lower row.

[0062] 8. Repeat the above steps to complete the process of spreading the egg and the first polar body. Figure 1 shown.

[0063] 9. Leave the porous glass slide containing the eggs and the corresponding first polar body at room temperature and air-dry overnight.

[0064] 10. The next day, wash the porous slides for the first time with TBST solution at room temperature. Wash three times, being as gentle as possible during the washing process.

[0065] 11. Treat with PBS blocking solution containing 30g / LBSA at room temperature for 1h.

[0066] 12. Add the primary antibody used to mark the chromosome centromere structure to the porous slide, cover it with a sealing film to ensure that each small hole on the porous slide is fully in contact with the primary antibody and prevent the antibody from volatilizing, and perform the first incubation at room temperature overnight. The primary antibody is Human Antibody Against Centromere, ImmunoVision, HCT-0100, diluted 1:1000 with blocking solution; the total amount of the primary antibody added is 100 μL.

[0067] 13. The next day, wash the multi-well slides for a second time with TBST solution, and wash three times.

[0068] 14. Add the corresponding secondary antibody for marking the chromosome centromere structure to the porous slide, and cover it with a sealing film to ensure that each hole on the porous slide is fully in contact with the secondary antibody and prevent the antibody from volatilizing. Perform the second incubation at room temperature and away from light for 1 hour. The secondary antibody is Goatanti-HumanIgG555, Invitrogen, A21433, diluted 1:1000 with blocking solution; the total amount of the secondary antibody added is 100 μL.

[0069] 15. Wash the porous slide for the third time with TBST solution, and wash it three times. Keep it away from light during the operation.

[0070] 16. Use DAPI solution to stain the chromosomes on the slide.

[0071] 17. Carefully cover with a coverslip, taking care not to create bubbles as much as possible.

[0072] 18. Use transparent nail polish to seal the slide and then observe and analyze under a fluorescence microscope. Figure 2 , Figure 3 .

[0073] The method of the present invention not only more clearly marks the chromosomes (grey) and their centromere structures (red), but also simultaneously analyzes the egg and its corresponding first polar body. Figure 2 It can be seen that Figure 2 The analysis results of the 19 pairs of sister chromosomes (marked with white numbers) in the egg and the 21 pairs of sister chromosomes (marked with white numbers; the analysis mainly focused on the number of centromere pairs) in the corresponding first polar body can accurately determine that the egg is aneuploid.

[0074] In addition, the method of the present invention can not only accurately analyze the chromosome ploidy of the egg, but also detect whether there is an abnormal phenomenon of premature separation of sister chromosomes. Figure 3 It can be seen that Figure 3A in the figure is an egg with normal chromosome ploidy, containing 20 pairs of sister chromosomes. Figure 3 Figure B shows an egg with premature separation of sister chromosomes, with the arrow marking a single chromosome that has separated prematurely.

[0075] Comparative Example 1

[0076] A preparation method similar to Example 1, except that step 3 is omitted and there is no Monastrol treatment process. The test results are shown in Figure 4 .

[0077] The chromosomes in mature eggs are concentrated on the equatorial plate in the cell. The small molecule Monastrol, as an inhibitor of the kinesin Eg5, can induce the egg to form a uniplanetary spindle after treatment, which is beneficial to the unfolding of chromosomes in the subsequent chromosome spreading process. Figure 4 It can be seen that the chromosomes that have not been treated with Monastrol are aggregated together and cannot be well unfolded.

[0078] Comparative Example 2

[0079] A preparation method similar to Example 1, except that in step 5, the zona pellucida of the egg is not completely removed after the treatment in the Tyrode's acid solution for 5 seconds. The test results are shown in Figure 5 .

[0080] according to Figure 5 It can be seen that the residual zona pellucida will seriously affect the chromosome spreading and staining effects in the subsequent process, resulting in the inability to accurately detect the chromosome ploidy and structure in the egg.

[0081] Comparative Example 3

[0082] A preparation method similar to Example 1, except that 20 μL PFA solution was added to the small holes of the porous glass slide in step 6. The test results are shown in Figure 6 .

[0083] according to Figure 6 It can be seen that adding excessive PFA solution can cause excessive dispersion of egg chromosomes. Excessive dispersion not only affects the efficiency of observation and analysis, but also makes it easier for chromosomes to be lost during the expansion process.

[0084] Comparative Example 4

[0085] A preparation method similar to Example 1, except that bubbles are generated in the sample area of ​​the spread sheet during the antibody incubation process in step 12. The test results are shown in Figure 7 .

[0086] according to Figure 7As shown, the circular area is where the bubbles are present. If bubbles are present in the sample area during the primary antibody or secondary antibody or DAPI incubation process during immunofluorescence staining, the area will not be stained normally, and eventually the chromosome information in the egg will be partially lost, resulting in detection failure.

[0087] Comparative Example 5

[0088] A preparation method similar to that of Example 1 is as follows:

[0089] Steps 1 to 5 are carried out according to steps 1 to 5 in Example 1;

[0090] 6. Add 50 μL of fixative solution to the middle area of ​​a regular slide (26×26 mm).

[0091] 7. Under a stereomicroscope, transfer 20 eggs with the zona pellucida removed to the fixative in the middle of a glass slide.

[0092] 8. Leave the slides to air dry overnight at room temperature.

[0093] 9. The next day, wash the slides three times with PBS solution at room temperature. Be as gentle as possible during the washing process.

[0094] 10. Use DAPI solution to stain the egg chromosomes on the slide.

[0095] 11. Cover with a cover glass, seal with transparent nail polish, and then observe and analyze under a fluorescence microscope. Figure 8 .

[0096] Traditional methods usually only stain the marker chromosomes (grey) and only analyze the egg itself, e.g. Figure 8 As shown. The normal chromosome ploidy of mouse eggs is 20 pairs. Figure 8 The egg showed 19 pairs of sister chromosomes, according to Figure 8 The analysis results cannot determine whether the egg itself is aneuploid or whether it is due to the loss of a pair of chromosomes during the expansion or staining process.

[0097] In summary, the present invention can not only determine the chromosome ploidy of the egg, but also accurately detect the existence of sister chromosomes in the cell by spreading and immunofluorescence staining the chromosomes in the egg and its corresponding first polar body respectively, thereby realizing the simultaneous detection of the chromosome ploidy and chromosome structure of the egg, expanding the detection range, and effectively eliminating the errors from the detection technology sources that occur in the detection process of the prior art; each cell is detected independently to reduce cross contamination and improve the detection accuracy; in addition, the present invention adopts a multi-porous glass slide to realize multiple groups of simultaneous detection, effectively improving the detection and analysis efficiency.

[0098] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing a slide for evaluating the chromosome ploidy and structure of mammalian eggs, characterized in that: The following steps are involved: Placing the egg in a first embryo culture medium containing a kinesin Eg5 inhibitor to obtain a treated egg; the egg is placed in the first embryo culture medium containing a kinesin Eg5 inhibitor for 1 to 2 hours; Transferring the treated eggs to an acidic buffer solution to obtain eggs with zona pellucida removed and the corresponding first polar body; the pH of the acidic buffer solution is 2.2-2.8; The oocytes with the zona pellucida removed and the corresponding first polar bodies are respectively transferred to the fixatives in different wells of a multi-porous glass slide for spreading and air-drying to obtain air-dried multi-porous glass slides; The air-dried multi-well glass slide is subjected to immunofluorescence staining to obtain a glass slide for evaluating the chromosome ploidy and structure of mammalian eggs; Before transferring the treated eggs to the acidic buffer solution, the method further includes: transferring the treated eggs to a second embryo culture solution for a first static treatment to clean and remove the kinesin Eg5 inhibitor; The kinesin Eg5 inhibitor includes Monastrol; and the concentration of the kinesin Eg5 inhibitor in the first embryo culture medium is 100-200 μmol / L.

2. The preparation method according to claim 1, characterized in that: The egg is placed in the first embryo culture medium containing the kinesin Eg5 inhibitor for a treatment period of 1 to 1.5 hours.

3. The preparation method according to claim 1, characterized in that: The concentration of the kinesin Eg5 inhibitor in the first embryo culture medium is 120-180 μmol / L.

4. The preparation method according to claim 1, characterized in that: The diameter of the pores of the porous glass slide is 4-6 mm; the volume of the fixing solution in the porous glass slide is 5-10 μL / pore.

5. The preparation method according to claim 1 or 4, characterized in that: The fixing solution includes components with the following concentrations: PFA 10 g / L, TritonX-100 0.15 Vol % and DTT 3 mM; the pH of the fixing solution is 9.

2.

6. The preparation method according to claim 1, characterized in that: Before transferring the egg and the corresponding first polar body, the method further includes: transferring the egg with the zona pellucida removed and the corresponding first polar body to a third embryo culture medium for a second static treatment.

7. The preparation method according to claim 1, characterized in that: The volume ratio of the acidic buffer solution to the treated eggs is 20-50 μL: 1-6 eggs.

8. The preparation method according to claim 1, characterized in that: The immunofluorescence staining method comprises sequentially performing first washing, sealing, adding a primary antibody for marking the chromosome centromere structure, first incubation, second washing, adding a secondary antibody for marking the chromosome centromere structure, second incubation, third washing, staining and sealing on the air-dried porous slide.

9. The preparation method according to claim 8, characterized in that: The total amount of the primary antibody for marking the chromosome centromere structure added to the air-dried porous slide is 100-200 μL; the first incubation is an overnight incubation at room temperature; The total amount of the secondary antibody added to the air-dried porous slide to mark the chromosome centromere structure is 100-200 μL; the second incubation is incubated at room temperature in the dark for 1-2 hours.

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