Extraction method of mouse epididymis body

Abstract

The invention discloses a mouse epididymal body extraction method which comprises the following steps: S1, digesting mouse epididymal tissues: putting separated mouse epididymis into currently prepared mouse sperm insemination liquid, cutting into pieces, discarding sperms, and adding cell dissociation liquid to form single-cell suspension; s2, purification of mouse epididymis exosomes: filtering the single-cell suspension, and purifying, eluting and extracting the exosomes through an exoEasy Maxi Kit kit; s3, concentration of the epididymis bodies: performing ultracentrifugation on the eluent of the exosomes to concentrate the exosomes, discarding the supernatant to obtain precipitates, namely the high-purity epididymis bodies, and performing identification after subsequent resuspension. The method has the advantage that the mouse epididymis body with high purity, high yield and complete structure can be obtained.

Description

Technical Field

[0001] This invention relates to the field of bioextraction technology, and in particular to a method for extracting mouse epididymal corpuscles. Background Technology

[0002] Exosomes are produced in the intranuclear compartments of most eukaryotic cells, and most cells can secrete exosomes. They have been found in extracellular fluids such as plasma, urine, saliva, and semen. Epididymal exosomes, also known as epididymal corpora, are exosomes within the lumen of the epididymis and are released by epididymal epithelial cells. Epididymal corpora play a particularly important role in sperm maturation, promoting sperm maturation and motility. Epididymal corpora contain hundreds of different functional proteins, and the protein composition varies depending on the epididymal segment from which they originate. For example, epididymal corpora from the head of the bull epididymis contain 555 proteins, while those from the tail contain 438 proteins. A total of 1640 proteins have been found in epididymal corpora from the head, body, and tail of the mouse epididymis. These proteins participate in multiple biological processes, including protein transport, redox processes, metabolic processes, spermatogenesis, sperm-zona pellucida binding, and fertilization. Currently, many proteins acquired during epididymal transport and required for sperm fertilization have been identified in epididymal bodies, including ZP family proteins and ADAM family proteins involved in sperm binding to the zona pellucida. In addition, epididymal bodies contain a large amount of non-coding RNA, particularly sRNAs. The epididymal body-mediated transfer of proteins and RNA from the epididymal epithelium to sperm cells is an important mechanism promoting sperm maturation.

[0003] Given the crucial role of epididymal corpora in sperm maturation and fertilization, and the high complexity of their contents, a thorough understanding of their specific mechanisms of action is essential for comprehending male reproductive biology. However, the prerequisite for effectively conducting these studies is the ability to obtain bioactive epididymal corpora intact and in high purity. Current epididymal corpora extraction techniques suffer from systemic deficiencies. First, epididymal tissue digestion typically employs traditional enzymatic methods (trypsin combined with collagenase), requiring a 37°C shaker. This step easily leads to cell membrane rupture, releasing RNase and subsequently degrading functional RNA within the epididymal corpora. Additionally, mechanical homogenization is also used for tissue disruption, but the resulting shear force directly damages the integrity of the vesicle membrane. In the separation and purification stage, traditional ultracentrifugation, due to repeated centrifugation to remove tissue fragments, is time-consuming, causes vesicle compression and deformation, and has low purification efficiency. Furthermore, the lack of strict temperature control and the absence of protective agents during the process leads to the inactivation of the epididymal corpuscle marker protein CD9, vesicle aggregation, and RNA degradation, severely limiting its application value in sperm maturation mechanism research. Summary of the Invention

[0004] The purpose of this invention is to provide a high-purity, high-yield, and structurally intact method for extracting mouse epididymal corpora, in order to solve the problems of vesicle damage, impurity residue, and cumbersome operation in the prior art.

[0005] The above-mentioned technical objective of the present invention is achieved through the following technical solution:

[0006] A method for extracting mouse epididymal corpuscles, characterized by comprising the following steps:

[0007] S1, Digestion of mouse epididymal tissue: The separated mouse epididymis was placed in the fertilized fluid of freshly bred mouse sperm, minced, the sperm was discarded, and cell dissociation solution was added to form a single-cell suspension;

[0008] S2, Purification of mouse epididymal exosomes: Exosomes were extracted by filtration of single-cell suspension and purification elution using the exoEasy Maxi Kit.

[0009] S3, Epididymal corpuscle concentration: The exosome eluent is concentrated by ultracentrifugation, the supernatant is discarded, and the precipitate is the high-purity epididymal corpuscle, which is then resuspended and identified.

[0010] Preferably, the mouse sperm fertilization solution comprises the following components: 93.8 mM NaCl, 4.69 mM KCl, 0.2 mM MgSO4, 0.37 mM KH2PO4, 2.04 mM CaCl2, 21.4 mM lactic acid, 2.78 mM glucose, 21 mM HEPES, 4 mM NaHCO3, pH adjusted to 7.4, and before use, 330 mM Na-Pyruvate and HSA are added at a volume ratio of 1:1000 to the initial solution, and the final concentration of NaHCO3 is adjusted to 21 mM NaHCO3.

[0011] Preferably, the cell dissociation solution is Accumax cell dissociation solution, which is purchased from Invitrogen, catalog number 00-4666-56, and the addition ratio of Accumax cell dissociation solution is 1 ml of dissociation solution per 20 mg of epididymal tissue.

[0012] Preferably, step S1 specifically involves: after isolating the mouse epididymis, cutting it into small pieces of 1-2 mm in freshly fertilized mouse sperm, incubating at 37°C for 10 min, discarding the sperm, weighing the epididymis, and transferring it to a centrifuge tube, adding cell dissociation solution to the centrifuge tube, gently shaking on a shaker at room temperature for 2.5 h, and aspirating the tissue up and down 20 times every 30 min. After digestion, filtering the sample through a 40 μm cell filter, adding the same volume of 1×PBS to stop dissociation, centrifuging for 10 min at 10000 rpm, and collecting the supernatant to obtain exosomes.

[0013] Preferably, the exoEasy Maxi Kit is purchased from QIAGEN, catalog number 76064, and the kit contains the following reagents and consumables: XE elution buffer, XWP wash buffer, XBP binding buffer, and exo Easy centrifuge column.

[0014] Preferably, step S2 specifically involves: filtering the sample obtained in step S1 using a 0.8 μm cell filter, adding the same volume of XBP binding buffer to the sample, inverting the test tube 5 times to mix thoroughly, transferring the sample to an exo Easy centrifuge column, adding 16 ml each time, centrifuging at 500g for 1 min, repeating until all samples pass through the exo Easy centrifuge column, adding 10 ml of XWP washing buffer to the collection tube, centrifuging at 3000g for 5 min, discarding the buffer, transferring the centrifuge column to a new centrifuge tube, then adding 1 ml of XE elution buffer to the centrifuge column membrane, incubating for 1 min, centrifuging at 500g for 5 min to elute the sample, adding 1 ml of XE elution buffer to the column membrane again, incubating for 1 min, centrifuging at 500g for 5 min to elute the sample.

[0015] Preferably, step S3 specifically involves: centrifuging the exosomes at 100,000g for 90 minutes at 4°C, and the precipitate is high-purity epididymal corpuscles.

[0016] Preferably, the centrifuge tubes must be precisely leveled before the ultracentrifugation, with a weight difference of ≤0.01g between the two, and a fixed-angle rotor is used. After centrifugation, the rotor is slowly braked to a stop.

[0017] Preferably, the identification method in step S3 includes detecting the marker protein CD9 by Western blot, observing the morphology by scanning electron microscopy, and analyzing the particle size by dynamic light scattering.

[0018] Preferably, the dynamic light scattering method analyzes the diameter distribution of epididymal corpuscles to be 30–150 nm, with the main peak located around 70 nm.

[0019] In summary, this invention offers the following advantages: It develops a three-step synergistic operating system of "ACCUMAX gentle enzymatic hydrolysis + exoEasy MaxiKit pre-purification + ultracentrifugation concentration," which completely solves the systemic defects in tissue exosome extraction through a novel technological combination. First, the ACCUMAX complex enzyme gently dissociates the tissue at room temperature, selectively degrading the extracellular matrix without damaging the cell membrane, preventing the release of intracellular RNase, and maintaining the integrity of exosomal RNA (especially sRNAs) and membrane proteins (such as CD9). Then, the exoEasy Maxi Kit is used for pre-purification, efficiently removing impurities and avoiding the mechanical damage of traditional ultracentrifugation, shortening the purification time to less than 1 hour and significantly improving the epididymal body yield. Finally, a single short ultracentrifugation and concentration of exosomes in a low-temperature environment avoids vesicle rupture caused by repeated centrifugation, maximizing the preservation of epididymal body activity. This invention provides a breakthrough technological platform for epididymal body extraction and exosome therapy. Attached Figure Description

[0020] Figure 1 The expression of CD9, a marker protein of mouse epididymal corpuscles extracted in this invention;

[0021] Figure 2 The surface morphology of mouse epididymal corpuscles extracted in this invention;

[0022] Figure 3 This is the particle size distribution of mouse epididymal corpora extracted in this invention. Detailed Implementation

[0023] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings. This description does not limit the scope of the invention in any way. Any modifications or substitutions made to the methods, steps, or conditions without departing from the core spirit and essence of the invention are within the scope of protection of the present invention. In the embodiments, if specific conditions are not specified for the experimental methods or the formulations of reagents are not detailed, conventional methods are assumed.

[0024] The mouse sperm fertilization solution (HTF) used in this invention has the following formulation: 93.8 mM NaCl, 4.69 mM KCl, 0.2 mM MgSO4, 0.37 mM KH2PO4, 2.04 mM CaCl2, 21.4 mM lactic acid, 2.78 mM glucose, 21 mM MEPES, 4 mM NaHCO3, pH adjusted to 7.4. Before use, 330 mM Na-Pyruvate and HSA are added at a volume ratio of 1:1000 to the initial solution, and the final NaHCO3 concentration is adjusted to 21 mM NaHCO3.

[0025] Accumax cell dissociation solution was purchased from Invitrogen, catalog number 00-4666-56.

[0026] The exoEasy Maxi Kit was purchased from QIAGEN, catalog number 76064. The kit includes the following reagents and consumables: XE elution buffer, XWP wash buffer, XBP binding buffer, and exo Easy centrifuge column.

[0027] Step 1, Digestion of mouse epididymal tissue:

[0028] a. Isolate the epididymis of 10 56-day-old mice.

[0029] b. Place the separated epididymis in a 100mm culture dish containing HTF and cut it into small pieces of 1-2mm. Then incubate at 37℃ for 10 minutes to allow the sperm to swim out.

[0030] c. Discard the sperm, weigh the epididymis, and transfer it to a 50ml centrifuge tube.

[0031] d. Add 1 ml of dissociation solution to each 20 mg of epididymal tissue in a centrifuge tube and gently shake on a shaker at room temperature for 2.5 h.

[0032] e. Move the tissue up and down 20 times every 30 minutes to maximize single-cell yield.

[0033] f. After digestion, filter the sample through a 40μm cell filter.

[0034] g. Add the same volume of 1×PBS to stop dissociation.

[0035] h. Centrifuge for 10 min at 10000 rpm and collect the supernatant.

[0036] Step 2, purification of mouse epididymal exosomes

[0037] a. Filter the sample obtained in the previous step using a 0.8 μm cell filter.

[0038] b. Add the same volume of XBP binding buffer to the sample, and invert the test tube 5 times to mix thoroughly.

[0039] c. Transfer the sample to a centrifuge column, adding 16 ml each time, and centrifuge at 500g for 1 min.

[0040] d. Repeat until all samples have passed through the exo Easy centrifuge column.

[0041] e. Add 10 ml of XWP washing buffer to the collection tube, centrifuge at 3000 g for 5 min, and discard the buffer.

[0042] f. Transfer the centrifuge column to a new centrifuge tube, then add 1 ml of XE elution buffer to the centrifuge column membrane and incubate for 1 min.

[0043] Centrifuge at 500g for 5 minutes to elute the sample.

[0044] h. Add the eluent back onto the column membrane and incubate for 1 min. Centrifuge at 500 g for 5 min to elute the sample.

[0045] Step 3, Epididymal corpuscle concentration:

[0046] a. Transfer the eluent containing epididymal bodies into an ultracentrifuge tube.

[0047] b. Weigh the items and balance them with PBS. The weight difference between the two should be ≤0.01g.

[0048] c. Centrifuge at 100,000g for 90 minutes at 4℃. Note that the ultracentrifuge should be started at the fastest speed and stopped at the slowest speed at the end of the centrifugation.

[0049] d. After centrifugation, discard the supernatant. At this point, epididymal corpuscles can be seen at the bottom of the tube, which indicates high-purity epididymal corpuscles.

[0050] Next, add 20 μl of protein lysis buffer (RIPA), thoroughly pipette and place on ice, let stand for 30 min to fully lyse, centrifuge at 12000g for 30 min, and determine the protein concentration by BCA method.

[0051] Identification methods:

[0052] Western blot detection of epididymal corpuscle marker protein (CD9)

[0053] a. Conventional glue preparation: 12% separating glue, 5% stacking glue.

[0054] b. Electrophoresis, set constant current 25mA, voltage 250V, electrophoresis time 72min.

[0055] c. Standard film transfer, 22V constant voltage, 90min.

[0056] The results are as follows Figure 1 The CD9 marker protein is highly expressed in the epididymal corpuscles.

[0057] Scanning electron microscopy of epididymal corpuscle morphology

[0058] a. Epididymal small weight suspension

[0059] Take the epididymal corpuscle precipitate and suspend it in 20 μl PBS (recommended concentration range 1-5 mg / ml). Excessive amount can easily lead to vesicle stacking and affect subsequent observation.

[0060] b. Carrier fixation

[0061] Transfer 10 μl of suspension to the surface of the sealing membrane, and invert the copper mesh covering the Formvar film to contact the droplet for 10 seconds to capture the exosomes. Tilt the filter paper at 45° to remove the residual liquid, avoiding contact with the membrane surface throughout the process.

[0062] c. Enhanced negative staining contrast

[0063] Filter the 2% uranium acetate staining solution through a 0.22 μm filter membrane, add 10 μl dropwise to cover the copper mesh, and control the reaction time to within 30 seconds. Tilt the sample to allow the staining solution to flow down naturally, then transfer it to a desiccator for 20 minutes in the dark.

[0064] d. Conductivity optimization

[0065] The stained copper mesh was placed on the sample stage and a 5-10 nm gold layer was sputtered to eliminate the charge accumulation effect.

[0066] e. Electron microscopy observation

[0067] Optimize scanning electron microscope parameters and perform microscopic imaging analysis of the samples. See the morphology of the epididymal corpuscles. Figure 2 It consists of disc-shaped vesicles with a diameter of 30–150 nm.

[0068] Epididymal corpuscle size determination (dynamic light scattering, DLS)

[0069] Instrument calibration:

[0070] a. Turn on the DLS instrument and its associated computer, and allow it to warm up for at least 30 minutes.

[0071] b. Calibrate using a standard latex particle suspension provided by the instrument manufacturer. Ensure the measured particle size is within the nominal tolerance range.

[0072] c. Clean the calibration sample cell.

[0073] d. Sample preparation:

[0074] Epididymal body samples: fully resuspended in filtered sterile PBS buffer (avoid using buffers containing particles or fluorescent interference).

[0075] e. Instrument initialization:

[0076] Open the instrument software, select the Dynamic Light Scattering (DLS) mode, and set the measurement parameters.

[0077] f. Background measurement:

[0078] Add filtered pure PBS buffer (volume same as subsequent samples) to a clean sample cell. Carefully place the sample cell into the sample chamber, ensuring no air bubbles adhere to the light path.

[0079] g. Sample measurement:

[0080] Carefully inject the optimized concentration of epididymal corpuscle-PBS sample into the sample cell (avoiding the generation of air bubbles).

[0081] Gently close the cell lid and place the sample cell into the sample chamber (note the orientation).

[0082] h. Start "Sample Measurement" in the software.

[0083] i. Clean the sample cell: Rinse thoroughly with pure water several times, then rinse with ethanol, and finally blow dry.

[0084] j. Data review: Check whether the autocorrelation function curve of each measurement is smooth and well-fitted.

[0085] k. Check the Z-Average particle size and PDI value.

[0086] Check the particle size distribution chart and observe the position of the main peak, whether the peak shape is symmetrical, and whether there are shoulder peaks or small peaks (which may indicate the presence of aggregation or impurities).

[0087] The results are as follows Figure 3 As shown, the particle size of epididymal corpuscles is concentrated in the range of 30–150 nm. In this experiment, the main peak of vesicle particle size is around 70 nm, and its diameter is consistent with the characteristics of exosomes.

[0088] This invention develops a three-step synergistic operation system of "ACCUMAX gentle enzymatic hydrolysis + exoEasy Maxi Kit pre-purification + ultracentrifugation concentration". This novel technological combination completely solves the systemic defects in tissue exosome extraction. First, the ACCUMAX complex enzyme gently dissociates the tissue at room temperature, selectively degrading the extracellular matrix without damaging the cell membrane, preventing the release of intracellular RNase, and maintaining the integrity of exosomal RNA (especially sRNAs) and membrane proteins (such as CD9). Then, the exoEasyMaxi Kit is used for pre-purification, efficiently removing impurities and avoiding the mechanical damage of traditional ultracentrifugation, shortening the purification time to less than 1 hour and significantly improving the epididymal body yield. Finally, a single short ultracentrifugation and concentration of exosomes at low temperature avoids vesicle rupture caused by repeated centrifugation, maximizing the preservation of epididymal body activity. This invention provides a breakthrough technological platform for epididymal body extraction and exosome therapy.

[0089] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art can make various modifications or equivalent substitutions to the present invention within the scope of its essence and protection. Such modifications or equivalent substitutions should also be considered to fall within the protection scope of the present invention.

Claims

1. A method for extracting mouse epididymal corpuscles, characterized in that, Includes the following steps: S1, Digestion of mouse epididymal tissue: The separated mouse epididymis was placed in the fertilized fluid of freshly bred mouse sperm, minced, the sperm was discarded, and cell dissociation solution was added to form a single-cell suspension; S2, Purification of mouse epididymal exosomes: Exosomes were extracted by filtration of single-cell suspension and purification elution using the exoEasy Maxi Kit. S3, Epididymal corpuscle concentration: The exosome eluent is concentrated by ultracentrifugation, the supernatant is discarded, and the precipitate is the high-purity epididymal corpuscle, which is then resuspended and identified.

2. The method for extracting mouse epididymal corpuscles according to claim 1, characterized in that: The mouse sperm fertilization solution comprises the following components: 93.8 mM NaCl, 4.69 mM KCl, 0.2 mM MgSO4, 0.37 mM KH2PO4, 2.04 mM CaCl2, 21.4 mM lactic acid, 2.78 mM glucose, 21 mM HEPES, 4 mM NaHCO3, pH adjusted to 7.

4. Before use, 330 mM Na-Pyruvate and HSA are added at a volume ratio of 1:1000 to the initial solution, and the final NaHCO3 concentration is adjusted to 21 mM NaHCO3.

3. The method for extracting mouse epididymal corpuscles according to claim 2, characterized in that: The cell dissociation solution was Accumax cell dissociation solution, which was purchased from Invitrogen, catalog number 00-4666-56. The addition ratio of Accumax cell dissociation solution was 1 ml of dissociation solution per 20 mg of epididymal tissue.

4. The method for extracting mouse epididymal corpuscles according to claim 1, characterized in that: Step S1 specifically involves: after isolating the mouse epididymis, cutting it into small pieces of 1-2 mm in freshly fertilized mouse sperm, incubating at 37°C for 10 min, discarding the sperm, weighing the epididymis, and transferring it to a centrifuge tube, adding cell dissociation solution to the centrifuge tube, gently shaking on a shaker at room temperature for 2.5 h, and aspirating the tissue up and down 20 times every 30 min. After digestion, filtering the sample through a 40 μm cell filter, adding the same volume of 1×PBS to stop dissociation, centrifuging for 10 min at 10000 rpm, and collecting the supernatant to obtain exosomes.

5. The method for extracting mouse epididymal corpuscles according to claim 1, characterized in that: The exoEasyMaxi Kit was purchased from QIAGEN, catalog number 76064. The kit contains the following reagents and consumables: XE elution buffer, XWP wash buffer, XBP binding buffer, and exo Easy centrifuge column.

6. The method for extracting mouse epididymal corpuscles according to claim 5, characterized in that: Step S2 specifically involves: filtering the sample obtained in step S1 using a 0.8 μm cell filter, adding the same volume of XBP binding buffer to the sample, inverting the test tube 5 times to mix thoroughly, transferring the sample to an exo Easy centrifuge column, adding 16 ml each time, centrifuging at 500g for 1 min, repeating until all samples pass through the exo Easy centrifuge column, adding 10 ml of XWP washing buffer to the collection tube, centrifuging at 3000g for 5 min, discarding the buffer, transferring the centrifuge column to a new centrifuge tube, then adding 1 ml of XE elution buffer to the centrifuge column membrane, incubating for 1 min, centrifuging at 500g for 5 min to elute the sample, adding 1 ml of XE elution buffer to the column membrane again, incubating for 1 min, centrifuging at 500g for 5 min to elute the sample.

7. The method for extracting mouse epididymal corpuscles according to claim 1, characterized in that: Step S3 specifically involves centrifuging the exosomes at 100,000g for 90 minutes at 4°C, and the precipitate is high-purity epididymal corpuscles.

8. The method for extracting mouse epididymal corpuscles according to claim 7, characterized in that: Before the ultracentrifugation, the centrifuge tubes must be precisely balanced, with a weight difference of ≤0.01g. A fixed-angle rotor is used, and the rotor is slowly braked to a stop after centrifugation.

9. The method for extracting mouse epididymal corpuscles according to claim 1, characterized in that: The identification methods in step S3 include detecting the marker protein CD9 by Western blot, observing the morphology by scanning electron microscopy, and analyzing the particle size by dynamic light scattering.

10. The method for extracting mouse epididymal corpuscles according to claim 1, characterized in that: The dynamic light scattering method analysis showed that the diameter distribution of epididymal corpuscles ranged from 30 to 150 nm, with the main peak located near 70 nm.

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