Porcine epididymal cell-derived extracellular vesicle as well as preparation method and application thereof

Abstract

The invention discloses a porcine epididymal cell-derived extracellular vesicle as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. By optimizing the preparation method of the extracellular vesicles, the extracellular vesicles with high purity and standard shape are successfully obtained. Furthermore, by virtue of a proteomics technology, the composition characteristics of rich key functional proteins related to oxidation resistance, movement regulation and fertilization are systematically clarified for the first time. Functional experiments prove that the vesicle can be effectively attached to sperms, the sperm motility is remarkably improved under the low-temperature preservation condition, the oxidation resistance is enhanced, membrane damage, mitochondrial dysfunction and DNA damage are relieved, and the specific protection effect on the sperms is shown. The invention provides a reliable material basis and theoretical basis for developing a novel sperm preservative and an assisted reproduction technology based on extracellular vesicles.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a porcine epididymal cell-derived extracellular vesicle and a preparation method and application thereof. BACKGROUND

[0002] In the prior art, the preparation and separation method of extracellular vesicles is relatively mature, however, there is still a significant gap in the systematic collection, analysis and functional verification system of extracellular vesicles derived from porcine epididymal epithelial cell lines. Specifically, at present, there is no stable method for large-scale preparation and systematic characterization of extracellular vesicles from this specific cell line, especially lacking systematic analysis and deep functional annotation of its protein composition. In addition, existing research has not fully verified the specific role and mechanism of such vesicles in sperm cryopreservation, motility, antioxidant stress and DNA integrity maintenance. Therefore, there is an urgent need in the art for a technical solution that can stably obtain porcine epididymal epithelial cell-derived extracellular vesicles, determine its protein composition, and scientifically verify its role in sperm protection and function enhancement. SUMMARY

[0003] The purpose of the present application is to provide a porcine epididymal cell-derived extracellular vesicle and a preparation method and application thereof to solve the problems existing in the prior art. The extracellular vesicle is obtained by low-speed centrifugation, which is rich in antioxidant proteins, movement regulating factors and sperm-egg recognition molecules and other key functional proteins. Functional experiments have confirmed that the extracellular vesicle can effectively adhere to sperm under 17℃ cryopreservation conditions, significantly improve sperm motility and antioxidant levels, and reduce membrane damage, mitochondrial dysfunction and DNA damage, and the protective effect is time-dependent. The present application proves that the extracellular vesicle preparation system has reliable component superiority and clear functional effectiveness, providing an important research foundation and application prospect for the development of new sperm preservatives and assisted reproductive technology.

[0004] To achieve the above-mentioned purpose, the present application provides the following solutions:

[0005] In a first aspect, the present application provides a preparation method of a porcine epididymal cell-derived extracellular vesicle, comprising the following steps:

[0006] The porcine epididymal cells are pretreated, the pretreated sample is collected and filtered to obtain a filtrate, the filtrate is centrifuged to collect a supernatant, the supernatant is centrifuged again and the centrifugal supernatant is collected, the centrifugal supernatant is centrifuged to collect a precipitate, the precipitate is resuspended and centrifuged, and the porcine epididymal cell-derived extracellular vesicle is obtained.

[0007] Preferably, the pretreatment method is as follows: the porcine epididymal epithelial cells are cultured in an exosome-free serum medium containing testosterone and 5α-dihydrotestosterone for 24-48h, and the culture solution is collected to obtain the pretreated sample.

[0008] Preferably, the filtrate is centrifuged at 2000g for 20min to collect the supernatant.

[0009] Preferably, the supernatant is centrifuged at 10000g for 60min twice, and the centrifugal supernatant is collected.

[0010] Preferably, the centrifugal supernatant is centrifuged at 100000g for 2h to collect the precipitate.

[0011] Preferably, the precipitate is resuspended and centrifuged at 120000g for 70min to obtain the porcine epididymal cell-derived extracellular vesicles.

[0012] In a second aspect, the present application also provides the porcine epididymal cell-derived extracellular vesicles prepared by the preparation method.

[0013] In a third aspect, the present application also provides the use of the porcine epididymal cell-derived extracellular vesicles in the preparation of sperm preservation solution.

[0014] In a fourth aspect, the present application also provides a sperm preservation solution, wherein the sperm preservation solution comprises the porcine epididymal cell-derived extracellular vesicles.

[0015] The present application discloses the following technical effects:

[0016] The present application fills the technical gap of the porcine epididymal epithelial cell-derived extracellular vesicles in the aspects of systematic preparation, composition analysis and function verification. By optimizing the preparation method of extracellular vesicles, the present application successfully obtains extracellular vesicles with high purity and standard morphology. Further, by means of proteomics technology, the present application first systematically clarifies the composition characteristics of the vesicles rich in antioxidant, exercise regulation and key functional proteins related to fertilization. The function experiment proves that the vesicles can effectively adhere to sperm, significantly improve sperm motility, enhance antioxidant capacity, and reduce membrane damage, mitochondrial dysfunction and DNA damage under low-temperature preservation conditions, and show a clear protective effect on sperm. The present application provides a reliable material basis and theoretical basis for the development of new sperm preservation agents and assisted reproductive technology based on extracellular vesicles. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0018] Figure 1 The technical process flow chart of the present application;

[0019] Figure 2 Results of the analysis of the nanosize distribution and the motion characteristics of the extracellular vesicles evaluated by the Nanosight 300;

[0020] Figure 3 Results of the analysis of the ultrastructure morphology of the extracellular vesicles evaluated by the scanning electron microscope;

[0021] Figure 4 Results of the analysis of the tracing by the fluorescent dye Dil;

[0022] Figure 5 Results of the analysis of the involvement of the extracellular vesicles in EVs-transport, assembly, budding and phagocytosis regulation;

[0023] Figure 6 Results of the annotation analysis of the involvement of the proteins carried by the extracellular vesicles in the antioxidant function;

[0024] Figure 7 Results of the analysis of the involvement of the proteins carried by the extracellular vesicles in the mitochondrial function;

[0025] Figure 8 Results of the analysis of the involvement of the proteins carried by the extracellular vesicles in the fertilization and the maturation of the spermatozoa epididymal epithelium function;

[0026] Figure 9 Results of the influence of the extracellular vesicles on the antioxidant capacity of the spermatozoa; *P<0.05, **P<0.01, ***P<0.001, ns: no significant difference;

[0027] Figure 10 Results of the influence of the extracellular vesicles on the redox balance and the motility of the spermatozoa; scale bar: 100 pm; *P<0.05, **P<0.01, ***P<0.001, ns: no significant difference;

[0028] Figure 11 Results of the influence of the extracellular vesicles on the survival rate of the spermatozoa; scale bar: 100 pm; *P<0.05, ns: no significant difference;

[0029] Figure 12 Results of the influence of the extracellular vesicles on the mitochondrial function of the spermatozoa; scale bar: 100 pm; *P<0.05, **P<0.01, ns: no significant difference;

[0030] Figure 13 Results of the influence of the extracellular vesicles on the DNA damage of the spermatozoa; scale bar: 100 pm; *P<0.05, **P<0.01, ns: no significant difference. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be illustrative of certain aspects, features and embodiments of the present application, but not a limitation thereof.

[0032] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, for example, the upper limit and the lower limit of the range are not included in the range unless the context clearly indicates otherwise. Any intermediate value and any other stated or intervening value of any element, process or inhibitor described herein are expressly contemplated as being within the scope of the present application. The upper and lower limits of these smaller ranges can independently be included or excluded in the range.

[0033] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict, the content of the present specification will control.

[0034] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0035] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", and the like are open-ended terms that are intended to denote the inclusion of elements or steps, but not to exclude any other elements or steps.

[0036] The present application establishes a complete technical solution from preparation, characterization to function verification, and provides a reliable method basis for sperm preservation and assisted reproductive application based on porcine epididymal epithelial cell-derived extracellular vesicles (see flow chart Figure 1 ).

[0037] Example 1 Preparation method of extracellular vesicles

[0038] The present application provides a method for efficiently and stably collecting extracellular vesicles from a porcine epididymal epithelial cell line. The extraction of extracellular vesicles adopts an optimized differential centrifugation combined with washing and purification strategy.

[0039] The specific procedure is as follows: first, pig epididymal epithelial cells (pEECs) are added to an exosome-free serum-free medium containing 2 μM testosterone and 0.1 μM 5α-dihydrotestosterone for culture for 24 h and 48 h, the pEECs culture solution obtained in the two time periods is collected, the collected sample is filtered at 40 μm to remove cell clumps, the filtrate is retained, then the filtrate is centrifuged at 2000 g for 20 min to remove dead cells, the supernatant is centrifuged at 10000 g for 60 min, the operation is repeated twice to remove cell debris, and the supernatant is centrifuged at 100000 g for 2 h, and the precipitate is retained, at this time, the precipitate contains free proteins and extracellular vesicles, the precipitate is resuspended with ddH2O, and is centrifuged at 120000 g for 70 min to finally obtain extracellular vesicles (EVs).

[0040] Example 2 Structural characterization of extracellular vesicles

[0041] 1. Nanosight 300 evaluation of extracellular vesicle nanoparticle size distribution and motion characteristics analysis

[0042] The NanoSight NS300 based on Brownian motion and light scattering principle is used to physically characterize the extracellular vesicles separated from the pig epididymal epithelial cell conditioned medium. This technology tracks the Brownian motion trajectory of individual vesicles in solution in real time, and determines the particle size distribution, particle concentration (expressed in particle number / mL) and motion characteristics of the vesicle population. The particle size distribution of the separated extracellular vesicles is characterized by Nanosight 300.

[0043] The analysis results show that (Fig. 1) Figure 2 ), the obtained vesicles are mainly distributed in the typical exosome size range of 30-200 nm, and the particle size distribution curve is unimodal, indicating that the sample has good uniformity. The data confirms that the extracellular vesicles prepared by the method of the present application have the typical extracellular vesicle (especially exosome) ultrastructure characteristics defined by the International Society for Extracellular Vesicles (ISEV) guidelines, providing a reliable quality control basis for subsequent functional research.

[0044] 2. Scanning electron microscope evaluation of extracellular vesicle ultrastructure morphology analysis

[0045] The scanning electron microscope is used to analyze the ultrastructure morphology of the separated extracellular vesicle sample. After standard sample preparation process (including fixation, dehydration and gold spraying treatment), observation is carried out under high vacuum condition.

[0046] The results show that (Fig. 2) Figure 3),the prepared sample can be seen a large number of clear boundary, morphological integrity of spherical or spherical vesicle structure, the diameter is mainly distributed in the nanometer scale range, the membrane structure is continuous and no significant rupture, in line with the typical ultrastructure of extracellular vesicles. The results from the intuitive morphological level further confirmed the structural integrity of the product obtained by the method of the application.

[0047] 3. Fluorescent dye Dil labeling tracing analysis

[0048] The lipophilic membrane fluorescent probe Dil (DiIC 18 (3) was used to label the extracellular vesicles. The dye has weak fluorescence in the unbound state, and when embedded in the lipid bilayer of the vesicle membrane, it can be excited by 549 nm excitation light to emit strong orange-red fluorescence (peak at about 565 nm), thereby realizing the visualization tracing of the vesicles.

[0049] After the labeled vesicles were co-incubated with sperm, clear fluorescence signal distribution on the surface of sperm was observed by fluorescence microscopic imaging system, indicating that the extracellular vesicles can effectively transfer and stably bind with the sperm membrane structure (Fig. 3). Figure 4 The results directly prove that the extracellular vesicles have biological activity of interacting with sperm.

[0050] 4. Protein spectrum and deep learning analysis of extracellular vesicles

[0051] Protein spectrum determination was performed on the extracted extracellular vesicles, and bioinformatics analysis was combined to deeply analyze the protein composition, and to reveal the role of these proteins in vesicle biogenesis, potential functional contribution to germ cells and other biological processes.

[0052] Through non-targeted proteomics and systematic bioinformatics analysis, the protein composition of the extracellular vesicles derived from pig epididymal epithelial cells was sequenced and functionally annotated. Based on the analysis of the Gene Ontology (https: / / www.geneontology.org / ) database, it was shown that the proteins carried by the extracellular vesicles (EVs) played a crucial role in their biogenesis and function. The vesicle proteome was significantly enriched in the vesicle transport and membrane dynamics related pathways ( Figure 5 ), and was widely involved in key steps such as cargo sorting, membrane shaping, budding release and targeted delivery of vesicles, regulating the whole process of EVs generation, secretion and uptake. Some proteins are involved in the regulation of endosome sorting complex (ESCRT) dependent or independent pathways, directly affecting the budding and release efficiency of vesicles; other proteins mediate the recognition and endocytosis between EVs and recipient cells, and regulate their phagocytosis or internalization process.

[0053] Meanwhile, KEGG (Kyoto Encyclopedia of Genes and Genomes, https: / / www.kegg.jp / ) pathway enrichment analysis confirmed that it has the typical molecular characteristics of extracellular vesicles. Further annotation results show that the proteome plays an important role in the antioxidant defense system, including superoxide scavenging, active oxygen metabolism and cell oxidative stress response processes (https: / / www.kegg.jp / kegg / kegg.html) Figure 6 In addition, its function is also related to the key link of mitochondrial energy metabolism, such as the regulation of ATP synthesis and membrane potential homeostasis ( Figure 7 ), which is specifically manifested in promoting the process of proton-driven ATP synthesis and negatively regulating the depolarization of mitochondrial membrane potential.

[0054] Protein-protein interaction network analysis by STRING database (https: / / string-db.org / ) shows that these vesicle proteins have extensive interactions and are involved in cholesterol transport and maintenance of epithelial cell function. Importantly, functional annotation reveals that the proteome contains multiple members directly related to the reproductive process, involving key steps such as sperm and zona pellucida binding, sperm-egg recognition and fertilization (https: / / www.kegg.jp / kegg / kegg.html) Figure 8 Its function focuses on directly affecting the stability of sperm membrane, capacitation process and acrosome reaction by regulating cholesterol metabolism (including transport and efflux), thereby promoting sperm and zona pellucida binding, sperm-egg recognition and fertilization efficiency. At the same time, the protein group is also involved in regulating epithelial cell migration, apical / basal polarity establishment and differentiation, indicating that it also has an important regulatory role in maintaining the normal structure and function of epididymal tissue.

[0055] The functional annotation results of the above system show that the extracellular vesicles obtained by the present application have clear and multi-level functional characteristics in terms of molecular composition, providing a solid molecular basis for their biological functions in supporting sperm maturation, maintaining epididymal epithelial function and promoting the fertilization process.

[0056] The proteome of the extracellular vesicles described in the present application systematically realizes its biological function prediction through the following multi-level strategies:

[0057] Vesicle generation and delivery strategy: through the specific protein components it carries, it actively regulates the cargo loading, membrane budding and release process of the vesicle, and mediates the specific recognition and endocytosis of the vesicle with the target cell (such as sperm) surface, thereby completing the directional delivery of active ingredients.

[0058] Epididymal microenvironment support strategy: the vesicle proteins regulate the establishment of epithelial cell polarity, differentiation and migration, provide structural and functional support for the epididymal tissue, and maintain the local microenvironment conducive to sperm maturation.

[0059] Sperm function enhancement strategy: vesicle proteins directly enhance sperm membrane stability and capacitation state by regulating cholesterol metabolism, thereby improving sperm motility, movement, and binding capacity with the zona pellucida, laying the foundation for the subsequent fertilization process.

[0060] Fertilization potential improvement strategy: by integrating the above multiple optimizations of sperm motility, membrane function, and recognition ability, the success rate of sperm-egg recognition and fertilization is ultimately systematically improved.

[0061] In summary, the extracellular vesicles, through the synergistic effect of their proteome, follow a cascade strategy of "biogenesis → targeted delivery → functional regulation", achieving a multi-level and systematic improvement of epididymal function, sperm quality, and fertilization efficiency.

[0062] Example 3 Improvement of sperm function by extracellular vesicles during sperm storage

[0063] The obtained extracellular vesicles were added to the pig sperm preservation solution at a concentration of 1 μg / mL, and an equal amount of PBS buffer was added to the pig sperm preservation solution as a control group. The sperm preservation solution was evaluated for its improvement of sperm function, including sperm antioxidant capacity, motility, plasma membrane redox balance, mitochondrial function, survival rate, and DNA integrity, at 1, 3, and 5 days of preservation.

[0064] 1. Effect of extracellular vesicles on sperm antioxidant capacity and mitochondrial membrane potential damage

[0065] (1) ATP content detection

[0066] A detection system based on luciferase-luciferin chemiluminescence was used. After cell lysis, ATP in the supernatant participated in the luminescence reaction under the catalysis of luciferase, and the luminescence intensity was proportional to the ATP concentration. The chemiluminescence instrument was used to determine and calculate the ATP content through the standard curve.

[0067] (2) Total antioxidant capacity (T-AOC) determination

[0068] Based on the principle of ABTS free radical scavenging. Antioxidants in cell lysate can inhibit the generation of ABTS + , by measuring the change of absorbance at 734 nm, and using Trolox as a standard, the total antioxidant capacity of the sample was calculated.

[0069] (3) Superoxide dismutase (SOD) activity detection

[0070] WST-1 method was used. SOD can inhibit the reaction of superoxide radicals with WST-1 to generate methylene, by detecting the inhibition rate of absorbance at 450 nm, and combining with the protein concentration of the sample, the SOD enzyme activity was calculated.

[0071] (4) Glutathione Peroxidase (GPx) Activity Assay

[0072] Based on glutathione-coupled enzymatic reaction system. GPx catalyzes the reduction of glutathione (GSH) peroxide, while consuming NADPH. By monitoring the rate of decrease in NADPH absorbance at 340 nm, GPx activity is calculated.

[0073] (5) Catalase (CAT) Activity Assay

[0074] Using the property of CAT to decompose H2O2, the rate of decrease in absorbance of the reaction system at 240 nm over time is directly measured to calculate the CAT enzyme activity.

[0075] (6) Malondialdehyde (MDA) Content Determination

[0076] TBA method is used. MDA and TBA generate a red product under acidic heating conditions, and the absorbance is measured at 532 nm. The MDA content is calculated by the standard curve to evaluate the degree of lipid peroxidation.

[0077] (7) Glutathione-S-Transferase (GST) Activity Assay

[0078] Standard method based on spectrokinetics is used. This method is based on the principle that GST catalyzes the reaction of glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) to form GS-DNB conjugate. The rate of increase in absorbance of this product at 340 nm over time is monitored by a microplate reader to calculate the enzyme activity. The specific operation includes: preparing cell lysate supernatant and determining protein concentration; mixing the sample with reaction buffer containing GSH and CDNB, and starting the reaction at 37°C; continuously measuring the absorbance change value of the reaction system; according to the absorbance change rate, the molar extinction coefficient of the product, the reaction time and the protein concentration of the sample, the specific activity of GST in the sample (U / mg protein) is calculated.

[0079] (8) Evaluation of Cellular Reactive Oxygen Species (ROS) Level

[0080] Fluorescent probe method is used. This method is based on the principle that a specific fluorescent probe (DCFH-DA) can be oxidized by intracellular reactive oxygen species to generate strong fluorescent substances, and the intracellular ROS level is quantitatively reflected by detecting the fluorescence intensity. The specific steps include: co-incubation of cells with fluorescent probe to allow the probe to enter the cells; after washing to remove excess probe, fluorescence detection is carried out under suitable conditions; the fluorescence signal intensity is measured using a fluorescence microplate reader or flow cytometer, and quantitative analysis is performed. After standardization of experimental data, it is used to compare the differences in intracellular ROS levels between different treatment groups.

[0081] (9) JC-1 detection of mitochondrial membrane potential

[0082] JC-1 (CBIC2 (3) ) is a fluorescent lipophilic carbocyanine dye used to measure mitochondrial membrane potential. When the mitochondrial membrane potential is high, JC-1 aggregates in the matrix to form polymers, which can produce red fluorescence (λex=585 nm, λem=590 nm). When the mitochondrial membrane potential is low, JC-1 cannot aggregate in the mitochondrial matrix and exists as a monomer to produce green fluorescence (λex=514 nm, λem=529 nm). The detection method is a conventional method, which will not be described here.

[0083] All quantitative data were processed using professional statistical and drawing software, and the results were expressed as mean ± standard error, and appropriate significance test was performed.

[0084] The experimental results show that (9) JC-1 detection of mitochondrial membrane potential Figure 9 ), after adding the prepared extracellular vesicles to the pig sperm preservation solution, the biological active substances carried by the extracellular vesicles can significantly improve the antioxidant function of the sperm. Specifically, it can effectively reduce the level of reactive oxygen species (ROS) and lipid peroxidation product malondialdehyde (MDA) in sperm; JC-1 detection of mitochondrial membrane potential aggregation state / monomer experiment results show that extracellular vesicles can increase the mitochondrial aggregation state / monomer ratio, indicating that extracellular vesicles can reduce mitochondrial structure and function damage and reduce mitochondrial membrane potential damage; at the same time, it can significantly improve the total antioxidant capacity (T-AOC), adenosine triphosphate (ATP) content, and the activity of key antioxidant enzymes such as glutathione peroxidase (Gpx), superoxide dismutase (SOD) and glutathione S-transferase (GST).

[0085] 2. Effect of extracellular vesicles on sperm plasma membrane redox balance and motility

[0086] The sperm plasma membrane is rich in polyunsaturated fatty acids, which is prone to lipid peroxidation damage during in vitro preservation, leading to a decline in membrane structure integrity and function, and thus seriously affecting the motility of sperm and its subsequent fertilization ability. In order to objectively evaluate the protective effect of the extracellular vesicles (EVs) described in the present application on the sperm plasma membrane, the present application systematically monitors the changes in the redox state and motility parameters of the sperm plasma membrane during preservation. The oxidation state of the plasma membrane is dynamically detected using a fluorescent probe sensitive to lipid peroxidation, and the degree of membrane lipid oxidation is reflected by the change in fluorescence signal; the sperm motility is quantitatively tracked and evaluated at multiple time points by a computer-aided sperm analysis system.

[0087] The system evaluation found that the addition of EVs can significantly maintain the redox homeostasis of sperm plasma membrane, effectively inhibit the accumulation of reactive oxygen species and the progress of lipid peroxidation during the preservation process, thereby stabilizing the structure and function of sperm plasma membrane, and ultimately helping to maintain the sustained motility of sperm during in vitro preservation. This confirms that EVs have a clear and positive role in protecting sperm plasma membrane and delaying oxidative damage, providing experimental evidence for its application in sperm preservation and assisted reproduction. Figure 10 ).

[0088] 3. Effect of extracellular vesicles on sperm survival rate

[0089] By using Calcein AM and propidium iodide (PI) for live / dead double staining, sperm was stained and fluorescence microscopic imaging analysis was performed under specific conditions, so as to realize the accurate quantification of sperm survival state.

[0090] The results are shown in Figure 11 . The addition of EVs in the preservation solution can effectively maintain the survival rate of sperm, and the protective effect shows a clear time-dependent characteristic. With the extension of in vitro preservation time, the survival rate of sperm in the EVs treatment group is always significantly higher than that in the untreated control group. This confirms that the EVs described in the present application have a positive and stable effect in prolonging the in vitro preservation time of sperm and slowing down the preservation-related damage, providing a key functional basis for its application in assisted reproductive technology.

[0091] 4. Effect of extracellular vesicles on sperm mitochondrial function

[0092] The experiment used Rhodamine 123 fluorescent probe to specifically label sperm mitochondria, and the changes of mitochondrial membrane potential and structural integrity during the preservation process were evaluated by dynamic fluorescence imaging technology. The probe can enter the mitochondrial matrix depending on the mitochondrial membrane potential, and its fluorescence intensity is positively correlated with the membrane potential level. After staining and washing, the fluorescence signal was observed and quantitatively analyzed by fluorescence microscope to evaluate the mitochondrial function state.

[0093] The present application proves through in vitro preservation experiments that the provided extracellular vesicles (EVs) have a significant protective effect on sperm mitochondrial function, and the protective effect is time-dependent. The results show that Figure 12 , with the extension of preservation time, the fluorescence signal intensity and distribution stability of sperm mitochondria in the EVs treatment group are significantly better than those in the control group, reflecting better mitochondrial membrane potential maintenance ability and slower functional decline rate. This proves that EVs not only effectively slow down the functional damage of mitochondria in the early preservation period, but also provide sustained protection during the extended preservation period, thereby helping to maintain the energy metabolism homeostasis of sperm and its motility, providing an important basis for its application in reproductive medicine.

[0094] 5. The effect of extracellular vesicles on sperm DNA damage

[0095] The oxidative damage to sperm DNA was localized and quantified by immunofluorescence detection of 8-hydroxydeoxyguanosine (8-OHdG).

[0096] The results showed that ( Figure 13 The addition of extracellular vesicles (EVs) significantly reduced oxidative damage to sperm head DNA in the early stages of preservation (days 1 to 3), indicating a clear protective effect on sperm genetic material. However, this protective effect gradually weakened over time, becoming statistically insignificant by day 5. These results confirm that the protective effect of EVs on sperm DNA has a clear time-dependent effect, providing an important basis for optimizing their use in sperm preservation systems.

[0097] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing extracellular vesicles derived from porcine epididymal cells, characterized in that, Includes the following steps: Porcine epididymal cells were pretreated, and the pretreated sample was collected and filtered to obtain filtrate. The filtrate was centrifuged and the supernatant was collected. The supernatant was centrifuged again and the centrifuged supernatant was collected. The centrifuged supernatant was centrifuged and the precipitate was collected. The precipitate was resuspended and centrifuged to obtain the extracellular vesicles derived from porcine epididymal cells.

2. The preparation method according to claim 1, characterized in that, The pretreatment method is as follows: porcine epididymal epithelial cells are cultured in an exosome-free serum culture medium containing testosterone and 5α-dihydrotestosterone for 24-48 hours, and the culture medium is collected to obtain the pretreated sample.

3. The preparation method according to claim 1, characterized in that, The filtrate was centrifuged at 2000g for 20 minutes to collect the supernatant.

4. The preparation method according to claim 1, characterized in that, The supernatant was centrifuged again at 10000g for 60 min, and this process was repeated twice. The supernatant was then collected.

5. The preparation method according to claim 1, characterized in that, The supernatant was centrifuged at 100,000g for 2 hours to collect the precipitate.

6. The preparation method according to claim 1, characterized in that, The precipitate was resuspended and centrifuged at 120,000g for 70 minutes to obtain the extracellular vesicles derived from porcine epididymal cells.

7. Extracellular vesicles derived from porcine epididymal cells prepared by the preparation method according to any one of claims 1-6.

8. The use of extracellular vesicles derived from porcine epididymal cells according to claim 7 in the preparation of sperm preservation solution.

9. A sperm preservation solution, characterized in that, The sperm preservation solution includes extracellular vesicles derived from porcine epididymal cells as described in claim 7.

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