A method for applying electromagnetic pulses to in vitro cultured oocytes

Abstract

This application belongs to the field of electromagnetic biology, and specifically discloses a method for applying electromagnetic pulses to in vitro cultured oocytes. The method includes applying electromagnetic pulses with a frequency of 1-100 Hz, an intensity of 1-10 mT, and a time of 5-20 min to the oocytes in the germinal vesicle stage. This application first applies pulsed magnetic fields to oocytes, establishing a new way to improve the maturation and developmental ability of oocytes, which can significantly improve the maturation and developmental ability of oocytes, thereby improving the normal development of follicles and generally improving the problem of decreased ovarian reserve function; in addition, this application uses electromagnetic fields, a physical therapy, which is a non-invasive technique, greatly increasing the safety and effectiveness of its application.

Description

Technical Field

[0001] This application belongs to the field of electromagnetic biology, and more specifically, relates to a method for applying electromagnetic pulses to in vitro cultured oocytes. Background Art

[0002] Exploring female fertility preservation strategies is currently a hot and difficult topic in the field of reproduction. On the one hand, the follicle pool of female patients with declining fertility is depleted, and only a very small number of resting primordial follicles in the ovarian cortex cannot grow and develop naturally in vivo, but immature oocytes are difficult to mature in vitro. On the other hand, for young female cancer patients, freezing ovarian tissue before radiotherapy and chemotherapy and obtaining immature oocytes for in vitro maturation (IVM) after thawing is the preferred method for fertility preservation. Improving the maturation rate of human oocytes in vitro culture, the quality of oocytes, and the subsequent embryonic development potential is a crucial link in improving the success rate of assisted reproductive outcomes.

[0003] The follicle is the core functional unit of the reproductive system of women of childbearing age, and the ultimate goal of its development is to produce a fertile egg to complete the processes of fertilization and embryonic development. In modern society, due to environmental factors, mental stress, etc., the primordial follicle pool is depleted prematurely, resulting in premature ovarian function decline, ovarian reserve function decline, premature ovarian insufficiency, and ultimately premature ovarian failure, which seriously affects women's reproductive ability. The decline in fertility has brought a heavy physical and psychological burden to the female population, so maintaining the normal development of follicles is particularly important for female reproduction. The development of follicles is a complex process involving multiple cells and multiple stages. Among them, granulosa cells and oocytes are important cells that make up the follicle, and the formation of follicles requires close interaction between granulosa cells and oocytes. Oocytes and granulosa cells regulate the normal development of follicles through a series of autocrine or paracrine methods. In vitro maturation (IVM) of oocytes refers to obtaining immature oocytes from the ovary, culturing them in vitro, and making them mature and capable of fertilization and supporting embryonic development to full-term delivery. There are many problems in the current stage of IVM technology, such as the lack of certain components in the in vitro culture medium, which is not sufficient to support the development of oocytes, etc. The existing methods for regulating the in vitro maturation and development ability of oocytes are very limited. Therefore, new treatment strategies are urgently needed. For the decline in ovarian reserve function caused by ovarian aging in elderly women, the current main clinical solutions are to advocate a healthy lifestyle, control weight, psychological counseling, contraception, and sexual health guidance, etc. Although there are certain effects, these improvement methods are difficult to achieve a definite therapeutic effect in the target organ, and only a very low proportion of patients achieve complete remission. Therefore, new treatment strategies are urgently needed.

[0004] In modern science, pulsed strong magnetic field is listed as one of the most important extreme conditions for scientific experiments. As the focus and frontier of strong magnetic field research, pulsed strong magnetic field is now receiving more and more attention. Pulsed electromagnetic field (PEMF) therapy has some potential advantages, including non-invasiveness, safety, and the possibility of being used in combination with other available therapies. PEMF is a magnetic field effect with pulsed intermittents generated by current passing through the Helmholtz coil. The PEMF device mainly consists of three parts. The first part is the signal generator, which can generate a voltage signal with a specific waveform and frequency; the second part is the signal amplifier, which can generate a current output to supply the electromagnetic field generator. Adjusting the output of the amplifier can change the magnitude of the field strength. The third part is the electromagnetic field generator, which is mainly composed of a coil. PEMF can act on biological systems non-invasively through capacitance and inductance. The former requires the relative electrodes to be placed around the tissue of interest and in direct contact with the skin, while the latter does not require contact with the skin. As a non-invasive physical intervention method, pulsed electromagnetic field was first used in the treatment of bone nonunion and delayed fracture healing. As a representative of low-frequency electromagnetic fields, low-frequency pulsed electromagnetic field (LF-PEMF) refers to a low-frequency, low-intensity magnetic field with a frequency of 1 to 100 Hz and a peak magnetic field of <10 mT. Pulsed electromagnetic field (PEMF) therapy has been proven to have some potential advantages, including non-invasiveness, safety, non-toxicity to non-cancerous cells, and the possibility of being used in combination with other available therapies. In recent years, it has been increasingly used in the treatment of neurological diseases and tumors, including skin cancer, breast cancer, prostate cancer, hepatocellular carcinoma, lung cancer, ovarian cancer, pancreatic cancer, bladder cancer, thyroid cancer, and colon cancer. Low-frequency pulsed strong magnetic fields can selectively kill tumor tissues and inhibit tumor growth, and regulate the immune system function of organisms. However, its effects on the reproductive system are still unclear. Summary of the invention

[0005] So can electromagnetic pulses be used to improve the in vitro maturation and development of oocytes so that they can play a longer role and have a good effect after being transplanted into the body? Through this application, we hope to regulate the in vitro maturation and development of oocytes through electromagnetic pulses, thereby providing a reference for clinical research on the reproductive system.

[0006] To achieve the above-mentioned purpose, the present application provides a method for applying electromagnetic pulses to oocytes cultured in vitro, comprising: applying electromagnetic pulses with a frequency of 1 to 100 Hz, an intensity of 1 to 10 mT, and a time of 5 to 20 min to the oocytes in the germinal vesicle stage.

[0007] Preferably, the culture density of the in vitro cultured oocytes is 300 - 600 cells / ml.

[0008] Preferably, the number of times of electromagnetic pulse application is 1 - 10 times, and the application interval is 30 - 60 ms.

[0009] Preferably, the oocytes are mouse oocytes.

[0010] As a further preference, the strain of the mouse is C57 strain, ICR strain or KM strain.

[0011] As a further preference, the age of the mouse is 8 - 40 weeks.

[0012] As a further preference, the culture medium used for in vitro culture is M16 medium.

[0013] Another object of the present application is to provide oocytes applied with electromagnetic pulses by the above method.

[0014] Generally speaking, compared with the prior art through the above technical solutions conceived by the present application, the following technical advantages are mainly possessed:

[0015] 1. The present application first applies pulsed magnetic field to oocytes, establishing a new way to improve the maturation and developmental ability of oocytes, which can significantly improve the maturation and developmental ability of oocytes, and then improve the normal development of follicles, and generally improve the problem of decreased ovarian reserve function; in addition, the present application uses electromagnetic field, a physical therapy, which is a non-invasive technology, greatly increasing the safety and effectiveness of its application;

[0016] 2. It has been verified that after the action of the magnetic field, various indexes of 40-week-old aged mice have been improved, such as the fertilization rate is increased, the mitochondrial membrane potential is increased, the number of mitochondria is increased, the proportion of abnormal spindle morphology is decreased, the reactive oxygen species are reduced, and the lysosome vesicle density is decreased; among them, the in vitro blastocyst formation rate and apoptosis situation reach the level close to that of 8-week-old mice. Description of the Drawings

[0017] Figure 1a is the fertilization rate of in vitro fertilization in the examples and comparative examples of the present application;

[0018] Figure 1b is the percentage statistical chart of development to the blastocyst stage after in vitro fertilization in the examples and comparative examples of the present application;

[0019] Figure 1c is the in vitro blastocyst formation rate of the complex after in vitro fertilization in the examples and comparative examples of the present application; almost

[0020] Figure 2aIt shows the mitochondrial membrane potential of oocytes by JC-1 staining in the examples and comparative examples of this application;

[0021] Figure 2b It is the statistical graph of mitochondrial membrane potential provided in the examples and comparative examples of this application;

[0022] Figure 3a It shows the mitochondrial distribution and quantity in oocytes by Mito-Tracker staining provided in the examples and comparative examples of this application;

[0023] Figure 3b It is the statistical graph of mitochondrial fluorescence intensity provided in the examples and comparative examples of this application;

[0024] Figure 4a It is the schematic diagram of spindle morphology in oocytes provided in the examples and comparative examples of this application;

[0025] Figure 4b It is the statistics of the ratio of abnormal spindle morphology in oocytes provided in the examples and comparative examples of this application;

[0026] Figure 5a It is the detection graph of reactive oxygen species level in oocytes provided in the examples and comparative examples of this application;

[0027] Figure 5b It is the statistical result of reactive oxygen species level provided in the examples and comparative examples of this application;

[0028] Figure 6a It is the detection graph of oocyte apoptosis by Annexin staining provided in the examples and comparative examples of this application;

[0029] Figure 6b It is the statistical result of oocyte apoptosis provided in the examples and comparative examples of this application;

[0030] Figure 7 It is the detection picture of lysosomes by LysoTracker Green staining provided in the examples and comparative examples of this application;

[0031] Figure 8 It is the statistical result of lysosome vesicle density in the examples and comparative examples of this application. Detailed implementation manners

[0032] In order to make the purpose, technical solutions and advantages of this application clearer, the following further elaborates on this application in combination with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not used to limit this application.

[0033] In the description of the present application, it should be understood that the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. In the description of the present application, "a plurality of" means two or more, unless otherwise specifically defined.

[0034] In addition, the reference to "an embodiment" throughout this specification; the language such as "an embodiment", "an example" or the like means that the specific features, structures or characteristics described in connection with the embodiment are included in at least one embodiment of the present application. Therefore, the appearances of the phrase "in an embodiment;" and the like of "in an embodiment" throughout this specification may or may not all refer to the same embodiment.

[0035] The present application provides a method for applying electromagnetic pulses to in vitro cultured oocytes, including: applying electromagnetic pulses with a frequency of 1 - 100 Hz, an intensity of 1 - 10 mT, and a time of 5 - 30 min to the oocytes in the germinal vesicle stage; in some embodiments, the frequency is preferably 30 - 60 Hz, the intensity is preferably 5 - 10 mT, and the time length is preferably 7 - 15 min. In some embodiments, the culture density of the in vitro cultured oocytes is 300 - 600 cells / ml; in other embodiments, the number of times of applying the electromagnetic pulses is 1 - 10 times, and the interval between applications is 30 - 60 ms.

[0036] In some embodiments, the oocytes are mammalian oocytes, such as mouse oocytes of C57 line, ICR line or KM line, etc., preferably female small oocytes at 8 - 40 weeks of age; the culture medium used during culture is preferably M16 medium.

[0037] The oocytes to which electromagnetic pulses have been applied by the above method have significantly improved maturation and development capabilities.

[0038] The following is an example:

[0039] Comparative Example 1 Acquisition and IVM (In Vitro Maturation) Culture of Mouse Oocytes

[0040] The oocytes involved in this example are germinal vesicle (GV) stage oocytes, which are obtained by in vitro culture, and the specific steps are as follows:

[0041] S1. Mouse superovulation: After newly introduced 40 - week - old C57 female mice adapt to the environment, according to their body weight, 5 - 10 IU of pregnant mare serum gonadotropin (PMSG) is injected intraperitoneally.

[0042] S2. Preparation and pre-incubation of culture medium droplets: Prepare the droplet dish in advance and incubate it in the incubator for more than 6 hours. Prepare one M2 washing dish and one oocyte-puncturing dish for each mouse (2 ovaries) on average. Make 20 μL droplets of the IVM culture medium and cover them with mineral oil to prevent evaporation. Prepare one dish for each group for the IVM culture dish, and reserve one extra IVM culture dish for backup. Drop 10 μL of the M2 culture medium containing 2.5 μM milrinone on the lid of the injection micro-droplet dish per drop and cover it with mineral oil.

[0043] S3. Mouse ovarian dissection: Sacrifice the mouse after 46 - 48 hours, cut the skin and muscle through the abdomen or lateral position of the back, find the ovarian fat pad under the kidney, pull out the ovarian tissue, puncture the cyst, and separate the ovarian tissue and put it into the pre-warmed M2 solution.

[0044] S4. Obtaining cumulus-oocyte complexes (COCs): After washing off the adherent blood on the surface, use a sterile syringe to puncture the follicles under the stereomicroscope to release the COCs like poached eggs.

[0045] S5. Obtaining GV-stage oocytes: Melt the capillary pipette and pull it to an appropriate diameter (about 100 μm), and manually break the needle. The broken needle needs to be flat for use to avoid scratching the oocytes. Repeatedly aspirate and blow the COCs by the method of mouth pipette to remove the granulosa cells, and then obtain the GV-stage oocytes. Store them in a solution containing sodium bicarbonate: 2.101 g / L, phenol red: 0.0106 g / L, sodium pyruvate: 0.0363 g, dextran glucose: 1.0 g / L, and adjust the cell density to 500 cells / ml.

[0046] Comparative Example 2

[0047] Repeat Comparative Example 1 with the same steps, except that in step S1, newly arrived 8-week-old C57 female mice are used.

[0048] Example 1

[0049] Repeat Comparative Example 1 with the same steps, except that after step S5, apply a magnetic field to the GV-stage oocytes once under the pulsed magnetic field conditions of 1 mT and 10 min.

[0050] In this example, the magnetic field facility uses the pulsed magnetic field experimental device of Huazhong University of Science and Technology. The device mainly includes: self-resetting fuse F; rectifier bridge (D1, D2, D3, D4); filter capacitor C1; energy storage capacitor C2; filter inductor L1; DC control switches S1, S2, S3, S4; freewheeling diodes D5, D6; magnet (R m is the magnet resistance, L m is the magnet inductance, C mMagnet parasitic capacitance); Current sensor CT; Voltage sensor VT; Temperature sensor TP; Bidirectional transient suppression diode D8; Controller.

[0051] Experimental result verification

[0052] Hereinafter, Example 1, Comparative Example 1, and Comparative Example 2 are verified in terms of developmental ability, mitochondrial membrane potential, etc. In all the drawings, Comparative Example 1 is represented by Old, Comparative Example 2 is represented by Young, and Example 1 is represented by Old+PMF.

[0053] Example 2

[0054] Repeat Example 1 with the same steps, except that after step S5, the intensity of the pulsed magnetic field is 10 mT.

[0055] Experimental result verification

[0056] Hereinafter, Example 1, Comparative Example 1, and Comparative Example 2 are verified in terms of developmental ability, mitochondrial membrane potential, etc. In all the drawings, Comparative Example 1 is represented by Old, Comparative Example 2 is represented by Young, and Example 1 is represented by Old+PMF.

[0057] Verification Example 1 Effect of pulsed magnetic field on the developmental ability of immature oocytes of aged mice

[0058] To observe the effect of pulsed magnetic field on the developmental ability of immature oocytes of aged mice, we selected two indicators: fertilization rate and blastocyst rate. The oocytes of Example 1, Example 2, Comparative Example 1, and Comparative Example 2 were taken for in vitro culture and in vitro fertilization respectively. The specific steps of in vitro fertilization are as follows:

[0059] S1. Fertilization and preparation and pre-incubation of culture medium droplets: Prepare the droplet dish in advance and incubate it in the incubator for more than 6 h. The sperm activation droplet is made into a 50 μL droplet with HTF solution. A minimum pipette tip is used to draw a line between the two droplets to form a dumbbell shape, and mineral oil is covered on it to prevent evaporation. The HTF fertilization solution and KSOM embryo culture solution are made into 20 μL droplets, and mineral oil is covered on them to prevent evaporation.

[0060] S2. Sperm activation: Select 12-week-old sexually mature and robust C57 male mice, sacrifice them by cervical dislocation, open the abdomen and pull out the testicles, find the epididymis and the tail of the epididymis. Cut off all the tails of the epididymis with scissors and put them into one side of the sperm activation droplet, and place them in a 37 °C incubator for 1 h. It can be seen that the activated sperm swim to the other side of the HTF droplet.

[0061] S3. Adding sperm: Use the mouth pipette method to suck the activated sperm and add it to the oocyte droplets of Example 1, Comparative Example 1, and Comparative Example 2. The amount of sperm added should be such that the sperm can push the oocytes to move. After adding sperm, put the culture dish into the incubator.

[0062] S4. Egg washing: 5 h after sperm addition, it can be seen that the granulosa cells around the oocytes have been digested and shed. After slightly washing the sperm around the oocytes, they were transferred into KSOM embryo culture medium (K+Simplex Optimised Medium) for further culture.

[0063] S5. Embryo culture: The formation rate of two-cell embryos was observed the next day, and the development of oocytes was observed every day.

[0064] The results of in vitro fertilization are shown in Figure 1. Among them, Figure 1a the abscissa is the experimental grouping, the ordinate is the percentage, and the value columns from left to right are the fertilization rate statistics of the control group 2, the control group 1, and the treatment group of Example 1; Figure 1b is the percentage statistical chart of the development of three groups of oocytes to the blastocyst stage after fertilization; Figure 1c is the in vitro blastocyst formation rate of three groups of oocyte complexes. It can be clearly observed that after being affected by the magnetic field, the fertilization rate and blastocyst formation rate of immature oocytes of old mice are significantly improved. The specific values are shown in Table 1:

[0065] Table 1 Effects of pulsed magnetic fields with different intensities on the developmental ability of immature oocytes of young mice

[0066]

[0067] Verification Example 2 Effect of pulsed magnetic field on mitochondrial membrane potential of oocytes of old mice

[0068] The mitochondrial membrane potential of the oocytes in Example 1, Control Group 1, and Control Group 2 was detected by JC-1 staining method. The brief steps are as follows:

[0069] S1. Solution preparation and incubation: Add 200 μL of JC-1 staining solution (the composition is JC-1 (200×): ultrapure water: JC-1 staining buffer (5×) mixed at a volume ratio of 1:160:40,...) to the first well of a 5-well plate, and add JC-1 washing solution (the composition is JC-1 staining buffer (5×): distilled water mixed at a volume ratio of 1:4) to the second and third wells.

[0070] S2. JC-1 staining: Collect the oocytes at the end of culture with a mouth pipette, transfer them into the JC-1 staining solution, incubate at 37 °C for 20 min, and wash twice in the JC-1 washing solution to remove the dye particles adhering to the zona pellucida of the oocytes.

[0071] S3. Microscopic examination: Prepare a 5 μL microdrop in a confocal dish with IVM medium and cover it with mineral oil to prevent evaporation. Transfer the oocytes into the microdrop and observe and take pictures under a confocal microscope.

[0072] Figure 2aTo show the mitochondrial membrane potential of oocytes by JC-1 staining, where J-aggregates represent the staining effect of JC-1 monomers after aggregation to form polymers, which can produce red fluorescence; J-monomer represents the staining effect of unaggregated JC-1 monomers, which can produce green fluorescence; Merge represents the merged fluorescence image of JC-1 polymers, monomers and nuclear staining. Figure 2b It is a statistical chart of mitochondrial membrane potential; it can be seen that compared with Comparative Example 1, the mitochondrial membrane potential of the oocytes in Example 1 increased.

[0073] Verification Example 3 Effect of pulsed magnetic field on the number of mitochondria in aged mouse oocytes

[0074] The mitochondrial distribution of the oocytes in Example 1, Comparative Example 1 and Comparative Example 2 was detected by the mitochondrial fluorescent probe method. The specific steps are as follows:

[0075] S1. Solution preparation and incubation: Add mitochondrial fluorescent probe staining solution to the first well of a 5-well plate.

[0076] S2. Probe staining: Collect the oocytes after the culture ends with a mouth pipette and transfer them into the mitochondrial fluorescent probe staining solution, and incubate at 37 °C for 20 - 30 min.

[0077] S3. Microscopic examination: Prepare a 5 μL microdrop with IVM medium in a confocal dish and cover it with mineral oil to prevent evaporation. Transfer the oocytes into the microdrop and observe and take pictures under a confocal microscope.

[0078] Figure 3a It is the Mito-Tracker staining showing the distribution and number of mitochondria in oocytes, where Mitotracker represents the mitochondrial fluorescent staining image of oocytes, Hoechst represents the nuclear fluorescent staining image, and Merge represents the merged fluorescence image of mitochondria and nucleus; Figure 3b It is the statistical chart of mitochondrial fluorescence intensity provided by the embodiment of the present application; it can be seen from the figure that MitoTracker Red staining of mitochondria shows that mitochondria in young mouse oocytes are evenly distributed in the cytoplasm and accumulate around the spindle. However, aging destroys this distribution pattern of mitochondria, showing fading of the signal in the cytoplasm and loss of aggregation around the chromosomes, which can be rescued by magnetic field to a certain extent.

[0079] Compared with the young mice in Comparative Example 2, a higher proportion of disorganized spindles, misaligned chromosomes, and incorrect attachment of centromeres and microtubules exist in the oocytes of the aged mice in Comparative Example 1. After the action of the magnetic field, the abnormal rate of the spindle in Example 1 is significantly reduced.

[0080] Verification Example 4 Effect of pulsed magnetic field on the spindle of aged mice

[0081] The spindle morphology of the oocytes in Example 1, Comparative Example 1 and Comparative Example 2 was detected by immunofluorescence. All steps of oocyte immunofluorescence staining (IF) were completed under a stereomicroscope. The oocytes were placed in a 96-well plate or a 5-well culture dish, and the oocytes were transferred by mouth pipette. The specific operation steps are as follows:

[0082] S1. Fixation: The oocytes at each stage were fixed in 4% PFA fixative and incubated at room temperature for 10 min.

[0083] S2. Membrane permeabilization and blocking: According to the different antibodies to be incubated subsequently, the cells were incubated in a cell membrane permeabilization solution for 1 - 2 h and washed 3 times in a blocking solution.

[0084] S3. Primary antibody incubation: The oocytes were transferred into the primary antibody and incubated overnight at 4°C.

[0085] S4. Secondary antibody incubation: After washing the oocytes 3 times with the blocking solution in the dark room, they were transferred into the fluorescent secondary antibody and incubated at room temperature for 1 h.

[0086] S5. DAPI counterstaining: After washing the oocytes incubated with the secondary antibody 3 times, they were transferred into the DAPI solution and incubated for 5 - 10 min. Anti - quenching agent droplets were prepared in a confocal dish, about 5 μL / drop, and the oocytes were transferred into the droplets and covered with 1 mL of mineral oil.

[0087] Figure 4a It is a schematic diagram of the spindle morphology in the oocytes provided by the embodiment of the present application. Among them, α - tubin represents the fluorescence staining of the oocyte spindle, DAPI represents the fluorescence staining of chromatin, and Merge represents the merged fluorescence image; Figure 4b It is the statistical chart of the abnormal spindle ratio in three groups of oocytes provided by the embodiment of the present application; It can be seen from the figure that by analyzing the fluorescence intensity after staining with the reactive oxygen species probe, the results show that the magnetic field rescued the increase in the reactive oxygen species level of oocytes caused by aging.

[0088] Verification Example 5 Detection of Reactive Oxygen Species Level in Oocytes

[0089] The reactive oxygen species (ROS) levels of the oocytes in Example 1, Comparative Example 1 and Comparative Example 2 were detected. The specific steps are as follows:

[0090] S1. Solution preparation and incubation: Add 0.5 mL of DMEM to the first, second, fourth, and fifth wells of a 5 - well plate, and add 200 μL of reactive oxygen species staining solution to the third well.

[0091] S2. Reactive oxygen species staining: Collect the oocytes at the end of culture with a mouth pipette, wash them twice with DMEM, transfer them into the reactive oxygen species staining solution, incubate at 37°C for 20 min, wash twice in DMEM, and remove the excess probe.

[0092] S3. Microscopic examination: Prepare 5 μL of microdroplets with IVM medium in a confocal dish and cover with mineral oil to prevent evaporation. Transfer the oocytes into the microdroplets and observe and photograph them under a confocal microscope.

[0093] Figure 5a This is the detection of the reactive oxygen species (ROS) level in oocytes provided by the embodiments of the present application. Among them, ROS represents the ROS staining in oocytes, Brightfield represents the nuclear staining, and Merge represents the merged fluorescence picture. Figure 5b This is the statistics of the ROS level provided by the embodiments of the present application. It can be seen from the figure that the ROS content in the oocytes of the young group in Comparative Example 2 is relatively low, the ROS in the oocytes of the aged group in Comparative Example 1 increases significantly, and the ROS level in Example 1 is significantly restored after the magnetic field treatment.

[0094] Detection of the early apoptosis level of oocytes in Verification Example 6

[0095] The Annexin V-FITC staining method was used to detect the oocytes of Example 1, Comparative Example 1, and Comparative Example 2. The brief steps are as follows:

[0096] S1. Solution preparation and incubation: Add 0.5 mL of DPBS to the first, second, fourth, and fifth wells of a 5-well plate, and add 200 μL of Annexin V-FITC staining solution to the third well.

[0097] S2. Staining: Collect the oocytes after the culture ends with a pipette, wash them twice with DPBS, transfer them into the staining solution, and incubate at room temperature in the dark for 20 min.

[0098] S3. Microscopic examination: Wash the stained oocytes twice with DPBS. Prepare 5 μL of microdroplets with AnnexinV-FITC binding solution in a confocal dish and cover with mineral oil to prevent evaporation. Transfer the oocytes into the microdroplets and observe and photograph them under a confocal microscope.

[0099] Figure 6a This is the detection of the apoptosis of oocytes by Annexin staining provided by the embodiments of the present application. Among them, Annexin-V represents the Annexin-FITC fluorescence picture of oocytes, Brightfield represents the nuclear fluorescence picture, and Merge represents the merged fluorescence picture. Figure 6b This is the statistics of the apoptosis of oocytes provided by the embodiments and comparative examples of the present application. It can be seen from the figure that compared with Comparative Example 1, Example 1 after the magnetic field treatment reduces the early apoptosis level to a certain extent.

[0100] Detection of stained lysosomes in Verification Example 7

[0101] Figure 7This is the detection of lysosomes stained with LysoTracker Green provided by the embodiments of the present application. Among them, Lyso-Tracker represents the fluorescence image of oocyte lysosomes, Hoechst represents the fluorescence image of cell nuclei, and Merge represents the merged fluorescence image; Figure 8 is the fluorescence intensity of Lyso-Tracker; it can be seen from the figure that the magnetic field significantly reduces the density of lysosomal vesicles in oocytes of aged mice.

[0102] Through the above verification examples, it can be seen that after the magnetic field action, various indicators of 40-week-old aged mice have been improved. For example, the fertilization rate increases, the mitochondrial membrane potential increases, the number of mitochondria increases, the proportion of abnormally shaped spindles decreases, the reactive oxygen species decreases, and the density of lysosomal vesicles decreases; among them, the in vitro blastocyst formation rate and apoptosis situation reach the level close to that of 8-week-old mice. Adjusting the electromagnetic pulse frequency between 1 and 100 Hz, the intensity between 1 and 10 mT, and the duration between 5 and 30 min can all achieve the above improvement effects; at the same time, we adjust the culture density of oocytes between 300 and 600 cells / ml and try to apply 2 to 10 electromagnetic pulses to the same group of oocytes, with an interval of 30 to 60 ms each time, all of which can achieve the above improvement effects, and the above improvement effects exist for mice aged 8 to 40 weeks.

[0103] The test of verification examples 2-7 was carried out on Example 2, and the same effect as that of Example 1 was also obtained.

[0104] Those skilled in the art can easily understand that the above are only the preferred embodiments of the present application and are not used to limit the present application. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for applying electromagnetic pulses to GV stage oocytes cultured in vitro, characterized in that: include: An electromagnetic pulse with a frequency of 1 to 100 Hz, an intensity of 1 mT and a duration of 5 to 20 min is applied to the oocyte in the germinal vesicle stage, and the electromagnetic pulse is applied once.

2. The method according to claim 1, characterized in that The culture density of the in vitro cultured oocytes is 300-600 oocytes / ml.

3. The method according to claim 1, characterized in that The oocyte is a mouse oocyte.

4. The method according to claim 3, characterized in that The strain of the mice is C57, ICR or KM.

5. The method according to claim 3, characterized in that The age of the mice ranged from 8 to 40 weeks.

6. The method according to claim 3, characterized in that The culture medium used for the in vitro culture is M16 culture medium.

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