Application of sulfydryl modified dendrobine in oocyte in-vitro maturation culture

Abstract

The invention belongs to the field of cell in-vitro culture, and particularly discloses an application of sulfydryl modified dendrobine in oocyte in-vitro maturation culture, a sulfydryl (-SH) functional group is introduced on a dendrobine molecular structure through chemical synthesis to obtain modified dendrobine, the modified dendrobine is added into an IVM culture medium, and the oocyte in-vitro maturation culture is obtained. A newly introduced sulfydryl (-SH) functional group can be directly subjected to redox reaction with ROS (such as superoxide anions and hydroxyl radicals), so that the free radical scavenging capacity is enhanced, and the ROS is reduced; the GSH level can be obviously improved; the oxidative stress of porcine oocytes can be effectively relieved, the early cell apoptosis level is reduced, the mitochondrial membrane potential is maintained, and the blastocyst rate is increased.

Description

Technical Field

[0001] The present invention relates to the field of in vitro cell culture, and particularly to the application of mercapto-modified dendrobine in the in vitro maturation culture of oocytes. Background Art

[0002] The biological definition of in vitro maturation (IVM) of oocytes is to take immature GⅤ-stage oocytes out of follicles and culture them in an appropriate culture system to promote their in vitro maturation to the MⅡ stage. Compared with in vivo embryo production, in vitro embryo production (IVEP) has many advantages, including efficiently selecting excellent genetics for transfer or genetic modification to quickly obtain animals with ideal traits.

[0003] The culture conditions during in vitro embryo production may have a certain impact on the developmental potential of early embryos, but before the start of this process, the quality of oocytes is the key factor determining the normal development of oocytes into blastocysts. However, during in vitro maturation culture, due to increased oxygen tension, interference such as light and temperature, and the lack of maternal antioxidant protection, oocytes will generate a higher level of oxidative stress, which has an adverse impact on subsequent development. Therefore, supplementing antioxidants at the IVM stage is an effective means to improve the quality of oocytes.

[0004] Currently, researchers have conducted experiments by adding various antioxidant substances during IVM to ensure a balanced intracellular redox state and good oocyte quality. Multiple experiments have confirmed that adding antioxidants (such as thiols, polyphenolic compounds, melatonin, carotenoids, resveratrol, etc.) to the IVM medium can improve oocyte quality and reduce the damage caused by excessive ROS exposure. Natural antioxidants have many advantages such as wide sources, good antioxidant effects, and low toxicity and side effects, and are currently a research hotspot.

[0005] "Influence of Dendrobine on the Quality of Mouse Oocytes and the Developmental Potential of Early Embryos" published by E Zhiqiang discloses that dendrobine (DNE) is the main bioactive alkaloid isolated from the raw material dendrobium of Dendrobium, and can inhibit oxidative stress and apoptosis. Research shows that DNE can significantly improve the in vitro maturation rate of mouse oocytes and promote the development of early embryos. After adding 10 μM of DNE, the ROS level of mouse oocytes decreases, the glutathione level increases, the mitochondrial membrane potential increases, and it can also increase the cleavage rate and blastocyst rate of early embryos, reduce the apoptosis rate, and maintain the balance of the redox system, thereby promoting development.

[0006] However, since dendrobine is an alkaloid with significant antioxidant activity, its mechanism of reducing ROS (reactive oxygen species) mainly involves directly scavenging free radicals, regulating the activity of antioxidant enzymes, and inhibiting pro-inflammatory pathways. When dendrobine is added to the IVM medium for in vitro maturation culture of oocytes, the addition amount is relatively large, and the effect of reducing ROS is generally average, and there is an urgent need for improvement. Summary of the Invention

[0007] To solve the above technical problems, the present invention provides an application of mercapto-modified dendrobine in in vitro maturation culture of oocytes.

[0008] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0009] The first technical solution provided by the present invention is an application of mercapto-modified dendrobine in in vitro maturation culture of oocytes, wherein mercapto-modified dendrobine with a concentration of 5 μM - 20 μM is added to the in vitro maturation medium for porcine oocytes; wherein, the preparation method of the mercapto-modified dendrobine is as follows:

[0010] Take 1 mol of dendrobine and dissolve it in N,N-dimethylformamide to obtain a mixed solution. Under stirring, add 1.2 - 1.5 mol of mercaptoacetic acid, 0.05 mol of 4-dimethylaminopyridine, and 0.1 mol of N,N'-dicyclohexylcarbodiimide to the mixed solution, react at 40 °C for 8 - 12 hours, filter to collect the filtrate, remove the organic solvent by reduced pressure distillation, and purify to obtain mercapto-modified dendrobine by silica gel column chromatography. The eluent for silica gel column chromatography is an ethyl acetate / petroleum ether system, and the volume ratio of ethyl acetate / petroleum ether is 5% - 20%.

[0011] Further, the preparation method of the in vitro maturation medium for porcine oocytes is as follows:

[0012] 1) Take 10 μL of L-Cysteine, 500 μL of FBS, 10 μL of EGF, 1 mL of PEF, and 100 μL of Pen-Strep (100x), mix them, and make up the volume to 10 mL with M199 medium to prepare the in vitro maturation medium for oocytes. After preparation, shake well, filter and sterilize with a 0.22 μm filter, seal and store at 4 °C for later use;

[0013] 2) Take 9.6 mL of the in vitro maturation medium for oocytes, and add 100 μL of Glu-max (100x), 250 μL of PG600, and 50 μL of hCG to prepare 10 mL of the working solution of the in vitro maturation medium for oocytes;

[0014] 3) Add mercapto-modified dendrobine to the working solution of the in vitro maturation medium for oocytes so that the final concentration of mercapto-modified dendrobine is 5 μM - 20 μM.

[0015] The second technical solution provided by the present invention is a method for in vitro maturation culture of oocytes, comprising the following steps:

[0016] S1. After obtaining freshly slaughtered porcine ovarian tissue, immediately place it in 9% sterile physiological saline at 38°C, then first remove the excess connective tissue and ovarian ligament on the ovary; subsequently, quickly rinse the ovary with 75% medical alcohol preheated to 38.5°C and gently squeeze the ovary by hand to facilitate the extrusion of blood impurities adhering to the ovarian surface, and quickly rinse the ovary three times with physiological saline preheated to 38.5°C and gently squeeze the ovary by hand to facilitate the extrusion of blood impurities adhering to the ovarian surface. Place it in a water bath at 38.5°C and use a syringe needle with the bevel facing down to aspirate the follicular fluid in follicles with a diameter of 3 - 8 mm. After pulling out the needle, transfer it to a 50 mL centrifuge tube for natural sedimentation. The centrifuge tube is placed in the water bath to maintain the temperature and the natural sedimentation time is not less than 20 min; after the oocytes in the follicular fluid are fully sedimented, use a syringe to aspirate the excess supernatant and retain 5 mL of sediment, then add 25 mL of egg washing solution, and perform natural sedimentation again for 15 min. After sufficient sedimentation, discard the supernatant and retain the sediment. Gently shake the sediment and aspirate 5 mL of sediment and drop it onto a 100 mm marked culture dish, and add an appropriate amount of egg washing solution and gently shake it; start the heating stage equipped with a stereomicroscope, and use a glass pipette to screen out porcine oocytes with clear cytoplasmic edges, uniform wrapping states, and overall integrity within the field of view of the stereomicroscope;

[0017] S2. The selected porcine oocytes are washed three times in the egg washing solution in sequence, then washed three times in the prepared porcine oocyte in vitro maturation culture solution, and then 500 μL of porcine oocyte in vitro maturation culture solution is added to each well in a four-well plate, and 50 - 60 porcine oocytes are added to each well; finally, place the four-well plate in a cell incubator at 38.5°C and 5% CO2 for 42 h.

[0018] Furthermore, the preparation method of the egg washing solution is as follows:

[0019] Add 1.000 g of PVA to 50 mL of ultrapure water, put in pre-sterilized magnetic beads, place the beaker on a magnetic stirrer, set the temperature to 55°C and the stirring rate to 400 r / min, continuously stir for 30 min, cool and then add 9.550 g of DPBS, continue to stir and dissolve with the magnetic stirrer, and then add ultrapure water to make the volume of the solution up to 1000 mL; subsequently, collect the clear supernatant and filter it to obtain the egg washing solution, and store it in a refrigerator at 4°C.

[0020] Compared with the prior art, in the present invention, a mercapto (-SH) functional group is introduced into the molecular structure of dendrobine through chemical synthesis to obtain modified dendrobine. By adding modified dendrobine to the IVM medium, the newly introduced mercapto (-SH) functional group can directly undergo redox reactions with ROS (such as superoxide anions and hydroxyl radicals), enhancing the free radical scavenging ability, reducing ROS; and can significantly increase the level of GSH; it can effectively alleviate the oxidative stress of porcine oocytes, reduce the level of early apoptosis, maintain the mitochondrial membrane potential, and increase the blastocyst rate. Brief Description of the Drawings

[0021] Figure 1 Shows the cumulus cell expansion after adding 10 μM dendrobine and 10 μM mercapto-modified dendrobine.

[0022] Figure 2 Shows the effects of adding 10 μM dendrobine and 10 μM mercapto-modified dendrobine on the oxidative stress level of porcine oocytes.

[0023] Figure 3 Shows the mitochondrial activity of porcine oocytes after adding 10 μM dendrobine and 10 μM mercapto-modified dendrobine.

[0024] Figure 4 Shows the effects of adding 10 μM dendrobine and 10 μM mercapto-modified dendrobine on embryonic development. Detailed Embodiment

[0025] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in combination with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0026] In the following embodiments, the raw materials, reagents, and instruments used are self-prepared or commercially purchased unless otherwise specified.

[0027] Among them, the main reagents and instruments are shown in Table 1 and Table 2.

[0028] Table 1

[0029]

[0030] Table 2

[0031]

[0032] Main solutions:

[0033] (1) Preparation of oocyte washing solution:

[0034] First, take a 100 mL beaker and add approximately 50 mL of ultrapure water. Then add 1.000 g of PVA, put in the pre-sterilized magnetic beads, place the beaker on a magnetic stirrer, set the temperature to 55 °C and the stirring rate to 400 r / min, and continuously stir for about 30 minutes. After cooling, add 9.550 g of DPBS and continue to stir and dissolve with the magnetic stirrer. Finally, add ultrapure water to make the volume of the solution up to 1000 mL. Subsequently, perform filtration sterilization using a filter, and then aliquot into 50 mL sterile centrifuge tubes. After aliquoting, store it in a 4 °C refrigerator and ensure it is used within one week.

[0035] (2) Preparation of in vitro maturation medium for porcine oocytes:

[0036] 1) Take 10 μL of L-Cysteine, 500 μL of FBS, 10 μL of EGF, 1 mL of PEF, and 100 μL of Pen-Strep (100x), mix them, and make up to 10 mL with M199 medium to prepare the in vitro maturation medium for oocytes. After mixing well, filter and sterilize with a 0.22 μm filter, seal, and store at 4 °C for later use;

[0037] 2) Take 9.6 mL of the in vitro maturation medium for oocytes and add 100 μL of Glu-max (100x), 250 μL of PG600, and 50 μL of hCG to prepare 10 mL of the working solution of the in vitro maturation medium for oocytes;

[0038] 3) Add exosomes from dominant follicles to the working solution of the in vitro maturation medium for oocytes to make the final concentration of exosomes from dominant follicles 10 ng / mL - 200 ng / mL.

[0039] (3) Preparation of concentrated stock solutions of required reagents:

[0040] a. The concentrated stock solution of EGF is 10 μg / mL;

[0041] b. The concentrated stock solution of L-Cysteine is 0.1 g / mL;

[0042] c. PG600: Dissolve the powder in each bottle with 1 mL of M199 medium and aliquot into 250 μL / tube;

[0043] d. hCG: Dissolve the powder in each bottle with 2.5 mL of M199 medium and aliquot into 50 μL / tube.

[0044] All of the above concentrated stock solutions need to be stored in a -80 °C refrigerator environment.

[0045] (4) Preparation of 0.1% hyaluronidase: Weigh 0.1 g of hyaluronidase and add it to 100 mL of DPBS. Stir well until it is completely dissolved. Subsequently, perform filtration sterilization. After sterilization, aliquot the solution into 1.5 mL centrifuge tubes and store it at -20 °C for future use.

[0046] (5) Preparation of fusion solution: 0.01 g of PVA, 5.1016 g of mannitol, 0.0012 g of MgSO4, 0.0007 g of CaCl2, and make up to 100 mL with Sigma water. Filter and aliquot, store at -20 °C.

[0047] (6) Preparation of embryo development medium (PZM-3): First, weigh 0.048 g of KH2PO4, 0.048 g of MgSO4, 0.22 g of sodium pyruvate, and 0.0436 g of calcium lactate and place them in separate 1.5 mL EP tubes. Add 1 mL of Sigma water to each tube and pipette to mix well for standby. Mix 250 mg of bovine serum albumin with 1.374 mL of Sigma water and aliquot into tubes of 305 μL each. Weigh 0.6312 g of NaCl, 0.2106 g of NaHCO3, 0.0746 g of KCl, 0.0146 g of L-glutamine, 0.05 g of inositol, 0.004 g of gentamicin, 0.01 g of phenol red, and 0.3 g of BSA into a 100 mL beaker, and add half a beaker of Sigma water and stir to dissolve. Pipette 100 μL of each of the KH2PO4, MgSO4, and sodium pyruvate solutions into the beaker and pipette 3 times. Then add 1 mL of calcium lactate, 300 μL of bovine serum albumin, 1 mL of MEM (100x), and 2 mL of BME (50x), and stir to mix well. Transfer the liquid to a 100 mL volumetric flask, rinse the beaker with Sigma water and make up to 100 mL, filter, aliquot into tubes of 10 mL each, and store at -80 °C.

[0048] (7) Preparation of chemical assisted activation solution: Dissolve 5 mg of cytochalasin B (CB) in 1 mL of DMSO to make a 1000x concentrated stock solution, and CHX is also a 1000x concentrated stock solution. Mix 9.980 mL of PZM-3 + 10 μL of CB + 10 μL of CHX thoroughly to prepare 10 mL of chemical assisted activation solution.

[0049] (8) In vitro operation solution (T2): M199 (containing Hepes) with 2% FBS

[0050] (9) Mercapto-modified dendrobine: Dissolve 1 mol of dendrobine in N,N-dimethylformamide to obtain a mixed solution. While stirring, add 1.2 - 1.5 mol of mercaptoacetic acid, 0.05 mol of 4-dimethylaminopyridine, and 0.1 mol of N,N'-dicyclohexylcarbodiimide to the mixed solution, and react at 40 °C for 8 - 12 hours. Filter to collect the filtrate, remove the organic solvent by vacuum distillation, and purify to obtain mercapto-modified dendrobine by silica gel column chromatography. The eluent for silica gel column chromatography is an ethyl acetate / petroleum ether system, and the volume ratio of ethyl acetate to petroleum ether is 5% - 20%.

[0051] Example 1

[0052] 1) After obtaining the freshly slaughtered porcine ovarian tissue, immediately place it in 9% sterile physiological saline at 38 °C. First, remove the excess connective tissue and ovarian ligament on the ovary; then quickly rinse the ovary with 75% medical alcohol preheated to 38.5 °C and gently squeeze the ovary by hand to facilitate the extrusion of blood impurities adhering to the ovarian surface. Then quickly rinse the ovary three times with physiological saline preheated to 38.5 °C and gently squeeze the ovary by hand to facilitate the extrusion of blood impurities adhering to the ovarian surface. Place it in a water bath at 38.5 °C and use a syringe needle with the bevel facing down to extract the follicular fluid from follicles with a diameter of 3 - 8 mm. After removing the needle, transfer it to a 50 mL centrifuge tube for natural precipitation. The centrifuge tube is placed in the water bath to maintain the temperature and the natural sedimentation time is not less than 20 min; after the oocytes in the follicular fluid are fully sedimented, use a syringe to aspirate the excess supernatant and retain 5 mL of the precipitate, then add 25 mL of egg washing solution, and let it sediment naturally for another 15 min. After sufficient sedimentation, discard the supernatant and retain the precipitate. Gently shake the precipitate and then aspirate 5 mL of the precipitate and drop it onto a 100 mm marked culture dish, and add an appropriate amount of egg washing solution and gently shake it; start the heating stage equipped with the stereomicroscope, and use a glass pipette to screen out porcine oocytes with clear cytoplasmic edges, uniform wrapping states, and overall integrity within the field of view of the stereomicroscope.

[0053] 2) The selected porcine oocytes are washed three times successively in the egg washing solution, then washed three times in the porcine oocyte in vitro maturation culture medium prepared in claim 2, and then add 500 μL of porcine oocyte in vitro maturation culture medium containing different concentrations (0 (as the control group), 5 μM, 10 μM, 20 μM) of mercapto-modified dendrobine to each well of a four-well plate, and add 50 - 60 porcine oocytes to each well; finally, place the four-well plate in a cell culture incubator at 38.5 °C and 5% CO2 for 42 h.

[0054] Comparative Example 1

[0055] The difference from Example 1 is that in step 2), 500 μL of porcine oocyte in vitro maturation culture medium containing different concentrations (5 μM, 10 μM, 20 μM) of dendrobine is added.

[0056] Since the cumulus cell expansion is an important indicator. Inside antral follicles, oocytes rely on the metabolic support of surrounding somatic cells, and these metabolisms play a key role in the developmental competence of oocytes (oocyte quality). Secondly, the first polar body extrusion rate is an important indicator for evaluating the in vitro maturation quality of oocytes, which can reflect the meiotic process, maturation quality, chromosomal integrity of oocytes and their subsequent fertilization and developmental potential. To deeply explore the effect of LFF-Exo on the in vitro maturation of porcine oocytes, after 42 h of in vitro culture, the maturation of oocytes was reflected by evaluating indicators such as cumulus expansion, first polar body extrusion rate, ROS and GSH.

[0057] After 42 h of in vitro maturation culture of porcine oocytes in Example 1 and Comparative Document 1, hyaluronic acid was used to digest cumulus cells, and the digested oocytes were transferred to T2 in vitro manipulation solution, and the oocytes with the extrusion of the first polar body were selected by prodding the oocytes.

[0058] The calculation method of the in vitro maturation rate is: (the number of oocytes extruding the first polar body ÷ the total number of cultured oocytes) × 100%.

[0059] The in vitro maturation rate of porcine oocytes is affected by the concentrations of dendrobine and mercapto-modified dendrobine as shown in Table 3 and Table 4.

[0060] Table 3

[0061]

[0062] Note: Different letter markings represent significant differences (P < 0.05).

[0063] Table 4

[0064]

[0065] Note: Different letter markings represent significant differences (P < 0.05).

[0066] As can be seen from Table 3 and Table 4, compared with unmodified dendrobine, mercapto-modified dendrobine significantly increased the first polar body extrusion rate; when the addition concentration of dendrobine was 10 μM, the first polar body extrusion rate reached 86.53 ± 1.99%, significantly higher than 71.84 ± 0.21% of the control group. When the addition concentration of mercapto-modified dendrobine was 10 μM, the first polar body extrusion rate reached 89.95 ± 2.46%, significantly higher than 71.84 ± 0.21% of the control group. The results suggest that appropriately increasing the concentration of mercapto-modified dendrobine can effectively improve the in vitro maturation efficiency of porcine oocytes. Therefore, in the subsequent examples, the addition concentration of dendrobine or mercapto-modified dendrobine at 10 μM was the optimal addition amount. The principle is that the newly introduced mercapto (-SH) functional group can directly undergo redox reactions with ROS (such as superoxide anion and hydroxyl radical), enhance the free radical scavenging ability, and reduce ROS.

[0067] After 42 h of in vitro maturation culture of porcine oocytes, photos were taken using a microscope Leica DMi8. With the aid of ImageJ software, the total expanded area of cumulus cells was measured. For a single oocyte, the calculation method of its expansion degree was: dividing the total expanded area of cumulus cells by the number of oocytes. The situation of cumulus cell expansion with the addition of 10 μM dendrobine and 10 μM mercapto-modified dendrobine was as Figure 1 shown, and it can be seen from Figure 1 that compared with the control, when adding 10 μM mercapto-modified dendrobine, the diffusion area of cumulus cells was significantly higher than that of the control group and the group with the addition of 10 μM dendrobine.

[0068] In addition, during the in vitro maturation of oocytes, reactive oxygen species (ROS) and glutathione (GSH), as two key biomolecules, have a significant impact on the maturation, quality, and fertilization ability of oocytes. Therefore, the levels of ROS and GSH were detected after 42 hours of in vitro culture.

[0069] First, hyaluronidase was used to remove cumulus cells. On this basis, oocytes that had extruded the first polar body were carefully selected, with the number in each group controlled at about 15. Subsequently, the selected oocytes were washed 3 times with PBS-PVA solution, aiming to thoroughly clean the oocytes to avoid residual substances interfering with the subsequent staining effect. After washing, these oocytes were placed separately in PBS-PVA solution containing DCFH-DA dye. The solution containing oocytes was placed in a cell incubator and incubated for 30 minutes under light-proof conditions. After incubation, the oocytes were transferred to a PBS-PVA droplet and washed 3 times again to remove the unbound dye and prevent it from affecting the staining result. During the experiment, microdroplets were prepared with PBS-PVA and paraffin oil was covered on the surface of the microdroplets. The purpose of covering paraffin oil was to prevent the liquid in the microdroplets from volatilizing. After completing the above preparations, each group of oocytes was transferred into separate microdroplets, and finally photographed and recorded under a fluorescence microscope.

[0070] The effects of adding 10 μM dendrobine and 10 μM thiol-modified dendrobine on the oxidative stress level of porcine oocytes are as Figure 2 shown. Compared with the control, the ROS fluorescence intensity significantly decreased after adding 10 μM thiol-modified dendrobine and 10 μM dendrobine, and the ROS fluorescence intensity after adding 10 μM thiol-modified dendrobine was significantly less than that after adding 10 μM dendrobine; compared with the control, the GSH fluorescence intensity significantly increased after adding 10 μM thiol-modified dendrobine and 10 μM dendrobine, and the GSH fluorescence intensity after adding 10 μM thiol-modified dendrobine was significantly higher than that after adding 10 μM dendrobine. It can be Figure 2 seen that adding 10 μM thiol-modified dendrobine can effectively alleviate the oxidative stress of porcine oocytes.

[0071] After the oocytes added with 10 μM dendrobine and 10 μM thiol-modified dendrobine matured, 0.1% hyaluronidase was gently used to remove cumulus cells. After washing the oocytes three times with PBS-PVA, they were put into JC-1 dye diluted 1:1000 and incubated in the incubator for 30 min under light-proof conditions. After incubation, they were washed 3 times with PBS-PVA, and red and green fluorescence signals were captured by photographing with a fluorescence microscope. The ratio of red fluorescence intensity to green fluorescence intensity was analyzed and calculated using ImageJ.

[0072] The effects of adding 10 μM dendrobine and 10 μM thiol-modified dendrobine on the mitochondrial activity of porcine oocytes are as Figure 3 shown. From Figure 3It can be seen that the mitochondrial membrane potential of the control group was significantly lower than that of the groups supplemented with 10 μM dendrobine and 10 μM mercapto-modified dendrobine, and the mitochondrial membrane potential after adding 10 μM mercapto-modified dendrobine was significantly higher than that after adding 10 μM dendrobine. The above results indicate that the addition of 10 μM mercapto-modified dendrobine alone or in combination in the IVM medium significantly improves the mitochondrial activity of porcine oocytes.

[0073] In this example, to verify the effect of the addition of mercapto-modified dendrobine on subsequent embryo development, the required liquids were preheated in a 38.5 °C water bath. At least 4 h before parthenogenetic activation, the chemical assisted activation solution and the embryo development solution need to be equilibrated in advance. Add 500 μL of the assisted activation solution or the embryo development solution to each well of the four-well plate, and cover it with 250 μL of paraffin oil to prevent evaporation. The in vitro mature COCs were digested with 0.1% hyaluronidase to remove cumulus cells, and the oocytes that extruded the first polar body were picked out with a self-made mouth pipette under a stereomicroscope. The picked mature oocytes were washed 3 times with the in vitro operation solution and set aside. Select 20 oocytes, wash them 3 times with the fusion solution to allow them to come into full contact with the fusion solution. At the same time, the metal electrodes were pre-washed 3 times with the fusion solution, and then 40 μL of the fusion solution was added between the electrodes. Subsequently, these 20 oocytes were transferred between metal electrodes with a width of 1 mm, ensuring that the oocytes were arranged in a line in the center between the two poles and avoiding their direct contact with the electrodes. The distance between the oocytes was about the distance of 10 oocytes. Immediately afterwards, a pulsed voltage with a voltage of 130 V and a duration of 80 μs was applied once. After applying the pulsed voltage, pause for about 5 s, transfer the cells to the chemical assisted activation solution and wash them 3 times. After completing the above operations, transfer the oocytes to the pre-equilibrated chemical assisted activation solution for a 4-hour chemical assisted activation process. After the activation treatment is completed, take out the oocytes, wash them 3 times with the PZM-3 culture solution, and then transfer them to the pre-equilibrated PZM-3 culture solution for continuous culture. When cultured to 48 hours, observe and count the cleavage rate. The cleavage rate is obtained through the following formula: (the number of oocytes that have cleaved ÷ the total number of parthenogenetically activated oocytes) × 100%; when cultured to 168 hours, observe the blastocyst rate. The calculation formula of the blastocyst rate is: (the number of blastocysts formed ÷ the number of oocytes that have cleaved) × 100%. Among them, the detection method of the total number of cells in the blastocyst is as follows:

[0074] (1) Wash the blastocysts 3 times in PBS-PVA;

[0075] (2) Fix with 4% paraformaldehyde (PFA) and store at 4 °C;

[0076] (3) Wash the fixed blastocysts 3 times with PBS-PVA;

[0077] (4) Permeabilize in PBS-PVA containing 0.5% Triton X-100 for 1 h;

[0078] Wash three times with PBS-PVA;

[0079] Block with 3% BSA at room temperature for 2 h;

[0080] Wash three times with PBS-PVA, 5 min each time;

[0081] Mount the slides with mounting medium containing DAPI;

[0082] Take pictures with a fluorescence microscope;

[0083] Analyze and count the number of cells using ImageJ software.

[0084] The effects of adding 10 μM dendrobine and 10 μM mercapto-modified dendrobine on embryonic development are as Figure 4 shown. It can be Figure 4 seen that the cleavage rate of eggs added with 10 μM dendrobine and 10 μM mercapto-modified dendrobine is significantly higher than that of the control group, and the cleavage rate of eggs added with 10 μM mercapto-modified dendrobine is significantly higher than that of the group treated with 10 μM dendrobine; the blastocyst rate of eggs added with 10 μM dendrobine and 10 μM mercapto-modified dendrobine is significantly higher than that of the control group, and the blastocyst rate of eggs added with 10 μM mercapto-modified dendrobine is significantly higher than that of the group treated with 10 μM dendrobine; the total cell number rate of blastocysts of eggs added with 10 μM dendrobine and 10 μM mercapto-modified dendrobine is significantly higher than that of the control group, and the total cell number of blastocysts of eggs added with 10 μM mercapto-modified dendrobine is significantly higher than that of the group treated with 10 μM dendrobine.

[0085] In summary, in the present invention, a mercapto (-SH) functional group is introduced into the molecular structure of dendrobine through chemical synthesis to obtain modified dendrobine. By adding modified dendrobine to the IVM medium, the newly introduced mercapto (-SH) functional group can directly undergo a redox reaction with ROS (such as superoxide anion and hydroxyl radical), enhancing the free radical scavenging ability and reducing ROS; and it can significantly increase the level of GSH; it can effectively relieve the oxidative stress of porcine oocytes, reduce the level of early apoptosis, maintain the mitochondrial membrane potential, and increase the blastocyst rate.

[0086] The technical solution of the present invention is not limited to the limitations of the above specific embodiments. Any technical deformation made according to the technical solution of the present invention falls within the protection scope of the present invention.

Claims

1. Application of mercapto-modified dendrobine in in vitro maturation culture of oocytes, characterized in that, The in vitro maturation medium for porcine oocytes is supplemented with thiol-modified dendrobine at a concentration of 5 μM - 20 μM; wherein, the preparation method of the thiol-modified dendrobine is as follows: Take 1 mol of dendrobine and dissolve it in N,N-dimethylformamide to obtain a mixed solution. Under stirring, add 1.2 - 1.5 mol of mercaptoacetic acid, 0.05 mol of 4-dimethylaminopyridine, and 0.1 mol of N,N'-dicyclohexylcarbodiimide to the mixed solution, and react at 40 °C for 8 - 12 h. Filter and collect the filtrate, remove the organic solvent by vacuum distillation, and purify the thiol-modified dendrobine by silica gel column chromatography. The eluent for silica gel column chromatography is an ethyl acetate / petroleum ether system, and the volume ratio of ethyl acetate to petroleum ether is 5% - 20%.

2. Use of the mercapto-modified dendrobine according to claim 1 in in vitro maturation culture of oocytes, characterized in that, The preparation method of the in vitro maturation medium for porcine oocytes is as follows: 1) Take 10 μL of L-Cysteine, 500 μL of FBS, 10 μL of EGF, 1 mL of PEF, and 100 μL of Pen-Strep(100x), mix them, and make up to 10 mL with M199 medium to prepare the in vitro maturation medium for oocytes. After preparation, shake well, filter and sterilize with a 0.22 μm filter, seal and store at 4 °C for later use; 2) Take 9.6 mL of the in vitro maturation medium for oocytes, and add 100 μL of Glu-max(100x), 250 μL of PG600, and 50 μL of hCG to prepare 10 mL of the working solution of the in vitro maturation medium for oocytes; 3) Add thiol-modified dendrobine to the working solution of the in vitro maturation medium for oocytes to make the final concentration of thiol-modified dendrobine 5 μM - 20 μM.

3. An in vitro maturation culture method for oocytes, characterized in that, It includes the following steps: S1. After obtaining the freshly slaughtered porcine ovarian tissue, immediately put it into 9% sterile physiological saline at 38 °C, and then first remove the excess connective tissue and ovarian ligament on the ovary; subsequently, quickly rinse the ovary with 75% medical alcohol preheated to 38.5 °C and gently squeeze the ovary by hand to facilitate the extrusion of blood impurities adhering to the ovarian surface. Then quickly rinse the ovary three times with physiological saline preheated to 38.5 °C and gently squeeze the ovary by hand to facilitate the extrusion of blood impurities adhering to the ovarian surface. Put it into a water bath at 38.5 °C and use a syringe needle with the bevel facing down to extract the follicular fluid from follicles with a diameter of 3 - 8 mm. After pulling out the needle, transfer it to a 50 mL centrifuge tube for natural precipitation. The centrifuge tube is placed in a water bath to maintain the temperature and the natural sedimentation time is not less than 20 min; after the oocytes in the follicular fluid are fully sedimented, use a syringe to aspirate the excess supernatant and retain 5 mL of the precipitate, then add 25 mL of egg washing solution, and sediment naturally for another 15 min. After full sedimentation, discard the supernatant and retain the precipitate. Gently shake the precipitate and then aspirate 5 mL of the precipitate and drop it onto a 100 mm marked culture dish, and add an appropriate amount of egg washing solution and shake gently; start the heating stage equipped with a stereomicroscope, and use a glass pipette to screen out porcine oocytes with clear cytoplasmic edges, uniform wrapping states, and overall integrity within the field of view of the stereomicroscope. S2. The selected porcine oocytes are washed three times successively in the oocyte washing solution, then washed three times in the porcine oocyte in vitro maturation culture medium prepared in claim 2. After that, 500 μL of the porcine oocyte in vitro maturation culture medium is added to each well of a four-well plate, and 50 - 60 porcine oocytes are added to each well. Finally, the four-well plate is placed in a cell incubator at 38.5 °C and 5% CO2 for 42 h.

4. The in vitro maturation culture method of oocytes according to claim 3, characterized in that, The preparation method of the oocyte washing solution is as follows: 1.000 g of PVA is added to 50 mL of ultrapure water, and pre-sterilized magnetic beads are put in. The beaker is placed on a magnetic stirrer, the temperature is set at 55 °C, the stirring rate is set at 400 r / min, and stirring is continued for 30 min. After cooling, 9.550 g of DPBS is added, and stirring is continued with the magnetic stirrer until dissolution. Then, ultrapure water is added to make the volume of the solution up to 1000 mL. Subsequently, the clear supernatant is collected and filtered to obtain the oocyte washing solution, which is stored in a refrigerator at 4 °C.

Images