Application of cordycepin in aspect of improving in-vitro early embryonic development potential

Abstract

The invention discloses an application of cordycepin (Cordycepin, Cor) in the aspect of improving the development potential of an in-vitro early embryo. The inventor finds that Cor with a proper concentration is added into a culture medium in the in-vitro maturation process of the porcine oocytes, the Cor can promote in-vitro maturation of the porcine oocytes and improve the quality of the oocytes, and the Cor has a promoting effect on the development potential of early embryos after parthenogenetic activation. Therefore, the inventor constructs a porcine oocyte and early embryo in-vitro culture system, optimizes an in-vitro maturation culture solution formula and a Cor concentration, and further establishes a method for improving the development potential of the in-vitro early embryo, that is, the porcine oocyte is subjected to in-vitro maturation culture, parthenogenetic activation and early embryo in-vitro culture; in-vitro maturation culture liquid containing cordycepin is adopted in the in-vitro maturation culture of the porcine oocytes. In a word, the invention provides a research basis for developing a safe and effective natural small molecule medicament capable of being used for improving the reproductive performance of the sow, provides a reference for in-vitro mass production of high-quality livestock embryos, and has important practical significance in the fields of IVM and IVC.

Description

Technical Field

[0001] The invention belongs to the technical field of embryo development research, and in particular relates to the application of cordycepin in improving the development potential of early embryos in vitro. Background Art

[0002] In Vitro Embryo Production (IVP) refers to the use of biotechnology to cultivate and develop embryos in vitro. It is usually used in assisted reproductive technology, such as in vitro fertilization, embryo transfer, and egg and embryo cryopreservation technology. There is a great difference in quality between embryos developed in vitro and those produced in vivo. In vitro maturation of oocytes (IVM) and in vitro development of early embryos (IVC) are key steps in IVP technology. IVM aims to develop oocytes from the primitive state of cumulus-oocyte complex into mature MⅡ stage oocytes, thereby laying a solid foundation for improving the developmental potential of IVC embryos. The key to IVP technology is to use in vitro culture medium to simulate the microenvironment in vivo, improve the quality of in vitro oocytes, and promote the maturation of immature oocytes at the same time, which is crucial for the successful development of pre-implantation embryos. Although pig IVP technology has made significant progress at home and abroad, there are still problems such as relatively low in vitro maturation efficiency of the existing culture system, low embryo development potential, certain differences in developmental ability compared with in vivo, and low in vitro embryo yield.

[0003] IVC technology is inseparable from IVM technology, and IVM technology has high requirements for culture conditions. The instability of culture conditions may change the metabolism of oocytes, affect the quality of oocytes and the subsequent development of embryos, such as culture environment, culture time, culture medium composition and proportion, etc. Porcine oocytes are rich in lipids, which are particularly sensitive to reactive oxygen species (ROS). High levels of ROS may lead to lipid peroxidation, mitochondrial dysfunction, DNA breakage, and oxidative modification of proteins, thereby impairing the maturation of oocytes and ultimately reducing the potential for early embryonic development. Although a variety of antioxidants and other drugs have been added to in vitro culture media, such as resveratrol, melatonin, and sodium selenite, they are not widely used in in vitro culture of oocyte maturation and early embryos because they may have toxic side effects on cells and are expensive. Studies have shown that some natural Chinese herbal extracts have the characteristics of low toxicity, no residue, and drug resistance. Their main active ingredients show excellent antioxidant capacity and can play an important role as natural small molecules in the treatment of various diseases.

[0004] Cordycepin (Cor) is a natural active substance extracted from Cordyceps sinensis or Cordyceps militaris. It is a nucleotide composed of adenosine and deoxypentose with carbon branches. It is also a nucleoside antibiotic and belongs to alkaloids. According to research reports, cordycepin has anti-inflammatory, antiviral, anti-tumor, antibacterial, antioxidant, and immunomodulatory functions, and plays an important regulatory role in a variety of inflammatory diseases and oxidative stress injury diseases. However, there are no reports on whether Cor can promote porcine oocyte meiosis, improve in vitro oocyte quality and maturation efficiency, improve early embryonic development potential, and be used for parthenogenetic activation. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an application of cordycepin in improving the development potential of early embryos in vitro.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] Application of cordycepin in improving the developmental potential of early embryos in vitro.

[0008] The embryos were derived from porcine oocytes.

[0009] Improving the developmental potential of early embryos in vitro means increasing the embryo's cleavage rate, blastocyst rate, and blastocyst cell proliferation activity.

[0010] Application of cordycepin in the preparation of in vitro maturation culture medium for porcine oocytes.

[0011] Porcine oocyte in vitro maturation culture medium, containing cordycepin.

[0012] The above-mentioned porcine oocyte in vitro maturation culture medium is based on conventional culture medium and added with cordycepin.

[0013] The concentration of cordycepin was 2.5 μM.

[0014] The method for improving the development potential of early embryos in vitro is to perform in vitro maturation culture, parthenogenetic activation and in vitro culture of early embryos on pig oocytes, and the pig oocytes are cultured in vitro using an in vitro maturation culture medium containing cordycepin.

[0015] The in vitro maturation culture medium is based on conventional culture medium and supplemented with cordycepin.

[0016] The concentration of cordycepin was 2.5 μM.

[0017] When studying the application of cordycepin (Cor) in the field of pig IVP, the inventors found that adding an appropriate concentration of Cor to the culture medium of the in vitro maturation process of pig oocytes would promote the in vitro maturation of pig oocytes and promote the development potential of early embryos after parthenogenetic activation. To this end, the inventors constructed an in vitro culture system for pig oocytes and early embryos, optimized the formula of in vitro maturation culture medium and the concentration of Cor, and then established a method for improving the development potential of early embryos in vitro, that is, in vitro maturation culture, parthenogenetic activation and in vitro culture of early embryos of pig oocytes, and in vitro maturation culture medium containing cordycepin was used for in vitro maturation culture of pig oocytes. In summary, the present invention performs in vitro maturation and culture of porcine oocytes, parthenogenetic activation, and in vitro culture of early embryos, observes the ability of oocytes to reach the MII stage and the quality of subsequent embryos, and reveals the role of Cor in improving the developmental potential of early embryos after in vitro maturation and parthenogenetic activation of porcine oocytes. This provides a research basis for the development of safe and effective natural small molecule agents that can be used to improve the reproductive performance of sows, and provides a reference for the in vitro mass production of high-quality livestock embryos, which has important practical significance in the field of IVM and IVC. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a diagram showing the effect of Cor on the expansion of the cumulus and the first polar body of the oocyte. In the figure: A is a statistical diagram of the expansion of the cumulus in COCs of the Con group and the Cor (1.25, 2.5, 5 and 10 μM) group; B is a statistical diagram of the first polar body extrusion rate of oocytes in the Con group and the Cor (1.25, 2.5, 5 and 10 μM) group; C is a typical bright field diagram of the expansion of the cumulus in COCs of the Con group and the optimal concentration Cor 2.5 μM group; D is a typical bright field diagram of the first polar body extrusion of oocytes in the Con group and the optimal concentration Cor 2.5 μM group. (Scale bar is 100 μm)

[0019] Figure 2 This is a diagram showing the effect of Cor on the oxidative stress level of oocytes. In the figure: A is a typical diagram of ROS content in oocytes in the Con group and the Cor group; B is a statistical diagram of ROS fluorescence intensity in oocytes in the Con group and the Cor group. (Scale bar is 100μm)

[0020] Figure 3 This is the effect of Cor on the lipid droplet content of oocytes. In the figure: A is a typical diagram of lipid droplet content in oocytes of Con group and Cor group; B is a statistical diagram of lipid droplet fluorescence intensity in oocytes of Con group and Cor group. (Scale bar is 20μm)

[0021] Figure 4This is a diagram showing the effect of Cor on the subsequent development rate of oocytes. In the figure: A is a typical bright field image of parthenogenetic activated embryo cleavage, blastocyst and hatched embryo in the Con group and the Cor group; B is a statistical graph of cleavage rate; C is a statistical graph of blastocyst rate; D is a statistical graph of blastocyst hatching rate. (Scale bar is 100μm)

[0022] Figure 5 This is a diagram showing the effect of Cor on the subsequent developmental potential of oocytes. In the figure: A is a typical diagram of blastocyst proliferation cells and blastocyst cells in the Con group and the Cor group; B is a statistical diagram of the number of blastocyst cells in the Con group and the Cor group; C is a statistical diagram of the activity (EdU / DNA%) of blastocyst proliferation cells in the Con group and the Cor group. (Scale bar is 100μm)

[0023] Note: In the figure, Con is the control group, and 2.5μm is the experimental group; * indicates that there is a difference at P<0.05, ** indicates that there is an extremely significant difference at P<0.01, *** indicates that there is an extremely significant difference at P<0.001, and **** indicates that there is an extremely significant difference at P<0.0001; data are expressed as Mean±SEM; AU represents arbitrary units. DETAILED DESCRIPTION

[0024] 1. Oocyte Collection

[0025] The ovaries were obtained from the slaughterhouse and transported to the laboratory in 34℃ sterile saline. The ovaries were repeatedly washed in a beaker with sterile saline preheated at 37.5℃ until no blood and water ooze out, and saline was added to cover the ovaries. The beaker containing the ovarian material was transferred to a 37.5℃ water bath between embryos. The follicular fluid from the 2-8mm antral follicles on the surface of the ovary was aspirated with a 10mL syringe into a 50mL centrifuge tube, which was placed in a 37.5℃ water bath to fully settle for 20 minutes and then the supernatant was discarded. The precipitate was aspirated into a 60mm culture dish with a pipette, diluted with egg washing solution, and placed under a stereo microscope with a 37.5℃ hot stage. The cumulus-oocyte complexes (COCs) with intact cytoplasm, uniform size, and densely wrapped cumulus were selected with a glass capillary egg aspiration needle, and transferred to the preheated oocyte washing solution and in vitro maturation solution, and the COCs were washed until there was no cell debris. After washing and collecting the COCs, they were ready for the next step of culture.

[0026] The oocyte washing solution composition is shown in Table 1. After adjusting the pH with NaOH, the solution was filtered through a 0.22 μM filter and stored at 4°C for later use.

[0027] Table 1 Oocyte cleaning solution composition

[0028]

[0029]

[0030] Screening of 2Cor concentration and in vitro maturation of oocytes

[0031] Cor was dissolved in DMSO (final concentration not exceeding 0.1%), prepared into a stock solution with a concentration of 100mM and stored at -20°C. Different concentrations of Cor (0, 1.25, 2.5, 5 and 10μM) were added to the maturation solution to prepare in vitro maturation solution. 400μL of in vitro maturation solution was added to each well of a four-well plate, covered with 200μL of mineral oil, and balanced in an incubator for more than 6h. The selected COCs were transferred to them according to the experimental groups, 100 COCs were placed in each well, and cultured in a 38.5°C, 5% CO2 incubator for 44h until the oocytes matured. After 44h of culture, the oocytes were digested with 0.1% (w / v) hyaluronidase until all the surrounding cumulus cells were peeled off. The expansion of the oocyte cumulus and the discharge of the first polar body were observed under a stereomicroscope, and the optimal concentration of Cor was screened according to the relative expansion area of ​​the cumulus and the polar body discharge rate for subsequent experiments.

[0032] The components of the in vitro maturation culture medium are shown in Tables 2 and 3. The pH of the experimental group culture medium and the control group culture medium was adjusted with NaOH, filtered with a 0.22 μM filter, and stored at 4° C. for later use.

[0033] The purity of Cor (source: Guangdong Rongda Biological Co., Ltd.) used in the experimental research of the present invention is 99.4%.

[0034] Table 2 Composition of the culture medium for in vitro maturation of porcine oocytes in the experimental groups

[0035]

[0036]

[0037] Table 3 Composition of in vitro maturation medium of porcine oocytes in the control group

[0038]

[0039] 3. Oocyte parthenogenetic activation

[0040] The mature oocytes in each group were parthenogenetically activated by electrical stimulation. After 40-44h maturation culture of COCs, the oocytes were digested with 0.1% (w / v) hyaluronidase until all the surrounding cumulus cells were peeled off. The oocytes were observed under a stereomicroscope, and the mature oocytes containing the first polar body were selected from the cells cultured in the control group and the experimental group. 100μL of electrofusion solution FA was added to the electrofusion tank, and the selected oocytes were placed in the electro-activation solution FA for balancing three times and then moved to the electrofusion tank. The fusion instrument was started and electro-activated with 2 direct current pulses of 30μs and 1.3kv / cm. The activated oocytes were thoroughly cleaned of residual FA solution with balanced PZM-3 culture solution. After cleaning and collection, they were prepared for the next step of culture.

[0041] The composition of the FA activation solution is shown in Table 4. After adjusting the pH with NaOH, the solution was filtered with a 0.22 μM filter and stored at 4° C. for later use.

[0042] Table 4 FA Fusion Activation Solution Composition

[0043]

[0044]

[0045] 4 Early embryo culture in vitro

[0046] Prepare in vitro culture droplets in advance, prepare 7 50μL PZM-3 culture droplets in a 30mm culture dish, cover with 2.5mL mineral oil, and equilibrate in the incubator for more than 6 hours. Transfer the activated embryos to each droplet according to the experimental group, 20 embryos per droplet, and carry out subsequent culture in a 38.5℃, 5% CO2 incubator. After 48h and 168h of culture, check the cleavage rate and blastocyst rate respectively.

[0047] The components of the PZM-3 embryo culture medium are shown in Table 5. The pH of the PZM-3 embryo culture medium was adjusted with NaOH, filtered through a 0.22 μM filter, and stored at 4° C. for later use.

[0048] Table 5 PZM-3 embryo culture medium composition

[0049]

[0050] 5. Detection of Oocyte Reactive Oxygen Species Level

[0051] According to the instructions of the reactive oxygen detection kit. After 44 hours of in vitro maturation, the COCs were fully digested to obtain oocytes, which were washed three times in 0.1% PVA-DPBS. Using the DCFH-DA detection kit, the oocytes were transferred to 10μM DCFH-DA dye diluted in 0.1% PVA-DPBS and incubated in a 38.5℃ incubator for 30min. After incubation, they were washed three times with 0.1% PVA-DPBS to remove unbound fluorescent probes. The oocytes were then immediately placed under a fluorescence microscope to obtain fluorescence images and calculate fluorescence intensity.

[0052] 6. Detection of oocyte lipid droplet content

[0053] The neutral lipid droplet fluorescent probe BODIPY was used to detect the lipid droplet content of oocytes. After 44 hours of in vitro maturation, COCs were fully digested to obtain oocytes, which were washed three times in 0.1% PVA-DPBS. Transferred to a dye solution in which the BODIPY probe was diluted to 10 μg / mL with maturation solution and placed in an incubator for incubation for 30 minutes. After incubation, it was washed three times with 0.1% PVA-DPBS to remove unbound fluorescent probes. Immediately afterwards, the oocytes were placed in a confocal dish and placed under a confocal laser scanning microscope to obtain fluorescence images and calculate the fluorescence intensity.

[0054] 7. Embryo quality testing after parthenogenetic activation

[0055] The quality of embryos was tested using the EdU-488 cell proliferation detection kit. After 168 hours of embryonic development, the expanding blastocysts were taken for EdU staining under a microscope. Prepare the EdU labeling solution in advance, dilute the EdU labeling solution with pzm3 culture medium to a final concentration of 10 μM working solution, make culture droplets, cover with mineral oil, and balance in the incubator for 3 hours. After the embryos were washed three times with 0.1% PVA-DPBS, they were transferred to the EdU labeling droplets and incubated at 38.5°C for 3 hours. Fix the embryos with 4% PFA at room temperature for 15 minutes, and wash them three times with PBS containing 3% BSA after fixation, each for 3 minutes. Permeabilize with PBS containing 0.3% Triton X-100 at room temperature for 20 minutes, and wash three times with PBS containing 3% BSA after permeabilization, each for 3 minutes. Prepare the Click reaction solution according to the instructions, incubate the embryos in the reaction solution at room temperature in the dark for 30 minutes, and wash them three times with PBS containing 3% BSA after incubation, each for 3 minutes. Finally, the embryos were stained with Hoechst33342 for 15 min at room temperature, rinsed three times with 0.1% PVA-DPBS, fixed on a slide coated with glycerol, and covered with a cover glass with lanolin. Finally, images were acquired with a fluorescence microscope and the fluorescence intensity was calculated.

[0056] 8 Experimental results

[0057] (1) After 44 hours of in vitro maturation, the cumulus expansion area and the first polar body extrusion rate of COCs were statistically analyzed. Figure 1 As shown, the oocyte in vitro maturation medium containing 2.5μM Cor significantly increased the COCs cumulus expansion area compared with the Con group (1.47±0.04VS1.0±0.05), the COCs cumulus expansion area in the 1.25μM Cor group had no significant difference compared with the Con group, while the COCs cumulus expansion area in the 5μM and 10μM Cor groups was significantly reduced compared with the Con group. In addition, the first polar body extrusion rate of oocytes was significantly increased after 2.5μM Cor treatment (85.90±1.69%VS 72.89±1.99%), the first polar body extrusion rate of oocytes in the 1.25μM and 5μM Cor groups had no significant difference compared with the Con group, while the first polar body extrusion rate of oocytes in the 10μM Cor group was significantly reduced compared with the Con group. The results show that the addition of 2.5μM Cor can effectively improve the maturation rate of oocytes and promote oocyte meiosis. Therefore, in subsequent experiments, 2.5μM concentration of Cor was selected for research.

[0058] (2) Oocytes are prone to oxidative stress and are very sensitive to ROS. The effect of Cor on the oxidative stress level of in vitro matured oocytes was investigated by using the DCFH-DA probe to label ROS in oocytes. Figure 2 As shown, compared with the control oocyte in vitro maturation culture medium, the oocyte in vitro maturation culture medium containing 2.5μM Cor can significantly reduce the fluorescence intensity of DCFH-DA in oocytes (42.43±1.2VS 31.85±1.0). The results show that adding 2.5μM Cor can significantly reduce the level of reactive oxygen and improve the quality of oocytes.

[0059] (3) Lipid droplets are an important form of energy storage in oocytes, providing energy for oocyte maturation and early embryonic development. Oocytes were stained with BODIPY493 / 503 fluorescent probe to detect the lipid droplet content in oocytes. Figure 3 As shown, compared with the control oocyte in vitro maturation culture medium, the oocyte in vitro maturation culture medium containing 2.5μM Cor can significantly increase the BODIPY493 / 503 fluorescence intensity (15.32±0.2VS23.82±0.2). The results show that the addition of 2.5μM Cor can significantly increase the lipid droplet content of oocytes, promote the lipid droplet synthesis of porcine oocytes in vitro maturation, and improve the quality of oocytes.

[0060] (4) The quality of oocytes directly affects the developmental capacity of early embryos. In order to study the effect of Cor on the cleavage rate and blastocyst rate of parthenogenetically activated porcine embryos, oocytes cultured in the oocyte in vitro maturation medium of the experimental group and the control group were parthenogenetically activated and cultured to blastocysts. The cleavage rate was checked after 48 hours, the blastocyst rate was checked after 144 hours, and the blastocyst hatching rate was calculated after 168 hours. Figure 4 As shown, compared with the control group, adding 2.5 μM Cor to the culture medium can significantly promote the cleavage rate (75.0±1.8% VS 83.3±1.7%), blastocyst rate (31.0±1.0% VS 38.0±1.2%) and hatching rate (8.0±1.23% VS 14.0±1.0%) of subsequent parthenogenetic activated embryos. The results show that adding 2.5 μM Cor can significantly promote the subsequent embryonic development of oocytes and improve the potential of early embryonic development in vitro.

[0061] (5) The number and proliferation activity of cells in blastocysts are important indicators of embryo quality. In order to study the effect of Cor on the number and proliferation activity of blastocysts after parthenogenetic activation of oocytes, blastocysts were stained with Hoechst33342 and EdU after 144 hours of in vitro culture. Figure 5 As shown, the number of blastocyst cells (44.45±0.8VS55.41±0.7) and the ratio of EdU positive cells to Hoechst positive cells (EdU / DNA%), i.e., the proliferation activity of blastocyst cells (33.25±0.8%VS47.41±1.0%), after treatment with 2.5μM Cor were significantly increased compared with the control group. The results showed that the addition of 2.5μM Cor can significantly increase the number of embryonic cells, improve cell proliferation activity, improve the quality of in vitro embryos, and enhance the potential of early embryonic development.

Claims

1. Application of cordycepin in improving the potential of early embryonic development in vitro.

2. The use according to claim 1, Features: The embryos were derived from porcine oocytes.

3. The use according to claim 2, Features: The improvement of the in vitro early embryo development potential is to improve the embryo's cleavage rate, blastocyst rate, and blastocyst cell proliferation activity.

4. Application of cordycepin in the preparation of in vitro maturation culture medium for porcine oocytes.

5. A porcine oocyte in vitro maturation culture medium, Features Contains cordycepin.

6. The porcine oocyte in vitro maturation culture medium according to claim 5, Features Cordycepin is added based on conventional culture medium.

7. The porcine oocyte in vitro maturation culture medium according to claim 6, Features: The concentration of cordycepin is 2.5 μM.

8. A method for improving the developmental potential of early embryos in vitro, Features: The porcine oocytes are subjected to in vitro maturation culture, parthenogenetic activation and in vitro early embryo culture, and the porcine oocytes are subjected to in vitro maturation culture using an in vitro maturation culture medium containing cordycepin.

9. The method for improving the developmental potential of early embryos in vitro according to claim 8, Features: The in vitro maturation culture medium is based on conventional culture medium and cordycepin is added.

10. The method for improving the developmental potential of early embryos in vitro according to claim 9, Features: The concentration of cordycepin is 2.5 μM.

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