Application of fisetin in preparation of products for promoting embryonic development

Abstract

The invention discloses application of fisetin in preparation of a product for promoting embryonic development, and belongs to the technical field of in-vitro embryos. The invention provides application of fisetin in preparation of a product for promoting embryonic development. The fisetin is added into the in-vitro culture medium, so that the fisetin is proved to be capable of improving the blastocyst formation rate of in-vitro embryo culture, increasing the total cell number of the embryo, improving the mitochondrial function of the in-vitro embryo, improving the multiplication capacity of the in-vitro embryo and relieving the endoplasmic reticulum stress level of the in-vitro embryo, thereby effectively improving the quality and development capacity of the in-vitro embryo.

Description

Technical Field

[0001] The invention relates to the field of in vitro embryo technology, in particular to application of fisetin in preparing products for promoting embryo development. Background Art

[0002] In recent years, in vitro embryo production (IVEP) is an important technology of great significance to the genetic improvement of animals. It can be used in agricultural production, assisted reproductive technology, and early embryonic development of female animals. It is considered an important tool for genetic improvement through the maternal line, so the quality of female eggs selected is no less than that of high-standard sperm in artificial insemination. Data show that the total number of bovine embryos cultured in vitro in 2021 was 1,499,136, almost four times the number of embryos in vivo. However, despite the continuous optimization of in vitro maturation and development medium formulas, it is still difficult to simulate the optimal in vitro culture environment for embryos, and the quality and subsequent development of embryos are still low, especially in the in vitro embryo production system for pigs. Therefore, the continuous development and optimization of these culture systems is of great significance to improving the quality of early pig embryos.

[0003] Embryo in vitro culture refers to artificially simulating the developmental microenvironment of the embryo in the maternal fallopian tube and uterus, including sufficient nutrients and pH, suitable culture medium, temperature, humidity and gas environment, so that the early embryos obtained by in vitro fertilization or intracytoplasmic sperm injection continue to develop in an in vitro environment. In this process, the embryos are often implanted into the mother in the form of blastocysts, which can achieve a higher embryo implantation rate. However, because the oxygen content in the in vitro environment is higher than that in vivo, embryos operated in vitro are susceptible to oxidative stress, and early embryos are more sensitive to damage caused by oxidative stress. Therefore, it is considered to add natural products with antioxidant activity to the in vitro culture medium of early embryos to reduce the level of oxidative stress in the embryo. Existing studies have shown that adding substances with antioxidant activity such as anethole, kaempferol and melatonin to the in vitro culture medium can reduce the level of oxidative stress in the embryo, thereby improving the development rate and quality of the embryo. Due to the similarities between pig oocytes and humans, this study used pig oocytes as a research model for subsequent exploration.

[0004] Fisetin is a bioactive flavonol widely found in various fruits and vegetables, such as strawberries, apples, persimmons, grapes, onions and cucumbers. Several studies have shown that Fisetin is a promising new antioxidant. It can extend the lifespan of animals and has biological properties such as neuroprotection. In addition, Fisetin can be used as an anti-aging drug to delay cell senescence, thereby improving arterial function in aged mice. Not only that, Fisetin can also reduce the senescence of cultured human endothelial cells. Some researchers have used aged mice as a research model to prove that FIS can delay the aging of postovulatory oocytes. Fisetin can also be used as a dietary supplement as a PPAR-γ agonist to reduce myocardial infarction area in mice and restore myocardial ischemia-reperfusion models in mice. In addition, due to its multiple biological activities, FIS is considered a health-promoting factor preparation, and there are already several dietary supplements containing Fisetin on the market. However, to date, the effects of Fisetin on the reproductive system of mammals and the related mechanisms remain unclear. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes the use of fisetin in the preparation of a product for promoting embryo development, which can effectively promote the embryo development ability and quality.

[0006] According to a first aspect of the present invention, the use of fisetin in preparing a product for promoting embryo development is proposed.

[0007] According to a preferred embodiment of the present invention, at least the following beneficial effects are achieved:

[0008] Fisetin (FIS), a natural flavonol compound, can be used in the study of diseases such as cancer and nerve damage. It has the following chemical structure:

[0009]

[0010] The present invention discloses for the first time the application of fisetin in the preparation of a drug for improving the in vitro embryo development ability. Previously, there has been no report that fisetin is related to the in vitro embryo development ability, which expands the application scope of fisetin.

[0011] The present invention applies fisetin to the field of embryo technology for the first time, and in particular to the in vitro culture technology of preimplantation embryos of mammals. The present invention finds that fisetin can increase the blastocyst formation rate of embryos and the total number of cells in embryos during preimplantation embryo development; the component can also increase the mitochondrial function and cell proliferation of early embryo cells; the component can also reduce the level of endoplasmic reticulum stress marker protein GRP78, prevent embryos from experiencing more oxidative stress, thereby improving the quality of in vitro embryo culture and developmental ability, and is beneficial to in vitro embryo culture.

[0012] Fisetin is a flavonol compound that can also be used in medicine. It has no toxic side effects on mammalian cells, is widely available, and is easy to obtain. Adding fisetin to the culture medium is simple to operate, easy to control, and has significant effects.

[0013] Fisetin can be used to prepare pharmaceutical compositions, culture media or kits for promoting embryonic development, and has good application prospects.

[0014] According to some embodiments of the present invention, the product is a kit, a culture medium or a drug.

[0015] According to some embodiments of the present invention, the promoting embryo development comprises at least one of improving embryo quality or improving embryo development ability.

[0016] According to some embodiments of the present invention, the promoting embryo development includes at least one of increasing the embryo blastocyst rate, increasing the total number of embryo cells, improving the embryo proliferation ability, enhancing the mitochondrial function of embryonic cells and reducing the level of endoplasmic reticulum stress marker protein.

[0017] Fisetin can effectively improve the development quality and developmental capacity of in vitro embryos by increasing the blastocyst formation rate, increasing the total cell number, effectively protecting mitochondrial function, and promoting embryo proliferation ability, while alleviating the endoplasmic reticulum stress marker GRP78 level.

[0018] According to some embodiments of the present invention, the quality and developmental capacity of in vitro embryos are improved by increasing the blastocyst formation rate of in vitro embryos.

[0019] As verified by the in vitro embryo blastocyst formation rate test, compared with the case where the product of the present invention is not administered, the administration of the product of the present invention increases the in vitro embryo blastocyst formation rate by 32.32±15.28%.

[0020] According to some embodiments of the present invention, the quality and developmental capacity of in vitro embryos are improved by increasing the total cell number of in vitro embryos.

[0021] The increase in the total number of cells in the embryo represents an improvement in the level of embryonic division, which in turn reflects the quality of the in vitro embryo.

[0022] As verified by the in vitro embryo total cell number test, compared with the case where the product of the present invention is not administered, the administration of the product of the present invention increases the in vitro embryo total cell number by 17.07±0.19%.

[0023] According to some embodiments of the present invention, the quality and developmental capacity of in vitro embryos are improved by enhancing the mitochondrial function of in vitro embryonic cells.

[0024] Mitochondria are the center of cellular metabolic activities, participating in the energy generation of adenosine triphosphate (ATP) and many biosyntheses, maintaining intracellular calcium ion homeostasis, and regulating the cellular levels of various enzyme metabolites, amino acids, and cofactors. Mitochondria mainly generate energy through oxidative phosphorylation reactions, and then use tricarboxylic acid cycle enzymes to generate electron carriers, deliver electrons to the electron transport chain, and regulate redox reactions. However, mitochondria are also the main site for the production of reactive oxygen species. Excessive reactive oxygen species can cause mitochondrial dysfunction in oocytes, damage DNA, lipid membranes, and proteins, and stop cell division.

[0025] As verified by the in vitro embryo mitochondrial function test, compared with the case where the product of the present invention is not administered, the administration of the product of the present invention improves the in vitro embryo mitochondrial function by 14.00±1.29%.

[0026] According to some embodiments of the present invention, the quality and developmental capacity of in vitro embryos are improved by improving the cell proliferation capacity of in vitro embryos.

[0027] Preferably, the proliferation level of the in vitro embryo is evaluated by calculating the proliferation rate by detecting / calculating the ratio of the number of EDU-positive cells in the in vitro embryo to the total number of cells.

[0028] Cell proliferation is an important life characteristic of organisms. Cells proliferate by dividing, which is the basis of growth, development, reproduction and inheritance of organisms. Cell proliferation refers to a series of division processes in which cells undergo complex reactions such as DNA replication, RNA transcription and protein synthesis under the action of cycle regulatory factors, among which the replication and doubling of nuclear DNA is an important feature of the whole process. Cell proliferation can be used to assess the health of cells, measure the response to toxic damage, or be used as a prognostic and diagnostic tool for a variety of stimuli. Available markers usually depend on DNA levels or synthesis, cell metabolism or proliferation-specific proteins. By overexpressing or interfering with a gene in cells, the effect of a gene on cell proliferation ability can be studied, the function of the gene can be further studied, or the cells can be treated with drugs to study the effect of drugs on proliferation.

[0029] As verified by the in vitro embryo proliferation ability test, compared with the case where the product of the present invention is not administered, the administration of the product of the present invention increases the in vitro embryo proliferation level by 27.56±0.09%.

[0030] According to some embodiments of the present invention, the quality and developmental capacity of in vitro embryos are improved by reducing the level of GRP78, an endoplasmic reticulum stress marker in in vitro embryos.

[0031] When cells are stimulated by the external environment, as an endoplasmic reticulum molecular chaperone and calcium-binding protein, overexpressed GRP78 binds to the hydrophobic residue ends of misfolded or unfolded proteins and recognizes calcium ions, reducing the accumulation of misfolded or unfolded proteins on the endoplasmic reticulum and maintaining calcium ion balance, thereby restoring the physiological function of the endoplasmic reticulum; in addition, GRP78, as an endoplasmic reticulum stress marker protein, can bind to pro-apoptotic receptors activated by endoplasmic reticulum stress and inhibit their signal expression, thereby protecting cells and maintaining the body's internal environmental homeostasis.

[0032] As verified by the in vitro embryo endoplasmic reticulum stress GRP78 level test, the administration of the product of the present invention reduced the in vitro embryo GRP78 level by 8.00±0.13% compared with the case where the product of the present invention was not administered.

[0033] According to the second aspect of the present invention, a product for promoting embryonic development is also provided, wherein the raw materials of the product include fisetin.

[0034] According to some embodiments of the present invention, the product is a kit, a culture medium or a drug.

[0035] According to some embodiments of the invention, the embryo comprises a mammalian in vitro embryo.

[0036] Preferably, the mammals include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sports animals, pet animals (such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, camels, bison, cattle, cows), primates (such as apes, monkeys, gorillas and chimpanzees), canines (such as dogs and wolves), felines (such as cats, lions and tigers), equines (such as horses, donkeys and zebras), food animals (such as cows, pigs and sheep), ungulates (such as deer and giraffes), and rodents (such as mice, rats, hamsters and guinea pigs). In a preferred embodiment, the mammals include pigs.

[0037] According to some embodiments of the present invention, the mammalian embryo comprises at least one of a mammalian in vitro parthenogenetic activated embryo and a mammalian in vitro fertilized embryo.

[0038] Preferably, the mammalian in vitro embryos include parthenogenetically activated oocytes, fertilized oocytes, and any time point of the 2-cell, 4-cell, 8-cell, 16-cell, morula and blastocyst stages developed from parthenogenetically activated early embryos and / or fertilized oocytes.

[0039] According to some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable excipient.

[0040] According to some embodiments of the present invention, the adjuvant includes a pharmaceutically acceptable diluent, excipient, carrier, adhesive, lubricant, suspending agent, coating agent, solubilizing agent. The medicine can be in various forms, such as liquid, semisolid and solid dosage forms. In other embodiments of the present invention, the form of the medicine includes a liquid solution (such as an injectable and infusible solution), a spray, a dispersion or a suspension.

[0041] According to some embodiments of the invention, the drug is a liquid solution.

[0042] According to some embodiments of the present invention, the culture medium comprises at least one of a basal culture medium, inorganic salts, a solvent, antibiotics and nutritional functional factors that are beneficial to in vitro embryo development.

[0043] According to some embodiments of the present invention, the basic culture medium includes TCM-199 culture medium, PZM-5 culture medium, CR1-aa culture medium, Ham's F10 culture medium, Ham's F12, NCSU-37 culture medium, and NCSU-23 culture medium.

[0044] According to some embodiments of the present invention, the inorganic salt includes at least one of sodium chloride, potassium chloride, potassium dihydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, and sodium bicarbonate.

[0045] According to some embodiments of the present invention, the antibiotic includes at least one of gentamicin, penicillin, streptomycin and kanamycin.

[0046] According to some embodiments of the present invention, the nutritional functional factors that are beneficial to in vitro embryo development include at least one of hormones, energy substrates, amino acids, and growth factors.

[0047] According to some embodiments of the invention, the hormones include follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), pregnant mare serum gonadotropin (PMSG), and estradiol (E2).

[0048] According to some embodiments of the invention, the energy substrate comprises glucose, pyruvate, lactate.

[0049] According to some embodiments of the present invention, the amino acids include essential amino acids and non-essential amino acids. According to some specific embodiments of the present invention, the amino acids include L-cysteine, L-isoleucine, L-leucine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-arginine hydrochloride, L-cystine dihydrochloride, L-histidine hydrochloride monohydrate, and L-lysine hydrochloride.

[0050] According to some embodiments of the present invention, the growth factors include epidermal growth factor (EGF), insulin-like growth factor (IGF), basic fibroblast growth factor (bFGF), and nerve growth factor (NGF).

[0051] The third aspect of the present invention provides the use of fisetin in a product for promoting the embryonic development of porcine oocytes in vitro.

[0052] According to some embodiments of the present invention, there is provided a use of fisetin in improving the blastocyst formation rate of in vitro embryos.

[0053] According to some embodiments of the present invention, there is provided a use of fisetin in promoting in vitro embryos and increasing the total cell number of in vitro embryos.

[0054] According to some embodiments of the present invention, there is provided a use of fisetin in improving the mitochondrial function of embryos in vitro.

[0055] According to some embodiments of the present invention, there is provided a use of fisetin for increasing the proliferation capacity of in vitro embryos.

[0056] According to some embodiments of the present invention, there is provided a use of fisetin for reducing endoplasmic reticulum stress levels of embryos in vitro.

[0057] According to some embodiments of the invention, the in vitro embryo comprises a mammalian in vitro embryo.

[0058] According to some embodiments of the present invention, the in vitro embryo comprises an in vitro parthenogenetically activated mammalian embryo.

[0059] According to some embodiments of the present invention, the in vitro embryo comprises an in vitro parthenogenetically activated pig embryo.

[0060] A fourth aspect of the present invention provides a method for promoting porcine oocyte embryonic development in vitro, the method comprising the following steps:

[0061] S1. Apply fisetin to embryos in vitro;

[0062] S2. Cultivate in vitro embryos until the blastocyst stage.

[0063] The method can be used for non-therapeutic purposes. Specifically, the method can be used for scientific research, such as in vitro embryo culture research and pre-implantation embryo development mechanism research.

[0064] According to some embodiments of the present invention, the method comprises the following steps:

[0065] S1. adding an effective amount of fisetin to the in vitro embryo culture medium;

[0066] S2. Cultivate in vitro embryos until the blastocyst stage.

[0067] According to some embodiments of the present invention, the effective amount of fisetin is 0.01 to 1 μmol / L.

[0068] Preferably, the effective amount of fisetin is 0.05-0.8 μmol / L.

[0069] More preferably, the effective amount of fisetin is 0.1 μmol / L.

[0070] According to some embodiments of the present invention, in step S1, the fisetin can be applied at any time point during the in vitro embryo development to the blastocyst stage, including the formation of early embryos after parthenogenetic activation of oocytes, after sperm and egg fertilization, and development to the 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst stages.

[0071] According to some embodiments of the present invention, in step S1, the fisetin may be applied once, but more preferably applied multiple times.

[0072] According to some embodiments of the invention, the fisetin may be administered from once a day to once a week.

[0073] Specifically, fisetin can be administered once a day, once every two days, once every three days, once every four days, once every five days, once every six days, or once a week.

[0074] The term "effective amount" used in the present invention refers to the amount of the compound applied to provide the expected effect compared to the case where the compound is not applied, which is calculated by the mass of the applied fisetin and the volume of the applied liquid environment (e.g., culture medium); the expected effect includes significantly improving the blastocyst formation rate of in vitro embryos, and / or significantly increasing the total number of cells in vitro embryos, improving the mitochondrial function of in vitro embryos, increasing the proliferation capacity of in vitro embryos, and / or significantly reducing the endoplasmic reticulum stress level of in vitro embryos. Significantly improving the blastocyst formation rate of in vitro embryos means that the blastocyst formation rate of in vitro embryos is increased by 32.32±15.28% compared to the case where the compound is not applied. Significantly promoting in vitro embryo proliferation means that the total number of in vitro embryo cells is increased by 17.07±0.19% compared to the case where the compound is not applied. Significantly improving the mitochondrial function of in vitro embryos means that the mitochondrial function of in vitro embryos is increased by 14.00±1.29% compared to the case where the compound is not applied. Significantly increasing the proliferation capacity of in vitro embryos means that the proliferation level of in vitro embryos is increased by 27.56±0.09% compared to the case where the compound is not applied. Significantly reducing the endoplasmic reticulum stress level of in vitro embryos means that the fluorescence intensity of the endoplasmic reticulum stress marker (GRP78) of in vitro embryos is reduced by 8.00±0.13% compared with the case where the compound is not administered.

[0075] According to some embodiments of the present invention, in step S2, the culture temperature is 37-39°C.

[0076] According to some embodiments of the present invention, in step S2, the culturing time is 120 to 168 hours.

[0077] In the context of the numerical value of the effect of improving the in vitro embryo development ability, the numerical value of "increased to" used is the measured value when the product of the present invention is applied. In the context of the numerical value of the effect of improving the in vitro embryo development ability, the numerical value of "increased" used is calculated in the following manner: [(measured value of applying the product of the present invention)-(measured value of not applying the product of the present invention)] / (measured value of not applying the product of the present invention).

[0078] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0080] Figure 1 The effect of 0.01, 0.1 and 1 μM concentrations of fisetin on the blastocyst formation rate in vitro in Example 2 is shown; data are expressed as maximum-minimum; n≥3 independent parallel experiments; significant differences are expressed as: ***P<0.001.

[0081] Figure 2 The total number of in vitro embryonic cells in the control group and the experimental group is shown; the data are expressed as maximum-minimum; n≥3 independent parallel experiments; significant differences are expressed as: ***P<0.001.

[0082] Figure 3 The effect of fisetin on in vitro embryonic mitochondrial function in Example 3 is shown, and the levels of mitochondrial function of embryos in the control group and the experimental group are shown; the data are expressed as mean ± standard deviation; n≥3 independent parallel experiments; significant differences are expressed as: ***P<0.001.

[0083] Figure 4 The effect of fisetin on the proliferation ability of in vitro embryos in Example 4 is shown. The proliferation levels of in vitro embryos in the control group and the experimental group are shown, and the data are expressed as maximum-minimum; n≥4 independent parallel tests; significant differences are expressed as: **P<0.01.

[0084] Figure 5The effect of fisetin on the antioxidant level of embryos in vitro in Example 5 is shown, and the level of endoplasmic reticulum marker GRP78 in embryos of the control group and the experimental group is shown; the data are expressed as maximum-minimum; n≥5 independent parallel experiments; significant differences are expressed as: ***P<0.001. DETAILED DESCRIPTION

[0085] The embodiments of the present invention are described in detail below, and the same or similar reference numerals throughout the embodiments represent the same or similar elements or elements with the same or similar functions. The embodiments described below are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0086] In the description of the present invention, if there is a description of first, second, etc., it is only for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0087] The words "preferably", "more preferably", etc. in the present invention refer to embodiments of the present invention that may provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0088] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0089] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of the present invention.

[0090] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0091] Example 1 A method for promoting porcine oocyte embryonic development in vitro.

[0092] A method for promoting porcine oocyte embryonic development in vitro comprises the following steps:

[0093] 1. Collection of porcine oocytes:

[0094] Ovaries from prepubertal gilts were collected from slaughterhouses and transported to the laboratory in 0.9% saline at 37°C. The ovarian follicles with a diameter of 3-8 mm were aspirated with a syringe, and the oocyte-cumulus complexes (COCs) and follicular fluid were placed in a 15 ml shake tube and allowed to stand in a 38°C water bath for 15 min until the COCs precipitated to the bottom of the tube, and the supernatant was aspirated. The precipitate was resuspended by adding an appropriate amount of HEPES buffer (SIGMA, H6147) containing 0.1% (w / v) polyvinyl alcohol (PVA, SIGMA, P8136) and 0.05 g / L gentamicin (SIGMA, E003632), and the supernatant was aspirated after standing for 15 min. This step was repeated three times. The COCs precipitate was transferred to a culture dish, an appropriate amount of HEPES buffer was added, and COCs with uniform oocyte cytoplasm and three or more layers of dense cumulus cells surrounding the oocyte were selected with a glass pipette under a stereomicroscope.

[0095] 2. In vitro maturation of porcine oocytes:

[0096] The selected COCs were washed three times with maturation medium IVM; the maturation medium IVM was: 0.1g / L sodium pyruvate (SGMA, P4562), 0.6mmol / L L-cysteine ​​(SIGMA, C7477), 10ng / mL epidermal growth factor (SIGMA, E4127), 10% porcine follicular fluid, 10IU / mL luteinizing hormone (Ningbo Second Hormone Factory), 10IU / mL follicle stimulating hormone (Ningbo Second Hormone Factory) were added to TCM-199 (GIBCO, 11150-059). 500μL maturation medium IVM was added to a 4-well culture dish, the culture medium was covered with 500μL mineral oil, and then the COC washed three times was placed in the maturation medium IVM. The 4-well culture dish was placed in a 38.5℃, 5% CO2, humidity-saturated incubator for 44-46h, and the oocytes matured after 44-46h and reached the MⅡ stage.

[0097] 3. Parthenogenetic activation of early pig embryos:

[0098] COCs reaching the MⅡ stage were transferred to 0.1% hyaluronidase using a pipette, and the mixture was repeatedly blown about 30 times to remove the cumulus cells around the oocytes to obtain naked oocytes. The naked oocytes were transferred to a 297mmol / L mannitol (pH 7.2, SIGMA, M9546) solution containing 0.1mmol / L CaCl2 (SIGMA, C7902), 0.05mmol / L MgSO4 (SIGMA, M1880), 0.01% (w / v) PVA, and 0.5mmol / L HEPES. The naked oocytes were parthenogenetically activated by direct current pulses with an electric field strength of 1.2kV / cm for 60μs each time, twice in total.

[0099] 4. The parthenogenetically activated early embryos were cultured in a porcine embryo in vitro culture medium (PZM-5 medium, which contains 7.5 mg / mL cytochalasin B) for 3-5 hours. The PZM-5 medium consists of the following ingredients: 108 mmol / L (mM) sodium chloride (sigma, S5886), 10 mM potassium chloride (sigma, P5405), 0.35 mM potassium dihydrogen phosphate (sigma, P5655), 0.4 mM magnesium sulfate (sigma, M2643), 25 mM sodium bicarbonate (sigma, S5761), 0.2 mM sodium pyruvate (sigma, P5280), 2.0 mM calcium lactate (sigma, C 8356), 2.0mM glutathione (sigma, G8540), 5.0mM hypotaurine (sigma, H1384), 20ml / L essential amino acids (sigma, B6766), 10mL / L non-essential amino acids (sigma, M7145), 25mg / mL gentamicin (sigma, B6766), 4mg / mL bovine serum albumin (sigma, A8806), add ultrapure water to a certain volume, and adjust the pH to 7.2-7.4.

[0100] 5. Remove the culture medium and apply a porcine embryo in vitro culture medium (PZM-5 culture medium) containing fisetin to the parthenogenetically activated early embryos. Set up an experimental group including a PZM-5 culture medium containing 0.1 μmol / L (μM) fisetin; and a control group, in which a PZM-5 culture medium not containing fisetin is applied to the parthenogenetically activated early embryos.

[0101] 6. The early embryos are cultured in a 38.5°C, 5% CO2, saturated humidity incubator for 3-7 days without changing the culture medium.

[0102] Example 2 Application of Fisetin in Improving the Blastocyst Formation Rate in In Vitro

[0103] Blastocyst formation rate test of pig in vitro parthenogenetic embryos: The early embryos of Example 1 were cultured in vitro until day 7, and the blastocyst formation rates of the control group and the experimental group were observed. Blastocyst formation rate = (number of blastocysts formed / number of all cultured early parthenogenetic embryos) × 100%.

[0104] Compared with the control group, the addition of 0.1 μmol / L (μM) fisetin to the porcine embryo in vitro culture medium (PZM-5 medium) can significantly increase the blastocyst formation rate of early parthenogenetic pig embryos. The blastocyst formation rate of the control group was 36.70±7.07%, and the blastocyst formation rate of the medium with 0.1 μM fisetin was 48.56±8.15%, which was significantly higher than that of the control group (P<0.001). Figure 1 shown.

[0105] Adding an effective concentration (0.1 μM) of fisetin to the culture medium can help promote the smooth development of parthenogenetically activated early embryos into blastocysts. Whether the oocytes can successfully develop into blastocysts after activation is the key to in vitro embryo culture. Only early embryos with good developmental ability and developmental quality can develop into blastocysts, and the subsequent use of blastocysts for in vivo transplantation can increase the embryo implantation rate. It can be seen that fisetin improves the developmental ability and quality of early embryos before implantation, laying a good foundation for embryo implantation. It also shows that fisetin has good application prospects in the preparation of products that improve the blastocyst formation rate of in vitro embryos.

[0106] Example 3 Application of Fisetin in Improving Mitochondrial Function of Embryos in Vitro

[0107] Mitochondrial function test of pig in vitro parthenogenetic embryos: The mitochondrial function of 4-cell early parthenogenetic pig embryos was tested using JC-1 mitochondrial probe (JC-1, Beyotime, Shanghai, China):

[0108] The early embryos of the experimental group (the amount of fisetin added in Example 1 was 0.1 μM) and the control group (fisetin was not added in Example 1) were cultured in vitro to the 4-cell stage of early parthenogenetic pig embryos, and the 4-cell embryos were washed 5 times with PBS buffer, incubated in the dark at 38.5°C and 5% CO2 for 17 h in 10 μg / mL PBS-PVA (JC-1, Beyotime, Shanghai, China), and washed 4 times with PBS-PVA. The stained oocytes were placed in 3 μg / mL PBS-PVA. Images were taken using a fluorescence microscope (Nikon, Tokyo, Japan). The fluorescence intensity was analyzed using Image J software, and the evaluation index was the ratio of the JC-1 fluorescence intensity of different colors (red / green), and the relative fluorescence intensity of the treatment group relative to the control group was calculated.

[0109] The mitochondrial membrane potential of 4-cell embryos was measured by JC-1. The mitochondrial level of the fisetin treatment group (0.1μM) was significantly higher than that of the control group (P<0.001). The control group was 0.99±0.14, and the treatment group was 1.13±0.18, which was 14.00±1.29% higher than that of the control group. Therefore, the above results show that 0.1μM fisetin can effectively increase mitochondrial membrane potential and protect mitochondrial function, prevent mitochondrial damage and defects, and thus improve the developmental quality and developmental capacity of early embryos. It also shows that fisetin has good application prospects in the preparation of products for improving mitochondrial function of in vitro embryos. The results are as follows Figure 3 shown.

[0110] Example 4 Application of Fisetin in Improving the Proliferation Ability of In Vitro Embryos

[0111] Testing of endoplasmic reticulum stress capacity of porcine in vitro parthenogenetic embryos: Cell proliferation was assessed using the BeyoClick EDU-647 cell proliferation detection kit (Beyotime, Jiangsu, China):

[0112] The early embryos of the experimental group (the amount of fisetin added in Example 1 was 0.1 μM) and the control group (fisetin was not added in Example 1) were cultured in vitro until the 7th day, and the pig parthenogenetic activated in vitro embryos were fixed with 3.7% paraformaldehyde for 30 minutes, then permeabilized with 0.3% Triton X-100 for 30 minutes, and then cleaned with PBS-PVA, and incubated with BeyoClick additive solution at room temperature in the dark for 1 hour. Finally, the embryos were incubated with 10 μg / ml Hoechst 33342 at room temperature for 8 minutes to mark the cell nucleus. The number of EDU-positive cells and the total number of cells were analyzed using a fluorescence microscope (Nikon, Tokyo, Japan) and Image J software. The proliferation capacity of the blastocyst was evaluated by the ratio of the number of EDU-positive cells to the total number of cells.

[0113] The proliferation rate of the fisetin-treated group (0.1 μM) was significantly higher than that of the control group (P<0.01), and the proliferation rate of the fisetin-treated group increased by 27.56±0.09% relative to that of the control group. Figure 4 B shows that the total number of embryonic cells in the fisetin treatment group (0.1 μM) was significantly higher than that in the control group (P<0.001), where the control group was 42.41±12.32 and the treatment group was 49.65±12.51, which was 17.07±0.19% higher than that in the control group. Figure 4 shown.

[0114] The above results show that fisetin can effectively improve the proliferation ability of porcine parthenogenetic activated in vitro embryos, thereby improving the development quality and development ability of in vitro embryos. It also shows that fisetin has good application prospects in the preparation of products that improve the proliferation ability of in vitro embryos.

[0115] Example 5 Application of Fisetin in Reducing Endoplasmic Reticulum Stress Levels in Embryos in Vitro

[0116] Test of the antioxidant level of pig in vitro parthenogenetic embryos: Anti-GRP78 antibody (Abcam, catalog number Ab21685) was used to detect the fluorescence intensity of GRP78, a marker of endoplasmic reticulum stress in pig in vitro parthenogenetic embryos:

[0117] The early embryos of the experimental group (the amount of fisetin added in Example 1 was 0.1 μM) and the control group (fisetin was not added in Example 1) were cultured in vitro until the 7th day, and the pig parthenogenetic activated in vitro embryos were fixed with 3.7% paraformaldehyde for 30 minutes, then permeabilized with 0.3% Triton X-100 for 30 minutes, and then blocked with 3% BSA at 37°C for 1 hour. After washing with PBS-PVA 5 times, the pig in vitro embryos were incubated with rabbit anti-GRP78 primary antibody additive solution in a 4°C refrigerator in the dark overnight. The next day, they were taken out, washed 5 times with PBS-PVA, and then incubated in the dark for 1 hour at room temperature with goat anti-rabbit secondary antibody (Abcam, ab150077). After 1 hour, they were washed 5 times with PBS-PVA, and the pig in vitro embryos were incubated with 10 μg / mL DNA fluorescent dye Hoechst 33342 at room temperature in the dark for 8 minutes to mark the nucleus. Images were taken using a fluorescence microscope (Nikon, Tokyo, Japan). The images were analyzed using Image J software, and the fluorescence intensity of the endoplasmic reticulum stress marker GRP78 and the total number of cells in porcine in vitro embryos were counted and calculated. The weaker the fluorescence, the lower the level of endoplasmic reticulum stress in the embryo.

[0118] The relative fluorescence intensity of GRP78, a marker of endoplasmic reticulum stress, in the fisetin treatment group (0.1 μM) was significantly higher than that in the control group (P<0.001), with the control group at 1.00±0.08 and the treatment group at 0.92±0.07, which was 8.00±0.13% lower than that in the control group. The above results indicate that fisetin can effectively alleviate the endoplasmic reticulum stress level of pig parthenogenetic activated in vitro embryos, thereby improving the developmental quality and developmental capacity of in vitro embryos. Figure 5 shown.

[0119] Data analysis: Examples 2-5 were statistically analyzed using SPSS software, and the independent sample t-test was used to compare the data between the two groups.

[0120] Example 6

[0121] This embodiment provides a culture medium for promoting the development of porcine oocytes in vitro, which includes 0.1 μM fisetin, a basic culture medium, inorganic salts, a solvent, antibiotics, and nutritional functional factors that are beneficial to in vitro embryo development.

[0122] In summary, the present invention proves that fisetin can improve the blastocyst formation rate of in vitro embryo culture, increase the total number of embryo cells, protect the mitochondrial function of in vitro embryos, improve the proliferation ability of in vitro embryos, and alleviate the endoplasmic reticulum stress level of in vitro embryos, thereby effectively improving the quality and developmental ability of in vitro embryos.

[0123] Fisetin has good market application prospects in the preparation of products for improving the quality and developmental capacity of in vitro embryos.

[0124] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Application of fisetin in the preparation of products for promoting embryonic development.

2. The use according to claim 1, characterized in that: The product is a kit, a culture medium or a medicine.

3. The use according to claim 1, characterized in that: The promoting embryo development includes at least one of improving embryo quality or improving embryo development ability.

4. The use according to claim 3, characterized in that: The promoting of embryo development comprises at least one of increasing the embryo blastocyst rate, increasing the total number of embryo cells, improving the embryo proliferation ability, enhancing the mitochondrial function of embryo cells and reducing the level of endoplasmic reticulum stress marker protein.

5. A product for promoting embryonic development, characterized in that: The raw materials of the product include fisetin.

6. The product according to claim 5, characterized in that The product is a kit, a culture medium or a medicine.

7. Use of fisetin in products for promoting porcine oocyte embryonic development in vitro.

8. A method for promoting porcine oocyte embryonic development in vitro, characterized in that: The method comprises the following steps: S1. Apply fisetin to embryos in vitro; S2. Cultivate in vitro embryos until the blastocyst stage.

9. The use according to claim 8, characterized in that The effective amount of fisetin is 0.01-1 μmol / L; preferably, the effective amount of fisetin is 0.05-0.8 μmol / L; more preferably, the effective amount of fisetin is 0.1 μmol / L.

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