CN116515738A - Application of yellow angelica alcohol in preparation of product for promoting embryo development - Google Patents
Application of yellow angelica alcohol in preparation of product for promoting embryo development Download PDFInfo
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- CN116515738A CN116515738A CN202310423588.0A CN202310423588A CN116515738A CN 116515738 A CN116515738 A CN 116515738A CN 202310423588 A CN202310423588 A CN 202310423588A CN 116515738 A CN116515738 A CN 116515738A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0604—Whole embryos; Culture medium therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
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- Biotechnology (AREA)
- Reproductive Health (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Developmental Biology & Embryology (AREA)
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Abstract
The invention discloses application of yellow angelica alcohol in preparing a product for promoting embryo development. The yellow angelica alcohol is a natural compound extracted from angelica keiskei, belongs to flavonoid substances, has the functions of resisting oxidation, resisting apoptosis, resisting depression and the like, is applied to the technical field of embryo engineering for the first time, and is found to improve the blastula rate of in-vitro embryo culture, protect the mitochondrial function of in-vitro embryo cells and improve the proliferation capacity of in-vitro embryo cells, thereby effectively improving the quality and the development capacity of in-vitro embryos.
Description
Technical Field
The invention relates to the technical field related to biological medicine, in particular to application of yellow angelica alcohol in preparation of a product for promoting embryo development.
Background
Embryo engineering refers to a variety of micromanipulation and handling techniques for early embryos or gametes, including in vitro fertilization, embryo transfer, embryo segmentation transfer, embryonic stem cell culture, and the like. In vitro embryo production is a key link of embryo engineering technology, and is a key for research of animal husbandry, human assisted reproduction technology and embryo development mechanism before implantationTechniques for [1] . In vitro embryo production mainly comprises in vitro fertilization and in vitro embryo culture. In vitro embryo culture is the process of culturing activated oocytes into blastocysts in vitro [2] 。
Embryo quality for in vitro embryo production is affected by factors such as: the culture solution, osmotic pressure, pH, humidity, oxygen, carbon dioxide and other physical and chemical factors are very easy to influence, so that the embryo can generate excessive active oxygen, and as the in vitro embryo production technology is mostly operated in vitro, the in vivo antioxidant systems such as enzyme (superoxide dismutase, catalase and the like) and non-enzyme (melatonin) and the like are lack to resist the excessive active oxygen, thereby causing oxidative stress and reducing the development and quality of the embryo.
In order to solve this problem and to simulate the microenvironment of embryo development in vivo as much as possible, studies have shown that the addition of some natural antioxidants (e.g. melatonin etc.) during embryo culture in vitro may achieve good results, but all have different drawbacks, such as melatonin is related to seasonal reproduction, unsuitable for administration by lactating women, etc. Therefore, it is one of the hot spots of current research to find a new, more effective, safer natural antioxidant to improve the developmental capacity and quality of in vitro embryos.
[1]Kruip T,Bevers M M,Kemp B.Environment of oocyte and embryo determines health of IVP offspring[J].Theriogenology,2000,53(2):611-618.
[2] Wang Na, li Jiao, wang Xiaowu, etc. factors affecting the efficiency of bovine in vitro embryo production [ J ]. Today's zoo veterinarian, 2018, 34 (11): 4.
disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, the invention provides the application of the yellow angelica alcohol in preparing the product for promoting embryo development, and the component is derived from natural plants and can effectively develop the in vitro embryo.
According to one aspect of the invention, there is provided the use of yellow angelic alcohol in the preparation of a product for promoting embryonic development.
According to a preferred embodiment of the invention, there is at least the following advantageous effect: the invention relates to a method for preparing Xanthoangelol (Xanthoangelol), which is a natural compound extracted from Angelica keiskei Koidz, and belongs to flavonoid substances, wherein the flavonoid substances have the functions of resisting oxidation, resisting apoptosis, resisting depression and the like.
In some embodiments of the invention, the chemical structural formula of the xanthoangelol is as follows:
in some embodiments of the invention, the promoting embryo development comprises at least one of improving embryo quality or improving embryo developmental capacity.
In some preferred embodiments of the invention, the product comprises at least one of a kit, a medium, or a medicament.
In some more preferred embodiments of the invention, the promoting embryo development comprises at least one of increasing embryo blastocyst rate, increasing mitochondrial membrane potential, or increasing mitochondrial distribution of embryo cells and expression levels of genes associated therewith. The yellow angelic alcohol can improve the blastula rate of in-vitro embryo culture, protect the mitochondrial function of in-vitro embryo cells and improve the proliferation capacity of in-vitro embryo cells, thereby effectively improving the quality and the development capacity of in-vitro embryos.
In some embodiments of the invention, the quality and developmental capacity of an embryo is enhanced by increasing the mitochondrial membrane potential of the embryo cell. Mitochondria are the main sites for animal and plant cells to produce ATP, and are important organelles that promote cellular energy conversion and participate in apoptosis. The mitochondria store electrochemical potential energy in the inner mitochondrial membrane when energy is generated, and the asymmetric distribution of the concentration of protons and other ions on both sides of the inner mitochondrial membrane forms mitochondrial membrane potential. Normal mitochondrial membrane potential is a prerequisite for maintaining oxidative phosphorylation of mitochondria and production of adenosine triphosphate, and stabilization of mitochondrial membrane potential is beneficial for maintaining normal physiological function of cells; the decrease in mitochondrial membrane potential is one of the manifestations of decreased mitochondrial function, and unhealthy mitochondria have their membrane potential decreased or lost, and thus mitochondrial membrane potential is one of the important indicators for evaluating mitochondrial function of cells.
In some embodiments of the invention, embryo quality and developmental capacity are enhanced by increasing mitochondrial distribution of embryo cells and their associated genes, mitochondria being an important subcellular target of Reactive Oxygen Species (ROS) -induced cell damage. Four-cell stage embryos were stained by Mito-Tracker red probe (200 mmol/L, beyotidme). Normal mitochondrial distribution is uniform, and accumulation distribution, peripheral distribution and semi-peripheral Zhou Fenbu are considered abnormal mitochondrial distribution. The research proves that the improvement of the expression level of genes such as TFAM, NRF1, NRF2 and the like can also prove the good functional state of mitochondria, and the expression level of the three genes is improved by the angelicin, so that the functional state of the mitochondria is improved, and the embryo development capacity and quality are further improved.
In some embodiments of the invention, the promoting embryo development is to increase embryo blastocyst rate in vitro. Preferably, administration of the product of the invention results in an improvement in vitro embryo blastocyst rate of 10% -35% relative to the in vitro embryo blastocyst rate as demonstrated by the in vitro embryo blastocyst rate test.
Specifically, in some embodiments of the invention, administration of the product of the invention increases embryo blastocyst rate in vitro by 10% to 35% relative to the case where the product of the invention is not administered. In some embodiments of the invention, administration of the products of the invention increases embryo blastocyst rate in vitro by at least about 10%. In other embodiments of the invention, administration of the products of the invention increases embryo blastocyst rate in vitro to 10-45%; preferably, administration of the product of the invention increases embryo blastocyst rate in vitro to 15-50%, more preferably at least to about 51%.
In some embodiments, the promoting embryo development increases mitochondrial membrane potential of the embryo cells in vitro. Preferably, administration of the product of the invention increases the mitochondrial membrane potential of the in vitro embryonic cells by 25% -40% relative to the in vitro embryonic cell mitochondrial membrane potential test as demonstrated by the in vitro embryonic cell mitochondrial membrane potential test.
Specifically, in some embodiments of the invention, administration of the product of the invention increases mitochondrial membrane potential of embryonic cells in vitro by 30-45% relative to the absence of administration of the product of the invention. In some embodiments of the invention, administration of the product of the invention increases mitochondrial membrane potential of embryonic cells in vitro by at least about 46%.
In some embodiments of the invention, the promoting embryo development is to increase mitochondrial distribution of embryo cells in vitro. Preferably, administration of the product of the invention increases the total number of cells of the in vitro embryo by 12% -23% relative to the case where the product of the invention is not administered, as demonstrated by the in vitro embryo total number test.
Specifically, in some embodiments of the invention, administration of the product of the invention increases mitochondrial distribution of embryonic cells by 13% -25% in vitro relative to the absence of administration of the product of the invention. In some embodiments of the invention, administration of the products of the invention increases mitochondrial distribution of embryonic cells in vitro by at least about 26%.
In some embodiments of the invention, the promoting embryo development is to increase gene levels associated with mitochondrial distribution in embryonic cells in vitro. Preferably, administration of the product of the invention increases the level of gene associated with mitochondrial distribution in vitro by 11% -21% relative to the level of gene associated with mitochondrial distribution in vitro in embryonic cells as demonstrated by the level of gene associated with mitochondrial distribution in vitro.
Specifically, in some embodiments of the invention, administration of the product of the invention increases gene levels associated with mitochondrial distribution in embryonic cells by 11% to 19% in vitro relative to the absence of administration of the product of the invention. In some embodiments of the invention, administration of the products of the invention increases gene levels associated with mitochondrial distribution in embryonic cells in vitro by at least about 12%. In some embodiments of the invention, administration of the products of the invention increases the level of genes associated with mitochondrial distribution in cells in vitro to 15% -20%; preferably, administration of the product of the invention increases the in vitro embryo cell mitochondrial distribution related gene levels to 13% -25%, more preferably at least to about 26%.
In the context of values for the effect of improving the developmental capacity of an embryo in vitro, the value "to improve" is used as a measure for the case of administration of the product according to the invention. In the context of values for the effect of improving the developmental capacity of an embryo in vitro, the value of "improvement" used is calculated by: [ (measured values for the product of the invention) - (measured values for the product of the invention) v ] (measured values for the product of the invention were not applied).
According to another aspect of the invention, the use of yellow angelic alcohol in the preparation of a product for improving embryo blastocyst rate is also provided.
According to a further aspect of the invention, there is also provided the use of xanthoangelol in the preparation of a product for increasing mitochondrial membrane potential of embryonic cells.
According to a further aspect of the invention, there is also provided the use of xanthoangelol in the preparation of a product for increasing mitochondrial distribution in embryonic cells and their associated gene levels.
According to a further aspect of the invention, there is also provided a product for promoting embryo development, the starting material of the product comprising xanthoangelol.
In some embodiments of the invention, the product comprises at least one of a drug, a medium, or a kit.
In some embodiments of the invention, the embryo comprises a mammalian in vitro embryo. Preferably, the mammal includes, but is not limited to, humans, domesticated animals, farm animals, zoo animals, sports animals, pet animals (e.g., dogs, cats, guinea pigs, rabbits, rats, mice, horses, camels, bison, cows), primates (e.g., apes, monkeys, gorillas, and chimpanzees), canines (e.g., dogs and wolves), felines (e.g., cats, lions, and tigers), equines (e.g., horses, donkeys, and zebras), eating animals (e.g., cows, pigs, and sheep), ungulates (e.g., deer and giraffes), and rodents (e.g., mice, rats, hamsters, and guinea pigs). In some embodiments of the invention, the mammal comprises a pig.
In some embodiments of the invention, the mammalian in vitro embryo comprises a mammalian in vitro parthenogenesis activated embryo, a mammalian in vitro fertilization embryo. Preferably, the mammalian in vitro embryo comprises a parthenogenetic activated oocyte, a fertilized egg cell, and a 2-cell, 4-cell, 8-cell, 16-cell, morula and blastocyst developed from the parthenogenetic activated oocyte and/or fertilized egg cell.
In some embodiments of the invention, the medicament further comprises a pharmaceutically acceptable excipient. Preferably, the auxiliary materials comprise pharmaceutically acceptable diluents, excipients, carriers, binders, lubricants, suspending agents, coating agents and solubilizers. The drug may be in a variety of forms, such as liquid, semi-solid, and solid dosage forms. In other embodiments of the invention, the pharmaceutical forms include liquid solutions (e.g., injectable and infusible solutions), sprays, dispersions, or suspensions. In some embodiments of the invention, the drug is a liquid solution.
Preferably, the culture medium further comprises basal medium, solvent, antibiotics, nutritional functional factors that facilitate in vitro embryonic cell development.
Preferably, the basal medium comprises TCM-199 medium, PZM-5 medium, CR1-aa medium, ham' S F medium, NCSU-37 medium, NCSU-23 medium.
Preferably, the inorganic salt includes sodium chloride, potassium dihydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, sodium bicarbonate.
Preferably, the antibiotics include gentamicin, penicillin, streptomycin, and the like.
Preferably, the nutritional functional factors that facilitate in vitro embryonic cell development include hormones, energy substrates, amino acids, growth factors.
According to some embodiments of the inventionThe hormone comprises Follicle Stimulating Hormone (FSH), luteinizing Hormone (LH), human chorionic gonadotropin (hCG), pregnant Mare Serum Gonadotropin (PMSG), estradiol (E) 2 )。
According to some embodiments of the invention, the energy substrate comprises glucose, pyruvate, lactate.
According to some embodiments of the invention, the amino acid comprises an essential amino acid, an optional amino acid. According to some embodiments of the invention, the amino acid comprises 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, L-lysine hydrochloride.
According to some embodiments of the invention, the growth factor comprises Epidermal Growth Factor (EGF), insulin-like growth factor (IGF), basic fibroblast growth factor (bFGF), nerve Growth Factor (NGF).
According to a further aspect of the invention, there is also provided the use of xanthoangelol in promoting embryo development.
According to a further aspect of the invention, there is also provided the use of xanthoangelol in promoting the quality of embryo development.
According to a further aspect of the invention, there is also provided the use of xanthoangelol in promoting embryo development.
According to a further aspect of the invention, there is also provided the use of xanthoangelol in increasing embryo blastocyst rate.
According to a further aspect of the invention, there is also provided the use of xanthoangelol for increasing mitochondrial membrane potential of embryonic cells.
According to a further aspect of the invention, there is also provided the use of xanthoangelol for increasing the mitochondrial distribution of embryonic cells and their associated gene expression levels.
Preferably, the embryo comprises a mammalian in vitro embryo. Preferably, the in vitro embryo comprises a mammalian in vitro parthenogenesis activated embryo. More preferably, the in vitro embryo comprises a pig in vitro parthenogenesis activated embryo. More preferably, the in vitro embryo comprises a pig in vitro parthenogenesis activated oocyte.
According to yet another aspect of the present invention, there is also provided a method for improving the ability of an embryo to develop in vitro, comprising the steps of: applying the yellow angelic alcohol to the in vitro embryo.
The method may be used for non-therapeutic purposes. In particular, the method can be used for scientific research, such as in vitro cell culture research, in vitro embryo culture research, pre-implantation embryo development mechanism research, and the like.
According to a further aspect of the present invention there is also presented a method for promoting porcine embryo development in vitro comprising the steps of: applying the yellow angelic alcohol to the in vitro embryo.
According to some embodiments of the invention, the method comprises the steps of:
s1, applying yellow angelic alcohol to an in-vitro embryo;
s2, culturing the in-vitro embryo until the blastula stage.
Preferably, the xanthoangelol may be applied in the form of a product made according to an embodiment of the first aspect of the present invention or according to an embodiment of the fifth aspect.
Preferably, the xanthoangelol in S1 may be applied at any point during the in vitro embryo development to the blastocyst stage, including after parthenogenesis of the oocyte, after fertilisation of the sperm egg, and at any point during development to the 2-cell, 4-cell, 8-cell, 16-cell, morula, blastocyst stage.
Preferably, the xanthoangelol in S1 may be applied once, but more preferably applied multiple times. According to some embodiments of the invention, the xanthoangelol may be administered from once daily to once weekly. Specifically, the xanthoangelol may 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, once a week.
Preferably, the effective amount of xanthoangelol in S1 is 0.1-2.5. Mu. Mol/L, more preferably 0.2-1. Mu. Mol/L, most preferably 0.3-0.5. Mu. Mol/L. The term "effective amount" as used in the present specification and in the claims means an amount of a compound sufficient to provide the desired effect, as compared to the case where the compound is not administered, based on the mass of the xanthoangelol administered versus the volume of the liquid environment (e.g., culture medium) administered; the expected effect comprises significantly increasing in vitro embryo blastula rate, and/or significantly increasing in vitro embryo cell mitochondrial membrane potential, and/or significantly increasing in vitro embryo cell mitochondrial distribution and expression of its associated gene level. Significantly increasing the blastocyst rate of an in vitro embryo means that the blastocyst rate of an in vitro embryo is increased by 10% -35%, preferably by 10% -45%, more preferably by at least about 51% as compared to the in vitro embryo without administration of the compound. Significantly increasing the mitochondrial membrane potential of an in vitro embryonic cell means that the mitochondrial membrane potential of an in vitro embryonic cell is increased by 25% to 40%, preferably by 35% to 45%, more preferably by at least about 41% as compared to the case without the administration of the compound. Significantly increasing mitochondrial distribution of in vitro embryonic cells means an increase in total cell number of the in vitro embryo of 12% -23%, preferably 13% -25%, more preferably at least about 26% as compared to the case without administration of the compound; and/or the expression of the gene level associated with mitochondrial distribution in vitro in the embryo is increased by 11% -29%, preferably by 13% -25%, more preferably by at least about 19%.
Preferably, the cultivation temperature in S2 is 36-40℃and the cultivation time is 40-50 hours.
The beneficial effects of the invention are as follows:
the invention discloses the application of the yellow angelica alcohol in preparing the medicine for improving the in-vitro embryo development capability for the first time, the correlation between the yellow angelica alcohol and the in-vitro embryo development capability is not reported before, and the application range of the yellow angelica alcohol is expanded.
The yellow angelic alcohol can effectively protect mitochondrial functions and improve mitochondrial distribution by improving blastula rate, thereby effectively improving the development quality and development capacity of in vitro embryos.
The yellow angelica alcohol is a natural extract of angelica keiskei koidzumi, has no toxic or side effect on mammalian cells, is wide in source and easy to obtain, and has obvious effect, and the yellow angelica alcohol is added into a culture medium, so that the operation is simple, the control is easy.
The yellow angelica alcohol can be used for preparing a pharmaceutical composition, a culture medium or a kit for promoting embryo development and has good industrial application prospect.
Additional features and advantages of the 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 invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 shows the effect of varying concentrations of yellow angelic alcohol on embryo blastocyst rate in vitro in example 2 of the present invention; data are expressed as mean ± standard error; n=6 independent parallel experiments; the significance difference is expressed as: * P <0.05, < P <0.001.
FIG. 2 is a graph showing the effect of xanthoangelol on mitochondrial function of in vitro embryos in example 3 of the present invention.
FIG. 3 is a bar graph showing the effect of xanthoangelol on mitochondrial function of in vitro embryos in example 3 of the present invention, showing quantification of JC-1 relative fluorescence intensity levels (red/green fluorescence) of control and experimental group embryos; data are expressed as mean ± standard error; n=3 independent parallel experiments; the significance difference is expressed as: * P <0.001.
FIG. 4 shows the effect of xanthoangelol on in vitro embryonic cell mitochondrial distribution in example 4 of the present invention; data are expressed as mean ± standard error; n=5 independent parallel experiments; the significance difference is expressed as: * P <0.01, P <0.001.
FIG. 5 shows the effect of xanthoangelol on the expression level of mitochondrial related genes of in vitro embryo cells in example 4 of the present invention; data are expressed as mean ± standard error; n=5 independent parallel experiments; the significance difference is expressed as: * P <0.01, P <0.001.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention. The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available. Unless otherwise indicated, the same parameter is the same in each embodiment. The following examples are illustrative only and are not to be construed as limiting the invention.
In the description of the present invention, the descriptions of the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
Example 1
The embodiment is the application of the yellow angelica alcohol in preparing the product for promoting embryo development. The method specifically comprises the following steps: the yellow angelic alcohol is added into the pig embryo in vitro culture medium according to the effective amount of 0.5, 2.5 and 25 mu mol/L (mu M).
The method for using the culture medium to promote the development of porcine oocyte embryos in vitro comprises the following steps:
1. collection of pig oocytes:
ovaries from prepubertal gilts were collected from the slaughterhouse, placed in 0.9% physiological saline at 37 ℃ and transported to the laboratory. The cumulus-oocyte complex (COC) and follicular fluid in 3-6mm diameter follicles in the ovaries were aspirated with a syringe, placed in a shaking tube, and allowed to stand in a water bath at 38 ℃ for 15min until COC precipitated to the bottom of the tube, and the supernatant was discarded. The precipitate was resuspended in an appropriate amount of HEPES buffer (SIGMA, H6147) containing 0.1% (w/v) polyvinyl alcohol (PVA, SIGMA, P8136) and 0.05g/L gentamicin (SIGMA, E003632), left to stand for 15min, the supernatant was discarded, and the procedure was repeated three times. Transferring the COC precipitate to a culture dish, adding appropriate amount of 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) buffer, and selecting COC with uniform cytoplasm of oocyte and three or more layers of dense cumulus cells around the oocyte under a split microscope by using a glass pipette.
2. In vitro maturation of porcine oocytes:
washing the selected COC three times with maturation medium IVM (in vitro maturation); the maturation medium IVM was: to TCM-199 (GIBCO, 11150-059) were added 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 works), 10IU/mL follicle stimulating hormone (Ningbo second hormone works). 500mL of maturation medium IVM was added to a 4-well dish, the medium was covered with 500mL of mineral oil, and then the washed three times COC was placed in the maturation medium IVM. The 4-well plates were placed at 38.5℃with 5% CO 2 Culturing in a humidity saturated incubator for 44h, and maturing oocytes after 44h to reach MII stage.
3. Parthenogenesis of porcine oocytes:
and (3) transferring the COC reaching the MII stage to 0.125% hyaluronidase by using a pipette, repeatedly blowing, and removing cumulus cells around the oocyte to obtain the naked oocyte. The naked oocytes were transferred to 297mmol/L (mM) mannitol (pH 7.2, SIGMA, M9546) solution containing 0.1mM CaCl 2 (SIGMA，C7902)、0.05mM MgSO 4 (SIGMA, M1880), 0.01% (w/v) PVA, 0.5mM HEPES. The parthenogenetic activation of the naked oocyte is carried out by carrying out two times in total by a direct current pulse with the electric field intensity of 1.2kV/cm of 60 mu s each time.
4. Applying the xanthoangelol to the pig in vitro parthenogenesis embryo and culturing the in vitro embryo:
1) Parthenogenesis activated oocytes were cultured in porcine embryo in vitro medium (containing 7.5mg/mL cytochalasin B) for 3-4h. Wherein, the pig embryo in vitro culture medium consists of the following components: 108mmol/L (mM) sodium chloride (sigma, S5886), 10mM potassium chloride (sigma, P5405), 0.35mM potassium dihydrogen phosphate (sigma, P5655), 0.4mM magnesium sulfate (sigma, M2643), 25mM sodium bicarbonate (sigma, S5761), 0.2mM sodium pyruvate (sigma, P5280), 2.0mM calcium lactate (sigma, C8356), 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), 25 mg/gentamycin (sigma, B6766), 4mg/mL bovine serum albumin (sigma, A8806), and ultrapure water was added to fix the volume to 1L.
2) Removing the culture medium, and applying porcine embryo in vitro culture medium containing the xanthoangelol to parthenogenesis activated oocytes (adding the xanthoangelol to the culture medium formula of the step 1) according to (0.5, 2.5 and 25 mu mol/L (mu M)) to serve as an experimental group; 1 control group was set up and porcine embryo in vitro medium containing no xanthoangelol was applied to parthenogenetically activated oocytes.
3) Oocytes were placed at 38.5℃with 5% CO 2 The culture was performed in an incubator saturated with humidity for 7 days without changing the medium.
Example 2
The embodiment is the application of the yellow angelica alcohol in preparing the product for improving the embryo blastocyst rate in vitro. The method specifically comprises the following steps: the yellow angelica alcohol is added into the pig embryo in vitro culture medium according to the effective amount of 0.1, 0.5 and 2.5 mu mol/L (mu M).
The medium was used for pig in vitro embryo culture by the same procedure as in example 1, and the quality and development ability of in vitro embryos were evaluated by detecting and calculating the blastocyst rate in the culture medium of the experimental group and the control group at day 7, and the results are shown in fig. 1. Wherein, blastula ratio= (number of blastula formed/number of all early parthenogenetic embryos cultured) ×100%.
As can be seen from FIG. 1, compared with the control group, the addition of 0.5 mu mol/L (mu M) of the yellow angelol into the pig embryo in-vitro culture medium can obviously improve the blastula rate of the pig in-vitro parthenogenesis activated embryo, wherein the blastula rate of the control group is 34.96%; when the concentration of the yellow angelica alcohol is 0.1 mu M, the blastula rate is 39.96%, which is improved by 2% compared with the control group; when the concentration is 0.5 mu M, the blastula rate can reach 45.1 percent, which is obviously higher than that of the control group (P < 0.01) and is improved by 10.14 percent compared with the control group. However, when the concentration of xanthoangelol was 2.5 μm, the blastula rate was 37.7%, 2.74% higher than the control group.
The results show that adding the yellow angelica alcohol into the culture medium helps promote parthenogenesis of the activated oocyte to the blastocyst. The activation of oocyte to develop smoothly to blastocyst is the key of in vitro embryo culture, and only early embryo with good development capacity and development quality can develop to blastocyst, and then embryo implantation rate can be improved by using blastocyst for in vivo transplantation. Therefore, the yellow angelica alcohol can improve the development capacity and the development quality of the embryo at the early stage before implantation, lays a good foundation for embryo implantation, and simultaneously shows that the yellow angelica alcohol has good application prospect in preparing the product for improving the embryo blastula rate of the embryo in vitro.
Example 3
The embodiment is the application of the yellow angelica alcohol in preparing a product for improving the mitochondrial membrane potential of the in vitro embryo cells. The method specifically comprises the following steps: the yellow angelic alcohol is added into the pig embryo in vitro culture medium according to the effective amount of 0.5 mu mol/L (mu M).
The above-described medium was used in pig in vitro embryo culture by the same procedure as in example 1, and the quality and development ability of the in vitro embryo were evaluated by detecting and calculating the mitochondrial membrane potential of the embryo cells in each medium when cultured to the 4-cell stage of the pig early parthenogenetic embryo. 4-cells were washed 3 times with PBS buffer, placed in a medium containing 0.5mmol/L (mM) of 5,5', 6' -tetrachloro-1, 1', 3' -tetraethyliminocarbonyl cyanine iodide (JC-1) at 38℃in 5% CO 2 Is incubated for 30min in the incubator.
Images were taken using a fluorescence microscope (Nikon, tokyo, japan). Images were analyzed with Image J software and membrane potential was calculated as the ratio of red fluorescence (corresponding to active mitochondria (J aggregates)) to green fluorescence (corresponding to low active mitochondria (J monomers)). The fluorescence intensity of the control group was set to 1, and the relative fluorescence intensity of the experimental group with respect to the control group was calculated.
As shown in fig. 2 and 3, JC-1 staining of 4-cell stage embryos measured mitochondrial membrane potential, which was significantly higher than that of the control group (P < 0.001), specifically 1.33 times that of the control group, increased by 33% relative to the control group; wherein the mitochondrial membrane potential is one of the important indexes for evaluating mitochondrial function, the reduction of the mitochondrial membrane potential is one of the manifestations of mitochondrial function reduction, and unhealthy mitochondria can have the membrane potential reduced or lost. Therefore, the results show that the addition of 0.5 mu M of the yellow angelica alcohol into the culture medium can effectively improve the mitochondrial membrane potential and protect the mitochondrial function, thereby improving the development quality and the development capacity of early embryo. Meanwhile, the yellow angelica alcohol has good application prospect in preparing the product for improving the mitochondrial membrane potential of the in vitro embryo cells.
Example 4
The embodiment is the application of the yellow angelica alcohol in preparing a product for improving the mitochondrial distribution of in vitro embryo cells and the expression level of related genes. The method specifically comprises the following steps: the yellow angelic alcohol is added into the pig embryo in vitro culture medium according to the effective amount of 0.5 mu mol/L (mu M).
The medium was used in pig in vitro embryo culture by the same procedure as in example 1, and quality and development ability of in vitro embryos were evaluated by detecting mitochondrial distribution of pig blasts and expression levels of related genes (TFAM gene, NRF1 gene, NRF2 gene):
when cultured until the 4-cell stage of the pig early parthenogenetic embryo, the 4-cells were stained with Mito-Tracker red probe (200 mmol/L, beyotime) for 45min. After washing, observations were made under a fluorescence microscope (Olympus). 20-30 oocyte mitochondrial distributions were examined.
Oocytes of the experimental group and the control group (without adding yellow angelic alcohol in example 1) were cultured in vitro to the blastocyst stage of the pig early parthenogenetic embryo (after 7 days of in vitro culture of the oocytes), and blasts were collected and lysed in 50 μl of lysis buffer. The extraction of mRNA from blasts was performed on ice using a kit (DynaBeads mRNA Direct Kit) according to the manufacturer's instructions and cDNA was synthesized by reverse transcription of mRNA. ThenKit for use (KAPA)FAST) was subjected to real-time fluorescent quantitative PCR procedures according to the instructions and qPCR reactions were performed using a CFX Connect Optics Module PCR instrument. The PCR reaction system was 20. Mu.L of cDNA, 1. Mu.L of upstream primer, 1. Mu.L of downstream primer, SYBR Green 10. Mu.L, ddH 2 O7. Mu.L. The names and sequences of the primers are shown in Table 1, in which GAPDH gene is used as a reference gene.
TABLE 1 names and sequences of the primers used in qPCR reactions
As a result, as shown in fig. 4 and 5, fig. 4 shows that the mitochondrial distribution level of the experimental group (yellow angelol 0.5 μm) was significantly higher than that of the control group (P < 0.001), wherein the control group was 0.99, the experimental group was 1.27, and the mitochondrial distribution of the experimental group was increased by 0.28 relative to the control group. FIG. 5 shows that the expression level of embryo cell related genes of the yellow angelica alcohol treatment group (0.5 mu M) is obviously higher than that of the control group (P < 0.001), and the expression of mitochondrial transcription factor A (TFAM gene), nuclear respiration factor 1 (NRF 1 gene) and nuclear erythrocyte related factor 2 (NRF 2 gene) are respectively 2.67 times, 1.41 times and 1.67 times that of the control group, and the addition of 0.5 mu M yellow angelica alcohol into the culture medium can improve the expression level of in vitro embryo cell oxidative stress genes, and also shows that the yellow angelica alcohol has good application prospect in preparing products for improving the proliferation capacity of in vitro embryo cells.
In conclusion, the invention proves that the yellow angelica alcohol can improve the blastula rate of in-vitro embryo culture, protect the mitochondrial function of in-vitro embryo cells and improve the proliferation capacity of in-vitro embryo cells, thereby effectively improving the quality and development capacity of in-vitro embryos. The yellow angelic alcohol has good market application prospect in preparing products for improving the quality and the development ability of in-vitro embryos.
Data statistics: examples 2-4 were all statistically analyzed using SPSS software, wherein student's t-test was used to compare two sets of data. One-way analysis of variance (ANOVA) was used to analyze three or more averages.
The embodiments of the present invention have been described in detail, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present invention.
Claims (10)
1. Application of yellow angelic alcohol in preparing embryo development promoting product is provided.
2. The use according to claim 1, characterized in that: the product is a kit, a culture medium or a medicament.
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 developmental capacity.
4. Application of yellow angelol in preparing products for improving embryo blastula rate, improving mitochondrial membrane potential and/or improving mitochondrial distribution of embryo cells and expression level of related genes thereof.
5. A product for promoting embryo development, characterized in that: the raw materials of the product comprise yellow angelica alcohol.
6. The product according to claim 5, wherein: the product is a kit, a culture medium or a medicament.
7. A method for improving the developmental capacity of an embryo in vitro, comprising: the method comprises the following steps: applying the yellow angelic alcohol to the in vitro embryo.
8. A method for promoting porcine embryo development in vitro, which is characterized in that: the method comprises the following steps: applying the yellow angelic alcohol to the in vitro embryo.
9. The method according to claim 8, wherein: the method comprises the following steps:
s1, applying yellow angelic alcohol to an in-vitro embryo;
s2, culturing the in-vitro embryo until the blastula stage.
10. The method according to claim 8 or 9, characterized in that: the effective amount of the yellow angelica alcohol is 0.1-2.5 mu mol/L; preferably 0.2 to 1. Mu. Mol/L, most preferably 0.3 to 0.5. Mu. Mol/L.
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