CN117264878A - Culture solution added with echinacoside for improving porcine oocyte aging and application thereof - Google Patents
Culture solution added with echinacoside for improving porcine oocyte aging and application thereof Download PDFInfo
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- CN117264878A CN117264878A CN202311212065.8A CN202311212065A CN117264878A CN 117264878 A CN117264878 A CN 117264878A CN 202311212065 A CN202311212065 A CN 202311212065A CN 117264878 A CN117264878 A CN 117264878A
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- FSBUXLDOLNLABB-ISAKITKMSA-N echinacoside Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](OC(=O)\C=C\C=2C=C(O)C(O)=CC=2)[C@@H](CO[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)O[C@@H](OCCC=2C=C(O)C(O)=CC=2)[C@@H]1O FSBUXLDOLNLABB-ISAKITKMSA-N 0.000 title claims abstract description 121
- 210000000287 oocyte Anatomy 0.000 title claims abstract description 113
- NJYVDFDTLLZVMG-UHFFFAOYSA-N echinacoside Natural products CC1OC(OC2C(O)C(OCCc3ccc(O)c(O)c3)OC(COC4OC(CO)C(O)C(O)C4O)C2OC(=O)C=Cc5cc(O)cc(O)c5)C(O)C(O)C1O NJYVDFDTLLZVMG-UHFFFAOYSA-N 0.000 title claims abstract description 91
- 230000032683 aging Effects 0.000 title claims abstract description 39
- 239000000243 solution Substances 0.000 claims abstract description 121
- WQZGKKKJIJFFOK-SVZMEOIVSA-N (+)-Galactose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-SVZMEOIVSA-N 0.000 claims abstract description 108
- 230000035800 maturation Effects 0.000 claims abstract description 51
- 210000001733 follicular fluid Anatomy 0.000 claims abstract description 44
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims abstract description 29
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- 101800003838 Epidermal growth factor Proteins 0.000 claims abstract description 24
- 229940116977 epidermal growth factor Drugs 0.000 claims abstract description 24
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 claims abstract description 24
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 20
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- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims abstract description 19
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 9
- 210000000349 chromosome Anatomy 0.000 claims description 9
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- 210000001672 ovary Anatomy 0.000 claims description 9
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- 102000011022 Chorionic Gonadotropin Human genes 0.000 claims description 7
- 108010062540 Chorionic Gonadotropin Proteins 0.000 claims description 7
- 229940015047 chorionic gonadotropin Drugs 0.000 claims description 7
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- 239000004201 L-cysteine Substances 0.000 claims description 5
- 235000013878 L-cysteine Nutrition 0.000 claims description 5
- GZCGUPFRVQAUEE-KCDKBNATSA-N aldehydo-D-galactose Chemical compound OC[C@@H](O)[C@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-KCDKBNATSA-N 0.000 claims description 5
- 238000011160 research Methods 0.000 claims description 5
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- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-UHFFFAOYSA-N 0.000 description 2
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- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 1
- GZCGUPFRVQAUEE-UHFFFAOYSA-N 2,3,4,5,6-pentahydroxyhexanal Chemical compound OCC(O)C(O)C(O)C(O)C=O GZCGUPFRVQAUEE-UHFFFAOYSA-N 0.000 description 1
- 241000336291 Cistanche deserticola Species 0.000 description 1
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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
- C12N5/0608—Germ cells
- C12N5/0609—Oocytes, oogonia
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
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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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/34—Sugars
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Abstract
The invention discloses a culture solution for improving aging of porcine oocytes by adding echinacoside and application thereof, and the culture solution comprises a basal culture solution, D-galactose and echinacoside, wherein the basal culture solution is based on an M199 culture solution which does not contain HEPES and has the volume fraction of 82.9%, 10% of porcine follicular fluid and 5% of fetal bovine serum are added, and meanwhile, 1% of L cysteine with the volume fraction of 6.9mg/mL, 0.1% of epidermal growth factor with the volume fraction of 10 mug/mL, 1% of gonadotropin with the volume fraction of 10IU/mL and chorionic gonadotrophin with the volume fraction of 10IU/mL are added, the addition of D-galactose is 9mg/mL, and the addition of the echinacoside is 39.337 mug/mL or 78.673 mug/mL. According to the invention, a damage model is built by adding 9 mg/mLD-galactose into a basic culture solution, and 78.673 mug/mL of echinacoside is added on the basis, so that the maturation rate of oocytes can be remarkably improved.
Description
Technical Field
The invention relates to a culture solution for in vitro maturation of porcine oocytes, in particular to a culture solution for improving aging of porcine oocytes by adding echinacoside and application thereof, belonging to the field of application of livestock breeding technology.
Background
In vitro maturation culture (IVM) of porcine oocytes is an important element in livestock breeding technology as one of the ways to produce porcine embryos, and the quality of in vitro mature oocytes affects a number of technical elements including in vitro fertilization, parthenogenesis and embryo cloning. Because the existing in-vitro culture system can not completely reduce the in-vivo microenvironment, the in-vitro mature oocyte has obvious difference in maturation rate, maturation quality and development potential compared with the in-vivo mature oocyte, so that the in-vitro culture system with higher maturation rate of the oocyte is established to be more stable.
At present, it is not clear what core factors beneficial to oocyte development are added in the in vitro maturation process, and factors which are widely considered to contribute to oocyte maturation include hormones, growth factors, cytokines and other components.
Therefore, providing a swine oocyte in vitro maturation culture solution and application thereof is a problem to be solved by those skilled in the art.
D-galactose is an aldohexose, a reducing sugar, which is naturally found in the body and many foods, and at high levels, it can be converted to aldose and hydrogen peroxide under the catalysis of galactose oxidase, thereby generating Reactive Oxygen Species (ROS). Oxidative stress, inflammation, mitochondrial dysfunction and apoptosis may then result. The medicine is a most favored aging model building medicine because of convenience, small side effect and high survival rate.
Echinacoside is a natural phenethanoside and is a main active ingredient in cistanche deserticola, and researches show that Echinacoside has beneficial effects in the aspects of neuroprotection, anti-tumor, anti-aging, myocardial remodeling, antioxidation, anti-inflammatory and anti-apoptosis. At present, no study on the combined addition of echinacoside in the in-vitro maturation process of porcine oocytes exists. The method is significant in exploring whether the addition of echinacoside can obviously improve the in-vitro maturation efficiency of the porcine oocyte, thereby improving the quality of the porcine oocyte.
Disclosure of Invention
The invention aims to solve the problems and provide a culture solution for improving the aging of porcine oocytes by adding echinacoside and application thereof.
The invention achieves the above object by the following technical scheme, a culture solution for improving aging of porcine oocytes by adding echinacoside, comprising basal culture solution, D-galactose (Sigma, G5388) and echinacoside (MCE, HY-N0020), wherein the basal culture solution is based on M199 culture solution (Sigma, M4530) which does not contain HEPES and has 82.9% volume fraction, 10% volume fraction of porcine follicular fluid and 5% volume fraction of fetal bovine serum (Cellmax, SA 311.01) are added, 1% volume fraction of L-cysteine (Sigma, C7880), 0.1% volume fraction of epidermal growth factor (Sigma, E4127), 1% volume fraction of gonadotropin and mixed solution of chorionic gonadotropin (Ningbo three) which has 10IU/mL are added, the addition amount of D-galactose is 9mg/mL, the addition amount of echinacoside is 39.337 mug or 84 mug/mL,
the preparation method of the culture solution for improving the aging of the porcine oocyte by adding echinacoside comprises the following steps:
SA, adding 9mg/mL of D-galactose into the basic culture solution, and establishing an aging in-vitro culture solution model of the porcine oocyte;
SB, dissolving 5mg of echinacoside in 127.108 mu L of DMSO to obtain 50mM concentrated stock solution, and respectively diluting the 50mM concentrated stock solution to 78.673 mu g/mL and 39.337 mu g/mL by using the aging in vitro culture solution of the porcine oocyte prepared in SA;
and SC, adding 39.337 mug/mL or 78.673 mug/mL echinacoside into the aging in vitro culture solution model of the pig oocyte prepared in SA, and finally obtaining the culture solution for improving the aging of the pig oocyte by adding the echinacoside.
Preferably, the addition amount of the echinacoside in the culture solution for improving the aging of the porcine oocyte is 78.673 mug/mL.
Preferably, the echinacoside has a formula of C 35 H 45 O 20 The molecular weight is 786.73, and the structural formula is as follows:
preferably, the preparation process of the pig follicular fluid comprises the following steps:
collecting fresh ovary of pig, placing in 38.5deg.C normal saline thermos bottle, extracting follicular fluid of ovarian surface follicle with 18G needle syringe, collecting the extracted follicular fluid in 15mL centrifuge tube, standing in 38.5deg.C incubator for 30min, centrifuging the upper follicular fluid at 3000rpm for 30min, centrifuging the lower layer to obtain follicle granulosa Cell Oocyte Complex (COCs), filtering the centrifuged supernatant with 0.22 μm filter, packaging the filtered follicular fluid, and storing at 20deg.C.
Preferably, follicles with a diameter of 2 to 8mm are selected during the follicular fluid extraction.
The invention also discloses an application of the culture solution for improving the aging of the porcine oocyte by adding the echinacoside, in particular to an application of the echinacoside in improving the maturation rate and the development quality of the injured porcine oocyte, which comprises the research on the expansion degree of the porcine oocyte cumulus, the first polar body in the MII period of the porcine oocyte, and the spindle and chromosome morphology after the maturation of the porcine oocyte, the experimental design is as follows,
control group: in vitro maturation culture solution without D-galactose and echinacoside
Based on the M199 culture solution without HEPES, 10% of pig follicular fluid and 5% of fetal bovine serum are added, and a mixture of 1% of L cysteine with 6.9mg/mL of L cysteine, 0.1% of epidermal growth factor with 10 mug/mL of epidermal growth factor, 1% of gonadotropin with 10IU/mL of chorionic gonadotrophin is added in a supplementary manner;
d-galactose control group: in vitro maturation culture solution containing 9mg/mL D-galactose
Based on the M199 culture solution without HEPES, which is 82.9% in volume fraction, 10% in volume fraction of pig follicular fluid and 5% in volume fraction of fetal bovine serum are added, and 1% in volume fraction of L cysteine, 0.1% in volume fraction of 10 mug/mL of epidermal growth factor, 1% in volume fraction of a mixed solution of gonadotropin with chorionic gonadotrophin with a volume fraction of 10IU/mL and 9mg/mL of D-galactose are added in a supplementary manner;
echinacoside group 1: in vitro maturation culture solution containing 39.337 mug/mL echinacoside
Based on the M199 culture solution without HEPES, 10% of pig follicular fluid and 5% of fetal bovine serum are added, and 1% of L cysteine with the volume fraction of 6.9mg/mL, 0.1% of epidermal growth factor with the volume fraction of 10 mu g/mL, a mixed solution of gonadotropin with the volume fraction of 1% of 10IU/mL and chorionic gonadotrophin with the volume fraction of 10IU/mL, 9mg/mL of D-galactose and 39.337 mu g/mL of echinacoside are added in a supplementary manner;
echinacoside group 2: in vitro maturation culture solution containing 78.673 mug/mL echinacoside
Based on the M199 culture solution without HEPES with 82.9% volume fraction, 10% volume fraction of pig follicular fluid and 5% volume fraction of fetal bovine serum were added, while 1% volume fraction of L-cysteine with 6.9mg/mL, 0.1% volume fraction of epidermal growth factor with 10. Mu.g/mL, 1% volume fraction of a mixture of gonadotropin with 10IU/mL and chorionic gonadotropin with 10IU/mL, 9mg/mL of D-galactose, 78.673. Mu.g/mL of echinacoside were supplemented.
Preferably, in the research test of the expansion degree of the porcine oocyte cumulus and the first polar body in the MII period of the porcine oocyte, a control group, a D-galactose control group, an echinacoside group 1 and an echinacoside group 2 are arranged, three independent repeated tests are carried out, 60 oocytes in the MII period are repeatedly detected each time, and the culture is carried out for 42-48 hours; in the study test of spindle and chromosome morphology after pig oocyte maturation, echinacoside group 1 is canceled, a reserved control group, a D-galactose control group and Echinacoside group 2 are reserved, three independent repetitions are arranged in each group of test, 15-20 MI-phase oocytes are detected each time, the culture is carried out for 24-28 hours, and the result difference statistics are analyzed by using GraphPad Prism statistical software.
The beneficial effects of the invention are as follows: compared with the prior art, the culture solution for improving the aging of the porcine oocyte by adding the echinacoside and the application thereof are provided, a damage model is built by adding 9 mg/mLD-galactose into a basic culture solution, 78.673 mug/mL of the echinacoside is added on the basis, the maturation rate of the oocyte can be obviously improved, the maturation rate is improved by approximately 20%, the normal proportion of a spindle body is improved by approximately 26%, a large number of high-quality oocytes can be effectively obtained by the scheme, the support is provided for the technologies of in vitro fertilization, parthenogenesis, preparation of cloned embryos by nuclear transfer and the like, and the culture solution has important significance in the aspects of propagation of improved stock, animal model construction, transgenic animal production and the like.
Drawings
FIG. 1 shows the degree of expansion of the cumulus expansion of a porcine oocyte with or without echinacoside added to the culture medium during in vitro maturation of the porcine oocyte according to the present invention: sequentially comprises a control group, a D-galactose control group, a echinacoside group 1 (39.337 mug/mL) and a echinacoside group 2 (78.673 mug/mL); the original picture scale is 200 μm; the scale of the magnified image was 50. Mu.m.
FIG. 2 shows the first extreme exotic condition of the pig oocyte MII period with or without echinacoside added to the culture solution during the in vitro maturation process of the pig oocyte;
wherein, A is the first polar body of the pig oocyte in MI phase of the control group, the D-galactose control group, the echinacoside group 1 (39.337 mug/mL echinacoside is added in the basic culture solution), the echinacoside group 2 (78.673 mug/mL echinacoside is added in the basic culture solution); the original picture scale is 200 μm; the scale of the magnified image was 50. Mu.m.
B is the first polar body discharge percentage of the pig oocytes in MI phase of the control group, the D-galactose control group, the echinacoside group 1 (39.337 mug/mL echinacoside is added into the basic culture solution) and the echinacoside group 2 (78.673 mug/mL echinacoside is added into the basic culture solution).
FIG. 3 shows the status of spindle and chromosome after maturation of porcine oocytes with or without echinacoside added to the culture solution during in vitro maturation of porcine oocytes in accordance with the present invention;
wherein A is the situation of the shapes of the spindle bodies of the pig oocytes in MI phase of a control group, a D-galactose control group and echinacoside group 2 (78.673 mug/mL echinacoside is added into a basic culture solution), and a confocal microscope is used for photographing; the scale bar is 20 mu m;
b is the normal proportion percentage of the shapes of the spindle bodies of the pig oocytes in MI phase of a control group, a D-galactose control group and echinacoside group 2 (78.673 mu g/mL echinacoside is added into a basic culture solution);
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The reagents used in the present invention are as follows:
d-galactose: purchased from Sigma, model number: g5388;
echinacoside: purchased from MCE, model number: HY-N0020;
m199 culture: purchased from Sigma, model number: m4530;
fetal bovine serum at 5% volume fraction: purchased from Cellmax, model number: SA311.01;
l-cysteine: purchased from Sigma, model number: c7880;
epidermal growth factor: purchased from Sigma, model number: e4127;
mixed liquor of gonadotrophin and chorionic gonadotrophin: purchased from Ningbo Sansheng;
hyaluronidase solution: purchased from Sigma, model number: h3506;
anti-alpha-tubulin-FITC: purchased from Sigma, model number: f2168 A) is provided;
fixing solution: purchased from Sigma, model number: 818715;
permeabilization liquid: purchased from solebao, model number: 9002-93-1;
sealing liquid: purchased from Sigma, model number: a1933;
washing buffer: purchased from Biyundian, model P0106;
hoechst 33342: purchased from zemoeid under model 2189158.
The instrument and equipment used in the invention are as follows:
a split microscope: purchased from olynbas, model SZX7;
confocal microscopy: purchased from Olympus, model FV3000.
Example 1 culture solution for improving aging of porcine oocytes by adding Echinacoside and preparation method thereof
A culture solution for improving aging of porcine oocytes by adding echinacoside, which comprises a basal culture solution, D-galactose and echinacoside, wherein the basal culture solution is based on an M199 culture solution which does not contain HEPES and has the volume fraction of 82.9%, 10% of porcine follicular fluid and 5% of fetal bovine serum, and is supplemented with a mixture of 1% of L cysteine with the volume fraction of 6.9mg/mL, 0.1% of epidermal growth factor with the volume fraction of 10 mug/mL, 1% of gonadotropin with the volume fraction of 10IU/mL and chorionic gonadotropin with the volume fraction of 10IU/mL, the addition of D-galactose is 9mg/mL, and the addition of the echinacoside is 39.337 mug/mL or 78.673 mug/mL.
The preparation method of the culture solution for improving the aging of the porcine oocyte by adding echinacoside comprises the following steps:
SA, adding 9mg/mL of D-galactose into the basic culture solution, and establishing an aging in-vitro culture solution model of the porcine oocyte;
SB, dissolving 5mg of echinacoside in 127.108 mu L of DMSO to obtain 50mM concentrated stock solution, and respectively diluting the 50mM concentrated stock solution to 78.673 mu g/mL and 39.337 mu g/mL by using the aging in vitro culture solution of the porcine oocyte prepared in SA;
and SC, adding 39.337 mug/mL or 78.673 mug/mL echinacoside into the aging in vitro culture solution model of the pig oocyte prepared in SA, and finally obtaining the culture solution for improving the aging of the pig oocyte by adding the echinacoside.
In the above embodiment, the echinacoside has a formula of C 35 H 45 O 20 The molecular weight is 786.73, and the structural formula is as follows:
in the above embodiment, the preparation process of the pig follicular fluid specifically comprises:
collecting fresh ovaries of pigs, placing the ovaries into a physiological saline vacuum bottle at 38.5 ℃, extracting follicular fluid of follicles on the surfaces of the ovaries by using an 18G needle syringe, selecting follicles with the diameter of 2-8 mm in the process of extracting the follicular fluid, collecting the extracted follicular fluid into a 15mL centrifuge tube, placing the centrifuge tube in a constant temperature box at 38.5 ℃ for standing for 30min, collecting upper follicular fluid, centrifuging at 3000rpm for 30min, collecting the upper layer of the follicular fluid which is a cumulus granulosa Cell Oocyte Complex (COCs), filtering the supernatant after centrifugation by using a 0.22 mu m filter, and subpackaging the obtained pig follicular fluid after filtration and preserving at 20 ℃.
EXAMPLE 2 application of culture solution for improving aging of porcine oocytes by adding Echinacoside
2.1 design of experiments
Control group: in vitro maturation culture solution without D-galactose and echinacoside
Based on the M199 culture solution without HEPES, 10% of pig follicular fluid and 5% of fetal bovine serum are added, and a mixture of 1% of L cysteine with 6.9mg/mL of L cysteine, 0.1% of epidermal growth factor with 10 mug/mL of epidermal growth factor, 1% of gonadotropin with 10IU/mL of chorionic gonadotrophin is added in a supplementary manner;
d-galactose control group: in vitro maturation culture solution containing 9mg/mL D-galactose
Based on the M199 culture solution without HEPES, which is 82.9% in volume fraction, 10% in volume fraction of pig follicular fluid and 5% in volume fraction of fetal bovine serum are added, and 1% in volume fraction of L cysteine, 0.1% in volume fraction of 10 mug/mL of epidermal growth factor, 1% in volume fraction of a mixed solution of gonadotropin with chorionic gonadotrophin with a volume fraction of 10IU/mL and 9mg/mL of D-galactose are added in a supplementary manner;
echinacoside group 1: in vitro maturation culture solution containing 39.337 mug/mL echinacoside
Based on the M199 culture solution without HEPES, 10% of pig follicular fluid and 5% of fetal bovine serum are added, and 1% of L cysteine with the volume fraction of 6.9mg/mL, 0.1% of epidermal growth factor with the volume fraction of 10 mu g/mL, a mixed solution of gonadotropin with the volume fraction of 1% of 10IU/mL and chorionic gonadotrophin with the volume fraction of 10IU/mL, 9mg/mL of D-galactose and 39.337 mu g/mL of echinacoside are added in a supplementary manner;
echinacoside group 2: in vitro maturation culture solution containing 78.673 mug/mL echinacoside
Based on the M199 culture solution without HEPES with 82.9% volume fraction, 10% volume fraction of pig follicular fluid and 5% volume fraction of fetal bovine serum were added, while 1% volume fraction of L-cysteine with 6.9mg/mL, 0.1% volume fraction of epidermal growth factor with 10. Mu.g/mL, 1% volume fraction of a mixture of gonadotropin with 10IU/mL and chorionic gonadotropin with 10IU/mL, 9mg/mL of D-galactose, 78.673. Mu.g/mL of echinacoside were supplemented.
2.2 study of the extent of expansion of the cumulus of porcine oocytes and the first polar in vitro discharge of porcine oocytes during MII period
2.2.1 pretreatment of porcine oocytes
Collecting fresh ovaries of pigs, placing the fresh ovaries in a physiological saline vacuum bottle at 38.5 ℃, extracting follicular fluid of follicles on the surfaces of the ovaries by using an 18G needle head injector, selecting follicles with the diameter of 2-8 mm in the process of extracting follicular fluid, collecting the extracted follicular fluid in a 15mL centrifuge tube, placing the follicular fluid in a constant temperature box at 38.5 ℃ for 30min, standing to obtain follicular fluid on the upper layer, collecting oophoroma granulosa Cell Oocyte Complexes (COCs) on the lower layer, discarding the follicular fluid on the upper layer, leaving COCs on the lower layer, adding DPBS with the same volume as a sediment as an operation liquid of the oophoroma cells, reversing and uniformly mixing, pouring the mixture into a 60mm culture dish, selecting the oophoroma cells with three layers or more by using a 500 mu m glass needle, washing the oophoroma cells with three layers or more of COCs in an in vitro maturation culture liquid without echinacoside and D-galactose for maturation culture.
2.2.2 in vitro maturation culture of porcine oocytes
Control group: adding in vitro maturation culture solution without echinacoside and D-galactose into 4-well plate, adding 500 μl of culture solution into each well, coating paraffin oil 300 μl, and heating at 38.5deg.C and 5% CO 2 Preheating in a saturated humidity incubator for more than 5 hours, placing selected and washed COCs in mature holes, and culturing for 42-48 hours with 60 COCs in each hole.
D-galactose control group: adding 9mg/mL in vitro maturation culture solution containing D-galactose into 4-well plate, adding 500 μl of culture solution into each well, coating paraffin oil 300 μl, and heating at 38.5deg.C and 5% CO 2 Preheating in a saturated humidity incubator for more than 5 hours, placing selected and washed COCs in mature holes, and culturing for 42-48 hours with 60 COCs in each hole.
Echinacoside group 1: adding in vitro maturation culture solution containing 9mg/mL D-galactose and 39.337 μg/mL echinacoside into 4-well plate, adding 500 μl culture solution into each well, coating paraffin oil 300 μl, and heating at 38.5deg.C and 5% CO 2 Preheating in a saturated humidity incubator for more than 5 hours, placing selected and washed COCs in mature holes, and culturing for 42-48 hours with 60 COCs in each hole.
Echinacoside group 2: adding in vitro maturation culture solution containing 9mg/mL D-galactose and 78.673 μg/mL echinacoside into 4-well plate, adding 500 μl culture solution into each well, and coating paraffin oil 300 μl, 38.5deg.C and 5% CO 2 Preheating in a saturated humidity incubator for more than 5 hours, placing selected and washed COCs in mature holes, and culturing for 42-48 hours with 60 COCs in each hole.
2.2.3MII porcine oocyte collection
Preparing hyaluronidase solution with concentration of 1mg/mL by using TCM-199 as solvent, sucking cultured mature COCs by using a glass needle with caliber of 200 mu m, putting into 200 mu L of 1mg/mL hyaluronidase solution, lightly blowing by using a 100 mu L pipette until oocytes which are fully removed from cumulus particles are visible under a split microscope, transferring the oocytes into the TCM-199 solution by using the 200 mu m glass needle for three times of washing, wherein each washing is required to be performed in clean TCM-199, and then selecting oocytes of a first polar body by using the 200 mu m glass needle under the split microscope, namely mature oocytes in the MII stage.
The above method is applicable to the control group, D-galactose control group, echinacoside group 1 and Echinacoside group 2.
Maturation rate = number of oocytes discharged from first polar body (MII stage)/total number of oocytes.
TABLE 1 MII phase oocyte maturation rates in different test groups
As shown in the results of figures 1 and 2, when 78.673 mug/mL echinacoside is added to the culture solution, the maturation rate is remarkably improved, the expansion degree of the cumulus cells is more sufficient, and the first polar body in the MII period of the oocyte is more obviously discharged outside compared with the D-galactose control group.
The test result shows that the effect of adding 78.673 mug/mL of echinacoside into the basic culture solution is better, and the echinacoside in the echinacoside group 2 is added in the subsequent test.
2.3 study of spindle and chromosome morphology after maturation of porcine oocytes
2.3.1 test design
Control group: in vitro maturation culture solution without D-galactose and echinacoside
Based on the M199 culture solution without HEPES, 10% of pig follicular fluid and 5% of fetal bovine serum are added, and a mixture of 1% of L cysteine with 6.9mg/mL of L cysteine, 0.1% of epidermal growth factor with 10 mug/mL of epidermal growth factor, 1% of gonadotropin with 10IU/mL of chorionic gonadotrophin is added in a supplementary manner;
d-galactose control group: in vitro maturation culture solution containing 9mg/mL D-galactose
Based on the M199 culture solution without HEPES, which is 82.9% in volume fraction, 10% in volume fraction of pig follicular fluid and 5% in volume fraction of fetal bovine serum are added, and 1% in volume fraction of L cysteine, 0.1% in volume fraction of 10 mug/mL of epidermal growth factor, 1% in volume fraction of a mixed solution of gonadotropin with chorionic gonadotrophin with a volume fraction of 10IU/mL and 9mg/mL of D-galactose are added in a supplementary manner;
echinacoside group 2: in vitro maturation culture solution containing 78.673 mug/mL echinacoside
Based on the M199 culture solution without HEPES with 82.9% volume fraction, 10% volume fraction of pig follicular fluid and 5% volume fraction of fetal bovine serum were added, while 1% volume fraction of L-cysteine with 6.9mg/mL, 0.1% volume fraction of epidermal growth factor with 10. Mu.g/mL, 1% volume fraction of a mixture of gonadotropin with 10IU/mL and chorionic gonadotropin with 10IU/mL, 9mg/mL of D-galactose, 78.673. Mu.g/mL of echinacoside were supplemented.
Three sets of repetition are arranged in the control group, the D-galactose control group and the echinacoside group 2, 15-20 MI oocytes are detected in each set of repetition, and the result difference statistics are analyzed by using GraphPad Prism statistical software.
2.3.2 in vitro maturation culture of porcine oocytes
Reference is made to example 2.2.2 above.
2.3.3MI oocyte collection
Preparing a hyaluronidase solution with a concentration of 1mg/mL by using TCM-199 as a solvent, sucking COCs with a caliber of 200 mu m for culturing for 24-28h by using a glass needle, putting the COCs into 200 mu L of the hyaluronidase solution with a concentration of 1mg/mL, lightly blowing the COCs by using a 100 mu L pipette until oocytes with fully removed cumulus particles can be seen under a lens, transferring the oocytes into the TCM-199 solution by using the glass needle for three times, and washing in clean TCM-199 for each time, wherein the obtained cells are the oocytes in the MI period.
The above method was applied to the control group, D-galactose control group and echinacoside group 2.
2.3.4 pig oocyte spindle and chromosome morphology detection
And (3) detecting spindle bodies and chromosome morphology of the mature oocytes of pigs, staining the mature oocytes by using anti-alpha-tubulin-FITC, respectively standing the MI-phase oocytes obtained in a control group, a D-galactose control group and an echinacoside group 2 in a fixing solution at room temperature for 30min, washing the washing solution for three times, and transferring the washing solution into a permeabilization solution at room temperature for 8h. Transferring into a sealing solution, standing at room temperature for 1h, respectively placing the MI-phase oocytes in a control group, a D-galactose control group and an echinacoside group 2 after standing at room temperature into a solution containing anti-alpha-tubulin-FITC (1:800), incubating overnight at 4 ℃ in a wet box, washing the MI-phase oocytes with a washing buffer three times each for not less than 3min, staining cell nuclei by Hoechst 33342, and observing under a confocal microscope. The results are shown in FIG. 3.
As can be seen from fig. 3, the spindle normal rate in the control group is 70.07 ± 2.754%; the spindle normal rate in the D-galactose control group is 30.60+/-7.002 percent; and compared with the D-galactose control group, when 78.673 mug/mL echinacoside is added into the porcine oocyte basic culture solution, the spindle normal rate in the echinacoside group 2 is 57.13 +/-7.150 percent, the spindle normal rate is obviously improved, the spindle normal rate is improved by about 26 percent, and the chromosome arrangement disorder degree is reduced.
In summary, the invention provides a culture solution for improving the aging of porcine oocytes by adding echinacoside and application thereof, a damage model is built by adding 9 mg/mLD-galactose into a basic culture solution, 78.673 mug/mL of echinacoside is added on the basis, the maturation rate of oocytes can be obviously improved, the maturation rate is improved by approximately 20%, the normal proportion of spindles is improved by approximately 26%, a large number of high-quality oocytes can be effectively obtained by adopting the scheme, the support is provided for the technologies of in vitro fertilization, parthenogenesis, preparation of cloned embryos by nuclear transfer and the like, and the culture solution has important significance in the aspects of propagation of improved stock, animal model construction, transgenic animal production and the like.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.
Claims (9)
1. A culture solution for improving aging of porcine oocytes by adding echinacoside, which is characterized by comprising a basal culture solution, D-galactose and echinacoside, wherein the basal culture solution is based on an M199 culture solution which does not contain HEPES and has a volume fraction of 82.9%, 10% of porcine follicular fluid and 5% of fetal bovine serum are added, and a mixture of L cysteine with a volume fraction of 6.9mg/mL, epidermal growth factor with a volume fraction of 10 μg/mL of 0.1% and gonadotropin with a volume fraction of 10IU/mL and chorionic gonadotropin with a volume fraction of 10IU/mL is supplemented, the addition amount of D-galactose is 9mg/mL, and the addition amount of echinacoside is 39.337 μg/mL or 78.673 μg/mL.
The preparation method of the culture solution for improving the aging of the porcine oocyte by adding echinacoside comprises the following steps:
SA, adding 9mg/mL of D-galactose into the basic culture solution, and establishing an aging in-vitro culture solution model of the porcine oocyte;
SB, dissolving 5mg of echinacoside in 127.108 mu L of DMSO to obtain 50mM concentrated stock solution, and respectively diluting the 50mM concentrated stock solution to 78.673 mu g/mL and 39.337 mu g/mL by using the aging in vitro culture solution of the porcine oocyte prepared in SA;
and SC, adding 39.337 mug/mL or 78.673 mug/mL echinacoside into the aging in vitro culture solution model of the pig oocyte prepared in SA, and finally obtaining the culture solution for improving the aging of the pig oocyte by adding the echinacoside.
2. The culture solution for improving aging of porcine oocytes by adding echinacoside according to claim 1, wherein the culture solution is characterized in that: the addition amount of the echinacoside in the culture solution for improving the aging of the porcine oocyte is 78.673 mug/mL.
3. A culture broth for improving aging of porcine oocytes by adding echinacoside according to claim 1 or claim 2, wherein: the molecular formula of the echinacoside is C 35 H 45 O 20， The molecular weight is 786.73, and the structural formula is as follows:
4. the culture solution for improving aging of porcine oocytes by adding echinacoside according to claim 1, wherein the culture solution is characterized in that: the preparation method of the pig follicular fluid specifically comprises the following steps:
collecting fresh ovary of pig, placing in 38.5deg.C normal saline thermos bottle, extracting follicular fluid of ovarian surface follicle with 18G needle syringe, collecting the extracted follicular fluid in 15mL centrifuge tube, standing in 38.5deg.C incubator for 30min, collecting upper follicular fluid, centrifuging at 3000rpm for 30min, collecting lower layer to obtain cumulus granulosa cell oocyte complex, filtering the centrifuged supernatant with 0.22 μm filter, packaging the filtered follicular fluid, and storing at 20deg.C.
5. The culture solution for improving aging of porcine oocytes by adding echinacoside according to claim 4, wherein the culture solution is characterized in that: and selecting the follicles with the diameter of 2-8 mm in the process of extracting the follicular fluid of the follicles on the surface of the ovary.
6. The use of a culture broth for improving aging of porcine oocytes by adding echinacoside according to claim 1.
7. The use of a culture broth for improving aging of porcine oocytes with echinacoside as claimed in claim 6, wherein: in particular to application of echinacoside in improving the maturation rate and the development quality of injured pig oocytes.
8. The use of a culture broth for improving aging of porcine oocytes with echinacoside as claimed in claim 7, wherein: comprises the research of the expansion degree of a porcine oocyte cumulus, the first polar body exogenesis of the porcine oocyte in the MII period and the spindle and chromosome morphology of the mature porcine oocyte, and the experimental design is that,
control group: in vitro maturation culture solution without D-galactose and echinacoside
Based on the M199 culture solution without HEPES, 10% of pig follicular fluid and 5% of fetal bovine serum are added, and a mixture of 1% of L cysteine with 6.9mg/mL of L cysteine, 0.1% of epidermal growth factor with 10 mug/mL of epidermal growth factor, 1% of gonadotropin with 10IU/mL of chorionic gonadotrophin is added in a supplementary manner;
d-galactose control group: in vitro maturation culture solution containing 9mg/mL D-galactose
Based on the M199 culture solution without HEPES, which is 82.9% in volume fraction, 10% in volume fraction of pig follicular fluid and 5% in volume fraction of fetal bovine serum are added, and 1% in volume fraction of L cysteine, 0.1% in volume fraction of 10 mug/mL of epidermal growth factor, 1% in volume fraction of a mixed solution of gonadotropin with chorionic gonadotrophin with a volume fraction of 10IU/mL and 9mg/mL of D-galactose are added in a supplementary manner;
echinacoside group 1: in vitro maturation culture solution containing 39.337 mug/mL echinacoside
Based on the M199 culture solution without HEPES, 10% of pig follicular fluid and 5% of fetal bovine serum are added, and 1% of L cysteine with the volume fraction of 6.9mg/mL, 0.1% of epidermal growth factor with the volume fraction of 10 mu g/mL, a mixed solution of gonadotropin with the volume fraction of 1% of 10IU/mL and chorionic gonadotrophin with the volume fraction of 10IU/mL, 9mg/mL of D-galactose and 39.337 mu g/mL of echinacoside are added in a supplementary manner;
echinacoside group 2: in vitro maturation culture solution containing 78.673 mug/mL echinacoside
Based on the M199 culture solution without HEPES with 82.9% volume fraction, 10% volume fraction of pig follicular fluid and 5% volume fraction of fetal bovine serum were added, while 1% volume fraction of L-cysteine with 6.9mg/mL, 0.1% volume fraction of epidermal growth factor with 10. Mu.g/mL, 1% volume fraction of a mixture of gonadotropin with 10IU/mL and chorionic gonadotropin with 10IU/mL, 9mg/mL of D-galactose, 78.673. Mu.g/mL of echinacoside were supplemented.
9. The use of a culture broth for improving aging of porcine oocytes with echinacoside as claimed in claim 8, wherein: in the research test of the expansion degree of the porcine oocyte cumulus and the first polar in vitro row of the porcine oocyte MII period, setting a control group, a D-galactose control group, a echinacoside group 1 and a echinacoside group 2, carrying out three independent repeated tests, repeatedly detecting 60 MII-period oocytes each time, and culturing for 42-48 hours; in the study test of spindle and chromosome morphology after pig oocyte maturation, echinacoside group 1 is canceled, a reserved control group, a D-galactose control group and Echinacoside group 2 are reserved, three independent repetitions are arranged in each group of test, 15-20 MI-phase oocytes are detected each time, the culture is carried out for 24-28 hours, and the result difference statistics are analyzed by using GraphPad Prism statistical software.
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