CN110760473A - Porcine oocyte in-vitro maturation culture solution and preparation method and application thereof - Google Patents
Porcine oocyte in-vitro maturation culture solution and preparation method and application thereof Download PDFInfo
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Abstract
A porcine oocyte in-vitro maturation culture solution and a preparation method and application thereof belong to the technical field of oocyte in-vitro maturation culture. The invention provides a porcine oocyte in-vitro maturation culture solution and a preparation method and application thereof, aiming at solving the problem that the in-vitro maturation rate and the development rate of the conventional porcine oocyte are low. The culture solution consists of basic culture solution TCM-199, penicillin, streptomycin and NaHCO34-hydroxyethyl piperazine ethanesulfonic acid, polyvinyl alcohol, sodium pyruvate, insulin, cysteine, epidermal growth factor, porcine follicular fluid, pregnant mare serum gonadotropin, human chorionic gonadotropin and tannic acid. The culture solution can improve the in-vitro maturation quality of the oocyte, the cumulus cell diffusion index, the embryo sac rate and the total cell number of the blastocyst of the parthenogenetic embryo, the cleavage rate of the in-vitro fertilization embryo, the blastocyst rate and the total cell number of the blastocyst.
Description
Technical Field
The invention belongs to the technical field of oocyte in-vitro maturation culture. In particular to a porcine oocyte in vitro maturation culture solution and a preparation method and application thereof.
Background
Modern biological and biomedical research shows that the biological and genetic characteristics of pigs have quite high homology with those of human beings, and are considered as the first choice animals of human xenogeneic organ sources, so the pigs are important model animals in biomedical research. The effective combination of in vitro culture technology and the gene editing technology which is developed rapidly at present not only promotes the development and application of embryo engineering, but also drives the development of the medical field. Oocyte In Vitro Maturation (IVM) is an important part of embryo In Vitro Production (IVP), and the standard for in vitro maturation culture (IVM) of porcine oocytes is also increasing today. Despite the great improvements of the porcine oocyte IVM system, the in vitro maturation rate of porcine oocytes is still lower than in vivo.
With the development of animal embryo engineering, the demand for oocytes in the aspects of scientific research and production is increasing. The traditional method for obtaining mature oocytes through superovulation has far failed to meet the current requirements for oocytes, and the in vitro maturation of mammalian oocytes is always a key step for researching the in vitro culture, in vitro fertilization, embryo development and the like of the oocytes and is also a research hotspot and difficulty. Compared with the in vivo maturation of the oocyte, the in vitro maturation of the oocyte has the problems of uncoordinated and asynchronous maturation of nucleus and cytoplasm, which directly leads to the hindered development potential of the oocyte, and is expressed by low in vitro fertilization embryo development rate, poor embryo quality and the like, and the result shows that the in vitro maturation culture system of the oocyte has stronger correlation with the fertilization rate and the blastocyst development rate. Therefore, the method deeply explores the maturation rule of the oocyte, improves the culture environment of the oocyte in vitro maturation, establishes a complete oocyte in vitro maturation system, and is one of the important contents of the current embryo engineering research.
Disclosure of Invention
The invention provides a porcine oocyte in-vitro maturation culture solution and a preparation method and application thereof, aiming at solving the problem that the in-vitro maturation rate and the development rate of the conventional porcine oocyte are low. The in vitro maturation culture solution for the porcine oocytes contains tannic acid.
Preferably, the oocyte in vitro maturation culture solution is prepared from basic culture solution TCM-199, penicillin and streptomycin，NaHCO34-hydroxyethyl piperazine ethanesulfonic acid Hepes, polyvinyl alcohol, sodium pyruvate, insulin, cysteine, epidermal growth factor, porcine follicular fluid, pregnant mare serum gonadotropin PMSG, human chorionic gonadotropin hCG and tannic acid.
Preferably, the oocyte in-vitro maturation culture solution is calculated by taking TCM-199 as a reference, the mass concentration of penicillin is 0.048-0.066g/L, the mass concentration of streptomycin is 0.08-0.12g/L, and NaHCO is used3The concentration of the peptide is 1.95-2mmol/L, the concentration of Hepes is 9.5-10mmol/L, the mass concentration of polyvinyl alcohol is 2.5-3.5g/L, the concentration of sodium pyruvate is 0.9-0.95mmol/L, the mass concentration of insulin is 8-12ng/mL, the concentration of cysteine is 0.5-0.7mmol/L, the mass concentration of epidermal growth factor is 8-12ng/mL, the volume concentration of pig follicle fluid is 75-125mL/L, the concentration of PMSG is 8-12IU/mL, the concentration of hCG is 8-12IU/mL, and the concentration of tannic acid is 1-50 mug/mL.
Preferably, the oocyte in-vitro maturation culture solution is calculated by taking TCM-199 as a reference, the mass concentration of penicillin is 0.06g/L, the mass concentration of streptomycin is 0.1g/L, and NaHCO is used3The concentration of (A) is 1.99mmol/L, the concentration of Hepes is 9.8mmol/L, the mass concentration of polyvinyl alcohol is 3g/L, the concentration of sodium pyruvate is 0.91mmol/L, the mass concentration of insulin is 10ng/mL, the concentration of cysteine is 0.6mmol/L, the mass concentration of epidermal growth factor is 10ng/mL, the volume concentration of pig follicle fluid is 100mL/L, the concentration of PMSG is 10IU/mL, the concentration of hCG is 10IU/mL, and the concentration of tannic acid is 10 mug/mL.
The invention also provides a preparation method of the in vitro maturation culture solution for the porcine oocytes, which comprises the following specific steps:
(1) obtaining the pig follicular fluid: extracting liquid from the follicle of the pig ovary, centrifuging the liquid, reserving supernatant, and filtering and removing impurities from the supernatant to obtain required pig follicle liquid;
(2) mixing TCM-199, penicillin, streptomycin and NaHCO3Hepes, polyvinyl alcohol, sodium pyruvate, insulin, cysteine, epidermal growth factor, PMSG, hCG, tannic acid and the pig follicle fluid obtained in the step (1) are mixed to obtain a culture fluid crudeAnd (4) liquid.
(3) And (3) filtering and sterilizing the crude culture solution obtained in the step (2) to obtain the in-vitro maturation culture solution of the porcine oocytes.
Preferably, the temperature of the pig ovary in the step (1) is 28-35 ℃.
Preferably, the centrifugation in step (1) is carried out at 12000-14000r/min and 30-60min at 4-6 ℃.
Preferably, the step (1) of removing impurities by filtration is carried out by filtration with a 0.45 μm filter.
Preferably, the filter sterilization in step (3) is performed by filtration through a 0.22 μm filter.
The invention also provides application of the in vitro maturation culture solution for the porcine oocytes in vitro culture of the porcine oocytes.
Advantageous effects
The tannin-containing oocyte in-vitro maturation culture solution can improve the in-vitro maturation quality of oocytes; improving cumulus cell diffusion index; the embryo sac rate and the total cell number of the blastocyst of the parthenogenetic embryo are improved; improve the cleavage rate, the blastula rate and the total cell number of the blastula of the in vitro fertilization embryo.
Detailed Description
All test reagents according to the invention were purchased from Sigma (USA) with the exception of the special instructions: TCM-199(Invitrogen), 100X ITS-A (Invitrogen), penicillin (Invitrogen), streptomycin (Invitrogen), tannic acid (APExBIO), PZM-5(Funakoshi Corporation), H2DCFDA (Invitrogen), CellTrackerBlue CMF2HC (Invitrogen).
The oocyte in-vitro maturation culture solution is prepared from basic culture solution TCM-199, penicillin, streptomycin and NaHCO34-hydroxyethyl piperazine ethanesulfonic acid (Hepes), polyvinyl alcohol, sodium pyruvate, insulin, cysteine, epidermal growth factor, porcine follicular fluid, Pregnant Mare Serum Gonadotropin (PMSG), human chorionic gonadotropin (hCG) and tannic acid. The present invention will be further described with reference to the following specific examples, but the present invention is not limited to these examples.
Example 1 oocyte in vitro maturation medium.
The oocyte in vitro maturation culture solution described in this example comprises the following specific components:
based on TCM-199, the mass concentration of penicillin is 0.048g/L, the mass concentration of streptomycin is 0.12g/L, and NaHCO is used3The concentration of (A) is 1.95mmol/L, the concentration of Hepes is 10mmol/L, the mass concentration of polyvinyl alcohol is 2.5g/L, the concentration of sodium pyruvate is 0.95mmol/L, the mass concentration of insulin is 12ng/mL, the concentration of cysteine is 0.5mmol/L, the mass concentration of epidermal growth factor is 8ng/mL, the volume concentration of pig follicle fluid is 125mL/L, the concentration of PMSG is 12IU/mL, the concentration of hCG is 8IU/mL, and the concentration of tannic acid is 1 mug/mL.
The preparation method of the in vitro maturation culture solution for the porcine oocytes comprises the following specific steps:
(1) obtaining the pig follicular fluid: extracting liquid from the follicle of the pig ovary at 30 ℃, centrifuging the liquid at 4 ℃ for 40min at 12000r/min, retaining the supernatant, and filtering the supernatant with a 0.45 mu m filter to remove impurities to obtain the required pig follicle liquid;
(2) mixing TCM-199, penicillin, streptomycin and NaHCO3Mixing Hepes, polyvinyl alcohol, sodium pyruvate, insulin, cysteine, epidermal growth factor, PMSG, hCG, tannic acid and the pig follicle fluid obtained in the step (1) to obtain a crude culture fluid.
(3) Filtering and sterilizing the crude culture solution obtained in the step (2) by using a 0.22 mu m filter to obtain the in-vitro maturation culture solution of the porcine oocytes.
Example 2 oocyte in vitro maturation medium.
The oocyte in vitro maturation culture solution described in this example comprises the following specific components:
based on TCM-199, the mass concentration of penicillin is 0.06g/L, the mass concentration of streptomycin is 0.1g/L, and NaHCO is added3The concentration of the amino acid is 1.99mmol/L, the concentration of Hepes is 9.8mmol/L, the mass concentration of polyvinyl alcohol is 3g/L, the concentration of sodium pyruvate is 0.91mmol/L, the mass concentration of insulin is 10ng/mL, the concentration of cysteine is 0.6mmol/L, and the epidermal growth factor isThe mass concentration of the active ingredients is 10ng/mL, the volume concentration of the pig follicle fluid is 100mL/L, the concentration of PMSG is 10IU/mL, the concentration of hCG is 10IU/mL, and the concentration of tannic acid is 10 mug/mL.
The preparation method of the in vitro maturation culture solution for the porcine oocytes comprises the following specific steps:
(1) obtaining the pig follicular fluid: extracting liquid from the follicle of the pig ovary at 28 ℃, centrifuging the liquid at 13000r/min for 30min at 5 ℃, retaining the supernatant, and filtering the supernatant by using a 0.45-micrometer filter to remove impurities to obtain the required pig follicle liquid;
(2) mixing TCM-199, penicillin, streptomycin and NaHCO3Mixing Hepes, polyvinyl alcohol, sodium pyruvate, insulin, cysteine, epidermal growth factor, PMSG, hCG, tannic acid and the pig follicle fluid obtained in the step (1) to obtain a crude culture fluid.
(3) Filtering and sterilizing the crude culture solution obtained in the step (2) by using a 0.22 mu m filter to obtain the in-vitro maturation culture solution of the porcine oocytes.
Example 3 oocyte in vitro maturation medium.
The oocyte in vitro maturation culture solution described in this example comprises the following specific components:
based on TCM-199, the mass concentration of penicillin is 0.066g/L, the mass concentration of streptomycin is 0.08g/L, and NaHCO is used3The concentration of (A) is 2mmol/L, the concentration of Hepes is 9.5mmol/L, the mass concentration of polyvinyl alcohol is 3.5g/L, the concentration of sodium pyruvate is 0.9mmol/L, the mass concentration of insulin is 8ng/mL, the concentration of cysteine is 0.7mmol/L, the mass concentration of epidermal growth factor is 12ng/mL, the volume concentration of pig follicle fluid is 75mL/L, the concentration of PMSG is 8IU/mL, the concentration of hCG is 12IU/mL, and the concentration of tannic acid is 50 mu g/mL.
The preparation method of the in vitro maturation culture solution for the porcine oocytes comprises the following specific steps:
(1) obtaining the pig follicular fluid: extracting liquid from the follicle of pig ovary at 35 ℃, centrifuging the liquid at 14000r/min for 60min at 6 ℃, retaining the supernatant, and filtering the supernatant by using a 0.45 mu m filter to remove impurities to obtain the required pig follicle liquid;
(2) mixing TCM-199, penicillin, streptomycin and NaHCO3Mixing Hepes, polyvinyl alcohol, sodium pyruvate, insulin, cysteine, epidermal growth factor, PMSG, hCG, tannic acid and the pig follicle fluid obtained in the step (1) to obtain a crude culture fluid.
(3) Filtering and sterilizing the crude culture solution obtained in the step (2) by using a 0.22 mu m filter to obtain the in-vitro maturation culture solution of the porcine oocytes.
Comparative example oocyte in vitro maturation medium without tannic acid addition.
The oocyte in vitro maturation culture solution described in this example comprises the following specific components:
based on TCM-199, the mass concentration of penicillin is 0.06g/L, the mass concentration of streptomycin is 0.1g/L, and NaHCO is used3The concentration of the compound is 1.99mmol/L, the concentration of Hepes is 9.8mmol/L, the mass concentration of polyvinyl alcohol is 3g/L, the concentration of sodium pyruvate is 0.91mmol/L, the mass concentration of insulin is 10ng/mL, the concentration of cysteine is 0.6mmol/L, the mass concentration of epidermal growth factor is 10ng/mL, the volume concentration of porcine follicle fluid is 100mL/L, the concentration of PMSG is 10IU/mL, and the concentration of hCG is 10 IU/mL.
Comparative example the procedure of preparing a culture solution for in vitro maturation of porcine oocytes described in example 1 was repeated except that tannic acid was not added in step (2).
The effect of culturing porcine oocytes in vitro maturation culture medium without tannin described in examples 1-3, and comparative example was verified:
1. collection and in vitro maturation of porcine oocytes
Pig ovaries were harvested from a local slaughterhouse and transported back to the laboratory in 30 ℃ normal saline. Cumulus Oocyte Complexes (COCs) in follicles 3-6mm in diameter were aspirated using a syringe fitted with an 18G needle. The COCs were washed in the oocyte in vitro maturation medium described in examples 1 to 3 and comparative examples, and then transferred to the oocytes in vitro maturation medium described in examples 1 to 3 and comparative examplesCulturing in culture medium at 38.5 deg.C and 5% CO respectively2Then the oocytes were transferred to the oocytes in vitro maturation culture medium of the groups of examples 1 to 3 and the comparative example, to which PMSG and hCG were not added, respectively, and cultured for a further 21 hours.
After culturing the oocytes of all groups for 42 hours, cumulus cells were eluted using hyaluronidase containing 0.1%, and the oocytes were observed under a microscope. As can be seen from Table 1, the maturation effect of the groups of examples 1 to 3 was similar to that of the comparative example in the oocyte maturation rate after 42 hours of IVM culture.
TABLE 1 Effect of the culture solutions of examples 1-3 and comparative examples on in vitro maturation of porcine oocytes
2. Pig cumulus cell diffusion degree determination and classification
After the in vitro culture of the oocyte is finished, evaluating the diffusion condition of the cumulus cell: grade 0, cumulus cells do not have any diffusion; grade 1, only the outermost layer of cumulus cells slightly diffuses; grade 2, cumulus cell diffusion of the outer layer; grade 3, except the radioactive crown, the remaining cumulus cells are diffused; grade 4, all cumulus cells including the corona radiata achieved the greatest degree of diffusion. The Cumulus cell diffusion Index (CEI) is calculated, i.e. the weighted arithmetic mean of the Cumulus cell diffusion at each level is calculated and used as an Index for measuring the Cumulus cell diffusion.
As can be seen from Table 2, there are significant differences between the groups of examples 2-3 and the comparative example.
The diffusion degree and the diffusion index of the cumulus cells of the example 2 and the example 3 are better compared with those of the comparative example, and the diffusion degree and the diffusion index of the cumulus cells of the example 1 are slightly inferior to those of the examples 2 and 3 but superior to those of the comparative example.
TABLE 2 Effect of the culture solutions of examples 1-3 and comparative examples on the spreading of porcine cumulus cells
3. Parthenogenetic activation
Placing the naked egg eluted by hyaluronidase into activating solution (0.28mol/L mannitol, 0.1mmol/L CaCl)2，0.1mmol/L MgSO40.5mmol/L Hepes), activated using BTX ECM 2001 under 1.5KV/cm unipolar dc pulses at 60 μ s. The activated oocytes were cultured in PZM-5 medium for 7 days.
As can be seen from Table 3, there was no significant difference in cleavage rate and total blastula cell count among the groups; the groups of examples 2-3 were significantly higher in blastocyst rate than the group of example 1 and the group of comparative example, and the group of example 1 was also better in blastocyst rate than the group of comparative example.
TABLE 3 Effect of the culture solutions of examples 1-3 and comparative examples on in vitro development of porcine parthenogenetic embryos
4. In Vitro Fertilization (IVF)
Each group selected 15 MII stage mature oocytes randomly placed in mTris-buffered media (mTBM) droplets (40. mu.L) and covered with pre-warmed mineral oil. Fresh semen transported to the laboratory within 2h from the local pig farm was washed twice with DPBS containing 1mL/L BSA, centrifuged at 1000g for 2 min, and the pellet obtained after centrifugation was resuspended with mTBM. After resuspension, 10. mu.L of the semen resuspension was added to 40. mu.L of mTBM droplets to achieve a sperm-to-egg ratio of 2000: 1. Sperm viability > 80% is ensured before fertilization proceeds. mTBM droplet containing oocytes and sperm at 39 deg.C, 5% CO2Was incubated for 20 minutes under the conditions of (1). After 20 minutes incubation, the oocytes were gently blown off of unattached, tight sperm on the zona pellucida. Oocytes were washed with mTBM and placed in sperm-free mTBM at 39 ℃ with 5% CO2Culturing for 5-6 hours under the conditions of (1). After incubation, gametes were washed with PZM-5 medium and subsequently placed in 500. mu.L four-well plates (50 gametes/well) at 39 ℃ with 5% CO2，90％N2The culture was continued for 168 hours.
As can be seen from table 4, the example 2 group was significantly higher than the example 1 group, the example 3 group and the comparative example group in blastocyst rate, and there was no significant difference between the example 1 group and the example 3 group and the comparative example.
TABLE 4 Effect of the culture solutions of examples 1-3 and comparative examples on the development of in vitro fertilized embryos in pigs
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. The in vitro maturation culture solution for the porcine oocytes is characterized by comprising tannic acid.
2. The in vitro maturation culture medium for porcine oocytes according to claim 1, wherein the culture medium is selected from the group consisting of TCM-199, penicillin, streptomycin, NaHCO34-hydroxyethyl piperazine ethanesulfonic acid Hepes, polyvinyl alcohol, sodium pyruvate, insulin, cysteine, epidermal growth factor, porcine follicular fluid, pregnant mare serum gonadotropin PMSG, human chorionic gonadotropin hCG and tannic acid.
3. The in vitro maturation culture medium for porcine oocytes according to claim 2, wherein the in vitro maturation culture medium for oocytes comprises penicillin in a mass concentration of 0.048-0.066g/L, streptomycin in a mass concentration of 0.08-0.12g/L and NaHCO, based on TCM-1993The concentration of the compound is 1.95-2mmol/L, the concentration of Hepes is 9.5-10mmol/L, the mass concentration of polyvinyl alcohol is 2.5-3.5g/L, the concentration of sodium pyruvate is 0.9-0.95mmol/L, the mass concentration of insulin is 8-12ng/mL, the concentration of cysteine is 0.5-0.7mmol/L, and the mass concentration of epidermal growth factor is 8-12ng/mL, the volume concentration of pig follicle fluid is 75-125mL/L, the concentration of PMSG is 8-12IU/mL, the concentration of hCG is 8-12IU/mL, and the concentration of tannic acid is 1-50 mug/mL.
4. The in vitro maturation culture medium for porcine oocytes according to claim 3, wherein the in vitro maturation culture medium for oocytes comprises penicillin at a mass concentration of 0.06g/L, streptomycin at a mass concentration of 0.1g/L, NaHCO, based on TCM-1993The concentration of (A) is 1.99mmol/L, the concentration of Hepes is 9.8mmol/L, the mass concentration of polyvinyl alcohol is 3g/L, the concentration of sodium pyruvate is 0.91mmol/L, the mass concentration of insulin is 10ng/mL, the concentration of cysteine is 0.6mmol/L, the mass concentration of epidermal growth factor is 10ng/mL, the volume concentration of pig follicle fluid is 100mL/L, the concentration of PMSG is 10IU/mL, the concentration of hCG is 10IU/mL, and the concentration of tannic acid is 10 mug/mL.
5. The method for preparing the in vitro maturation culture solution of porcine oocytes according to any one of claims 1 to 4, comprising the following steps:
(1) obtaining the pig follicular fluid: extracting liquid from the follicle of the pig ovary, centrifuging the liquid, reserving supernatant, and filtering and removing impurities from the supernatant to obtain pig follicle liquid;
(2) mixing TCM-199, penicillin, streptomycin and NaHCO3Mixing Hepes, polyvinyl alcohol, sodium pyruvate, insulin, cysteine, epidermal growth factor, PMSG, hCG, tannic acid and the pig follicle fluid obtained in the step (1) to obtain a crude culture fluid;
(3) and (3) filtering and sterilizing the crude culture solution obtained in the step (2) to obtain the in-vitro maturation culture solution of the porcine oocytes.
6. The method of claim 5, wherein the temperature of the pig ovary in step (1) is 28-35 ℃.
7. The method as claimed in claim 5, wherein the centrifugation in step (1) is performed at 4-6 ℃ and at 12000-14000r/min for 30-60 min.
8. The method according to claim 5, wherein the step (1) of removing impurities by filtration is carried out by filtration using a 0.45 μm filter.
9. The process according to claim 5, wherein the step (3) of filter sterilization is carried out by filtration through a 0.22 μm filter.
10. Use of the in vitro maturation culture medium of porcine oocytes according to any one of claims 1 to 4 for in vitro culturing of porcine oocytes.
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| CN113174366A (en) * | 2021-03-29 | 2021-07-27 | 广西壮族自治区畜牧研究所 | In-vitro maturation system of porcine oocyte containing butylbenzohydroxy acid and application thereof |
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| CN113174366B (en) * | 2021-03-29 | 2023-04-11 | 广西壮族自治区畜牧研究所 | In-vitro maturation system of porcine oocyte containing butylbenzohydroxy acid and application thereof |
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