CN104928236A - Nutrient solution and culturing method for maturation of oocyte in vitro - Google Patents
Nutrient solution and culturing method for maturation of oocyte in vitro Download PDFInfo
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- CN104928236A CN104928236A CN201510389988.XA CN201510389988A CN104928236A CN 104928236 A CN104928236 A CN 104928236A CN 201510389988 A CN201510389988 A CN 201510389988A CN 104928236 A CN104928236 A CN 104928236A
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Abstract
The invention discloses a nutrient solution for maturation of oocyte in vitro and a method for culturing oocyte using the nutrient solution, and relates to a nutrient solution for nutrient solution for oocyte and a method for culturing the oocyte with the nutrient solution. The nutrient solution and the culturing method using the nutrient solution solve the problem that the viability of oocyte culturing in vitro is low. The nutrient solution for maturation of oocyte in vitro is composed of normal nutrient solution of oocyte, vitamin B12, fetal calf serum, FSH, LH, 17 betas estradiol, sodium pyruvate, EGF, penicillin and streptomycin sulphate. The oocyte is placed into the utrient solution for maturation of oocyte in vitro, the maturation culture on the oocyte lasts for 20-24 hours in the environment in which the volume concentration of CO2 is 5%, and a mature oocyte is acquired. The nutrient solution for maturation of oocyte in vitro can decrease stress injury caused by the external environment, the cytoplasm maturing of the oocyte is promoted, and the parthenogenetic activation of the matured oocyte matured in vitro and the embryos developmental potential after being fertilized are improved.
Description
Technical field
The present invention relates to a kind of ovocyte nutrient solution and cultural method.
Background technology
Obtain prerequisite and key that high-quality Oocytes Maturation In vitro is the Embryo engineering technology success or not such as animal embryo produced in vitro, transgene clone.Although oocyte in vitro maturation technology achieves significant progress in recent years, but mature oocyte ectogenesis ability is still much lower compared with ovocyte in body.By the impact of external environment in oocyte in vitro maturation process, be very easy to stress damage occurs, cause developmental potency to decline.
Vitamins B
12being cobalami again, is the VITAMIN of unique containing metal element, vitamins B
12it is the one found up to now in vitamin B group the latest.The vitamins B of occurring in nature
12be all Microbe synthesis, high animals and plants can not manufacture vitamins B
12.Vitamins B
12be that only one needs intestinal secretion thing (castle's intrinsic factor) to help the absorbed VITAMIN of ability, its residence time in enteron aisle is long, approximately needs three hours (most of water-soluble vitamins only needs a few second) to be absorbed.Vitamins B
12be a kind of polycyclic system compound containing 3 valency cobalts, the pyrrole ring of 4 reduction connects together and becomes 1 large ring of corrin (similar to porphyrin), is vitamins B
12the core of molecule.So be all called as corrinoid containing the compound of this ring.Vitamin B12 is ruddy needle crystal, soluble in water and ethanol, the most stable under pH value 4.5 ~ 5.0 mild acid conditions, decompose in strong acid (pH<2) or basic solution, heat can have to a certain degree to be destroyed, but the high-temperature sterilization of short period of time loss is little, chance high light or ultraviolet are easily destroyed.Vitamins B
12main Physiological Function be participate in manufacture erythrocyte, prevent pernicious anemia; Cerebral nerve is prevented to be damaged; But vitamins B at present
12be all enter in life entity again after being absorbed by stomach to be utilized, not yet have vitamins B
12directly act on the report of cell in vitro especially ovocyte.
Summary of the invention
The present invention solves the low problem of existing Oocyte in Vitro developmental potency, provides a kind of oocyte in vitro maturation culture solution and cultural method.
Oocyte in vitro maturation culture solution of the present invention is by ovocyte cellar culture liquid and vitamins B
12, foetal calf serum, FSH, LH, 17 beta estradiols, Sodium.alpha.-ketopropionate, EGF, penicillin and Vetstrep composition; Vitamins B in oocyte in vitro maturation culture solution
12concentration be 2mg/ml, the concentration of serum is 0.1ml/ml, the concentration of follicular stimulating hormone is 0.05IU/ml, the concentration of lutropin is 0.05IU/ml, the concentration of 17 beta estradiols is 1 μ g/ml, the concentration of Sodium.alpha.-ketopropionate is 24.2mg/L, the concentration of Urogastron is 10ng/ml, the concentration of penicillin is 100IU/ml, the concentration of Vetstrep is 100 μ g/ml.
Above-mentioned oocyte in vitro maturation culture solution is utilized to cultivate the method for ovocyte:
Ovocyte is put into the oocyte in vitro maturation culture solution of preheating, be then placed in CO
2volumetric concentration be 5% environment in maturation culture 20 ~ 24 hours, namely obtain ripe ovocyte.
Oocyte in vitro maturation culture solution In-vitro maturation mouse of the present invention and bovine oocyte effectively can improve the early embryonic development rate after fertilization or lonely female activation; IVF of Oocyte in Bovine blastocyst rate reaches more than 36%, and lonely female activation blastocyst rate reaches more than 22%; Oocytes Fertilized in vitro blastocyst rate reaches more than 55%, and lonely female activation blastocyst rate reaches more than 34%.
Oocyte in vitro maturation culture solution of the present invention can significantly improve Embryo viability after oocyte in vitro maturation, and with low cost, is convenient to extensive popularization.
Oocyte in vitro maturation culture solution of the present invention can reduce the stress damage that external environment is brought, and promotes the Cytoplasmic maturation of ovocyte, improves the lonely female activation of ovocyte of maturation in vitro and the Embryo viability of after fertilization.
Oocyte IVM method of the present invention is simple, and effective, the cycle is short.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: present embodiment oocyte in vitro maturation culture solution is by ovocyte cellar culture liquid and vitamins B
12, foetal calf serum, FSH, LH, 17 beta estradiols, Sodium.alpha.-ketopropionate, EGF, penicillin and Vetstrep composition; Vitamins B in oocyte in vitro maturation culture solution
12concentration be 2mg/ml, the concentration of serum is 0.1ml/ml, the concentration of follicular stimulating hormone (FSH) is 0.05IU/ml, the concentration of lutropin (LH) is 0.05IU/ml, the concentration of 17 beta estradiols is 1 μ g/ml, the concentration of Sodium.alpha.-ketopropionate is 24.2mg/L, the concentration of Urogastron (EGF) is 10ng/ml, the concentration of penicillin is 100IU/ml, the concentration of Vetstrep is 100 μ g/ml.
Embodiment two: the difference of present embodiment and embodiment one is: ovocyte cellar culture liquid is DMEM nutrient solution, TCM199 nutrient solution or MEM nutrient solution.Other step and parameter identical with embodiment one.
Embodiment three: the difference of present embodiment and embodiment one or two is: serum is calf serum, foetal calf serum or new-born calf serum.Other step and parameter identical with embodiment one or two.
Embodiment four: present embodiment oocyte in vitro maturation culture solution cultivates ovocyte according to the following steps:
Ovocyte is put into the oocyte in vitro maturation culture solution of preheating, be then placed in CO
2volumetric concentration be 5% environment in maturation culture 24 hours, namely obtain ripe ovocyte.
Embodiment five: the difference of present embodiment and embodiment four is: ovocyte is GV phase ovocyte.Other step and parameter identical with embodiment four.
Embodiment six: the difference of present embodiment and embodiment four or five is: oocyte in vitro maturation culture solution preheating 1 ~ 3 hour.Other step and parameter identical with embodiment four or five.
Embodiment seven: the difference of one of present embodiment and embodiment four to six is: oocyte in vitro maturation culture solution is placed in the environment preheating of 35 ~ 40 DEG C.Other step and parameter identical with one of embodiment four to six.
Embodiment eight: the difference of one of present embodiment and embodiment four to seven is: cultivate bovine oocyte, oocyte in vitro maturation culture solution is placed in the environment preheating of 38.5 DEG C.Other step and parameter identical with one of embodiment four to seven.
Embodiment nine: the difference of one of present embodiment and embodiment four to eight is: cultivate oocyte of mouse, oocyte in vitro maturation culture solution is placed in the environment preheating of 37 DEG C.Other step and parameter identical with one of embodiment four to eight.
Embodiment ten: the difference of one of present embodiment and embodiment four to nine is: put into 30 μ l ovocytes in the oocyte in vitro maturation culture solution of 100 μ l.Other step and parameter identical with one of embodiment four to nine.
Embodiment 1
In vitro maturation:
One, oocyte in vitro maturation culture solution is prepared: accurately take vitamins B
12100mg, the TCM199 nutrient solution (purchased from GIBCO company) of 25ml is first used to dissolve, then in nutrient solution, add 5ml foetal calf serum and 5000IU penicillin and 5000 μ g Streptomycin sulphates, gentle agitation mixes, then adds FSH 2.5IU, LH 2.5IU, 17 beta estradiol 50 μ g, Sodium.alpha.-ketopropionate 1.21mg and EGF 500ng, mix gently, leave standstill after 2 ~ 3 hours, after the strainer suction filtration sterilization of 0.22 μm, be dispensed in 1.5ml centrifuge tube, store at 4 DEG C, for subsequent use.
Two, vitro culture: mouse GV phase ovocyte is put into 37 DEG C of preheatings oocyte in vitro maturation culture solution of 2 hours, be then placed in CO
2volumetric concentration be 5% environment in maturation culture 24 hours; Ox GV phase ovocyte is put into 38.5 DEG C of preheatings oocyte in vitro maturation culture solution of 2 hours, be then placed in CO
2volumetric concentration be 5% environment in maturation culture 24 hours.
Three, in vitro fertilization or lonely female activation, embryo continues cultivation 7 days, cultivates 48 hour record 2-cell stages and 4-cell stage ratio:
Wherein, lonely female activation: oocyte of mouse adopts SrCl
2activate, mouse GV phase ovocyte is first containing 10mM SrCl
2with CB cultivate 2.5h without in calcium CZB, then cultivate 3.5h in the calcic CZB containing CB, be transferred to afterwards in CZB and continue to cultivate, cultivate 48 little being transferred in sugary CZB up to the 4-cell stage phase and be cultured to blastaea;
Bovine oocyte adopts inomycin to activate, ox GV phase ovocyte first activates 5 minutes in containing the CR1 of 5 μMs of inomycin, cultivation is continued 4 hours again in the nutrient solution of the CR1 containing 6-DMAP, be transferred in CR1 afterwards and cultivate, cultivate and within 48 hours, be developed to 4-cell stage stage phase and change to again in CR1 and continue to be cultured to blastaea.
In vitro fertilization: to choose the male mouse of sexual maturity (10 ~ 12 week age) Kunming white, prove that it has fertility by mating test, cervical dislocation is put to death, sperm is collected from cauda epididymidis and vas deferens, the sperm agglomerate of collection is inserted 1ml containing in the T6 drop of 10mg/mlBSA, blow and beat gently with mouth suction pipe, spermatium is scatter, in 37 DEG C, 5%CO
2and the CO of saturated humidity
2hatch 1.5 hours in incubator, carry out capacitation; Sperm concentration is detected during this period with cell counting count board; Again the ovocyte of 14 hours after 12 hours or IVM after hCG is being washed 3 times by seminal fluid (T6+20mg/mlBSA), during the fertilization moving into equilibrate overnight is dripped (20 pieces/40 μ l), add the capacitated sperm of appropriate volume, make sperm concentration 1 × 10
6left and right.Cultivate after 6 hours, wash away around ovocyte with sugar-free CZB liquid and adhere to sperm, choose the zygote containing two protokaryons and second polar body, be placed in sugar-free CZB and cultivate;
Freezing for breeding oxen straw semen (purchased from animal improvement station, the Inner Mongol) is thawed in 37 DEG C of water-baths, with the BO liquid centrifuge washing containing 10mM caffeine twice, after second time is centrifugal, leave standstill 2 ~ 3min, careful absorption floating sperm, the fertilization B liquid (BO+3mg/mLBSA+8 μ l/ml heparin) adding equal volume makes sperm suspensions, cell counting under light microscopic, make final concentration reach × 10
6/ ml.Do fertilization droplet (100 μ l) with sperm suspensions, every droplet puts into the ovocyte after 15 pieces of maturation culture; Remove ovarian cumulus after fertilization 6 ~ 7h, zygote is proceeded to Development culture liquid; Development culture liquid adopts SOF+6%BSA, and every 40 μ l Development culture liquid droplets are put into 15 ~ 20 pieces of zygotes and cultivated.
Embodiment 2
In vitro maturation:
One, oocyte in vitro maturation culture solution is prepared: accurately take vitamins B
12100mg, the MEM nutrient solution (purchased from GIBCO company) of 25ml is first used to dissolve, then in nutrient solution, add 5ml foetal calf serum and 5000IU penicillin and 5000 μ g Streptomycin sulphates, gentle agitation mixes, then adds FSH 2.5IU, LH 2.5IU, 17 beta estradiol 50 μ g, Sodium.alpha.-ketopropionate 1.21mg and EGF 500ng, mix gently, leave standstill after 2 ~ 3 hours, after the strainer suction filtration sterilization of 0.22 μm, be dispensed in 1.5ml centrifuge tube, store at 4 DEG C, for subsequent use.
Two, vitro culture: mouse GV phase ovocyte is put into 37 DEG C of preheatings oocyte in vitro maturation culture solution of 2 hours, be then placed in CO
2volumetric concentration be 5% environment in maturation culture 24 hours; Ox GV phase ovocyte is put into 38.5 DEG C of preheatings oocyte in vitro maturation culture solution of 2 hours, be then placed in CO
2volumetric concentration be 5% environment in maturation culture 24 hours.
Three, in vitro fertilization or lonely female activation, embryo continues cultivation 7 days, cultivates 48 hour record 2-cell stages and 4-cell stage ratio:
Wherein, lonely female activation: oocyte of mouse adopts SrCl
2activate, Oocytes Maturation In vitro is first containing 10mM SrCl
2with CB cultivate 2.5h without in calcium CZB, then cultivate 3.5h in the calcic CZB containing CB, be transferred to afterwards in CZB and continue to cultivate, cultivate 48 little being transferred in sugary CZB up to the 4-cell stage phase and be cultured to blastaea;
Bovine oocyte adopts inomycin to activate, ox GV phase ovocyte first activates 5 minutes in containing the CR1 of 5 μMs of inomycin, cultivation is continued 4 hours again in the nutrient solution of the CR1 containing 6-DMAP, be transferred in CR1 afterwards and cultivate, cultivate and within 48 hours, be developed to the 4-cell stage phase and change to again in CR1 and continue to be cultured to blastaea.
In vitro fertilization: to choose the male mouse of sexual maturity (10 ~ 12 week age) Kunming white, prove that it has fertility by mating test, cervical dislocation is put to death, sperm is collected from cauda epididymidis and vas deferens, the sperm agglomerate of collection is inserted 1ml containing in the T6 drop of 10mg/mlBSA, blow and beat gently with mouth suction pipe, spermatium is scatter, in 37 DEG C, 5%CO
2and the CO of saturated humidity
2hatch 1.5 hours in incubator, carry out capacitation; Sperm concentration is detected during this period with cell counting count board; Again by the ovocyte of 14 hours after IVM by washing 3 times in seminal fluid (T6+20mg/mlBSA), during the fertilization moving into equilibrate overnight is dripped (20 pieces/40 μ l), add the capacitated sperm of appropriate volume, make sperm concentration 1 × 10
6left and right.Cultivate after 6 hours, wash away around ovocyte with CZB liquid and adhere to sperm, choose the zygote containing two protokaryons and second polar body, be placed in CZB and cultivate;
Freezing for breeding oxen straw semen is thawed in 37 DEG C of water-baths, with the BO liquid centrifuge washing containing 10mM caffeine twice, after second time is centrifugal, leave standstill 2 ~ 3min, careful absorption floating sperm, the fertilization B liquid (BO+3mg/mLBSA+8 μ l/ml heparin) adding equal volume makes sperm suspensions, cell counting under light microscopic, make final concentration reach × 10
6/ ml.Do fertilization droplet (100 μ l) with sperm suspensions, every droplet puts into the ovocyte after 15 pieces of maturation culture; Remove ovarian cumulus after fertilization 6 ~ 7h, zygote is proceeded to Development culture liquid; Development culture liquid adopts SOF+6%BSA, and every 40 μ l Development culture liquid droplets are put into 15 ~ 20 pieces of zygotes and cultivated.
Embodiment 3
Controlled trial group
Prepare 2 groups of contrast nutrient solutions, difference that is corresponding and embodiment 1 is only in nutrient solution not containing vitamins B
12, other all identical with 2 with embodiment 1.
Experimental data as shown in Table 1 and Table 2.
Table 1
Table 2
Result confirms, oocyte in vitro maturation culture solution In-vitro maturation mouse of the present invention and bovine oocyte effectively can improve the early embryonic development rate after fertilization or lonely female activation.
Claims (10)
1. an oocyte in vitro maturation culture solution, is characterized in that oocyte in vitro maturation culture solution is by ovocyte cellar culture liquid and vitamins B
12, foetal calf serum, FSH, LH, 17 beta estradiols, Sodium.alpha.-ketopropionate, EGF, penicillin and Vetstrep composition; Vitamins B in oocyte in vitro maturation culture solution
12concentration be 2mg/ml, the concentration of serum is 0.1ml/ml, the concentration of follicular stimulating hormone is 0.05IU/ml, the concentration of lutropin is 0.05IU/ml, the concentration of 17 beta estradiols is 1 μ g/ml, the concentration of Sodium.alpha.-ketopropionate is 24.2mg/L, the concentration of Urogastron is 10ng/ml, the concentration of penicillin is 100IU/ml, the concentration of Vetstrep is 100 μ g/ml.
2. oocyte in vitro maturation culture solution according to claim 1, is characterized in that ovocyte cellar culture liquid is DMEM nutrient solution, TCM199 nutrient solution or MEM nutrient solution.
3. oocyte in vitro maturation culture solution according to claim 1, is characterized in that serum is calf serum, foetal calf serum or new-born calf serum.
4. utilize claim 1 oocyte in vitro maturation culture solution to cultivate the method for ovocyte, it is characterized in that ovocyte oocyte in vitro maturation culture solution is cultivated according to the following steps:
Ovocyte is put into the oocyte in vitro maturation culture solution of preheating, be then placed in CO
2volumetric concentration be 5% environment in maturation culture 20 ~ 24 hours, namely obtain ripe ovocyte.
5. oocyte in vitro maturation culture solution according to claim 4 cultivates the method for ovocyte, it is characterized in that ovocyte is GV phase ovocyte.
6. oocyte in vitro maturation culture solution according to claim 4 cultivates the method for ovocyte, it is characterized in that oocyte in vitro maturation culture solution preheating 1 ~ 3 hour.
7. the oocyte in vitro maturation culture solution according to claim 4 or 6 cultivates the method for ovocyte, it is characterized in that oocyte in vitro maturation culture solution is placed in the environment preheating of 35 ~ 40 DEG C.
8. oocyte in vitro maturation culture solution according to claim 7 cultivates the method for ovocyte, it is characterized in that cultivating bovine oocyte, and oocyte in vitro maturation culture solution is placed in environment preheating and the cultivation of 38.5 DEG C.
9. oocyte in vitro maturation culture solution according to claim 7 cultivates the method for ovocyte, it is characterized in that cultivating oocyte of mouse, and oocyte in vitro maturation culture solution is placed in environment preheating and the cultivation of 37 DEG C.
10. oocyte in vitro maturation culture solution according to claim 4 cultivates the method for ovocyte, it is characterized in that putting into 1 piece of ovocyte in the oocyte in vitro maturation culture solution of 3 μ l.
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Cited By (10)
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| CN107841484A (en) * | 2016-09-19 | 2018-03-27 | 上海市计划生育科学研究所 | A kind of external egg mother cell cultivating system and its application |
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| CN111518748A (en) * | 2019-12-09 | 2020-08-11 | 赵义清 | CNP-based human immature oocyte two-phase in vitro maturation technology |
| CN113025564A (en) * | 2021-03-17 | 2021-06-25 | 金华市人民医院 | Preparation method of oocyte in-vitro maturation culture solution |
| CN113308435A (en) * | 2021-07-15 | 2021-08-27 | 山东农业大学 | Culture solution and culture method for improving in-vitro maturation quality of animal oocyte |
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