CN104342400B - A kind of method for improving sheep oocyte ectogenesis ability - Google Patents
A kind of method for improving sheep oocyte ectogenesis ability Download PDFInfo
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Abstract
A kind of method for raising sheep oocyte ectogenesis ability that the present invention is provided; the matter time BCB egg mother cells that separation is obtained will be dyed; maturation in vitro, rear use in vitro fertilization contain 200nMol/L DNA methylase inhibitor inhibitor Trichostatin A (TSA) nutrient solution cultures 10h; continue to cultivate with the nutrient solution for not containing TSA; cleavage rates are counted after culture 48h; cultivate and blastocyst rate is counted after 7d; another to wash the BCB+ egg mother cells for dyeing separation acquisition with maturation culture solution 23 times, culture is directly utilized to 24h.The method is external to sheep oocyte to be utilized, with important practical value.
Description
Technical field
The present invention relates to DNA methylase inhibitor inhibitor Trichostatin A (TSA), to the quality after BCB is screened
The method that the negative egg mother cells of bad BCB are cultivated, can significantly improve sheep oocyte extracorporeal embryo development rate.
Background technology
TSA is a kind of noncompetitive reversible acetylation of histone inhibitor, and it derives from the metabolite of streptomysin, it
Main function be strengthen histone Acetylation Level.TSA can specific inhibition of histone deacetylase activity, drop
Low DNA methylation level and the expression for activating imprinted gene and house-keeping gene.And the high Acetylation Level of histone can strengthen
The effect of transcription factor and nucleosome, is conducive to the startup of genetic transcription.The embryo when acetylation of histone effect peaks
Genome be activated, it is and consistent with the increase of gene expression dose.Therefore, DNA methylase inhibitor inhibitor TSA may
Influence can be produced on the developmental potency of sheep oocyte and embryo in vitro.
In a kind of screening and culturing method (patent No. external for sheep oocyte of early-stage Study：
ZL200910113567.9 found in), it is female thin that the negative oocyte maturation rate pole after BCB is screened is substantially less than positive ovum
Born of the same parents, thus, negative Oocyte quality how is improved as urgent problem to be solved.Literature search is disclosed：1.Reprod Fert
Dev, 2007,19 (1), 169, author：Zhang Y H etc., are delivered《The clone embryos culture system in vitro of TSA optimization processings
It can obtain cloning piggy》Article show that 50nM TSA can improve the blastocyst rate of porcine clone embryos in vitro culture.2.Zygote,
2009,17,209-215, author：Shuntaro Ikeda etc., are delivered《Pass through during the Oocytes in Vitro Fertilization of ox
TSA increase acetylation of histone levels influence the inner cell mass number of blastaea》Article is drawn, in the Oocytes in Vitro Fertilization mistake of ox
After being handled in journey through TSA, blastaea inner cell mass number is significantly improved.3.Reprod Dev, 2011,57 (1)：34-42, author：
Lee M J etc., are delivered《TSA improves the development of nuclear transfer of bovine somatic cells embryo》Article show that TSA handles Niu Kelong embryos
Tire, can significantly improve the efficiency of nuclear transfer of bovine somatic cells.
The country has what the people of Institute of Animal Sciences, Chinese Academy of Agricultural Sciences Liu Xiao etc. 8 delivered《Trichostatin A is to pig
The influence of oocyte in vitro maturation and zona-free oocytes developmental potency》, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences Lee
Delivered to the people of minister etc. 5《TSA is to effect of the sheep known for its fine thick wool fibroblast as donorcells》, Northeast Agricultural University's life science
What 5 people such as institute's Huang ripple delivered《TSA concentration and processing time influence on pig nucleus transplantation vitro Development of Embryos》.
TSA is combined the point of penetration as this method by the present invention, and methodology and the application of expansion are learned
Research, sheep oocyte ectogenesis ability can be greatly enhanced, with important practice significance.
The content of the invention
The mesh of the present invention is：To improve the quality of sheep oocyte, i.e., screening is first dyed using BCB, then to screening
Ropy egg mother cell afterwards carries out suppression culture with TSA, and effect is fairly obvious, and this method is simple to operation, practicality pole
By force.
The object of the present invention is achieved like this：A kind of method for improving sheep oocyte ectogenesis ability, substep
Implement；Step 1：The matter time BCB- egg mother cell that separation is obtained will be dyed, washed 2-3 times with maturation culture solution, by 25-30 pieces/
Drop, during the volumes that have balanced of 2h are the maturation culture solution of 55-78 μ l/ drops before being put into, is put into CO2Case, in 5%CO2, 95% sky
Cultivated under the conditions of gas；Step 2：Step 1 maturation in vitro 23-25h egg mother cell is taken out, at 0.1% hyaluronidase
30-60s is managed, through pressure-vaccum, the cumulus cell around egg mother cell is sloughed；Washed 2-4 times with liquid in vitro fertilization, by 25-30 pieces/drop
Density add before in the 50-70 μ l drops in vitro fertilization that have balanced of 2h；Water-bath defrosting seminal fluid, the dress that 2h has been balanced before moving into
There is the test tube of liquid in vitro fertilization, be put into CO2Case, upstream 20-25min, takes supernatant, under the conditions of 1500rpm, centrifuges 4-5min,
Abandoning supernatant, by remaining Sperm pellets, by 2-4 × 106Individual/ml density adds drop in vitro fertilization, with handling well
Egg mother cell is incubated jointly；Step 3：By step 2 be fertilized 20-23h ovum take out, move to the external training containing TSA balanced
In nutrient solution, through pressure-vaccum, slough cumulus cell and stick sperm on ovum, wash 3-4 time, move into and put down by the density in 50-70 pieces/hole
10h is cultivated in the in vitro culture liquid containing TSA in the 500 μ l/ holes weighed, then 2-3 is washed with the in vitro culture liquid without TSA
It is secondary, it is positioned in the in vitro culture liquid without TSA and cultivates 7d, counts blastocyst rate；
The wherein acquisition of BCB- egg mother cells：The Sheep Ovary slaughtered in 30min is won, temperature is inserted at 30-35 DEG C, contains
In the physiological saline of 1000IU/ml penicillin and 1000IU/ml streptomysins, cleaned 3-4 times, had with suction with physiological saline in 3h
1ml takes out ovum liquid, the ovarian follicle of the 10ml syringes suction Ovarian surface 2-8mm with No. 9 syringe needles, the ovarian follicle that will be reclaimed in syringe
Liquid is beaten in 35-60mm ware, and egg mother cell is picked under the microscope, is put into the BCB dyes that addition concentration is 25-30 μM of ol/L
Color liquid, is placed in 35-39 DEG C of water-bath and dyes 80-90min, under the microscope separates BCB+ and BCB- egg mother cells, wherein will
The BCB+ egg mother cells of separation are dyed, are washed 2-3 times with maturation culture solution, culture to 24h is directly utilized；
Step 4：The preparation of experimental liquid：
Take out ovum liquid：TCM-199hepes+1mg/ml PVA+0.7mg/ml liquaemins；
Brilliant cresyl blue dyeing liquor：PBS+26 μM of ol/L brilliant cresyl blue+5mg/ml BSA；
Maturation culture solution：TCM-199HCO3+ 10%FBS+0.05IU/ml FSH+0.05IU/ml LH+1 μ g/ml
Estradiol+24.2 μ g/ml Sodium Pyruvate+0.1mM/L cysteine+10ng/ml EGF+100IU/ml are dual anti-；
SOF：6.29mg/ml NaCL+0.534mg/ml KCL+0.162mg/ml KH2PO4+ 0.6 μ l/ml sodium lactates+
0.089mg/ml MgSO4+2.1mg/mlNaHCO3+ 0.0357mg/ml Sodium Pyruvates+0.299mg/mlCaCL2·2H2O；
Liquid in vitro fertilization：SOF+20%FBS+6IU/ml liquaemin+100IU/ml gentamicin sulphates；
In vitro culture liquid：SOF+3mg/mlBSA；
The liquid of in vitro culture containing TSA：SOF+200nMol/L TSA+3mg/mlBSA.
The present invention is in the research of early stage, it has been verified that brilliant cresyl blue dyeing (the BCB) (patent No.：
ZL200910113567.9 it) can be used for determining G6PDH activity, according to the difference of G6PDH activity, BCB dyeing is presented different degrees of
Coloring；And it was found that the maturing rate of BCB- oocyte IVMs, cleavage rates, blastocyst rate are substantially less than BCB+ groups, therefore
It is combined using TSA with BCB triage techniqueses；It is exactly the nutrient solution effect 10h by BCB- egg mother cells using addition TSA, can carries
High BCB- Oocyte qualities, and BCB+ Oocyte qualities are not affected, so as to improve egg mother cell overall development
Efficiency.
The design and research mechanism of the present invention shows that addition Trichostatin A can significantly improve the matter after being screened through BCB time
BCB- oocyte IVM blastocyst rates.It is scientific and reasonable with the technology path that BCB triage techniqueses are combined using TSA,
Combined method has reached innovation effect, and has one's own knack, and shows technological progress.
Embodiment
The present invention is described further in conjunction with the embodiments.
Embodiment, the matter time BCB- egg mother cell that separates and obtain will be dyed, washed with maturation culture solution 3 times, by 28 pieces/
Drop, during the volumes that have balanced of 2h are the maturation culture solution of 60 μ L/ drops before being put into, is put into CO2Case, in 5%CO2, 95% air bar
Cultivated under part；Maturation in vitro 24h egg mother cell is taken out, with 0.1% hyaluronic acid ferment treatment 45s, through pressure-vaccum, portion is sloughed
Divide cumulus cell；Washed with liquid in vitro fertilization 3 times, the 60 μ L's that 2h has been balanced before being added by the density of 28 pieces/drop is in vitro fertilization
In drop；Water-bath defrosting seminal fluid, the test tube equipped with liquid in vitro fertilization that 2h has been balanced before moving into, is put into CO2Case, upstream 23min,
Supernatant is taken, under the conditions of 1500rpm, 5min, abandoning supernatant, by remaining Sperm pellets, by 3 × 10 is centrifuged6Individual/mL's is close
Degree adds drop in vitro fertilization, is incubated jointly with the egg mother cell handled well；The ovum for the 21h that is fertilized is taken out, moves to what is balanced
In in vitro culture liquid containing TSA, through pressure-vaccum, slough whole cumulus cells and stick sperm on ovum, wash 4 times, by 60 pieces/
The density in hole moves into and 10h is cultivated in the in vitro culture liquid containing TSA in 500 μ L/ holes balance, then with being free of the external of TSA
Nutrient solution is washed 3 times, is positioned in the in vitro culture liquid without TSA and is cultivated 7d, counts blastocyst rate；Wherein BCB- egg mother cells
Obtain：The Sheep Ovary slaughtered in 30min is won, temperature is inserted at 32 DEG C, penicillin containing 1000IU/ml and 1000IU/ml chains
In the physiological saline of mycin, cleaned in 3h with physiological saline 3 times；There is 1mL to take out ovum liquid with suction, the 10mL injections with No. 9 syringe needles
Device suction Ovarian surface 6mm ovarian follicle, the liquor folliculi reclaimed in syringe is beaten in 45mm ware, ovum is picked under the microscope
Mother cell, is put into the BCB dyeing liquors that addition concentration is 30 μM of ol/L, as dyeing 90min in 36 DEG C of water-baths；Under the microscope will
BCB+ and BCB- egg mother cells are separated, and are cultivated respectively to 24h, then carry out respectively it is in vitro fertilization, the BCB- ovum for the 22h that is fertilized is female thin
Born of the same parents take out, and move in the in vitro culture liquid containing TSA balanced, through pressure-vaccum, slough whole cumulus cells and stick on ovum
Sperm, is washed 4 times, is moved into by the density in 60 pieces/hole in the nutrient solution containing TSA in the 500 μ L/ holes balanced and is cultivated 10h, then
Washed with the nutrient solution without TSA 3 times, enter and count statistics blastocyst rate after cleavage rates, 7d in nutrient solution after culture 48h；
The BCB+ egg mother cells for wherein dyeing separation are directly utilized；
The wherein configuration of experimental liquid：
Take out ovum liquid：TCM-199hepes+1mg/mLPVA+0.7mg/ml liquaemins；
Brilliant cresyl blue dyeing liquor：PBS+26 μM of ol/L brilliant cresyl blues+5mg/mLBSA；
Maturation culture solution：TCM-199HCO3+ 10%FBS+0.05IU/mL FSH+0.05IU/mL LH+1 μ g/mL
Estradiol+24.2 μ g/mL Sodium Pyruvate+0.1mM/L cysteine+10ng/mL EGF+100IU/mL are dual anti-；
SOF：6.29mg/ml NaCL+0.534mg/mL KCL+0.162mg/mL KH2PO4+ 0.6 μ l/mL sodium lactates+
0.089mg/mL MgSO4+2.1mg/mL NaHCO3+ 0.0357mg/mL Sodium Pyruvate+0.299mg/mL CaCL2·2H2O；Body
Outside by seminal fluid：SOF+20%FBS+6IU/mL liquaemin+100IU/mL gentamicin sulphates；
The liquid of in vitro culture containing TSA：SOF+200nMol/L TSA+3mg/mLBSA.
In vitro culture liquid：SOF+3mg/mLBSA；
This method using microscope detect Oocyte quality, cytoplasm not blueness BCB- Oocyte qualities it is notable
Less than the BCB+ egg mother cells that kytoplasm blueness.
This method experiment select TCM-199hepes, PVA, liquaemin, brilliant cresyl blue, PBS, BSA, FBS, FSH, LH,
Estradiol, EGF, cysteine, TSA be purchased from SIGMA companies, dual anti-purchased from the prosperous limited public affairs of biotechnology of Beijing ancient cooking vessel state
Department.
Experimental result and data verification：With one group of data verification, it uses TSA to dye the effective of the method being combined with BCB
Property.TSA is combined with BCB dyeing, Oocyte in Vitro developmental potency can be improved to greatest extent.
Table 1
Note：Data are analyzed through statistics software SPSS, same column mark different lowercases for significant difference (P<0.05).
Learnt by upper table：The BCB- egg mother cells acted on through TSA, although not influenceed on cleavage rates after in vitro fertilization,
But blastocyst rate is significantly higher than the BCB- egg mother cells not acted on through TSA.
Claims (1)
1. a kind of method for improving sheep oocyte ectogenesis ability, it is characterised in that：Implement step by step：
Step 1, the matter time BCB- egg mother cell that separates and obtain will be dyed, washed 2-3 times with maturation culture solution, by 25-30 pieces/
Drop, during the volumes that have balanced of 2h are the maturation culture solution of 55-78 μ L/ drops before being put into, is put into CO2Case, in 5%CO2, 95% sky
Cultivated under the conditions of gas；
Step 2, step 1 maturation in vitro 23-25h egg mother cell taken out, with 0.1% hyaluronic acid ferment treatment 30-60s,
Through pressure-vaccum, the cumulus cell around egg mother cell is sloughed；Washed 2-4 times, added by the density of 25-30 pieces/drop with liquid in vitro fertilization
Before entering in the drop in vitro fertilization for the 50-70 μ L that 2h has been balanced；Water-bath defrosting seminal fluid, move into before 2h balanced equipped with vitro by
The test tube of seminal fluid, is put into CO2Case, upstream 20-25min, takes supernatant, under the conditions of 1500rpm, centrifuges 4-5min, supernatant discarding
Liquid, by remaining Sperm pellets, by 2-4 × 106Individual/mL density adds drop in vitro fertilization, with the egg mother cell handled well
It is common to be incubated；
Step 3, the ovum for 20-23h that step 2 is fertilized take out, and move in the in vitro culture liquid containing TSA balanced, through pressure-vaccum,
Slough cumulus cell and stick sperm on ovum, wash 3-4 time, the 500 μ L/ holes balanced by the density immigration in 50-70 pieces/hole
The in vitro culture liquid containing TSA in cultivate 10h, then washed 2-3 times, be positioned over without TSA with the in vitro culture liquid without TSA
In vitro culture liquid in culture 7d, count blastocyst rate；
The wherein acquisition of BCB- egg mother cells：The Sheep Ovary slaughtered in 30min is won, temperature is inserted at 30-35 DEG C, contains
In the physiological saline of 1000IU/ml penicillin and 1000IU/ml streptomysins, cleaned 3-4 times, had with suction with physiological saline in 3h
1mL takes out ovum liquid, the ovarian follicle of the 10ml syringes suction Ovarian surface 2-8mm with No. 9 syringe needles, the ovarian follicle that will be reclaimed in syringe
Liquid is beaten in 35-60mm ware, and egg mother cell is picked under the microscope, is put into the BCB dyes that addition concentration is 25-30 μM of ol/L
Color liquid, is placed in 35-39 DEG C of water-bath and dyes 80-90min, under the microscope separates BCB+ and BCB- egg mother cells, wherein will
The BCB+ egg mother cells of separation are dyed, are washed 2-3 times with maturation culture solution, culture to 24h is directly utilized；
The preparation of step 4 experimental liquid：
Take out ovum liquid：TCM-199hepes+1mg/mL PVA+0.7mg/mL liquaemins；
Brilliant cresyl blue dyeing liquor：PBS+26 μM of ol/L brilliant cresyl blue+5mg/mL BSA；
Maturation culture solution：TCM-199HCO3+ 10%FBS+0.05IU/mL FSH+0.05IU/mL LH+1 μ g/mL estradiol
+ 24.2 μ g/mL Sodium Pyruvate+0.1mM/L cysteines+10ng/mLEGF+100IU/mL are dual anti-；
SOF：6.29mg/ml NaCL+0.534mg/mL KCL+0.162mg/ml KH2PO4+ 0.6 μ l/mL sodium lactates+
0.089mg/mL MgSO4+2.1mg/mL NaHCO3+ 0.0357mg/mL Sodium Pyruvate+0.299mg/mL CaCL2·2H2O；
Liquid in vitro fertilization：SOF+20%FBS+6IU/mL liquaemin+100IU/mL gentamicin sulphates；
In vitro culture liquid：SOF+3mg/mL BSA；
The liquid of in vitro culture containing TSA：SOF+200nMol/L TSA+3mg/mL BSA.
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| TSA对绵羊卵母细胞体外成熟及孤雌胚胎发育的影响;杨楠 等;《西北农业学报》;20140326;第23卷(第3期);第9页摘要 * |
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