CN104886040A - Method for efficiently protecting mitochondrial function of vitrified frozen bovine oocyte - Google Patents
Method for efficiently protecting mitochondrial function of vitrified frozen bovine oocyte Download PDFInfo
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- CN104886040A CN104886040A CN201510119500.1A CN201510119500A CN104886040A CN 104886040 A CN104886040 A CN 104886040A CN 201510119500 A CN201510119500 A CN 201510119500A CN 104886040 A CN104886040 A CN 104886040A
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Abstract
The invention discloses a method for efficiently protecting the mitochondrial function of vitrified frozen bovine oocytes. According to the method, 10-9M melatonin (MT) is added into a bovine oocyte in vitro maturation medium and a vitrification solution. There is no significant difference between bovine oocytes frozen by using this method and fresh oocytes with respect to ATP content and in vitro development. Application of the method of the invention can improve the developmental capacity and application scope of frozen oocytes. The method of the invention is simple in execution and low in cost, and will play a great role in cryopreservation of bovine oocyte.
Description
Technical field
The present invention relates to the method for the freezing bovine oocyte mitochondrial function of a kind of efficient cover glassization, thus greatly improve function and the developmental potency thereof of freezing egg mother cell Mitochondria.
Background technology
Along with the sharp increase of world population and the day by day serious of environmental pollution, mammalian genetic resources reserve sharply reduces, and the freezen protective of egg mother cell becomes the effective means of protection species resource diversity and rescue animals on the brink of extinction.Simultaneously, it is also the important component part accelerated livestock animal breeding, set up animal gene storehouse and embryo transplantation industrialization, and provide abundant test material for modern biotechnologies such as clone, transgenosiss, make the supply of egg mother cell and use by the restriction of Time and place.
But although mammal and the research of human oocyte freezen protective are progressively carried out and obtains remarkable progress, the research of egg mother cell freezen protective is far away not as good as the level of embryo or sperm cryopreservation.Research shows, for the law of procedure is freezing, the effect of vitrificated cryopreserration egg mother cell is wanted better, but the developmental potency after oocyte vitrification freezen protective still obviously declines, such as: carry out in vitro fertilization after bovine oocyte vitrification is freezing, blastocyst rate (17.50%) comparatively fresh egg mother cell (32.06%) have dropped about 50%.
Mitochondria is the organelle of rich content in cell, is also the energy plants of cell, and every vital movement that it is cell by oxidative phosphorylation provides ATP, and it is again apoptotic regulation and control maincenter and important calcium storehouse simultaneously.Much research is verified, and mitochondrial distribution, film potential etc. have very important impact to the developmental potentiality of egg mother cell.At present, about glass freezing to mitochondria freezing cause damage, adopt which kind of method or measure can alleviate this damage, all also lack further investigation.
Summary of the invention
For above-mentioned deficiency, the invention provides the method for a kind of efficient cover glassization freezing bovine oocyte Mitochondria function.
The inventive method is in bovine oocyte vitrification refrigerating process, adds MT, efficiently protect the function of bovine oocyte Mitochondria in bovine oocyte in vitro maturation liquid, glass freezing liquid.
Preferably, the interpolation concentration of described MT is 10
-7~ 10
-10m.More preferably, the interpolation concentration of described MT is 10
-9m.
The present invention is also provided for the glass freezing liquid of cover glassization freezing bovine oocyte Mitochondria function, and this freezing liquid contains MT, and the content of MT is preferably 10
-7~ 10
-10m, is more preferably 10
-9m.In embodiments of the present invention, glass freezing liquid is that EDFSF40:EG, DMSO and FSF liquid mixes according to volume ratio (v/v) 2 ﹕ 2 ﹕ 6, wherein FSF liquid: the DPBS solution containing 30% (w/v) ficoll Ficoll 70,0.5M sucrose and 20%FBS.
In addition, the present invention also provides the application of MT in the freezing bovine oocyte mitochondria of cover glassization.
The present invention's research shows, glass freezing significance can improve ROS level in bovine oocyte, and the abnormal ROS level raised can cause mitochondrial potential abnormal (as degradation under film potential), MT process can significance reduce ROS level in freezing egg mother cell and with fresh control group there was no significant difference.Further research shows; glass freezing can make ATP content in bovine oocyte significantly reduce (fresh control group 0.86pmol/ egg mother cell; frozen control group 0.58pmol/ egg mother cell); and when adding MT in bovine oocyte in vitro maturation liquid and glass freezing liquid; can available protecting egg mother cell Mitochondria function, especially in interpolation 10
-9remarkable result is shown, its ATP output and fresh egg mother cell there was no significant difference (MT freezing group of 0.84pmol/ egg mother cell, fresh control group 0.86pmol/ egg mother cell) during MT.Illustrate and add 10 in ripe liquid and glass freezing liquid in vitro
-9m MT, can maintain mitochondrial function by available protecting.Find after lonely female activation is carried out to freezing egg mother cell, cleavage rates (85.63% after the lonely female activation of MT freezing group of egg mother cell, 137/160), the equal significance of blastocyst rate (44.53%, 61/137) is higher than frozen control group (54.30%, 101/186; 26.73%, 37/101), and with fresh control group (88.79%, 95/107; 46.32%, 44/95) there was no significant difference, illustrates the function of MT by efficient protection egg mother cell Mitochondria, and then facilitates the developmental potency of freezing egg mother cell.
Advantage of the present invention, simple to operate, nontoxic to egg mother cell, the mitochondrial function of the freezing egg mother cell of energy available protecting and developmental potency thereof, this, for the range of application improving freezing egg mother cell, has important theoretical and practical significance.
Processing method of the present invention is simple to operate, expense is not high, the egg mother cell superfreeze research field ox is played a great role by the present invention.
Accompanying drawing explanation
Fig. 1 is ROS colored graph scale=20 μm in egg mother cell;
Fig. 2 is that MT process, CsA process are on the comparison of ROS level impact in glass freezing bovine oocyte.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.
Embodiment 1, MT, cyclosporin (CsA) are to the comparison of glass freezing bovine oocyte mitochondrial function defencive function
1, the collection of egg mother cell and maturation in vitro
Bovine oocyte is collected from local slaughter house, be kept in the 35 DEG C of physiological saline being added with 75 μ g/mL penicillin and 50 μ g/mL streptomycins and send laboratory back in 2h, be extract cumulus oocytes complesxes out the ovarian follicle of 2-8mm from diameter, select the fine and close cumulus cell that at least haves three layers complex washing after for maturation in vitro, what about 50 cumulus oocytes complesxes one group were placed on that the ripe liquid of 500 μ L make be coated with in 4 orifice plates of mineral oil 38.5 DEG C, 5%CO
2cultivate under maximal humidity condition.Ripe formula of liquid: M-199 (Gibco BRL company, Grand Island, New York, the U.S.) and the follicular stimulating hormone (FSH) of 10 μ g/mL, 10 μ g/mL lutropins (LH), 1 μ g/mL estradiol, hyclone (FBS, the Gibco BRL Division of 10%, Grand Island, New York, the U.S.), the heparin of 10 μ g/mL.
2, the preparation of OPS
According to the method that Vajta etc. introduces, make a little preparation OPS that changes and manage, the plastic tube (I.M.V, Lai Gele, France) of 0.25mL, high temperature deliquescing descendant is for elongating, and make tip exterior diameter be 0.23mm with microforge measurement, thickness of pipe wall is about 0.02mm.
3, pretreatment and glass freezing liquid
Pretreatment fluid: the DPBS solution containing the sub-wind (DMSO) of 10% ethylene glycol (EG)+10% dimethyl.
Glass freezing liquid EDFSF40:EG, DMSO and FSF liquid mixes according to volume ratio (v/v) 2 ﹕ 2 ﹕ 6
FSF liquid: the DPBS solution containing 30% (w/v) ficoll Ficoll 70,0.5M sucrose and 20%FBS
4, egg mother cell freezing with thaw
After In-vitro maturation 22-24h, with the hyaluronidase digestion 1-2min of 0.1%, digest the cumulus cell in compound, pick out first polar body and the uniform egg mother cell of kytoplasm for experiment.Egg mother cell is hatched 30s and is proceeded in EPFSF40 and cultivate 25s in ED, then loads in OPS pipe frozen in direct plunge into Liquid Nitrogen.
When thawing, OPS pipe is taken out from liquid nitrogen, the part that egg mother cell is housed is dissolved in rapidly in 0.25M, the sucrose of 38.5 DEG C, with mouth suction pipe, egg mother cell is put in 1min the sucrose of 0.25M from the blowout of OPS pipe, 5min in the sucrose of 0.15M, then wash twice with ripe liquid.The integrality of observing oocyte membrane and oolemma judges its survival condition.
5, active oxygen (ROS) level determination
Egg mother cell thaws and cultivates after 1h, among PBSs washes 3 time after taking out from culture fluid, then puts into 10 μm of DCFH-DA dyeing liquors and to dye 20min at 37 DEG C of incubators.In PBS, wash 3 times after taking-up, then put into PBS and drip gather image under fluorescence microscope, carry out Fluorescence Intensity Assays with EZ-C1freeviewer software.
6, ATP content analysis
By the method that Van Blerkom etc. introduces, make a little change ATP Analysis number, utilize ATP dependence fluorescein-luciferase, carry out fluorimetric assay for biological materials (ATP fluorimetric assay for biological materials, Roche Diagnistics limited company of Kit HS II Switzerland, Mannheim, Germany), measure fluorescence generation quantitative assay ATP number.
Be added in the 0.5mL centrifuge tube of 20 egg mother cells by 20 μ L cell cracking agents, piping and druming mixing makes these cells make it dissolve equably inside.In solution, the concentration of ATP is raised to 10.0pM from 0.01, and each concentration analysis once, draws a calibration curve.ATP liquid to be measured is put in 96 orifice plates and balances 3-5min, subsequently the light that sends of analytical standard liquid and sample, just can record with in photometer (InfiniteM200, Tecan Group Co., Ltd, Austria) 10s.Draw out a calibration curve with the relative light intensity of serial dilution, from calibration curve, just can find out the concentration of sample ATP like this.
7, lonely female activation
Egg mother cell first processes 5min 5 μMs of A23187 (calcium ion carrier A 23187), is then placed in 2 μMs of 6-DMAP and cultivates 4h, add the BSA of 10% after activation again to culture fluid, egg mother cell at 38.5 DEG C, 5%CO
2continue under condition to cultivate 48h, add 10%FBS in the most backward culture fluid again and cultivate 5 days, within every 2 days in whole culture period, change a culture fluid, record cleavage rates and blastocyst rate.
8, experimental design
Experimentally design, egg mother cell is divided into four groups, (1) MT group: distinguish each interpolation 10 in maturation in vitro liquid and glass freezing liquid
-7, 10
-8, 10
-9, 10
-10m MT; (2) Ciclosporin A (CsA) group: add 40mg/mLCsA in glass freezing liquid; (3) frozen control group: all do not contain MT or CsA in maturation in vitro liquid and glass freezing liquid; (4) fresh control group: maturation in vitro liquid does not add MT or CsA.After oocyte in vitro maturation, glass freezing thaw, the egg mother cell selecting survival carries out lonely female activation, compares the difference between ROS level between four groups, ATP content, developmental potency.
9. data statistics
Adopt SAS software to analyze experimental data, after anyway rotary, adopt variance analysis when percentage compares, result represents according to standard deviation with mean, and P<0.05 is significance of difference standard.Each processed group at least repeats more than 3 times.
10, experimental result
Experiment 1.MT process, CsA process are on the comparison of ROS level impact in glass freezing bovine oocyte
ROS dyes classical picture as shown in Figure 1.ROS result shows (Fig. 2), and glass freezing significance improves the level (P<0.05) of ROS in egg mother cell.In MT processed group, 10
-9in M MT process egg mother cell, the horizontal significance of ROS is lower than 10
-7m, 10
-8m, 10
-10m MT (P<0.05), and with fresh control group there was no significant difference (P>0.05).The horizontal significance of CsA processed group ROS is lower than frozen control group (P<0.05) but higher than 10
-9m MT processed group (P<0.05) and fresh control group (P<0.05).
Experiment 2:MT process, CsA process are on the comparison of ATP content impact in glass freezing bovine oocyte
As shown in table 1, glass freezing significance reduces the content (0.86pmol vs.0.58pmol, P<0.05) of ATP in bovine oocyte.In MT processed group, in ox maturation in vitro liquid and freezing liquid, add 10
-9aTP content the highest (0.84pmol vs.0.69pmol, 0.71pmol, 0.73pmol in egg mother cell after M MT; P<0.05), and with fresh control group there was no significant difference (0.84pmol vs.0.86pmol, P>0.05).In CsA processed group egg mother cell, ATP content (0.72pmol) significance is higher than frozen control group (0.58pmol, P<0.05), but lower than fresh control group (P<0.05) and 10
-9m MT processed group (P<0.05).
Table 1MT process is on the impact of ATP content in glass freezing bovine oocyte
The subscript that a, b, c are different represents numerical value significant difference (P<0.05) in file, and following form represents too.
The comparison that experiment 3:MT process, CsA process affect glass freezing bovine oocyte developmental potency
As shown in table 2, the cleavage rates (54.30%) of glass freezing group egg mother cell, blastocyst rate (26.73%) significance are lower than fresh control group (88.79%, 46.32%; P<0.05).In MT processed group, 10
-9the cleavage rates (85.63%) of M MT, blastocyst rate (44.53%) are significantly all higher than freezing 10
-7m MT (71.95%, 37.29%), 10
-8m MT (72.80%, 35.16%), 10
-10m MT (73.94%, 33.33%; P<0.05) and with fresh control group there was no significant difference (P>0.05).The cleavage rates (74.17%) of CsA processed group, blastocyst rate (34.82%) significance higher than frozen control group (P<0.05), but lower than fresh control group (P<0.05) and 10
-9m MT processed group (P<0.05).
Table 2MT process is on the impact of glass freezing bovine oocyte developmental potency
Conclusion, can be drawn by above experimental result, compares, 10 with CsA process
-9m MT process can efficient protective wire mitochondria function, and then improves the developmental potency of glass freezing bovine oocyte.
Claims (7)
1. the application of epiphysin in the freezing bovine oocyte mitochondria of cover glassization.
2. a method for cover glassization freezing bovine oocyte Mitochondria function, the method is in bovine oocyte vitrification refrigerating process, in bovine oocyte in vitro maturation liquid, glass freezing liquid, add MT.
3. method according to claim 2, is characterized in that, the interpolation concentration of described MT is 10
-7~ 10
-10m.
4. method according to claim 2, is characterized in that, the interpolation concentration of described MT is 10
-9m.
5. a glass freezing liquid, it contains MT.
6. freezing liquid according to claim 5, is characterized in that, described MT content is 10
-7~ 10
-10m.
7. freezing liquid according to claim 6, is characterized in that, described MT content is 10
-9m.
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Cited By (5)
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| CN107047539A (en) * | 2017-03-28 | 2017-08-18 | 中国农业科学院北京畜牧兽医研究所 | A kind of method of calcium ion concentration in regulation and control glass freezing bovine oocyte |
| CN108588011A (en) * | 2018-05-08 | 2018-09-28 | 中国农业科学院北京畜牧兽医研究所 | A method of improving glass freezing Oocytes in Vitro Fertilization ability |
| CN109294978A (en) * | 2018-11-10 | 2019-02-01 | 四川农业大学 | A method of improving glass freezing mature oocyte parthenogenetic development potentiality |
| CN110547291A (en) * | 2019-09-27 | 2019-12-10 | 安徽医科大学 | efficient antioxidant human oocyte cryoprotectant |
| CN114540283A (en) * | 2022-01-27 | 2022-05-27 | 中国农业科学院北京畜牧兽医研究所 | High-efficiency vitrification freezing method for bovine in-vitro embryo production |
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Cited By (8)
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| CN107047539A (en) * | 2017-03-28 | 2017-08-18 | 中国农业科学院北京畜牧兽医研究所 | A kind of method of calcium ion concentration in regulation and control glass freezing bovine oocyte |
| CN107047539B (en) * | 2017-03-28 | 2020-05-12 | 中国农业科学院北京畜牧兽医研究所 | Method for regulating and controlling calcium ion concentration in vitrified frozen bovine oocyte |
| CN108588011A (en) * | 2018-05-08 | 2018-09-28 | 中国农业科学院北京畜牧兽医研究所 | A method of improving glass freezing Oocytes in Vitro Fertilization ability |
| CN108588011B (en) * | 2018-05-08 | 2022-04-05 | 中国农业科学院北京畜牧兽医研究所 | Method for improving in vitro fertilization capability of vitrified frozen oocyte |
| CN109294978A (en) * | 2018-11-10 | 2019-02-01 | 四川农业大学 | A method of improving glass freezing mature oocyte parthenogenetic development potentiality |
| CN110547291A (en) * | 2019-09-27 | 2019-12-10 | 安徽医科大学 | efficient antioxidant human oocyte cryoprotectant |
| CN114540283A (en) * | 2022-01-27 | 2022-05-27 | 中国农业科学院北京畜牧兽医研究所 | High-efficiency vitrification freezing method for bovine in-vitro embryo production |
| CN114540283B (en) * | 2022-01-27 | 2023-10-20 | 中国农业科学院北京畜牧兽医研究所 | Efficient vitrification freezing method for bovine in-vitro embryo production |
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