CN107047539A - A kind of method of calcium ion concentration in regulation and control glass freezing bovine oocyte - Google Patents
A kind of method of calcium ion concentration in regulation and control glass freezing bovine oocyte Download PDFInfo
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Abstract
The method that the present invention provides calcium ion concentration in a kind of regulation and control glass freezing bovine oocyte, it is to be placed in maturation in vitro 22h bovine oocyte in the maturation in vitro liquid of the AM containing BAPTA to be incubated 2h, then OPS technologies are used, freezed with the glass freezing liquid containing 10 μM of BAPTA AM and 1 μM of RR, recover 30min in the maturation in vitro liquid containing RR after defrosting, the cleavage rates, blastocyst rate, blastomere number of glass freezing bovine oocyte, and ability in vitro fertilization, developmental potency can be made to obtain conspicuousness raising.This method has simple to operate; cost is low; it is nontoxic to egg mother cell; the mitochondrial function and its developmental potency of freezing egg mother cell can effectively be protected; this freezes the application of egg mother cell for expanding; with important theoretical and practical significance, it will be played a great role in bovine oocyte superfreeze research field.
Description
Technical field
The present invention relates to bovine oocyte superfreeze technical field, specifically, it is related to a kind of regulation and control vitrifying cold
Freeze the method for calcium ion concentration in bovine oocyte.
Background technology
The freezen protective of mammal ovocyte is the effective hand for protecting species resource diversity and rescue animals on the brink of extinction
Section, or the embryo biotechnology such as in vitro fertilization, intracytoplasmic sperm injection and body-cell neucleus transplanting provides abundant experiment material,
Make the supply of egg mother cell and use not limited by time and space.Therefore, the freezen protective of egg mother cell has weighs very much
The theory significance and practical value wanted, are always the study hotspot and focus in cryobiology field.At present, because vitrifying is cold
Freeze with cooling rate is fast, freezing injury is low, simple operation and other advantages, have become the effective means of egg mother cell freezen protective
(Larman and Gardner,2014)。
Although the vitrificated cryopreserration of mammal ovocyte obtains remarkable progress (Vajta et al., 1998；
Dinnyés et al.,2000；Hou et al.,2005；Zhao et al., 2011), but it still much can not meet herding
The need for industry is produced and embryo biotechnology is fast-developing.To find out its cause, mainly due to glass freezing ovum can be caused female thin
Ca in born of the same parents2+Concentration abnormality is raised, and then causes the release in advance of cortical granule, oolemma hardening, rate of fertilization reduction (Larman
et a1.,2006,2007；Tian et al.,2007；Bogliolo et al.,2012；Nikiforaki et al.,
2014) glass freezing egg mother cell application, has been had a strong impact on.At present, glass freezing causes Ca in egg mother cell2+It is dense
Spend elevated mechanism also not fully aware of, more lacking being capable of Ca in Effective Regulation glass freezing egg mother cell2+Concentration, improves glass
Glassization freezes the effective measures of bovine oocyte fertility.
The content of the invention
It is an object of the invention to provide a kind of method of calcium ion concentration in Effective Regulation glass freezing bovine oocyte,
And then improve the in vitro fertilization and developmental potency of glass freezing bovine oocyte.
In order to realize the object of the invention, present invention firstly provides a kind of vitrifying for bovine oocyte freezen protective is cold
Freeze and contain 5-20 μM of BAPTA-AM (calcium chelating agent) and 0.5-2 μM of RR (mitochondrial calcium uniporter in liquid, the freezing liquid
Inhibitor, ammoniated ruthenium oxychloride), prepared with EDFSF40 liquid.Wherein, the EDFSF40 liquid is by EG (ethylene glycol), DMSO (dimethyl sulfoxide (DMSO))
Constituted with FSF liquid according to the ﹕ 6 of 2 ﹕ of volume ratio 2.FSF liquid be containing 30w/v% ficolls 70 (Ficoll 70), 0.5M sucrose and
20%FBS (hyclone) DPBS solution, not calcium-magnesium-containing.
Preferably, BAPTA-AM concentration is 5 μM, 10 μM, 20 μM in glass freezing liquid of the invention, and RR concentration is
0.5μM、1μM、2μM.It is highly preferred that BAPTA-AM concentration is 10 μM, RR concentration is 1 μM.
The present invention also provides a kind of agent combination for bovine oocyte freezen protective, including maturation in vitro liquid, contains
BAPTA-AM maturation in vitro liquid, pretreatment fluid, glass freezing liquid, thawing solution and the maturation in vitro liquid containing RR.
The maturation in vitro liquid is to contain 10 μ g/mL FSH (follicular stimulating hormone), 10 μ g/mL LH (lutropin), 1 μ
The M199 nutrient solutions of g/mL estradiol and 10%FBS.
The maturation in vitro liquid containing BAPTA-AM is the maturation in vitro liquid containing 5-20 μM of BAPTA-AM.Preferably,
BAPTA-AM concentration is 5 μM, 10 μM, 20 μM.It is highly preferred that BAPTA-AM concentration is 10 μM.
The pretreatment fluid is the DPBS solution (not calcium-magnesium-containing) containing 10%EG and 10%DMSO.
The thawing solution is the DPBS solution (not calcium-magnesium-containing) containing 0.15-0.25M sucrose.Preferably, the thawing solution
For the DPBS solution containing 0.15M or 0.25M sucrose.
The maturation in vitro liquid containing RR is the maturation in vitro liquid containing 0.5-2 μM of RR.Preferably, RR concentration is 0.5
μM、1μM、2μM.It is highly preferred that RR concentration is 1 μM.
The present invention also provides the agent combination in the kit for vitrificated cryopreserration bovine oocyte is prepared
Application.
The present invention also provides BAPTA-AM and RR in regulation and control glass freezing bovine oocyte intracellular Ca2+, mitochondria Ca2+
Level, improves the application in cleavage rates, blastocyst rate, fertilization and the developmental potency of glass freezing bovine oocyte.
The present invention further provides it is a kind of regulate and control glass freezing bovine oocyte in calcium ion concentration method, its be by
Maturation in vitro 22h bovine oocyte is placed in the maturation in vitro liquid containing BAPTA-AM and is incubated 2h, is then carried out using OPS methods cold
Freeze, 30min is recovered in the maturation in vitro liquid containing RR after defrosting.
It the described method comprises the following steps：
1) cumulus oocytes complesxes (COCs) are placed in maturation in vitro liquid and cultivate 22h, then use hyaluronidase
Cumulus cell is sloughed, MII phase egg mother cells is collected, is transferred in the maturation in vitro liquid containing BAPTA-AM and is incubated 2h；
2) egg mother cell is incubated to 30s in pretreatment fluid, then is transferred in glass freezing liquid and handles 25s, is then charged into
Frozen in OPS pipes in direct plunge into Liquid Nitrogen；
3) when thawing, OPS pipes are taken out from liquid nitrogen, the part that will be equipped with egg mother cell is immediately placed in 38.5 DEG C and contained
In the DPBS solution of 0.25M sucrose, egg mother cell is blown out to the DPBS for being put in 38.5 DEG C of sucrose containing 0.25M with suction pipe from OPS pipes
1min in solution, is then transferred to 5min in the DPBS solution of 38.5 DEG C of sucrose containing 0.15M, is thawed；
4) after thawing, egg mother cell is recovered into 30min in the maturation in vitro liquid containing RR.
In the specific embodiment of the present invention, it the described method comprises the following steps：
S1, from slaughterhouse collect ox ovary, be stored in 35 DEG C of physiology containing 75 μ g/mL penicillin and 50 μ g/mL streptomysins
Send laboratory in salt solution in 2h back to, cumulus oocytes complesxes are extracted out from a diameter of 2-8mm ovarian follicle, select at least 3
It is used for maturation in vitro after the complex cleaning of the fine and close cumulus cell of layer, about 50 cumulus oocytes complesxes are one group and are placed in
Fill 500 μ L maturation in vitro liquid and 4 orifice plates of the surface covered with mineral oil (contain about 50 in i.e. every ripe liquid of 500 μ L
COCs in), in 38.5 DEG C, 5%CO222h is cultivated in incubator, cumulus cell is then sloughed with hyaluronidase, the MII phases are collected
Egg mother cell, is transferred in the maturation in vitro liquid containing BAPTA-AM in 38.5 DEG C, 5%CO2Incubator in be incubated 2h；
S2, egg mother cell is incubated to 30s in pretreatment fluid, then is transferred in glass freezing liquid and handles 25s, be then charged into
Frozen in OPS pipes in direct plunge into Liquid Nitrogen；
S3, when thawing, OPS pipes are taken out from liquid nitrogen, the part that will be equipped with egg mother cell is immediately placed in 38.5 DEG C and contained
In the DPBS solution of 0.25M sucrose, egg mother cell is blown out to the DPBS for being put in 38.5 DEG C of sucrose containing 0.25M with suction pipe from OPS pipes
1min in solution, is then transferred to 5min in the DPBS solution of 38.5 DEG C of sucrose containing 0.15M, is thawed；
After S4, defrosting, egg mother cell is recovered into 30min in the maturation in vitro liquid containing RR.
The OPS used in the present invention is managed as both ends open formula plastic tube (I.M.V, Lai Gele, France), thickness of pipe wall
0.02mm, volume 0.25mL；One end of the OPS pipes is manually drawn and attenuated, and for holding egg mother cell, draws and attenuates one end OPS pipes
Top overall diameter is 0.23mm.
Research shows that glass freezing meeting conspicuousness improves intracellular Ca in bovine oocyte2+Level, mitochondria Ca2+Water
Flat, RR, BAPTA-AM processing can significantly reduce freezing egg mother cell Mitochondria Ca2+Level and intracellular Ca2+Level, and with
There was no significant difference for fresh control group.Further study showed that, glass freezing can make CG abnormal distributions in bovine oocyte,
ATP contents are reduced.Bovine oocyte is used after the glass freezing liquid freezing containing 1 μM of RR, freezing containing 1 μM of RR
Recover 30min in maturation in vitro liquid, can effectively maintain glass freezing bovine oocyte Mitochondria Ca2+Level, ATP contents
Normally, and then the developmental potency after the lonely female activation of glass freezing bovine oocyte is provided.And maturation in vitro 22h ox ovum is female
Cell is put into 10 μM of BAPTA-AM and is incubated 2h, is then freezed using the glass freezing liquid containing 10 μM of BAPTA-AM,
Intracellular Ca in glass freezing bovine oocyte can effectively be maintained2+Level, CG distributions are normal, and then improve glass freezing ox
Cleavage rates after Oocytes in Vitro Fertilization.Finally, maturation in vitro 22h bovine oocyte is put into 10 μM of BAPTA-AM to incubate
2h is educated, is then freezed using containing 10 μM of BAPTA-AM and 1 μM of RR glass freezing liquid, containing 1 μM of RR after defrosting
Recover 30min in maturation in vitro liquid, the cleavage rates, blastocyst rate, blastomere number of glass freezing bovine oocyte can be made, and
Ability in vitro fertilization, developmental potency obtain conspicuousness raising.
The glass freezing bovine oocyte method that the present invention is provided has simple to operate, and cost is low, to egg mother cell without
Poison is harmless, can effectively protect the mitochondrial function and its developmental potency of freezing egg mother cell, this is for expanding freezing egg mother cell
Application, with important theoretical and practical significance, will bovine oocyte superfreeze research field play it is huge
Effect.
Brief description of the drawings
Fig. 1 is bovine oocyte mitochondria Ca in the embodiment of the present invention 12+Colored graph.Wherein, A：Fresh egg mother cell center line
Plastochondria Ca2+Level；B：Freeze egg mother cell Mitochondria Ca2+Level.
Fig. 2 is RR processing in the embodiment of the present invention 1 to glass freezing bovine oocyte mitochondria Ca2+Influence.Wherein,
Shoulder mark difference lowercase letter indication difference is significantly (P ＜ 0.05).
Fig. 3 is the standard curve in ATP measurement process in the embodiment of the present invention 1.
Fig. 4 is influence of the RR processing to glass freezing bovine oocyte ATP contents in the embodiment of the present invention 1.
Fig. 5 is egg mother cell intracellular Ca in ovum embodiment 1 of the present invention2+Colored graph.Wherein, A：Ox ovum is female thin under normal circumstances
Born of the same parents' intracellular Ca2+Concentration is relatively low；B：Intracellular Ca in glass freezing egg mother cell2+Concentration is significantly raised.
Fig. 6 is glass freezing in the embodiment of the present invention 1 to intracellular born of the same parents intracellular Ca in bovine oocyte2+Influence.
The colored graph that Fig. 7 is CG in bovine oocyte in the embodiment of the present invention 1.Wherein, A：Normal distribution, CGs is distributed in
Kytoplasm cortical area, distribution annular in shape.Arrow：Annular distribution；B：There are a large amount of distributions, no annular distribution in spatial abnormal feature, kytoplasm.
Bar=20 μm.
Fig. 8 is bovine oocyte Sperm penetration of ova colored graph in the embodiment of the present invention 1.Wherein, A：Formed after normal fertilization it is female,
Male pronucleus；B：After abnormal fertilization in addition to female, male first protokaryon, there is the sperm of non-depolymerization.Bar=20 μm.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment
In the conventional meanses that are well known to those skilled in the art of used technological means, raw materials used is commercial goods.
M199 nutrient solutions are purchased from Gibco BRL companies (Grand Island, New York, the U.S.) in the present invention, and hyclone is purchased from
Gibco BRL Division companies (Grand Island, New York, the U.S.).
Influence of embodiment 1 RR, BAPTA-AM processing to glass freezing IVF of Oocyte in Bovine ability
1st, the collection of egg mother cell and maturation in vitro
Bovine oocyte is collected from local slaughterhouse, is stored in 35 containing 75 μ g/mL penicillin and 50 μ g/mL streptomysins
Laboratory is sent back in 2h, extract cumulus oocytes complesxes out from a diameter of 2-8mm ovarian follicle, select in DEG C physiological saline
At least have three layers fine and close cumulus cell complex cleaning after be used for maturation in vitro, 50 cumulus oocytes complesxes are one group
It is placed in and fills 500 μ L maturation in vitro liquid, and in 4 orifice plates of the surface covered with mineral oil, in 38.5 DEG C of 5%CO2Incubator in
22-24h is cultivated, cumulus cell is then sloughed with hyaluronidase, MII phase egg mother cells are collected, for subsequent experimental..
Maturation in vitro liquid：M199 cultures containing 10 μ g/mL FSH, 10 μ g/mL LH, 1 μ g/mL estradiol and 10%FBS
Liquid.
2nd, the preparation of OPS pipes
According to method disclosed in Vajta etc. (1998), slightly modified preparation OPS pipes, 0.25mL plastic tube (I.M.V, Lay
Ge Le, France), high temperature is manually elongated after softening, and it is 0.23mm that top overall diameter is made with microforge measurement, and thickness of pipe wall is about
0.02mm。
3rd, pretreatment and glass freezing liquid
Pretreatment fluid：DPBS solution (not calcium-magnesium-containing) containing 10%EG and 10%DMSO.
EDFSF40 liquid for preparing glass freezing liquid：The ﹕ 6 of 2 ﹕ 2 are mixed EG, DMSO and FSF liquid by volume.Wherein,
FSF liquid：DPBS solution containing 30w/v% ficolls 70,0.5M sucrose and 20%FBS, not calcium-magnesium-containing.
Thawing solution：DPBS solution containing 0.15M or 0.25M sucrose.
4th, the freezing and defrosting of egg mother cell
Egg mother cell, which is incubated 30s in pretreatment fluid and is transferred in glass freezing liquid, handles 25s, is then charged into OPS pipes
Frozen in direct plunge into Liquid Nitrogen.
During defrosting, OPS pipes are taken out from liquid nitrogen, the part that will be equipped with egg mother cell is dissolved in rapidly 38.5 DEG C containing 0.25M
In the DPBS solution of sucrose, egg mother cell is blown out to the DPBS solution for being put in 38.5 DEG C of sucrose containing 0.25M with mouth suction pipe from OPS pipes
Middle 1min, is then transferred to 5min in the DPBS solution of 38.5 DEG C of sucrose containing 0.15M.Observe the complete of oocyte membrane and oolemma
Property judges its survival condition.Subsequent experimental is used for according to the egg mother cell that survival is selected in experimental design.
5th, kytoplasm Ca2+Level is detected
Egg mother cell is placed in 10 μM of Fluo-3/AM 45min is incubated at 38.5 DEG C, then washed with DBPS/BSA liquid
Wash, finally in fluorescence microscopy Microscopic observation.Reflected using Nikon EZ-C1FreeViewer analysis of fluorescence intensity in kytoplasm
Ca2+Level.All operations are both needed to carry out in darkroom.
6th, mitochondrial calcium Ca2+Concentration mensuration
Mitochondrial calcium detection kit is purchased from Jie Mei companies of the U.S..Mitochondria Ca2+Concentration mensuration step is as follows：Maturation in vitro
Liquid cleaning egg mother cell 3 times, takes 300 μ l A liquid to be put into 1.5ml centrifuge tubes, and dyeing work is made in 30 μ l dyeing liquors of addition
Liquid；Egg mother cell after cleaning is put into dyeing working solution, room temperature (25 DEG C) lucifuge is incubated 30min, places into 37 DEG C of cultures
30min is incubated in case；Cell surface remnants dyeing working solutions are washed away with maturation in vitro liquid, it is at once glimmering in laser co-focusing after mounting
Observed under light microscope, excitation wavelength 550nm, distribute wavelength 590nm, mitochondria Ca2+It is presented red.Using graphical analysis
Software (EZ-C1FreeViewer) carries out fluorescence intensity quantitative analysis.
7th, ATP content analysis
Using method disclosed in Van Blerkom etc., slightly modified ATP Analysis number, using ATP dependences fluorescein-
Luciferase, progress fluorimetric assay for biological materials (ATP fluorimetric assay for biological materials, limited company of Roche Diagnistics of Kit HS II Switzerland,
Mannheim, Germany), determine fluorescence yield quantitative determination ATP numbers.
20 μ L cell cracking agents are added in the 0.5mL centrifuge tubes of 20 egg mother cells, piping and druming mixing makes these thin
Born of the same parents are equably dissolved.ATP concentration rises to 10.0pM from 0.01pM in solution, and each concentration analysis once, draws a standard
Curve.ATP prepare liquids, which are put into 96 orifice plates, balances 3-5min, the light that subsequent analytical standard liquid and sample are sent, and uses photometer
It can be measured in (InfiniteM200, Tecan Group Co., Ltd, Austria) 10s.With the relative light intensity of serial dilution
A standard curve is drawn out, sample ATP concentration is can be seen that from standard curve.
8th, Sperm penetration of ova is detected
Sperm penetration of ova is detected：Egg mother cell is incubated after 12h in containing sperm by seminal fluid, in the transparent of 50 μ l 0.1%
It is clean with the piping and druming sperm removing of mouth suction pipe in matter acid enzyme.Egg mother cell 4 DEG C of fixed 48h in fixer, are dyed with H33342
9min, dyeing liquor is sloughed with destainer, uses nail sheet for oil seal.Cell nuclear state is determined in phase contrast microscope.
9th, it is in vitro fertilization
It is in vitro fertilization：Freeze 37 DEG C of water-baths of essence to thaw, be placed in 7ml after defrosting and wash in seminal fluid, gently mix, 1800rpm centrifugations
5min 2 times.Supernatant is sucked after centrifugation, is 5 × 10 with by seminal fluid adjustment sperm concentration6Individual/ml.Draw 20 μ l seminal fluid and add and contain
There are 80 μ l of 20 pieces of egg mother cells by seminal fluid, in 38.5 DEG C, 5%CO2Carried out in incubator in vitro fertilization.Be fertilized 18-20h
Afterwards, it is placed in CR1aa and cultivates, nutrient solution (CR1aa containing 10%FBS) is changed after culture 48h.After in vitro culture 7d, body is obtained
Outer fertilization blastaea.
10th, experimental design
(1) BAPTA-AM processing is to glass freezing bovine oocyte intracellular Ca2+Level, CG distributions and ability in vitro fertilization
Influence
During COCs maturation in vitro, it is sloughed to cumulus cell in 22h with hyaluronidase, selection kytoplasm is equal
Even MII phase egg mother cells, egg mother cell are moved to respectively the maturation in vitro liquid relaying containing 5 μM, 10 μM, 20 μM BAPTA-AM
Continuous culture 2h, then carries out glass freezing (first by ovum mother carefully with the freezing liquid containing 5 μM, 10 μM, 20 μM BAPTA-AM respectively
Born of the same parents are incubated 30s in pretreatment fluid, then are transferred in glass freezing liquid and handle 25s, are then charged into direct plunge into Liquid Nitrogen in OPS pipes
In freeze), recover 30min after defrosting, bovine oocyte intracellular Ca carried out respectively2+Level, CG distributions and Sperm penetration of ova ability, body
The detection of outer fertilized embryo developmental potency.And then BAPTA-AM processing is studied to bovine oocyte intracellular Ca2+, CG distributions and external
The influence of fertility.
(2) RR processing is to glass freezing bovine oocyte Mitochondria Ca2+Level, ATP contents and lonely female activation development
The influence of ability
Cumulus cell is sloughed with hyaluronidase after COCs maturation in vitro 24h, the uniform MII phases ovum of selection kytoplasm is female thin
Born of the same parents, carry out glass freezing with the freezing liquid containing 0.5 μM, 1 μM, 2 μM RR respectively and (first incubate egg mother cell in pretreatment fluid
30s is educated, then is transferred in glass freezing liquid and handles 25s, is then charged into OPS pipes and is frozen in direct plunge into Liquid Nitrogen), after defrosting point
Do not recover 30min in the maturation in vitro liquid containing 0.5 μM, 1 μM, 2 μM RR, and then study RR processing to mitochondria Ca2+Level,
The influence of ATP contents and lonely female activation developmental potency.
(3) RR and influence of the BAPTA-AM Combined Treatments to glass freezing IVF of Oocyte in Bovine ability
During COCs maturation in vitro, it is sloughed to cumulus cell in 22h with hyaluronidase, selection kytoplasm is equal
Even MII phase egg mother cells, egg mother cell is moved into the maturation in vitro liquid containing 10 μM of BAPTA-AM and continues to cultivate 2h, so
Freezed afterwards with the glass freezing liquid containing 10 μM of BAPTA-AM and 1 μM of RR, the body containing 1 μM of RR is put into after defrosting
Recover 30min in outer ripe liquid, the detection of IVF of Oocyte in Bovine ability is then carried out respectively.
13rd, data statistics
Experimental data is analyzed using SAS softwares, variance analysis is used when percentage compares after anyway rotary,
As a result represented with average according to standard deviation, P<0.05 is significance of difference standard.Each treatment group is at least repeated 3 times the above.
Experimental result：
(1) RR processing is to glass freezing bovine oocyte Mitochondria Ca2+The influence of level
(1) various concentrations RR processing is to glass freezing bovine oocyte Mitochondria Ca2+The influence of level
Bovine oocyte mitochondria Ca2+Dyeing picture is shown in Fig. 1, fresh bovine oocyte (Figure 1A) Mitochondria Ca2+It is dense
Degree is relatively low, and glass freezing control group egg mother cell (Figure 1B) Mitochondria Ca2+Concentration is higher.As shown in Fig. 2 vitrifying is cold
Jelly group bovine oocyte mitochondria Ca2+Level is significantly higher than fresh group (P ＜ 0.05)；RR treatment group mitochondrias Ca2+Level shows
Write and be less than glass freezing group (P ＜ 0.05), wherein 0.5 μM and 1 μM for the treatment of group are still significantly higher than fresh group (P ＜ 0.05), and 2
μM group and fresh group of difference not significantly (P>0.05).
(2) RR handles the influence to bovine oocyte ATP contents after defrosting
The standard curve drawn during ATP assays is as shown in figure 3, gained normal equation is：Y=0.0049x+
0.2133, calibration curve coefficient correlation (R2) it is 0.9991.ATP contains in sample value divided by sample number, as each egg mother cell
Amount.As shown in figure 4, glass freezing significantly reduces ATP contents in bovine oocyte (P ＜ 0.05), RR treatment group ATP contents
Substantially less than fresh control group (P ＜ 0.05), but be significantly higher than frozen control group (P ＜ 0.05), and between each treatment group mutually
There is significant difference (P ＜ 0.05).
(3) influence of the RR processing to the lonely female activation developmental potentiality of bovine oocyte after defrosting
As shown in table 1, after the lonely female activation of egg mother cell glass freezing group cleavage rates (72.22%), blastocyst rate
(23.08%) it is substantially less than fresh control group (84.30%, 41.18%；P ＜ 0.05), 1 μM of RR treatment group cleavage rates
(88.20%), blastocyst rate (42.25%) and fresh control group (84.30%, 41.18%) are compared to there was no significant difference but notable
Higher than 0.5 μM RR treatment group (77.18%, 27.83%).1 μM of RR treatment groups cleavage rates (88.20%) conspicuousness is higher than 2 μM
There was no significant difference between RR treatment groups (79.29%) but two groups of blastocyst rate (42.25%vs.34.23%).1 μM of RR processing
Effect is higher than 2 μM of RR, and reason is probably because 2 μM of RR concentration are higher, and then caused by the toxic action produced.
Influence of the RR of table 1 processing to glass freezing bovine oocyte developmental potentiality
Note:With column data shoulder mark, different lowercase letter indication differences are notable (P ＜ 0.05).
(2) BAPTA-AM processing is to glass freezing bovine oocyte intracellular Ca2+Level, CG distributions and energy in vitro fertilization
The influence of power
(1) BAPTA-AM processing is to glass freezing bovine oocyte intracellular Ca2+The influence being distributed with CG
Intracellular Ca in egg mother cell2+Dyeing as shown in figure 5, bovine oocyte intracellular Ca under normal circumstances2+Concentration is relatively low
Intracellular Ca in (Fig. 5 A), glass freezing egg mother cell2+Concentration is significantly raised (Fig. 5 B).As shown in fig. 6, fresh control group intracellular
Ca2+Concentration conspicuousness is less than glass freezing control group, BAPTA-AM each group intracellulars Ca2+It is cold that level is substantially less than vitrifying
Jelly group (P ＜ 0.05), wherein 5 μM for the treatment of groups are still significantly higher than fresh group (P ＜ 0.05), and 10 μM, 20 μM for the treatment of groups with it is fresh
Not notable (the P of group difference>0.05).
Egg mother cell CG dyeing such as Fig. 7 shows that CG is distributed in kytoplasm cortical area and annular distribution (figure is presented under normal circumstances
7A), CG is discharged into kytoplasm (Fig. 7 B) in advance from cortical area under abnormal conditions.As shown in table 2, glass freezing group CG is normal
Distributive law (56.25%) conspicuousness is less than fresh control group (86.11%), and CG normal distribution rates are notable after BAPTA-AM processing
Lifted (65.85%, 75.56%, 84.21%, 86.11%), and 10 μM of BAPTA-AM freezings groups (75.56%), 20 μM
There was no significant difference with fresh control group (86.11%) for BAPTA-AM freezings group (84.21%) CG normal distributions rate.
The influence that the BAPTA-AM of table 2 processing is distributed to glass freezing bovine oocyte CG
Note:With column data shoulder mark, different lowercase letter indication differences are notable (P ＜ 0.05).
(2) BAPTA-AM handles the influence to glass freezing bovine oocyte Sperm penetration of ova ability
Sperm penetration of ova is dyed as shown in figure 8, form female, male pronucleus (Fig. 8 A) after normal fertilization in egg mother cell, it is abnormal by
After essence in egg mother cell in addition to female, male pronucleus, there is the sperm (Fig. 8 B) of non-depolymerization.As shown in table 3, glass freezing group is normal
Rate of fertilization (60%) is substantially less than fresh control group (87.5%；P ＜ 0.05), 10 μM of RR freezing group normal fertilization rates
(84.85%) be significantly higher than 5 μM of RR freezings groups (63.33%) and 20 μM of RR freezings groups (70.97%), and with fresh control group
(87.5%) there was no significant difference.
Influence of the BAPTA-AM of table 3 processing to glass freezing IVF of Oocyte in Bovine ability
Note:With column data shoulder mark, different lowercase letter indication differences are notable (P ＜ 0.05).
(3) BAPTA-AM handles the influence to glass freezing IVF of Oocyte in Bovine efficiency
As shown in table 4, frozen control group spilting of an egg rate (41.63%) in vitro fertilization, blastocyst rate (12.37%) show experimental result
Write and be less than fresh control group (75%, 34.09%).In RR treatment groups, 10 μM of RR freezing group cleavage rates (65.73%) are significantly high
In frozen control group (41.63%), 5 μM of RR freezings groups (52.54%), 20 μM of RR freezings groups (49.73%), but with it is fresh right
According to group (75%), there was no significant difference.There was no significant difference (11.96%-15.71%), equal conspicuousness is low for blastocyst rate between each group
In fresh control group (34.09%).
Influence of the BAPTA-AM of table 4 processing to glass freezing IVF of Oocyte in Bovine ability
Note:With column data shoulder mark, different lowercase letter indication differences are notable (P ＜ 0.05).
(3) RR and influence of the BAPTA-AM Combined Treatments to glass freezing IVF of Oocyte in Bovine ability
By RR, BAPTA-AM previous experiments, determine that 1 μM of RR and 10 μM of BAPTA-AM carries out Combined Treatment experiment.It is real
Test result as shown in table 5, cleavage rates (58.24%), the blastocyst rate (10.53%) of frozen control group are substantially less than fresh control group
(83.55%, 34.72%；P ＜ 0.05), cleavage rates (79.42%), the blastocyst rate of RR, BAPTA-AM Combined Treatment group
(29.09%) conspicuousness is higher than frozen control group (58.24%%, 10.53%%；P ＜ 0.05)
The RR of table 5 and influence of the BAPTA-AM Combined Treatments to glass freezing IVF of Oocyte in Bovine efficiency
Note:With column data shoulder mark, different lowercase letter indication differences are notable (P ＜ 0.05).
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
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Claims (10)
1. a kind of glass freezing liquid for bovine oocyte freezen protective, it is characterised in that contain 5- in the freezing liquid
20 μM of BAPTA-AM and 0.5-2 μM of RR, are prepared with EDFSF40 liquid；
Wherein, the EDFSF40 liquid is made up of EG, DMSO and FSF liquid according to the ﹕ 6 of 2 ﹕ of volume ratio 2；FSF liquid is to contain 30w/v%
The DPBS solution of ficoll 70,0.5M sucrose and 20%FBS, not calcium-magnesium-containing.
2. freezing liquid according to claim 1, it is characterised in that in the freezing liquid BAPTA-AM concentration be 5 μM, 10
μM, 20 μM, RR concentration is 0.5 μM, 1 μM, 2 μM；Preferably, BAPTA-AM concentration is 10 μM, and RR concentration is 1 μM.
3. a kind of agent combination for bovine oocyte freezen protective, it is characterised in that including maturation in vitro liquid, containing BAPTA-
AM maturation in vitro liquid, pretreatment fluid, freezing liquid, thawing solution and the maturation in vitro liquid containing RR described in claim 1 or 2；
The maturation in vitro liquid is the M199 trainings containing 10 μ g/mL FSH, 10 μ g/mL LH, 1 μ g/mL estradiol and 10%FBS
Nutrient solution；
The maturation in vitro liquid containing BAPTA-AM is the maturation in vitro liquid containing 5-20 μM of BAPTA-AM；
The pretreatment fluid is the DPBS solution containing 10%EG and 10%DMSO, not calcium-magnesium-containing；
The thawing solution is the DPBS solution containing 0.15-0.25M sucrose, not calcium-magnesium-containing；
The maturation in vitro liquid containing RR is the maturation in vitro liquid containing 0.5-2 μM of RR.
4. agent combination according to claim 3, it is characterised in that the maturation in vitro liquid containing BAPTA-AM be containing
5 μM, 10 μM, 20 μM BAPTA-AM maturation in vitro liquid；
The thawing solution is the DPBS solution containing 0.15M, 0.25M sucrose；
The maturation in vitro liquid containing RR is the maturation in vitro liquid containing 0.5 μM, 1 μM, 2 μM RR.
5. agent combination according to claim 4, it is characterised in that the maturation in vitro liquid containing BAPTA-AM be containing
10 μM of BAPTA-AM maturation in vitro liquid；The maturation in vitro liquid containing RR is the maturation in vitro liquid containing 1 μM of RR.
6.BAPTA-AM and RR is in regulation and control glass freezing bovine oocyte intracellular Ca2+, mitochondria Ca2+Level, improves vitrifying
Freeze the application in cleavage rates, blastocyst rate, fertilization and the developmental potency of bovine oocyte.
7. a kind of method of calcium ion concentration in regulation and control glass freezing bovine oocyte, it is characterised in that by maturation in vitro 22h
Bovine oocyte be placed in the maturation in vitro liquid containing BAPTA-AM and be incubated 2h, then freezed using OPS methods, after defrosting
Recover 30min in maturation in vitro liquid containing RR；
The reagent used in methods described is from the agent combination described in claim any one of 3-5.
8. method according to claim 7, it is characterised in that comprise the following steps：
1) cumulus oocytes complesxes are placed in maturation in vitro liquid and cultivate 22h, it is thin then to slough ovarian cumulus with hyaluronidase
Born of the same parents, collect MII phase egg mother cells, are transferred in the maturation in vitro liquid containing BAPTA-AM and are incubated 2h；
2) egg mother cell is incubated to 30s in pretreatment fluid, then is transferred in glass freezing liquid and handles 25s, OPS pipes are then charged into
Frozen in middle direct plunge into Liquid Nitrogen；
3) when thawing, OPS pipes are taken out from liquid nitrogen, the part that will be equipped with egg mother cell is immediately placed in 38.5 DEG C of sugarcanes containing 0.25M
In the DPBS solution of sugar, egg mother cell is blown out from OPS pipes with suction pipe in being put in the DPBS solution of 38.5 DEG C of sucrose containing 0.25M
1min, is then transferred to 5min in the DPBS solution of 38.5 DEG C of sucrose containing 0.15M, is thawed；
4) after thawing, egg mother cell is recovered into 30min in the maturation in vitro liquid containing RR.
9. method according to claim 8, it is characterised in that comprise the following steps：
S1, from slaughterhouse collect ox ovary, be stored in 35 DEG C of physiological saline containing 75 μ g/mL penicillin and 50 μ g/mL streptomysins
Laboratory is sent back in middle 2h, cumulus oocytes complesxes are extracted out from a diameter of 2-8mm ovarian follicle, selects the cause that at least haves three layers
It is used for maturation in vitro after the complex cleaning of close cumulus cell, 50 cumulus oocytes complesxes are placed in for one group and fill 500
In μ L maturation in vitro liquid, and 4 orifice plates of the surface covered with mineral oil, in 38.5 DEG C, 5%CO2Incubator in cultivate 22h, so
Cumulus cell is sloughed with hyaluronidase afterwards, MII phase egg mother cells is collected, is transferred in the maturation in vitro liquid containing BAPTA-AM
38.5 DEG C, 5%CO2Incubator in be incubated 2h；
S2, egg mother cell is incubated to 30s in pretreatment fluid, then is transferred in glass freezing liquid and handles 25s, be then charged into OPS
Frozen in pipe in direct plunge into Liquid Nitrogen；
When S3, defrosting, OPS pipes are taken out from liquid nitrogen, the part that will be equipped with egg mother cell is immediately placed in 38.5 DEG C of sugarcanes containing 0.25M
In the DPBS solution of sugar, egg mother cell is blown out from OPS pipes with suction pipe in being put in the DPBS solution of 38.5 DEG C of sucrose containing 0.25M
1min, is then transferred to 5min in the DPBS solution of 38.5 DEG C of sucrose containing 0.15M, is thawed；
After S4, defrosting, egg mother cell is recovered into 30min in the maturation in vitro liquid containing RR.
10. method according to claim 8 or claim 9, it is characterised in that the OPS pipes are both ends open formula plastic tube, tube wall
Thick 0.02mm, volume 0.25mL；One end of the OPS pipes is manually drawn and attenuated, for holding egg mother cell, draws and attenuates one end OPS pipes
Top overall diameter be 0.23mm.
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