CN108588007A - The method for improving ox embryo in vitro preparation efficiency - Google Patents
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Abstract
The present invention provides a kind of method improving ox embryo in vitro preparation efficiency, including the external acquisition of bovine oocyte, the in vitro fertilization and ectogenesis culture of the In-vitro maturation of cumulus oocyte complex, bovine oocyte.Wherein, contain 0.01 70 μM of C in culture solution used in ectogenesis culture35H49F2N7O4·CF3CO2H(MM102).By under in vitro conditions, choosing optimal ox system in vitro fertilization, after ox development in vitro fertilization 48 hours, blastomere is placed in the development liquid added with various concentration MM102, the results show that MM102 has facilitation to the fertilized eggs ectogenesis of ox, higher blastocyst rate can be obtained.The method can be used for improving efficiency prepared by ox embryo in vitro.The present invention can provide certain theoretical foundation for Livestock in Vitro Fertilization technical research, and can further apply in the fields such as livestock reproduction and domestic animal heredity conservation.
Description
Technical field
The present invention relates to Celluar and Molecular Biology fields, specifically, being related to a kind of raising ox embryo in vitro
The method of preparation efficiency.
Background technology
Ox technology in vitro fertilization include Oocyte in Vitro collect with ripe, sperm microcytotoxicity and smart egg's external fertilization,
And the processes such as fertilized eggs in vitro culture.1977, Iritane and Niwa obtained the first ox in vitro fertilization in the world.The body in China
Outer fertilization research starts from the later stage eighties, and on the basis of forefathers study, China makes much progress.1989, the rising sun did institute
The successful incubations such as scholar go out China's first case ox in vitro fertilization.Hereafter, Fan Biqin, Lu Kehuan, Shi De along etc. research teams also successively
Obtain ox in vitro fertilization.So far, due to the extensive utilization of embryo IVC technology, the offspring of most of domestic animals
It has successfully obtained.Though ox is the animal more early studied in Livestock in Vitro Fertilization, its production efficiency is unsatisfactory.
Mixed stocker leukaemia (MLL1) is a kind of H3K4 transmethylases, and the activity of MLL1 enzymes is inhibited to merge egg for MLL1
Acute leukemia is a kind of new therapeutic strategy caused by white.When WDR/MLL protein-interactings, MLL1 enzymes, which just have, lives
Property.MM102(C35H49F2N7O4·CF3CO2H) be WDR/MLL protein-interactings inhibitor.It is expressed in leukaemia cell
When MLL1-AF9 fusions, inhibit the table of the activity of MLL1 transmethylases and HoxA9 the and Meis-1 genes of MLL1 inductions
It reaches.Currently, MM102 is in terms of improving embryo development rate, there are no researchs.
Invention content
The object of the present invention is to provide a kind of methods improving ox embryo in vitro preparation efficiency.Pass through after fertilization in vitro
MM102 is added in embryo development procedure, probes into influences of the MM102 to ox embryonic development, is Livestock in Vitro Fertilization and raising blastaea
Rate provides reference.
In order to realize the object of the invention, a kind of method improving ox embryo in vitro preparation efficiency provided by the invention, including
The external acquisition of bovine oocyte, the In-vitro maturation of cumulus oocytes complesxes, bovine oocyte it is in vitro fertilization with
And ectogenesis culture and etc..
Wherein, contain 0.01-70 μM of C in culture solution used in ectogenesis culture (IVC-2 liquid)35H49F2N7O4·
CF3CO2H.Preferably, 30 μM, 50 μM or 70 μM of C is contained in the IVC-2 liquid35H49F2N7O4·CF3CO2H, more preferable 50 μ
M。
Method above-mentioned, when containing 0 μM of MM102 in IVC-2 liquid, blastocyst rate 24.33%.
Method above-mentioned, when containing 30 μM of MM102 in IVC-2 liquid, blastocyst rate 25.47%.
Method above-mentioned, when containing 50 μM of MM102 in IVC-2 liquid, blastocyst rate 32.61% is higher than other systems.
Method above-mentioned, when containing 70 μM of MM102 in IVC-2 liquid, blastocyst rate 29.65%.
Method above-mentioned, when MM102 concentration is when between 0-50 μM, with the increase of MM102 concentration, on blastocyst rate is gradual
It rises, and blastaea form is normal.
Method above-mentioned, when MM102 concentration reaches 70 μM, blastocyst rate declines, but blastaea form is still normal.
The immunofluorescence dyeing that multipotency gene is carried out to the blastaea turned out under above-mentioned developmental condition finds indifference.
Preferably, the formula of the IVC-2 liquid is：
Wherein, the formula of the SOF liquid is：
The formula of the liquid storage A is：
The formula of the liquid storage B is：
Water 10mL
NaHCO3 0.21g
The formula of the liquid storage C is：
Water 10mL
Sodium Pyruvate 0.08g
The formula of the liquid storage D is：
Water 10mL
CaCl2·2H2O 0.262g
Method above-mentioned, the used formula of liquid of cultivating of the In-vitro maturation of cumulus oocytes complesxes are：
Method above-mentioned, it is in vitro fertilization to be carried out in A liquid and B liquid successively, wherein A formula of liquid is：Liquid storage in vitro fertilization+
10mM caffeines, B formula of liquid are：Liquid storage+20mg/mL bovine serum albumin(BSA)s+1IU/mL heparin in vitro fertilization；
Wherein, the formula of the liquid storage in vitro fertilization is：
Method above-mentioned, it is preferred to use syringe extraction method collects cytoplasm uniformly and has the ovum of multilayer whole grain cell
Mound-oocyte complex, In-vitro maturation 22-24h.
Method above-mentioned, bovine oocyte it is in vitro fertilization in, using breeding oxen freeze essence, by the sperm washed and body
Cumulus oocytes complesxes after outer maturation are put by sperm, and after fertilization removes granular cell, is placed in IVC-1 liquid and trains
48h is supported, is then transferred in IVC-2 liquid, ectogenesis 7-9d, blastocyst rate is counted.
Wherein, the formula of the IVC-1 liquid is：
In the present invention, condition of in vitro culture is：38.5 DEG C, 5%C02, saturated humidity incubator.
The present invention also provides a kind of ectogenesis culture solution IVC-2 for improving ox embryo in vitro preparation efficiency, wherein
Contain 0.01-70 μM of C in IVC-2 liquid35H49F2N7O4·CF3CO2H.Preferably, 30 μM, 50 μM are contained in the IVC-2 liquid
Or 70 μM of C35H49F2N7O4·CF3CO2H, more preferable 50 μM.
By above-mentioned technical proposal, the present invention at least has following advantages and advantageous effect：
The present invention is small in ox development 48 in vitro fertilization by under in vitro conditions, choosing optimal ox system in vitro fertilization
Blastomere is placed in the development liquid added with various concentration MM102 by Shi Hou, the results show that MM102 external to the fertilized eggs of ox
Development has facilitation, and higher blastocyst rate can be obtained.The method can be used for improving efficiency prepared by ox embryo in vitro.The present invention can
Certain theoretical foundation is provided for Livestock in Vitro Fertilization technical research, and livestock reproduction and domestic animal heredity guarantor can be further applied
In the fields such as kind.
Description of the drawings
Fig. 1 is the 7th day external spermatheca embryo form under various concentration MM102 cultivating systems in the embodiment of the present invention 3.
The blastaea turned out when Fig. 2 is in IVC-2 liquid in the embodiment of the present invention 3 containing 0 μM of MM102, carries out multipotency base
The immunofluorescence dyeing result of cause.
The blastaea turned out when Fig. 3 is in IVC-2 liquid in the embodiment of the present invention 3 containing 30 μM of MM102, carries out multipotency base
The immunofluorescence dyeing result of cause.
The blastaea turned out when Fig. 4 is in IVC-2 liquid in the embodiment of the present invention 3 containing 50 μM of MM102, carries out multipotency base
The immunofluorescence dyeing result of cause.
The blastaea turned out when Fig. 5 is in IVC-2 liquid in the embodiment of the present invention 3 containing 70 μM of MM102, carries out multipotency base
The immunofluorescence dyeing result of cause.
In Fig. 1-Fig. 5, scale is 100 μm in figure.
Specific implementation mode
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.
The external acquisition of 1 bovine oocyte of embodiment and In-vitro maturation
The ovary for now butchering ox is collected from slaughterhouse, is stored in the clean insulation barrel for filling 0.9% sterile saline,
In transporting laboratory in 2-4h back.After ovary is sent to laboratory, it is placed in 20-25 DEG C of 0.9% sterile saline and cleans at least 3
Secondary, the acquisitions of ox Oocytes uses syringe extraction method, with the ovarian follicle of syringe 2-7mm a diameter of to Ovarian surface into
Row extracts.The liquor folliculi of extraction is collected into cytoplasm uniformly under stereomicroscope and has ovarian cumulus-ovum of multilayer whole grain cell
Mother cell complex (COCs), after washing 3 times with In-vitro maturation liquid, COCs is collected in ripe liquid rapidly carry out in vitro at
Ripe culture.All COCs carry out In-vitro maturation in four orifice plates.Cultivating system is 700 holes μ L/ of In-vitro maturation liquid,
Liquid layer surface paving plus 300 holes μ L/ of paraffin oil；Condition of culture is 50 pieces/hole, 38.5 DEG C, 5%CO2, saturated humidity incubator.
Wherein, the formula of In-vitro maturation liquid is：
2 bovine oocyte of embodiment it is in vitro fertilization
After In vitro maturation 22-24h, Oocytes in Vitro Fertilization is carried out.The bull semen of purchase, is pressed
Ovum number and sperm pipe number 100：1 ratio row defrosting sperm is in the 15mL sterile centrifugation tubes containing 8mL A liquid in vitro fertilization.37 DEG C,
It 4000 revs/min, centrifuges 5 minutes.It washes twice, after sucking supernatant, slowly gently adds 1-2mL in vitro fertilization on the surface layer of Sperm pellets
Culture solution, float 3-5min in 37 DEG C of water-baths, and it is isometric in the centrifuge tube of 1.5mL, being added to draw the muddy part in upper layer
B liquid in vitro fertilization, after mixing, be added fertilization drop, often drop fertilization drop sperm concentration be 1 × 106-5×106A/mL.It will
After COCs after In-vitro maturation washs 3 times with A+B liquid in vitro fertilization (A, B liquid are in equal volume than mixing), by 15-20 pieces
It moves into fertilization drop.Culture dish is put into incubator and carries out culture in vitro fertilization.Cultivating system is culture solution in vitro fertilization, often
Drip 100 μ L, liquid layer surface paving plus paraffin oil；38.5 DEG C of condition of culture, 5%CO2, saturated humidity incubator.
Wherein, A formula of liquid in vitro fertilization is：Liquid storage+10mM caffeines in vitro fertilization, B formula of liquid in vitro fertilization are：In vitro
Fertilization liquid storage+20mg/mL bovine serum albumin(BSA)+1IU/mL heparin.
The formula of the liquid storage in vitro fertilization is：
The ectogenesis culture of 3 fertilized bovine oocytes of embodiment
Sperm is with after Oocytes in Vitro Fertilization culture 6h, and fertilized eggs move into ectogenesis culture solution, with 100 μ L liquid reliefs
Device is gently blown and beaten repeatedly, to take off granular cell and remove the sperm being attached on fertilized eggs, is washed 3 times with IVC-1 liquid.It will be by
Smart ovum moves into ectogenesis IVC-1 drops respectively, 20 pieces/drop, culture dish is put into incubator and carries out Development culture 48h.Training
The system of supporting is that ectogenesis culture solution often drips 50 μ L, liquid layer surface paving plus paraffin oil, and CO is put into before ectogenesis2It is put down in incubator
Weigh 5h or more；It is cultivated in vitro to 48h, spilting of an egg number is counted, by IVC-2 liquid of the embryo of the spilting of an egg containing various concentration MM102
It washes 3 times, is put into corresponding development IVC-2 drops and continues to cultivate.The 7-9d days statistics blastocyst rates (table 1).Condition of culture is
38.5 DEG C, 5%CO2, saturated humidity incubator.
Wherein, the formula of the IVC-1 liquid is：
The formula of the IVC-2 liquid is：
Wherein, the formula of the SOF liquid is：
The formula of the liquid storage A is：
The formula of the liquid storage B is：
Water 10mL
NaHCO3 0.21g
The formula of the liquid storage C is：
Water 10mL
Sodium Pyruvate 0.08g
The formula of the liquid storage D is：
Water 10mL
CaCl2·2H2O 0.262g
Method above-mentioned, the used formula of liquid of cultivating of the In-vitro maturation of cumulus oocytes complesxes are：
Blastocyst rate under 1 various concentration MM102 cultivating systems of table
The 7th day external spermatheca embryo form is shown in Fig. 1 under various concentration MM102 cultivating systems.When MM102 concentration is at 0-50 μM
Between when, with the increase of MM102 concentration, blastocyst rate is gradually increasing, and blastaea form is normal.When MM102 concentration reaches 70 μM
When, blastocyst rate declines, but blastaea form is still normal.
The blastaea turned out when in IVC-2 liquid containing 0 μM, 30 μM, 50 μM, 70 μM of MM102 carries out exempting from for multipotency gene
As a result Fig. 2-Fig. 5 is shown in epidemic disease fluorescent staining respectively.The MM102 that can be seen that addition various concentration from Fig. 2-Fig. 5 is more to ox blastaea
The no significant impact of expression of energy gene.
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 is 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.
Claims (10)
1. a kind of method improving ox embryo in vitro preparation efficiency, which is characterized in that the external acquisition including bovine oocyte, ovum
The step of In-vitro maturation of mound-oocyte complex, the in vitro fertilization of bovine oocyte and ectogenesis culture；
Wherein, contain 0.01-70 μM of C in culture solution used in ectogenesis culture35H49F2N7O4·CF3CO2H。
2. according to the method described in claim 1, it is characterized in that, containing 30 μ in culture solution used in ectogenesis culture
M, 50 μM or 70 μM of C35H49F2N7O4·CF3CO2H, preferably 50 μM.
3. according to the method described in claim 1, it is characterized in that, culture formula of liquid used in ectogenesis culture is：
Wherein, the formula of the SOF liquid is：
The formula of the liquid storage A is：
The formula of the liquid storage B is：
4. according to the method described in claim 1, it is characterized in that, the In-vitro maturation institute of cumulus oocytes complesxes
The culture formula of liquid used is：
5. according to the method described in claim 1, it is characterized in that, in vitro fertilization carry out in A liquid and B liquid successively, wherein A
Formula of liquid is：Liquid storage+10mM caffeines in vitro fertilization, B formula of liquid are：Liquid storage+20mg/mL bovine serum albumin(BSA)s in vitro fertilization+
1IU/mL heparin；
Wherein, the formula of the liquid storage in vitro fertilization is：
6. according to the method described in claim 1, it is characterized in that, in vitro by collected cumulus oocytes complesxes
Maturation culture 22-24h.
7. according to the method described in claim 1, it is characterized in that, bovine oocyte it is in vitro fertilization in, using breeding oxen
Freeze essence, the cumulus oocytes complesxes after the sperm washed and maturation in vitro are put by sperm, after fertilization removal
Granulocyte is placed in IVC-1 liquid and cultivates 48h, is then transferred in IVC-2 liquid, ectogenesis 7-9d, counts blastocyst rate；
Wherein, the formula of the IVC-1 liquid is：
The formula of the IVC-2 liquid is the same as described in claim 3.
8. according to claim 1-7 any one of them methods, which is characterized in that condition of in vitro culture is：38.5 DEG C, 5%C02、
Saturated humidity incubator.
9. a kind of ectogenesis culture solution for improving ox embryo in vitro preparation efficiency, which is characterized in that in the culture solution
Contain 0.01-70 μM of C35H49F2N7O4·CF3CO2H。
10. culture solution according to claim 9, which is characterized in that contain 30 μM, 50 μM or 70 μM in the culture solution
C35H49F2N7O4·CF3CO2H, preferably 50 μM.
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