CN103898048A - In vitro maturation culture method of denuded oocytes of mice - Google Patents
In vitro maturation culture method of denuded oocytes of mice Download PDFInfo
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- CN103898048A CN103898048A CN201410120868.5A CN201410120868A CN103898048A CN 103898048 A CN103898048 A CN 103898048A CN 201410120868 A CN201410120868 A CN 201410120868A CN 103898048 A CN103898048 A CN 103898048A
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Abstract
The invention discloses an in vitro maturation culture method of denuded oocytes of mice. The method comprises the following steps: (1) performing in vitro maturation culture on the denuded oocytes; (2) performing parthenogenetic activation on matured denuded oocytes; (3) performing in vitro fertilization on the matured denuded oocytes. According to the method, an environment for artificially simulating natural maturation of oocytes can be created, so that the denuded oocytes can efficiently maturate and grow in vitro and can be subjected to in vitro fertilization and embryonic development as same as normally growing oocytes after in vitro maturation culture. The practice proves that the in vitro maturation culture efficiency of the denuded oocytes of the system is high, the system is mature and stable, and the embryonic development quality of the denuded oocytes is high after in vitro natural fertilization, so that the application and popularization of the denuded oocytes in reproductive health of human beings and genetic breeding of modern livestock can be greatly facilitated. In addition, the operation of obtaining blastulae from the denuded oocytes is simplified and the denuded oocytes serving as ovum sources can be conveniently put into actual production.
Description
Technical field
The present invention relates to a kind of in-vitro maturation culture method of mouse denuded oocyte.
Background technology
In normal reproductive development, ovocyte periphery is enclosed with a large amount of cumulus cells, and when spontaneous ovulation, ovocyte enters in uterine tube and waits for sperm combination together with peripheral cumulus cell.In the time carrying out the application of embryo engineering operation or auxiliary procreation technology, ovocyte periphery is enclosed with a large amount of cumulus cells, and to have into heat efficiency high, and the ovocyte after maturation has same sperm natural combination and form embryo's function.But, in the ovocyte directly extracting from ovary in clinical application, there is ovocyte over half not wrapped up by cumulus cell completely around, such oocyte maturation efficiency is low, allow to maturation, also the ability that does not possess same sperm combination after maturation, in clinical application, such ovocyte is often directly discarded.In clinical application, conventionally the ovocyte not wrapped up by cumulus cell is completely called to ova nuda.In the scientific research of reproductive development, ova nuda is that a kind of important cell resource is studied for reproductive development, and an important potential ovum source healthy as human ancillary reproductive and that modern domestic animal heredity is bred.
Because ovocyte does not have cumulus cell or less cumulus cell parcel around, therefore, ova nuda is the important tests material that the ripe mechanism of research ovocyte reproductive development and artificial egg parent cell occur.Before this, in the ectogenesis maturation of world's ova nuda, conventional culture system can cause ovocyte to carry out maturation growing, but ripe ovocyte does not have same sperm in conjunction with the ability of being fertilized, and externally can not further grow the blastaea stage that reaches uterus implantation.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of in-vitro maturation culture method of mouse denuded oocyte.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is: the in-vitro maturation culture method of mouse denuded oocyte, comprises the following steps:
Get mouse ovarian, use under the microscope syringe needle head, puncture ovarian follicle on ovary, ovocyte in ovarian follicle is discharged in the TCM199 Oocyte in Vitro operation liquid containing HEPES and 10%FBS, with picking up ovum pin at random after external ova nuda washing, the oocyte maturation nutrient solution of adding containing 50 μ M forskolin for random packet
in-Vitro Maturation Media cultivates after 3h, shifts the oocyte maturation nutrient solution of adding containing 2.5 μ M PD166285 for ova nuda
in-Vitro Maturation Media continues vitro culture 16h;
Wherein TCM199 is Therma company product, nutrient solution product article No. SH30233;
in-Vitro Maturation Media is Sage company of U.S. product, and nutrient solution product article No. is ART-1600;
(2) the female activation of the orphan of ripe ova nuda
The post-mature of learning from else's experience is cultivated the ova nuda of discharging first polar body, shifts ova nuda to containing 10mM SrCl
2cZB nutrient solution in activate and cultivate 2.5h, then activate the ova nuda processed and cultivate to continuing in G-1PLUS nutrient solution, in the time that lonely female activation ova nuda is developed to the 8-cell stage stage, shifts embryo and continue cultivation to G-2PLUS nutrient solution;
Wherein the component of CZB nutrient solution is: 81.62mM sodium-chlor+4.83mM Repone K+1.18mM potassium primary phosphate+1.18mM magnesium sulfate+1.71mM calcium chloride+25mM sodium bicarbonate+31.3mM Sodium.alpha.-hydroxypropionate+1mM glutamine+0.1mM EDTA+5mg/mlBSA;
G-1PLUS nutrient solution and G-2PLUS nutrient solution are Vitrolife Sweden AB product, and G-1PLUS nutrient solution article No. is that 10128, G-2PLUS nutrient solution article No. is 10132;
(3) ripe ova nuda is in vitro fertilization
The post-mature of learning from else's experience is cultivated the ova nuda of discharging first polar body, with being subject to after semen washing, transfer in HTF fertilization drop, shift the sperm of capacitation to the HTF fertilization drop of ripe ova nuda simultaneously, externally jointly hatch cultivation, ripe ova nuda is hatching after cultivation through 6h jointly with capacitated sperm, select under the microscope the zygote of discharging second polar body or forming female-male pronucleus, after washing in G-1PLUS nutrient solution, be transferred in G-1PLUS nutrient solution and continue, in the time that the ova nuda of being fertilized is developed to the 8-cell stage stage, shifts embryo and continue to cultivate to G-2PLUS nutrient solution;
Wherein HTF is subject to the component of seminal fluid to be: 101.6mM sodium-chlor+4.69mM Repone K+0.37mM potassium primary phosphate+0.2mM magnesium sulfate+2.04mM calcium chloride+25mM sodium bicarbonate+21.4mM Sodium.alpha.-hydroxypropionate+0.33mM Sodium.alpha.-ketopropionate+2.78mM glucose+4mg/ml BSA.
The invention has the beneficial effects as follows:
Find by research, body series can be created the environment of manual simulation's ovocyte naturally-occurring maturation, make that ova nuda is in vitro can be efficiently ripe grows, and after ova nuda vitro maturation, possess that the ovocyte of same normal development is the same can in vitro fertilization and fetal development.Practice confirms, this system denuded oocyte in vitro maturation growth efficiency is high, and system is mature and stable, and after the external natural fertilization of ova nuda, fetal development quality is high, therefore, this invention can greatly promote application and the popularization of ova nuda in human reproduction's health and modern domestic animal genetic breeding.Simplify the operation that obtains blastaea by ova nuda, put into actual production for ova nuda as ovum source and provide convenience.
Embodiment
1 experimental technique
The maturation in vitro of 1.1 ova nudas is cultivated
Get mouse ovarian, use under the microscope syringe needle head, puncture ovarian follicle on ovary, the ovocyte in ovarian follicle is discharged in the TCM199 Oocyte in Vitro operation liquid containing HEPES and 10%FBS, wherein TCM199 is Therma company product, product article No. SH30233; With picking up ovum pin at random after external ova nuda washing, random packet is put into the oocyte maturation nutrient solution containing 50 μ M forskolin
in In-Vitro MaturationMedia, ova nuda is containing cultivating after 3h in the oocyte maturation nutrient solution of 50 μ M forskolin, ova nuda at the oocyte maturation nutrient solution containing 2.5 μ M PD166285
in In-Vitro Maturation Media, after washing, be transferred to the oocyte maturation nutrient solution containing 2.5 μ M PD166285
in In-Vitro Maturation Media, continue vitro culture 16h.Wherein
in-Vitro Maturation Media is Sage company of U.S. product, and nutrient solution product article No. is ART-1600.
The female activation of orphan of 1.2 ripe ova nudas
The post-mature of learning from else's experience is cultivated the ova nuda of discharging first polar body, shifts ova nuda to containing 10mM SrCl
2cZB nutrient solution in activate and cultivate 2.5h, then activate the ova nuda processed and cultivate to continuing in G-1PLUS nutrient solution, in the time that lonely female activation ova nuda is developed to the 8-cell stage stage, shifts embryo and continue cultivation to G-2PLUS nutrient solution.Wherein G-1PLUS nutrient solution and G-2PLUS nutrient solution are Vitrolife Sweden AB product, and G-1PLUS nutrient solution article No. is that 10128, G-2PLUS nutrient solution article No. is 10132; The component of CZB nutrient solution is in addition: 81.62mM sodium-chlor+4.83mM Repone K+1.18mM potassium primary phosphate+1.18mM magnesium sulfate+1.71mM calcium chloride+25mM sodium bicarbonate+31.3mM Sodium.alpha.-hydroxypropionate+1mM glutamine+0.1mM EDTA+5mg/ml BSA.
1.3 ripe ova nudas in vitro fertilization
The post-mature of learning from else's experience is cultivated the ova nuda of discharging first polar body, with being subject to after semen washing, transfer in HTF fertilization drop, shift the sperm of capacitation to the HTF fertilization drop of ripe ova nuda simultaneously, externally jointly hatch cultivation, ripe ova nuda is hatching after cultivation through 6h jointly with capacitated sperm, select under the microscope the zygote of discharging second polar body or forming female-male pronucleus, after washing in G-1PLUS nutrient solution nutrient solution, be transferred in G-1PLUS nutrient solution nutrient solution and continue, in the time that the ova nuda of being fertilized is developed to the 8-cell stage stage, shift embryo continues to cultivate to G-2PLUS nutrient solution nutrient solution.
2 results
Mouse denuded oocyte is respectively through cultivating after 16h containing cultivating 3h in the oocyte maturation nutrient solution of 50 μ M forskolin and containing maturation in the oocyte maturation nutrient solution of 2.5 μ M PD166285, mouse denuded oocyte presents germinal vesicle and breaks, first polar body is discharged, ripe ova nuda sharpness of border, cell cytoplasm is even, and form is good.Denuded oocyte in vitro maturation rate is 75.71%.
After the lonely female activation of ripe ova nuda, ova nuda can continue to grow formation embryo, and fetal development form is normal, and embryo's blastomere cell homogeneous is consistent.The lonely female activity ratio of ova nuda is 42.65%, activates and grows the embryo who forms after continuation is cultivated, and can grow formation blastaea, and the blastaea rate of parthenogenetic embryo tire is 18.33%.In the control group of experiment, not through containing cultivating 3h and also can lonely female activation containing the ripe ova nuda of cultivating in the oocyte maturation nutrient solution of 2.5 μ MPD166285 in the oocyte maturation nutrient solution of 50 μ M forskolin, its lonely female activity ratio is 36.19%, but the ova nuda developmental potency after lonely female activation is poor, do not have the ova nuda after lonely female activation can grow formation blastaea.
Cultivate ripe ripe ova nuda through drug treating, can, with mouse sperm combination, form zygote in vitro, zygote can normal development form embryo.The embryo of after fertilization is good in each etap cultivation form, blastomere cell sharpness of border symmetrically and evenly, and ova nuda rate in vitro fertilization is 17.03%.The zygote that derives from ova nuda continues to cultivate after 4 days in vitro, can grow formation blastaea from the embryo of ova nuda, and blastaea rate is 5.65%.In the control group of experiment, process is containing cultivating 3h and the ova nuda containing ripe cultivation in the oocyte maturation nutrient solution of 2.5 μ M PD166285 in the oocyte maturation nutrient solution of 50 μ M forskolin, can be ripe and discharge first polar body, but in vitro can not be with sperm combination and fertilization in fertilization process.
Above-described embodiment of the present invention, does not form limiting the scope of the present invention.Any amendment of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in claim protection domain of the present invention.
Claims (1)
1. the in-vitro maturation culture method of mouse denuded oocyte, is characterized in that comprising the following steps:
Get mouse ovarian, use under the microscope syringe needle head, puncture ovarian follicle on ovary, ovocyte in ovarian follicle is discharged in the TCM199 Oocyte in Vitro operation liquid containing HEPES and 10%FBS, with picking up ovum pin at random after external ova nuda washing, random packet is cultivated after 3h with adding containing the oocyte maturation nutrient solution In-Vitro Maturation Media of 50 μ M forskolin, shifts the ova nuda oocyte maturation nutrient solution In-Vitro Maturation Media adding containing 2.5 μ M PD166285 and continues vitro culture 16h;
(2) the female activation of the orphan of ripe ova nuda
The post-mature of learning from else's experience is cultivated the ova nuda of discharging first polar body, shifts ova nuda to containing 10mM SrCl
2cZB nutrient solution in activate and cultivate 2.5h, then activate the ova nuda processed and cultivate to continuing in G-1PLUS nutrient solution, in the time that lonely female activation ova nuda is developed to the 8-cell stage stage, shifts embryo and continue cultivation to G-2PLUS nutrient solution; The component of described CZB nutrient solution is: 81.62mM sodium-chlor+4.83mM Repone K+1.18mM potassium primary phosphate+1.18mM magnesium sulfate+1.71mM calcium chloride+25mM sodium bicarbonate+31.3mM Sodium.alpha.-hydroxypropionate+1mM glutamine+0.1mM EDTA+5mg/ml BSA;
(3) ripe ova nuda is in vitro fertilization
The post-mature of learning from else's experience is cultivated the ova nuda of discharging first polar body, with being subject to after semen washing, transfer in HTF fertilization drop, the sperm that simultaneously shifts capacitation is hatched cultivation jointly to external in the HTF fertilization drop of ripe ova nuda, ripe ova nuda is hatching after cultivation through 6h jointly with capacitated sperm, select under the microscope the zygote of discharging second polar body or forming female-male pronucleus, after washing in G-1PLUS nutrient solution, be transferred in G-1PLUS nutrient solution and continue, in the time that the ova nuda of being fertilized is developed to the 8-cell stage stage, shifts embryo and continue to cultivate to G-2PLUS2 nutrient solution; Described HTF is subject to the component of seminal fluid to be: 101.6mM sodium-chlor+4.69mM Repone K+0.37mM potassium primary phosphate+0.2mM magnesium sulfate+2.04mM calcium chloride+25mM sodium bicarbonate+21.4mM Sodium.alpha.-hydroxypropionate+0.33mM Sodium.alpha.-ketopropionate+2.78mM glucose+4mg/ml BSA.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104726397A (en) * | 2015-03-16 | 2015-06-24 | 安徽农业大学 | Breeding method of denuded oocyte mice |
| CN105861424A (en) * | 2016-04-27 | 2016-08-17 | 华东师范大学 | Rat external fertilization nutrient fluid and application thereof |
| CN107201343A (en) * | 2017-07-14 | 2017-09-26 | 阜阳师范学院 | A kind of goat cells clone embryos nutrient solution and cultural method |
| CN108251353A (en) * | 2018-01-22 | 2018-07-06 | 深圳韦拓生物科技有限公司 | A kind of Human embryo in vitro culture liquid and culture systems |
| 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 |
| CN110527663A (en) * | 2019-09-12 | 2019-12-03 | 河南牧业经济学院 | A kind of external fertilization method of oocyte of mouse |
| CN112048467A (en) * | 2020-09-11 | 2020-12-08 | 江苏集萃药康生物科技有限公司 | Application of KSOM-AA culture solution in-vitro culture of NOD (non-specific oligonucleotide) background mouse embryos |
| CN114686423A (en) * | 2022-04-29 | 2022-07-01 | 南京优而生物科技发展有限公司 | Preparation method of HTF culture solution for improving sperm motility of mice |
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| CN101560494A (en) * | 2009-05-31 | 2009-10-21 | 山东农业大学 | Mouse denuded oocyte in vitro maturation technology |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104726397A (en) * | 2015-03-16 | 2015-06-24 | 安徽农业大学 | Breeding method of denuded oocyte mice |
| CN105861424A (en) * | 2016-04-27 | 2016-08-17 | 华东师范大学 | Rat external fertilization nutrient fluid and application thereof |
| CN105861424B (en) * | 2016-04-27 | 2019-06-14 | 华东师范大学 | A kind of rats in vitro fertilization nutrient solution and its application |
| CN107201343A (en) * | 2017-07-14 | 2017-09-26 | 阜阳师范学院 | A kind of goat cells clone embryos nutrient solution and cultural method |
| CN107201343B (en) * | 2017-07-14 | 2021-05-11 | 阜阳师范学院 | Goat cell clone embryo culture solution and culture method |
| CN108251353A (en) * | 2018-01-22 | 2018-07-06 | 深圳韦拓生物科技有限公司 | A kind of Human embryo in vitro culture liquid and culture systems |
| CN108251353B (en) * | 2018-01-22 | 2020-12-08 | 深圳韦拓生物科技有限公司 | Human embryo in-vitro culture solution and culture system |
| 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 |
| CN110527663A (en) * | 2019-09-12 | 2019-12-03 | 河南牧业经济学院 | A kind of external fertilization method of oocyte of mouse |
| CN112048467A (en) * | 2020-09-11 | 2020-12-08 | 江苏集萃药康生物科技有限公司 | Application of KSOM-AA culture solution in-vitro culture of NOD (non-specific oligonucleotide) background mouse embryos |
| CN114686423A (en) * | 2022-04-29 | 2022-07-01 | 南京优而生物科技发展有限公司 | Preparation method of HTF culture solution for improving sperm motility of mice |
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