CN101591637B - Novel bovine oocyte in vitro maturation culture solution - Google Patents
Novel bovine oocyte in vitro maturation culture solution Download PDFInfo
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- CN101591637B CN101591637B CN2008101125916A CN200810112591A CN101591637B CN 101591637 B CN101591637 B CN 101591637B CN 2008101125916 A CN2008101125916 A CN 2008101125916A CN 200810112591 A CN200810112591 A CN 200810112591A CN 101591637 B CN101591637 B CN 101591637B
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Abstract
The invention relates to a novel bovine oocyte in vitro maturation (IVM) culture solution, belonging to the field of embryo biotechnolory. In the invention, the routine IVM culture solution is added with amniotic epithelia or the supernatant of the culture solution thereof to establish a novel bovine immature oocyte IVM culture system, so as to improve the quality and maturation rate of the IVM bovine oocyte, thus solving the problem of low fertilization rate and low formation rate of blastula in the existing culture method.
Description
Technical field
The present invention relates to the embryo engineering field, particularly relate to bovine oocyte in vitro maturation liquid.
Background technology
Bovine oocyte in vitro maturation culture technique as one of basis of embryo's biotechnology research, from the thirties in last century so far, many science researchers are in the bovine oocyte growth in vitro, do a lot of work in the growth field, the bovine oocyte culture condition has been carried out extensively and in depth research, and obtained bigger progress, present bovine oocyte in vitro maturation (invitro matrue, IVM) the culture technique system is set up, but because oocyte maturation mechanism, ripe regulation and control aspect remains many not to be illustrated and some problem demanding prompt solutions as yet, so that problems such as current methods exists ovocyte specification of quality height at present, and rate in vitro fertilization and blastaea rate of formation are low.
Amnion is formed at gastrula fertilization before the 8th day, the plasticity-of gastrula embryonic cell before the amnion tissue cell maintains, the main origin of amnion tissue comes from ectodermic amnion epithelium (amniotic epithelial cells, AECs) and derive from mesoblastic amnion mesenchymal (amniotic mesenchyme cells, AMCs) 2 class cells are formed (Whittle WL, Gibb W, ChallisJR.:The characterization of human amnion epithelial and mesenchymal cells:the cellularexpression, activity and glucocorticoid regulation of prostaglandin output.Placenta.2000May; 21 (4): 394-401.), amniotic epithelial cells has the differentiation potential of three kinds of germ confluent monolayer cells, entoderm (liver, pancreas), mesoderm (myocardial cell) and ectoderm (neurocyte) (Miki T, Lehmann T, Cai H, et al.:Stem CellCharacteristics of Amniotic Epithelial Cells.Stem Cells.2005Aug 4; [Epub ahead of print]).Amniotic epithelial cells has synthetic catecholamine (Elwan MA.:Synthesis ofdopamine fromL-3, the 4-dihydroxyphenylalanine by human amniotic epithelial cells.Eur J Pharmacol.1998Jul31 of discharging; 354 (1): R1-2; Elwan MA, Ishii T, SakuragawaN.et al.:Characterization ofthe dopaminetransporter gene expression and binding sites in cultured human amniotic epithelial cells.NeurosciLett.2003May 15; 342 (1-2): 61-4.), vagusstoff (Horikoshi T, Fujii T, Kawashima K, et al.:Acetylcholine increase in amniotic fluid of experimental rats for intrauterine growth retardation.Life Sci.2003Mar 28; 72 (18-19): 2145-9; Uchida S, Suzuki Y, Araie M, Kashiwagi K, et al.:Factors secreted by human amniotic epithelial cells promote the survival of rat retinal ganglion cells.Neurosci Lett.2003Apr 24; 341 (1): 1-4.) the neurobiology function of neurotransmitter, biologically active substances such as all right secretory nerve nutritional factor have neurotrophic function.Detected Brain Derived Neurotrophic Factor (brain-derivedneurotrophic factor at present, BDNF), NT-3, nerve growth factor (nerve growth factor, NGF) and ciliary neurotrophic factor (ciliary neurotrophic factor, CNTF) (Uchida S, Suzuki Y, Araie M, Kashiwagi K, et al.:Factors secreted by human amniotic epithelial cells promote the survival of rat retinal ganglion cells.Neurosci Lett.2003Apr 24; 341 (1): 1-4; Marvin KW, Keelan JA, Eykholt RL, et al.:Expression ofangiogenic and neurotrophic factors in the human amnion and choriodecidua.Am J Obstet Gynecol.2002Sep; 187 (3): 728-34.), the prompting amnion tissue discharges neurotrophic factor by secretion and enters amniotic fluid, the neurodevelopmental commitment of embryo had important regulating effect (Uchida S, Inanaga Y, Kobayashi M, et al.:Neurotrophicfunction of conditioned medium from human amniotic epithelial cells.J Neurosci Res.2000Nov15; 62 (4): 585-90.).In addition, amniotic epithelial cells can also be secreted somatomedins such as synthetic TGF, EGF, bFGF and HGF, therefore should use the differentiation of cell amnion stroma as trophoderm promotion extracorporeal culturing embryo stem cell, utilization amnion tissue extract promotes immature egg cells in vitro maturation.In sum, amniotic epithelial cells can be secernent known and some unknown active substance, can forward regulate the biological characteristics of culturing cell.
Summary of the invention
Low and the low problem of blastaea rate of formation at the bovine oocyte rate of fertilization, characteristics in conjunction with amniotic epithelial cells, the invention provides a kind of novel bovine oocyte in vitro maturation culture solution, improved bovine oocyte maturing rate, oocyte fertilization rate and blastaea rate of formation.
Novel bovine oocyte in vitro maturation culture solution is characterized in that: add amniotic epithelial cells or/and the supernatant liquor of amniotic epithelial cells nutrient solution in the IVM substratum.
The supernatant liquor of described amniotic epithelial cells nutrient solution is for cultivating the supernatant liquor of 5-7 days amniotic epithelial cells.
The add-on of the supernatant liquor of described amniotic epithelial cells nutrient solution is the supernatant liquor that adds 2-20ml amniotic epithelial cells nutrient solution in the 100ml IVM substratum.
Described amniotic epithelial cells is the 1-7 amniotic epithelial cells in generation.
The amniotic epithelial cells of described amniotic epithelial cells behaviour or ox.
Described IVM substratum is M199+10%FBS+10 μ g/ml FSH+0.1 μ g/ml LH+1 μ g/ml E
2
Utilize epithelial characteristic, we separate the supernatant liquor that obtains amniotic epithelial cells and amniotic epithelial cells nutrient solution at last at the vitro culture amniotic epithelial cells, it are added in the IVM nutrient solution constitute novel bovine oocyte in vitro maturation culture solution.The supernatant liquor of amniotic epithelial cells and amniotic epithelial cells nutrient solution carries out forward to the bovine oocyte maturation to be regulated, the secretion of amnion epithelium, synthetic bovine oocyte in vitro maturation biologically active substance, thereby improve the bovine oocyte maturing rate, experimental results show that: compare with former IVM nutrient solution, novel bovine oocyte in vitro maturation culture solution can improve oocyte fertilization rate and blastaea rate of formation.
The present invention adds amniotic epithelial cells or its culture supernatant in the IVM of routine nutrient solution, set up ox immature egg parent cell IVM and cultivate new system, to improve IVM bovine oocyte quality and maturing rate, solve the low and low problem of blastaea rate of formation of rate of fertilization in the present cultural method.
Description of drawings
Fig. 1 vitro culture bovine oocyte becomes the method flow synoptic diagram of mature egg cell
Embodiment
The present invention is described in further detail below by embodiment.
Embodiment 1
1, in-vitro maturation culture method for oocyte (supernatant liquor of amniotic epithelial cells nutrient solution is as the IVM medium additives)
1.1 amniotic epithelial cells culture supernatant-maturation in vitro (AECS-IVM) additive and medium preparation
1.1.1 about 1mm
3Ox or people's amnion be cut into fragment, at the D-hank ' s that contains 0.125%Trypsins (pancreatin), 37 ℃ jolt 30min, rotating speed 400～600rpm;
1.1.2 filter suspension with the cell sieve, collect amniotic epithelial cells;
12.1.3 (cell count is 10 for concentration routinely
5～10
6Individual/bottle) inoculate amniotic epithelial cells in 75cm
2Culturing bottle;
1.1.4 the vitro culture amniotic epithelial cells is got the culture supernatant in 5-7 days;
1.1.5 centrifugal cell and the cell debris of going, 4 ℃, 1500-2500rpm, 15-30 minute;
1.1.6 get supernatant liquor, remove throw out, 0.22 μ m strainer filtering supernatant, packing is stored in and deposits-80 ℃ of profound hypothermia refrigerators, as standby.
1.1.7AECS-IVM medium preparation: every 100ml IVM substratum (M199+10%FBS+10 μ g/ml FSH+0.1 μ g/mlLH+1 μ g/ml E
2), adding and cultivate amniotic epithelial cells supernatant liquor 2-20ml, 1N NaOH regulates pH7.4-7.8, and 4 ℃ of storages are stand-by.
1.2 the maturation in vitro of prematurity ox ovum is cultivated
1.2.1 conventional collection, collection bovine oocyte;
1.2.2 abundant balance 2 hours, give a baby a bath on the third day after its birth time with the AECS-IVM substratum that contains HEPES in the bovine oocyte AECS-IVM substratum, the AECS-IVM substratum that does not contain HEPES is washed twice.
1.3 maturation in vitro (IVM)
1.3.1 in the above-mentioned AECS-IVM substratum of the bovine oocyte of handling well, put it into CO more together
2Cultivated 22 hours culture condition in the incubator: 38.5 ℃, 4.5%CO
2Saturated humidity.
1.3.2 the entire operation process is tried one's best the shortening time, is no more than 30 minutes from ovoscopy to going into training, with 60 pieces of calculating.
Identify the mature oocyte situation 1.3.3 cultivate to detect after 22 hours, tangible diffusion phenomena can appear in the granulosa cell of ovocyte periphery, and can see first polar body.
(IVF) 1.4 in vitro fertilization
1.4.1 the conventional seminal fluid of preparing;
1.4.2 conventional processing IVM ox ovum, selective maturation bovine oocyte are put in the IVM substratum, and be in vitro fertilization standby.
1.4.3 the time of fertilization is put into the IVF culture plate with the seminal fluid of ripe bovine oocyte and preparation, puts into CO
2Incubator was cultivated 19-32 hour.Culture condition: 38.5 ℃, 4.5%CO
2Saturated humidity.
1.5 vitro culture (IVC)
Fertilized oocyte in the IVF drop is picked, put into the IVC drop, put into the CO2 incubator and cultivate.Changed nutrient solution once every 48 hours.
Cultivate and occurred the 2-cell stage in first day
Cultivate and occurred the 4-cell stage in second day
Cultivate and occurred the 8-cell stage on the 3rd day
Cultivate and occurred the 16-cell stage on the 4th day
Cultivate and occurred the 32-cell stage on the 5th day
Cultivate and occurred late mulberry and early stage blastaea on the 6th day
Cultivate and occurred blastaea on the 7th day.
Embodiment 2
1, in-vitro maturation culture method for oocyte (amniotic epithelial cells is as the IVM medium additives)
1.1 cultivate amniotic epithelial cells as the IVM nurse cell
1.1.1 about 1mm
3Amnion be cut into fragment, containing 0.125%Trypsins D-hank ' s, 37 ℃ jolt 30min, rotating speed 400～600rpm
1.1.2 filter suspension with the cell sieve, collect amniotic epithelial cells
1.1.3 concentration inoculation amniotic epithelial cells is in 75cm routinely
2Culturing bottle
1.1.4 vitro culture ox or people's amniotic epithelial cells, inclining supernatant liquor, adds 100ml IVM substratum (M199+10%FBS+10 μ g/ml FSH+0.1 μ g/ml LH+1 μ g/ml E
2), as amniotic epithelial cells-maturation in vitro (AEC-IVM) culture system
1.2 the vitro culture of prematurity ox ovum
1.2.1 conventional collection, collection bovine oocyte;
1.2.2 abundant balance 2 hours, give a baby a bath on the third day after its birth time with the AEC-IVM substratum that contains HEPES in the bovine oocyte AEC-IVM substratum, the AEC-IVM substratum that does not contain HEPES is washed twice.
1.3 maturation in vitro (IVM)
1.3.1 the bovine oocyte of handling well is put into the AEC-IVM substratum, puts into CO more together
2Cultivated 22 hours culture condition in the incubator: 38.5 ℃, 4.5%CO
2Saturation concentration.
1.3.2 the entire operation process is tried one's best the shortening time, is no more than 30 minutes from ovoscopy to going into training; With 60 pieces of calculating.
Identify ripe ox ovum 1.3.3 cultivate to detect after 22 hours, the mature oocyte situation, tangible diffusion phenomena can appear in the granulosa cell of ovocyte periphery, but also can see first polar body (wrapping up tighter being not easy sees clearly).
(IVF) 1.4 in vitro fertilization
1.4.1 the conventional seminal fluid of preparing;
1.4.2 conventional processing IVM ox ovum, selective maturation bovine oocyte are put in the IVM substratum, and be in vitro fertilization standby.
1.4.3 the time of fertilization is put into CO with the IVF culture plate
2Incubator was cultivated 19-32 hour.
1.5 vitro culture (IVC)
Fertilized oocyte in the IVF drop is picked, put into the IVC drop, put into the CO2 incubator and cultivate.Changed nutrient solution once every 48 hours.
Cultivate and occurred the 2-cell stage in first day
Cultivate and occurred the 4-cell stage in second day
Cultivate and occurred the 8-cell stage on the 3rd day
Cultivate and occurred the 16-cell stage on the 4th day
Cultivate and occurred the 32-cell stage on the 5th day
Cultivate and occurred late mulberry and early stage blastaea on the 6th day
Cultivate and occurred blastaea on the 7th day.
Control group: with above-mentioned IVM substratum, do not add any other material, cultural method and operation and embodiment 1 are together.Experimental result sees Table 1
Table 1
Last table is checked with t and is carried out statistical study.Have subscript letter a, b inequality in the same column number value and represent significant difference (P＜0.05) as can be seen from the above table, embodiment 1 has added the IVM substratum that has added amniotic epithelial cells among the IVM substratum of cultivating the amniotic epithelial cells supernatant liquor and the embodiment 2, more conventional IVM substratum maturing rate improves greatly, and there were significant differences on statistical significance (P＜0.05), and also there were significant differences on rate of fertilization (P＜0.05); And the blastaea rate does not have difference (P＞0.05) statistically, but numerically is greatly improved.
Claims (5)
1. novel bovine oocyte in vitro maturation culture solution, it is characterized in that: add amniotic epithelial cells or/and the supernatant liquor of amniotic epithelial cells nutrient solution in the IVM substratum, described IVM substratum is M199+10% FBS+10 μ g/mlFSH+0.1 μ g/ml LH+1 μ g/ml E
2
2. novel bovine oocyte in vitro maturation culture solution according to claim 1, the supernatant liquor of described amniotic epithelial cells nutrient solution is for cultivating the supernatant liquor of 5-7 days amniotic epithelial cells.
3. novel bovine oocyte in vitro maturation culture solution according to claim 2, the add-on of the supernatant liquor of described amniotic epithelial cells nutrient solution are the supernatant liquor that adds 2-20ml amniotic epithelial cells nutrient solution in the 100ml IVM substratum.
4. novel bovine oocyte in vitro maturation culture solution according to claim 1, described amniotic epithelial cells are the 1-7 amniotic epithelial cells in generation.
5. novel bovine oocyte in vitro maturation culture solution according to claim 1, the amniotic epithelial cells of described amniotic epithelial cells behaviour or ox.
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| CN104928236A (en) * | 2015-07-06 | 2015-09-23 | 中国农业科学院特产研究所 | Nutrient solution and culturing method for maturation of oocyte in vitro |
| CN107460161B (en) * | 2017-07-21 | 2020-07-21 | 中国农业大学 | Culture medium for promoting in-vitro maturation of immature oocyte and application thereof |
| CN107475181B (en) | 2017-09-30 | 2021-03-02 | 中国农业大学 | In-vitro maturation culture solution for immature oocyte and application thereof |
| CN109456972A (en) * | 2018-12-07 | 2019-03-12 | 河南省农业科学院畜牧兽医研究所 | It is a kind of promote bovine oocyte maturation miRNA and its application |
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| US20040166578A1 (en) * | 2001-09-26 | 2004-08-26 | National Institute Of Agrobiological Sciences | Culture medium for in vitro culture of in vitro-produced porcine empryo and method for in vitro production of porcine embryo using the culture medium |
| CN1904038A (en) * | 2006-07-28 | 2007-01-31 | 浙江大学 | Cow ovum cell in vitro ripening culturing liquid containing tea polyphenol and its culturing method |
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| US20040166578A1 (en) * | 2001-09-26 | 2004-08-26 | National Institute Of Agrobiological Sciences | Culture medium for in vitro culture of in vitro-produced porcine empryo and method for in vitro production of porcine embryo using the culture medium |
| CN1904038A (en) * | 2006-07-28 | 2007-01-31 | 浙江大学 | Cow ovum cell in vitro ripening culturing liquid containing tea polyphenol and its culturing method |
Non-Patent Citations (2)
| Title |
|---|
| 李喜春等."牛卵母细胞体外成熟培养研究进展".《中国牛业科学》.2006,第32卷(第4期),41-45页. |
| 阎萍等."白牦牛卵母细胞体外成熟的研究".《中国草食动物》.2006,第26卷(第4期),7-9页. |
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