CN102250831A - Culture medium for enhancing ectogenesis of ox preantral follicle and application thereof - Google Patents
Culture medium for enhancing ectogenesis of ox preantral follicle and application thereof Download PDFInfo
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- CN102250831A CN102250831A CN 201110194207 CN201110194207A CN102250831A CN 102250831 A CN102250831 A CN 102250831A CN 201110194207 CN201110194207 CN 201110194207 CN 201110194207 A CN201110194207 A CN 201110194207A CN 102250831 A CN102250831 A CN 102250831A
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Abstract
The invention discloses a culture medium for enhancing ectogenesis of ox preantral follicle and application thereof. A method for preparing the culture medium for facilitating ectogenesis of isolated mammal preantral follicle comprises the following step of: mixing insulin, transferrin, sodium selenite, sodium pyruvate, glutamine, hypoxanthine, blood serum, follicle-stimulating hormone, luteotropin, estrogen, a PTEN (Pentaerythritol Tetranitrate) inhibitor with an alpha-MEN culture medium to obtain the culture medium. As proved by an experiment, ox preantral follicle is firstly separated from cortex ovarii, the obtained follicle is cultured in vitro in a culture medium and a control culture medium provided by the invention, and the diameter accretion and the cavitation rate of the follicle are observed. A result indicates that the cavitation rate and the diameter accretion value of the preantral follicle obtained by culturing with the culture medium provided by the invention are higher than those of the preantral follicle obtained with the control culture medium, and a necessary culturing system is provided for further excavation of a female reproductive resource.
Description
Technical field
The present invention relates to Animal husbandry (animal reproduction), relate in particular to ectogenetic substratum of ovarian follicle and application thereof before a kind of raising ox chamber.
Background technology
Tens thousand of primordial follicles are arranged on the ox ovary, in its growth and maturity process, a considerable amount of ovarian follicles degenerations, locking are arranged, the follicular development that only a few is only arranged is to the stage of maturity, until ovulation.In the female reproduction resource, embryo biotechnologies such as ovocyte is that in vitro fertilization, embryo transfer, sex control, embryo are cut apart, animal cloning and transgenosis are researched and developed requisite material, the deficient principal element that becomes these Research progress of restriction in ovocyte source, so the exploitation of the preceding ovarian follicle of ovarian cavity solves the effective way of this difficult problem beyond doubt.
With the maturation in vitro cultivation comparison of common somatic cell culture and the follicular oocyte of having the chamber stage by oneself, the vitro culture difficulty of ovarian follicle is bigger before the Mammals chamber.However, ovarian follicle before the two-step approach vitro culture mouse chamber such as Eppig in 1989 obtains mature oocyte and successfully gives birth to normal offspring, and people have successively begun the research of ovarian follicle vitro culture before the chamber on other animal since then.Particularly research is more aspect domestic animals such as ox, sheep, pig, and has all obtained some progress on research contents, means and method.But up to the present, do not set up the system of ovarian follicle vitro culture before the cover ox chamber yet.Therefore, further optimize and set up the ox chamber before ovarian follicle vitro culture system seem particularly important.
Summary of the invention
An object of the present invention is to provide a kind of method for preparing the ectogenetic substratum of ovarian follicle before the Mammals chamber of promote exsomatizing.
Method provided by the invention, comprise the steps: Regular Insulin, Transferrins,iron complexes, Sodium Selenite, Sodium.alpha.-ketopropionate, glutamine, xanthoglobulin, serum, follitropin, lutropin, oestrogenic hormon, PTEN inhibitor, water and α-MEM substratum is mixed, obtain substratum, the concentration of described Regular Insulin in described substratum is 5ug/ml, the concentration of described Transferrins,iron complexes in described substratum is 5ug/ml, and the concentration of described Sodium Selenite in described substratum is 5ng/ml; The concentration of described Sodium.alpha.-ketopropionate in described substratum is (0.21-0.29) mmol/l, the concentration of described glutamine in described substratum is (1.5-2.0) mmol/l, the concentration of described xanthoglobulin in described substratum is (1.8-2.5) mmol/l, the concentration of described serum in described substratum is 5%-10% (volumn concentration), and the concentration of described follitropin in described substratum is 0.25 μ gml
-1-0.5 μ gml
-1, the concentration of described lutropin in described substratum is 5IU ml
-1-10IU ml
-1, the concentration of described oestrogenic hormon in described substratum is 0.5 μ gml
-1-1.0 μ gml
-1, the concentration of described PTEN inhibitor in described substratum is 100nmol/L-10 μ mol/L, the described α-concentration of MEM substratum in described substratum is 10.1g/L.
The concentration of described Sodium.alpha.-ketopropionate in described substratum is 0.21mmol/l, 0.23mmol/l or 0.29mmol/l; The concentration of described glutamine in described substratum is 1.5mmol/l, 1.8mmol/l or 2.0mmol/l, the concentration of described xanthoglobulin in described substratum is 1.8mmol/l, 2mmol/l or 2.5mmol/l, the concentration of described serum in described substratum is 5%, 7.5% or 10% (volumn concentration), and the concentration of described follitropin in described substratum is 0.25 μ gml
-1, 0.4 μ gml
-1Or 0.5 μ gml
-1, the concentration of described lutropin in described substratum is 5IU ml
-1, 8IU ml
-1Or 10IU ml
-1, the concentration of described oestrogenic hormon in described substratum is 0.5 μ gml
-1, 0.8 μ gml
-1Or 1.0 μ gml
-1, the concentration of described PTEN inhibitor in described substratum is 100nmol/L, 1 μ mol/L or 10 μ mol/L.
Described serum is foetal calf serum;
Described oestrogenic hormon is oestrogenic hormon E2;
Described PTEN inhibitor is PTEN inhibitor pic.
Described Mammals is an ox.
The substratum of described method preparation also is a scope of protection of the invention.
Described substratum is the application in the ovarian follicle ectogenesis before promoting stripped Mammals chamber.
Described Mammals is an ox.
The ovarian follicle ectogenesis is by increasing FD and/or improving ovarian follicle coelosis rate and embody before the described stripped Mammals chamber.
Described α-MEM substratum is commercially available, also can be prepared as follows: 200.00mg/L calcium chloride (anhydrous), 400.00mg/L Repone K, 98.00mg/L magnesium chloride (anhydrous), 6,800.00mg/L sodium-chlor, 140.00mg/L one hypophosphite monohydrate disodium hydrogen, 1,000.00mg/L D-glucose, 0.20mg/L Thioctic Acid, 10.00mg/L are phenol red, 110.00mg/L Sodium.alpha.-ketopropionate, 25.00mg/L L-L-Ala; 127.00mg/L L-arginine monohydrochloride, 50.00mg/LL-aspartic acid monohydrate, 30.00mg/L L-aspartic acid, 31.00mg/L L-Gelucystine dihydrochloride, 100.00mg/L L-cysteine hydrochloride monohydrate, 75.00mg/L L-L-glutamic acid, 292.00mg/L L-glutaminate, 50.00mg/L L-glycine, 42.00mg/L L-L-Histidine hydrochloride monohydrate, 52.00mg/L L-Isoleucine, 52.00mg/L L-leucine, 73.00mg/L L lysine HCL, 15.00mg/L L-methionine(Met), 32.00mg/LL-phenylalanine, 40.00mg/L L-proline(Pro), 25.00mg/L L-Serine, 48.00mg/L L-Threonine, 10.00mg/L L-tryptophane, 52.00mg/L L-tyrosine (disodium salt), 46.00mg/L L-Xie Ansuan, 50.00mg/L L-xitix, 0.10mg/L vitamin H, 1.00mg/L calcium pantothenate, 1.00mg/L choline chloride 60,1.00mg/L folic acid, 2.00mg/L meso-inositol, 1.00mg/L niacinamide, 1.00mg/L pyridoxal hydrochloride, 0.10mg/L riboflavin, 1.00mg/L thiamine hydrochloride, 1.40mg/L vitamins B
12Mix with water, obtain substratum.
Another object of the present invention provides the method that a kind of vitro culture acquisition has the method for chamber ovarian follicle.
Method provided by the invention comprises the steps: ovarian follicle before the stripped Mammals chamber is cultivated in described substratum, has obtained the chamber ovarian follicle.
The temperature of described cultivation is 39 ℃, and described cultivation is at 5%CO
2Carry out under the condition, the time of described cultivation is 21 days.
Of the present invention experimental results show that, the present invention at first separates ovarian follicle before the ox chamber from ovary cortex, subsequently the ovarian follicle that obtains is carried out vitro culture in substratum provided by the invention and control medium, observe the coelosis rate of its diameter accretion and ovarian follicle, found that, culture medium culturing provided by the invention obtains the coelosis rate and the increasing diameter long value all is higher than contrast, provides necessary culture system for further excavating the female reproduction resource.By the present invention, can be that the researchist of follicular development provides a cover the complete system of vitro culture effectively before the research ox early stage chamber, lay the foundation for further studying its oocyte in vitro maturation mechanism.The present invention is simple and direct, efficient, be convenient to promote.
Description of drawings
Fig. 1 be the pic of different concns to the ox chamber before the influence of ovarian follicle coelosis rate
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The acquisition of embodiment 1, new culture system
1, the obtaining of ovarian follicle before the chamber: the stripped ox ovary that the slaughterhouse is obtained places the glass dish of 60mm diameter, remove unnecessary mesentery and corpus luteum, subsequently ovary is cut in half along ovarian hilum, remove medullary substance, at substratum (M199 substratum, GIBCO, article No.: after cleaning 2 times 31100-035), draw from ovary cortex portion according to horizontal and vertical both direction with cutter and to get ovarian follicle, the ovarian follicle and the substratum that obtain are blown and beaten with dropper, blown and beaten 80～l00 time with the suction pipe of diameter 1mm then, filter through 200 μ m mesh screens, again with diameter 0.5mm suction pipe piping and druming 80-l00 time, filter with 100 μ m mesh screens,, remove supernatant liquor with the centrifugal 5min of 1500r/min, its precipitation adds parting liquid dilute suspension, microscopy ovarian follicle under the stereoscopic microscope, counting is also measured diameter;
The result is an ovarian follicle before each ovary 120-140 piece chamber for follicle number, and FD is 60-80um.
2, the foundation of ovarian follicle vitro culture system before the chamber:
1) substratum preparation
Substratum 1: with ITS (ITS solid specification is 25mg Regular Insulin, 25mg Transferrins,iron complexes, 25ug Sodium Selenite, available from Sigma, article No.: I1884; Be diluted with water to final concentration when using and be 5ug/ml Regular Insulin, final concentration 5ug/ml Transferrins,iron complexes, final concentration 5ng/ml Sodium Selenite), Sodium.alpha.-ketopropionate (Sigma, article No.: P4562), glutamine (Sigma, article No.: G3126), xanthoglobulin (Sigma, article No.: H9636), foetal calf serum (GIBCO, article No.: 16000), follitropin (FSH, Sigma, article No.: F2293), lutropin (LH, Sigma, article No.: L9773), oestrogenic hormon E2 (Sigma, E2257), the PTEN inhibitor Pic (inhibitor of cancer suppressor gene PTEN, ALEXIS, L22757), water and α-MEM substratum (GIBCO, 12000) mix, obtain substratum, the concentration of Regular Insulin in described substratum is 5ug/ml, the concentration of Transferrins,iron complexes in described substratum is 5ug/ml, and the concentration of Sodium Selenite in described substratum is 5ng/ml; The concentration of Sodium.alpha.-ketopropionate in described substratum is 0.23mmol/l, the concentration of glutamine in described substratum is 1.5mmol/l, the concentration of xanthoglobulin in described substratum is 2mmol/l, the concentration of foetal calf serum in described substratum is 7.5% (volumn concentration), and the concentration of follitropin in described substratum is 0.25 μ gml
-1, the concentration of lutropin in described substratum is 5IU ml
-1, the concentration of oestrogenic hormon E2 in described substratum is 0.5 μ gml
-1, the concentration of PTEN inhibitor Pic in described substratum is 10 μ mol/L.
Substratum 2: composition and final concentration thereof and substratum 1 are basic identical,
Different is that the concentration of PTEN inhibitor Pic in described substratum is 1 μ mol/L.
Substratum 3: composition and final concentration thereof and substratum 1 are basic identical, and different is that the concentration of PTEN inhibitor Pic in described substratum is 100nmol/L.
Control medium: composition and final concentration thereof and substratum 1 are basic identical, and different is not add PTEN inhibitor Pic.
2) optimal concentration of PTEN inhibitor Pic screening
Ovarian follicle is cultivated respectively in above-mentioned substratum 1, substratum 2 and substratum 3 before the chamber that step 1 is obtained, and 39 ℃, 5%CO
2, cultivated 21 days.
3) ovarian follicular growth situation
Statistics FD increasing value and ovarian follicle coelosis rate;
The coelosis rate is follicle number/total follicle number * 100% that survives of cultivating of coelosis;
The numerical value that the diameter of ovarian follicle obtains when once observing before the diameter of ovarian follicle deducted when the increasing diameter long value was once observed after being.
The FD result is as follows:
Table 1 be the pic of different concns to the ox chamber before the influence of FD
The pic of different concns to the ox chamber before ovarian follicle coelosis rate influence the result as shown in Figure 1, control is a control medium, as seen from the figure, in the ovarian follicle of the substratum 3 of the Pic that contains 100nM, 1uM, 10uM, substratum 2, substratum 1 ovarian follicle coelosis rate such as following table 2 15 days, 17 days, 19 days, 21 days (doing the 0th day) from adding the substratum note:
Table 2 is the ovarian follicle coelosis rate of the pic of different concns
By the comparison of FD growth and coelosis rate, discovery contains the promoter action maximum of 1 pair of ovarian follicular growth of substratum of 10umol/LPic.
Embodiment 2, in new culture system ovarian follicle before the vitro culture chamber
Ovarian follicle is divided into before the chamber ovarian follicle and chamber ovarian follicle, coelosis are arranged is that ovarian follicle is grown up to the chamber state is arranged from state growth in no chamber before the chamber.
Method 1:
Ovarian follicle was in the substratum 1 of embodiment 1 before the step 1 of embodiment 1 obtained the chamber, and 39 ℃, 5%CO
2, cultivated 21 days.With the control medium of embodiment 1 in contrast.
The ovarian follicle ectogenesis obtains the coelosis rate of the ovarian follicle of coelosis before the statistics chamber, experiment triplicate, results averaged or mean+SD.
The result is as follows:
The diameter average increment value of the ovarian follicle after substratum 1 is cultivated 21d is 167.8 ± 35.7um, and the coelosis rate is 34.38%;
The diameter average increment value of the ovarian follicle in control medium behind the cultivation 21d is 133.6 ± 22.3um, and the coelosis rate is 17%.
Method 2:
Basic identical with method 1, different is to adopt substratum 4 to cultivate,
Substratum 4 is basic identical with the composition of substratum 1, and different is that the concentration of described Sodium.alpha.-ketopropionate in described substratum is 0.21mmol/l; The concentration of described glutamine in described substratum is 1.8mmol/l, the concentration of described xanthoglobulin in described substratum is 1.8mmol/l, the concentration of described serum in described substratum is 5% (volumn concentration), and the concentration of described follitropin in described substratum is 0.4 μ gml
-1, the concentration of described lutropin in described substratum is 8IU ml
-1, the concentration of described oestrogenic hormon in described substratum is 0.8 μ gml
-1
The ovarian follicle ectogenesis obtains the coelosis rate of the ovarian follicle of coelosis, result and substratum 1 no significant difference before the statistics chamber.
Method 3:
Basic identical with method 1, different is to adopt substratum 5 to cultivate,
Substratum 5 is basic identical with the composition of substratum 1, and different is that the concentration of described Sodium.alpha.-ketopropionate in described substratum is 0.29mmol/l; The concentration of described glutamine in described substratum is 2.0mmol/l, the concentration of described xanthoglobulin in described substratum is 2.5mmol/l, the concentration of described serum in described substratum is 10% (volumn concentration), and the concentration of described follitropin in described substratum is 0.5 μ gml
-1, the concentration of described lutropin in described substratum is 10IU ml
-1, the concentration of described oestrogenic hormon in described substratum is 1.0 μ gml
-1
The ovarian follicle ectogenesis obtains the coelosis rate of the ovarian follicle of coelosis, result and substratum 1 no significant difference before the statistics chamber.
Claims (10)
1. method for preparing the ectogenetic substratum of ovarian follicle before the Mammals chamber of promote exsomatizing, for Regular Insulin, Transferrins,iron complexes, Sodium Selenite, Sodium.alpha.-ketopropionate, glutamine, xanthoglobulin, serum, follitropin, lutropin, oestrogenic hormon, PTEN inhibitor, water and α-MEM substratum is mixed, obtain substratum, the concentration of described Regular Insulin in described substratum is 5ug/ml, the concentration of described Transferrins,iron complexes in described substratum is 5ug/ml, and the concentration of described Sodium Selenite in described substratum is 5ng/ml; The concentration of described Sodium.alpha.-ketopropionate in described substratum is (0.21-0.29) mmol/l, the concentration of described glutamine in described substratum is (1.5-2.0) mmol/l, the concentration of described xanthoglobulin in described substratum is (1.8-2.5) mmol/l, the concentration of described serum in described substratum is 5%-10% (volumn concentration), and the concentration of described follitropin in described substratum is 0.25 μ gml
-1-0.5 μ gml
-1, the concentration of described lutropin in described substratum is 5IU ml
-1-10IUml
-1, the concentration of described oestrogenic hormon in described substratum is 0.5 μ gml
-1-1.0 μ gml
-1, the concentration of described PTEN inhibitor in described substratum is 100nmol/L-10 μ mol/L, the described α-concentration of MEM substratum in described substratum is 10.1g/L.
2. method according to claim 1 is characterized in that:
The concentration of described Sodium.alpha.-ketopropionate in described substratum is 0.21mmol/l, 0.23mmol/l or 0.29mmol/l; The concentration of described glutamine in described substratum is 1.5mmol/l, 1.8mmol/l or 2.0mmol/l, the concentration of described xanthoglobulin in described substratum is 1.8mmol/l, 2mmol/l or 2.5mmol/l, the concentration of described serum in described substratum is 5%, 7.5% or 10% (volumn concentration), and the concentration of described follitropin in described substratum is 0.25 μ gml
-1, 0.4 μ gml
-1Or 0.5 μ gml
-1, the concentration of described lutropin in described substratum is 5IU ml
-1, 8IU ml
-1Or 10IU ml
-1, the concentration of described oestrogenic hormon in described substratum is 0.5 μ gml
-1, 0.8 μ gml
-1Or 1.0 μ gml
-1, the concentration of described PTEN inhibitor in described substratum is 100nmol/L, 1 μ mol/L or 10 μ mol/L.
3. method according to claim 1 and 2 is characterized in that:
Described serum is foetal calf serum;
Described oestrogenic hormon is oestrogenic hormon E2;
Described PTEN inhibitor is PTEN inhibitor pic.
4. according to arbitrary described method among the claim 1-3, it is characterized in that: described Mammals is an ox.
5. the substratum of arbitrary described method preparation among the claim 1-4.
6. the described substratum of claim 5 application in the ovarian follicle ectogenesis before promoting stripped Mammals chamber.
7. application according to claim 6 is characterized in that: described Mammals is an ox.
8. according to claim 6 or 7 described application, it is characterized in that:
The ovarian follicle ectogenesis is by increasing FD and/or improving ovarian follicle coelosis rate and embody before the described stripped Mammals chamber.
9. a vitro culture obtains to have the method for chamber ovarian follicle, comprises the steps: ovarian follicle before the stripped Mammals chamber is cultivated in the described substratum of claim 5, has obtained the chamber ovarian follicle.
10. method according to claim 9 is characterized in that:
The temperature of described cultivation is 39 ℃, and described cultivation is at 5%CO
2Carry out under the condition, the time of described cultivation is 21 days.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146643A (en) * | 2013-03-22 | 2013-06-12 | 孔凡锋 | Culture method for in-vitro human oocyte preantral follicles |
| CN114164169A (en) * | 2021-11-30 | 2022-03-11 | 吉林医药学院 | Application of astaxanthin in promoting development of preantral follicles of mice and preparation |
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| CN101302497A (en) * | 2008-06-27 | 2008-11-12 | 华中农业大学 | Method for cloning embryo by nuclear transfer of bovine somatic cells |
| US20100191040A1 (en) * | 2009-01-23 | 2010-07-29 | The Board Of Trustees Of The Leland Stanford Junior University | Manipulation of ovarian primordial follicles |
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| CN101302497A (en) * | 2008-06-27 | 2008-11-12 | 华中农业大学 | Method for cloning embryo by nuclear transfer of bovine somatic cells |
| US20100191040A1 (en) * | 2009-01-23 | 2010-07-29 | The Board Of Trustees Of The Leland Stanford Junior University | Manipulation of ovarian primordial follicles |
Non-Patent Citations (2)
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146643A (en) * | 2013-03-22 | 2013-06-12 | 孔凡锋 | Culture method for in-vitro human oocyte preantral follicles |
| CN103146643B (en) * | 2013-03-22 | 2015-01-21 | 孔凡锋 | Culture method for in-vitro human oocyte preantral follicles |
| CN114164169A (en) * | 2021-11-30 | 2022-03-11 | 吉林医药学院 | Application of astaxanthin in promoting development of preantral follicles of mice and preparation |
| CN114164169B (en) * | 2021-11-30 | 2022-09-13 | 吉林医药学院 | Application of astaxanthin in promoting development of preantral follicles of mice and preparation |
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Application publication date: 20111123 |