CN102258005B - Method for maintaining meiotic arrest of oocytes by storing/culturing oocytes in vitro - Google Patents
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Abstract
The invention provides a method for maintaining the meiotic arrest of oocytes by storing/culturing the oocytes in vitro. In the method, the oocytes are stored/cultured in vitro by whole serum serving as preserving fluid or culture solution for storing/culturing the oocytes in vitro at the temperature of between 20 and 30 DEG C to maintain the meiotic arrest of the oocytes. When the method is used, the utilization rate of ovarian oocytes in scientific research and the production of animal husbandry can be improved greatly, the time limit is relieved for the utilization of ovaries of slaughterhouses in the scientific research, and the arrangement of in-vitro operating time is facilitated; by storing/culturing the oocytes during the germinal vesicle period at normal temperature, the in-vitro mature quality of the oocytes is improved and the synchronization of nucleus mature and cytoplasm mature is promoted; therefore, the method is an optimal method for controlling the process of the nucleus mature physiologically without depending on inhibitors, and has the important significance on the study and application of animal embryo engineering and the assisted reproductive technology of human.
Description
Technical field
The invention belongs to the biology of reproduction field, be that a kind of physiological composition that utilizes is kept the germinal vesicle phase (Germinal Vesicle, GV) Oocyte Meiosis is stagnated, inhibition ovocyte germinal vesicle breaks (Germinal Vesicle Breakdown, method GVBD).
Background technology
The ovocyte developmental potency of maturation in vitro significantly is lower than the ovocyte of cylinder mature.The kytoplasm maturation takes place in ovocyte when examining maturation, thereby supporting successfully to grow subsequently expires.A series of event takes place and comprises transcribing of meiosis prophase and translate kytoplasm ripe gradually in ovocyte in the body, and kytoplasm is ripe reduction division just takes place in advance to be recovered yet the external ovocyte that obtains from ovarian follicle does not also obtain after in changing nutrient solution over to.
In order to improve the quality of maturation in vitro ovocyte, mainly take pharmacological method to keep reduction division at present and stagnate, mainly comprise and use protein synthesis inhibitor or phosphorylation inhibitor (Fulka etc., Mol Reprod Dev, 29:379-384 (1991); Lonergan etc., J Reprod Fertil, 109:355-365 (1997); Avery etc., Mol Reprod Dev, 50:334-344 (1998); Li etc., Acta Zoologial Sinica, 47 (6): 684-690 (2001); Le Beux etc., Theriogenology, 60:1049-1058 (2003)) and cAMP transduction inhibitor (Sirard etc., Theriogenology, 49:483-497 (1998)).Yet, though medicine can effectively suppress the ovocyte germinal vesicle break (GVBD) take place, often ovocyte is had certain toxicity.Cultivate under the low temperature in theory and can keep the Oocyte Meiosis stagnation.Yet the formation (Wu etc., Mol Reprod Dev, 54:388-395 (1999)) of the positive eumeiosis spindle body of the remarkable reduction of lower temperature and the ovocyte of cryopreservation are through remarkable reduction rate of fertilization in vitro fertilization.We preserve ovocyte at the temperature condition that need seek an appropriateness thus, and can keep its reduction division stagnant condition.Be reported in and add the reduction division stagnation that part liquor folliculi (Leibfried and First, Bio reprod, 23:699-704 (1980)) can be used for keeping ovocyte in the nutrient solution, but it is very short to hold time.
Therefore, also lack the effective ways that a kind of reduction division that can keep ovocyte is in a long time stagnated at present.
Summary of the invention
In order to solve the problems of the technologies described above, an object of the present invention is to provide method and the application thereof of a kind of external preservation/cultivation ovocyte.Another object of the present invention provides the application in the preserving in Oocyte in Vitro/cultivating of whole serum.Another purpose of the present invention provides preparation and the application thereof that contains whole serum.
The objective of the invention is to be achieved through the following technical solutions.
On the one hand, the invention provides the method for a kind of external preservation/cultivation ovocyte, preservation liquid or nutrient solution that described method adopts whole serum to preserve/cultivate as Oocyte in Vitro are stagnated to keep Oocyte Meiosis.
Preferably, described preservation/culture temperature of keeping the Oocyte Meiosis stagnation is 20-30 ℃, more preferably 27-28 ℃.
Preferably, 1～10 ovocyte is preserved/cultivated to per 10～100 μ l whole serums in the described method; Be preferably per 10 μ l whole serums and preserve/cultivate 1 ovocyte.
Preferably, described method also is included in whole serum and preserves/cultivate before the ovocyte, with TCM-199 nutrient solution rinsing ovocyte, and the step of cleaning ovocyte with whole serum.
Preferably, described whole serum is selected from foetal calf serum and new-born calf serum.
Preferably, described ovocyte is selected from the porcine oocytes of germinal vesicle phase.
On the other hand, the invention provides aforesaid method in the external preservation/cultivation of ovocyte, in the control of oocyte in vitro maturation and the application in optimization and the improvement humans and animals auxiliary procreation technology.
Preferably, the ovocyte in the described application is selected from porcine oocytes, is preferably the porcine oocytes of germinal vesicle phase.
In addition, the present invention also provides whole serum conduct in external preservation/cultivation ovocyte to preserve liquid or nutrient solution, and to keep the application that Oocyte Meiosis is stagnated, wherein said whole serum is selected from tire ox blood and new-born calf serum.
Another aspect the invention provides a kind of preparation for the preservation ovocyte, and described preparation comprises whole serum.
Preferably, described whole serum is selected from foetal calf serum and new-born calf serum.
The present invention also provides the application of described preparation in preserving ovocyte.
Preferably, described ovocyte is the porcine oocytes of germinal vesicle phase.
In sum, the present invention has attempted adopting fully foetal calf serum or new-born calf serum to preserve and cultivate porcine oocytes as preserving liquid in conjunction with different temperature first, has obtained very gratifying result, can effectively suppress porcine oocytes GVBD and take place.
Compared with prior art, the present invention has the following advantages at least:
1, method of the present invention can improve the utilization ratio of ovary ovocyte in scientific research and livestock industry production greatly, has removed temporal restriction for utilizing the slaughterhouse ovary, is convenient to the arrangement of manipulation in vitro time;
2, method of the present invention can be preserved germinal vesicle (GV) phase ovocyte at normal temperatures and cultivate, help to improve the oocyte in vitro maturation quality, promote that nuclear is ripe and kytoplasm is synchronously ripe, be a kind of method of the ripe process of physiology control nuclear that does not rely on inhibitor of the best, for animal embryo engineering and the research of human auxiliary procreation technology with use all significant;
3, foetal calf serum or new-born calf serum need not be added other any compositions as the normal temperature nutrient solution of porcine oocytes, and efficient, cheap, convenient, source does not have toxicity easily and to cell, can obtain the good clinical effect;
4, the method for the present invention Oocyte in Vitro that also can be used for other species is preserved or control and the optimization of maturation in vitro, to cell without any toxicity.
Foetal calf serum or new-born calf serum are as the porcine oocytes room-temperature extender, do not add other any compositions, temperature control can effectively suppress the part ovocyte in the GV phase between 20 to 30 ℃, 27.5 ℃ inhibition is best, preserve three days most of ovocytes and be stuck in the GV phase, and first polar body is discharged in the ripe back of cultivating, and maturing rate is a little less than fresh ovocyte.
Embodiment
Below in conjunction with embodiment the present invention is further described in detail.Be to be understood that the embodiment that provides only for to preparation with use ad hoc approach of the present invention and product to describe, rather than in order to limit the scope of the invention.
If no special instructions, among the embodiment employed reagent all available from Sigma (St.Louis, MO) company.
The obtaining and preserving of embodiment 1:GV phase porcine oocytes
The sow pig at 3-5 the monthly age that selection Beijing the 5th meat processing combine has just butchered obtains ovary immediately, and does not have corpus luteum on definite ovary after slaughtering.Ovary is contained in the 25-30 ℃ of physiological saline that contains 75 μ g/ml penicillin and 50 μ g/ml Streptomycin sulphates, in 2h, transported to the laboratory.
(purifying grade is 100 grades at JJ-CJ-1FD type clean bench, Wujiang treating plant head factory is produced) on, adopt the 20ml disposable syringe (going up dried shrimps Sha Wayike Industrial Co., Ltd produces) that No. 18 syringe needles are fixedly arranged in the ovarian follicle of intermediate diameters (2-6mm), to extract liquor folliculi, and after sedimentation, inhale and remove supernatant, the precipitation of sedimentation is cleaned twice with TCM-199, namely comprise ovarian cumulus ovocyte complex body in the precipitation.Under C-DS type microscope (production of Nikon company), select cumulus cell to wrap up ovarian cumulus-ovocyte complex body (COCs) tight, ovocyte kytoplasm even particle distribution with mouthful suction pipe, with TCM-199 rinsing 3 times, then at foetal calf serum (FCS, available from Gibco company, article No. 1600-044) gives a baby a bath on the third day after its birth in time.
A. adopt the foetal calf serum cultivation to drip with TCM-199 and cultivate the contrast experiment who drops in the porcine oocytes of preservation GV phase under the differing temps
Preparing FCS with the amount of 200 μ l foetal calf serums/drip cultivates and drips; Preparation TCM-199, the amount that the TCM-199/ for preparing with 200 μ l drips prepares the TCM-199 contrast culture drips, and then two kinds is cultivated to drip to be placed on to shift to an earlier date preheating 2 hours in 20-30 ℃ of biochemical incubator of SPX-50 type (production of the south of the River, Ningbo instrument plant).
Ready ovarian cumulus-ovocyte complex body is put into two kinds of cultivations respectively drip, each drips and puts into about 20 COCs.The cultivation that will contain ovarian cumulus-ovocyte complex body is dripped and is placed culture dish, is positioned over then in the Hera Cell 150 type cell culture incubators (production of Thermo company), preserves 3 days under differing temps.
Preserve and detect the situation that ovarian cumulus-reduction division of ovocyte complex body is suppressed after 3 days.Ovarian cumulus-ovocyte complex body placed on the slide glass and at the stationary liquid (acetic acid: methyl alcohol=1: 3) of new preparation, under the room temperature fixedly 48-72 hour, cell after fixing dyeed 5 minutes in 1% (w/v) orcein (being dissolved in 45% acetic acid), observed under 400 * phase microscope then.Whether complete according to nuclear membrane, ovocyte is divided into germinal vesicle phase (GV) and germinal vesicle break the phase (GVBD).The results are shown in Table 1.
Table 1: preserved GV phase porcine oocytes 3 days under the differing temps
The experiment of this representativeness optimizes foetal calf serum (FCS) and can suppress GV phase porcine oocytes germinal vesicle at 20-30 ℃ and break, and it is the highest further to optimize 27.5 ℃ of inhibiting rates, and TCM-199 is control group.
The applicant adopt same method validation new-born calf serum also can be used for suppressing GV phase porcine oocytes germinal vesicle and break.
B.27.5 preserve GV phase porcine oocytes with different time under ℃ condition
Under 27.5 ℃ of conditions, preserve GV phase porcine oocytes, got cell at the 1st day, the 2nd day and the 3rd day that preserves respectively and carry out the GV phase and check, the results are shown in Table 2.
Show to preserve under the 2:27.5 ℃ of condition different time GV phase porcine oocytes
Preserve GV phase porcine oocytes with FCS and have time-dependent manner, prolong effect in time and descend, decline in second day is the most obvious, preserves the GV of the maintaining phase over half that still had in three days.
The applicant adopt same method validation new-born calf serum can preserve porcine oocytes at least 3 days equally.
Embodiment 2: the check of oocyte in vitro maturation
The porcine oocytes complex body of preserving among the embodiment 13 days is taken out, in TCM-199 operation liquid, give a baby a bath on the third day after its birth together time with fresh ovocyte, in maturation culture solution, give a baby a bath on the third day after its birth then time, the maturation of putting into preheating (38.5 ℃ of preheating temperatures) is in advance cultivated and is dripped, cultivation in the Hera Cell 150 type cell culture incubators (productions of Thermo company) of 38.5 ℃, 5%CO2 and 100% humidity.Wherein, maturation culture solution is that (Gibco, Grand Island NY) have added 75 μ g/ml penicillin to improved TCM-199,50 μ g/ml Streptomycin sulphates, 0.57mM halfcystine, 0.5 μ g/ml FSH, 0.5 μ g/ml LH and 10ng/mlEGF; The ripe droplet that drips the 100 μ l that make for maturation culture solution of cultivating is cultivated 25 pieces of ovocytes for every.
Under stereomicroscope, observe ovocyte and whether discharge first polar body.Because the porcine oocytes polar body is minimum, stirs with suction pipe at 40 * microscopically, makes ovocyte rotate to each surface, thereby finds first polar body.The result shows that cultivating 44 hours ovocytes discharges first polar body, reaches ripe.The results are shown in Table 3.
Table 3: preserve oocyte maturation rate after 3 days
Among this embodiment, ovocyte is preserved in FCS and was kept most potentiality of development in three days, can discharge polar body, reaches ripe.
Embodiment 3: the detection of ovocyte microfilament percentage of head rice and GSH content
1, the detection of ovocyte microfilament percentage of head rice
Detect the microfilament percentage of head rice of preserving 3 days ovocyte among fresh ovocyte and the embodiment 1.Ovocyte removes zona pellucida with acid tyrode's solution (pH2.5), with the fixing 30min at least of 4% Paraformaldehyde 96 (pH7.4PBS dilution) room temperature.1%Triton X-10037 ℃ of following permeable membrane spent the night, and blocking-up liquid is the PBS that contains 1%BSA, block 1 hour, and the Phalloidine (1 μ g/ml is diluted in the confining liquid) that then changes the FITC coupling of diluting at 1: 100 over to was incubated at room 2 hours.The PBS that contains 0.1%Tween 20 and 0.01%Triton X-100 gives a baby a bath on the third day after its birth time, and each 5min uses PI (10 μ g/ml in PBS) to dye nuclear 10min afterwards.Ovocyte is encapsulated in and uses laser confocal scanning microscope (Zeiss LSM 510META) to observe on the slide glass after will fully cleaning at last.The results are shown in Table 4.
Table 4: preserve ovocyte microfilament percentage of head rice after 3 days
Among this embodiment, preserving in FCS after three days with the microfilament is that the cytoskeleton of representative remains intact, and does not have tangible difference with fresh GV phase ovocyte, and preserves three days in control group TCM-199, and the microfilament integrity is seriously damaged.
2, the detection of GSH content
Detect the glutathione content of preserving 3 days ovocyte among fresh ovocyte and the embodiment 1.30 ovocytes are transferred in the 1.5ml centrifuge tube that contains 5 μ l distilled water, add 5 μ l1.25M phosphoric acid.Sample multigelation three times under-70 ℃ and room temperature condition is stored in-70 ℃ then up to analyzing subsequently.GSH content in the ovocyte is used the DTNB-GSSG reductase enzyme and is analyzed.Briefly be described below: the phosphoric acid buffer that 700 μ l is contained the 0.2M of 0.33mg/ml NADPH (contains 10mM EDTA, PH 7.2), 100 μ l contain 6mM 5,5 ' dithio-bis (2-nitrobenzoic acid) phosphoric acid buffer and the adding of 190 μ l distilled water (DTNB) contains in the centrifuge tube of sample, adds the GSH reductase enzyme initial action of 10 μ l 250IU/ml behind the mixing.Sample 412nm absorbance value using visible light spectrophotometer detects, and per half a minute record data change, and record altogether 3 minutes.Different concns GSH standard model (0,0.01,0.02,0.05,0.1,0.2,0.5and 1.0mM) is analyzed equally, and determines each ovocyte GSH content in the experimental group sample according to this.The results are shown in Table 5.
Table 5: preserve ovocyte GSH content after 3 days
Reduced form GSH (gsh) is most important anti-oxidation stress material in the cell, it is a most important index weighing the kytoplasm quality, among this embodiment, preserve in FCS after three days that GSH content is significantly higher than the ovocyte of preserving among the control group TCM-199 three days in the ovocyte kytoplasm.
Claims (8)
1. the method for external preservation/cultivation ovocyte, wherein, preservation liquid or nutrient solution that described method adopts whole serum to preserve/cultivate as Oocyte in Vitro, stagnate to keep Oocyte Meiosis, described preservation/culture temperature of keeping the Oocyte Meiosis stagnation is 27.5 ℃, described whole serum is selected from foetal calf serum and/or new-born calf serum, and described ovocyte is the porcine oocytes of germinal vesicle phase.
The method of claim 1, wherein in the described method per 10～100 μ l whole serums preserve/cultivate 1～10 ovocyte.
3. method as claimed in claim 2, wherein, 1 ovocyte is preserved/cultivated to per 10 μ l whole serums in the described method.
4. the method for claim 1, wherein described method also is included in whole serum and preserves/cultivate before the ovocyte, with TCM-199 nutrient solution rinsing ovocyte, and the step of cleaning ovocyte with whole serum.
In the claim 1 to 4 each described method in the external preservation/cultivation of ovocyte, in control and the optimization of oocyte in vitro maturation and improve application in the humans and animals auxiliary procreation technology, wherein, described ovocyte is the porcine oocytes of germinal vesicle phase.
6. liquid or nutrient solution are preserved in whole serum conduct in external preservation/cultivation ovocyte, to keep the application that Oocyte Meiosis is stagnated, described preservation/culture temperature of keeping the Oocyte Meiosis stagnation is 27.5 ℃, described whole serum is selected from foetal calf serum and/or new-born calf serum, and described ovocyte is the porcine oocytes of germinal vesicle phase.
One kind be used for to preserve/cultivate the preparation of ovocyte, described preparation is made up of whole serum, described whole serum is selected from foetal calf serum and/or new-born calf serum, described ovocyte is the porcine oocytes of germinal vesicle phase.
8. as the application of preparation as described in the claim 7 in preserving ovocyte, wherein, described ovocyte is the porcine oocytes of germinal vesicle phase.
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| CN101429492A (en) * | 2008-12-12 | 2009-05-13 | 深圳华大基因科技有限公司 | Mature method for in vitro culture of porcine oocyte |
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