CN108004204B - Method for high-throughput screening of high-quality oocytes - Google Patents
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Abstract
The invention provides a method for screening high-quality oocytes in high flux, which comprises the steps of firstly enabling the oocytes to be mature in an in vitro culture environment through maturation liquid I and maturation liquid II, then carrying out cumulus cell removal through a cumulus cell removal solution, and simultaneously carrying out primary selection on the integrity of a cell membrane structure of the oocytes, wherein the oocytes with incomplete semi-permeable membrane structures do not have perivitelline gaps, and a part of inferior oocytes can be eliminated in the process; and finally checking in an isotonic hormone-free solution, and if the cell membrane of the primarily selected oocyte cannot be restored to the state before the treatment of the cumulus-removing solution, the oocyte must be eliminated, so that the high-quality oocyte can be quickly screened in high flux for developing the productive cloning work.
Description
Technical Field
The invention belongs to the technical field of cell engineering, and particularly relates to a method for high-throughput screening of high-quality oocytes through an osmotic pressure treatment technology.
Background
Currently, in nuclear transfer studies, only two methods are used to screen oocytes: the first is blind selection, which is done directly no matter what quality of oocyte; and the second method is to dye the oocytes by using brilliant cresyl blue, the oocytes dyed with colors are judged to be good in quality, and the oocytes not dyed with colors are judged to be poor in quality. Literature search reveals: theriogenology, 68(2007)1299-1304 authors: manjunatha et al published article "selection of buffalo oocytes with developmental potential with brilliant cresyl blue before maturation" in which oocytes were placed in BCB staining solution for 90 minutes, compared the selection rates of different concentrations of BCB for oocytes; comparing the difference of the diameters of the selected negative and positive oocytes; the maturation rate and blastocyst rate of different experimental groups (control group, blank group, positive group and negative group) were compared, and the result positive group was higher than the other groups. Theriogenology, 63(2005)2194-2205, authors: alm et al, the article "influence of pre-maturation selection of oocytes with brilliant cresyl blue capable of detecting G6PDH activity on the rate of blastocyst development in vitro in cattle" examined the difference in G6PDH activity between BCB-stained negative and positive oocytes, and the negative group had higher G6PDH activity than the positive and experimental groups. However, the in vitro maturation rate and blastocyst rate of the positive group were higher than those of the other groups. Theriogenology, 57(2002) 1397-: the article of 'selecting lamb goat oocytes with brilliant cresol blue' published by Rodriguez-Gonzalez et al contains that the selection of lamb goat oocytes by BCB staining solution with different concentrations is researched, the in vitro fertilization rate and the maturation rate of the selected oocytes are compared, and the staining solution with the concentration of 26 mu M is found to be suitable for the selection of lamb goat oocytes. The selected positive oocyte is obviously higher than the negative oocyte in the aspects of cell diameter, in vitro development rate and the like. Theriogenology, 61(2004)735-744, authors: MarcPujol et al published article "selection of oocytes of heifers with developmental potential by Brilliant Cresyl Blue (BCB)" the content of the article is to study the selection of BCB staining solutions on oocytes of heifers for in vitro embryo production. The development potential and oocyte diameter difference between the positive oocytes and the control group and between the positive oocytes and the adult cows are compared, and the result shows that: the selection rates of oocytes in different grades are different, the diameter of the positive oocyte is larger than that of the negative oocyte, and the development rate of the positive embryo blastocyst is higher than that of the negative group and the control group but lower than that of the adult cow group. In 8 people published by Beijing institute of zootechnics and veterinary medicine, Qiaolimin and the like of China academy of agricultural sciences, the influence of brilliant cresyl blue staining on bovine oocyte in-vitro maturation and in-vitro fertilization is also applied to screening of bovine oocytes by using the brilliant cresyl blue staining solution. Obviously, the second method requires addition of dye to the culture medium and culturing for 90min, and is complicated in operation, low in screening efficiency, and not favorable for mass-production cloning work.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide a method for screening high-quality oocytes with high throughput, which is simple and convenient to operate and is used for solving the problem of low efficiency of screening the oocytes at present.
In order to achieve the purpose of the invention, the inventor combines scientific research experiences for many years and does not research and make efforts, finally, the oocytes are quickly matured in an in vitro culture environment by sequentially using different osmotic pressure solutions in the embryo operation process, and meanwhile, the high-throughput screening of high-quality oocytes is quickly realized by utilizing the reaction of a cell semipermeable membrane to osmotic pressure. Specifically, the technical scheme of the invention is summarized as follows:
a method for screening high-quality oocytes in high throughput is characterized by comprising the following steps:
(1) transferring the selected cumulus oocyte complexes into a maturation solution I for culturing, wherein the maturation solution I comprises the following components: TCM199+ 2.90-3.15 mM glucose + 0.85-0.95 mM sodium pyruvate + 0.52-0.60 mML-cysteine + 90-110 IU/mL penicillin + 90-110 mug/mL streptomycin sulfate + 9-11% follicular fluid + 9-11 IU/mL PMSG + 9-11 IU/mLhCG;
(2) transferring the cumulus oocyte complex cultured in the step (1) into a maturation liquid II for culturing, wherein the maturation liquid II comprises the following components: TCM199+ 2.90-3.15 mM glucose + 0.85-0.95 mM sodium pyruvate + 0.52-0.60 mM L-cysteine + 90-110 IU/mL penicillin + 90-110 μ g/mL streptomycin sulfate + 9-11% follicular fluid;
(3) transferring the cumulus oocyte complex cultured in the step (2) into a cumulus removal solution, oscillating for 0.5-2min, and then transferring oocytes with perivitelline gaps for the next operation, wherein the cumulus removal solution comprises the following components: PVA-TL-Hepes + 15-25 mg/mL hyaluronidase + 9-15 mg mannitol + 0.15-0.30 mg BSA;
(4) and (3) transferring the oocytes primarily selected in the step (2) into an isotonic hormone-free solution for washing, and eliminating oocytes whose plasma membranes cannot be recovered, wherein the isotonic hormone-free solution comprises the following components: TCM199+ 2.90-3.15 mM glucose + 0.85-0.95 mM sodium pyruvate + 0.52-0.60 mML-cysteine + 90-110 IU/mL penicillin + 90-110 ug/mL streptomycin sulfate + 19-11% follicular fluid + 7-9 mg/mLHepes.
Further preferably, the method for high-throughput screening of oocytes with good quality as described above, wherein the culture conditions of step (1) and step (2) are: 5% CO2Saturated humidity, time 16-28h, temperature 38-39 deg.C.
Further preferably, the method for high-throughput screening of oocytes with good quality as described above, wherein the composition of maturation liquid I in step (1) is: TCM199+3.05mM glucose +0.91mM sodium pyruvate +0.57mM L-cysteine +100IU/mL penicillin + 100. mu.g/mL streptomycin sulfate + 10% follicular fluid +10IU/mLPMSG +10 IU/mhCG.
Further preferably, the method for high-throughput screening of oocytes with good quality as described above, wherein the composition of maturation liquid II in step (2) is: TCM199+3.05mM glucose +0.91mM sodium pyruvate +0.57mM L-cysteine +100IU/mL penicillin + 100. mu.g/mL streptomycin sulfate + 10% follicular fluid.
Further preferably, the method for high-throughput screening of high-quality oocytes is as described above, wherein oocytes with a perivitelline gap width of 4-6 μm after the treatment in step (3) are selected for further operation.
Further preferably, the method for high-throughput screening of high-quality oocytes as described above, wherein the cumulus removal solution in step (3) consists of: PVA-TL-Hepes +20mg/mL hyaluronidase +12mg mannitol +0.2mg BSA.
Further preferably, the method for high-throughput screening of high-quality oocytes as described above, wherein the composition of the isotonic hormone-free solution in step (4) is: TCM199+3.05mM glucose +0.91mM sodium pyruvate +0.57mM L-cysteine +100IU/mL penicillin + 100. mu.g/mL streptomycin sulfate + 10% follicular fluid +7.5 mg/mLHepes.
Compared with the prior art, the in-vitro maturation efficiency of the oocytes is improved through the maturation liquid I and the maturation liquid II, so that the oocytes are rapidly matured in an in-vitro culture environment, then the cumulus cells are removed through the cumulus cell removing solution, meanwhile, the integrity of the cell membrane structure of the oocytes is initially selected, perivitelline gaps cannot occur in the oocytes with incomplete semi-permeable membrane structures, and a part of inferior oocytes can be eliminated in the process; and finally checking in an isotonic hormone-free solution, and if the cell membrane of the primarily selected oocyte cannot be restored to the state before the treatment of the cumulus-removing solution, the oocyte must be eliminated, so that the high-quality oocyte can be quickly screened in high flux for developing the productive cloning work.
Detailed Description
The following examples are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and all simple modifications, equivalent changes and modifications made to the above examples according to the technical spirit of the present invention still fall within the protection scope of the technical solution of the present invention.
Example 1: preparation of the solution
The method for judging the quality of the oocyte by treating the porcine oocyte through osmotic pressure and the solution formula group method thereof have five formulas of sequential hypertonic solution and isotonic solution, which are respectively as follows:
the mature liquid I comprises the following components in percentage by weight: TCM199+3.05mM glucose +0.91mM sodium pyruvate +0.57mM L-cysteine +100IU/mL penicillin + 100. mu.g/mL streptomycin sulfate + 10% follicular fluid +10IU/mL pregnant horse serum gonadotropin (PMSG) +10IU/mL human chorionic gonadotropin (hCG).
The formula of the mature liquid II is as follows: TCM199+3.05mM glucose +0.91mM sodium pyruvate +0.57mM L-cysteine +100IU/mL penicillin + 100. mu.g/mL streptomycin sulfate + 10% follicular fluid.
The formula of the cumulus removing solution is as follows: PVA-TL-Hepes +20mg/mL hyaluronidase +12mg mannitol +0.2mg BSA.
The formula of the isotonic hormone-free solution is as follows: TCM199+3.05mM glucose +0.91mM sodium pyruvate +0.57mM L-cysteine +100IU/mL penicillin + 100. mu.g/mL streptomycin sulfate + 10% follicular fluid +7.5 mg/mLHepes.
The formula of the hypertonic Hepes buffer solution is as follows: 9.5g TCM199, 0.05g NaHCO30.75g of hepes, 0.05g of penicillin, 0.06g of streptomycin, 1.755g of NaCL, 3g of BSA, and deionized water were added to prepare 100 mL.
Example 2: high throughput screening of porcine oocytes
The test procedure was as follows:
(1) a vacuum pump and its accompanying equipment were prepared, and the negative pressure value was adjusted to 0.02 atm. And (4) extracting follicles with the diameter of 3-6 mm on the ovary by using a 16-gauge needle. Microscopic picking Cumulus Oocyte Complex (COCs) with complete and uniform cytoplasm wrapped by granulocytes, placing the selected COCs into a maturation solution I for culturing for 24h under the conditions of 39 ℃ and 5% CO2And saturation humidity. After 24h, the medium was changed to maturation solution II and then cultured for 16-24 h.
(2) After culturing for 40-48 h, transferring the oocyte into a cumulus removal solution, treating for 1min on a vortex oscillator, and then transferring the oocyte with perivitelline space by using an egg transferring needle for the next operation. It should be noted that if the cytoplasm of the oocyte is unable to shrink, this indicates that the membrane structure of the oocyte membrane is incomplete and the oocyte has died; if the cytoplasm of the oocyte is too contracted, the cytoplasm of the oocyte is too little and the quality is not good; if the cytoplasm of the oocyte is contracted after the treatment so that the width of the perivitelline space between the cell membrane and the zona pellucida is about 5 μm, the quality of the oocyte is optimal.
(3) And (3) transferring the optimized high-quality pig oocytes into an isotonic hormone-free solution for washing for 3 times, and eliminating eggs with unrecoverable oocyst plasma membranes.
(4) The porcine oocytes with restored cytoplasmic membranes were treated in a hypertonic Hepes buffer for 30min and then observed under a microscope.
The test results are as follows:
(1) the non-shrinkable oocytes were subjected to parthenogenetic activation, and 1000 oocytes were treated in total, and no 1 oocyte was able to undergo cleavage.
(2) The oocytes which can not recover elasticity are subjected to parthenogenetic activation, and 1000 oocytes are treated in total. About 70% of oocytes can be cracked, but most of oocytes are unequally cracked, and one electrical activation needs to be supplemented, and the time lag of the cleavage is about 30-60 min.
(3) The oocytes that failed to recover their elasticity were subjected to enucleation study for a total of 1000 oocytes. There is no way to determine how much cytoplasm is removed during enucleation, which causes great difficulty for the operator.
(4) After the oocytes which can not recover elasticity are placed in the maturation liquid II and are observed after 30min of recovery, 30% of the oocytes still can recover elasticity, and the cytoplasmic mass of the remaining 70% of the oocytes which can not recover is poor.
(5) The oocytes that had recovered their elasticity later were used as nuclear receptors for cloning studies, and a total of 100 were treated. No cleavage was seen when embryo transfer was performed.
(6) The selected oocytes with better quality are used as nuclear receptors for clone research, and 2000 oocytes are processed in total. At the time of embryo transfer, more than 93% of the reconstructed embryos had already been cleaved.
Claims (6)
1. A method for screening high-quality oocytes in high throughput is characterized by comprising the following steps:
(1) transferring the selected cumulus oocyte complexes into a maturation solution I for culturing, wherein the maturation solution I comprises the following components: TCM199+ 2.90-3.15 mM glucose + 0.85-0.95 mM sodium pyruvate + 0.52-0.60 mM L-cysteine + 90-110 IU/mL penicillin + 90-110 ug/mL streptomycin sulfate + 9-11% follicular fluid + 9-11 IU/mL PMSG + 9-11 IU/mL hCG;
(2) transferring the cumulus oocyte complex cultured in the step (1) into a maturation liquid II for culturing, wherein the maturation liquid II comprises the following components: TCM199+ 2.90-3.15 mM glucose + 0.85-0.95 mM sodium pyruvate + 0.52-0.60 mM L-cysteine + 90-110 IU/mL penicillin + 90-110 μ g/mL streptomycin sulfate + 9-11% follicular fluid;
(3) transferring the cumulus oocyte complex cultured in the step (2) into a cumulus removal solution, oscillating for 0.5-2min, and then transferring oocytes with perivitelline gaps for the next operation, wherein the cumulus removal solution comprises the following components: PVA-TL-Hepes + 15-25 mg/mL hyaluronidase + 9-15 mg mannitol + 0.15-0.30 mg BSA;
(4) and (3) transferring the oocytes primarily selected in the step (2) into an isotonic hormone-free solution for washing, and eliminating oocytes whose plasma membranes cannot be recovered, wherein the isotonic hormone-free solution comprises the following components: TCM199+ 2.90-3.15 mM glucose + 0.85-0.95 mM sodium pyruvate + 0.52-0.60 mM L-cysteine + 90-110 IU/mL penicillin + 90-110 ug/mL streptomycin sulfate + 19-11% follicular fluid + 7-9 mg/mL Hepes;
and (4) selecting the oocytes with the perivitelline gap width of 4-6 mu m after the treatment of the step (3) to carry out the next operation.
2.The method for high-throughput screening of high-quality oocytes according to claim 1, wherein the culture conditions of the step (1) and the step (2) are as follows: 5% CO2Saturated humidity, time 16-28h, temperature 38-39 deg.C.
3.The method for high-throughput screening of high-quality oocytes according to claim 1, wherein the composition of the maturation solution I in step (1) is as follows: TCM199+3.05mM glucose +0.91mM sodium pyruvate +0.57mM L-cysteine +100IU/mL penicillin + 100. mu.g/mL streptomycin sulfate + 10% follicular fluid +10IU/mL PMSG +10IU/mL hCG.
4.The method for high-throughput screening of high-quality oocytes according to claim 1, wherein the composition of the maturation liquid II in the step (2) is as follows: TCM199+3.05mM glucose +0.91mM sodium pyruvate +0.57mM L-cysteine +100IU/mL penicillin + 100. mu.g/mL streptomycin sulfate + 10% follicular fluid.
5. The method for high-throughput screening of high-quality oocytes according to claim 1, wherein the cumulus removal solution in step (3) consists of: PVA-TL-Hepes +20mg/mL hyaluronidase +12mg mannitol +0.2mg BSA.
6. The method for high-throughput screening of high-quality oocytes according to claim 1, wherein the composition of the isotonic hormone-free solution in step (4) is as follows: TCM199+3.05mM glucose +0.91mM sodium pyruvate +0.57mM L-cysteine +100IU/mL penicillin + 100. mu.g/mL streptomycin sulfate + 10% follicular fluid +7.5mg/mL Hepes.
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