CN111004775B - Method for physically removing oocyte zona pellucida - Google Patents
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- CN111004775B CN111004775B CN202010019282.5A CN202010019282A CN111004775B CN 111004775 B CN111004775 B CN 111004775B CN 202010019282 A CN202010019282 A CN 202010019282A CN 111004775 B CN111004775 B CN 111004775B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0608—Germ cells
- C12N5/0609—Oocytes, oogonia
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
The invention discloses a method for physically removing oocyte zona pellucida, which is characterized in that oocyte to be removed is placed in micromanipulation liquid microdroplet, sterile normal saline or sterile PBS solution is sucked into an inner cavity of an injection needle tip, so that the oocyte, an egg holding needle and the injection needle are positioned at the same horizontal line, injection pressure and balance pressure of a microinjection instrument in a cleaning mode are preset, the ovum is fixed at the front end of the egg holding needle, the injection needle is inserted into the oocyte zona pellucida, a cleaning button is pressed, the solution is quickly injected into peri-oval gaps of the oocyte, and cytoplasm of the oocyte is caused to jump out from zona pellucida holes penetrated by the injection needle, and the oocyte for removing zona pellucida is obtained. The invention has the advantages of quick zona pellucida removal, high efficiency, complete and smooth zona pellucida removal, better maintenance of the activity of oocytes and avoidance of the influence of enzymes on the oocytes.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for physically removing oocyte zona pellucida.
Background
Zona Pellucida (ZP) is an extracellular matrix which is wrapped on the surface layer of a mammalian oocyte and is mainly secreted by the oocyte, and is broken after the Zona pellucida is formed and grows up, and the blastocyst is hatched. The zona pellucida plays a vital role in fertilization, and its structure is mainly composed of three glycoproteins (ZP 1, ZP2, ZP 3). Wherein ZP3 recognizes and binds to sperm of the same species, which in turn induces a spermatogenic acrosome reaction, initiating the fertilization process. Thus playing a critical role in the developmental fertilization of ova and the development of pre-implantation embryos.
Along with the development of related application technologies of oocytes and embryos, the zona pellucida-free oocytes have better application value in the aspects of nuclear transfer, embryo development mechanism, transgenic research and the like, and most of traditional zona pellucida removing methods adopt acidic table fluid or 0.5% pronase to digest zona pellucida, but in actual operation, the method is found to be easy to cause oocyte loss due to improper operation, such as blown oocytes are dissolved or stuck on the suction tube wall due to slightly larger mouth blowing force; in addition, when the zona pellucida is digested by pronase, the zona pellucida is strictly controlled by time and can be dissolved in liquid at a slightly slow speed, so that too many oocytes cannot be digested at one time, and the efficiency is low.
Furthermore, a physical zona pellucida removing method is proposed, for example, the invention patent application with the application number of 201810455757.8 discloses a piezoelectric ultrasonic cutting system and a method for the zona pellucida of the oocyte, an oocyte ultrasonic cutting surgical knife is adopted to cut the zona pellucida from the tangential direction of the surface layer of the oocyte, and meanwhile, the piezoelectric ultrasonic cutting system is provided with a special oocyte holding device and a bottom oocyte pose adjusting chip, so that the auxiliary supporting function on the oocyte can be achieved. The operation equipment in the prior art is complex, a special device and a chip are also required to be added, the operation method is not easy to master, the successful removal rate is low, the activity of oocytes is difficult to maintain, the time consumption is long, and the efficiency of removing zona pellucida is low.
Therefore, there is a need for a method of removing zona pellucida from oocytes that is physically intact to the oocytes, and that is efficient and retains oocyte activity.
Disclosure of Invention
In order to solve the above problems, the present invention provides a method for physically removing zona pellucida from an oocyte, which does not damage the oocyte, can maintain the activity of the oocyte, and can rapidly and effectively remove the zona pellucida from the oocyte.
The invention is realized by the following technical scheme: a method for physically removing oocyte zona pellucida, comprising the steps of:
(1) Preparation of micromanipulation fluid droplets: adding 50uL of serum albumin into 10mL of TL-hepes solution, adding 50uL of diabody, and uniformly mixing to obtain a micromanipulation solution; placing the prepared micromanipulation liquid into operation discs, and moving 15-20 mu L of micromanipulation liquid into each operation disc to obtain microdroplets;
(2) Placing oocytes from which zona pellucida is to be removed in microscopic operating fluid droplets of an operating panel, and transferring 1-20 oocytes once in each droplet;
(3) Placing an operation panel on a micromanipulator, sucking 5-10uL of sterile normal saline or sterile PBS solution into an inner cavity at the tip end of an injection needle, mounting an egg holding needle and the injection needle on a fixed rod of the micromanipulator, adjusting a screw, and observing in the visual field of an inverted microscope to ensure that the oocyte, the egg holding needle and the injection needle are positioned at the same horizontal line;
(4) The method comprises the steps of presetting the injection pressure Pi of the microinjection instrument to be 120-200hPa under a cleaning mode, setting the balance pressure Pc to be 220-250hPa, connecting an egg holding needle with a pneumatic manual microinjection instrument, fixing an egg at the front end of the egg holding needle by adjusting a knob of the pneumatic manual microinjection instrument, then penetrating the injection needle into an oocyte zona pellucida, enabling a needle point to stay in a peri-oval gap, at the moment, pressing a cleaning button, enabling sterile normal saline or sterile PBS solution to be rapidly injected into the peri-oval gap of the oocyte by the microinjection instrument through the pressure pumped by the injection needle, so that cytoplasm of the oocyte is jumped out from a zona pellucida hole penetrated by the injection needle, and enabling the complete cytoplasm to stay in a microdroplet after the zona pellucida is removed, thus obtaining the oocyte with the zona pellucida removed. And then transferred to the culture solution for further culture or directly used for subsequent study.
The droplets of step (1) are covered with a layer of mineral oil.
The zona pellucida-removed oocytes of step (2) include oocytes in the GV, MI, MII or prokaryotic phase.
The invention adopts a conventional commercially available product micromanipulator to remove the transparent belt, wherein the micromanipulator comprises an inverted microscope/stereoscopic microscope, a micromanipulator and a microinjection instrument. Generally, microinjection apparatus is used to directly inject a donor (gene, nucleus or other component of a cell) into a host cell (enucleated oocyte, embryonic cell or cell cultured in vitro), and the method of using microinjection apparatus to remove zona pellucida from oocyte has not been reported yet. The conventional injection operation is performed by using an automatic key or an injection key, and the cleaning (Clean) key of the microinjection instrument is used for cleaning, wherein the Clean key can be pressed for a long time when an injection needle is blocked, the injection pressure can be adjusted according to the severity of the blocking, the adjustable pressure range is larger, the pressure is too small, cytoplasm can not come out, the pressure is too large, and the cytoplasm can be blown off and dissolved in liquid, so the inventor can completely realize the removal of oocyte transparent belt within a certain pressure range through numerous experiments, and the success rate and the survival rate are very high.
The invention has the advantages and effects that: the method is a physical method for removing oocyte zona pellucida, the removing efficiency is more than 99 percent, and the method is suitable for oocytes of non-human primates and rodent mice, and is very worth popularizing.
1. The operation equipment is relatively simple, and a special device is not required to be added;
2. the operation process is simple, and a simple micromanipulation technician can be mastered; the phenomenon of sticking to the bottom of the dish can not be generated;
3. the oocyte can be well kept active, the oocyte does not need to be transferred into a special container or other liquid and is still finished in a daily oocyte culture dish and in-vitro operation liquid of the oocyte, and the oocyte is quite fragile and sensitive, so that the invention is beneficial to keeping the activity of the oocyte in vitro;
4. the zona pellucida is fast, the efficiency is high, the zona pellucida removing time of each oocyte is 1 second (800-1000 ms), and a plurality of zona pellucida removing operations can be completed in a short time;
5. the transparent belt removing rate is high and can reach 99.9 percent of success rate;
6. the operation of removing the zona pellucida from a plurality of oocytes can be completed under a micromanipulator, so that the difficulty that the oocytes can be dissolved or lost when the operation is slightly negligent by using a table fluid is avoided;
7. the method belongs to the physical method for removing the zona pellucida, avoids the influence of enzyme on oocytes, and is applicable to oocytes of various animals, and further, is also applicable to laboratory single-cell RNA extraction research without zona pellucida and early event research related to embryo development.
In a word, the invention can complete the operation of removing the zona pellucida under the micromanipulator, and along with the more and more research work on oocytes and embryos in the research field of life science, the more and more requirements are required for the operation of removing the zona pellucida of the oocytes and the embryos, the invention is convenient for rapid popularization and application.
Drawings
FIG. 1 is a schematic view of the operation device of example 1; in the figure, 1. Oocytes; 2. an ovum holding needle; 3. an injection needle; 4 and 5 are needle holders; cellTram Air pneumatic manual microinjection instrument; 7. CellTram Air pneumatic manual microinjection instrument end knob; 8. eppendorf FemtoJet microinjection instrument; 9. eppendorf FemtoJet microinjection instrument adjusts the knob of the injection pressure (Pi); 10.Eppendorf FemtoJet a knob for microinjection instrument adjustment Time (Time); 11. eppendorf FemtoJet a knob for adjusting the equilibrium pressure (Pc).
FIG. 2 is a microscopic image of the cynomolgus monkey oocyte of example 1 after zona pellucida removal;
FIG. 3 is a microscopic image of the C57 mouse oocyte of example 2 after zona pellucida removal.
Detailed Description
The present invention will be described in further detail with reference to the following examples, but the scope of the present invention is not limited to the above description, and the reagents and methods used in the examples, unless otherwise specified, employ conventional reagents and conventional methods.
Example 1
Main instrument equipment: microinjection apparatus (Eppendorf, femtoJet 4i, germany); nikon stereoscopic microscope (japan); pneumatic manual microinjection apparatus (brand: eppendorf, model: cellTram Air, origin: germany); injection needles (MIC-35, ORIGIO, U.S.); egg holding needle (MPH-MED-35, ORIGIO, USA); petri dishes, pasteur pipettes, available from Falcon, inc., USA; pipettes were purchased from Eppendorf corporation, germany.
Collecting oocytes of female cynomolgus monkeys according to a conventional ovulation promoting method, and removing zona pellucida from the cynomolgus monkey oocytes by a physical method, wherein the method comprises the following specific steps of:
(1) Preparation of micromanipulation fluid droplets: adding 50uL serum albumin (BSA, sigma, USA) into 10mL TL-hepes solution (Caisson, USA), adding 50uL double antibody (Gibco, USA), and mixing uniformly to obtain a micromanipulation solution; placing the prepared micromanipulation liquid in an operation panel, equally dividing the dish into 5 areas (an area in the middle, an area on the upper, lower, left and right sides and in a cross shape) in a plastic dish operation panel with the specification of 35mm, transferring 15-20 mu L of micromanipulation liquid into the dish in each area by using a liquid-transferring gun, wherein the center is called a center droplet, the operation of removing the transparent belt is mainly completed in the droplet, the peripheral droplet is mainly used for washing oocytes, and a layer of mineral oil is covered on the center droplet;
(2) Oocytes are placed in microdrops: placing oocytes (including GV stage, MI stage, MII stage and prokaryotic stage) from which zona pellucida is to be removed in the peripheral microdroplet of the step (1), washing 5 times by using an egg sucking needle under a stereoscopic microscope, and then placing central microdroplets covered with mineral oil, wherein each central microdroplet is transferred into 1-20 oocytes once;
(3) Placing the operation panel on a micromanipulator, sucking 5-10uL of sterile normal saline into the inner cavity of the tip of the injection needle by using a pipette, mounting an egg holding needle and the injection needle on a fixed rod of the micromanipulator, adjusting a screw, and observing in the visual field of an inverted microscope to ensure that the oocyte, the egg holding needle and the injection needle are positioned at the same horizontal line, as shown in figure 1;
(4) The method comprises the steps of presetting the injection pressure Pi of the microinjection instrument to be 150hPa and the balance pressure Pc to be 230hPa in a cleaning mode, connecting an egg holding needle with a pneumatic manual microinjection instrument, fixing an ovum at the front end of the egg holding needle by adjusting a knob of the pneumatic manual microinjection instrument, lightly penetrating the injection needle into a zona pellucida of the oocyte, keeping a needle point at a peri-oval gap, pressing a cleaning (Clean) button for 800 milliseconds at the moment, enabling sterile normal saline to be quickly injected into the peri-oval gap of the oocyte through the pressure pumped by the injection needle by the microinjection instrument, and enabling cytoplasm of the oocyte to instantly jump out of a zona pellucida hole penetrated by the injection needle due to the fact that the sterile normal saline quickly occupies a cytoplasmic space, and enabling complete cytoplasm to be in a microdroplet after the complete cytoplasm is stripped, so that the oocyte is obtained.
After that, another oocyte is fixed and the zona pellucida is removed in the same manner. After all oocytes are operated, transferring the oocytes into peripheral microdroplets by using an egg sucking needle for 3 times, and transferring the oocytes into a culture solution for continuous culture or directly using the oocytes for subsequent researches.
The removal rate of this example reaches 100% and the cell death rate is 0.1%.
Example 2
Oocytes of female C57 mice are collected according to a conventional ovulation promoting method, zona pellucida is removed from the oocytes of the C57 mice by a physical method, and the method comprises the following specific steps:
(1) As in example 1;
(2) As in example 1;
(3) Placing an operation panel on a micromanipulator, sucking 5-10uL of sterile PBS solution into an inner cavity at the tip end of an injection needle by using a pipette, mounting an egg holding needle and the injection needle on a fixed rod of the micromanipulator, adjusting a screw, and observing in the visual field of an inverted microscope to ensure that the oocyte, the egg holding needle and the injection needle are positioned at the same horizontal line;
(4) The method comprises the steps of presetting the injection pressure Pi of a microinjection instrument to be 120hPa and the balance pressure Pc to be 220hPa in a cleaning mode, connecting an egg holding needle with a pneumatic manual microinjection instrument, fixing an ovum at the front end of the egg holding needle by adjusting a knob of the pneumatic manual microinjection instrument, lightly penetrating the injection needle into a zona pellucida of the oocyte, keeping a needle point at a periegg gap, pressing a cleaning (Clean) button for 1 second at the moment, enabling a sterile PBS solution to be quickly injected into the periegg gap of the oocyte by the microinjection instrument through the pressure pumped by the injection needle, and enabling cytoplasm of the oocyte to be instantly and suddenly discharged from a zona pellucida hole penetrated by the injection needle due to the fact that the sterile PBS solution rapidly occupies a cytoplasmic space, and remaining in a droplet after complete cytoplasm is stripped of the pellucida, so that the oocyte with the zona pellucida is removed is obtained.
After that, another oocyte is fixed and the zona pellucida is removed in the same manner. After all oocytes are operated, transferring the oocytes into peripheral microdroplets by using an egg sucking needle for 3 times, and transferring the oocytes into a culture solution for continuous culture or directly using the oocytes for subsequent researches.
The removal rate in this example was 98.5% and the cell death rate was 0%.
Replacing the injection pressure Pi, the equilibrium pressure Pc and the time in step (4) to obtain the following table results:
example 3
The oocyte of female buffalo is collected according to the conventional ovulation promoting method, and zona pellucida is removed from the buffalo oocyte by a physical method, and the method comprises the following specific steps:
(1) As in example 1;
(2) As in example 1;
(3) Placing an operation panel on a micromanipulator, sucking 5-10uL of sterile PBS solution into an inner cavity at the tip end of an injection needle by using a pipette, mounting an egg holding needle and the injection needle on a fixed rod of the micromanipulator, adjusting a screw, and observing in the visual field of an inverted microscope to ensure that the oocyte, the egg holding needle and the injection needle are positioned at the same horizontal line;
(4) The method comprises the steps of presetting the injection pressure Pi of a microinjection instrument to be 200hPa and the balance pressure Pc to be 250hPa in a cleaning mode, connecting an egg holding needle with a pneumatic manual microinjection instrument, fixing an ovum at the front end of the egg holding needle by adjusting a knob of the pneumatic manual microinjection instrument, lightly penetrating the injection needle into a zona pellucida of the oocyte, keeping a needle point at a periegg gap, pressing a cleaning (Clean) button for 1 second at the moment, enabling a sterile PBS solution to be quickly injected into the periegg gap of the oocyte by the microinjection instrument through the pressure pumped by the injection needle, and enabling cytoplasm of the oocyte to be instantly and suddenly discharged from a zona pellucida hole penetrated by the injection needle due to the fact that the sterile PBS solution rapidly occupies a cytoplasmic space, and remaining in a droplet after complete cytoplasm is stripped of the pellucida, so that the oocyte with the zona pellucida is removed is obtained.
After that, another oocyte is fixed and the zona pellucida is removed in the same manner. After all oocytes are operated, transferring the oocytes into peripheral microdroplets by using an egg sucking needle for 3 times, and transferring the oocytes into a culture solution for continuous culture or directly using the oocytes for subsequent researches.
The removal rate in this example was 99.9% and the cell death rate was 0%.
Claims (3)
1. A method for physically removing oocyte zona pellucida, which is characterized by comprising the following steps:
(1) Preparation of micromanipulation fluid droplets: adding 50uL of serum albumin into 10mL of TL-hepes solution, adding 50uL of diabody, and uniformly mixing to obtain a micromanipulation solution; placing the prepared micromanipulation liquid into operation discs, and moving 15-20 mu L of micromanipulation liquid into each operation disc to obtain microdroplets;
(2) Placing oocytes from which zona pellucida is to be removed in microscopic operating fluid droplets of an operating panel, and transferring 1-20 oocytes once in each droplet;
(3) Placing an operation panel on a micromanipulator, sucking 5-10uL of sterile normal saline or sterile PBS solution into an inner cavity at the tip end of an injection needle, mounting an egg holding needle and the injection needle on a fixed rod of the micromanipulator, adjusting a screw, and observing in the visual field of an inverted microscope to ensure that the oocyte, the egg holding needle and the injection needle are positioned at the same horizontal line;
(4) The method comprises the steps of presetting the injection pressure Pi of the microinjection instrument to be 120-200hPa under a cleaning mode, setting the balance pressure Pc to be 220-250hPa, connecting an egg holding needle with a pneumatic manual microinjection instrument, fixing an egg at the front end of the egg holding needle by adjusting a knob of the pneumatic manual microinjection instrument, then penetrating the injection needle into an oocyte zona pellucida, enabling a needle point to stay in a peri-oval gap, at the moment, pressing a cleaning button, enabling sterile normal saline or sterile PBS solution to be rapidly injected into the peri-oval gap of the oocyte by the microinjection instrument through the pressure pumped by the injection needle, so that cytoplasm of the oocyte is jumped out from a zona pellucida hole penetrated by the injection needle, and enabling the complete cytoplasm to stay in a microdroplet after the zona pellucida is removed, thus obtaining the oocyte with the zona pellucida removed.
2. The method of physically removing oocyte zona pellucida according to claim 1, wherein: the droplets of step (1) are covered with a layer of mineral oil.
3. The method of physically removing oocyte zona pellucida according to claim 1, wherein: the zona pellucida-removed oocytes of step (2) include oocytes in the GV, MI, MII or prokaryotic phase.
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