CN110904034A

CN110904034A - A method of removing the nucleus of an egg cell

Info

Publication number: CN110904034A
Application number: CN201911120764.3A
Authority: CN
Inventors: 王巨龙; 萧晟; 黎婷; 张海燕; 刘宽辉; 李恒
Original assignee: Wuhu Institute of Technology
Current assignee: Wuhu Institute of Technology
Priority date: 2019-11-15
Filing date: 2019-11-15
Publication date: 2020-03-24

Abstract

The invention discloses a method for removing the nucleus of an egg cell. Using a mouse as a model, pronase is used to treat the MII-stage egg cell to digest the zona pellucida, then a cutting knife is used to cut the egg cell in half, and 1/3 to 1/3 of the polar body is discarded. 1/2 of the cytoplasm, the cell body containing the polar body was stained with Hoechst stain and examined under a fluorescence microscope to detect the enucleation rate; the method of manually removing the egg cell nucleus can significantly increase the egg cell enucleation efficiency, which is suitable for large-scale cloning. Animal production becomes possible.

Description

Method for removing egg cell nucleus

Technical Field

The invention relates to the technical field of cell biology, in particular to a method for removing egg cell nucleuses.

Background

Since the 21 st century, life sciences, especially animal cloning techniques, have made enormous efforts to genetic breeding, making great contributions to human agriculture and life health. Since the birth of cloned sheep multiple benefits, animal cloning technology is rapidly developed. The mammalian cloning technology comprises the procedures of enucleation of oocyte, implantation of cloned animal somatic cell into enucleated oocyte, activation and reconstruction of oocyte, in vitro development, implantation into surrogate mother, and the like, and the oocyte enucleation technology is one of the key technologies in the animal cloning procedure.

The prior oocyte enucleation technology mainly comprises two main types of chemical enucleation methods and micromanipulation enucleation methods. The chemical enucleation method utilizes chemical drugs to prevent normal separation of oocyte spindles to achieve the enucleation purpose, often the chemical enucleation efficiency is low, and the drug action is not beneficial to the development of eggs. The requirement for operating instruments is high for the core removal by micromanipulation, and a laboratory cannot bear expensive micromanipulation equipment.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for removing egg cell nuclei, which improves the enucleation operability and enucleation efficiency.

Based on the aim, the invention provides a method for removing oocyte nuclei, which comprises the steps of obtaining mature oocytes in vitro, removing zona pellucida of the oocytes, cutting the oocytes and removing the nuclei.

Optionally, the in vitro mature oocyte retrieval comprises the following steps: selecting a healthy female mouse which is 5-8 weeks old before estrus, taking out the ampulla of the oviduct through aseptic surgery, rinsing the female mouse with M2 for 2-3 times, clamping the oviduct, scratching the ampulla of the oviduct through a syringe needle of a microscope, collecting a cumulus oophorus-oocyte compound, transferring the compound into hyaluronidase, and slowly blowing to remove granular cells; collecting the MII-stage oocyte, washing the MII-stage oocyte 3-4 times by using M2, and placing the obtained MII-stage oocyte in an incubator.

Optionally, the oocyte zona pellucida is removed, the oocyte in the MII stage is treated by pronase until the zona pellucida is digested, and then the oocyte is transferred into FBS fetal bovine serum solution to stop digestion, and the oocyte with the zona pellucida removed is placed in an incubator.

Optionally, the concentration of pronase is 0.3-1.0%.

Optionally, the concentration of the FBS fetal bovine serum is 5-15%.

Optionally, the oocyte enucleation cutting comprises the following steps: and transferring the digested oocyte of the zona pellucida into a cutting drop, quickly cutting the oocyte under a microscope, and removing cytoplasm containing the polar bodies 1/3-1/2 after cutting.

Optionally, the cutting drop is 2.0-3.0 ug/mL cytochalasin B.

Optionally, the temperature of the incubator is 35-40 ℃, and CO is₂The content is 3-8%.

From the above, it can be seen that the method for removing the nucleus of the egg cell provided by the invention does not need expensive microscopic equipment or medicament action by using the manual method for removing the nucleus of the egg cell, and the nucleus removal can be completed only by using a common tool under a microscope. The manual removal of oocyte nucleus not only needs no expensive micromanipulation instrument but also has the advantages of simple enucleation operation and high enucleation efficiency.

The method for manually removing the nucleus of an oocyte obtains a large number of enucleated oocytes in a short time. The method can provide sufficient material sources for cloning animals, has the characteristics of small damage to egg cells due to denucleation and low toxicity, and is simple to operate, thereby becoming a prerequisite for popularization and application.

Drawings

FIG. 1 is a process flow diagram of an embodiment of the present invention;

FIG. 2 is a schematic diagram of an embodiment of the present invention showing the protease treatment of MII stage oocytes;

FIG. 3 is a schematic view of the zona pellucida of egg cell total elimination according to the embodiment of the present invention.

Detailed Description

In the following description of the embodiments, the detailed description of the present invention, such as the manufacturing processes and the operation and use methods, will be further described in detail to help those skilled in the art to more fully, accurately and deeply understand the inventive concept and technical solutions of the present invention.

In order to solve the defect of enucleation of a chemical enucleation method and a micromanipulation enucleation method in the prior art, the method for removing oocyte nuclei provided by the invention comprises the steps of obtaining oocytes matured in vitro, removing zona pellucida of the oocytes, cutting the oocytes and removing the nuclei.

In order to operate conveniently and quickly and improve the enucleation rate, the in vitro mature oocyte acquisition method comprises the following steps: selecting a healthy female mouse which is 5-8 weeks old before estrus, taking out the ampulla of the oviduct through aseptic surgery, rinsing the female mouse with M2 for 2-3 times, clamping the oviduct, scratching the ampulla of the oviduct through a syringe needle of a microscope, collecting a cumulus oophorus-oocyte compound, transferring the compound into hyaluronidase, and slowly blowing to remove granular cells; collecting oocytes in the MII stage, washing the oocytes with M2 for 3-4 times, and preferably placing the obtained oocytes in the MII stage in an incubator, wherein the in vitro mature oocyte obtaining comprises the following steps: selecting a healthy female mouse which is 5-8 weeks old before estrus, taking out the ampulla of the oviduct through aseptic surgery, rinsing the female mouse with M2 for 2-3 times, clamping the oviduct, scratching the ampulla of the oviduct through a syringe needle of a microscope, collecting a cumulus oophorus-oocyte compound, transferring the compound into hyaluronidase, and slowly blowing to remove granular cells; collecting the MII-stage oocyte, washing the MII-stage oocyte 3-4 times by using M2, and placing the obtained MII-stage oocyte in an incubator.

In order to operate conveniently and quickly, improve the denucleation rate and remove the zona pellucida of the oocyte, the oocyte in the MII stage is treated by pronase until the zona pellucida is digested, then the oocyte is transferred into FBS fetal bovine serum solution to stop the digestion, and the oocyte with the zona pellucida removed is placed in an incubator.

In order to operate conveniently and quickly and improve the denucleation rate, the concentration of the pronase is 0.3-1.0%.

In order to operate conveniently and rapidly and improve the denucleation rate, the concentration of FBS fetal bovine serum is 5-15%.

In order to operate conveniently and quickly and improve the denucleation rate, the oocyte with the digested zona pellucida is transferred into a cutting drop, the oocyte is cut quickly under a microscope, and cytoplasm containing polar bodies 1/3-1/2 is discarded after cutting.

In order to operate conveniently and quickly and improve the denucleation rate, the cutting drop is 2.0-3.0 ug/mL cytochalasin B.

In order to operate conveniently and quickly and improve the denucleation rate, the temperature of the incubator is 35-40 ℃, and CO is used₂The content is 3-8%.

The manual removal of the egg cell nucleus does not require expensive microscopic equipment nor pharmaceutical action, and the enucleation can be completed under a microscope only by using a common tool. The manual removal of oocyte nucleus not only needs no expensive micromanipulation instrument but also has the advantages of simple enucleation operation and high enucleation efficiency. Compared with the prior enucleation technology, the egg cells are cut into two halves under a microscope by using a common cutting tool, wherein one half does not contain cell nucleuses, and the egg cells are selected by nuclear staining. The invention can greatly improve the efficiency of enucleating egg cells. The method for manually removing the nucleus of an oocyte obtains a large number of enucleated oocytes in a short time. The method can provide sufficient material sources for cloning animals, the denucleation and low toxicity have little damage to egg cells, and the operation is simple, thus becoming the prerequisite for popularization and application.

Specifically, the method for removing egg cell nuclei provided in embodiment 1 of the present invention includes the following steps:

1. in vitro mature oocyte retrieval

Taking the oocyte of a mouse as an enucleation model, selecting a healthy female mouse which is at 10:00pm at the early stage of estrus and is 5-8 weeks old, and injecting 10IU PMSG intraperitoneally after 48 h. And (5) killing the female mice by cervical dislocation after HCG 14-16 h injection. The ampulla of the fallopian tube is taken out through aseptic surgery and rinsed 2-3 times with M2. Another sterile 35mm petri dish was used to aspirate egg picking solution (10% FBS DMEM) into a strip-shaped droplet using a 200uL gun. The oviduct is clamped by forceps, the ampulla of the oviduct is lacerated by a 1mL syringe needle of a stereomicroscope, and a cumulus-oocyte compound is collected. The complex was transferred to hyaluronidase using a mouth-pipette and gently aspirated to remove the granulocytes. MII stage oocytes were collected using a mouth pipette and washed three times with M2. The harvested MII stage oocytes were placed at 37 ℃ in 5% CO₂An incubator.

2. Zona pellucida of ova

The influence of the pronase concentration on the development of later-stage cloned embryos is influenced, the too low pronase concentration usually eliminates the zona pellucida of the oocytes for too long time, and the too high concentration has certain damage to the oocyte membranes, so that the oocytes die. After the fertilized eggs are treated by pronase with different concentrations to remove the zona pellucida,transferring into normal embryo culture solution, and culturing at 37 deg.C with 5% CO₂And (3) incubating in an incubator, detecting the numbers of secondary cells, quaternary cells, morula and blastocysts in 48h, 56h, 72h and 96h respectively, wherein the in vitro culture blastocyst development rate is shown in table 1:

TABLE 1 Effect of pronase treatment on in vitro embryonic development in mice

Experiments show that the elimination effect is optimal when the concentration of pronase is 0.3-1.0%, and the influence on the development of later-stage cloned embryos is small.

In the embodiment 1 of the invention, 0.5% pronase is adopted to treat the oocytes in the MII stage as shown in figure 1, the oocytes are immediately transferred into 10% FBS fetal bovine serum solution after 10 seconds to stop digestion, and the zona pellucida of all the oocytes is eliminated as shown in figure 2.

3. Manual oocyte cutting

The zona pellucida digested oocytes were transferred into 20. mu.L cutting drops (2.5ug/mL cytochalasin B). The pole body is placed at the position of 6:00 or 12:00 by a cutting knife. The operation under a microscope requires that the egg cells are cut quickly and accurately, and cytoplasm containing polar bodies 1/3-1/2 is discarded after cutting. Several enucleated oocytes were randomly selected and passed through an active Hoechst stain and examined under a fluorescent microscope. The enucleation efficiency of oocytes was counted, and the enucleation rate was the number of enucleations/number of oocytes, and the influence of different treatment methods on the enucleation efficiency is shown in table 2.

TABLE 2 Effect of different treatment regimes on denucleation efficiency

The method for manually removing the nucleus of an oocyte obtains a large number of enucleated oocytes in a short time. The method can provide sufficient material sources for cloned animals, has the advantages of simple operation and short time consumption, and becomes a prerequisite for popularization and application.

Meanwhile, in the embodiment of the invention, the pronase with the concentration of 0.3-1.0% is adopted to treat the egg cells, a plurality of enucleated egg cells are randomly selected to pass through an active Hoechst stain, and the enucleated egg cells are detected in a fluorescence microscope. The enucleation efficiency of oocytes was counted, and the enucleation rate was defined as the number of enucleations/number of oocytes, and the influence of pronase of different concentrations on the enucleation efficiency is shown in table 3.

TABLE 3 Effect of varying concentrations of pronase on efficiency of enucleation

The pronase with different concentration has certain influence on the enucleation rate of the oocyte, but the enucleation rate after the pronase treatment is obviously higher than that without the pronase treatment.

Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to these examples; within the idea of the invention, also features in the above embodiments or in different embodiments may be combined, steps may be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.

The embodiments of the invention are intended to embrace all such alternatives, modifications and variances that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, substitutions, improvements and the like that may be made without departing from the spirit and principles of the invention are intended to be included within the scope of the invention.

Claims

1. A method for removing the nucleus of an egg cell, characterized in that it comprises the steps of obtaining mature oocytes in vitro, removing the zona pellucida of the egg cell, and cutting the oocyte to remove the nucleus.

2. The method for removing the nucleus of an egg cell according to claim 1, wherein the acquisition of the matured oocyte in vitro comprises the steps of: selecting a healthy, 5-8 week old female mouse in the proestrus stage, and removing the oocyte by aseptic surgery. Take the ampulla of the fallopian tube, rinse it with M2 for 2 to 3 times, clip the fallopian tube and cut the ampulla of the fallopian tube with a syringe needle under the microscope, collect the cumulus-oocyte complex, transfer the complex to hyaluronidase and slowly pipette The granulosa cells were removed; the MII-stage oocytes were collected and washed 3-4 times with M2, and the obtained MII-stage oocytes were placed in an incubator.

3. the method for removing egg cell nucleus according to claim 1, is characterized in that, described removing egg cell zona pellucida, the oocyte of described MII stage adopts pronase treatment to after zona pellucida digestion, is transferred into FBS Digestion was terminated in fetal bovine serum solution, and the zona pellucida-depleted oocytes were placed in an incubator.

4 . The method for removing the nucleus of an egg cell according to claim 3 , wherein the concentration of the pronase is 0.3-1.0%. 5 .

5 . The method of claim 3 , wherein the concentration of the FBS fetal bovine serum is 5-15%. 6 .

6. The method for removing the nucleus of an egg cell according to claim 1, wherein the cutting and removing the nucleus of the oocyte comprises the steps of: moving the digested oocyte of the zona pellucida into a cutting drop, and quickly cutting it under a microscope For egg cells, the cytoplasm containing 1/3 to 1/2 of the polar body was discarded after cutting.

7 . The method of claim 6 , wherein the cutting drop is 2.0-3.0 ug/mL cytochalasin B. 8 .

8 . The method for removing egg cell nucleus according to claim 2 or 3 , wherein the temperature of the incubator is 35-40° C., and the CO ₂ content is 3-8%. 9 .