CN113444683A - Additive for improving oocyte in-vitro maturation quality, culture method and application - Google Patents
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Abstract
The invention discloses an additive for improving the in vitro maturation quality of oocytes, which improves the in vitro culture quality of the oocytes by adding a PF4 protein additive into an in vitro oocyte culture solution. The method is further applied to the fields of oocyte in-vitro maturation, somatic cell cloning technology and parthenogenetic embryo by improving the quality of oocyte in-vitro culture. On one hand, the in vitro maturation of oocytes of various species can be promoted, in vitro fertilized embryos and cloned embryos with better quality can be prepared, and the in vitro maturation culture of oocytes for human assisted reproduction is facilitated; on the other hand, the prepared high-quality embryo and the animal produced by the high-quality embryo are used for research in the fields of medicine, life science, animal husbandry and the like, and the development of production practice is further promoted.
Description
Technical Field
The invention relates to an oocyte in-vitro culture technology, in particular to an additive for improving oocyte in-vitro maturation quality, a culture method and application.
Background
In Vitro Maturation (IVM) of oocytes refers to taking immature oocytes from ovarian follicles, culturing the immature oocytes in vitro maturation culture medium, and simulating the environment of the ovarian follicles in vivo as much as possible to mature the oocytes and obtain fertilization ability. The technology has very important application value in the fields of life science, medicine, human assisted reproduction, animal husbandry and the like, and is a basic technology capable of obtaining a large number of mature oocytes. In actual production of animal husbandry, mature oocytes are used as recipient cells, somatic cells of animals with excellent characters are used as donor cells, and then a large number of excellent individuals with the same genetic information are obtained through a somatic cell nuclear transfer technology, so that a large number of high-quality breeding stocks can be provided for animal breeding. In addition, with the development of gene editing technology and the application of embryo transfer technology, somatic cell nuclear transfer technology and their combination can prepare bioreactors capable of producing medicines and animal models of human diseases for research. In addition, the in vitro fertilization embryo, parthenogenetic activation embryo and clone embryo are very important research materials, and can be used for researching and exploring life processes and mechanism mechanisms of human and various species, such as life occurrence, embryo development, organ differentiation and the like. However, mature porcine oocytes are used as a raw material, and have high acquisition cost, great acquisition technical difficulty and limited acquisition quantity, which greatly limits the wide application of somatic cell nuclear transfer technology in production application and scientific research. Therefore, IVM technology that can obtain large amounts of mature oocytes using low-cost and difficult methods is becoming increasingly important. Indeed, oocytes matured in vitro are much less of a quality than oocytes matured in vivo, which is also one of the important reasons limiting the broad and efficient use of somatic cell nuclear transfer technology. The deep layer is caused by the fact that the mechanism of normal maturation of the oocyte in vivo is not clear, and factors and substances for promoting maturation of the oocyte in the follicle are not thoroughly researched so far, so that the lack of the substances in vitro maturation culture solution is caused, and the better developmental growth of the oocyte is not supported sufficiently.
Platelet factor 4(PF4) is a protein stored in platelets, capable of binding to heparin and promoting blood coagulation and thrombosis, and has the effects of inhibiting hematopoiesis, promoting inflammation, promoting arteriosclerosis and angiogenesis.
Disclosure of Invention
The invention aims to provide an additive for improving the in vitro maturation quality of oocytes, a culture method and application of the additive in the fields of in vitro maturation of oocytes, somatic cell cloning, parthenogenetic embryos and the like.
In one aspect of the invention, the invention provides an additive for improving the in vitro maturation quality of oocytes, and the additive is PF4 protein.
In certain embodiments, the concentration of PF4 protein is 5-250 ng/mL.
In certain embodiments, the concentration of PF4 protein is 125 ng/mL.
In another aspect of the invention, a method for improving the in vitro maturation quality of the oocyte by adding a PF4 protein additive into an oocyte culture solution or a method for improving the in vitro maturation quality of the oocyte by adding a PF4 protein additive with the concentration of 5-250ng/mL into the oocyte culture solution or a method for improving the in vitro maturation quality of the oocyte by adding a PF4 protein additive with the concentration of 125ng/mL into the oocyte culture solution is provided.
In some embodiments, the methods for improving the quality of oocyte maturation in vitro employ culture medium compositions as follows: 0.1IU/ml human chorion, gonadotropin, 0.1IU/ml pregnant mare serum gonadotropin, 0.6mM cysteine, 10% pig follicular fluid, 10% fetal bovine serum and PF4 protein are added into the TCM-199 culture medium.
In some embodiments, the methods for improving the quality of oocyte maturation in vitro employ culture medium compositions as follows: adding 0.1IU/mL human chorion, gonadotropin, 0.1IU/mL pregnant mare serum gonadotropin, 0.6mM cysteine, 10% pig follicular fluid, 10% fetal calf serum and 5-250ng/mL PF4 protein into TCM-199 culture medium.
In some embodiments, the methods for improving the quality of oocyte maturation in vitro employ culture medium compositions as follows: 0.1IU/mL human chorion, gonadotropin, 0.1IU/mL pregnant mare serum gonadotropin, 0.6mM cysteine, 10% pig follicular fluid, 10% fetal bovine serum and 125ng/mL PF4 protein are added into the TCM-199 culture medium.
The third aspect of the invention provides an application of an additive for improving the in vitro maturation quality of oocytes in vitro maturation culture of oocytes, wherein the additive is PF4 protein; preferably, the additive is 5-250ng/mL PF4 protein; more preferably, the additive is 125ng/mL PF4 protein.
The fourth aspect of the invention provides the application of the additive for improving the in vitro maturation quality of the oocyte in the technical field of somatic cell cloning, wherein the additive is PF4 protein; preferably, the additive is 5-250ng/mL PF4 protein; more preferably, the additive is 125ng/mL PF4 protein.
The fifth aspect of the invention provides an application of an additive for improving the in vitro maturation quality of oocytes in the technical field of parthenogenetic embryos, wherein the additive is PF4 protein; preferably, the additive is 5-250ng/mL PF4 protein; more preferably, the additive is 125ng/mL PF4 protein.
The sixth aspect of the invention provides an application of the additive for improving the in vitro maturation quality of the oocyte in vitro maturation culture of the oocyte of a pig, a mouse, a cow, a sheep, a human or other mammals, wherein the additive is PF4 protein; preferably, the additive is 5-250ng/mL PF4 protein; more preferably, the additive is 125ng/mL PF4 protein.
The invention has the beneficial effects that: the quality of the oocyte in vitro culture is improved by adding PF4 protein into the oocyte culture solution in vitro. The method is further applied to the fields of oocyte in-vitro maturation, somatic cell cloning technology and parthenogenetic embryo by improving the quality of oocyte in-vitro culture. On one hand, the in vitro maturation of oocytes of various species can be promoted, in vitro fertilized embryos and cloned embryos with better quality can be prepared, and the in vitro maturation culture of oocytes for human assisted reproduction is facilitated; on the other hand, the prepared high-quality embryo and the animal produced by the high-quality embryo are used for research in the fields of medicine, life science, animal husbandry and the like, and the development of production practice is further promoted.
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FIG. 1 is a graph showing the relative levels of PF4 protein in follicular fluid at the in Vivo Maturation (VMF) and immature in Vivo (VIF) stages.
Detailed Description
The present invention will be described in further detail with reference to examples.
1. Quantitative proteomics detection of follicular fluid
The follicular fluid before and after in vivo maturation is detected by using quantitative proteomic mass spectrometry so as to find out which factors are significantly changed in the follicular maturation process. Collecting in-vivo mature follicular fluid in the ovary of the oestrous sow and in-vivo immature follicular fluid in the ovary of the oestrous sow by a surgical method, wherein the immature follicles are selected from follicles with the diameter of 3-8 mm. Then removing the high-abundance proteins in the two follicular fluids by using a high-abundance protein kit, preparing an upper computer sample, and performing quantitative proteomics mass spectrometry detection, wherein the result shows that: the expression level of platelet factor 4(PF4) was very significantly increased, and the relative expression level of PF4 protein in mature follicular fluid in vivo was 4 times greater than that in immature follicular fluid in vivo (the results are shown in fig. 1). Furthermore, the mature follicle is 3-5 times the diameter of the immature follicle, i.e., the absolute level of PF4 in the follicle differs by at least 100 times, so it is reasonable to assume that PF4 protein is a substance that plays an important role in the maturation of oocytes.
2. Preparation of pig oocyte in vitro maturation culture solution
To further investigate the role of the PF4 protein in the in vitro maturation of oocytes, the following culture media were prepared:
a. basic maturation culture solution: 0.1IU/ml human chorion, gonadotropin, 0.1IU/ml pregnant mare serum gonadotropin, 0.6mM cysteine, 10% (V/V) slaughterhouse derived pig follicular fluid and 10% (V/V) fetal bovine serum are added into the TCM-199 culture medium.
PF4 maturation medium: the basal maturation solution was supplemented with PF4 protein at 5ng/mL, 25ng/mL, 125ng/mL, and 250ng/mL, respectively.
3. In vitro maturation culture of oocytes
Selecting follicle with diameter of 3-8mm, extracting oocyte with syringe and needle, screening oocyte, selecting cumulus cell-oocyte complex with more than three layers of cumulus cells and with black round oocyte cytoplasm, uniform oocyte cytoplasm and clear cytoplasm boundary, adding cumulus cell-oocyte complex into the basic maturation culture solution prepared in step 2 or PF4 maturation culture solution containing PF4 protein with different concentrations, and adding 5% CO2And cultured in an incubator at 38.5 ℃ for 44 hours. After 44 hours of culture, cumulus cell-oocyte complexes were removed, adherent cumulus cells were removed with hyaluronidase, and then polar-depleted and cytoplasm-homogeneous oocytes were picked up for use in embryosAnd (5) building a tire.
4. Construction of pig parthenogenetic embryo and cloned embryo and embryo in vitro culture
a. Construction of a pig parthenogenetic embryo: adding the preheated electrical activation liquid into a fusion tank, placing the mature oocytes obtained in the step 3 into the electrical activation liquid for balancing for 2min, then placing the oocytes into the fusion tank, and activating the oocytes by using 510V, 60 mu s and 2 DC. The electrically activated oocytes were washed 3 times with PZM embryo culture fluid and then transferred to four well plates with PZM embryo culture fluid added for culture. The PZM culture solution comprises the following components: 108.00mM NaCl, 10.00mM KCl, KH2PO40.35 mM、MgSO4·7H2O 0.40mM、NaHCO325.07 mM, sodium Pyruvate (Na-Pyruvate)0.20mM, calcium lactate 2.00mM, taurine (Hypotaurin) 5.00mM, L-glutamine 1.00mM, essential amino acid 20mL/L, and non-essential amino acid 10 mL/L.
b. Construction of porcine cloned embryos: preparing donor cells of a 1-plate 6cm culture dish in advance, removing culture solution when the cells grow to 80-90%, gently cleaning the donor cells with DPBS for 2 times, adding the donor cells into preheated trypsin digestion solution, adding 1mL of cell culture solution into the wells in an ultraclean workbench, blowing the bottom of the culture dish for several times by using a pipette gun, transferring cell suspension into a 15mL centrifuge tube, and centrifuging the cell suspension for 5min at 800rpm by using a centrifuge. The supernatant was discarded, 1mL of the culture medium was added, and the cells were resuspended by pipetting with a pipette.
Removing the nucleus of the oocyte obtained in the step 3, then injecting donor cells with better shapes into the perivitelline space of the oocyte, after all the enucleated oocytes are injected, cleaning the electrofusion groove by using fusion-activating liquid, adding 500 mu L of the fusion-activating liquid, placing 10 nucleus-injected eggs into the electrofusion groove, enabling the axis of an egg receptor-nucleus donor to be vertical to an electrode by rotating a glass fine needle, and carrying out fusion activation by using 510V, 50 mu s and 2 DC.
c. Embryo in vitro culture: after the embryos were constructed, the embryos were placed in four-well plates containing PZM embryo medium and placed in 5% CO2And culturing in an incubator at 38.5 ℃, and recording the cleavage number, blastocyst number and other data after culturing for 48h and 168h respectively.
5. Effect of supplementing PF4 protein in maturation culture solution during IVM on oocyte quality
5.1 Experimental results of parthenogenetic embryos prepared from oocytes treated with PF4 protein
Effect of supplementation of PF4 protein in maturation medium on oocyte quality during IVM. Compared with a control group, the parthenogenetic embryo prepared from the oocyte treated by the PF4 protein is added with a PF4 protein treatment group of 125ng/mL to form an optimal concentration group, the cleavage rate is improved by 3.72%, and the blastocyst rate is improved by 40.15%; in addition, supplementing other concentrations of PF4 protein also increased embryo cleavage and blastocyst rates to varying degrees. The detailed results are shown in table 1 below:
table 1: effect of different concentrations of PF4 protein on parthenogenetic embryos
From the results in the above table, the 125ng/mL PF4 protein-treated group was the optimal concentration group, and therefore, this example demonstrated the effect of 125ng/mL PF4 protein-treated oocytes on their parthenogenetic embryos.
5.2 Experimental results of cloned embryos prepared from oocytes treated with PF4 protein
Since the best treatment effect of 125ng/ml PF4 protein was obtained from parthenogenetic embryos, cloned embryos were prepared by treating oocytes with 125ng/ml PF4 protein. Finally, compared with a control group, the cleavage rate of the cloned embryo of the 125ng/mL PF4 protein treated group is improved by 8.48%, the 4-cell stage rate is improved by 3.91%, and the blastocyst rate is improved by 52.62%. The detailed results are shown in table 2 below:
table 2: effect of different concentrations of PF4 protein on cloned embryos
5.3 Experimental results of adding PF4 protein into in vitro maturation culture solution to prepare mature oocytes
The oocyte maturation rate is one of the important indicators for measuring the quality of oocytes, especially in the context of in vitro maturation of oocytes. Oocyte maturation rate refers to the rate at which the nucleus of the oocyte matures and exits the first polar body. The experimental result of the part is also obtained, the mature rate of the oocytes in the treatment group with the optimal concentration (125ng/mL PF4 protein treatment group) is increased by 11.87% compared with that in the control group by supplementing PF4 protein into the mature culture solution in the IVM process, and the mature rate of the oocytes in the other concentration treatment groups is increased to different degrees. The detailed results are shown in table 3 below:
table 3: oocyte maturation rate results of different concentrations of PF4 protein-treated group
In conclusion, the oocyte quality can be effectively improved by supplementing the PF4 protein in the oocyte in-vitro maturation solution. By improving and innovating technology, on one hand, the in-vitro maturation of oocytes of various species can be promoted, in-vitro fertilized embryos and cloned embryos with better quality can be prepared, and the in-vitro maturation culture of the oocytes for human assisted reproduction is facilitated; on the other hand, the prepared high-quality embryo and the animal produced by the high-quality embryo are used for research in the fields of medicine, life science, animal husbandry and the like, and the development of production practice is further promoted.
What has been described above is but some implementations of the invention. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the inventive concept thereof, and these changes and modifications can be made without departing from the spirit and scope of the invention.
Claims (10)
1. An additive for improving the in vitro maturation quality of oocytes, wherein the additive is PF4 protein.
2. The additive for improving oocyte in vitro maturation quality according to claim 1, wherein the concentration of PF4 protein is 5-250 ng/mL.
3. The additive for improving oocyte in vitro maturation quality according to claim 2, wherein the concentration of PF4 protein is 125 ng/mL.
4. A method for improving the quality of oocyte maturation in vitro by adding an additive according to any one of claims 1 to 3 to the oocyte culture fluid.
5. The method for improving the quality of oocyte maturation in vitro according to claim 4, wherein the method uses the following culture medium components: 0.1IU/ml human chorion, gonadotropin, 0.1IU/ml pregnant mare serum gonadotropin, 0.6mM cysteine, 10% pig follicular fluid, 10% fetal bovine serum and PF4 protein are added into the TCM-199 culture medium.
6. The method for improving the quality of oocyte maturation in vitro according to claim 5, wherein the concentration of PF4 protein is 5-250 ng/mL;
preferably, the concentration of PF4 protein is 125 ng/mL.
7. Use of an additive according to any one of claims 1 to 3 for improving the quality of in vitro maturation of oocytes in the culture of in vitro maturation of oocytes.
8. Use of the additive according to any one of claims 1 to 3 for improving the quality of oocyte maturation in vitro in the field of somatic cell cloning technology.
9. The use of the additive according to any one of claims 1 to 3 for improving the quality of in vitro maturation of oocytes in the field of parthenogenetic embryo technology.
10. Use of the additive according to any one of claims 1 to 3 for improving the quality of oocyte in vitro maturation in the culture of oocytes of pigs, mice, cattle, sheep, humans or other mammals in vitro maturation.
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| CN114214269A (en) * | 2021-12-17 | 2022-03-22 | 浙江大学 | Porcine embryo culture solution and application |
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