CN116286616A - Additive for improving oocyte in-vitro maturation quality and application thereof - Google Patents

Additive for improving oocyte in-vitro maturation quality and application thereof Download PDF

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CN116286616A
CN116286616A CN202310357405.XA CN202310357405A CN116286616A CN 116286616 A CN116286616 A CN 116286616A CN 202310357405 A CN202310357405 A CN 202310357405A CN 116286616 A CN116286616 A CN 116286616A
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anserine
oocyte
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oocytes
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李紫聪
李亚楠
梁雅琳
张宇星
招华兴
张贤君
吴珍芳
蔡更元
洪林君
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South China Agricultural University
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Abstract

The invention discloses an additive Anserine (Anserine) for improving the in-vitro maturation quality of oocytes, which can effectively improve the in-vitro maturation quality of oocytes by adding Anserine (Anserine) into an in-vitro culture solution of oocytes, thereby improving the in-vitro maturation rate of oocytes and the development capability of subsequent embryos. Through the continuous development and progress of the technology, the quality of oocytes of human beings, cattle, sheep, mice and the like can be improved, and the in-vitro maturation and the development of subsequent embryos can be effectively promoted, so that the production and the application of the in-vitro embryos can be carried out; and a foundation is laid for improving the existing oocyte in-vitro maturation culture system in the auxiliary reproduction field, and promoting the commercialized application and biomedical development of the oocyte in-vitro maturation culture system.

Description

Additive for improving oocyte in-vitro maturation quality and application thereof
Technical Field
The invention relates to the technical field of cell culture, in particular to an additive for improving oocyte in-vitro maturation quality and application thereof.
Background
The in vitro maturation (in vivo maturation, IVM) technology of oocytes aims at culturing immature oocytes into mature oocytes with fertilization and embryo development capabilities through in vitro suitable culture conditions, and is an important link for preparing cloned embryos, in vitro fertilized embryos and parthenogenetic embryos. Oocyte in vitro maturation culture can be used for producing a large amount of high-quality embryos in vitro in animal husbandry, so that the production cost is reduced, excellent breeding animals are efficiently propagated, and the variety improvement is accelerated. The method can fully utilize undeveloped biological resources in human assisted reproduction, and can assist in treating ovarian hyperstimulation syndrome (OHSS) so as to improve fertility. In addition, in scientific research, the oocyte in-vitro maturation culture can be used for researching the mechanism of oocyte aging and embryo dysplasia and the reprogramming mechanism of somatic cells.
At present, although oocyte in vitro maturation culture technology is widely applied in the fields of livestock raising, assisted reproduction and life science, in vitro culture is difficult to simulate the microenvironment of in vivo follicles, and oocytes are easily affected by oxidative stress in the in vitro culture process, so that the quality and in vitro maturation rate of oocytes in vitro culture are inferior to those of in vivo oocytes. Oocyte quality is a critical factor in determining in vitro maturation and subsequent embryo development capacity. In addition, the lack of antioxidant enzymes and ROS scavengers during in vitro maturation culture of oocytes, the in vitro cultured oocytes are relatively weak in their ability to resist oxidative stress, which affects the quality of the oocytes. Therefore, the method has great significance in deeply exploring the oocyte maturation rule, improving the oocyte in-vitro maturation culture condition, and improving the oocyte in-vitro maturation quality and the subsequent embryo development efficiency.
Anserine (Anserine, beta-alanyl-1-methyl L-histidine) is a highly stable water-soluble dipeptide that naturally occurs in skeletal muscle tissue and brain tissue of vertebrates, with PK values of about 7.1, and remains intact at low pH (< 3.0), by promoting uric acid excretion, alleviating hyperuricemia.
Disclosure of Invention
The invention aims to provide an additive for improving the in-vitro maturation quality of oocytes and application thereof, so as to improve the maturation quality of in-vitro oocyte culture.
According to a first aspect of the present invention there is provided an additive for improving the quality of oocyte maturation in vitro, the additive being anserine. Therefore, by adding the anserine into the oocyte in-vitro culture solution, the in-vitro maturation quality of the oocyte can be improved.
According to a second aspect of the present invention, there is provided the use of anserine as an additive in the preparation of a product having the effect of improving the quality of oocyte maturation in vitro. Therefore, the product containing the anserine can improve the in-vitro maturation quality of the oocyte in the in-vitro maturation culture of the oocyte.
According to a third aspect of the present invention there is provided an oocyte in vitro maturation broth comprising the components of: 0.6mM cysteine, 0.1IU/Ml human chorionic gonadotropin, 0.1IU/Ml equine chorionic gonadotropin, 10% foetal calf serum, 10% follicular fluid and anserine were added to TCM199 medium. Therefore, the culture solution can improve the maturation quality of oocytes cultured in vitro.
In certain embodiments, the anserine concentration is 0.01-10mM.
According to a fourth aspect of the present invention there is provided the use of an oocyte in vitro maturation medium for improving the quality of oocyte in vitro maturation.
According to a fifth aspect of the present invention there is provided the use of an oocyte in vitro maturation broth for increasing the mitochondrial membrane potential level of an oocyte.
According to a sixth aspect of the present invention there is provided the use of an oocyte in vitro maturation medium for reducing ROS levels in porcine oocytes.
According to a seventh aspect of the present invention there is provided the use of an oocyte in vitro maturation medium in the culture of parthenogenetic embryos.
According to an eighth aspect of the invention, there is provided the use of anserine for improving the quality of oocyte maturation in vitro.
According to a ninth aspect of the invention, there is provided the use of anserine in the culture of parthenogenetic embryos.
According to a tenth aspect of the present invention there is provided the use of anserine for increasing the mitochondrial membrane potential level of an oocyte.
According to an eleventh aspect of the present invention there is provided the use of anserine for reducing ROS levels in porcine oocytes.
The invention has the beneficial effects that: by adding Anserine (Anserine) into the oocyte in-vitro culture solution, the in-vitro maturation quality of the oocyte can be effectively improved, and the in-vitro maturation rate of the oocyte and the development capability of subsequent embryos are further improved. Through the continuous development and progress of the technology, the quality of oocytes of human beings, cattle, sheep, mice and the like can be improved, and the in-vitro maturation and the development of subsequent embryos can be effectively promoted, so that the production and the application of the in-vitro embryos can be carried out; and a foundation is laid for improving the existing oocyte in-vitro maturation culture system in the auxiliary reproduction field, and promoting the commercialized application and biomedical development of the oocyte in-vitro maturation culture system.
Drawings
FIG. 1 is a graph showing the relative levels of Anserine in porcine mature follicles (MFF) and Immature Follicular Fluid (IFF);
FIG. 2 is a graph showing the results of mitochondrial membrane potential levels in oocytes of control and Anserine-treated groups;
FIG. 3 is a graph showing the results of ROS levels in oocytes in the control and Anserine treated groups.
Detailed Description
The invention is described in further detail below with reference to the accompanying drawings.
1. Non-targeted metabonomics detection of follicular fluid
In order to gain insight into the in vitro maturation process of oocytes, non-targeted metabonomic detection analysis was performed on in vivo mature follicular fluid and immature follicular fluid. Collecting ovaries of oestrus sows by an operation method to obtain in-vivo mature follicular fluid, collecting 3-8 mm follicles in ovaries of non-oestrus sows in vitro to obtain the immature follicular fluid, preparing an on-machine sample, and performing mass spectrometry detection. In the metabonomics test results, the relative levels of Anserine (Anserine, beta-alanyl-1-methyl L-histidine) in mature follicular fluid in vivo were found to be significantly higher than in immature follicular fluid, approximately 2.5 times that in immature follicular fluid (as shown in FIG. 1). Anserine is presumed to play an important role in oocyte maturation.
2. Preparation method of in vitro maturation culture solution of porcine oocytes
a. Preparation method of basal maturation culture solution (control group): TCM199 Medium was supplemented with 0.6mM cysteine, 0.1IU/Ml human chorionic gonadotropin, 0.1IU/Ml equine chorionic gonadotropin, 10% (V/V) fetal bovine serum, 10% (V/V) slaughterhouse derived porcine follicular fluid.
b. Preparation method of Anserine maturation culture solution (Anserine treatment group): anserine (Anserine) is added into the basic maturation culture solution in a concentration of 0.01mM, 0.1mM, 1mM, 5mM and 10mM to prepare the Anserine maturation culture solutions with different concentrations.
3. Oocyte in vitro maturation culture
a. Collection of oocytes
The collected pig ovaries were soaked in physiological saline (penicillin, streptomycin sulfate) at 25-35 ℃ and returned to the laboratory within 3 hours. Washing pig ovary with normal saline preheated to 37deg.C for 3-5 times, taking out follicular fluid from 2-6mm follicle with 10mL syringe (with 18-gauge needle), naturally precipitating for 15-20min, removing supernatant, re-suspending precipitate with DPBS containing double antibody, standing for 3-5min, removing supernatant again, and repeating for 3 times. High quality cumulus-oocyte complexes (COCs) with at least 3 layers of cumulus cells packed were picked under a stereoscopic microscope, and the high quality COCs were washed 3 times with DPBS containing a double antibody. The treated COCs are respectively put into the basic maturation prepared in the step 2 in equal quantityIn culture (control) or Anserine maturation culture (Anserine treated) containing different concentrations of Anserine, then at 38.5℃in 5% CO 2 Culturing in a saturated humidity incubator for 42-44h.
b. Picking up MII-stage oocytes with polar bodies
Will already be at 38.5℃with 5% CO 2 Placing oocytes cultured in an incubator with saturated humidity for 42-44h into a 1.5mL centrifuge tube containing 1mg/mL hyaluronidase, repeatedly blowing a pipette gun for 70-90 times to remove surrounding cumulus cells, washing with operating fluid for 6 times until the cumulus cells are washed clean, selecting oocytes containing a first polar body under a stereoscope for standby, recording the number of the oocytes containing the first polar body, and calculating the first polar body discharge rate, namely the maturation rate, of the oocytes of a control group and an Anserine treatment group, wherein the first polar body discharge rate, namely the maturation rate, can be used as an important index for evaluating the quality of the oocytes.
The results are shown in Table 1: compared with a control group, the in vitro maturation rate of oocytes can be improved to different degrees by adding Anserine (Anserine) with different concentrations into the in vitro maturation culture solution:
anserine with the concentration of 0.01mM is added, so that the in-vitro maturation rate of the oocyte is improved by 10.70%;
anserine with the concentration of 0.1mM is added, so that the in-vitro maturation rate of the oocyte is improved by 9.07%;
anserine with the concentration of 5mM is added, so that the in-vitro maturation rate of the oocyte is improved by 22.17%;
anserine with the concentration of 10mM is added, and the in-vitro maturation rate of the oocyte is improved by 30.31 percent.
TABLE 1 influence of Anserine addition on in vitro maturation rate of porcine oocytes in vitro maturation culture
Figure BDA0004163782870000041
Note that: the same column data shoulder marks are obviously different in lower case letters (P < 0.05)
4. Preparation and in vitro maturation culture of pig parthenogenetic embryo
The control group and Anserine were obtained separatelyThe mature oocytes containing the first polar body in the group are put into activating solution for washing 3-6 times. Opening a power supply of a fusion instrument, placing a fusion tank, electrifying the fusion tank, placing 10-15 mature oocytes into the fusion tank each time by using a hand sucking needle, enabling the oocytes to be parallel to an electrode and not contact with the wall of the fusion tank, activating the arranged oocytes under the direct current pulse of 80v/mm, 80us and 2DC, and repeating the steps until all the mature oocytes containing the first polar body are activated. Incubating the activated oocytes in a CB-assisted activation solution prepared in advance, transferring into a four-well plate added with PZM embryo culture solution after 4 hours, and placing the four-well plate at 38.5deg.C and 5% CO 2 Culturing in an incubator with saturated humidity. PZM the culture medium comprises the following components: naCl 108.00mM, KCl 10.00mM, KH 2 PO 4 0.35mM、MgSO 4 ·7H 2 O 0.40mM、NaHCO 3 25.07mM, sodium Pyruvate (Na-Pyruvate) 0.20mM, calcium lactate 2.00mM, taurine (Hypotaurine) 5.00mM, L-glutamine 1.00mM, essential amino acids 20mL/L, and nonessential amino acids 10mL/L.
The day of parthenogenesis (0 d) was started, the number of embryos that had been split was observed and recorded at 2d, the number of embryos that had developed to blasts was observed and recorded at 6d, and the rates of split and blasts were calculated for the control and Anserine treated groups. In addition, blastocysts were subjected to nuclear staining in host 33342, and observed and photographed under a fluorescence microscope, and the number of blastoblasts was counted.
The results are shown in table 2: in Anserine treated group relative to control group
Anserine with the concentration of 0.01mM is added, so that the parthenogenetic embryo cleavage rate of the oocyte is improved by 5.70%, and the blastula rate is improved by 13.96%.
Anserine with the concentration of 0.1mM is added, so that the parthenogenetic embryo cleavage rate of the oocyte is improved by 8.95%, and the blastula rate is improved by 21.45%.
Anserine with the concentration of 1mM is added, and the parthenogenetic embryo blastula rate of the oocyte is improved by 3.61 percent.
Anserine with the concentration of 5mM is added, so that the parthenogenetic embryo cleavage rate of the oocyte is improved by 13.18%, and the blastula rate is improved by 41.03%.
Anserine with the concentration of 10mM is added, so that the parthenogenetic embryo cleavage rate of the oocyte is improved by 15.03%, and the blastula rate is improved by 23.92%.
TABLE 2 influence of Anserine addition on parthenogenetic embryo development efficiency in vitro maturation culture of porcine oocytes
Figure BDA0004163782870000051
Note that: the same column of data shoulder marks are obviously different in lower case letters (P < 0.05); the shoulder marks have extremely remarkable difference in the capital letters (P < 0.01)
Based on the results in tables 1 and 2, a concentration set of 5mM anserine (anserine) was selected for further experiments in the in vitro maturation medium.
5. Effect of addition of Anserine (Anserine) at 5mM concentration to oocyte mitochondrial membrane potential in vitro maturation medium.
Mitochondrial membrane potential levels, i.e., JC-1 levels, are often used as an indicator of oocyte quality, and the ratio of red fluorescence to green fluorescence is often used to indicate the functional status of mitochondria. FIG. 2 shows that Anserine treated oocytes have a red fluorescence intensity higher than that of the control group and a red-green fluorescence ratio (i.e., JC-1 level) significantly higher than that of the control group. This indicates that an Anserine concentration of 5mM significantly increases the mitochondrial membrane potential level of the oocyte, thereby improving the quality of the oocyte.
6. Results regarding the effect of addition of Anserine (Anserine) at a concentration of 5mM to the in vitro maturation broth on the ROS levels of oocytes.
ROS levels are also a key indicator of oocyte quality, with stronger green fluorescence representing higher ROS levels. FIG. 3 shows that the green fluorescence intensity of Anserine treated oocytes was very significantly reduced compared to the control group. This indicates that Anserine at 5mM concentration significantly reduced ROS levels in porcine oocytes, enhancing the ability of porcine oocytes to resist oxidative stress, and thus improving oocyte quality.
In conclusion, the quality of the oocyte can be effectively improved by adding Anserine into the oocyte in-vitro maturation culture solution, so that the in-vitro maturation rate of the oocyte and the development capability of a subsequent embryo are improved. Through the continuous development and progress of the technology, the quality of oocytes of human beings, cattle, sheep, mice and the like can be improved, the in-vitro maturation and the development of subsequent embryos can be effectively promoted, and thus the production and the application of the in-vitro embryos can be carried out, and the foundation is laid for improving the existing oocyte in-vitro maturation culture system, promoting the commercialization application and the biomedical development in the auxiliary reproduction field.
What has been described above is merely some embodiments of the present invention. It will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the spirit of the invention.

Claims (10)

1. An additive for improving the in vitro maturation quality of oocytes, wherein the additive is anserine.
2. Use of an additive as claimed in claim 1 in the preparation of a product having the effect of improving the quality of oocyte maturation in vitro.
3. An oocyte in vitro maturation medium comprising the additive of claim 1, wherein the components of the medium include: 0.6mM cysteine, 0.1IU/Ml human chorionic gonadotropin, 0.1IU/Ml equine chorionic gonadotropin, 10% foetal calf serum, 10% follicular fluid and anserine were added to TCM199 medium.
4. The culture broth of claim 3, wherein the anserine concentration is 0.01-10mM.
5. Use of the culture broth of claim 3 or 4 for improving the quality of oocyte maturation in vitro;
preferably, the use in increasing the mitochondrial membrane potential level of an oocyte;
preferably, the use for reducing ROS levels in porcine oocytes.
6. Use of the culture solution according to claim 3 or 4 for the culture of parthenogenetic embryos.
7. Application of anserine in improving oocyte in-vitro maturation quality.
8. Application of anserine in parthenogenetic embryo culture is provided.
9. Application of anserine in improving oocyte mitochondrial membrane potential level is provided.
10. Use of anserine for reducing ROS levels in porcine oocytes.
CN202310357405.XA 2023-04-06 2023-04-06 Additive for improving oocyte in-vitro maturation quality and application thereof Pending CN116286616A (en)

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