KR101808934B1 - Method for preventing of oocyte aging and enhancing developmental rate utilizing melatonin - Google Patents
Method for preventing of oocyte aging and enhancing developmental rate utilizing melatonin Download PDFInfo
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- KR101808934B1 KR101808934B1 KR1020160002128A KR20160002128A KR101808934B1 KR 101808934 B1 KR101808934 B1 KR 101808934B1 KR 1020160002128 A KR1020160002128 A KR 1020160002128A KR 20160002128 A KR20160002128 A KR 20160002128A KR 101808934 B1 KR101808934 B1 KR 101808934B1
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Abstract
The present invention relates to a method for preventing aging of an oocyte and improving an embryo development rate using the melatonin, and more particularly, to a method for improving an aging oocyte by treating melatonin to reduce reactive oxygen species (ROS) And to a method for preventing aging of the oocyte by increasing the expression of actin filaments of senescent oocyte and improving the embryo development rate.
The present invention relates to a method for improving the quality of oocytes by preventing melatonin from aging of oocytes matured over time and improving the quality of the in vitro culture embryo and prolonging the culture period in vitro, Thereby improving the development rate.
Description
The present invention relates to a method for preventing aging of an oocyte and improving an embryo development rate using the melatonin, and more particularly, to a method for improving an aging oocyte by treating melatonin to reduce reactive oxygen species (ROS) And to a method for preventing aging of the oocyte by increasing the expression of actin filaments of senescent oocyte and improving the embryo development rate.
In vitro maturation of oocytes is the basis for transgenic cloned embryos. Therefore, a good in vitro maturation system can be regarded as the basis for transgenic cloned embryos. In particular, it is an important task to prevent aging of oocytes because transformed nuclear transfer embryos take time in micro manipulation process. In addition, it is very difficult to synchronize the maturation of oocytes in the in vitro maturation process of pig ova, and it causes the aging process of the oocyte to be easily induced, thereby reducing or losing developmental capacity. The best in vitro maturation system of the egg is still under development.
Melatonin is a molecule related to the cycle of biological response, also known as N-acetyl-5-methoxy tryptamine. In mammals, melatonin is synthesized from the pineal gland present in the brain and secreted into the blood. It adjusts the rhythm of the body and makes us sleep at night. Depression occurs when the amount of synthesis is too much or works in vivo for a long time, and when the amount of synthesis is low, insomnia is caused. Melatonin is derived from tryptophan and may be synthesized in the same way as the bone marrow cell lymphocyte epithelial cells. Melatonin is known to regulate the West cadadian rhythm, such as photoperiod, by its secreted secretory properties in the dark, to regulate the cell cycle in mammals, and to show strong resistance to oxidative stress and apoptosis.
In vitro matured oocytes develop aging in comparison with matured oocytes in the body, poor quality, and low fertility after fertilization. This aged oocyte also affects the developmental ability of fertilized and nuclear-substituted embryos, which is fatal in the early pregnancy and is also likely to be abnormal in birth. Aging of ova is a very complex physiological process, which is also related to cytology and molecular biology. Currently known information is known to cause the accumulation of oxidative shock in the cell, which leads to such inhibition of development. Recently, melatonin has been shown to be multifunctional and has the highest antioxidant effect, so it has been found that melatonin is used when mammalian eggs are matured in vitro.
It is an object of the present invention to provide a method for preventing aging of mammalian in vitro matured oocytes and a method for improving mammalian embryo development rate using a medium containing melatonin.
Another object of the present invention is to provide a medium composition for preventing aging of mammalian in vitro matured oocytes containing melatonin and a medium composition for improving mammalian embryo development rate.
In order to accomplish the object of the present invention, the present invention provides a method for preparing an oocyte, comprising: (a) preparing an in vitro maturated aged oocyte of a mammal other than a human; And (b) culturing the in vitro maturated aged oocyte in a medium containing melatonin. The present invention also provides a method of preventing oocyte maturation in mammals other than humans.
In one embodiment of the present invention, the concentration of the melatonin contained in the medium in the step (b) may be 10 -6 M (1 uM) to 10 -3 M (1 mM), but is not limited thereto.
In one embodiment of the present invention, in step (a), the mammal other than the human may be a pig, a sheep, a dog, a horse, or a goat, but is not limited thereto.
In one embodiment of the present invention, reactive oxygen species (ROS) may be reduced or DNA damage may be reduced in the in vitro maturated aged oocytes.
Also, in order to achieve the above object, the present invention provides a method for preparing an oocyte, comprising: (a) preparing an in vitro maturated aged oocyte of a mammal other than a human; (b) culturing said in vitro maturated aged oocyte in a medium containing melatonin; (c) in vitro fertilization of said in vitro maturated aged oocytes to produce embryos; And (d) culturing the embryo. The present invention also provides a method for improving the mammalian embryo development rate.
In one embodiment of the present invention, the concentration of the melatonin contained in the medium in the step (b) may be 10 -6 M (1 uM) to 10 -3 M (1 mM), but is not limited thereto.
In one embodiment of the present invention, in step (a), the mammal other than the human may be a pig, a sheep, a dog, a horse, or a goat, but is not limited thereto.
In one embodiment of the present invention, the embryo development rate may be the rate of development of the embryo and the blastocyst.
In addition, to achieve the object of the present invention, there is provided a medium composition containing melatonin, wherein the medium composition for preventing aging of mammalian in vitro matured oocytes excluding humans is provided.
In one embodiment of the present invention, the concentration of melatonin may be 10 -6 M (1 uM) to 10 -3 M (1 mM), but is not limited thereto.
In addition, to achieve the object of the present invention, there is provided a culture composition containing melatonin, which comprises a medium composition for improving the mammalian embryo development rate except for human.
In one embodiment of the present invention, the concentration of melatonin may be 10 -6 M (1 uM) to 10 -3 M (1 mM), but is not limited thereto.
The present invention relates to a method for improving the quality of oocytes and improving the quality of in vitro culture embryos by preventing melatonin from aging of oocytes matured over time, thereby prolonging the incubation period in vitro, Can be improved.
In addition, the present invention can be applied to in vitro fertilization, oocyte sperm microinjection, somatic cell nuclear exchange, and development of an optimal culture medium for in vitro maturation and in vitro culture.
Figure 1 shows the developmental rate of the in vitro maturation embryo according to the concentration of melatonin.
FIG. 2 shows the developmental stages of the in vitro maturation embryo according to the melatonin treatment.
Figure 3 shows changes in the ROS of oocytes matured after melatonin treatment.
FIG. 4 shows the degree of DNA damage of oocytes matured in vitro when treated with melatonin.
Figure 5 shows that when melatonin is treated, the ratio of abnormal oocytes is reduced during in vitro maturation.
Figure 6 shows the effect of melatonin on the expression of actin filaments, formation of abnormal microtubules and release of surface granules of aged oocytes.
Figure 7 shows the effect of melatonin on the in vitro development rate of oocytes aged and cell death in blastocysts.
Hereinafter, a method for preventing aging of mammalian in vitro matured oocytes according to an embodiment of the present invention and a media composition for preventing aging of mammalian in vitro matured oocytes will be described in detail.
Further, a method for improving mammalian embryo development rate and a medium composition for improving mammalian embryo development rate according to an embodiment of the present invention will be described in detail.
The present invention relates to a method for preventing aging of mammalian in vitro matured oocytes, excluding humans, comprising the following steps.
(a) preparing an in vitro maturated aged oocyte of a mammal other than a human; And
(b) culturing said in vitro maturated aged oocyte in a medium containing melatonin.
The present invention also relates to a method for enhancing mammalian embryo development rates, excluding humans, comprising the steps of:
(a) preparing an in vitro maturated aged oocyte of a mammal other than a human;
(b) culturing said in vitro maturated aged oocyte in a medium containing melatonin;
(c) in vitro fertilization of said in vitro maturated aged oocytes to produce embryos; And
(d) culturing the embryo.
The present invention also relates to a culture composition containing melatonin, which comprises a medium composition for preventing aging of mammalian in vitro vitro matured oocytes excluding human, or a medium composition for improving mammalian embryo development rate excluding humans.
The term "in vitro maturation of oocytes" as used herein means a process of culturing in vitro to mature immature oocytes.
As used herein, the term " aging of the oocyte "refers to a stage where the immature oocyte ages after undergoing the maturation process.
As used herein, the term "anti-aging of the oocyte" means retarding or inhibiting the aging of the oocyte during incubation for in vitro maturation of immature oocytes.
In the present invention, the mammal is preferably a mammal other than a human. The mammals other than humans are preferably cows, pigs, sheep, including pigs, sheep, dogs, cows, horses, and chlorine, and most preferably pigs.
The medium in the present invention preferably uses a basic medium used for culturing mammalian oocytes. The basic medium varies depending on the species of the mammal, but includes any one or more selected from the group consisting of inorganic salts, carbon sources, amino acids, bovine serum albumin, and coadjuvants, and includes all conventional media known to those skilled in the art. Examples of the inorganic salts include NaCl, KCl, and NaHCO 3. Examples of the carbon source include glucose, sodium, pyruvate, and calcium lactate. The amino acids include essential amino acids such as glutamine, Other auxiliaries may be other trace elements and buffers. The basal medium used for the in vitro maturation culture may further comprise antibiotics.
The concentration of melatonin contained in the medium of the present invention is not particularly limited, and a person skilled in the art can select and determine an appropriate concentration for preventing aging of mammalian in vitro matured oocytes or improving mammalian embryo development rate. The concentration of melatonin contained in the medium is 10 -6 M (1 uM) to 10 -3 M (1 mM).
Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are provided to aid understanding of the present invention, and the scope of the present invention is not limited to the following examples.
[ Example 1] The effect of melatonin concentration on in vitro maturation embryo Development rate Research
To determine the developmental rate of melanocytes to blastocysts in vitro, we used 10 -15 M (1 fM), 10 -12 M (1 pM), and 10 -9 M ), 10 -6 M (1 uM), and 10 -3 M (1 mM) of melatonin to induce in vitro maturation. After 24 hours, only mature-stage MII-stage oocytes with the first polar body released were inoculated with 1 × 10 6 / mL spermatozoa to induce in vitro maturation. After 8 days of incubation, developmental rates to blastocysts were examined. The results are shown in FIG. .
1, the developmental rates of melatonin 10 -6 M (1 uM) and 10 -3 M (1 mM) to the blastocyst significantly increased compared to the other treatment groups and the control group.
[ Example 2] Stages of In Vitro Maturation Embryos Treated with Melatonin Developmental Research
In order to confirm the developmental stage of each embryo development stage, in vitro maturation stage of the oocyte culture, melatonin treated in vitro, and in vitro fertilized embryos were cultured in the in vitro culture medium, and on
2, there was no significant difference between the melatonin-treated group and the control group in the 2-cell stage and 4-cell stage, but the development rate of the blastocyst and blastocyst was significantly increased in the melatonin-treated group compared to the control group.
[ Example 3] Anti-aging and embryo development rate of in vitro maturation oocytes treated with melatonin
In vitro maturation of melanoma was induced by in vitro maturation of 1 oM melatonin, and melatonin was treated for 22, 30, and 44 hours for the in vitro maturation of oocytes. The anti-aging mechanism was investigated, and the results are shown in Figs. 3 to 7.
FIG. 3 shows changes in ROS of oocytes matured after melatonin treatment, FIG. 4 shows the degree of DNA damage of oocytes matured in vitro when melatonin was treated, and FIG. , Indicating a reduction in the percentage of abnormal oocytes during in vitro maturation. FIG. 6 shows the effect of melatonin on actin filament expression, abnormal microtubule formation and surface granule release of aged oocytes, and FIG. 7 shows the effect of melatonin on the development of oocytes in vitro and cell death in blastocysts .
Melatonin reduced ROS in in vitro maturation aged oocytes (see FIG. 3), and reduced DNA damage in vitro maturation aged oocytes (see FIG. 4), reducing the rate of abnormal oocytes during in vitro maturation of oocytes (See Fig. 5). In addition, melatonin increased actin filament expression in senescent oocytes, decreased the formation of abnormal microtubules, and reduced the release of superficial granules (see FIG. 6). In addition, melatonin improved the in vitro development rate of aged oocytes and decreased cell death in blastocysts (see Fig. 7).
Thus, it can be seen that melatonin prevents aging of in vitro maturation oocytes and improves embryo development rate.
Claims (12)
(a) preparing an immature oocyte of a mammal other than a human;
(b) culturing the immature oocyte in an in vitro maturation culture medium containing 10 -6 M (1 uM) to 10 -3 M (1 mM) of melatonin for 24 hours;
(c) recovering only matured MII-stage oocytes in which the first polar body has been released after culturing for 24 hours, and in vitro fertilization with 1 × 10 6 / mL sperm; And
(d) culturing the in vitro fertilized embryo.
Wherein the step (d) is performed for 5 days or more.
Wherein the mammal except for the human is a bovine.
The oocyte of step (c) has decreased DNA damage, increased actin filament expression, decreased abnormal microtubule formation, and reduced surface granule release compared with in vitro matured oocytes cultured in a culture without melatonin treatment Lt; RTI ID = 0.0 > blastocysts, < / RTI >
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| CN117925511A (en) * | 2018-01-23 | 2024-04-26 | 车医科学大学校产学协力团 | Method for improving efficiency of embryo development or embryogenesis of somatic replicated ovum |
| KR20190089782A (en) * | 2018-01-23 | 2019-07-31 | 차의과학대학교 산학협력단 | Composition and Method for enhancing developmental rate of embryos using melatonin |
| CN109294978A (en) * | 2018-11-10 | 2019-02-01 | 四川农业大学 | A method of improving glass freezing mature oocyte parthenogenetic development potentiality |
| KR102176125B1 (en) | 2018-12-12 | 2020-11-09 | 충북대학교 산학협력단 | method of Improving embryo quality and implantation rate using melatonin |
| KR102183228B1 (en) * | 2019-06-28 | 2020-11-25 | 대구대학교 산학협력단 | Composition for in vitro oocyte maturation and early embryonic development comprising Mito-TEMPO and culture method using the same |
| KR20210081533A (en) | 2019-12-24 | 2021-07-02 | 충북대학교 산학협력단 | Method of delaying oocyte aging using Ubiquinol 10 |
| CN112877280B (en) * | 2021-01-28 | 2022-08-16 | 华东师范大学 | In-vitro fertilization nutrient solution for oocytes of large-week-old female mice and application of nutrient solution |
| CN113403263A (en) * | 2021-05-15 | 2021-09-17 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | Method for improving oocyte in vitro and in vivo maturation quality and application |
| CN113564103B (en) * | 2021-05-17 | 2023-01-06 | 南京医科大学 | Application of 4,4' -dimethoxy chalcone in delaying in-vitro and in-vivo aging of oocyte |
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