KR102158946B1 - Peroxisome dynamics in oocytes affirmed through phytanic acid treatment - Google Patents

Abstract

The present application relates to a method of inducing activation of peroxisomes in a process of in vitro maturation from immature oocytes to mature oocytes. In particular, since alpha oxidation, which is lipid metabolism of peroxisomes, is a key component in the in vitro maturation process, a medium to which phytanic acid is added is used for this purpose.

Description

Role of peroxisomes in oocytes through phytanic acid {Peroxisome dynamics in oocytes affirmed through phytanic acid treatment}

The present application relates to the role of peroxisomes in the in vitro maturation process from immature oocytes to mature oocytes, and in the in vitro culture process including embryo development after fertilization of mature oocytes. In particular, since alpha oxidation, which is the lipid metabolism of peroxisomes, is a key component in the in vitro culture process, it relates to a medium containing phytanic acid and its use.

The technology for producing cloned animals is important not only for commercial utility, but also for biomedical and biological research. The industrial application field of cloned animal production technology is very wide ranging from production of high-quality livestock food, production of high value-added pharmacologically active substances, production of animals with improved bioresistance against various pathogens, production of disease model animals, and gene therapy.

Among the various uses of cloned animals, interest in developing new systems for disease treatment using cloned pigs having physiological similarities with humans is increasing. It is already known that studies of in vitro maturation systems using pigs are an ideal candidate for patients with polycystic ovary syndrome without ovulation. Many studies are being conducted in the direction of increasing the maturity rate in the process of in vitro maturation and consequently increasing the embryonic development rate. However, studies on specific organelles of oocytes, particularly accurate metabolism and mechanism studies, have not been conducted yet. Therefore, in the present application, research was conducted by paying attention to peroxisome, one of the organelles.

The structure of the peroxisome is surrounded by a single membrane and has an almost round shape. Often, enzyme molecules are highly concentrated in granules.

The name of peroxisome is a compound word of peroxide and ~some, which makes peroxide hydrogen peroxide (H ₂ O ₂ ) a by-product, and in the meaning of ~some protein, it is named because it has an enzyme that transfers hydrogen from various substances to oxygen. As the name suggests, the oxidation reaction of peroxisomes is responsible for various functions in the body.

Looking at the function of the peroxisome 1. It has oxidative enzymes, so it can beta-oxidize some fatty acids. 2. It has a catalase enzyme and uses this enzyme reaction to oxidize (detoxify) toxic substances such as phenol, formaldehyde, and alcohol. 3. Produces bile salts. 4. Hydrogen peroxide generated in the oxidation process is also used as an oxidizing agent in some oxidation reactions, but if it becomes excessive, it decomposes hydrogen peroxide using catalase. Finally 5. It can be used for alpha oxidation of phytanic acid.

Fatty acid metabolism in cells is very important for energy acquisition and storage, and cholesterol synthesis. Fatty acid oxidation is the process of gaining energy by breaking down fatty acids. In the case of animal cells, long fatty acids above 16C or branched fatty acids are first decomposed in peroxisomes, and then small fatty acids below 16C are oxidized in the mitochondria to obtain energy.

Specifically, peroxysomes beta-oxidize fatty acids into small molecules and send them to mitochondria, providing an energy source for cellular respiration. When it is decomposed into triglycerol, a triglyceride, and three fatty acid molecules, it is converted into an organic acid by beta oxidation of the fatty acid produced at this time, and flows into the TCA circuit in this state and is used to produce ATP.

As for the alpha oxidation performed only in peroxisomes, it is known that only phytanic acid is metabolized by alpha oxidation. Phytanic acid is a branched chain fatty acid that can be obtained from dairy products, ruminant fat, and certain fish. Unlike most fatty acids, phytanic acid is not metabolized by β-oxidation. Instead, phytanic acid undergoes α oxidation in peroxisomes, and one carbon is removed to convert to pristanic acid. Pristanic acid forms medium-chain fatty acids through β-oxidation several times in peroxisomes, and medium-chain fatty acids can be converted into carbon dioxide and water in the mitochondria. In this process, ATP is created.

In addition, peroxysomes also serve to eliminate toxicity by transferring the hydrogen portion of alcohol or other new substances to oxygen. At this time, hydrogen peroxide is produced by peroxisomes. Hydrogen peroxide itself is toxic, but in this organelle is the enzyme Catalase, which converts hydrogen peroxide into water. Therefore, hydrogen peroxide generated in the fat oxidation process is used as an oxidizing agent in some oxidation reactions, and when excessively increased, it also serves to decompose hydrogen peroxide using catalase.

In vitro maturation studies related to fat oxidation are known to have a negative effect on embryonic development by reducing nuclear maturation when blocking fatty acid beta oxidation in mice. In addition, the importance of lipid metabolism is emerging in the maturation of oocytes in vitro. The reason is that recently, in the production of ATP, it is attracting attention that fat oxidation is more effective than glucose oxidation. However, as of yet, the role of fatty acid alpha oxidation of peroxisomes in the in vitro culture process of eggs has not been elucidated. Therefore, the present applicants confirmed that efficient maturation and development can be achieved through alpha oxidation, lipid metabolism of peroxisomes, using a medium treated with phytanic acid in the process of in vitro maturation and embryo development of oocytes, and completed the present invention. I did.

Chinese Patent Publication CN105695398A (2016-06-22)

ATP is an essential element in the in vitro culture of eggs. In terms of the ATP production rate, it is known that the ATP production rate by lipid metabolism is much higher than the ATP production rate by glucose. However, the specific organelles or mechanisms of the lipid metabolism have not been identified yet.

Therefore, the purpose of the present application is to confirm the importance of lipid metabolism in the in vitro culture of oocytes.

Another object of the present application is to confirm that peroxisome is a key component in the lipid metabolism, and to provide a mechanism technology. Another object of the present application is to provide an appropriate concentration of phytanic acid required for alpha oxidation in the lipid metabolism activation process.

Another object of the present application is to confirm that mitochondria are a key component in the production of ATP through phytanic acid.

Another object of the present application is to provide a method of increasing the efficiency in the in vitro maturation process of immature oocytes by a culture method using a medium treated with phytanic acid.

Another object of the present application is to provide a method of increasing the efficiency by metabolizing lipids of phytanic acid in embryo development after in vitro fertilization of the mature oocytes.

In addition, another object of the present application is to apply to infertility and infertility patients, in particular patients with peroxisome-related diseases, by controlling the activity of peroxisomes during in vitro culture of eggs.

In order to solve the problems of the present application described above, according to an aspect of the present application, a medium for in vitro maturation of immature oocytes is provided.

In this case, the medium is a cell culture minimum medium (CCMM) containing sugar, amino acids and water; And phytanic acid.

In this case, the medium may contain phytanic acid at a concentration of 30-70 μM.

The minimum cell culture medium is TCM-199 (tissue culture medium-199), DMEM (Dulbecco modified eagle medium), M-199 (medium-199), Ham's F-1 (Nutrient mixture F-10 (HAM)), NCSU (North Carolona State University) 23, NCSU37, Porcine zygote medium (PZM)-5, and may be any one selected from the group consisting of PZM-3.

The medium may further include any one or more of a gonadotropin and a growth factor.

The gonadotropin is follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), equine chorionic gonadotropin: eCG) and pregnant mare serum gonadotropin (PMSG) may be any one or more selected from the group consisting of.

The growth factors include insulin-like growth factor (IGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), and transforming growth factor (IGF). factor, TGF) may be any one or more selected from the group consisting of.

In addition, the medium may further contain one or more of sodium pyruvate, insulin-transferrin-selenium solution (ITS-A), and cysteine.

Specifically, in the present application, sodium pyruvate, insulin-transferrin-selenium solution (ITS-A), cysteine, epidermal growth factor: EGF in the medium. ), human chorionic gonadotropin (hCG), equine chorionic gonadotropin (eCG), porcine follicular fluid, and M-199 (tissue culture medium- containing phytanic acid) 199) based medium can be used.

In order to solve another problem of the present application described above, according to another aspect of the present application, in the process of in vitro maturation of immature oocytes, a phytanic acid compound is treated to induce activation of the alpha oxidation of peroxisomes. A method for producing mature oocytes is provided.

The method for producing mature oocytes provided by the present application may use the medium. At this time, using the medium

It may include the step of maturing the immature oocytes in vitro for 20-22 hours under conditions of 38-39° C., 5% CO ₂ , and humidity.

In addition, it may include a step of further culturing for 20 to 22 hours under conditions in which the gonadotropin is removed from the medium after the step.

The phytanic acid in the in vitro maturation medium provided by the present application can produce mature oocytes in which alpha oxidation of peroxisomes is activated.

In order to solve another problem in the present application, according to another aspect of the present application, there is provided a method for in vitro development of an embryo, characterized in that the embryo is developed in vitro using the medium.

The in vitro development method provided by the present application includes the step of forming a blastocyst from the initial embryonic development of a fertilized egg through a difficult period. At this time, the method may be cultured for 7 days under the conditions of 38-39° C., 5% CO ₂ , 5% O _{2 and} 90% N ₂ .

The present application provides a medium treated with phytanic acid in the production of ATP through lipid metabolism during in vitro maturation of oocytes and embryonic development. Through the medium, mature oocytes with improved expression of genes and metabolites related to the alpha oxidation mechanism of peroxisomes are provided. In addition, through the medium, in vitro fertilized eggs can be stably produced by increasing the rate of blastocyst formation and the number of final cells in the development of fertilized embryos.

Therefore, the present application can contribute to breeding, preservation, transplantation of xenogeneic organs, production of disease model animals, and the like of superior breeds of animals. In addition, through the regulation of peroxisome activity, it is a technology applicable to infertility and infertility patients, especially in patients with diseases such as polycystic ovarian syndrome, for collecting and culturing eggs. You will be able to contribute to development. Furthermore, it is applied to patients with peroxisome-related diseases.

1 is a graph showing the rate of immature, degeneration, maturation, and degree of expansion of cumulus after culturing porcine oocytes in vitro in a medium containing phytanic acid at a concentration of 0, 20, 40, and 80 μM.
Figure 2 is a graph showing the expression levels of oocyte maturation-related nuclear maturation genes and cumulus cell expansion-related genes in an in vitro culture medium containing phytanic acid at a concentration of 0, 20, 40, and 80 μM.
3 is a graph showing an image and fluorescence analysis of the expression of PHYH, a peroxisome activating gene, of mature oocytes in a medium containing and without phytanic acid at a concentration of 40 μM using immunostaining.
4 is a graph showing an image and fluorescence analysis of the expression of PEX19, a peroxisome activating gene, of mature oocytes in a medium containing and without phytanic acid at a concentration of 40 μM using immunostaining.
5 is a graph confirming the expression of peroxisome activating genes PHYH, PEX3, and PEX5 of mature oocytes in a medium containing and without phytanic acid at a concentration of 40 μM.
6 is a graph confirming the expression of peroxisome activating genes PEX12, PEX19, PPARα, and PPARγ of mature oocytes in a medium containing and without phytanic acid at a concentration of 40 μM.
7 is a graph confirming the expression of the peroxisome activating genes PHYH, PEX3, and PEX5 of the cumulus cells in a medium containing and without phytanic acid at a concentration of 40 μM.
8 is a graph confirming the expression of peroxisome activating genes PEX12, PEX19, PPARα, and PPARγ of cumulus cells in a medium containing and without phytanic acid at a concentration of 40 μM.
9 is an image and graph showing fat droplets and fatty acids of mature oocytes in a medium containing and without phytanic acid at a concentration of 40 μM with a fluorescent probe.
10 is a graph confirming the expression of lipid metabolism genes of mature oocytes in a medium containing and without phytanic acid at a concentration of 40 μM.
11 is a graph confirming the expression of lipid metabolism genes in mature cumulus cells in a medium containing and without phytanic acid at a concentration of 40 μM, respectively.
FIG. 12 is a graph showing an image obtained by staining the mitochondrial membrane potential of oocytes matured in a medium containing and without phytanic acid at a concentration of 40 μM with a fluorescent probe and analyzing the fluorescence ratio.
13 is an image and graph showing ATP produced in mature oocytes in a medium containing and without phytanic acid at a concentration of 40 μM, respectively, with a fluorescent probe.
14 is a graph confirming the expression of genes related to mitochondrial biosynthesis of mature oocytes in a medium containing and not containing phytanic acid at a concentration of 40 μM.
15 is a graph confirming the expression of genes related to mitochondrial biosynthesis in mature cumulus cells in a medium containing and without phytanic acid at a concentration of 40 μM.
16 is an image and graph showing the amount of GSH and ROS of mature oocytes in a medium containing and without phytanic acid at a concentration of 40 μM, respectively, with a fluorescent probe.
17 is a graph confirming the expression of genes related to antioxidant or apoptosis of mature oocytes in a medium containing and without phytanic acid at a concentration of 40 μM.
18 is a graph confirming the expression of genes related to antioxidant or apoptosis of mature cumulus cells in a medium containing and without phytanic acid at a concentration of 40 μM, respectively.
FIG. 19 is a graph showing the percentage of embryos developed in the medium containing and not containing phytanic acid at a concentration of 40 μM, the blastocyst formation rate, and the total number of blastocysts.
FIG. 20 is an image of nuclear staining in order to confirm the total number of blastocysts of each medium containing and without phytanic acid at a concentration of 40 μM.

In order to describe the content disclosed herein, various terms will be defined herein. In addition to these terms, other terms are defined elsewhere in this specification as necessary. Unless clearly defined otherwise herein, industry terms used herein will have industry-accepted meanings. In case of conflict, it may be interpreted by the definitions herein.

The term “culture” as used in this application refers to growing an organism or part of an organism (such as organ tissue cells) in an appropriately artificially controlled environmental condition. In this case, external conditions such as temperature, humidity and gaseous composition (partial pressure of carbon dioxide or oxygen) are important. In addition, the most important direct influence on the organism to be cultured is the medium (incubator), which is the direct environment of the organism and the supply of various nutrients necessary for survival or proliferation.

The term'in vitro culture' as used in the present application refers to a series of laboratory processes in which cells are cultured in a laboratory incubator under conditions similar to the internal environment in a manner that distinguishes them from growing in the body. In the present application, in vitro culture is performed during the maturation process of immature oocytes and the stage of embryo development after fertilization of mature oocytes.

The term'in vitro maturation' (IVM) as used in the present application means that cells and the like are cultured in a laboratory incubator under conditions similar to the internal environment to mature immature cells. In the present application, it means that immature oocytes and oocyte-cumulus cell complexes are matured in a special culture medium in vitro to mature into oocytes of the second metaphase II. In addition to in vitro maturation, the present application also describes the stage of embryo development after fertilization.

The term'medium' used in the present application refers to a mixture for growth and proliferation of cells, etc. in vitro containing essential elements such as sugar, amino acids, various nutrients, serum, growth factors, minerals, etc. . In particular, the medium of the present application may be a medium containing a basic medium and phytanic acid as a medium for in vitro maturation of immature oocytes and egg-cumulus cell complexes or for embryo development after in vitro fertilization.

The term'egg' as used in the present application refers to the germ cell of a female animal, and is produced in the gonad called the ovary. In mammals, all eggs already have from birth, and they mature through the process of egg formation. The egg of the present application includes a first oocyte, a second oocyte, a first polar body, or a second polar body.

The term "embryo" used in this application is an early stage of development, where zygote is formed through cell division and differentiation, and when development proceeds further, it is called a fetus. In the present application, the embryo is defined as the fertilized egg starts the first cell division until it becomes a fetus. Embryo development refers to the stage of formation of blastocysts after fertilization, including the stage of differentiation of a fertilized egg into an embryo.

The term "lipid metabolism" as used in this application refers to the process of synthesizing and decomposing lipids. Beta oxidation, for example, is metabolic fixation that breaks down in mitochondria or peroxisomes to produce acetyl-CoA.

The term "mitochondria" used in the present application has a function involved in regulating energy metabolism and cell death, and is an organelle present in eukaryotic cells. One of the main functions of mitochondria of the present application is oxidative phosphorylation via an electron transport system. Energy ATP derived from metabolism of fuel substances such as glucose or fatty acids is produced through the oxidative phosphorylation process.

The term "membrane potential" as used in this application is an aerobic glycolysis process in the mitochondria, the TCA cycle, to produce NADH required for energy generation. NADH transports hydrogen ions (H+) in the mitochondria to the outside while transferring electrons through the electron transport system. As a result, a high electrochemical potential, a mitochondrial membrane potential (Ψm) of about -150mV is formed in the inner and outer mitochondria due to the difference in hydrogen ion concentration. In this application, the depolarization of the membrane potential means that a lot of cell death has occurred.

Mature oocytes are required for in vitro fertilization or the generation of cloned animals for the purpose of treatment of infertile patients through in vitro fertilization. However, in the process of selecting and obtaining mature oocytes in the body, there is a problem that the follicles grow at different rates, and the maturity when harvested is different.

Therefore, for the above purpose, a method of harvesting an immature oocyte and maturing it in vitro is mainly used.

However, this also has a relatively low rate of development into mature oocytes, and in particular, in the case of humans and pigs, there is a problem that the in vitro oocyte maturation rate and embryo development rate are very low.

Eventually, in order to obtain efficient in vitro mature oocytes, in vitro maturation conditions are important, so the present application uses lipid metabolism in supplying energy to increase the maturation rate. A specific example of the lipid metabolism is the alpha metabolism of peroxisomes. Accordingly, the present application includes a culture medium for activation of the alpha metabolism and a culture method using the same, for example, a culture medium containing phytanic acid and a culture method using the same.

As a known in vitro maturation study related to fat oxidation, it has been reported that when fatty acid beta oxidation in mice is blocked, nuclear maturation decreases and has a negative effect on embryonic development. Other studies have also emphasized the importance of fat oxidation in nuclear maturation and embryonic development.

However, existing studies on fat oxidation have been mainly focused on the fat oxidation of mitochondria. This is because the major fat oxidation in cells consists of beta oxidation, and beta oxidation occurs inside the mitochondria.

Fatty acid metabolism in cells is very important for energy acquisition and storage, and cholesterol synthesis. In peroxisomes, fatty acids are beta-oxidized to make small molecules and send them to the mitochondria, providing an energy source for cellular respiration. When the triglyceride is decomposed into three molecules of glycerol and fatty acids, the fatty acids produced at this time are converted into organic acids by beta oxidation, and flow into the TCA circuit in this state to produce ATP.

So far, studies on alpha oxidation of peroxisomes during fat oxidation are insufficient. In addition, the role of alpha oxidation of peroxisomes in the process of in vitro culture of eggs has not been revealed. Accordingly, the present applicants emphasized the importance of alpha oxidation, which is the lipid metabolism of peroxisomes, in the process of in vitro maturation and embryonic development of oocytes. Specifically, it was confirmed that efficient maturation and development can be achieved through the alpha metabolism, and the present application was completed.

The present application relates to activation of peroxisomes by alpha oxidation and use thereof in the process of in vitro maturation of oocytes.

According to one aspect, the present application provides a method for inducing peroxisome activation in in vitro maturation of oocytes.

In another aspect, there is provided a method of inducing alpha oxidation in peroxisomes for the activation.

According to another aspect, there is provided a culture medium inducing alpha oxidation of peroxisomes.

In one embodiment, the culture medium is provided with a composition containing pisanthan in the cell culture minimal medium.

According to another aspect, the present application provides a method of activating peroxisomes by alpha oxidation in embryonic development after in vitro fertilization of mature oocytes.

The general technical content of each step may be understood by referring to a method for preparing mature oocytes using conventional in vitro maturation techniques known in the art.

Hereinafter, the in vitro maturation method of immature oocytes using the alpha oxidation activation of peroxisomes of the present application will be described in detail.

Specifically, a method of metabolizing lipids for in vitro maturation of oocytes disclosed by the present application and a medium and maturation for embryonic development and activation of peroxisomes in the embryonic process will be described in more detail.

One. In vitro maturation method of immature oocytes

The maturation process of oocytes is largely divided into nuclear maturation and cytoplasmic maturation. Nuclear maturation refers to the process of resuming meiosis and progressing to the second metaphase II (MII) stage. Cytoplasmic maturation refers to all changes except for changes in the nucleus for meiosis.

In vitro maturation of oocytes is the process of maturing immature oocytes in vitro until the second intermediate meiosis phase. In the industry, it is difficult to recover oocytes matured in the body, and in vitro maturation technology is used due to the limitation of the number of recovered oocytes.

In one aspect of the present application, there is provided a culture method for in vitro maturation of immature oocytes.

The culture method is characterized by inducing activation of the alpha oxidation of peroxisomes during in vitro maturation.

The oocytes produced through the above method are characterized by having a high maturation rate, a low degeneration rate, and a high egg cell expansion rate.

1-1. Obtaining immature oocytes

A typical method of in vitro culture of oocytes includes the steps of collecting immature oocytes from the body; And maturing the oocytes in an in vitro maturation medium.

The method of obtaining mature oocytes through in vitro culture of the present application includes the steps of: (a) collecting immature oocytes from the body; And (b) maturing the oocytes in the in vitro maturation medium until the second intermediate meiosis stage.

In one embodiment, the immature oocyte of step (a) is a first oocyte.

In another embodiment, the immature oocyte of the step (a) may be an egg-cum cell complex.

In the present application, the method of culturing immature oocytes for in vitro maturation may include the step of obtaining immature oocytes.

More specifically, the method of obtaining mature oocytes through in vitro culture is

(1) recovering the ovary of the animal;

(2) recovering immature eggs from the recovered ovaries;

(3) culturing the immature oocytes through the medium; And

(4) It may include the step of selecting the mature oocytes.

As an embodiment, in the present application, a pig was used as the animal of (1).

In particular, the present application relates to a culture medium and a culture method used in the step of culturing (3) immature oocytes among the above methods, and when the medium or culture method of the present application is used, the mature egg is improved in quantity and quality. It can provide blast cells.

1-2. In vitro maturation

In vitro maturation can generally be achieved through the maturation stage by recovering immature oocytes from the ovary.

In vitro maturation of the egg in the present application may include all stages of maturation of the egg.

In one embodiment of the present application, by culturing the egg cell-oocyte complex, it can be matured in vitro until the second intermediate meiosis phase (Metaphase II).

In another embodiment of the present application, the isolated immature oocytes may be matured in vitro until the second intermediate meiosis phase (Metaphase II).

In another embodiment of the present application, the isolated cumulus cells may be cultured and expanded in vitro.

The cumulus cell is a cell attached to the zona pellucida of an egg, differentiated from granulosa cells, and has an expanded form by culturing the cumulus cell-oocyte complex.

1-3. Alpha oxidation of peroxisomes in in vitro maturation

In another aspect of the present application, it relates to a method of increasing the maturation rate by activation of lipid metabolism of peroxisomes during maturation of immature oocytes.

The lipid metabolism is the alpha oxidation of peroxisomes.

The alpha oxidation may be induced by phytanic acid.

It is known that only phytanic acid metabolism in peroxisome is metabolized by alpha oxidation. Phytanic acid is a branched chain fatty acid that can be obtained when humans consume dairy products, ruminant fat, and certain fish. Unlike most fatty acids, it is not metabolized by β-oxidation.

Since the alpha oxidation of the peroxisome is lipid metabolism in the peroxisome, the expression of genes related to lipid metabolism may increase.

The present application relates to a method for finally generating ATP, an energy source, by lipid metabolism of the activated peroxisome.

As a known technique, ATP is an essential element in promoting embryonic development, and it is known that it can be efficiently supplied by lipid metabolism. Furthermore, a number of researchers have studied the essential link between lipid metabolism and -ATP production in early embryonic development. In addition, the mitochondria of mature oocytes are known as a key organelle for ATP production.

1-4. Antioxidant function of peroxisomes

In addition, the present application relates to the use of an oxidative stress defense mechanism, one of the functions of peroxisomes, in the maturation process of immature oocytes.

As a known technique, the mechanism of defense against oxidative stress of peroxisomes is known.

The catalase enzyme in peroxisome breaks down hydrogen peroxide into water and oxygen to protect cells from oxidative stress. In addition, GSH, a representative component of antioxidants, may also be included in the antioxidant mechanism of peroxisomes.

Hereinafter, the medium used in the maturation method will be specifically presented.

In another aspect of the present application, a medium used for in vitro maturation of immature oocytes is provided.

The in vitro maturation medium of the present application is characterized in that it essentially contains a cell culture basic medium and phytanic acid.

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The present application relates to a maturation medium for in vitro maturation, and may have the following composition.

The culture medium of the present application is characterized in that it contains phytanic acid.

The culture medium of the present application is a cell culture minimal medium.

In one embodiment of the present application, the phytanic acid may be included in a concentration of 1 to 80 μM in the medium according to the present application.

In another embodiment of the present application, the phytanic acid may be included in a concentration of 30 to 70 μM in the medium according to the present application.

In another embodiment of the present application, the phytanic acid may be included in the medium according to the present application at a concentration of 40 μM.

Looking more specifically about the phytanic acid, it can be represented by the structural formula of [Chemical Formula 1].

[Formula 1]

The phytanic acid represented by Formula 1 is a branched chain fatty acid that can be obtained when humans consume dairy products, ruminant fat, and certain fish.

Uniquely, unlike most fatty acids, phytanic acid is not metabolized by β-oxidation. Instead, phytanic acid is converted to pristanic acid by removing one carbon from the peroxisome through α oxidation. Pristanic acid is known to form medium-chain fatty acids through several β-oxidations in peroxisomes, and medium-chain fatty acids are known to be converted into carbon dioxide and water in the mitochondria.

The compound represented by Formula 1 includes all of the compound itself, analogs, and acceptable salt forms.

The compound of the present application may include all solvates exhibiting the same effect and the like that may be prepared therefrom within the scope of the present application.

The phytanic acid and its analogs may be prepared using standard techniques known to those skilled in the field of organic chemical synthesis, purchased, or prepared by referring to related documents.

As an example, the compound can be prepared by a conventional synthetic method, and for example, a derivative of the compound is dissolved in an organic solvent such as methanol, ethanol, acetone, dichloromethane, acetonitrile, etc., and an organic acid or an inorganic acid is added to form a precipitate. It can be prepared by filtration and drying, or it can be prepared by distilling a solvent and an excess of acid under reduced pressure and then drying to crystallize under an organic solvent.

The basic medium that can be used in the present application is a culture medium for in vitro maturation of eggs, and may be a cell culture minimum medium (CCMM) containing only a carbon source, a nitrogen source, and a trace element component. That is, it is a mixture containing essential sugars, amino acids, water, etc. necessary for cells to live, and is a basic medium excluding serum, nutrients, and various growth factors.

As an energy source, polysaccharides, monosaccharides, organic acids, amino acids, alcohols, polycyclic compounds, cellurose, and hemicellulose (hemicellurose), lignin, and other carbon sources can be used. Examples of polysaccharides include dextrin and starch, disaccharides include sucrose, maltose, trehalose, and mannitol, and monosaccharides include glucose, mannose, fructose, and galactose.

The cell culture minimum medium (CCMM) of the present application may be artificially synthesized and used, or a commercially prepared medium may be used.

Commercially prepared medium is TCM-199 (tissue culture medium-199), DMEM (dulbecco modified eagle medium), M-199 (Medium-199) Ham's F-1 (Nutrient Mixture F-10 (HAM), NCSU ( North Carolina State University) 23, NCSU37, Porcine zygote medium (PZM)-5, or PZM-3, and preferably M-199 medium.

In addition to the above components, the medium of the present application may further include other components as necessary.

As a generally known technique, the medium composition used for in vitro maturation of an egg includes fetal bovine serum (FBS), follicular fluid, gonadotropin (FSH, LH, E2), growth in a completely synthetic medium or simple medium. It may include any one or a combination thereof, such as a growth factor, but is not limited thereto.

For example, the culture medium may contain 10 (v/v)% of the total volume of the follicular fluid.

As an example, the culture medium may further contain sex hormones.

The gonadotropin is produced in the anterior lobe of the pituitary gland and is secreted and refers to a hormone having a function of stimulating the gonads (ovaries or testis).

The gonadotropin is follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), equine chorionic gonadotropin: eCG) and pregnant mare serum gonadotropin (PMSG) may be any one or more selected from the group consisting of.

In one embodiment of the present application, the gonadotropin may be a human chorionic gonadotropin and an equine chorionic gonadotropin.

As another example, in the present application, growth hormone may be further included in the medium.

The growth promoting factor is a generic term for a polypeptide that promotes various cell division, growth, and differentiation, and is involved in the signal transduction system of cells.

The growth promoting factors are insulin-like growth factor (IGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), nerve growth factor, NGF), transforming growth factor (TGF), platelet-derived growth factor (PDGF), and bone-derived growth factor (BDF) selected from the group consisting of It can be any one or more.

In one embodiment of the present application, the growth promoting factor may be an epidermal growth factor.

Preferably, the maturation medium of the present application essentially contains phytanic acid in the cell culture minimal medium and was used for in vitro maturation of immature oocytes using a medium containing follicular fluid, gonadotropin, and growth promoting factor.

In addition, the present application may further include sodium pyruvate, insulin-transferrin-selenium solution (ITS-A), and cysteine as optional ingredients in the maturation medium. .

For example, an antibiotic may be further included as an optional component in the medium.

As another example, an anti-aggregating agent may be further included as an optional component in the medium.

In the present application, by using the maturation medium, the immature oocyte may be matured in vitro through two steps.

The primary culture during the culture may be, for example, incubated for 20 to 22 hours under conditions of 39° C., 5% CO ₂ , and 95% humidity.

After the first culture under the above conditions, the culture medium may be cultured for 20 to 22 hours a second time in the absence of hormones.

That is, after the first culture, after washing with a hormone-free culture medium, the second culture process can be added by transferring to a hormone-free culture medium.

Both of the media for the first and second culture processes contain phytanic acid.

Accordingly, the present application can obtain mature oocytes by maturing immature oocytes in vitro by the above method.

Oocytes matured using the culture medium are in a form in which peroxisomes are activated by phytanic acid in the culture medium.

In addition, by using the culture method of the present application, the mature oocyte is in a form in which peroxisomes are activated by phytanic acid in the culture medium.

In one embodiment, the mature oocytes may increase expression of genes related to peroxisome activation by phytanic acid contained in the medium.

The mature oocytes may have increased expression of any one or more genes selected from PHYH, PEX3, PEX5, PEX12, PEX19, PPARα, and PPARγ.

Through the result of the increase in the expression of the peroxisome activating gene of the present application, it was confirmed that the alpha oxidation of the peroxisome was activated with phytanic acid contained in the medium.

In one embodiment, the cultured mature oocytes may have increased expression of genes related to lipid metabolism.

The lipid metabolism may include lipogenesis.

The cultured mature oocytes may have increased expression of any one or more genes selected from ATGL, HSL, MGLL, CGI58, and PLIN2.

In another embodiment, the cultured cumulus cells may have reduced expression of genes related to lipid metabolism.

The cultured cumulus cells may have reduced expression of any one or more genes selected from HSL, MGLL, CGI58, and PLIN2.

Oocytes matured by the phytanic acid-treated medium and culture method of the present application have activated peroxisomes and lipid metabolism. As an exception, this does not apply to mature cumulus cells.

In another embodiment, the generation of lipid droplets may be increased by lipid metabolism activated by peroxisomes by phytanic acid in the mature oocyte.

In addition, in another embodiment of the present application, fatty acid levels may be increased by lipid metabolism activated by peroxisomes by phytanic acid in the mature oocytes.

Applicants have confirmed that phytanic acid increases ATP production in mitochondria.

In one embodiment, ATP in the mitochondria of mature oocytes treated with phytanic acid may be increased.

In another embodiment, in the cultured mature oocyte, depolarization of the mitochondrial membrane potential may be reduced by phytanic acid.

In another embodiment, the cultured mature oocytes may increase expression of genes related to mitochondrial biogenesis by phytanic acid.

The phytanic acid-treated, mature oocytes may have increased expression of any one or more genes selected from PGC-1α, NRF1, and TFAM.

The present application may include a method in which oocytes matured by the phytanic acid-treated medium and culture method are activated peroxisomes and lipid metabolism, and thus ATP increases are generated in mitochondria.

The present application can utilize peroxisome's oxidative stress defense mechanism in the maturation process of immature oocytes.

Therefore, although oxidative stress occurs due to the activation of phytanic acid alpha oxidation of peroxisomes, it is possible to confirm a form in which oxidative stress is rather reduced because the defense mechanism by the activation of the antioxidant system within itself operates.

In one embodiment, GSH of the mature oocytes treated with phytanic acid may increase.

In another embodiment, oxidative stress (ROS) of the mature oocytes treated with phytanic acid may be reduced.

In another embodiment, catalase of the mature oocytes treated with phytanic acid may increase.

In the present application, cell death according to the oxidative stress defense mechanism was confirmed.

In another embodiment, the mature oocyte may have reduced expression of genes related to cell death.

The cultured mature oocytes may have a reduced rate of expression of Bax or Bcl2 genes.

It was confirmed that the phytanic acid-treated mature oocytes cultured according to the present application can also increase the survival rate of oocytes by phytanic acid.

2. Oocyte

It is possible to obtain mature oocytes using the culture medium according to the present application.

In addition, mature oocytes may be obtained by using the culture method of the present application.

In addition, the mature oocyte is a form in which the cytoplasm is mature.

In mature oocytes, the percentage of oocytes in the second intermediate meiosis stage is increased.

The mature oocyte may contain 80% or more of the percentage of oocytes in the second intermediate meiosis stage.

In one embodiment, the cultured mature oocytes have increased expression of genes related to egg development by phytanic acid contained in the medium.

Mature oocytes have increased expression of one or more genes selected from GDF9 (Growth differentiation factor 9), BMP15 (Bone morphogenetic protein 15), and POU5F1 (POU domain, class 5, transcription factor 1).

In another embodiment, the expression of genes related to cumulus cell expansion may be increased by phytanic acid contained in the medium.

Mature oocytes have increased expression of at least one gene selected from HAS2 (Hyaluronic acid synthase 2), PTGS2 (Prostaglandin G/H synthase 2 precursor), and TNFAIP6 (Tumor necrosis factor alpha induced protein 6).

By the medium and culture method of the present application, the effect of expansion of cumulus cells and maturation of eggs can be achieved.

When the mature oocyte of the present application is used, it is excellent in the generation of embryos and blastocyst generation efficiency, and thus can be used with high efficiency for the production of fertilized eggs in vitro or nuclear transfer eggs. In addition, the in vitro fertilized eggs or nuclear transfer eggs may be usefully used in the production of cloned animals using disease animal models and somatic cell nuclear transfer methods.

Accordingly, the present application includes a method for producing a fertilized egg in vitro using the mature oocyte and a method for developing the same.

3. In vitro fertilization

The present application can produce and cultivate in vitro fertilized eggs using oocytes matured in vitro by the maturation medium and culture method of the present application.

The in vitro fertilized egg refers to a fertilized egg produced by fertilizing an egg collected from an ovary by mixing it with sperm in a test tube.

The in vitro fertilized egg culture method of the present application,

(a) recovering immature oocytes from the collected ovaries;

(b) culturing the immature oocytes in an in vitro culture medium containing phytanic acid for in vitro maturation;

(c) selecting the mature oocytes and fertilizing them with frozen-thawed semen in vitro; And

(d) the step of developing the fertilized egg in a medium containing phytanic acid.

The present application may include embryos obtained by the above method.

The medium containing phytanic acid of the present application may also be used in the process of developing a fertilized embryo. The medium can be used for in vitro development with an increased blastocyst formation rate and an increased number of final cells. Therefore, it is effective in producing stable in vitro fertilized eggs. Accordingly, the present application may include various types of inventions for desired functions such as animal cloning, in addition to in vitro maturation and embryo development through a medium and method containing phytanic acid.

The basic medium that can be used in this step is a culture medium for embryonic development of fertilized eggs after in vitro fertilization, and may be artificially synthesized and used, or a commercially prepared medium may be used.

The embryo development medium of the present application may preferably be Porcine zygote medium (PZM)-5.

As a specific example, the medium is a completely synthetic medium as a culture medium for pig culture embryo development. Therefore, since the medium contains components necessary for cultivation of fertilized embryos, it can be used without additional configuration.

The culture medium for embryo development of the present application is characterized in that it contains phytanic acid.

For example, the phytanic acid may be included in a concentration of 1 to 80 μM in the medium according to the present application.

For example, the phytanic acid may be included in a concentration of 30 to 60 μM in the medium according to the present application.

For example, the phytanic acid may be included in the medium according to the present application at a concentration of 40 μM.

As an example, the medium may be cultured for 7 days under hypoxic culture conditions (5% O ₂ , 5% CO ₂ , and 90% N ₂ ) at a temperature of 39°C.

In vitro fertilized eggs and embryos produced by the above method may be used in various fields such as animal models for disease treatment.

As such, the present application can provide mature oocytes with improved both quantitative and qualitative expression of related genes and metabolites by using phytanic acid as a representative material for improving the alpha oxidation mechanism of peroxisomes. All cases in which these technical features are used without limitation in the description of the present application may be included in the scope of the present application.

[Example]

[Example 1] Obtaining immature oocytes and in vitro maturation medium and method of oocytes

Pig ovaries were obtained from pre-pubescent sows in slaughterhouses and transferred to a laboratory at 28-32°C.

Immature oocytes are 10 mM N-piperazine-N'-2-ethanesulfonic acid (HEPES) 0.3% polyvinyl alcohol (PVA), 5 mM sodium In M-199 (tissueculture medium-199; Invitrogen, Carlsbad, CA, USA) medium containing hydroxide, 2 mM sodium bicarbonate, and 1% Pen-Strep (Invitrogen) Washed 3 times.

After that, 10 ng/mL epidermal growth factor (EGF), 0.91 mM sodium pyruvate, 10 μl/mL insulin-transferrin-selenium solution: ITS-A, 100X ( Invitrogen)), 0.57 mM cysteine, 10 IU/mL human chorionic gonadotropin (hCG), 10 IU/mL equine chorionic gonadotropin (eCG) and 10% porcine follicular fluid (porcine follicular fluid: vol/vol) was added to an IVM medium containing M-199 medium.

Immature oocytes in the medium were cultured for 20-22 hours at 39° C., 5% CO ₂ , and 95% humidity for in vitro maturation.

Thereafter, the immature oocytes were washed twice with hormone-free in vitro mature medium and then cultured for an additional 20-22 hours in hormone-free in vitro mature medium.

[Example 2] Confirmation of oocyte maturation ratio and cumulus cell expansion

(1) Confirmation of the maturation ratio of oocytes

In order to confirm the maturation of the oocyte, the nuclear maturity of the pig egg was observed under a microscope and this was repeated 4 times.

After 42-44 hours of in vitro maturation, mature oocytes were removed by light pipetting with 0.1 (v/v)% hyaluronidase in Tyrode's albumin lactate pyruvate (TALP) medium, and then transferred to TALP medium. Rinse 3 times.

The peeled mature oocytes were fixed with 4% formaldehyde for 1 hour and stained with 5 μg/mL Hoechst-33342 for 8 minutes. After washing, it is mounted on a slide glass to evaluate the mature oocytes under a microscope.

The classification criteria are degenerate, immature (if there is no protrusion of polar body), and maturity (intermediate secondary meiosis).

(2) Confirmation of cumulus cell expansion

The rate of expansion of the cumulus cells can be classified from the shape of the cumulus cell-oocyte complex as it appears at the end of in vitro maturation, 42-44 hours.

The degree of expansion of cumulus cells was divided from 0 to 4 and expressed as a cumulus cell expansion index (CEI).

At 0, it is in the form of flat, single-layered fibroblasts without expansion. If you have an old model without an extended form, it can be classified as 1. In 2, the expansion shape of the egg bulb is visible only in the outermost layer. In 3, most of the layers have an enlarged egg bulb. However, the corona radiator at the center is excluded. Finally, in 4, the entire cumulus cell is expanded from the center.

1 is a result of classification according to classification criteria when immature oocytes are matured in a maturation medium treated with 0, 20, 40, or 80 μM of phytanic acid. As can be seen from the results, it was confirmed that the immature ratio was the lowest in the mature medium treated with 40 μM phytanic acid. In addition, the rate of degeneration was significantly lower in the maturation medium treated with 40 μM phytanic acid. It was confirmed that the maturation ratio and the cumulus expansion ratio were statistically significantly higher in the maturation medium treated with 40 μM phytanic acid. Therefore, according to the results of FIG. 1, it was found that the maturation medium treated with 40 μM phytanic acid was the most effective maturation medium for maturing immature oocytes.

[Example 3] Immunofluorescence staining of mature oocytes

Mature oocytes were stripped of somatic cells using 0.1% hyaluronidase in TALP medium. Mature oocytes were washed 3 times with PBS containing 1% PVA, and fixed with 4% (w/v) paraformaldehyde/PBS for 1 hour. Mature oocytes were exposed to 1% Triton X-100 /DW(v/v) 39°C for 1 hour in a well-permeable environment.

After washing 4 times with 1% PVA, non-specific sites were blocked with 2% bovine serum albumin (BSA) in PBS for 2 hours. Mature oocytes were incubated overnight at 4° C. with primary antibodies PHYH (1:400; MBS3212923; MyBioSource, San Diego, USA) or PEX19 (1:400; MBS9605735; MyBioSource, San Diego, USA).

Then, the secondary antibody (1:200, ab6717, Abcam. Cambridge, UK) was treated at 37°C for 2 hours. After staining the nuclei, the mature oocytes were placed on a glass slide and observed with a fluorescence microscope (TE2000-S; Nikon) under the same exposure time and conditions. The intensity of PHYH and PEX19 was measured by analyzing with Image J software (version 1.46r; National Institutes of Health, USA).

3 and 4 show the above immunofluorescent staining image and an analyzed graph.

In FIGS. 3 and 4, it was confirmed that the fluorescence of PHYH or PEX19 indicating the activation of peroxisomes in the oocytes matured by treatment with 40 μM phytanic acid was stronger than that of mature oocytes without phytanic acid treatment. It was confirmed that the graph of the results measured by analyzing the image also increased statistically significantly.

That is, it was confirmed that the peroxisomes were activated in the oocytes matured with the mature medium treated with 40 μM phytanic acid.

[Example 4] Gene expression analysis using quantitative real-time PCR

(1) Gene expression analysis using quantitative real-time PCR

Mature oocytes were peeled off using 0.1% hyaluronidase in TALP medium. Quantitative real-time PCR was performed by a conventional method. RNA was extracted using the RNAqueousTM Micro Kit (Invitrogen, Vilnius, Lithuania) according to the manufacturer's protocol, and then the RNA concentration was quantified using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Complementary DNA (cDNA) was prepared using amfiRivert cDNA Synthesis Platinum Master Mix 0 (GenDEPOT, Houston, TX, USA).

For quantitative real-time PCR, the mixture of each reaction is 1 μL cDNA, 0.4 μL (10 pmol/μL) forward primer, 0.4 μL (10 pmol/μL) reverse primer, 10 μL SYBR Premix Ex Taq (TaKaRa, Otsu, Japan) and 8.2 μL of Nuclease-free water (NFW; Ambion, Austin, TX, USA) to prepare a PCR plate (Micro-Amp Optical 96-Well Reaction Plate, Singapore), and then StepOneTM Real-Time PCR System (Applied Biosystems, Waltham, MA, USA).

The amplification protocol included an initial denaturation step at 95° C. for 10 minutes, followed by 40 cycles including denaturation at 95° C. for 15 seconds, annealing at 60° C. for 1 minute, and expansion at 72° C. for 1 minute.

The expression of each target gene was compared with the expression of the internal regulatory gene (GAPDH).

R = 2 ^{-[ΔCt sample-ΔCt control]}

It was quantified using the equation. For ease of comparison, the average expression level of each gene in the control group was set to 1.

(2) Gene expression patterns related to egg development and egg cell expansion

The expression of genes GDF9, BMP15, and POU5F1 related to egg development according to the treatment concentration of phytanic acid in mature oocytes was examined.

The effect on the cumulus cell expansion-related genes (HAS2, TNFAIP6, and PTGS2) according to the treatment concentration of phytanic acid in mature oocytes was evaluated.

After maturing the immature oocytes in a medium treated with 0, 20, 40, or 80 μM of phytanic acid in the maturation medium of immature oocytes, the results are shown in FIG. 2.

In FIG. 2, it was confirmed that, in the case of BMP15 and POI5F1 among the genes related to egg development, oocytes matured in a maturation medium treated with 40 μM phytanic acid had the highest expression. However, in the case of GDF9, the expression rate of mature oocytes was highest in the maturation medium treated with 80 μM of phytanic acid.

In addition, in the case of TNFAIP6 and PTGS2 among the cumulus cell expansion-related genes of FIG. 2, it was confirmed that the expression rate of oocytes matured in the maturation medium treated with 40 μM phytanic acid was highest. HAS2 had the highest expression rate in mature oocytes in the maturation medium treated with 20 μM phytanic acid.

When the results of Figures 1 and 2 are summarized, it can be confirmed that the medium that most effectively matures immature oocytes is a mature medium treated with 40 μM phytanic acid. Therefore, in a later experiment, the most effective phytanic acid was performed in a medium treated with 40 μM.

(3) Gene expression pattern related to peroxisome activation

Expression of genes PHYH, PEX3, PEX5, PEX12, PEX19, PPARα, and PPARγ related to the activation of peroxisomes according to the treatment of phytanic acid in mature oocytes were confirmed.

In FIGS. 3 and 4, it was confirmed that the activation of peroxisomes was increased in the oocytes matured in the mature medium treated with 40 μM phytanic acid by immunofluorescence staining, compared to the oocytes matured in the mature medium not treated with phytanic acid. Additionally, the gene expression results related to peroxisome activation are shown in FIGS. 5 and 6.

Oocytes matured in the maturation medium treated with phytanic acid were found to significantly increase the expression of peroxisome activation-related genes PHYH, PEX3, PEX5, PEX12, PEX19, PPARα, and PPARγ compared to the group that did not.

However, as shown in FIGS. 7 and 8, when phytanic acid was treated with 40 μM of phytanic acid, only the expression of the PHYH gene was increased, and PEX3, PEX5, PEX12, and PPARα did not differ in control and gene expression. In addition, it was confirmed that the expression of PEX19 and PPARγ was rather reduced when phytanic acid was treated with 40 μM. Therefore, it was confirmed that only the mature oocytes in the mature medium treated with 40 μM phytanic acid had peroxisomes activated.

In other words, it was confirmed that the oocytes matured in the mature medium treated with 40 μM phytanic acid had increased activation of peroxisomes compared to the mature oocytes in the mature medium without phytanic acid. It could be seen that there was no effect.

(4) Gene expression patterns related to lipid metabolism

Expression of lipid metabolism-related genes PLIN2, ATGL, CGI58, HSL, and MGLL according to the treatment of phytanic acid in mature oocytes was confirmed.

As shown in FIG. 10, oocytes matured in a medium containing phytanic acid at a concentration of 40 μM were expressed in all of the PLIN2, ATGL, CGI58, HSL, and MGLL genes than oocytes matured in a medium not containing phytanic acid. I was able to confirm this high.

From the above results, it was found that the activation of peroxisomes and lipid metabolism were activated by the phytanic acid contained in the medium.

In addition, when compared with the expression of lipid metabolism-related factors in the cumulus cells in FIG. 11, different from mature oocytes, the cumulus cells of the medium containing phytanic acid are almost similar to or rather reduced to the oocytes of the medium not containing phytanic acid. I was able to confirm that it was done.

Therefore, it was found that the activation of lipid metabolism by phytanic acid is limited to mature oocytes and is effective.

(5) Mitochondrial biosynthesis related gene expression pattern

Expression of the factors NRF1, PGC-1α, and TFAM related to mitochondrial biogenesis according to the treatment of phytanic acid in mature oocytes was confirmed.

14, the expression of NRF1 and PGC-1α was significantly increased in mature oocytes of the medium treated with phytanic acid compared to the control group. In the case of TRAM, there was no change.

On the other hand, comparing in FIG. 15, it was confirmed that NRF1 was not increased in expression in the case of phytanic acid-treated cumulus cells, and in the case of the TFAM gene, it was confirmed that it was decreased in the phytanic acid-treated cumulus cells.

Therefore, as a result of this, it was found that peroxisomes are activated in oocytes matured in a medium treated with phytanic acid, and lipid metabolism is increased by alpha oxidation according to the activation of peroxisomes. The final result of the increase in lipid metabolism is the production of ATP in mitochondria, and as a result, it was confirmed that the expression of genes related to mitochondrial biosynthesis was also increased.

(6) Peroxisome's Antioxidant Function and Apoptosis-related Gene Expression Pattern

Expression of genes CAT, BCL-2, and BAX related to antioxidant function of peroxisomes according to the treatment of phytanic acid in mature oocytes were confirmed.

17, it can be seen that the expression of the Catalase gene was increased in the oocytes matured in the medium containing phytanic acid compared to the control group. As such, it was found that BCL-2, a cell death-related gene, increased by phytanic acid and decreased BAX.

On the other hand, it can be seen from FIG. 18 that the cumulus cells are not similar to the results of mature oocytes.

As a result, it was confirmed that phytanic acid was also involved in the mechanism of defense against oxidative stress, and as a result, it was found that it also affects the survival rate of oocytes.

[Example 5] Lipid drop staining and analysis of fatty acids

Before staining with 10 mg BODIPY-LD (D3922; Molecular Proves, Eugene, OR), BODIPY (BODIPY 493/503) or BODIPY 558/568 C12 (BODIPY-FA; D3835; Molecular Probes, Eugene, OR, USA) Washed with 1% PVA/PBS. Then, 10 μg/mL BODIPY-LD, BODIPY-FA, or 6 μM BODIPY 558/568 C12 were added in PBS, and incubated for 2 hours at room temperature in the dark.

The oocytes from which the oocytes were removed were fixed at room temperature for 2 hours with 4% PBS (paraformaldehyde-phosphate buffered saline; Invitrogen), and after staining, the oocytes were washed 3 times with PBS and placed on a glass slide and gently compressed with a coverslip. The image of each egg was captured using a fluorescence microscope (TE2000-S; Nikon). The fluorescence intensity of fat droplets and the fluorescence intensity of similar fatty acids were measured using Image J software (version 1.46r; National institutes of Health, USA).

Mature oocytes stained with a fluorescent probe were imaged under a fluorescence microscope, and the fluorescence intensity was analyzed and shown in FIG. 9 as a graph.

As shown in FIG. 9, the oocytes matured in the medium containing phytanic acid had higher fluorescence intensity of fat droplets compared to the control group. In addition, it was confirmed that the fluorescence intensity of the fatty acid was also significantly increased.

As a result, it was found that the oocytes matured in the medium containing phytanic acid had lipid metabolism activated compared to the control group.

[Example 6] JC-1 mitochondrial membrane potential measurement

In each group experiment, 30-40 oocytes were used. Mature oocytes were peeled off somatic cells using 0.1% hyaluronidase in TALP medium, washed three or more times with PVA/PBS, and fixed with 4% PFA for 1 hour.

After fixation, it was cultured by treating JC-1 in PZM-5 medium at 37°C for 30 minutes. After dyeing, washing was performed three or more times, and then mounted on a slide glass. The fluorescence ratio was measured and analyzed with the total JC-1 and the unit JC-1 (590nm/530nm, respectively).

The image obtained by the following method and the analysis result of the expression ratio according to the wavelength are shown in FIG. 12.

As shown in FIG. 12, it was confirmed that the fluorescence of the aggregate of JC-1 was increased compared to the control in mature oocytes in the medium containing phytanic acid.

As a result, when the fluorescence intensity of aggregate/monomer was analyzed as a ratio, it was found that the mitochondrial membrane potential was significantly increased in mature oocytes in the medium containing phytanic acid.

According to this result, it was confirmed that phytanic acid had an effect on the mitochondrial membrane potential.

[Example 7] ATP amount analysis

Mature oocytes from which somatic cells were removed were washed three times with 1% PVA, and then fixed with 4% formaldehyde at room temperature for 2 hours. After fixation, after washing 3 times with 1% PVA, 0.5 μM of BODIPY FL ATP (BODIPY-ATP; A12410; Molecular Probes, Eugene, OR, USA) was treated in PBS treated for 1 hour at room temperature.

Stained mature oocytes were washed with 1% PVA and mounted on a slide glass. Images obtained by fluorescence microscopy were analyzed through software.

13, it can be seen that the intensity of fluorescence measured by ATP was increased in the mature oocytes in the medium containing phytanic acid than in the mature oocytes in the medium not containing phytanic acid.

As a result, it was found that phytanic acid increases the ATP production rate during the oocyte maturation process.

[Example 8] Measurement of oxidative stress in mature oocytes

For three replicate experiments, a total of 62 oocytes were used. 42-44 hours after in vitro fertilization, mature oocytes were peeled off using 0.1% hyaluronidase in TALP medium.

To measure the levels of GSH and ROS in oocytes, cells were stained with CellTracker Blue (4-chloromethyl-6.8-difluoro-7-hydroxycoumarin;CMF2HC; Invitrogen) and H2DCFDA (2', 7'-dichlorodihydrofluorescein diacetate; Invitrogen). I did.

After washing, mature oocytes were cultured in TALP medium treated with 10 μM of CellTracker Blue or 10 μM of H2DCFDA for 30 minutes. Stained mature oocytes were transferred to a slide glass and observed with an epifluorescence microscope (TE2000-S; Nikon, Tokyo, Japan). Images were acquired using UV filters (370 nm for GSH and 460 nm for ROS) and analyzed using Image J software. Analysis was carried out by setting the measurement standard to 1.

Referring to FIG. 16, it was confirmed that the fluorescence of GSH, which is an oxidative stress defense mechanism, increased in the mature oocytes of the medium containing phytanic acid compared to the control. As a result, it was found that the fluorescence intensity of the oxidative stress ROS was weakened.

As a result, it could be confirmed that phytanic acid was also related to the oxidative stress defense mechanism.

[Example 9] Statistical Analysis

Each experiment was reproduced at least 3 times. For statistical analysis, the program GraphPad PRISM 5.01 (PRISM 5, GraphPad Software, Inc., San Diego, CA 92108, USA) was used.

To determine the obvious differences between each group in the experiment, data were expressed as mean ± S.E.M. It was analyzed by univariate analysis variance (ANOVA) using the independent sample T test and Tukey's Multiple Comparison Test. Different alphabets indicate that there is statistical significance (P<0.05).

[Example 10] In vitro fertilization method

After 42-44 hours of in vitro maturation, somatic cells were gently peeled off using a pipette with 0.1% hyaluronidase and washed three times in TALP medium. 3 mM KCl, 113.1 mM NaCl, 11 mM glucose, 7.5 mM CaCl ₂ , 1 mM in a 35x10mm Petri dish covered with mineral oil in advance by selecting mature oocytes with a second polar body, clean cell membrane, and uniform cytoplasm. It was placed in a 40 μL mTBM (modified Tris-buffered medium) solution consisting of caffeine, 20 mM Tris, 0.57 mM L-cystein, and 8% Bovine Serum Albumin (BSA) (w/v).

A liquid semen sample supplied by DARBY Corporation (Anseong, Gyeonggi-do) was centrifuged at 1000 g for 2 minutes and released with PBS to which 1% BSA was added. The released semen sample was centrifuged at 1000 g for 2 minutes. Only >90% of sperm movement was used for fertilization.

The sperm suspension was added to 40 µl of a medium solution (mTBM) to set the final sperm concentration to ⁵ ×10 ⁵ cells/mL. Oocytes were cultured in mTBM for 6 hours at 39° C. in humid conditions of 5% CO ₂ and 95% air with sperm.

After in vitro fertilization, the fertilized eggs were selected together with the second polar body, washed with mTBM, and cultured in 25 μL microdrops (10 gametes/drop) of PZM-5 medium. Embryos cultured in the solution were incubated for 7 days at 39°C in humid conditions of 5% O ₂ , 5% CO ₂ and 90% N ₂ .

[Example 11] Embryo development and blastocyst formation after in vitro fertilization

The day when the fertilized egg was transferred to the second pole body and PZM-5 is called the 0th day. From 2 days 48 hours after in vitro fertilization to 7 days 168 hours after in vitro fertilization, the divided embryos are observed under a microscope. The blastocyst formation rate and final cell number were analyzed. 0.5% pronase was used to remove the sperm (Spermatozoa) zona pellucida. The blastocyst from which the zona pellucida was removed was stained with 5 μg/mL Hoechst 33342 and observed by a fluorescence microscope.

The results observed by the above method were analyzed and shown in FIG. 19, and the obtained fluorescence image is shown in FIG. 20.

Referring to FIG. 19, it was confirmed that the rate of blastocyst formation was increased when a medium containing phytanic acid was used during embryo development after in vitro fertilization. As a result, it was found that the number of final blastocysts increased.

Through the above results, it was confirmed that phytanic acid exerts a positive effect on embryo development even after in vitro fertilization and can increase the number of finally produced blastocysts.

<110> Seoul national university <120> Peroxisome dynamics in oocytes affirmed through phytanic acid treatment. <130> CP19-211 <160> 50 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward primer <400> 1 gtcggttgtg gatctgacct 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 2 ttgacgaagt ggtcgttgag 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> POU5F1 forward primer <400> 3 ggtggaggaa gctgacaaca 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> POU5F1 reverse primer <400> 4 tctccaggtt gcctctcact 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BMP15 forward primer <400> 5 ctgggcttgc ctgtttgttc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BMP15 reverse primer <400> 6 gcccagttcc catcacttca 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GDF9 forward primer <400> 7 atccttcagc ccctagtggt 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GDF9 reverse primer <400> 8 ggctgccaga agagtcatgt 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PTGS2 forward primer <400> 9 ccagcacttc acccatcagt 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PTGS2 reverse primer <400> 10 gagtgtcttt ggctgtcgga 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HAS2 forward primer <400> 11 atgtacacgg ccttcagagc 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HAS2 reverse primer <400> 12 atctcctccg acacctccaa 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNFAIP6 forward primer <400> 13 gctcacggat ggggattcaa 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNFAIP6 reverse primer <400> 14 aatggggtag ccaactctgc 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CAT forward primer <400> 15 agggagaggc ggtttattgc 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CAT reverse primer <400> 16 ggactcgttg gtgaagctca 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BCL2 forward primer <400> 17 aatgtctcag agcaaccggg 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BCL2 reverse primer <400> 18 ggggcctcag ttctgttctc 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BAX forward primer <400> 19 catgaagaca ggggcccttt 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BAX reverse primer <400> 20 catcctctgc agctccatgt 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PHYH forward primer <400> 21 cccttcaggc ccagcaataa 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PHYH reverse primer <400> 22 gcctttgtga gttcctggga 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PEX3 forward primer <400> 23 aatgcatctt cctggggacg 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PEX3 reverse primer <400> 24 atactgtcgt cgtgcttggg 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PEX5 forward primer <400> 25 caggcggaga atgagcaaga 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PEX5 reverse primer <400> 26 ggactcgttg gtgaagctca 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PEX12 forward primer <400> 27 ctcctaaact cgatcgcccc 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PEX12 reverse primer <400> 28 cggttccgat ctctctctgc 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PEX19 forward primer <400> 29 ctcaatctat cgggcccacc 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PEX19 reverse primer <400> 30 tagacgacac tcctgcctca 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PPARalpha forward primer <400> 31 aggtcacgct gctgaagtac 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PPARalpha reverse primer <400> 32 cgcaccaaat gatagcagcc 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PPARgamma forward primer <400> 33 ccattcccga gagctgatcc 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PPARgamma reverse primer <400> 34 tttatcccca cagacacggc 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PLIN2 forward primer <400> 35 ctaaaggggc tgtgactggg 20 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PLIN2 reverse primer <400> 36 cacttccggt cactgcttct 20 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ATGL forward primer <400> 37 gacggtggca tctcagacaa 20 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ATGL reverse primer <400> 38 tggatgttgg tggagctgtc 20 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HSL forward primer <400> 39 gcctttcctg cagaccatct 20 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HSL reverse primer <400> 40 cactggtgaa gagggagctg 20 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MGLL forward primer <400> 41 accccacaga gtgtcccata 20 <210> 42 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MGLL reverse primer <400> 42 gggtgtagct gagggtttcc 20 <210> 43 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CGI58 forward primer <400> 43 tcttgctggg acacaacctg 20 <210> 44 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CGI58 reverse primer <400> 44 ccaaagggtc ctgcaatcct 20 <210> 45 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PGC-1alpha forward primer <400> 45 cacggacaga actgagggac 20 <210> 46 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PGC-1alpha reverse primer <400> 46 acctgcgcaa agtgtatcca 20 <210> 47 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NRF1 forward primer <400> 47 cagcaagtac agcaggtcca 20 <210> 48 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NRF1 reverse primer <400> 48 atgaggccgt ttccgtttct 20 <210> 49 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TFAM forward primer <400> 49 gctctccgtt cagttttgcg 20 <210> 50 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TFAM reverse primer <400> 50 acctgccagt ctgccctata 20

Claims (16)

As a medium for in vitro maturation of immature oocytes,
The medium is
Cell culture minimum medium (CCMM) containing sugar, amino acid and water; And oocyte in vitro maturation medium comprising phytanic acid
The medium for in vitro maturation of oocytes according to claim 1, wherein the phytanic acid is contained in a concentration of 30-70 μM.
The method of claim 1, wherein the cell culture minimal medium is
TCM-199 (tissue culture medium-199), DMEM (Dulbecco modified eagle medium), M-199 (medium-199), Ham's F-1 (Nutrient mixture F-10 (HAM)), NCSU (North Carolona State University) 23, NCSU37, Porcine zygote medium (PZM)-5, and oocyte in vitro maturation medium, characterized in that any one selected from the group consisting of PZM-3
The method of claim 1,
The medium is a medium for in vitro maturation of oocytes further comprising any one or more of a gonadotropin and a growth factor
The method of claim 4,
The gonadotropin is follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), equine chorionic gonadotropin: eCG) and pregnant mare serum gonadotropin (PMSG), characterized in that any one or more selected from the group consisting of oocytes in vitro maturation medium
The method of claim 4,
The growth factors include insulin-like growth factor (IGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), and transforming growth factor (IGF). factor, TGF) for in vitro maturation of oocytes, characterized in that at least one selected from the group consisting of
The method of claim 1,
The medium is an oocyte, characterized in that it further comprises at least one of sodium pyruvate, insulin-transferrin-selenium solution (ITS-A), and cysteine. In vitro maturation medium
The method of claim 1,
The medium is
Sodium pyruvate,
Insulin-transferrin-selenium solution (ITS-A),
Cysteine,
Epidermal growth factor (EGF),
Human chorionic gonadotropin (hCG),
Equine chorionic gonadotropin (eCG),
A medium for in vitro maturation of oocytes, characterized in that it is a medium based on M-199 (tissue culture medium-199) containing porcine follicular fluid and phytanic acid
In the process of in vitro maturation of immature oocytes,
A method for producing mature oocytes, characterized by treating a phytanic acid compound to induce the activation of alpha oxidation of peroxisomes
The method of claim 9,
The method is a method for producing mature oocytes, characterized in that the medium of any one of claims 1 to 8 is used.
The method of claim 9,
A method for producing mature oocytes, characterized in that immature oocytes are matured in vitro for 20-22 hours under conditions of 38-39℃ 5% CO ₂ and humidity
The method of claim 11,
A method for producing mature oocytes, characterized in that further culturing for 20 to 22 hours under conditions in which the gonadotropin is removed from the medium
The method of claim 9,
The method for producing mature oocytes is a method for producing mature oocytes, characterized in that phytanic acid in a medium for in vitro maturation activates alpha oxidation of peroxisomes in oocytes.
Of claims 1 to 8,
In vitro development method of an embryo, characterized in that the fertilized egg is developed in vitro using the medium of any one
The method of claim 14,
The in vitro development method of embryonic development in vitro, characterized in that it comprises the step of forming a blastocyst from the initial embryonic development of the fertilized egg through the hardship.
The method of claim 14,
The method is a method for in vitro development of embryos, characterized in that cultured for 7 days under the conditions of 38-39° C., 5% CO ₂ , 5% O _{2 and} 90% N ₂

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