WO2023176017A1

WO2023176017A1 - Oocyte maturation promoter and uses thereof

Info

Publication number: WO2023176017A1
Application number: PCT/JP2022/033031
Authority: WO
Inventors: 山本徳則; 村瀬哲磨; 日巻武裕
Original assignee: 山本徳則
Priority date: 2022-03-18
Filing date: 2022-09-01
Publication date: 2023-09-21
Also published as: WO2023175930A1

Abstract

Provided is an oocyte maturation promoter that can be used in the fields of animal husbandry and assisted reproductive technology, and to uses thereof. An oocyte maturation promoter according to the present invention contains a filtrate of a disrupted adipose tissue-derived stem cell solution., as an active ingredient.

Description

Oocyte maturation accelerator and its uses

The present invention relates to technology that can be used in the field of animal husbandry and assisted reproductive medicine. Specifically, the present invention relates to an oocyte maturation promoter and its uses.

BACKGROUND ART In the field of livestock farming and assisted reproductive medicine, a technique is used in which sperm and eggs are fertilized outside the body (in vitro fertilization), and the fertilized eggs (embryos) are then implanted into the uterus of another female animal. The quality and condition of the oocytes are important factors that affect the transplant results, as they affect the quality of the fertilized eggs.
In particular, since immature eggs cannot be fertilized by sperm, egg maturation is a necessary condition for successful in vitro fertilization. However, the condition of the egg cannot be determined by ultrasound or hormone levels, but only by collecting the egg, and fertilization problems due to poor egg maturation are often encountered during in vitro fertilization. Therefore, methods for maturing collected eggs are being explored as a way to increase the pregnancy success rate.

By the way, the present inventors conducted research under the hypothesis that adipose tissue-derived stem cells have the role of feeder cells and also act actively in reproductive processes, and found that adipose tissue-derived stem cells and their crushed products were in vitro. discovered that it has the effect of activating sperm and eggs, and has filed a patent application (see Patent Documents 1 to 3). For example, Patent Document 1 describes a method of activating sperm by co-cultivating sperm with cells such as adipose tissue-derived stem cells or bone marrow-derived stem cells; It has been described that eggs can be activated by co-culturing them. Furthermore, for example, Patent Document 3 describes a sperm activator containing a crushed product such as adipose tissue-derived stem cells as an active ingredient.

JP2015-82987A Unexamined Japanese Patent Publication No. 2016-7161 International Publication No. 2018/038180

In the fields of livestock farming and assisted reproductive medicine, there is a need to increase conception efficiency and pregnancy success rates.
For example, in the field of livestock farming such as cattle, producing a large number of offspring with excellent genetic traits leads to an increase in the added value of livestock products and improves the profitability of livestock farming. Although the number of cases of in vitro fertilization in livestock other than cows, such as pigs, has increased in recent years, it is still small compared to cases of in vitro fertilization in cattle. In pigs, polyspermic fertilization, in which two or more sperm fuse with the egg cytoplasm, occurs at a high rate, so this does not lead to an improvement in the single fertilization rate, which is normal fertilization. Improving the success rate of in vitro fertilization in humans is expected to be a means of halting Japan's extremely low birthrate and aging population. In this way, methods for improving conception rates and fertilization rates are being studied in the livestock industry and assisted reproductive medicine fields.

However, in the inventions described in Patent Documents 1 and 2, sperm and eggs are activated by co-culturing them with adipose tissue-derived stem cells, but since both use cells, It was necessary to start cell culture at the right time, and it lacked ease of adjustment and operation. There are also concerns about the occurrence of adverse events due to the use of living cells. The invention described in Patent Document 3 describes the activation of spermatozoa using a crushed adipose tissue-derived stem cell, but does not describe the activation of eggs.

In order to solve the above problems, the present inventors focused on the filtrate of adipose tissue-derived stem cell disruption solution and as a result of repeated studies, they discovered that it has the effect of promoting egg maturation in vitro. It was completed. That is, the present invention has the following configuration.

[1] An oocyte maturation promoter containing a filtrate of adipose tissue-derived stem cell disruption solution as an active ingredient.
[2] The oocyte maturation promoter according to [1], which is used for in vitro fertilization.
[3] The oocyte maturation promoter according to [1] or [2], wherein the biological species of the adipose tissue-derived stem cells and the biological species of the eggs are the same.
[4] The oocyte maturation promoter according to [1], wherein the biological species of the adipose tissue-derived stem cells is a non-human mammal.
[5] The oocyte maturation promoter according to [1], wherein the biological species of the adipose tissue-derived stem cells is human.
[6] The oocyte maturation promoter according to [1], which has a ROS value of less than 1 when the control is set to 1.
[7] A method for producing an oocyte maturation promoter, including the following steps.
(1) A step of disrupting adipose tissue-derived stem cells.
(2) A step of filtering the crushed liquid obtained in step (1) or the supernatant obtained by centrifuging the crushed liquid to obtain a filtrate.
(3) A step of formulating the filtrate obtained in step (2).
[8] A method for producing mature eggs, which comprises culturing eggs in the presence of the egg maturation promoter according to [1].
[9] An in vitro fertilization method, in which an egg treated with the egg maturation promoter according to [1] is allowed to coexist with sperm in vitro.
[10] An in vitro fertilization method in which eggs and sperm are allowed to coexist in vitro in the presence of the egg maturation promoter according to [1].
[11] An embryo transfer method comprising culturing a fertilized egg produced by the in vitro fertilization method described in [8] or [9], and injecting the grown blastocyst into the uterus of a mammal.
[12] Use of the oocyte maturation accelerator according to [1] as an oocyte quality improving agent.

The egg maturation promoter of the present invention can mature eggs. More specifically, in vitro fertilization uses collected eggs, but since the eggs collected during egg collection are immature eggs, they cannot be fertilized even if they are inseminated with sperm. By using the oocyte maturation promoter of the present invention, immature oocytes can be matured, making it easier for the fertilization process to proceed. Fertilization rates are improved, and the quality of fertilized eggs and their ability to develop into blastocysts are promoted, resulting in improved implantation rates and, ultimately, pregnancy rates. In this specification, maturation refers to nuclear maturation and cytoplasmic maturation of an egg.
Furthermore, since the oocyte maturation promoting agent of the present invention uses a filtrate, adverse events caused by cell administration can be avoided. For example, when cells are generally administered, the cells being administered are alive, so even if pathogens are present, they can hardly be inactivated or removed, posing a risk of infection.
Furthermore, when administering cells, it is necessary to culture the cells according to the patient's schedule. Frozen and thawed cells are generally not used for treatment. This is because cell activity may decrease or cells may die. On the other hand, since the oocyte maturation promoter of the present invention is a cell-free liquid, it can be stored by cryopreservation and can be used for treatment by thawing it when necessary. Therefore, there is no need to time the cell culture for use or increase the number of cells required for treatment, and therefore preparation and handling are easy, and the clinical advantage is extremely large.

In the area comparison test of cumulus cell-oocyte complexes (COCs), the area of COCs before maturation culture (A) and the area of COCs after culture (B) Mitochondria distribution status in the egg (A: Type I, in which mitochondria are distributed uniformly throughout the cytoplasm, B: Type II, in which mitochondria are distributed around the cell membrane and also unevenly distributed inside the cell, C: Type III, in which mitochondria are distributed only around the cell membrane)

TECHNICAL FIELD The present invention relates to an oocyte maturation promoter. More specifically, it contains a crushed filtrate of adipose tissue-derived stem cells as an active ingredient. In addition, in this specification, "containing as an active ingredient" means containing the filtrate of the crushed solution of adipose tissue-derived stem cells in an effective amount for treatment.
<Egg maturation accelerator>

In the present invention, a mature egg refers to an egg that has undergone nuclear and cytoplasmic maturation. A mature egg can be fertilized with a sperm and, after fertilization, can develop into a blastocyst. Note that an immature egg is an egg that cannot be fertilized even if it is inseminated with a sperm.
Since the oocyte maturation promoter of the present invention can be used to promote oocyte maturation, it is easy to obtain mature oocytes. Mature eggs can be fertilized with sperm, which can improve the fertilization rate and also have a positive effect on development after fertilization. This in turn improves the implantation rate and pregnancy rate.
In the present invention, whether or not an egg is mature can be determined as "matured" if acceleration of egg maturation can be confirmed by at least one of the following methods. Among them, reactive oxygen species (ROS) are also related to the aging of eggs, so they can be considered an indicator of pregnancy rates, especially in elderly patients.
(1) Area comparison of cumulus cell-oocyte complexes (COCs)

In fertilization, the cumulus cell layer is an important factor. Generally, when the egg is immature, the cumulus cells are small, and as the egg matures, the cumulus cells grow and expand. Since the degree of swelling of cumulus cells is considered to be an indicator of oocyte maturation, in this specification, we measured the area of COCs, and if the area increased compared to the control, it was determined that the oocyte had matured. to decide.
The specific test method is shown. Prepare a maturation medium drop (10 μL) containing the oocyte maturation promoter of the present invention. One immature oocyte is placed in each drop, and the COCs are photographed with a digital camera before maturation culture begins, and then cultured in a CO ₂ incubator (37-40°C, 5% CO ₂ . The same applies hereinafter). The COCs after culture are photographed in the same way, and the area of COCs before and after maturation culture is compared. When A is the area of COCs after maturation culture divided by the area of COCs before maturation culture, if A is increased compared to the control, the oocyte is judged to be "mature". It is preferable that A is 1.05 times or more compared to the control, and more preferably 1.10 times or more. Further, it is preferable that A is 2.00 times or less as compared to the control.

The maturation medium is not particularly limited as long as it is a medium commonly used for oocyte maturation culture, but includes NCSU medium such as NCSU37 medium and NCSU23 medium, Medium 199, Dulbecco's modified Eagle medium (D-MEM) (Nacalai Tesque stock) DMEM/F12 (SIGMA, Gibco, etc.), synthetic fallopian tube fluid (SOF), etc. can be used. Two or more types of media may be used in combination. If necessary, serum, plasma, serum albumin, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), antibiotics, 2-mercaptoethanol, amino acids, vitamins, inorganic salts, etc. may be added to the medium. The cells are preferably cultured using a culture container such as a culture dish.
The culture conditions may be those commonly used in oocyte maturation culture. For example, an environment of 37°C to 40°C and 5% CO ₂ is preferable.
The camera used for photographing is not particularly limited as long as it can appropriately measure the area of COCs, but it is preferable to take photographs by installing a digital camera on an inverted microscope.
(2) Evaluation of reactive oxygen species (ROS) and glutathione (GSH) content

Oxidative stress is said to be deeply involved in the development of oocytes into eggs. Active oxygen that causes oxidative stress is known to be a factor that causes various diseases, and oxidative stress caused by accumulation of active oxygen is particularly a cause of aging. As the egg ages, the normally perfectly round egg may become oval or distorted, making it difficult for it to be fertilized. Additionally, even after fertilization occurs, cells may become difficult to divide or the embryo may stop growing. The decline in pregnancy rates, especially for women in their late 30s and above, is often due to the aging of eggs. It is also said that reduced glutathione protects eggs from active oxygen toxicity, and the amount thereof is used as an indicator of cytoplasmic maturation of eggs. Therefore, in this specification, the amount of reactive oxygen species (ROS) and reduced glutathione (GSH) is measured, and when the following requirements are met, it is determined that the egg has "matured".
The specific test method is shown. 30 to 40 immature eggs are introduced as a group into 200 μL of a maturation medium supplemented with the oocyte maturation promoter of the present invention and cultured. After maturation culture, 0.1% (w/v) Hepes-Tyrode-Lactate-Pyruvate-polyvinylalcohol (hereinafter also referred to as Hepes-TLP-PVA) supplemented with hyaluronidase was added, and the cumulus cells were detached by pipetting to remove the first polar body. Oocytes confirmed to have been released are selected.
Immerse it in 500 μL of Hepes-TLP-PVA supplemented with 10 μL of 2',7'-Dichlorofluorecein diacetate and 5 μL of Cell Tracker Blue CMF ₂ HC, and stain for 30 minutes in a CO ₂ incubator. After washing the stained eggs, record fluorescence images using a fluorescence microscope using UV filters of 460 nm for ROS and 365 nm for GSH. When the control is set as "1", the egg is judged to be "mature" when ROS is less than 1 or GSH is greater than 1. ROS is preferably 0.9 or less, more preferably 0.8 or less, and even more preferably 0.7 or less. Further, ROS is preferably 0.1 or more, and more preferably 0.2 or more. GSH is preferably 1.0 or more, more preferably 1.1 or more, and even more preferably 1.3 or more. Further, GSH is preferably 2.0 or less, and preferably 1.8 or less. For the medium and culture conditions, the conditions used when comparing the areas of COCs can be used.
(3) Distribution of mitochondria

Mitochondria are organelles that produce energy in living organisms, and are thought to be involved in the aging of eggs. Mitochondrial replication is suppressed after oocyte maturation until the morula stage, so the quality of mitochondria already present in mature oocytes is important for early embryonic development. Therefore, the present inventors thought that evaluating the distribution of mitochondria could be used as an index for predicting cytoplasmic maturation. In this specification, the distribution of mitochondria is evaluated, and an egg is determined to be "mature" when the following requirements are met.
Refer to the method described in Sha Wei et al. (2010) “Effect of gonadotropins on oocyte maturation in vitro: an animal model.” Fertil Steril, Mar. 15;93(5):1650-61. Specifically, using the Mito Tracker® Red CMXRos (Invitrogen) kit, amikamycin (100 mg titer) (Meiji Seika Pharma) was added to 1 μL of Stock A prepared according to the instructions Medium 199 ( Gibco) Add 149 μL and mix. Furthermore, mix 1850 μL of Medium 199 (Gibco) to create multiple 150 μL microdroplets. Egg selection is performed by the method described above.

After washing the selected eggs, about 10 eggs are transferred into the microdroplet as a group, and then left standing in a CO ₂ incubator for 30 minutes to stain mitochondria. The eggs are then washed and photographed using a confocal laser scanning microscope using a Rhodamine filter for image analysis.
Type I is when mitochondria are distributed evenly throughout the cytoplasm, type II is when mitochondria are distributed around the cell membrane and unevenly distributed inside the cell membrane, and type II is when mitochondria are distributed only around the cell membrane. The ones that exist are classified as type III. The ratio of type I to the number of test eggs is calculated, and when that of the control is set as "1", if the ratio is greater than 1, the eggs are judged to have "matured". It is preferably 1.3 times or more, and more preferably 1.5 times or more. Further, it is preferable that the ratio is 5 times or less.
(4) Mitochondrial activity evaluation

Since mitochondrial activity is known to be important for the maturation and quality of pig oocytes and subsequent embryonic development, we believe that assessing mitochondrial activity can predict cytoplasmic maturation. We thought that it could be used as an indicator for In this specification, mitochondrial activity is evaluated, and an egg is determined to be "mature" when the following requirements are met.
Based on the method described in Lee SK et al. (2014) “The association of mitochondrial potential and copy number with pig oocyte maturation and developmental potential.”, J Reprod Dev. 2014 Apr 24;60(2):128-35 do. Specifically, using MitoProbe (registered trademark) JC-1 Assay Kit (Invitrogen), amikamycin (100 mg titer) (Meiji Seika Pharma) was added to 57.5 μL of Stock A prepared according to the instructions. Add 1092.5 μL of 199 (Gibco) and mix to create multiple 80 μL microdroplets. Egg selection is performed by the method described above.
After washing the selected eggs, about 10 eggs are transferred into the microdroplet as a group, and then left standing in a CO ₂ incubator for 50 minutes to stain mitochondria. After washing the eggs, the eggs are photographed using a confocal laser scanning microscope using a Rhodamine filter and a FITC filter, and the images are analyzed. The membrane potential difference is calculated by dividing the fluorescence intensity of red, which shows high activity, by the fluorescence intensity of green, which shows low activity, and is used as mitochondrial activity. When the control value is set to 1, if the mitochondrial activity is higher than that value, the egg is judged to be ``mature.'' It is preferably 1.01 or more, and more preferably 1.02 or more. Further, the value is preferably 1.10 or less, more preferably 1.07 or less.
<Adipose tissue-derived stem cells>

Adipose-derived stem cells (ASC, Adipose-derived regeneration cells: ADSC, Adipose-derived mesenchymal stem cells: AT-MSC, AD-MSC, etc.) are mesenchymal stem cells.Hereinafter, simply referred to as “ (sometimes referred to as "ADSC") is a type of somatic stem cell, and is a stem cell contained in adipose tissue. Adipose tissue-derived stem cells also have self-renewal and multipotency, and can differentiate into a variety of cells, including not only fat but also bone, cartilage, nerves, muscle, cardiac muscle, blood vessels, hepatocytes, and pancreatic islet cells. It is known that it is possible.
The inventors of the present invention have discovered that the filtrate of the disrupted solution of the stem cells, rather than the adipose tissue-derived stem cells themselves, has the effect of promoting egg maturation.

The origin of the ADSC used in the oocyte maturation promoter of the present invention, that is, the biological species, is not particularly limited. The biological species may be determined in consideration of the origin of the eggs used and the use of the eggs (mature eggs) obtained by the method of the present invention. As long as the desired effect is achieved, that is, the egg is matured, the biological species of ADSC used in the egg maturation promoter and the biological species of the egg may be different, but they are preferably the same. In order to avoid the problem of immune rejection, it is preferable to collect ADSCs from the same individual as the subject (recipient) to which the egg maturation promoter is applied and use them as the egg maturation promoter.

The origin (species) of ADSCs includes humans and non-human mammals. Non-human mammals include cows, pigs, horses, goats, sheep, monkeys, dogs, cats, mice, rats, guinea pigs, hamsters, and the like. Among non-human mammals, cows, pigs, horses, goats, and sheep are preferable because livestock farming can be carried out efficiently. In addition, examples of the origin (species) of eggs include humans, non-human mammals, birds, fish, and the like. Non-human mammals include the animals mentioned above.
<Method for preparing adipose tissue-derived stem cells>

In the present invention, ADSCs also include cells obtained by culturing (including subculture) the somatic stem cells, as long as they maintain pluripotency. Typically, ADSCs are prepared in an "isolated state" using adipose tissue separated from a living body as a starting material and as cells constituting a cell population (including cells other than ADSCs derived from adipose tissue). The "isolated state" here refers to a state that has been removed from its original environment (i.e., a state that forms part of a living body), that is, a state that exists in a state different from its original state of existence due to artificial manipulation. It means there is.
Incidentally, ADSC in the present invention may be prepared according to a conventional method. Since ADSC is widely used for various purposes, those skilled in the art can also prepare it with reference to literature and books. Cells distributed from public cell banks or commercially available cells may also be used. Hereinafter, as an example of the cell preparation method, a method (one example) for preparing adipose tissue-derived stem cells will be described.

ADSCs are prepared through steps such as separating stem cells from adipose matrix, washing, concentrating, and culturing. The method for preparing ADSC is not particularly limited. For example, known methods (Fraser JK et al. (2006), Fat tissue: an under appreciated source of stem cells for biotechnology. Trends in Biotechnology; Apr; 24(4): 150-4. Epub 2006 Feb 20. Review.; Zuk PA et al.(2002), Human adipose tissue is a source of multipotent stem cells. Molecular Biology of the Cell; Dec;13(12):4279-95.; Zuk PA et al.(2001), Multilineage cells from ADSCs can be prepared according to human adipose tissue: implications for cell-based therapies. Tissue Engineering; Apr; 7(2): 211-28. Additionally, devices for preparing ADSCs from adipose tissue (e.g., Celution® device (Cytori Therapeutics, Inc., San Diego, USA)) are commercially available, and ADSCs can be prepared using this device. You can decide. Using this device, a cell population including ADSCs can be separated from adipose tissue (K. Lin. Et al. Cytotherapy (2008) Vol. 10, No. 4, 417-426). A specific example of the method for preparing ADSC is shown below.
(1) Preparation of cell populations from adipose tissue

Adipose tissue is collected from humans and non-human mammals by excision, suction, or other means. The non-human mammal may be any of the animals mentioned above, including pet animals, livestock, and experimental animals. Furthermore, the age and sex of the creature are not particularly limited. In order to avoid the problem of immune rejection, it is preferable to collect adipose tissue (autologous adipose tissue) from the same individual as the subject (recipient) to whom the oocyte maturation promoter is applied. However, this does not preclude the use of adipose tissue from animals of the same species (allogeneic) or adipose tissue from animals of different species.
In addition, in humans, ADSCs can also be prepared from tissue pieces aspirated during liposuction surgery during cosmetic surgery, or from excised adipose tissue contained in tissue excised from a living body during surgery or the like. Because ADSCs exist around large blood vessels, more can be obtained from excised adipose tissue than from lipoaspirate fluid. On the other hand, preparing stem cells from liposuction fluid results in smaller surgical scars and less burden on the donor.

Examples of adipose tissue include subcutaneous fat, visceral fat, intramuscular fat, and intermuscular fat. Among these, subcutaneous fat can be harvested very easily under local anesthesia, so the burden on the donor during collection is low, and it can be said to be a preferable cell source. Usually one type of adipose tissue is used, but it is also possible to use two or more types of adipose tissue in combination. Alternatively, adipose tissue collected in multiple batches (not necessarily the same type of adipose tissue) may be mixed and used for subsequent operations.

The amount of adipose tissue to be collected can be determined by taking into account the type of donor, the type of tissue, or the amount of ADSC required, and is, for example, about 0.5 to 500 g. However, considering the burden on the donor, it is preferable to limit the amount collected at one time to about 10 to 20 g or less. The collected adipose tissue is subjected to the following enzymatic treatment after removing blood components adhering thereto and cutting it into pieces as necessary. Note that blood components can be removed by washing the adipose tissue in an appropriate buffer or culture solution.

Enzyme treatment is performed by digesting adipose tissue with enzymes such as collagenase, trypsin, and dispase. Such enzymatic treatments may be carried out using techniques and conditions known to those skilled in the art (see, for example, R. I. Freshney, Culture of Animal Cells: A Manual of Basic Technique, 4th Edition, A John Wiley & Sones Inc., Publication). . The cell population obtained by the above enzyme treatment includes pluripotent stem cells, endothelial cells, stromal cells, hemocyte cells, and/or their precursor cells. The type and ratio of cells constituting the cell population depend on the origin and type of adipose tissue used.
(2) Obtaining sedimented cell populations (SVF fractions: stromal vascular fractions)

The cell population is then subjected to centrifugation. The sediment obtained by centrifugation is collected as a sedimented cell population (herein also referred to as "SVF fraction"). The conditions for centrifugation vary depending on the type and amount of cells, but are, for example, 800 to 1,500 rpm for 1 to 10 minutes. Note that, prior to centrifugation, it is preferable to subject the enzyme-treated cell population to filtration or the like to remove enzyme-undigested tissue and the like contained therein.
The "SVF fraction" obtained here contains ADSCs. Note that the type and ratio of cells constituting the SVF fraction depend on the origin and type of adipose tissue used, the conditions of enzyme treatment, etc. Further, the characteristics of the SVF fraction are shown in International Publication No. 2006/006692A1 pamphlet.
(3) Selective culture of adherent cells (ADSC) and cell collection

In addition to ADSCs, the SVF fraction contains other cellular components (endothelial cells, stromal cells, hemocyte cells, their precursor cells, etc.). Therefore, in one embodiment of the present invention, the following selective culture is performed to remove unnecessary cell components from the SVF fraction. The resulting cells are then used as ADSCs in the present invention.
First, the SVF fraction is suspended in an appropriate medium, then seeded onto a culture dish and cultured overnight. Remove floating cells (non-adherent cells) by replacing the medium. Thereafter, culture is continued while changing the medium as appropriate (for example, once every 2 to 4 days). Perform subculture as necessary. Although the number of passages is not particularly limited, from the viewpoint of maintaining pluripotency and proliferation ability, it is not preferable to repeat passages excessively (it is preferable to limit passages to about 5 passages). Note that a normal culture medium for animal cell culture can be used as the culture medium. For example, Dulbecco's modified Eagle's Medium (DMEM) (Nissui Pharmaceutical Co., Ltd., etc.), α-MEM (Dainippon Pharmaceutical Co., Ltd., etc.), DMEM:Ham's F12 mixed medium (1:1) (Dainippon Pharmaceutical Co., Ltd., etc.) , Ham's F12 medium (Dainippon Pharmaceutical Co., Ltd., etc.), MCDB201 medium (Functional Peptide Institute), etc. can be used. A medium supplemented with serum (fetal bovine serum, human serum, sheep serum, etc.) or serum replacement (Knockout serum replacement (KSR), etc.) may also be used. The amount of serum or serum substitute added can be set, for example, within the range of 5 to 30% (v/v).

Adhesive cells selectively survive and proliferate by the above operations. Subsequently, the proliferated cells are collected. The recovery operation may be carried out in accordance with a conventional method, and for example, the cells can be easily recovered by peeling off the cells after enzyme treatment (trypsin or dispase treatment) with a cell scraper, pipette, or the like. Furthermore, when sheet culture is performed using a commercially available temperature-sensitive culture dish, it is also possible to collect the cells directly in the form of a sheet without enzyme treatment. By using the cells (ADSC) collected in this way, a cell population containing ADSC in high purity can be prepared.
(4) Low serum culture (selective culture in low serum medium) and cell collection

In one aspect of the present invention, the following low serum culture is performed instead of or after the operation (3) above. The resulting cells are then used as ADSCs in the present invention.
In low-serum culture, the SVF fraction (if this step is performed after (3), use the cells collected in (3)) is cultured under low-serum conditions, and the desired pluripotent stem cells (i.e., ADSCs) are cultured under low-serum conditions. ) to selectively grow. Since the low-serum culture method requires only a small amount of serum, when ADSCs obtained by the method of the present invention are used for therapeutic purposes, it is possible to use the subject's (patient's) own serum. That is, culture using autologous serum becomes possible. Here, "low serum conditions" refers to conditions in which the medium contains 5% (v/v) or less of serum. Cells are preferably cultured in a culture solution containing 2% (v/v) or less serum. More preferably, the cells are cultured in a culture solution containing 2% (v/v) or less of serum and 1 to 100 ng/mL of fibroblast growth factor-2 (bFGF).

The serum is not limited to fetal bovine serum, but human serum, sheep serum, etc. can be used. When the activated sperm obtained by the method of the present invention is used for human treatment, preferably human serum, more preferably serum of the subject to be treated (ie, autologous serum) is used.
As the medium, a normal medium for animal cell culture can be used, provided that the amount of serum contained during use is low. For example, Dulbecco's modified Eagle's Medium (DMEM) (Nissui Pharmaceutical Co., Ltd., etc.), α-MEM (Dainippon Pharmaceutical Co., Ltd., etc.), DMEM: Ham's F12 mixed medium (1:1) (Dainippon Pharmaceutical Co., Ltd., etc.), Ham's F12 medium (Dainippon Pharmaceutical Co., Ltd., etc.), MCDB201 medium (Functional Peptide Institute), etc. can be used.

By culturing with the above method, ADSCs can be selectively proliferated. Furthermore, since ADSCs that proliferate under the above culture conditions have high proliferative activity, the number of cells required for the present invention can be easily prepared by subculturing. In addition, International Publication No. 2006/006692A1 pamphlet shows the characteristics of cells that selectively proliferate by culturing the SVF fraction in low serum.
Subsequently, cells selectively proliferated by the above-described low serum culture are collected. The recovery operation may be performed in the same manner as in the case (3) above. By using the collected ADSCs, a cell population containing highly purified ADSCs can be obtained.

In the above method, cells grown by culturing the SVF fraction in low serum are used. Cells grown by low-serum culture may also be used as ADSCs. That is, in one embodiment of the present invention, cells that proliferate when a cell population obtained from adipose tissue is cultured in low serum are used as ADSCs. Alternatively, instead of the pluripotent stem cells obtained by selective culture ((3) and (4) above), the SVF fraction (containing adipose tissue-derived mesenchymal stem cells) may be used as is. Here, "used as is" means used in the present invention without undergoing selective culture.
<Method for preparing filtrate of adipose tissue-derived stem cell disruption solution>

The oocyte maturation promoter of the present invention contains as an active ingredient a filtrate (sometimes referred to as ADSC filtrate) obtained by filtering a crushed solution obtained by crushing ADSC.
ADSCs can be disrupted using a general cell disruption method. For example, processing methods using freeze-thaw (processing of freezing and then thawing), ultrasonic waves, a French press, a mortar, a homogenizer, glass beads, etc. can be used. Furthermore, the cells to be subjected to the crushing treatment are not limited to live cells, but dead cells or damaged cells may also be used. Among the above-mentioned crushing treatments, freeze-thaw treatment and ultrasonic treatment are preferred. In particular, freezing and thawing treatment is particularly preferable because it is simple, avoids contamination due to contact between machines and cells, and is hygienic. When crushing by freezing and thawing, preferred conditions described below can be used. When disrupting by ultrasonication, it is preferable to use unfrozen cells, but since the heat emitted from the equipment often causes protein denaturation and aggregation, cell suspension It is preferable to repeatedly perform short-term treatments while cooling in ice. Specifically, it is preferable to repeat crushing for 5 to 15 seconds with an output of 200 to 300 W and a pause of 10 to 30 seconds multiple times.

Note that freezing and thawing is preferably repeated in order to sufficiently lyse cells, because cells expand and form ice crystals during the freezing process, and the ice crystals destroy the cells and are thawed during thawing. Specifically, it is preferable to repeat the freezing and thawing process 1 to 5 times. Freezing conditions in the freeze-thaw treatment are not particularly limited, but it is preferable to freeze at -20 to -196°C, for example. The melting conditions are also not particularly limited. For example, melting by leaving overnight in a refrigerator at 5°C or lower, melting in a hot water bath (for example, 35 to 40°C), melting at room temperature, etc. can be employed.
The concentration of the cell suspension used for disruption is preferably 1×10 ⁴ to 1×10 ⁷ ADSC cells/mL. More preferably, ADSC is 10×10 ⁴ to 500×10 ⁴ cells/mL. The concentration is easy to work with, and a sufficient amount of filtrate can be obtained in one operation.

The oocyte maturation promoter of the present invention is prepared by filtering the cell disruption solution and using the obtained filtrate. Unwanted components can be removed by filter filtration. Furthermore, if a filter with an appropriate pore size is used, removal of unnecessary components and sterile filtration can be performed at the same time. The material of the filter used for the filter treatment is not particularly limited, but cellulose acetate and metal filters, which do not easily adsorb proteins, are preferred. Particularly preferred is cellulose acetate. The filter pore size is preferably 0.1 to 0.45 μm. More preferably 0.15 to 0.3 μm. If sterile filtration is also performed at the same time, a pore size of 0.2 μm is preferred.
Note that the disruption solution may be centrifuged in advance, and the resulting supernatant may be filtered. By centrifuging the crushed solution before filtering, nuclei and the like can be removed and clogging of the filter can be prevented, allowing efficient filtering. If centrifugation is performed in advance, it is preferable to centrifuge at 100 to 1,500 xg for 3 to 10 minutes after ADSC disruption. Furthermore, the temperature during centrifugation is not particularly limited.

If the filtrate is not used immediately for treatment, it can be stored frozen until use. Preferably, it is stored at -100 to -60°C. Generally, repeated freezing and thawing of cells tends to reduce cell activity and increase the number of dead cells, but the oocyte maturation promoter of the present invention is a filtrate and does not contain stem cells, so No matter how many times it is stored and thawed, its quality remains the same. In the present invention, the obtained filtrate is used as an oocyte maturation promoter.
<Production method of oocyte maturation accelerator>

The oocyte maturation promoter in the present invention can be produced by the following steps. The ADSC culture method, crushing treatment method, centrifugation treatment method, and filter filtration method were described above.
(1) A step of disrupting adipose tissue-derived stem cells.
(2) A step of filtering the crushed liquid obtained in step (1) or the supernatant obtained by centrifuging the crushed liquid to obtain a filtrate.
(3) A step of formulating the filtrate obtained in step (2).
The oocyte maturation promoter of the present invention can also be used in the form of a composition when used for various purposes described below. Specifically, fetal serum may be included for the purpose of protecting cells as long as its effectiveness is not lost. Furthermore, the obtained filtrate contains other pharmaceutically acceptable ingredients, such as carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, It may also be formulated into a formulation containing physiological saline or the like.
The oocyte maturation promoter of the present invention is a cell suspension containing ADSCs of 1×10 ⁴ to 1×10 ⁷ cells/mL, preferably a cell suspension containing ADSCs of 10×10 ⁴ to 500×10 ⁴ cells/mL. crush. The resulting filtrate does not actually contain cells, but since the cell suspension at the above concentration is disrupted, the filtrate contains cells equivalent to 1×10 ⁴ to 1×10 ⁷ cells/mL. . It is preferably equivalent to 10×10 ⁴ to 500×10 ⁴ cells/mL. The preferable concentration of the cell suspension varies depending on the animal species, but the lower limit is preferably 10×10 ⁴ cells/mL or more, 30×10 ⁴ cells/mL or more, and 50×10 ⁴ cells/mL or more. The upper limit of the concentration of cell suspension is 450×10 ⁴ cells/mL or less, 400×10 ⁴ cells/mL or less, 380×10 ⁴ cells/mL or less, 300×10 ⁴ cells/mL or less, 250×10 ⁴ cells/mL or less, preferably 200×10 ⁴ cells/mL or less. The concentration range of the cell suspension can be determined as any combination of these upper and lower limits. In addition, for pigs, 30×10 ⁴ to 150×10 ⁴ cells/mL is more preferable, and 40×10 ⁴ to 130×10 ⁴ cells/mL is even more preferable. For cattle, 60×10 ⁴ to 450×10 ⁴ cells/mL is more preferable, and 70×10 ⁴ to 400×10 ⁴ cells/mL is even more preferable.
<Applications of oocyte maturation accelerator>

The egg maturation promoter of the present invention is useful for in vitro fertilization, treatment and improvement of infertility caused by decreased egg function, livestock breeding, breeding and maintenance of species (e.g. maintenance of endangered species, maintenance of pet lines, etc.). crossbreeding) etc.
Since the oocyte maturation promoter of the present invention can reduce the amount of ROS, it improves the effect of improving the quality of oocytes due to aging. Therefore, the oocyte maturation accelerator of the present invention not only matures oocytes, but can also be used as an oocyte quality improving agent, and is expected to have the effect of improving pregnancy rates in elderly human patients.
Hereinafter, in vitro fertilization will be explained in detail among the uses of the oocyte maturation promoter of the present invention. Note that, except for the conditions characteristic of the present invention, that is, the use of the oocyte maturation promoter of the present invention, conventional methods may be followed (for example, the livestock artificial fertilization course text (Livestock internally fertilized eggs, livestock in vitro fertilization (Egg Transplantation Edition) (Japan Livestock Artificial Insemination Technicians Association) etc. may be helpful).
<Method for promoting egg maturation in in vitro fertilization>

Examples of methods for promoting egg maturation include culturing eggs in the presence of the egg maturation promoter of the present invention. In other words, the method for producing mature eggs includes culturing eggs in the presence of the egg maturation promoter of the present invention.
The eggs to be cultured may be eggs collected from an ovary or cryopreserved eggs. The biological species of the egg and the biological species derived from the egg maturation promoter are preferably the same. Mention may be made of the human or non-human mammals mentioned above.

For culturing, conditions suitable for culturing oocytes can be adopted, but for example, culturing in an environment of 37 to 40°C and 5% CO ₂ is preferable. However, considering the animal species of the egg, it is preferable to set the temperature close to the animal's body temperature. For example, when using eggs from livestock such as pigs and cows, it is recommended to set the temperature to a relatively high temperature (38-39°C). Furthermore, in the case of human eggs, a temperature condition of around 37°C is preferable. As the medium, the previously mentioned NCSU medium, Medium 199, etc. can be used. Two or more types of media may be used in combination. Components that can be added to the medium are also as described above. Typically, cells are cultured using a culture container such as a culture dish.

The culturing method is not particularly limited as long as the oocytes can be exposed to the oocyte maturation accelerator, such as by culturing the oocyte maturation accelerator and the oocytes together. For example, eggs can be placed, suspended, or immersed in a liquid medium containing an egg maturation promoter. The order in which the oocyte maturation promoter and oocytes are added is not particularly limited, and they may be added at the same time and cultured, or may be added and cultured at regular intervals. Specifically, it is preferable to add eggs to a medium containing an egg maturation promoter.
It is preferable to add 10 to 70 eggs to 10 to 50 μL of the egg maturation promoter, which is a filtrate obtained by crushing a cell suspension with a concentration of 10× ^{10 4} to 500×10 ⁴ cells/mL.
After administration of the oocyte maturation promoter, the oocytes are preferably cultured for 1 hour to 4 days. If the culture period is too short, the eggs will not mature sufficiently, so 1 to 3 days is more preferable.
Once it is confirmed that the eggs have matured using one of the methods mentioned above, the eggs can be collected and used for in vitro fertilization, infertility treatment due to decreased egg function, livestock mating/breeding, breeding, species maintenance, etc. I can do it.

One embodiment of the in vitro fertilization method using the egg maturation promoter of the present invention includes (i) a method in which eggs treated with the egg maturation promoter coexist with sperm in vitro; (ii) in the presence of the egg maturation promoter. One method is to allow eggs and sperm to coexist outside the body. Coexistence here refers to the existence of two or more things together; for example, in (i), an egg maturation promoter is mixed with an egg to form a mature egg, and then sperm is added thereto; in (ii), , an egg maturation accelerator, a method of mixing eggs and sperm, etc.

Sperm used in the above-mentioned in vitro fertilization include sperm collected by sperm collection, intratesticular sperm collection, microscopic epididymal sperm collection, percutaneous epididymal sperm collection, sperm concentration washing method, etc. , sperm which have been pre-cultured, and sperm which have been thawed after cryopreservation. Preferably, the spermatozoa are diluted with the same medium and prepared at a concentration of 50×10 ⁴ to 4000×10 ⁴ sperm/mL. In order to increase fertilization efficiency, it is preferable to use highly motile sperm.
In addition, the biological species of eggs and sperm are, in principle, the same. The biological species from which eggs or sperm are derived include the aforementioned humans and non-human mammals. However, the biological species of the egg and the sperm may be different as long as the combination allows fertilization.
Culture conditions for eggs and sperm, or mature eggs and sperm, can follow conventional methods for in vitro fertilization.

In in vitro fertilization, when eggs matured by the egg maturation promoter of the present invention are used, fertilization becomes easier and the fertilization rate improves. When a fertilized egg develops normally, it undergoes repeated somatic cell division. Since the subsequent embryo differentiation is also good, the rate of development into morula and blastocyst is improved. Examples of uses for blastocysts include embryo transfer methods in which the grown blastocyst is injected into the uterus of a mammal. This leads to improved implantation and pregnancy rates. Note that embryo transfer is not limited to blastocyst transfer.

EXAMPLES The present invention will be explained in detail with reference to Examples below, but the present invention is not limited to these Examples in any way. The method for preparing the ADSC filtrate used in the experiment, the model animal, and the measurement method are described below. In this example, experiments were conducted using animals of the same species.
<Preparation of ADSC filtrate (freeze-thaw)>
(1) Porcine adipose tissue-derived stem cells (ADSC)

Approximately 4 g of umbilical cord subcutaneous fat from a slaughtered pig was cut out and subcultured using the following method. The subcultured ADSCs were dispensed into cell storage tubes and stored frozen at -80°C.

The cryopreserved cells in the tube were thawed in a 38.5°C water bath and transferred to a 15 mL centrifuge tube. Next, an appropriate amount of PBS (-) supplemented with 10% FCS was added and centrifuged washing was performed several times. After aspirating the supernatant, add 1 mL of selective culture solution (25 mL of Mesen PRO (Gibco) to 25 mL of 2% penicillin-streptomycin, 500 μL of Growth Supply (Gibco), and 250 μL of 200 mM L-Glutamin (Nacalai Tesque)). The cells were resuspended, seeded into flasks, and cultured in a CO ₂ incubator (38.5° C., 5% CO ₂ ) until confluent. Confluent adipose stem cells were detached with 0.25% (w/v) trypsin/1 mmol/l EDTA and collected in a 15 mL centrifuge tube. Furthermore, the inside of the flask was rinsed with PBS (-) supplemented with 10% FCS, and the collected cell solution and rinsing solution were mixed and centrifuged (200 g, 5 minutes). After centrifugation, the supernatant was removed by suction, and 300 μL of PBS (-) was added to resuspend. Thereafter, PBS (-) was added to the cell suspension to adjust the concentration, dispensed into cryotubes, and then frozen in a -25°C freezer to disrupt the cells.
After freezing for at least one night, the cells were thawed at room temperature, centrifuged (1,000 g, 3 minutes), and the supernatant was sterilized by filtration using a 0.2 μm membrane filter (Advantec). Thereafter, PBS(-) was added to the filtrate to adjust the concentration, and the oocyte maturation promoter of the present invention was obtained. The oocyte maturation accelerator was stored at -25°C until use and thawed at room temperature when used.
(2) Bovine adipose tissue-derived stem cells (ADSC)
Subcutaneous fat from the umbilical region of the bovine carcass was collected. An oocyte maturation promoter was prepared using the same procedure as for pigs.
<Collection of eggs>

(1) Collection of ovum from pigs Ovaries were collected from slaughtered female three-way crossbred pigs (crossbred pigs of Landrace, Large Yorkshire, and Duroc breeds) before the start of spring. The collected ovaries were immediately immersed in a 0.85% (w/v) sodium chloride solution (physiological saline) containing 100 μg/mL kanamycin sulfate (Meiji Seika Pharma Co., Ltd.) in a 50 mL centrifuge tube. The centrifuge tube was kept at 33-36°C, and after washing the ovary with physiological saline, COCs were collected along with the follicular fluid from a follicle with a diameter of approximately 1-5 mm on the ovarian surface. The collected COCs were washed with Hepes-TLP-PVA, and only morphologically normal COCs with sufficient adhesion of cumulus cells were selected under a stereomicroscope (OLYMPUS) and used for experiments.
(2) Collection of bovine eggs Ovaries were collected from slaughtered fattened cows (Japanese Black cattle) aged 29 to 30 months. The procedure was the same as for pig oocyte collection, except that COCs were collected by suction along with follicular fluid from follicles approximately 2 to 8 mm in diameter on the ovarian surface.
<Preparation of sperm>

(1) Preparation of pig sperm Semen for artificial insemination of the Greater Yorkshire breed (Ibaraki Farm, Livestock Breeding Center, Independent Administrative Institution) was used. Semen for artificial insemination was collected 3 days before insemination. 9 mL of sperm washing solution (NaCl 9 μg/mL, BSA 1 μg/m, Kanamycin 0.1 μL) was added to 1 mL of semen for artificial insemination, and centrifugal washing was performed (1,800 rpm, 5 minutes). After centrifugal washing, remove the supernatant and add in vitro fertilization medium (PFM, Research Institute for the Functional Peptides, Co., Ltd.) pre-warmed to 38.5°C to dilute to a sperm concentration of 1.0 × 10 ⁶ A sperm suspension was prepared at a concentration of sperm per mL.
(2) Preparation of bovine sperm Frozen straw semen for artificial insemination of Japanese black cattle (provided by Hida Cattle Research Department, Gifu Prefectural Livestock Research Institute) was used. Frozen bovine semen straws filled in straws and stored in liquid nitrogen were thawed by immersing them in hot water at 38°C for 20 seconds, and BO _- Theophylline (BO-medium (Brackett Washing and centrifugation (1,800 rpm, 5 minutes) were performed twice using 10 mM Theophylline (10 mM Theophylline). After centrifugation, dilute with BO-Theophylline and BO-BSA-Heparin (BO-medium, 1% Bovine serum albumin, 2.5 IU/ml Heparin) so that the sperm concentration at the time of insemination is 2.0 × 10 ⁷ sperm/ml. , and a sperm suspension.

The sperm vitality of the prepared sperm suspension was confirmed using an inverted microscope (OLYMPUS). The most active forward movement is +++, the most active forward movement is ++, the slow forward movement is +, the turning or pendulum movement is ±, and there is no movement at all -. The ratio of each component to the total was calculated by substituting it into the following formula. In order to appropriately judge the effect of the oocyte maturation promoter of the present invention, highly motile sperm, specifically, a sperm suspension with a sperm vitality of 70 or more, was used in the subsequent experiments.

In the case of pigs, various concentrations of the present invention were added to a 180 μL microdroplet of in vitro maturation medium (Medium 199 supplemented with 1 g/mL PVA, 10 IU/mL eCG, 10 IU/mL hCG, 0.01 μg/mL EGF, and 10% pig follicular fluid). 20 μL each of oocyte maturation accelerator was added. A microdroplet of 200 μL of in vitro maturation medium served as a control.
In the case of cattle, the same procedure as for pigs was performed except that an in vitro maturation medium (Medium 199 supplemented with 10% fetal bovine serum) was used.
<Area comparison test of cumulus cell-oocyte complexes (COCs)>

Prepare 10 in vitro maturation medium drops (10 μL) containing various concentrations of the oocyte maturation promoter of the present invention, place one immature oocyte in each drop, and invert all COCs before starting the maturation culture. After photographing with a digital camera (Canon) installed on a microscope (OLYMPUS), the cells were cultured in a CO ₂ incubator (38.5°C, 5% CO ₂ ) (pig: 42 hours, cow: 22 hours). Thereafter, COCs in each addition area were photographed in the same manner. Using the image analysis software Image J (National Institutes of Health), the area of COCs in each added area before and after maturation culture was measured and the average values were compared (n=8). Analysis was performed using a one-way analysis of variance test method, and the Tukey-Kramer method was used to compare each addition interval.
The results of the area comparison test are shown in FIG. 1 and Tables 1A (pig) and 1B (cow). Note that X in Table 1 is the area of COCs after mature culture divided by the area of COCs before mature culture.

FIG. 1 is a diagram when 100×10 ⁴ cells/mL of the pig-derived oocyte maturation promoter of the present invention was added. A is COCs before in vitro maturation culture, and B is COCs after in vitro maturation culture. Table 1 shows the quantitative measurement results of the area of COCs in each added area. Tables 1A and 1B show that the addition of the oocyte maturation promoter of the present invention increased the area of COCs and caused the cumulus cells to swell. In pigs, there was a significant concentration-dependent increase especially in the 50×10 ⁴ and 100×10 ⁴ cells/mL categories. In cattle, significantly higher values were shown compared to controls, especially in the 100×10 ⁴ cells/mL and 200×10 ⁴ cells/mL categories.
Swelling of cumulus cells is considered an indicator of cytoplasmic maturation and is said to be involved in oocyte maturation, sperm entry, and fertilization. It has also been revealed that significant cumulus cell swelling has a positive effect on the maturation of pig and bovine oocytes and their subsequent embryonic development potential. As described above, the swelling of cumulus cells is thought to play a very important role in the maturation of oocytes and the subsequent ability to develop embryos. The above result that the number of cumulus cells increased significantly with the addition of the oocyte maturation promoter of the present invention indicates that the oocyte maturation promoter of the present invention contributed to improving oocyte maturation.
<Measurement of reactive oxygen species (ROS) and reduced glutathione (GSH) levels>

30 to 40 immature eggs were added as a group to 200 μL of in vitro maturation medium supplemented with various concentrations of the oocyte maturation promoter of the present invention, and incubated in a CO ₂ incubator (38.5°C, 5% CO ₂ ) for 42 hours. Cultured.
After in vitro maturation culture, Hepes-TLP-PVA supplemented with 0.1% (w/v) hyaluronidase (Sigma-Aldrich) was added, and all cumulus cells were detached by mechanical pipetting. Thereafter, eggs in which release of the first polar body was confirmed were selected. The oocytes were immersed in 500 μL of Hepes-TLP-PVA supplemented with 10 μL of 2',7'-Dichlorofluorecein diacetate (Sigma-Aldrich) and 5 μL of Cell Tracker Blue CMF ₂ HC (Invitrogen), and placed in a CO ₂ incubator. The cells were stained for 30 minutes. After washing the stained eggs with Hepes-TLP-PVA, fluorescence images were taken and recorded using a fluorescence microscope (OLYMPUS) equipped with a digital camera (Canon) using UV filters of 460 nm for ROS and 365 nm for GSH. . The obtained fluorescence images were converted into black and white and analyzed using the image analysis software Image J (National Institutes of Health), and from the obtained fluorescence brightness, the relative ROS and GSH contents of each addition group were calculated with respect to the control. (n=3). Analysis was performed using a one-way analysis of variance test method, and the Tukey-Kramer method was used to compare each addition interval.
The results for pigs are shown in Table 2A, and the results for cattle are shown in Table 2B.

As is clear from Tables 2A and 2B, the addition of the oocyte maturation promoter of the present invention reduces activated oxygen species (ROS) and increases glutathione (GSH). Particularly in pigs, the ROS content significantly decreased in a concentration-dependent manner in the 50×10 ⁴ and 100×10 ⁴ cells/mL categories, and the GSH content significantly increased in a concentration-dependent manner. In cattle, the ROS content showed significantly lower values in a concentration-dependent manner in the 100×10 ⁴ cells/mL and 200×10 ⁴ cells/mL categories. Furthermore, the GSH content showed higher values in the categories of 100×10 ⁴ cells/mL, 200×10 ⁴ cells/mL, and 400×10 ⁴ cells/mL compared to the control. Therefore, it can be seen that the oocyte maturation promoter of the present invention suppressed oxidative stress and improved the degree of oocyte cytoplasmic maturation. In particular, the decrease in ROS content indicates that the decline in oocyte quality caused by oxidative stress was suppressed and improved.
<Distribution status of mitochondria in mature oocytes>

The method described in Sha Wei et al. (2010) mentioned above was used as reference. Pig oocytes were selected by the method described above.
Stock A was prepared using the Mito Tracker® Red CMXRos (Invitrogen) kit according to the instructions. To 1 μL of Stock A, 149 μL of Amikamycin (100 mg titer) (Meiji Seika Pharma) supplemented Medium 199 (Gibco) was added and mixed. Furthermore, 1850 μL of Medium 199 (Gibco) was mixed to create multiple 150 μL microdroplets.
Pig eggs selected from each addition group were washed with 0.3% (w/v) PVA-PBS and then transferred into the microdroplets in groups of about 10 eggs. Thereafter, the cells were allowed to stand for 30 minutes in a CO ₂ incubator, and the mitochondria were stained. After washing the mature eggs with 0.3% (w/v) PVA-PBS, they were photographed using a confocal laser scanning microscope (Carl Zeiss, Oberkochen) using a Rhodamine filter and image analysis software (Carl Zeiss; ZEN). analyzed (n=4). A one-way analysis of variance test was performed, and multiple comparison tests were performed using the Tukey-Kramer method to compare each addition interval.
The results are shown in FIG. 2 and Tables 3A and 3B.
Type I has mitochondria distributed uniformly throughout the cytoplasm (Figure 2A);
Type II is the type in which mitochondria are distributed around the cell membrane and is also unevenly distributed inside the cell membrane (Figure 2B).
Type III cells were defined as those in which mitochondria were distributed only around the cell membrane (Fig. 2C), and the distribution of mitochondria was evaluated by calculating the respective proportions.

From Table 3A, it was found that the proportion of type I increased compared to the control, especially in the 50×10 ⁴ and 100×10 ⁴ cells/mL categories. From Table 3B, especially in the 50×10 ⁴ and 100×10 ⁴ cells/mL categories, the ratio of type I to the number of test eggs is greater than that of the control, so it can be said that the eggs have matured.
In pig oocytes, which are immature eggs, mitochondria are mainly distributed near the cell membrane, and then, as nuclear maturation and cytoplasmic maturation proceed, they are said to be uniformly distributed and diffused throughout the cytoplasm. It has also been reported that uniform mitochondrial distribution improves oocyte fertilization and early embryonic development. Therefore, it seems that the addition of the oocyte maturation promoter of the present invention promoted each maturation and cytoplasmic maturation, thereby improving the distribution of mitochondria.
<Evaluation of mitochondrial activity>

The method described in Lee SK et al. (2014) mentioned above was used as reference. Pig oocytes were selected by the method described above.
Stock A was prepared using MitoProbe (registered trademark) JC-1 Assay Kit (Invitrogen) according to the instructions. 1092.5 μL of Medium 199 (Gibco) supplemented with amikamycin (100 mg titer) (Meiji Seika Pharma) was added to 57.5 μL of Stock A and mixed to produce multiple 80 μL microdroplets.
Pig eggs selected from each addition group were washed with 0.3% (w/v) PVA-PBS and then transferred into the microdroplets in groups of about 10 eggs. Thereafter, the cells were allowed to stand for 50 minutes in a CO ₂ incubator, and the mitochondria were stained. After staining, mature oocytes were immediately washed with 0.3% (w/v) PVA-PBS, photographed using a confocal laser scanning microscope (Carl Zeiss) using Rhodamine filters and FITC filters, and image analysis software (Carl Zeiss). ;ZEN).
The fluorescence intensity of the image was measured using image J software, and the membrane potential difference was calculated by dividing the fluorescence intensity of red, which shows high activity, by the fluorescence intensity of green, which shows low activity, and mitochondrial activity was evaluated (n = 3). Analysis was performed using a one-way analysis of variance test method, and the Tukey-Kramer method was used to compare each addition interval.
The results are shown in Table 4. Table 4 shows the relative membrane potential difference of each addition group using the control as a control.

From the results in Table 4, the addition of the oocyte maturation promoter of the present invention significantly increased mitochondrial activity compared to the control, especially in the 100×10 ⁴ cells/mL category. Mitochondrial activity is known to be important for the maturation and quality of pig oocytes and subsequent embryonic development, and the results in Table 4 demonstrate that the oocyte maturation promoter of the present invention improves mitochondrial function. I understand. Furthermore, since it is known that low mitochondrial activity causes apoptosis, improvement of mitochondrial activity by the oocyte maturation promoter of the present invention also leads to suppression of early apoptosis. As described above, the oocyte maturation promoter of the present invention matures the cytoplasm of the oocyte, thereby sufficiently maintaining the subsequent embryonic development potential, which in turn contributes to improving the fertilization rate, implantation rate, and pregnancy rate. Guessed.
<In vitro fertilization>

In order to confirm whether or not eggs using the oocyte maturation promoter of the present invention are successfully fertilized in vitro, COCs were cultured for in vitro maturation in an in vitro maturation medium supplemented with various concentrations of the oocyte maturation promoter of the present invention. , performed in vitro fertilization.
Specifically, several dozen immature eggs (pigs: 30 to 40, cows: 20 to 35) are added to 200 μL of in vitro maturation medium supplemented with 20 μL of the oocyte maturation promoter of the present invention at various concentrations. and cultured in a CO ₂ incubator (38.5°C, 5% CO ₂ ) (pig: 42 hours, cow: 22 hours). Thereafter, Hepes-TLP-PVA supplemented with 0.1% (w/v) hyaluronidase (Sigma-Aldrich) was sprinkled onto the COCs, and the remaining cumulus cells were peeled off using a glass pipette, leaving about 2 or 3 layers of cumulus cells. Then, wash multiple times with 80 μL of microdroplets of IVF medium (PFM, Research Institute for the Functional Peptides, Co., Ltd.), and add 90 to 180 μL of IVF for each group of 20 to 40 eggs. microdroplets of medium for use. 10 to 20 μL of sperm suspension was added thereto for insemination. The final sperm concentration was adjusted to 1.0×10 ⁶ animals/mL for pigs and 2.0×10 ⁷ animals/mL for cows. Thereafter, the eggs were co-cultured in a CO ₂ incubator (38.5° C., 5% CO ₂ ) for 6 hours after insemination, and then the eggs were transferred to a development medium and cultured for 6 hours. Thereafter, the in vitro fertilization status, maturation rate, and normal fertilization rate were evaluated according to the following method, and a comparative study was performed in each addition period. Analysis was performed using a one-way analysis of variance test method, and a multiple comparison test was performed using the Tukey-Kramer method for comparison between each addition interval.
(1) Confirmation of IVF status

After 12 hours of insemination, some eggs from each addition group were randomly removed from the development medium and fixed with Carnoy's solution (ethanol:acetic acid=3:1) under a stereomicroscope. After 48 hours of fixation, the nuclei were stained with 1% (w/v) orcein acetate solution, and the fertilization status was observed under an upright microscope (OLYMPUS), and judgments were made as follows (n = 5).
(i) Maturation rate: Percentage of eggs in which the first polar body and second meiotic metaphase chromosomes were observed among the test eggs.
(ii) Sperm penetration rate: Percentage of eggs in which a sperm head or one or more male pronuclei are observed relative to mature eggs.
(iii) Normal fertilization rate: Percentage of eggs in which the first and second polar bodies have been released and both male and female pronuclei have been observed, compared to the eggs that have been penetrated by sperm.
(iv) Polyspermic fertilization rate (common in pigs): Percentage of eggs in which the first and second polar bodies have been released and three or more pronuclei have been observed for the eggs that have been penetrated by sperm (v) Fertilization efficiency: The ratio of normally fertilized eggs to mature eggs The above results for pigs are shown in Table 5A. The results for maturation rate in cattle are shown in Table 5B, and the results for normal fertilization rate are shown in Table 5C.

As is clear from Table 5A, in pigs, the maturation rate, sperm penetration rate, normal fertilization rate, and fertilization efficiency increased particularly in the 50×10 ⁴ and 100×10 ⁴ cells/mL categories. On the other hand, the polyspermic fertilization rate decreased particularly in the 50×10 ⁴ and 100×10 ⁴ cells/mL categories. As is clear from Tables 5B and 5C, in cattle, the maturation rate and normal fertilization rate particularly increase in the 100×10 ⁴ cells/mL and 200×10 ⁴ cells/mL, especially in the 200×10 ⁴ cells/mL category. mL had the highest value.
Cumulus cell secreted factors include important factors that induce meiosis in oocytes, and are said to be involved in the progression of nuclear maturation of oocytes during the in vitro maturation culture process. As is clear from Table 1, when the oocyte maturation promoting agent of the present invention is added, the swelling of cumulus cells is remarkable, so it is likely that many maturation-related factors are secreted from the cumulus cells by the oocyte maturation promoting agent of the present invention. It is presumed that this stimulated the nuclear maturation of oocytes. Similarly, it is speculated that the swelling of cumulus cells contributed to the increased sperm penetration rate. Furthermore, since decondensation of sperm nuclei and formation of male pronuclei are said to depend on the state of egg maturation, fertilization efficiency can be improved by using eggs to which the oocyte maturation promoter of the present invention has been added. It can be said that it is natural that it will increase.

On the other hand, incomplete cytoplasmic maturation is said to delay the polyspermic fertilization rejection mechanism, thereby increasing the polyspermic fertilization rate. Pigs in particular have a higher incidence of polyspermic fertilization than other animal species. Eggs fertilized by polysperm do not develop normally, and this is one of the factors hindering the efficiency of IVF embryo production in pigs. However, as is clear from the results in Table 5A, the oocyte maturation promoter of the present invention probably improves the cytoplasmic maturation situation, and as a result, the multisperm fertilization rejection mechanism functions normally, improving the normal fertilization rate and increasing the multisperm fertilization rate. It is thought that the sperm fertilization rate has decreased.
(2) Confirmation of cleavage rate and blastocyst formation status

Even if fertilization occurs normally, if the subsequent somatic cell division (cleavage) does not occur properly, the embryo will not develop into a good blastocyst. Furthermore, even if cleavage is performed appropriately, if the egg does not develop and differentiate normally thereafter, the implantation rate will not improve even if the egg is returned to the uterus. Therefore, when in vitro fertilization was performed using the oocyte maturation promoter of the present invention, the cleavage rate and subsequent blastocyst formation status were measured by the following method.
Specifically, in vitro fertilization was performed on pigs and cows using the above method, and two days after insemination, the cleavage status was observed under an inverted microscope (OLYMPUS). The rate was calculated. Seven days after insemination, the blastocyst formation status was observed, and the blastocyst formation rate, which is the percentage of embryos in which the blastocyst cavity was confirmed, was calculated. Furthermore, the total number of blastocyst cells was measured using Hoechst 33342 staining of the obtained blastocysts, and the percentage of apoptotic cells relative to the total number of blastocyst cells was measured using TUNEL staining, and the results were compared for each addition interval (n=3). . Analysis was performed using a one-way analysis of variance test method, and a multiple comparison test was performed using the Tukey-Kramer method for comparison between each addition interval.
The results of the cleavage rate and cleavage status in pigs up to the 4-cell stage are shown in Table 6A, the formation status from then on to the blastocyst stage is shown in Table 6B, and the results of the apoptotic cell ratio are shown in Table 6C. Table 7A shows the cleavage status and blastocyst formation rate up to the 2-cell stage in cattle, and Table 7B shows the results of the total number of blastocyst cells and the percentage of apoptotic cells.

From the results in Table 6A, it can be seen that when in vitro fertilization is performed using matured eggs by adding the oocyte maturation promoter of the present invention, fertilized eggs in the 50 x 10 ⁴ and 100 x 10 ⁴ cells/mL categories in pigs. The incidence rate increased significantly, indicating an improvement in the rate of occurrence. In general, embryos at the early stage of development are controlled by factors such as maternal mRNA and proteins accumulated in the oocyte cytoplasm during the maturation culture process. The present results are thought to be due to the addition of the oocyte maturation promoter of the present invention during the maturation culture process, which improved cytoplasmic maturation and accumulated more maternal factors within the cells, thereby improving early embryonic development ability.

Furthermore, from the results in Tables 6B and 6C, a significant increase in the number of blastocysts at each developmental stage was observed in pigs during subsequent development. This is presumed to be due to the favorable cleavage condition. Furthermore, a decrease in the apoptotic cell index was also observed.
In cattle, as is clear from Table 7A, the addition of the oocyte maturation promoter of the present invention improved the cleavage rate in all categories. In particular, the 200×10 ⁴ cells/mL category had the highest value. The blastocyst formation rate also showed a significantly higher value in the 200×10 ⁴ cells/mL section compared to the control, and was the highest value among each addition section. Furthermore, morphologically normal blastocysts were obtained in all experimental groups.
In addition, as is clear from Table 7B, the value was significantly higher than that of the control, and the total number of blastocyst cells was the highest value in each addition interval in the 200 × 10 ⁴ cells/mL category. became. The number of apoptotic cells and the ratio of apoptotic cells to the total number of blastocyst cells showed a tendency to decrease compared to the control in all oocyte maturation promoter addition groups, especially at 200×10 ⁴ and 400×10 ⁴ cells/ A significant effect was observed in the mL category.
<Blastocyst quality evaluation>

From Tables 6C and 7B, it was found that the addition of the oocyte maturation promoter of the present invention significantly improved the total blastocyst cell number formation rate. However, there are also grades of blastocysts, and it is necessary to select and transfer blastocysts of a high grade, that is, those with a high possibility of implantation. The grade of a blastocyst is evaluated based on the number of cells in the part that will become a fetus (inner cell mass = ICM) and the part that will become a placenta (trophectoderm = TE) (Gardner classification).

Therefore, the differentiation rate of ICM and TE in blastocysts when the oocyte maturation promoting agent of the present invention was added was evaluated by the following method.
In the case of pigs, in vitro fertilization was performed using the above method, and the morphologically recognized blastocysts obtained after 7 days of insemination were immersed in 6 μL of 0.5% (w/v) pronase solution. , dissolved the zona pellucida. Washed with Hepes-TLP-PVA, added reaction solution A (10 μL of Anti-Pig Serum antibody produced in rabbit (Sigma-Aldrich) and 50 μL of PBS(-)), and stained in a CO ₂ incubator for 1 hour. . After that, it was washed with Hepes-TLP-PVA, reaction solution B (5 μL of Complement sera from guinea pig (Sigma-Aldrich) and 50 μL of PI-Hoechst 33342-PBS stock) was added, and the mixture was incubated again in the CO ₂ incubator for 1 hour. Stained. After staining, the cells were washed with Hepes-TLP-PVA, and fluorescence images were recorded using a 365 nm UV filter using a fluorescence microscope (Olympus) equipped with a digital camera. The stained nuclei were counted, and those with 32 or more cells were considered to be blastocysts, and the total number of blastocyst cells was calculated. In addition, ICM cells stained with blue fluorescence and TE cells stained with red fluorescence were each counted, and the proportion of ICM cells and TE cells relative to the total number of blastocyst cells was calculated.
In the case of cattle, in vitro fertilization was performed using the above method, and the morphologically recognized blastocysts obtained 7 days after insemination were incubated in 500 μL of 4% (w/v) paraformaldehyde. It was immersed and fixed for 30 minutes at room temperature and protected from light. Fixed embryos were washed several times with 0.3% PVA-PBS and permeabilized by immersion in PVA-PBS supplemented with 1% (w/v) Triton X-100 (Sigma-Aldrich) for 30 minutes at room temperature, protected from light. . After permeabilization, wash several times with 0.3% PVA-PBS and store in PVA-PBS supplemented with 0.5% BSA and 10% Horse serum (16050-130, Gibco, Thermo Fisher Scientific) for 2 hours at 4°C, protected from light. Nonspecific binding of antibodies was blocked by immersion. After blocking, the cells were immersed in primary antibody CDX2 (D11D10) Rabbit mAb (Cell Signaling Technology, Danvers, MA, USA) diluted 1:50 with 0.3% PVA-PBS at 4°C for 24 hours in the dark. After the primary antibody reaction, the embryos were washed several times with 0.3% PVA-PBS, and the secondary antibody Anti-rabbit IgG (H＋L), F (ab')2 Fragment ( The cells were immersed in Alexa Fluor (registered trademark) 555 Conjugate; Cell Signaling Technology) solution at 4°C for 1 hour in the dark, to stain TE cells (red fluorescence). Next, the nuclei were stained by immersion in 0.3% PBS-PVA containing 10 μg/mL Hoechst 33342 at 4 °C in the dark for 15 minutes, mounted on a glass slide, covered with a cover glass, and covered with a top coat. did. Fluorescence images of the prepared whole-mount specimens were recorded and observed using a fluorescence microscope (OLYMPUS) equipped with a digital camera (Canon) using UV filters of 365 nm for Hoechst 33342 and 546 nm for Alexa Fluor (registered trademark) 555. . From the obtained fluorescence image, nuclei stained with blue and red fluorescence were counted using image analysis software Image J (National Institutes of Health), and the total cell number and TE cell number were counted. In addition, the number of ICM cells was calculated by subtracting the number of TE cells from the total number of cells.
The results for pigs are shown in Table 8A and the results for cattle are shown in Table 8B. ICM cell percentage and TE cell percentage indicate the ratio of the number of ICM cells and the number of TE cells to the total number of cells.

From the results in Table 8A, it can be seen that the addition of the oocyte maturation promoter of the present invention increases the cell number in all cases. In pigs, the total cell number, ICM cell number, and ICM cell percentage significantly increased, especially at concentrations of 50×10 ⁴ cells/mL and 100×10 ⁴ cells/mL.
In cattle, the total cell number was significantly higher than the control, especially in the 200 × 10 ⁴ cells/mL category, and the ICM and TE cell counts were also higher than the control in all experimental categories. It became the value. Furthermore, the results of the ratio of the number of ICM cells to the total number of blastocyst cells showed a tendency for the rate of differentiation into ICM to increase in all experimental categories.
Evaluation of the number of these cells in a blastocyst-stage embryo is an indicator for measuring the quality of the embryo, and it has been shown that ICM cells in particular are involved in implantation after embryo transfer and normal development of the fetus, so the present invention It can be seen that high quality blastocysts were produced by adding the oocyte maturation promoter. It is presumed that it has the effect of improving the implantation rate and pregnancy rate after transplantation.

According to the oocyte maturation promoter of the present invention, oocyte maturation can be promoted, thereby improving the fertilization rate. The present invention is effective for use in crossbreeding and breeding of livestock (improving the success rate and efficiency of embryo transfer), breeding and maintenance of species (for example, maintenance of endangered species, maintenance of pet lines, or crossbreeding), etc. . It is also effective in treating infertility caused by functional decline such as poor egg maturation. It can also improve the quality of aged eggs.

Claims

An oocyte maturation promoter containing as an active ingredient the filtrate of adipose tissue-derived stem cell disruption solution.
The oocyte maturation promoter according to claim 1, which is used for in vitro fertilization.
The egg maturation promoter according to claim 1 or 2, wherein the biological species of the adipose tissue-derived stem cells and the biological species of the egg are the same.
The oocyte maturation promoter according to claim 1, wherein the biological species of the adipose tissue-derived stem cells is a non-human mammal.
The oocyte maturation promoter according to claim 1, wherein the biological species of the adipose tissue-derived stem cells is human.
The oocyte maturation promoter according to claim 1, which has a ROS value of less than 1 when the control value is 1.
A method for producing an oocyte maturation promoter, including the following steps.
(1) A step of disrupting adipose tissue-derived stem cells.
(2) A step of filtering the crushed liquid obtained in step (1) or the supernatant obtained by centrifuging the crushed liquid to obtain a filtrate.
(3) A step of formulating the filtrate obtained in step (2).
A method for producing mature eggs, which comprises culturing eggs in the presence of the egg maturation promoter according to claim 1.
An in vitro fertilization method, which comprises causing an egg treated with the egg maturation promoter according to claim 1 to coexist with sperm in vitro.
An in vitro fertilization method, which comprises causing eggs and sperm to coexist in vitro in the presence of the egg maturation promoter according to claim 1.
An embryo transfer method, which comprises culturing a fertilized egg produced by the in vitro fertilization method according to claim 8 or 9, and injecting the grown blastocyst into the uterus of a mammal.
Use of the oocyte maturation promoter according to claim 1 as an oocyte quality improving agent.