JP2012105585A - Composition for artificial insemination - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a stable composition for artificial insemination of constant quality, safe with no concern for being subject to infection.SOLUTION: The composition for artificial insemination characterized by including sericin is provided. Using this composition, embryos, sperms and ova are cultured and preserved.

Description

  In the assisted reproductive technology (ART), the present invention is a composition for artificial fertilization, in particular, a composition used for culture or storage of an embryo during in vitro fertilization, treatment of sperm or ovum, and an embryo using the composition. And a method for treating sperm and ovum.

  In assisted reproductive technology (ART), 5-20% (V / V) human serum (HS) or human serum albumin (HSA) is generally added for ovum and embryo culture and cryopreservation thereof. Medium is used. Serum often contains growth factors in addition to the five essential nutrients of proteins, carbohydrates, lipids, vitamins and minerals, which are essential factors, and thus is often added when culturing animal-derived cells. However, HS or HSA always has the risk of being contaminated with viruses and the like, and it is difficult to eliminate these risks. In addition, since the biological activity differs depending on the lot, a great deal of labor is required to select an excellent lot (Non-Patent Documents 1, 2, 3, 4).

  On the other hand, commercially available artificial serum contains many unknown components. Similarly, since the biological activity differs depending on the lot, a great deal of labor is required to select an excellent lot. For example, Polyvinylpyrrolidone (PVP), which is widely used in serum-free media, is a polymer compound, and its toxicity to the embryo is unknown, which is an obstacle to use in the field of ART (Non-patent Document 5). .

  Furthermore, bovine fetuses derived from in vitro cultured embryos tend to be larger than fetuses derived from in vivo embryos, and it is pointed out that this may be caused by serum added to the culture medium (Non-patent Document 6). ).

Sericin, a protein derived from silkworm cocoons, is rich in hydrophilic amino acids such as serine and threonine and is not derived from mammals. Therefore, it is expected that the risk of infection due to contamination with viruses and the like is extremely low.
It is known that sericin has a cell growth promoting action and a cell death inhibiting action and can be used as a medium additive for animal cell culture (Patent Document 1, Non-Patent Documents 7 and 8). Moreover, it is known that the composition for cell cryopreservation containing sericin has a good cell viability (Patent Document 2, Non-Patent Document 9).

  At the time of artificial insemination, particularly in vitro fertilization, an embryo culture solution, a frozen solution or a thawed solution satisfying conditions such as “safe and free of concern for infection” and “constant and stable quality” are expected. However, no effective substance is known for ART such as in vitro fertilization to replace serum that satisfies the above-mentioned conditions.

International Publication No. 2002/086133 Pamphlet JP 2006-115837 A

Kazuhiko Kaji, et al. “Serum-free cell culture manual” (Tadao Ohno, Hiroki Murakami) p1-45, Kodansha Scientific, 1989 Akita Suzuki “ART Laboratory for new development of infertility treatment” Medical View, July 1, 2000, first edition, p76-79 Hiroshi Konan “Cell culture, I see,” Yodosha January 1, 2004, p92-96 Fertil Steril. 1996 Mar; 65 (3): p614-619, Setting standards for the levels of endotoxin in the embryo culture media of human in vitro fertilization and embryo transfer. (Nagata Y, Shirakawa K.) Theriogenology, 72: p624-635, 2009, Kato Y and Nagao Y, Effect of PVP on sperm capacitation status and embryonic development in cattle. Biol. Reprod., 67: p765-775, 2002, Lazzari G et al., Cellular and molecular deviations in bovine in-vitro produced embryos are related to the large offspring syndrome. Cytotechnology 40: p3-12, 2002 J Hepatobiliary Pancreat Surg (2009) 16: p223-228 Biotechnol. Appl. Biochem. (2005) 42: p183-188

  Artificial fertilization composition used in ART for egg and sperm processing, embryo culture, egg, sperm and embryo preservation, for safe, constant quality and stable fertilization without concern for infection A composition is provided.

  The inventors have intensively studied that embryos grew smoothly and thawed after cryopreservation by using sericin without using mammalian-derived components such as serum in the culture and storage of fertilized embryos. The inventors have found that embryos also develop and have completed the present invention.

That is, the present invention is as follows.
[1] An artificial fertilization composition comprising sericin.
[2] The composition according to [1], which does not contain serum.
[3] The composition according to [1] or [2], wherein the concentration of sericin is 0.001 to 10% (w / v).
[4] The composition according to any one of [1] to [3], wherein the artificial fertilization is in vitro fertilization.
[5] The composition according to any of [1] to [4], which is used for embryo culture.
[6] The composition according to any one of [1] to [4], which is used for embryo preservation.
[7] The composition according to [6], wherein the preservation of the embryo is freezing and / or thawing of the embryo.
[8] The composition according to any one of [1] to [4], which is used for treatment or storage of sperm and / or eggs.
[9] An embryo preservation kit comprising the composition according to any one of [1] to [4].
[10] A method for culturing an embryo, characterized by using sericin.
[11] A method for freezing and / or thawing an embryo characterized by using sericin.
[12] A method for treating sperm, characterized by using sericin.
[13] The composition according to [1], which is a frost damage preventive agent used when freezing eggs, sperm, and embryos.

Since the composition for artificial fertilization of the present invention does not contain a mammal-derived component, it can reduce the risk of virus contamination.
Since sericin is resistant to high pressure and high temperature, the composition for artificial fertilization of the present invention can be subjected to dry heat sterilization and autoclave sterilization treatment, and can greatly reduce the risk of infection.
Moreover, since sericin has a low quality over time and a constant quality, the composition for artificial fertilization of the present invention can maintain a stable and constant quality.
Moreover, since sericin contains abundant hydrophilic amino acids such as serine and threonine and can be easily dissolved, the composition for artificial fertilization of the present invention can be easily prepared.
Furthermore, since sericin is a high-molecular substance, it can maintain the osmotic pressure of cells in the same way as serum and serum albumin.
Due to these advantageous effects, embryo development can be promoted and qualitatively stabilized, and embryos, sperm, and eggs can be stored for a long period of time, and safe and stable artificial insemination can be easily performed.

Morphology of blastocyst stage in vitro fertilized embryo developed in medium supplemented with BSA, PVP or sericin. A newborn mouse born by transplantation of a blastocyst developed in a medium supplemented with BSA or sericin to a recipient. Embryonic morphology after thawing a frozen two-cell stage embryo with a melt containing HSA or sericin and culturing for 72 hours.

The present invention is a composition for artificial fertilization characterized by containing sericin (hereinafter sometimes referred to as the composition of the present invention).
In the present specification, “artificial insemination” refers to a technique for insemination including artificial insemination and in vitro fertilization.

The sericin used in the present invention may be derived from a natural product or synthesized by a conventional chemical and / or genetic engineering technique. The sericin may be a hydrolyzate of sericin hydrolyzed with acid, alkali, enzyme or the like.
Naturally derived sericin can be obtained by extraction from straw or raw silk by a conventional extraction method. Specifically, for example, sericin can be obtained by using, for example, silkworm, raw silk, or silk fabric as a raw material, a method of partially hydrolyzing and extracting it with hot water, acid, alkali, enzyme, etc. Further, a product purified to a high purity is preferable for obtaining a stable culture and storage state with a constant quality.
Furthermore, sericin may be a powder or granular solid, or a liquid dissolved or suspended in water or a buffer.

  In the present invention, the average molecular weight of sericin is not particularly limited, but is preferably 5,000 to 100,000, more preferably 10,000 to 50,000.

In the composition of the present invention, it is preferable to use less serum, and a composition containing no serum (also referred to as a serum-free composition of the present invention) is more preferable.
In the present invention, serum means serum derived from humans, cows, and the like and serum-derived factors such as serum albumin, and the serum-free composition of the present invention may be in a form that does not substantially contain serum. It does not exclude contamination.
Since the serum-free composition of the present invention does not use serum that is difficult to completely eliminate the possibility of infection, the risk of virus infection and the like can be avoided.
Moreover, since the serum-free composition of the present invention does not lose its activity due to high temperature and high pressure treatment, sericin can be subjected to dry heat sterilization and autoclave sterilization, thereby preventing virus and bacteria contamination.

  In the composition of the present invention, the concentration of sericin is usually 0.001 to 10% (w / v), preferably 0.01 to 1.0% (w / v), more preferably 0.06 to 0.8% (w / v) with respect to the entire composition. w / v), particularly preferably 0.1 to 0.3% (w / v). Stable artificial insemination is possible at concentrations in the above range, but if it exceeds 10% (w / v), the possibility of cell survival decreases, and if it falls below 0.001% (w / v), the desired effect cannot be obtained. .

  In the present invention, the concentration of sericin means the final concentration of sericin, and the final concentration of sericin refers to the concentration in the final form such as a medium, a frozen solution, a thawed solution, or a washing solution, as will be described later. means. In the case of a composition which is a solid such as powder and does not contain a liquid, it means that the composition is blended so as to have the above-mentioned concentration in the form of actual use.

In the composition of the present invention, as long as it contains sericin, other components are not particularly limited as long as they are safe components used for artificial fertilization.
Examples include amino acids, carbohydrates, electrolytes, vitamins, trace metal elements, hormones, cell growth factors, lipids or constituents thereof, carrier proteins, extracellular matrix components (adhesion factors), reducing substances, buffers, and the like.

As amino acids, L-phenylalanine, L-tryptophan, L-lysine, L-threonine, L-valine, L-methionine, L-isoleucine, L-leucine, L-proline, glycine, L-alanine, L-tyrosine, Examples include L-histidine, L-arginine, taurine, L-aspartic acid, L-serine, L-glutamic acid, L-cystine, L-glutamine, L-hydroxyproline, L-asparagine and salts thereof. Preferably, amino acids that are used alone or appropriately combined to meet a desired purpose are included.
In the composition of the present invention, the final total amino acid concentration is generally 0.005 to 10 mM, preferably 0.01 to 3 mM, more preferably 0.02 to 1 mM, based on the entire composition.

Examples of the saccharide include glucose, maltose, sucrose, fructose, xylitol, sorbitol, trehalose and the like. Preferably, saccharides which are used alone or appropriately combined to meet a desired purpose can be mentioned.
In the composition of the present invention, the final carbohydrate concentration is usually 0.1 to 5 mM, preferably 0.1 to 3 mM, more preferably 0.2 to 2.78 mM with respect to the entire composition.

  The electrolytes include sodium chloride, sodium acetate, sodium citrate, potassium chloride, calcium chloride, calcium gluconate, magnesium chloride, magnesium sulfate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, hydrogen carbonate Examples thereof include sodium, sodium pyruvate, sodium lactate, calcium lactate, hydrates and inorganic salts thereof.

Examples of vitamins include vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, nicotinic acid, biotin, and folic acid.
Examples of trace metal elements include zinc, iron, manganese, copper, iodine, selenium, and cobalt.

Examples of the hormone include insulin, hydrocortisone, dexamethasone, triiodothyronine and the like.
Cell growth factors include epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, insulin-like growth factor, growth hormone and the like.

Examples of lipids or components thereof include essential oils such as mineral oil, oleic acid, linoleic acid, and linolenic acid, cholesterol, ethanolamine, choline, and egg yolk.
Examples of the carrier protein include serum albumin and transferrin.
Examples of extracellular matrix components (adhesion factors) include fibronectin, collagen, gelatin and the like.
Examples of the reducing substance include 2-mercaptoethanol (ME), dithiothreitol, and reduced glutathione.

  Examples of the buffer include phosphates (for example, PBS), BES, TES, acetamide glycine, glycinamide, glycylglycine, TRICINE, Tris, trisethanolamine, veronal, and HEPES.

  In addition, antibiotics such as penicillin, streptomycin, kanamycin, gentamicin, and erythromycin, antifungal agents such as amphotericin B and nystatin, fertilizers such as metalloprotease inhibitors, cyclophosphamide, and casein peptides may be added as appropriate.

The composition of the present invention is particularly preferably used for in vitro fertilization.
The target of in vitro fertilization is not particularly limited as long as it is a mammal, and examples thereof include humans, monkeys, cows, horses, mice, and other mammals. Of these, humans and mice are preferable, and humans are particularly preferable.

  The composition of the present invention can be used for embryo culture. The composition that can be used for embryo culture means an artificial fertilization medium composition that can be used as a culture medium for embryos obtained by artificial fertilization.

  The compositions of the present invention can be used to culture any mammalian embryo. Preferably, it is suitable for culturing embryos of humans, monkeys, cows, mice, rats and the like, more preferably suitable for humans and mice.

In the present invention, the embryo may be an embryo at any stage such as a pronuclear stage embryo, an early stage embryo, or a blastocyst. Of these, early embryos and blastocysts are preferable.
The composition of the medium may vary depending on the stage of the embryo, and the medium may be cultured in a medium having the same composition throughout all stages.

The medium composition for artificial fertilization is a medium or the like containing sericin and capable of culturing an embryo collected by in vivo artificial insemination or produced by in vitro fertilization. Note that a medium that can culture egg cells and a medium that can be used for fertilization of egg cells outside the body are also included in the composition.
The medium to which sericin is added in the present invention includes HTF medium, m-HTF medium, ham medium, ham F-10 medium, MEM medium, 199 medium, BME medium, CMRL 1066 medium, McCoy-5A medium, Weymouth medium, and Trowell. T-8 medium, Leibovitz L-15 medium, NCTC medium, Williams-E medium, Kane and Foote medium, MCDB104 medium, Brinster medium, m-Tyrode medium, BWW medium, Whitten medium, TYH medium, Hoppes & Pitts medium, m-KRB medium, BO medium, T6 medium, GPM medium, synthetic oviduct fluid (SOF), Charles Ronsenkrans medium, K-SOM Examples thereof include a medium or a modified medium thereof. Among these, HTF medium, m-HTF medium, ham medium, ham F-10 medium, MEM medium, and the like are preferable.

  In the medium, amino acids, carbohydrates, electrolytes, vitamins, trace metal elements, hormones, cell growth factors, lipids or constituents thereof, carrier proteins, extracellular matrix components (adhesion factors) as described above are included as necessary. Further, reducing substances, antibiotics, antifungal agents, buffering agents and the like may be added.

  The medium can be produced by blending the components by a conventional method, and may be a liquid medium, a solid medium or a semi-solid medium. Preferably, the medium of the present invention is used in the form of a sterile solution, a use-diluted sterile concentrate, or a use-dissolving sterile lyophilizer by conventional methods. Under the present circumstances, you may use pharmaceutical additives, such as a pH adjuster, a stabilizer, and an excipient | filler by a well-known method as needed. Hydrochloric acid, acetic acid, sodium hydroxide, etc. as pH adjusting agents, HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid), sodium sulfite, sodium bisulfite, pyrosulfurous acid as stabilizers Sodium and the like are exemplified, and conventional excipients can be used. Further, a pH indicator such as phenol red may be added.

In order to avoid blending changes due to the interaction of the constituent components, a kit-shaped medium in which a part of the blending components is filled in another container is also an aspect of the present application.
Moreover, the form is not ask | required if a sericin exists in a culture medium at the time of culture | cultivation, For example, the form which adds a sericin to a culture medium at the time of use is also 1 aspect of this application.

  In the composition of the present invention, the concentration of sericin is usually 0.001 to 10% (w / v), preferably 0.01 to 1.0% (w / v), more preferably 0.06 to 0.8% (w / v) with respect to the entire composition. w / v), particularly preferably 0.1 to 0.3% (w / v). A sericin concentration in the above range is preferable because the embryo develops smoothly.

  The total amount of amino acids contained in the medium is usually 0.005 to 10 mM, preferably 0.01 to 3 mM, more preferably 0.02 to 1 mM, based on the whole medium, from the viewpoint of embryo growth.

  When the composition of the present invention is used as a medium for in vitro fertilization, the amino acid concentration is about 1/3 to 2/3, preferably about 1/2 of the total amount, depending on the concentration of sericin. It is effective for the growth of the embryo.

The composition of the present invention can be used for embryo preservation.
Embryo preservation includes freezing and thawing of the embryo.
Embryo freezing includes not only freezing the embryo, but also freezing the embryo after equilibration, and freezing can stop the division of the embryo and maintain its function.
The embryo may be an embryo at any stage such as a pronuclear stage embryo, an early stage embryo, or a blastocyst. Of these, early embryos such as 2-cell stage embryos, blastocysts and the like are preferable.
Examples of the freezing method include a slow method and a vitrification method.

  Embryo thawing includes not only thawing and thawing a frozen embryo, but also a treatment such as subjecting it to an optimal culture solution to resume division.

The composition of the present invention used for freezing embryos may vary in composition depending on the type of embryo and the freezing method.
Usually, a conventional medium such as HTF medium or mHTF medium is added to amino acids, carbohydrates, electrolytes, vitamins, trace metal elements, hormones, cell growth factors, lipids or components thereof, carrier proteins, extracellular as necessary. A substrate component (adhesion factor), a reducing substance, a polyhydric alcohol, a carrier for density gradient centrifugation, a buffering agent, etc. are added.

Examples of the polyhydric alcohol include ethylene glycol, propylene glycol, glycerin and the like.
Examples of the carrier for density gradient centrifugation include various Ficolls.
Specific examples of the other components are as described above.

  When the composition of the present invention is used for embryo freezing or thawing, the final concentration of sericin is usually 0.001 to 10% (w / v), preferably 0.01 to 1.0, based on the weight of the whole composition. % (W / v), more preferably 0.06 to 0.8% (w / v), particularly preferably 0.3 to 0.5% (w / v). When the sericin concentration is in the above range, the survival rate of the embryo is high, but if it exceeds 10% (w / v), the survival rate of the embryo is low, and if it falls below 0.001% (w / v), the desired effect cannot be obtained. .

The composition of the present invention used for thawing the embryo may vary in composition depending on the type of embryo and the freezing method.
Usually, an amino acid, carbohydrate, electrolyte, vitamins, trace metal element, hormone, cell growth factor, lipid or its component, carrier protein, extracellular matrix component (if necessary) in a conventional medium such as mHTF medium Adhesion factor), reducing substance, polyhydric alcohol, density gradient centrifugation carrier, buffer, etc. are added. Specific examples are as described above.

  As an example of the above composition, when early embryos are rapidly frozen and thawed by vitrification, there is no particular limitation as long as sericin is contained, mHTF medium, polyhydric alcohol, density gradient centrifugation carrier, carbohydrate And the like containing a frozen solution or a thawing solution.

  In addition, when the early embryo is frozen / thawed by a slow method, it is not particularly limited as long as it contains sericin, but examples thereof include a freezing / thawing solution containing an mHTF medium, a polyhydric alcohol, a carbohydrate and the like.

  Furthermore, in the case of rapidly freezing and thawing the embryos at each stage from the early embryo to the blastocyst by vitrification, there is no particular limitation as long as it contains sericin, but mHTF medium, DMSO (dimethyl sulfoxide) and polyhydric alcohol And a frozen / thawed solution containing carbohydrates.

Embryo preservation kits comprising the composition of the present invention are also encompassed by the present invention.
An embryo preservation kit comprises a composition for use in freezing an embryo and a composition for thawing the embryo.
A composition for use in freezing an embryo and a composition for thawing an embryo have the above-described compositions, respectively, and are prepared by a conventional method as a sterile solution, a use-diluted sterile concentrate, or a use-dissolving type. Used in the form of a sterile lyophilizer.

The composition of the present invention can be used for the treatment and storage of sperm and eggs.
Sperm treatment is the treatment or pretreatment of sperm at the time of semen collection or mutilation. Examples of sperm treatment include preparation and washing of the obtained sperm, and examples of sperm pretreatment include promotion of sperm momentum and sperm fertility acquisition.

  Oocyte processing is the processing or pretreatment of an ovum. Examples of ovum treatment include egg collection and washing after egg collection, and examples of ovum pretreatment include culturing before fertilization.

  The preservation of sperm and ovum is to preserve it until it is subjected to artificial insemination or in vitro fertilization. The storage period is not limited, but in the case of long-term storage, frozen storage is preferable. In addition, thawing after cryopreservation is also included in the present invention.

The above-mentioned sericin concentration is applied to the composition used for the treatment and storage of sperm and eggs, and the medium and other necessary components are added as appropriate.
For example, necessary components are appropriately added to a known medium or the like, and sericin is usually 0.001 to 10% (w / v), preferably 0.01 to 1.0% (w / v), more preferably 0.06 to 0.8% (w / v). v), and particularly preferably, 0.1 to 0.3% (w / v) added sperm and egg washing solution, preservation solution, cryopreservation solution, thawing solution and the like. Specifically, an ovum or sperm washing solution obtained by adding 0.1 to 0.3% (w / v) of sericin to a culture solution obtained by removing phosphoric acid and / or Glucose from an mHTF medium or mHTF medium.

  An embryo culture method characterized by using sericin is also included in the present invention.

In the present invention, particularly in embryo culture after in vitro fertilization, an embryo culture method characterized by using sericin is performed as follows.
The collected immature ovum is added to a sericin-added or non-added medium and cultured for egg cells. Then, generally at 37 ° C., 5% (v / v) carbon dioxide gas / 95% (v / v) in air or 5% (v / v) carbon dioxide gas / 5% (v / v) oxygen / 90% (v / v v) Fertilization is performed by adding sperm in a humidified environment in nitrogen (measuring) and culturing in the medium for about 6 to 10 hours. A fertilized egg obtained by in vitro fertilization is cultured in a medium containing sericin. The preferred concentration of sericin and the composition of the medium are as described above. Change the medium every 2-3 days. A medium that does not require replacement may be used. The above culture is usually carried out at 37 ° C. in a humidified environment in 5% (v / v) carbon dioxide gas / 5% (v / v) oxygen / 90% (v / v) nitrogen. Usually, after 96 hours of culture from fertilization, the fertilized egg reaches the blastocyst.
The definitions of sericin, embryo, etc. are as defined above.

A method for freezing and / or thawing an embryo characterized by using sericin is also included in the present invention.
The method for freezing and / or thawing an embryo after in vitro fertilization in the present invention is performed as follows.

Specifically, in the case of freezing a two-cell embryo with a freezing solution containing sericin, the concentration and components of each component are the freezing solution containing sericin, culture solution, sucrose, ethylene glycol, Ficoll, etc. Equilibrate sequentially with different frozen solutions. After that, it is poured into a straw, rapidly cooled near the liquid nitrogen surface, and then poured into liquid nitrogen for storage. Storage is performed at -196 ° C.
Alternatively, slow freezing may be performed using a program freezer after injection into the straw.

For thawing frozen embryos, a straw is taken out of liquid nitrogen and thawed with warm water at 30 ° C. for 5 to 10 seconds. Thereafter, the dilution is sequentially diluted with a melting solution containing sericin, for example, a melting solution containing a culture solution or sucrose, and transferred to a medium for culturing the embryo.
The definitions of sericin, embryo and freezing method are the same as above.

A sperm treatment method characterized by using sericin is also included in the present invention.
Sperm treatment is as described above.
Specifically, after collecting semen, semen is laminated on percoll or the like heated to room temperature, and centrifuged to remove dead sperm, leukocytes, deformed cells, etc. in the semen. The collected sperm is treated with a washing solution containing sericin. Further, when storing sperm, sperm is added to a frozen solution containing sericin, glycerol, HEPES, etc., placed in a straw or cryotube, cooled to 2-5 ° C. and then put into liquid nitrogen.

The composition for artificial fertilization characterized by containing sericin is used as a freezing protection agent when freezing eggs, sperm and embryos.
The frost damage protective agent in the present invention may be sericin alone or may contain other components. The freezing damage preventive agent according to the present invention is added to a freezing solution or a melting solution at the time of freezing or thawing to prevent destruction of cell membranes of eggs, sperm, and embryos, and is stored frozen for a long period of time while maintaining the morphology and function of cells. Can do.

  The composition of the present invention can be used as a reagent such as an ART research reagent or a test tissue culture reagent.

  Next, the present invention will be described more specifically with reference to examples.

Experimental material
Animals: MCH (ICR) male mice 8-12 weeks old (1 mouse), female mice over 10 weeks old (10 animals)
reagent:
1. Culture medium: Only-One Medium (Nippon Medical Instrumentation Co., Ltd.), HTF medium (Nippon Medical Instrumentation Co., Ltd.)
2. Sericin (Pure Sericin, Wako Pure Chemical Industries, Ltd.), BSA (bovine serum albumin; Sigma Inc.), PVP (Polyvinylpyrrolidone (MW.40,000), Sigma Inc.)
3. Mineral oil (Nippon Medical Instruments Co., Ltd.)
4). Vitrification Freezing & Thawing Medium Kit (Nippon Medical Instruments Co., Ltd.)

1. Efficacy of sericin in serum-free medium
Experimental method (1) Equilibrium of culture solution:
Cover a 200 μl drop of serum-free HTF (containing 0.1% (w / v) sericin) medium for semen collection and maturation with mineral oil, 5% (v / v) carbon dioxide, 95% (v / v) Equilibrated overnight in an incubator set at 37 ° C in air.

(2) Oocyte preparation:
As superovulation treatment, 7.5 IU / mouse of PMSG (pregnant mare serum gonadotropin: cellotropin, Asuka Pharmaceutical) was administered into the abdominal cavity of female mice, and 48 hours later, hCG (human chorionic gonadotropin: gonatropin, asuka) Was administered intraperitoneally at 7.5 IU / animal. Ovulation ova were collected from the oviducts 14 to 16 hours after hCG administration and used for in vitro fertilization (IVF).

(3) Preparation of sperm suspension:
200 μl of semen obtained from one male mouse epididymis was collected and placed in a culture medium drop of serum-free HTF (containing 0.1% (w / v) sericin) equilibrated as described above. The IVF semen was prepared by pre-culturing for 1 to 2 hours.

(4) Hosei and culture:
Add approximately 70-110 eggs to a 200 μl drop of serum-free HTF for serum (containing 0.1% (w / v) sericin) equilibrated on the previous day using the method described above, and prepare as described above. 0.9 μl of the semen (experimental example) was added to perform sperm (final sperm concentration: 5 × 10 ⁵ / ml).
After 6-10 hours, fertilized eggs were confirmed by observing the formation of pronuclei under a microscope.
Confirmed fertilized eggs are randomly grouped, and 50 μl drops of 3 types of Only-One Medium each containing 0.1% (w / v) PVP, 0.1% (w / v) sericin or 0.3% (w / v) BSA And cultured for 96 hours, and the embryo development rate was examined from the pronuclear stage to the blastocyst stage.

(5) Embryo transfer:
Female mice were mated with males ligated with vagina to serve as recipients.
In vitro fertilized eggs obtained by the method of (4) above, 0.1% (w / v) sericin-added Only-One Medium and 0.3% (w / v) BSA-added Only-One Medium (no amino acid) medium as a control The blastocyst stage embryo was cultured from the pronuclear stage to the blastocyst stage, and the resulting blastocyst stage embryo was transplanted under general anesthesia to the uterus of a recipient mouse on the fifth day of pseudopregnancy.
To prevent food loss, a caesarean section was performed on the 16th day of pseudopregnancy, and the fetal implantation marks were confirmed. The newborn mouse was then fed and fostered to a foster parent prepared in advance.

Culture solution composition (1) HTF medium + 0.1% (w / v) sericin (2) Only-one medium + 0.1% (w / v) sericin

Statistical processing test was performed by the X-square independent test method, and P <0.05 was determined to be statistically significant.

Experimental results (1) Evaluation of fertilization rate
0.1% (w / v) sericin was added to the HTF medium (HTF medium; for semen collection and maze), and 0.3% (w / v) BSA was added to the HTF medium as a comparative control. After fertilization for 6 to 10 hours, the pronucleus-forming eggs were confirmed with a microscope, and the fertilization rate was calculated from the ratio of pronucleus-forming eggs per fertilized egg. As shown in Table 2, the fertilization rate in the HTF medium supplemented with 0.1% (w / v) sericin was as high as that of the HTF medium supplemented with 0.3% (w / v) BSA.

(2) Evaluation of embryo development rate in blastocysts Three types of media, (1) 0.1% (w / v) PVP-added Only-One Medium, (2) 0.1% (w / v) sericin-added Only-One Medium, (3) The rate at which fertilized eggs cultured in 0.3% (w / v) BSA-added Only-One Medium developed to the blastocyst was calculated as the embryo development rate (the same experiment was repeated three times). .
As shown in Table 3, the embryo development rate in the sericin-added medium was very high with no difference from the PVP- or BSA-added medium.
The morphology of the embryo was observed under a microscope (× 200). As for the sericin-added medium, morphologically normal blastocyst stage embryos were confirmed as in the case of BSA or PVP-added medium (FIG. 1).

(3) Evaluation of embryo transfer
Obtained by transplanting blastocyst stage embryos generated in 0.1% (w / v) Sericin-added Only-One Medium or 0.3% (w / v) BSA-added Only-One Medium (without amino acid) as a control to recipients There were no significant differences in the number of pups produced and the rate of birth (Table 4).

As a result of comparing the serum-free culture medium containing sericin with the one containing BSA and PVP, the embryo development rate from the nymph to the blastocyst stage is 90% or more in any medium, and the significant difference is It was not seen (Table 3).
When the morphology of the blastocyst was observed under a microscope, the inner cell mass, the trophectoderm and the blastocoel were confirmed, and no abnormality was observed (FIG. 1).
It was found that a serum-free culture medium using sericin is effective in the early development after in vitro fertilization.

On the other hand, when a mouse embryo that has developed up to the blastocyst stage using serum-free sericin medium is transplanted into the uterus, it is compared with embryos grown in a medium (control) supplemented with conventional BSA. However, the efficiency of implantation and pups did not change (Table 4). Furthermore, all pups born by caesarean section developed normally (FIG. 2). Therefore, it was shown that culture using a sericin-added medium is safe.
Conventionally, PVP, which has been used without serum, is a high molecular compound, and its toxicity to the embryo is unknown, which has been an obstacle to use in the field of ART. It can be expected that this problem can be overcome by using sericin.

2. Examination of the effectiveness of sericin in vitrification freezing of mouse 2-cell embryos with serum-free vitrification and thawing solution
Experimental method (vitrification freezing method)
(1) Freezing method Two-cell stage embryos were separated from the rapid freezing kit (Table 5) Sol. B, Sol. A, Sol. C, Sol. Sequential equilibration with D followed by freezing of the embryos.
Sol. Among the compositions of A to D, a solution was prepared by adding 0.3% (w / v) sericin in place of 0.3% (w / v) human serum albumin (HSA), and using these, the two-cell phase was similarly used. Embryo equilibration and freezing were performed.

(2) Thawing method Frozen two-cell stage embryos were prepared from Sol. A and Sol. B was sequentially used and thawed, and the number of viable 2-cell embryos immediately after thawing was confirmed, and then cultured to the blastocyst stage in the above-described sericin-added culture medium. The incidence to the blastocyst stage was observed as described above.

Freezing and thawing kit composition (HSA included)

Experimental results When a 2-cell embryo was frozen and thawed using a Vitrification Freezing & Thawing Medium Kit (Nippon Medical Instruments Co., Ltd., Cat. No. 2002-030) containing sericin, the survival rate immediately after thawing was HSA. It was 100% equivalent to the freezing and thawing kit contained. Two-cell embryos thawed with a melt containing HSA or sericin were cultured at 37 ° C. in 5% (v / v) carbon dioxide / 95% (v / v) air until the blastocyst stage. The development rate of thawing embryos in the blastocyst stage was as high as 80% or more (Table 6). When observed under a microscope (× 200), all blastocysts showed normal morphology (FIG. 3).

  Based on the above, the serum-free freezing and thawing solution containing sericin is the freezing and thawing number of blastocysts and the embryo development rate up to the blastocyst stage. showed that.

Claims (13)

  An artificial fertilization composition comprising sericin.   The composition according to claim 1, which does not contain serum.   The composition according to claim 1 or 2, wherein the concentration of sericin is 0.001 to 10% (w / v).   The composition according to any one of claims 1 to 3, wherein the artificial fertilization is in vitro fertilization.   The composition according to any one of claims 1 to 4, which is used for embryo culture.   The composition according to any one of claims 1 to 4, which is used for preservation of an embryo.   The composition according to claim 6, wherein preservation of the embryo is freezing and / or thawing of the embryo.   The composition according to any one of claims 1 to 4, which is used for treatment or storage of sperm and / or eggs.   An embryo preservation kit comprising the composition according to any one of claims 1 to 4.   A method for culturing an embryo, characterized by using sericin.   A method for freezing and / or thawing an embryo characterized by using sericin.   A method for treating sperm, characterized by using sericin.   The composition according to claim 1, which is a frost damage protective agent used when freezing eggs, sperm and embryos.