JP6977999B2 - Embryo culture medium and embryo culture method - Google Patents
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この発明は、胚培養培地、及び胚培養方法に関し、特に、ピルビン酸を含む胚培養培地、及び胚培養方法に関する。 The present invention relates to an embryo culture medium and an embryo culture method, and more particularly to an embryo culture medium containing pyruvate and an embryo culture method.
近年、生殖医療や再生医療の進歩に伴い、体外培養による胚の発生を母体内での胚の発生に近似させるための研究が広く行なわれ、新たな培養方法が各種報告されている。 In recent years, with the progress of reproductive medicine and regenerative medicine, researches have been widely conducted to approximate the development of embryos by in vitro culture to the development of embryos in the mother's body, and various new culture methods have been reported.
例えば、非特許文献１では、ＫＳＯＭａａ培地にヒト血清アルブミンを添加することが、マウス１細胞期胚の胚盤胞への発生率、及び該胚盤胞の着床率・産仔率の向上に有効であることが報告されている。 For example, in Non-Patent Document 1, addition of human serum albumin to KSOMaa medium improves the incidence of mouse 1-cell stage embryos in blastocysts and the implantation rate and offspring rate of the blastocysts. It has been reported to be valid.
また、非特許文献２では、マウス初期胚の体外発生は、ピルビン酸に依存すること、また、培地にピルビン酸を添加しない場合には、細胞膜の透過性を付与したジメチルαケトグルタル酸により補完できることが報告されている。 Further, in Non-Patent Document 2, in vitro development of early mouse embryos depends on pyruvic acid, and when pyruvic acid is not added to the medium, it can be complemented by dimethyl α-ketoglutaric acid which imparts permeability to the cell membrane. Has been reported.
このジメチルαケトグルタル酸については、ヒストン脱メチル化酵素やＴＥＴ（ｔｅｎ‐ｅｌｅｖｅｎ ｔｒａｎｓｌｏｃａｔｉｏｎ）タンパク質が酵素活性を発揮する上で不可欠であること（非特許文献３）、マウスの胚性幹細胞（ＥＳ）細胞の培地に４ｍＭを添加すると、ヒストンとＤＮＡの脱メチル化が促進されて自己複製が促進される一方で分化が抑制されること（非特許文献４、非特許文献５）、ミトコンドリア代謝とエピジェネティックの制御を介してヒト多能性幹細胞の分化が促進されること（非特許文献６）等が報告されている。 Regarding this dimethyl α-ketoglutaric acid, histone demethylase and TET (ten-eleven differentiation) protein are indispensable for exerting enzymatic activity (Non-Patent Document 3), and mouse embryonic stem cell (ES) cells. When 4 mM is added to the medium, histone and DNA demethylation is promoted and self-renewal is promoted while differentiation is suppressed (Non-Patent Documents 4 and 5), mitochondrial metabolism and epigenetic. It has been reported that the differentiation of human pluripotent stem cells is promoted through the control of (Non-Patent Document 6).
ところが、本発明者が実施したＩＣＲ系マウスについての実験によれば、後述するように、ＦＩ（Ｃ５７ＢＬ／６Ｊ Ｘ Ｃ３ＨＥ）系マウスに係る非特許文献２の報告とは異なり、ピルビン酸不添加のＫＳＯＭ培地では、ジメチルαケトグルタル酸を添加しても１細胞期胚から胚盤胞への発生が見られないという結果が得られ、ジメチルαケトグルタル酸を培地に添加しても、初期胚の発生に有効でない場合の有ることが明らかになった。
本発明は、上記課題に鑑みてなされたものであり、胚の発生に対し、より有効にジメチルαケトグルタル酸が作用して、効率よく胚を発生させることが可能な胚培養培地、及び胚培養方法の提供を目的とする。
However, according to the experiment on the ICR-based mouse carried out by the present inventor, as will be described later, unlike the report of Non-Patent Document 2 relating to the FI (C57BL / 6JX C3HE) -based mouse, pyruvate-free is added. In the KSOM medium, the results showed that development from 1-cell stage embryos to blastocysts was not observed even when dimethyl α-ketoglutaric acid was added, and development of early embryos was obtained even when dimethyl α-ketoglutaric acid was added to the medium. It became clear that there are cases where it is not effective.
The present invention has been made in view of the above problems, and is an embryo culture medium capable of efficiently developing an embryo by the action of dimethyl α-ketoglutaric acid more effectively on the development of an embryo, and an embryo culture. The purpose is to provide a method.
上記課題を解決するためになされた発明は、ヒトを含む哺乳動物の胚を培養する胚培養培地であって、ピルビン酸と、ジメチルαケトグルタル酸とを含み、ジメチルαケトグルタル酸の添加量が１ｍＭ以上８ｍＭ未満であり、ピルビン酸の添加量が０．０１ｍＭ以上０．２ｍＭ以下である。
ジメチルαケトグルタル酸の添加量は、１ｍＭ以上４ｍＭ以下であることが好ましい。
Invention was made to solve the aforementioned problems is a embryo culture medium for culturing a mammalian embryo, including humans, and pyruvate, see containing and dimethyl α-ketoglutarate, the addition amount of dimethyl α-ketoglutarate It is 1 mM or more and less than 8 mM, and the amount of pyruvic acid added is 0.01 mM or more and 0.2 mM or less .
The amount of dimethyl α-ketoglutaric acid added is preferably 1 mM or more and 4 mM or less.
本発明の胚培養培地は、ピルビン酸の添加量が０．０１ｍＭ以上０．２ｍＭ以下であり、０．０２ｍＭ以上０．２ｍＭ以下であることが、好ましい。こうすることで、胚をより効率よく発生させることができる。胚培養培地に、ジメチルαケトグルタル酸の存在下で、０．０１ｍＭ以上のピルビン酸を添加することで、効率よく胚の胚盤胞への発生を行うことができ、０．０２ｍＭ以上のピルビン酸を添加することで、さらに効率よく胚の胚盤胞への発生を行うことができる。 Embryo culture media of the present invention, the amount of pyruvate is that at 0.2mM or less than 0.01 mM, that is, preferably 0.2mM or less than 0.02 mM. By doing so, embryos can be developed more efficiently. By adding 0.01 mM or more of pyruvic acid to the embryo culture medium in the presence of dimethyl α-ketoglutaric acid, it is possible to efficiently develop embryos into blastocysts, and 0.02 mM or more of pyruvic acid. By adding the above, embryos can be more efficiently developed into blastocysts.
また、本発明は、ヒトを除く哺乳動物用の胚を培養する胚培養方法であって、ピルビン酸と、ジメチルαケトグルタル酸とを添加した培地を用いて胚の培養を行い、ジメチルαケトグルタル酸の添加量が１ｍＭ以上８ｍＭ未満であり、ピルビン酸の添加量が０．０１ｍＭ以上０．２ｍＭ以下である胚培養方法を含む。 Further, the present invention provides a embryo culture method of culturing embryos for mammals other than humans, have rows embryo culture using a pyruvate, a medium supplemented with dimethyl α-ketoglutarate, dimethyl α-ketoglutaric Includes an embryo culture method in which the amount of acid added is 1 mM or more and less than 8 mM, and the amount of pyruvic acid added is 0.01 mM or more and 0.2 mM or less.
このように、本発明の胚培養培地、及び胚培養方法では、ピルビン酸と、ジメチルαケトグルタル酸の両方を添加することで、より効率的に、胚を発生させることができる。 As described above, in the embryo culture medium and the embryo culture method of the present invention, embryos can be generated more efficiently by adding both pyruvic acid and dimethyl α-ketoglutaric acid.
以下、本発明の一の実施形態について詳述する。ただし、本発明は、以下の実施形態に限られるものではない。また、以下の説明において、ピルビン酸を添加した培地、添加しない培地を、それぞれ＋Ｐ培地、−Ｐ培地ともいうものとする。 Hereinafter, one embodiment of the present invention will be described in detail. However, the present invention is not limited to the following embodiments. Further, in the following description, the medium to which pyruvic acid is added and the medium to which pyruvic acid is not added are also referred to as + P medium and −P medium, respectively.
本実施形態に係る胚培養培地は、基礎培地に、ピルビン酸とジメチルαケトグルタル酸（ｄｍ−αＫＧ）が主に添加される他、着床前胚の体外培養で発生を支持する支持物質（以下、「支持物質」という）が添加されている。
ここで、「基礎培地」とは、ピルビン酸の添加の有無にかかわらず、ｄｍ−αＫＧを含まない全ての培地をいい、それだけで哺乳動物の胚を培養可能なものも、培養できないものも含むものとする。
In the embryo culture medium according to the present embodiment, pyruvic acid and dimethyl α-ketoglutaric acid (dm-αKG) are mainly added to the basal medium, and a supporting substance that supports development in in vitro culture of pre-implantation embryos (hereinafter referred to as “)”. , "Supporting material") is added.
Here, the "basal medium" refers to all media containing no dm-αKG regardless of the addition or absence of pyruvic acid, and includes those capable of culturing mammalian embryos and those that cannot be cultivated by themselves. It shall be a waste.
「哺乳動物」は、特に限定するものではないが、ヒト、マウス、ウシ、ウサギ、アカゲザル、ブタ、又はラット等が例示される。 The "mammal" is not particularly limited, and examples thereof include humans, mice, cows, rabbits, rhesus monkeys, pigs, and rats.
基礎培地として用いられる培地としては、一般に着床前胚の体外発生培地として用いられている培地が好ましいが、特に限定されず、それ以外の培地を用いてもよい。
着床前胚の体外発生培地としては、マウス着床前胚の体外発生培地、ラット・ウサギ・ハムスター着床前胚の体外発生培地、ウシ・ブタ着床前胚の体外発生培地、又はヒト着床前胚の体外発生培地が挙げられる。
The medium used as the basal medium is generally preferably a medium used as an in vitro development medium for pre-implantation embryos, but is not particularly limited, and other media may be used.
As the in vitro development medium of the pre-implantation embryo, the in vitro development medium of the mouse pre-implantation embryo, the in vitro development medium of the rat / rabbit / hamster pre-implantation embryo, the in vitro development medium of the bovine / pig pre-implantation embryo, or the human arrival. Examples include in vitro developmental media of prebed embryos.
マウス着床前胚の体外培養培地としては、例えば、ＫＭＯＳ培地，ＫＭＯＳａａ培地（ＫＳＯＭ培地にアミノ酸溶液を添加したもの），Ｍ１６培地，Ｍ１２培地，ＣＺＢ培地，ＭＴＦ培地，ＢＷＷ培地，Ｗｈｉｔｔｅｎ’ｓ培地，ＢＭＯＣ−ＩＩＩ培地が挙げられ、ＫＭＯＳ培地，ＫＭＯＳａａ培地が好ましい。
尚、ＫＳＯＭ培地は、一般にＥＤＴＡ（エチレンジアミン四酢酸）を含むが、非生理物質であるため、省略してもよい。
Examples of the in vitro culture medium for mouse pre-implantation embryos include KMOS medium, KMOSaa medium (KSOM medium with an amino acid solution added), M16 medium, M12 medium, CZB medium, MTF medium, BWW medium, and Whiten's medium. , BMOC-III medium, and KMOS medium and KMOSaa medium are preferable.
The KSOM medium generally contains EDTA (ethylenediaminetetraacetic acid), but since it is a non-physiological substance, it may be omitted.
ラット・ウサギ・ハムスター着床前胚の体外培養培地としては、例えば、ＨＥＣＭ１培地、ＨＥＣＭ３培地，Ｒ１ＥＣＭ培地，ｍＫＲＢ培地，Ｋａｎｅ’ｓ培地が挙げられる。 Examples of the in vitro culture medium for rat / rabbit / hamster pre-implantation embryos include HECM1 medium, HECM3 medium, R1ECM medium, mKRB medium, and Kane's medium.
ウシ・ブタ着床前胚の体外培養培地としては、例えば、ｍＳＯＦ培地，ｍＳＯＦａａ培地，ＣＲ１ａａ培地，ＢＥＣＭ培地，ＰＺＭ培地（ＰＺＭ５培地）が挙げられ、ＣＲ１ａａ培地が好ましい。 Examples of the in vitro culture medium for bovine / porcine pre-implantation embryos include mSOF medium, mSOFaa medium, CR1aa medium, BECM medium, and PZM medium (PZM5 medium), and CR1aa medium is preferable.
ヒト着床前胚の体外培養培地としては、例えば、ＨＴＦ培地、ＱＡ Ｃｌｅａｖａｇｅ培地，Ｃｏｍｐｌｅｔｅ Ｂｌａｓｔｏｃｙｓｔ培地，ＱＡ Ｂｌａｓｔｏｃｙｓｔ培地，オンリーワン培地（株式会社日本医化器械製作所製），Ｅａｒｌｙ ｃｕｌｔｕｒｅ培地（株式会社日本医化器械製作所製）、ｇｌｏＢａｌ培地（ｌｉｆｅ ｇｌｏｂａｌ社製）が挙げられる。 Examples of the in vitro culture medium for human pre-implantation embryos include HTF medium, QA Cleavage medium, Complete Blastocyst medium, QA Blastocyst medium, Only One medium (manufactured by Nippon Ika Kikai Seisakusho Co., Ltd.), and Early culture medium (Japan Co., Ltd.). Examples include (manufactured by Ika Kikai Seisakusho) and global medium (manufactured by life global).
胚培養培地に対するピルビン酸の添加濃度は、特に限定されないが、０．０１ｍＭ以上０．２ｍＭ以下が好ましい。こうすることで、ピルビン酸による胚の発生効率の上昇をさらに向上させることができる。
また、当該ピルビン酸の添加濃度は、０．０２ｍＭ以上０．２ｍＭ以下であることが特に好ましい。こうすることで、ピルビン酸が０．０２ｍＭ未満の場合に比べて、より一層、胚の発生効率を向上できる。
The concentration of pyruvic acid added to the embryo culture medium is not particularly limited, but is preferably 0.01 mM or more and 0.2 mM or less. By doing so, it is possible to further improve the increase in embryogenesis efficiency due to pyruvic acid.
Further, the addition concentration of the pyruvic acid is particularly preferably 0.02 mM or more and 0.2 mM or less. By doing so, the embryogenesis efficiency can be further improved as compared with the case where pyruvic acid is less than 0.02 mM.
胚培養培地に対するｄｍ−αＫＧの添加濃度は、特に限定されないが、８ｍＭ未満が好ましい。当該濃度が８ｍＭ以上であると、胚の発生効率を低下させる虞がある。 The concentration of dm-αKG added to the embryo culture medium is not particularly limited, but is preferably less than 8 mM. If the concentration is 8 mM or more, the embryonic development efficiency may be reduced.
支持物質としては、例えば、組換えヒト血清アルブミン，ＰＶＡ（ポリビニルアルコール），ＰＶＰ（ポリビニルピロリドン），脱イオン化ウシ血清アルブミン（ｄＢＳＡ），ＦＣＳ（Ｆｅｔａｌ ｃａｌｆ ｓｅｒｕｍ（ウシ胎仔血清））が挙げられ、脱イオン化ウシ血清アルブミン，ＦＣＳが好ましい。 Examples of the supporting substance include recombinant human serum albumin, PVA (polyvinyl alcohol), PVP (polyvinylpyrrolidone), deionized bovine serum albumin (dBSA), and FCS (Fetal calf serum). Ionized bovine serum albumin and FCS are preferred.
次に、本発明の実施例に係る培地を用いた実験を通じて本発明をさらに詳述する。ただし、本発明は、以下の実施例に限定されるものではない。 Next, the present invention will be described in more detail through experiments using the medium according to the examples of the present invention. However, the present invention is not limited to the following examples.
［マウス胚の培養実験］
マウス１細胞期胚の発生に及ぼすジメチルαケトグルタル酸（ｄｍ−αＫＧ）の効果を明らかにすべく、下記の実施例、比較例に示した培地を用いて、下記の要領で培養実験を行った。
（実施例１）
０．６％脱イオン化ウシ血清アルブミン（ｄＢＳＡ、Ｓｉｇｍａ−Ａｌｄｒｉｃｈ社製）を添加したＫＳＯＭ（ＥＤＴＡ不含、ピルビン酸（Ｓｉｇｍａ−Ａｌｄｒｉｃｈ社製）不含）−Ｐ培地に、０．０１ｍＭピルビン酸を添加して＋Ｐ培地とし、さらに、ｄｍ−αＫＧ（東京化成工業社製）を１ｍＭ添加して、実施例１とした。尚、実施例１、及び後述する実施例２、３においては、当該−Ｐ培地が基礎培地となる。
[Mouse embryo culture experiment]
In order to clarify the effect of dimethyl α-ketoglutaric acid (dm-αKG) on the development of mouse 1-cell stage embryos, culture experiments were carried out in the following manner using the media shown in the following Examples and Comparative Examples. ..
(Example 1)
0.01 mM pyruvic acid in KSOM (EDTA-free, pyruvic acid (Sigma-Aldrich) -free) -P medium supplemented with 0.6% deionized bovine serum albumin (dBSA, manufactured by Sigma-Aldrich). It was added to prepare a + P medium, and 1 mM of dm-αKG (manufactured by Tokyo Chemical Industry Co., Ltd.) was further added to obtain Example 1. In Example 1 and Examples 2 and 3 described later, the −P medium is the basal medium.
（実施例２）
ピルビン酸を０．０２ｍＭとした他は、実施例１と同様にして培地を作成し、実施例２とした。
(Example 2)
A medium was prepared in the same manner as in Example 1 except that pyruvic acid was 0.02 mM, and the medium was used as Example 2.
（実施例３）
ピルビン酸を０．２ｍＭとした他は、実施例１と同様にして培地を作成し、実施例３とした。
(Example 3)
A medium was prepared in the same manner as in Example 1 except that pyruvic acid was 0.2 mM, and the medium was used as Example 3.
（比較例１）
ｄｍ−αＫＧ無添加の−Ｐ培地を比較例１とした。
(Comparative Example 1)
Comparative Example 1 was a −P medium to which dm-αKG was not added.
（比較例２）
ｄｍ−αＫＧを１ｍＭ添加した−Ｐ培地を比較例２とした。
(Comparative Example 2)
Comparative Example 2 was a −P medium supplemented with 1 mM of dm-αKG.
（比較例３〜比較例５）
ｄｍ−αＫＧ無添加で、ピルビン酸を０．０１ｍＭ、０．０２ｍＭ，０．２ｍＭを添加した（＋Ｐ）培地をそれぞれ比較例３、比較例４、比較例５とした。
(Comparative Example 3 to Comparative Example 5)
The (+ P) medium to which 0.01 mM, 0.02 mM, and 0.2 mM of pyruvic acid was added without adding dm-αKG was designated as Comparative Example 3, Comparative Example 4, and Comparative Example 5, respectively.
＜実験１＞
・方法
実施例１〜実施例３、及び比較例１〜比較例５の培地を用い、ＩＣＲ系マウス（Ｋｗｌ：ＩＣＲ、紀和実験動物研究所製）から採取した１細胞期胚を３７℃、５％体積ＣＯ2の空気中で培養し、２細胞期胚、４細胞期胚への発生数と９６時間後における胚盤胞発生数を計数し、供試胚数に対する発生率を求めた。
<Experiment 1>
-Method: Using the media of Examples 1 to 3 and Comparative Examples 1 to 5, 1-cell stage embryos collected from ICR-based mice (Kwl: ICR, manufactured by Kiwa Experimental Animal Research Institute) were collected at 37 ° C. and 5 ° C. % Volume CO 2 was cultured in air, and the number of embryos developed into 2-cell stage embryos and 4-cell stage embryos and the number of blastocysts developed after 96 hours were counted, and the incidence rate with respect to the number of test embryos was determined.
・結果
実験１の結果を表１、及び図１に示す。
ＩＣＲ系マウス胚を用いた当該実験においても、ＦＩ系マウスを用いた上記非特許文献２の報告と同様に、ｄｍ−αＫＧ無添加の−Ｐ培地（比較例１）では、ほとんどの胚が２細胞期で発生を停止し、胚盤胞へは全く発生しなかった。
また、ｄｍ−αＫＧ無添加の＋Ｐ培地（０．０１ｍＭ〜０．２ｍＭのピルビン酸を添加、比較例３〜比較例５）では、１細胞期胚を有意の割合で胚盤胞へ発生させることができた。その割合は、ピルビン酸濃度の上昇につれて増加し、０．２ｍＭのピルビン酸を添加した比較例５で最も高い割合（７８．５％）を示した。比較例５の割合は、非特許文献２における割合と同様であった。
ところが、１ｍＭのｄｍ−αＫＧを添加した−Ｐ培地（比較例２）では、ＦＩ系マウス胚を用いた非特許文献２の報告とは異なり、胚盤胞への発生は全く改善されず、ｄｍ−αＫＧがピルビン酸の効果を補完しない場合の有ることが明らかとなった。
In the experiment using ICR-based mouse embryos, most of the embryos were 2 in the -P medium (Comparative Example 1) to which dm-αKG was not added, as in the report of Non-Patent Document 2 using FI-based mice. It stopped developing at the cellular stage and never developed into blastocysts.
In addition, in + P medium without dm-αKG (added 0.01 mM to 0.2 mM pyruvic acid, Comparative Examples 3 to 5), 1-cell stage embryos should be generated in blastocysts at a significant rate. Was completed. The ratio increased with increasing pyruvic acid concentration, and showed the highest ratio (78.5%) in Comparative Example 5 to which 0.2 mM pyruvic acid was added. The ratio of Comparative Example 5 was the same as the ratio in Non-Patent Document 2.
However, unlike the report in Non-Patent Document 2 using FI mouse embryos, the −P medium supplemented with 1 mM dm-αKG (Comparative Example 2) did not improve the development to blastocysts at all, and dm. It has become clear that there are cases where -αKG does not complement the effects of pyruvate.
一方で、１ｍＭのｄｍ−αＫＧを添加した＋Ｐ培地（実施例１〜実例例３）では、１細胞期胚から胚盤胞への発生率が、ｄｍ−αＫＧ無添加の＋Ｐ培地（比較例３〜比較例５）と比べても、有意に高くなった（図１の実線と破線による折線参照）。
以上の結果から、ｄｍ−αＫＧは、ピルビン酸の存在下において発生培地に添加することで、胚の発生効率を大きく上昇させることが明らかになった。ピルビン酸とは異なる作用機構によって、マウス初期胚の胚盤胞への発生を相乗的に促進すると推測される。
On the other hand, in the + P medium supplemented with 1 mM dm-αKG (Examples 1 to Example 3), the incidence from 1-cell stage embryos to blastocysts was high in the + P medium supplemented with no dm-αKG (Comparative Example 3). It was significantly higher than that in Comparative Example 5) (see the solid line and the broken line in FIG. 1).
From the above results, it was clarified that dm-αKG greatly increases the developmental efficiency of embryos when added to the development medium in the presence of pyruvic acid. It is speculated that it synergistically promotes the development of early mouse embryos into blastocysts by a mechanism of action different from that of pyruvate.
また、図１に実線の折線で示すように、＋Ｐ培地においては、ピルビン酸を０．０２ｍＭ以上添加すると、１細胞期胚の胚盤胞への発生率が、急に上昇することが明らかになった。 In addition, as shown by the solid line in FIG. 1, it is clear that the incidence of 1-cell stage embryos in blastocysts increases sharply when pyruvic acid is added at 0.02 mM or more in + P medium. became.
［マウス産仔の発生実験］
マウスの産仔発生に及ぼすｄｍ−αＫＧの効果を明らかにすべく、上記の実施例、比較例に示した培地を用いて、下記の要領で実験２を行った。
[Development experiment of mouse offspring]
In order to clarify the effect of dm-αKG on the development of offspring of mice, Experiment 2 was carried out in the following manner using the media shown in the above Examples and Comparative Examples.
＜実験２＞
・方法
実験１において、実施例１の培地（＋Ｐ培地、ｄｍ−αＫＧ１ｍＭ添加）、比較例５（＋Ｐ培地、ｄｍ−αＫＧ無添加）の培地により発生した胚盤胞と、交配後４日目のマウス子宮から採取した胚盤胞（Ｉｎ ｖｉｖｏ区）を偽妊娠２．５日目のマウスの子宮内に非外科的方法により移植し、妊娠１９．５日目の着床率と産仔率を求めた。
<Experiment 2>
-Method In Experiment 1, the blastocysts generated by the medium of Example 1 (+ P medium, dm-αKG 1 mM added) and Comparative Example 5 (+ P medium, dm-αKG not added) and the blastocysts generated on the 4th day after mating. Blastocysts (Invivo plot) collected from the mouse uterus were transplanted into the uterus of mice on the 2.5th day of gestation by a non-surgical method, and the implantation rate and the birth rate on the 19.5th day of gestation were measured. I asked.
・結果
実験２により得られた結果を表２に示す。
実施例１の培地（＋Ｐ培地、ｄｍ−αＫＧ添加）で発生した胚盤胞の移植による着床率、及び産仔への発生率は、表２に示すように、比較例５の培地（＋Ｐ培地、ｄｍ−αＫＧ無添加）の胚盤胞に比べていずれも高く、さらに、その値は、Ｉｎ ｖｉｖｏ区胚盤胞に類似した値であった。
従って、ｄｍ−αＫＧは、体外培養で発生する胚盤胞への産仔への発生能を生体の卵管内で発生した胚の能力まで高める効果を有することが示唆される。
As shown in Table 2, the implantation rate by transplantation of blastocysts generated in the medium of Example 1 (+ P medium, addition of dm-αKG) and the rate of occurrence in offspring are the medium of Comparative Example 5 (+ P). Both were higher than the blastocysts of the medium and dm-αKG-free), and the values were similar to those of the Invivo-group blastocysts.
Therefore, it is suggested that dm-αKG has an effect of increasing the ability of embryos to develop into blastocysts generated by in vitro culture to the ability of embryos developed in the oviduct of a living body.
以上、実験１、実験２の結果から、ｄｍ−αＫＧのマウス初期胚に及ぼす効果については、これまで−Ｐ培地での発生停止を解除して＋Ｐ培地での胚盤胞発生率まで発生を高める効果の有ることが唯一報告されている（非特許文献２）が、＋Ｐ培地においてもｄｍ−αＫＧは、初期胚の胚盤胞、及び産仔への発生を促進する効果の有ることを初めて示すことができた。 From the results of Experiment 1 and Experiment 2, regarding the effect of dm-αKG on early mouse embryos, the developmental arrest in -P medium has been canceled and the blastocyst development rate in + P medium has been increased. Although only reported to have an effect (Non-Patent Document 2), it is shown for the first time that dm-αKG has an effect of promoting the development of early embryos into blastocysts and offspring even in + P medium. I was able to.
また、ｄｍ−αＫＧを添加した＋Ｐ培地初期胚を培養することによって、卵管、及び子宮内で発生する胚に類似した発生の（胚質）を有する胚盤胞を高率に産生できることが示唆される。 It is also suggested that blastocysts with development (embryo) similar to embryos that develop in the oviduct and uterus can be produced at a high rate by culturing early embryos in + P medium supplemented with dm-αKG. Will be done.
［ウシ胚の培養実験］
次に、ウシ体外受精胚の発生に及ぼすｄｍ−αＫＧの効果を明らかにすべく、下記の実施例、比較例に示した培地を用いて、下記の要領で実験３を行った。
[Culturing experiment of bovine embryo]
Next, in order to clarify the effect of dm-αKG on the development of bovine in vitro fertilized embryos, Experiment 3 was carried out in the following manner using the media shown in the following Examples and Comparative Examples.
（実施例４）
５体積％のＦＣＳ（Ｆｅｔａｌ ｃａｌｆ ｓｅｒｕｍ、ウシ胎仔血清, ＧＩＢＣＯ（登録商標））と０．２ｍＭのピルビン酸とを添加したＣＲ１ａａ培地に、さらに、４ｍＭのｄｍ−αＫＧを添加して形成した培地を実施例４とした。
(Example 4)
A medium formed by adding 4 mM dm-αKG to a CR1aa medium supplemented with 5% by volume FCS (Fetal bovine serum, fetal bovine serum, GIBCO®) and 0.2 mM pyruvic acid was added. It was referred to as Example 4.
（比較例６）
５体積％のＦＣＳを添加したＣＲ１ａａ培地を比較例６とした。
(Comparative Example 6)
The CR1aa medium supplemented with 5% by volume FCS was designated as Comparative Example 6.
（比較例７、比較例８）
５体積％のＦＣＳを添加したＣＲ１ａａ培地に、１ｍＭ、４ｍＭのｄｍ−αＫＧを添加し、それぞれ比較例７、及び比較例８の培地とした。
(Comparative Example 7, Comparative Example 8)
1 mM and 4 mM dm-αKG were added to CR1aa medium supplemented with 5% by volume FCS to prepare the media of Comparative Example 7 and Comparative Example 8, respectively.
（比較例９）
５体積％のＦＣＳ、及び０．２ｍＭのピルビン酸を添加したＣＲ１ａａ培地を比較例９の培地とした。
(Comparative Example 9)
The CR1aa medium supplemented with 5% by volume FCS and 0.2 mM pyruvic acid was used as the medium of Comparative Example 9.
（比較例１０）
５体積％のＦＣＳと０．２ｍＭのピルビン酸とを添加したＣＲ１ａａ培地に、さらに、８ｍＭのｄｍ−αＫＧを添加して形成した培地を比較例１０とした。
(Comparative Example 10)
Comparative Example 10 was a medium formed by further adding 8 mM dm-αKG to a CR1aa medium supplemented with 5% by volume FCS and 0.2 mM pyruvic acid.
＜実験３＞
・方法
屠体ウシ卵巣の直径２〜６ｍｍの卵胞から吸引採取した卵子卵丘細胞複合体を成熟培地（１０体積％ＦＣＳ、及び０．０２ＡＵ ＦＳＨ（前葉性卵胞刺激ホルモン、共立製薬社製）を添加したＭ１９９培地、Ｓｉｇｍａ−Ａｌｄｒｉｃｈ社製)にて２２時間、５体積％ＣＯ2の空気中、３８．５℃の条件下で体外成熟させた。
尚、ここで「成熟」とは、卵母細胞を受精可能な減数***第２***中期まで進行させることをいう。
<Experiment 3>
-Method: A mature medium (10% by volume FCS and 0.02AU FSH (anterior follicle-stimulating hormone, manufactured by Kyoritsu Pharmaceutical Co., Ltd.) was applied to the ovarian cumulus cell complex aspirated from a follicle having a diameter of 2 to 6 mm in a sacrificial bovine ovary. In vitro maturation was carried out in the added M199 medium (manufactured by Sigma-Aldrich) for 22 hours in the air of 5% by volume CO 2 under the condition of 38.5 ° C.
Here, "maturation" means that the oocyte progresses to the metaphase of meiosis 2nd division where fertilization is possible.
一方で、京都大学で液体窒素中に凍結保存されているウシ***の入ったストローを３５℃の温湯中で融解し、保温したＩＶＦ１００溶液（株式会社機能性ペプチド研究所製）で融解した***を洗浄処理した。次に、***濃度を１×１０7個／ｍｌとなるようにＩＶＦ１００溶液で調整した。この***液５０μｌに等量のＩＶＦ１００溶液を加えて最終***濃度が５×１０6個／ｍｌとなるように調整し、この１００μｌの***液のドロップ中に、上述の体外成熟させた卵子卵丘細胞複合体を移し（この操作を「媒精」という）、体外受精を行った。体外受精開始後約６時間で受精が完了した。
体外受精胚は、９６ｗｅｌｌプレート（住友ベークライト株式会社製２００μｌ／ｗｅｌｌ、Ｕ底、ＰｒｉｍｅＳｕｒｆａｃｅ（登録商標））の各ｗｅｌｌに満たした２００μｌの体外培養液（胚培養培地）中で、５体積％ＣＯ2、５体積％Ｏ2、９０体積％Ｎ2、３８．５℃の低酸素条件下で体外培養を行った。媒精開始時を０日目とし、２日目に卵割率そして８日目に胚盤胞への発生率を算出した。
以下、実験３を胚培養培地に対するピルビン酸の添加の有無により下記の２つの実験に分けて説明する。
On the other hand, straws containing bovine sperm cryopreserved in liquid nitrogen at Kyoto University were thawed in warm water at 35 ° C, and the sperms thawed in a warm IVF100 solution (manufactured by Functional Peptide Research Institute Co., Ltd.) were melted. It was washed. Next, the sperm concentration was adjusted with IVF100 solution so as to be 1 × 10 7 pieces / ml. An equal amount of IVF100 solution was added to 50 μl of this sperm solution to adjust the final sperm concentration to 5 × 10 6 cells / ml, and the above-mentioned in vitro matured egg cumulus oophorus was added to the drop of 100 μl of sperm solution. The cell complex was transferred (this operation is called "medium fertilization") and in vitro fertilization was performed. Fertilization was completed about 6 hours after the start of in vitro fertilization.
The in vitro fertilized embryo is 5% by volume CO 2 in 200 μl of in vitro culture medium (embryo culture medium) filled in each well of a 96-well plate (200 μl / well manufactured by Sumitomo Bakelite Co., Ltd., U-bottom, Prime Surface (registered trademark)). In vitro culture was performed under low oxygen conditions of 5% by volume O 2 , 90% by volume N 2, and 38.5 ° C. The cleavage rate was calculated on the 2nd day and the blastocyst development rate on the 8th day, with the start of insemination as the 0th day.
Hereinafter, Experiment 3 will be described separately in the following two experiments depending on the presence or absence of addition of pyruvic acid to the embryo culture medium.
・実験３−１
ピルビン酸無添加の胚培養培地におけるｄｍ−αＫＧ濃度の胚培養に及ぼす効果を比較例６〜比較例８の培地を用いて調べた。ただし、比較例６〜比較例８の培地には、ＦＣＳに由来する微量（０．０１〜０．０３ｍＭ）のピルビン酸が含まれている。
・ Experiment 3-1
The effect of the dm-αKG concentration on the embryo culture in the embryo culture medium without addition of pyruvic acid was investigated using the media of Comparative Examples 6 to 8. However, the mediums of Comparative Examples 6 to 8 contain a trace amount (0.01 to 0.03 mM) of pyruvic acid derived from FCS.
・実験３−２
ピルビン酸（０．２ｍＭ）を含む胚培養培地におけるｄｍ−αＫＧの胚培養に及ぼす効果を実施例４、比較例９、及び比較例１０を用いて調べた。
・ Experiment 3-2
The effect of dm-αKG on embryo culture in an embryo culture medium containing pyruvic acid (0.2 mM) was investigated using Example 4, Comparative Example 9, and Comparative Example 10.
・結果
実験３（３−１，３−２）により得られた結果を表３に示す。
・実験３−１の結果
比較例６〜比較例８の卵割率（体外受精による供試卵数に対する卵割胚の割合）は、７８〜８０％であり、各比較例間でｄｍ−αＫＧの濃度が異なることによる有意差はなかった。また、当該供試卵から胚盤胞への発生率は、ｄｍ−αＫＧ無添加区の比較例６では、８．４％であり、殆どの胚は、８〜１６細胞期で発生を停止した。しかし、ｄｍ−αＫＧを１ｍＭ、あるいは４ｍＭを加えた比較例７、８の場合、それぞれの胚盤胞の発生率は２５．１％、２９．２％と有意に上昇した。
-Results of Experiment 3-1 The cleavage ratio (ratio of cleavage embryos to the number of test eggs by in vitro fertilization) of Comparative Examples 6 to 8 was 78 to 80%, and dm-αKG was observed between the comparative examples. There was no significant difference due to the different concentrations of. The incidence from the test egg to the blastocyst was 8.4% in Comparative Example 6 of the dm-αKG-free group, and most embryos stopped developing at the 8 to 16 cell stage. .. However, in the case of Comparative Examples 7 and 8 to which 1 mM or 4 mM of dm-αKG was added, the incidence of blastocysts increased significantly to 25.1% and 29.2%, respectively.
・実験３−２の結果
卵割率は、実験３−１の結果と同様の値であった。胚盤胞への発生率は、ｄｍ−αＫＧ無添加区（比較例９）では、３７．８％であったのに対し、４ｍＭのｄｍ−αＫＧを加えた実施例４では、５６．５％と有意に上昇した。しかし、ｄｍ−αＫＧを８ｍＭ加えた比較例１０では、３７．８％と、実施例４に比べて有意に低下した。
以上の結果から、マウス体外受精胚と同様に、ウシ体外受精胚においても、ピルビン酸の存在下においてｄｍ−αＫＧは、胚盤胞への発生を促進することが明らかとなった。
-Results of Experiment 3-2 The cleavage rate was the same as the result of Experiment 3-1. The incidence on blastocysts was 37.8% in the dm-αKG-free group (Comparative Example 9), whereas it was 56.5% in Example 4 to which 4 mM dm-αKG was added. And increased significantly. However, in Comparative Example 10 to which 8 mM of dm-αKG was added, it was 37.8%, which was significantly lower than that of Example 4.
From the above results, it was clarified that dm-αKG promotes the development into blastocysts in the presence of pyruvic acid in bovine in vitro fertilized embryos as well as in mouse in vitro fertilized embryos.
以上、本発明の胚培養培地は、上記の実施形態に限られず、例えば、ピルビン酸の添加濃度は、０．０１ｍＭ未満であってもよいし、０．２ｍＭを越えてもよい。ｄｍ−αＫＧの添加濃度は、８ｍＭ以上であってもよい。 As described above, the embryo culture medium of the present invention is not limited to the above-mentioned embodiment, and for example, the concentration of pyruvic acid added may be less than 0.01 mM or more than 0.2 mM. The concentration of dm-αKG added may be 8 mM or more.
Claims (3)
ピルビン酸と、ジメチルαケトグルタル酸とを含み、
ジメチルαケトグルタル酸の添加量が１ｍＭ以上８ｍＭ未満であり、
ピルビン酸の添加量が０．０１ｍＭ以上０．２ｍＭ以下である胚培養培地。 An embryo culture medium for culturing mammalian embryos including humans.
And pyruvic acid, and dimethyl α-ketoglutarate seen including,
The amount of dimethyl α-ketoglutaric acid added is 1 mM or more and less than 8 mM.
An embryo culture medium in which the amount of pyruvic acid added is 0.01 mM or more and 0.2 mM or less.
ピルビン酸と、ジメチルαケトグルタル酸とを添加した培地を用いて胚の培養を行い、
ジメチルαケトグルタル酸の添加量が１ｍＭ以上８ｍＭ未満であり、
ピルビン酸の添加量が０．０１ｍＭ以上０．２ｍＭ以下である胚培養方法。 An embryo culture method for culturing embryos for mammals other than humans.
There lines cultured embryo with a pyruvate, a medium supplemented with dimethyl α-ketoglutarate,
The amount of dimethyl α-ketoglutaric acid added is 1 mM or more and less than 8 mM.
An embryo culture method in which the amount of pyruvic acid added is 0.01 mM or more and 0.2 mM or less.
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