JP2020028248A - 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 pyruvic acid 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, research for approximating embryo development by in vitro culture to embryo development in the mother has been widely conducted, 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, the addition of human serum albumin to KSOMaa medium improves the incidence of mouse 1-cell stage embryos in blastocysts, and the implantation rate and litter rate of the blastocysts. 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。 In addition, in Non-Patent Document 2, the in vitro development of the mouse early embryo depends on pyruvate, and when pyruvate is not added to the medium, it can be complemented by dimethyl α-ketoglutarate that imparts cell membrane permeability. Have 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-ketoglutarate, that histone demethylase and TET (ten-eleven translocation) protein are indispensable for exerting the enzyme activity (Non-patent document 3), mouse embryonic stem cells (ES) cells When 4 mM is added to the culture medium, the demethylation of histone and DNA is promoted to promote self-renewal 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 an experiment on an ICR mouse performed by the present inventors, as described later, unlike the report of Non-Patent Document 2 on the FI (C57BL / 6JXC3HE) mouse, pyruvate-free mice were added, as described later. In the KSOM medium, the result that no development from the one-cell stage embryo to the blastocyst was observed even when dimethyl α-ketoglutarate was added, and even when dimethyl α-ketoglutarate was added to the medium, the early embryo development was observed. It is clear that some cases are not valid.
The present invention has been made in view of the above problems, and an embryo culture medium capable of efficiently generating embryos by dimethyl α-ketoglutaric acid acting more effectively on embryo development, and 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む胚培養培地である。 The invention made to solve the above problems is an embryo culture medium for culturing embryos of mammals including humans, which is an embryo culture medium containing pyruvic acid and dimethyl α-ketoglutarate.
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以下であることが好ましく、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、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。 In the embryo culture medium of the present invention, the amount of pyruvate added is preferably 0.01 mM or more and 0.2 mM or less, particularly preferably 0.02 mM or more and 0.2 mM or less. By doing so, embryos can be generated more efficiently. By adding 0.01 mM or more pyruvate to the embryo culture medium in the presence of dimethyl α-ketoglutarate, the embryo can be efficiently developed into blastocysts, and pyruvate of 0.02 mM or more can be obtained. The embryo can be more efficiently developed into a blastocyst by adding the compound.
また、本発明は、ヒトを含む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う胚培養方法を含む。 The present invention also relates to an embryo culture method for culturing embryos for mammals including humans, including a method for culturing an embryo using a medium to which pyruvate and dimethyl α-ketoglutarate are added. .
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, the embryo can be more efficiently generated by adding both pyruvate and dimethyl α-ketoglutarate.
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. In the following description, a medium to which pyruvic acid is added and a 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。
The embryo culture medium according to the present embodiment includes a basal medium to which pyruvic acid and dimethyl α-ketoglutarate (dm-αKG) are mainly added, and a support substance (hereinafter, referred to as “support”) that supports development in an in vitro culture of a pre-implantation embryo. , "Support material").
Here, the term “basal medium” refers to any medium that does not contain dm-αKG, regardless of whether pyruvate is added or not, including those that can culture mammalian embryos by themselves and those that cannot. Shall be considered.
「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。 “Mammal” is not particularly limited, and examples include human, mouse, cow, rabbit, rhesus monkey, pig, rat, and the like.
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 preferably a medium generally used as an in vitro development medium for pre-implantation embryos, but is not particularly limited, and another medium may be used.
Examples of the culture medium for the pre-implantation embryo include the culture medium for the mouse pre-implantation embryo, the culture medium for the rat / rabbit / hamster pre-implantation embryo, the culture medium for the bovine / porcine pre-implantation embryo, or the human development medium. In vitro developmental media for pre-bed 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 preimplantation embryos include KMOS medium, KMOSaa medium (KSOM medium with amino acid solution added), M16 medium, M12 medium, CZB medium, MTF medium, BWW medium, Whitten's medium , BMOC-III medium, and KMOS medium and KMOSaa medium are preferable.
The KSOM medium generally contains EDTA (ethylenediaminetetraacetic acid), but may be omitted because it is a non-physiological substance.
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 embryo 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 and swine preimplantation embryos include mSOF medium, mSOFaa medium, CR1aa medium, BECM medium, and PZM medium (PZM5 medium), with CR1aa medium being preferred.
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 Clearage medium, Complete Blastocyst medium, QA Blastocyst medium, Only One medium (manufactured by Nippon Medical Chemical Co., Ltd.), and Early culture medium (Japan Co., Ltd.). 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 pyruvate 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 efficiency of embryo development by pyruvate.
Further, it is particularly preferable that the concentration of pyruvic acid to be added is 0.02 mM or more and 0.2 mM or less. By doing so, the embryo development 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 embryo 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 support substance include recombinant human serum albumin, PVA (polyvinyl alcohol), PVP (polyvinyl pyrrolidone), deionized bovine serum albumin (dBSA), and FCS (Fetal calf serum (fetal calf serum)). Ionized bovine serum albumin, FCS is 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 an experiment using a medium according to an example 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。
[Culture experiment of mouse embryo]
In order to clarify the effect of dimethyl α-ketoglutarate (dm-αKG) on the development of mouse one-cell stage embryos, culture experiments were performed using the media shown in the following Examples and Comparative Examples in the following manner. .
(Example 1)
0.01 mM pyruvate was added to a KSOM (does not contain EDTA, pyruvate (containing Sigma-Aldrich))-P medium supplemented with 0.6% deionized bovine serum albumin (dBSA, manufactured by Sigma-Aldrich). This was used as a + P medium, and 1 mM of dm-αKG (manufactured by Tokyo Kasei Kogyo Co., Ltd.) was added to prepare Example 1. In Example 1 and Examples 2 and 3 described below, the -P medium is a 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 the amount of pyruvic acid was changed to 0.02 mM.
（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 changed to 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 without dm-αKG.
（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 containing 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 Examples 3 to 5)
(+ P) mediums without addition of dm-αKG and 0.01 mM, 0.02 mM, and 0.2 mM pyruvate were 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 medium of Examples 1 to 3 and Comparative Examples 1 to 5, 1-cell stage embryos collected from ICR mice (Kwl: ICR, manufactured by Kiwa Laboratory Animal Research Institute) at 37 ° C, 5 ° C. The cells were cultured in air at% volume CO 2 , and the number of embryos generated at the 2-cell stage embryo and the 4-cell stage embryo and the number of blastocysts after 96 hours were counted, and the incidence relative 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 the ICR mouse embryo, most of the embryo was 2 in the dm-αKG-free medium (Comparative Example 1) as in the report of Non-patent Document 2 using the FI mouse. It stopped developing at the cell stage and did not develop into blastocysts at all.
In addition, in the + P medium without dm-αKG (0.01 mM to 0.2 mM pyruvate was added, Comparative Examples 3 to 5), a one-cell stage embryo was generated at a significant ratio into blastocysts. Was completed. The ratio increased as the pyruvic acid concentration increased, and the highest ratio (78.5%) was obtained in Comparative Example 5 in which 0.2 mM pyruvic acid was added. The ratio of Comparative Example 5 was similar to the ratio in Non-Patent Document 2.
However, in the -P medium supplemented with 1 mM dm-αKG (Comparative Example 2), unlike the report of Non-patent Document 2 using the FI mouse embryo, the development to blastocysts was not improved at all, and dm It has been clarified that -αKG may not complement the effect 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 to which 1 mM dm-αKG was added (Examples 1 to 3), the rate of occurrence from the one-cell stage embryo to the blastocysts was + P medium without dm-αKG (Comparative Example 3). ~ Comparative Example 5), significantly increased (see the solid line and the broken line in FIG. 1).
From the above results, it has been clarified that dm-αKG greatly increases embryo development efficiency when added to a development medium in the presence of pyruvic acid. It is presumed that the mechanism of action different from that of pyruvate synergistically promotes the development of mouse early embryos into blastocysts.
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 broken line in FIG. 1, in the + P medium, the addition rate of pyruvic acid to 0.02 mM or more clearly shows that the incidence of blastocysts in 1-cell stage embryos sharply increases. 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 offspring development of mice, Experiment 2 was performed using the media shown in the above Examples and Comparative Examples in the following manner.
＜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 in the medium of Example 1 (+ P medium, dm-αKG added with 1 mM) and the medium of Comparative Example 5 (+ P medium, dm-αKG not added) were mixed with the blastocysts on day 4 after mating. A blastocyst (In vivo section) collected from the mouse uterus was implanted into the uterus of a mouse on day 2.5 of pseudopregnancy by a non-surgical method, and the implantation rate and litter rate on day 19.5 of gestation were determined. 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 of blastocysts generated in the medium of Example 1 (+ P medium and dm-αKG added) and the rate of occurrence in offspring were as shown in Table 2. The blastocysts were higher than those of the blastocysts in the medium (without addition of dm-αKG), and their values were similar to those of the blastocysts in In vivo.
Therefore, it is suggested that dm-αKG has an effect of increasing the developmental ability of a blastocyst generated in an in vitro culture to a litter to the ability of an embryo generated in a fallopian tube 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。 As described above, from the results of Experiments 1 and 2, regarding the effect of dm-αKG on early mouse embryos, the developmental arrest was released so far in the -P medium to increase the blastocyst development rate in the + P medium. Although it is only reported that it has an effect (Non-Patent Document 2), dm-αKG has the effect of promoting the development of blastocysts in early embryos and pups even in + P medium for the first time. 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。 In addition, it is suggested that culturing blastocysts having developmental (embryonic) similar to embryos occurring in fallopian tubes and uterus can be produced at a high rate by culturing + P medium early embryos supplemented with dm-αKG. Is 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。
[Cultivation experiment of bovine embryo]
Next, in order to clarify the effects of dm-αKG on the development of bovine in vitro fertilized embryos, Experiment 3 was performed using the media shown in the following Examples and Comparative Examples in the following manner.
（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 further adding 4 mM of dm-αKG to CR1aa medium to which 5% by volume of FCS (Fetal calf serum, fetal calf serum, GIBCO (registered trademark)) and 0.2 mM of pyruvate was added. Example 4 was used.
（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 of FCS was used 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 of dm-αKG were added to CR1aa medium supplemented with 5% by volume of FCS, to obtain mediums of Comparative Examples 7 and 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 of FCS and 0.2 mM of pyruvate 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 of dm-αKG to a CR1aa medium to which 5% by volume of FCS and 0.2 mM of pyruvate were added.
＜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分裂第２分裂中期まで進行させることをいう。
<Experiment 3>
Method The oocyte cumulus cell complex aspirated from a follicle having a diameter of 2 to 6 mm of a carcass bovine ovary was subjected to a maturation medium (10% by volume FCS and 0.02 AU FSH (frontal follicle stimulating hormone, manufactured by Kyoritsu Pharmaceutical Co., Ltd.)). In vitro maturation was performed in an added M199 medium (Sigma-Aldrich) for 22 hours at 38.5 ° C. in the air of 5% by volume CO 2 .
Here, “maturation” means that the oocyte is allowed to progress to the second metaphase of meiosis at which 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精子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）で融解した精子を洗浄処理した。次に、精子濃度を１×１０7個／ｍｌとなるようにＩＶＦ１００溶液で調整した。この精子液５０μｌに等量のＩＶＦ１００溶液を加えて最終精子濃度が５×１０6個／ｍｌとなるように調整し、この１００μｌの精子液のドロップ中に、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, a straw containing bovine sperm frozen and stored in liquid nitrogen at Kyoto University is thawed in hot water at 35 ° C., and the sperm thawed with a warmed IVF100 solution (manufactured by Functional Peptide Research Institute Co., Ltd.) It was washed. Next, the sperm concentration was adjusted with an IVF100 solution to 1 × 10 7 / ml. An equal volume of IVF100 solution was added to 50 μl of this sperm solution to adjust the final sperm concentration to 5 × 10 6 / ml, and during the drop of this 100 μl sperm solution, the above-mentioned extracorporeally matured egg cumulus The cell complex was transferred (this operation is referred to as “semen”), and in vitro fertilization was performed. Fertilization was completed about 6 hours after the start of in vitro fertilization.
In vitro fertilized embryos were prepared in a 200-well in vitro culture medium (embry culture medium) filled in each well of a 96-well plate (200 μl / well, U-bottom, PrimeSurface (registered trademark) manufactured by Sumitomo Bakelite Co., Ltd.), and 5% by volume CO 2. 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 start of insemination was defined as day 0, the cleavage rate on day 2 and the incidence on blastocysts on day 8 were calculated.
Hereinafter, Experiment 3 will be described by dividing it into the following two experiments depending on whether pyruvic acid is added 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 pyruvic acid was examined using the culture media of Comparative Examples 6 to 8. However, the medium of Comparative Examples 6 to 8 contains 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 pyruvate (0.2 mM) was examined 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 rate of Comparative Examples 6 to 8 (the ratio of cleavage embryos to the number of eggs tested by in vitro fertilization) was 78 to 80%, and dm-αKG was determined between Comparative Examples. There was no significant difference due to the different concentrations of In addition, the rate of occurrence from the test eggs to blastocysts was 8.4% in Comparative Example 6 in the dm-αKG-free group, and most embryos stopped developing at the 8 to 16 cell stage. . However, in 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%.
・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 value as the result of Experiment 3-1. The incidence of blastocysts in the dm-αKG-free group (Comparative Example 9) was 37.8%, whereas that in Example 4 to which 4 mM dm-αKG was added was 56.5%. And increased significantly. However, in Comparative Example 10 in which dm-αKG was added at 8 mM, the value was 37.8%, which was significantly lower than that in Example 4.
From the above results, it was revealed that dm-αKG promotes the development of blastocysts in the presence of pyruvate 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 embodiment. For example, the concentration of pyruvic acid added may be less than 0.01 mM or may exceed 0.2 mM. The addition concentration of dm-αKG may be 8 mM or more.
Claims (4)
ピルビン酸と、ジメチルαケトグルタル酸とを含む胚培養培地。 An embryo culture medium for culturing embryos of mammals including humans,
An embryo culture medium containing pyruvic acid and dimethyl α-ketoglutarate.
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う胚培養方法。 An embryo culture method for culturing embryos for mammals including humans,
An embryo culture method in which an embryo is cultured using a medium to which pyruvic acid and dimethyl α-ketoglutaric acid are added.
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