体外受精中の短期的な温度上昇は胚の収量に影響を与え、ウシの卵母細胞、積体細胞、胚盤胞の遺伝子発現パターンを変化させる

Short term temperature elevation during IVM affects embryo yield and alters gene expression pattern in oocytes, cumulus cells and blastocysts in cattle.

• Konstantina Stamperna
• Themistoklis Giannoulis
• Ioannis Nanas
• Maria Kalemkeridou
• Katerina Dadouli
• Katerina Moutou
• Georgios S Amiridis
• Eleni Dovolou

抄録

熱ストレスは牛の不妊の原因となり、ステロイド産生能、卵胞機能、排卵障害の変化を引き起こし、最終的には卵子の質と胚の生存に悪影響を及ぼす。ここでは、体外受精中の短時間の中等度の温度上昇が胚の収量および様々な遺伝子の発現に及ぼす影響を評価した。8回のレプリケートにおいて、積乱卵母細胞複合体（COC）を、IVMの2時間目から8時間目まで39℃で24時間（対照n=605）、または41℃で24時間熟成させ（処置、n=912）、受精させ、推定接合体を39℃で9日間培養した。開裂率と胚形成率は、受精後48時間目と7、8、9日目にそれぞれ評価した。また、5 レプリケートの積雲細胞、卵母細胞、胚盤胞をスナップ凍結し、代謝、熱・酸化ストレス応答、アポトーシス、胎盤形成に関連する遺伝子の相対発現解析を行った。処理群では、開裂率と胚形成率が対照群に比べて統計的に有意に低かった（開裂率86.7％対74.2％、胚盤胞：7日目29.9％対19.7％、8日目34.2％対22.9％、9日目35.9％対24.5％）。積算細胞の3遺伝子（HSP90AA1、CPT1B、G6PD）と胚盤胞の3遺伝子（DNMT3A、PLAC8、GPX1）の相対的なmRNAの発現量は、群間で有意に異なることを示した（p<0.05）、卵母細胞のG6PD、SOD2、GXP1、胚盤胞のPTGS2の発現は群間で異なる傾向があった（0.05<p<0.08）。熱ストレスは、卵母細胞（G6PD、GPX1、CCNB1）、積層細胞（LDH、CCNB1）、胚盤胞（AKR1B1、PLAC8）のHSPと他の遺伝子の発現の相関を変化させた（p<0.05）。これらの結果は、わずか 6 時間でも卵母細胞を高温に曝露すると、卵母細胞の発達能力が低下し、胚盤胞の収量が抑制され、遺伝子発現の協調パターンが大きく変化することを示唆しています。

Heat stress causes subfertility in cattle by inducing alterations in steroidogenic capacity, follicular function and ovulation defects, which eventually negatively affect oocyte quality and embryo survival. Here, the effects of short, moderate temperature elevation during IVM, on embryo yield, and on the expression of various genes was evaluated. In 8 replicates, cumulus oocyte complexes (COCs) were matured for 24 h at 39 °C (controls n = 605) or at 41 °C from hour 2 to hour 8 of IVM (treated, n = 912), fertilized, and presumptive zygotes were cultured for 9 days at 39 °C. Cleavage and embryo formation rates were evaluated 48 h post insemination and on days 7, 8, 9 respectively. Cumulus cells, oocytes and blastocysts from 5 replicates were snap frozen for the relative expression analysis of genes related to metabolism, thermal and oxidative stress response, apoptosis, and placentation. In treated group, cleavage and embryo formation rates were statistically significantly lower compared with the control (cleavage 86.7% vs 74.2%; blastocysts: day 7, 29.9% vs 19.7%, day 8, 34.2% vs 22.9% and day 9 35.9% vs 24.5%). Relative mRNA abundance of three genes in cumulus cells (HSP90AA1, CPT1B, G6PD) and three genes in blastocysts (DNMT3A, PLAC8, GPX1) indicated significantly different expression between groups (p < 0.05)., The expression of G6PD, SOD2, GXP1 in oocytes and PTGS2 in blastocysts tended to differ among groups (0.05<p < 0.08). Heat stress altered (p < 0.05) the correlation of expression between HSPs and other genes in oocytes (G6PD, GPX1, CCNB1), cumulus cells (LDH, CCNB1) and blastocysts (AKR1B1, PLAC8). These results imply that exposure of oocytes to elevated temperature, even for only 6 h, disrupts the developmental competence of the oocytes, suppresses blastocyst yield and significantly alters the coordinated pattern of gene expressions.

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