Kojima T, Udagawa K, Onishi A, Iwahashi H, Komatsu Y
Department of Animal Reproduction, National Institute of Animal Industry, Ibaraki, Japan.
Mol Reprod Dev. 1996 Apr;43(4):452-7. doi: 10.1002/(SICI)1098-2795(199604)43:4<452::AID-MRD7>3.0.CO;2-O.
The present study was conducted (1) to examine the effect of an acute increase in ambient temperature on the development of porcine day 6 embryos in culture and after transfer to recipient gilts, and (2) to analyze intracellular production of heat shock proteins (hsps). The viability of porcine day 6 embryos following a temporary acute elevation in ambient temperature (at 42 degrees-45.5 degrees C and for 10-180 min) was examined. Synthesis of 70 kDa hsp (hsp70) and 90 kDa hsp (hsp90) was determined by SDS-PAGE and Western blot analysis in porcine day 6 embryos subjected to heat stresses. Nonheat-stressed embryos were considered as control. Significantly higher numbers of viable nuclei were observed in treatment groups of 42 degrees C-10 min (236.6 +/- 71.4; P < 0.05) and 43 degrees C-30 min (276.8 +/- 89.4; P < 0.005) compared to control (173.9 +/- 53.9). The 42 degrees C-180 min group (158.0 +/- 27.1 microns) had a greater increase in diameter after 24 hr in culture following heat stress compared to control (82.5 +/- 47.3 microns), while heat stress with 43 degrees C for > or = 60 min, 44 degrees-44.5 degrees C for > or = 30 min, or 45 degrees-45.5 degrees C for > or = 10 min impaired their survival, as assessed by differences in number of viable nuclei. The embryos subjected to heat stresses under the conditions of 42 degrees C-180 min, 43 degrees C-10 min, 43 degrees C-30 min, 44 degrees C-10 min, or 45 degrees C-10 min developed to normal piglets after transfer to recipient gilts. Overall pregnancy rate was 75% (6/8), and farrowing rate 62.5% (5/8). Of heat-stressed embryos transferred, 59% (36/61) developed to normal piglets. Heat-stress conditions of 42 degrees C for 180 min, 43 degrees C for 30 min, 44 degrees C for 10 min, and 45 degrees C for 10 min were determined as critical with respect to the in vitro and in vivo survival of porcine embryos. Porcine day 6 embryos constitutively synthesized hsp70 even without heat stress, while hsp90 was detected only at trace level. Neither hsp70 nor hsp90 levels increased in the embryos subjected to heat stresses. In conclusion, porcine day 6 embryos could continue to develop in vivo or during in vitro culture after exposure to acute and temporary rise in temperature. However, no increase of hsp70 and hsp90 was observed in the heat-stressed porcine embryos, while hsp70 was detected in the nonheat-stressed porcine embryos. The precise mechanism of the thermotolerance was unclear.
（1）检测环境温度急性升高对体外培养及移植到受体母猪后的第6天猪胚胎发育的影响；（2）分析热休克蛋白（hsps）的细胞内产生情况。检测了环境温度暂时急性升高（42℃ - 45.5℃，持续10 - 180分钟）后第6天猪胚胎的活力。通过SDS - PAGE和蛋白质免疫印迹分析，测定了热应激处理的第6天猪胚胎中70 kDa热休克蛋白（hsp70）和90 kDa热休克蛋白（hsp90）的合成情况。未受热应激的胚胎作为对照。与对照组（173.9±53.9）相比，42℃ - 10分钟处理组（236.6±71.4；P < 0.05）和43℃ - 30分钟处理组（276.8±89.4；P < 0.005）观察到存活细胞核数量显著更多。热应激后在培养24小时后，42℃ - 180分钟组（158.0±27.1微米）的直径比对照组（82.5±47.3微米）有更大增加，而43℃持续≥60分钟、44℃ - 44.5℃持续≥30分钟或45℃ - 45.5℃持续≥10分钟的热应激损害了胚胎的存活，这通过存活细胞核数量的差异来评估。在42℃ - 180分钟、43℃ - 10分钟、43℃ - 30分钟、44℃ - 10分钟或45℃ - 10分钟条件下受热应激的胚胎移植到受体母猪后发育成了正常仔猪。总体妊娠率为75%（6/8），产仔率为62.5%（5/8）。移植的受热应激胚胎中，59%（36/61）发育成了正常仔猪。就猪胚胎的体外和体内存活而言，确定42℃持续180分钟、43℃持续30分钟、44℃持续10分钟和45℃持续10分钟的热应激条件是临界的。第6天猪胚胎即使在无热应激时也组成性合成hsp70，而hsp90仅在痕量水平被检测到。受热应激的胚胎中hsp70和hsp90水平均未增加。总之，第6天猪胚胎在暴露于急性和暂时的温度升高后能够在体内或体外培养期间继续发育。然而，在受热应激的猪胚胎中未观察到hsp70和hsp90增加，而在未受热应激的猪胚胎中检测到了hsp70。热耐受性的确切机制尚不清楚。
