Animal Production Research Centre Nitra, 95141 Luzianky, Nitra, Slovak Republic.
Anim Reprod Sci. 2010 May;119(1-2):130-6. doi: 10.1016/j.anireprosci.2009.12.008. Epub 2009 Dec 16.
The aim of the study was to determine a role of Hsp70 in the response of rabbit preimplantation embryos to hyperthermia (HT) in vitro. The embryos were cultured at standard (ST, 37.5 degrees C, control) or elevated (HT, 41.5 degrees C) temperature for 6h. In half of the embryos from both groups, Hsp70 was blocked by the addition of an antibody against Hsp70 into the medium at stages either prior to (<or= 8 cell) or after (morula) developmental block (DB). After 6h, the HT embryos were post-cultured at 37.5 degrees C for 16-20 h and then evaluated for developmental stages, apoptosis (TUNEL, T), cell number and quality of actin cytoskeleton. The exposure of embryos to 41.5 degrees C either before or after DB did not affect blastocysts (Bl) rate, T-index and embryo cell number. Blockage of Hsp70 before DB resulted in no embryos at the Bl stage. From the Hsp70-blocked embryos after DB, only 27.6% (ST + anti-Hsp70) and 22.6% (HT + anti-Hsp70) of blastocysts were formed, versus 71.4% (ST) and 69.7% (HT) in the non-Hsp70-blocked group. T-index of Hsp70-blocked embryos prior to DB increased from 6.8% (ST) to 28.6% (ST + anti-Hsp70) and from 6.1% (HT) to 29.6% (HT + anti-Hsp70), whilst the cell number dropped from 111 +/- 8.1 (ST) to 20.5 +/- 2.42 (ST + anti-Hsp70) and from 106.8 +/- 8.4 (HT) to 15.4 +/- 1.34 (HT + anti-Hsp70). In contrast, in the Hsp70-blocked embryos after DB, neither T-index nor cell number was changed comparing to the non-blocked. Damages to the actin cytoskeleton, like intracytoplasmic clumps or loss of actin filaments, were only minimally caused by hyperthermia but mostly were a result of Hsp70 blockage. In conclusion, the embryos at stages before the genome activation (GA) appear to be more sensitive to HT. The resistance of pre-GA embryos to HT is related to Hsp70, whilst after GA, Hsp70 is probably not the only factor of thermotolerance and the existence of an additional protective mechanism(s) is hypothesized.
本研究旨在确定热休克蛋白 70（Hsp70）在兔体外受精胚胎对高温（HT）反应中的作用。胚胎在标准（ST，37.5°C，对照）或升高（HT，41.5°C）温度下培养 6 小时。在两组胚胎的一半中，在发育阻滞（DB）之前（<或=8 细胞）或之后（桑葚胚）的阶段，通过在培养基中添加针对 Hsp70 的抗体来阻断 Hsp70。6 小时后，将 HT 胚胎在 37.5°C 下后培养 16-20 小时，然后评估发育阶段、细胞凋亡（TUNEL，T）、细胞数和肌动蛋白细胞骨架的质量。胚胎在 DB 之前或之后暴露于 41.5°C 不会影响囊胚（Bl）率、T 指数和胚胎细胞数。在 DB 之前阻断 Hsp70 会导致没有处于 Bl 阶段的胚胎。来自 DB 后阻断 Hsp70 的胚胎中，仅 27.6%（ST+抗-Hsp70）和 22.6%（HT+抗-Hsp70）形成囊胚，而非阻断 Hsp70 的组中为 71.4%（ST）和 69.7%（HT）。DB 前阻断 Hsp70 的胚胎的 T 指数从 6.8%（ST）增加到 28.6%（ST+抗-Hsp70）和从 6.1%（HT）增加到 29.6%（HT+抗-Hsp70），而细胞数从 111+/-8.1（ST）下降到 20.5+/-2.42（ST+抗-Hsp70）和从 106.8+/-8.4（HT）下降到 15.4+/-1.34（HT+抗-Hsp70）。相比之下，在 DB 后阻断 Hsp70 的胚胎中，与未阻断的胚胎相比，T 指数或细胞数没有变化。细胞内团块或肌动蛋白丝丢失等肌动蛋白细胞骨架损伤主要是由热休克引起的，而不是由 Hsp70 阻断引起的。总之，基因组激活（GA）前阶段的胚胎似乎对 HT 更敏感。GA 前胚胎对 HT 的抵抗力与 Hsp70 有关，而在 GA 后，Hsp70 可能不是耐热性的唯一因素，并假设存在额外的保护机制。
