Tseng J K, Tang P C, Ju J C
Department of Animal Science, National Chung Hsing University, Taichung, Taiwan, ROC.
Theriogenology. 2006 Sep 15;66(5):1073-82. doi: 10.1016/j.theriogenology.2006.03.003. Epub 2006 Apr 19.
The precise physiological causes that result in reduced development of oocytes after heat shock (HS) are not clear. In this study, apoptosis, heat shock protein70 (hsp70), and in vitro development of porcine oocytes were evaluated after HS. Porcine cumulus-oocyte complexes (COCs) were subjected to in vitro maturation for 42 h. The matured oocytes were then heated at 41.5 degrees C for 0 h (control, C0h), 1 h (HS1h), 2 h (HS2h), or 4 h (HS4h). An additional group of oocytes was cultured for 4 h without HS (control, C4h). In Experiment 1, expression of hsp70 was detected by Western-blotting and no difference between controls and HS groups was observed. In Experiment 2, apoptosis of matured oocytes after HS was examined by Annexin V-FITC and TUNEL. No significant TUNEL-positive signals were detected in the heated oocytes compared to the controls, but the intensity of Annexin V-FITC labeling among different groups increased with length of HS and in vitro culture (P<0.05). Oocytes were parthenogenetically activated by an electric pulse plus 6-DMAP (Experiment 3). Mean (+/-S.E.M.) embryonic development in HS2h (cleavage: 42+/-29%; blastocyst: 11+/-10%) and HS4h (cleavage: 36+/-28%; blastocyst: 11+/-8%) were decreased when compared to those in C0h (cleavage: 63+/-12%; blastocyst: 24+/-14%) and C4h (cleavage: 66+/-8%; blastocyst: 21+/-11%). Numbers of blastocysts with TUNEL-positive signals were similar among groups, but the signals increased before the eight-cell stage in HS groups (P<0.05). In conclusion, developmental competence of matured pig oocytes was compromised after heat shock, but it was not closely associated with the expression of oocyte hsp70. However, there may be a link between apoptosis and developmental competence of porcine oocytes.
热休克（HS）后导致卵母细胞发育减少的确切生理原因尚不清楚。在本研究中，对热休克后猪卵母细胞的凋亡、热休克蛋白70（hsp70）和体外发育情况进行了评估。猪卵丘-卵母细胞复合体（COCs）进行体外成熟培养42小时。然后将成熟卵母细胞在41.5℃下加热0小时（对照组，C0h）、1小时（HS1h）、2小时（HS2h）或4小时（HS4h）。另一组卵母细胞在无热休克条件下培养4小时（对照组，C4h）。在实验1中，通过蛋白质免疫印迹法检测hsp70的表达，未观察到对照组和热休克组之间存在差异。在实验2中，通过膜联蛋白V-异硫氰酸荧光素（Annexin V-FITC）和脱氧核糖核苷酸末端转移酶介导的缺口末端标记法（TUNEL）检测热休克后成熟卵母细胞的凋亡情况。与对照组相比，在加热的卵母细胞中未检测到显著的TUNEL阳性信号，但不同组间膜联蛋白V-异硫氰酸荧光素标记强度随热休克时间和体外培养时间的延长而增加（P<0.05）。通过电脉冲加6-二甲基氨基嘌呤（6-DMAP）对卵母细胞进行孤雌激活（实验3）。与C0h组（卵裂率：63±12%；囊胚率：24±14%）和C4h组（卵裂率：66±8%；囊胚率：21±11%）相比，HS2h组（卵裂率：42±29%；囊胚率：11±10%）和HS4h组（卵裂率：36±28%；囊胚率：11±8%）的平均（±标准误）胚胎发育率降低。各组中具有TUNEL阳性信号的囊胚数量相似，但热休克组在八细胞期之前信号增加（P<0.05）。总之，热休克后成熟猪卵母细胞的发育能力受损，但与卵母细胞hsp70的表达没有密切关系。然而，猪卵母细胞的凋亡与发育能力之间可能存在联系。
