Kose M, Kaya M S, Aydilek N, Kucukaslan I, Bayril T, Bademkiran S, Kiyma Z, Ozyurtlu N, Kayis S A, Guzeloglu A, Atli M O
Faculty of Veterinary Medicine, Department of Obstetrics and Gynaecology, Dicle University, Diyarbakir, Turkey.
Faculty of Veterinary Medicine, Department of Physiology, Dicle University, Diyarbakir, Turkey.
Theriogenology. 2016 Apr 1;85(6):1161-6. doi: 10.1016/j.theriogenology.2015.11.032. Epub 2015 Dec 10.
Early and efficient detection of embryonic death (ED) has a valuable impact as important as early pregnancy diagnosis in ruminants. Among early pregnancy diagnosis methods, detection of the expression of interferon tau-stimulated genes (ISGs) in peripheral blood leukocytes (PBLs) is well documented in cows and ewes. Therefore, we hypothesized that the expression profile of ISGs in PBLs might also be useful for detecting ED in these animals. For this purpose, pregnant ewes were used as an experimental model. Pregnancy was detected on Day 18 after mating by transrectal ultrasonography. Pregnant ewes were divided into a control group (sham injection on Day 18, n = 10) and ED group (treated with 75 μg synthetic PGF2α on Day 18, n = 12). PBLs and plasma were collected on Days 0 (mating day), 15, 18, 19, 20, 21, 23, and 25 by jugular venipuncture. Total RNA was isolated from PBLs. ISGs expression levels were determined by real-time polymerase chain reaction in triplicate. Electrochemiluminescence immunoassay was used to measure progesterone (P4) levels in plasma. In the ED group, the P4 level declined to less than 1 ng/mL on Day 19 and remained at a low level until the end of the study. Compared with that on Day 0, receptor transporter protein 4 (RTP4) and ISG15 expression was upregulated on Day 15 and remained high until Day 21 in both groups, and RTP4 and ISG15 mRNA levels were attenuated on Days 23 and 25 only in the ED group (P < 0.001). Myxovirus resistance 1 expression was upregulated on Day 15 and remained high until Day 23 in both groups, but was attenuated on Day 25 in the ED group (P < 0.05). The B2-microglobulin mRNA level did not change significantly during the study in either group. These results indicate that the decline in P4 concentration was an immediate response to PGF2α and that the embryo may have survived longer than the CL on the basis of the extended period of ISGs expression. This suggests that the absence of P4 could be the reason for ED rather than a direct effect of PGF2α. In conclusion, the expression of ISGs, including ISG15, RTP4, and myxovirus resistance 1, but not B2-microglobulin, in PBLs may serve as a marker of ED.
早期且高效地检测胚胎死亡（ED）在反刍动物中具有与早期妊娠诊断同等重要的宝贵意义。在早期妊娠诊断方法中，检测外周血白细胞（PBLs）中干扰素τ刺激基因（ISGs）的表达在奶牛和母羊中已有充分记载。因此，我们推测PBLs中ISGs的表达谱也可能有助于检测这些动物的胚胎死亡。为此，使用怀孕母羊作为实验模型。在交配后第18天通过经直肠超声检查检测妊娠情况。怀孕母羊被分为对照组（第18天进行假注射，n = 1）和胚胎死亡组（第18天用75μg合成前列腺素F2α处理，n = 12）。在第0天（交配日）、15、18、19、20、21、23和25天通过颈静脉穿刺采集PBLs和血浆。从PBLs中分离总RNA。通过实时聚合酶链反应一式三份测定ISGs表达水平。采用电化学发光免疫分析法测定血浆中孕酮（P4）水平。在胚胎死亡组中，P4水平在第19天降至低于1ng/mL，并在研究结束前一直维持在低水平。与第0天相比，两组中受体转运蛋白4（RTP4）和ISG15在第15天表达上调，并在第21天前一直保持高水平，且仅在胚胎死亡组中，RTP4和ISG15 mRNA水平在第23天和25天减弱（P < 0.001）。两组中抗黏液病毒1在第15天表达上调，并在第23天前一直保持高水平，但在胚胎死亡组中第25天减弱（P < 0.05）。在研究期间，两组中β2 -微球蛋白mRNA水平均无显著变化。这些结果表明，P4浓度下降是对前列腺素F2α的即时反应，并且基于ISGs表达的延长时间，胚胎可能比黄体存活时间更长。这表明P4的缺乏可能是胚胎死亡的原因，而非前列腺素F2α的直接作用。总之，PBLs中包括ISG15、RTP4和抗黏液病毒1但不包括β2 -微球蛋白在内的ISGs表达可能作为胚胎死亡的标志物。
