Ticianelli J S, Emanuelli I P, Satrapa R A, Castilho A C S, Loureiro B, Sudano M J, Fontes P K, Pinto R F P, Razza E M, Surjus R S, Sartori R, Assumpção M E O A, Visintin J A, Barros C M, Paula-Lopes F F
Department of Pharmacology, Institute of Biosciences, São Paulo State University (UNESP), 18618-970 Botucatu, Brazil.
Department of Animal Reproduction and Veterinary Radiology, School of Veterinary Medicine and Animal Science (FMVZ), São Paulo State University (UNESP), Prof. Dr. Walter Mauricio Correra Street, 18618-970 Botucatu, Brazil.
Reprod Fertil Dev. 2017 Sep;29(9):1787-1802. doi: 10.1071/RD16154.
The present study determined the transcriptome profile in Nelore and Holstein oocytes subjected to heat shock during IVM and the mRNA abundance of selected candidate genes in Nelore and Holstein heat-shocked oocytes and cumulus cells (CC). Holstein and Nelore cows were subjected to in vivo follicle aspiration. Cumulus-oocyte complexes were assigned to control (38.5°C, 22h) or heat shock (41°C for 12h, followed by 38.5°C for 10h) treatment during IVM. Denuded oocytes were subjected to bovine microarray analysis. Transcriptome analysis demonstrated 127, nine and six genes were differentially expressed between breed, temperature and the breed×temperature interaction respectively. Selected differentially expressed genes were evaluated by real-time polymerase chain reaction in oocytes and respective CC. The molecular motor kinesin family member 3A (KIF3A) was upregulated in Holstein oocytes, whereas the pro-apoptotic gene death-associated protein (DAP) and the membrane trafficking gene DENN/MADD domain containing 3 (DENND3) were downregulated in Holstein oocytes. Nelore CC showed increased transcript abundance for tight junction claudin 11 (CLDN11), whereas Holstein CC showed increased transcript abundance for antioxidant metallothionein 1E (MT1E) . Moreover, heat shock downregulated antioxidant MT1E mRNA expression in CC. In conclusion, oocyte transcriptome analysis indicated a strong difference between breeds involving organisation and cell death. In CC, both breed and temperature affected mRNA abundance, involving cellular organisation and oxidative stress.
本研究确定了在体外成熟（IVM）过程中遭受热休克的内洛尔牛和荷斯坦牛卵母细胞的转录组图谱，以及内洛尔牛和荷斯坦牛热休克卵母细胞和卵丘细胞（CC）中选定候选基因的mRNA丰度。对荷斯坦牛和内洛尔牛进行体内卵泡抽吸。在IVM期间，将卵丘-卵母细胞复合体分配至对照（38.5°C，22小时）或热休克（41°C处理12小时，随后38.5°C处理10小时）处理组。对裸卵进行牛微阵列分析。转录组分析表明，分别在品种、温度以及品种×温度交互作用之间有127个、9个和6个基因差异表达。通过实时聚合酶链反应对卵母细胞和相应的CC中选定的差异表达基因进行评估。分子运动蛋白驱动蛋白家族成员3A（KIF3A）在荷斯坦牛卵母细胞中上调，而促凋亡基因死亡相关蛋白（DAP）和膜转运基因含DENN/MADD结构域3（DENND3）在荷斯坦牛卵母细胞中下调。内洛尔牛CC中紧密连接蛋白claudin 11（CLDN11）的转录本丰度增加，而荷斯坦牛CC中抗氧化金属硫蛋白1E（MT1E）的转录本丰度增加。此外，热休克下调了CC中抗氧化剂MT1E的mRNA表达。总之，卵母细胞转录组分析表明品种之间在组织和细胞死亡方面存在显著差异。在CC中，品种和温度均影响mRNA丰度，涉及细胞组织和氧化应激。
