123 Effect of heat stress on oocyte developmental competence and global gene expression dynamics in Bos taurus crossbred beef cows

Abstract

It is known that animals under the effect of heat stress present reduced fertility. We aimed to investigate the effect of heat stress on the developmental competence and global gene expression profile of oocytes through the transition from spring to summer under Louisiana conditions. Oocytes were collected from 6 crossbred, non-lactating cows once a month from May to July. Temperature and humidity indexes for May and July were 72.48 and 78.06, respectively. An index above 75 indicates that cows are under heat stress. All cows underwent dominant follicle removal, and then 7 days later, ovum pickup was performed to aspirate germinal vesicle (GV) oocytes. Half of the oocytes were processed for RNA-seq as GV, and half were matured in vitro to metaphase II (MII). Smart-seq protocol was followed to prepare RNA sequencing libraries from a pool of 4 oocytes (GV n=6; MII n=6). Sequencing reads were pre-filtered and aligned to the bovine genome, and gene expression values were calculated as transcripts per million. Genes were deemed differentially expressed between different conditions if they showed a false discovery rate P-value<0.05 using DESEqn 2 package. DAVID (https://david.ncifcrf.gov) and IPA (ingenuity pathway analysis) were used to reveal the gene ontology and pathways, respectively. The RNA sequencing showed that a total of 212 genes were differentially expressed as a result of heat stress at the GV stage, with 94 and 118 genes up- and down-regulated, respectively. Gene ontology analysis indicated significant over-representation of elements involved in steroid biosynthetic process, oxidation reduction, and mitophagy in response to mitochondrial depolarization. Several pathways were influenced by heat stress, including glucocorticoid biosynthesis, apoptosis signalling, and HIPPO signalling. At the MII oocyte stage, only 93 genes (19 up-regulated and 74 down-regulated) were significantly differentially expressed in oocytes between July and May groups. Oocytes retrieved on different collection days, from the same cows under the same treatments, showed no difference on maturation rates, suggesting that the in vitro maturation process equalizes the expression of several genes. The primary biological processes significantly affected in MII oocytes were regulation of MAPK cascade, melanosome organisation, and negative regulation of transcription. In addition, we found that UBE2I, a gene involved in phosphorylation-dependent sumoylation of heat shock factor 1 (HSF1), was significantly up-regulated in July compared with May in MII oocytes. Interestingly, only 5 common genes were significantly affected by heat in both GV and MII oocytes: E2F8, GATAD2B, BHLHE41, FBXO44, and RAB39B. Our findings provide new insight into the molecular mechanisms of detrimental conditions (heat stress) on bovine oocytes, which may help to reveal master regulators controlling oocyte competence.