145  Functional genomics uncovers the impact of heat stress in a mouse model of oocyte growth

Heat stress (HS) is an inability to thermoregulate under exposure to elevated temperature. To explore the impact of HS on oogenesis, we developed a novel in vivo model that exposes mice to HS during the first wave of oocyte growth. Here, we describe the impact of such HS on the transcriptional landscape of fully grown mouse oocytes (FGO). Swiss mice (EPM2 substrain, UNIFESP, Brazil) at 6–8 weeks were randomly mated and placed in individual cages before delivery. Lactating females with litters were randomly allocated to control (CTL; 21°C/24 h) or HS (35°C/12 h/light and 21°C/12 h/dark; 60% relative humidity) from postnatal day 10 (P10) until weaning at P21. The HS was carried out in an automated environmental chamber (Alesco, Brazil) inside the rodent facility. Weaned HS and CTL females were kept at 21°C until puberty at P35. Females were subjected to intraperitoneal injection of 10 IU PMSG and killed 46 h post-injection at P35–42 to collect oocytes. Pools of 200 FGO were subject to total RNA extraction and RNA-sequencing (RNA-seq). RNA libraries were prepared with the Zymo-SEqn 3′ mRNA Library Kit. Initial quality control of sequencing results was performed with FastQC (v0.11.9). Sequencing errors were identified with UMI-tools (v1.1.1). Initial sequencing analysis was conducted with Trim Galore! (v0.6.6), and mapping to the mouse genome (GRCm38) was performed with STAR (v2.6.1d). Processing read alignments was carried out with SAMtools (v1.9). The estimation of the complexity of genomic sequencing libraries was performed with Preseq (v2.0.3). The quality control of alignment sequencing data was done with RSeQC (v4.0.0) and Qualimap (v2.2.2-dev). Reads were summarised by featureCounts (v2.0.1). The differential expression was determined by DEsEqn 2 (v1.28.0). Differently expressed genes (DEG; log₂ fold change cutoff of 0.585) were enriched by Gene Ontology (GO) and KEGG pathway analysis. The functional enrichment was done with g:Profiler (FDR cutoff 0.05). A total of 16 379 and 20 664 unique transcripts were mapped in CTL and HS groups, respectively. A total of 85 transcripts were differentially expressed between groups, of which 41 were up-regulated and 44 down-regulated under HS. The up-regulated genes in HS were mostly ribosome transcripts, and components of inhibin and oestradiol pathways. A published single-cell RNA-seq dataset of mouse oocytes revealed that up-regulated ribosomal transcripts (8/13; 53.33%) are enriched in non-growing and early growing oocytes, thus suggesting an accumulation at the FGO stage due to translation blockade. The down-regulated genes in HS did not enrich for specific pathways (or biological process) and may be due to the nonsense-mediated RNA decay (predicted by g:Profiler) or stochasticity. These results demonstrate the potential of mice as models of HS and further reveal novel susceptibility gene transcripts in FGO exposed to HS.