186 A step toward understanding direct impacts of a higher estrus-associated temperature: transcript-level changes in cumulus–oocyte complexes directly exposed to acute elevated temperature

A higher estrus-associated temperature (HEAT) is a hallmark feature in the sexually receptive female bovid. In some cases, body temperature may reach 41.0°C and persist for a few hours. To determine the extent to which an acute elevated temperature directly affects the cumulus–oocyte complex (COC) soon after it has resumed meiosis, the abundance of 47 transcripts whose gene products are known to be involved in steroidogenesis or oocyte maturation or affected by elevated temperature was evaluated. On six different collection dates, COCs underwent IVM for 2, 4, 6, 10, or 24 h (hIVM) at 38.5°C. Subsets of other COCs were directly exposed to 41°C for 2, 4, or 6 hIVM or were exposed to 41°C for 6 hIVM before transfer to 38.5°C for an additional 4 h (10 hIVM total) or 18 h (24 hIVM total). All COCs, kept separate by treatment origin, were removed from culture and washed before lysis in extraction buffer. Lysates were stored at −80°C until RNA isolation. Primer sets confirmed to span exon-exon junction were used. Targeted RNA sequencing was performed using an Illumina MiSeq device with a 2 × 150 configuration using pools of COCs (n = 35 COCs for each temperature and time combination). With this 2 × 5 factorial treatment arrangement, maturation temperature, maturation time, and the respective interaction were included in the statistical model with COC collection date as a random effect. Transcript abundance for the majority evaluated (43 out of 47 total) differed depending on hIVM and IVM temperature (hIVM × IVM temperature interaction; P < 0.05). Most of the 47 transcripts (34, 72.3%) were affected by an acute exposure to 41°C, with effects noted as early as 2 hIVM for 19 and by 4 hIVM for the remaining 15. During these first few hours of IVM, an elevated temperature had direct effects on the abundance of transcripts with described roles in progesterone production or signaling, cellular signaling, the β-catenin complex, the cell cycle, and cumulus expansion. Temperature impacts were also noted on Src family kinase transcripts and others with less defined roles in the COC but have been shown to be affected by heat in cumulus cells, oocytes, or in follicular fluid. Use of multidimensional scaling plots showed that samples originating from COCs directly exposed to 41°C for 4 hIVM overlapped with those matured at 38.5°C for 6 hIVM. Study outcomes document direct effects of a physiologically relevant elevated temperature on transcripts in the COC. These efforts represent important steps toward providing novel insight into the dynamic physiology of the COC in estrual female bovids, both during a short period of HEAT and after return to baseline.

This work was supported by Agriculture and Food Research Initiative Competitive Grant no. 2022-67015-36374 from the USDA National Institute of Food and Agriculture, UT Institute of Agriculture, AgResearch and the Genomics Center for the Advancement of Agriculture, the Department of Animal Science, and the USDA National Institute of Food and Agriculture, Multistate Project No. NE2227.