(155) Transient Mild Scrotal Hyperthermia Affects Porcine Epididymal Extracellular Vesicles and Sperm

Supipi Mirihagalle, Kankanit Doungkamchan, David Miller

Department of Animal Sciences, College of Agricultural, Consumer and Environmental Sciences, University of Illinois at Urbana-Champaign

Abstract Text:

Elevated environmental temperatures reduce reproductive performance in mammals. In swine, the scrotum needs to be maintained 2-8°C below abdominal temperature for males to produce high-quality semen. The mechanisms of how scrotal hyperthermia affects sperm production are not clear. In preliminary experiments, insulated and control sacks were attached to the scrotum of 3 boar littermate pairs for 48 hr to elevate the scrotal skin temperature by 3°C and semen was collected before and 3 times/week after the insulation removal. Based on proteomic analysis of collected sperm, the abundance of 4 proteins (SPATA18, TOMM34, VDAC1, and GPX4) was increased 2-5 fold by scrotal hyperthermia compared to pre-insulation abundance as early as 6 days after insulation removal and before significant deterioration of sperm motility and morphology were observed. Sperm collected 6 days after insulation removal were in the epididymis during scrotal insulation, suggesting that epididymal maturation was affected by the scrotal hyperthermia but there are few studies on the heat sensitivity of the epididymis. Epididymal extracellular vesicles (EVs) fuse with sperm and add proteins as they move through the epididymis, allowing sperm to gain the ability to be motile and fertile. All 4 proteins that are elevated following hyperthermia are transmembrane proteins and may be secreted by the epididymis via EVs and added to sperm. Therefore, we hypothesized that the change in sperm protein profile following hyperthermia may be due to heat stress on the epididymis, affecting the secretion of EVs, causing alterations in EV protein content, or affecting the fusion ability of EVs to sperm. We collected caput and cauda epididymal fluid and isolated EVs by ultracentrifugation. The average diameter of the caput and cauda EVs was 133.1±47.3 nm and 105.3±40.5 nm and they contained the EV marker proteins CD81 and CD9. After characterizing the EVs, we performed western blots for EV and epididymal sperm protein lysates and found that all 4 proteins that were elevated following hyperthermia are present in EVs and sperm collected from both that caput and cauda epididymis. This suggests that those 4 proteins are added to the sperm via epididymal EVs. Quantitative analysis of the EV and sperm proteins showed no significant difference in the abundance of the 4 proteins in caput vs cauda epididymis. A mixture of caput and cauda EVs were labeled with FM4-64FX dye and incubated with caput sperm to observe EV-sperm fusion in-vitro which resulted in EV fusion to sperm head and mid-piece area. To understand the effects of scrotal hyperthermia on epididymal EVs and sperm, scrotal insulation experiments were carried out and epididymal fluid and sperm were collected at 3-days post insulation. Proteomics analysis showed differential expressions of proteins including upregulation of HSP90B1 in both EVs and sperm. Also, there was upregulation of CAVIN1 in epididymal fluid which suggests an increase in EV secretion in response to heat. Analysis of the effects of heat stress on sperm glycolysis and oxidative phosphorylation using a Seahorse FXe96 analyzer did not show significant effects on sperm metabolism. Future work will include proteomic and metabolomic analysis at additional times post insulation and analyzing the effects of hyperthermia on epididymal EV secretion and fusion with sperm to obtain a broad understanding of the effects of hyperthermia on epididymal EVs and sperm. This work was supported by USDA.