Effect of increased testicular temperature on seminal plasma proteome of the ram

The present study evaluated the effects of heat stress on the ram seminal plasma proteome. Six Morada Nova rams were scrotal insulated for 8 days. Scrotal circumference, sperm parameters, and seminal fluid proteins were evaluated before (Day 0) and twice during scrotal insulation (Days 4 and 8), and weekly until semen parameters returned to preinsulation values (normal). Seminal proteins were analyzed by two-dimensional SDS-PAGE and mass spectrometry. Scrotal circumference decreased from 30 ± 0.4 cm on Day 0 to 22.6 ± 0.6 cm on Day 36 (P < 0.05) and became equivalent to preinsulation values on Day 71. Motile sperm became nearly absent from Day 8 to Day 64 but returned to normal on Day 113. Percentage of normal sperm changed similarly and returned to normal on Day 106. Rams were azoospermic between Days 29 and 64, and sperm concentration came back to normal on Day 92. The number of spots/two-dimensional gel reduced from 256 ± 31 on Day 0 to 104 ± 14 on Day 29 (when rams were azoospermic) and then increased to 183 ± 9 on Day 113 (P < 0.05), similar to spot counts before insulation. The intensities of 24 spots, referring to 17 seminal plasma proteins, were affected by treatment (P < 0.05). After insulation, seminal plasma had greater expression of actin (two isoforms), albumin, heat shock protein 70 kDa, protein DJ-1, HRPE773-like, C-reactive protein precursor, bodhesin-2 (one isoform), spermadhesins. Most protein spots had the greatest intensity between Days 8 and 29, returning to preinsulation values on Day 113 (when many sperm criteria returned to normal). Proteins downregulated after scrotal insulation included dipeptidyl peptidase 3, isoforms of heat shock protein 90 kDa, RSVP22, MMP2 and of Bdh2. In this case, RSVP22 was reduced on Day 113 and all others, on Day 134. Expression of MMP2 and HSP90.1 was reduced throughout the study. Integrin β5, V-type H+-ATPase subunit A, ZBTB 42-like protein, isoforms of Bdh2, PSP-I, and RSVP22 were upregulated after testis insulation. Intensities of these spots were maximum (P < 0.05) 8 days after insulation started or on Day 29. Expression of most of such proteins returned to normal on Day 113. In conclusion, scrotal insulation affected testis and sperm parameters of rams, indicating alterations in both spermatogenesis and sperm maturation. Changes of seminal plasma proteome were coincidental with variations in semen parameters. Proteins affected by heat challenge are potentially involved in sperm protection, maturation, and fertilization.