Presence and distribution pattern(s) of sperm protein at 22 kDa (SP22) on ejaculated and caudal epididymal equine spermatozoa prior to and following heat-induced testicular degeneration

Abstract

SP22 is highly correlated with fertility in rats (Klinfelter et al., J Androl 1997;18:139-150) and has been identified on equine spermatozoa (Miller et al., Biol Reprod 2003;68:166-167). Environmental damage to spermatozoa affects localization pattern(s) of SP22, but it is unknown if testicular degeneration (TD) alters expression of SP22. We hypothesized that expression and localization of SP22 on ejaculated and epididymal spermatozoa are altered by heat induced TD. To test this hypothesis, a total of three ejaculates were collected every other day from each of five stallions. Eighty-four days following hemi-castration to recover epididymal sperm, the remaining testis was insulated for 14 days. The insulation was achieved using diapers, cotton, and elastikon in a sling-like fashion. Semen was collected on days 6, 8, 10, and 13 of insulation. The remaining testes were removed on day 14, and epididymal spermatozoa were recovered. Ejaculates were evaluated by spectrophotometer and computer assisted semen analyzer (Sperm Vision, Minitube of America, Verona, WI). The cauda epididymides were flushed with air to collect spermatozoa. The samples underwent centrifugation (600xg), before resuspension in 1X PBS. Ejaculated and epididymal spermatozoa were prepared for Western blot analysis and immunocytochemistry (ICC). Tissues from the castrated testes (preand post-insulation) were sectioned, placed in Zamboni’s fixative, embedded, and stained with hematoxylin and eosin. SP22 staining patterns were determined on 100 spermatozoa from each