Characterisation of seminal plasma-derived mediators involved in PMN evasion of ram spermatozoa in vitro

Seminal plasma (SP) is a complex immune modulator within the female reproductive tract of several species. In sheep, it can restore the fertility of cryopreserved sperm and is necessary for the fertility of epididymal sperm following cervical artificial insemination. We have also shown it capable of protecting cryopreserved sperm from polymorphonuclear leukocyte (PMN) binding in vitro . Thus, we hypothesize SP, and its components confer immune protection to sperm following deposition in the ovine cervix and that sperm cryopreservation disrupts this delicate sperm-female interaction. To delineate the contributions of soluble and extracellular vesicle (EV) associated SP components, frozen-thawed ram spermatozoa were incubated with whole SP, EV-depleted SP (DSP) and enriched EVs. All SP treatments significantly reduced binding (FTSP: 20.7 ± 3.39%; FTDSP: 24.3 ± 3.64%; FTEV: 19.8 ± 3.63%) compared to the control (36.3 ± 3.63%; p -5 ; 100 kDa). Two fractions, F3 (30 -5 0kDa) and F5 (>100kDa) reduced PMN binding (22.44 ± 2.18%, 21.67 ± 1.85%, respectively) compared to the control (FT: 58.83 ± 4.54%; p 0.05), indicating these fractions contain key immunoregulatory factors. Proteomic analysis identified 2,161 proteins in whole SP, with SP-EVs contributing 795 unique proteins. Among protective fractions (SP, DSP, EV, F3, F5), 588 proteins were shared, including a core signature of PITPNB, RPLP2, SLC9A3R1, PTTG1IP, CKAP4, and FKBP11, absent in non-protective fractions. Complement regulators (CD46, CD59, CFH, CLU) and glycocalyx-associated proteins (CRISPs, DEFB124) were enriched in protective fractions, supporting layered immunomodulation via both soluble and vesicular delivery. Functional analyses demonstrated that SP modulates PMN binding and activation while maintaining sperm viability, highlighting a structured, multi-layered immunoregulatory system. These findings provide the most comprehensive characterization of ram SP and SP-EVs to date, integrating functional assays with proteomic profiling, and reveal conserved protein signatures that underpin sperm immune tolerance. This work establishes a foundation for future studies targeting EV-mediated delivery and glycocalyx-associated immunoregulators to improve fertility outcomes and assisted reproductive technologies.