Successful reproduction requires sperm to traverse an immune-active female reproductive tract while avoiding clearance by innate leukocytes, particularly polymorphonuclear neutrophils (PMNs). In sheep, artificial insemination with frozen–thawed (FT) sperm results in markedly lower fertility than fresh semen, attributed in part to altered sperm surface properties and loss or dilution of seminal plasma (SP). SP contains a complex mixture of soluble proteins and extracellular vesicles (EVs) that can modulate neutrophil responses and preserve sperm. This study aimed to identify SP components and delivery modes that prevent PMN binding to ram sperm in vitro by combining functional PMN binding assays with comprehensive proteomic profiling.
Two complementary studies were performed. Study 1 compared the protective effect of whole SP with EV-depleted SP (DSP) and enriched EVs on FT ram sperm. Study 2 fractionated SP by molecular weight into five fractions (F1–F5: <10 kDa, 10–30 kDa, 30–50 kDa, 50–100 kDa, >100 kDa) to localize inhibitory activity. A pooled composite SP sample was generated from ejaculates collected from 24 mature Merino rams across multiple collections. Ejaculates were clarified by sequential centrifugation, pooled by matched ejaculate characteristics, aliquoted, and stored at −80 °C until use. For PMN binding assays, sperm were incubated with each SP treatment and assessed for PMN binding and sperm viability.
EVs were enriched from the SP pool using sequential centrifugation and ultracentrifugation, followed by a discontinuous OptiPrep density gradient to isolate visually enriched EV fractions. EV pellets were washed and resuspended in PBS for downstream assays. EV preparations were validated according to MISEV2023-aligned methods: nanoparticle tracking analysis determined particle size distribution and concentration, and transmission electron microscopy provided structural confirmation. The study reports that SP-derived EVs contributed 795 unique proteins to the total SP proteome, underscoring a substantial vesicular cargo.
To resolve functional mediators, SP was fractionated using sequential Amicon centrifugal filters to yield five molecular-weight fractions: F1 (<10 kDa), F2 (10–30 kDa), F3 (30–50 kDa), F4 (50–100 kDa), and F5 (>100 kDa). In PMN binding experiments, all SP preparations (whole SP, DSP, and EV preparations) significantly reduced PMN binding compared to the FT control: FT control binding measured 36.3 ± 3.63% versus FT+SP 20.7 ± 3.39%, FT+DSP 24.3 ± 3.64%, and FT+EV 19.8 ± 3.63% (p < 0.0001). Fractionation localized protective activity primarily to F3 (30–50 kDa) and F5 (>100 kDa), which reduced PMN binding to 22.44 ± 2.18% and 21.67 ± 1.85%, respectively, compared to the FT control value in that fractionation experiment (58.83 ± 4.54%; p < 0.0001). F3 and F5 effects were comparable to whole SP, indicating these molecular-weight windows contain key immunoregulatory factors.
Mass spectrometry analysis identified 2,161 proteins across whole SP. Comparative proteomics integrating data-dependent and data-independent acquisition approaches revealed that among the protective fractions (SP, DSP, EV, F3, F5), 588 proteins were shared. A conserved core signature present in protective but absent in non-protective fractions included PITPNB, RPLP2, SLC9A3R1, PTTG1IP, CKAP4, and FKBP11. Enrichment analysis flagged multiple complement regulators (CD46, CD59, CFH, CLU) and glycocalyx-associated proteins (including CRISPs and DEFB124) within protective fractions. These findings support a model of layered immunoregulation in which both soluble proteins and EV cargo deliver complementary factors to modulate PMN activity and preserve sperm.
Functional assays showed that SP treatments modulate PMN binding and activation while maintaining sperm viability, indicating that SP restores elements of the tolerogenic environment necessary for sperm survival in the female tract. The presence of complement regulators within protective fractions suggests suppression or modulation of complement-mediated opsonization as one mechanism. Glycocalyx-associated proteins and CRISP/defensin family members indicate roles in masking or stabilizing sperm surface antigens and in direct neutrophil modulation. The identification of substantial EV-unique cargo (795 proteins) implies EV-mediated delivery is an important route for transferring immunoregulatory molecules to sperm or interacting leukocytes.
This study provides an integrated functional and proteomic characterization of ram seminal plasma and seminal plasma EVs, identifying specific molecular-weight fractions (30–50 kDa and >100 kDa) and a conserved protein signature associated with protection from PMN binding. The results establish candidate targets—including complement regulators, glycocalyx-associated proteins, and EV cargo—that can be examined in future mechanistic studies. The authors note these data create a foundation for exploring EV-mediated delivery and glycocalyx-related immunoregulators as strategies to improve the fertility of cryopreserved sperm and assisted reproductive technologies in sheep and other species.