---
title: "Seminal plasma and EV mediators that prevent PMN binding to ram sperm: proteomic and functional ch"
id: "frontiers-in-immunology-11-characterisation-of-seminal-plasma-derived-mediators-involved-in-pmn-evasion-of"
canonical_url: "https://medichelpline.com/clinical-feed/frontiers-in-immunology-11-characterisation-of-seminal-plasma-derived-mediators-involved-in-pmn-evasion-of"
content_type: "clinical_feed_article"
specialty: "Infectious Disease"
source_name: "Frontiers in Immunology"
source_url: "https://www.frontiersin.org/articles/10.3389/fimmu.2026.1891264"
published_at: "2026-09-03T00:00:00.000Z"
evidence_level: "Journal Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# Seminal plasma and EV mediators that prevent PMN binding to ram sperm: proteomic and functional ch
## Provenance & Clinical Metadata
- **Canonical URL:** https://medichelpline.com/clinical-feed/frontiers-in-immunology-11-characterisation-of-seminal-plasma-derived-mediators-involved-in-pmn-evasion-of
- **Specialty:** [Infectious Disease](https://medichelpline.com/clinical-feed/infectious-disease.md)
- **Primary Source:** Frontiers in Immunology
- **Source URL:** [Original Journal Publication](https://www.frontiersin.org/articles/10.3389/fimmu.2026.1891264)
- **Published At:** 2026-09-03T00:00:00.000Z
- **Evidence Rating:** Journal Feed
## Executive GIST (TL;DR)
- This study examined how **seminal plasma (SP)** and its components protect frozen–thawed (FT) ram spermatozoa from polymorphonuclear neutrophil (**PMN**) binding in vitro, comparing whole SP, EV-depleted SP (DSP), enriched extracellular vesicles (EVs), and five molecular-weight fractions (F1–F5). - All SP preparations (SP, DSP, EV) significantly reduced PMN binding relative to FT control (FT: 36.3 ± 3.63%; FT+SP: 20.7 ± 3.39%; FT+DSP: 24.3 ± 3.64%; FT+EV: 19.8 ± 3.63%; p 100 kDa), which reduced PMN binding to 22.44 ± 2.18% and 21.67 ± 1.85%, respectively, compared to FT control (58.83 ± 4.54% in that assay; p < 0.0001) and were comparable to whole SP. - Mass spectrometry identified 2,161 proteins in whole SP; SP-derived EVs contributed 795 unique proteins. Among protective fractions (SP, DSP, EV, F3, F5), 588 proteins were common. - A conserved core protein signature present in protective fractions and absent in non-protective fractions included PITPNB, RPLP2, SLC9A3R1, PTTG1IP, CKAP4, and FKBP11. - Protective fractions were enriched for **complement regulators** (CD46, CD59, CFH, CLU) and **glycocalyx-associated proteins** (CRISPs, DEFB124), suggesting layered immunoregulation via soluble proteins and EV cargo. - Functional assays showed SP modulates PMN binding and activation while maintaining sperm viability, supporting a structured multi-layered immunoregulatory system in ram SP. - The study provides the most comprehensive proteomic characterization of ram SP and SP-EVs to date and identifies candidate mediators and delivery modes (soluble vs EV) for future work to improve post-thaw fertility and ART outcomes.
## Clinical Analysis & Structured Key Points
Frontiers | Characterisation of seminal plasma-derived mediators involved in PMN evasion of ram spermatozoa in vitro 100 kDa). Two fractions, F3 (30-50kDa) 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."> ORIGINAL RESEARCH article Front. Immunol. , 03 September 2026 Sec. Molecular Innate Immunity Volume 17 - 2026 | https://doi.org/10.3389/fimmu.2026.1891264 Published in Frontiers in Immunology Molecular Innate Immunity 7 impact factor 11.3 citescore Editor & Reviewers Edited by U K Uday Kishore Reviewed by M C Madalitso Chelenga R Z Renzheng Zhang Outline Figures and Tables Figure 1 View in article Figure 2 View in article Figure 3 View in article Figure 4 View in article Figure 5 View in article Figure 6 View in article Table 1 Summary of the top 10 most abundant proteins quantified across SP, DSP, EV, and isolated fractions; 100 kDa, using data-independent acquisition (DIA) proteomics. View in article ORIGINAL RESEARCH article Front. Immunol. , 03 September 2026 Sec. Molecular Innate Immunity Volume 17 - 2026 | https://doi.org/10.3389/fimmu.2026.1891264 Characterisation of seminal plasma-derived mediators involved in PMN evasion of ram spermatozoa in vitro S W Sophie Warr 1 * L M Lydia Mikhael 1 E T Elizabeth Torres-Arce 2,3 J S John Schjenken 2,3 T P Taylor Pini 4 S P Simon P. de Graaf 1 J P Jessica P. Rickard 1 1. School of Life and Environmental Sciences, Faculty of Science, The University of Sydney, Sydney, NSW, Australia 2. Centre for Reproductive Science, School of Science, College of Engineering, Science and Environment, University of Newcastle, University Drive, Callaghan, NSW, Australia 3. Reproductive and Family Health Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW, Australia 4. School of Veterinary Science, Faculty of Science, The University of Queensland, Gatton, QLD, Australia See more Article metrics View details Abstract 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 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. Introduction Reproductive success relies on the ability of spermatozoa to traverse the immunologically complex female reproductive tract (FRT), negotiating anatomical barriers, mucosal secretions, and active immune surveillance ( 1 ). During natural mating, sperm are remarkably equipped to meet this challenge: the female immune system balances effective pathogen defense with permissiveness toward paternal gametes, creating a transient state of immune tolerance. How this selective quiescence is achieved remains poorly understood, representing a critical knowledge gap in early male-female reproductive interactions and our conceptualization of the ‘fertile sperm.’ The fragility of this mechanism is perhaps best highlighted in the sheep, where cervical artificial insemination (AI) with frozen-thawed (FT) ram spermatozoa results in fertility rates of 75% with fresh semen ( 2 ). This discrepancy is attributed primarily to impaired sperm transit through the cervix, rather than overt deficits in classical sperm parameters such as motility, or viability ( 3 – 5 ). Cryopreservation induces phenotypic alterations that extend beyond these conventional metrics, including modification of sperm membrane-associated proteins ( 6 ), which leads to the exposure of paternal antigens ( 7 ) on the sperm surface. Additionally, it contributes to dilution or removal of seminal plasma (SP), a fluid known to influence sperm transport and survival ( 8 , 9 ). Seminal plasma-naïve epididymal sperm also fail to traverse the ovine cervix, further supporting a role for SP in mediating fertility ( 9 ). Collectively, cryopreservation and loss of SP produce a sperm phenotype that is molecularly and immunogenically distinct from fresh, seminal plasma exposed ‘fertile’ sperm, leading to a potential failure in permissive mechanisms and increased clearance by the female immune system. Across mammalian species, semen deposition triggers a rapid and highly conserved inflammatory response in the female reproductive tract, characterized by epithelial receptor activation ( 10 ), cytokine and chemokine release ( 11 ), fluid influx ( 12 ), and leukocyte recruitment ( 13 – 15 ), that together regulate microbial defense, sperm clearance, and reproductive tract conditioning for conception ( 16 ). Central to this response is the rapid influx of polymorphonuclear neutrophils (PMNs), which migrate into the cervical and uterine lumen within minutes of semen exposure and interact directly with spermatozoa, a phenomenon documented across multiple species including women ( 17 ), mice ( 18 ), and a wide range of livestock species, including cows ( 19 ), sheep ( 20 ), pigs ( 21 ) and horses ( 22 ). PMN influences early sperm clearance in the mucosa and lumen, where they deploy a combination of phagocytosis, degranulation, and neutrophil extracellular trap (NET) formation to remove spermatozoa and pathogens ( 20 , 23 , 24 ). Complement activation, inflammatory cytokine release, and chemokine-mediated recruitment further coordinate this response ( 25 – 27 ). While PMNs are essential for immune protection, their potent effector functions can compromise sperm survival. In vitro studies across bovine ( 28 , 29 ), equine ( 30 , 31 ), porcine ( 32 , 33 ), donkey ( 34 ), and human ( 35 ) species have documented sperm-PMN interactions, showing sperm elicit potent NET and phagocytic responses, also highlighting the significance of female-derived serum in this function ( 36 ). Studies in sheep have been limited ( 37 , 38 ). Despite robust physiological investigation, the molecular determinants of selective sperm clearance, particularly how sperm evade immune recognition, remain poorly defined across human and livestock models. Across these studies, seminal plasma has emerged as a key regulator of this early immune dialogue, suppressing sperm-PMN binding and modulating neutrophil activity ( 36 ). Studies in domestic species have demonstrated that SP can suppress neutrophil activation, reduce NET formation, and protect viable spermatozoa through antioxidant activity and immunoregulatory proteins ( 33 , 39 – 41 ). These findings suggest that SP components function to fine-tune post-mating inflammation, balancing antimicrobial defense with the preservation of fertilization-competent sperm. In the ram the responsible SP protective factors appear to be heat-labile, indicating a pivotal role for proteins ( 38 ). SP is highly complex, containing in excess of 700 proteins, lipids, glycans, and extracellular nucleic acids, capable of modulating sperm function and the female immune environment ( 42 – 44 ). Within SP, proteins exist as soluble factors or are packaged within extracellular vesicles (EVs); lipid-bound nanostructures (30–200 nm) that carry proteins, lipids, and nucleic acids and provide a protected mode of delivery to transcriptionally silent sperm or FRT cells ( 45 , 46 ). EVs are increasingly recognized as modulators of reproductive immunity, capable of delivering bioactive molecules that modify immune environments and support sperm survival (reviewed by ( 46 )). Seminal EVs, including prostasomes and epididymosomes, have been reported to suppress inflammatory responses by reducing reactive oxygen species (ROS) production, modulating cytokine signaling, and transferring immunoregulatory cargo to leukocytes, including PMNs (reviewed by ( 47 , 48 )). Through these mechanisms, EVs are proposed to limit excessive PMN activation and sperm binding while preserving antimicrobial defense. However, the contribution of EV-associated factors to SP-mediated sperm protection, particularly in the ram, remains largely unexplored. Despite clear evidence that SP protects spermatozoa from PMN attack, the specific molecular factors, their mode of delivery (soluble versus EV-associated), and their functional characteristics are unknown. Moreover, the interplay between SP proteins and the innate immune system, including PMN responses, complement regulation, and inflammatory signaling, remains undefined. Identifying these factors has direct relevance for understanding key mechanism of sperm immune tolerance within the female tract and how this could be manipulated to direct strategies to improve the fertility of cryopreserved sperm following cervical AI in sheep and more widely enhancing assisted reproductive technologies (ARTs) for livestock and wildlife. The present study builds on previous findings demonstrating SP-mediated protection of frozen-thawed (FT) ram sperm against PMN binding in vitro ( 38 ). Here, we aim to isolate and characterize the specific SP components responsible for this protective effect, examining first the role of soluble proteins or EV-associated proteins, then attempting to correlate individual molecular weight-defined fractions to PMN protection. By combining PMN binding assays with proteomic analyses, this work seeks to define the molecular mechanisms through which SP restores permissive sperm immunity, providing insight into the molecular dialogue between semen and the female reproductive tract and identifying targets to improve post-thaw sperm survival and fertility outcomes. Materials and methods Experimental design Two complementary studies were designed to identify mediators in seminal plasma that could be responsible for the protection of frozen–thawed ram spermatozoa from PMN binding. Study 1 compared the protective effect of whole seminal plasma (SP) to its soluble, EV-depleted (DSP) and extracellular vesicle (EV) fractions. Building on these outcomes, Study 2 resolved SP into unique molecular-weight fractions ( 100 kDa) to localise functional activity and refine the search for candidate mediators. To qualitatively assess the protein components underpinning these effects and integrate findings across both studies, all treatments were analyzed by mass spectrometry using both data-dependent acquisition (DDA) and data-independent acquisition (DIA), enabling presence/absence assessment alongside broader proteomic profiling. Extracellular vesicles were characterised in accordance with MISEV2023 guidelines ( 49 ). Chemicals All chemicals were purchased from Sigma-Aldrich (Melbourne, Australia) unless otherwise stated. Animal use and ethical approval Merino ewes ( n = 2) used for blood collection were kept on a chaff-based diet supplemented with lupins in an animal house at the University of Sydney, Camperdown, NSW, Australia. Mature Merino rams used for seminal plasma collection and preparation ( n = 24) were kept on pasture at the Sheep Unit, University of Sydney, Camden Campus, Cobbitty. All work was approved by the University of Sydney Animal Ethics Committee (Project No: 2023/2277 and 2023/AE002277). Seminal plasma collection and preparation Seminal plasma was obtained from ejaculates ( n = 9 per ram) collected from mature Merino rams ( n = 24) in the presence of a teaser ewe during the breeding season 2024/25. The average ejaculate volume across all rams was 1.25 ± 0.44mL, with a wave motion of 4.65 ± 0.33 across all collections. Accompanying ejaculate characteristics per ram, performed per ejaculate prior to inclusion in the pool can be found in Supplementary Table 1 . Following initial subjective assessment of mass motility, samples scoring below 3 (scale 1–5) were excluded prior to clarification. Each ejaculate was processed immediately following collection and centrifuged at 12,000 × g, 4 °C for 10 min. The supernatant was aspirated and centrifuged again at 12,000 × g, 4 °C for a further 10 min to remove any remaining spermatozoa or cellular debris. Based on ejaculate characteristics ( Supplementary Table 1 ) and final seminal plasma volume, equal volumes of seminal plasma from rams with equivalent profiles were pooled to generate a single composite seminal plasma sample for downstream analyses. This pooled sample was aliquoted and stored at −80 °C until use. For each experiment, a single aliquot was thawed on ice immediately prior to use. Isolation of Ram seminal plasma extracellular vesicles (EVS) An aliquot of the seminal plasma pool was sequentially centrifuged with increasing velocity at 4 °C to eliminate contaminating cells and cellular debris (500 x g, 5 min; 2,000 x g, 5 min; 2,000 x g, 5 min; 8,000 x g, 5 min; 17,000 x g, 20 min; and repeat centrifugations at 17,000 x g for 10 min until a pellet was no longer visible). Extracellular vesicles were then enriched as previously described by ( 44 , 45 ). Samples were centrifuged at 100, 000 x g for 90 min in a fixed rotor (Hi-tachi CP100NX Ultracentrifuge). Following ultracentrifugation, the non-pelleted supernatant fraction was collected as extracellular vesicle–depleted seminal plasma (DSP). The pellet was washed with 12 ml of phosphate-buffered saline (PBS; 137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1 mM KH2PO4, pH 7.4, mOsm 295+/ -5 ) and centrifuged again at 100,000 x g for 90 min. The resulting pellet was resuspended in 1 ml of PBS, and layered onto a discontinuous density gradient modified from previous studies (comprising 50%, 25%, 12.5%, and 6.25%) OptiPrep (Sigma Aldrich, Melbourne, Australia) suspension diluted in a solution of 0.25 M sucrose, 10 mM Tris HCl, pH 7.5) and centrifuged at 108,300 x g for 16 h at 4 °C. After centrifugation, 12 equivalent fractions (1mL per fraction) were collected. A visually enriched EV band corresponding to fractions 9 and 10, was pooled and retained for downstream analyses. Pooled fractions were washed in PBS by ultracentrifugation (100,000 × g, 120 min). Pellets were solubilized in PBS as required for downstream application. Validation of EV characteristics Nanoparticle tracking analysis was used to determine particle size distribution and concentration of seminal plasma-derived EV samples, as previously described ( 50 ). Briefly, EV suspensions were diluted 1:10,000 in 0.1 µM filtered PBS before introduction into the chamber of a ZetaView x30 (Particle Matrix. Meerbusch, Germany). Settings were kept constant between samples (488 nm laser; sensitivity 80; shutter 100), capturing 11 × 60-second videos per sample. Particle size distribution and concentration were determined using ZetaView (version 8.05.16 SP3). For transmission electron microscopy, purified EV suspensions were fixed in 4% paraformaldehyde (1:1 v/v; 15 min on ice), and 15 µL was applied onto carbon-coated copper grids (ProSciTech, Kirwan, QLD, Australia) and incubated at room temperature for 1 hour. Grids were rinsed three times in 0.1 µm-filtered PBS and post-fixed in 1% glutaraldehyde for 5 min at room temperature, followed by three washes in ultrapure water. Grids were stained with methyl cellulose–uranyl acetate for structural contrast (10 min at RT), washed and air-dried overnight. Samples were imaged using a JEOL JEM-1200EXII transmission electron microscope (JEOL Ltd, Tokyo, Japan) at 50–250 kV. Fractionation of seminal plasma SP fractionation was adapted from ( 51 ). Briefly, an aliquot of the seminal plasma pool was sequentially centrifuged with increasing velocity at 4 °C to eliminate contaminating cells and cellular debris (500 x g, 5 min; 2,000 x g, 5 min; 2,000 x g, 5 min; 8000 x g, 5 min; 17,000 x g, 20 min; and repeat centrifugations at 17,000 x g for 10 min until a pellet was no longer visible). The sample was then fractionated based on molecular weight using Amicon Ultra centrifugal filters (Merck, Darmstadt, Germany), following the manufacturer’s instructions. Briefly, samples were centrifuged at 4,000 × g for 10 minutes at 20 °C through a filter with a 10 kDa molecular weight cutoff (MWCO) to collect the 100 kDa [F5]. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-page), silver staining, and immunoblotting Proteins from SP, DSP and EV samples (Study 1), and from each MW-fractionated SP treatment
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