{"paper_id":"103fd01d-e25c-4bc5-9e7e-3de7773a4194","body_text":"Tnfr1-associated Signaling Proteins in Mature Human Spermatozoa | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Tnfr1-associated Signaling Proteins in Mature Human Spermatozoa Arcangelo Barbonetti, Camilla Tonni, Vittoria Donatelli, Chiara Castellini, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8939920/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract Tumor necrosis factor-alpha (TNF-α) is a pleiotropic cytokine that activates the extrinsic apoptotic pathway through TNFR1, leading to recruitment of adaptor proteins and caspase activation in most somatic cell types. Although TNF-α has been implicated in inflammation-associated sperm dysfunction, the expression and functional competence of TNFR1 in mature human spermatozoa remain poorly defined. Here, we report that human spermatozoa express TNFR1 and that the receptor localizes along the flagellum, where it colocalizes with caveolin-1 within membrane microdomains. However, exposure to TNF-α across a wide concentration range did not affect sperm motility or mitochondrial membrane potential and failed to induce activation of caspase-8 or caspase-3. Molecular analyses revealed markedly reduced expression of the adaptor proteins TRADD and FADD and an absence of detectable pro-caspase-8 in spermatozoa compared with Jurkat cells. These findings indicate that, despite preserved receptor expression and membrane compartmentalization, the downstream extrinsic apoptotic machinery is profoundly limited in mature sperm cells. Our data support a model in which death receptor expression and downstream signaling competence become developmentally uncoupled during terminal differentiation, reflecting selective remodeling of apoptotic signaling pathways. Biological sciences/Cell biology/Cell signalling/Extracellular signalling molecules Biological sciences/Molecular biology/Protein folding/Protein aggregation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Tumor necrosis factor alpha (TNF-α) is a pleiotropic pro-inflammatory cytokine primarily produced by activated monocytes and macrophages. It plays a key role in inflammation, immunity, and apoptosis, and its overexpression is associated with chronic inflammatory and autoimmune disorders (refs. 1,2). TNF-α induces apoptosis by binding to its type 1 receptor (TNFR1), which activates a series of adaptor proteins, including TNFR-associated death domain (TRADD) and FAS-associated death domain (FADD). This process triggers the activation of caspase-8 and subsequently downstream caspase-3 (refs. 3–5). This extrinsic pathway can intersect with the intrinsic one, thanks to the ability of caspase-8 to cleave Bid, a member of the pro-apoptotic Bcl-2 family. This truncated form can translocate to the mitochondria and initiate the intrinsic pathway by promoting the release of cytochrome c (ref. 6). In the male genital tract, TNF-α can be detected in seminal plasma, particularly during inflammatory conditions (refs. 7,8) and has been implicated in sperm dysfunction. Although leukocytospermia and elevated pro-inflammatory cytokines have long been suspected contributors to male infertility, clinical findings remain inconclusive (refs. 9–14). In vitro studies investigating the effects of TNF-α on sperm have yielded conflicting results. Some studies report no impact on sperm motility or viability (refs. 15–18), while others indicate mitochondrial depolarization, DNA fragmentation, and phosphatidylserine externalization consistent with apoptosis (refs. 19–22). Despite these findings, the expression and functional competence of TNFR1 in mature human spermatozoa have not been clearly defined. Here, we demonstrate that human spermatozoa express TNFR1, which colocalizes with caveolin-1 in the flagellum. However, our findings indicate that TNFR1 does not efficiently engage the apoptotic cascade under the conditions tested, likely due to defective expression of TRADD, FADD, and pro-caspase-8. These data provide new insights into the apoptotic resistance of spermatozoa in inflammatory environments and suggest that the functional impact of TNF-α in mature spermatozoa may differ from classical receptor-mediated apoptotic responses. RESULTS Exposure to TNF-α does not affect sperm motility or mitochondrial membrane potential Human spermatozoa, isolated using the swim-up technique, were exposed to increasing concentrations of TNF-α (ranging from 0.5 to 100 ng/mL) for 2 hours. Sperm motility parameters were evaluated using computer-aided semen analysis (CASA). No significant differences in total motility or kinematic parameters (VCL, VSL, and VAP) were observed after the exposure to TNF-α (Fig. 1 A). Given that the effect of TNF through TNFR1 activation typically induces receptor-mediated apoptosis via caspase-8 activation and downstream mitochondrial involvement, we further examined the mitochondrial membrane potential (ΔΨm) using the JC-1 fluorescent probe. In line with the motility findings, TNF-α exposure did not significantly alter ΔΨm in spermatozoa, as evidenced by the stable red-to-green fluorescence ratio across all tested concentrations (Fig. 1 B). In contrast, treatment with rotenone (1 µM), used as a positive control, led to a marked loss of ΔΨm, confirming the sensitivity of the assay. These results indicate that human spermatozoa do not exhibit functional responses to TNF-α under the experimental conditions tested. TNFR1 is expressed in human spermatozoa and colocalizes with caveolin-1 Western blot analysis of sperm lysates from six normozoospermic donors revealed a single immunoreactive band at ~ 50 kDa, consistent with TNFR1 expression (Fig. 2 ), as previously reported in other cell types (refs. 23–24). TNFR1 localization was further investigated by confocal immunofluorescence analysis. The TNFR1 staining appeared to be diffuse and punctate, with predominant localization along the tail and, to a lesser extent, in the head (Fig. 3 B). A similar staining pattern was observed for caveolin-1 (Cav-1) (Fig. 3 C). Merged images demonstrated substantial colocalization of TNFR1 and Cav-1, particularly along the principal piece of the tail (Fig. 3 D). Quantitative colocalization was confirmed using Pearson’s correlation coefficient and Manders’ overlap coefficient. No staining was observed in negative controls where primary antibodies were omitted (Fig. 3 F). Disruption of the TNFR1 downstream signaling cascade in spermatozoa Despite confirming TNFR1 expression, the lack of functional response to TNF-α prompted us to explore the expression of downstream apoptotic mediators. Western blot analysis revealed significantly lower levels of TRADD in spermatozoa compared to Jurkat cells used as positive control (Fig. 4 A). Similarly, the expression of FADD was almost undetectable, with only a faint band observed at the expected 27 kDa (Fig. 4 B). These findings indicate a disruption of the TNFR1 signaling cascade at the level of adaptor protein recruitment. To further characterize this defect, the expression of pro-caspase-8 in spermatozoa exposed to TNF-α (10 ng/mL) or staurosporine (10 µM) was evaluated. While Jurkat cells exhibited a progressive reduction in pro-caspase-8 levels in response to pro-apoptotic stimuli, human spermatozoa displayed no detectable expression of this protein under any condition (Fig. 5 ). Lack of caspase activation in response to TNF-α We next assessed the activation of caspase-8 and caspase-3 in live human sperm cells using FITC-conjugated fluorogenic inhibitors (IETD-FMK for caspase-8 and DEVD-FMK for caspase-3). Our experiments showed that exposure to increasing concentrations of TNF-α failed to induce significant activation of either caspase-8 or caspase-3 (Fig. 6 A and B). As expected, caspase-3 activation was strongly induced by staurosporine. However, caspase-8 activation remained negligible even under this condition, confirming the extremely limited expression and activation potential of this caspase initiator in human spermatozoa. DISCUSSION This study demonstrates that, although human spermatozoa express TNFR1, the receptor fails to engage the canonical extrinsic apoptotic cascade under the experimental conditions tested. These findings refine current assumptions about cytokine-induced damage to sperm and suggest a functional uncoupling between TNFR1 expression and apoptotic signaling in mature male gametes. More broadly, these findings suggest that death receptor expression and signaling competence may become developmentally uncoupled during terminal differentiation, reflecting a selective remodeling of apoptotic pathways in highly specialized cells. This dissociation between receptor presence and downstream signaling competence raises broader questions regarding the structural and functional plasticity of apoptotic pathways in terminally differentiated cells. While death receptor signaling is typically considered a conserved and tightly regulated cascade, our findings suggest that selective loss or attenuation of key adaptor components may represent a physiological mechanism to preserve cellular specialization in post-mitotic cells. In this context, spermatozoa provide a unique model to explore how canonical death pathways can be developmentally reconfigured without complete elimination of receptor expression. TNFR1 is a canonical death receptor that triggers receptor-mediated apoptosis upon ligand binding. This process involves the sequential recruitment of TRADD and FADD, which subsequently activate caspase-8 and the executioner caspase-3 (ref. 25). This pathway also intersects with the mitochondrial apoptotic axis via tBID-mediated inhibition of BCL2 and activation of BAX/BAK, resulting in mitochondrial outer membrane permeabilization, ΔΨm loss, and cytochrome c release—culminating in caspase-9 and caspase-3 activation. To investigate whether this pathway is functional in spermatozoa, we assessed mitochondrial membrane potential and motility in sperm treated with TNF-α. We found that neither parameter was significantly altered, suggesting that TNF-α fails to initiate death signaling in these cells. This is further supported by the absence or markedly low expression of adaptor proteins TRADD and FADD, and the undetectable levels and activation of pro-caspase-8, reinforcing the notion of an incomplete or functionally constrained TNFR1-mediated apoptotic axis in human sperm. It should be noted that TNF-α signals through two distinct receptors, TNFR1 and TNFR2, which mediate partially overlapping but functionally divergent pathways. While TNFR1 contains a death domain and is directly linked to extrinsic apoptotic signaling, TNFR2 primarily activates non-apoptotic pathways, including NF-κB–dependent responses. The present study specifically addressed the competence of the TNFR1-mediated apoptotic axis; therefore, potential contributions of TNFR2 to non-apoptotic TNF-α signaling in mature spermatozoa warrant further investigation. Although previous studies reported mild adverse effects of TNF-α on sperm motility and DNA integrity, these changes were limited and only evident after prolonged exposures (refs. 20–22). Furthermore, these effects were reversed by TNF-α antagonists such as infliximab or etanercept. Importantly, these earlier studies did not directly demonstrate the expression of TNFR1 in human spermatozoa. Our Western blot analysis confirms the presence of TNFR1 in human sperm as a distinct ~ 50 kDa band, consistent with reports in somatic cells (refs. 23–24). TNFR1 signaling has been shown to originate in caveolae-like membrane microdomains which are rich in cholesterol and sphingolipids and anchored by caveolin-1 (refs. 26–28). Confocal microscopy revealed that TNFR1 is localized along the sperm tail, specifically colocalizing with caveolin-1 in the principal piece. Quantitative image analysis confirmed this colocalization, indicating a membrane environment that is conducive to receptor signaling. However, despite this spatial organization, no functional signaling occurred. Molecular analyses indicated markedly reduced expression of TRADD and FADD, along with a near-complete absence of pro-caspase-8, as determined by Western blot and flow cytometry. The activation of caspase-8 remained undetectable even following staurosporine treatment, consistent with reports describing the inability of mature sperm to undergo receptor-mediated apoptosis (refs. 29–32). Although some studies have reported the presence and activity of caspase-8 in ejaculated sperm (ref. 33), the prevailing evidence supports the idea that the extrinsic apoptotic pathway is inactive in these terminally differentiated cells. In contrast, the intrinsic, mitochondria-driven apoptotic machinery is intact and functionally relevant (refs. 34–36). Indeed, oxidative stress appears to be the dominant mechanism of sperm injury in leukocytospermia and genital tract inflammation, rather than cytokine-mediated signaling (ref. 37). The question then arises: why do spermatozoa express TNFR1 at all? TNFR1 has been proposed to play a physiological role during spermatogenesis, particularly within the seminiferous epithelium. It is highly expressed in intratubular germ cells, including spermatocytes and round spermatids, which also secrete TNF-α (refs. 38–40). TNF-α has been shown to modulate the expression of aromatase in germ cells, promoting estrogen biosynthesis in pachytene spermatocytes and inhibiting it in round spermatids (ref. 41). More recently, TNF-α signaling has been implicated in immunosuppressive interactions between spermatozoa and the female reproductive tract, such as the downregulation of pro-inflammatory cytokine expression in human fallopian tube epithelial cells (ref. 42). Taken together, our findings support a model in which TNFR1 fulfills physiological functions during earlier stages of germ cell development but becomes progressively uncoupled from its pro-apoptotic signaling machinery during the final phases of sperm differentiation. In mature spermatozoa, persistent receptor expression may reflect selective protein retention or alternatively serve non-apoptotic roles, potentially including modulation of extracellular TNF-α within the reproductive tract. More broadly, our data reveal a dissociation between receptor presence and downstream signaling competence, consistent with selective remodeling of the extrinsic apoptotic pathway during terminal differentiation. Although TNFR1 remains expressed and localized within caveolin-1–enriched membrane domains, the marked reduction or absence of TRADD, FADD, and pro-caspase-8 prevents engagement of the canonical death cascade. These observations suggest that the functional consequences of inflammatory cytokine signaling in mature spermatozoa may operate through mechanisms distinct from classical receptor-mediated apoptosis. MATERIALS AND METHODS Semen samples Ejaculates of healthy donors were collected by masturbation following an abstinence period of 3–7 days. Donors were students or post-graduate students from the University of L’Aquila, who had no known prior male reproductive pathologies including varicocele and infection. All samples were normozoospermic according to the World Health Organization (ref. 43) and showed no evidence of leukocytospermia. After collecting, samples were left for at least 30 min to liquefy before processing. The study was approved by the local Institutional Review Board, and all subjects signed an informed consent statement. The study was performed in accordance with the Declaration of Helsinki. In vitro exposure of donor spermatozoa to TNF-α Motile sperm suspensions were obtained by swim-up procedure from six different donors. Briefly, spermatozoa were washed twice (700 × g for 7 min) in Biggers, Whitten, and Wittingham (BWW) with 0.1% human serum albumin (HSA) fraction V (Sigma-Aldrich S.r.l., Milan, Italy). After the second centrifugation, supernatants were removed by aspiration, leaving approximately 0.5 ml of medium on the pellet and, after an incubation time of 30 min, supernatants containing highly concentrated motile sperms were carefully aspirated and the sperm concentration was properly adjusted. In different settings, aliquots of the same motile sperm suspensions were exposed for 2 h to scalar concentrations of TNF-α (from 0.5 to 100 ng/ml) (EuroClone, Pero, Italy) and then subjected to the assessment of motility, sperm mitochondrial membrane potential (ΔΨm) and activated caspase-8 and 3, as described below. Evaluation of sperm motility Sperm motility was evaluated by Computer-Aided Semen Analysis (CASA), using Hamilton Thorne (EOS srl, Firenze, Italy). Ten microliters of each sperm sample were placed into a pre-warmed (37°C) Makler counting chamber (Sefi Medical Instruments, Haifa, Israel). At least 200 spermatozoa were evaluated for each sample. Setting parameters were the following: analysis duration of 1 s (30 frames); minimum contrast, 80; minimum size, 3; low size gate, 0.7; high size gate, 2.6; low intensity gate, 0.34; light intensity gate, 1.40. Spermatozoa exhibiting an average pathway velocity > 5 µm/s were categorized by the software as motile spermatozoa. Flow cytometric evaluation of ΔΨm The fluorescent lipophilic cationic dye 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benimidazolyl carbocyanine iodide (JC-1) (Sigma-Aldrich S.r.l., Milan, Italy) was used to evaluate trans-membrane potential of sperm mitochondria, as previously described (refs. 44,45). This probe possesses the unique ability to differentially label mitochondria with low and high ΔΨm. In mitochondria with high ΔΨm, JC-1 forms multimeric aggregates that emit orange light (wavelength of 590 nm) when excited at 488 nm. In mitochondria with low ΔΨm, JC-1 forms monomers that emit green light (525–530 nm) when excited at 488 nm. Motile sperm suspensions, each containing 5 × 10 6 spermatozoa were diluted in 1 mL of PBS to give a final sperm concentration of 1.5-2.0 × 10 6 /mL and then stained with 0.5 µL of JC-1 stock solution (3 mM in DMSO). Samples were incubated at 37°C in the dark for 60 min and then analyzed at the flow cytometer equipped with a 15-mW argon-ion laser for excitation. For each sample 10 000 events were recorded at a flow rate of 200–300 cells/s. Based on the light scatter characteristics of swim up selected spermatozoa, debris and aggregates were gated out by establishing a region around the population of interest in the forward scatter/side scatter dot plot on a log scale. Compensation between FL1and FL2 was carefully adjusted according to the manufacturer’s instructions. Green and orange-red fluorescence was measured in FL-1 and FL-2 channel, respectively. The percentage of positive cells was evaluated using the flow cytometer System II Version 3.0 software (Beckman Coulter, Inc.). Western blot analysis 50 × 10 6 motile sperm, obtained by swim-up procedure, were stimulated with 10 ng/ml of recombinant TNF-α or with staurosporine (10 µM) for 2 hours at 37°C in an atmosphere of 5%CO 2 /95% air. The motile spermatozoa were washed in phosphate-buffered saline (PBS) and homogenized and lysed in ice-cold RIPA buffer (Merck KGaA, Darmstadt, Germany) containing 100 mM protease inhibitor cocktail (Sigma-Aldrich, Saint Louis, MO, USA). The protein amount was evaluated with DC Protein Assay (Bio-Rad, Hercules, CA, USA) using BSA as standard. Total cell lysates (25 µg protein) were mixed with sample buffer; samples were denatured (at 100°C for 5 min), subjected to 12% SDS polyacrylamide gel, and electroblotted onto 0.45 µm nitrocellulose membrane sheets (Bio-Rad) for 1 hour at 4°C at 70 V using a Mini Trans-Blot Cell apparatus (Bio-Rad). Non-specific binding sites were blocked with 5% non-fat dry milk for 1 h at room temperature and membranes were incubated overnight at 4°C with primary antibodies (Table 1 ). As secondary antibodies, peroxidase-conjugated anti-rabbit and anti-mouse IgG antibodies (dilution 1:2 000) were acquired from Sigma-Aldrich. The immunoreactive bands were visualized by enhanced chemiluminescent (ECL, Amersham Pharmacia Biotech, Buckinghamshire, UK) according to the manufacturer instructions and acquired by UVItec Alliance (Cambridge, UK). As positive control, homogenates of Jurkat cells (human T cell lymphoblast-like cell line from leukemia) were used under the same experimental conditions. Table 1 List of primary antibodies used in the present study. Primary antibody Dilution Company Rabbit monoclonal anti-TNF-R1/CD120a 1:1000 Invitrogen Corporation, Waltham, MA, USA Rabbit monoclonal anti-TRADD 1:1000 Invitrogen Corporation, Waltham, MA, USA Mouse monoclonal anti-FADD 1:2000 Invitrogen Corporation, Waltham, MA, USA Mouse monoclonal anti-pro-caspase 8 1:1000 Immunological Sciences, Rome, Italy Mouse monoclonal anti-α-tubulin 1:4000 Sigma-Aldrich, Saint Louis, MO, USA Full-length uncropped western blots corresponding to all figures are provided in Supplementary Figure S1 . TNFR1/Cav-1 co-localization in human spermatozoa: immunofluorescence staining in confocal microscopy Motile sperm suspensions, obtained by swim-up procedure, were fixed with ice-cold formaldehyde (1%, v/v) in PBS, pH 7.4 for 30 min at 4ºC and then permeabilized with 0.1%, v/v Triton X-100 (Bio-Rad, Hercules, CA, USA) in PBS for 10 min at room temperature. Non-specific binding sites were blocked with 4% bovine serum albumin (BSA) (Sigma-Aldrich S.r.l., Milan, Italy) for 10 minutes at room temperature. Sperm suspensions were then incubated with mouse anti-human TNF-R1/CD120a mAb (dilution 1:100) in blocking buffer for 1 h at room temperature. Co-localization of TNFR1 with Cav-1 was also explored using a rabbit anti-human Cav-1 mAb (ThermoFisher Scientific, Monza, Italy; dilution 1:100) in blocking buffer for 1 h at room temperature. In control samples, primary antibodies were omitted. After multiple washes by centrifugations (700 × g , 7 min), spermatozoa were incubated with the DyLight® 594 conjugated donkey anti-mouse (Aurogene S.r.l. Roma, Italy; dilution 1:2 000) and/or a Alexa Fluor® 488 conjugated goat anti-rabbit (ThermoFisher Scientific, Monza, Italy; dilution 1:2 000) for 1 h at room temperature, washed again as above and subsequently smeared on slides. Slides were mounted with PBS–glycerol to be examined by confocal microscopy (Leica TCS SP5 II, Wetzlar, Germany). In the merged images, the degree of colocalization within a specific region of interest (ROI) was estimated by using two quantitative coefficients: Pearson’s Correlation Coefficient (PCC) and Manders’ overlap coefficient (MOC). The former describes the correlation of the intensity distribution between channels and ranges from − 1 (fully negative correlation) to + 1 (completely positive correlation), with zero standing for no correlation; the latter indicates an actual overlap of the signals and ranges from 0 (non-overlapping images) to + 1 (100% colocalization) (refs. 47–48). Flow cytometric assessment of activated caspases Caspase-8 and caspase-3 activation was evaluated using Apoptosis CaspGLOW Fluorescein Active Staining Kits for Caspase-8 and Caspase-3 (BioVision, Inc., Mountain View, CA, USA). These assays employ FITC-conjugated inhibitors—IETD-FMK for caspase-8 and DEVD-FMK for caspase-3—as fluorescent markers. These cell-permeable, non-toxic peptides covalently bind to activated caspase-8 and caspase-3, serving as in situ markers for apoptosis. After a 2 h exposure to increasing concentration of TNF-α or to staurosporine (10 µM) (Sigma-Aldrich S.r.l., Milan, Italy), as positive control, 1 µL of FITC-IETD-FMK or FITC-DEVD-FMK was added to 300 µL of sperm suspension containing 0.3 × 10 6 spermatozoa. After 1 h incubation at 37°C in an atmosphere of 5%CO 2 /95% air, samples were centrifuged three times (1 000 × g for 4 min) and analyzed by flow cytometry. For each sample 10 000 events were recorded at a flow rate of 200–300 cells/s. Statistical analysis Statistical analysis was performed using the R statistical software (version 4.5.1, 2025, The R Foundation for Statistical Computing, Vienna, Austria). The Student’s t-test was also performed where indicated. After assessing the distribution of data with the Shapiro-Wilk test, the Wilcoxon rank-sum test or ANOVA followed by post hoc comparisons by the Tukey’s studentized range-honestly significant difference (HSD) test were used, as appropriate. Data were expressed as mean ± SD of independent experiments performed in triplicate and statistical significance was accepted when p ≤ 0.05. Declarations Acknowledgments: The authors thank Prof. Maria Grazia Cifone for her precious and valuable advice and for reviewing manuscript. Conflict of interest: The authors declare no conflict of interest. Author contributions: A.B. conceived the study, performed the statistical analysis, and critically revised the manuscript; C.T. and V.D. contributed to the statistical analysis and the drafting of the first version of the manuscript; C.C., F.R.A., and P.P. contributed to the experimental procedures and data collection; C.M. and D.T. contributed to the statistical analysis and the critical revision of the manuscript; B.C. supervised the experiments and contributed to the critical revision of the manuscript. In Figure 1 C.C. performed CASA analysis and flow cytometric assessment of mitochondrial potential. In Figure 2, Figure 4 and Figure 5 C.C. processed semen samples, F.R.A. and P.P. performed western blot assays. In Figure 3 C.C. performed immunofluorescence evaluation. In Figure 6 C.C. performed flow cytometric caspase assays. Ethics Approval and Consent to Participate: The study was approved by the local Institutional Review Board, and all subjects signed an informed consent statement. The study was performed in accordance with the Declaration of Helsinki. Funding: The authors received no specific funding for this work. Data Availability Statement: The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request. References Idriss HT & Naismith JH. TNF alpha and the TNF receptor superfamily: structure-function relationship(s). Microsc Res Tech 50, 184–195 (2000). Apostolaki M, Armaka M, Victoratos P & Kollias G. Cellular mechanisms of TNF function in models of inflammation and autoimmunity. Curr Dir Autoimmun 11, 1–26 (2010). Hsu H, Shu HB, Pan MG & Goeddel DV. TRADD-TRAF2 and TRADD-FADD interactions define two distinct TNF receptor 1 signal transduction pathways. Cell 84, 299–308 (1996). Schütze S, Tchikov V & Schneider-Brachert W. Regulation of TNFR1 and CD95 signalling by receptor compartmentalization. Nat Rev Mol Cell Biol 9, 655–662 (2008). Heinrich M. et al. Cathepsin D links TNF-induced acid sphingomyelinase to Bid-mediated caspase-9 and – 3 activation. Cell Death Differ 11, 550–563 (2004). Mustafa M. et al. Apoptosis: A Comprehensive Overview of Signaling Pathways, Morphological Changes, and Physiological Significance and Therapeutic Implications. Cells 13, 1838 (2024). Bronson R, Cooper G & Rosenfeld D. Sperm antibodies: their role in infertility. Fertil Steril 42, 171–183 (1984). Gruschwitz MS, Brezinschek R & Brezinschek HP. Cytokine levels in the seminal plasma of infertile males. J Androl 17, 158–163 (1996). Wolff H. The biologic significance of white blood cells in semen. Fertil Steril 63, 1143–1157 (1995). Ochsendorf FR. Infections in the male genital tract and reactive oxygen species. Hum Reprod Update 5, 399–420 (1999). Whittington K & Ford WC. Relative contribution of leukocytes and of spermatozoa to reactive oxygen species production in human sperm suspensions. Int J Androl 22, 229–235 (1999). Henkel R. et al. Effect of reactive oxygen species produced by spermatozoa and leukocytes on sperm functions in non-leukocytospermic patients. Fertil Steril 83, 635–642 (2005). Agarwal A. et al. Reactive oxygen species and sperm DNA damage in infertile men presenting with low level leukocytospermia. Reprod Biol Endocrinol 12, 126 (2014). Castellini, C. et al. Relationship between leukocytospermia, reproductive potential after assisted reproductive technology, and sperm parameters: a systematic review and meta-analysis of case-control studies. Andrology 8, 125–135 (2020). Wincek TJ, Meyer TK, Meyer MR & Kuehl TJ. Absence of a direct effect of recombinant tumor necrosis factor-alpha on human sperm function and murine preimplantation development. Fertil Steril 56, 332–339 (1991). Haney AF, Hughes SF & Weinberg JB. The lack of effect of tumor necrosis factor-alpha, interleukin-1-alpha, and interferon-gamma on human sperm motility in vitro. J Androl 13, 249–253 (1992). Fedder J & Ellerman-Eriksen S. Effect of cytokines on sperm motility and ionophore-stimulated acrosome reaction. Arch Androl 35, 173–185 (1995). Lewis SE, Donnelly ET, Sterling ES, Kennedy MS, Thompson W & Chakravarthy U. Nitric oxide synthase and nitrite production in human spermatozoa: evidence that endogenous nitric oxide is beneficial to sperm motility. Mol Hum Reprod 2, 873–878 (1996). Estrada LS et al. Effect of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on human sperm motility, viability and motion parameters. Int J Androl 20, 237–242 (1997). Said TM, Agarwal A, Falcone T, Sharma RK, Bedaiwy MA & Li L. Infliximab may reverse the toxic effects induced by tumor necrosis factor alpha in human spermatozoa: an in vitro model. Fertil Steril 83, 1665–1673 (2005). Perdichizzi A. et al. Effects of tumour necrosis factor-alpha on human sperm motility and apoptosis. J Clin Immunol 27, 152–162 (2007). Pascarelli NA, Fioravanti A, Moretti E, Guidelli GM, Mazzi L & Collodel G. The effects in vitro of TNF-α and its antagonist 'etanercept' on ejaculated human sperm. Reprod Fertil Dev 29, 1169–1177 (2017). Loetscher H, Schlaeger EJ, Lahm HW, Pan YC, Lesslauer W & Brockhaus M. Purification and partial amino acid sequence analysis of two distinct tumor necrosis factor receptors from HL60 cells. J Biol Chem 265, 20131–20138 (1990). Schall TJ et al. Molecular cloning and expression of a receptor for human tumor necrosis factor. Cell 61, 361–370 (1990). Hotchkiss RS, Strasser A, McDunn JE & Swanson PE. Cell death. N Engl J Med 361, 1570–1583 (2009). Ko YG, Lee JS, Kang YS, Ahn JH & Seo JS. TNF-alpha-mediated apoptosis is initiated in caveolae-like domains. J Immunol 162, 7217–7223 (1999). D'Alessio A, Al-Lamki RS, Bradley JR & Pober JS. Caveolae participate in tumornecrosis factor receptor 1 signaling and internalization in a human endothelial cell line. Am J Pathol 166, 1273–1282 (2005). Dargelos E, Renaud V, Decossas M, Bure C, Lambert O & Poussard S. Caveolae-mediated effects of TNF-α on human skeletal muscle cells. Exp Cell Res 370, 623–631 (2018). Lachaud C, Tesarik J, Cañadas ML & Mendoza C. Apoptosis and necrosis in human ejaculated spermatozoa. Hum Reprod 19, 607–610 (2004). Koppers AJ, Mitchell LA, Wang P, Lin M & Aitken RJ. Phosphoinositide 3-kinase signalling pathway involvement in a truncated apoptotic cascade associated with motility loss and oxidative DNA damage in human spermatozoa. Biochem J 436, 687–698 (2011). Grunewald S, Sharma R, Paasch U, Glander HJ & Agarwal A. Impact of caspase activation in human spermatozoa. Microsc Res Tech 72, 878–888 (2009). Weil M, Jacobson MD & Raff MC. Are caspases involved in the death of cells with a transcriptionally inactive nucleus? Sperm and chicken erythrocytes. J Cell Sci 111, 2707–2715 (1998). Paasch U, Grunewald S, Agarwal A & Glandera HJ. Activation pattern of caspases in human spermatozoa. Fertil Steril 81 Suppl 1, 802–809 (2004). Aitken RJ & Koppers AJ. Apoptosis and DNA damage in human spermatozoa. Asian J Androl 13, 36–42 (2011). Aitken RJ, Findlay JK, Hutt KJ & Kerr JB. Apoptosis in the germ line. Reproduction 141, 139–150 (2011). Aitken RJ & Baker MA. Causes and consequences of apoptosis in spermatozoa; contributions to infertility and impacts on development. Int J Dev Biol 57, 265–272 (2013). Aitken RJ & De Iuliis GN. On the possible origins of DNA damage in human spermatozoa. Mol Hum Reprod 16, 3–13 (2010). De SK, Chen HL, Pace JL, Hunt JS, Terranova PF & Enders GC. Expression of tumor necrosis factor-alpha in mouse spermatogenic cells. Endocrinology 133, 389–396 (1993). Bialas M, Fiszer D, Rozwadowska N, Kosicki W, Jedrzejczak P & Kurpisz M. The role of IL-6, IL-10, TNF-α and its receptors TNFR1 and TNFR2 in the local regulatory system of normal and impaired human spermatogenesis. Am J Reprod Immunol 62, 51–59 (2009). Lysiak JJ. The role of tumor necrosis factor-alpha and interleukin-1 in the mammalian testis and their involvement in testicular torsion and autoimmune orchitis. Reprod Biol Endocrinol 2, 9 (2004). Bourguiba S, Chater S, Delalande C, Benahmed M & Carreau S. Regulation of aromatase gene expression in purified germ cells of adult male rats: Effects of transforming growth factor β, tumor necrosis factor α, and cyclic adenosine 3',5'-monosphosphate. Biol Reprod 69, 592–601 (2003). Mousavi SO et al. Immunological response of fallopian tube epithelial cells to spermatozoa through modulating cytokines and chemokines. J Reprod Immunol 146, 103327 (2021). World Health Organization, WHO laboratory manual for the examination and processing of human semen , 6th edn. (WHO Press; Geneva, Switzerland, 2021). Barbonetti A. et al. Soluble products of Escherichia coli induce mitochondrial dysfunction-related sperm membrane lipid peroxidation which is prevented by lactobacilli. PLoS One 8, e83136 (2013) Castellini C. et al. Effects of bisphenol S and bisphenol F on human spermatozoa: An in vitro study. Reprod Toxicol 103, 58–63 (2021). Bolte S & Cordelières FP. A guided tour into subcellular colocalization analysis in light microscopy. J Microsc 224, 213–232 (2006). Zinchuk V & Grossenbacher-Zinchuk O. Recent advances in quantitative colocalization analysis: focus on neuroscience. Prog Histochem Cytochem 44, 125–172 (2009). Adler J & Parmryd I. Quantifying colocalization by correlation: the Pearson correlation coefficient is superior to the Mander's overlap coefficient. Cytometry A 77, 733–742 (2010). Additional Declarations There is no conflict of interest Supplementary Files SupplementaryFigureS1WesternBlots.pdf Supplentary Figure S1 Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: revise 16 Apr, 2026 Review # 3 received at journal 09 Apr, 2026 Review # 2 received at journal 01 Apr, 2026 Review # 1 received at journal 24 Mar, 2026 Reviewer # 3 agreed at journal 16 Mar, 2026 Reviewer # 2 agreed at journal 16 Mar, 2026 Reviewer # 1 agreed at journal 15 Mar, 2026 Reviewers invited by journal 15 Mar, 2026 Submission checks completed at journal 12 Mar, 2026 Editor assigned by journal 10 Mar, 2026 First submitted to journal 10 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-8939920\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Article\",\"associatedPublications\":[],\"authors\":[{\"id\":606461098,\"identity\":\"c86b2926-10ca-4411-8d66-e8a08bd2dc1c\",\"order_by\":0,\"name\":\"Arcangelo Barbonetti\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABLUlEQVRIie3Pv0rDQBzA8QuBuPzSWxtS2lc4KRSKUV+lIXCTloAguCUUMkWzVnyJunUz4aAuZ3fRJRQ6ORS6RC3oXQsVyWUXzHc4uDs+9wehuro/XAshQEgLdrN0tZ2KBtUEfoiho2y8J9XmN9Fhv1Em+I7la//DAYzMbH01dYa4k2TsOHptkUc2QXlRIs057dq3NxSsoOFZT5xeWJGO2Hm0BMKpr3wYB2SbMQOSArHCiLmT2ZYwccgZUZEOB/3TjL8k6b5L8iBJX5LkTUkIB8OGIpWkt7tFfJ9pguCm+pZDbvSOzMADa9Sg/ZBTdzzzSBbPJVn66YCWSJvrixfYnLTxwTV7DqeOm4yyxaq4ZKcG9u7zwil/X6ZFYtAVGyRVA9GmYp1Uirq6urr/1DcMLGqz3lsbRQAAAABJRU5ErkJggg==\",\"orcid\":\"https://orcid.org/0000-0002-6888-9585\",\"institution\":\"University of L'Aquila\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Arcangelo\",\"middleName\":\"\",\"lastName\":\"Barbonetti\",\"suffix\":\"\"},{\"id\":606461099,\"identity\":\"930214bb-47bc-40d9-9069-a4bc9b594e92\",\"order_by\":1,\"name\":\"Camilla Tonni\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of L'Aquila\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Camilla\",\"middleName\":\"\",\"lastName\":\"Tonni\",\"suffix\":\"\"},{\"id\":606461100,\"identity\":\"8d0d5210-ec29-4d09-a85a-7e82ba932a33\",\"order_by\":2,\"name\":\"Vittoria Donatelli\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of L'Aquila\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Vittoria\",\"middleName\":\"\",\"lastName\":\"Donatelli\",\"suffix\":\"\"},{\"id\":606461101,\"identity\":\"5e5cb7d6-abb3-4998-b6af-602b42f9d3b5\",\"order_by\":3,\"name\":\"Chiara Castellini\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of L'Aquila\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Chiara\",\"middleName\":\"\",\"lastName\":\"Castellini\",\"suffix\":\"\"},{\"id\":606461102,\"identity\":\"030436c8-40c6-493b-9381-c4cc8c770305\",\"order_by\":4,\"name\":\"Carolina Moretto\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of L'Aquila\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Carolina\",\"middleName\":\"\",\"lastName\":\"Moretto\",\"suffix\":\"\"},{\"id\":606461103,\"identity\":\"74f81aea-b080-4a32-be86-e80940de0399\",\"order_by\":5,\"name\":\"Daniele Tienforti\",\"email\":\"\",\"orcid\":\"https://orcid.org/0000-0002-9359-7955\",\"institution\":\"University of L'Aquila\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Daniele\",\"middleName\":\"\",\"lastName\":\"Tienforti\",\"suffix\":\"\"},{\"id\":606461104,\"identity\":\"39df633a-bf65-4ac9-9b78-55dfb0898cd7\",\"order_by\":6,\"name\":\"Francesca Augello\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of L'Aquila\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Francesca\",\"middleName\":\"\",\"lastName\":\"Augello\",\"suffix\":\"\"},{\"id\":606461105,\"identity\":\"fec33e06-cad5-490a-bdc6-73f6f36db464\",\"order_by\":7,\"name\":\"Benedetta Cinque\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of L'Aquila\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Benedetta\",\"middleName\":\"\",\"lastName\":\"Cinque\",\"suffix\":\"\"},{\"id\":606461106,\"identity\":\"8208ddb8-165a-44c3-9485-dd42e5db4d53\",\"order_by\":8,\"name\":\"Paola Palumbo\",\"email\":\"\",\"orcid\":\"https://orcid.org/0000-0001-7027-2691\",\"institution\":\"University of L’Aquila\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Paola\",\"middleName\":\"\",\"lastName\":\"Palumbo\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2026-02-22 15:00:19\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-8939920/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-8939920/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":104827609,\"identity\":\"47df04e9-7067-4b99-b9c1-ee29fb9dc436\",\"added_by\":\"auto\",\"created_at\":\"2026-03-17 15:52:02\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":3552883,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eEffect of increasing concentrations of TNF-α on sperm motility and mitochondrial membrane potential (ΔΨm). (A) Motility parameters were assessed using Computer-Aided Semen Analysis (CASA), including VCL (curvilinear velocity, μm/s), VSL (straight-line velocity, μm/s), and VAP (average path velocity, μm/s). (B) Mitochondrial membrane potential (ΔΨm) was evaluated by flow cytometry following JC-1 staining. Means ± SD of five independent experiments from different donors. No significant differences in motility parameters were observed across TNF-α concentrations (ANOVA, p = 0.3). B: *p \\u0026lt; 0.05 vs. all the others (Tukey’s HSD test).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8939920/v1/fc835b7a9c0927f2b5fb9073.png\"},{\"id\":104835151,\"identity\":\"33022fbf-838b-460a-91c0-64393787abc5\",\"added_by\":\"auto\",\"created_at\":\"2026-03-17 17:41:05\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":8482358,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eWestern blot analysis of TNFR1 expression in motile sperm suspensions isolated from ejaculates of six different donors. The presence of the immunoreactive band for TNFR1 was ascertained with a specific monoclonal antibody able to detect a single band in positive controls (Jurkat cells). Β-actin was used as a loading control. The expected molecular masses for TNFR1 and β-actin are approximately 50 kDa and 43 kDa, respectively. Densitometric analysis of TNFR1 bands normalized to β-actin is shown in the lower panel. Data are presented as mean ± SD of three experiments. Full-length blots are shown in Supplementary Figure S1.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8939920/v1/fd79e6661c3753c04935a622.png\"},{\"id\":104827614,\"identity\":\"210cf9d3-5205-4cec-91b8-56aa052c09e5\",\"added_by\":\"auto\",\"created_at\":\"2026-03-17 15:52:02\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":13033933,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eColocalization of TNFR1 and caveolin-1 (Cav-1) in human spermatozoa. The panel on the upper left side (A) is a representative phase contrast photomicrograph of a spermatozoon from a sample stained with (B) anti-TNFR1 monoclonal antibody, detected by DyLight® 594-labeled secondary antibody, and (C) anti-Cav-1 monoclonal antibody, detected by Alexa Fluor® 488-labeled secondary antibody, as described in materials and methods. In the double immunofluorescent labeling (D), the red signal, representing TNFR1, coincided with the green signal, representing Cav-1, on large areas of the sperm principal piece, as indicated by the yellowish color. TNFR1/Cav-1 colocalization was confirmed by the high values of Pearson’s correlation coefficient (PCC) and Manders’ overlap coefficient (MOC) in the region of interest (ROI). In negative control (E-F), primary antibodies were omitted.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8939920/v1/00cba407b473a7867f30678d.png\"},{\"id\":104827613,\"identity\":\"213d7f90-4786-489c-abcc-03e8d93bb267\",\"added_by\":\"auto\",\"created_at\":\"2026-03-17 15:52:02\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":3670090,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eWestern Blot analysis for TNFR1-associated death-domain (TRADD) (A), and FAS-associated death-domain (FADD) (B) was performed on protein extracts of human motile sperm suspensions and in Jurkat cells (positive control). Densitometric analysis is presented normalizing vs α-tubulin. Representative images from three independent experiments are presented. Data are expressed as mean ± SEM. *p\\u0026lt; 0.05 with Student’s t-test. Full-length blots are shown in Supplementary Figure S1.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8939920/v1/e8bde6b0d2d1d9d74712aceb.png\"},{\"id\":104835424,\"identity\":\"62ea172c-d9c1-4f9d-a59c-b1536c70fa9c\",\"added_by\":\"auto\",\"created_at\":\"2026-03-17 17:44:44\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":3708796,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eWestern blot assay for pro-caspase-8 was performed on protein extracts of human motile sperm suspensions and in Jurkat cells (positive control), either untreated (NT), or treated with TNF-α (10 ng/ml), or staurosporine (10 μM). Densitometric analysis is presented normalizing vs α-tubulin. Representative blots and quantification are shown. Full-length blots are shown in Supplementary Figure S1.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8939920/v1/22b7c4bada4e796f1493db84.png\"},{\"id\":104827610,\"identity\":\"dedba853-ebbf-46eb-bf21-7f308cb3d340\",\"added_by\":\"auto\",\"created_at\":\"2026-03-17 15:52:02\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":4359046,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eEffect of TNF-αon caspase-8 and caspase-3 activation in human spermatozoa. (A) Caspase-8 and (B) caspase-3 activity were assessed by flow cytometry using FITC-conjugated fluorogenic inhibitors (FITC-IETD-FMK and FITC-DEVD-FMK, respectively). Spermatozoa were treated with TNF-α (0.5–100 ng/mL) or staurosporine (10 μM, positive control). Data are expressed as mean ± SD of five independent experiments using different donor samples. For comparative analysis, one-way ANOVA followed by Tukey’s post hoc test was used (*p \\u0026lt; 0.05).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8939920/v1/9115a3c5f7cdac041f429b8e.png\"},{\"id\":104836147,\"identity\":\"a3ccd29d-5499-42b4-86c3-8fb6b8474b58\",\"added_by\":\"auto\",\"created_at\":\"2026-03-17 17:51:44\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":33632354,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8939920/v1/a443e13c-7ac0-4ddc-b2bf-e502be58ebfb.pdf\"},{\"id\":104827608,\"identity\":\"8064116c-3372-417c-9ab5-46305206d0ab\",\"added_by\":\"auto\",\"created_at\":\"2026-03-17 15:52:02\",\"extension\":\"pdf\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":474209,\"visible\":true,\"origin\":\"\",\"legend\":\"Supplentary Figure S1\",\"description\":\"\",\"filename\":\"SupplementaryFigureS1WesternBlots.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8939920/v1/8b1d1b556c7ebd1310216bdc.pdf\"}],\"financialInterests\":\"There is no conflict of interest\",\"formattedTitle\":\"\\u003cp\\u003eTnfr1-associated Signaling Proteins in Mature Human Spermatozoa\\u003c/p\\u003e\",\"fulltext\":[{\"header\":\"INTRODUCTION\",\"content\":\"\\u003cp\\u003eTumor necrosis factor alpha (TNF-α) is a pleiotropic pro-inflammatory cytokine primarily produced by activated monocytes and macrophages. It plays a key role in inflammation, immunity, and apoptosis, and its overexpression is associated with chronic inflammatory and autoimmune disorders (refs. 1,2). TNF-α induces apoptosis by binding to its type 1 receptor (TNFR1), which activates a series of adaptor proteins, including TNFR-associated death domain (TRADD) and FAS-associated death domain (FADD). This process triggers the activation of caspase-8 and subsequently downstream caspase-3 (refs. 3\\u0026ndash;5). This extrinsic pathway can intersect with the intrinsic one, thanks to the ability of caspase-8 to cleave Bid, a member of the pro-apoptotic Bcl-2 family. This truncated form can translocate to the mitochondria and initiate the intrinsic pathway by promoting the release of cytochrome c (ref. 6).\\u003c/p\\u003e \\u003cp\\u003eIn the male genital tract, TNF-α can be detected in seminal plasma, particularly during inflammatory conditions (refs. 7,8) and has been implicated in sperm dysfunction. Although leukocytospermia and elevated pro-inflammatory cytokines have long been suspected contributors to male infertility, clinical findings remain inconclusive (refs. 9\\u0026ndash;14). \\u003cem\\u003eIn vitro\\u003c/em\\u003e studies investigating the effects of TNF-α on sperm have yielded conflicting results. Some studies report no impact on sperm motility or viability (refs. 15\\u0026ndash;18), while others indicate mitochondrial depolarization, DNA fragmentation, and phosphatidylserine externalization consistent with apoptosis (refs. 19\\u0026ndash;22).\\u003c/p\\u003e \\u003cp\\u003eDespite these findings, the expression and functional competence of TNFR1 in mature human spermatozoa have not been clearly defined.\\u003c/p\\u003e \\u003cp\\u003eHere, we demonstrate that human spermatozoa express TNFR1, which colocalizes with caveolin-1 in the flagellum. However, our findings indicate that TNFR1 does not efficiently engage the apoptotic cascade under the conditions tested, likely due to defective expression of TRADD, FADD, and pro-caspase-8. These data provide new insights into the apoptotic resistance of spermatozoa in inflammatory environments and suggest that the functional impact of TNF-α in mature spermatozoa may differ from classical receptor-mediated apoptotic responses.\\u003c/p\\u003e\"},{\"header\":\"RESULTS\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eExposure to TNF-α does not affect sperm motility or mitochondrial membrane potential\\u003c/h2\\u003e \\u003cp\\u003eHuman spermatozoa, isolated using the swim-up technique, were exposed to increasing concentrations of TNF-α (ranging from 0.5 to 100 ng/mL) for 2 hours. Sperm motility parameters were evaluated using computer-aided semen analysis (CASA). No significant differences in total motility or kinematic parameters (VCL, VSL, and VAP) were observed after the exposure to TNF-α (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eGiven that the effect of TNF through TNFR1 activation typically induces receptor-mediated apoptosis via caspase-8 activation and downstream mitochondrial involvement, we further examined the mitochondrial membrane potential (ΔΨm) using the JC-1 fluorescent probe. In line with the motility findings, TNF-α exposure did not significantly alter ΔΨm in spermatozoa, as evidenced by the stable red-to-green fluorescence ratio across all tested concentrations (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB). In contrast, treatment with rotenone (1 \\u0026micro;M), used as a positive control, led to a marked loss of ΔΨm, confirming the sensitivity of the assay.\\u003c/p\\u003e \\u003cp\\u003eThese results indicate that human spermatozoa do not exhibit functional responses to TNF-α under the experimental conditions tested.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eTNFR1 is expressed in human spermatozoa and colocalizes with caveolin-1\\u003c/h3\\u003e\\n\\u003cp\\u003eWestern blot analysis of sperm lysates from six normozoospermic donors revealed a single immunoreactive band at ~\\u0026thinsp;50 kDa, consistent with TNFR1 expression (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e), as previously reported in other cell types (refs. 23\\u0026ndash;24).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e TNFR1 localization was further investigated by confocal immunofluorescence analysis. The TNFR1 staining appeared to be diffuse and punctate, with predominant localization along the tail and, to a lesser extent, in the head (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB). A similar staining pattern was observed for caveolin-1 (Cav-1) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC). Merged images demonstrated substantial colocalization of TNFR1 and Cav-1, particularly along the principal piece of the tail (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD). Quantitative colocalization was confirmed using Pearson\\u0026rsquo;s correlation coefficient and Manders\\u0026rsquo; overlap coefficient. No staining was observed in negative controls where primary antibodies were omitted (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eF).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\\n\\u003ch3\\u003eDisruption of the TNFR1 downstream signaling cascade in spermatozoa\\u003c/h3\\u003e\\n\\u003cp\\u003eDespite confirming TNFR1 expression, the lack of functional response to TNF-α prompted us to explore the expression of downstream apoptotic mediators. Western blot analysis revealed significantly lower levels of TRADD in spermatozoa compared to Jurkat cells used as positive control (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA). Similarly, the expression of FADD was almost undetectable, with only a faint band observed at the expected 27 kDa (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB). These findings indicate a disruption of the TNFR1 signaling cascade at the level of adaptor protein recruitment.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eTo further characterize this defect, the expression of pro-caspase-8 in spermatozoa exposed to TNF-α (10 ng/mL) or staurosporine (10 \\u0026micro;M) was evaluated. While Jurkat cells exhibited a progressive reduction in pro-caspase-8 levels in response to pro-apoptotic stimuli, human spermatozoa displayed no detectable expression of this protein under any condition (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\\n\\u003ch3\\u003eLack of caspase activation in response to TNF-α\\u003c/h3\\u003e\\n\\u003cp\\u003eWe next assessed the activation of caspase-8 and caspase-3 in live human sperm cells using FITC-conjugated fluorogenic inhibitors (IETD-FMK for caspase-8 and DEVD-FMK for caspase-3). Our experiments showed that exposure to increasing concentrations of TNF-α failed to induce significant activation of either caspase-8 or caspase-3 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eA and B). As expected, caspase-3 activation was strongly induced by staurosporine. However, caspase-8 activation remained negligible even under this condition, confirming the extremely limited expression and activation potential of this caspase initiator in human spermatozoa.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\"},{\"header\":\"DISCUSSION\",\"content\":\"\\u003cp\\u003eThis study demonstrates that, although human spermatozoa express TNFR1, the receptor fails to engage the canonical extrinsic apoptotic cascade under the experimental conditions tested. These findings refine current assumptions about cytokine-induced damage to sperm and suggest a functional uncoupling between TNFR1 expression and apoptotic signaling in mature male gametes. More broadly, these findings suggest that death receptor expression and signaling competence may become developmentally uncoupled during terminal differentiation, reflecting a selective remodeling of apoptotic pathways in highly specialized cells.\\u003c/p\\u003e \\u003cp\\u003eThis dissociation between receptor presence and downstream signaling competence raises broader questions regarding the structural and functional plasticity of apoptotic pathways in terminally differentiated cells. While death receptor signaling is typically considered a conserved and tightly regulated cascade, our findings suggest that selective loss or attenuation of key adaptor components may represent a physiological mechanism to preserve cellular specialization in post-mitotic cells. In this context, spermatozoa provide a unique model to explore how canonical death pathways can be developmentally reconfigured without complete elimination of receptor expression.\\u003c/p\\u003e \\u003cp\\u003eTNFR1 is a canonical death receptor that triggers receptor-mediated apoptosis upon ligand binding. This process involves the sequential recruitment of TRADD and FADD, which subsequently activate caspase-8 and the executioner caspase-3 (ref. 25). This pathway also intersects with the mitochondrial apoptotic axis via tBID-mediated inhibition of BCL2 and activation of BAX/BAK, resulting in mitochondrial outer membrane permeabilization, ΔΨm loss, and cytochrome c release\\u0026mdash;culminating in caspase-9 and caspase-3 activation. To investigate whether this pathway is functional in spermatozoa, we assessed mitochondrial membrane potential and motility in sperm treated with TNF-α. We found that neither parameter was significantly altered, suggesting that TNF-α fails to initiate death signaling in these cells. This is further supported by the absence or markedly low expression of adaptor proteins TRADD and FADD, and the undetectable levels and activation of pro-caspase-8, reinforcing the notion of an incomplete or functionally constrained TNFR1-mediated apoptotic axis in human sperm.\\u003c/p\\u003e \\u003cp\\u003eIt should be noted that TNF-α signals through two distinct receptors, TNFR1 and TNFR2, which mediate partially overlapping but functionally divergent pathways. While TNFR1 contains a death domain and is directly linked to extrinsic apoptotic signaling, TNFR2 primarily activates non-apoptotic pathways, including NF-κB\\u0026ndash;dependent responses. The present study specifically addressed the competence of the TNFR1-mediated apoptotic axis; therefore, potential contributions of TNFR2 to non-apoptotic TNF-α signaling in mature spermatozoa warrant further investigation.\\u003c/p\\u003e \\u003cp\\u003eAlthough previous studies reported mild adverse effects of TNF-α on sperm motility and DNA integrity, these changes were limited and only evident after prolonged exposures (refs. 20\\u0026ndash;22). Furthermore, these effects were reversed by TNF-α antagonists such as infliximab or etanercept. Importantly, these earlier studies did not directly demonstrate the expression of TNFR1 in human spermatozoa. Our Western blot analysis confirms the presence of TNFR1 in human sperm as a distinct\\u0026thinsp;~\\u0026thinsp;50 kDa band, consistent with reports in somatic cells (refs. 23\\u0026ndash;24).\\u003c/p\\u003e \\u003cp\\u003eTNFR1 signaling has been shown to originate in caveolae-like membrane microdomains which are rich in cholesterol and sphingolipids and anchored by caveolin-1 (refs. 26\\u0026ndash;28). Confocal microscopy revealed that TNFR1 is localized along the sperm tail, specifically colocalizing with caveolin-1 in the principal piece. Quantitative image analysis confirmed this colocalization, indicating a membrane environment that is conducive to receptor signaling. However, despite this spatial organization, no functional signaling occurred.\\u003c/p\\u003e \\u003cp\\u003eMolecular analyses indicated markedly reduced expression of TRADD and FADD, along with a near-complete absence of pro-caspase-8, as determined by Western blot and flow cytometry. The activation of caspase-8 remained undetectable even following staurosporine treatment, consistent with reports describing the inability of mature sperm to undergo receptor-mediated apoptosis (refs. 29\\u0026ndash;32). Although some studies have reported the presence and activity of caspase-8 in ejaculated sperm (ref. 33), the prevailing evidence supports the idea that the extrinsic apoptotic pathway is inactive in these terminally differentiated cells. In contrast, the intrinsic, mitochondria-driven apoptotic machinery is intact and functionally relevant (refs. 34\\u0026ndash;36). Indeed, oxidative stress appears to be the dominant mechanism of sperm injury in leukocytospermia and genital tract inflammation, rather than cytokine-mediated signaling (ref. 37).\\u003c/p\\u003e \\u003cp\\u003eThe question then arises: why do spermatozoa express TNFR1 at all? TNFR1 has been proposed to play a physiological role during spermatogenesis, particularly within the seminiferous epithelium. It is highly expressed in intratubular germ cells, including spermatocytes and round spermatids, which also secrete TNF-α (refs. 38\\u0026ndash;40). TNF-α has been shown to modulate the expression of aromatase in germ cells, promoting estrogen biosynthesis in pachytene spermatocytes and inhibiting it in round spermatids (ref. 41). More recently, TNF-α signaling has been implicated in immunosuppressive interactions between spermatozoa and the female reproductive tract, such as the downregulation of pro-inflammatory cytokine expression in human fallopian tube epithelial cells (ref. 42).\\u003c/p\\u003e \\u003cp\\u003eTaken together, our findings support a model in which TNFR1 fulfills physiological functions during earlier stages of germ cell development but becomes progressively uncoupled from its pro-apoptotic signaling machinery during the final phases of sperm differentiation. In mature spermatozoa, persistent receptor expression may reflect selective protein retention or alternatively serve non-apoptotic roles, potentially including modulation of extracellular TNF-α within the reproductive tract.\\u003c/p\\u003e \\u003cp\\u003eMore broadly, our data reveal a dissociation between receptor presence and downstream signaling competence, consistent with selective remodeling of the extrinsic apoptotic pathway during terminal differentiation. Although TNFR1 remains expressed and localized within caveolin-1\\u0026ndash;enriched membrane domains, the marked reduction or absence of TRADD, FADD, and pro-caspase-8 prevents engagement of the canonical death cascade. These observations suggest that the functional consequences of inflammatory cytokine signaling in mature spermatozoa may operate through mechanisms distinct from classical receptor-mediated apoptosis.\\u003c/p\\u003e\"},{\"header\":\"MATERIALS AND METHODS\",\"content\":\"\\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eSemen samples\\u003c/h2\\u003e \\u003cp\\u003eEjaculates of healthy donors were collected by masturbation following an abstinence period of 3\\u0026ndash;7 days. Donors were students or post-graduate students from the University of L\\u0026rsquo;Aquila, who had no known prior male reproductive pathologies including varicocele and infection. All samples were normozoospermic according to the World Health Organization (ref. 43) and showed no evidence of leukocytospermia. After collecting, samples were left for at least 30 min to liquefy before processing.\\u003c/p\\u003e \\u003cp\\u003e The study was approved by the local Institutional Review Board, and all subjects signed an informed consent statement. The study was performed in accordance with the Declaration of Helsinki.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eIn vitro exposure of donor spermatozoa to TNF-α\\u003c/h3\\u003e\\n\\u003cp\\u003e \\u003cdiv class=\\\"BlockQuote\\\"\\u003e \\u003cp\\u003eMotile sperm suspensions were obtained by swim-up procedure from six different donors. Briefly, spermatozoa were washed twice (700 \\u0026times; g for 7 min) in Biggers, Whitten, and Wittingham (BWW) with 0.1% human serum albumin (HSA) fraction V (Sigma-Aldrich S.r.l., Milan, Italy). After the second centrifugation, supernatants were removed by aspiration, leaving approximately 0.5 ml of medium on the pellet and, after an incubation time of 30 min, supernatants containing highly concentrated motile sperms were carefully aspirated and the sperm concentration was properly adjusted. In different settings, aliquots of the same motile sperm suspensions were exposed for 2 h to scalar concentrations of TNF-α (from 0.5 to 100 ng/ml) (EuroClone, Pero, Italy) and then subjected to the assessment of motility, sperm mitochondrial membrane potential (ΔΨm) and activated caspase-8 and 3, as described below.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/p\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eEvaluation of sperm motility\\u003c/h2\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"BlockQuote\\\"\\u003e \\u003cp\\u003eSperm motility was evaluated by Computer-Aided Semen Analysis (CASA), using Hamilton Thorne (EOS srl, Firenze, Italy). Ten microliters of each sperm sample were placed into a pre-warmed (37\\u0026deg;C) Makler counting chamber (Sefi Medical Instruments, Haifa, Israel). At least 200 spermatozoa were evaluated for each sample. Setting parameters were the following: analysis duration of 1 s (30 frames); minimum contrast, 80; minimum size, 3; low size gate, 0.7; high size gate, 2.6; low intensity gate, 0.34; light intensity gate, 1.40. Spermatozoa exhibiting an average pathway velocity\\u0026thinsp;\\u0026gt;\\u0026thinsp;5 \\u0026micro;m/s were categorized by the software as motile spermatozoa.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eFlow cytometric evaluation of ΔΨm\\u003c/h2\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"BlockQuote\\\"\\u003e \\u003cp\\u003eThe fluorescent lipophilic cationic dye 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benimidazolyl carbocyanine iodide (JC-1) (Sigma-Aldrich S.r.l., Milan, Italy) was used to evaluate trans-membrane potential of sperm mitochondria, as previously described (refs. 44,45). This probe possesses the unique ability to differentially label mitochondria with low and high ΔΨm. In mitochondria with high ΔΨm, JC-1 forms multimeric aggregates that emit orange light (wavelength of 590 nm) when excited at 488 nm. In mitochondria with low ΔΨm, JC-1 forms monomers that emit green light (525\\u0026ndash;530 nm) when excited at 488 nm. Motile sperm suspensions, each containing 5 \\u0026times; 10\\u003csup\\u003e6\\u003c/sup\\u003e spermatozoa were diluted in 1 mL of PBS to give a final sperm concentration of 1.5-2.0 \\u0026times; 10\\u003csup\\u003e6\\u003c/sup\\u003e/mL and then stained with 0.5 \\u0026micro;L of JC-1 stock solution (3 mM in DMSO). Samples were incubated at 37\\u0026deg;C in the dark for 60 min and then analyzed at the flow cytometer equipped with a 15-mW argon-ion laser for excitation. For each sample 10 000 events were recorded at a flow rate of 200\\u0026ndash;300 cells/s. Based on the light scatter characteristics of swim up selected spermatozoa, debris and aggregates were gated out by establishing a region around the population of interest in the forward scatter/side scatter dot plot on a log scale. Compensation between FL1and FL2 was carefully adjusted according to the manufacturer\\u0026rsquo;s instructions. Green and orange-red fluorescence was measured in FL-1 and FL-2 channel, respectively. The percentage of positive cells was evaluated using the flow cytometer System II Version 3.0 software (Beckman Coulter, Inc.).\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eWestern blot analysis\\u003c/h2\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"BlockQuote\\\"\\u003e \\u003cp\\u003e50 \\u0026times; 10\\u003csup\\u003e6\\u003c/sup\\u003e motile sperm, obtained by swim-up procedure, were stimulated with 10 ng/ml of recombinant TNF-α or with staurosporine (10 \\u0026micro;M) for 2 hours at 37\\u0026deg;C in an atmosphere of 5%CO\\u003csub\\u003e2\\u003c/sub\\u003e/95% air. The motile spermatozoa were washed in phosphate-buffered saline (PBS) and homogenized and lysed in ice-cold RIPA buffer (Merck KGaA, Darmstadt, Germany) containing 100 mM protease inhibitor cocktail (Sigma-Aldrich, Saint Louis, MO, USA). The protein amount was evaluated with DC Protein Assay (Bio-Rad, Hercules, CA, USA) using BSA as standard. Total cell lysates (25 \\u0026micro;g protein) were mixed with sample buffer; samples were denatured (at 100\\u0026deg;C for 5 min), subjected to 12% SDS polyacrylamide gel, and electroblotted onto 0.45 \\u0026micro;m nitrocellulose membrane sheets (Bio-Rad) for 1 hour at 4\\u0026deg;C at 70 V using a Mini Trans-Blot Cell apparatus (Bio-Rad). Non-specific binding sites were blocked with 5% non-fat dry milk for 1 h at room temperature and membranes were incubated overnight at 4\\u0026deg;C with primary antibodies (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). As secondary antibodies, peroxidase-conjugated anti-rabbit and anti-mouse IgG antibodies (dilution 1:2 000) were acquired from Sigma-Aldrich. The immunoreactive bands were visualized by enhanced chemiluminescent (ECL, Amersham Pharmacia Biotech, Buckinghamshire, UK) according to the manufacturer instructions and acquired by UVItec Alliance (Cambridge, UK). As positive control, homogenates of Jurkat cells (human T cell lymphoblast-like cell line from leukemia) were used under the same experimental conditions.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eList of primary antibodies used in the present study.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"3\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003ePrimary antibody\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eDilution\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eCompany\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eRabbit monoclonal\\u003c/p\\u003e \\u003cp\\u003eanti-TNF-R1/CD120a\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e1:1000\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eInvitrogen Corporation, Waltham, MA, USA\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eRabbit monoclonal anti-TRADD\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e1:1000\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eInvitrogen Corporation, Waltham, MA, USA\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eMouse monoclonal anti-FADD\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e1:2000\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eInvitrogen Corporation, Waltham, MA, USA\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eMouse monoclonal\\u003c/p\\u003e \\u003cp\\u003eanti-pro-caspase 8\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e1:1000\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eImmunological Sciences, Rome, Italy\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eMouse monoclonal anti-α-tubulin\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e1:4000\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eSigma-Aldrich, Saint Louis, MO, USA\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003eFull-length uncropped western blots corresponding to all figures are provided in Supplementary Figure \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eTNFR1/Cav-1 co-localization in human spermatozoa: immunofluorescence staining in confocal microscopy\\u003c/h2\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"BlockQuote\\\"\\u003e \\u003cp\\u003eMotile sperm suspensions, obtained by swim-up procedure, were fixed with ice-cold formaldehyde (1%, v/v) in PBS, pH 7.4 for 30 min at 4\\u0026ordm;C and then permeabilized with 0.1%, v/v Triton X-100 (Bio-Rad, Hercules, CA, USA) in PBS for 10 min at room temperature. Non-specific binding sites were blocked with 4% bovine serum albumin (BSA) (Sigma-Aldrich S.r.l., Milan, Italy) for 10 minutes at room temperature. Sperm suspensions were then incubated with mouse anti-human TNF-R1/CD120a mAb (dilution 1:100) in blocking buffer for 1 h at room temperature. Co-localization of TNFR1 with Cav-1 was also explored using a rabbit anti-human Cav-1 mAb (ThermoFisher Scientific, Monza, Italy; dilution 1:100) in blocking buffer for 1 h at room temperature. In control samples, primary antibodies were omitted. After multiple washes by centrifugations (700 \\u0026times; \\u003cem\\u003eg\\u003c/em\\u003e, 7 min), spermatozoa were incubated with the DyLight\\u0026reg; 594 conjugated donkey anti-mouse (Aurogene S.r.l. Roma, Italy; dilution 1:2 000) and/or a Alexa Fluor\\u0026reg; 488 conjugated goat anti-rabbit (ThermoFisher Scientific, Monza, Italy; dilution 1:2 000) for 1 h at room temperature, washed again as above and subsequently smeared on slides. Slides were mounted with PBS\\u0026ndash;glycerol to be examined by confocal microscopy (Leica TCS SP5 II, Wetzlar, Germany). In the merged images, the degree of colocalization within a specific region of interest (ROI) was estimated by using two quantitative coefficients: Pearson\\u0026rsquo;s Correlation Coefficient (PCC) and Manders\\u0026rsquo; overlap coefficient (MOC). The former describes the correlation of the intensity distribution between channels and ranges from \\u0026minus;\\u0026thinsp;1 (fully negative correlation) to +\\u0026thinsp;1 (completely positive correlation), with zero standing for no correlation; the latter indicates an actual overlap of the signals and ranges from 0 (non-overlapping images) to +\\u0026thinsp;1 (100% colocalization) (refs. 47\\u0026ndash;48).\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eFlow cytometric assessment of activated caspases\\u003c/h2\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"BlockQuote\\\"\\u003e \\u003cp\\u003eCaspase-8 and caspase-3 activation was evaluated using Apoptosis CaspGLOW Fluorescein Active Staining Kits for Caspase-8 and Caspase-3 (BioVision, Inc., Mountain View, CA, USA). These assays employ FITC-conjugated inhibitors\\u0026mdash;IETD-FMK for caspase-8 and DEVD-FMK for caspase-3\\u0026mdash;as fluorescent markers. These cell-permeable, non-toxic peptides covalently bind to activated caspase-8 and caspase-3, serving as in situ markers for apoptosis. After a 2 h exposure to increasing concentration of TNF-α or to staurosporine (10 \\u0026micro;M) (Sigma-Aldrich S.r.l., Milan, Italy), as positive control, 1 \\u0026micro;L of FITC-IETD-FMK or FITC-DEVD-FMK was added to 300 \\u0026micro;L of sperm suspension containing 0.3 \\u0026times; 10\\u003csup\\u003e6\\u003c/sup\\u003e spermatozoa. After 1 h incubation at 37\\u0026deg;C in an atmosphere of 5%CO\\u003csub\\u003e2\\u003c/sub\\u003e/95% air, samples were centrifuged three times (1 000 \\u0026times; g for 4 min) and analyzed by flow cytometry. For each sample 10 000 events were recorded at a flow rate of 200\\u0026ndash;300 cells/s.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStatistical analysis\\u003c/h2\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"BlockQuote\\\"\\u003e \\u003cp\\u003eStatistical analysis was performed using the R statistical software (version 4.5.1, 2025, The R Foundation for Statistical Computing, Vienna, Austria). The Student\\u0026rsquo;s t-test was also performed where indicated. After assessing the distribution of data with the Shapiro-Wilk test, the Wilcoxon rank-sum test or ANOVA followed by post hoc comparisons by the Tukey\\u0026rsquo;s studentized range-honestly significant difference (HSD) test were used, as appropriate. Data were expressed as mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SD of independent experiments performed in triplicate and statistical significance was accepted when p\\u0026thinsp;\\u0026le;\\u0026thinsp;0.05.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgments:\\u003c/strong\\u003e The authors thank Prof. Maria Grazia Cifone for her precious and valuable advice and for reviewing manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConflict of interest:\\u003c/strong\\u003e The authors declare no conflict of interest.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contributions:\\u003c/strong\\u003e A.B. conceived the study, performed the statistical analysis, and critically revised the manuscript; C.T. and V.D. contributed to the statistical analysis and the drafting of the first version of the manuscript; C.C., F.R.A., and P.P. contributed to the experimental procedures and data collection; C.M. and D.T. contributed to the statistical analysis and the critical revision of the manuscript; B.C. supervised the experiments and contributed to the critical revision of the manuscript. In Figure 1 C.C. performed CASA analysis and flow cytometric assessment of mitochondrial potential. In Figure 2, Figure 4 and Figure 5 C.C. processed semen samples, F.R.A. and P.P. performed western blot assays. In Figure 3 C.C. performed immunofluorescence evaluation. In Figure 6 C.C. performed flow cytometric caspase assays.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics Approval and Consent to Participate:\\u003c/strong\\u003e The study was approved by the local Institutional Review Board, and all subjects signed an informed consent statement. The study was performed in accordance with the Declaration of Helsinki.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding:\\u003c/strong\\u003e The authors received no specific funding for this work.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData Availability Statement:\\u003c/strong\\u003e The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eIdriss HT \\u0026amp; Naismith JH. TNF alpha and the TNF receptor superfamily: structure-function relationship(s). \\u003cem\\u003eMicrosc Res Tech\\u003c/em\\u003e 50, 184\\u0026ndash;195 (2000).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eApostolaki M, Armaka M, Victoratos P \\u0026amp; Kollias G. Cellular mechanisms of TNF function in models of inflammation and autoimmunity. \\u003cem\\u003eCurr Dir Autoimmun\\u003c/em\\u003e 11, 1\\u0026ndash;26 (2010).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHsu H, Shu HB, Pan MG \\u0026amp; Goeddel DV. TRADD-TRAF2 and TRADD-FADD interactions define two distinct TNF receptor 1 signal transduction pathways. \\u003cem\\u003eCell\\u003c/em\\u003e 84, 299\\u0026ndash;308 (1996).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSch\\u0026uuml;tze S, Tchikov V \\u0026amp; Schneider-Brachert W. Regulation of TNFR1 and CD95 signalling by receptor compartmentalization. \\u003cem\\u003eNat Rev Mol Cell Biol\\u003c/em\\u003e 9, 655\\u0026ndash;662 (2008).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHeinrich M. et al. Cathepsin D links TNF-induced acid sphingomyelinase to Bid-mediated caspase-9 and \\u0026ndash;\\u0026thinsp;3 activation. \\u003cem\\u003eCell Death Differ\\u003c/em\\u003e 11, 550\\u0026ndash;563 (2004).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMustafa M. et al. Apoptosis: A Comprehensive Overview of Signaling Pathways, Morphological Changes, and Physiological Significance and Therapeutic Implications. \\u003cem\\u003eCells\\u003c/em\\u003e 13, 1838 (2024).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBronson R, Cooper G \\u0026amp; Rosenfeld D. Sperm antibodies: their role in infertility. \\u003cem\\u003eFertil Steril\\u003c/em\\u003e 42, 171\\u0026ndash;183 (1984).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGruschwitz MS, Brezinschek R \\u0026amp; Brezinschek HP. Cytokine levels in the seminal plasma of infertile males. \\u003cem\\u003eJ Androl\\u003c/em\\u003e 17, 158\\u0026ndash;163 (1996).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWolff H. The biologic significance of white blood cells in semen. \\u003cem\\u003eFertil Steril\\u003c/em\\u003e 63, 1143\\u0026ndash;1157 (1995).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eOchsendorf FR. Infections in the male genital tract and reactive oxygen species. \\u003cem\\u003eHum Reprod Update\\u003c/em\\u003e 5, 399\\u0026ndash;420 (1999).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWhittington K \\u0026amp; Ford WC. Relative contribution of leukocytes and of spermatozoa to reactive oxygen species production in human sperm suspensions. \\u003cem\\u003eInt J Androl\\u003c/em\\u003e 22, 229\\u0026ndash;235 (1999).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHenkel R. et al. Effect of reactive oxygen species produced by spermatozoa and leukocytes on sperm functions in non-leukocytospermic patients. \\u003cem\\u003eFertil Steril\\u003c/em\\u003e 83, 635\\u0026ndash;642 (2005).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAgarwal A. et al. Reactive oxygen species and sperm DNA damage in infertile men presenting with low level leukocytospermia. \\u003cem\\u003eReprod Biol Endocrinol\\u003c/em\\u003e 12, 126 (2014).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eCastellini, C. et al. Relationship between leukocytospermia, reproductive potential after assisted reproductive technology, and sperm parameters: a systematic review and meta-analysis of case-control studies. \\u003cem\\u003eAndrology\\u003c/em\\u003e 8, 125\\u0026ndash;135 (2020).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWincek TJ, Meyer TK, Meyer MR \\u0026amp; Kuehl TJ. Absence of a direct effect of recombinant tumor necrosis factor-alpha on human sperm function and murine preimplantation development. \\u003cem\\u003eFertil Steril\\u003c/em\\u003e 56, 332\\u0026ndash;339 (1991).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHaney AF, Hughes SF \\u0026amp; Weinberg JB. The lack of effect of tumor necrosis factor-alpha, interleukin-1-alpha, and interferon-gamma on human sperm motility in vitro. \\u003cem\\u003eJ Androl\\u003c/em\\u003e 13, 249\\u0026ndash;253 (1992).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eFedder J \\u0026amp; Ellerman-Eriksen S. Effect of cytokines on sperm motility and ionophore-stimulated acrosome reaction. \\u003cem\\u003eArch Androl\\u003c/em\\u003e 35, 173\\u0026ndash;185 (1995).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLewis SE, Donnelly ET, Sterling ES, Kennedy MS, Thompson W \\u0026amp; Chakravarthy U. Nitric oxide synthase and nitrite production in human spermatozoa: evidence that endogenous nitric oxide is beneficial to sperm motility. \\u003cem\\u003eMol Hum Reprod\\u003c/em\\u003e 2, 873\\u0026ndash;878 (1996).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eEstrada LS et al. Effect of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on human sperm motility, viability and motion parameters. \\u003cem\\u003eInt J Androl\\u003c/em\\u003e 20, 237\\u0026ndash;242 (1997).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSaid TM, Agarwal A, Falcone T, Sharma RK, Bedaiwy MA \\u0026amp; Li L. Infliximab may reverse the toxic effects induced by tumor necrosis factor alpha in human spermatozoa: an in vitro model. \\u003cem\\u003eFertil Steril\\u003c/em\\u003e 83, 1665\\u0026ndash;1673 (2005).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePerdichizzi A. et al. Effects of tumour necrosis factor-alpha on human sperm motility and apoptosis. \\u003cem\\u003eJ Clin Immunol\\u003c/em\\u003e 27, 152\\u0026ndash;162 (2007).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePascarelli NA, Fioravanti A, Moretti E, Guidelli GM, Mazzi L \\u0026amp; Collodel G. The effects in vitro of TNF-α and its antagonist 'etanercept' on ejaculated human sperm. \\u003cem\\u003eReprod Fertil Dev\\u003c/em\\u003e 29, 1169\\u0026ndash;1177 (2017).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLoetscher H, Schlaeger EJ, Lahm HW, Pan YC, Lesslauer W \\u0026amp; Brockhaus M. Purification and partial amino acid sequence analysis of two distinct tumor necrosis factor receptors from HL60 cells. \\u003cem\\u003eJ Biol Chem\\u003c/em\\u003e 265, 20131\\u0026ndash;20138 (1990).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSchall TJ et al. Molecular cloning and expression of a receptor for human tumor necrosis factor. \\u003cem\\u003eCell\\u003c/em\\u003e 61, 361\\u0026ndash;370 (1990).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHotchkiss RS, Strasser A, McDunn JE \\u0026amp; Swanson PE. Cell death. \\u003cem\\u003eN Engl J Med\\u003c/em\\u003e 361, 1570\\u0026ndash;1583 (2009).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKo YG, Lee JS, Kang YS, Ahn JH \\u0026amp; Seo JS. TNF-alpha-mediated apoptosis is initiated in caveolae-like domains. \\u003cem\\u003eJ Immunol\\u003c/em\\u003e 162, 7217\\u0026ndash;7223 (1999).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eD'Alessio A, Al-Lamki RS, Bradley JR \\u0026amp; Pober JS. Caveolae participate in tumornecrosis factor receptor 1 signaling and internalization in a human endothelial cell line. \\u003cem\\u003eAm J Pathol\\u003c/em\\u003e 166, 1273\\u0026ndash;1282 (2005).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eDargelos E, Renaud V, Decossas M, Bure C, Lambert O \\u0026amp; Poussard S. Caveolae-mediated effects of TNF-α on human skeletal muscle cells. \\u003cem\\u003eExp Cell Res\\u003c/em\\u003e 370, 623\\u0026ndash;631 (2018).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLachaud C, Tesarik J, Ca\\u0026ntilde;adas ML \\u0026amp; Mendoza C. Apoptosis and necrosis in human ejaculated spermatozoa. \\u003cem\\u003eHum Reprod\\u003c/em\\u003e 19, 607\\u0026ndash;610 (2004).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKoppers AJ, Mitchell LA, Wang P, Lin M \\u0026amp; Aitken RJ. Phosphoinositide 3-kinase signalling pathway involvement in a truncated apoptotic cascade associated with motility loss and oxidative DNA damage in human spermatozoa. \\u003cem\\u003eBiochem J\\u003c/em\\u003e 436, 687\\u0026ndash;698 (2011).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGrunewald S, Sharma R, Paasch U, Glander HJ \\u0026amp; Agarwal A. Impact of caspase activation in human spermatozoa. \\u003cem\\u003eMicrosc Res Tech\\u003c/em\\u003e 72, 878\\u0026ndash;888 (2009).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWeil M, Jacobson MD \\u0026amp; Raff MC. Are caspases involved in the death of cells with a transcriptionally inactive nucleus? Sperm and chicken erythrocytes. \\u003cem\\u003eJ Cell Sci\\u003c/em\\u003e 111, 2707\\u0026ndash;2715 (1998).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePaasch U, Grunewald S, Agarwal A \\u0026amp; Glandera HJ. Activation pattern of caspases in human spermatozoa. \\u003cem\\u003eFertil Steril\\u003c/em\\u003e 81 Suppl 1, 802\\u0026ndash;809 (2004).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAitken RJ \\u0026amp; Koppers AJ. Apoptosis and DNA damage in human spermatozoa. \\u003cem\\u003eAsian J Androl\\u003c/em\\u003e 13, 36\\u0026ndash;42 (2011).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAitken RJ, Findlay JK, Hutt KJ \\u0026amp; Kerr JB. Apoptosis in the germ line. \\u003cem\\u003eReproduction\\u003c/em\\u003e 141, 139\\u0026ndash;150 (2011).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAitken RJ \\u0026amp; Baker MA. Causes and consequences of apoptosis in spermatozoa; contributions to infertility and impacts on development. \\u003cem\\u003eInt J Dev Biol\\u003c/em\\u003e 57, 265\\u0026ndash;272 (2013).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAitken RJ \\u0026amp; De Iuliis GN. On the possible origins of DNA damage in human spermatozoa. \\u003cem\\u003eMol Hum Reprod\\u003c/em\\u003e 16, 3\\u0026ndash;13 (2010).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eDe SK, Chen HL, Pace JL, Hunt JS, Terranova PF \\u0026amp; Enders GC. Expression of tumor necrosis factor-alpha in mouse spermatogenic cells. \\u003cem\\u003eEndocrinology\\u003c/em\\u003e 133, 389\\u0026ndash;396 (1993).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBialas M, Fiszer D, Rozwadowska N, Kosicki W, Jedrzejczak P \\u0026amp; Kurpisz M. The role of IL-6, IL-10, TNF-α and its receptors TNFR1 and TNFR2 in the local regulatory system of normal and impaired human spermatogenesis. \\u003cem\\u003eAm J Reprod Immunol\\u003c/em\\u003e 62, 51\\u0026ndash;59 (2009).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLysiak JJ. The role of tumor necrosis factor-alpha and interleukin-1 in the mammalian testis and their involvement in testicular torsion and autoimmune orchitis. \\u003cem\\u003eReprod Biol Endocrinol\\u003c/em\\u003e 2, 9 (2004).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBourguiba S, Chater S, Delalande C, Benahmed M \\u0026amp; Carreau S. Regulation of aromatase gene expression in purified germ cells of adult male rats: Effects of transforming growth factor β, tumor necrosis factor α, and cyclic adenosine 3',5'-monosphosphate. \\u003cem\\u003eBiol Reprod\\u003c/em\\u003e 69, 592\\u0026ndash;601 (2003).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMousavi SO et al. Immunological response of fallopian tube epithelial cells to spermatozoa through modulating cytokines and chemokines. \\u003cem\\u003eJ Reprod Immunol\\u003c/em\\u003e 146, 103327 (2021).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWorld Health Organization, \\u003cem\\u003eWHO laboratory manual for the examination and processing of human semen\\u003c/em\\u003e, \\u003cem\\u003e6th edn.\\u003c/em\\u003e (WHO Press; Geneva, Switzerland, 2021).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBarbonetti A. et al. Soluble products of Escherichia coli induce mitochondrial dysfunction-related sperm membrane lipid peroxidation which is prevented by lactobacilli. \\u003cem\\u003ePLoS One\\u003c/em\\u003e 8, e83136 (2013)\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eCastellini C. et al. Effects of bisphenol S and bisphenol F on human spermatozoa: An in vitro study. \\u003cem\\u003eReprod Toxicol\\u003c/em\\u003e 103, 58\\u0026ndash;63 (2021).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBolte S \\u0026amp; Cordeli\\u0026egrave;res FP. A guided tour into subcellular colocalization analysis in light microscopy. \\u003cem\\u003eJ Microsc\\u003c/em\\u003e 224, 213\\u0026ndash;232 (2006).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZinchuk V \\u0026amp; Grossenbacher-Zinchuk O. Recent advances in quantitative colocalization analysis: focus on neuroscience. \\u003cem\\u003eProg Histochem Cytochem\\u003c/em\\u003e 44, 125\\u0026ndash;172 (2009).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAdler J \\u0026amp; Parmryd I. Quantifying colocalization by correlation: the Pearson correlation coefficient is superior to the Mander's overlap coefficient. \\u003cem\\u003eCytometry A\\u003c/em\\u003e 77, 733\\u0026ndash;742 (2010).\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"cell-death-discovery\",\"isNatureJournal\":false,\"hasQc\":false,\"allowDirectSubmit\":false,\"externalIdentity\":\"cddiscovery\",\"sideBox\":\"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)\",\"snPcode\":\"41420\",\"submissionUrl\":\"https://mts-cddiscovery.nature.com/\",\"title\":\"Cell Death Discovery\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"ejp\",\"reportingPortfolio\":\"Nature AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-8939920/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-8939920/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eTumor necrosis factor-alpha (TNF-α) is a pleiotropic cytokine that activates the extrinsic apoptotic pathway through TNFR1, leading to recruitment of adaptor proteins and caspase activation in most somatic cell types. Although TNF-α has been implicated in inflammation-associated sperm dysfunction, the expression and functional competence of TNFR1 in mature human spermatozoa remain poorly defined.\\u003c/p\\u003e \\u003cp\\u003eHere, we report that human spermatozoa express TNFR1 and that the receptor localizes along the flagellum, where it colocalizes with caveolin-1 within membrane microdomains. However, exposure to TNF-α across a wide concentration range did not affect sperm motility or mitochondrial membrane potential and failed to induce activation of caspase-8 or caspase-3. Molecular analyses revealed markedly reduced expression of the adaptor proteins TRADD and FADD and an absence of detectable pro-caspase-8 in spermatozoa compared with Jurkat cells.\\u003c/p\\u003e \\u003cp\\u003eThese findings indicate that, despite preserved receptor expression and membrane compartmentalization, the downstream extrinsic apoptotic machinery is profoundly limited in mature sperm cells. Our data support a model in which death receptor expression and downstream signaling competence become developmentally uncoupled during terminal differentiation, reflecting selective remodeling of apoptotic signaling pathways.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Tnfr1-associated Signaling Proteins in Mature Human Spermatozoa\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2026-03-17 15:51:52\",\"doi\":\"10.21203/rs.3.rs-8939920/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"revise\",\"date\":\"2026-04-16T10:35:30+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"This content is not available.\",\"date\":\"2026-04-09T04:59:28+00:00\",\"index\":3,\"fulltext\":\"This content is not available.\"},{\"type\":\"editorInvitedReview\",\"content\":\"This content is not available.\",\"date\":\"2026-04-01T13:22:08+00:00\",\"index\":2,\"fulltext\":\"This content is not available.\"},{\"type\":\"editorInvitedReview\",\"content\":\"This content is not available.\",\"date\":\"2026-03-24T09:53:36+00:00\",\"index\":1,\"fulltext\":\"This content is not available.\"},{\"type\":\"reviewerAgreed\",\"content\":\"This content is not available.\",\"date\":\"2026-03-17T02:18:19+00:00\",\"index\":3,\"fulltext\":\"This content is not available.\"},{\"type\":\"reviewerAgreed\",\"content\":\"This content is not available.\",\"date\":\"2026-03-16T08:43:34+00:00\",\"index\":2,\"fulltext\":\"This content is not available.\"},{\"type\":\"reviewerAgreed\",\"content\":\"This content is not available.\",\"date\":\"2026-03-16T01:25:43+00:00\",\"index\":1,\"fulltext\":\"This content is not available.\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2026-03-15T21:49:20+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2026-03-12T14:38:37+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2026-03-10T19:17:10+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Cell Death Discovery\",\"date\":\"2026-03-10T19:17:09+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"cell-death-discovery\",\"isNatureJournal\":false,\"hasQc\":false,\"allowDirectSubmit\":false,\"externalIdentity\":\"cddiscovery\",\"sideBox\":\"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)\",\"snPcode\":\"41420\",\"submissionUrl\":\"https://mts-cddiscovery.nature.com/\",\"title\":\"Cell Death Discovery\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"ejp\",\"reportingPortfolio\":\"Nature AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"72a23011-b2d4-40ef-9b3f-363a9196039b\",\"owner\":[],\"postedDate\":\"March 17th, 2026\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"in-revision\",\"subjectAreas\":[{\"id\":64660706,\"name\":\"Biological sciences/Cell biology/Cell signalling/Extracellular signalling molecules\"},{\"id\":64660707,\"name\":\"Biological sciences/Molecular biology/Protein folding/Protein aggregation\"}],\"tags\":[],\"updatedAt\":\"2026-04-16T10:43:04+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2026-03-17 15:51:52\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-8939920\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-8939920\",\"identity\":\"rs-8939920\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}