Epididymal epithelial cells facilitate NEU1 loading to modulate sperm α-2,6 sialylation, enhance maturation and motility

Abstract

Background

Male infertility is a prevalent reproductive disorder worldwide, with decreased sperm quality—particularly reduced motility and fertilization capacity—being one of its common causes. Sperm maturation is a complex process involving multiple molecular mechanisms, the specific pathways regulating sperm motility remain unclear and require further investigation. Among these mechanisms, sialylation serves as an important glycosylation modification during sperm maturation and capacitation, playing a crucial role in these processes. Nonetheless, the specific function of Neuraminidase-1 (NEU1), a kind of sialidase, in sperm maturation and function remains poorly understood. This study aims to investigate the origin and function of NEU1 in sperm, as well as its impact on sperm motility and fertilization potential, providing new insights into its role in male infertility.

Methods

A combination of computational prediction and experimental validation was used to assess the expression profile of sialylation-related enzymes in the male reproductive tract, identifying NEU1 as a candidate for further investigation. Immunofluorescence, Western blotting, and flow cytometry were performed to analyze the expression pattern of NEU1 in human and murine sperm. Functional studies were conducted by inhibiting NEU1 activity to examine its effects on sperm motility and fertilization capacity. Additionally, NEU1 expression was compared between asthenozoospermic and normozoospermic individuals, and in vitro fertilization (IVF) assays were performed to evaluate its role in fertilization. Furthermore, a murine model was used to explore the origin of NEU1 during sperm maturation, particularly focusing on whether it is secreted and transferred onto sperm by epididymal epithelial cells.

Results

This study demonstrated that NEU1 is highly expressed in both human and murine sperm, and its expression level is closely associated with sperm motility and fertilization capacity. Sperm with higher motility exhibited significantly elevated NEU1 expression, which positively correlated with sperm kinematic parameters such as velocity and linearity. Inhibition of NEU1 activity resulted in a marked decline in sperm motility and fertilization potential. Furthermore, NEU1 is secreted by epididymal epithelial cells and subsequently transferred to the sperm surface. It regulates α-2,6 sialylation, thereby influencing sperm maturation, energy metabolism, and capacitation. IVF assays further confirmed a significant correlation between NEU1 expression and fertilization success.

Conclusion

This study identifies NEU1 as a key regulatory enzyme on the sperm surface, directly influencing sperm motility and fertilization capacity. NEU1 is secreted by epididymal epithelial cells and transferred onto sperm, mediating sialylation modifications and regulating sperm capacitation and metabolic processes. These findings provide novel insights into the functional role of sialidases in sperm maturation and function, offering potential biomarkers and therapeutic targets for the diagnosis and treatment of male infertility.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00018-025-05909-0.

Introduction

Statistical data indicate that male factors contribute to approximately 50% of infertility cases [1]. Male infertility remains a longstanding and critical challenge in the field of assisted reproduction, with its underlying mechanisms yet to be fully elucidated. Impaired sperm quality, particularly reduced motility and fertilization capacity, is a major contributing factor [1]. Sperm quality not only influences the quality of fertilized oocytes but also impacts embryo development and ultimate live birth rates [2]. However, current semen analysis methods exhibit significant overlap in distinguishing fertile from infertile men [3, 4], underscoring the limitations of existing diagnostic approaches. Notably, decreased sperm motility represents one of the most common yet mechanistically unclear challenges in male infertility [5, 6]. Sperm motility is a key parameter in clinical assessments of male fertility [7], yet the underlying mechanisms of reduced motility remain unclear in many patients, necessitating further research to uncover the underlying mechanisms.

In recent years, post-translational modifications (PTMs) have been recognized as closely associated with sperm quality and male infertility [8]. Among these, glycosylation modifications, particularly sialylation of terminal monosaccharides, play a crucial role in sperm capacitation and fertilization [9, 10]. Sialylation is involved in sperm recognition [8] and motility [11], positioning it as a potential biochemical marker for evaluating male infertility [12, 13]. The dynamic regulation of sialylation relies on two classes of key enzymes: sialyltransferases, which catalyze the addition of sialic acid, and hydrolytic enzymes, such as sialidases, which remove sialic acid modifications. Previous studies have shown that sperm sialyltransferases mediate sialic acid modifications, enabling sperm to evade immune clearance [14–16]. However, the numerous and functionally diverse sialyltransferases contrast with the only four known types of neuraminidases (NEU) in mammals [17, 18]. Given the fewer isoforms and their more defined enzymatic roles [19], we focused our subsequent research on NEU. Moreover, our preliminary research has linked deficiencies in sperm NEU to idiopathic male infertility [15]. Nevertheless, the specific roles of NEU in sperm motility, capacitation, and fertilization remain poorly understood.

Additionally, sperm maturation is a highly dynamic process that involves a series of protein modifications and functional changes in the epididymis, ultimately leading to the acquisition of motility and fertilization capacity [20], with sialylation playing a crucial role in this process [21]. The epididymal fluid and secretions are rich in both free and conjugated sialic acids [22], which are dynamically incorporated into sperm as they transit through the elongated epididymal duct and undergo progressive maturation [23, 24]. These modifications not only influence the stability and surface charge of the sperm membrane but also affect sperm survival in the female reproductive tract and subsequent fertilization [25]. For instance, the polysialylation of neural cell adhesion molecule (NCAM) is integrated into the acrosomal posterior region during epididymal sperm maturation, facilitating the fertilization process [14]. Given this, sperm maturation is a highly dynamic process, and investigating the sialylation-related mechanisms in the epididymis not only provides insights into the pathophysiological basis of male infertility but also offers new perspectives for future diagnostic and therapeutic strategies.

Given that current semen analysis methods struggle to accurately assess sperm function [26], and clinical interventions for reduced sperm motility lack specificity, there is a pressing need to explore the molecular basis of sperm motility defects and identify reliable biochemical markers. This would significantly advance diagnostic and therapeutic strategies for male infertility. Building on our observation of reduced sialidase expression in low-motility sperm, this study integrates clinical sample analysis and mouse models to investigate the origin and function of NEU in sperm maturation, while further evaluating its impact on sperm capacitation and fertilization capacity. By elucidating the dynamic changes of NEU during sperm maturation and its role in maintaining sperm motility, this research aims to uncover the functional significance of sialylation in sperm maturation and male fertility, providing novel directions for the molecular diagnosis and potential therapeutic strategies for male infertility.

Materials and methods

Biological materials and ethical approval

This study used 8-week-old adult KM wild-type male mice purchased from Beijing Hua Fukang Biological Technology Co., Ltd., with a minimum of three mice analyzed per experiment. All animal experiments were approved by the Ethics Committee of the Laboratory Animal Center at West China Second University Hospital, Sichuan University (Approval No. 2022-050), and conducted in accordance with the guidelines set forth by the Institutional Animal Care and Use Committee (IACUC) of Peking University for animal welfare and experimental standards.

Normal human testicular and epididymal tissue paraffin sections were obtained from Renji Hospital, affiliated with Shanghai Jiao Tong University School of Medicine, with ethical approval granted by the hospital’s Ethics Committee (approval number: KY2020-193). Human sperm samples were sourced from the Human Sperm Bank at West China Second Hospital, Sichuan University. Prior to sample collection, participants abstained from ejaculation for 3–7 days. Semen samples were collected via masturbation and delivered to the sperm bank within 1 h, where they underwent liquefaction at 37 °C with gentle agitation. Following a 30-minute liquefaction period, routine semen analysis was performed to exclude samples with inflammation, liquefaction difficulties, or advanced age. The remaining fresh semen samples were selected based on diagnostic criteria for oligoasthenoteratozoospermia (OAT) and infertility. These samples were collected via centrifugation tubes for subsequent experiments. All participants provided informed consent, and the study was approved by the Medical Research Ethics Committee of West China Second Hospital, Sichuan University (approval numbers: 2019-050, 2023-231).

Immunofluorescence staining (IF) and paraffin section immunohistochemistry

Following sperm extraction, samples were washed with PBS to remove seminal plasma and residual culture medium. Centrifugation at 400 g for 5 min at 4 °C yielded sperm pellets. Samples were smeared onto adhesive slides, air-dried at 37 °C in an oven to prepare sperm smears, and encircled with an immunohistochemistry pen (Biosharp). A 4% paraformaldehyde (PFA) fixative (Biosharp) was applied, followed by incubation at room temperature (RT) for 10 min. After three PBS washes, 0.3% Triton X-100 diluted in PBS was added for permeabilization at RT for 15 min, followed by three additional 5-minute PBS washes. Excess liquid was removed, and slides were blocked with 3% bovine serum albumin (BSA, BIOFROXX) at RT for 1 h. Primary antibodies were then applied, including anti-NEU1 (1:10, GenScript), anti-NEU2 (1:400, GenScript), anti-NEU3 (1:250, GenScript), anti-NEU4 (1:100, GenScript), anti-CK8 (1:500, Abcam), mouse monoclonal [RV202] to anti-Vimentin (1:500, Abcam), anti-CD9 (1:25, Santa Cruz), and anti-E-cadherin (1:500, Cell Signaling), and incubated overnight at 4 °C. The next day, slides were washed three times with PBST, and secondary antibodies (goat anti-rabbit IgG H&L [Alexa Fluor^® 568], 1:1000, Abcam; goat anti-mouse IgG H&L [Alexa Fluor^® 488], 1:1000, Abcam) were added and incubated at RT for 1 h. After three additional PBST washes, DAPI (Beyotime) was applied for 5 min at RT. Excess liquid was removed, followed by three PBST washes and mounting with an anti-fade mounting medium (Beyotime). Slides were sealed with coverslips using nail polish and imaged using a Leica DMi8 Fully Motorized Inverted Fluorescence Microscope or a Leica Stellaris Laser Scanning Confocal Microsystem.

Paraffin sections were incubated at 60 °C for 2 h, followed by automated dewaxing and rehydration using an automated dewaxing instrument (10 min each in eco-friendly dewaxing solutions I and II, 5 min in anhydrous ethanol, 2 min each in 95%, 90%, and 80% ethanol, and 2 min in H₂O). Sections were then placed in a staining jar containing EDTA citrate buffer (pH 8.0) and microwaved for 4 min until boiling, followed by cooling to RT in an ice bath. Tissue areas were encircled with an immunohistochemistry pen (Biosharp), permeabilized with 0.3% Triton X-100 in PBS at RT for 15 min, and washed. Subsequent blocking, primary antibody incubation (anti-NEU1 [1:10, GenScript], anti-NEU2 [1:1000, GenScript], anti-NEU3 [1:250, GenScript], anti-NEU4 [1:100, GenScript]), secondary antibody incubation, and imaging procedures were performed as described above.

Protein extraction and Western blot

Samples were lysed using strong RIPA buffer (Beyotime) supplemented with 1% Protease Inhibitor Cocktail and 1% Phosphatase Inhibitor (100×, CWBIO), incubated at RT for 15 min, and centrifuged at 13,000 g at 4 °C to obtain protein lysates. Protein concentrations were determined using a BCA kit (Biosharp), and samples were denatured with 5× SDS loading buffer (Biosharp) at 95 °C for 10 min. Proteins were separated by SDS-PAGE (80 V for 40 min, followed by 120 V for 60 min) on prepared gels, transferred to membranes at 300 A for 1 h, and blocked with 3% BSA in TBST at RT for 1 h. Primary antibodies diluted in 3% BSA (anti-NEU1 [1:100, GenScript], anti-NEU3 [1:1000, GenScript], anti-GAPDH [1:5000, ABclonal], anti-NEU1 [1:500, Santa Cruz], anti-NEU1 [1:2000, Abcam], anti-CD63 [1:500, Santa Cruz], anti-β-Gal [1:500, Santa Cruz], Cathepsin A Polyclonal antibody [anti-PPCA, 1:1500, Proteintech], anti-Caveolin-1 [1:1500, Proteintech], anti-EGFR [1:1000, ABclonal], anti-LAMP1 [1:1500, Santa Cruz], anti-TLR2 [1:1500, Abways], anti-NCAM [1:500, Zen BioScience], anti-Phosphotyrosine [1:1000, Abcam]) were incubated overnight at 4 °C. The following day, membranes were washed with TBST, and secondary antibodies (HRP Goat Anti-Rabbit IgG [1:5000, ABclonal], HRP Goat Anti-Mouse IgG [1:5000, ABclonal]) diluted in 3% BSA in TBST were applied and incubated at RT for 1 h. After thorough TBST washes, membranes were incubated with an equal mixture of ECL chemiluminescent substrate A and B solutions (Biosharp) and imaged using the G: BOX Chemi XRQ chemiluminescence imaging system.

Computer-assisted sperm analysis (CASA) and statistical analysis

Semen samples were analyzed using a sperm quality analyzer (Puhua and Suijia) to assess multiple parameters, including sperm concentration, total sperm count, round cell concentration, percentage of grade A (rapid progressive motility), hyperactivation, grade B (slow progressive motility), grade C (non-progressive motility), grade D (immotile), percentage of progressive motility (A + B), curvilinear velocity (VCL), straight-line velocity (VSL), amplitude of lateral head displacement (ALH), linearity, straightness, average path velocity, and beat cross frequency. Analyses adhered to the 5th edition of World Health Organization (WHO) standards. A 10 µL aliquot of each sample was loaded onto a sperm counting chamber, placed on the instrument slide holder, and analyzed. The following modulators were employed in this research: DANA (N-Acetyl-2,3-dehydro-2-deoxyneuraminic acid) at 50 µM, 500 µM, and 5 mM (Sigma); PD98059 at 5 µM and 50 µM; SCH772984 at 5 µM and 50 µM; Butein at 10 µM; LM22B-10 at 10 µM; Metformin at 500 µM; Ouabain at 500 µM; ATP at 200 µM, 1 mM, and 2 mM; and AICAR (Acadesine) at 20 µM and 200 µM (all from TargetMol, except where specified as Sigma for DANA).

All semen parameters obtained from CASA were included in the statistical analysis. Normality was first assessed; if the data did not follow a normal distribution, the Wilcoxon signed-rank test was used [27]. For normally distributed data, homogeneity of variance was tested: if variances were equal, a t-test was applied; otherwise, a corrected t-test was used. Statistical significance was set at p < 0.05. All analyses were conducted using R statistical software version 4.2.0 with packages car, openxlsx, plotROC, pROC, dplyr, and verification. Statistical figures were generated using GraphPad Prism 9.

Flow cytometry (FACS)

Post-extraction, samples were washed with PBS and treated with red blood cell lysis buffer (Biosharp) for erythrocyte removal. Samples were fixed with 4% PFA (Biosharp) and incubated at RT for 10 min with gentle rotation. After centrifugation and PBS washing, permeabilization was performed with 0.1% saponin in PBS for 10 min, followed by another PBS wash. Blocking was conducted with 3% BSA (BIOFROXX) in permeabilization buffer at RT for 1 h with rotation. Primary antibodies (anti-NEU1 [1:100, GenScript], anti-NEU3 [1:1000, GenScript], Biotinylated Maackia Amurensis Lectin I [anti-MAL-I, 1:50, Vector Labs], Biotinylated Maackia Amurensis Lectin II [anti-MAL-II, 1:50, Vector Labs], biotinylated Sambucus Nigra Lectin [anti-SNA, 1:50, Vector Labs], biotinylated Wheat Germ Agglutinin [anti-WGA, 1:50, Vector Labs], Peanut Agglutinin [anti-PNA, 1:100], anti-Jacalin [1:300, Vector Labs], Alexa Fluor 594 anti-biotinylated Phosphotyrosine [1:200, Biolegend]), anti-EGFR [1:300, ABclonal], anti-Phospho-EGFR [1:100, ABclonal], JC-1 [1:200, Beyotime], DCFH-DA [1:50, Beyotime] were incubated overnight at 4 °C. The next day, samples were centrifuged, washed with PBST, and incubated with secondary antibodies (PE Donkey anti-rabbit IgG [1:1000, Biolegend], FITC Streptavidin [1:500, Biolegend], DyLight 488 Streptavidin [1:500, Biolegend]) in permeabilization buffer at RT for 1 h. After PBST washes, samples were stained with DAPI (Beyotime) and analyzed using a CytoFLEX flow cytometer.

Immunoelectron microscopy (IEM)

Sperm samples were spread onto poly-L-lysine-coated ACLAR^® 33 C films (Electron Microscopy Sciences) and fixed at room temperature for 15 min in 4% paraformaldehyde (PFA, Sigma-Aldrich) and 0.1% glutaraldehyde (Sigma-Aldrich) in 0.1 M phosphate buffer (PB, pH 7.4), followed by overnight storage at 4 °C in 2% PFA. The samples were washed with PBS containing 50mM glycine for 5 min to quench free aldehydes, then permeabilized with 0.1% saponin in PBS for 30 min. Blocking was performed with 3% bovine serum albumin (BSA) and 1% normal goat serum in PBS for 1 h.

After blocking, the samples were incubated overnight at 4 °C with primary antibodies (anti-NEU1, 1:50, GenScript; anti-NEU3, 1:1000, GenScript). Following PBS washes, they were incubated overnight at 4 °C with goat anti-rabbit Fab’ conjugated to 1.4 nm gold particles (1:200, Nanoprobes). After multiple PBS washes, the samples were fixed in PBS containing 2% glutaraldehyde and 0.3% tannic acid (Electron Microscopy Sciences), followed by gold enhancement for 2 min using the GoldEnhance™ EM Plus kit (Nanoprobes). They were then washed with 1% sodium thiosulfate and ultrapure water before post-fixation in 1% osmium tetroxide (OsO4) in 0.1 M PB for 30 min and stained with 1% uranyl acetate for 30 min.

The samples underwent graded ethanol dehydration (30%, 50%, 70%, 85%, 95%, and 2 × 100%, each for 5 min), followed by infiltration and embedding in EMbed 812 resin (SPI), which was polymerized overnight at 65 °C. Ultrathin Sect. (75 nm) were cut using an ultramicrotome (Leica UC7) with a diamond knife (Ultra 35°, Diatome) and mounted onto single-slot copper grids. The sections were stained with uranyl acetate and lead citrate, then examined using a transmission electron microscope (FEI and HITACHI) and imaged with a digital camera (Orius 832, Gatan).

RNA extraction and qPCR

Workstation was thoroughly cleaned with a solid-phase decontaminant (Solarbio). Samples were lysed with an appropriate volume of TRIzol Reagent (Ambion) at 4 °C for 30 min. One-fifth volume of chloroform was added, mixed thoroughly, and centrifuged at 13,000 g at 4 °C for 10 min. The supernatant was collected, mixed with an equal volume of isopropanol, and precipitated overnight at −20 °C. The following day, centrifugation at 13,000 g at 4 °C for 10 min yielded a white RNA pellet, which was washed with 75% ethanol, air-dried, and resuspended in DEPC water. RNA concentration was quantified using a Nanodrop spectrophotometer. Genomic DNA was removed using gDNA Eraser Premix (Takara) at 42 °C for 2 min. cDNA was synthesized using the PrimeScript RT Reagent Kit (Takara) at 37 °C for 10 min, with termination at 85 °C for 5 s. qPCR was performed using the TB Green Fast qPCR Kit (Takara) and amplified/detected on a BIO-RAD CFX Connect system (95 °C for 30 s; 40 cycles: 95 °C for 5 s, 60 °C for 10 s).

Sialidase (Neu) activity assay

Each well of a 96-well black clear-bottom plate received 70 µL of assay buffer (Beyotime). Samples were resuspended in assay buffer, quantified by cell counting, and added to each well (final volume adjusted to 10 µL with buffer). Blank wells received 10 µL of assay buffer, while positive control wells received 10 µL of neuraminidase. A 10 µL aliquot of sialidase fluorescent substrate was added to each well, mixed by shaking for 1 min, and incubated at 37 °C for 1 h. Fluorescence was measured using a PE multifunctional microplate reader with excitation at 322 nm and emission at 450 nm.

Mouse in vitro fertilization (IVF) and DANA treatment

Twelve-week-old female mice were intraperitoneally injected with 10 IU PMSG (Solarbio), followed by 10 IU HCG (Sansheng) after 48 h. Oocytes were collected 14–16 h post-HCG injection. Male mice were sacrificed, and epididymal tail sperm were extracted. The control group was capacitated in TYH medium (AibeiBio) at 37 °C in a CO₂ incubator for 1 h. The experimental group was treated with 5 µM and 50 µM DANA (Sigma) diluted in TYH medium. Female mice were sacrificed, and adipose tissue was separated to extract oviducts into MII medium (AibeiBio). Oviduct ampullae were incised with a syringe needle to release oocytes, which were washed with HTF fertilization medium (AibeiBio) to remove HEPES, and sperm were added for fertilization. After 4–6 h, oocytes were retrieved, washed to remove residual sperm, and cultured in KSOM medium (AibeiBio) for embryo development.

Mouse sperm and exosome extraction

Mice were sacrificed, and adipose tissue was removed to isolate testes and epididymides. Epididymides were gently incised with a syringe needle to release sperm into PBS, incubated at 37 °C in a CO₂ incubator for 15 min to facilitate sperm release, and filtered through 150- and 300-mesh sieves to remove tissue debris. Centrifugation at 400 g at RT for 5 min separated sperm. A 5 µL aliquot was examined under a microscope to assess sperm purity and viability.

The remaining liquid post-sperm extraction was centrifuged at 3,000 rpm for 10 min to remove residual cells. The supernatant was further centrifuged at 2,000 g for 20 min to eliminate cell debris, and the resulting supernatant was centrifuged at 10,000 g for 20 min. Exosomes were extracted from the final supernatant using an exosome isolation kit (Invitrogen). One-half volume of PBS was added for mixing, followed by one-fifth total volume of Exosome Precipitation Reagent, mixed thoroughly, and incubated at RT for 10 min. Centrifugation at 10,000 g at RT for 5 min yielded the exosome pellet.

Following established protocols for surface membrane protein biotinylation [28], exosome pellets were obtained and subjected to multiple washes with PBS buffer containing 0.5 mM Mg²⁺ and 1 mM Ca²⁺. After resuspension, the exosomes were incubated with 0.04 mM EZ-Link Sulfo-NHS-SS-Biotin (Invitrogen) or EZ-Link Sulfo-NHS-LC-Biotin (TargetMol) at 4 °C for 20 min. The reaction was quenched by adding a stop buffer containing 50 mM glycine, followed by ultrahigh-speed centrifugation to recover exosome membrane proteins. After thorough washing, the exosomes were lysed using RIPA lysis buffer, and the biotinylated proteins were enriched using streptavidin magnetic beads (MedChemExpress), yielding the final biotin-labeled membrane protein fraction.

Mouse epididymal epithelial primary extraction and siRNA transfection

Post-sacrifice, epididymides were dissected into head, body, tail, and vas deferens regions. Epididymal epithelial tissue was segmented into small pieces, incubated at 37 °C in a CO₂ incubator to release sperm, and filtered through a 300-mesh sieve. Tissue was minced on ice and digested with 0.25% Trypsin-EDTA (Invitrogen) at 37 °C with shaking for 30 min. Culture medium (serum-free IMDM supplemented with 10% FBS, 1 nM 5α-dihydrotestosterone [DHT], and 1% antibiotic-antimycotic) was prepared. Digestion was terminated with BSA, and centrifugation at 400 g removed trypsin. Samples were further digested with type I collagenase (Sigma, 0.1–5 mg/mL) and DNase I (Roche-Sigma, 0.01%) for 30 min. Cells were resuspended in culture medium, seeded in 10 cm dishes, and subjected to differential adhesion for 1 h. Non-adherent cells were collected, reseeded in 6-well plates, and cultured until reaching 60–70% confluence.

Prior to transfection, cells were refreshed with medium. Transfection reagent was prepared by mixing 5 µL Lipofectamine 3000 with 125 µL serum-free medium and incubated at RT for 5 min. siRNA was diluted in serum-free medium to 50 µM and 100 µM (total volume 130 µL), incubated at RT for 5 min, and combined with the transfection reagent. The mixture was incubated at RT for 15 min, and 250 µL of transfection solution was added per well. Cells were harvested at 48 and 72 h post-transfection.

Computational prediction of sialylation-related enzymes in the male reproductive tract

To preliminarily identify sialylation-related enzymes in the male reproductive tract, this study builds upon our laboratory’s previous comprehensive analysis, which encompassed the expression changes of 24 sialylation-related enzymes and 14 receptors during human and murine sperm development. By integrating multiple publicly available GEO datasets, we evaluated the expression profiles of all sialylation-related enzymes and selected specific sialidases for in-depth study. In addition, the GlycoMaple online tool (https://glycosmos.org/glycomaple/index) was used to predict glycosylation modifications in the transcriptomic data from the human and murine male reproductive tracts.

Co-immunoprecipitation (Co-IP)

Membrane proteins were extracted from samples using 2% DDM (BluePlus) at 4 °C for 2 h, supplemented with 1% Protease Inhibitor Cocktail and 1% Phosphatase Inhibitor (100×, CWBIO), and centrifuged at 13,800 rpm for 15 min to obtain protein supernatant. A portion was reserved as the input group, and protein concentration was determined using a BCA kit (Biosharp). The remaining sample was divided into two aliquots: the IP group was incubated with 10 µg NEU1 antibody (Abcam) overnight at 4 °C with rotation, while the control group received 10 µg rabbit IgG (Beyotime). The next day, 20 µL Dynabeads™ Protein G (Invitrogen) was added, and incubation continued at 4 °C for 3 h. Samples were placed on a magnetic rack, supernatant was discarded, and beads were washed five times with 0.02% DDM. After the final wash, samples were resuspended in loading buffer, denatured, centrifuged at 14,000 g at 4 °C for 8 min, and supernatant was collected for subsequent Western blot analysis.

Seahorse energy metabolism analysis

The sensor cartridge was hydrated one day before the experiment by incubating it overnight at 37 °C in a non-CO₂ incubator. The wells of the cell culture plate designated for measurement were coated with 100 µL of 0.5 mg/mL Concanavalin A (ConA, Sigma) and incubated overnight at 37 °C in a CO₂ incubator [29]. A 50 mL low-HEPES Seahorse TYH buffer [30] was prepared (135 mM NaCl, 4.7 mM KCl, 1.7 mM CaCl₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 5.6 mM glucose, 1 mM HEPES), adjusted to pH 7.4 with NaOH at 37 °C, and sterile-filtered using a 0.22 μm filter (Millipore).

On the following day, drugs were prepared at stock concentrations and diluted according to the Seahorse XF Cell Mito Stress Test Kit (Agilent) protocol, with final concentrations of 30 µM oligomycin (OM), 10 µM FCCP, and 10 µM rotenone/antimycin A (Rot/AA). Drugs were added to detection wells, and calibration was performed. Meanwhile, male mice were euthanized, and sperm were collected from the cauda epididymis. The coating solution in the measurement wells was removed, and the wells were washed with assay medium. Equal amounts of sperm were added to each well, followed by centrifugation at 200 g for 1 min at room temperature to allow sperm adhesion. The supernatant was carefully removed, and Seahorse assay medium was gently added. The sperm status was examined under a microscope before proceeding with the measurement using the Seahorse XFe24 Analyzer.

Proteomic detection and analysis

Semen samples from asthenozoospermic and normal groups were centrifuged and repeatedly washed with PBS to remove seminal plasma, yielding sperm pellets. Proteins were extracted using 300 µL of 8 M urea solution supplemented with 10% protease inhibitor, centrifuged at 14,100 g for 20 min to obtain protein supernatant, and concentrations were determined using the Bradford method. A 100 µg aliquot of protein was reduced with DTT solution at 37 °C with shaking for 1 h, diluted fourfold with 25 mM ammonium bicarbonate (ABC) buffer, digested overnight at 37 °C with 2% trypsin, and the reaction was terminated with formic acid. Samples were lyophilized for storage. Proteomic analysis was performed using 4D-DIA and LC-MS/MS (timsTOF Pro and Q Exactive HF-X) to assess protein expression differences between asthenozoospermic and control groups. Samples were gradient-eluted using a C18 column (wash buffer: 100% acetonitrile with 0.1% formic acid; elution buffer: 70% acetonitrile), combined, lyophilized, and stored at −80 °C. Data were processed with Spectronaut software using the Homo sapiens UniProt database for protein identification and quantification (FDR < 1%). Data normalization was performed, and differentially expressed proteins were identified using log₂ fold change (log₂FC) and p-value (p < 0.05, |log₂FC| >1). Results were visualized with volcano plots for differential protein distribution and heatmaps for expression pattern clustering (red for upregulation, blue for downregulation). KEGG and GO database analysis was conducted for gene set enrichment, calculating Normalized Enrichment Scores (NES) and identifying significant pathways (p < 0.05). Finally, Venn diagrams were employed to analyze the intersection of NEU1-interacting proteins and differentially expressed proteins, identifying potential NEU1-interacting differential genes.

Results

NEU1 is the predominantly expressed Sialidase in human spermatozoa

The neuraminidase (NEU) family plays a critical role in various biological processes in humans [31], yet its expression in the male reproductive tract and spermatozoa remains poorly characterized. To investigate the distribution of NEU in reproductive tissues, we first analyzed GEO datasets of human testis and epididymis [32], followed by immunofluorescence detection. Single-cell data (GSE159713) revealed that Neu1 is highly expressed across all segments of the mouse epididymis, with particularly prominent expression in the principal cells of the epididymal epithelium (Supplemental Fig. 1A-B). Immunofluorescence experiments further demonstrated that NEU1 and NEU3 are primarily localized to the testicular interstitium and epididymal epithelium, whereas NEU2 and NEU4 were undetectable in these tissues (Fig. 1A). To further explore NEU expression in spermatozoa, we conducted immunofluorescence analysis of different NEU family members in human sperm. The findings demonstrated expression of NEU1 and NEU3, but not NEU2 or NEU4 (Fig. 1B). Western blot analysis confirmed that NEU1 expression in sperm significantly exceeds that of NEU3 (Fig. 1C, D), suggesting that NEU1 is the predominant sialidase in human spermatozoa and likely plays a key role in sperm maturation and functional regulation.

Fig. 1.

NEU1 expression in human sperm and its association with motility and fertility outcomes (A) Immunofluorescence analysis showing NEU1 and NEU3 localization in testicular interstitium and epididymal epithelium, while NEU2 and NEU4 are undetectable. (B) Immunofluorescence detection of NEU family members in human spermatozoa, confirming NEU1 and NEU3 expression. (C) Western blot analysis demonstrating that NEU1 is the predominant sialidase in human spermatozoa. (D) Flow cytometry analysis revealing reduced NEU1 expression in sperm from asthenozoospermic and infertile men compared to normozoospermic and fertile controls. (E) Correlation analysis between sperm NEU1 expression and motility parameters, including straight-line velocity (VSL) and progressive motility percentage. (F) ROC curve analysis showing that sperm NEU1 expression predicts asthenozoospermia and infertility with higher sensitivity and specificity than seminal plasma NEU1 or sperm NEU3

Sperm NEU1 expression correlates with motility and fertility outcomes

Given the prominent expression of NEU1 in human spermatozoa but its reduced levels in asthenozoospermic patients, this study further explored its association with sperm quality and clinical reproductive outcomes. Flow cytometry analysis of clinical patient samples revealed that NEU1 expression in spermatozoa from asthenozoospermic patients was significantly lower than in the normozoospermic group, while no notable differences were observed in seminal plasma NEU1 or sperm NEU3 levels between the groups (Fig. 1E). Similarly, infertile men exhibited significantly reduced sperm NEU1 expression compared to fertile controls (Fig. 1E). Further analysis demonstrated a positive correlation between sperm NEU1 expression and multiple motility parameters, including straight-line velocity (VSL) and the percentage of progressively motile sperm (Fig. 1F, Supplemental Fig. 1 C). Receiver operating characteristic (ROC) curve analysis indicated that sperm NEU1 expression offers higher sensitivity and specificity in predicting asthenozoospermia and infertility compared to seminal plasma NEU1 or sperm NEU3 (Fig. 1G). These findings suggest that the expression level of NEU1 in sperm may be associated with sperm motility and reproductive outcomes.

NEU1 expression patterns in mouse spermatozoa resemble those in humans and increase during maturation

To determine whether mice are a suitable model for studying NEU1 in human spermatozoa, we first examined NEU1 localization in human and mouse sperm using immunoelectron microscopy. The results showed that NEU1 is predominantly localized to the nuclear region of the sperm head and the mitochondrial membrane of the tail in both species, indicating a highly conserved spatial distribution (Fig. 2A). To explore dynamic changes in NEU1 expression during sperm maturation, we combined prior data analysis with new findings, revealing a progressive increase in NEU1 expression in human and mouse spermatozoa during epididymal maturation [32]. Immunofluorescence analysis of mouse sperm confirmed that NEU1 expression in the sperm head intensifies from the testis to the epididymis (Fig. 2B). Notably, in the caput epididymis, NEU1 fluorescence was particularly strong in the sperm tail (Fig. 2B), suggesting dynamic regulation of its distribution during epididymal transit.

Fig. 2.

NEU1 localization and dynamic expression in human and mouse sperm during epididymal maturation (A) Immunoelectron microscopy showing NEU1 localization in the sperm head nuclear region and tail mitochondrial membrane in both human and mouse sperm. (B) Immunofluorescence analysis of mouse sperm demonstrating increasing NEU1 expression from testis to epididymis. (C) Flow cytometry analysis of mouse sperm from different reproductive tract regions showing a progressive increase in NEU1 expression from the testis to vas deferens. (D) Immunofluorescence of paraffin-embedded mouse testicular and epididymal sections confirming NEU1 upregulation. (E) Changes in human sperm motility after capacitation

Flow cytometry analysis of mouse spermatozoa from different reproductive tract regions further corroborated that NEU1 expression increases progressively from the testis through the caput and cauda epididymis to the vas deferens (Fig. 2C), aligning with our hypothesis. Immunofluorescence of paraffin-embedded mouse testicular and epididymal sections provided additional evidence of this upward trend during sperm maturation (Fig. 2D). Collectively, these results demonstrate that mice not only share similar spatial NEU1 distribution with humans but also exhibit comparable expression dynamics during sperm maturation, supporting their use as a model for studying human sperm NEU1.

NEU1 enriches on sperm membranes post-capacitation, and its inhibition impairs motility and fertilization

Sperm capacitation is a critical process for achieving fertilization competence [33]. To investigate NEU1’s role in this process, we subjected human spermatozoa to capacitation treatment, which significantly enhanced motility parameters, including linearity, percentage of grade A sperm, hyperactivated sperm, progressive motility, and straight-line velocity (Fig. 2E, Supplemental Fig. 2B). Immunofluorescence analysis revealed a marked increase in NEU1 expression on the sperm surface post-capacitation, predominantly in the tail region (Fig. 3A). A similar trend was observed in mouse spermatozoa, with surface phosphorylation levels rising over time during capacitation (Supplemental Fig. 2 A), suggesting intracellular NEU1 translocation in response to capacitation stimuli [34].

Fig. 3.

NEU1 translocation to the sperm membrane post-capacitation and its effect on motility (A) Immunofluorescence showing increased NEU1 expression on the sperm surface, particularly in the tail region, post-capacitation. (B) Comparison of NEU1 expression in sperm stratified by motility using the swim-up method, showing higher NEU1 levels in highly motile sperm. (C) Enzyme activity assay showing decreased sperm NEU1 activity after DANA treatment (D, E) Impact of NEU1 inhibition on sperm motility parameters in human and mouse sperm

To further examine the relationship between NEU1 and sperm motility, we stratified capacitated human spermatozoa using the swim-up method. Spermatozoa with higher motility exhibited significantly greater NEU1 expression than those with lower motility (Fig. 3B). Treatment with the NEU1 inhibitor 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) [35] significantly reduced NEU1 activity (Fig. 3C), leading to a notable decline in motility in both human (Fig. 3D) and mouse spermatozoa (Fig. 3E). These results indicate a strong correlation between NEU1 expression and sperm motility, with reduced NEU1 expression or activity impairing motility.

We next assessed NEU1’s impact on fertilization capacity. In vitro fertilization experiments in mice demonstrated that inhibition of NEU1 activity significantly reduced embryo fertilization rates (Fig. 4A). Lectin-based flow cytometry experiments showed a gradual decrease in SNA signals during sperm maturation, consistent with NEU1 expression patterns (Fig. 4B), reinforcing NEU1’s importance in sperm maturation and function. In contrast, MAL-I, MAL-II, and WGA signals increased (Fig. 4B), potentially reflecting changes in sialyltransferase during maturation (Supplemental Fig. 2 C). In summary, sperm NEU1 activity is implicated in mouse embryo fertilization and may also play a role in regulating sperm α−2,6 sialylation.

Fig. 4.

NEU1 inhibition reduces fertilization rates and alters sperm sialylation during maturation (A) In vitro fertilization assay showing a significant decline in embryo fertilization rates following NEU1 inhibition. (B) Analysis of sperm sialylation modifications during maturation, showing decreased SNA signals and increased MAL-I, MAL-II, and WGA signals

NEU1 is transported from epididymal epithelium to spermatozoa to promote maturation

As the epididymis is a key site for sperm maturation, we further investigated the expression and localization of NEU1 in the epididymal epithelium. Single-cell RNA sequencing (scRNA-seq) analysis of the epididymis (GSE159713) revealed a progressive increase in NEU1 expression during sperm maturation, with elevated levels in epididymal epithelial cells (Supplemental Fig. 3 A). Confocal microscopy confirmed that NEU1 is primarily localized to the cytoplasm and plasma membrane of epididymal epithelial cells (Fig. 5A). Primary epididymal epithelial cells from mice were cultured, with the fastest growth observed in cells from the caput epididymis (Supplemental Fig. 3B). Cell purity was verified using E-cadherin staining (Supplemental Fig. 3 C), and enzyme activity assays indicated the highest activity in caput epithelium (Supplemental Fig. 3D), guiding our selection of this region for subsequent experiments. Mouse epididymal fluid exhibited high sialidase activity and NEU1 expression (Supplemental Fig. 3E). These findings suggest that epididymal epithelial cells may secrete active NEU1, which may subsequently be loaded onto sperm.

Fig. 5.

Epididymal epithelial cells transfer NEU1 to sperm (A) Confocal microscopy showing NEU1 localization in the cytoplasm and plasma membrane of epididymal epithelial cells. (B) Co-incubation of testicular sperm with epididymal fluid results in increased sperm NEU1 levels. (C) siRNA-mediated knockdown of NEU1 in epididymal epithelial cells leads to reduced NEU1 transfer to sperm. (D) siRNA co-incubation model is associated with impaired sperm motility. (E) Sialylation analysis showing increased sialylation levels in siRNA co-incubation model. (F) Capacitation marker analysis revealing reduced tyrosine phosphorylation (PY20) and elevated PNA levels in siRNA co-incubation model

To investigate the role of NEU1 in epididymal sperm, this study co-incubated mouse testicular spermatozoa with epididymal fluid, resulting in a significant increase in sperm NEU1 content (Fig. 5B). Using three designed siRNAs (siRNA1: CTACAGCCTTCATCGTAGA; siRNA2: GCTCAGGCATTCAGAAACA; siRNA3: GATCGGCTCTGTAGACACT), we transfected primary mouse epididymal epithelial cells to knock down NEU1 expression, optimizing conditions (100 nM siRNA3, 72-hour transfection) to establish a low-NEU1-expression model (Supplemental Fig. 4A-F). Co-incubation of these cells with mouse spermatozoa (siRNA co-incubation model) led to a significant reduction in sperm NEU1 content (Fig. 5C). Reduced NEU1 expression in spermatozoa was associated with decreased motility (Fig. 5D), increased sialylation levels (Fig. 5E), reduced capacitation markers (tyrosine phosphorylation, PY20), and elevated PNA levels (Fig. 5F). These findings suggest that diminished NEU1 acquisition from epididymal epithelium impairs sperm motility, sialylation regulation, and capacitation, thereby hindering maturation.

Epididymal epithelium secretes active NEU1 via exosomes to mediate sperm maturation

The epididymis serves as a critical site for sperm maturation, providing an essential environment for the acquisition of motility and fertilization capacity [36]. Exosomes, as key mediators of bioactive molecule transport between sperm and the epididymis [37], play a pivotal role in the process of sperm maturation. In muscle connective tissue, NEU1 is transported and secreted via exosome [38]. However, its secretion mechanism in epididymal epithelial cells remains unclear. Therefore, this study further explored whether NEU1 could be transported between the epididymis and sperm via exosomes. First, exosomes were isolated from epididymal fluid, and NEU1 expression was detected (Fig. 6A). Transmission electron microscopy (TEM) verified the exosomes’ typical morphology (Fig. 6B), and NEU1-containing exosomes were also detected in human seminal plasma (Fig. 6C), suggesting exosomal secretion and transport of NEU1. Immunoprecipitation experiments revealed that exosomal NEU1 forms complexes with β-Gal and Caveolin1, though no interaction with PPCA was detected (Fig. 6D). Immunoelectron microscopy localized NEU1 to the membrane of epididymal fluid exosomes in mice (Fig. 6E). Surface membrane protein biotinylation experiments further confirmed that NEU1 is predominantly localized to the membrane of mouse exosomes (Supplemental Fig. 4G). In situ immunoelectron microscopy of mouse epididymal tissue captured the exosome secretion process from epithelial cells (Fig. 6F), with NEU1 enzyme activity detected in the fluid (Fig. 6G).

Fig. 6.

NEU1 is secreted via epididymal exosomes and transferred to sperm (A) NEU1 detection in exosomes isolated from epididymal fluid. (B) Transmission electron microscopy (TEM) verifying the morphology of epididymal exosomes. (C) Detection of NEU1-containing exosomes in human seminal plasma. (D) Immunoprecipitation revealing NEU1 interaction with β-Gal and Caveolin1 in exosomes. (E) Immunoelectron microscopy localizing NEU1 to epididymal exosome membranes. (F) In situ immunoelectron microscopy capturing exosome secretion from epididymal epithelial cells. (G) NEU1 enzyme activity detected in epididymal fluid

To investigate NEU1’s interactions in spermatozoa, immunoprecipitation analysis of human sperm showed that NEU1, present in membrane proteins, complexes with β-Gal, Caveolin1, and additional proteins including EGFR [39, 40], LAMP1 [41], TLR2 [42, 43], and NCAM [44] (Supplemental Fig. 5A-B). In a NEU1-knockout HeLa cell model, LAMP1 expression and molecular weight increased significantly (Supplemental Fig. 5 C), suggesting LAMP1 as a potential downstream target of NEU1 in spermatozoa [45].

Inhibition of NEU1 activity impairs sperm capacitation and energy metabolism

The findings from the aforementioned studies indicate a correlation between NEU1 expression, sperm motility, and epididymal sperm maturation. To assess NEU1’s specific effects on sperm function, we inhibited NEU1 activity and evaluated physiological changes. Inhibition increased PNA levels, impaired recovery post-capacitation, and significantly suppressed tyrosine phosphorylation during capacitation (Fig. 7A). The extracellular signal-regulated kinase (ERK) pathway, a well-documented phosphorylation cascade activated during sperm capacitation [46, 47], is modulated by the sialidase inhibitor DANA [15]. Treatment with ERK inhibitors PD098059 [46, 48] and SCH772984 [49, 50] resulted in diminished sperm motility following capacitation (Supplemental Fig. 6 A), consistent with previous reports [51]. In contrast, the addition of ERK activators Butein and LM22B-10 mitigated the inhibitory effects of DANA on sperm motility (Supplemental Fig. 6B). Similarly, ATP inhibitors Metformin and Ouabain reduced sperm motility (Supplemental Fig. 6 C), whereas ATP pathway activators AICAR and exogenous ATP rescued the suppressive effects of DANA (Supplemental Fig. 6D). These results suggest that NEU1 enhances post-capacitation sperm motility through activation of the ERK and ATP signaling pathways.

Fig. 7.

NEU1 inhibition disrupts sperm capacitation and energy metabolism (A) Increased PNA levels and suppressed tyrosine phosphorylation following NEU1 inhibition, indicating impaired capacitation and membrane stability. (B) Seahorse metabolic analysis showing reduced oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) upon NEU1 inhibition. (C) Flow cytometry analysis revealing increased α−2,6 sialylation (SNA signals) following NEU1 inhibition. (D) Western blot analysis showing reduced EGFR membrane translocation and phosphorylation post-NEU1 inhibition

To investigate the effects of NEU1 inhibition on sperm energy metabolism, we employed the Seahorse XF analyzer to evaluate mitochondrial function. In the mitochondrial stress test, sperm from the experimental group exhibited a significant reduction in oxygen consumption rate (OCR) and a correspondingly lower extracellular acidification rate (ECAR) (Fig. 7B, Supplemental Fig. 7A-H). The OCR decline indicates impaired mitochondrial function and energy metabolism, while the lower ECAR suggests diminished glycolytic activity, possibly linked to altered membrane glycosylation. Flow cytometry further confirmed that DANA treatment reduced mitochondrial membrane potential (Supplemental Fig. 8 A) and elevated reactive oxygen species (ROS) levels (Supplemental Fig. 8B).

Fig. 8.

Proteomic Analysis of Asthenozoospermia vs. Normal Group (A) Volcano plot showing differentially expressed proteins between the asthenozoospermia and the control group, with log₂(fold change) on the x-axis and -log₁₀(p-value) on the y-axis. Red and blue dots indicate significantly upregulated and downregulated proteins, respectively. (B) Heatmap of differentially expressed proteins across the two groups, with red indicating upregulation and blue indicating downregulation. (C) Gene Set Enrichment Analysis (GSEA) based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for differentially expressed proteins, with the x-axis showing the Normalized Enrichment Score (NES). Red bars indicate pathways significantly upregulated in asthenozoospermia, and blue bars indicate downregulated pathways. (D, E) Circos plot visualizing top differentially expressed proteins associated with downregulated pathways in asthenozoospermia. (F) Venn diagram showing the overlap between NEU1-interacting proteins in the human sperm membrane proteome and the differentially expressed proteins identified in this study

Subsequent flow cytometry analysis of sperm membrane sialylation showed upregulated SNA signals post-NEU1 inhibition, reflecting increased α−2,6 sialylation (Fig. 7C). Downstream signaling analysis revealed reduced EGFR membrane translocation and phosphorylation, affecting its localization during capacitation (Fig. 7D).

In summary, NEU1 is critical for sperm capacitation and energy metabolism. Its inhibition disrupts membrane sialylation, impairs mitochondrial function and glycolysis, and interferes with capacitation-related signaling, potentially compromising fertilization capacity.

Proteomic analysis of asthenozoospermia vs. normal group

Proteomic analysis identified a total of 6,008 proteins, among which 504 exhibited significant differential expression between the asthenozoospermia group and the control group (p < 0.05, |log₂FC| >1). The volcano plot (Fig. 8A) revealed that 71 proteins were significantly upregulated (depicted in red) and 106 were significantly downregulated (depicted in blue). The heatmap further elucidated the clustering patterns of expression profiles across samples (Fig. 8B). Gene Set Enrichment Analysis (GSEA) indicated significant downregulation of pathways related to Extracellular Matrix Organization and Metabolic Diseases (such as Glycosphingolipid Metabolism and Sphingolipid Metabolism) in asthenozoospermic patients. Conversely, pathways including Cellular Senescence and Apoptosis were significantly upregulated (Fig. 8C). GO enrichment analysis of the top differentially expressed proteins in asthenozoospermia further demonstrated significant enrichment in pathways such as glycosaminoglycan phospholipase binding and ganglioside lysosome organization metabolic processes (Fig. 8D). Circos plot visualization highlighted key proteins within significantly altered pathways, consistently focusing on lysosomal function and the key molecule NEU1 within Glycosphingolipid metabolism pathways. These findings align with and corroborate our previous research (Figs. 8E). Additionally, an intersection analysis with NEU1-interacting proteins from the human sperm membrane proteome and significantly downregulated proteins in asthenozoospermia was performed to identify NEU1-associated proteins relevant to the disease, and 17 overlapping genes were identified (Fig. 8F). Notably, membrane proteins interacting with NEU1, including RAB27B, RAB3D, RAB27A, and ITLN1, were all downregulated in asthenozoospermia in conjunction with NEU1. Western blot analysis confirmed these findings, showing significantly reduced expression levels of NEU1, RAB3D, and RAB27A proteins in the sperm of patients with asthenozoospermia (Supplemental Fig. 8 C).

Discussion

Sperm maturation and fertilization represent intricate and highly orchestrated processes critical to male fertility, wherein glycosylation modifications, particularly sialylation, play a pivotal role in regulating sperm function [32]. However, the precise biological significance of these modifications and their dynamic changes during sperm maturation remain underexplored. The sialidase family member NEU1 has been implicated in glycoprotein modification, signal transduction, and intercellular interactions across various cell types [52, 53]. Previous studies have reported that NEU1 deficiency in male mice leads to infertility and, in some cases, genital prolapse [54], underscoring its essential role in male reproductive function. Yet, its expression in spermatozoa and its impact on sperm motility and fertilization capacity have not been systematically investigated. Thus, this study aims to elucidate the dynamic changes of NEU1 in human spermatozoa and its potential regulatory role in sperm function.

Previous studies have reported decreased NEU1 expression levels in idiopathic infertile men [15]; however, the relationship between NEU1, sperm motility, and reproductive outcomes has not been thoroughly investigated. To delineate NEU1’s role in spermatozoa, we initially analyzed clinical semen samples, revealing a significant correlation between NEU1 expression levels and both sperm motility and fertility outcomes. This observation aligns with our prior findings [15] and supports the hypothesis that NEU1 may serve as a potential biomarker for male infertility. However, given the limited sample size in this study, this conclusion requires validation in larger, independent cohorts. Furthermore, we employed siRNA co-incubation models and inhibitor treatments to inversely confirm NEU1’s motility-enhancing effects on sperm. These results suggest that NEU1 may directly or indirectly influence sperm motility and capacitation processes. Future investigations could leverage genetically engineered mouse models to further dissect the specific molecular mechanisms by which NEU1 regulates sperm motility.

Sperm undergo significant functional and biochemical maturation during epididymal transit, with a marked improvement in motility [55]. However, the precise role of NEU1 in sperm motility and epididymal maturation remains unclear. To explore NEU1’s dynamics during sperm maturation, we established an in vitro co-incubation model of epididymal epithelial cells and spermatozoa, discovering that NEU1 is transported to the sperm surface via epididymosomes, thereby promoting maturation. This finding is consistent with prior studies indicating the role of epididymosomes in sperm protein modification [56, 57]. Inhibition of this transport process significantly impaired sperm motility and capacitation responses, suggesting that epididymal NEU1 may be a critical determinant of sperm maturation. To further clarify NEU1’s regulatory mechanisms in this context, subsequent studies will develop conditional knockout mouse models targeting epididymal epithelial cells. This approach will assess the impact of impaired NEU1 loading on sperm function and associated signaling pathways, refining and expanding our proposed framework.

Furthermore, previous studies have shown that disruptions in sperm energy metabolism can significantly impair motility and fertilization capacity [58–61]. In this study, Seahorse metabolic analysis revealed that inhibition of NEU1 activity led to mitochondrial dysfunction, reduced energy consumption, and a decline in overall metabolic activity in sperm. Notably, NEU1 expression has been associated with oxidative stress and mitochondrial energy metabolism disorders [62]; however, its downstream targets remain unclear. Future studies should employ glycoproteomics analysis to identify potential downstream targets of NEU1 and further validate their functional impact on sperm physiology through targeted functional assays.

Overall, this study integrates clinical sample analysis and animal experiments to demonstrate a robust positive correlation between NEU1 and sperm motility, proposing NEU1 as a potential biomarker and therapeutic target for male infertility. During sperm maturation, NEU1 is delivered to the sperm surface via epididymal epithelial exosomes, subsequently contributing to capacitation, metabolic regulation, and fertilization. Mouse model studies further confirmed NEU1’s role in modulating sperm α−2,6 sialylation, potentially influencing fertilization capacity through energy metabolism regulation. Collectively, these findings underscore NEU1’s critical involvement in sperm maturation and fertilization, offering novel avenues for the diagnosis and management of male infertility. Future research should focus on elucidating the precise molecular mechanisms underlying NEU1’s regulation of sperm function, providing deeper scientific insights into enhancing sperm maturation and male reproductive potential.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Shiqi Yi conceptualized and designed the study, conducted experimental assays, prepared figures, drafted the manuscript, and revised the content. Ruohan Wang established the co-incubation model and provided expert guidance on animal experiments. Yingchun Hu provided professional guidance on the preparation, imaging, and data analysis of human sperm samples for electron microscopy. Shukai Wang assisted with animal experiments. Linyu Zhang, Xinrui Sun, Xin Yuan, Jiajie Li and Shuangling Zou organized the results. Hongxiang Wang provided human testicular and epididymal tissue sections and contributed to the ethical approval process. Fang Ma reviewed and edited the manuscript. All authors approved the final version and take responsibility for its content.

Funding

This research was supported by the National Natural Science Foundation of China (Grant No. 31470797) and the Sichuan Provincial Administration of Traditional Chinese Medicine (Grant No. 2023MS036).

Data availability

This study leverages publicly available data sourced from previously published databases. Proteomic data are available from the corresponding author upon reasonable request.

Declarations

Conflict of interest

The authors declare that no commercial or financial relationships exist that could be perceived as potential conflicts of interest in relation to this study.

Ethics approval

All animal experiments were approved by the Ethics Committee of the Laboratory Animal Center at West China Second University Hospital, Sichuan University (Approval No. 2022-050), and conducted in accordance with the guidelines set forth by the Institutional Animal Care and Use Committee (IACUC) of Peking University for animal welfare and experimental standards.

Normal human testicular and epididymal tissue paraffin sections were obtained from Renji Hospital, affiliated with Shanghai Jiao Tong University School of Medicine, with ethical approval granted by the hospital’s Ethics Committee (Approval No. KY2020-193). Human sperm samples were sourced from the Human Sperm Bank at West China Second Hospital, Sichuan University. All participants provided informed consent, and the study was approved by the Medical Research Ethics Committee of West China Second Hospital, Sichuan University (Approval No. 2019-050, 2023 − 231).

Informed consent

Prior informed consent for participation and publication have been obtained from all individuals included in the study.