Both protein and non-protein components in extracellular vesicles of human seminal plasma improve human sperm function via CatSper-mediated calcium signaling

Abstract

STUDY QUESTION

What is the significance and mechanism of human seminal plasma extracellular vesicles (EVs) in regulating human sperm functions?

SUMMARY ANSWER

EV increases the intracellular Ca²⁺ concentrations [Ca²⁺]_i via extracellular Ca²⁺ influx by activating CatSper channels, and subsequently modulate human sperm motility, especially hyperactivated motility, which is attributed to both protein and non-protein components in EV.

WHAT IS KNOWN ALREADY

EVs are functional regulators of human sperm function, and EV cargoes from normal and asthenozoospermic seminal plasma are different. Pre-fusion of EV with sperm in the acidic and non-physiological sucrose buffer solution could elevate [Ca²⁺]_i in human sperm. CatSper, a principle Ca²⁺ channel in human sperm, is responsible for the [Ca²⁺]_i regulation when sperm respond to diverse extracellular stimuli. However, the role of CatSper in EV-evoked calcium signaling and its potential physiological significance remain unclear.

STUDY DESIGN, SIZE, DURATION

EV isolated from the seminal plasma of normal and asthenozoospermic semen were utilized to investigate the mechanism by which EV regulates calcium signal in human sperm, including the involvement of CatSper and the responsible cargoes in EV. In addition, the clinical application potential of EV and EV protein-derived peptides were also evaluated. This is a laboratory study that went on for more than 5 years and involved more than 200 separate experiments.

PARTICIPANTS/MATERIALS, SETTING, METHODS

Semen donors were recruited in accordance with the Institutional Ethics Committee on human subjects of the Affiliated Hospital of Nantong University and Jiangxi Maternal and Child Health Hospital. The Flow NanoAnalyzer, western blotting, and transmission electron microscope were used to systematically characterize seminal plasma EV. Sperm [Ca²⁺]_i responses were examined by fluorimetric measurement. The whole-cell patch-clamp technique was performed to record CatSper currents. Sperm motility parameters were assessed by computer-assisted sperm analysis. Sperm hyperactivation was also evaluated by examining their penetration ability in viscous methylcellulose media. Protein and non-protein components in EV were analyzed by liquid chromatography-mass spectrum. The levels of prostaglandins, reactive oxygen species, malonaldehyde, and DNA integrity were detected by commercial kits.

MAIN RESULTS AND THE ROLE OF CHANCE

EV increased [Ca²⁺]i via an extracellular Ca²⁺ influx, which could be suppressed by a CatSper inhibitor. Also, EV potentiated CatSper currents in human sperm. Furthermore, the EV-in [Ca²⁺]_i increase and CatSper currents were absent in a CatSper-deficient sperm, confirming the crucial role of CatSper in EV induced Ca²⁺ signaling in human sperm. Both proteins and non-protein components of EV contributed to the increase of [Ca²⁺]_i, which were important for the effects of EV on human sperm. Consequently, EV and its cargos promoted sperm hyperactivated motility. In addition, seminal plasma EV protein-derived peptides, such as NAT1-derived peptide (N-P) and THBS-1-derived peptide (T-P), could activate the sperm calcium signal and enhance sperm function. Interestingly, EV derived from asthenozoospermic semen caused a lower increase of [Ca²⁺]_i than that isolated from normal seminal plasma (N-EV), and N-EV significantly improved sperm motility and function in both asthenozoospermic samples and frozen-thawed sperm.

LARGE SCALE DATA

N/A.

LIMITATIONS, REASONS FOR CAUTION

This was an in vitro study and caution must be taken when extrapolating the physiological relevance to in vivo regulation of sperm.

WIDER IMPLICATIONS OF THE FINDINGS

Our findings demonstrate that the CatSper-mediated-Ca²⁺ signaling is involved in EV-modulated sperm function under near physiological conditions, and EV and their derivates are a novel CatSper and sperm function regulators with potential for clinical application. They may be developed to improve sperm motility resulting from low [Ca²⁺]_i response and/or freezing and thawing.

STUDY FUNDING/COMPETING INTEREST(S)

This research was supported by the National Natural Science Foundation of China (32271167), the Social Development Project of Jiangsu Province (BE2022765), the Nantong Social and People's Livelihood Science and Technology Plan (MS22022087), the Basic Science Research Program of Nantong (JC22022086), and the Jiangsu Innovation and Entrepreneurship Talent Plan (JSSCRC2021543). The authors declare no conflict of interest.

seminal plasma extracellular vesicles, calcium signal, CatSper, sperm function, hyperactivation, asthenozoospermia

Introduction

Extracellular vesicles (EVs), the membrane-encapsulated particles that carry active cargos, are shed from most cell types and exist in a wide variety of body fluids. Increasing evidence indicates that EV are important mediators of intercellular communication, rather than non-specific waste from cells (Mathieu et al., 2019; Tai et al., 2019). EV can be taken up via specific membrane surface receptors or can release certain signal molecules to regulate recipient cell functions (Singh et al., 2016; Chen et al., 2018). Mammalian seminal plasma contains membranous EV (also called exosomes) mainly produced by the epididymis and prostate, traditionally termed epididymosomes or prostasomes, which contain proteins, nucleic acids, and a high content of cholesterol and sphingomyelin (Arvidson et al., 1989; Thimon et al., 2008; Ronquist et al., 2012; Vojtech et al., 2014). Previous studies have demonstrated that seminal plasma EV and their cargos have antibacterial, antioxidant, and immunosuppressive properties, and may be involved in several biological processes indirectly influencing sperm function (Ronquist et al., 2012; Noda and Ikawa, 2019). For example, EV reduce reactive oxygen species (ROS) overproduction and attenuate oxidative stress in sperm (Saez et al., 1998), and transfer Ca²⁺ signaling factors to regulate progesterone-induced sperm motility after the EV fuse with sperm in advance under acidic and non-physiological buffers (Park et al., 2011). Directly interacting with sperm, human seminal plasma EV may play pivotal roles in sperm maturation in the epididymis, as well as sperm function and fertilization (Bechoua et al., 2011; Du et al., 2016; Machtinger et al., 2016; Saez and Sullivan, 2016). Interestingly, EV isolated from donors with oligoasthenozoospermia exhibit different miRNA and protein profiles than EV from normozoospermic samples (Abu-Halima et al., 2016; Lin et al., 2019). Furthermore, normal human EV promote sperm motility and capacitation, while this effect is impaired in EV derived from donors with asthenospermia, in Biggers–Whitten–Whittingham noncapacitating medium (Murdica et al., 2019a). Overall, these studies indicate that EV act as functional regulators of male fertility, and also suggest that dysfunction of EV and its components may contribute to male infertility. However, the underlying mechanisms by which EV modulate sperm functions have not been fully explored.

Intracellular calcium homeostasis is a key component in the control of sperm motility, and Ca²⁺ influx is important for the maintenance of progressive motility and sperm function (Alasmari et al., 2013a; Pereira et al., 2017), especially for the acrosome reaction and hyperactivation (Darszon et al., 2005; Navarrete et al., 2015). All these processes are indispensable for sperm fertility. Consistent with roles in the regulation of sperm function, prostasomes have been showed to increase the [Ca²⁺]_i of human sperm through direct fusion between prostasomes and sperm (Palmerini et al., 1999). However, those results were obtained in the acidic and non-physiological sucrose buffer solution. To date, there is no convincing report concerning the precise mechanism of EV actions in the sperm [Ca²⁺]_i response under near physiological buffers.

Cation channel of sperm (CatSper), a unique cation channel protein family exclusively expressed in sperm, is composed of at least eleven subunits and controls Ca²⁺ influx to regulate sperm functions, such as capacitation, chemotaxis, acrosome reaction, and hyperactivation (Lishko et al., 2012; Lin et al., 2021). CatSper channel is essential for male fertility. For example, CatSper-deficient male mice and men with mutations in CatSper genes exhibit infertility due to lack of sperm hyperactivation (Lishko and Mannowetz, 2018). In addition, CatSper can be activated by many physiological substances, such as progesterone (P4), prostaglandins, and certain chemicals (Brenker et al., 2012; Mannowetz et al., 2017). Thus, it has been considered as a polymodal chemosensor for sperm function and fertility regulation (Brenker et al., 2012). However, the role of CatSper in EV-induced [Ca²⁺]_i remains unknown. Also, investigation is required to determine the EV components and their derivates that exert regulatory roles on human sperm function.

The present study investigated the mechanism by which EV regulate Ca²⁺ signaling in human sperm, with a focus on the involvement of CatSper. In addition, the functional and pathological significance of [Ca²⁺]_i responses evoked by EV and their derivates-evoked were also examined to provide insight into the potential clinical application of EV and EV protein-derived peptides.

Materials and methods

Reagents

Human tubal fluid (HTF) medium was purchased from Nanjing Aibei Biotechnology Co., Ltd (Nanjing, China). Fluo-4 AM and Pluronic F-127 were obtained from Molecular Probes (Eugen, OR, USA). Triton X-100 and Proteinase K (Pro K) were obtained from Solarbio (Beijing, China). Mibefradil (Mi), P4, 1,2-bis (o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA), EGTA were purchased from Sigma-Aldrich (St Louis, MO, USA). CD9 and CD63 antibodies were obtained from proteintech (Wuhan, China). Prostaglandin E1 (PGE1) and E2 (PGE2) were obtained from Aladdin (Shanghai, China) and TCI Development Co., Ltd (Shanghai, China), respectively. Ionomycin was acquired from MCE (MedChemExpress, NJ, USA).

Sperm sample preparation

The collection of semen samples and experiments in this study were approved by the Institutional Ethics Committees on human subjects of the Affiliated Hospital of Nantong University and Jiangxi Maternal and Child Health Hospital. Informed consent documents were signed by the donors. Semen viscosity and volume, sperm motility, and sperm concentration were assessed according to the WHO laboratory manual for the examination and processing of human semen (Fifth Edition, WHO, 2010). Samples were subjected to high-saline solution (HS, 135 mM NaCl, 5 mM KCl, 1 mM MgSO₄, 2 mM CaCl₂, 20 mM HEPES, 5 mM glucose, 10 mM lactic acid, and 1 mM Na-pyruvate, adjusted to pH 7.4 with NaOH) for washing or a swim-up purification method for the experiments, as described in previous studies (Luo et al., 2015; Brenker et al., 2018). Briefly, 2 ml semen were added to 4 ml HS for 30 min at 37°C. The upper sperm suspension (5 ml) was harvested and washed two times with HS at 1000 g, 5 min. The prepared sperm was used for the next experiments.

Extraction of EV

Seminal plasma EV were isolated as previous study with some modification (Abu-Halima et al., 2016). After semen was centrifuged at 7850g for 30 min at 4°C, the supernatant was collected and stored at −80°C until use. Before isolation, seminal plasma was filtered successively with 0.45 and 0.22 μm MF-Millipore™ membranes (Millipore, MA, USA). The filtered seminal plasma was centrifuged at 10 000g for 30 min at 4°C to remove cell debris and other impurities. Remaining supernatant was centrifuged at 68 000g for 90 min at 4°C. Then, the pellets were suspended in 35 ml of 0.32 M sucrose and centrifuged at 100 000g for 90 min at 4°C. Next, the pellets were washed with phosphate buffer saline (PBS) at 100 000g for 90 min at 4°C. Finally, the EV pellets were resuspended in 3.5 ml HS and stored at −80°C for further study. Here, 3.5 ml EV were isolated from the 35 ml seminal plasma, so that the stored concentration of EV was about 10 times of the physiological concentration. Accordingly, when the stored EV was 1:10 diluted, the experimental EV concentration was defined as 1:1 to match the physiological concentration. In order to isolate the supernatant from pellets of EV, the EV were treated with 0.02% Triton X-100 as described previously (Osteikoetxea et al., 2015). Then the supernatant and precipitates were separated at 100 000g for 90 min at 4°C.

Nanoflow analysis

The diameter range and concentration of EV were analyzed with a Flow NanoAnalyzer N30 (NanoFCM Inc, Xiamen, China). Marker protein expression in EV was analyzed by nanofluidic methods. The EV were labeled by CD63 or IgG antibodies (1:100) and followed by the corresponding secondary antibody (1:5000, Invitrogen, CA, USA). During the experiment, primary or secondary antibodies alone were added to samples as controls. In addition, CD63 antibody with PBS was used as background controls. After a final dilution (1:5000), each sample was analyzed by a Flow NanoAnalyzer N30 (NanoFCM Inc, Xiamen, China).

Transmission electron microscopy

The EV were observed with a transmission electron microscopy (TEM) (JEM-1200EX, Japan Electronics Co., Ltd). First, 5–10 μl EV were sedimented on copper mesh for 3 min and liquid absorbed with filter paper. After the sample was rinsed with PBS, it was dyed with phosphotungstic acid. Finally, it was dried at room temperature for 2 min and images were captured on the TEM (electron microscopy operating voltage 80–120 kV).

Metabolomics of EV non-protein components

After EV were extracted from seminal plasma, their cargos were obtained using ultrasonic crushing and ultrafiltration with molecular weight cut off 3 kDa, respectively. Then, non-protein components were further refined by methanol precipitation to remove the proteins, and the remaining parts were used as samples for EV metabolomics analysis by Liquid Chromatography-Mass Spectrometry (LC-MS) (Thermo, Ultimate 3000LC, Q Exactive, Waltham, MA, USA) with Hyper gold C18 column (100×4.6 mm 1.9 μm). The parameters for electron spray ionization (ESI) +  and ESI− ion modes were as follows: column temperature: 45°C; flow rate: 0.3 ml/min. ESI + : mobile phase A: water + 5% acetonitrile + 0.1% formic acid; mobile phase B: acetonitrile + 0.1% formic acid. ESI−: mobile phase A: water + 5% acetonitrile + 0.05% acetic acid; mobile phase B: acetonitrile + 0.05% acetic acid; automatic injector temperature: 4°C. The data were analyzed using feature extraction and preprocessed with Compound Discoverer software (Thermo Scientific, Sunnyvale, CA, USA).

Computer-aided sperm analysis system

To evaluate the effect of EV on sperm motility, seminal plasma and EV-free seminal plasma were prepared. EV-free seminal plasma was prepared as follows: cell debris and sperm were removed from intact seminal plasma by centrifugation and >220 nm vesicles were filtered with a 0.22 μm MF-Millipore™ membrane (Millipore, MA, USA). The filtered seminal plasma was centrifuged at 100 000g for 90 min twice, and then the supernatant was regarded as the EV-free seminal plasma. Sperm was resuspended in seminal plasma and EV-free seminal plasma, and then placed in a 5% carbon dioxide incubator (SANYO Electric Co., Ltd, Osaka, Japan) at 37°C for different time periods. Sperm parameters were measured by computer-aided sperm analysis system (CASA) (Hamilton Thorne, Inc., FL, USA). Total motility, progressive motility (PR), curvilinear velocity (VCL), straight-line (rectilinear) velocity (VSL), average path velocity (VAP), linearity (LIN), and other sperm parameters were recorded and analyzed.

Evaluation of the ability of sperm to penetrate viscous media

The environment of the female reproductive tract was simulated with 1% methylcellulose, because sperm complete fertilization of an oocyte after migration through the female reproductive tract. After sperm were selected by a swim-up purification procedure (30 min, 37°C), the sperm in the upper fractions were washed twice in HS buffer and adjusted to a concentration of 2.0 × 10⁷ cells/ml. EV or other drugs were added after capacitation for 2 h (progesterone was added after 2.5 h). After 3 h, 20 μl of 1% methylcellulose was drawn into a glass capillary tube, and the capillary was placed in the mixed sperm. The number of sperm at 3 cm (from the base) of the capillary tube was recorded after 1 h.

Evaluation of the acrosome reaction

Acrosomal reactions of human sperm were detected by chlorotetracycline (CTC) staining as described in the previous study (Luo et al., 2019). To assess whether N-P and T-P can induce acrosomal reactions like P4, human sperm were first cultured in HTF medium for 3.5 h, then incubated for 30 min with N-P, T-P, and P4, respectively, and stained with CTC. After staining, human sperm were imaged using a ZEISS fluorescence microscope (ZEISS, Oberkochen, Germany). The acrosomal reaction of sperm is characterized by the absence of fluorescence in the head. The ratio of acrosome reaction was evaluated by counting at least 1000 sperm.

Assessment of sperm hyperactivation

Sperm were capacitated in HTF medium for 3 h and treated with EV or drugs for 1 h at 37°C. Then 8 μl of sperm samples were used to assess motion characteristics via the CASA system (Hamilton Thorne, Inc., FL, USA) and at least 200 spermatozoa were counted for each assay. Aliquots of 8 μl non-capacitated sperm were used as a non-hyperactivated control. The ALH (lateral head displacement), LIN (linearity), VCL (curvilinear velocity), and other parameters were recorded. Hyperactivation was defined as cells with VCL ≥150 μm/s, LIN <50%, and ALH ≥7 μm, as described previously (Alasmari et al., 2013b). The percentage of hyperactivated sperm was calculated as the number of hyperactivated sperm/all motile sperm.

Assessment of intracellular calcium levels

Intracellular calcium levels in sperm were monitored with Fluo-4 AM dye and pluronic F127 (F127). In brief, the sperm were washed three times with HS and incubated with Fluo-4 AM (2 μM) and F127 (0.1%) in HS at 37°C for 30 min in darkness. After washing the cells with HS two times, sperm was exposed to EV or drugs and fluorescence intensity was assessed by the EnSpire^® Multimode Plate Reader (PerkinElmer, Waltham, MA, USA) at 37°C with excitation and emission wavelengths of 488 and 525 nm, respectively. To determine changes in the calcium signal, the sperm suspension was added to a 96-well plate, then the base fluorescence intensity (F₀) was measured. After the drug was added, the fluorescence (F) was measured, and the change in sperm [Ca²⁺]_i was calculated by ΔF/F₀ (%)=(F−F₀)/F₀×100%. The statistical analysis of the effects of EV or other drugs on sperm [Ca²⁺]_i at the time frame of peak values is indicated in bar charts.

Single-cell calcium imaging technology

The samples were purified by a 50% percoll solution (Percoll: HSA: EBSS: ddH20 = 5:1:1:3). After centrifugation at 1000 g for 15 min, the sperm was collected, washed once with HS, and resuspended in HS. The sperm was incubated with Fluo-4 AM and F127 in a 5% CO₂ incubator at 37°C for 30 min. After excess dye was washed off with HS, the sperm was adjusted to a concentration of 2.0 × 10⁷ cells/ml. Then 200 μl of sperm suspension was placed evenly into a dish bottom coated with poly-lysine. The sample was adhered to the wall and kept away from light for 30 min. After excess fluid was poured off, sperm in the dish were washed with HS. Finally, [Ca²⁺]_i was recorded in a single-cell calcium imaging system (OLYMPUS, Tokyo, Japan) with excitation and emission wavelengths of 488 and 525 nm, respectively, before and after EV treatment. The Hamamatsu Digital Camera C13440 (Tokyo, Japan) was equipped to capture the calcium changes and the frame rate is 60 Hz in our study. Regions of interest (ROIs) of different contrast were mapped on the head of each sperm using ImageJ (NIH, Bethesda, MD). After subtracting the background fluorescence intensity, the change in average fluorescence intensity for 10–15 sperm cells were calculated using ΔF/F₀ (%)=(F − F₀)/F₀ × 100%. F0 represents the base average fluorescence intensity of Fluo-4 AM at 525 nm over a period of 30 s. F was the fluorescence intensity of Fluo-4 AM at 525 nm after the sperm was treated by EV or other drugs. ΔF = F − F₀ indicates the relative changes of fluorescence intensity after the drug treatment.

Sperm patch-clamp recordings

The whole-cell patch-clamp technique was applied to record human sperm CatSper currents as previously described (Zeng et al., 2011). Seals were formed in the sperm cytoplasmic droplet or the neck region using a 15–30 MΩ pipette. The pipette solution for recording CatSper currents contained 135 mM Cs-Mes, 10 mM HEPES, 10 mM EGTA, and 5 mM CsCl, adjusted to pH 7.2 with CsOH. The transition to the whole-cell mode involved the application of short (1 ms) voltage pulses (400–650 mV) combined with light suction. The currents were stimulated for 1 s by voltage ramps from −100 to +100 mV from a holding potential of 0 mV. For recording the monovalent current of CatSper, a divalent-free (DVF) solution (150 mM NaCl, 20 mM HEPES, and 5 mM EDTA, pH 7.4) was used to record basal CatSper monovalent currents. Following this, different concentrations of EV and P4 were perfused to record CatSper currents at 15 s intervals about 30–60 s. The currents were analyzed with a Clampfit (Axon, Gilze, Netherlands) and figures were plotted with GraphPad 8 software (Golden Software, Inc., Golden, CO, USA).

Western blotting

The extracted EV were added to 5 × protein loading buffer and boiled at 95°C for 5 min to denature proteins for later use. Each protein sample was subjected to SDS-PAGE and transferred to a PVDF membrane, which was then blocked with 3% BSA for 1 h. Next, the membrane was incubated with CD63 and CD9 antibody (1:1000) at 4°C overnight, and then washed three times with Tris-buffered saline with Tween-20 (TBST, 20 mM Tris, 137 mM NaCl, 0.1% Tween-20, pH 7.6). The secondary antibody was added and incubated for 1 h. The PVDF membrane was then washed three times with TBST for 5 min, then exposed to ECL chemical reagent, and examined by a gel imaging system (Bio-Rad, CA, USA).

ELISA

The content of prostaglandins in EV was detected with PGE1 and PGE2 enzyme-linked immunosorbent assay kits from Wuhan Shenke Experimental Technology Co., Ltd (Wuhan, China) and Nanjing Jiancheng Bioengineering Institute (Nanjing, China), respectively, according to the operating instructions.

Sperm freezing and thawing procedure

The liquefied semen samples were diluted (1:1) with human sperm cryopreservation medium (Yuanye Bio-Technology, Shanghai, China) in the absence or presence of EV. Then it was transferred into 2.0 ml sterile plastic straws and placed in a Nalgene Mr Frosty Freezing Container (Thermo Scientific, Sunnyvale, CA, USA) to freeze from RT to −80°C at a rate of −1°C/min. After 5 h, the sample was thawed quickly at 37°C for 5 min. Semen was washed, treated, and detected according to the different experimental design procedures as above-mentioned methods.

Determination of sperm ROS levels and DNA integrity

Sperm were harvested and suspended in HS buffer. Then the cells were incubated with 10 μM 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA, Beyotime, Shanghai, China) at 37°C for 30 min. After washing of cells with HS three times, the ROS level of the sperm were evaluated by fluorescence intensity with DCFH-DA (excitation, 488 nm; emission, 525 nm) using an EnSpire^® Multimode Plate Reader (PerkinElmer, Waltham, MA, USA). Sperm DNA integrity was evaluated by a commercially available kit (Huakang Biomedical, Shenzhen, China) based on the chromatin diffusion principle according to the manufacturer's manual and a previous study (Zheng et al., 2018).

Identification of EV proteins and synthesis of peptides

Seven samples were used to extract EV protein by 8 M urea containing 1% protease inhibitor. The products of trypsin enzymolysis were submitted into a high performance liquid chromatography-mass spectrum system (HPLC-MS) to analyze and identify EV proteins using a 4D label-free quantification technique (Jingjie Biotechnology Co., Ltd, Hangzhou, China). The designed and screened EV protein-related peptides were synthesized by Synthbio Co., Ltd (Synthbio, Hefei, China). At least 85% pure peptides were used to conduct calcium signal and sperm function detection experiments.

Statistical analysis

In the present study, all sperm samples from the same person or pool were equally divided into several aliquots for experiments. Differences between the controls and treated samples were assessed with the paired Student’s t-test using the statistical software GraphPad Prism (version 5.01, GraphPad Software, San Diego, CA, USA) unless otherwise stated. Data are expressed as the mean ± SD. P < 0.05 was regarded as statistically significant. All experiments were performed at least three times unless otherwise stated.

Results

Identification of EV from human semen

EV were isolated from 15 different donors by ultracentrifugation and had an average diameter of 94.9 ± 21.8 nm (Fig. 1A). The EV size ranged from 50 to 200 nm, with most <100 nm and more than 4.67 × 10¹³ particles/ml were examined by the Flow NanoAnalyzer N30 (Fig. 1B). After treatment with Triton X-100, particle numbers decreased by about 18-fold to 2.66 × 10¹² particles/ml and many particles became smaller (Fig. 1C). The Flow NanoAnalyzer and western blotting analysis showed the presence of universal EV markers, CD63 and CD9, in the isolated EV (Fig. 1D–F). TEM further confirmed that the EV contained a single lipid bilayer and appeared as round particles in the expected size range of EV (Fig. 1G).

Figure 1.

Characteristics of human seminal plasma EV. (A) The diameter range of EV. After EV were isolated by ultracentrifugation, they were analyzed with Flow NanoAnalyzer. (B and C) The characteristic and concentration of particles. Intact and Triton X-100-treated (1%, 30 min) EV were detected by the Flow NanoAnalyzer. (D and E) Expression of CD63 protein in EV stained with IgG or CD63 antibody, respectively. Intact EV were subjected to immunofluorescence staining with antibodies directed against IgG or CD63 and then measured with Flow NanoAnalyzer. (F) Expression of CD63 and CD9 proteins, EV markers, in EV. EV from two different donors were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis with an antibody directed against CD63 or CD9. (G) Morphology of EV observed by transmission electron microscope. All experimental results, except for the morphology of EV, were repeated at least three times.

EV elevated [Ca²⁺]_i signals via extracellular Ca²⁺ influx

Since sperm plasma EV can regulate different aspects of sperm function and Ca²⁺ signaling is the major pathway to modulate sperm functions, here we investigate whether EV could activate calcium signaling in near physiological conditions. The effect of isolated EV on human sperm [Ca²⁺]_i was examined by utilizing a Ca²⁺ response fluorescent dye, and EV around physiological concentrations was utilized in this study unless specifically noted. Adding 10 μl normalized EV to 90 μl sperm suspension meant that the ratio of EV: sperm was 1:1 and the details are described in the EV extraction section of Materials and Methods. EV application increased human sperm [Ca²⁺]_i within 1 min in a dose-dependent manner in a pH 7.4 HS buffer (Fig. 2A and B). Next, the EV-evoked Ca²⁺ response was further confirmed by single-sperm [Ca²⁺]_i imaging technology (Fig. 2C–E). To determine whether the EV-induced increase in [Ca²⁺]_i was due to Ca²⁺ influx or the mobilization of Ca²⁺ stores, the [Ca²⁺]_i of sperm was measured in a Ca²⁺-free medium containing Ca²⁺ chelators BAPTA or EGTA. Under these conditions, EV had no effect on [Ca²⁺]_i (Fig. 2F and G), indicating that the EV-induced human sperm [Ca²⁺]_i resulted from an extracellular Ca²⁺ influx.

Figure 2.

EV induced the [Ca²⁺]_i increase via extracellular Ca²⁺ influx. (A) Examples of [Ca²⁺]_i change were illustrated with the time frame. The ratio of EV: sperm=1:1 indicated that the concentration of applied EV was close to physiological conditions (10 μl of 10× concentrated EV was added to 90 μl sperm suspension). Sperm [Ca²⁺]_i was monitored after loading cells with Fluo-4 AM (2 μM) and pluronic F127 (0.1% w/v) and the fluorescence intensity of the sperm was detected by microplate reader before and after adding the different concentrations of EV. (B) Statistical analysis of the effects of different concentrations of EV on sperm [Ca²⁺]_i. (C) Single-sperm [Ca²⁺]_i imaging indicated the [Ca²⁺]_i response in sperm after HS or EV treatment. Ionmycin treatment indicated that sperm were alive and had positive calcium responses. (D and E) The traces of imaging data came from the average fluorescence intensity of 12 high-saline solution (HS) and 10 (EV) sperm within the time frame. (F) Effect of EV on sperm [Ca²⁺]_i in Ca²⁺-free buffer. Sperm [Ca²⁺]_i was determined before and after adding 1:1 EV in the HS buffer with presence or absence of BAPTA (12 mM) or EGTA (5 mM). (G) Statistical analysis of the amplitude ΔF/F₀ = ((F − F₀)/F₀ × 100%) of [Ca²⁺]_i change from the peak values of F. Sperm [Ca²⁺]_i were recorded according to above-mentioned methods. Data were presented as mean±SD (n≥3). *P < 0.05, **P < 0.01, ***P < 0.001, ns (no significant) versus HS group.

CatSper mediated the EV-induced [Ca²⁺]_i response

In human spermatozoa, CatSper is the predominant Ca²⁺ channel responsible for Ca²⁺ influx (Lishko and Mannowetz, 2018). Hence, we examined whether CatSper is involved in the EV-induced human sperm [Ca²⁺]_i burst via extracellular Ca²⁺ influx. The CatSper blocker, Mi (Strünker et al., 2011), significantly impaired the EV-activated Ca²⁺ response, suggesting that CatSper was involved in the EV-induced increase in sperm [Ca²⁺]_i (Fig. 3A and B). To further verify the role of CatSper, the whole-cell patch-clamp technique was applied to examine the activation effect of EV on sperm CatSper. The results showed that EV potentiated CatSper currents in normal sperm samples (Fig. 3C and D), supporting the role of CatSper in the EV-induced increase of [Ca²⁺]_i. To clarify the overall role of CatSper in mediating EV-induced Ca²⁺ influx, we took advantage of an infertile sperm sample lacking the CatSper2 subunit, and consequently, lacking the CatSper current (Luo et al., 2019). The results showed that EV had no amplification effect on both CatSper current and [Ca²⁺]_i in the CatSper-deficient sample (Fig. 3E and F), confirming the requirement of CatSper for EV-induced effects in human sperm. Collectively, these results support that CatSper is required for mediating EV-induced [Ca²⁺]_i responses in human sperm.

Figure 3.

CatSper mediated the EV-induced [Ca²⁺]_i increase. (A) The CatSper blocker, mibefradil (Mi), impaired the EV-activated [Ca²⁺] response. Sperm [Ca²⁺]_i was monitored after loading sperm with Fluo-4 AM (2 μM) and pluronic F127 (0.1% w/v) and the fluorescence intensity of the sperm was detected by microplate reader after treating sperm with EV in the presence or absence of Mi (30 μM). (B) Statistical analysis of the peak [Ca²⁺]_i amplitude changes from (A). (C and D) EV activated CatSper current in normal human sperm. CatSper currents were examined by the whole-cell clamp technique from −100 to +100 mV as described in Materials and Methods section. Different concentrations of EV diluted in a sodium-based divalent free (DVF) buffer were perfused to record EV-induced CatSper currents. DVF solutions were used to record baseline monovalent CatSper currents. The pA (Picoampere) number represented the magnitude of the recorded current. Difference analysis between DVF and EV- or progesterone (P4)-treated samples in Figure 3D were assessed with the unpaired Student’s t-test. (E) EV did not potentiate currents in CatSper-deficient sperm. (F) [Ca²⁺]_i in CatSper-deficient sperm had no response to EV or EV protein-derived peptides stimulation. Sperm [Ca²⁺]_i was detected with microplate reader before and after EV (sperm: EV = 1:1) and/or P4 (1 μM). Ionmycin (1 μM), a Ca²⁺ signal activator independent of CatSper, served as a positive control. Data were presented as mean±SD (n ≥ 5). **P < 0.01, ***P < 0.001 versus high-saline solution (HS) or DVF group. ^##P < 0.01 versus EV group.

Both protein and non-protein components contained in EV provoked Ca²⁺ signaling

To investigate the specific EV cargos that control the calcium signals in human sperm, we obtained supernatants and precipitates from 0.02% Triton X-100-treated EV using ultracentrifugation (Osteikoetxea et al., 2015). Only the supernatant components induced the sperm [Ca²⁺]_i signaling (Fig. 4A and B), suggesting that the soluble molecules but not the membrane embedded components may be responsible for the above effects. Consistent with this idea, ultrasonic-disrupted EV had a stronger stimulatory effect of inducing the [Ca²⁺]_i signal compared with intact EV (Fig. 4C and D). To further investigate whether the EV-bearing proteins mediated the Ca²⁺ effects, EV supernatant treated with pro K at 58°C overnight (18 h) was applied, exhibiting a reduction of sperm [Ca²⁺]_i signal compared with that evoked by an unheated supernatant component (Fig. 4E and F). Furthermore, EV supernatant with high temperature denaturation also reduced the elevation of [Ca²⁺]_i (Fig. 4G and H). However, in both cases, the remaining elevation of [Ca²⁺]_i was still significant (Fig. 4E–H), indicating that non-protein components contributed to EV cargos-induced calcium response.

Figure 4.

Protein and non-protein components contained in EV provoked [Ca²⁺]_i signal. (A and B) Effect of EV components on sperm [Ca²⁺]_i. (C and D) Effect of ultrasonication on the EV-induced Ca²⁺ level increase. (E and F) Effect of proteinase (Pro) K on the EV supernatant-induced Ca²⁺ influx. (G and H) Effect of heating on the EV supernatant-induced Ca²⁺ increase. B, D, F, and H were the statistical analysis of the [Ca²⁺]_i amplitude changes from A, C, E, and G, respectively. Triton X-100 (0.02%)-penetrated EV were ultra-centrifuged to obtain supernatants and precipitates. After stimulating sperm with EV, supernatants, precipitates, ultrasonic treated-EV, heating- or Pro K-treated EV supernatants, sperm [Ca²⁺]_i was monitored by a microplate reader with Fluo-4 AM (2 μM) staining. Data were presented as mean ± SD (n ≥ 4). *P < 0.05, **P < 0.01, ***P < 0.001 versus high-saline solution (HS) group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus EV group. ^&P < 0.05, ^&&P < 0.01 versus supernatant group.

Consistent with the result that the EV-evoked [Ca²⁺]_i increase was dependent on extracellular calcium influx, there was also no Ca²⁺ response to pro K-treated EV and EV supernatant in a Ca²⁺-free medium (Fig. 5A–D). To screen the non-protein components responsible for Ca²⁺ signaling regulation, as described in the Materials and Methods section, the LC-MS technology was applied, and a larger number of small molecules with the potential to regulate calcium signaling were identified (Supplementary Table S1). Among these molecules, prostaglandins, PGE1 and PGE2, have been reported to induce sperm calcium signaling (Shimizu et al., 1998). Here we confirmed the existence of PGE1 and PGE2 in EV as non-protein components by ELISA (Fig. 5E), and both of them could increase sperm [Ca²⁺]_i (Fig. 5F–I). However, the amounts of PGE1 and PGE2 were lower than the concentration required to induce calcium signals, suggesting that other calcium signaling regulatory molecules were also involved in this effect. These data indicated that both EV proteins and other non-protein components promoted the [Ca²⁺]_i increase via extracellular Ca²⁺ influx.

Figure 5.

The dependence on an extracellular Ca²⁺ influx for the EV induced-[Ca²⁺]_i increase was confirmed by EV cargos. (A) The dependence on extracellular Ca²⁺ influx for the proteinase (Pro) K treated EV-induced [Ca²⁺]_i increase. (B) Statistical analysis of the peak [Ca²⁺]_i amplitude changes from A. (C) The dependence on extracellular Ca²⁺ influx for the EV supernatant-induced [Ca²⁺]_i increase. (D) Statistical analysis of the peak [Ca²⁺]_i amplitude changes from C. Sperm [Ca²⁺]_i were recorded according to the above-mentioned methods. (E) The concentrations of prostaglandin E1 (PGE1) and prostaglandin E2 (PGE2) were examined by ELISA kits according to the instructions. (F–I) Sperm [Ca²⁺]_i were recorded after stimulation with to PGE1 or PGE2 according to above-mentioned microplate reader method. Data were presented as mean±SD (n ≥ 3). *P < 0.05, **P < 0.01, ***P < 0.001 versus HS group, ^###P < 0.001 versus EV group.

EV protein-derived peptides activated calcium signaling and improved sperm function

Next, in order to further dissect the specific protein or its derivate activating sperm calcium signaling, EV proteins were digested by Glu-C, Lys-C or Factor Xa (Xa) protease. The results showed that the EV proteins-induced calcium level was impaired due to the above protease digestion (Fig. 6A–F), which not only verified the contribution of proteins in the sperm calcium signal regulation but also indicated the feature of the EV protein or protein degradation products with the Ile-(Glu/Asp)-Gly-Arg sequence. Particularly, the cutting site of the Xa protease is usually located after arginine residues in the Ile-(Glu/Asp)-Gly-Arg (IDGR or IEGR) sequence. Next, five proteins, ADAM10, Syntenin-2, Syntenin-3, NAT1, and THBS-2, with the IDGR or IEGR sequence were screened in mass spectrometry identified EV proteins (Fig. 6G and H), to design and synthesize five peptides for investigating their roles in sperm function regulation (Fig. 6H). As shown in Fig. 6I–L, N-P and T-P have strong activation effects on Ca²⁺ signals in normal sperm but did not induce Ca²⁺ signaling in a CatSper-deficient sample (Fig. 6M), which was in line with the EV effects on sperm (Fig. 3F). These results suggested that seminal vesicle EV proteins with IDGR or IEGR sequences and their derived peptides might be responsible for sperm functions under physiological conditions.

Figure 6.

EV protein-derived peptides activated sperm calcium signaling. Sperm [Ca²⁺]_i were detected after stimulated to EV protein (EV Pro) or Glu-C (A and B), Lys-C (C and D) or Factor Xa protease (E and F) digested protein components, respectively, according to above-mentioned Flou-4 AM staining with a microplate reader. (G) Screening of vesicle protein derivatives that activate sperm calcium signaling. (H) Sequence information of synthetic EV protein-related peptides. (I–L) Single-sperm [Ca²⁺]_i imaging indicated a [Ca²⁺]_i response in sperm after NAT1-derived peptide (N-P, 100 μM) and THBS-1-derived peptide (T-P, 100 μM) treatment. The fluorescence intensity of the sperm was visualized and detected with a single-cell calcium imaging system before and after adding N-P and T-P, and then adding progesterone (P4) as a positive control. (M) [Ca²⁺]_i in CatSper-deficient sperm had no response to EV protein-derived peptides stimulation. Sperm [Ca²⁺]_i was detected before and after adding N-P (100 μM), T-P (100 μM). Ionomycin (1 μM) served as a CatSper independent Ca²⁺ signal activator to indicate alive sperm. Data were presented as mean±SD (n ≥ 3). *P < 0.05, **P < 0.01 versus EV Pro group.

EV, EV cargos, and EV protein-derived peptides facilitated sperm motility and function

In view the recent result showing that CatSper mainly regulates sperm hyperactivation (Ernesto et al., 2015; Ferreira et al., 2021), we examined the effect of the EV-induced and CatSper-mediated [Ca²⁺]_i increase upon sperm motility and hyperactivation. The effects of EV on sperm motility parameters in seminal plasma were investigated by a CASA system. Sperm total motility, progressive motility (PR), VAP, VCL, and VSL parameters were markedly higher in normal seminal plasma than in EV-free seminal plasma isolated after ejaculation (Fig. 7A–F). Next, the effects of EV on human sperm hyperactivation were examined. As shown in Fig. 8A, EV indeed significantly promoted human sperm hyperactivation. In addition, the ability of human spermatozoa to penetrate artificial viscous media, which reflects the hyperactivated motility of sperm, was enhanced by EV, and this effect could be attenuated but by the CatSper inhibitor Mi (Fig. 8B). Treatment of EV supernatant with Pro K or heating significantly impaired the hyperactivated sperm count and their ability of sperm to pass through viscous media (Fig. 8C–F). However, with either way of removing the effect of protein components, the treated solutions still showed the ability to increase sperm hyperactivation, suggesting that EV-induced sperm hyperactivation resulted from both EV protein and non-protein components. In addition, consistent with their ability to increase the [Ca²⁺]_i of human sperm, EV protein-derived peptides N-P and T-P could dramatically improve sperm functions including the ability of sperm to pass through viscous media and the acrosome reaction (Fig. 8G and H).

Figure 7.

EV improved sperm motility in the seminal plasma. Human sperm were incubated in seminal plasma or EV-free seminal plasma at 37°C and 5% CO₂ incubator for 1 h. The motility parameters, (A) total motility, (B) progressive motility (PR), (C) average path velocity (VAP), (D) curvilinear velocity (VCL), (E) straight-line velocity (VSL), (F) linearity (LIN), were analyzed by a computer-aided sperm analysis system (CASA). A minimum of 200 spermatozoa were counted for each assay. The effects of human tubal fluid (HTF) on sperm motility were evaluated as the positive control. Data were presented as mean ± SD (n = 9). *P < 0.05, **P < 0.01, ***P < 0.001 versus the EV-free seminal plasma.

Figure 8.

EV, EV cargos, and EV protein-derived peptides promoted human sperm hyperactivation. (A) The effect of EV on human sperm hyperactivation (HA). (B) Mibefradil (Mi) impaired the EV-induced ability of sperm to penetrate into viscous medium. (C and D) The EV supernatant-induced HA and the ability of sperm to penetrate into artificial viscous media were attenuated by proteinase (Pro) K treatment. (E and F) The EV supernatant-induced HA and the ability of sperm to penetrate into artificial viscous media were also attenuated by heating. HA was assessed via a computer-aided sperm analysis system and 200 spermatozoa were counted at least for each assay. HA was defined as those cells with a curvilinear velocity (VCL) ≥150 μm/s, linearity (LIN) <50%, and amplitude of lateral head displacement (ALH) ≥7 μm as described in Materials and Methods section. The percentage of hyperactivated sperm was calculated as the number of hyperactivated sperm amongst all motile sperm. The ability of sperm to penetrate into artificial viscous media was evaluated as described in the Methods section. (G) Effect of NAT1-derived peptide (N-P) and THBS-1-derived peptide (T-P) on the ability of sperm to penetrate into artificial viscous media. (H) Effect of N-P and T-P on acrosome reaction of sperm. Progesterone (P4) stimulation served as a positive control. Data were presented as mean ± SD (n ≥ 5). *P < 0.05, **P < 0.01, versus HTF, ^#P < 0.05, ^##P < 0.01 versus EV-treated group, ^@P < 0.05 versus supernatant group, ^&P < 0.05 versus Pro K group.

EV enhanced sperm function in frozen-thawed sperm

Next, we confirmed that the frozen-thaw procedure compromised sperm motility (Fig. 9A and B) (Donnelly et al., 2001) and the P4-induced calcium signal (Fig. 9C and D) (Rossato et al., 2000), suggesting that a disorder of calcium signaling in frozen-thawed sperm might contribute to lowered motility and sperm dysfunction. EV application could improve sperm hyperactivated motility and the ability of spermatozoa to penetrate artificial viscous media in post-cryopreserved resuscitated sperm (Fig. 9E–H) in our study. Furthermore, pre-treatment of semen with EV dramatically protected sperm from the freeze-thaw procedure-induced sperm function impairments, indicated by enhanced the sperm motility (Fig. 10A–D) and reduced ROS and DNA damage levels (Fig. 10E and F). Collectively, the factors relating to calcium signal response and antioxidation derived from EV could benefit the frozen-thawed sperm. These results are expected to expand the application of EV, and its cargos have the potential be developed into regulators of sperm function in the future.

Figure 9.

EV improved the hyperactivation of frozen-thawed sperm. (A and B) The freeze and thaw procedure reduced sperm total motility and progressive motility (PR), respectively. The total motility and PR parameters of the fresh, 37°C persevered (Control) and frozen and thawed (FT) semen samples were assessed by a computer-aided sperm analysis system. (C) The progesterone (P4)-induced calcium level was compromised by the freeze-thaw process. Sperm [Ca²⁺]_i was monitored after loading cells with Fluo-4 AM (2 μM) and pluronic F127 (0.1% w/v) and the fluorescence intensity of the normal sperm was detected by a microplate reader before and after adding P4. (D) Statistical analysis of the amplitude of the Ca²⁺ changes from C. (E and F) EV facilitated sperm hyperactivated motility (HA) in frozen-thawed sperm. (G and H) EV promoted the ability of sperm to penetrate into artificial viscous media in frozen-thawed sperm. Sperm HA and the ability of sperm to penetrate into artificial viscous media were determined by above-mentioned methods. P4 stimulation served as a positive control. Data were presented as mean ± SD (n ≥ 5). *P < 0.05, **P < 0.01, ***P < 0.001 versus fresh or human tubal fluid (HTF) groups. ^##P < 0.01 versus control (Con) group.

Figure 10.

Pre-treatment of EV improved sperm functions and lowered reactive oxygen species (ROS) levels and DNA damage in frozen-thawed sperm. EV at a ratio of 2:1 were added into the cryoprotectant to freezing sperm. The thawed sperm total motility (A), progressive motility (PR) (B), curvilinear velocity (VCL) (C), and straight-line velocity (VSL) (D) were determined by a computer-aided sperm analysis system. (E) Sperm ROS levels were detected by 2',7'-dichlorodihydrofluorescein diacetate staining. (F) DNA integrity (DFI) of the fresh and post-cryopreserved sperm was evaluated with a commercial kit. Data were presented as mean ± SD (n = 7). *P < 0.05, **P < 0.01, *** P < 0.01 versus no treatment group (Fresh), ^#P < 0.05, ^##P < 0.01 versus frozen-thawed (F-T) group.

EV improved asthenozoospermic sperm motility

Considering that EV facilitate sperm motility (Murdica et al., 2019a) and function via calcium signals, we further investigated a potential clinical application of EV. The elevation of [Ca²⁺]_i in the normal sperm was significantly lower for EV derived from semen samples from men with asthenozoospermia than that from normal seminal plasma (Fig. 11A and B). Similarly, EV, especially those derived from normozoospermic semen, promoted the penetration of asthenozoospermic sperm into viscous medium and improved asthenozoospermic sperm progressive motility (Fig. 11C and D).

Figure 11.

EV improved sperm motility and [Ca²⁺]_i signalling in asthenozoospermic sperm. (A) Effect of EV from normozoospermic semen samples (N-EV) and asthenozoospermic semen samples (A-EV) on normal sperm [Ca²⁺]_i. Sperm [Ca²⁺]_i was monitored after loading cells with Fluo-4 AM (2 μM) and pluronic F127 (0.1% w/v) and the fluorescence intensity of the normal sperm was detected by microplate reader before and after adding the equivalent concentrations of EV extracted from normozoospermic or asthenozoospermic patients. (B) Statistical analysis of the amplitude of the Ca²⁺ changes from (A). (C) Effect of N-EV and A-EV on the ability of sperm to penetrate into artificial viscous media in asthenozoospermic sperm. The ability of sperm to penetrate into artificial viscous media was evaluated by the above-mentioned methods. P4 stimulation served as a positive control. (D) Effect of N-EV and A-EV on progressive motility (PR) in asthenozoospermic sperm. The PR% in spermatozoa derived from donors was determined by a computer-aided sperm analysis system before or after treatment with EV isolated from N-EV or A-EV. HSA (human serum albumin) stimulation served as a positive control. Data were presented as mean ± SD (n ≥ 3). *P < 0.05, **P < 0.01, ***P < 0.001 versus no treatment group (high-saline solution (HS) or human tubal fluid (HTF)). ^#P < 0.05, ^##P < 0.01 versus N-EV treated group.

Discussion

In this study, we for the first time demonstrated that under near physiological ionic and pH conditions, EV themselves could induce a significant increase of [Ca²⁺]_i in human sperm, and the EV-evoked [Ca²⁺]_i increase resulted from an extracellular Ca²⁺ influx. Furthermore, the EV-induced elevation of [Ca²⁺]_i was controlled by CatSper activation, confirmed by the result that EV no longer evoked the [Ca²⁺]_i increase or potentiated the CatSper current in CatSper-deficient sperm. EV, EV cargos, and EV protein-derived peptides promoted sperm function, especially hyperactivation. In addition, EV improved sperm motility and function in both asthenozoospermic samples and frozen-thawed sperm.

This study used various methods, such as the Flow NanoAnalyzer, western blotting, and TEM, to systematically identify and characterize EV isolated from human seminal plasma. Our data showed that the obtained EV showed similar vesicle structural characteristics to those described in a previous study (Aalberts et al., 2013). The EV diameters (94.9 ± 21.8 nm) were consistent with past reports (Murdica et al., 2019a; Vojtech et al., 2014).

CD63 protein expression was regarded as the classical marker of EV. We detected the existence of CD63 in human seminal plasma EV by western blotting. However, we firstly found that the ratio of CD63 positive EV was ∼18.5% in the samples by the Flow NanoAnalyzer, suggesting that not all EV expressed CD63 and there might be some non-classical markers-labeled seminal plasma EV as in EVs from other cells (Aalberts et al., 2013; Höög and Lötvall, 2015; Jeppesen et al., 2019). For example, it has been reported that chicken seminal fluid lacks the classical CD9- and CD44-bearing EVs (Alvarez-Rodriguez et al., 2020).

It was previously reported that EV may contribute to elevated Ca²⁺ concentrations in sperm via the release of calcium contained in EV during fusion with sperm, a result obtained under ion-free and acidic pH conditions (Palmerini et al., 1999). Here we found that, in a pH 4.4 HS buffer, the EV-induced [Ca²⁺]_i increase was much higher than in a pH 7.4 HS buffer (Supplementary Fig. S1A and B); this is probably due to more fusion of sperm with EV and transfer of Ca²⁺ signaling tools under acidic conditions. In addition, we further confirmed that EV contain free calcium ions even when they are incubated in a Ca²⁺-free extracellular solution (Supplementary Fig. S1C). However, EV failed to increase [Ca²⁺]_i in CatSper-deficient sperm, suggesting that the contribution of direct EV-sperm fusion to the observed [Ca²⁺]_i increase in this study should be minimal. In fact, our results were obtained under pH 7.4 and near physiological ions, a condition would largely prevent the fusion of EV with sperm (Palmerini et al., 1999). The results suggest that the EV-induced sperm [Ca²⁺]_i response was determined by an extracellular Ca²⁺ influx via CatSper. We proposed that, near physiological ions and pH conditions, EV may act as a ‘bridge’ between its cargos and sperm to activate CatSper-mediated calcium signaling. Specifically, calcium signaling was induced\by various EV cargos resulting from the low-level fusion of EV with sperm in the present study, which subsequently affected the CatSper channel directly or indirectly. The further results that EV-induced calcium signal response was inhibited by a CatSper blocker Mi and EV had no immediate enhancement effects on calcium signal and currents in CatSper-deficient samples verified our above assumption.

EV reportedly contains proteins, nucleic acid, lipids, and some small molecule compounds, such as prostaglandins (Oliw et al., 1993; Ronquist et al., 2011; Sullivan and Saez, 2013) and ATPs (Guo et al., 2019). Our results showed that Triton X-100 and ultrasonic-treated EV conferred a stronger stimulatory effect on the [Ca²⁺]_i signaling response compared with intact EV. In contrast, the precipitates isolated from Triton X-100-treated EV had no effect on sperm [Ca²⁺]_i, consistent with the idea that the soluble molecules inside sperm are responsible for CatSper activation. We speculated that both EV protein and non-protein components might exert Ca²⁺ signal activation in human sperm. In this study, we at first confirmed the contribution of protein components by examining the effects of Pro K- or heat-treated EV supernatants on [Ca²⁺]_i increase (Fig. 4). Furthermore, we tried to dissect the specific EV proteins by digesting them with Glu-C, Lys-C or Factor Xa (Xa) protease, and found five proteins, ADAM10, Syntenin-2, Syntenin-3, NAT1, and THBS-2 as the EV protein candidates (or their derivates) which may activate calcium signaling in human sperm (Fig. 6G and H). Regarding the non-protein components, we at first confirmed the existence of PGE1 and PGE2 (Fig. 5E), since it had been reported that nanomole levels of PGE1 and PGE2 could be detected from intact EV (Oliw et al., 1993). However, the detected concentrations of PGE1 and PGE2 in EV were much lower than those capable of evoking an observable [Ca²⁺]_i increase, implying that other major non-protein molecules or the combination of diverse non-protein components contributed to the EV-induced [Ca²⁺]_i increase. Indeed, by LC-MS analysis, 21 small molecules holding evidence to modulate calcium signaling in diverse cell types could be detected in human sperm plasma EV (Supplementary Table S1). For instance, arachidonic acid can increase the cytosolic free-calcium concentration depending on the existence of external calcium rat basophilic leukemia cells (Teshima et al., 2007). Methyl Jasmonate activates L-type calcium channel-mediated calcium signaling (Hemati et al., 2021). D-(+)-tryptophan induces a transient increase of intracellular calcium level in human neutrophils (Irukayama-Tomobe et al., 2009). Surprisingly, some non-physiological compounds with calcium regulation potential were also identified, reminding us that environmental pollutants might influence sperm function through EV. Overall, since there are a substantial number of protein and non-protein molecules that maybe involved in the [Ca²⁺]i provoking effect of EV, whether a specific molecule can be identified as the major component in EV to activate CatSper definitely requires further investigation.

Based on the effect of EV on CatSper activation and [Ca²⁺]_i increase, what role may EV play physiologically? EV from diverse resources come in contact with sperm in the male reproductive tract prior to ejaculation, then when sperm enter into the cervix and uterus, where is a viscous environment requiring progressive and hyperactivated motility of sperm. In fact, our results demonstrated that EV facilitated sperm hyperactivation and augmented sperm penetration into a methylcellulose medium (Fig. 8), suggesting that EV may enhance the ability of sperm to pass through the viscous female reproductive tract. Furthermore, our data indicated that physiological concentrations of EV promoted sperm total and progressive motility (Fig. 7), confirming previous reports showing that EV promoted sperm motility in an artificial buffer (Stegmayr and Ronquist, 1982; Oliw et al., 1993; Baskaran et al., 2020). Thus, we speculate that EV play a significant physiological role for sperm to penetrate the viscous female environment during the early stage of fertilization.

Is there a major molecule in EV responsible for regulating progressive and hyperactivated motility of human sperm? Based on the calcium-dependence feature of the progressive and hyperactivated motility, any EV components which could affect CatSper and [Ca²⁺]_i may contribute to progressive and hyperactivated motility regulation, as evidenced by that the findings that EV, EV cargos, and EV protein-derived peptides all could promote human sperm hyperactivation (Fig. 8). Other reports also offered some candidates. For example, EV protein including CRISP1, CD38, and cSrc kinases are thought to confer sperm hypermotility (Krapf et al., 2012; Kim et al., 2015; Murdica et al., 2019b). In addition, extracellular ATPs produced in seminal plasma EV can also promote sperm motility (Guo et al., 2019). Here we demonstrated that EV protein-derived peptides, T-P and N-P, could enhance [Ca²⁺]_i and improve sperm function (Figs 6 and 8), suggesting that their parental proteins, such as NAT1 and THBS-2, or peptides or degradation products of these parental proteins with the IDGR or IDER amino acid sequences, might contribute to sperm function regulation under physiological conditions. Nevertheless, at present no specific component in EV has been identified as the major molecule responsible for sperm motility regulation.

Consistent with the idea that EV regulate sperm progressive and hyperactivated motility during fertilization, a recent study reported that EV derived from normozoospermic but not from asthenozoospermic individuals improved sperm motility (Stegmayr and Ronquist, 1982). In addition, spermatozoa from asthenozoospermic patients exhibited reduced calcium responsiveness to progesterone (Espino et al., 2009). In this study, we validated that EV isolated from semen samples from men with severe asthenozoospermia (PR < 15%) have a markedly weaker effect on [Ca²⁺]_i activation than normal EV, and that the motility of asthenozoospermic sperm could be elevated by EV isolated form normal samples (Fig. 11). Taken together, the dysfunction or dyssecretosis of EV maybe a considerable factor in male infertility.

In addition, to improve the quality of asthenozoospermic sperm, EV have a potential application in protect sperm during post-cryopreservation. We showed that EV application improved sperm motility and functions post-cryopreservation and allowed them to resist oxidative stress indicating by the lowered ROS levels and DNA fragmentation index (Figs 9 and 10), which was consistent with the recent report that the antioxidant capacity of EV might be contributed by reduced ROS levels due to glutathione S-transferase mu2 (Mahdavinezhad et al., 2022; Wang et al., 2022). In other mammalians, such as in Canine, Red Wolves, and Cheetahs, post-thaw sperm quality and function have benefited from non-semen derived exosomes (Qamar et al., 2019; De Almeida Monteiro Melo Ferraz et al., 2020).

Many researchers have focused on finding a drug to treat male infertility and improve the clinical outcomes of assisted reproduction (Campbell et al., 2021; Gruber et al., 2022). Furthermore, there is a substantial need for fertility preservation. Our results suggest that the people seeking fertility preservation might be able to use their own EV to protect or improve sperm function, which would avoid possible health risks and ethical issues. Hence, we propose that high quality endogenous or engineering-modified EV or EV components-derived peptides may be developed as an efficient and noninvasive therapeutic agent to treat the reduced sperm motility caused by disorders in calcium signal responses (Vilanova-Perez et al., 2020).

Conclusions

In summary, we for the first time demonstrated that under near physiological ionic and pH conditions human seminal plasma EV could activate CatSper to induce the increase of [Ca²⁺]_i in human sperm via an extracellular Ca²⁺ influx, which contributed to EV-induced sperm hyperactivated motility. EV provided cargos that allow sperm to mobilize and manage a CatSper-regulated Ca²⁺ signaling mechanism rather than calcium ions in EV entering the sperm directly through fusion. The improvement of sperm motility and functions of both asthenozoospermic and post-cryopreserved sperm caused by EV might offer an opportunity for a potential clinical application. Meanwhile, seminal plasma EV protein-derived peptides have also been shown to induce an increase in [Ca²⁺]_i and improve human sperm function. This study provides insight into the role and mechanisms of human seminal plasma EV in sperm function, and reveals novel CatSper regulators for further examination in cases of sperm dysfunction and male infertility caused by [Ca²⁺]_i dysregulation. We also present potential options or candidate peptide drugs for clinical trials to improve sperm function in vitro.

Data availability

The data underlying this article will be shared on reasonable request to the corresponding authors.

Acknowledgements

The authors thank all the participants who took part in this study, as well as all medical staff who helped with human semen collection in the Affiliated Hospital of Nantong University. We thank Charles Allan, PhD, from Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.

Authors’ roles

X.Z., X.Z., and H.K. conceived, designed, and supervised the research. M.L., D.S., R.H., C.C., X.L., and J.Z. performed the experiments, analyzed the data, and created the figures. H.C., Q.W., X.S., and J.S. collected and prepared clinical samples. X.Z. and D.S. wrote the manuscript, and X.Z. revised the manuscript. All authors read and approved the final manuscript.

Funding

This research was supported by the National Natural Science Foundation of China (32271167), the Social Development Project of Jiangsu Province (BE2022765), the Nantong Social and People's Livelihood Science and Technology Plan (MS22022087), the Basic Science Research Program of Nantong (JC22022086), and the Jiangsu Innovation and Entrepreneurship Talent Plan (JSSCRC2021543).

Conflict of interest

All the authors declare no competing financial or non-financial interests.

Author notes

Xiaoning Zhang, Min Liang and Dandan Song contributed equally to this work.