Abstract
We studied the participation of the ubiquitin-proteasome system (UPS) in spermadhesin release during in vitro capacitation (IVC) of domestic boar spermatozoa. At ejaculation, boar spermatozoa acquire low molecular weight (8–16 kDa) seminal plasma proteins, predominantly spermadhesins, aggregated on the sperm surface. Due to their arrangement, such aggregates are relatively inaccessible to antibody labeling. As a result of de-aggregation and release of the outer layers of spermadhesins from the sperm surface during IVC, antibody labeling becomes feasible in the capacitated spermatozoa. In vivo, the capacitation-induced shedding of spermadhesins from the sperm surface is associated with the release of spermatozoa from the oviductal sperm reservoir. We took advantage of this property to perform image-based flow cytometry to study de-aggregation and shedding of boar spermadhesins (AQN, AWN, PSP protein families) and boar DQH (BSP1) sperm surface protein which induces higher fluorescent intensity in capacitated vs ejaculated spermatozoa. Addition of a proteasomal inhibitor (100 µM MG132) during IVC significantly reduced fluorescence intensity of all studied proteins (P < 0.05) compared to vehicle control IVC. Western blot detection of spermadhesins did not support their retention during IVC with proteasomal inhibition (P > 0.99) but showed the accumulation of DQH (P = 0.03) during IVC, compared to vehicle control IVC. Our results thus demonstrate that UPS participates in the de-aggregation of spermadhesins and DQH protein from the sperm surface during capacitation, with a possible involvement in sperm detachment from the oviductal sperm reservoir and/or sperm-zona pellucida interactions. The activity of sperm UPS modulates de-aggregation of boar spermadhesins and DQH sperm surface protein/binder of sperm1 (BSP1) during the sperm capacitation.
Introduction
Mammalian seminal plasma is a mixture of secretions mainly produced by the accessory organs of the male reproductive tract (seminal vesicles, prostate and Cowper’s glands), with a carryover of fluids from the testis and epididymis. Proteins of the seminal plasma come in contact with spermatozoa at ejaculation and bind to their surface to participate in the formation of sperm surface protein complexes. Seminal plasma proteins can be separated into three groups according to their structural characteristics. These are (1) spermadhesins; (2) proteins containing fibronectin type II (Fn2) domains or binder of sperm proteins; and (3) proteins exhibiting either enzymatic or inhibitory activities (reviewed in Jonakova et al. 2010).
Boar spermadhesins (AQN, AWN, PSP protein families) and boar DQH sperm surface protein/binder of sperm1 (BSP1) modulate binding properties of spermatozoa whereas boar proteinase inhibitor, sperm surface associated acrosin inhibitor (AI/SPINK2), is thought to protect spermatozoa from proteolytic degradation (Jonakova et al. 1991, 1992, 1998, 2010, Manaskova et al. 2007, Manaskova & Jonakova 2008, Davidova et al. 2009).
The proteins of AQN, AWN and PSP families are secreted mainly by the seminal vesicles, but also by prostate and epididymis (Veselsky et al. 1992, 1999, Manaskova & Jonakova 2008). Protein structure, biochemical features and binding properties of spermadhesins have been described in detail (reviewed in Topfer-Petersen et al. 1998, Jonakova & Ticha 2004, Jonakova et al. 2007, 2010). The AQN1 spermadhesin on the sperm surface has been implicated in sperm interactions with the oviductal epithelium in the pig (Ekhlasi-Hundrieser et al. 2005, Liberda et al. 2006). Another boar sperm surface protein, DQH (BSP1), may also assist in the formation of the sperm reservoir in the porcine oviduct (Manaskova et al. 2007). Sperm-oocyte binding test and other experimental data demonstrated that intact AQN1, AWN and DQH proteins on the sperm surface are required for the primary sperm-ZP binding (Veselsky et al. 1992, Dostalova et al. 1995, Ensslin et al. 1995, Rodriguez-Martinez et al. 1998, Veselsky et al. 1999, Manaskova et al. 2000, Manaskova et al. 2007).
Under physiological conditions, the boar seminal plasma proteins form variable aggregates (homo- and hetero-oligomers) differing in relative molecular mass, number of individual spermadhesins and Fn2 domain proteins, and interaction properties (Calvete et al. 1997, Jonakova et al. 2000, Manaskova et al. 2000, Jelinkova et al. 2003, Manaskova et al. 2003). The aggregated forms of boar seminal plasma proteins AQN, AWN, PSP-II, DQH and proteinase inhibitors bound to the sperm surface are formed during ejaculation. The interaction of aggregated forms with polysaccharides of glycosaminoglycans of the oviductal epithelial cells is an event leading to sperm capacitation. Significant to boar sperm capacitation, the aggregates of DQH, AQN and AWN proteins interact with cholesterol (Jonakova et al. 2000, Manaskova et al. 2000). Most recent studies of seminal plasma proteins are focused on their application in sperm-based biotechnologies (Caballero et al. 2012).
The ubiquitin-proteasome system (UPS) is a complex enzymatic machinery responsible for protein degradation and turnover in all living organisms including animals and plants. Research on UPS involvement in mammalian fertilization has brought to light compelling evidence on the participation of UPS in sperm capacitation (reviewed in Kerns et al. 2016), including but not limited to UPS role in the redistribution and turnover of protein species implicated in the formation of sperm-oviductal epithelium reservoir (MFGE8, ADAM5), sperm-ZP binding (MFGE8) and sperm plasma membrane fusion with the oolema in mice (ADAM5) (prior to publishing). Yet other sperm surface/seminal plasma proteins are thought to be processed by UPS during sperm capacitation (Yi et al. 2012, Miles et al. 2013), while some subunits of the 26S proteasome become post-translationally modified themselves during sperm capacitation (Zigo et al. 2018). Both AQN1 and AI/SPINK2 have been found to be ubiquitinated and to associate with sperm proteasomal subunits, and such interactions suggest the involvement of AQN1, SPINK2 and acrosomal proteasomes in various events of fertilization that include acrosomal surface remodeling, sperm capacitation, acrosomal exocytosis and sperm-ZP interaction (Yi et al. 2007, 2010a ,b ). The involvement of the sperm proteasomes with acrosomal function starts as early as sperm capacitation and may influence sperm detachment from the oviductal sperm reservoir (Yi et al. 2010a ).
The objective of this study is to broaden the understanding of the sperm capacitation by further elucidating the role of UPS in the de-aggregation and shedding of seminal plasma proteins from the sperm surface during IVC, with focus on spermadhesins and DQH, and implications for sperm-oviductal epithelium detachment.
Materials and methods
Statement of scientific rigor
All experiments in this article have been replicated with appropriate controls and consistent results between replicates. Readers are strongly encouraged to peruse Supplementary data (see section on supplementary data given at the end of this article) and contact corresponding author should concerns arise about the repeatability.
Reagents and antibodies used
Proteasomal inhibitor MG132 (cat # BML-PI102) was purchased from ENZO Life Sciences (Farmingdale, NY, USA). HaltTM Protease and Phosphatase inhibitor (cat # 78443) was purchased from Thermo Fisher Scientific. PAGErTM Gold gels (cat # 59545) and ProSieve protein colored markers were purchased from Lonza Rockland Inc. (Rockland, ME, USA). PVDF Immobilon Transfer Membrane and Luminata Crescendo Western HRP Substrate were bought from Millipore Sigma (Burlington, MA, USA). All other chemicals used in this study were purchased from Sigma-Aldrich.
Mouse monoclonal anti-β-tubulin antibody, clone E7 (Antibody Registry ID: AB_2315513) was bought from Developmental Studies Hybridoma Bank of University of Iowa, Iowa City, IA, USA. Rabbit polyclonal anti-AQN1, anti-AWN, anti- PSP-I, and PSP-II antibodies and mouse monoclonal anti-DQH antibody were raised in-house (Jonakova et al. 1998, Manaskova et al. 2003). Goat anti-mouse IgG (cat # 31430) and goat anti-rabbit IgG (cat # 31460), HRP secondary antibodies were purchased from Thermo Fisher Scientific. Goat anti-mouse IgG (cat # A10524) and goat anti-rabbit IgG (cat # A10523), Cyanine5 (Cy5) secondary antibodies; lectin PNA, AF488 and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen.
Semen collection and processing
All studies involving vertebrate animals were completed under the strict guidance of an Animal Care and Use protocol approved by the Animal Care and Use Committee (ACUC) of the University of Missouri. The sperm-rich fraction of fresh boar semen was collected on a weekly basis from three healthy, non-transgenic fertile boars used for routine in vitro fertilization trials with high blastocyst yield and as a positive control for somatic cell nuclear transfer experiments. Concentration and motility of ejaculates were evaluated by conventional spermatological methods under a light microscope. Sperm concentration was measured by hemocytometer (Thermo Fisher Scientific) and ranged from 250 to 350 million/mL. Good morphology spermatozoa with progressive motility were counted out of a total 200 spermatozoa per sample and only ejaculates with >80% motile spermatozoa and <20% morphological abnormalities were used in the study. Ejaculates were free of contaminants other than the expected minimal content of cytoplasmic droplets, thus not necessitating gradient purification. Ejaculates were centrifuged (2000 RPM, 10 min; IEC Centra-CL2 with 221 6 × 15 mL Economy swinging-bucket rotor, Thermo Fisher Scientific) to separate seminal plasma from spermatozoa. Portions of spermatozoa were used fresh directly for capacitation studies, as described below. Remaining spermatozoa were divided in halves, and the first half was washed three times with warm phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2HPO4, pH = 7.4), fixed in 2.0% formaldehyde for 45 min at room temperature and stored at 4°C for further use. The second half of the remaining spermatozoa was washed three times with warm Tris buffered saline (TBS; 50 mM TRIS∙HCl, pH = 7.4, 137 mM NaCl) and used for protein extraction, as described below.
Collection and processing of cauda epididymal spermatozoa
Cauda epididymal spermatozoa were collected from epididymides of a slaughtered healthy, non-transgenic fertile boar (National Swine Resource and Research Center; NSRRC, Columbia, MO) used for routine in vitro fertilization trials with high blastocyst yield. Epididymal fluid with cauda epididymal spermatozoa was gently aspirated with a transfer pipette and pelleted. The pellet was resuspended in warm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered Tyrode lactate medium (TL-HEPES, containing 10 mM Na-lactate, 0.2 mM Na-pyruvate, 2 mM NaHCO3, 2 mM CaCl2, 0.5 mM MgCl2; pH = 7.4, t = 37°C) and centrifuged at 1600 RPM (Thermo Fisher Scientific) 10 min and the spermatozoa containing supernatant was filtered through a 15 µm mesh. Sperm fraction was confirmed under a light microscope for lack of contamination. Cauda epididymal spermatozoa were divided in halves, and the first half was washed three times with warm PBS, fixed in 2.0% formaldehyde for 45 min at room temperature and stored at 4°C for further use. The second half was washed three times with warm TBS and used for protein extraction, as described below.
Sperm capacitation with proteasomal modulation
Fresh spermatozoa separated from seminal plasma were immediately washed three times in warm TL-HEPES supplied with 0.01% (w/v) polyvinyl alcohol (TL-HEPES-PVA). After the final wash, spermatozoa were resuspended in TL-HEPES-PVA medium supplemented with 2% (w/v) bovine serum albumin (BSA) to final concentration not exceeding 107 spermatozoa/mL and transferred in 15 mL Falcon tubes. Three treatment groups were initiated: (i) without proteasomal inhibition; (ii) with proteasomal inhibition, 100 μM MG132 (475.63 µg/10 mL of capacitation medium; 475.63 g/mol) in Ethanol (EtOH); and (iii) vehicle control, 0.2% (v/v) EtOH. All three treatment groups were capacitated for 4 h at 37°C and 5% (v/v) CO2. Sperm samples after capacitation were washed from BSA and used for protein extraction and flow cytometric quantification, described below.
Sample preparation and labeling for flow cytometric analysis
Fresh, capacitated spermatozoa with or without proteasomal inhibitors or vehicle solutions were washed three times with warm PBS, and fixed in 2.0% formaldehyde for 45 min at room temperature. Approximately 10 million of washed, 2.0% formaldehyde fixed capacitated spermatozoa with/without proteasomal inhibition including vehicle control, as well as washed, 2.0% formaldehyde-fixed cauda epididymal and ejaculated spermatozoa (described above) were permeabilized in 0.1% TrX-100 in PBS (PBST) for 45 min at room temperature. Spermatozoa were blocked with 5% normal goat serum (NGS) PBST for 30 min at room temperature. All the antibodies used for flow cytometric studies were previously characterized as cited. Primary antibodies used were as follows: anti-AQN1 (Jonakova et al. 1998) (1:400 dilution), anti-AWN (Jonakova et al. 1998) (1:600 dilution), anti-PSP-I (Manaskova et al. 2003) (1:200 dilution), anti-PSP-II (Manaskova et al. 2003) (1:400 dilution), anti-DQH, clones G12/G6 (Manaskova et al. 2007) (1:200 dilution); all diluted in 1% NGS PBTS. Primary antibodies were added to sperm sample tubes, and incubated overnight at 4°C. For negative controls, cauda epididymal spermatozoa were used, as these never came to contact with seminal plasma proteins. Further negative controls included non-immune mouse and rabbit sera of comparable globulin concentrations that were reported earlier (Zigo et al. 2018). The following morning, spermatozoa were washed twice with 1% NGS PBST, and appropriate species-specific secondary antibodies such as goat anti-mouse and goat anti-rabbit conjugated to Cyanine5 (GaM-Cy5, GAR-Cy5; both Invitrogen) were diluted 1:200–1:400 in PBST with 1% NGS, and allowed to incubate for 40 min at room temperature. For acrosome integrity assessment, peanut agglutinin lectin conjugated to Alexa Fluor 488 (PNA-AF488, 1:2500 dilution; Molecular Probes) was used, and 4′,6-diamidino-2-phenylindole dilactate (DAPI), a DNA stain (1:1500 dilution; Molecular Probes) was used as reference and nuclear contrast stain (Zigo et al. 2018). Both PNA-AF488 and DAPI were mixed and coincubated with secondary antibodies. After incubation with secondary antibodies, spermatozoa were washed twice with 1% NGS PBST. Prior to performing flow cytometry, the sample aliquots were checked for fluorescence labeling under a Nikon Eclipse 800 epifluorescence microscope (Nikon Instruments, Melville, NY, USA). After positive labeling was confirmed, the final concentration was adjusted to 10 million spermatozoa per 50 μL.
Image-based flow cytometry
The fluorescently labeled samples were measured with an Amnis FlowSight imaging flow cytometer (EMD Millipore Corp.) fitted with a 20X microscope objective (numerical aperture of 0.5) with an imaging rate up to 2000 events/s. The sheath fluid was PBS, free of Ca2+ or Mg2+. The flow-core size and speed was 10 μm diameter and 66 mm/sec, respectively. Raw images were acquired using INSPIRE® software (Amnis-Millipore). The camera was set to 1.0 μm per pixel of the charged-coupled device. The image display dimension for field of view was 60 μm and 8 µm depth of field. Samples were analyzed using four lasers concomitantly: a 405-nm line with intensity set to 50 mW; 488-nm line with intensity set to 50 mW; 642-nm line with intensity ranging from 5 to 10 mW and a 785-nm line (side scatter) with intensity set to 5 mW. A total of 10,000 events were collected per sample. Data analysis of the raw images was accomplished using IDEAS® software (Version 6.2.64.0; Amnis-Millipore), where the electronic images were compensated for channel crossover by using single-color controls (i.e., DAPI-only; AF488-only; and Cy5-only labeling of spermatozoa) that were merged to generate a multi-color matrix. The compensation matrix file was then applied to an experimental raw-image file (.rif), yielding a color-compensated image file (.cif). Displaying spermatozoa using Gradient RMS for the bright field channel allowed the gating of focused spermatozoa. Single cell events were gated by combining Area × Aspect Ratio scatter plot of the bright field in the first step, and Area × Aspect Ratio scatter plot of the DAPI channel in the following step. Single cell population gate was used for histogram display of mean pixel intensities by frequency for the following channels: AF488 (channel 2), DAPI (channel 7) and Cy5 (channel 11). Intensity histograms of individual channels were then used for drawing regions of subpopulations with varying intensity levels and visual confirmation. Intensity of DAPI (channel 7) was used for histogram normalization among different treatment groups. For more detailed analysis, a scatter plot of each single gated object was drawn from mean pixel intensities of AF488 (channel 2) vs Cy5 (channel 11). Appropriate masks were applied to all relevant channels to exclude fluorescently positive debris from features’ calculation. The Feature Finder tool was utilized to identify the most relevant optical/morphometric feature of subpopulations difference, where mean pixel intensities were not sufficiently distinctive. Negative controls of epididymal spermatozoa are included in the figures, normal mouse and normal rabbit sera are shown in a related article by Zigo et al. (2018).
Extraction of sperm proteins
After capacitation, all three sperm treatment groups (proteasomally inhibited, non-inhibited and vehicle control) were washed three times with TBS, at the same time with washed cauda epididymal and ejaculated spermatozoa (described above), and used for protein extractions.
Approximately 100 million spermatozoa were mixed with 100 µL of SDS-PAGE loading buffer (Laemmli 1970) supplemented with 50 mM dithiothreitol (DTT; Sigma-Aldrich) and protease and phosphatase inhibitors (Thermo Fisher Scientific). Spermatozoa were left to denature/reduce in loading buffer at 80°C, 10 min. Spermatozoa were spun off, and extracts were stored in −25°C for further analysis.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting (WB)
Total protein equivalent of 10 million spermatozoa was loaded per single lane. SDS-PAGE was carried out on 4–20% gradient gels (Lonza) as previously described (Laemmli 1970, Miles et al. 2013, Zigo et al. 2015). The molecular masses of separated proteins were estimated using prestained ProSieve protein colored markers (Lonza) run in parallel. After SDS-PAGE, proteins were electrotransferred onto a PVDF Immobilon Transfer Membrane (Millipore Sigma) using an Owl wet transfer system (Thermo Fisher Scientific) at a constant 50 V for 4 h for immunodetection (Zimmerman et al. 2011, Miles et al. 2013), according to the method described by Towbin et al. (1979). Residual gels after electrotransfer were stained with Coomassie Brilliant Blue (CBB) R-250 for protein load control.
Protein immunodetection
The PVDF membranes (Millipore Sigma) with the transferred proteins were blocked with 10% (w/v) non-fat milk in TBS with 0.05% (v/v) Tween 20 (TBST; Sigma-Aldrich) and incubated with the following primary antibodies: anti-AQN1 (Jonakova et al. 1998) (1:5,000,000 dilution), anti-AWN (Jonakova et al. 1998) (1:2,000,000 dilution), anti-PSP-I (Manaskova et al. 2003) (1:40,000 dilution), anti-PSP-II (Manaskova et al. 2003) (1:25.000 dilution), anti-DQH, clones G12/G6 (Manaskova et al. 2007) (1:100 dilution), overnight. For protein load normalization purposes, anti-β-tubulin antibody (1:4000, Developmental Studies Hybridoma Bank) was coincubated with each primary antibody. The next day, the membranes were incubated for 40 min with an appropriate species-specific secondary antibody, such as the HRP-conjugated goat anti-rabbit (GAR-IgG-HRP) and/or anti-mouse (GAM-IgG-HRP) antibodies (1:10,000 dilution; Invitrogen). The membranes were reacted with chemiluminescent substrate (Luminata Crescendo Western HRP Substrate; Millipore Sigma) and blots were screened with ChemiDoc Touch Imaging System (Bio-Rad), to record the protein bands, and analyzed by Image Lab Touch Software (Bio-Rad). Where not specified, procedures were carried out at room temperature. Membranes were stained with CBB R-250 after chemiluminescence detection for protein load control.
Statistical analysis
At least four independent replicates were conducted for flow cytometry and eight replicates for Western blotting to ensure scientific rigor. Numbers of replicates are also denoted in the figure legends. Each data point is presented as the mean ± s.d. Datasets were tested for normal distribution by using Shapiro–Wilk normality test, and processed using one-way analysis of variance (ANOVA) using the GraphPad Prism 7.03 (GraphPad Software, Inc.) in a completely randomized design. Sidak’s multiple comparison test was used to compare mean values of individual treatments with 95% confidence interval. A value of P < 0.05 was considered statistically significant.
Results
Validation of IVC system and proteasome modulation during boar sperm capacitation
We previously reported that spermatozoa before in vitro capacitation (IVC) and after IVC, using our established IVC system, differed significantly in protein tyrosine phosphorylation and proacrosin/acrosin activation (Kerns et al. 2018), both of which are hallmarks of boar sperm capacitation (Flesch et al. 1999, Tardif et al. 2001, Ded et al. 2010). Furthermore, we reported that IVC of boar spermatozoa induces outer acrosomal membrane (OAM) remodeling evidenced by the presence of four distinct sperm populations differing in area and intensity of PNA lectin labeling (Kerns et al. 2018). Addition of proteasomal inhibitors to capacitation media hindered OAM remodeling, causing statistically significant differences between individual sperm populations. Consequently, we used PNA labeling to monitor the progress of sperm capacitation and the efficacy of proteasomal inhibition during the IVC in presented study. As a result, the IVC induced OAM remodeling changes parted the treated spermatozoa into four distinct populations, and the addition of 100 µM MG132 resulted in significant differences (P < 0.05) in the relative abundance of populations 1 and 3 when compared to vehicle control (Supplementary Fig. 1).
The UPS contributes to seminal plasma protein de-aggregation during sperm capacitation
To study seminal plasma protein de-aggregation during IVC, we performed flow cytometric analysis of ejaculated and IVC capacitated spermatozoa with and without proteasomal inhibition (100 µM MG132) including vehicle control (0.2% EtOH). The aggregation of seminal plasma proteins on the sperm surface during ejaculation prevents their detection by antibodies when processed in a suspension. Antibody labeling becomes feasible as a result of de-aggregation and release of the outer layers of spermadhesins from the sperm surface during capacitation, which enables higher fluorescent labeling intensity in capacitated vs ejaculated spermatozoa. We also included PNA lectin labeling to monitor the association of this de-aggregation with OAM remodeling and acrosome integrity. Complementarily, we used WB detection of seminal plasma proteins to monitor whether the de-aggregation is linked to their release during sperm capacitation.
Flow cytometric histograms of all studied seminal plasma proteins showed a similar trend, where two distinct sperm populations with differential labeling intensity were present (Figs 1, 2, 3, 4 and 5A). The population with lower fluorescence intensity, where the majority of ejaculated spermatozoa were present, is the population with aggregated seminal plasma proteins. The presence of these aggregates on the surface of ejaculated spermatozoa completely masks its epitopes, and ejaculated spermatozoa appear similar to cauda epididymal spermatozoa that lack seminal plasma proteins on their surface (Figs 1, 2, 3, 4 and 5A). After IVC, the second population with higher fluoresce intensity arose as a result of seminal plasma proteins de-aggregation, making its epitopes available to antibody labeling. De-aggregation was significantly hindered when spermatozoa were capacitated with proteasomal inhibition in all studied proteins compared to vehicle controls (P = 0.0142 for AQN-1, P < 0.0001 for AWN, P = 0.0014 for PSP-I, P = 0.0086 for PSP-II and P = 0.0059 for DQH). Numerical values for seminal plasma protein de-aggregation are summarized in Table 1.
(A) Flow cytometric measurements of AQN-1 de-aggregation during in vitro capacitation under proteasome permissive/inhibiting conditions (100 µM MG132) and vehicle control (0.2% EtOH), combined with epifluorescence imaging of AQN-1 localization (B–B″). Every flow cytometric run represents 10,000 events. (C) Western blot detection of AQN-1 spermadhesin in the ejaculated and capacitated spermatozoa under proteasome permissive/inhibiting conditions (100 μM MG132) including vehicle control, with densitometric analysis (lower insert) and β-tubulin for protein normalization purposes (upper insert); (C′) the PVDF membrane stained with CBB after chemiluminescence detection shows comparable protein loads per lane, (C″) residual gel after electrotransfer for protein normalization purposes. Proteins were extracted with Laemmli loading buffer supplemented with 50 mM DTT, resolved on a 4–20% gradient gel and protein equivalent of 10 million spermatozoa was loaded per single lane. Results are presented as mean ± s.d. of five and eight independent replicates for flow cytometry and Western blot, respectively. Differences in proteasomal inhibition with statistical significance (P < 0.05) are highlighted in red. Cauda epididymal spermatozoa were used as a negative control for both flow cytometric measurements and WB detection.
Citation: Reproduction 157, 3; 10.1530/REP-18-0413
(A) Flow cytometric measurements of AWN de-aggregation during in vitro capacitation under proteasome permissive/inhibiting conditions (100 µM MG132) and vehicle control (0.2% EtOH), combined with epifluorescence imaging of AWN localization (B–B″). Every flow cytometric run represents 10,000 events. (C) Western blot detection of AWN spermadhesin in the ejaculated and capacitated spermatozoa under proteasome permissive/inhibiting conditions (100 μM MG132) including vehicle control, with densitometric analysis (lower insert) and β-tubulin for protein normalization purposes (upper insert); (C′) the PVDF membrane stained with CBB after chemiluminescence detection shows comparable protein loads per lane, (C″) residual gel after electrotransfer for protein normalization purposes. Proteins were extracted with Laemmli loading buffer supplemented with 50 mM DTT, resolved on a 4–20% gradient gel, and protein equivalent of 10 million spermatozoa was loaded per single lane. Results are presented as mean ± s.d. of five and eight independent replicates for flow cytometry and Western blotting, respectively. Differences in proteasomal inhibition with statistical significance (P < 0.05) are highlighted in red. Cauda epididymal spermatozoa were used as a negative control for both flow cytometric measurements and WB detection.
Citation: Reproduction 157, 3; 10.1530/REP-18-0413
(A) Flow cytometric measurements of PSP-I de-aggregation during in vitro capacitation under proteasome permissive/inhibiting conditions (100 µM MG132) and vehicle control (0.2% EtOH), combined with epifluorescence imaging of PSP-I localization (B–B″). Every flow cytometric run represents 10,000 events. (C) Western blot detection of PSP-I spermadhesin in the ejaculated and capacitated spermatozoa under proteasome permissive/inhibiting conditions (100 μM MG132) including vehicle control, with densitometric analysis (lower insert) and β-tubulin for protein normalization purposes (upper insert); (C′) the PVDF membrane stained with CBB after chemiluminescence detection shows comparable protein loads per lane, (C″) residual gel after electrotransfer for protein normalization purposes. Proteins were extracted with Laemmli loading buffer supplemented with 50 mM DTT, resolved on a 4–20% gradient gel, and protein equivalent of 10 million spermatozoa was loaded per single lane. Results are presented as mean ± s.d. of four and eight independent replicates for flow cytometry and Western blotting, respectively. Differences in proteasomal inhibition with statistical significance (P < 0.05) are highlighted in red. Cauda epididymal spermatozoa were used as a negative control for both flow cytometric measurements and WB detection.
Citation: Reproduction 157, 3; 10.1530/REP-18-0413
(A) Flow cytometric measurements of PSP-II de-aggregation during in vitro capacitation under proteasome permissive/inhibiting conditions (100 µM MG132) and vehicle control (0.2% EtOH), combined with epifluorescence imaging of PSP-II localization (B–B″). Every flow cytometric run represents 10,000 events. (C) Western blot detection of PSP-II spermadhesin in the ejaculated and capacitated spermatozoa under proteasome permissive/inhibiting conditions (100 μM MG132) including vehicle control, with densitometric analysis (lower insert) and β-tubulin for protein normalization purposes (upper insert); (C′) the PVDF membrane stained with CBB after chemiluminescence detection shows comparable protein loads per lane, (C″) residual gel after electrotransfer for protein normalization purposes. Proteins were extracted with Laemmli loading buffer supplemented with 50 mM DTT, resolved on a 4–20% gradient gel, and protein equivalent of 10 million spermatozoa was loaded per single lane. Results are presented as mean ± SD of four and eight independent replicates for flow cytometry and Western blotting, respectively. Differences in proteasomal inhibition with statistical significance (P < 0.05) are highlighted in red. Cauda epididymal spermatozoa were used as a negative control for both flow cytometric measurements and WB detection.
Citation: Reproduction 157, 3; 10.1530/REP-18-0413
(A) Flow cytometric measurements of DQH (pB1) de-aggregation during in vitro capacitation under proteasome permissive/inhibiting conditions (100 µM MG132) and vehicle control (0.2% EtOH), combined with epifluorescence imaging of DQH localization (B–B″). Every flow cytometric run represents 10,000 events. (C) Western blot detection of DQH spermadhesin in the ejaculated and capacitated spermatozoa under proteasome permissive/inhibiting conditions (100 μM MG132) including vehicle control, with densitometric analysis (lower insert) and β-tubulin for protein normalization purposes (upper insert); (C′) the PVDF membrane stained with CBB after chemiluminescence detection shows comparable protein loads per lane, (C″) residual gel after electrotransfer for protein normalization purposes. Upper and lower inserts in C were cropped from the identical membrane and only differ in exposure times. Proteins were extracted with Laemmli loading buffer supplemented with 50 mM DTT, resolved on a 4–20% gradient gel, and protein equivalent of 10 million spermatozoa was loaded per single lane. Results are presented as mean ± s.d. of four and eight independent replicates for flow cytometry and Western blotting, respectively. Differences in proteasomal inhibition with statistical significance (P < 0.05) are highlighted in red. Cauda epididymal spermatozoa were used as a negative control for both flow cytometric measurements and WB detection.
Citation: Reproduction 157, 3; 10.1530/REP-18-0413
A summary of seminal plasma protein turnover during sperm capacitation under proteasomal permissive and inhibiting conditions.
| Sperm population | Flow cytometric analysis | Densitometric analysis of WB | |||
|---|---|---|---|---|---|
| No labeling | De-aggregated | Acrosomal lab. | Whole sperm. lab. | ||
| AQN-1 | |||||
| Ejaculated | 79.02% ± 6.92% | 13.41% ± 6.01% | 3.36% ± 2.03% | 9.22% ± 6.45% | 2.12 ± 0.79 |
| Capacitated, no 26S inhibition | 27.86% ± 10.82% | 69.86% ± 8.71% | 38.26% ± 6.86% | 22.70% ± 5.61% | Reference (1.00) |
| Capacitated + 100 µM MG132 | 40.68% ± 4.47% | 50.82% ± 5.09% | 23.44% ± 2.91% | 20.46% ± 5.35% | 0.83 ± 0.27 |
| Capacitated + 0.2% EtOH | 32.52% ± 5.65% | 64.44% ± 4.66% | 33.90% ± 2.93% | 22.36% ± 6.62% | 0.74 ± 0.17 |
| AWN | |||||
| Ejaculated | 78.12% ± 5.10% | 12.77% ± 5.73% | 2.78% ± 1.70% | 8.14% ± 4.13% | 2.06 ± 0.64 |
| Capacitated, no 26S inhibition | 21.46% ± 4.39% | 74.04% ± 5.80% | 40.76% ± 7.68% | 22.42% ± 3.32% | Reference (1.00) |
| Capacitated + 100 µM MG132 | 40.36% ± 1.94% | 54.72% ± 3.65% | 25.04% ± 0.35% | 21.12% ± 1.06% | 0.92 ± 0.26 |
| Capacitated + 0.2% EtOH | 24.32% ± 5.34% | 71.56% ± 4.47% | 37.74% ± 7.50% | 24.60% ± 4.23% | 0.80 ± 0.16 |
| PSP-I | |||||
| Ejaculated | 84.43% ± 4.36% | 6.56% ± 2.01% | 5.22% ± 1.81% | 4.15% ± 1.76% | 2.28 ± 0.49 |
| Capacitated, no 26S inhibition | 24.28% ± 6.92% | 77.78% ± 7.28% | 41.78% ± 1.52% | 18.88% ± 2.46% | Reference (1.00) |
| Capacitated + 100 µM MG132 | 43.10% ± 7.38% | 58.25% ± 8.25% | 27.18% ± 4.42% | 17.73% ± 3.62% | 1.02 ± 0.28 |
| Capacitated + 0.2% EtOH | 28.48% ± 8.08% | 73.65% ± 6.74% | 37.88% ± 4.29% | 20.63% ± 2.87% | 0.94 ± 0.36 |
| PSP-II | |||||
| Ejaculated | 85.40% ± 7.39% | 7.15% ± 2.85% | 4.69% ± 1.40% | 5.41% ± 4.01% | 2.35 ± 0.28 |
| Capacitated, no 26S inhibition | 27.33% ± 5.60% | 71.78% ± 6.84% | 42.25% ± 3.46% | 22.20% ± 5.41% | Reference (1.00) |
| Capacitated + 100 µM MG132 | 45.93% ± 8.99% | 50.55% ± 6.92% | 25.20% ± 3.13% | 23.30% ± 5.94% | 1.01 ± 0.20 |
| Capacitated + 0.2% EtOH | 31.03% ± 10.28% | 66.75% ± 10.64% | 37.80% ± 3.04% | 25.58% ± 4.42% | 1.03 ± 0.26 |
| DQH | |||||
| Ejaculated | 78.63% ± 8.40% | 17.80% ± 7.89% | 4.22% ± 1.64% | 9.26% ± 5.02% | 1.60 ± 0.36 |
| Capacitated, no 26S inhibition | 20.93% ± 4.87% | 76.78% ± 6.06% | 44.33% ± 4.87% | 22.20% ± 5.97% | Reference (1.00) |
| Capacitated + 100 µM MG132 | 36.78% ± 9.17% | 59.43% ± 9.92% | 28.95% ± 5.04% | 21.30% ± 4.77% | 1.50 ± 0.41 |
| Capacitated + 0.2% EtOH | 20.95% ± 6.67% | 77.13% ± 7.40% | 41.63% ± 4.96% | 24.05% ± 3.92% | 1.04 ± 0.22 |
Table 1 is compiled from individual tables from Figs 1, 2, 3, 4 and 5. Statistical significance is denoted in bold letters.
To further study seminal plasma proteins’ labeling after de-aggregation, we plotted it against PNA labeling, and the representative density scatterplots for ejaculated and all three treatment groups of IVC spermatozoa are presented in Figs 1, 2, 3, 4 and 5, tables and Supplementary Figs 2, 3, 4, 5 and 6, respectively. Similarly, as in fluorescent histograms, we observed three distinct labeling patterns for all studied seminal plasma proteins. From Supplementary Figs 2, 3, 4, 5 and 6, it is evident that most of ejaculated spermatozoa are only PNA positive (Figs 1, 2, 3, 4 and 5B) as a result of seminal plasma protein aggregation. After IVC, spermatozoa with de-aggregated seminal plasma proteins were labeled in acrosomal region only (Figs 1, 2, 3, 4 and 5B′) and the relative abundance of spermatozoa with this labeling pattern differed depending on proteasomal inhibition. We observed significant differences in the sperm head labeling for all studies seminal plasma proteins in proteasomally inhibited IVC spermatozoa compared to vehicle control (P = 0.0490 for AQN-1, P = 0.0003 for AWN, P = 0.0319 for PSP-I, P = 0.0371 for PSP-II, and P = 0.0419 for DQH). As above, these numerical values are summarized in Table 1. Another observed labeling pattern was the whole spermatozoon labeling (Figs 1, 2, 3, 4 and 5B″), the abundance of which did not change after proteasomal inhibition in all studied seminal plasma proteins (P = 0.9971 for AQN-1, P = 0.8053 for AWN, P = 0.9669 for PSP-I, P = 0.9964 for PSP-II and P = 0.9911 for DQH; Table 1).
The WB detection of seminal plasma proteins in SDS extracts from cauda epididymal, ejaculated and capacitated spermatozoa (Figs 1, 2, 3, 4 and 5C) showed the presence of all corresponding proteins in all of the above treatments except epididymal spermatozoa. AQN1 was detected as multiple bands ranging from 9.5 to 14 kDa under both reducing (Fig. 1C) and non-reducing conditions (results not shown); AWN was detected as typical multiple bands ranging from 9 to 16 kDa under both reducing (Fig. 2C) and non-reducing conditions (results not shown); PSP-I was detected as multiple bands ranging from <9 to 13 kDa under both reducing (Fig. 3C) and non-reducing conditions (results not shown); PSP-II was detected as multiple bands ranging from <9 to 14.5 kDa under both reducing (Fig. 4C) and non-reducing conditions (results not shown), and finally DQH migrated at molar weight of 11.48 ± 0.08 kDa under reducing conditions (Fig. 5C), and at 12.13 ± 0.15 kDa under non-reducing conditions (Supplementary Fig. 7). A unique DQH band was detected in all three treatment groups of capacitated spermatozoa migrating at 14.03 ± 0.05 kDa under reducing conditions (Fig. 5C), and at 19.97 ± 0.33 kDa under non-reducing conditions (Supplementary Fig. 7A). The abundances of all studied seminal plasma proteins were significantly decreased in all treatment groups of capacitated spermatozoa when compared to ejaculated spermatozoa (P < 0.001 for AQN-1, AWN and PSPs, and P = 0.002 for DQH; Figs 1, 2, 3, 4, 5C and Table 1). No accumulation of studied spermadhesins (AQN1, AWN, and PSP family proteins) was observed in capacitated spermatozoa with proteasomal inhibition when compared to vehicle control (P > 0.99, Table 1). The accumulation of DQH was observed in capacitated spermatozoa with proteasomal inhibition compared to vehicle control (P = 0.03) and no difference in DQH abundance was observed between capacitated spermatozoa with proteasomal inhibition and ejaculated spermatozoa (P = 0.99; Fig. 5C, Table 1, Supplementary Fig. 7B, respectively).
Discussion
This study aims to further elucidate the role of the UPS in boar sperm capacitation, and specifically its role in the regulation of seminal plasma protein de-aggregation and release during sperm capacitation. Seminal plasma proteins in boars include spermadhesins (AQN, AWN and PSP families), fibronectin type II domain-containing proteins (also known as binder of sperm proteins – BSP) and proteins exhibiting enzymatic inhibitory and other activities (Jonakova et al. 2010). Even though the functions of individual seminal plasma proteins are well known, the fact that seminal plasma composition is very variable not just among species or individuals, but also between fractions of the same ejaculate has made it difficult to fully understand its effect in sperm function (Bedford 2015).
To validate our IVC system and proteasomal inhibition, we utilized lectin PNA as an indicator of sperm capacitation state, as it specifically binds to glycan moieties uniquely present on the OAM and exposed during IVC, (Supplementary Fig. 1). We previously reported that OAM remodeling during the progression of sperm capacitation is closely linked to protein tyrosine phosphorylation (Kerns et al. 2018). In agreement with our previous results, we observed the presence of four differentially PNA-labeled sperm populations, of which the relative abundance was altered by UPS inhibition.
Capacitation is an important step in post-testicular maturation of mammalian spermatozoa, essential for sperm detachment from the oviductal sperm reservoir (Suarez 2016). Spermahesin AQN1 being a case in point, displayed the strongest interaction with oviductal epithelium, making them prime candidates for sperm interactions with the oviductal epithelium in pig (Ekhlasi-Hundrieser et al. 2005, Liberda et al. 2006). We previously detected ubiquitinated forms of AQN1 and AI/SPINK2 which suggest that the activity and turnover of both AQN1 and AI/SPINK2 and thus the efficiency of the sperm-oocyte recognition process may be controlled by the UPS. With this evidence in mind, we further explored the engagement of proteasome in the turnover of sperm-bound seminal plasma proteins. We observed a significantly hindered de-aggregation of all major seminal plasma proteins (Figs 1, 2, 3, 4, 5A and Table 1) on the surface of proteasomally inhibited, capacitated spermatozoa. However, we were unable to observe accumulation of any of the studied spermadhesins (Figs 1, 2, 3, 4C and Table 1) in spermatozoa capacitated under proteasome-inhibiting conditions. The possible explanation of our findings can be interpreted in the light of spermadhesin binding to sperm surface and therefore their functions. Apart from PSP family proteins (non-heparin binding), which were found to have immunosuppressive function (Jonakova et al. 2000, Veselsky et al. 2000) and likely play only a marginal role in fertilization events; it is primarily non-aggregated AWN1 and AQN3 rather than their aggregated forms that are able to interact with a higher affinity to phosphorylethanolamine matrices (a major constituent of boar sperm membrane phospholipids) (Dostalova et al. 1995, Manaskova et al. 2000), suggesting that this nascent spermadhesin subpopulation can bind directly to the lipids of the sperm membrane thus forming the first layer of spermadhesins responsible for sperm-oocyte interaction (Dostalova et al. 1994). Aggregated spermadhesins could then migrate to the top of this first layer, serving as stabilizing factors that protect the acrosomal membranes from premature fusion/exocytosis as well as participating in the formation of the oviductal sperm reservoir. The majority of AQN and AWN spermadhesins coating the ejaculated sperm surface are released during capacitation (Figs 1C, 2C and Table 1). This indicates that large populations of individual spermadhesin are loosely associated with the sperm surface and may function as decapacitation factors, which is in agreement with previous studies (Dostalova et al. 1994). Sperm surface-bound AQN1 has been implicated in sperm interactions with the oviductal epithelium in pig (Ekhlasi-Hundrieser et al. 2005, Liberda et al. 2006). The removal of this protein from the surface of spermatozoa might be necessary for the disruption of the sperm-oviductal epithelium interaction.
Inhibition of proteasome during IVC hinders the remodeling of OAM (Supplementary Fig. 1) that adds to our previous findings (Yi et al. 2012). The activity of the ubiquitin-activating enzyme E1 (UBA1), the principal enzyme of the first step of protein ubiquitination is required for sperm capacitation, namely for correct remodeling of OAM. It is therefore reasonable to speculate that UPS plays an indirect, regulatory role in spermadhesins de-aggregation via modulation of OAM remodeling during capacitation, rather than being the proteolytic agent of spermadhesin shedding. Furthermore, there seems to be no particular reason for UPS to directly regulate the release of top sperm-coating spermadhesin layers as these are only loosely associated to the sperm surface, also supported by the WB detection of spermadhesins (Figs 1C, 2, 3, 4C and Table 1). Our results, however, do not exclude the possibility that the first layer of AWN and AQN responsible for sperm-oocyte interaction could be directly regulated by UPS-dependent proteolysis. As reported previously (Dostalova et al. 1994), up to 75% of the AQN and 90% of AWN are released from ejaculated spermatozoa during capacitation. This leaves a very small pool of AQN and AWN to study for UPS targeted degradation, and would require a different methodological approach. Nevertheless, this was not the aim of the presented study.
Similar to previously studied spermadhesins, we found de-aggregation of DQH/BSP1 to be hindered in spermatozoa capacitated under proteasomal inhibition (Fig. 5A and Table 1). Most likely, this de-aggregation event is directly linked to UPS regulation of OAM remodeling during capacitation, as in the case of spermadhesins. Furthermore, we found DQH to be covalently modified during IVC (Fig. 5C, Supplementary Fig. 7), and the apparent molar weight of modified DQH differed upon reducing conditions of SDS-PAGE. We found that DQH, or at least its covalently modified species are being accumulated in spermatozoa capacitated under proteasomal inhibition (Fig. 5C, Table 1, Supplementary Fig. 7). This suggests direct UPS modulation of DQH during sperm capacitation. It can be postulated that UPS may directly regulate seminal plasma protein de-aggregation during capacitation through modulation of DQH protein. DQH was previously found to be the major component of high-molecular-weight (>100 kDa) aggregates (Manaskova et al. 2000), therefore the specific targeting of this molecule seems logical in order to initiate the de-aggregation process during capacitation. This is not unlike the house of cards collapsing after one of the bottom cards is drawn from it. Nevertheless, this finding inspires further investigation.
In summary, the above studies further extend the knowledge about UPS participation in the process of sperm capacitation. Seminal plasma protein de-aggregation on the sperm surface was confirmed to be modulated by UPS, most probably via OAM remodeling and DQH targeting. The nature of DQH covalent modification, as well as the implication on direct UPS regulation of DQH protein, requires further studies.
Supplementary data
This is linked to the online version of the paper at https://doi.org/10.1530/REP-18-0413.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2015-67015-23231 from the USDA National Institute of Food and Agriculture (NIFA; (P S)), seed funding from the Food for the 21st Century (F21C) Program of the University of Missouri (P S), project BIOCEV CZ.1.05/1.1.00/02.0109 from the European Regional Development Fund (ERDF) BIOCEV grant CZ.1.05/1.1.00/02.0109 (M Z, P M-P, V J), the Czech Academy of Sciences (RVO: 86652036) ( M Z, P M-P, V J), USDA NIFA Graduate Fellowship award no. 2017-67011-26023 (K K) and CIGA20182006 (PP), Grant Agency of the Czech Republic No. GA-18-11275S (PP).
Acknowledgements
We thank the staff of National Swine Resource and Research Center (NSRRC) for boar semen and boar epididymis collections; Kathy Craighead for editorial and administrative assistance, and Miriam Sutovsky for technical and logistic assistance.
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