Capacitation Of Boar Spermatozoa Research Articles

Bicarbonate and BSA increase the capacitation pattern and acrosomal exocytosis in boar sperm after 120 min of incubation.

Sperm capacitation is a crucial step towards the acquisition of fertilizing capacity. Despite the attempts to mimic the invivo situation, there is still a lack of standardization invitro techniques. Bicarbonate and serum albumin (BSA) are routinely used, although controversial results are reported regarding the optimal concentration of each compound. In addition, whether caffeine is needed on invitro capacitation media in boar sperm remains to be elucidated. Here, 18 boar commercial artificial insemination doses were used to test different concentrations of bicarbonate (19, 37 or 56 mM) in experiment 1, BSA (1.5, 3, 4.5 mg/mL) in experiment 2 and the presence or absence of caffeine (5.15 mM) experiment 3. We analysed at 0, 30 and 120 min of incubation at 38.5°C, 5% CO2 : Total motility (TMOT), membrane integrity (VIAB), acrosomal exocytosis (rAcro; H33342/PI/PNA), capacitation status (chlortetracycline staining CTC) and mitochondrial membrane potential (JC-1). The higher concentrations of bicarbonate (37 and 56 mM) decreased TM and VIAB (p < .01) but increased rAcro (p < .01) after 120 min of incubation compared to the fresh control. In contrast, only the BSA concentration of 3 mg/mL reduced the VIAB at 120 min, but all the concentrations tested increased the average of JC-1 and decreased TM (p < .01) throughout incubation compared to the fresh control. Finally, in experiment 3, when boar sperm were incubated in the capacitating media with bicarbonate, BSA and with or without caffeine, the capacitated pattern measured by the CTC technique and rAcro increased after 120 min of incubation (p < .01) compared to fresh control, either in the presence or in the absence of caffeine. In summary, our results suggested that the combination of capacitating components, like bicarbonate and BSA, contributed to increasing the proportion of capacitated boar spermatozoa, mitochondrial membrane potential as well as acrosomal exocytosis. However, caffeine did not significantly influence invitro sperm capacitation in this species.

Effect of glucose and reactive oxygen species on boar sperm induced-acrosome exocytosis

Ejaculated boar spermatozoa can be liquid preserved for several days and be easily activated to produce physiological changes. One of the major changes is acrosome exocytosis that is physiologically related to capacitation. Glycolysis and reactive oxygen species (ROS) were studied regarding several boar sperm functions, but data available about their effect on boar sperm acrosome exocytosis are scarce. The objective of this work was to evaluate the effect of glucose and ROS on boar sperm acrosome exocytosis. We evaluated acrosome exocytosis by progesterone induction of capacitated sperm and assess viability, kinematics parameters, ROS levels, ATP content and Protein Kinase A activity in media with or without glucose and hydrogen peroxide or potassium chromate, as source of ROS. Our results show that glucose has no effect on acrosome exocytosis and also, it is not necessary for boar sperm capacitation, although it has a positive effect in the presence of ROS. On the other hand, ROS effects are related to spontaneous acrosome reaction. We conclude that glycolysis may function as a metabolic pathway that provides sustain but is not directly involved in boar sperm acrosome exocytosis and capacitation. Also, ROS do not promote capacitation in boar sperm, but affect spontaneous acrosome exocytosis.

The Ubiquitin-Proteasome System Participates in Sperm Surface Subproteome Remodeling during Boar Sperm Capacitation.

Sperm capacitation is a complex process endowing biological and biochemical changes to a spermatozoon for a successful encounter with an oocyte. The present study focused on the role of the ubiquitin-proteasome system (UPS) in the remodeling of the sperm surface subproteome. The sperm surface subproteome from non-capacitated and in vitro capacitated (IVC) porcine spermatozoa, with and without proteasomal inhibition, was selectively isolated. The purified sperm surface subproteome was analyzed using high-resolution, quantitative liquid chromatography-mass spectrometry (LC-MS) in four replicates. We identified 1680 HUGO annotated proteins, out of which we found 91 to be at least 1.5× less abundant (p < 0.05) and 141 to be at least 1.5× more abundant (p < 0.05) on the surface of IVC spermatozoa. These proteins were associated with sperm capacitation, hyperactivation, metabolism, acrosomal exocytosis, and fertilization. Abundances of 14 proteins were found to be significantly different (p < 0.05), exceeding a 1.5-fold abundance between the proteasomally inhibited (100 µM MG132) and vehicle control (0.2% ethanol) groups. The proteins NIF3L1, CSE1L, NDUFB7, PGLS, PPP4C, STK39, and TPRG1L were found to be more abundant; while BPHL, GSN, GSPT1, PFDN4, STYXL1, TIMM10, and UBXN4 were found to be less abundant in proteasomally inhibited IVC spermatozoa. Despite the UPS having a narrow range of targets, it modulated sperm metabolism and binding by regulating susceptible surface proteins. Changes in CSE1L, PFDN4, and STK39 during in vitro capacitation were confirmed using immunocytochemistry, image-based flow cytometry, and Western blotting. The results confirmed the active participation of the UPS in the extensive sperm surface proteome remodeling that occurs during boar sperm capacitation. This work will help us to identify new pharmacological mechanisms to positively or negatively modulate sperm fertilizing ability in food animals and humans.

Sperm capacitation as a predictor of boar fertility.

Prediction of a boar's fertility level has great economic importance for sow herds. After standard sperm morphology and motility metrics are met, approximately 25% of boars have less than 80% conception rates. Due in part to the many factors involved in the fertilization process, a multifactorial model incorporating multiple relevant sperm physiology factors will likely lead to increased understanding of boar fertility. Here we review the current literature on boar sperm capacitation as a predictor of boar fertility. While limited, several studies have provided correlations between the percentage of sperm in an ejaculate that are capable of undergoing sperm capacitation in a chemically defined media and artificial insemination field fertility as well as proteome and other methods. Work summarized here underscores the need for further understanding of boar fertility.

The CASR antagonist NPS2143 induces loss of acrosomal integrity and proteolysis of SPACA1 in boar spermatozoa.

The mechanism by which p32 protein increases during capacitation in boar spermatozoa is unknown. This manuscript shows a new mechanism of induction of p32 in boar spermatozoa: the proteolysis of the phosphorylated and glycosylated form of SPACA1. Protein tyrosine phosphorylation (PY) induction is associated with sperm capacitation. We previously showed that calcium-sensing receptor (CASR) inhibition by NPS2143 induces the 32 kDa tyrosine-phosphorylated protein (p32) in boar spermatozoa. We showed that NPS2143 induced an increase in p32 and loss of acrosomal integrity in live and dead spermatozoa in capacitating conditions (Tyrode's complete medium); the p32 rise occurred in dead spermatozoa, as shown by flow cytometry sorting. EGTA addition blunted the increase in p32, the loss of acrosomal integrity, and the increase in dead spermatozoa induced by NPS2143, indicating that the effects of NPS2143 are calcium-dependent. Mass spectrometry was used to identify which tyrosine-phosphorylated proteins were induced by NPS2143, but only serine/threonine-phosphorylated proteins were found; among these, SPACA1 was identified with different molecular weights (18, 32, and 35-45 kDa). We confirmed tyrosine phosphorylation of SPACA1 at 32 and 35-45 kDa by immunoprecipitation and co-localization of PY and SPACA1 in the presence of NPS2143 by immunofluorescence. The molecular weight of SPACA1 (35-45 kDa) decreased after treatment with peptide-N-glycosidase F, indicating that this protein is N-glycosylated. The soybean trypsin inhibitor (STI), a serine protease inhibitor, suppressed the appearance of p32 and SPACA1 (30 and 32 kDa) induced by NPS2143. Also, 8-Br-cAMP and A23187 treatments induced an increase in p32 and SPACA1 (30-32 kDa) and a parallel induction of the acrosome reaction. These findings suggest that CASR inhibition induces loss of acrosomal integrity and proteolysis of the glycosylated and phosphorylated SPACA1 (35-45 kDa) resulting in a SPACA1 rise at 32 kDa (p32).

Hexane fraction of Prunus japonica thunb. Seed extract enhances boar sperm motility via CatSper ion channel

IntroductionMammalian sperm motility is facilitated by flagellar beating, which depends on active ion movement through ion channels and their regulation. Prunus japonica Thunb., also known as oriental bush cherry, is a widely used traditional medicinal plant. However, its significance in improving fertility and sperm quality has not been fully elucidated yet. One of our previous reports revealed that P. japonica seed extract (PJE) can improve human sperm motility through intracellular pH modulation. Aim of the studyThe present study was designed to investigate the effects of PJE on boar spermatozoa and potential underlying mechanisms. Materials and methodsSperm motility changes were examined using a computer-assisted sperm analysis (CASA) system under both capacitated and non-capacitated conditions. Intracellular calcium concentration was measured using either confocal microscopy or a fluorescent microplate reader with Fluo-4AM calcium fluorescent dye. Sperm capacitation-related proteins were analyzed using western blotting. ResultsA significant increase in rapid motility, velocity, and linear displacement of sperm was observed in PJE-treated capacitated boar sperm, whereas the effect was insignificant in the non-capacitated counterparts. Intracellular calcium levels were significantly elevated upon PJE treatment (20–100 μg/L) in a concentration-dependent manner. The increase in intracellular calcium levels was inhibited when the sperm were treated with a CatSper (cation channel of sperm) channel inhibitor, 10 μM Mibefradil, indicating the involvement of the ion channel in the PJE modulatory mechanism. In addition, western blotting revealed an increased level of protein phosphorylation (p-tyrosine and p-PKA), which is a hallmark of sperm capacitation. ConclusionsPJE treatment resulted in a combination of increased motility, intracellular calcium concentration, and capacitation, thereby indicating its potential to ameliorate sperm motility parameters and induce capacitation of boar spermatozoa as a result of intracellular calcium elevation via the CatSper channel. Our observations further elaborate ion channel-related underlying mechanisms and show putative implications of the seed extract of traditionally used P. japonica Thunb. in ameliorating sperm quality.

Odorant Receptor OR2C1 Is an Essential Modulator of Boar Sperm Capacitation by Binding with Heparin

Heparin, a class of glycosaminoglycans (GAGs), is widely used to induce sperm capacitation and fertilization. How heparin induces sperm capacitation remains unclear. Olfactory receptors (ORs) which are G protein-coupled receptors, have been proposed to be involved in sperm capacitation. However, the interaction between ORs and odor molecules and the molecular mechanism of ORs mediating sperm capacitation are still unclear. The present study aimed to explore the underlying interaction and mechanism between heparin and ORs in carrying out the boar sperm capacitation. The results showed that olfactory receptor 2C1 (OR2C1) is a compulsory unit which regulates the sperm capacitation by recognizing and binding with heparin, as determined by Dual-Glo Luciferase Assay and molecular docking. In addition, molecular dynamics (MD) simulation indicated that OR2C1 binds with heparin via a hydrophobic cavity comprises of Arg3, Ala6, Thr7, Asn171, Arg172, Arg173, and Pro287. Furthermore, we demonstrated that knocking down OR2C1 significantly inhibits sperm capacitation. In conclusion, we highlighted a novel olfactory receptor, OR2C1, in boar sperm and disclosed the potential binding of heparin to Pro287, a conserved residue in the transmembrane helices region 7 (TMH7). Our findings will benefit the further understanding of ORs involved in sperm capacitation and fertilization.

Analysis of differential proteins between non-capacitated and capacitated boar sperm and verification of the effect of phosphofructokinase on capacitation

The objective of this study was to analyze the differences in the proteins in non-capacitated and capacitated boar sperm and to identify the functions of the differential proteins and key capacitation proteins of boar sperm before and after capacitation. Transwell chambers were used to separate capacitated sperm proteins using a unique polycarbonate membrane. Meanwhile, isotopic tags for relative and absolute quantification combined with LC‒MS/MS analysis were used for quantitative determination of differential proteins. Through the comparative analysis of different databases, 475 different proteins were identified in non-capacitated sperm and capacitated sperm, of which 303 were significantly upregulated and 172 were significantly downregulated. These differentially-expressed proteins are mainly involved in redox processes, cell biosynthesis processes and cell aromatic compound metabolism biological processes. They also participate in the signaling pathways of phosphorylation, ketone synthesis and degradation, most of which interact to varying degrees. Among these differentially-expressed proteins, phosphofructokinase attracted our attention as a potential capacitated protein. We further verified that phosphofructokinase can promote boar sperm capacitation by immunoblotting.

Inhibition of 26S proteasome enhances AKAP3-mediated cAMP-PKA signaling during boar sperm capacitation

This study investigates the effects of the ubiquitin–proteasome pathway (UPP) on porcine sperm capacitation and its interactions with the cAMP-PKA pathway. The semen of adult Landrace boars was divided into four groups: non-capacitated, capacitated, 10 μM/mL MG132, and 10 μM/mL DMSO groups. We characterized the parameters related to sperm dynamics using a computer-assisted sperm analysis system. The level of sperm protein tyrosine phosphorylation was detected using Western blotting, and the change of zinc ion signal was detected via flow cytometry. The relationship between A-kinase-anchor protein 3 (AKAP3), ubiquitin (Ub), and protein kinase A (PKA) was assessed by co-precipitation assays; to evaluate the interactions between the UPP and cAMP-PKA pathway, threonine, serine, and tyrosine phosphorylation were detected using Western blotting to evaluate the interaction between the UPP and cAMP-PKA pathway; Hoechst staining was used to detect the sperm-egg binding state and evaluate the effects of UPP inhibition. During capacitation, the levels of protein tyrosine, serine, and threonine phosphorylation and ubiquitination of porcine sperm increased, and sperm-egg binding was inhibited (P < 0.05). AKAP3 was degraded by UPP, and after inhibiting the 26 S proteasome, ubiquitinated AKAP3 accumulated in large quantities. Our findings indicate that, after the 26 S proteasome was inhibited, PKA was uncoupled from AKAP3 and degraded by UPP; the level of tyrosine phosphorylation induced by PKA-AKAP3 was reduced, the level of serine threonine phosphorylation increased, and the ubiquitination pathway interacted with the phosphorylation pathway and was involved in sperm capacitation.

Extracellular vesicle-encapsulated miR-21-5p in seminal plasma prevents sperm capacitation via Vinculin inhibition

To penetrate the zona pellucida before sperm-egg binding, sperm must undergo highly time-controlled capacitation and acrosome reaction in the female reproductive tract. Our previous study demonstrated that miR-21-5p is the most abundant miRNA in boar seminal plasma (SP)-derived extracellular vesicles (EVs) and can target Vinculin (VCL) gene, which may participate in boar sperm capacitation. Thus, this study aims to explore the potential role of miR-21-5p from SP-derived EVs in preventing sperm capacitation and its underlying mechanism. We observed that sperm could incorporate miR-21-5p from SP-derived EVs. The roles of SP-derived EVs miR-21-5p in sperm capacitation were then determined using gain- and loss-of-function analyses. In addition, the expression levels of miR-21-5p, VCL, and VCL protein in liquid-preserved boar sperm following transfection were determined using RT-qPCR and Western blotting. Our results revealed that miR-21-5p overexpression inhibited sperm capacitation and acrosome reaction. Similarly, miR-21-5p expression was significantly lower (P < 0.05) in capacitated sperm than un-capacitated sperm. However, the protein level of VCL was also significantly lower (P < 0.05) in capacitated sperm than un-capacitated sperm. Furthermore, immunofluorescence analysis showed that VCL protein mainly located in sperm head and sperm capacitation was inhibited after treating with VCL protein inhibitor (Chrysin). In conclusion, our study provides reasonable evidence that miR-21-5p expression in SP-derived EVs could prevent sperm capacitation via VCL inhibition.

Bicarbonate-Triggered In Vitro Capacitation of Boar Spermatozoa Conveys an Increased Relative Abundance of the Canonical Transient Receptor Potential Cation (TRPC) Channels 3, 4, 6 and 7 and of CatSper-γ Subunit mRNA Transcripts.

Simple SummaryThe detection of sub-fertile boars has been a difficult task, and despite their prevalence being low, its impact is very significant because it implies economic drawbacks for artificial insemination (AI) centers and farms. Unfortunately, some crucial reproductive processes fall beyond the routine analysis performed in the porcine model, such as sperm capacitation, which is a necessary event for fertilization. A synergistic action of bicarbonate (HCO3−) with calcium (Ca2+) is needed to achieve capacitation. The transport of Ca2+ is mediated by CatSper channels and Canonical Transient Potential Channels (TRPC). We quantified mRNA transcripts of different subunits of CatSper (β, γ and δ) and TRPC (1, 3, 4, 6 and 7) before and after in vitro capacitation by HCO3− ions. Our results showed that in vitro capacitation using HCO3− increases the relative abundance of mRNA transcripts of almost all subunits of Ca2+ channels, except CatSper-δ and TRPC1, which were significantly reduced. More studies are needed to elucidate the specific roles of the TRPC channels at a physiological and functional level.Sperm capacitation is a stepwise complex biochemical process towards fertilization. It includes a crucial early calcium (Ca2+) transport mediated by CatSper channels and Canonical Transient Potential Channels (TRPC). We studied the relative abundance of mRNA transcripts changes of the CatSper β, γ and δ subunits and TRPC-channels 1, 3, 4, 6 and 7 in pig spermatozoa, after triggering in vitro capacitation by bicarbonate ions at levels present in vivo at the fertilization site. For this purpose, we analyzedfive5 ejaculate pools (from three fertile adult boars) before (control-fresh samples) and after in vitro exposure to capacitation conditions (37 mM NaHCO3, 2.25 mM CaCl2, 2 mM caffeine, 0.5% bovine serum albumin and 310 mM lactose) at 38 °C, 5% CO2 for 30 min. In vitro capacitation using bicarbonate elicits an increase in the relative abundance of mRNA transcripts of almost all studied Ca2+ channels, except CatSper-δ and TRPC1 (significantly reduced). These findings open new avenues of research to identify the specific role of each channel in boar sperm capacitation and elucidate the physiological meaning of the changes on sperm mRNA cargo.

Evaluation of boar and bull sperm capacitation and the acrosome reaction using flow cytometry

Flow cytometry can be used to evaluate many sperm attributes and Dr. Duane Garner was influential in developing assays to understand sperm physiology and function. We review some of Dr. Garner’s work and describe experiments that evaluate sperm capacitation using Dr. Garner’s philosophy. In exploratory experiments, boar sperm were cryopreserved in lactose egg yolk (LEY) or Beltsville Freezing Extender 5 (BF5) and incubated in one capacitating medium. In another experiment, frozen-thawed bull sperm were incubated in TALP-Ca or CFDM1 capacitating media. In both experiments, sperm viability and capacitation were evaluated using multiple probes. Boar sperm frozen in LEY had greater survival rates (38%) than sperm frozen in BF5 (22%; P < 0.05) but did not capacitate as effectively as sperm in BF5 (P < 0.05). In Experiment 2, bull sperm survived to a greater extent when incubated in TALP-Ca than in CFDM1 (P < 0.05) and had greater capacitation for most parameters (P < 0.05). Of particular interest, 77% of sperm incubated in TALP-Ca had activated second messenger systems involved in capacitation, compared with < 5% of sperm incubated in CFDM1. The results indicate different freezing and capacitating media induce different responses to sperm capacitation and functions. If only sperm viability and acrosomal integrity were evaluated, these results would be interpreted very differently. Dr. Garner’s philosophy of evaluating multiple sperm parameters was an impetus to determine unique treatment differences which help in understanding sperm capacitation, and design further experiments to determine how media content causes sperm physiology differences.

Transwell isolation and difference analysis of capacitated boar sperm proteins based on the iTRAQ technique

During capacitation, proteins in boar sperm are released to maintain the stability of their own state and membrane structure. No studies have analyzed the differences between retained proteins and released proteins during sperm capacitation. In the present study, a Transwell chamber and polycarbonate membrane were used to separate the proteins of boar sperm and their released proteins. Isotopically labeled relative and absolute quantification (iTRAQ) was used to analyze each compartment protein. A total of 108 differential proteins were identified in the upper and lower chambers of the Transwell, among which 27 were significantly upregulated (p-value≤0.05 and |log2 (fold change)|≥1) and 81 were significantly downregulated (p-value≤0.05 and |log2 (fold change)|≤1). These differential proteins were mainly involved in biological processes (e.g., the regulation of cysteine peptidase activity, transmembrane transportation, ion transportation and ATP synthesis) and major signaling pathways (e.g., glutathione/galactose metabolism, cellular adhesion and PI3K-Akt), and most of them interacted with each other to some extent. In conclusion, retained proteins and released proteins of capacitated sperm were effectively separated using a Transwell chamber, and differential proteins were successfully identified from among the proteins. Bioinformatics analysis suggested that these differential proteins affect sperm capacitation mainly by adjusting sperm energy metabolism, motion characteristics and acrosome membrane status.

Boar sperm incubation with reduced glutathione (GSH) differentially modulates protein tyrosine phosphorylation patterns and reorganization of calcium in sperm, in vitro fertilization, and embryo development depending on concentrations

The sperm in the female's reproductive tract undergo changes to fertilize the oocyte (sperm capacitation). These changes are regulated by redox system. However, some assisted reproductive technologies require sperm capacitation under in vitro conditions, though this increases the generation of ROS. Therefore, the aim of this study was to evaluate the effect of GSH as an antioxidant agent during the capacitation of boar sperm [evaluated by calcium compartmentalization, tyrosine phosphorylation (Tyr-P), motility, viability, and acrosomal integrity], in vitro fertilization (evaluated by penetration, monospermy, and efficiency %), and later embryo development (evaluated by cleavage and blastocyst rates, total number of cells per blastocyst and blastocyst diameter). Four experimental groups with different GSH concentrations (0-control, 0.5, 1, and 5 mM) were formed. When 1-GSH was added to the medium, the percentage of capacitated sperm increased after 4 h of incubation; the localization of Tyr-P was modified at 1 h and 4 h of incubation depending on the GSH concentration. Percentages of total and progressive sperm motility also increased at 4 h of incubation, but only in the 5-GSH group compared to control. Viability, acrosomal integrity, and general Tyr-P (Western blot) not differ among the experimental groups. The addition of GSH during gamete interaction increased penetration, monospermy, and efficiency rates in the 1-GSH group compared to the others. However, the effect of GSH was not observed in cleavage and blastocyst rates compared to the control. In conclusion, adding GSH modulates sperm capacitation (by means of calcium compartmentalization and tyrosine phosphorilation pattern) depending on its concentration, and improves IVF output at 1-GSH during gamete interaction.

AQP3 has a key role during in vitro capacitation of boar spermatozoa

AQP3 has a key role during in vitro capacitation of boar spermatozoa

Bisphenol A Diglycidyl Ether (BADGE) and Progesterone Do Not Induce Ca2+ Signals in Boar Sperm Cells

AimExposure of boar sperm cells to Bisphenol A diglycidyl ether (BADGE) has been shown to lead to reproductive failure in sows, however, the mode of action is unknown. As we have recently shown that BADGE can interfere with Ca2 + signaling in human sperm cells through an action on CatSper, and as CatSper has been shown to be expressed in boar sperm cells, we hypothesized that a similar mechanism in the boar sperm cells could be responsible for the reproductive failure.MethodsDirect effects of BADGE and the endogenous ligand of human CatSper, progesterone, on Ca2+ signaling in human and boar sperm cells were evaluated side-by-side using a Ca2+ fluorimetric assay measuring changes in intracellular Ca2+. Effects of BADGE on Ca2+ signaling in boar sperm were furthermore assessed by flow cytometry by an independent laboratory.ResultsThe exact same solutions of BADGE and progesterone induced transient biphasic Ca2+ signals in human sperm cells, but failed to do so in both non-capacitated and capacitated boar sperm cells. BADGE also failed to induce transient biphasic Ca2+ signals in boar sperm cells in the flow cytometric assay.ConclusionBADGE and progesterone failed to induce Ca2+ signals in boar sperm cells. This indicates that the signaling mechanisms leading to activation of CatSper differs between human and boar sperm cells, and suggests that the mode of action by which exposure of boar sperm cells to BADGE can lead to reproductive failure in sows does not involve effects on Ca2+ signaling.

Identification of lipid raft glycoproteins obtained from boar spermatozoa.

The surface of the spermatozoa is coated with glycoproteins the redistribution of which during in vitro capacitation plays a key role in the subsequent fertilization process. Lipid rafts are membrane microdomains involved in signal transduction through receptors and include or recruit specific types of proteins and glycoproteins. Few studies have focused on identifying glycoproteins resident in the lipid rafts of spermatozoa. Proteins associated with lipid rafts modify their localization during capacitation. The objective of the study was to identify the glycoproteins associated with lipid rafts of capacitated boar spermatozoa through a lectin-binding assay coupled to mass spectrometry approach. From the proteomic profiles generated by the raft proteins extractions, we observed that after capacitation the intensity of some bands increased while that of others decreased. To determine whether the proteins obtained from lipid rafts are glycosylated, lectin blot assays were performed. Protein bands with a good resolution and showing significant glycosylation modifications after capacitation were analyzed by mass spectrometry. The bands of interest had an apparent molecular weight of 64, 45, 36, 34, 24, 18 and 15kDa. We sequenced the 7 bands and 20 known or potential glycoproteins were identified. According to us, for ten of them this is the first time that their association with sperm lipid rafts is described (ADAM5, SPMI, SPACA1, Seminal plasma protein pB1, PSP-I, MFGE8, tACE, PGK2, SUCLA2, MDH1). Moreover, LYDP4, SPAM-1, HSP60, ZPBP1, AK1 were previously reported in lipid rafts of mouse and human spermatozoa but not in boar spermatozoa. We also found and confirmed the presence of ACR, ACRBP, AWN, AQN3 and PRDX5 in lipid rafts of boar spermatozoa. This paper provides an overview of the glycosylation pattern in lipid rafts of boar spermatozoa before and after capacitation. Further glycomic analysis is needed to determine the type and the variation of glycan chains of the lipid rafts glycoproteins on the surface of spermatozoa during capacitation and acrosome reaction.

Elucidating the Role of K+ Channels during In Vitro Capacitation of Boar Spermatozoa: Do SLO1 Channels Play a Crucial Role?

This study sought to identify and localize SLO1 channels in boar spermatozoa by immunoblotting and immunofluorescence, and to determine their physiological role during in vitro sperm capacitation. Sperm samples from 14 boars were incubated in a capacitation medium for 300 min in the presence of paxilline (PAX), a specific SLO1-channel blocker, added either at 0 min or after 240 min of incubation. Negative controls were incubated in capacitation medium, and positive controls in capacitation medium plus tetraethyl ammonium (TEA), a general K+-channel blocker, also added at 0 min or after 240 min of incubation. In all samples, acrosome exocytosis was triggered with progesterone after 240 min of incubation. Sperm motility and kinematics, integrity of plasma and acrosome membranes, membrane lipid disorder, intracellular calcium levels and acrosin activity were evaluated after 0, 60, 120, 180, 240, 250, 270 and 300 min of incubation. In boar spermatozoa, SLO1 channels were found to have 80 kDa and be localized in the anterior postacrosomal region and the mid and principal piece of the tail; their specific blockage through PAX resulted in altered calcium levels and acrosome exocytosis. As expected, TEA blocker impaired in vitro sperm capacitation, by altering sperm motility and kinematics and calcium levels. In conclusion, SLO1 channels are crucial for the acrosome exocytosis induced by progesterone in in vitro capacitated boar spermatozoa.

Fluorescent analysis of boar sperm capacitation process in vitro

BackgroundCapacitation involves physiological changes that spermatozoa must undergo in the female reproductive tract or in vitro to obtain the ability to bind, penetrate and fertilize the egg. Up to date, several methods have been developed to characterize this complex biological process. The goal of the presented study is to mutually compare several fluorescent techniques, check their ability to detect changes in molecular processes during the capacitation progress and determine their ability to predict the percentage of acrosome reacted (AR) sperm after the exposure to solubilized zona pellucida (ZP). The capacitation process was analyzed using four fluorescent techniques: 1. chlortetracycline (CTC) staining, 2. anti-acrosin antibody (ACR.2) assay, 3. anti-phosphotyrosine (pY) antibody assay, 4. fluorescein isothiocyanate-conjugated phalloidin (FITC-phall) assay. All these methods were tested using fluorescent microscopy and flow cytometry.ResultsAll selected methods are capable to detect the capacitation progress of boar sperm in vitro, but there are significant differences in their outcome when using fluorescent microscopy or flow cytometry experimental arrangements and subsequent statistical analysis (KW-ANOVA). Also, the ability to predict the absolute numbers of sperm which will undergo ZP-induced AR differ significantly (CTC and ACR.2 gave the best predictions).ConclusionsOur study compared four largely used methods used to characterize capacitation process, highlighted their differences and showed that all are able to detect capacitation progress, CTC and ACR.2 are furthermore able to accurately predict the percentage of AR sperm after ZP-induced AR.

The calcium-sensing receptor regulates protein tyrosine phosphorylation through PDK1 in boar spermatozoa.

Regulation of protein tyrosine phosphorylation is required for sperm capacitation and oocyte fertilization. The objective of the present work was to study the role of the calcium-sensing receptor (CaSR) on protein tyrosine phosphorylation in boar spermatozoa under capacitating conditions. To do this, boar spermatozoa were incubated in Tyrode's complete medium for 4 hr and the specific inhibitor of the CaSR, NPS2143, was used. Also, to study the possible mechanism(s) by which this receptor exerts its function, spermatozoa were incubated in the presence of specific inhibitors of the 3-phosphoinositide dependent protein kinase 1 (PDK1) and protein kinase A (PKA). Treatment with NPS2143, GSK2334470, an inhibitor of PDK1 and H-89, an inhibitor of PKA separately induced an increase in tyrosine phosphorylation of 18 and 32 kDa proteins, a decrease in the serine/threonine phosphorylation of the PKA substrates together with a drop in sperm motility and viability. The present work proposes a new signalling pathway of the CaSR, mediated by PDK1 and PKA in boar spermatozoa under capacitating conditions. Our results show that the inhibition of the CaSR induces the inhibition of PDK1 that blocks PKA activity resulting in a rise in tyrosine phosphorylation of p18 and p32 proteins. This novel signalling pathway has not been described before and could be crucial to understand boar sperm capacitation within the female reproductive tract.