Involvement of Complexin 2 in Docking, Locking and Unlocking of Different SNARE Complexes during Sperm Capacitation and Induced Acrosomal Exocytosis

Acrosomal exocytosis (AE) is an intracellular multipoint fusion reaction of the sperm plasma membrane (PM) with the outer acrosomal membrane (OAM). This unique exocytotic event enables the penetration of the sperm through the zona pellucida of the oocyte. We previously observed a stable docking of OAM to the PM brought about by the formation of the trans-SNARE complex (syntaxin 1B, SNAP 23 and VAMP 3). By using electron microscopy, immunochemistry and immunofluorescence techniques in combination with functional studies and proteomic approaches, we here demonstrate that calcium ionophore-induced AE results in the formation of unilamellar hybrid membrane vesicles containing a mixture of components originating from the two fused membranes. These mixed vesicles (MV) do not contain the earlier reported trimeric SNARE complex but instead possess a novel trimeric SNARE complex that contained syntaxin 3, SNAP 23 and VAMP 2, with an additional SNARE interacting protein, complexin 2. Our data indicate that the earlier reported raft and capacitation-dependent docking phenomenon between the PM and OAM allows a specific rearrangement of molecules between the two docked membranes and is involved in (1) recruiting SNAREs and complexin 2 in the newly formed lipid-ordered microdomains, (2) the assembly of a fusion-driving SNARE complex which executes Ca2+-dependent AE, (3) the disassembly of the earlier reported docking SNARE complex, (4) the recruitment of secondary zona binding proteins at the zona interacting sperm surface. The possibility to study separate and dynamic interactions between SNARE proteins, complexin and Ca2+ which are all involved in AE make sperm an ideal model for studying exocytosis

Role of Carbonic Anhydrase IV in the Bicarbonate-Mediated Activation of Murine and Human Sperm

HCO3− is the signal for early activation of sperm motility. In vivo, this occurs when sperm come into contact with the HCO3− containing fluids in the reproductive tract. The activated motility enables sperm to travel the long distance to the ovum. In spermatozoa HCO3− stimulates the atypical sperm adenylyl cyclase (sAC) to promote the cAMP-mediated pathway that increases flagellar beat frequency. Stimulation of sAC may occur when HCO3− enters spermatozoa either directly by anion transport or indirectly via diffusion of CO2 with subsequent hydration by intracellular carbonic anhydrase (CA). We here show that murine sperm possess extracellular CA IV that is transferred to the sperm surface as the sperm pass through the epididymis. Comparison of CA IV expression by qRT PCR analysis confirms that the transfer takes place in the corpus epididymidis. We demonstrate murine and human sperm respond to CO2 with an increase in beat frequency, an effect that can be inhibited by ethoxyzolamide. Comparing CA activity in sperm from wild-type and CA IV−/− mice we found a 32.13% reduction in total CA activity in the latter. The CA IV−/− sperm also have a reduced response to CO2. While the beat frequency of wild-type sperm increases from 2.86±0.12 Hz to 6.87±0.34 Hz after CO2 application, beat frequency of CA IV−/− sperm only increases from 3.06±0.20 Hz to 5.29±0.47 Hz. We show, for the first time, a physiological role of CA IV that supplies sperm with HCO3−, which is necessary for stimulation of sAC and hence early activation of spermatozoa

Assessment of sperm quality traits in relation to fertility in boar semen

<p>Abstract</p> <p>Background</p> <p>Several studies have been published where sperm plasma membrane integrity correlated to fertility. In this study we describe a simple fluorometer-based assay where we monitored the fluorescence intensity of artificially membrane-ruptured spermatozoa with a fixed time staining with fluorescent DNA dyes.</p> <p>Methods</p> <p>Membrane-impermeant fluorescent dyes Hoechst 33258 (H258) and propidium iodide (PI) were used to measure the fluorescence of the nucleus in artificially membrane ruptured spermatozoa and membrane-permeant dye Hoechst 33342 (H342) was used to measure fluorescence of intact spermatozoa. The concentration of spermatozoa in insemination doses varied from 31.2 × 10⁶/ml to 50 × 10⁶/ml and the average value was 35 × 10⁶/ml. Each boar was represented by three consecutive ejaculates, collected at weekly intervals. Nonreturn rate within 60 days of first insemination (NR %) and litter size (total number of piglets born) of multiparous farrowings were used as fertility measures.</p> <p>Results</p> <p>Sperm fluorescence intensity of H258 and H342, but not the fluorescence intensity of PI-stained spermatozoa correlated significantly with the litter size of multiparous farrowings, values being r = - 0.68 (P < 0.01) for H258, r = - 0.69 (P < 0.01) for H342 and r = - 0.38, (P = 0.11) for PI.</p> <p>Conclusions</p> <p>The increase in fluorescence values of membrane-ruptured H258 and unruptured H342-stained spermatozoa in boar AI doses can be associated with smaller litter size after AI. This finding indicates that the fluorescence properties of the sperm nucleus could be used to select for AI doses with greater fertilizing potential.</p

Cytokine and Protein Markers of Leprosy Reactions in Skin and Nerves: Baseline Results for the North Indian INFIR Cohort

Leprosy affects skin and peripheral nerves. Although we have effective antibiotics to treat the mycobacterial infection, a key part of the disease process is the accompanying inflammation. This can worsen after starting antibacterial treatment with episodes of immune mediated inflammation, so called ‘reactions’. These reactions are associated with worsening of the nerve damage. We recruited a cohort of 303 newly diagnosed leprosy patients in North India with the aim of understanding and defining the pathological processes better. We took skin and nerve biopsies from patients and examined them to define which molecules and mediators of inflammation were present. We found high levels of the cytokines Tumour Necrosis Factor alpha, Transforming Growth Factor beta and inducible Nitric Oxide Synthase in biopsies from patients with reactions. We also found high levels of bacteria and inflammation in the nerves. These experiments tell us that we need to determine which other molecules are present and to explore ways of switching off the production of these pro-inflammatory molecules

Effects of Hepatitis B Virus S Protein Exposure on Sperm Membrane Integrity and Functions

Background: Hepatitis B is a public health problem worldwide. Viral infection can affect a man’s fertility, but only scant information about the influence of hepatitis B virus (HBV) infection on sperm quality is available. The purpose of this study was to investigate the effect of hepatitis B virus S protein (HBs) on human sperm membrane integrity and functions. Methods/Principal Findings: Reactive oxygen species (ROS), lipid peroxidation (LP), total antioxidant capacity (TAC) and phosphatidylserine (PS) externalization were determined. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays and flow cytometric analyses were performed. (1) After 3 h incubation with 25 mg/ml of HBs, the average rates of ROS positive cells, annexin V–positive/propidium iodide (PI)-negative cells, Caspases-3,-8,-9 positive cells and TUNEL-positive cells were significantly increased in the test groups as compared to those in the control groups, while TAC level was decreased when compared with the control. The level of malondialdehyde (MDA) in the sperm cells exposed to 50 mg/ml of HBs for 3 h was significantly higher than that in the control (P,0.05–0.01). (2) HBs increased the MDA levels and the numbers of ROS positive cells, annexin V–positive/PI-negative cells, caspases-3,-8,-9 positive cells and TUNEL-positive cells in a dose-dependent manner. (3) HBs monoclonal antibody (MAb) and N-Acetylcysteine (NAC) reduced the number of ROS-positive sperm cells. (4) HBs decreased the TAC levels in sperm cells in a dose-dependent manner. Conclusion: HBs exposure could lead to ROS generation, lipid peroxidation, TAC reduction, PS externalization, activation o

Type-1 Cannabinoid Receptors Reduce Membrane Fluidity of Capacitated Boar Sperm by Impairing Their Activation by Bicarbonate

Background Mammalian spermatozoa acquire their full fertilizing ability (so called capacitation) within the female genital tract, where they are progressively exposed to inverse gradients of inhibiting and stimulating molecules. Methodology/Principal Findings In the present research, the effect on this process of anandamide, an endocannabinoid that can either activate or inhibit cannabinoid receptors depending on its concentration, and bicarbonate, an oviductal activatory molecule, was assessed, in order to study the role exerted by the type 1 cannabinoid receptor (CB1R) in the process of lipid membrane remodeling crucial to complete capacitation. To this aim, boar sperm were incubated in vitro under capacitating conditions (stimulated by bicarbonate) in the presence or in the absence of methanandamide (Met-AEA), a non-hydrolysable analogue of anandamide. The CB1R involvement was studied by using the specific inhibitor (SR141716) or mimicking its activation by adding a permeable cAMP analogue (8Br-cAMP). By an immunocytochemistry approach it was shown that the Met-AEA inhibits the bicarbonate-dependent translocation of CB1R from the post-equatorial to equatorial region of sperm head. In addition it was found that Met-AEA is able to prevent the bicarbonate-induced increase in membrane disorder and the cholesterol extraction, both preliminary to capacitation, acting through a CB1R-cAMP mediated pathway, as indicated by MC540 and filipin staining, EPR spectroscopy and biochemical analysis on whole membranes (CB1R activity) and on membrane enriched fraction (C/P content and anisotropy). Conclusions/Significance Altogether, these data demonstrate that the endocannabinoid system strongly inhibits the process of sperm capacitation, acting as membrane stabilizing agent, thus increasing the basic knowledge on capacitation-related signaling and potentially opening new perspectives in diagnostics and therapeutics of male infertility