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

How Pig Sperm Prepares to Fertilize: Stable Acrosome Docking to the Plasma Membrane

Background: Mammalian sperms are activated in the oviduct. This process, which involves extensive sperm surface remodelling, is required for fertilization and can be mimicked under in vitro fertilization conditions (IVF). Methodology/Principal Findings: Here we demonstrate that such treatments caused stable docking and priming of the acrosome membrane to the apical sperm head surface without the emergence of exocytotic membrane fusion. The interacting membranes could be isolated as bilamellar membrane structures after cell disruption. These membrane structures as well as whole capacitated sperm contained stable ternary trans-SNARE complexes that were composed of VAMP 3 and syntaxin 1B from the plasma membrane and SNAP 23 from the acrosomal membrane. This trans-SNARE complex was not observed in control sperm. Conclusions/Significance: We propose that this capacitation driven membrane docking and stability thereof is a preparative step prior to the multipoint membrane fusions characteristic for the acrosome reaction induced by sperm-zona binding. Thus, sperm can be considered a valuable model for studying exocytosis

Urine hemojuvelin in cats with naturally occurring kidney disease

Background: Soluble-type hemojuvelin in serum and urine has been shown to be a biomarker in humans for chronic kidney disease (CKD) and acute kidney injury (AKI). No similar research has been conducted on cats. Objective: Urine hemojuvelin (u-hemojuvelin) can be used as a clinical indicator for cats with various renal diseases. Animals: Eighteen healthy cats, 10 cats with AKI, 21 cats with acute-on-chronic kidney injury (ACKI), and 45 cats with CKD were enrolled. Methods: The expression profile of u-hemojuvelin was assessed by Western blot analysis, whereas the u-hemojuvelin concentration was measured using an in-house sandwich ELISA. Each cat's u-hemojuvelin-to-creatinine ratio (UHCR) also was determined. Results: Significant differences were found in both u-hemojuvelin concentration and UHCR between the control cats and the other cats (AKI, CKD, ACKI). Both u-hemojuvelin and UHCR had high areas under the receiver operator curve (AUROC) for diagnoses of AKI (u-hemojuvelin, 0.885; UHCR, 0.982), CKD (hemojuvelin, 0.869; UHCR, 0.959), and ACKI (hemojuvelin, 0.910; UHCR, 1). Late stage (International Renal Interest Society, IRIS stages 3 and 4) CKD cats had significantly higher u-hemojuvelin concentration and UHCR than did early stage cats (IRIS stages 1 and 2). Both u-hemojuvelin and UHCR were significantly correlated with high blood urea nitrogen, plasma creatinine, and plasma phosphate concentrations and with low hematocrit (Hct), red blood cell (RBC) count, and plasma albumin concentration. The UHCR values were also significantly correlated with white blood cell count in blood

Counteracting Cisplatin-Induced Testicular Damages by Natural Polyphenol Constituent Honokiol

Cisplatin, despite its anti-cancer ability, exhibits severe testicular toxicities when applied systemically. Due to its wide application in cancer treatment, reduction of its damages to normal tissue is an imminent clinical need. Here we evaluated the effects of honokiol, a natural lipophilic polyphenol compound, on cisplatin-induced testicular injury. We showed in-vitro and in-vivo that nanosome-encapsulated honokiol attenuated cisplatin-induced DNA oxidative stress by suppressing intracellular reactive oxygen species production and elevating gene expressions of mitochondrial antioxidation enzymes. Nanosome honokiol also mitigated endoplasmic reticulum stress through down regulation of Bip-ATF4-CHOP signaling pathway. Additionally, this natural polyphenol compound diminished cisplatin-induced DNA breaks and cellular apoptosis. The reduced type I collagen accumulation in the testis likely attributed from inhibition of TGFβ1, αSMA and ER protein TXNDC5 protein expression. The combinatorial beneficial effects better preserve spermatogenic layers and facilitate repopulation of sperm cells. Our study renders opportunity for re-introducing cisplatin to systemic anti-cancer therapy with reduced testicular toxicity and restored fertility

Secretory mouse quiescin sulfhydryl oxidase 1 aggregates defected human and mouse spermatozoa in vitro and in vivo

A flavin-dependent enzyme quiescin Q6 sulfhydryl oxidase 1 (QSOX1) catalyzes the oxidation of thiol groups into disulfide bonds. QSOX1 is prominently expressed in the seminal plasma. However, its role in male reproduction is elusive. Here, we purified the secreted form of QSOX1, i.e., QSOX1c, from mouse seminal vesicle secretions and revealed for the first time its function involved in sperm physiology. Exogenous addition of QSOX1c time-dependently promoted the in vitro aggregation of thiol-rich, oxidative stressed, and apoptotic mouse and human sperm cells. Also, in vivo aggregated sperm cells collected from mouse uterine and human ejaculates also showed high levels of QSOX1c, intracellular reactive oxygen species, annexin V, and free thiols. In summary, our studies demonstrated that QSOX1c could agglutinate spermatozoa susceptible to free radical attack and apoptosis. This characteristic may provide an opportunity to separate defective sperm cells and improve sperm quality before artificial insemination in humans and animals

LC-MALDI MS-based identification of proteins from membrane cavitates from capacitated porcine sperm.

<p>Presented are subsets of those proteins identified that correspond to previously identified sperm proteins relevant to sperm-oocyte interactions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032603#pone.0032603-VanGestel2" target="_blank">[29]</a>. SNARE-related protein identifications are highlighted in bold. In addition one proteins with a putative function in the recruitment of SNARE proteins into membrane rafts <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032603#pone.0032603-Ackermann2" target="_blank">[26]</a> is shown in red. Number signs indicate proteins that have been previously found in membrane cavitates of porcine sperm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032603#pone.0032603-VanGestel2" target="_blank">[29]</a>. Asterisks indicate proteins that were identified in the MV preparations (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032603#pone-0032603-t001" target="_blank">Tables 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032603#pone-0032603-t002" target="_blank">2</a>). Protein ID was considered to be conclusive when two or more peptides were identified (e<0.05) (all proteins above the line).</p><p>Protein ID was considered conclusive when two or more peptides were identified (e<0.05, all proteins above the line).</p><p>Highlighted in bold: SNARE proteins or SNARE interacting proteins.</p><p>Highlighted in red: Protein with putative function in the recruitment of SNARE proteins into the membrane raft area.</p>#<p>: indicate proteins which are routinely found in the apical plasma membrane cavitates of porcine sperm.</p>*<p>: indicate proteins that are recovered as plasma membrane proteins in the mixed vesicles.</p