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

Co-incubation of human spermatozoa with Chlamydia trachomatis serovar E causes premature sperm death

The aim of this work was to investigate the effect of elementary bodies (EB) of Chlamydia trachomatis serovars E and LGV on sperm motility, viability and acrosomal status. Highly motile preparations of spermatozoa from normozoospermic patients were co-incubated for 6 h with 0.54×106 EB per ml. At 1, 3 and 6 h of incubation, sperm motility was determined by computer-assisted semen analysis (CASA) and the proportion of dead cells determined by the hypo-osmotic swelling (HOS) test. Acrosomal status was also examined using a standard monoclonal antibody assay. In the absence of EB, the percentage of motile spermatozoa remained >69% over the 6h incubation and the proportion of dead spermatozoa at <12%. However, during the incubation with EB of serovar E there was a significant decline in the percentage of motile spermatozoa (P < 0.05), and a corresponding increase in the proportion of dead spermatozoa (P < 0.05) at all time-points. However, following incubation with serovar LGV, only the percentage of dead spermatozoa after 6 h incubation was significantly different from the control (P < 0.05). The amount of acrosome-reacted spermatozoa remained unchanged (<16%) in all incubations at all time-points. Dose-response experiments indicated that increasing the concentration of EB to 2.5×106 per ml did not significantly alter the results. Furthermore, co-incubation of spermatozoa with dead EB (killed by heat treatment) abolished the chlamydia-mediated response, indicating that the effect is a result of the live organism and not soluble components or membrane elements. These data suggest that a detrimental effect on sperm function by some serovars may be an as yet unrecognized component of infertility problems

Identification of the site of attachment of the glycolipid anchor in porcine membrane dipeptidase

A significant number of cell-surface proteins m anchored in the plasma membrane by covalently attached glycosyl-phosphatidylinositol(glycolipid) and to date over 100 examples have been described from many stages of eukaryotic evolution [reviewed in 131. As well as having an N-terminal signal sequence to direct the protein to the ER, glycolipid-anchored proteins also have encoded in their cDNA a C-terminal hydrophobic region of amino acids. This sequence is believed to hold the nascent protein in the membrane prior to anchor addition and is cleaved off posttranslationally with concomitant addition of the glycolipid anchor. Anchor attachment occurs on the terminal residue present after cleavage which may be one of six small side-chain residues (Ala, Asn, Asp, Cys, Gly or, most commonly, Ser) [ 11. It has also recently been shown that a depe of specificity, again for small amino acids, exists in the two adjacent C-terminal amino acid positions, in particular the residue next but one to the attachment residue may only be Ala, Gly or Ser [3]. Here we report the determination of the site of glycolipid anchor attachment of porcine membrane dipeptidase(dehydropeptidase-I; EC3.4.13.11)

Activation of the glycosyl-phosphatidylinositol-anchored membrane dipeptidase upon release from pig kidney membranes by phospholipase C

Incubation of pig kidney microvillar membranes with Bacillus thuringiensis or Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) resulted in the release of a number of glycosyl-phosphatidylinositol (GPI)-anchored hydrolases, including alkaline phosphatase (EC 3.1.3.1), amino-peptidase P (EC 3.4.11.9), membrane dipeptidase (EC 3.4.13.19), 5'-nucleotidase (EC 3.1.3.5) and trehalase (EC 3.2.1.28). Of these five ectoenzymes only for membrane dipeptidase was there a significant (approx. 100%) increase in enzymic activity upon release from the membrane. Maximal activation occurred at a PI-PLC concentration 10-fold less than that required for maximal release. In contrast solubilization of the membranes with n-octyl beta-D-glucopyranoside had no effect on the enzymic activity of membrane dipeptidase. A competitive e.l.i.s.a. with a polyclonal antiserum to membrane dipeptidase indicated that the increase in enzymic activity was not due to an increase in the amount of membrane dipeptidase protein. Although PI-PLC cleaved the GPI anchor of the affinity-purified amphipathic form of pig membrane dipeptidase there was no concurrent increase in enzymic activity. In the absence of PI-PLC, membrane dipeptidase in the microvillar membranes hydrolysed Gly-D-Phe with a Km of 0.77 mM and a Vmax. of 602 nmol/min per mg of protein. However, in the presence of a concentration of PI-PLC which caused maximal release from the membrane and maximal activation of membrane dipeptidase the Km was decreased to 0.07 mM while the Vmax. remained essentially unchanged at 624 nmol/min per mg of protein. Overall these results suggest that cleavage by PI-PLC of the GPI anchor on membrane dipeptidase may relax conformational constraints on the active site of the enzyme which exist when it is anchored in the lipid bilayer, thus resulting in an increase in the affinity of the active site for substrate

Identification of the site of attachment of the glycolipid anchor in porcine membrane dipeptidase

A significant number of cell-surface proteins m anchored in the plasma membrane by covalently attached glycosyl-phosphatidylinositol(glycolipid) and to date over 100 examples have been described from many stages of eukaryotic evolution [reviewed in 131. As well as having an N-terminal signal sequence to direct the protein to the ER, glycolipid-anchored proteins also have encoded in their cDNA a C-terminal hydrophobic region of amino acids. This sequence is believed to hold the nascent protein in the membrane prior to anchor addition and is cleaved off posttranslationally with concomitant addition of the glycolipid anchor. Anchor attachment occurs on the terminal residue present after cleavage which may be one of six small side-chain residues (Ala, Asn, Asp, Cys, Gly or, most commonly, Ser) [ 11. It has also recently been shown that a depe of specificity, again for small amino acids, exists in the two adjacent C-terminal amino acid positions, in particular the residue next but one to the attachment residue may only be Ala, Gly or Ser [3]. Here we report the determination of the site of glycolipid anchor attachment of porcine membrane dipeptidase(dehydropeptidase-I; EC3.4.13.11)

Sperm maturation in vitro: co-culture of spermatozoa and epididymal epithelium

Sperm maturation involves an intimate interaction between spermatozoa and the epididymal epithelium. Aspects of this relationship can be examined by co-incubating epididymal spermatozoa with epididymal epithelium in vitro. Plaques of epididymal epithelium from a variety of species (for example rodents, dogs, humans) can be maintained in culture medium supplemented with growth factors and androgens. When co-incubated with these epithelial cultures, immature epididymal spermatozoa undergo maturation changes that lead to the acquisition of progressive motility, zona binding and, in some instances, fertilizing capacity in vitro. The use of such co-culture techniques for the understanding of sperm maturation in vitro and in vivo is reviewed with reference to recent experiments

Membrane peptidase expression by confluent cultures of Caco-2 cells

Contluent cultures of Caco-2 cells, a human colonic carcinoma cell line, undergo differentiation into enterocyte-like cells and are widely used as a model system to study the expression of membrane peptidases on the apical brush border [I-41 Indirect irnniunofluorescent staining has previously shown dipeptidylpeptidase IV (DPPIV, EC 3 4 14 5) to be homogeneously distributed on the surface of Caco-2 cells [ I ] whereas endopeptidase-24 1 1 (E-24 I I , EC 3 4 24 I I ) was found on the surface of some cells but not on others [4] This observation has also been reported by Jalal e/ d. [ S ] who have shown that E-24 1 1 is expressed by all cells but in some is confined to an intracellular pool In the present study the cellsurface and intracellular expression of five membrane peptidases on confluent cultures of Caco-2 cells has been investigated using indirect immunofluorescent staining The membrane peptidases studied were DPPIV, E-24 I I , angiotensin converting enzyme (ACE, EC 3 4 15 I). aminopeptidase N (AP-N, EC 3 4 1 I 2) and aminopeptidase W (AP-W, EC 3 4 11 16)

Membrane peptidase expression by confluent cultures of Caco-2 cells

Contluent cultures of Caco-2 cells, a human colonic carcinoma cell line, undergo differentiation into enterocyte-like cells and are widely used as a model system to study the expression of membrane peptidases on the apical brush border [I-41 Indirect irnniunofluorescent staining has previously shown dipeptidylpeptidase IV (DPPIV, EC 3 4 14 5) to be homogeneously distributed on the surface of Caco-2 cells [ I ] whereas endopeptidase-24 1 1 (E-24 I I , EC 3 4 24 I I ) was found on the surface of some cells but not on others [4] This observation has also been reported by Jalal e/ d. [ S ] who have shown that E-24 1 1 is expressed by all cells but in some is confined to an intracellular pool In the present study the cellsurface and intracellular expression of five membrane peptidases on confluent cultures of Caco-2 cells has been investigated using indirect immunofluorescent staining The membrane peptidases studied were DPPIV, E-24 I I , angiotensin converting enzyme (ACE, EC 3 4 15 I). aminopeptidase N (AP-N, EC 3 4 1 I 2) and aminopeptidase W (AP-W, EC 3 4 11 16)