Role of protein kinase C in the acrosome reaction of mammalian spermatozoa.
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ABSTRACT:
SUBMITTER: Breitbart H
PROVIDER: S-EPMC1130709 | biostudies-other | 1992 Jan
REPOSITORIES: biostudies-other
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Project description:Cytoskeleton-related proteins are essential for cell shape maintenance and cytoskeleton remodeling. The spermatozoa of Eriocheir sinensis (Chinese mitten crab) have a unique cellular structure, and the mechanism of spermatozoal metamorphosis during the acrosome reaction is not well understood. In this study, the E. sinensis spermatozoa were induced using calcium ionophore A23187 to undergo the acrosome reaction in vitro, and the acrosome-reacting and fresh (non-reacting) spermatozoa were collected separately. The differential expression of cytoskeleton-related protein genes in acrosome-reacting and fresh spermatozoa of E. sinensis was analyzed by whole transcriptome sequencing and bioinformatics analysis, and PPI network and miRNA-mRNA regulation network were constructed to analyze their possible function and regulation mechanism. The results showed that numerous differentially expressed cytoskeleton-related protein genes, miRNAs and lncRNAs were found in acrosome-reacting and fresh spermatozoa of E. sinensis; 27 cytoskeleton-related protein genes were down regulated and 687 miRNAs were up regulated in acrosome-reacting spermatozoa; 147 miRNAs target these 27 cytoskeleton-related protein genes. In the PPI networks, RAC1, BCAR1, RDX, NCKAP1, EPS8, CDC42BPA, LIMK1, ELMO2, GNAI1 and OCRL were identified as hub proteins. These proteins are mainly involved in the regulation of cytoskeleton organization, actin cytoskeleton organization, microtubule skeleton organization and small GTPase-mediated signal transduction and other biological processes, and play roles in pathways such as actin cytoskeletal regulation and axon guidance. miR-9, miR-31 and two novel miRNAs in the miRNA-mRNA regulatory network are the core miRNAs targeting cytoskeleton-related protein genes. miR-9 targets and regulates OBSCN, CDC42BPA, ELMO2, BCAS3, TPR and OCRL; while miR-31 targets and regulates CDC42BPA and TPR. This study provides a theoretical basis for revealing the mechanism of acrosome reaction under the special spermatozoa morphology of E. sinensis.
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Project description:The functional relationship between the formation of hundreds of fusion pores during the acrosome reaction in spermatozoa and the mobilization of calcium from the acrosome has been determined only partially. Hence, the second messenger NAADP, promoting efflux of calcium from lysosome-like compartments and one of its potential molecular targets, the two-pore channel 1 (TPC1), were analyzed for its involvement in triggering the acrosome reaction using a TPCN1 gene-deficient mouse strain. The present study documents that TPC1 and NAADP-binding sites showed a colocalization at the acrosomal region and that treatment of spermatozoa with NAADP resulted in a loss of the acrosomal vesicle that showed typical properties described for TPCs: Registered responses were not detectable for its chemical analogue NADP and were blocked by the NAADP antagonist trans-Ned-19. In addition, two narrow bell-shaped dose-response curves were identified with maxima in either the nanomolar or low micromolar NAADP concentration range, where TPC1 was found to be responsible for activating the low affinity pathway. Our finding that two convergent NAADP-dependent pathways are operative in driving acrosomal exocytosis supports the concept that both NAADP-gated cascades match local NAADP concentrations with the efflux of acrosomal calcium, thereby ensuring complete fusion of the large acrosomal vesicle.
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Project description:An investigation was made of the modifications in phospholipids that occur during the exocytotic event known as the 'sperm acrosome reaction'. Phospholipids were prelabelled with 32P, and exocytosis was induced with Ca2+ and the ionophore A23187. When incubated with [32P]Pi in various media suitable for supporting sperm survival or fertilization in vitro, spermatozoa from all five species examined (ram, boar, guinea pig, mouse and human) incorporated 32P rapidly into the components of the phosphoinositide cycle. There were differences both between species and between media with respect to the actual rate of incorporation of label, and also between species with respect to other phospholipids labelled. Treatment of spermatozoa with Ca2+ and A23187 to induce the acrosome reaction resulted in a rapid breakdown of phosphatidylinositol 4, 5-bisphosphate and phosphatidylinositol 4-phosphate, which was complete within 3 min; there was also a great increase in labelling of phosphatidate. Occurrence of acrosome reactions in the sperm population was only observed after 5-10 min and reached a maximum response of greater than 90% after more than 30 min. The phosphoinositide breakdown was related to subsequent exocytosis: after EGTA/ionophore treatment, neither inositide breakdown nor exocytosis took place; however, later addition of Ca2+ resulted in immediate inositide breakdown, and exocytosis followed, with a delay relative to Ca2+ addition exactly similar to that following standard Ca2+/ionophore treatment. Neomycin inhibited both inositide breakdown and subsequent exocytosis provided it was added together with Ca2+ and ionophore; however, if the drug was added 3 min after Ca2+ and ionophore (by which time inositide breakdown was already complete), exocytosis was not inhibited. Ca2+ seemed to have several consecutive roles in the acrosome reaction. Low (micromolar) levels of free Ca2+ were needed both for phosphoinositide breakdown and for an event downstream of this breakdown; no other bivalent cation could substitute for Ca2+ in either event, and inositide breakdown was actually inhibited by Mg2+. In addition, millimolar levels of Ca2+ were needed for later stages of exocytosis, although this requirement could be satisfied by Sr2+. We conclude that breakdown of polyphosphoinositides is an essential early process after Ca2+ entry in the chain of events that lead to exocytosis in the mammalian sperm acrosome reaction.
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Project description:The binding properties of seminal polyamines to ram spermatozoa and their possible role in sperm capacitation and the acrosome reaction were studied. Binding and release of [14C]spermine from ram spermatozoa occurred at a rate faster than in somatic cells and were not energy-dependent. Release of bound spermine was further facilitated by heparin, a constituent of the female reproductive tract which was reported to induce capacitation and the acrosome reaction. High- and low-affinity polyamine-binding sites were identified, of which the high-affinity site was specific to polyamines with three or more amino groups. We also found that spermine inhibited the acrosome reaction and propose that it is the major seminal decapacitating factor. Since precise timing of capacitation and the acrosome reaction are critical for successful fertilization, it is suggested that the role of seminal spermine is to prevent premature capacitation and the acrosome reaction.
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