Isolation and Proteomic Characterization of the Mouse Sperm Acrosomal Matrix
Ontology highlight
ABSTRACT:
SUBMITTER:
PROVIDER: S-EPMC3945919 | biostudies-literature | 2014 Mar
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:A critical step during fertilization is the sperm acrosome reaction in which the acrosome releases its contents allowing the spermatozoa to penetrate the egg investments. The sperm acrosomal contents are composed of both soluble material and an insoluble material called the acrosomal matrix (AM). The AM is thought to provide a stable structure from which associated proteins are differentially released during fertilization. Because of its important role during fertilization, efforts have been put toward isolating the AM for biochemical study and to date AM have been isolated from hamster, guinea pig, and bull spermatozoa. However, attempts to isolate AM from mouse spermatozoa, the species in which fertilization is well-studied, have been unsuccessful possibly because of the small size of the mouse sperm acrosome and/or its fusiform shape. Herein we describe a procedure for the isolation of the AM from caput and cauda mouse epididymal spermatozoa. We further carried out a proteomic analysis of the isolated AM from both sperm populations and identified 501 new proteins previously not detected by proteomics in mouse spermatozoa. A comparison of the AM proteome from caput and cauda spermatozoa showed that the AM undergoes maturational changes during epididymal transit similar to other sperm domains. Together, our studies suggest the AM to be a dynamic and functional structure carrying out a variety of biological processes as implied by the presence of a diverse group of proteins including proteases, chaperones, hydrolases, transporters, enzyme modulators, transferases, cytoskeletal proteins, and others.
| S-EPMC3434786 | biostudies-literature
Project description:The acrosome is an exocytotic vesicle located on the apical tip of the sperm head. In addition to having different morphological regions, two biochemically distinct compartments can be defined within the acrosome: a particulate acrosomal matrix and a soluble partition. The domains within the acrosome participate in the release of acrosomal proteins from the sperm during exocytosis, depending on whether the proteins partition into either the soluble or matrix compartments of the acrosome. We have examined the mechanism of differential release by evaluating the solubilization of acrosomal matrix protein ZP3R (sp56) from mouse sperm during the course of spontaneous acrosomal exocytosis. Using indirect immunofluorescence and immunoblotting, we found that the ZP3R monomer is processed from 67,000 M(r) to 43,000 M(r) by proteases coincident with release from the acrosome. Sperm require a maturational step, termed capacitation, before they are competent for acrosomal exocytosis and the processing of ZP3R is dramatically reduced under non-capacitating conditions. The cleavage probably takes place in complement control protein domain (CCP) 6 or the bridge region between CCP6 and CCP7, which is not present in the guinea pig orthologue AM67. The cleaved form of ZP3R does not bind to unfertilized eggs. We have incorporated these structural considerations into a model to explain the functional consequences of acrosomal exocytosis on sperm-zona interactions.
| S-EPMC2729263 | biostudies-literature
Project description:Identifying inducers of sperm acrosomal exocytosis (AE) to understand sperm functionality is important for both mechanistic and clinical studies in mammalian fertilization. Epifluorescence microscopy methods, while reproducible, are laborious and incompatible for high throughput screening. Flow cytometry methods are ideal for quantitative measurements on large numbers of samples, yet typically rely on the use of lectins that can interfere with physiologic AE-inducers. Here, we present an optimized triple stain flow cytometric method that is suitable for high-throughput screening of AE activation by glycopolymers. SYTO-17 and propidium iodide (PI) were used to differentiate cells based on their membrane integrity or viability, and membrane impermeable soybean trypsin inhibitor (SBTI) was used to monitor acrosome exocytosis. The SBTI/PI/SYTO-17 combination provides a positive screen for viability and AE of live sperm cells with minimal noise or false positives. A scattering gate enables the use of samples that may be contaminated with non-cellular aggregates, e.g., cryopreservation agents. This assay format enabled detailed analysis of glycopolymer dose response curves. We found that fucose polymer has a narrow effective dose range (EC50 = 1.6 μM; IC50 = 13.5 μM); whereas mannose polymer and β-N-acetylglucosamine polymer have broader effective dose ranges (EC50 = 1.2 μM and 3.4 μM, respectively). These results highlight the importance of testing inducers over a large concentration range in small increments for accurate comparison.
| S-EPMC4886651 | biostudies-literature
Project description:As a prerequisite to mammalian fertilization, the sperm acrosomal vesicle fuses with the plasma membrane and the acrosome contents are exocytosed. Induction occurs through engagement of the sperm receptors by multiple sugar residues. Multivalent polymers displaying mannose, fucose, or GlcNAc are effective synthetic inducers of mouse sperm acrosomal exocytosis (AE). Each carbohydrate is proposed to have a distinct binding site on the sperm cell surface. To determine the role of the scaffold structure in the efficiency of AE induction, different polymer backbones were employed to display the different activating sugar residues. These glycopolymers were prepared by ruthenium-catalyzed ring-opening metathesis of 5-substituted norbornene or cyclooctene. The conformations of the glycopolymers were characterized by small-angle X-ray scattering. Polynorbornene displaying mannose, fucose, or GlcNAc forms flexible cylinders in aqueous solution. However, polycyclooctenes displaying any of these same sugars are much more flexible and form random coils. The flexible polycyclooctenes displaying fucose or GlcNAc were less effective inducers of AE than their norbornene counterparts. In contrast, polycyclooctene displaying mannose was the most effective AE inducer and had a more collapsed spherelike structure. Our results suggest that the AE efficacy of fucose, GlcNAc, and mannose polymers relies on a relatively rigid polymer that can stabilize receptor signaling complexes.
| S-EPMC5464008 | biostudies-literature
Project description:Protein kinase A (PKA) is a broad-spectrum Ser/Thr kinase involved in the regulation of several cellular activities. Thus, its precise activation relies on being localized at specific subcellular places known as discrete PKA signalosomes. A-Kinase anchoring proteins (AKAPs) form scaffolding assemblies that play a pivotal role in PKA regulation by restricting its activity to specific microdomains. Because one of the first signaling events observed during mammalian sperm capacitation is PKA activation, understanding how PKA activity is restricted in space and time is crucial to decipher the critical steps of sperm capacitation. Here, we demonstrate that the anchoring of PKA to AKAP is not only necessary but also actively regulated during sperm capacitation. However, we find that once capacitated, the release of PKA from AKAP promotes a sudden Ca2+ influx through the sperm-specific Ca2+ channel CatSper, starting a tail-to-head Ca2+ propagation that triggers the acrosome reaction. Three-dimensional super-resolution imaging confirmed a redistribution of PKA within the flagellar structure throughout the capacitation process, which depends on anchoring to AKAP. These results represent a new signaling event that involves CatSper Ca2+ channels in the acrosome reaction, sensitive to PKA stimulation upon release from AKAP.
| S-EPMC6005427 | biostudies-literature
Project description:Zona pellucida binding protein 1 (ZPBP1), a spermatid and spermatozoon protein that localizes to the acrosome, was originally identified in pigs and named for its binding to the oocyte zona pellucida. In an in silico search for germ cell-specific genes, Zpbp1 and its novel paralog, Zpbp2, were discovered and confirmed to be expressed only in the testes in both mice and humans. To study the in vivo functions of both ZPBP proteins, we disrupted Zpbp1 and Zpbp2 in mice. Males lacking ZPBP1 were sterile, with abnormal round-headed sperm morphology and no forward sperm motility. Ultrastructural studies demonstrated that absence of ZPBP1 prevents proper acrosome compaction, resulting in acrosome fragmentation and disruption of the Sertoli-spermatid junctions. Males null for ZPBP2 were subfertile, demonstrated aberrant acrosomal membrane invaginations, and produced dysmorphic sperm with reduced ability to penetrate zona pellucida. Molecular phylogenetic analysis of ZPBPs from amphibians, birds, and mammals suggests that these paralogous genes coevolved to play cooperative roles during spermiogenesis. Whereas ZPBP1 was discovered for an in vitro role in sperm-egg interactions, we have shown that both ZPBP proteins play an earlier structural role during spermiogenesis.
| S-EPMC2099232 | biostudies-literature
Project description:Recent evidence demonstrated that most fertilizing mouse sperm undergo acrosomal exocytosis (AE) before binding to the zona pellucida of the eggs. However, the sites where fertilizing sperm could initiate AE and what stimuli trigger it remain unknown. Therefore, the aim of this study was to determine physiological sites of AE by using double transgenic mouse sperm, which carried EGFP in the acrosome and DsRed2 fluorescence in mitochondria. Using live imaging of sperm during in vitro fertilization of cumulus-oocyte complexes, it was observed that most sperm did not undergo AE. Thus, the occurrence of AE within the female reproductive tract was evaluated in the physiological context where this process occurs. Most sperm in the lower segments of the oviduct were acrosome-intact; however, a significant number of sperm that reached the upper isthmus had undergone AE. In the ampulla, only 5% of the sperm were acrosome-intact. These results support our previous observations that most of mouse sperm do not initiate AE close to or on the ZP, and further demonstrate that a significant proportion of sperm initiate AE in the upper segments of the oviductal isthmus.
| S-EPMC4783209 | biostudies-literature
Project description:In recent years, the study of mammalian acrosomal exocytosis has produced some major advances that challenge the long-held, general paradigms in the field. Principally, the idea that sperm must be acrosome-intact to bind to the zona pellucida of unfertilized eggs, based largely on in vitro fertilization studies of mouse oocytes denuded of the cumulus oophorus, has been overturned by experiments using state-of-the-art imaging of cumulus-intact oocytes and fertilization experiments where eggs were reinseminated by acrosome-reacted sperm recovered from the perivitelline space of zygotes. In light of these results, this minireview highlights a number of unresolved questions and emphasizes the fact that there is still much work to be done in this exciting field. Future experiments using recently advanced technologies should lead to a more complete and accurate understanding of the molecular mechanisms governing the fertilization process in mammals.
| S-EPMC4076372 | biostudies-literature
Project description:A critical step during fertilization is the sperm acrosome reaction in which the acrosome releases its contents allowing the spermatozoa to penetrate the egg investments. The sperm acrosomal contents are composed of both soluble material and an insoluble material called the acrosomal matrix (AM). The AM is thought to provide a stable structure from which associated proteins are differentially released during fertilization. Mouse acrosomal matrices were isolated from 2 different populations of epididymal spermatozoa: AM_caput from caput epididymal spermatozoa (2 replicates) and AM_cauda from cauda epididymal spermatozoa (3 replicates). Proteins were separated by SDS-PAGE on a 15% polyacrylamide gel. After electrophoresis, proteins were stained with Coomassie blue. Gel lanes were cut into 8–9 slices and each slice placed into a 0.5 ml microcentrifuge tube. The gel pieces were washed to destain the gels. Reduction was performed with dithiothreitol solution. After reduction, the proteins were alkylated. The gel pieces were washed, dehydrated and air-dried. The digestion was proceed with trypsin and peptides extracted. The peptides obtained from in-gel digestion were analyzed by nano-flow nano-LC-MS/MS using an LTQ-XL ion trap mass spectrometer. To get the maximum number of publicly available sequences for analyses, the mouse sequences from Ensembl were merged with those from NCBI. The database was built using BioPerl modules. Specifically, from Ensembl two files were downloaded and merged including Mus_musculus.NCBIM37.64.pep.all.fa.gz (containing the superset of all translations resulting from Ensembl known or novel gene predictions) and Mus_musculus.NCBIM37.64.pep.abinitio.fa.gz (containing translations resulting from “ab initio” gene prediction algorithms such as SNAP and GENSCAN). From NCBI, the nonredundant (nr) file (on 11/2011) was downloaded and mouse sequences extracted (Taxonomy ID = 10090). This file was merged with the Ensembl sequences to create a file containing 356,881 sequences of which some were redundant. Duplicates were removed based on identical amino acid sequences to get 203,220 unique sequences used to build our blast database. Spectra obtained from the trypsin digestion products using the LTQ Orbitrap XL mass spectrometer were identified by the Proteome Discoverer (version 1.3) program, based on SEQUEST cluster as a search engine (University of Washington, licensed to Thermo Electron Corp., San Jose, CA) against our mouse database (203,220 nonredundant protein sequences). The search engine used the following parameters: precursor ion mass tolerance, 2.5 Da; fragment ion mass tolerance, 0.8 Da; fully tryptic enzyme specificity; two missed cleavages; dynamic modifications of cysteine carbamidomethylation and of methionine oxidation. The proportion of false positive assignations among the tentative peptide identifications, also called false discovery rate (FDR), has been estimated by using decoy databases constructed from the target database and was set at 1%.
2013-11-14 | PXD000575 | Pride
Project description:Some molecular and cellular mechanisms of fertilization are still not completely described. The role of the acrosomal matrix (AM), the particulate compartment within the acrosome, is one of them. Here, we proposed that amyloids, highly ordered protein aggregates, by their physical and chemical properties could contribute to AM roles in fertilization. Purified AM were exposed to a two-step extraction to sequentially strip off soluble proteins. The remaining insoluble material (core) was subjected to a mass spectrometry analysis. Preparation of samples for MS analysis: Three different approaches were used to optimize identification of peptides in the AM core. For in-gel digestion, core samples were resuspended in 13.2mM SA pH3 containing 8M urea and 100 mM DTT and incubated for 1 hr at RT. Proteins were separated on a hand casted 12% polyacrylamide Tris-glycine gel. After silver staining, visible bands were cut from the gel, destained and washed with ddH2O (2 X 5 min) then with 50% ethanol (2 X 5 min) before stored at -20 degrees C. Gel pieces were tryptically digested in 25mM triethylammonium bicarbonate buffer at 37 degrees C overnight with trypsin. For in-solution digestion, core samples were resuspended in 13.2mM SA pH3/ 8M urea/ 100mM DTT and incubated for 1 h at RT followed by 15 min at 70 degrees C. Iodoacetamide was added to 10mM and proteins were alkylated by incubation for 15 min at RT in the dark. Samples were added to a pre-rinsed spin filter and centrifuged at 14,000 X g. Samples were washed with 9M urea then with 25mM ammonium bicarbonate. Samples were digested with trypsin in 25mM ammonium bicarbonate overnight. After digestion, samples were spin at 14,000 X g and washed 2 times with 25mM ammonium bicarbonate. The retentate was transferred to a new tube and air dried. For on-membrane digestion, the samples were dotted on nitrocellulose membrane and digested by trypsin. Briefly, core samples were resuspended and treated as for in-solution digestion. Then, samples were dotted by gravity on the membrane. Wells were rinsed with 20mM SA pH3 followed by TBS. Dots were cut and air-dried. After the protein digestion with trypsin in 25mM ammonium bicarbonate the membranes were dissolved with acetone and the precipitated peptides were air-dried. All digested peptides were reconstituted in 2% acetonitrile/ 0.1% formic acid for MS analysis. MS data acquisition: Protein identification by liquid chromatography tandem mass spectrometry (LCMS/MS) analysis of peptides was performed using an LTQ Orbitrap Velos MS interfaced with a 2D nanoLC system. Protein and Peptide Identification: MS/MS spectra were extracted using the ProteoWizard Toolkit. The spectra were analyzed using the GPM Manager with X!Tandem to search against a home made mouse database containing 213,054 nonredundant protein sequences created by using mouse sequences from Ensembl database (files Mus_musculus.GRCm38.73.pep.all & Mus_musculus.GRCm38.73.pep.abinitio) and from NCBI database (file nr downloaded on 09/19/2013) . Search was performed using fully tryptic enzyme specificity (See deposited MS data for details). Peptides and proteins that have an expectation value of log10 (e) <= -2 were included in the results. Curated results were obtained by keeping only proteins with at least one mouse-specific matching peptide (peptide match = 100% identities and 100% coverage on a unique mouse protein and <100% identities and/or <100% coverage on human proteins). In addition, trypsin-like proteins were kept if at least one peptide was not matching on exogenous pig trypsins.
2014-05-07 | PXD000592 | Pride