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Isolation and proteomic characterization of the mouse sperm acrosomal matrix.

ABSTRACT: 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.

SUBMITTER: Guyonnet B

PROVIDER: S-EPMC3434786 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Isolation and proteomic characterization of the mouse sperm acrosomal matrix.

Guyonnet Benoit B Zabet-Moghaddam Masoud M SanFrancisco Susan S Cornwall Gail A GA

Molecular & cellular proteomics : MCP 20120615 9

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 ...[more]

PMID: 22707618

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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.

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%.

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Isolation and proteomic characterization of the mouse sperm acrosomal matrix.

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Isolation and proteomic characterization of the mouse sperm acrosomal matrix.

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