Isolation and Proteomic Characterization of the Mouse Sperm Acrosomal Matrix

Guyonnet, Benoît; Zabet-Moghaddam, Masoud; SanFrancisco, Susan; Cornwall, Gail A.

doi:10.1074/mcp.m112.020339

Cited by 53 publications

(27 citation statements)

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“…For instance, prion states of numerous proteins are stably heritable both through mitosis and through meiosis in budding yeast. While in mammals prion-mediated diseases such as Creutzfeldt-Jakob disease do not seem to be transmitted vertically, a number of other proteins capable of forming (potentially non-pathogenic) amyloids in vitro (von Horsten et al, 2007) have been identified associated with the sperm acrosome (Guyonnet et al, 2012). Beyond prions, other proteins such as transcription factors, or the abundant protamines (which, like histones, are subject to a wide variety of covalent modifications), present in sperm could conceivably alter the phenotype of offspring.…”

Section: Introductionmentioning

confidence: 99%

Daddy Issues: Paternal Effects on Phenotype

Rando

2012

Cell

261

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Section: Introductionmentioning

confidence: 99%

Daddy Issues: Paternal Effects on Phenotype

Rando

2012

Cell

261

220

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“…In previous studies, we showed that isolated mouse sperm AM contained a diverse group of proteins, including proteases, chaperones, hydrolases, transporters, enzyme modulators, cytoskeletal proteins, and others, suggesting a complex functional structure (16). In the present study, extraction with 1% SDS solubilized the majority of the AM proteins.…”

Section: Discussionmentioning

confidence: 77%

“…Briefly, following extraction with Triton X-100 to remove membranes, the spermatozoa were vortexed in buffer at pH 3 and released intact AM were separated from spermatozoa by low-speed centrifugation with the AM going into the supernatant (total AM) (16). In our previous studies, we used antibodies against known AM proteins, including proacrosin (ACR), ZAN, and ACR binding protein (ACRBP), in immunofluorescence and Western blot analyses to confirm the isolated material was indeed AM (16). Although PNA-positive structures were present in all of the samples, OC but not A11 immunostaining was detected in the AM from caput (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Functional Amyloids in the Mouse Sperm Acrosome

Guyonnet

Egge

Cornwall

2014

Molecular and Cellular Biology

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The acrosomal matrix (AM) is an insoluble structure within the sperm acrosome that serves as a scaffold controlling the release of AM-associated proteins during the sperm acrosome reaction. The AM also interacts with the zona pellucida (ZP) that surrounds the oocyte, suggesting a remarkable stability that allows its survival despite being surrounded by proteolytic and hydrolytic enzymes released during the acrosome reaction. To date, the mechanism responsible for the stability of the AM is not known. Our studies demonstrate that amyloids are present within the sperm AM and contribute to the formation of an SDS-and formic-acid-resistant core. The AM core contained several known amyloidogenic proteins, as well as many proteins predicted to form amyloid, including several ZP binding proteins, suggesting a functional role for the amyloid core in sperm-ZP interactions. While stable at pH 3, at pH 7, the sperm AM rapidly destabilized. The pH-dependent dispersion of the AM correlated with a change in amyloid structure leading to a loss of mature forms and a gain of immature forms, suggesting that the reversal of amyloid is integral to AM dispersion.A n essential step during fertilization is the sperm acrosome reaction (AR) in which the acrosome, an exocytotic vesicle overlying the sperm head, releases its contents, allowing the spermatozoon to penetrate the investments surrounding the oocyte. Point fusions between the outer acrosomal and plasma membranes result in membrane vesiculation, allowing the soluble contents to be released. The acrosome also includes an insoluble fraction called the acrosomal matrix (AM), which is defined as a membrane-free, electron-dense material that remains after spermatozoa are extracted with Triton X-100 (1). Functionally, the AM is thought to provide a stable scaffold that allows the controlled and sequential release of matrix-associated proteins during the AR, as well as to facilitate interactions between the sperm and oocyte (2, 3). While the mechanisms for the assembly and disassembly of the AM are not known, the self-assembly of proteins into a large complex has been proposed for its formation and disassembly is thought to be due to active proteases (1).The site of the AR has been controversial and was previously thought not to occur in the mouse until spermatozoa encounter the zona pellucida, the thick coat surrounding the oocyte (4, 5). However, recent studies with video imaging microscopy to follow individual mouse spermatozoa with enhanced green fluorescent protein expressed in their acrosomes showed that, in fact, the fertilizing spermatozoa underwent the AR much earlier during transit through the cumulus cells prior to encountering the zona pellucida (6). Further studies indicated that these acrosome-reacted spermatozoa remained capable of binding and penetrating the zona pellucida (7). Together, these studies suggest that the AM, instead of the soluble components of the acrosome, is required for binding and penetration of the zona pellucida. The presence of several zona pelluc...

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“…Five forms of lysozyme-like genes Lyzl2 , Lyzl3/Spaca3 , Lyzl4 , Lyzl5/Spaca5 and Lyzl6 have been identified in the testis of human and other mammalian species [16–19]. The lysozyme-like proteins (LYZL) along with commonly known lysozyme have been identified from sperm proteome of mouse and human [20,21]. LYZL4 protein has also been observed on the surface of human embryonic stem cells [22].…”

Section: Introductionmentioning

confidence: 99%

Structural, Functional and Phylogenetic Analysis of Sperm Lysozyme-Like Proteins

et al. 2016

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Sperm lysozyme-like proteins belonging to c-type lysozyme family evolved in multiple forms. Lysozyme-like proteins, viz., LYZL2, LYZL3 or SLLP1, LYZL4, LYZL5 and LYZL6 are expressed in the testis of mammals. Not all members of LYZL family have been uniformly and unambiguously identified in the genome and proteome of mammals. Some studies suggested a role of SLLP1 and LYZL4 in fertilization; however, the function of other LYZL proteins is unknown. We identified all known forms of LYZL proteins in buffalo sperm by LC-MS/MS. Cloning and sequence analysis of the Lyzl cDNA showed 38–50% identity at amino acid level among the buffalo LYZL paralogs, complete conservation of eight cysteines and other signature sequences of c-type lysozyme family. Catalytic residues in SLLP1, LYZL4 and LYZL5 have undergone replacement. The substrate binding residues showed significant variation in LYZL proteins. Residues at sites 62, 101, 114 in LYZL4; 101 in SLLP1; 37, 62, and 101 in LYZL6 were more variable among diverse species. Sites 63 and 108 occupied by tryptophan were least tolerant to variation. Site 37 also showed lower tolerance to substitution in SLLP1, LYZL4 and LYZL5, but more variable in non-testicular lysozymes. Models of LYZL proteins were created by homology modeling and the substrate binding pockets were analyzed in term of binding energies and contacting residues of LYZL proteins with tri-N-acetylglucosamine (NAG)3 in the A-B-C and B-C-D binding mode. Except LYZL6, LYZL proteins did not show significant difference in binding energies in comparison to hen egg white lysozyme in the A-B-C mode. (NAG)3 binding energy in the B-C-D mode was higher by 1.3–2.2 kcal/mol than in A-B-C mode. Structural analysis indicated that (NAG)3 was involved in making more extensive interactions including hydrogen bonding with LYZL proteins in B-C-D mode than in A-B-C mode. Despite large sequence divergence among themselves and with respect to c-type lysozymes, substrate binding residues as well as hydrogen bonding network between (NAG)3 and proteins were mostly conserved. LYZL5 in buffalo and other mammalian species contained additional 10–12 amino acid sequence at c-terminal that matched with ankyrin repeat domain-containing protein 27. Phylogenetic analysis indicated LYZL2 to be most ancient among all the LYZL proteins and that the evolution of LYZL proteins occurred through several gene duplications preceding the speciation of mammals from other vertebrates as distant as reptiles and amphibians.

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Isolation and Proteomic Characterization of the Mouse Sperm Acrosomal Matrix

Cited by 53 publications

References 86 publications

Daddy Issues: Paternal Effects on Phenotype

Daddy Issues: Paternal Effects on Phenotype

Functional Amyloids in the Mouse Sperm Acrosome

Structural, Functional and Phylogenetic Analysis of Sperm Lysozyme-Like Proteins

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