ORCID Profile
0000-0003-3904-5532
Current Organisation
University College London
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Publisher: Frontiers Media SA
Date: 18-02-2022
DOI: 10.3389/FCELL.2022.836208
Abstract: The perinuclear theca (PT) is a highly condensed, largely insoluble protein structure that surrounds the nucleus of eutherian spermatozoa. Recent reports have indicated that the PT unexpectedly houses several somatic proteins, such as core histones, which may be important post-fertilization during re-modelling of the male pronucleus, yet little is known regarding the overall proteomic composition of the PT. Here, we report the first in depth, label-free proteomic characterization of the PT of boar spermatozoa following the implementation of a long-established subcellular fractionation protocol designed to increase the detection of low abundance proteins. A total of 1,802 proteins were identified, a result that represents unparalleled depth of coverage for the boar sperm proteome and exceeds the entire annotated proteome of the Sus scrofa species so far. In the PT structure itself, we identified 813 proteins and confirmed the presence of previously characterized PT proteins including the core histones H2A, H2B, H3 and H4, as well as Ras-related protein Rab-2A (RAB2A) and Rab-2B (RAB2B) amongst other RAB proteins. In addition to these previously characterized PT proteins, our data revealed that the PT is replete in proteins critical for sperm-egg fusion and egg activation, including: Izumo family members 1–4 (IZUMO1-4) and phosphoinositide specific phospholipase ζ (PLCZ1). Through Ingenuity Pathway Analysis, we found surprising enrichment of endoplasmic reticulum (ER) proteins and the ER-stress response in the PT. This is particularly intriguing as it is currently held that the ER structure is lost during testicular sperm maturation. Using the String and Cytoscape tools to visualize protein-protein interactions revealed an intricate network of PT protein complexes, including numerous proteasome subunits. Collectively, these data suggest that the PT may be a unique site of cellular homeostasis that houses an abundance of protein degradation machinery. This fits with previous observations that the PT structure dissociates first within the oocyte post-fertilization. It remains to be explored whether proteasome subunits within the PT actively assist in the protein degradation of paternal cell structures post-fertilization and how aberrations in PT protein content may delay embryonic development.
Publisher: Proceedings of the National Academy of Sciences
Date: 05-11-2021
Abstract: Spatial organization of mitochondria is vital for cellular function. In many specialized cell types, mitochondria are immobilized at specific subcellular loci through interactions with the cytoskeleton. One of the most striking mitochondrial configurations occurs in mammalian sperm, where mitochondria wrap around the flagellum. Malformation of the mitochondrial sheath causes infertility, but the molecular structures underlying this intricate arrangement are unknown. Here, we analyzed the mitochondrial sheath in sperm from three mammalian species. We find that although mitochondrial dimensions and cristae architecture vary across species, molecular assemblies mediating intermitochondria and mitochondria–cytoskeleton interactions are conserved. These findings yield important insight into sperm physiology and evolution and are relevant for other polarized cell types, such as muscles, neurons, photoreceptors, and hair cells.
Publisher: Wiley
Date: 09-03-2023
DOI: 10.1111/ANDR.13413
Abstract: Previously, we reported that cysteine‐rich secretory protein 2 is involved in high molecular weight complexes in boar spermatozoa. These cysteine‐rich secretory protein 2protein complexes are formed at the last phase of sperm formation in the testis and play a role in sperm shaping and functioning. This study aimed to identify cysteine‐rich secretory protein 2 interacting partners. These binding partner interactions were investigated under different conditions, namely, non‐capacitating conditions, after the induction of in vitro sperm capacitation and subsequently during an ionophore A23187‐induced acrosome reaction. The incubated pig sperm s les were subjected to protein extraction. Extracted proteins were subjected to blue native gel electrophoresis and native immunoblots. Immunoreactive gel bands were excised and subjected to liquid chromatography–mass spectrometry (LC‐MS) analysis for protein identification. Protein extracts were also subjected to CRISP2 immunoprecipitation and analyzed by LC‐MS for protein identification. The most prominent cystein‐rich secretory protein 2 interacting proteins that appeared in both independent LC‐MS analyses were studied with a functional in situ proximity interaction assay to validate their property to interact with cystein‐rich secretory protein 2 in pig sperm. Blue native gel electrophoresis and native immunoblots revealed that cystein‐rich secretory protein 2 was present within a ∼150 kDa protein complex under all three conditions. Interrogation of cystein‐rich secretory‐protein 2‐immunoreactive bands from blue native gels as well as cystein‐rich secretory protein 2 immunoprecipitated products using mass spectrometry consistently revealed that, beyond cystein‐rich secretory protein 2, acrosin and acrosin binding protein were among the most abundant interacting proteins and did interact under all three conditions. Co‐immunoprecipitation and immunoblotting indicated that cystein‐rich secretory protein 2 interacted with pro‐acrosin (∼53 kDa) and Aacrosin binding protein under all three conditions and additionally to acrosin (∼35 kDa) after capacitation and the acrosome reaction. The colocalization of these interacting proteins with cystein‐rich secretory protein 2 was assessed via in situ proximity ligation assays. The colocalization signal of cystein‐rich secretory protein 2 and acrosin in the acrosome seemed dispersed after capacitation but was consistently present in the sperm tail under all conditions. The fluorescent foci of cystein‐rich secretory protein 2 and acrsin binding protein colocalization appeared to be redistributed within the sperm head from the anterior acrosome to the post‐acrosomal sheath region upon capacitation. These results suggest that CRISP2 may act as a scaffold for protein complex formation and dissociation to ensure the correct positioning of proteins required for the acrosome reaction and zona pellucida penetration.
Publisher: Cold Spring Harbor Laboratory
Date: 17-02-2021
DOI: 10.1101/2021.02.16.431372
Abstract: Mitochondria-cytoskeleton interactions modulate cellular physiology by regulating mitochondrial transport, positioning, and immobilization. However, there is very little structural information defining mitochondria-cytoskeleton interfaces in any cell type. Here, we use cryo-focused ion beam milling-enabled cryo-electron tomography to image mammalian sperm, where mitochondria wrap around the ciliary cytoskeleton. We find that mitochondria are tethered to their neighbors through inter-mitochondrial linkers and are anchored to the cytoskeleton through ordered arrays on the outer mitochondrial membrane. We use subtomogram averaging to resolve in-cell structures of these arrays from three mammalian species, revealing they are conserved across species despite variations in mitochondrial dimensions and cristae organization. We find that the arrays consist of boat-shaped particles anchored on a network of membrane pores whose arrangement and dimensions are consistent with voltage dependent anion channels. Proteomics and in-cell cross-linking mass spectrometry suggest that the conserved arrays are composed of glycerol kinase-like proteins. Ordered supramolecular assemblies may serve to stabilize similar contact sites in other cell types where mitochondria need to be immobilized in specific subcellular environments, such as in muscles and neurons.
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for RICCARDO ZENEZINI CHIOZZI.