The nuclear form of glutathione peroxidase 4 colocalizes and directly interacts with protamines in the nuclear matrix during mouse sperm chromatin assembly

Abstract

The testis-specific nuclear form of Phospholipid Hydroperoxide Glutathione Peroxidase (nGPx4) is associated with the nuclear matrix during spermiogenesis and is implicated in sperm chromatin condensation. In this study, we have addressed the question whether nGPx4 directly interacts with protamines by transiently sharing a nuclear matrix localization. We first expressed tagged protamine 1-myc and protamine 2-V5 in HeLa and COS-1 cells and showed by both confocal microscopy and immunoblotting analyses that protamines were produced in vitro and colocalized correctly to the nucleus. Co-transfection experiments demonstrated that protamine 1 was physically associated with flag-nGPx4 specifically at the level of nuclear matrix. The peculiar presence of protamines together with nGPx4 in this subnuclear compartment was also confirmed in mouse elongated spermatids by immunofluorescence, suggesting that nGPx4 is a physiological component of a novel protein complex relevant to chromatin assembly in condensing haploid cells. Also, in epididymal sperm, nGPx4 and protamine 1 co-immunoprecipitated, indicating that nGPx4, although localized to a subnuclear compartment different from that of protamines, represents a constant link between nuclear matrix and chromatin in mammalian male gamete.

Inactivity of Peptidase ClpP Causes Primary Accumulation of Mitochondrial Disaggregase ClpX with Its Interacting Nucleoid Proteins, and of mtDNA

“…It is known that a G4-repeat DNA structure resolvase named RHAU is essential for spermatogonia differentiation via c-Kit [ 76 ]. Furthermore, GRSF1 targets the G-repeat-containing mRNA of GPx4 [ 77 ], a crucial factor for protamine-mediated chromatin condensation during spermatogenesis [ 78 ]. Thus, it is conceivable that dysregulated GRSF1 abundance interferes with germ cell differentiation, meiosis, and sperm capacitation in PRLTS3.…”

Proteomic Signatures in Spermatozoa Reveal the Role of Paternal Factors in Recurrent Pregnancy Loss

“…GPx4 is an essential selenoenzyme that exists as a cytosolic form, mitochondrial form, and nuclear form necessary for early embryogenesis and cell viability because of its unqiue ability to prevent phospholipid oxidation. Being predominantly expressed in late spermatids and spermatozoa is also known to play a vital role in the formation of disulfide bridging intermediates [29]. While, GSTM5 is a member of the µ-class of glutathione S-transferases is abundantly expressed in the testis that protects the spermatozoa from oxidative stress through several events of phosphorylation/ dephosphorylation while its disruption leads to an increase in protein sulfhydryl oxidation [30].…”

Inactivity of Peptidase ClpP Causes Primary Accumulation of Mitochondrial Disaggregase ClpX With Its Interactors at the Nucleoid and RNA Granule

“…It is known that a G4-repeat DNA structure resolvase named RHAU is essential for spermatogonia differentiation via c-Kit [74]. Furthermore, GRSF1 targets the G-repeat containing mRNA of GPx4 [75], a crucial factor for protaminemediated chromatin condensation [76]. Thus, it is conceivable that dysregulated GRSF1 abundance interferes with germ cell differentiation, meiosis and sperm capacitation in PRLTS3.…”