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.

Keywords: heterologous expression; nGPx4; nuclear matrix; protamines; sperm chromatin assembly.

Figure 1. Protamine 1 and protamine 2 are expressed in vitro in a heterologous cell system. Confocal microscopy analysis of HeLa cells transfected for 48 h with (A) the vector pBUDCE4.1-PRM1myc-PRM2V5 and (B) the control empty vector. Cells were immunostained with anti-c-myc (green) and anti-V5 (red) antibodies and counterstained with TOTO-3 (blue). Note that the two protamines colocalized specifically to the nucleus showing an overlapping diffuse nucleoplasmic signal and a variable number of bright dots. (Scale bars: 20 µm.)

Figure 2. Western blot analysis of protamine 1 expressed in vitro by COS-1 cells transfected with pBUDCE4.1-PRM1myc vector for 72 h. Ponceau red staining of the immobilon membrane is shown to visualize the migration markers: histones, salmon sperm protamine (SP), and mouse sperm protamines. Filters were immunoblotted with either the anti-c-myc antibody, which recognized only the protein synthesized by COS-1 cells, or the anti-protamine 1 antibody, which recognized protamine 1 of both COS-1 cells and mouse sperm. Protamine 1 extracted from mouse epididymal sperm as described in Materials and Methods, was used as immunoblot positive control. The additional band having slower migration than PRM1 likely reflects a different degree of protamine denaturation on the AU-PAGE. The protein synthesized by COS-1 cells showed a different electrophoretic mobility compared with PRM1 extracted from mouse spermatozoa, likely due to the presence of the myc-tag and/or the inability of COS-1 cells to post-translationally modify protamines.

Figure 3. nGP4 localizes to the nucleus when expressed in vitro after transfection of HeLa cells with the vector pCMV-Flag-nGPx4. Cells were stained with anti-flag (green) and anti-lamin B (red) antibodies and analyzed at the confocal microscopy. Lamin B staining was limited to the nuclear periphery, whereas nGPx4 staining was within the nucleus. (Scale bars: 20 µm.)

Figure 4. nGPx4 colocalizes esclusively to the nucleus together with both protamine 1 and protamine 2 in a heterologous cell system. HeLa cells were double transfected with the vectors pBUDCE4.1-PRM1myc-PRM2V5 and pCMV-flag-nGPx4 for 48 h and double stained in (A) with anti-flag (green) and anti-c-myc (red) antibodies; in (B) with anti-flag (green) and anti-V5 (red) antibodies. DNA was stained with TOTO-3 (blue). (Scale bars: 20 µm.)

Figure 5. Protamine 1 is located to the nuclear matrix in a heterologous cell system. COS-1 cells were transfected with pBUDCE4.1-PRM1myc, spotted on a slide, and subjected to in situ extraction and DNA digestion as described in Materials and Methods, and finally double stained with anti-myc antibody (green), anti lamin B antibody (red), and Toto-3 (blue). Lamin B was used as marker of the nuclear matrix. (Scale bars: 10 µm.)

Figure 6. nGPx4 associates with protamine 1 at the level of the nuclear matrix in a heterologous cell system. COS-1 cells were co-transfected with pCMV-Flag-nGPx4 and pBUDCE4.1-PRM1myc, subjected to in situ nuclear matrix preparation as described in Materials and Methods, immunostained with anti-flag (green) and anti-myc (red antibodies), and analyzed by confocal microscopy. The removal of DNA was assessed by TOTO-3 staining (blue). (Scale bars: 20 µm.)

Figure 7. nGPx4 associates with protamine 2 at the level of nuclear matrix in sonication resistant spermatids (steps 12–16, SRS) isolated from adult mouse testis. Nuclear matrix was prepared from SRS as described in Materials and Methods and double immunofluorescence was performed to detect nGPx4 (green) and protamine 2 (red) (A). We used topoisomerase IIβ (topo IIβ) (green) to assess the accuracy of nuclear matrix preparation (B). The double staining with anti-nGPx4 and anti-TopoIIβ was not possible, because both antibodies had been generated in rabbit. (Scale bars: 10 µm.)

Figure 8. nGPx4 co-immunoprecipitates with protamine 1 in mouse epididymal spermatozoa. Whole sperm lysate (input) was subjected to immunoprecipitation (IP) with anti-protamine 1 antibody, as described in Materials and Methods, and then analyzed by AU-PAGE and western blotting using anti-protamine 1 (A) or anti-nGPx4 (B) antibodies. Protamines extracted from mouse sperm (Sperm PRM1/PRM2) and basic nuclear proteins extracted from COS-1 cells transfected with either pCMV-Flag-nGPx4 (COS-1 nGPx4) or empty vector (COS-1 mock) as described in Materials and Methods, were used as migration markers of protamine 1 and nGPx4, respectively.