Spatiotemporal Changes of Levels of a Moonlighting Protein, Phospholipid Hydroperoxide Glutathione Peroxidase, in Subcellular Compartments During Spermatogenesis in the Rat Testis

Abstract

We studied temporal changes in the subcellular localization and levels of a moonlighting protein, phospholipid hydroperoxide glutathione peroxidase (PHGPx), in spermatogenic cells and mature sperm of the rat by immunofluorescence and immunoelectron microscopy. The PHGPx signals were detected in chromatoid bodies, clear nucleoplasm, mitochondria-associated material, mitochondrial aggregates, granulated bodies, and vesicles in residual bodies in addition to mitochondria, nuclei, and acrosomes as previously reported. Within mitochondria, PHGPx moved from the matrix to the outermost membrane region in step 19 spermatid, suggesting that this spatiotemporal change is synchronized with the functional change of PHGPx in mitochondria. In the nucleus, PHGPx was associated with electron-lucent spots and with the nuclear envelope, and PHGPx in the latter region increased after step 16. In early pachytene spermatids, PHGPx signals were noted in the nuclear material exhibiting a very similar density to chromatoid bodies and in the intermitochondrial cement, supporting the previous proposal that chromatoid bodies originate from the nucleus and intermitochondrial cement. The presence of PHGPx in such various compartments suggested versatile roles for this protein in spermatogenesis. Quantitative immunoelectron microscopic analysis also revealed dynamic changes in the labeling density of PHGPx in different subcellular compartments as follows: 1). Total cellular PHGPx rapidly increased after step 5 and reached a maximum at step 18; 2). mitochondrial labeling density increased after step 1 and achieved a maximum in steps 15-17; 3). nuclear labeling density suddenly increased in steps 12-14 to a maximum; 4). in cytoplasmic matrix, the density remained low in all steps; and 5). the labeling density in chromatoid bodies gradually decreased from pachytene spermatocytes to spermatids at step 18. These spatiotemporal changes in the level of PHGPx during the differentiation of spermatogenic cells to sperm infer that PHGPx plays a diverse and important biological role in spermatogenesis.

Reversal of mitochondrial proteomic loss in Type 1 diabetic heart with overexpression of phospholipid hydroperoxide glutathione peroxidase

“…1B) consistent with reports suggesting that mPHGPx exists primarily at contact sites between the OMM and IMM (21,23). Previous data from our laboratory has indicated increased phospholipid hydroperoxide and hydrogen peroxide scavenging in mPHGPx transgenic mice (15).…”

Chromatoid Bodies: Aggresome-like Characteristics and Degradation Sites for Organelles of Spermiogenic Cells

“…As the differentiation of spermatids progressed, the labeling intensity in the CBs decreased (data not shown). Heavy signals for PHGPx were found in the CB core ( Figure 5C) as described previously (Haraguchi et al 2003). Their intensity also decreased as the differentiation of spermatids progressed.…”

“…Ubiquitin-specific antibody was purified by affinity chromatography using ubiquitin-coupled Sepharose. Antibodies to lysosomal proteins were described previously: rabbit anti-rat cathepsin H (Yokota and Kato 1987); rabbit anti-cathepsin B, D, and L (Yokota et al 1985(Yokota et al ,1988Yokota and Kato 1987); rabbit anti-PHGPx (Haraguchi et al 2003); rabbit anti-LAMP1 (Akasaki et al 1990); and rabbit anti-LAP antibodies (Haraguchi and Yokota 2002). Rabbit anti-LDH was a gift from Dr. Ohsumi (Department of Life Science, Himeji Institute of Technology, Hyogo, Japan).…”

Intermitochondrial cement (nuage) in a spermatocytic seminoma: comparison with classical seminoma and normal testis

“…Vasa protein, a class of proteins believed to act as RNA chaperones [55], has been detected in the nuage of animals ranging from nematodes to vertebrates [43]. More recently, tudor-related proteins [5,6,25] and a moonlighting protein, phospholipid hydroperoxide glutathione peroxidase [22], have been described in the germline nuage.…”