Testis-specific expression of the nuclear form of phospholipid hydroperoxide glutathione peroxidase (PHGPx).
The selenoprotein phospholipid hydroperoxide glutathione peroxidase (PHGPx) is present in at least three different isoforms in testis: as a cytosolic, as a mitochondrial, and as a nuclear protein. We have recently shown that a sperm nucleus-specific glutathione peroxidase (snGPx) is identical to the mitochondrial and cytosolic forms of PHGPx apart from its N-terminus. This arginine-rich N-terminus of snGPx, reminiscent of protamines, is encoded by an alternative exon located in the first intron of the PHGPx gene and is responsible for nuclear localisation and chromatin binding of snGPx [Pfeifer et al., FASEB J. 15 (2001), pp. 1236-1238]. By using a combination of techniques including selective cloning of mRNA 5'-ends, RT-PCR, and S1 analyses, we provide evidence that the transcript encoding the nuclear form is generated by transcription initiation at an alternative promoter and not by alternative splicing. We show that the major transcription start region is located at -12 to -14 upstream of the AUG translation initiation site of the sperm nucleus-specific exon and lacks a TATA box. Two minor TATA-less transcription initiation sites are located at around -30 and -45. We have shown by in situ hybridisation that snGPx expression in testis, like protamine expression, is restricted to late stages of spermatogenesis whereas PHGPx expression is only found in spermatocytes and early spermatids. These findings have to be taken into account when studying either the differential regulation of PHGPx and snGPx expression in testis or the impact of putative mutations in snGPx on male fertility in man.
Institute of Clinical Molecular Biology and Tumor Genetics, GSF Research Centre for Environment and Health, Marchioninistr. 25, D-81377 Munich, Germany.
Stéphanie G
SG
Moreno
Gerhard
G
Laux
Markus
M
Brielmeier
Georg W
GW
Bornkamm
Marcus
M
Conrad
D020413:3' Untranslated Regions / Q000235:genetics; D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism*; D004076:Digoxigenin; D004847:Epithelial Cells / Q000378:metabolism; D005979:Glutathione Peroxidase / Q000096:biosynthesis* / Q000235:genetics*; D017403:In Situ Hybridization; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009928:Organ Specificity; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015719:Single-Strand Specific DNA and RNA Endonucleases; D013737:Testis / Q000201:enzymology*
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D012333:RNA, Messenger; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D005979:Glutathione Peroxidase; D015719:Single-Strand Specific DNA and RNA Endonucleases; D004076:Digoxigenin