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Differential Splicing of the Phospholipid Hydroperoxide Glutathione Peroxidase Gene in Diploid and Haploid Male Germ Cells in the Rat¹

^a Department of Histology and Medical Embryology, University of Rome "La Sapienza," 00161 Rome, Italy ^b Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, 34127 Trieste, Italy

Phospholipid hydroperoxide glutathione peroxidase (PHGPx, 20 kDa) and sperm nuclei glutathione peroxidase (snGPx, 34 kDa) are two selenoproteins present in mammalian testis and epididymal spermatozoa. They originate from the differential splicing of the PHGPx gene and appear to play important roles in sperm physiology. To determine the stages of spermatogenesis in which they are present, we compared the expression pattern of these two enzymes in highly purified populations of germ cells during specific phases of differentiation. In Northern and Western blotting experiments, both PHGPx transcript and protein were markedly expressed in pachytene spermatocytes and round spermatids. In contrast, the testis-specific snGPx was detected at both the mRNA and protein level only in haploid round spermatids. Accordingly, the developmental analysis of testicular RNAs from rats of different ages first revealed the appearance of PHGPx and snGPx transcripts at Day 20 and Day 30, respectively. Furthermore, both meiotic and postmeiotic cells contained catalytically active PHGPx/snGPx, with higher activity in the haploid cells. The intracellular distribution of PHGPx in mitochondria and nuclei of meiotic cells was demonstrated by immunocytochemical electron microscopy and Western blotting. These findings provide evidence that the PHGPx gene is differentially spliced during the meiotic prophase and haploid cell phases of spermatogenesis.

¹ This work was supported by the following grants: MIUR cofin 1999 and 2001 to C.B. and E.P. and FVG Region to E.P.

² Correspondence: Carla Boitani, Department of Histology and Medical Embryology, University of Rome "La Sapienza," Via Scarpa 14, 00161 Rome, Italy. FAX: 39 06 4462854; carla.boitani{at}uniroma1.it

³ These authors contributed equally to this study