title: Immunocytochemical detection of mitochondrial dihydroorotate dehydrogenase in human spermatozoa.
abstract: In mammalian cells the requirement for pyrimidines is met by uridine phosphate (UMP) de novo synthesis and, to a greater or lesser extent, by salvage of free nucleosides. The fourth enzyme of the de novo synthesis, the mitochondrially bound dihydroorotate dehydrogenase (DHODH) was the focus of the present study. Rabbit anti-DHODH IgG, which was generated using an immunization protocol with truncated recombinant human DHODH protein and purified by an immunosorbent method, was used for immunocytochemical detection and localization of this enzyme in ejaculated human spermatozoa. The presence of DHODH protein was demonstrated by Western blotting of solubilized membrane fractions with peroxidase conjugated anti-rabbit IgG in combination with chemiluminescence detection. Indirect immunofluorescence microscopy, using Cy3-conjugated anti-rabbit IgG, revealed specific binding in the midpiece of spermatozoa. As these cells no longer have a demand for de novo biosynthesis of pyrimidines, we hypothesize that the pathway could serve a specialized function in nitrogen or zinc metabolism during the process of spermiogenesis and/or epididymal maturation.
authors.affiliation: Institute for Physiological Chemistry, School of Medicine, Philipps-University, Marburg, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Dietz
authors.forename: E
authors.initials: E
authors.lastname: Hinsch
authors.forename: M
authors.initials: M
authors.lastname: Löffler
mesh_terms: D000818:Animals; D000090722:Dihydroorotate Dehydrogenase; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008928:Mitochondria / Q000201:enzymology*; D010088:Oxidoreductases / Q000032:analysis*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011817:Rabbits; D013094:Spermatozoa / Q000201:enzymology*; D014407:Tumor Cells, Cultured; D020298:U937 Cells
chemical_list: D000090722:Dihydroorotate Dehydrogenase; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors