Immunocytochemical detection of mitochondrial dihydroorotate dehydrogenasein human spermatozoa

In mammalian cells the requirement for pyrimidines is met by uridine phosph ate (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 mitoch ondrially bound dihydroorotate dehydrogenase (DHODH) was the focus of the p resent study. Rabbit anti-DHODH IgG, which was generated using an immunizat ion protocol with truncated recombinant human DHODH protein and purified by an immunosorbent method, was used for immunocytochemical detection and loc alization of this enzyme in ejaculated human spermatozoa. The presence of D HODH protein was demonstrated by Western blotting of solubilized membrane f ractions with peroxidase conjugated anti-rabbit IgG in combination with che miluminescence detection. Indirect immunofluorescence microscopy, using Cy3 -conjugated anti-rabbit IgG, revealed specific binding in the midpiece of s permatozoa. 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 spermiogenesi s and/or epididymal maturation.