Immunocytochemical detection of mitochondrial dihydroorotate dehydrogenase in human spermatozoa
| Autor: | Monika Löffler, Carsten Dietz, Elvira Hinsch |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
Male
Oxidoreductases Acting on CH-CH Group Donors Urology Endocrinology Diabetes and Metabolism Dihydroorotate Dehydrogenase Mitochondrion Biology law.invention chemistry.chemical_compound Biosynthesis law Tumor Cells Cultured Animals Humans chemistry.chemical_classification U937 Cells Immunohistochemistry Spermatozoa Molecular biology Uridine Mitochondria Blot De novo synthesis Enzyme Reproductive Medicine chemistry Biochemistry Recombinant DNA Dihydroorotate dehydrogenase Rabbits Oxidoreductases |
| Zdroj: | International Journal of Andrology. 23:294-299 |
| ISSN: | 1365-2605 0105-6263 |
| DOI: | 10.1046/j.1365-2605.2000.00246.x |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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