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Rep 417 Carrey Reproduction (2002) 123, 757–768 Research Detection and location of the enzymes of de novo pyrimidine biosynthesis in mammalian spermatozoa E. A. Carrey1*, C. Dietz2, D. M. Glubb1, M. Löffler2, J. M. Lucocq3 and P. F. Watson4 1Department of Biochemistry and Molecular Biology, Royal Free and University College Medical School, Royal Free Campus, London NW3 2PF, UK; 2Institut für Physiologische Chemie, Philipps Universität Marburg, D-35033 Marburg, Germany; 3School of Life Sciences, WTB/MSI Complex, University of Dundee, Dundee DD1 5EH, UK; and 4Department of Veterinary Basic Sciences, Royal Veterinary College, London NW1 0TU, UK Enzymes of the pathway for de novo biosynthesis of respectively, were used, together with an affinity-purified pyrimidine nucleotides have been reported in spermato- antibody raised against dihydroorotate dehydrogenase zoa from fruitfly and mammals. The aim of the present (DHODH), the mitochondrial enzyme for step 4. Western study was to test the hypothesis that the enzymes for bio- blot analysis, immunofluorescent microscopy and immuno- synthesis of uridine monophosphate (UMP) are concen- electron microscopy confirmed that CAD and UMP trated near the mitochondria, which are segregated in the synthase are found in the cytoplasm around and outside mid-piece of spermatozoa. Baby hamster kidney fibro- the mitochondria; DHODH is found exclusively inside the blasts were compared with spermatozoa from rams, boars, mitochondria. CAD was also located in the nucleus, where bulls and men. Antibodies raised against synthetic peptides it has been reported in the nuclear matrix, and in the from sequences of the multienzyme polypeptides contain- cytoplasm, apparently associated with the cytoskeleton. It ing glutamine-dependent carbamyl phosphate synthetase, is possible that CAD in the cytoplasm has a role uncon- aspartate transcarbamylase and dihydroorotase (CAD) and nected with pyrimidine biosynthesis. UMP synthase, which catalyse reactions 1–3 and 5–6, uridine monophosphate synthase (UMPS; steps 5 and 6; Introduction comprising orotate phosphoribosyltransferase and orotidine All organisms have the capacity to synthesize nucleotides 5Ј monophosphate (OMP) decarboxylase), respectively. de novo and to salvage or recycle the components (bases or Drosophila strains that are mutant in any of these genes nucleosides); however, the balance between biosynthesis have defective wings, and the females are sterile because and recycling may differ in distinct types of cell and at they cannot provide the mRNA for the pyrimidine- crucial stages of development. In general, de novo bio- biosynthetic enzyme in the embryos of their offspring. How- synthesis is important in rapidly dividing cells in which ever, the immature and adult mutant fruitflies survive by there is an obvious requirement for new nucleic acids, synthesizing pyrimidine nucleotides from uracil and uridine and for uridine diphosphate (UDP)-sugars and cytidine obtained from the diet and from recycling, respectively diphosphate (CDP)-lipids that are used to make other (Nørby, 1970; Conner and Rawls, 1982). components of cells. Porter et al. (1995) and Yang et al. (1995) reported that The de novo pathway for the biosynthesis of the the r and dhod genes are transcribed abundantly (dhod from pyrimidine nucleotide uridine monophosphate (UMP) in all a special male-limited transcriptional start) during sperma- higher organisms involves six steps that in turn are found tocyte development in Drosophila. However, the proteins in only three polypeptides. The genetics of the pathway are not translated from mRNA until several days later, have been studied extensively in the fruitfly, Drosophila during spermatid differentiation, and thus DHODH and melanogaster. The proteins encoded by the Drosophila CAD are present in mature Drosophila spermatozoa. In genes r, dhod and r-l are glutamine-dependent carbamyl contrast, the r-l gene is transcribed for a much shorter phosphate synthetase, aspartate transcarbamylase and period, only during gonial cell divisions, and the UMPS dihydroorotase (CAD; catalysing steps 1–3 inclusive), protein is not detected in spermatids or spermatozoa. Thus, dihydroorotate dehydrogenase (DHODH; step 4) and only a truncated pathway for UMP synthesis is found in Drosophila sperm cells. *Correspondence address: Purine Research Unit, 5th Floor, Thomas DHODH protein has been detected in the mitochondria Guy House, Guy’s Hospital, London Bridge, London SE1 9RT, UK of ejaculated mature bull and human spermatozoa (Dietz Email: [email protected] et al., 2000) using immunocytochemistry, immunoblot © 2002 Society for Reproduction and Fertility 1470-1626/2002 Downloaded from Bioscientifica.com at 09/28/2021 12:53:21AM via free access 758 E. A. Carrey et al. attach to or become integrated in the membrane, because neither protein has a mitochondrial targeting sequence (Suttle et al., 1988; Guy and Evans, 1994) as has been shown for DHODH (Rawls et al., 2000). ATP is required for the biosynthesis of carbamyl phosphate and for the synthesis of 5-phosphoribosyl 1-pyrophosphate (PRibPP) DHODH for step 5, and would be conveniently provided from the 2e– mitochondrion. The mitochondrion may also be a source of bicarbonate ion for the synthesis of carbamyl phosphate. If PRibPP CAD and UMPS molecules are found near the mitochon- Orotate drion, small metabolites can be dedicated (channelled) to Dihydroorotate use in the biosynthetic pathway without leaching out into solution. Carbamyl If CAD were present near the mitochondria of sper- OMP aspartate + PPi matozoa, it would provide dihydroorotate as a substrate for DHODH to convert to orotate, also generating electrons that can be transferred via ubiquinone to the respiratory Carbamyl phosphate UMP electron transfer chain. Porter et al. (1995) suggested that (+ aspartate) + CO2 these electrons might have a downstream redox effect that is important in the motility of spermatozoa. Dietz et al. (2000) proposed that the production of orotate might play a role in NH (+ bicarbonate + 2ATP) 3 UMP synthase the maturation of spermatozoa, in sustaining morphology or in modulating motility, whereas the electrons may repay Glutamine some of the energy cost for biosynthesis. The presence of UMPS in spermatozoa would complete the biosynthetic pathway and allow the further use of orotate for synthesis of UMP; however, as there is no DNA or RNA synthesis in CAD mature spermatozoa, there is no apparent role for newly synthesized nucleotides. In this study, immunoblot analysis and immunocyto- Fig. 1. The de novo biosynthetic pathway for the pyrimidine chemistry were used to determine whether mammalian nucleotide uridine monophosphate (UMP) in mammals. Most of spermatozoa contain both CAD and UMPS. Mammalian the biosynthetic pathway is catalysed in the cytoplasm and is linked to the respiratory chain through the fourth enzyme, fibroblasts, in which enzymes for all six steps are present, ubiquinone-dependent dihydroorotate dehydrogenase (DHODH) were used to confirm methods of detection. in the inner mitochondrial membrane. The multi-enzyme poly- peptide glutamine-dependent carbamyl phosphate synthetase, aspartate transcarbamylase and dihydroorotase (CAD) contains the Materials and Methods enzyme activities for reactions 1–3. Reaction 1 includes the donation of ammonia from the obligate substrate glutamine (step Antibody production 1a) and the synthesis of carbamyl phosphate using two molecules Antibodies were generated against synthetic peptides of ATP (step 1b). The bifunctional polypeptide UMP synthase corresponding to the two sites in the multi-enzyme poly- contains the enzyme activities for steps 5 and 6: orotate phos- peptide CAD that are known to be phosphorylated by protein phoribosyltransferase and orotidine 5Ј monophosphate decarbo- kinase A. Peptides S1 and S2 correspond to the sequences xylase. PRibPP: 5-phosphoribosyl 1-pyrophosphate. of phosphorylation sites 1 and 2, respectively (Carrey and Hardie, 1988). These peptides, each of 16–18 amino acids, analysis of extracted proteins and specific enzyme activity, were synthesized by Bristol Peptides (G. Bloomberg, and by detecting the activity of the enzyme in spermatozoa Department of Biochemistry, School of Medical Sciences, in the seminiferous tubules (Löffler et al., 1996). The finding University of Bristol, Bristol) and inoculated into sheep that DHODH is located in the mitochondrial inner mem- (Fig. 2). The antibodies were separated by affinity chro- brane in fibroblasts (Chen and Jones, 1976; Rawls et al., matography on columns to which each peptide had been 2000) led us to compare UMP synthesis with the urea cycle, bound separately (Harlow and Lane, 1988). The sera in which channelling between the enzymes in the cyto- and antibody preparations are referred to as ‘anti-S1’ and plasm and mitochondria has already been demonstrated ‘anti-S2’. (Cheung et al., 1989; Watford, 1991) (Fig. 1). Because CAD A similar procedure was adopted using peptides corre- and UMPS are obtained in the cytosolic fraction when sponding to sequences in the other cytoplasmic enzyme prepared from cell extracts, they would be expected to activities of pyrimidine biosynthesis. A sequence from the cluster on the outer surface of mitochondria rather than aspartate transcarbamylase (ATCase) domain in CAD was Downloaded from Bioscientifica.com at 09/28/2021 12:53:21AM via free access Enzymes of pyrimidine biosynthesis in mammalian spermatozoa 759 Name Sequence Identity of epitope S1 CAD residues 1398–1414 (PKA site 1) S2
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