Isolated rat seminiferous tubules synthesize guanidoacetic acid and creatine from arginine

Creatine is synthesized in a two-stage process, involving transfer of the amidine group of arginine to glycine to form guanidoacetic acid, which is subsequently methylated to creatine. The first report of testicular creatine synthesis was made by Alekseeva & Arkhangel'skaya (1964) based upon experiments in which whole testicular tissue was incubated in vitro with [ 14C]glycine. These findings were corroborated by studies in vivo in which [ I4C]arginine or [14C]guanidoacetate was injected directly into the testes of anaesthetized rats (Koszalka, 1968). However, in these studies no consideration was given to the localization of creatine synthesis within the organ. Using cell-specific testicular toxins and monitoring urinary creatine levels, we have shown that testicular creatine in the rat is associated with the seminiferous epithelium, and with the germ cells in particulr (Rawcliffe et al., 1988; N. P. Moore, D. M. Creasy, T. J. B. Gray & J. A. Timbrell, unpublished work). This led us to consider that creatine metabolism may play a role in the normal function of the seminiferous epithelium. The seminiferous tubules are highly metabolically active structures (e.g. Grootegoed er d., 1985) and the purpose of this investigation was to determine whether they synthesize creatine, as a preliminary to the study of interand intracellular creatine metabolism. The testes from 1617-week-old Hsd/Ola: SpragueDawley rats (Olac) were decapsulated and the seminiferous tubules were separated from the interstitial tissue by collagenase digestion essentially as described by Jutte et al. ( 1982). Tubules, or the unpurified interstitial cell suspension, were incubated for 3 h at 32°C in a shaking water bath and in a 5% C02:95% 0, atmosphere. The incubation medium was Hanks balanced salt solution, supplemented with glycine (0.1 5 mg/ml) and L-methionine (0.298 mg/ml), and containing L-[guanido-"Clarginine (0.1 ,uCi/ml, 54.4 mCi/mmol; Amersham (U.K.). At the end of the incubation, the tissue was homogenized by sonication (MSE Soniprep 1 SO), boiled for 5 min and centrifuged at 2000 g for 10 min. A 5 0 pI aliquot of the supernatant was applied to a C , reverse-phase h.p.1.c. column (MCH10 ODs, Varian) fitted with a guard column, and maintained at ambient temperature. The mobile phase was an aqueous solution of Na2S04 (10 mM), H2S0, ( 5 mM) and sodium 1-hexanesulphonate ( 5 mM). Arginine and its metabolites were separated by a flow program [flow rate = 1 ml/min for 8 min, increased to 2 ml/min (duration 1 min) for 4 min, and increased to 4 ml/min (duration 2 min) for a further 16 min]. Fractions were collected every 0.4 min and the radioactivity was determined by liquid scintillation counting. A typical h.p.1.c. profile, obtained from the incubation of isolated seminiferous tubules with guanido-radiolabelled Larginine as described above, is shown in Fig. 1. The major peak has a retention time of 11.6 min and is due to unmetabolized arginine; the two earlier eluting peaks ( I and 11) have retention times of 6.3 and 7.2 min and co-chromatograph with standards of guanidoacetate and creatine, respectively. Under the conditions described, seminiferous tubules extensively incorporated the amidine group of arginine into both guanidoacetate and creatine, accounting for Arginine