The distribution and metabolism of creatine in the rat testis: In vivo and in vitro effects of testicular toxicants

Early work in this laboratory has shown that after testicular damage is induced in the rat there is an increase in the excretion of creatine in the urine. The aim of this project was to examine the distribution and metabolism of creatine within the rat testis, and to assess urinary creatine excretion as an index of testicular damage. Groups of rats were administered single doses of various cell-specific testicular toxicants: a germ cell toxicant (methoxyacetic acid, MAA), one of two Sertoli cell toxicants (di-n-pentyl phthalate, DPP; 1,3-dinitrobenzene, 1,3-DNB), or a Leydig cell toxicant (ethane-1,2-dimethane sulphonate, EDS). Urinary creatine and creatinine levels were monitored over the following 48 h, after which time the testes were removed, weighed and, after processing, sections were examined by light microscopy. All four treatments resulted in reductions in relative testis weight and produced morphological changes similar to those which have been widely reported in the literature. In addition, MAA, DPP and 1,3-DNB all caused large, transient elevations in urinary creatine excretion and the urinary creatine:creatinine ratio within 24 h. EDS had no such effect. It is inferred from these observations that creatine is associated with the seminiferous epithelium, and may represent a marker for damage to these cells in vivo. The distribution of creatine within the seminiferous epithelium was examined by the use of Sertoli and germ cells isolated from the testes of 4 week old rats. Both cell populations were found to contain creatine and N-phosphorylcreatine (PCr) pools, and to express creatine phosphoryltransferase (CPT) activity. Isolated seminiferous tubules synthesised creatine, and its biosynthetic precursor, guanidinoacetic acid (GAA), from L-arginine as determined by radio-metabolism studies using L-[guanidino-14C]arginine. A crude interstitial cell preparation synthesised neither creatine nor GAA. Cultured Sertoli cells incorporated radioactivity from both L-[guanidino-14C]arginine and [1-14C]glycine into creatine and GAA. Therefore, Sertoli cells have the capacity to carry out both stages of creatine synthesis; transamidination between arginine and glycine, with subsequent methylation of GAA to creatine. Germ cells did not exhibit this activity. Cultured Sertoli cells, maintained in a defined medium, secreted creatine into the overlying incubation medium, in a manner which was linear with time over at least 6 h, but which reached a plateau within 24 h. The secretion of creatine over 24 h was stimulated both by physiological and toxicological modulators of Sertoli cell function. Stimulation of creatine secretion by follicle-stimulating hormone (FSH) and N6,O-2'-dibutyl adenosine 3',5'-cyclic monophosphate (dbcAMP) was enhanced by the inhibition of adenosine 3',5'-cyclic monophosphate phosphodiesterase (cAMP PDE), indicating that creatine secretion is under the influence of intracellular cAMP levels. The secretion of creatine by cultured Sertoli cells, incubated over 4 h in BBSS, was independent of exogenous L-glutamine. However, the dbcAMP-induced stimulation of creatine secretion was dependent upon the presence of L-glutamine in the incubation medium, suggesting that increases in creatine secretion may occur as a consequence of stimulated glutamine oxidation. Isolated germ cells sequestered [1-14C]creatine in a time-dependent manner, which was linear over at least 3 h, by a two component process. One component had a high affinity for creatine (Km = 25 μM), was inhibited both at low temperature and by the absence of sodium ions from the medium, and probably represents an active uptake system. The other component, which had a low affinity for creatine and was independent of temperature and sodium, probably represents passive diffusion. It is concluded that an intercellular pathway for creatine metabolism may exist within the seminiferous epithelium of the rat, by which creatine is synthesised and secreted by the Sertoli cells and is taken up by the germ cells from the interstitial milieu. Creatine is phosphorylated within both the Sertoli and germ cells, but its role is as yet unclear