Molecular mechanisms of leptin action in adult rat testis: potential targets for leptin-induced inhibition of steroidogenesis and pattern of leptin receptor messenger ribonucleic acid expression

Leptin, the product of the ob gene, is a pivotal signal in the regulation o f neuroendocrine function and fertility. Although much of the action of lep tin in the control of the reproductive axis is exerted at the hypothalamic level, some direct effects of leptin on male and female gonads have also be en reported. Indeed, recent evidence demonstrated that leptin is able to in hibit testosterone secretion at the testicular level. However, the molecula r mechanisms behind this effect remain unclear. The focus of this study was twofold: (1) to identify potential targets for leptin-induced inhibition o f steroidogenesis, and (2) to characterize in detail the pattern of express ion and cellular distribution of leptin receptor (Ob-R) mRNA in adult rat t estis. In pursuit of the first goal, slices of testicular tissue from adult rats were incubated with increasing concentrations of recombinant leptin ( 10(-9)-10(-7) M) in the presence of human chorionic gonadotropin (hCG; 10 I U/ml). In this setting. testosterone secretion in vitro was monitored, and expression levels of mRNAs encoding steroidogenic factor 1 (SF-1), steroido genic acute regulatory protein (StAR), cytochrome P450 cholesterol sidechai n cleavage enzyme (P450 scc) and 17 beta -hydroxysteroid dehydrogenase type III (17 beta -HSD) were assessed by Northern hybridization, In pursuit of the second goal, the pattern of cellular expression of the Ob-R gene in adu lt rat testis was evaluated by in situ hybridization using a riboprobe comp lementary to all Ob-R isoforms. In addition, testicular expression levels o f the different Ob-R isoforms, previously identified in the hypothalamus, w ere analyzed by means of semi-quantitative PT-PCR. In keeping with our prev ious data, recombinant leptin significantly inhibited hCG-stimulated testos terone secretion. In this context, leptin, in a dose-dependent manner, was able to co-ordinately decrease the hCG-stimulated expression levels of SF-1 , StAR and P450 scc mRNAs, but it did not affect those of 17 beta -HSD type III. In situ hybridization analysis showed a scattered pattern of cellular expression of the Ob-P, gene within the adult rat testis, including Leydig and Sertoli cells. In addition, assessment of the pattern of expression of Ob-R subtypes revealed that the long Ob-Rb isoform was abundantly expresse d in adult rat testis. However, variable levels of expression of Ob-R,a, Ob -Re, and Ob-Rf mRNAs were also detected, whereas those of the Ob-P-c varian t were nearly negligible. In conclusion, our results indicate that decrease d expression of mRNAs encoding several up-stream elements in the steroidoge nic pathway may contribute, at least partially, to leptin-induced inhibitio n of testicular steroidogenesis. In addition, our data on the pattern of te sticular expression of Ob-R isoforms and cellular distribution of Ob-R mRNA may help to further elucidate the molecular mechanisms of leptin action in rat testis.