Opposing effects of D-aspartic acid and nitric oxide on tuning of testosterone production in mallard testis during the reproductive cycle.

UNLABELLED: D-Aspartic acid (D-Asp) and nitric oxide (NO) play an important role in tuning testosterone production in the gonads of male vertebrates. In particular, D-Asp promotes either the synthesis or the release of testosterone, whereas NO inhibits it. In this study, we have investigated for the first time in birds the putative effects of D-Asp and NO on testicular testosterone production in relation to two phases of the reproductive cycle of the adult captive wild-strain mallard (Anas platyrhynchos) drake. It is a typical seasonal breeder and its cycle consists of a short reproductive period (RP) in the spring (April-May) and a non reproductive period (NRP) in the summer (July), a time when the gonads are quiescent. The presence and the localization of D-Asp and NO in the testis and the trends of D-Asp, NO and testosterone levels were assessed during the main phases of the bird's reproductive cycle. Furthermore, in vitro experiments revealed the direct effect of exogenously administered D-Asp and NO on testosterone steroidogenesis. METHODS: By using immunohistochemical (IHC) techniques, we studied the presence and the distributional pattern of D-Asp and NO in the testes of RP and NRP drakes. D-Asp levels were evaluated by an enzymatic method, whereas NO content, via nitrite, was assessed using biochemical measurements. Finally, immunoenzymatic techniques determined testicular testosterone levels. RESULTS: IHC analyses revealed the presence of D-Asp and NO in Leydig cells. The distributional pattern of both molecules was in some way correlated to the steroidogenic pathway, which is involved in autocrine testosterone production. Indeed, whereas NO was present only during the NRP, D-Asp was almost exclusively present during the RP. Consistently, the high testosterone testicular content occurring during RP was coupled to a high D-Asp level and a low NO content in the gonad. By contrast, in sexually inactive drakes (NRP), the low testosterone content in the gonad was coupled to a low D-Asp content and to a relatively high NO level. Consequently, to determine the exogenous effects of the two amino acids on testosterone synthesis, we carried out in vitro experiments using testis sections deriving from both the RP and NRP. When testis slices were incubated for 60 or 120 min with D-Asp, testosterone was enhanced, whereas in the presence of L-Arg, a precursor of NO, it was inhibited. CONCLUSION: Our results provide new insights into the involvement of D-Asp and NO in testicular testosterone production in the adult captive wild-strain mallard drake. The localization of these two molecules in the Leydig cells in different periods of the reproductive cycle demonstrates that they play a potential role in regulating local testosterone production.

D-Aspartic acid and nitric oxide as regulators of androgen production in boar testis.

D-Aspartic acid (D-Asp) and nitric oxide (NO) are two biologically active molecules playing important functions as neurotransmitters and neuromodulators of nerve impulse and as regulators of hormone production by endocrine organs. We studied the occurrence of D-Asp and NO as well as their effects on testosterone synthesis in the testis of boar. This model was chosen for our investigations because it contains more Leydig cells than other mammals. Indirect immunofluorescence applied to cryostat sections was used to evaluate the co-localization of D-Asp and of the enzyme nitric oxide synthase (NOS) in the same Leydig cells. D-Asp and NOS often co-existed in the same Leydig cells and were found, separately, in many other testicular cytotypes. D-Asp level was dosed by an enzymatic method performed on boar testis extracts and was 40+/-3.6 nmol/g of fresh tissue. NO measurement was carried out using a biochemical method by NOS activity determination and expressed as quantity of nitrites produced: it was 155.25+/-21.9 nmol/mg of tissue. The effects of the two molecules on steroid hormone production were evaluated by incubating testis homogenates, respectively with or without D-Asp and/or the NO-donor L-arginine (L-Arg). After incubation, the testosterone presence was measured by immunoenzymatic assay (EIA). These in vitro experiments showed that the addition of D-Asp to incubated testicular homogenates significantly increased testosterone concentration, whereas the addition of L-Arg decreased the hormone production. Moreover, the inclusion of L-Arg to an incubation medium of testicular homogenates with added D-Asp, completely inhibited the stimulating effects of this enantiomer. Our results suggest an autocrine action of both D-Asp and NO on the steroidogenetic activity of the Leydig cell.