Since the 1950s, Leydig cells have been identified as the androgen source in vertebrate testes, and, thereafter, in the late 1960s and early 1970s, their endocrine activity was shown to be strictly dependent on the pituitary LH . In the last decades, however, new technical advances have made it possible to delve deeper into the intricate mechanisms underlying the regulation of steroidogenesis, particularly in mammalian testes. These studies have soon shown that a myriad of intratesticular agents present in Leydig cells can alter testosterone production by acting as autocrine , paracrine [3, 4], and endocrine [5, 6] excitatory or inhibitory factors.
In recent years, several studies in this burgeoning filed of research have attributed a great importance to the regulating properties of two molecules, viz, the amino acid D-aspartic acid (D-Asp) and the free radical Nitrogen Oxide (NO) in the reproductive systems of mammals [27, 28].
However, the putative role of D-Asp and NO in testosterone production in the male gonads of some seasonal breeder vertebrates also offers a good basic unmanipulated model to improve our understanding of reproductive biology. In these animals, the gonads are active only for a relatively short period each year, specifically in the spring. During this season, sex hormone plasma concentrations peak, gametes are produced and the secondary sexual characters (SSC) completely develop. By contrast, sex hormone production is very low throughout the rest of the year.
In the present study we report for the first time the involvement of D-Asp and NO in the testis steroidogenesis of the mallard A. platyrhynchos, a typical seasonal breeder. Such hypothesis was verified by assaying testicular testosterone production in relation to the presence and distribution of D-Asp and NO during the reproductive period (RP) and the non reproductive period (NRP). Moreover, to better evaluate the role of exogenous D-Asp and NO in testicular testosterone synthesis, in vitro experiments were carried out.
Immunocytological techniques have allowed scientists to localize D-Asp in Leydig and Sertoli cells , as well as in peritubular and several germinal epithelium elements . A strong correlation between D-Asp content and testosterone synthesis has been found in the testes of rats [7–12], boar , amphibians , and reptiles , where, indeed, D-Asp stimulates testosterone synthesis and release. In Leydig cells, D-Asp appears to act on genes since it induces the synthesis of the regulatory protein StAr involved in the synthesis of testosterone . Consistently, in the present study we found that free D-Asp was endogenously contained in the mallard testis and varied throughout the two sexual reproductive cycles, ie the reproductive period (RP) and non reproductive period (NRP). D-Asp concentration was higher during the RP (23.9 ± 2.9 nmol/g tissue) than during the NRP (11.0 ± 1.2 nmol/g tissue). Such discrepancy was also evident in IHC studies, in which, although no difference in the number of Leydig cells was found between the two phases of the reproductive cycle considered, a significant increase in D-Asp immunopositive Leydig cells was detected during the RP but not during the NRP.
Circumstantial evidence suggests that Nitric Oxide (NO) is normally synthesized in the mammalian testis. The enzyme nitric oxide synthase (NOS), which determines NO formation in tissues through the oxidation of L-arginine, has indeed been localized in several testicular components, including Leydig and Sertoli cells [13, 16, 30–33]. Intriguingly, our immunohistochemical observations only in part support such theories, for we found that NO was mostly localized in Leydig cells. Intriguingly, here we report for the first time that NO is far more expressed during the NRP than during the RP. Indeed, our morphometric analysis revealed a more intense NO-immunopositivity during the NRP, as compared to the RP. Moreover, NO concentration resulted inversely correlated to either D-Asp or testosterone in the two periods of the reproductive cycle. In fact, the highest NO concentrations were registered in the testes during the NRP (84.7 ± 11.8 nmol/g tissue). Interestingly, this increase was paralleled by minimum testosterone levels (0.87 ± 0.2 ng/g tissue) and by the regression of the testes. Thus considering that during this period the animal undergoes postnuptial molt , we could advance the hypothesis that highest levels of NO are somehow related to molt. Indeed, a recent study done on Gallus domesticus has emphasized that NO production increases after molt induction . Nonetheless, further studies are still required to validate this hypothesis in mallards.
In current literature, various functions have been ascribed to NO in the testis, such as the stimulation of germ cell metabolism/apoptosis, the relaxation of vascular myocytes, the regulation of peristalsis and permeability of the seminiferous tubule , the inhibition of sperm motility, the induction of peritubular myofibroblast contraction [14, 31, 35–39], and, lastly, the significant depression of testosterone synthesis in Leydig cells. The latter mechanism, however, is still poorly defined. So far, it has been speculated that NO could inhibit Leydig cell testosterone synthesis either directly or indirectly. More specifically, when NO is released within the testes by either Leydig cells or by intraorganic nerve fibres , it could activate soluble guanylyl cyclase (sGC) and, consequently, cyclic guanosin monophosphate (cGMP) [30, 38]. The latter, in turn, could induce the stimulation of a yet poorly defined cytoplasmic protein kinase . It is worthy of note that the sGC/cGMP pathway of cellular response to NO has been proposed not only for the Leydig cell but also for other testicular cytotypes including Sertoli, endothelial, peritubular, and germ cells .
Even though the literature on NO underscores its direct or indirect role in the regulation of gonadal functions, specific research on avian species is still limited. Only recently, in an interesting study carried out on Japanese quails, it has been suggested that NO plays a positive regulatory role in the physiology of gonadal and adrenal axis and photosexual responses . Remarkably, however, the data that we report in this study, despite appearing inconsistent with current findings, actually do highlight two different aspects of analysis with respect to NO involvement. For instance, although the study on sexually immature Japanese quails pinpoints several factors that can enhance the regulation of hypothalamo-hypophyseal-gonadal and -adrenal axis, it does not mention how NO could possibly regulate steroidogenesis in Leydig cells. These discrepancies could be ascribable to the differences among species, in particular with regard to their status of sexual maturity and/or regulation machinery considered.
Moreover, what makes this study particularly insightful is the evidence that both D-Asp and NO displayed a local and direct action on Leydig cells that was strictly related to the two different phases of the sexual cycle. Indeed, it emerged that the two phases of the reproductive cycle were characterized by different biochemical pathways. More specifically, we observed that during the spring, whereas NO content was relatively low, highest concentrations of testosterone and D-Asp occurred in the testes of sexually active drakes. Conversely, in the testes of non reproductive males, testosterone and D-Asp levels declined markedly, whereas NO potential synthesis was relatively high. These intriguing findings were further corroborated by parallel results obtained from our in vitro experiments. The testosterone release from testis slices of reproductive ducks was stimulated when we added exogenous D-Asp to the incubation medium. By contrast, it was inhibited when we incubated the sections with an NO generating system.
In conclusion, these results provide substantial support to the hypothesis that D-Asp and NO play important regulatory roles in testicular testosterone production. Thus, given their intrinsic capability to balance hormonal production in Leydig cells, they ought to be considered putative opposite regulators of local testosterone synthesis. Equally important, our findings suggest that these factors could also be implicated in Leydig cell endocrine activity, as it occurs in seasonally reproducing vertebrates. Finally, we hope that the results of the present study, in addition to having deepened our understanding of the complex mechanisms involved in the reproductive system of vertebrates, may also make a significant contribution to the already enlightening data on breeding biology.