Maria Maddalena Di Fiore

Secretion of D-aspartic acid by the rat testis and its role in endocrinology of the testis and spermatogenesis

The D‐isomer of aspartic acid (D‐Asp) has been found in rat testes. In the present study, samples of testicular venous blood plasma, rete testis fluid, interstitial extracellular fluid, luminal fluid from the seminiferous tubules, testicular parenchymal cells, epididymal spermatozoa and peripheral blood plasma were collected and analyzed for D‐Asp by two methods, an enzymatic and a chromatographic HPLC method. The two methods gave very similar results for all samples. The highest concentrations of D‐Asp (about 120 nmol/ml) were found in testicular venous blood plasma, with slightly lower concentrations in rete testis fluid (95 nmol/ml) and epididymal spermatozoa (80 nmol/g wet weight). Lower levels were found in testicular parenchymal cells (which would comprise mostly spermatids and spermatocytes), luminal fluid from the seminiferous tubules and interstitial extracellular fluid (26, 23 and 11 nmol/ml respectively). However, these values were all higher than those for peripheral blood plasma (6 nmol/ml). It would appear that D‐Asp is being secreted by the testis mostly into the venous blood, passing thence into the rete testis fluid and being incorporated into the spermatozoa at the time or after they leave the testis. The distribution of D‐Asp is thus quite different from that of testosterone, and its role and the reason for its high concentration in the male reproductive tract remain to be elucidated.

Regional decreases of free d-aspartate levels in Alzheimer's disease

N-methyl-D-aspartate (NMDA) receptors have been shown to be involved in learning and memory processes. In Alzheimers disease, there is a reduction of NMDA receptors. Since D-aspartate is an endogenous agonist for the NMDA receptor, we hypothesised that if there are reduced levels of this amino acid in the Alzheimers brain, this could raise the reduction of NMDA receptor signal transduction system and contribute to the marked memory deficits seen in these patients. Therefore, using a chromatographic HPLC method, the regional distribution of free D-aspartate levels in post-mortem human brain samples from patients with Alzheimers disease (AD) (n = 5) and age-matched controls (n = 5) were determined. We found that the levels of D-aspartate are significantly lower in Alzheimers patients compared to controls (range: from -35 to -47%; P < 0.01). However, no differences were found in the cerebellum, a region spared from the neuropathological changes of AD. These data suggest that decreased levels of D-aspartate could contribute to a lower NMDA receptor function and consequently contribute to the memory deficits seen in AD.

Effects of short postcapture confinement on plasma reproductive hormone and corticosterone profiles in Rana esculenta during the sexual cycle

Summary The effects of short‐term confinement (24 hours) on the plasma levels of gonadal hormones and corticosterone have been evaluated in Rana esculenta, which is widely used in amphibian reproductive physiology studies. A 24‐h confinement has been chosen since this is the time that usually elapses from frog capture to utilization. Although short confinement does not influence the morphological aspect of gonads, genital tracts (oviduct in the female) and secondary sexual characters (thumbpads in the male), it causes a significant decrease of testosterone in both sexes, and of progesterone and estradiol in the female only. The confinement effects vary depending on the sexual cycle phase, but plasma hormonal patterns during the year are, on the whole, maintained. Plasma hormonal drop in captive frogs is coupled with an increase of circulating corticosterone, the administration of which strengthens the captivity effects. The results were considered to support the fact that, like in other vertebrates, plasm...

Occurrence and neuroendocrine role of D-aspartic acid and N-methyl-D-aspartic acid in Ciona intestinalis

Probes for the occurrence of endogenous D‐aspartic acid (D‐Asp) and N‐methyl‐D‐aspartic acid (NMDA) in the neural complex and gonads of a protochordate, the ascidian Ciona intestinalis, have confirmed the presence of these two excitatory amino acids and their involvement in hormonal activity. A hormonal pathway similar to that which occurs in vertebrates has been discovered. In the cerebral ganglion D‐Asp is synthesized from L‐Asp by an aspartate racemase. Then, D‐Asp is transferred through the blood stream into the neural gland where it gives rise to NMDA by means of an NMDA synthase. NMDA, in turn, passes from the neuronal gland into the gonads where it induces the synthesis and release of a gonadotropin‐releasing hormone (GnRH). The GnRH in turn modulates the release and synthesis of testosterone and progesterone in the gonads, which are implicated in reproduction.

Proliferative activity in the frog brain: A PCNA-immunohistochemistry analysis

By means proliferating cell nuclear antigen (PCNA) immunohistochemistry, we have provided a detailed neuroanatomical mapping of proliferative activity during development and adulthood in the frog (Rana esculenta) brain. Western blot analysis confirmed the presence of this protein in brain extracts from adults and tadpoles. Proliferative activity was observed in the ventricular and subventricular zones throughout the brain. The present study provides details as to which of the morphologically distinguishable brain region(s) has a long-lasting proliferative activity and in which region this activity undergoes a progressive decrease during development. In the subventricular zones of the third ventricle, PCNA-labeled cells were particularly abundant in the magnocellular preoptic nucleus and the ventromedial thalamic nucleus. It was observed that proliferation zones are present practically in all major subdivisions of the forebrain, midbrain and hindbrain, including the cerebellum in which PCNA-labeled cells were located in the outer granular layer and the inner molecular layer. The habenulae, epiphysis and isthmic nuclei never showed the presence of PCNA-immunoreactive nuclei. The widespread proliferative activity implies that the frog brain has a great potential for neurogenesis/gliogenesis not only during larval development but also in the adulthood.

Current knowledge of d-aspartate in glandular tissues

Free d-aspartate (d-Asp) occurs in substantial amounts in glandular tissues. This paper reviews the existing work on d-Asp in vertebrate exocrine and endocrine glands, with emphasis on functional roles. Endogenous d-Asp was detected in salivary glands. High d-Asp levels in the parotid gland during development suggest an involvement of the amino acid in the regulation of early developmental phases and/or differentiation processes. d-Asp has a prominent role in the Harderian gland, where it elicits exocrine secretion through activation of the ERK1/2 pathway. Interestingly, the increase in NOS activity associated with d-Asp administration in the Harderian gland suggests a potential capability of d-Asp to induce vasodilatation. In mammals, an increase in local concentrations of d-Asp facilitates the secretion of anterior pituitary hormones, i.e., PRL, LH and GH, whereas it inhibits the secretion of POMC/α-MSH from the intermediate pituitary and of oxytocin from the posterior pituitary. d-Asp also acts as a negative regulator for melatonin synthesis in the pineal gland. Further, d-Asp can stereo-specifically modulate the production of sex steroids, thus taking part in the endocrine control of reproductive activity. Although d-Asp receptors remain to be characterized, gene expression of NR1 and NR2 subunits of NMDAr responds to d-Asp in the testis.

Stimulation of androgen production by d-aspartate through the enhancement of StAR, P450scc and 3β-HSD mRNA levels in vivo rat testis and in culture of immature rat Leydig cells

Previous studies have shown a role of d-aspartic acid (d-Asp) in testicular steroidogenesis. Here, we evaluated the effects of d-Asp on androgen production and on expression levels of mRNAs encoding specific steroidogenic key molecules. d-Asp was endogenously present in adult rat testis and its content paralleled to serum luteinizing hormone (LH) and, local and circulating androstenedione and testosterone levels. In vivod-Asp administration induced serum LH release, causing an indirect increase of androstenedione and testosterone levels by enhancing steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase/D5-D4 isomerases (3β-HSD) mRNA levels. The direct endocrine role of d-Asp was evaluated using cultured immature Leydig cells (ILCs) obtained from 35days old rats. Cytoplasm and nucleus of ILCs localized d-Asp, while StAR marked the cytoplasm only. After 12h from d-Asp in vitro administration, ILCs resulted intensely d-Asp stained, and StaR protein level, evaluated by Western blotting, significantly increased. After 24h, significant androstenedione and testosterone syntheses were induced. At molecular level, d-Asp administration significantly increased StAR, P450scc and 3β-HSD mRNAs at 2, 4 and 12h, respectively. The temporal shift on relative mRNA expression levels indicated that d-Asp exerted its physiological role through sequential gene cascade activation of those molecules implicated in the synthesis of androgens. Conclusively, our findings demonstrated that d-Asp is a local messenger in testis and give a contribution in understanding the complexity of local endocrine regulation as well as the molecular events leading the acquisition to a steroidogenic competence by ILCs.

GnRH in the invertebrates: an overview.

Publisher Summary This chapter provides an overview of GnRH in the invertebrates. The gonadotropin-releasing hormone (GnRH), which was previously called luteinizing hormone-releasing hormone (LHRH), represents a pivotal peptide in animal reproduction. GnRH is a decapeptide originally isolated from the porcine and ovine hypothalamus and is characterized for its ability to enhance the release of pituitary gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH). All known forms of GnRH peptides have in common a pyroglutamyl residue at the amino terminus and an amidated glycine at the carboxy terminus. Except for the octoGnRH, all other known forms are decapeptides and differ by one or more amino acids; amino acids 4 and 9 are conserved in all forms known to date. Studies involving an evolutionary approach to GnRH have adopted one particular strategy; attention is on understudied invertebrate taxonomic groups. Invertebrate species that vary in their phylogenetic relationships are likely to reveal differences, as well as similarities, in GnRH form and function. Studies on invertebrate species are beginning to provide further insights on the structural and functional similarities and differences. The perspective that evolution has acted to shape the GnRH molecule effectively leads to investigations in a comparative approach.

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

BackgroundD-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 birds reproductive cycle. Furthermore, in vitro experiments revealed the direct effect of exogenously administered D-Asp and NO on testosterone steroidogenesis.MethodsBy 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.ResultsIHC 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.ConclusionOur 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.

The reproductive activity in the testis of Podarcis s. sicula involves d-aspartic acid: A study on c-kit receptor protein, tyrosine kinase activity and PCNA protein during annual sexual cycle

The current study provides substantial evidence that the pattern of synthesis of D-aspartic acid (D-Asp) in the testes of lizard Podarcis s. sicula throughout the reproductive cycle is in parallel with seasonal variations of testosterone, c-kit receptor protein, tyrosine kinase activity, and proliferating cell nuclear antigen (PCNA) protein. Although the trend is the same in all phases of the sexual cycle, the peaks of these three molecules are detectable only during the reproductive period. Using Western blot technique, we demonstrated that both polyclonal c-kit and PCNA antibodies specifically recognized bands with molecular mass of approximately 150 and approximately 36 kDa, respectively. By immunocytochemical methods, D-Asp immunopositivity appeared spread in the germinal epithelium as well as in the interstitial compartment of the testes. We also found specific c-kit labeling in I and II spermatogonia (SPG), in I and II spermatocytes (SPC), in the elongated spermatides, in spermatozoa, in Sertoli and Leydig cells. Like c-kit, PCNA positivity was located in the germinal epithelium pattern. Furthermore, we investigated the relationship between testosterone, c-kit receptor, tyrosine kinases activity and PCNA following treatment with D-Asp. In vivo experiments, entailing a single injection of D-Asp (2.0 micromol/g body weight), demonstrated that this amino acid significantly accumulated in the testes. After 3 h, its uptake was accompanied by an increase in testosterone levels and in the expression and intensity of immunostaining of c-kit receptor protein. Furthermore, at 6 h, exogenous D-Asp affected the phosphorylation of tyrosine kinases, whose activation was positively correlated with the temporal uptake of both D-Asp and testosterone detected in the testes. Thereafter, between 6 and 15 h, the expression of PCNA was induced and an increase in its immunolabeling intensity was observed. Taken all together, these results provide new insights into the testicular activity during the reproductive cycle of Podarcis s. sicula, suggesting that a sequential cascade of a functional relationship between testosterone levels, c-kit receptor protein, tyrosine kinase activity and PCNA could be partly mediated by D-aspartic acid.