The role and molecular mechanism of D-aspartic acid in the release and synthesis of LH and testosterone in humans and rats
© Topo et al; licensee BioMed Central Ltd. 2009
Received: 29 August 2009
Accepted: 27 October 2009
Published: 27 October 2009
D-aspartic acid is an amino acid present in neuroendocrine tissues of invertebrates and vertebrates, including rats and humans. Here we investigated the effect of this amino acid on the release of LH and testosterone in the serum of humans and rats. Furthermore, we investigated the role of D-aspartate in the synthesis of LH and testosterone in the pituitary and testes of rats, and the molecular mechanisms by which this amino acid triggers its action.
For humans: A group of 23 men were given a daily dose of D-aspartate (DADAVIT®) for 12 days, whereas another group of 20 men were given a placebo. For rats: A group of 10 rats drank a solution of either 20 mM D-aspartate or a placebo for 12 days. Then LH and testosterone accumulation was determined in the serum and D-aspartate accumulation in tissues. The effects of D-aspartate on the synthesis of LH and testosterone were gauged on isolated rat pituitary and Leydig cells. Tissues were incubated with D-aspartate, and then the concentration (synthesis) of LH and cGMP in the pituitary and of testosterone and cAMP in the Leydig cells was determined.
In humans and rats, sodium D-aspartate induces an enhancement of LH and testosterone release. In the rat pituitary, sodium D-aspartate increases the release and synthesis of LH through the involvement of cGMP as a second messenger, whereas in rat testis Leydig cells, it increases the synthesis and release of testosterone and cAMP is implicated as second messenger. In the pituitary and in testes D-Asp is synthesized by a D-aspartate racemase which convert L-Asp into D-Asp. The pituitary and testes possesses a high capacity to trapping circulating D-Asp from hexogen or endogen sources.
D-aspartic acid is a physiological amino acid occurring principally in the pituitary gland and testes and has a role in the regulation of the release and synthesis of LH and testosterone in humans and rats.
D-Aspartic acid (D-Asp) is an endogenous amino acid which has been found in the neuroendocrine tissues of both invertebrates and vertebrates . D-Asp was first found in the nervous system of marine mollusks  and subsequently in the nervous and endocrine tissues of many other animals, including humans . High levels of D-Asp occur transiently in the brain of chickens, rats and humans during the last stage of embryonic life, suggesting that it has a role in the development of the nervous system [3–6]. In addition, within the nervous system this amino acid is concentrated in the axon terminals (synaptosomes) and in synaptic vesicles together with L-Asp and L-Glu ; additionally, it is involved in visual activity , suggesting it has a role in neurotransmission.
In the endocrine system, high concentrations of D-Asp have been recorded in rat testes at birth as well as following sexual maturity . Further research involving rats showed the highest concentrations of D-Asp in testicular venous blood plasma, as well as in the rete testis, the epididimus, testicular parenchymal cells, seminiferous tubules, interstitial fluid and spermatozoa , suggesting that D-Asp is involved in spermatogenesis. A specific D-Asp localization was further observed in rat testes either in elongate spermatidits  or in Leydig cells . Several further studies have demonstrated that D-Asp is concentrated in the endocrine gland, particularly in the pineal gland, the pituitary and the testis [1, 12]. It has been observed that D-Asp in rats is capable of eliciting the release of the gonadotropin-releasing hormone (GnRH) from the hypothalamus, the luteinizing hormone (LH) and the growth hormone (GH) from the pituitary gland, and testosterone from the testes . In addition, D-Asp occurs in a high concentration in the pineal gland , where it modulates melatonin synthesis in rat pinealocytes , and is implicated in the α-melanocyte-stimulating hormone, GABA, and in dopamine release . In sheep, D-Asp is endogenously present in tissues and is electively stored in endocrine glands, such as the pituitary, and in the brain after its administration. NMDA and LH increased following D-Asp administration . Recently an in vitro study carried out on boar testes revealed that endogenous testicular D-Asp enhances gonad aromatase activity , the key enzyme that converts testosterone into 17β-estradiol. In addition, studies done on the testes and ovary of the lizard Podarcis s. sicula have shown similar findings, confirming that D-Asp is involved in the local production of estrogen [19, 20].
On the basis of the above findings, D-Asp seems to play a crucial role in reproduction either due to its suggested role of neuromodulator or because it is involved in biosynthesis and the release of sexual hormones. Recent studies analyzed the role of D-Asp in human reproduction in both females and males. In men, a lower D-Asp content was found in oligoasthenoteratospermic seminal fluid and spermatozoon, and a relationship exists between the amount and motility of semen and the content of D-Asp . In women, it has been found that D-Asp occurs in the follicular fluid as a physiological component, and interestingly, the concentration of D-Asp in the fluid is reduced in older women. In addition, the concentration of D-Asp in the follicular fluid is lower, as are the quality of the oocytes and the level of fertilization .
Although numerous studies have been conducted on this matter, no investigations have been done until now on the effects of D-Asp on the secretion of LH and testosterone in humans, and neither has the molecular mechanism by which D-Asp triggers it action in the synthesis and release of these hormones been investigated. This study aims to evaluate the effects of D-aspartate administration on LH and testosterone production in humans and rats and to understand the biochemical mechanisms by which D-Asp induces the synthesis and release of LH and testosterone, using rats as the model animal.
Determination of D-Asp by HPLC associate with D-AspO
The determination of D-Asp in serum and tissues was carried out using an HPLC enzymatic method combined with D-aspartate oxidase (D-AspO) according to our previously described method . For the serum, 0.4 ml serum was mixed with 3.6 ml of 0.2 M TCA and centrifuged at 10,000 g for 10 min. The supernatant was purified on cation exchange resin (AG 50WX8). The purified sample was dissolved in 0.4 ml H2O and then 50 μl were used for HPLC according to the described method . For solid tissues 10-100 mg tissues were homogenized at a ratio of 1:50 with 0.1 M TCA and centrifuged as above. The supernatant was purified as above and analyzed at HPLC as described . In order to quantify the content in amino acids in the sample, a standard curve consisting of 50 pmoles of D-Asp and 100 pmoles of each of the following amino acids: L-Asp, L-Glu, L-Ser, L-Thr, L-His, Gly, L-Arg, L-Ala, L-Val, L-Met, L-Tyr, L-Phe, L-Ile, L-Leu and L-Lys was carried out in the same assay condition. The method allowed the determination with high reliability of a minimum amount of D-Asp at a coefficient of variation (CV) of 5% as calculated from 10 repeated analyses of D-Asp from a sample in which a known amount of D-Asp has been added.
Effects of D-aspartate on LH and testosterone in serum release in humans and rats
The experiment using human subjects was carried out on two groups of healthy male volunteers aged between 27 and 37 years at the IVF (in vitro fertilization) Unit, Hospital "S. Luca", Vallo della Lucania, Italy. The first group was composed of 23 volunteers who constituted the experimental group; the second group was composed of 20 volunteer who constituted the placebo group. Informed consent was obtained from each participant and the procedure was approved by the Institutional Review Board of the Hospital. Every morning at breakfast for 12 consecutive days subjects in the first group were invited to consume, by mouth a solution of 10 ml of 2.0 M sodium D-aspartate (3.12 g/10 ml) supplemented with vitamin B6, folic acid and vitamin B12 and diluted in half a glass of water or fruit juice. This solution is marketed in Italy under the name DADAVIT® (Pharmaguida s.r.l., Italy) and used as a supplement to increase the quality of human seminal fluid. The participants belonging to the placebo group received 10 ml of a solution consisting of 2 M NaCl (1.12 g/10 ml in water) containing the same vitamins as above, and this solution was packaged as DADAVIT® solution (termed DADAVIT® placebo). Blood samples (8-10 ml) from each participant were taken before treatment, after 6 days of treatment, after 12 days of treatment, and 3 days after suspension of the treatment. The blood was taken in the morning between 9.30 and 10.30 a.m., a time when serum oscillations of LH and testosterone are at their minimum value . The determination of LH and testosterone in human blood was carried out using commercially available immunochemoluminescence kits for LH and testosterone purchased from Bayer HealthCare LLC, Subsidiary System (Bayer Corporation, USA). According to the manufacturing company that have prepared the kits for LH and testosterone, the sensitivity of the method was 0.1 mIU/ml for LH and 0.2 ng/ml for testosterone.
The experiments on rats were carried out as follows: Adult Wistar male rats (120 days old, 340 ± 20 g), purchased from Charles River laboratory (Italy), were housed 2 per cage in a controlled environmental animal facility with a 12-h light/dark cycle and fed with laboratory food pellets and water ad libitum until experimentation. All the procedures involving rats were in accordance with institutional guidelines. When ready, three groups of 10 animals each were prepared. Rats in the first group were allowed to drink a solution consisting of 20 mM of sodium D-aspartate for 12 days and then sacrificed. Rats in the second group were given to drink a solution of 20 mM sodium D-aspartate to drink for 12 consecutive days; then allowed to drink tap water for 3 days and then sacrificed. Rats in of the third group (control) were given to drink a solution of 20 mM NaCl to drink for 12 days and then sacrificed. After rats were sacrificed, the blood and solid tissues (frontal cortex, hippocampus, pituitary, testis, liver and kidney) were collected. The serum was obtained from the blood (after coagulation and centrifugation at 3,000 g for 30 min) and the concentration of LH and testosterone (hormone release) determined. The solid tissues were homogenized in PBS at a ratio of 1:20 and centrifuged at 10,000 g for 30 min. The supernatant was used for the analysis of D-Asp by HPLC.
The determination of LH in the rat serum was carried out using a reagent kit from Amersham Biosciences (EIA Biotrak™ System RPN 2562; Amersham Biosciences Europe GmbH, Cologno Monzese, Milano, Italy), whereas the concentration of testosterone in the rat serum was determined using the same kits and the same procedure that were used for the human serum.
Effects of D-aspartate on the rat pituitary in the synthesis of LH and cGMP: In vitro experiments
The pituitary was taken from an adult male rat (120 days old) and cut longitudinally into two portions, and then incubated with moderate shaking at 37°C for 60 min in 1.0 ml of Krebs-Ringer solution, pH 7.4, containing 50 μl of a cocktail of protease inhibitors (Sigma, P 8340) and also contained 1 mg/ml BSA in air/CO2 (19:1) and D-Asp at the concentration of 0.1 mM (10 μl of 10 mM). The same experiment was repeated using a second male rat, but D-Asp was at the concentration of 1.0 mM (10 μl of 100 mM). The same experiment was repeated on a third male rat, but without D-Asp (control). After incubation, each sample was homogenized in its incubation solution and divided into two equal portions. The first portion was centrifuged at 10,000 rpm for 5 min at 4°C, and the supernatant was used for LH determination (synthesis). The second portion was mixed with 25 μl of 1 M HCl and 25 μl of 1 M TCA, then homogenized and centrifuged. The supernatant was neutralized with 1 M NaOH and used for the determination of cGMP (cyclic guanosine monophosphate; 3',5'-cyclic guanosine monophosphate) and cAMP (cyclic adenosine monophosphate; 3',5'-cyclic adenosine monophosphate). These determinations were carried out using a radioimmunoassay based on the competition between unlabelled cAMP or cGMP and a fixed quantity of the tritium labeled compound (Amersham Biosciences, Buckinghamshire, England (cAMP: code TRK432; cGMP: code TRK500). This experiment was repeated five times on sample from different animals.
Effects of D-aspartate on rat Leydig cells in the synthesis of testosterone and cAMP: In vitro experiments
Leydig cells were prepared from the testes of 5 rats according to the described procedure . The purified Leydig cells were suspended at a concentration of 1 × 106 cells/ml in Krebs-Ringer solution containing a cocktail of protease inhibitors (Sigma, P 8340, diluted 1:100) and BSA 1 mg/ml. To 1 ml of this suspension were added, respectively, 10 μl of 10 mM of sodium D-aspartate and 10 μl of 100 mM of sodium D-aspartate (0.1 and 1.0 mM, respectively). For the control, 10 μl of H2O was used instead of D-Asp. The samples were incubated for 60 min at 37°C with moderate shaking. Then each sample was homogenized in its solution and divided into two equal portions. The first portion was centrifuged at 10,000 g for 5 min, and the supernatant was used for the testosterone determination. The second portion was mixed with 50 μl of 1 M HCl and 50 μl of 1 M TCA, then homogenized and centrifuged, and the supernatant was used for the determination of cAMP and cGMP. This experiment was repeated five independent times.
Biosynthesis of D-aspartate in rat tissues: D-aspartate racemase activity
Step 1: Rat tissues were homogenized at a ratio of 1:10 in 0.1 M Tris-HCl, pH 8.0, containing 10 mM EDTA, and centrifuged at 20,000 g for 20 min. Then, 500 μl of the supernatant was mixed with 500 μl of 0.2 M sodium L-aspartate and incubated at 37°C for 60 min. A blank was prepared as a sample, but incubated at 0°C (ice water) instead of 37°C, for 60 min. After incubation, 200 μl of 1.0 M TCA was added, and the solution was mixed and centrifuged for 10 min at 20,000 g.
Step 2: 1 ml of each supernatant (sample and blank) was neutralized with 80-90 μl of 2.0 M NaOH and mixed with 100 μl Tris-HCl 1 M, pH 8.2 and 5 μl of purified D-AspO (2 mg/ml; 300 U/ml), obtained by over-expression from beef kidney  or from the hepathopancreas of Octopus vulgaris  and incubated for 30 min at 37°C in order to oxidize the D-Asp in α-oxaloacetate. After that, 100 μl of 5.0 mM 2,4-dinithrophenylhydrazine (in 5 M HCl) was added, mixed and left at room temperature for 10 min. Finally, 200 μl of 5 M NaOH was added and mixed, and the absorbance of the sample was read against the blank at 445 nm. In order to determine the amount of D-Asp in the 1 ml of the above supernatant, a standard test of D-Asp was carried out. For this purpose, 1.0 ml of 0.01 mM of sodium D-aspartate was mixed with 100 μl of H2O, 100 μl Tris-HCl 1 M, pH 8.2 and 5 μl of purified D-AspO and the procedure was continued as sample. The absorbance of this standard was read against a blank in which 1.0 ml H2O was used instead of D-aspartate. One enzyme unity (EU) was defined as the amount of the enzyme capable of generating 1 nmol of D-Asp in 60 min of incubation at 37°C in the above assay conditions. The specific activity was defined as the EU/mg of the homogenate.
Results are presented as the mean ± SEM. LH and testosterone concentrations in human serum were analyzed by analysis of variance with repeated measurements (ANOVA, StatView). D-Asp storage in rat tissues and LH and testosterone concentration from in vivo and in vitro experiments on rats were analyzed by one-way ANOVA. Pairwise comparison of the means was made with Fisher's LSD test. Values of p < 0.05 were considered significant.
Specific determination of D-aspartic acid by HPLC-D-AspO
Effects of D-aspartate on LH and testosterone release in humans
In this study 23 participants took an oral dose of sodium D-aspartate (DADAVIT®) for 12 consecutive days and 20 participants took an oral dose of placebo (DADAVIT® placebo) for 12 consecutive days; the levels of LH and testosterone in the serum were monitored after 6 and 12 days of treatment and 3 days after suspension of the treatment (with D-aspartate or the placebo).
Effects of D-aspartate on LH and testosterone release in human serum
Basal levels treatment
After 6 days of D-Asp treatment
After 12 days of D-Asp
3 days after the suspension of D-Asp
LH (mIU/ml serum)
Subjects treated with with Na-D-aspartate
4.2 ± 0.5
4.5 ± 0.6
5.6 ± 0.9*
4.8 ± 0.8
Subjects treated with placebo
4.2 ± 0.4
4.3 ± 0.7
4.2 ± 0.6
4.1 ± 0.5
Testosterone (ng/ml serum)
Subjects treated with Na-D-aspartate
4.5 ± 0.6
5.2 ± 0.7
6.4 ± 0.8*
5.8 ± 0.6*
Subjects treated with placebo
4.6 ± 0.5
4.5 ± 0.7
4.7 ± 0.7
4.6 ± 0.7
Concerning the effect of D-Asp on the induction of testosterone release, after 12 days of D-Asp treatment, the levels of testosterone in the serum of the participants were significantly increased compared with basal levels. Out of 23 participants, 20 had increased testosterone. From a mean of 4.5 ± 0.6 ng/ml serum at zero time, it rose to 6.4 ± 0.8 ng/ml, a 42% increase (Table 1). Statistical analyses indicated a significant effect [ANOVA with repeated measures: treatment effect: F(1,82) = 7.724, p < 0.0082] and a significant interaction between treatment and days [F(2,82) = 32.599; P < 0.0001]. As with LH, so also with testosterone, the effect of D-aspartate was time dependent. When subjects were treated with sodium-D-aspartate for only 6 days, testosterone was found of 1.15-fold higher than basal levels, but this increase was not statistically significant (Table 1). Interestingly 3 days after the suspension of D-Asp treatment, testosterone was still increased 1.22-fold compared with the basal levels (5.8 ± 0.6 ng/ml against 4.5 ± 0.6 ng/ml). Fisher's post-hoc analysis also revealed a significant difference in the testosterone concentration in the serum 3 days after the end of the treatment (p < 0.01) (Table 1). One plausible explanation of this phenomenon is that since in rats ingested D-Asp remains accumulated in the testes in significant amounts until 3 days after the suspension of D-Asp treatment (see below), if it is assumed that in humans D-Asp also remains significantly increased in the testes 3 days after the suspension of D-Asp treatment, we can deduce that in humans as in rats, D-Asp had remained accumulated in significant amounts in the testes and consequently it continued to stimulate testosterone release.
Effects of D-aspartate on the release of LH and testosterone in rats
Effects of D-aspartate on LH and testosterone release in rat serum
Rats treated with placebo
Rats treated with Na-D-Aspartate for 12 days
Rat treated with Na-D-Asp for 12 days and then for 3 days with placebo
3.7 ± 0.3
5.6 ± 0.4*
4.1 ± 0.3
5.1 ± 0.4
10.4 ± 1.2*
6.1 ± 0.5*
Concerning testosterone, the effect of sodium D-aspartate on testosterone release in rat serum was to induce a 2.05-fold increase of this hormone; from a basal level of 5.1 ± 0.8 ng/ml in the control rats, testosterone reach the level of 10.4 ± 1.2 ng/ml in treated animals (p < 0.001) (Table 2). It is interesting to observe that, contrary to what occurred with LH, three days after the suspension of the treatment, testosterone levels still remained significantly increased compared with the control rats: a 1.27-fold increase (p < 0.01) (Table 2).
The effect of D-Asp on the increased release of testosterone in serum that occurs 3 days after suspension of the treatment may be due to two events: i) the increase of testosterone in serum is a consequence of the increased LH, which in turn stimulates the release of testosterone in the testes; and ii) D-Asp also has a direct action on the testes in enhancing testosterone release, as already demonstrated both previously [5, 13] and also by this study (see below). In addition, since the ingested D-Asp also still remains accumulated significantly in the testes 3 days after D-Asp suspension of treatment (this study), we deduce that the persistent increase of testosterone in rat blood is due to the accumulation of D-Asp that persists leading to stimulated testosterone production.
Occurrence and accumulation of D-aspartate in rat tissues following D-Asp treatment
Endogenous occurrence of D-aspartate in rat tissues and accumulation after treatment with sodium D-aspartate
Basal levels of D-Asp
Levels of D-Asp after 12 days of D-Asp treatment
Levels of D-Asp 3 days after the suspension of D-Asp treatment
129 ± 12
880 ± 45*
145 ± 12
109 ± 8
780 ± 45*
205 ± 14*
90 ± 6
350 ± 19*
110 ± 9
62 ± 5
108 ± 9*
78 ± 7
41 ± 4
85 ± 7*
55 ± 5
12 ± 3
24 ± 2*
11 ± 3
10 ± 2
45 ± 4*
13 ± 3
4 ± 1
8 ± 2*
3 ± 1
Effects of D-aspartate on the synthesis of LH and cGMP in the isolated rat pituitary
Effects of D-aspartate on the synthesis of testosterone and cAMP in rat Leydig cells
Biosynthesis of D-aspartate in rat tissues: D-aspartate racemase
In this study using a specific HPLC method combined with the use of D-AspO (Fig. 1) and specific immunoenzymatic methods for the determination of LH and testosterone as well as of the second messengers, cAMP and cGMP, we have demonstrated that D-aspartic acid plays a role in the release and synthesis of LH and testosterone in humans and rats. In humans we found that with the consumption of a daily dose of 10 ml of 2 M sodium D-aspartate solution (3.12 g) for 12 consecutive days, the levels of LH and testosterone in the serum were significantly increased, by 33% and 42% respectively. In 87% of the subjects who have been treated with sodium D-aspartate increased the concentration of LH and testosterone in the serum (p < 0.0001). After 6 days of treatment LH was already found to have increased, but this increase was not statistically significant (Table 1). Three days after sodium D-aspartate suspension, LH still was found higher than that of basal levels (a 1.14-fold increased), but this increase was not statistically significant (Table 1), indicating that the increase of LH was dose dependent. The consumption of sodium D-aspartate in humans also induced significant testosterone release in the serum. In fact, in the same subjects who took D-Asp for 12 days, the levels of testosterone increased significantly. ANOVA with repeated measurements indicated a significant value (p < 0.0001). As with LH, so also with testosterone, 6 days of treatment induced increased serum testosterone in the blood, but this increase was not significant. Interestingly, contrary to what occurred for LH, three days after suspension of the treatment, testosterone still remained increased in the serum 1.28-fold increase compared with the basal levels, and this increase was significant (p < 0.01) (Table 1). A possible explanation of this event is that the ingested D-Asp probably also remained accumulated in the testes 3 days after treatment was stopped, and it continued to stimulate the testosterone production in the testes. This hypothesis is supported by results obtained in rats. In fact, after rats had drunk a solution of 20 mM D-Asp for 12 days, this amino acid had accumulated in various tissues, especially the pituitary and the testes (Table 3). However, when D-Asp treatment was suspended, D-Asp that had accumulated in tissues diminished until it reached basal levels, except in the testes, where D-Asp still remained significantly increased 1.88 fold (Table 3).
In this study we used rats as model animals in order to understand the molecular mechanism by which D-Asp induce its action. Rats were allowed to drink a solution of sodium D-Asp for 12 days and after that the concentration of D-Asp that accumulated in tissues was determined along with the concentration of LH and testosterone. In rat as in humans, D-Asp also actively induced LH and testosterone release. In fact, when rats were treated with D-Asp, a significant increase in LH and testosterone was observed after the 12 days of treatment (Table 2), coinciding with the increased levels of D-Asp in the pituitary and testis respectively (Table 2). Thus, these data indicate that D-Asp is involved in the regulation of the above hormones.
In vitro experiments conducted on an isolated rat pituitary incubated with D-Asp demonstrated that D-Asp at the concentration of 0.1 mM in the medium is capable of inducing the synthesis of LH (Fig. 2, left panel) and testosterone (Fig. 3, left panel). In addition, when the pituitary gland was incubated with 0.1 mM D-Asp, a significant increase of cGMP occurred in the assay mixture (Fig 3, right panel) thus indicating that the release and synthesis of LH occurred under the intervention of the cGMP. These data are in agreement with previous results obtained by other investigators [27–29], who demonstrated that the molecule involved in the signal transduction for other metabolites in the rat pituitary was cGMP. Similarly, in vitro experiments conducted on isolated Leydig cells incubated in a medium containing 0.1 mM of sodium D-Asp demonstrated that this amino acid is capable of inducing the synthesis of testosterone (Fig. 3, left panel) and that this event is mediated by cAMP, which acts as the molecule involved in the signal transduction for testosterone synthesis from rat Leydig cells (Fig. 3, right panel). Also, this last result is in agreement with previous studies by other investigators in which it was demonstrated that the increase of testosterone synthesized by Leydig cells occurs under the intervention of the cAMP . The final concern of this study was to examine the biosynthesis of D-Asp in the pituitary and testis. The results demonstrated that rat tissues contain a racemase activity that is capable of converting L-Asp into D-Asp. We have termed this enzyme "D-aspartate racemase" and it is present in all the rat tissues we analyzed. The pituitary and the testis are the tissues with higher concentrations (Fig. 4). These data thus indicate that a relationship exists between the endogenous concentration of D-Asp and the concentration of the D-aspartate racemase in the tissues (Fig. 4).
In this study, we also investigated the action of L-Asp on hormone release in rats. A group of 10 male rats were treated with L-Asp instead of D-Asp at the same time and at the same concentration and then levels of LH and testosterone in the blood were determined. The results of this investigation indicated that the L-Asp does not induce any significant increase of serum LH or testosterone (data not shown), thus indicating thus that only the stereochemical form of D-Asp is active in the hormone release.
Here we demonstrated that D-aspartic acid, which occurs as a physiological compound in the mammalian pituitary and testis, has a role in the regulation of the release and synthesis of LH and testosterone. In humans and rats, sodium D-Asp treatment enhances the release of LH and testosterone. The experiments that we carried out on rats have permitted us to understand that this amino acid regulates the synthesis of LH and testosterone in the pituitary and the testis respectively. This action is mediated in the pituitary by cGMP and in the testis by cAMP, which act as the second messengers in the signal transduction in the pituitary and testes respectively. The pituitary and testis possesses a D-Aspartate racemase, which provides the necessary production of D-Asp.
High performance liquid chromatography
in vitro fertilization
Commercial integrator consisting of sodium D-aspartate and vitamins, produced by the 'Pharmaguida s.r.l., Italy'
cyclic guanosine monophosphate
5'-cyclic guanosine monophosphate
cyclic adenosine monophosphate
phosphate buffer saline.
This work was supported by the Department of Obstetrics and Gynecology, and Reproductive Medicine (IVF Unit), Hospital 'S. Luca', 84078 Vallo della Lucania, Italy; by the Zoological Station Anton Dohrn, Napoli, Italy, and by the Fondazione IRCCS-SDN, Via Gianturco 113, 80143 Napoli, Italy.
- D'Aniello A: D-Aspartic acid: An endogenous amino acid with an important neuroendocrine role. Brain Res Rev. 2007, 53: 215-234. 10.1016/j.brainresrev.2006.08.005.View ArticlePubMedGoogle Scholar
- D'Aniello A, Giuditta A: Identification of D-aspartic acid in the brain of Octopus vulgaris. J Neurochem. 1977, 29: 1053-1057. 10.1111/j.1471-4159.1977.tb06508.x.View ArticlePubMedGoogle Scholar
- Neidle A, Dunlop DS: Developmental changes of free D-aspartic acid in the chicken embryo and in the neonatal rat. Life Sci. 1990, 46: 1517-1522. 10.1016/0024-3205(90)90424-P.View ArticlePubMedGoogle Scholar
- Hashimoto A, Kumashiro S, Nishikawa T, Oka T, Takahashi K, Mito T, Takashima S, Doi N, Mizutani Y, Kaneco T, Ootomo E: Embryonic development and postnatal changes in free D-aspartate and D-serine in the human prefrontal cortex. J Neurochem. 1993, 61: 348-351. 10.1111/j.1471-4159.1993.tb03575.x.View ArticlePubMedGoogle Scholar
- D'Aniello A, Di Cosmo A, Di Cristo C, Annunziato L, Petrucelli L, Fisher GH: Involvement of D-aspartic acid in the synthesis of testosterone in rat testes. Life Sci. 1996, 59: 97-104. 10.1016/0024-3205(96)00266-4.View ArticlePubMedGoogle Scholar
- Wolosker A, D'Aniello A, Snyder SH: D-aspartate disposition in neuronal and endocrine tissues: ontogeny, biosynthesis and release. Neuroscience. 2000, 100: 183-189. 10.1016/S0306-4522(00)00321-3.View ArticlePubMedGoogle Scholar
- Spinelli P, Brown E, Ferrandino G, Branno M, Montarolo PG, D'Aniello E, Rastogi RK, D'Aniello B, Baccari G, Fisher G, D'Aniello A: D-Aspartic acid in the nervous system of Aplysia limacine: Possible Role in Neurotransmission. J Cell Physiol. 2006, 206: 672-681. 10.1002/jcp.20513.View ArticlePubMedGoogle Scholar
- D'Aniello S, Spinelli P, Ferrandino G, Peterson K, Tsesarskia M, Fisher GH, D'Aniello A: Cephalopod vision involves dicarboxylic amino acids: D-aspartate, L-aspartate and L-glutamate. Biochem J. 2005, 386: 331-340. 10.1042/BJ20041070.PubMed CentralView ArticlePubMedGoogle Scholar
- D'Aniello A, Di Fiore MM, D'Aniello G, Colin FE, Lewis G, Setchell BP: Secretion of D-aspartic acid by the rat testis and its role in endocrinology of the testis and spermatogenesis. FEBS Letters. 1998, 436: 23-27. 10.1016/S0014-5793(98)01087-4.View ArticlePubMedGoogle Scholar
- Sakai K, Homma H, Fukushima JA, Santa T, Tashiro T, Iwatsubo K, Imai K: Localization of D-aspartic acid in elongate spermatids in rat testis. Arch Biochem Biophys. 1998, 351: 96-105. 10.1006/abbi.1997.0539.View ArticlePubMedGoogle Scholar
- Nagata Y, Homma H, Lee JA, Imai K: D-Aspartate stimulation of testosterone synthesis in rat Leydig cells. FEBS Letters. 1999, 444: 160-164. 10.1016/S0014-5793(99)00045-9.View ArticlePubMedGoogle Scholar
- Takemtsu F, Homa H: Free D-aspartate in Mammals. Biol Pharm Bull. 2005, 28: 1566-1570. 10.1248/bpb.28.1566.View ArticleGoogle Scholar
- D'Aniello A, Di Fiore MM, Fisher GH, Milone A, Seleni A, D'Aniello S, Perna A, Ingrosso D: Occurrence of D-Aspartic acid and N-methyl-D-aspartic acid in rat neuroendocrine tissues and their role in the modulation of luteinizing hormone and growth hormone release. FASEB J. 2000, 14: 699-714.PubMedGoogle Scholar
- Imai K, Fukushima T, Hagiwara K, Santa T: Occurrence of D-aspartic acid in rat brain and pineal gland. Biomed Chromatogr. 1995, 9: 106-109. 10.1002/bmc.1130090211.View ArticlePubMedGoogle Scholar
- Ishio S, Yamada H, Hayashi M, Yatsushiro S, Noumi T, Yamaguchi A, Moriyama Y: D-Aspartate modulates melatonin synthesis in rat pinealocytes. Neurosci Lett. 1998, 249: 143-146. 10.1016/S0304-3940(98)00414-5.View ArticlePubMedGoogle Scholar
- Pampillo M, Scimonelli T, Bottin MC, Duvilanski BH, Rettori V, Seilicovich A, Lasaga M: The effect of D-aspartate on luteinizing hormone-releasing hormone, α-melanocyte-stimulating hormone, GABA and dopamine release. Neuroreport. 2002, 13: 2341-2344. 10.1097/00001756-200212030-00034.View ArticlePubMedGoogle Scholar
- Boni R, Santillo R, Macchia G, Spinelli P, Ferrandino G, D'Aniello A: D-Aspartate and reproductive activity in sheep. Theriogenology. 2006, 65: 1265-1278. 10.1016/j.theriogenology.2005.07.019.View ArticlePubMedGoogle Scholar
- Lamanna C, Assisi L, Botte V, Di Fiore MM: Involvement of D-Asp in P450 aromatase activity and estrogen receptors in boar testis. Amino acids. 2007, 32: 45-51. 10.1007/s00726-006-0351-9.View ArticlePubMedGoogle Scholar
- Raucci F, D'Aniello S, Di Fiore MM: Endocrine role of D-aspartic acid in the testes of lizard Podarcis s. sicula. J Endocrinol. 2005, 187: 347-59. 10.1677/joe.1.06115.View ArticlePubMedGoogle Scholar
- Assisi L, Botte V, D'Aniello A, Di Fiore MM: Enhancement of aromatase activity by D-aspartic acid in the ovary of the lizard Podarcis s. sicula. Reproduction. 2001, 121: 803-808. 10.1530/rep.0.1210803.View ArticlePubMedGoogle Scholar
- D'Aniello G, Ronsini S, Guida F, Spinelli P, D'Aniello A: Occurrence of D-aspartic acid in human spermatozoa: Possible role in reproduction. Fertil Steril. 2005, 84: 1444-1449. 10.1016/j.fertnstert.2005.05.019.View ArticlePubMedGoogle Scholar
- D'Aniello G, Grieco N, Di Filippo MA, Cappiello F, Topo E, D'Aniello E, Ronsini S: Reproductive implication of D-aspartic acid in human pre-ovulatory follicular fluid. Human Reprod. 2007, 22: 3178-3183. 10.1093/humrep/dem328.View ArticleGoogle Scholar
- Wilson JD: The testis. Metabolic control and Disease. Edited by: Bondy PK, Rosenberg LE. 1980, Philadelphia: Saunders WB, 1535-1578. 8Google Scholar
- Vasta V, Shimizu-Albergine M, Beavo JA: Modulation of Leydig Cell function by cyclic nucleotide phospfodiesterase 8A. Proc Natl Acad Sci USA. 2006, 113: 19925-19930. 10.1073/pnas.0609483103.View ArticleGoogle Scholar
- Yohda M, Okada H, Kumagai H: Molecular cloning and nucleotide sequencing of the aspartate racemase gene from lactic acid bacteria Streptococcus thermophilus. Biochem Biophys Acta. 1991, 1089: 234-240.PubMedGoogle Scholar
- Shibata K, Watanabe T, Yoshikawa H, Abe K, Takahashi S, Kera Y, Yamada RH: Purification and characterization of aspartate racemase from the bivalve mollusk Scapharca broughtonii. Comp Biochem Physiol B. 2003, 134: 307-314. 10.1016/S1096-4959(02)00267-1.View ArticlePubMedGoogle Scholar
- Snyder G, Naor Z, Fawcett CP, McCann SM: Gonadotropin release and cyclic nucleotides: Evidence for luteinizing hormone-releasing hormone induced elevation of guanosine 3',5'-monophosphate levels in gonadotrophs. Endocrinology. 1980, 107: 1627-1633. 10.1210/endo-107-5-1627.View ArticlePubMedGoogle Scholar
- Kawakami M, Kimura F: Stimulation of guanosine 3',5'-monophosphate accumulation in anterior pituitary glands in vivo by synthetic luteinizing hormone-releasing hormone. Endocrinology. 1980, 106: 626-630. 10.1210/endo-106-2-626.View ArticlePubMedGoogle Scholar
- Garrel G, Lozach A, Bachir LK, Laverriere JN, Counis R: Pituitary adenylate cyclase-activating polypeptide stimulates nitric-oxide synthase type I expression and potentiates the cGMP response to gonadotropin releasing hormone of rat pituitary gonadotrophs. J Biol Chem. 2002, 277: 46391-46401. 10.1074/jbc.M203763200.View ArticlePubMedGoogle Scholar
- Saez JM: Leydig cells: endocrine, paracrine, and autocrine regulation. Endocr Rev. 1994, 15: 574-626.View ArticlePubMedGoogle Scholar
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