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In the present study we report the occurrence of D-aspartic acid (D-Asp) in the ovary of the green frog Rana esculenta and its putative involvement in testosterone production by the gonad. In the ovary, D-Asp concentrations undergo significant variations during the main phases of the sexual cycle. In spawning females (March), its concentration was low (2.5 +/- 1.1 nmol/g ovary) and during the post-reproductive period (June) it increased and reached its peak level (58.0 +/- 10.1 nmol/g) in October. In that month, vitellogenesis occurs in a new set of ovarian follicles and continues until the next spring. The concentrations of D-Asp in the ovary and of testosterone in the ovary and in the plasma were inversely correlated during the reproductive cycle: when endogenous D-Asp was low (March), testosterone was high (36.9 +/- 4.8 ng/g ovary; 23.1 +/- 2.76 ng/ml plasma) and, in contrast, when the D-Asp concentration was high (October), the testosterone concentration was low (0.86 +/- 0.21 ng/g ovary and 5.0 +/- 1.3 ng/ml plasma). In vivo experiments, consisting of injection of D-Asp (2.0 mumol/g body weight) into the dorsal lymphatic sac of adult female frogs, demonstrated that this amino acid accumulates significantly in the ovary. After 3 h, moreover, it caused a decrease in testosterone level in the plasma of about 80%. This inhibition was reversible: within 18 h after the amino acid injection, as the D-Asp concentration in the ovary decreased, the testosterone titre was restored in both ovary and plasma. In vitro experiments, conducted in isolated ovarian follicles, confirmed this phenomenon and identified these gonadal components as the putative D-Asp targets. Other amino acids (L-Asp, D-Glu, L-Glu, D-Ala and L-Ala) used instead of D-Asp were ineffective. These findings indicate that D-Asp is involved in the control of androgen secretion by the ovary in this amphibian species, revealing a more complex system for control of this androgen synthesis than was previously believed to exist.

This study investigated the involvement of D-aspartic acid (D-Asp) in testicular steroidogenesis of the green frog Rana esculenta and its effect on stimulation of thumb pad morphology and glandular activity, a typical testosterone-dependent secondary sexual characteristic in this amphibian species. In the testis, D-Asp concentrations vary significantly during the reproductive cycle: they are low in pre- and post-reproductive periods, but reach peak levels in the reproductive period (140-236 nmol/g wet tissue). Moreover, the concentrations of D-Asp in the testis through the sexual cycle positively match the testosterone levels in the gonad and the plasma. The racemase activity evaluated during the cycle expresses its peak when D-Asp and testosterone levels are highest, that is, during the reproductive period, confirming the synthesis of D-Asp from L-Asp by an aspartate racemase. Short-term in vivo experiments consisting of a single injection of D-Asp (2.0 micro mol/g body weight) demonstrated that this amino acid accumulates significantly in the testis, and after 3 h its uptake is coupled with a testosterone increase in both testis and plasma. Moreover, within 18 h of amino acid administration, the D-Asp concentration in the testis decreased along with the testosterone titer to prestimulation levels. Other amino acids (L-Asp, D-Glu and L-Glu) used instead of D-Asp were ineffective, confirming that the significant increase in testicular testosterone was a specific feature of this amino acid. In long-term experiments, D-Asp had been administered chronically to frogs caught during the three phases of the reproductive cycle, inducing testosterone increase and 17beta-estradiol decrease in the gonad during the pre- and post-reproductive period, and vice versa during the reproductive period. The stimulatory effect of D-Asp on testosterone production by the testis is consistent with the stimulation of spermatogenesis and the maturation of thumb pads occurring in D-Asp-treated frogs. In these last animals, there was an increase of seminiferous ampoule area and a higher number of spermatids and sperm. Moreover, in spermatogonia I and II and in spermatocytes, a proliferating cell nuclear antigen (PCNA) intense immunopositivity was observed. In addition, the thumb pads of D-Asp-treated frogs compared with controls showed a significantly thicker epithelial lining, a wider area of their glands with taller secretion cells, and more numerous, PAS-positive-rich secretions. Finally, these results provide functional evidence for a biologic role of D-Asp in amphibian male steroidogenesis; therefore, this unusual amino acid could be considered a modulatory agent for reproductive processes.