Testicular endocrine activity is upregulated by D-aspartic acid in the green frog, Rana esculenta.
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.
Department of Life Sciences, Second University of Naples, Via Vivaldi 43, 81100 Caserta, Italy.
F
F
Raucci
L
L
Assisi
S
S
D'Aniello
P
P
Spinelli
V
V
Botte
M M
MM
Di Fiore
D000818:Animals; D001224:Aspartic Acid / Q000494:pharmacology*; D004958:Estradiol / Q000097:blood; D005835:Genitalia / Q000166:cytology / Q000187:drug effects; D008297:Male; D011893:Rana esculenta; D013091:Spermatogenesis / Q000187:drug effects; D013268:Stimulation, Chemical; D013737:Testis / Q000033:anatomy & histology / Q000378:metabolism*; D013739:Testosterone / Q000032:analysis / Q000096:biosynthesis* / Q000097:blood
D001224:Aspartic Acid; D013739:Testosterone; D004958:Estradiol