Journal of Chromatography B, 879 (2011) 3268–3276 Contents lists available at ScienceDirect Journal of Chromatography B jo u r n al hom epage: www.elsevier.com/locate/chromb d-Asp: A new player in reproductive endocrinology of the amphibian Rana esculenta  Franca Raucci, Maria Maddalena Di Fiore ∗ Department of Life Sciences, Second University of Naples (SUN), Via Vivaldi 43, 81100 Caserta, Italy a r t i c l e i n f o Article history: Received 30 November 2010 Accepted 1 April 2011 Available online 13 April 2011 Keywords: d-Aspartic acid Testosterone Seasonal breeders Oocyte Testis Frog a b s t r a c t We investigated the involvement of d-Aspartic acid (d-Asp) on ovarian and testicular morphology of the green frog, Rana esculenta, and its effect on the testosterone production. The study has been performed throughout the reproductive cycle. In both ovary and testis a substantial amount of d-Asp is endogenously present and its concentration varies as function of reproduction. In the frog, d-Asp content is differently correlated with gonadal and plasmatic levels of testosterone, depending on the sex. In fact, the amount of the d-Asp is inversely linked with that of the testosterone in the ovary, while this correlation directly matched in the testis. In vivo short-term experiments, consisting of a single intra-peritoneal injection of d- Asp (2.0 mol/g body weight), demonstrated that the enantiomer is significantly accumulated by both the ovary and testis, reaching after 3 h the highest uptake and thereafter decreasing to baseline values within 24 h. Furthermore, d-Asp influences the synthesis and/or the release of testosterone, causing a decrease of its level in the female, and an increase in the male, respectively. In vivo long-term experiments, d-Asp, chronically administered to the frogs of both sexes, enhances the maturation of both gonads, determining in the oocytes an higher accumulation of carbohydrate yolk plates in the ooplasm, and stimulating the spermatogenesis in the testis. Taken altogether, our results show that d-Asp operates differently in female and male frog gonads, indicating that it has different targets in the reproductive machinery depending on the sex. © 2011 Elsevier B.V. All rights reserved. 1. Introduction d-Aspartic acid (d-Asp) is an endogenous amino acid occurring as a free compound in several animal phyla [1]. In rat brain, this enantiomer has been localized in various neurons, including the hippocampus, but also in the hypothalamo-neurohypophyseal sys- tem suggesting its role in neurotransmission and neurosecretory activities [2–5]. However, the occurrence of d-Asp is not only restricted to the nervous tissues. Several evidences have established its involve- ment in endocrine system. In fact, the relationship between d-Asp and endocrine activity has been documented in the adenohypoph- ysis, in the pineal gland and in the testis, where d-Asp is involved in hormone synthesis and release [4–9]. Specifically to rat testis, developmental changes in the levels of d-Asp and testosterone parallel each other closely: they both increase to maximum lev- els at sexual maturity. Moreover, d-Asp is contained in spermatids  This paper is part of the special issue “Analysis and Biological Relevance of d- Amino Acids and Related Compounds”, Kenji Hamase (Guest Editor). ∗ Corresponding author. Tel.: +39 0823 274527; fax: +39 0823 274571. E-mail address: mariam.difiore@unina2.it (M.M. Di Fiore). [10] and in both Leydig [6,11] and Sertoli cells [6]. Experiments performed either in vitro on isolated testis [12] or in vivo, by intraperitoneal administration of d-Asp to adult male rats [4], showed that this d-amino acid is accumulated by the gonad, and also induces a significant increase in luteinizing hormone, testos- terone, progesterone [4] and prolactin [7] in the blood. These findings propose a novel role for d-Asp as a regulator of reproduc- tive activity, suggesting a specific implication in the local regulation of androgen production. Our research group has focused some stud- ies on the role of d-Asp in the gonads of the lizard, Podarcis sicula, a seasonal breeding vertebrate. In lizard female, d-Asp enhances follicular production of 17-estradiol by up-regulating the local aromatase activity [13] and also promotes the maturation of oocyte follicular epithelium [14]. Furthermore, it has been demonstrated that d-Asp is rapidly taken up by Leydig cells of injected lizards and its rise is coupled with a significant increase in testosterone levels with a consequent augment of mitotic activity of the germi- nal epithelium of the testis [15,16]. On the other hand, d-Asp may play a role in hormonal regulation, as it stimulates testosterone synthesis in the testis, increasing the mRNA level of a steroido- genic acute regulatory protein (StAR) [17]. In our previous papers, the role of d-Asp in endocrine control of reproduction has been also investigated in the gonads of Rana esculenta [18,19]. However, 1570-0232/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2011.04.007