Studio di sistemi molecolari coinvolti nella regolazione dell'attività riproduttiva in un modello sperimentale non-mammifero, l'anfibio anuro Rana esculenta

Endocannabinoids are a novel class of lipidic mediators. They derive from arachidonic acid and bind and activate specific membrane receptor as CB1 and CB2. The main compounds classified as "endocannabinoids" are arachidonoylethanolamide (AEA, anandamide) and 2-arachidonoyl glycerol (2-AG); these molecules have different affinity for the receptors. The effect of AEA via CB1 and CB2 depends on its extracellular concentration, which is controlled by uptake and degradation, respectively due to a specific AEA membrane transporter (AMT) and to a fatty acid amide hydrolase (FAAH). Generally, endogenous cannabinoids, cannabinoid receptors, FAAH and AMT are designed as "endocannabinoid system". Due to the great complexity of the system, the widespread distribution outside the nervous system and the high degree of evolutionary conservation, detailed CB1 molecular characterization may be an useful approach to better understand the endocannabinoids activity, at multiple levels. With this aim, we used as experimental model, the anuran amphibian Rana esculenta, a low vertebrate, whose reproductive activity is affected by environmental factors, mainly temperature and photoperiod, and various molecular factors that control many important endocrine, autocrine and paracrine relations along the hypothalamus-hypophysis-gonad. The typical organization of the testis allow to analyse, at the same time, different cellular types, included in special structures called "cysts". CB1 molecular cloning from R. esculenta brain and testis was the first part of our report. By RT-PCR approach, in combination with 3'RACE, we obtained a partial cDNA, 1586 bp long, from the frog brain. The cb1 nucleotide sequence, compared with those of other vertebrates, revealed a nucleotide identity ranging from 62.6 to 81.9%. A protein of 462 aa, with a predicted molecular mass of 51.89 kDa was deduced from the nucleotide sequence. Bioinformatic approach was, then, useful to characterize the protein domains, from the seven hydrophobic domains - typical of the coupled to G proteins receptors (GPCRs) - to extra and intracellular loops. Critical domains for CB1 functionality were also observed in the frog, from the potential sites of phosphorylation to the critical motifs for internalization, desensitization and interactions with G proteins. A fragment of 1384 bp was also cloned from frog testis; the alignment between brain and testis cDNA revealed two nucleotide differences, in correspondence of codons 186 and 252. To assess the possibility of post-transcriptional modifications, we cloned the whole coding region from genomic DNA preparations, obtained from the same samples used to prepare cDNAs. While brain cDNA was different from the genomic sequence in the codons 5, 30, 70, 186, 252, 408, testis cDNA differed in codons 5, 30, 70 and 408. The only nucleotide differences responsible to generate aminoacidic differences were observed in codons 70 and 408, important as glycosylation site and for the coupling with G proteins, respectively. By Northern blot analysis, we recognized in both brain and testis only a signal of 2.2 kb. On the other hand, the Southern blot analysis was useful to demonstrate that cb1 was a single copy gene. Our hypothesis was that an "editing" process, an elaborate and precise form of post-transcriptional RNA modification, took place. Furthermore, the secondary structure of the mRNA predicted from the genomic sequence was substantially different from the secondary structure of brain and testis mRNA. In this respect, we speculated that the nucleotide changes observed in this study may affect RNA folding and therefore its stability and turnover. In our experimental model, an analysis of cb1 expression in some tissues, as it has already done in other classes of vertebrates, may be an useful approach to receive more knowledge about the functionality of the receptor. With this aim, we analysed - by RT-PCR technique - the cb1 presence in different frog tissues. Higher levels of mRNA were found in the brain and in gonads. While in kidney, in liver and in muscle there was a significant amount of transcript, in pituitary, in heart and in spleen, the expression levels were very low. Using the seasonal reproduction of the frog, we analysed the receptor expression in the testis, from September to July. Cb1 levels were high in September with a peak in November, then they decreased during the winter stasis. Receptor expression increased again during the breeding season. The receptor showed a particolar pattern of expression also in the brain in toto, during the reproductive cycle: similar levels of expression were observed in September and October, they decreased in November and increased again from January to April; a significant decrease of the signal was observed in May and in June and a new increase was in July. To gain further information about this pattern of expression, mainly in relationship to the reproductive function, we dissected the whole brain in the isolated areas as well as the spinal cord in every tract and we used these samples for a second analysis of expression. Even if the receptor showed a particular pattern depending on the month considered, during the entire cycle, cb1 was more expressed in the forebrain and in the midbrain. A mismatching profile of cb1 between the testis and the whole brain may be the consequence of a negative control from the endocannabinoids on the GnRH secretion, the main controller of gonadal activity. To verify this hypothesis, we valued the neuroanatomical and functional relationship between the endocannabinoid system and the GnRH one, in the frog forebrain. The morphofunctional relationship between the endocannabinoid system and gonadotropin releasing hormone (GnRH) activity in the regulation of reproduction has poorly been investigated in vertebrates. Due to the anatomical features of lower vertebrate brain, in the present paper, we choose the frog R. esculenta (anuran Amphibian) as a suitable model to better investigate such aspects of the central control of the reproduction. By using double labelling immunofluorescence aided with a laser-scanning confocal microscope, we demonstrated that a subpopulation of the frog hypothalamic GnRH neurons possessed CB1 cannabinoid receptors. By means of semi-quantitative RT-PCR assay, we showed that the fluctuations of GnRHI clearly mismatched, during the annual sexual cycle, the expression profile of cb1 in the whole brain as well as in isolated areas such as the telencephalon and the diencephalon which are known to be mainly involved in GnRH release and control of the reproduction. Furthermore, we found that anandamide was able to inhibit GnRHI synthesis, and buserelin (a GnRH agonist), in turn, inhibited the synthesis of GnRHI and induced an increase of cb1 transcription. Our observations pointed out, for the first time in vivo, the occurrence of a morphofunctional anatomical basis to explain a reciprocal relationship between the endocannabinoid system and GnRH activity. Appendix A CB1 receptor, as the other members of the GPCRs superfamily, shows highly conserved domains, important for its functionality. In its seven transmembrane domain, we recognized the aminoacidic motif "NPxxY", known to regulate the internalization, the binding to the ligand and the interaction with the G proteins of many GPCRs. Starting from these informations, we studied the function of this motif on CB1 activity too. Using mutagenesis in vitro assay, we mutated the single residues of the motif to alanine and we used these mutated receptors to stably transfecte HEK cells. Internalization was the first activity that we analysed. HEK cells were treated with CP55940 ligand, at different concentrations and for different time points. The most significant effect concerned the asparagine 394 mutation that completely inhibited CB1 internalization. A deeper research allowed us to analyse the effect of these mutations on the recycle activity. The main effect concerned proline 395 mutant that seemed to recycle faster then the wild-type (WT). No significant change for both internalization and recycle was observed for the tyrosine 398 mutant that acted as the WT. For the internalization analysis as well as for the recycle, the data obtained using the on cell western approach were confirmed by confocal microscopy. Finally, we tested the ability of the WT and mutated receptors to activate MAPK, after interaction with G proteins. Whereas Y398A mutant behaved as the WT, N394A and P395A mutants showed lower peaks of activation then the control. With our experiments, we demonstrated that the "NPxxY" motif may affect the activity of CB1, as well as it was already observed for many other GPCRs. Appendix B Spermatogenesis is a complex event that allows to the spermatogonia to become a very specialized cell, with an acrosome and a flagellum, as the spermatozoa. Many are the molecular elements involved in the important interactions presented along the hypothalamus-hypophysis-goand axis. After the demonstration of an involvement of the endocannabinoid system in the regulation of the reproductive functionality - demonstrating a possible control at central level, on the GnRH production - we examined another molecular system involved in the spermatogenesis, the mUBPy/MSJ-1 system The remodelling of new proteins is a very important cellular function, mainly during the spermatogenesis, when novel structures develop and others are degraded. mUBPY presence was already showed in the mouse testis and brain, in relationship with MSJ-1 localization. Also in this case, our experimental model was useful to demonstrate that the presence of a molecular system in a low vertebrate reveals its important function, conserved along the evolution. By Western blot, we recognized a specific mUBPy signal in the frog testis, during the reproductive cycle. The pattern observed seemed opposite to the MSJ-1 one, analysed on the same samples. The enzyme was localized in spermatids cysts, by immunocytochemical analysis and formed a thin cytosolic rim around the nucleus, in the spermatozoa head, isolated from the cloaca. The enzyme presence in these cells was also confirmed by Western blot. A possible control from mUBPy of the proteinic folding efficiency during the spermatogenesis is hypothesised in R. esculenta. Further studies will be conducted for a molecular cloning of the enzyme and for the analysis of its profile of expression in the frog testis as well as in the central nervous system, during the annual reproductive cycle.