Silvia Fasano

Endocannabinoid System in Frog and Rodent Testis: Type-1 Cannabinoid Receptor and Fatty Acid Amide Hydrolase Activity in Male Germ Cells

Abstract N-arachidonoylethanolamide (anandamide [AEA]) is the main endocannabinoid described to date in the testis. It exerts its effects through the activation of G-protein coupled cannabinoid receptors (CNR). However, the activity of AEA in controlling male reproduction is still poorly known. Here we provide direct evidence on the presence of the “endocannabinoid system,” constituted by type-1 cannabinoid receptor (CNR1) and fatty acid amide hydrolase (FAAH), in the frog Rana esculenta testis demonstrating its expression in tubular compartment. In fact, during the annual reproductive cycle, both proteins increase in September, when the appearance of spermatids (SPT) occurs. Immunocytochemistry confirms their localization in germ cells and, in particular, in elongated SPT. Signals are still present in spermatozoa (SPZ), as demonstrated by Western blot analysis. Furthermore, the activation of CNR1 reduces sperm motility. Comparative research, carried out using mouse and rat SPZ, definitely indicates that the endocannabinoid system operates in SPZ of phylogenetically distant species. A conserved physiological role of endocannabinoid system in controlling the inhibition of sperm motility is suggested.

Immunoreactive GnRH in Hypothalamic and Extrahypothalamic Areas

Publisher Summary This chapter reviews the presence and the physiological significance of gonadotropin-releasing hormone (GnRH) inside and outside the brain of vertebrates; surveys comparative aspects that reveal similarities and differences, discussed from the evolutionary and adaptive viewpoint. Immunoreactive GnRH perikarya are widespread in the brain even outside the hypothalamus. Immunoreactive GnRH fibers project in many directions alongwith the Median Eminence (ME). In Elasmobranchs, unique among vertebrates, their fibers project in various directions except ME. The discharge into the ventricular system and from there into the venous system is also a primitive mechanism of transport observed in the lamprey and stickleback. The possibility of alternative routes of communication with pituitary or other targets is hypothesized in Chondrichthyes. The detection of ir GnRH in the blood of Chondrichthyes is attractive in explaining a puzzling situation in these fish, as much as the brain may communicate with the pituitary. The involvement of messengers other than cyclic adenosine monophosphate (cAMP) is a general feature of the mechanism of action of GnRH at pituitary and gonadal levels.

Global Gene Expression Profiling Of Human Pleural Mesotheliomas: Identification of Matrix Metalloproteinase 14 (MMP-14) as Potential Tumour Target

Background The goal of our study was to molecularly dissect mesothelioma tumour pathways by mean of microarray technologies in order to identify new tumour biomarkers that could be used as early diagnostic markers and possibly as specific molecular therapeutic targets. Methodology We performed Affymetrix HGU133A plus 2.0 microarray analysis, containing probes for about 39,000 human transcripts, comparing 9 human pleural mesotheliomas with 4 normal pleural specimens. Stringent statistical feature selection detected a set of differentially expressed genes that have been further evaluated to identify potential biomarkers to be used in early diagnostics. Selected genes were confirmed by RT-PCR. As reported by other mesothelioma profiling studies, most of genes are involved in G2/M transition. Our list contains several genes previously described as prognostic classifier. Furthermore, we found novel genes, never associated before to mesotheliom that could be involved in tumour progression. Notable is the identification of MMP-14, a member of matrix metalloproteinase family. In a cohort of 70 mesothelioma patients, we found by a multivariate Cox regression analysis, that the only parameter influencing overall survival was expression of MMP14. The calculated relative risk of death in MM patients with low MMP14 expression was significantly lower than patients with high MMp14 expression (P = 0.002). Conclusions Based on the results provided, this molecule could be viewed as a new and effective therapeutic target to test for the cure of mesothelioma.

Intratesticular feedback mechanisms in the regulation of steroid profiles in the frog, Rana esculenta.

Testosterone (T), 5 alpha-dihydrotestosterone (DHT), estradiol-17 beta (E), and progesterone (P) were measured in the plasma of the frog, Rana esculenta, during the annual cycle. Moreover, in vitro experiments were carried out in order to investigate the local regulation of steroidogenesis. Testosterone and DHT showed high values during autumn and early spring and had a T/DHT ratio which increased during summer, while E peaked in midspring, remaining at detectable values thereafter. Progesterone increased in autumn, winter, and spring. In vitro incubations of minced testes showed that E, stimulated by pituitary factors, inhibited androgen synthesis while T did not. Our results indicate that paracrine and/or autocrine mechanisms operate in the frog testis to regulate annual steroid profiles.

A Gradient of 2-Arachidonoylglycerol Regulates Mouse Epididymal Sperm Cell Start-Up

Abstract During transit through the epididymis, spermatozoa are normally kept immotile and do not attain the ability to become motile until they reach the caudal epididymis. This study was undertaken to determine whether endocannabinoids play a role in the epididymis and in particular in suppressing the ability of spermatozoa to become motile. We show that the levels of the endocannabinoid 2-arachidonoylglycerol (2-AG) are high in mouse spermatozoa isolated from the caput (head) of the epididymis, where these cells do not move (or possess sluggish and irregular motility) and decrease dramatically in spermatozoa isolated from the cauda (tail). The subsequent gradient regulates, via autocrine communication, the activity of cannabinoid receptor CNR1 (previously known as CB1) present on the sperm cell membrane and induces caudal spermatozoa to acquire the potential to become motile (“start-up”). Accordingly, the genetic or pharmacological inactivation of CNR1 increases number of motile spermatozoa in caput. Also, blockers of endocannabinoid cellular uptake inhibit the potential to move of spermatozoa and destroy the 2-AG gradient throughout the epididymis. This gradient-regulated mechanism may encourage further research for future therapies related to male infertility.

Molecular forms of immunoreactive gonadotropin-releasing hormone in hypothalamus and testis of the frog, Rana esculenta.

The hypothalamus and the testis of the frog, Rana esculenta, contain gonadotropin-releasing hormone (Gn-RH)-like peptides which are recognized by an antiserum raised against mammalian Gn-RH. Two molecular forms which coelute with synthetic chicken II and salmon Gn-RH from reverse-phase HPLC were distinguished in the hypothalamus. A single peak coeluting with synthetic chicken II Gn-RH was present in the testis.

Expression of Type-1 Cannabinoid Receptor During Rat Postnatal Testicular Development: Possible Involvement in Adult Leydig Cell Differentiation

Abstract Endocannabinoids are lipidic modulators able to bind cannabinoid receptors (CNRs). Two types of CNRs have been cloned, CNR1 (central) and CNR2 (peripheral). The objectives of the present study were to investigate the expression pattern of CNR1 in the rat testis during prepubertal development and to define the CNR1 spatiotemporal pattern. From 31 to 60 days of age, CNR1 was immunolocalized in round elongating spermatids and spermatozoa, suggesting an important role for this receptor in spermatogenesis. From 14 to 60 days of age, adult Leydig cells (ALCs) at different developmental stages were positive for CNR1. In particular, CNR1 expression in differentiating ALCs was negatively correlated to cell division. Bromodeoxyuridine uptake experiments on serial sections showed that immature Leydig cells in mitosis were negative for CNR1; in contrast, immature nonmitotic Leydig cells were positive for CNR1. A further observation of few ALCs in CNR1KO mice validates the role of CNR1 during proliferative activity involved in ALC differentiation. In addition, starting from 41 days of age, a faint CNR1 signal was also observed in Sertoli cells. Taken together, these results demonstrate the first clear evidence (to our knowledge) of CNR1 in mammalian germinal epithelium, ALCs, and Sertoli cells and indicate that differentiation of ALCs may depend on the endocannabinoid system.

The role of endocannabinoids in gonadal function and fertility along the evolutionary axis.

Endocannabinoids are natural lipids able to bind to cannabinoid and vanilloid receptors. Their biological actions at the central and peripheral level are under the tight control of the proteins responsible for their synthesis, transport and degradation. In the last few years, several reports have pointed out these lipid mediators as critical signals, together with sex hormones and cytokines, in various aspects of animal and human reproduction. The identification of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in reproductive cells and tissues of invertebrates, vertebrates and mammals highlights the key role played by these endogenous compounds along the evolutionary axis. Here, we review the main actions of endocannabinoids on female and male reproductive events, and discuss the interplay between them, steroid hormones and cytokines in regulating fertility. In addition, we discuss the involvement of endocannabinoid signalling in ensuring a correct chromatin remodeling, and hence a good DNA quality, in sperm cells.

Cannabinoid receptor 1 influences chromatin remodeling in mouse spermatids by affecting content of transition protein 2 mRNA and histone displacement.

Marijuana smokers and animals treated with Δ9-tetrahydrocannabinol, the principal component of marijuana, show alterations of sperm morphology suggesting a role for cannabinoids in sperm differentiation and/or maturation. Because the cannabinoid receptor 1 (CNR1) activation appears to play a pivotal role in spermiogenesis, the developmental stage where DNA is remodeled, we hypothesized that CNR1 receptors might also influence chromatin quality in sperm. We used Cnr1 null mutant (Cnr1-/-) mice to study the possible role of endocannabinoids on sperm chromatin during spermiogenesis. We demonstrated that CNR1 activation regulated chromatin remodeling of spermatids by either increasing Tnp2 levels or enhancing histone displacement. Comparative analysis of wild-type, Cnr1+/-, and Cnr1-/- animals suggested the possible occurrence of haploinsufficiency for Tnp2 turnover control by CNR1, whereas histone displacement was disrupted to a lesser extent. Furthermore, flow cytometry analysis demonstrated that the genetic loss of Cnr1 decreased sperm chromatin quality and was associated with sperm DNA fragmentation. This damage increased during epididymal transit, from caput to cauda. Collectively, our results show that the expression/activity of CNR1 controls the physiological alterations of DNA packaging during spermiogenesis and epididymal transit. Given the deleterious effects of sperm DNA damage on male fertility, we suggest that the reproductive function of marijuana users may also be impaired by deregulation of the endogenous endocannabinoid system.

The endocannabinoid system in vertebrate male reproduction: A comparative overview

Prevailing studies emphasize on endocannabinoid activity in brain. However, sporadic evidences hint that endocannabinoid system controls male reproduction ranging from invertebrates to vertebrates. Although N-arachidonoylethanolamine is described in rat testis, its activity is still poorly known. Type-1 cannabinoid receptor and fatty acid amide hydrolase are particularly expressed in elongating spermatids and spermatozoa suggesting that endocannabinoids affect spermiogenesis and sperm physiology. Aim of this paper is to provide an analysis of the information available in vertebrates on male germ cell progression and sperm maturation mediated by the endocannabinoid system.