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

BIOLOGY OF REPRODUCTION 79, 758–765 (2008) Published online before print 9 July 2008. DOI 10.1095/biolreprod.108.070128 Expression of Type-1 Cannabinoid Receptor During Rat Postnatal Testicular Development: Possible Involvement in Adult Leydig Cell Differentiation1 Giovanna Cacciola,3 Teresa Chioccarelli,3 Ken Mackie,4 Rosaria Meccariello,5 Catherine Ledent,6 Silvia Fasano,3 Riccardo Pierantoni,2,3 and Gilda Cobellis3 of D9-tetrahydrocannabinol, the primary psychoactive cannabinoid in marijuana (Cannabis sativa) [4]. This was followed many years later by the cloning of type-1 and type-2 CNRs (CNR1 and CNR2, respectively) [5, 6] and by the identification of their endogenous ligands, the endocannabinoids. Cannabinoid receptors are members of the superfamily of seven transmembrane-spanning (7-TM) receptors, having a protein structure defined by an array of seven membranespanning helices with intervening intracellular domains that can associate with Gi–Go proteins [7]. CNR1 is expressed primarily in the central nervous system and to a lesser extent in several peripheral tissues, including the testis; in contrast, CNR2 is distributed predominantly in immune cells [7]. All endocannabinoids are structurally distinct from plantderived compounds and synthetic cannabinoids. They are amides, esters, and ethers of long-chain polyunsaturated fatty acids and are able to bind both CNRs, although with different affinities and efficacies [8]. Furthermore, they are able to produce some of the peripheral and central effects of tetrahydrocannabinol [9]. To date, two main endocannabinoids have been described, arachidonoyletanolamide (AEA) [10] and 2-arachidonoylglycerol (2-AG) [11]. They are released enzymatically on demand from membrane phospholipids when cells are stimulated by depolarizing agents, neurotransmitters, and hormones [12]. Endocannabinoid signaling is efficiently limited by a two-step degradation process involving facilitated uptake from the extracellular space and intracellular catabolism mediated by specific enzymes. Cannabinoid receptors, endocannabinoids, and the machinery for their synthesis and degradation represent the elements of a novel endogenous signaling system (the endocannabinoid system) involved in a plethora of physiological functions [3]. In different species, CNRs have been found in testis [13–15] and isolated spermatozoa [16–18]. In testis, CNR1 has been observed in Leydig cells [14] and in several spermatogenic stages [15], as well as in the seminiferous epithelium of the amphibian frog Rana esculenta [17, 19]. In man and rodents, chronic exposure to or use of cannabinoids decreases testosterone production and secretion by Leydig cells [20, 21], depresses spermatogenesis [22], and reduces the weight of testes and accessory reproductive organs [23] (see review by Wang et al. [24]). The association between AEA (anandamide) injection and reduction in luteinizing hormone and testosterone levels in CD1 wild-type (WT) mice but not in CNR1 knockout (CNR1KO) mice confirms the importance of the adverse effects of cannabinoids on the testis [14]. The findings that male genital tract fluids contain endocannabinoids [25] and that the rat testis is able to synthesize AEA [26] suggest that these lipid-signaling 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. CNR1, endocannabinoid system, Leydig cells, prepubertal development, testis INTRODUCTION Cannabinoid receptors (CNRs) bind a class of lipid mediators, the endocannabinoids, that is involved in several physiological processes (e.g., food intake, memory, energy balance, immune system, and reproduction) [1–3]. In the past few decades, the increasing recreational use of cannabinoids such as marijuana and the potential negative health effects have reinforced public concern about these compounds. Cannabinoid research received an initial boost from the characterization 1 Supported by grants from PRIN and Regione Campania (legge 5) and by a fellowship from Istituto Nazionale Biostrutture e Biosistemi to G. Cacciola. 2 Correspondence: Riccardo Pierantoni, Dipartimento di Medicina Sperimentale, Seconda Università degli Studi di Napoli, 80138 Napoli, Italy. FAX: 39 081 566 7536; e-mail: Received: 21 April 2008. First decision: 11 May 2008. Accepted: 12 June 2008. Ó 2008 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org 758 Dipartimento di Medicina Sperimentale,3 Seconda Università degli Studi di Napoli, 80138 Napoli, Italy Department of Psychological and Brain Sciences,4 Indiana University, Bloomington, Indiana 47405 Dipartimento di Studi Delle Istituzioni Dei Sistemi Territoriali,5 Università ‘‘Parthenope’’ di Napoli, 80133 Napoli, Italy Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire,6 Université Libre de Bruxelles, Campus Erasme, 1050 Brussels, Belgium TYPE-1 CANNABINOID RECEPTOR IN RAT TESTIS MATERIALS AND METHODS Experimental Animals Sprague-Dawley rats (Rattus norvegicus) from our colony were housed in the Biotechnology Center A.O.R.N. Cardarelli (Naples, Italy) and were maintained on a standard pellet diet with free access to water. Males were killed at Postnatal Day 7, 14, 21, 31, 35, 41, 60, or 90, and the testes were rapidly removed. One testis per animal was frozen on dry ice for subsequent Western blot and RT-PCR analyses, whereas the contralateral testis was fixed for immunohistochemistry. CD1 WT and CNR1KO (official symbol, Cnr1tm1Map) mice (Mus musculus) (Charles River) [28] were killed at Postnatal Day 33 or 60. Testes were fixed in Bouins fluid according to standard procedures and were stained using (...truncated)