Giovanna Cacciola

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.

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.

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.

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.

Testicular Gonadotropin-releasing Hormone Activity, Progression of Spermatogenesis, and Sperm Transport in Vertebrates

Since the end of the 1970s, studies have shown that, besides the endocrine route, a chemical mediator may also act through autocrine and/or paracrine mechanisms. This has opened new frontiers for research as a result of a redefinition of what endocrinology represents. Apart from androgens within the male gonad, testicular gonadotropin‐releasing hormone, estrogens, molecular chaperones, proto‐oncogenes, and, very recently, the endocannabinoid system have been shown to play important roles. Their activities to regulate spermatogenesis, including spermiogenesis and sperm maturation, will be discussed from the comparative viewpoint to describe adaptive phenomena and to speculate on evolution.

The contribution of lower vertebrate animal models in human reproduction research.

Many advances have been carried out on the estrogens, GnRH and endocannabinoid system that have impact in the reproductive field. Indeed, estrogens, the generally accepted female hormones, have performed an unsuspected role in male sexual functions thanks to studies on non-mammalian vertebrates. Similarly, these animal models have provided important contributions to the identification of several GnRH ligand and receptor variants and their possible involvement in sexual behavior and gonadal function regulation. Moreover, the use of non-mammalian animal models has contributed to a better comprehension about the endocannabinoid system action in several mammalian reproductive events. We wish to highlight here how non-mammalian vertebrate animal model research contributes to advancements with implications on human health as well as providing a phylogenetic perspective on the evolution of reproductive systems in vertebrates.

Low 17beta-Estradiol Levels in Cnr1 Knock-Out Mice Affect Spermatid Chromatin Remodeling by Interfering with Chromatin Reorganization

ABSTRACT The type 1-cannabinoid receptor, CNR1, regulates differentiation of spermatids. Indeed, we have recently reported that the genetic inactivation of Cnr1 in mice influenced chromatin remodeling of spermatids, by reducing histone displacement and then sperm chromatin quality indices (chromatin condensation and DNA integrity). Herein, we have studied, at both central and testicular levels, the molecular signals potentially involved in histone displacement. In particular, investigation of the neuroendocrine axis involved in estrogen production demonstrated down-regulation of the axis supporting FSH/estrogen secretion in Cnr1-knockout male mice. Conversely, Cnr1-knockout male mice treated with 17beta-estradiol showed a weak increase of pituitary Fsh-beta subunit mRNA levels and a rescue of sperm chromatin quality indices demonstrating that estrogens, possibly in combination with FSH secretion, play an important role in regulating chromatin remodeling of spermatids.

CB1 activity in male reproduction: mammalian and nonmammalian animal models

The importance of the endocannabinoid system (ECBS) and its involvement in several physiological processes is still increasing. Since the isolation of the main active compound of Cannabis sativa, Delta(9)-THC, several lines of research have evidenced the basic roles of this signaling system mainly considering its high conservation during evolution. In this chapter the attention is focussed on the involvement of the ECBS in the control of male reproductive aspects at both central and local levels which are both considered from a comparative point of view.

The Endocannabinoid System: An Ancient Signaling Involved in the Control of Male Fertility

The effects of cannabinoids on human health have been known since the antiquities when the extract of the plant Cannabis sativa was used because of its psychoactivity. The scientific story of the cannabinoids started in the 1960s with the isolation and characterization of the active component of the plant. After the synthesis of cannabinoid analogues, the analysis of structure–effect relationships was implemented, and this had a similar effect to a positive “Pandoras box” opening. To date, numerous roles have been ascribed to the “endocannabinoid system.” Here we describe its involvement in the control of male reproduction, taking into consideration possible evolutionary speculations. Indeed, the endocannabinoid system is a very ancient signaling system, being clearly present from the divergence of the protostomian/deuterostomian.