Studio del ruolo dei protoncogeni e cannabinoidi nei meccanismi di rilascio ed acquisizione della motilità degli spermatozoi nei vertebrati

Rana esculenta is a seasonal breeder, characterized by a period of resumption of spermatogonial proliferation (late winter- early spring), a well defined period of mating (March- April) and a post-reproductive period (Rastogi et al., 1972). This animal model, particularly suitable to study the role of molecules related to testicular physiology (Pierantoni et al., 2002), was used to analyze protoncogenes (Cobellis et al., 2002; Cobellis et al., 2003) and endocannabinoid activity. Besides the demonstration of Fra1 presence in a non-mammalian vertebrate gonad, we report, for the first time in a vertebrate, experimental evidence showing that the expression of Fra1 is related to peritubular myoid cells (PMCs).We confirm the effect of hypophysis omogenate (PD) on sperm release (Minucci et al., 1989) and demonstrate that PD increases Fra1 signal through recruitment of new PMCs expressing Fra1. Recruitment happens really when PMCs propel SPZ toward tubules and ducts, thus suggesting a new role for Fra1 in the control of sperm release from the tubular compartment. Sperm release includes two events: spermiation and sperm transport; boths contribute to the sperm output from the testis. In vertebrates, few data are available on the signals responsible for sperm release. We demonstrate that hypophysis and estrogens together regulate sperm release, mainly affecting spermiation. We also show, for the first time in a vertebrate, that estrogens trigger Fra1 activity in PMCs and simultaneously influence sperm release. In particular, impairment of estrogen activity by ICI 182-780 reduces sperm release, PD induced, mainly affecting spermiation. Animal, in fact, treated with PD+ICI present low number of spermiated tubules and cloacal spermatozoa (SPZ). Finally we show that in cloaca endocannabinoids control percentage of motile SPZ by sperm motility inhibition. We suppose that they could keep the SPZ motility quiescent until mating, when sperm release dilute endocannabinoids and induces sperm motilty acquisition through CNR1 activity impairment. Linear dilution of cloacal fluid, in fact, enhances the percentage of motile SPZ, demonstrating that cloacal fluid substances control the percentage of motile SPZ depending on their concentrations. Treatment of SPZ with increasing concentrations of SR141716A unequivocally confers this control to endocannabinoids through CNR1 activation. The analysis of CNR1 Knock out male mice demonstrates that, in absence of CNR1, SPZ acquire motility precociously. In the epididymus of wild type (WT) mice, in fact, we find higher percentage of motile SPZ in cauda than in caput. In CNR1KO mice, instead, the percentage of motile SPZ in caput is dramatically increased, clearly demonstrating that, in absence of CNR1 signalling, SPZ precociously acquire motility.