In vitro effects of ghrelin and hexarelin on mouse sperm functional activity

LUQUE EM; BERTOLDI M.L.; VINCENTI LM; DESIMONE M.; RUIZ RD; FIOL DE CUNEO M; MARTINI AC

Córdoba

Jornada; Onceavas jornadas de investigación científica en el marco del bicentenario; 2010

Facultad de Ciencias Médicas, UNC

The presence of the growth hormone secretagogues receptor 1a (GHS-R1a) has been demonstrated in mammalian gametes. Because seminal plasma contains its natural ligand, ghrelin (Ghr), the objectives of the present study were to evaluate the functional activity of mouse spermatozoa incubated in vitro with growth hormone stimulating substances (GHSs). Functional activity was evaluated in caudal epididymal spermatozoa obtained from adult Albino swiss mice. Experiment 1: gametes were incubated for 3 and 20 min with hexarelin (HEX), a ghrelin analogue usually employed in human therapy, at concentrations 10-5, 10-7 or 10-9 M. Experiment 2: sperm were incubated for 3 and 40 min with Ghr (10-7 or 10-9 M) in the presence or absence of an antagonist (D-Lys3 GHRP-6, 10-5 M). Sperm motility were evaluated in a Makler counting chamber, viability with Hoechst 33258 and acrosome integrity with FITC-PSA. Exp 1: addition of HEX for 3 min diminished the percentage of motile spermatozoa; this reduction was statistically significant only at 10-9 M (M±SEM: control 71.8±1.5, n=6; HEX 10-5M 63.8±4.2, n=6; HEX 10-7M 61.4±4.1, n=5 and HEX 10-9M 53.1±4.4, n=5, p<0.05). Sperm functional activity at 20 min incubation were not modified. Exp 2: Ghr did not modified sperm functional activity at any concentration or incubation time evaluated. The antagonist per se did not affected sperm function either. Previous studies demonstrated an increase in intracelullar Ca++ after in vitro exposure of pituitary cells to Ghr or HEX, with a peak at 3-5 min of incubation and at the lower concentrations employed in the present study. Intracelullar calcium raise may be responsible for the detrimental effect detected on sperm motion, since these gametes require small concentrations of this ion to retain motility. At 20 min of incubation however, sperm compensatory mechanisms (ie. Ca++ arrest) may operate to preserve motility. In our experimental conditions the role of Ca++ must be confirmed, as well as the presence of GHSs receptors, different from the GHS-R1a, with selective or higher affinity to HEX rather than to Ghr.