Efeito da inibição da fosfolipase C pelo U73122 sobre o sêmen ovino

Abstract

The aim of this study was to evaluate the effect of U73122 on freezing ability of ram semen, since this reagent could prevent premature capacitation and acrosome reaction induced during the cryopreservation process. For this, ejaculates from 4 Santa Ines rams were obtained with the aid of artificial vagina and were used in two experiments. In experiment I, in natura sperm from 4 ejaculated were subjected to incubation at 37°C during 4h, supplemented or not with concentrations of 10, 20 or 30μM of U73122 in the presence or absence of glycerol. Kinetic parameters were evaluated during the incubation, in order to verify the behavior of sperm by the use of the reagent, and selecting doses to be used during cryopreservation. Sixteen ejaculates were used in the experiment II, which were cryopreserved with Botubov® extender only fraction increased or not U73122 reagent in concentrations of 10 and 20μM. Thawed samples were evaluated for sperm kinetics using the "Computer Assisted Sperm Analysis (CASA) system, and for membranes integrity by the association of fluorescent probes propidium iodide, PSA-FITC and JC-1. In addition, capacitation and acrosome reaction were evaluated using fluorescent probe hydrochloride chlortetracycline (CTC) before and after the cryopreserved semen samples undergoing induction in vitro capacitation for 4h of incubation at 37°C. The data were submitted to analysis of variance and least significant difference test (DMS) of Tukey at 5% probability. The treatment of in nature spermatozoa with different concentrations of U73122 for 4h incubation at 37°C, revealed a dose-effect and time dependent on the use of this reagent in spermatozoa. Furthermore, the presence of glycerol potentiated the toxic effect of U73122 on the cells. In cryopreserved sperm, the U73122 did not affect any of the kinetic parameters evaluated by CASA system, nor the number of sperm with the plasma membrane, acrosomal and mitochondrial you merge (PIAIC) or injured (PLALS). The evaluations carried out immediately after thawing showed that the use of 10 or 20μM of inhibitor reduced the percentage of capacitated sperm and acrosome reacted in the control group (p>0.05). After induction of in vitro capacitation, there was a decrease (p<0.05) in the number of uncapacitated sperm in all treatments suggesting a reversible effect of U73122 on sperm capacitation process and acrosome reaction. In conclusion, that the use of 10 uM of U73122 reagent does not alter the kinetics of sperm in natura and, 10 or 20 uM concentrations of this reagent can be used in diluents for cryopreservation, since they prevent premature capacitation and acrosome reaction induced during this process, without altering the kinetics and the integrity of the sperm membrane.