Avaliação funcional da neurotransmissão colinérgica no epidídimo, ducto eferente e ducto deferente de rato e seu envolvimento com a secreção de clusterina

Abstract

Previous studies from our laboratory have demonstrated the expression of acetylcholine M1, M2 and M3 muscarinic receptor subtypes in the efferent ducts and epididymis of rats, but their functions have not been fully clarified. Considering that clusterin (CLU) is an abundant protein in the epididymis and important for sperm maturation process, besides the involvement of autonomic innervation in protein secretion in the epididymis, the arm of this study was to evaluate the effects of cholinergic agonists and antagonists on CLU secretion in epididymis, efferent ducts and rat vas deferens. For that, adult male Wistar rats (CEUA-UFF 931/17) were randomized in 6 groups (n=6/group) and received 250μl (i.v.) containing saline (CO); carbacol (CA; 0.04 mg.k-1) in the absence or presence of antagonists: atropine (AT; 1.8 mg.k-1); pyrenzepine (PI; 0.5 mg.k-1) metoctramine (ME; 0,5 mg.k-1) or darifenacin (DA; 0.2 mg.k-1). The animals were kept under anesthesia and 2 hours after the treatments, the epididymis were removed and dissected. Homogenates were prepared for western blot assays or tissues, including efferent ducts and vas deferens, were embedded in paraffin for immunohistochemistry. Sperm evaluation (progressive motility, vigor, membrane integrity and hypo-osmotic test) was performed using sperm obtained from the epididymis cauda. Values are mean±SEM (one-way ANOVA, Newman-Keuls; P<0.05). The resuts showed that there was no statistical difference in CLU expression in the caput of epididymis among different experimental groups when compared to the control. In the proximal cauda of the epididymis, treatment with AT + CA, ME + CA and PI + CA induced an increase in CLU labeling in the apical and stereocilia region, with a more intense labeling with PI. DA + CA did not change the CLU stainning in this region. Such results are in agreement with results from western blot analysis, where PI + CA treatment considerably increased protein expression of CLU, more than AT and ME. In the vas deferens there was also increased expression of this protein with the same treatments and in the efferent ducts, pirenzepine, in particular, decreased the expression of CLU. Regarding the evaluation of sperm functionality, no parameter was changed in the different groups. In conclusion, our data suggest that blockade of M1 and M2 but not M3 subtype muscarinic receptors alters the expression of CLU in a variable manner, depending on the tissue and segment analyzed, suggesting the involvement of these receptors in the modulation of secretory processes and/or regulating protein expression and luminal fluid composition, important for male gamete maturation.