Muscarinic acetylcholine receptor subtypes in the male reproductive tract: expression and function in rat efferent ductules and epididymis.

In mammals, at least five different muscarinic acetylcholine receptor subtypes (mAChRs; M(1)-M(5)) are known to be widely expressed and distributed in different tissues from different species. They mediate distinct physiological functions according to their location and receptor subtype. Multiple events are associated with the regulation of intracellular signaling by mAChRs, and a coordinated balance of the molecular mechanisms governing receptor signaling, desensitization, resensitization, and mitogenic signaling is known to occur in various cell types. Most of the actions of acetylcholine (ACh) in the male reproductive tract are induced by its effects on mAChRs, but the role of specific mAChR subtypes on male reproductive function and fertility are still not well understood. The rat efferent ductules and epididymis are androgen-dependent tissues of the male reproductive tract, with important roles in the process to form a viable and fertile sperm. In the present study, aspects of the expression, localization, and potential function of mAChR subtypes in rat efferent ductules and epididymis are reviewed. Furthermore, evidences for the implication of mAChRs in the regulation of protein synthesis and secretion in these tissues are presented. Taken together, the studies contribute to our understanding about physiological aspects of mAChR and mechanisms by which the cholinergic system affects male reproduction.

Dual effect of agmatine in the bisected rat vas deferens.

The functional effects of the amine agmatine, the putative endogenous ligand for alpha(2)-adrenoceptors and imidazoline receptors, in rat vas deferens were investigated by using the epididymal and prostatic portions. Tissues were contracted by electrical stimulation or by exogenous drugs. In electrically stimulated portions, agmatine caused a dual effect on contractions. In the epididymal portion an inhibition on twitch contractions was observed, which was partially antagonised by idazoxan and yohimbine, indicating the involvement of at least a presynaptic alpha(2)-adrenoceptor-mediated mechanism, without the interference of imidazoline receptors. In the prostatic portion, agmatine enhanced the amplitude of twitches. In contractions induced by exogenous drugs, agmatine potentiated, only in the prostatic segment, the effects of noradrenaline (norepinephrine) or ATP; it also enhanced the effect of low concentrations of KCl and blocked the maximum effect of the higher concentrations. Effects induced by agmatine on the exogenous ATP in the prostatic portion were blocked by cromakalim, suggesting a blocking action on the postsynaptic K(+) channels, which explains, in part, the potentiation of the twitch amplitude. It was concluded that agmatine interferes with sympathetic neurotransmission, but the physiological relevance of this needs to be better understood because of the high doses employed to induce its effects.

Segment-specific decrease of both catecholamine concentration and acetylcholinesterase activity are accompanied by nerve refinement in the rat cauda epididymis during sexual maturation.

In the present work, histochemical and biochemical studies were conducted to analyze changes in the pattern of autonomic innervation during sexual maturation, using the rat epididymis as a model. Glyoxylic acid histochemistry and immunohistochemical studies against dopamine beta-hydroxylase (DbetaH) and acetylcholinesterase (AChE) indicated a reduction in the amount of catecholaminergic and AChE-positive neurons, fibers, and puncta detected in the cauda epididymis of adult rats (120 days old), when compared to immature (40 days) and young adult (60 days) animals. No obvious age-related variations were detected in the few catecholaminergic and AChE-positive fibers and puncta present in the caput region. AChE-positive fibers were found sorting out among epithelial cells and ending free upon the epithelial surface or into the tubular lumen of the cauda region of adult rats. Furthermore, a positive staining for AChE in epithelial cells was also detected in the caput and cauda epididymis in all ages studied. Biochemical analysis confirmed a significant decrease in noradrenaline concentration as well as AChE activity in the cauda epididymis with sexual maturation. Immunohistochemical studies against microtubule-associated protein 1B (MAP 1B), a neuronal cytoskeletal marker, further substantiated the quantitative changes observed in catecholaminergic and AChE-positive neuronal elements in the cauda epididymis. Thus, our results documented segment-specific variations in noradrenaline concentration and AChE activity during epididymal sexual maturation and suggest that such variations result, at least in part, from the refinement of the autonomic innervation pattern with age.

Rat epididymal sperm quantity, quality, and transit time after guanethidine-induced sympathectomy.

Guanethidine, a chemical that selectively abolishes peripheral noradrenergic nerves, was used to investigate the role of sympathetic innervation in the maintenance of epididymal sperm quantity and quality. Four groups of 10 adult male rats each were treated daily for 21 days, by i.p. injections, with either 0 (saline vehicle), 6.25, 12.5, or 25 mg/kg guanethidine. Norepinephrine content was reduced to undetectable levels in the cauda epididymidis in all guanethidine groups after 3 wk of treatment and was reduced to 7.4% of the control values after 1 wk of 6.25 mg/kg treatment. While body weight gain was significantly decreased at 12.5 and 25 mg/kg compared to that in controls, there was a significant increase in the weights of the seminal vesicles/coagulating glands in all treated groups. The number of homogenization-resistant spermatids per testis and the daily sperm production per testis remained unchanged. The weight of the epididymis was significantly increased at 6.25 and 12.5 mg/kg. Moreover, the number of cauda epididymal sperm and the transit time were increased significantly at 6.25 mg/kg (10.2 days) compared to values in the control cauda (6.3 days). Neither serum testosterone levels nor LH was affected in a dosage-related manner. There were no effects of guanethidine treatment on cauda epididymal sperm motility or morphology. A quantitative analysis of detergent-extracted cauda epididymal sperm proteins by SDS-PAGE revealed no differences, but there were diminutions in seven proteins in homogenates of caput/corpus tissue. Histologic analysis of testis and epididymis sections revealed no differences between control and denervated animals. In a subsequent experiment the lowest effective dosage (6.25 mg/kg) was given to rats for 1 wk, and an increased number of cauda epididymal sperm and a delay in sperm transit were observed. Our results indicate that low-dosage guanethidine exposure denervates the epididymis within 1 wk, thereby delaying epididymal transit; however, neither 1- nor 3-wk exposure produces qualitative changes in the sperm.

Fertility of rat epididymal sperm after chemically and surgically induced sympathectomy.

Guanethidine, a chemical that selectively blocks sympathetic noradrenergic neurons, was used to investigate the role of sympathetic innervation in the fertility of rat epididymal sperm, using both natural mating and in utero insemination protocols. This animal model correlates, at least in part, with spinal cord injury (SCI) in men. Adult male rats were treated daily by i.p. injections, for 21 or 42 days, with 0 or 6.25 mg/kg guanethidine. To compare the effects of guanethidine-induced sympathectomy with those following surgically induced sympathectomy, the inferior mesenteric ganglion and the proximal hypogastric nerves were removed in another group of rats. Both chemically and surgically induced sympathectomy increased the weight of the epididymis and seminal vesicles/coagulating glands as well as the number and the transit time of cauda epididymal sperm. Neither serum testosterone levels nor LH was affected by treatment with guanethidine. Using natural mating, no litters were produced by guanethidine-treated rats. Chemically denervated rats failed to produce copulatory plugs or ejaculate into the uterus. However, distal cauda epididymal sperm from chemically or surgically denervated rats displayed normal fertilization ability (80%) using in utero inseminations. In addition, the sperm of denervated rats did not show abnormal sperm chromatin structure using an assay that detects DNA damage. We conclude that sympathectomy delays the transit of sperm through the cauda epididymidis and produces ejaculatory dysfunction but does not compromise sperm quality in the distal cauda epididymidis. Moreover, these data provide compelling evidence that there is no association between the prolonged transit time of sperm within the epididymis, i.e., pre-ejaculatory sperm aging, and the fertility of those sperm, which has important implications for artificial insemination using sperm from men with SCI.

Effect of acute cold exposure on norepinephrine turnover rates in rat white adipose tissue.

The present studies were carried out to assess directly sympathetic activity in white adipose tissue in response to cold exposure. Norepinephrine (NE) content and NE turnover rates were determined in epididymal and retroperitoneal adipose tissue from rats exposed to cold (4 degrees C) and controls kept at ambient temperature. Parallel measurements were made in interscapular brown adipose tissue (IBAT), in which activation of catecholaminergic innervation by cold exposure is well known. Exposure to 4 degrees C for 4 h reduced the endogenous NE content by 50% in IBAT and by 30% in both epididymal and retroperitoneal adipose tissues. Compared to warm controls, average values of fractional rates of turnover and cf turnover rates, estimated with alpha-methyl-tyrosine, increased 5-fold in IBAT and 2.5-3-fold in epididymal and retroperitoneal tissues from rats exposed to cold. The present data provide the first direct evidence that white adipose tissue sympathetic activity is increased during acute cold exposure.

Morphometric evaluation of the rat testis, epididymis and vas deferens following chemical sympathectomy with guanethidine.

Selective chemical sympathectomy of the internal sex organs of adult male rats was undertaken by long term administration of low doses of guanethidine. The spermatogenic activity of the testis was unaffected by treatment. Examination of the vas deferens using morphometric methods revealed a marked increase in luminal area in contrast to a decrease in muscle layer area and in epithelial height. This is morphological evidence of sperm accumulation caused by a disorder in ductal contractile activity. No structural changes were observed in the epididymis. However, the concentration of spermatozoa in the sperm suspension stored in the cauda epididymidis was significantly increased in denervated rats. This result is discussed in terms of a sympathetic control of resorption mechanisms in the epididymis.