In Vitro Contraction of Isolated Cauda Epididymal Duct Smooth Muscle as a Complimentary Approach to Physiological, Pathological, Toxicological, and Pharmacological Studies on Epididymal Function.

Contraction of cauda epididymal duct (CE) smooth muscle is one of the very first events of the seminal emission phase of ejaculation. The contraction of CE smooth muscle is governed by a complex interaction of hormones, autacoids, and by the neurotransmitters released from the epididymal intramural nerve endings, and any impairment in the CE smooth muscle contraction has the potential to impair male fertility. Apart the obvious pathophysiological and toxicological importance of CE smooth muscle contraction, modulation of CE contraction has pharmaceutical interest offering a druggable target to development of drugs to improve/impair male fertility. The in vitro contraction experiments constitute a valuable approach to an in-depth evaluation of functional and molecular changes resulting from pathologies or drug exposure. Therefore, this chapter consists in a description of in vitro pharmacological reactivity contractility of the epididymal duct in a controlled medium, maintained at 30 °C of temperature and continuously bubbled with 95% O2 and 5% CO2 to obtain cumulative concentration-response curves that has been fundamental to some of our investigations on epididymal physiology, toxicology, and pharmacology.

Cardiac hyporesponsiveness in severe sepsis is associated with nitric oxide-dependent activation of G protein receptor kinase.

G protein-coupled receptor kinase isoform 2 (GRK2) has a critical role in physiological and pharmacological responses to endogenous and exogenous substances. Sepsis causes an important cardiovascular dysfunction in which nitric oxide (NO) has a relevant role. The present study aimed to assess the putative effect of inducible NO synthase (NOS2)-derived NO on the activity of GRK2 in the context of septic cardiac dysfunction. C57BL/6 mice were submitted to severe septic injury by cecal ligation and puncture (CLP). Heart function was assessed by isolated and perfused heart, echocardiography, and ß-adrenergic receptor binding. GRK2 was determined by immunofluorescence and Western blot analysis in the heart and isolated cardiac myocytes. Sepsis increased NOS2 expression in the heart, increased plasma nitrite + nitrate levels, and reduced isoproterenol-induced isolated ventricle contraction, whole heart tension development, and ß-adrenergic receptor density. Treatment with 1400W or with GRK2 inhibitor prevented CLP-induced cardiac hyporesponsiveness 12 and 24 h after CLP. Increased labeling of total and phosphorylated GRK2 was detected in hearts after CLP. With treatment of 1400W or in hearts taken from septic NOS2 knockout mice, the activation of GRK2 was reduced. 1400W or GRK2 inhibitor reduced mortality, improved echocardiographic cardiac parameters, and prevented organ damage. Therefore, during sepsis, NOS2-derived NO increases GRK2, which leads to a reduction in ß-adrenergic receptor density, contributing to the heart dysfunction. Isolated cardiac myocyte data indicate that NO acts through the soluble guanylyl cyclase/cGMP/PKG pathway. GRK2 inhibition may be a potential therapeutic target in sepsis-induced cardiac dysfunction.NEW & NOTEWORTHY The main novelty presented here is to show that septic shock induces cardiac hyporesponsiveness to isoproterenol by a mechanism dependent on nitric oxide and mediated by G protein-coupled receptor kinase isoform 2. Therefore, G protein-coupled receptor kinase isoform 2 inhibition may be a potential therapeutic target in sepsis-induced cardiac dysfunction.

Long-term adverse effects on reproductive function in male rats exposed prenatally to the glucocorticoid betamethasone.

Betamethasone is the drug of choice for antenatal treatment, promoting fetal lung maturation, decreasing the incidence of respiratory distress syndrome and neonatal mortality. Previous studies reported that prenatal treatment with this drug reduced testosterone levels, sperm quality and fertility in adult rats. We aimed to further evaluate the reproductive consequences of prenatal betamethasone exposure in male rats. Pregnant Wistar rats (n=13/group) were separated into two groups: control (vehicle) and betamethasone- treated (0.1mg/kg IM) and rats were injected on gestational days 12, 13, 18 and 19. Body weight, sexual behavior, reproductive organ weights, serum hormone levels, accessory glands contractility, sperm parameters, and fertility after in utero artificial insemination were evaluated. Our results showed that prenatal betamethasone exposure provoked a significant reduction in body weight at PND 01 and, at adulthood, decrease in FSH levels, sperm motility and production. Furthermore, seminal vesicle weight was decreased while testicular and ventral prostate weights were increased. Serum LH levels and the percentage of abnormal sperm were significantly increased. Although sexual behavior was not altered, a significant reduction in fertility in the adult rats exposed prenatally to betamethasone was noted. We concluded that prenatal betamethasone exposure leads to long-term reproductive impairment in male rats. These results may have important implications for humans, considering the use of this glucocorticoid in pregnant women.

Slimmer or fertile? Pharmacological mechanisms involved in reduced sperm quality and fertility in rats exposed to the anorexigen sibutramine.

Sperm acquire motility and fertility capacity during epididymal transit, under the control of androgens and sympathetic innervations. It is already known that the acceleration of epididymal sperm transit time can lead to lower sperm quality. In a previous work we showed that rats exposed to the anorexigen sibutramine, a non-selective serotonin-norepinephrine reuptake inhibitor, presented faster sperm transit time, lower epididymal sperm reserves and potentiation of the tension of epididymal duct to norepinephrine exposed acutely in vitro to sibutramine. In the present work we aimed to further investigate pharmacological mechanisms involved in these alterations and the impact on rat sperm quality. For this, adult male Wistar rats were treated with sibutramine (10 mg/kg/day) or vehicle for 30 days. Sibutramine decreased final body, seminal vesicle, ventral prostate and epididymal weights, as well as sperm transit time in the epididymal cauda. On the contrary of the in vitro pharmacological assays, in which sibutramine was added directly to the bath containing strips of distal epididymal cauda, the ductal tension was not altered after in vivo sub-chronic exposure to sibutramine. However, there is pharmacological evidence that the endogenous epididymal norepinephrine reserves were reduced in these animals. It was also shown that the decrease in prostate weight can be related to increased tension developed of the gland, due to sibutramine sympathomimetic effects. In addition, our results showed reduced sperm quality after in utero artificial insemination, a more sensitive procedure to assess fertility in rodents. The epididymal norepinephrine depletion exerted by sibutramine, associated with decreases in sperm transit time, quantity and quality, leading to reduced fertility in this experimental model, reinforces the concerns about the possible impact on fertility of man taking sibutramine as well as other non-selective serotonin-norepinephrine reuptake inhibitors, especially considering the lower reproductive efficiency of humans compared to males of other species.