Reference Gene U2 Enables Direct Comparison between Relative Gene Expression Levels of Vascular Smooth Muscle Cells in Tissue and Culture Using Real-Time Quantitative PCR.

In nearly every lab, real-time quantitative polymerase chain reaction (qPCR) is used to quantify gene expression. However, a comparison of different samples requires the careful selection of suitable reference genes (RGs), sometimes referred to as housekeeping genes. In the case of vascular smooth muscle cells (vSMCs), it is important to know under which conditions gene expression in isolated and cultured vSMCs can be compared with vSMCs in a healthy blood vessel. We isolated the vSMC-containing layer of the rat aorta (tunica media) and used one (longitudinal) half for direct RNA extraction, while the other half served to isolate and culture vSMCs prior to RNA extraction. First, the expression of the routinely used RGs beta-actin (Actb) and Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) is investigated in intact media and corresponding cultured vSMCs. Significant differences in their Ct values show that these RGs could not be used for such direct comparisons; therefore, we select 15 different RGs. Only the gene expression of the small ribonuclear protein (snRNP) U2 shows no significant differences between the absolute Ct values of cultured vSMCs and the intact media; moreover, no differences were found between male and female rats in our experimental setup. In conclusion, U2 was shown to be an appropriate (sex-independent) RG to compare relative expression levels of vSMCs in culture to those vSMCs within their physiological tissue environment.

Treatment of benign prostatic hyperplasia and abnormal ejaculation: live imaging reveals tamsulosin - but not tadalafil - induced dysfunction of prostate, seminal vesicles and epididymis.

In brief: One of the most commonly prescribed benign prostatic hyperplasia (BPH) pharmacotherapies, the alpha1-adrenergic blocker tamsulosin, is frequently discontinued, especially by younger patients due to ejaculatory disorders, often without feedback to the attending physician. Using a newly developed ex vivo system simulating sympathetic effects on the most relevant structures for the emission phase of ejaculation, that is seminal vesicles, prostate and the most distal part of the cauda epididymidis, we elucidated that tamsulosin fundamentally disturbed the obligatory noradrenaline-induced contractions in each of these structures which differed to an alternative pharmacotherapy, the PDE5 inhibitor tadalafil. Abstract: Structures responsible for the emission phase of ejaculation are the seminal vesicles, the most distal part of the cauda epididymidis and the newly characterized prostate excretory ducts. The emission phase is mainly regulated by the sympathetic nervous system through alpha1-adrenergic receptor activation by noradrenaline at the targeted organs. BPH treatment with alpha1A-adrenergic antagonists such as tamsulosin is known to result in ejaculation dysfunction, often leading to discontinuation of therapy. Mechanisms of this disturbance remain unclear. We established a rodent model system to predict drug responses in tissues involved in the emission phase of ejaculation. Imitating the therapeutic situation, prostate ducts, seminal vesicles and the distal cauda epididymal duct were pre-incubated with the smooth muscle cell-relaxing BPH drugs tadalafil, a novel BPH treatment option, and tamsulosin in an ex vivo time-lapse imaging approach. Afterwards, noradrenergic responses in the relevant structures were investigated to simulate sympathetic activation. Noradrenaline-induced strong contractions ultimately lead to secretion in structures without pre-treatment. Contractions were abolished by tamsulosin in prostate ducts and seminal vesicles and significantly decreased in the epididymal duct. Such effects were not observed with tadalafil pre-treatment. Data visualized a serious dysfunction of each organ involved in emission by affecting alpha1-adrenoceptors localized at the relevant structures but not by targeting smooth muscle cell-localized PDE5 by tadalafil. Our model system reveals the mechanism of tamsulosin resulting in adverse effects during ejaculation in patients treated for BPH. These adverse effects on contractility do not apply to tadalafil treatment. This new knowledge translates directly to clinical medicine.

Novel imaging of the prostate reveals spontaneous gland contraction and excretory duct quiescence together with different drug effects.

Prostate carcinoma and benign prostate hyperplasia (BPH) with associated lower urinary tract symptoms (LUTS) are among the most prevalent and clinically relevant diseases in men. BPH is characterized by an enlargement of prostate tissue associated with increased tone of smooth muscle cells (SMCs) which surround the single glands composing the prostate. Secretions of the glands leave the prostate through local excretory ducts during the emission phase of ejaculation. Pharmacological treatment of BPH suggests different local drug targets based on reduction of prostate smooth muscle tone as the main effect and disturbed ejaculation as a common side effect. This highlights the need for detailed investigation of single prostate glands and ducts. We combined structural and functional imaging techniques-notably, clear lipid-exchanged, acrylamide-hybridized rigid imaging/immunostaining/ in situ hybridization-compatible tissue-hydrogel (CLARITY) and time-lapse imaging-and defined glands and ducts as distinct SMC compartments in human and rat prostate tissue. The single glands of the prostate (comprising the secretory part) are characterized by spontaneous contractions mediated by the surrounding SMCs, whereas the ducts (excretory part) are quiescent. In both SMC compartments, phosphodiesterase (PDE)-5 is expressed. PDE5 inhibitors have recently emerged as alternative treatment options for BPH. We directly visualized that the PDE5 inhibitors sildenafil and tadalafil act by reducing spontaneous contractility of the glands, thereby reducing the muscle tone of the organ. In contrast, the ductal (excretory) system and thus the prostate's contribution to ejaculation is unaffected by PDE5 inhibitors. Our differentiated imaging approach reveals new details about prostate function and local drug actions and thus may support clinical management of BPH.-Kügler, R., Mietens, A., Seidensticker, M., Tasch, S., Wagenlehner, F. M., Kaschtanow, A., Tjahjono, Y., Tomczyk, C. U., Beyer, D., Risbridger, G. P., Exintaris, B., Ellem, S. J., Middendorff, R. Novel imaging of the prostate reveals spontaneous gland contraction and excretory duct quiescence together with different drug effects.

Time-lapse imaging as a tool to investigate contractility of the epididymal duct--effects of cGMP signaling.

The well orchestrated function of epididymal smooth muscle cells ensures transit of spermatozoa through the epididymal duct during which spermatozoa acquire motility and fertilizing capacity. Relaxation of smooth muscle cells is mediated by cGMP signaling and components of this pathway are found within the male reproductive tract. Whereas contractile function of caudal parts of the rat epididymal duct can be examined in organ bath studies, caput and corpus regions are fragile and make it difficult to mount them in an organ bath. We developed an ex vivo time-lapse imaging-based approach to investigate the contractile pattern in these parts of the epididymal duct. Collagen-embedding allowed immobilization without impeding contractility or diffusion of drugs towards the duct and therefore facilitated subsequent movie analyses. The contractile pattern was made visible by placing virtual sections through the acquired image stack to track wall movements over time. By this, simultaneous evaluation of contractile activity at different positions of the observed duct segment was possible. With each contraction translating into a spike, drug-induced alterations in contraction frequency could be assessed easily. Peristaltic contractions were also detectable and throughout all regions in the proximal epididymis we found regular spontaneous contractile activity that elicited movement of intraluminal contents. Stimulating cGMP production by natriuretic peptide ANP or inhibiting degradation of cGMP by the phosphodiesterase 5 inhibitor sildenafil significantly reduced contractile frequency in isolated duct segments from caput and corpus. RT-PCR analysis after laser-capture microdissection localized the corresponding molecules to the smooth muscle layer of the duct. Our time-lapse imaging approach proved to be feasible to assess contractile function in all regions of the epididymal duct under near physiological conditions and provides a tool to evaluate acute (side) effects of drugs and to investigate various signaling pathways.

Phosphodiesterase 5 (PDE5) inhibition, ANP and NO rapidly reduce epididymal duct contractions, but long-term PDE5 inhibition in vivo does not.

Contractility of the peritubular smooth muscle layer ensures the transit of immotile spermatozoa through the epididymal duct to acquire their fertilizing capacity. Atrial natriuretic peptide (ANP) and nitric oxide (NO) affect contractility via cGMP signals that are controlled by phosphodiesterases (PDEs). Sildenafil inhibits the cGMP-hydrolyzing PDE5 and thereby promotes relaxation of smooth muscle cells. While sildenafil is increasingly used in young patients for the treatment of pulmonary hypertension, virtually no knowledge exists about PDEs in the epididymis. Western blotting, immunohistochemistry and RT-PCR analyses after laser capture microdissection localized PDE5 to smooth muscle cells, but not to epithelial cells, of the epididymal duct in man and rat. Sildenafil, ANP and NO significantly slowed spontaneous contractions of rat epididymal duct segments in organ bath studies. Sildenafil effects were additive to ANP and NO. Long-term exposure to sildenafil in vivo did not change the PDE5 expression or the observed contractility pattern with the rapid relaxing response toward ANP, NO and sildenafil. Data demonstrate that PDE5 is an important member of cGMP signaling pathways regulating the finely orchestrated process of epididymal duct contractility and suggest, however, that in the epididymis side effects of therapeutically used sildenafil are unlikely.