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

Polysialylation of NCAM characterizes the proliferation period of contractile elements during postnatal development of the epididymis.

Polysialic acid (polySia) attached to the neural cell adhesion molecule (NCAM) regulates inter alia the proliferation and differentiation via the interactions with neurotrophins. Since in postnatal epididymis neurotrophins and their receptors like the Low-Affinity Nerve Growth Factor Receptor p75 and TrK B receptor are expressed, we wanted to analyze if the polysialylation of NCAM is also involved during the development of the epididymis. To this end, we monitored the developmental changes in the expression of the polysialyltransferases and NCAM polysialylation using murine epididymis at different time points during postnatal development. Our results revealed that during postnatal development of the epididymis both polysialyltransferases, ST8SiaII and ST8SiaIV, were expressed and that the expression levels dropped with increasing age. In agreement with the expression levels of the polysialyltransferases the highest content of polysialylated NCAM was present during the first 10 days after birth. Interestingly, proliferating smooth muscle cell populations prevalently expressed polysialylated NCAM. Furthermore, we observed that inverse to the decrease in polysialylation of smooth muscle cells a strong up-regulation of collagen takes place suggesting a functional relationship since collagen was recently described to induce the turnover of polysialylated NCAM via an induction of endocytosis in cellulo. The same time course of polySia and collagen synthesis was also observed in other regions of the male reproductive system e.g. vas deferens and tunica albuginea (testis). Together, we identified a spatio-temporal expression pattern of polySia-NCAM characterized by high proliferation rate of smooth muscle cells and low collagen content

Immunohistological localization of polySia in vas deferens of postnatal mice.

<p>Transversal paraffin-embedded serial sections of vas deferens of 7 day old mice were stained with a mAb against polySia (A). For negative control, tissue sections were pretreated with endoN to degrade polySia (B). For identification of smooth muscle cells mAB against smooth muscle actin (C) were used as well as Azan staining (D) was performed. 1 = inner layer, 2 = middle layer, 3 = outer layer of smooth muscle cells surrounding the vas deferens. Cutouts show a higher magnification of the inner longitudinal layer of the muscle cells surrounding the vas deferens. Tissues (A-C) were counterstained with Haemalaun. Scale bars representing 20 μm.</p

Visualization of transport of intraluminal contents.

<p>Snapshot of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092603#pone.0092603.s004" target="_blank">Movie S4</a> resulting from time-lapse imaging (1 frame/s) of a piece of the epididymal duct in the caput region (scale bar: 100 μm) showing net transport of intraluminal contents. In the cross section of the time stack, this net transport is indirectly visible when observing the pattern between the epididymis walls. The darker areas in the cross section do not remain constant over time indicating that intraluminal contents have moved out of the cross section.</p

Spontaneous contractions in caput and corpus epididymidis.

<p>Visualization of contractility derived from virtual sections through the corresponding time stacks (as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092603#pone-0092603-g001" target="_blank">Fig. 1</a>) in examples from duct segments originating from caput (A) and corpus (B) epididymidis of different individuals (samples 1, 2 and 3). All movies were captured at 1 frame/s. Regular spontaneous contractility is visible in all samples of the caput and corpus region.</p

Demonstration of GC-A, GC-B and sGC expression in smooth muscle cells of the rat epididymal duct by LCM+RT-PCR.

<p>Laser-assisted microdissection (A,B) combined with RT-PCR (C) was used to localize GC-A, GC-B and sGC mRNA within the epididymis. A,B: Examples of excised segments of the epididymal smooth muscle layer. Scale bar corresponds to 150 μm. C: Microdissected epididymal smooth muscle cells (M) were analyzed by RT-PCR. Preparation from whole epididymis tissue (ET) served as positive control. The ribosomal protein RPS18 served as loading control. “+”, “−” and “0” indicate lanes with reverse transcriptase, without reverse transcriptase and water control, respectively. GC-A (172 bp), GC-B (234 bp) and sGC (261 bp) transcripts could be detected in microdissected smooth muscle layer. The quality of microdissection was assessed using additional primers for SMA (148 bp) as marker for smooth muscle cells and TRPV6 (259 bp) as marker for epithelial cells, respectively, to exclude the contamination of dissected parts of the smooth muscle layer with epithelial cells.</p

Visualization of sildenafil effects on spontaneous contractile activity.

<p>Effects of sildenafil and the NO donor SNP on spontaneous contractility of caput segments of the epididymal duct (A–C). A: Visualization of contractility derived from virtual sections through the corresponding time stacks (scale bar: 100 μm) in examples of SNP and subsequent sildenafil treatment of a duct segment from the caput region. Enlarging the regions that surround the time of drug addition, indicated by colored frames, illustrate transient effects of the substances. B: Statistical analyses compared the contractile frequency during 2 minutes preceding and following the addition of the substances. A non-parametric one-way ANOVA for repeated measurements (Friedman's test for paired samples) was used followed by Dunn's test for multiple comparisons. Adjusted p-values for each comparison are given in the graphs with “*” indicating p<0.05 and “**” indicating p<0.01. Statistical analyses of SNP and sildenafil treatments show that sildenafil significantly reduced contractile activity whether given alone or after SNP. In contrast, SNP effects remained non significant. “Spont” indicates spontaneous contractile frequency. C: Visualization of sildenafil and SNP effects in another duct segment originating from caput (scale bar: 100 μm). In this example sildenafil results in a complete loss of contractility. When the NO donor SNP was added in this situation, it was without additional effect as expected. The addition of noradrenaline at the end of the experiment lead to a resumption of contractile activity indicating that the duct segment was still viable. Movies were captured at 1 frame/s.</p