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

Abstract

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.

Figure 1. Simultaneous demonstration of contractile activity in different regions of one epididymal duct segment.

Left side: Snapshot of the Movie S1 resulting from time-lapse digital photography of a piece of the epididymal duct in the caput region (scale bar: 100 μm). The different positions at which this time stack was virtually dissected are indicated by colored bars (A: green, B: yellow, C: red, D: black). Right side: Virtual sections through the time stack at indicated positions. Each contraction elicits a small movement of the epididymal duct with changes of its diameter resulting in a series of spikes (marked by vertical arrows). A–C: Virtual sections at different positions (1 frame/s). With the contractions spreading over the observed duct segment, the detection of contractions at different places yields equivalent results. D: Demonstration of contraction-derived pattern of spikes resulting from a movie captured at an accelerated rate of 7 frames/s providing more details. Contractile frequency in A–D is 7.5 beats/min.

Figure 2. Spontaneous contractions in caput and corpus epididymidis.

Visualization of contractility derived from virtual sections through the corresponding time stacks (as indicated in Fig. 1) 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.

Figure 3. Visualization of sildenafil effects on spontaneous contractile activity.

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.

Figure 4. Visualization of ANP effects on spontaneous contractile activity.

Effects of ANP and CNP on spontaneous contractility of corpus segments of the epididymal duct. A: Visualization of contractility derived from virtual sections through the corresponding time stacks in examples of ANP and subsequent CNP treatment of a duct segment from the corpus region (scale bar: 100 μm). Enlarging the regions that surround the time of drug addition, indicated by colored frames, illustrate transient effects of the substances. B: Statistical analyses compare the contractile frequency during 2 minutes preceding and following the addition of ANP and CNP. 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. ANP significantly decreased spontaneous contractile activity and had a significant effect when given after CNP. CNP effects were non significant. “Spont” indicates spontaneous contractile frequency. Movies were captured at 1 frame/s.

Figure 5. Demonstration of GC-A, GC-B and sGC expression in smooth muscle cells of the rat epididymal duct by LCM+RT-PCR.

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.

Figure 6. Visualization of transport of intraluminal contents.

Snapshot of Movie S4 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.