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

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

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

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

Immunohistological analysis of polySia⁺cells and cell proliferation status in postnatal vas deferens.

<p>Paraffin-embedded serial sections of 7 days old vas deferens were stained with a mAb against proliferating cell nuclear antigen (anti-PCNA) (A) and a mAb against polySia (B). For negative control tissue sections were pretreated with endoN to degrade polySia (data not shown). Tissues were counterstained with Haemalaun. Scale bars representing 20 μm.</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

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

<p>Left side: Snapshot of the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092603#pone.0092603.s001" target="_blank">Movie S1</a> 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.</p

Immunohistological analysis of polySia⁺cells and collagen status of the inner longitudinal layer of smooth muscle cells in early and late postnatal mice epididymis.

<p>Paraffin-embedded serial section of epididymis (cauda) at different time points (A-H). Sections of epididymis were stained with a mAb against polySia (A-D). For negative control, tissue sections were pretreated with endoN to degrade polySia (data not shown). An anti-SMA immunostaining was used to identify smooth muscle cells (data not shown). For collagen identification Azan staining was performed (E-H). * labels same positions of smooth muscle cells. Tissues were counterstained with Haemalaun (A-D). Scale bars representing 20 μm. (I) Illustration of the inverse correlation between polySia and collagen.</p

Immunohistological analysis of polySia⁺cells and cell proliferation status in early and late postnatal mice epididymis.

<p>Paraffin-embedded serial section of 4 days old (A-C) and 25 days old (D-F) mouse epididymis (cauda). Sections were stained with a mAb against proliferating cell nuclear antigen (anti-PCNA) for proliferating status (A,D) and additional stained with a mAb against polySia (B,E). For negative control, tissue sections were pretreated with endoN to degrade polySia (data not shown). For identification of smooth muscle cells a mAB against smooth muscle actin were used (C,F). * labels same positions of smooth muscle cells. Tissues were counterstained with Haemalaun. Scale bars representing 20 μm.</p

Immunohistological analysis of polySia⁺cells and collagen status of the tunica albuginea in early and late postnatal mice testis.

<p>Paraffin-embedded serial section of testis were stained with a mAb against polySia (A-D). For negative control, tissue sections were pretreated with endoN to degrade polySia (data not shown). For collagen visualization Azan staining was performed (E-H). Tissues were counterstained with Haemalaun (A-D). Scale bars representing 20 μm. (I) Illustration of the inverse correlation between polySia and collagen.</p