Nitric Oxide Regulates Estrus Cycle Dependent Colonic Motility in Mice

Abstract

Women are more susceptible to functional bowel disorders than men and the severity of their symptoms such as diarrhea, constipation, abdominal pain and bloating changes over the menstrual cycle, suggesting a role for sex hormones in gastrointestinal function. Nitric oxide (NO) is a major inhibitory neurotransmitter in the gut and blockade of nitric oxide synthase (NOS; responsible for NO synthesis) increases colonic motility in male mice ex vivo. We assessed the effects of NOS inhibition on colonic motility in female mice using video imaging analysis of colonic motor complexes (CMCs). To understand interactions between NO and estrogen in the gut, we also quantified neuronal NOS and estrogen receptor alpha (ERα)-expressing myenteric neurons in estrus and proestrus female mice using immunofluorescence. Mice in estrus had fewer CMCs under control conditions (6 ± 1 per 15 min, n = 22) compared to proestrus (8 ± 1 per 15 min, n = 22, One-way ANOVA, p = 0.041). During proestrus, the NOS antagonist N-nitro-L-arginine (NOLA) increased CMC numbers compared to controls (189 ± 46%). In contrast, NOLA had no significant effect on CMC numbers during estrus. During estrus, we observed more NOS-expressing myenteric neurons (48 ± 2%) than during proestrus (39 ± 1%, n = 3, p = 0.035). Increased nuclear expression of ERα was observed in estrus which coincided with an altered motility response to NOLA in contrast with proestrus when ERα was largely cytoplasmic. In conclusion, we confirm a cyclic and sexually dimorphic effect of NOS activity in female mouse colon, which could be due to genomic effects of estrogens via ERα.

Keywords: ERalpha and ERbeta; colonic motor complexes; enteric neural circuits; estrogen; estrus cycle; immunohistochemistry; neuroplasticity; nitric oxide synthase.

FIGURE 1

(A) Spatiotemporal maps showing the number of CMCs during 15 min recordings of estrus and proestrus colon under the control conditions and on exposure to NOLA (100 μM). (B) Females in estrus had fewer CMCs compared to proestrus (n = 23, unpaired t-test, p = 0.042). (C) Resting colonic diameters were similar between the groups under control conditions. (D) Proestrus colons showed a larger percentage increase in the number of CMCs on exposure to NOLA compared to colons from mice during estrus (n = 10, unpaired t-test, p = 0.003). (E) NOLA similarly decreased resting gut diameter in both estrus and proestrus groups (n = 10, unpaired t-test, p = 0.97). Horizontal scale bar = 60 s, vertical scale bar = 0.5 cm. **p < 0.01 and *p < 0.05.

FIGURE 2

(A) Immunofluorescence histochemical double-staining of the whole-mount preparations of the mouse colon. Estrus myenteric neurons had a significantly greater number of NOS expressing neurons compared to proestrus myenteric plexus (estrus 48 ± 2% and proestrus 39 ± 1%, n = 3, p = 042 one-way ANOVA). The pan neuronal marker Hu is shown in red and NOS expressing myenteric neurons in green. Scale bar = 20 μm. (B) Co-labeling of NOS neurons with estrogen receptor alpha in a randomly selected female mouse. Whole mounts of myenteric ganglia showing co-labeling of Hu (red), NOS (green), and ERα (magenta). ERα is expressed in the majority of nuclei of myenteric neurons (closed arrows). Arrowheads indicate myenteric neurons co-labeled with NOS and ERα. Scale bar = 30 μm. *p < 0.05.

FIGURE 3

Whole mount preparations of myenteric plexus expressing pan neuronal marker Hu in red and ERα/ERβ in green. Significantly higher numbers of myenteric neurons expressed ERα in the nucleus during estrus while having some cytoplasmic expression compared to proestrus females and male myenteric neurons expressing less nuclear and cytoplasmic ERα. ERβ expression was more cytoplasmic in all tissue samples and showed no significant difference in expression between groups. Scale bar = 20 μm (n = 3, One-way ANOVA, ***p < 0.001). (A) Localization of ERα in the myenteric plexus preparations of estrus, proestrus, and male colon: ERα labeling was observed in more than 90% of myenteric neurons in both male and female mice. Images of representative myenteric ganglia, demonstrating neurons stained for Hu, ERα, and merged images of both Hu and ERα. During estrus stage, ERα labeling is predominantly nuclear while during proestrus and in male mice both nuclear and cytoplasmic labeling was observed (arrows indicate nuclear labeling of ERα). Scale bars = 20 μm. Graph shows quantified data (n = 3) where there was significantly higher nuclear localization observed during estrus stage compared to both proestrus and male preparations (***p < 0.001). (B) Localization of ERβ in the myenteric plexus preparations of estrus, proestrus, and male colon: localization of the estrogen receptor beta (ERβ) is unchanged during estrus. ERβ labeling was observed in more than 90% of myenteric neurons in both male and female mice. Images of representative myenteric ganglia, demonstrating neurons stained for Hu, ERα, and merged images of Hu and ERα. No significant difference was observed among the groups with respect to nuclear labeling of ERβ (arrows indicate nuclear labeling of ERβ). Scale bars = 20 μm. The graph shows quantified data (n = 3) where there was no significant difference among the groups (p > 0.05). (C) Specificity of ERα PA1-308-antiserum was tested in a uterine tissue. Labeling was observed in the glandular epithelial cells of the uterus (frozen cross section). Scale bar = 20 μm.