Colitis causes delay in puberty in female mice out of proportion to changes in leptin and corticosterone

Abstract

Background: Inflammatory bowel disease that begins prior to puberty frequently causes a delay in puberty resulting in losses of growth, bone mineralization, and self esteem. A major cause of this pubertal delay is likely due in part to the effect of decreased levels of leptin on the function of the hypothalamic-pituitary-gonadal axis, though systemic inflammation is also thought to play a role.

Methods: To investigate further whether low leptin levels alone were responsible for delayed puberty in colitis, we induced colitis in 23-day-old female mice using 3% dextran sodium sulfate (DSS), resulting in 10 days of worsening colitis. These mice were compared to controls that were free-feeding and food-restricted (FR) mice that were given only enough food to keep them the same weight as the DSS group. All groups were followed for the timing of vaginal opening until 33 days old, when they were euthanized and their serum collected.

Results: DSS-treated mice exhibited later timing of vaginal opening relative to both of the other groups, as well as increased colonic inflammation by cytology and increased serum levels of interleukin (IL)-6 and tumor necrosis factor(TNF)-alpha. The difference in the timing of vaginal opening between the DSS and FR groups occurred despite equivalent serum levels of leptin between the groups and despite an increase in corticosterone in the FR group relative to both of the other groups.

Conclusions: We conclude that DSS colitis causes delay in puberty in sexually immature mice beyond what would be expected from decreases in weight and leptin levels.

Fig. 1

Weight, length, and food intake following colitis induction and food restriction. a Weight curve over the course of the experiment, day of life (DOL) 23–33. b Linear growth determined as follows: (length at DOL 33)/(length at DOL 23). c 10-Day cumulative food intake during experiment. *p < 0.05, **p < 0.01, ***p < 0.001, NS not significant (p > 0.05). DSS, dextran sodium sulfate; tx, treatment

Fig. 2

Colonic cytology, active inflammatory index, and chronic inflammatory index. Representative histological sections of colons from mice from the control. H&E, ×10 (a), DSS (b; arrow indicates ulceration of the mucosa. H&E, ×10), and food-restricted groups. H&E, 910 (c). d Active inflammatory index scores [35, 36]. e Chronic inflammatory index scores [35, 36]. ***p < 0.001

Fig. 3

Systemic inflammation. Serum measurement of cytokines at baseline, mid-points (days of life 27 and 30), and the end of the experiment (day of life 33), expressed as -fold change above control, calculated on a per-assay basis. Mid-point values were performed in separate groups of animals under the same protocol. See text for absolute serum values. a Interleukin-6 (IL-6). b Tumor necrosis factor-α (TNF -α). *p < 0.05, **p < 0.01, NS not significant (p > 0.05)

Fig. 4

Timing of vaginal opening. a Age of vaginal opening as assessed by daily examination. Animals that had not experienced vaginal opening by the end of the experiment (day of life 33) were assigned a value of 34 days old. b Kaplan–Meier curve showing the proportion of mice without vaginal opening over time. DSS vs control and food-restricted p < 0.05 as determined by logrank test. *p < 0.05

Fig. 5

Serum levels of leptin and corticosterone. a Serum levels of leptin at baseline, mid-points (days of life 27 and 30), and the end of the experiment (day of life 33). Mid-point values were performed in separate groups of animals under the same protocol, with equal food-restricted and DSS animal weights. b Serum levels of corticosterone at the end of the experiment (day of life 33). *p < 0.05 vs DSS; **p < 0.01 vs DSS; ***p < 0.001 vs. DSS; ^###p < 0.001 vs food-restricted, NS not significant (p > 0.05)