Improving the Dehydroepiandrosterone Induced PCOS Rat Model: Interplay of Age, High Fat Diet, and Treatment Regimen on Reproductive and Metabolic Phenotypes

Abstract

Polycystic ovary syndrome (PCOS) is a ubiquitous reproductive condition with triggering hallmarks such as glucose intolerance, hyperandrogenism, and dyslipidemia. Despite the existence of various PCOS animal models, an ideal model which could encompass all PCOS-specific phenotype is of dire need. Dehydroepiandrosterone (DHEA) induced PCOS rats are frequently employed; though, determining the superior model among pubertal and prepubertal rats, incorporation of high fat diet (HFD), and their sustainability remains uncertain. This study aims to examine the age factor, impact of HFD, and DHEA regimen in model development. Prepubertal and pubertal Sprague-Dawley rats were subcutaneously injected with DHEA (6 mg/kg and 60 mg/kg/day, respectively) with and without HFD up to 21 days. Serum testosterone, glucose, lipid profile, ovary morphology, and estrous cycle were evaluated. Following 21 days of treatment with DHEA, pubertal PCOS rats exhibited better reproductive phenotype than prepubertal rats. However, there was no significant difference in the lipid profile. Accordingly, both the age-group rats were concomitantly treated with DHEA and HFD for additional 3 weeks on alternate day basis after model development. The persistence of reproductive and metabolic features on treatment withdrawal were also simultaneously investigated by alienating the rats into continuous and stop dosing groups. The DHEA + HFD and DHEA treated pubertal rats in continuous dosing group showed significant PCOS features (p < 0.05) compared to stop dosing, prepubertal, and control groups. To conclude, continual dosing with DHEA on alternate days for 3 weeks is necessary to sustain metabolic and reproductive phenotypes of PCOS.

Keywords: Animal model; DHEA; High fat diet; Phenotype; Polycystic ovary syndrome.

Fig. 1

a Body weight, b blood glucose, c serum testosterone, d serum total cholesterol, e serum triglycerides, f serum HDL, g serum LDL, and h serum VLDL levels in DHEA treated and control groups of female Sprague Dawley rats. The data are presented as mean ± SD and analysed by one-way ANOVA with Bonferroni’s multiple comparison test, except for the body weight analysed by two-way ANOVA using Tukey’s multiple comparison test. (ns indicates not significant, **, ***, **** denotes levels of significance)

Fig. 2

Stages of estrous cycle a) proestrus stage, b) estrus stage, c) metestrus stage, and d) diestrus stage as observed under light microscope at 45 × magnification

Fig. 3

Percentage of days spent by the rats in each stage of the estrous cycle monitored up to 21 days of induction with DHEA

Fig. 4

Representative H and E staining of section of ovary tissue excised from pubertal control and DHEA treated rats. The images were taken at a magnification of 100 × and 400 × with 50 µm bars. Control group rats show abundant corpus luteum (CL) and growing follicles (GF). In DHEA treated rats, corpus luteum is reduced in count, while atretic (AF) and cystic follicles (CF) are seen

Fig. 5

Body weight of (a) prepubertal PCOS and control groups (b) pubertal PCOS and control groups. The data are presented as mean ± SD and analysed by two-way ANOVA with Tukey’s multiple comparison test

Fig. 6

Fasting blood glucose levels in (a) prepubertal rats and (b) pubertal rats on 21st day of induction to 3 weeks of monitoring. The data are presented as mean ± SD and analysed by two-way ANOVA with Tukey’s multiple comparison test

Fig. 7

Serum testosterone levels in (a) prepubertal rats, (b) pubertal rats on 21st day of induction to 3 weeks of monitoring. The data are presented as mean ± SD and analysed by two-way ANOVA with Tukey’s multiple comparison test

Fig. 8

Serum lipid profile a) total cholesterol, b) triglycerides, c) HDL-C, d) LDL-C, and e) VLDL-C in prepubertal rats on 21st day of induction to 3 weeks of monitoring. The data are presented as mean ± SD and analysed by two-way ANOVA with Tukey’s multiple comparison test

Fig. 9

Serum lipid profile a) total cholesterol, b) triglycerides, c) HDL-C, d) LDL-C, and e) VLDL-C in pubertal rats on 21st day of induction to 3 weeks of monitoring. The data are presented as mean ± SD and analysed by two-way ANOVA with Tukey’s multiple comparison test

Fig. 10

Percentage of days spent by (a) prepubertal rats (b) pubertal rats of various groups in each stage of the estrous cycle monitored from initial days of PCOS induction to 3 weeks after model development

Fig. 11

Representative H and E staining of section of ovary tissue excised from control, HFD, DHEA, and DHEA + HFD induced pubertal PCOS rats. The images were taken at a magnification of 10 × and 100 × with scale 1 mm bar. In control, growing follicles (GF), antral follicles (AF), and corpus luteum (CL) at various development stages can be seen while, cystic follicles are not apparent. In HFD group, degenerative follicles (CF) and luteal cysts (LC) are seen with several corpus luteum. Haemorrhagic fluid can be seen inside the corpus luteum. In DHEA group, few cystic follicles (CF) and corpus luteum are seen. In DHEA + HFD group, several cystic follicles (CF), luteal cysts (LC) as well as follicular atresia is evident with decreased count of corpus luteum

Fig. 12

Representative H and E staining of section of ovary tissue excised from control, DHEA, and DHEA + HFD induced prepubertal PCOS rats. The images were taken at a magnification of 40x, 100x, and 400 × with 50 µm bars. In control, growing follicles (GF) and corpus luteum (CL) at various development stages can be seen while, cysts are not evident. In treatment groups, cystic follicles (CF), atretic follicles (AF), and luteal cysts (LC) are seen. Haemorrhagic fluid can be seen inside the corpus luteum