Hypothalamic neuroendocrine functions in rats with dihydrotestosterone-induced polycystic ovary syndrome: effects of low-frequency electro-acupuncture

Abstract

Adult female rats continuously exposed to androgens from prepuberty have reproductive and metabolic features of polycystic ovary syndrome (PCOS). We investigated whether such exposure adversely affects estrous cyclicity and the expression and distribution of gonadotropin-releasing hormone (GnRH), GnRH receptors, and corticotrophin-releasing hormone (CRH) in the hypothalamus and whether the effects are mediated by the androgen receptor (AR). We also assessed the effect of low-frequency electro-acupuncture (EA) on those variables. At 21 days of age, rats were randomly divided into three groups (control, PCOS, and PCOS EA; n = 12/group) and implanted subcutaneously with 90-day continuous-release pellets containing vehicle or 5alpha-dihydrostestosterone (DHT). From age 70 days, PCOS EA rats received 2-Hz EA (evoking muscle twitches) five times/week for 4-5 weeks. Hypothalamic protein expression was measured by immunohistochemistry and western blot. DHT-treated rats were acyclic, but controls had regular estrous cycles. In PCOS rats, hypothalamic medial preoptic AR protein expression and the number of AR- and GnRH-immunoreactive cells were increased, but CRH was not affected; however, GnRH receptor expression was decreased in both the pituitary and hypothalamus. Low-frequency EA restored estrous cyclicity within 1 week and reduced the elevated hypothalamic GnRH and AR expression levels. EA did not affect GnRH receptor or CRH expression. Interestingly, nuclear AR co-localized with GnRH in the hypothalamus. Thus, rats with DHT-induced PCOS have disrupted estrous cyclicity and an increased number of hypothalamic cells expressing GnRH, most likely mediated by AR activation. Repeated low-frequency EA normalized estrous cyclicity and restored GnRH and AR protein expression. These results may help explain the beneficial neuroendocrine effects of low-frequency EA in women with PCOS.

Figure 1. Vaginal smears and estrous cycle patterns of control, PCOS, and PCOS EA rats.

A) Representative vaginal smears from a normal cycling control rat at different stages in the estrous cyclediestrus (top left), proestrus (top right), estrus (middle left), and metestrus (middle right). Representative hematoxylin eosin stained vaginal smear from a PCOS rat exhibiting predominantly leukocytes, the main cell type during diestrus stage (bottom left). Representative vaginal smear from a PCOS EA rat exhibiting epithelial kerotinocytes, the main cell type during estrus stage (bottom right). Scale bar, 100 µm (top left). B) Estrous cycle patterns at 70–95 days of age (i.e., 49–84 days after pellet implantation) in four representative rats from each group. P, proestrus; E, estrus; M, metestrus; and D, diestrus.

Figure 2. Western blot analysis of AR, p21, and c-Fos protein expression in the hypothalamus.

Regulation of AR, p21, and c-Fos protein expression in the hypothalamus in the control (n = 7), PCOS (n = 6), and PCOS EA (n = 6) groups. Top: total protein (50 µg) was isolated and used for western blot analysis. The blot is representative of each run with independent samples. Bottom: densitometric analysis of the levels of AR, p21, and c-Fos protein expression. Equal sample loading was confirmed by Coomassie blue staining. Relative levels of AR, p21, and c-Fos proteins were expressed as a ratio of densitometric value to whole proteins in Coomassie blue–stained gels. Values are mean±SEM of two independent experiments (n = 3 pools/group). ***p<0.001, **p<0.01 vs. control; ##p<0.05 vs. PCOS.

Figure 3. AR-ir cells in the medial preoptic area (MPO) and ventromedial hypothalamus (VMH).

A) Light micrographs of AR-ir cells, detected with polyclonal antibody as described in Materials and Methods. Scale bar, 100 µm. B) Quantification of AR-ir cells in the different regions in the control (n = 5), PCOS (n = 6), and PCOS EA (n = 6) groups. Values are mean±SEM. **p<0.01 vs. control; ##p<0.01 vs. PCOS, #p<0.05 vs. PCOS.

Figure 4. GnRH-ir cells in the rostral MS, MPO, and HDB of the hypothalamus.

A) Light micrographs of GnRH-ir cells, detected with polyclonal antibody as described in Materials and Methods. Scale bar, 100 µm. B) Quantification of GnRH-ir cells in the different regions in the control (n = 5), PCOS (n = 6), and PCOS EA (n = 6) groups. Values are mean±SEM. *p<0.05 vs. control; #p<0.05 vs. PCOS. C) Immunoprecipitation and western blot (WB) of GnRH protein in the hypothalamus.

Figure 5. GnRH-R-ir cells in the rostral MS, and nucleus of the HDB of the hypothalamus and pituitary (Pit).

A) Light micrographs of the GnRH-R-ir cells, detected with polyclonal antibodies as described in Materials and Methods. Scale bar, 100 µm. B) Quantification of GnRH-R cells in the control (n = 5), PCOS (n = 6), and PCOS EA (n = 6) groups. Values are mean±SEM. *p<0.05 vs. control; #p<0.05 vs. PCOS. C) Western blot of GnRH-R protein in the hypothalamus. Total protein (50 µg) was isolated and used for western blot analysis. The blot is representative of two essentially similar experiments, each run with independent samples. Densitometric analysis of GnRH-R protein expression in two independent experiments. Equal sample loading was confirmed by Coomassie blue staining. Relative levels of GnRH-R proteins were expressed as a ratio of densitometric value to whole proteins in Coomassie blue–stained gels. Data are expressed as ADU; values are the mean±SEM of two independent experiments (n = 3 pools/group). *p<0.05 vs. control.

Figure 6. CRH-ir cells in the PVN and MPO in control, PCOS and PCOS EA rats.

A) Light micrographs of CRH-ir cells, detected with polyclonal antibodies as described in Materials and Methods. Scale bar, 100 µm. B) Quantification of CRH-ir cells in the different regions in the control (n = 5), PCOS (n = 6), and PCOS EA (n = 6) groups. Values are mean±SEM. C) Protein samples were isolated from the hypothalamus of control, PCOS, and PCOS EA rats. Total protein (50 µg) was isolated and used for western blot analysis. The blot is representative of two essentially similar experiments, each run with independent samples. Densitometric analysis of CRH protein expression in two independent experiments. Equal sample loading was confirmed by Coomassie blue staining. Relative levels of CRH proteins were expressed as a ratio of densitometric value to whole proteins in Coomassie blue–stained gels. Data are expressed as ADU. Values are mean±SEM of two independent experiments (n = 3 pools/group).

Figure 7. Co-localization of AR, GnRH, and CRH in MPO neurons, determined by dual-fluorescence immunohistochemistry and confocal laser-scanning microscopy.

A) Main distribution of AR, GnRH, and CRH in control female rat brain (3v = third ventricle). Adapted from reference . B–D) Co-localization of AR, GnRH, CRH, and NeuN immunoreactivity in MPO neurons. E) Co-localization of rabbit polyclonal AR antibody and mouse monoclonal AR antibody in hypothalamic MPO. F and G) Co-localization of mouse monoclonal AR antibody with GnRH or CRH immunoreactivity in hypothalamic MPO neurons. In panels B–G, arrows indicate co-localization. Scale bars, 100 µm.