Hypothalamic Neuroendocrine Functions in Rats with Dihydrotestosterone-Induced Polycystic Ovary Syndrome: Effects of Low-Frequency Electro-Acupuncture

Control rats had 4–5-day estrous cycles, comprising diestrus, proestrus, estrus, and metestrus (Fig. 1A). Rats with DHT-induced PCOS had no dynamic change in estrous cycle, were constantly in diestrus, and exhibited predominantly leukocytes. During the first week of treatment, some rats in the PCOS EA group started to exhibit epithelial keratinocytes, the main cell type during estrus, indicating estrous cycle changes. After 4–5 weeks of low-frequency EA treatment, 11 of 12 rats (91.7%) in the PCOS EA group exhibited epithelial keratinocytes (Fig. 1A, 1B).

Hypothalamic AR protein expression was higher in PCOS rats than in controls (p<0.001) (Fig. 2), and so was AR immunoreactivity (AR-ir) in the medial preoptic area (MPO) (p<0.05) (Fig. 3A, 3B). Low-frequency EA decreased hypothalamic AR protein expression (p<0.05, Fig. 2) and AR-ir in the MPO and the ventromedial hypothalamus (VMH) to lower levels than in the PCOS group (Fig. 3A, 3B).

Functional androgen response elements are present in the promoter sequences of p21 [17], and the regulation of p21 expression is AR dependent [18]. To determine whether the AR was functionally active, we analyzed hypothalamic samples for AR activation in response to expression of p21, which regulates cell-cycle progression and correlates inversely with the levels of active AR. In both PCOS groups, p21 levels were lower than in controls by western blot analysis (Fig. 2). Western analysis revealed similar decreases in the expression of c-FOS, an early marker of neuronal activation [19], in the PCOS groups (Fig. 2). However, low-frequency EA did not elicit additional effects on p21 or c-FOS expression in PCOS rat hypothalamus.

GnRH-immunoreactive (GnRH-ir) cells are abundant in the MPO, rostral medial septum (MS), and nucleus of the horizontal limb of the diagonal band (HDB) of the hypothalamus [20]. PCOS rats had more GnRH-ir cells in the MPO and HDB than control rats (p<0.05) (Fig. 4A, 4B). After 4–5 weeks of EA treatment, PCOS EA rats had fewer GnRH-ir cells in those areas (p<0.05) (Fig. 4A, 4B) than the PCOS group. However, there was no obvious difference in the MS between the control, PCOS, and PCOS EA groups (Fig. 4A, 4B). Even though we detected GnRH-ir cells in hypothalamus, we failed to detect a GnRH band by western blot analysis, because of low GnRH expression. The low expression was confirmed by immunoprecipitation and western blot (Fig. 4C).

The GnRH-receptor (R) is mainly distributed in the pituitary gland and hypothalamus in the central nervous system. Continuous DHT exposure decreased both the number of GnRH-R-ir cells in the pituitary, MPO, and HDB (p<0.01) and hypothalamic GnRH-R protein expression (p<0.05 vs. controls) (Fig. 5A, 5B, 5C). There was no difference in the MS between groups (Fig. 5C). Low-frequency EA treatments did not affect the number of GnRH-R-ir cells or the level of GnRH-R expression.

Two main regions in the hypothalamus, the paraventricular nucleus (PVN) and MPO, showed CRH-immunoreactivity (CRH-ir). However, CRH-ir and CRH protein expression did not differ between PCOS and control rats or after 4–5 weeks of EA treatment (Fig. 6A, 6B, 6C).

Dual-fluorescence immunohistochemistry and confocal analysis showed co-localization of AR, GnRH, and CRH with the neuronal marker NeuN in controls. In addition, AR and GnRH expression in the MPO co-localized in the cytoplasm and nucleus, but few cytoplasmic ARs co-localized with CRH-expressing neurons (Fig. 7A–G).

Four Wistar dams, each with eight to nine female pups, were purchased from Charles River (Sulzfeld, Germany), raised with a lactating dam until 21 days of age, and then housed four to five per cage under controlled conditions (21–22°C, 55–65% humidity, 12-h light, 12-h dark cycle). Rats were fed commercial chow (Harlan Teklad Global Diet, 16% protein rodent diet nr 2016, Harlan Winkelmann Gmbh, Harlan, Germany) and tap water ad libitum. Animals were cared for in accordance with the principles of the Guide to the Care and Use of Experimental Animals (www.sjv.se↗). The study was approved by the Animal Ethics Committee of the University of Gothenburg.

At 21 days of age, rats were randomly divided into three experimental groups (control, PCOS, and PCOS EA; n = 12 per group) and implanted subcutaneously with 90-day continuous-release pellets (Innovative Research of America, Sarasota, FL) containing 7.5 mg of DHT (daily dose, 83 µg) or 7.5 mg of vehicle. In our previous study, this dose of DHT resulted in PCOS characteristics, including metabolic disturbances at adult age [46]. A microchip (AVID, Norco, CA) with an identification number was inserted in the neck along with the pellets. The control pellets were identical to the DHT pellets but without the bioactive molecule. All rats were weighed weekly from 21 days of age. Treatments started at 70 days of age, after 7 weeks of DHT exposure. The study was concluded after 12 weeks of DHT exposure, including 4–5 weeks of EA.

Low-frequency EA was given to conscious rats daily from Monday to Friday for 4–5 weeks (20–25 treatments in total). The treatment duration was 15 min in week 1, 20 min in weeks 2 and 3, and 25 min thereafter. Acupuncture needles were inserted in the rectus abdominis (stomach) [29] and in the triceps surae muscles (spleen) [6] bilaterally, in somatic segments that correspond to the innervation of the ovaries (i.e., from spinal levels T10 to L2 and at the sacral level). The needles (HEGU Svenska, Landsbro, Sweden) were inserted to a depth of 0.5–0.8 cm and attached to an electric stimulator (CEFAR ACU II; Cefar-Compex Scandinavia, Malmo, Sweden). The points were electrically stimulated with a low frequency of 2 Hz with 0.1-sec, 80-Hz burst pulses [12]–[16]. The intensity was adjusted to produce local muscle contractions and varied from 0.8–1.4 mA during the stimulation period. Because of receptor adaptation, the amplitude varied during each treatment. Most rats required higher amplitude at the end of the stimulation period.

Before handling or needle insertion, all rats were lightly anesthetized with isoflurane (2% in a 1∶1 mixture of oxygen and air; Isoba vet; Schering-Plough, Stockholm, Sweden) for 2–3 min. One investigator inserted all needles. During EA treatment, the rats were placed in a fabric harness and suspended above the desk. To avoid potential acute effects of EA, no treatment was performed 24 h before examinations and blood sampling. Rats in the control and PCOS groups were anesthetized, suspended in a harness, and handled in the same way as rats in the PCOS EA group but without needle insertion or electrical stimulation. All rats were conscious during handling and treatment.

The stage of cyclicity was determined by microscopic analysis of the predominant cell type in vaginal smears obtained daily from the onset of EA treatment at 70 days of age to the end of the experiment.

For 3, 3′-diaminobenzidine staining, five control rats and six rats each in the PCOS and PCOS EA groups were deeply anesthetized with thiobutabarbital sodium (130 mg/kg i.p.; Inactin, Sigma, St. Louis, MO) and perfused via the left cardiac ventricle with 4°C cold 0.9% sodium chloride (200 ml) and Histofix (Histolab, Gothenburg, Sweden) (100 ml) for rapid fixation. The brains were removed and postfixed in Histofix containing 20% sucrose for 48 h at 4°C and subsequently in 0.1 M PBS containing 30% sucrose for at least 24 h at 4°C. Serially frozen frontal sections (20 µm) were cut and stored in tissue culture wells containing 30% sucrose and 30% ethylene glycol in 0.1 M PBS, pH 7.4, at −20°C. The brain sections were taken from the hypothalamus (−0.40 and −3.60 mm from the bregma) [47], washed in 0.01 M PBS (Sigma) for GnRH and CRH experiments or in Tris-buffered saline (50 mM Tris, 0.9% NaCl, pH 7.5) for AR experiments; the endogenous peroxidase and nonspecific binding were removed by incubation with 3% H₂O₂ for 30 min and 0.5% Triton X-100 10 min at room temperature. The sections were then incubated with 10% normal horse serum for 1 h at 37°C and with primary antibody (Table 1) for 1 h at 37°C and then overnight at 4°C. Sections were stained with the avidin–biotinylated peroxidase complex detection system (ABC kit, Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions and treated for 1 min with 3, 3′-diaminobenzidine. Sections were examined with an Olympus DP50 microscope (Japan) under bright-field optics and photographed with Image-pro plus software (version 5.0, Media Cybernetics, Bethesda, MD) to count cells. Five sections were chosen in the same area to calculate the mean cell population. The positive cells were counted by up-down focusing.

For dual-fluorescence immunohistochemistry, hypothalamic sections were blocked in goat normal serum for 2 h at 37°C. Slides were incubated with two different primary antibodies in 1×TBS supplemented with 0.05% Triton X-100 (TBST) for 2 h at 37°C and overnight at 4°C. After five 10-min washes in TBST, sections were incubated with the first secondary antibody at 37°C for 1 h, washed five times with TBST for 10 min each, and incubated with the second secondary antibody at 37°C for 1 h. Sections were washed in TBST as above and mounted with fluorescent Vectashield with 4′, 6-diamidino-2-phenylindole (DAPI). All fluorescence images were acquired with an Axiovert 200/LSM 510 META laser-scanning confocal microscopy system (Zeiss, Jena, Germany) fitted with a Plan-Apochromat 63x/1.40 Oil DIC objective. Background settings were determined by examination of negative control specimens.

Images of positive staining were adjusted to make optimal use of the dynamic range of detection. All final immunohistochemistry was carried out in parallel under identical conditions. To control for nonspecific staining, adjacent sections were stained as above, except the primary antibody was replaced with TBST, normal rabbit IgG, or mouse IgG. TBST was used to control for nonspecific staining of the secondary antibody and to obtain the background level. Rabbit or mouse IgG was used to ensure that there was no cross-reactivity between the two staining sequences. Rat testis served as a positive control for AR. Rat placenta served as a positive control for GnRH and CRH. The immunohistochemical findings are representative of those observed in random sections from multiple animals. The staining was evaluated by two blinded observers.

The remaining rats (seven controls and six each from the PCOS and PCOS EA groups) were used to investigate AR, GnRH, and CRH protein expression by western blot. The target regions, including the mediobasal hypothalamus and suprachiasmatic-preoptic area were dissected (limited anteriorly by the optic chiasma, laterally by the hypothalamic fissures, posteriorly by the mammilary bodies, and in depth by the subthalamic sulcus). Tissue protein was prepared as described [48]. Protein concentrations were determined with the BCA protein assay (Pierce, Rockford, IL), using bovine serum albumin as the standard.

Expression of AR, p21, and c-FOS was detected by western blot analysis with a standard procedure [49]. Protein aliquots were pretreated with 4×sodium dodecyl sulfate (SDS) (1×50 mM Tris-HCl, 2% SDS, 10% glycerol, 10% β-mercaptoethanol, and 0.001% bromophenol blue) before loading and separated on 4–12% SDS-polyacrylamide gels (Novex, Invitrogen, Carlsbad, CA) with a Bis-Tris-MOPS buffer system under reducing conditions. The separated samples were electrophoretically transferred to polyvinyldifluoride membranes (Amersham International, Buckinghamshire, UK) and incubated with primary antibody (Table 1) in blocking buffer overnight at 4°C. The next day, the membranes were incubated with alkaline phosphatase–conjugated goat anti-rabbit or goat anti-mouse antibody and detected with CDP-Star substrate for alkaline phosphatase (Tropix, Bedford, MA). Immunoblotted signals were exposed and developed with ECL film (Amersham International) and directly from membranes by densitometry with ImageQuant software (version 5.0, Molecular Dynamics, Sunnyvale, CA). Signal intensities of the AR, p21, and c-FOS proteins were normalized to those of gels stained with Coomassie blue as ratios to produce arbitrary densitometric units (ADU) of relative abundance. Care was taken to ensure that the ADU of all bands considered was in the range of linearity previously assessed.

For immunoprecipitation experiments [50], tissues were extracted with ice-cold lysis buffer (25 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.5% Nonidet P-40, 1% SDS, 200 M sodium deoxycholate, 1 mM dithiothreitol, 5 mM EDTA, 0.5 mM phenylmethyl sulfonyl fluoride, and a cocktail of protease inhibitors (Roche Diagnostics, Mannheim, Germany). Specific antibodies against GnRH were added to 500 µg of protein extracts and incubated for 4 h at room temperature. Immune complexes were obtained by adding 50 µl of Pansorbin cells (Calbiochem, San Diego, CA). The resulting immobilized immune complexes were washed in RIPA buffer (50 mM Tris–HCl, pH 7.8, 150 mM NaCl, 15 mM MgCl₂, 0.5% Nonidet P-40, 0.3% Triton X-100, 0.5% sodium deoxycholate, 5 mM EDTA, 1 mM dithiothreitol, and a cocktail of protease inhibitors). The bound protein was eluted by boiling in 30 µl of SDS sample reducing/loading buffer (Novex) for 5 min. Immunoprecipitated complexes were loaded in the 4–12% SDS-polyacrylamide gels (Novex).

Data are expressed as mean±SEM of the number of independent experiments indicated in the figure legends. Multiple comparisons were performed with one-way ANOVA followed by correction of P values with Dunnett's posthoc test (SPSS, version 16.0; Chicago, IL). P<0.05 was set as the limit of statistical significance.