In rats with estradiol valerate-induced polycystic ovary syndrome, the acute blockade of ovarian β-adrenoreceptors improve ovulation

Newborn, female rats of the CII-ZV strain were kept with their mothers under controlled light conditions (lights on from 05:00 to 19:00 h) until weaning and were provided free access to food and water ad libitum under the same light conditions.

The animals were provided by the Facultad de Estudios Superiores-Zaragoza, UNAM, and the Bioethics Committee approved the experimental protocols. All procedures described in the present study were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the Mexican Council for Animal Care (NOM-062-ZOO-1999) and to the Guidelines for the Use of Animals in Neuroscience Research from the Society for Neuroscience. Every effort was made to minimize the number of animals in each experimental group and to ensure minimal discomfort and pain.

Ten-day-old female rats were intramuscularly injected with 2.0 mg EV (Sigma Chemical Co., St. Louis, Mo. USA) that had been dissolved in 0.1 mL sesame oil. The vehicle group (Vh) was injected with a single 0.1 mL dose of sesame oil. Vaginal smear was performed daily after the vaginal opening was first observed.

At 60 days of age, the animals in vaginal oestrus were randomly assigned to one of the following four experimental groups:

Following the methodology previously described [40], each of the rats underwent a bilateral laparotomy under general anesthesia, and the ovaries were exteriorized to enable injection of 20 μL of propranolol into each one, with the aid of a Nano-Injector, Stepper Motorized (Stoelting Co, USA) and a 100 μL micro syringe (Hamilton, USA) equipped with a 29-gauge needle; the injection rate was 4 μL/min. To prevent fluid leakage, the needle was kept in the ovarian bursa for 2 min. Subsequently, the ovaries were carefully cleaned, dried, and returned to the abdominal cavity, and the skin and muscle were sutured. The surgeries were performed between 9:00 and 11:00 A.M.

Animals from each group were deeply anesthetized with pentobarbital between 9:00 and 11:00 A.M. following confirmation of oestrus by vaginal smear after the surgery. Blood was obtained by intracardiac puncture; it was allowed to clot and was centrifuged for 15 min. at 3000 RPM. The serum was stored at − 20 °C until progesterone, testosterone and oestradiol levels were measured. The animals were then perfused with a 200 mL of saline solution followed by 200 mL of 4% paraformaldehyde dissolved in a phosphate buffered solution (PBS). At autopsy, the oviducts were dissected, the number of ova shed was counted with the aid of a stereomicroscope, and ovulation was corroborated by observing the presence of corpora lutea (CL).

The ovaries were dissected and kept in paraformaldehyde for 24 h, rinsed with saline and kept in a PBS solution with 30% sucrose until histochemical processing. The paraformaldehyde-perfused ovaries were sectioned with a cryostat (Microm HM 525) at temperatures of − 20 °C, and the 10-μm thick section were subsequently mounted on coated glass slides. Ovarian serially sections of five animals from each group were stained with hematoxylin-eosin and examined under a light microscope. All sections from each group were analyzed for the presence of fresh CL and follicular cysts with a Leica binocular microscope (DM750) coupled to a Leica camera (ICC50 HD). The criteria used to define fresh CL were healthy cells with large nuclei and the presence of blood vessels. The follicular cyst structures were defined according to Brawer et al., [30].

The ovarian sections of three animals taken at random from each experimental group (Vh, Vh + Pro, EV, and EV + Pro), were rinsed with PBS (pH 7.4) and were then rinsed twice with PBS with 0.5% Triton X-100. The nonspecific binding sites were blocked with IgG-free 2% bovine serum albumin (Sigma Chemical Co., USA). The sections were then incubated overnight at 4–8 °C with primary antibodies: polyclonal rabbit anti-TH antibody (1:200 sc-14,007 Santa Cruz Biotechnology Inc., USA) or polyclonal rabbit anti-DBH (1:200 sc- Santa Cruz Biotechnology Inc., USA), and the sections were subsequently incubated with a FITC-labelled goat anti-rabbit secondary antibody (Vector Labs Inc., USA). The slides were counterstained with Vectashield coupled with DAPI (Vector Labs Inc., USA) for nuclear staining. For negative controls, the primary antibody was substituted with PBS. Photomicrographs were taken using an Evolution VF Digital Camera (Media Cybernetics, Inc., USA) coupled with a fluorescence microscope (BX-41 Olympus Co.). From the ovarian sections of each animal, 10 ovarian follicles that exhibited the follicular antrum and the oocyte were selected, except in the cysts where the oocyte is absent (n = 3 animals per group with 10 pseudo-replicas per animal). Using the National Institutes of Health’s ImageJ software, the relative fluorescent to TH or DBH immunoreactivity was quantified following the methodology used previously [37, 40‐42] . The color micrographs were converted to 8-bit grayscale images, the criteria used to define the intensity settings were constant between all sections (the selection area in square pixels were equal for each ovarian follicle analyzed). The regions of interest were randomly selected based on the visualization; the fluorescence intensity was quantified in a constant area of each class of follicle evaluated.

The progesterone, testosterone and oestradiol serum levels were measured using a Radio-Immuno-Assay with kits purchased form Diagnostic Products (Los Angeles, CA). Progesterone results are expressed in ng/mL, and testosterone and oestradiol results are expressed in pg/mL. The intra- and interassay coefficients of variation were 8.35 and 9.45 for progesterone, 9.65 and 10.2 for testosterone, and 8.12 and 9.28 for oestradiol, respectively.

The results were expressed as the mean ± standard error (SE) for all experiments. The number of ova shed by ovulating rats was analyzed using Kruskal-Wallis tests, followed by a Mann-Whitney U-tests. The ovulation rate, expressed as the number of ovulating animals per number of treated animals, was analyzed using Fisher’s exact probability test. Hormone serum levels and immunoreactivity of TH or DBH were analyzed using one-way analysis of variance followed by Tukey test, with Graph Pad Software, Inc., (San Diego, CA, USA). A probability ≤5% was considered significant.

The animals injected with EV exhibited vaginal opening at 14 ± 0.0 days of age and were in oestrus according to the vaginal smear, which remained unchanged until the day of sacrifice. Animals injected with Vh exhibited vaginal opening at 35.1 ± 1.2 days of age and had 4-day oestrous cycles.

In the Vh group, all the animals ovulated regardless of whether or not they were injected with propranolol. However, the number of ova shed was smaller in the Vh plus propranolol group than in the Vh group (Table 1).

In the EV group, 1/8 animals ovulated, while in the EV plus propranolol group, 6/9 of the microinjected animals ovulated. The number of ova shed by the EV group microinjected with propranolol was smaller than the number observed in the Vh group (Table 1).

Microinjection of propranolol in both ovarian bursas of rats treated with Vh did not result in changed progesterone levels compared with the Vh group. Animals injected with EV exhibited higher concentrations of progesterone than the controls. The single injection of propranolol within the ovarian bursas in rats with EV resulted in lower progesterone levels than those observed in EV-injected rats (Fig. 1a).

In the Vh group, propranolol microinjection in both ovarian bursas did not modify testosterone levels compared with Vh-injected group. Testosterone levels in EV animals were higher than those in Vh-injected animals. In these animals, the microinjection of propranolol in both ovarian bursas resulted in lower testosterone levels than EV-treated group but higher testosterone levels than Vh-injected animals (Fig. 1b).

The microinjection of propranolol in Vh-treated animals did not change oestradiol levels compared with Vh-injected rats. The hormone levels in EV-treated animals were higher than those in Vh-treated animals. The microinjection of propranolol into both ovarian bursas resulted in lower oestradiol levels than EV-treatment group (Fig. 1c).

The ovaries of rats injected with Vh and microinjected or not with propranolol in both ovarian bursas presented growing follicles at different stages and CL (Fig. 2a and c). The ovaries of rats injected with EV presented follicular cysts, and only the ovaries of a single rat had CL (Fig. 2b). In the ovaries of EV-treated rats that were microinjected with propranolol in both ovarian bursas (Fig. 2d), CL were observed as in the Vh group.

The data had a normal distribution (TH fluorescence intensity of follicles with antrum: p value 0.9702 and cyst: p value 0.5176, Shapiro-Wilks normality test). TH and DBH immunoreactivity were found only in the interstitial tissue and theca cells of antral follicles. Compared to the Vh group, the TH immunoreactivity was not significantly different in the ovarian tissue of Vh- propranolol injection-treated rats. The highest intensity of TH immunoreactivity was observed in the theca cells of the ovarian follicles from the EV group. Propranolol injection into the ovarian bursas in EV-treated rats restored TH immunoreactivity, with respect to the EV group (Fig. 3).

The microinjection of propranolol did not modify the DBH immunoreactivity in the Vh group. The DBH immunoreactivity in the ovaries of rats injected with EV was higher with respect to the Vh group. Propranolol injection into the ovarian bursas of EV-treated rats restored DBH immunoreactivity in ovarian tissue with respect to the EV group (Fig. 4).