Animals and experiment design

The experiments involved 16-month-old (middle-aged) Wistar rats. They were bred in the Institute for Biological Research “Siniša Stanković”, Belgrade, Serbia; housed one per cage and maintained under constant laboratory conditions (22±2ºC, 12-12 h light-dark cycle), with free access to food (standard diet; the chemical composition given in Table 1) and water.

Table 1. Chemical composition of the rat diet used in the experiment

At the age of 15 months, experimental animals were randomly bilaterally orhidectomized (ORX; n=16 animals) under Ketamine anesthesia (15mg/kg b.m.; Richter Pharma, Wels, Austria). The recovery period lasted two weeks. Orchidectomized rats were then divided into two groups of eight animals (n=8) each. The first group was subcutaneously treated with testosterone propionate (Fluka Chemie AG, Buchs, Switzerland; ORX+T), in a dose of 5 mg/kg b.m. (19, 25-26) every day except on Sundays, for 3 weeks. The final injected volume was 0.5 ml per animal. The second orchidectomized group (ORX) was given the same volume (0.5 ml) of vehicle (sterile olive oil) alone, and they served as controls. Body mass, from these two groups after the treatment, was measured. All the animals (n=16) were sacrificed by decapitation under ether anesthesia (ether ad narcosis Ph. Iug. III., Lek, Ljubljana, Slovenia), 24 h after the last injection.

All experimental procedures were in compliance with Directive 2010/63/EU on the protection of animals used for experimental and other scientific purposes and was approved by the Ethical Committee for the Use of Laboratory Animals of the Institute for Biological Research “Siniša Stanković”, University of Belgrade, Serbia.

Hormonal analyses

Blood was collected from the trunk and separated sera samples of all animals were stored at the same time at -70°C until assayed. Serum testosterone concentrations were determined without dilution, using the competitive immunoenzymatic colorimetric method (EIAgen Testosterone Kit; Adaltis Italia S.p.A., Bologna, Italy), which sensitivity is 0.01 ng/ml, with an intra-assay coeficient of variation (CV) of 6.2%. Serum estradiol concentrations were measured without dilution, using the electrochemiluminescence immunoassay (ECLIA, Roche, Basel, Switzerland), which low detection limit is 5.0 pg/ml, with an intra-assay CV of 6.2%.

Light microscopy and immuno-histochemistry

After sacrificing, excised and weighed pituitaries were fixed in 4% paraformaldehyde for 24 h, dehydrated in a series of increasing concentrations of ethanol, enlightened in xylol and embedded in paraplast. Serial, 5-µm thick pituitary tissue sections were obtained using LEICA RM 2125RT rotational microtome (Leica Biosystems Nussloch GmbH, Nussloch, Germany), then deparaffinised in xylol and rehydrated in a series of decreasing concentrations of ethanol. For the light microscopy analysis, gonadotropic- (FSH- and LH- producing) cells were immunolabeled using the peroxidase-antiperoxidase complex (PAP) method (20, 21). After rehydration of the pituitary sections, the endogenous peroxidase activity was blocked by the incubation in 9 mmol (0.3%) hydrogen peroxide solution in methanol, for 15 min at 24°C. Before the application of the specific primary antisera, nonspecific background staining was prevented by incubation of the sections with nonimmune, i.e. normal swine serum, diluted (1:10) with phosphate buffered saline (PBS) pH 7.4, for 45 min at 24°C. Pituitary sections were then overlaid with the appropriate dilutions of the specific primary antibodies, namely polyclonal anti-rat βFSH (1:300) and βLH (1:500), for 24 h at 4°C. Antisera to rat βFSH (NIDDK-anti-rBeta FSH-IC1, Lot#AFP-7798 1289P) and βLH (NIDDK-anti-rBeta LH-IC, Lot#AFP-C697071P) were obtained from Dr. A. F. Parlow, National Hormone Peptide Program (NHPP), Harbor-UCLA Medical Centre, Carson, CA, USA. The specificities of these antisera were assessed by the NIDDK. After washing in PBS, the sections were incubated with secondary antibody polyclonal swine-anti-rabbit IgG/HRP (Dako A/S, Glostrup, Denmark). Binding sites were visualized using 0.05% diaminobenzidine (DAB, Serva, Heidelberg, Germany) and 0.03% hydrogen peroxide in 0.2M TRIS-HCl buffer, pH 7.4. The sections were counterstained with hematoxylin and mounted in Canada balsam (Molar Chemicals KFT, Budapest, Hungary). For the negative control pituitary sections, the primary antibody was omitted and replaced by PBS.

Digital images of the pituitary gland immunolabeled sections were taken using a LEITZ DM RB light microscope (Leica Mikroskopie & Systems GmbH, Wetzlar, Germany), a LEICA DFC320 CCD camera (Leica Microsystems Ltd., Heerbrugg, Switzerland) and the Leica DFC Twain Software (Leica, Germany).

Morphometry

Morphometrical measurements were performed as described previously (16-18). Namely, two pituitary sections (5-µm thick) from the superior, three from the middle and two from the inferior part (seven horizontal sections) of the rat pituitary glands were analysed. The point counting method was used at an overall magnification of x1000. The M₄₂ multipurpose test grid, inserted into the ocular of a Zeiss light microscope (Jena, Germany), was randomly positioned on the pituitary section at the beginning of counting. Counting was carried out on the following 50 test fields per section. Average values were calculated per pituitary i.e. per animal (7 sections, 350 test fields) and eight pituitaries were analysed per group. Cell volume (Vc, μm3) and relative volume density (percentage of immunopositive cells in mm3; V_VC, %) were determined for FSH- and LH-immunopositive cells.

The following parameters were counted:

Pn - number of points hitting on nuclei of immuno-histochemically labeled cells inside the test field

Ptc - number of points hitting on cytoplasm of immuno-histochemically labeled cells inside the test field

Nn - number of immuno-histochemically labeled cell nuclei inside the test field.

The formula for calculating the cell volume was:

Vc = 1/N_V , where

N_V is the numerical density of FSH and LH cells. Nuclei volume density (V_Vn) provides information about nuclei attendance in the estimated cells and is calculated as follows:

V_Vn = ΣPn/ΣPtc

Since rat FSH and LH cells are mononuclear, N_V corresponds to the number of cells per cubic millimetre, according to the formula:

N_V = (k/β) (N_A3/2 / V_Vn½)

On the basis of earlier karyometric studies (22), the shape coefficient β for pituitary cells was estimated to be 1.32, where k is a factor related to cell distribution according to their size (in the case of FSH and LH cells its value is 1) and N_A is the number of cells in the plane of the pituitary tissue section. N_A is calculated as follows:

N_A = ΣNn/ΣPtc·a ,

where a represents the rhombic area belonging to every point of the test system and is calculated using the formula:

a = d2 31/2/2 ,

where d is the test line length in the test system employed.

Relative volume density (V_VC) is calculated as the ratio of the sum of Pn and Ptc (Pn+Ptc) and the total number of points in the test system. Since a test system with 42 points was used and parameters were calculated using 50 test fields, the definite formula was:

V_VC = (Pn+Ptc)/ 50·42

Optical density measurements

The Windows based ImageJ program (Image J, version 1.50f) was used for the analysis of optical density that reflects the gonadotropic cell hormonal content. Namely, 30 unbiasedly captured images (the microscopic tool has already been described: 2088 x 1550 pixels, 63x objective magnification) per gonadotropic cell (FSH or LH) specific-immunostained pituitary per animal were analysed. Initially, the spectral deconvolution method of 3,3′-diaminobenzidine tetrahydrochloride (DAB)/Hematoxylin color spectra was performed, using optimized optical density vectors of the color deconvolution plug-in for adequate separation of the DAB color spectra. To determine the optical density for the red, green and blue (RGB) channel of Hematoxylin and DAB, we kept the protocol previously described by Ruifrok and Johnston (23) and Varghese et al. (24). Since the optical density is proportional to the concentration of the stain in gonadotropic cells, the amount of stain present is a factor determining the optical density (OD) at a wave length specific to the stain, according to the formula:

OD = – log10 (IC/I0.C),

IC is the intensity of detected light after passing through a specimen and I0.C refers to the intensity of light entering the specimen.

Statistical analysis

STATISTICA® version 7.0 (StatSoft, Inc) was used for the statistical analysis. Hormonal and morphometric data obtained from each group were statistically evaluated by the Student´s t-test. A probability value of 5% or less was considered statistically significant. The data are presented as means ± SD.

Body mass and pituitary weights

Data for the body mass and absolute/relative pituitary weights are summarized in Table 2. It is obvious that orchidectomy, as well as subsequent treatment with testosterone did not cause statistically significant changes in the measured parameters.

Table 2. Body mass, absolute and relative pituitary weights in orchidectomized (ORX) and testosterone treated orchidectomized (ORX+T) middle-aged rats

Hormonal data

The serum hormonal levels are shown in Figure 1. In ORX+T group, circulating testosterone and estradiol were significantly (p<0.05) increased by 24 and 2.7 fold respectively, compared to the ORX group values (Figure 1A and B).

Figure 1. Serum level of testosterone (A) and estradiol (B) in orchidectomized (ORX) and testosterone treated orchidectomized (ORX+T) middle-aged rats. All values are provided as the mean ± SD, n = 8 animals per group; *p<0.05 vs. ORX rats

Immuno-histochemical findings

Gonadotropic cells (FSH and LH) in ORX rats were widespread throughout the pars distalis, in groups or as single cells often in close contact with blood capillaries. They were hypertrophic, predominately oval in shape, with prominent eccentrically located nuclei. Strong immunopositivity of FSH and LH cells was observed. After the treatment with testosterone, gonadotropic cells were significantly smaller in size and less numerous (Figure 2A and B).

Figure 2. SH- (A) and LH- (B) immunoreactive cells in the pituitary pars distalis from orchidectomized (ORX) and testosterone treated orchidectomized (ORX+T) middle-aged rats (objective magnification 40x, bar = 25μm)

Morphometric and optical density-related results

The morphometric and optical density parameters observed for the FSH and LH cells in this study are represented in Figure 3. Namely, in ORX+T rats the volume of FSH and LH cells (Vc) was significantly (p<0.05) decreased by 51.3% and 56.6% respectively, in comparison with the ORX animals (Figure 3A). The relative volume density (Vvc) of FSH and LH cells in ORX+T animals was significantly (p<0.05) decreased by 54.0% and 72.8% respectively, compared to the ORX group (Figure 3B). Optical density of FSH and LH cells after orchidectomy and testosterone treatment was significantly (p<0.05) decreased by 25.7% and 16.2% respectively, in comparison with ORX rats (Figure 3C).

Figure 3. (A) Cellular volume (Vc, μm3), (B) relative volume density (V_vc, %) and (C) optical density of FSH and LH cells in orchidectomized (ORX) and testosterone treated orchidectomized (ORX+T) middle-aged rats. All values are provided as the mean ± SD, n = 8 animals per group. *p<0.05 vs. ORX rats