Genistein-Induced Histomorphometric and Hormone Secreting Changes in the Adrenal Cortex in Middle-Aged Rats

Abstract

The soybean phytoestrogen, genistein, is increasingly consumed as an alternative therapeutic for age-related diseases, namely cardiovascular conditions, cancer and osteoporosis. Besides estrogenic/antiestrogenic action, this isoflavone exerts a prominent inhibitory effect on tyrosine kinase and the steroidogenic enzyme families, thus affecting hormonal homeostasis. The aim of this study was to examine the effects of genistein on: histomorphometric features of the adrenal cortex, blood concentrations of aldosterone, corticosterone and dehydroepiandrosterone (DHEA) and adrenal tissue corticosterone content in orchidectomized middle-aged male rats. Sixteen-month-old Wistar rats were divided into sham-operated (SO), orchidectomized (Orx) and genistein-treated orchidectomized (Orx+G) groups. Genistein (30 mg/kg/day) was administered subcutaneously for three weeks, while the control groups received the vehicle alone. The adrenal cortex was analysed histologically and morphometrically. Circulating concentrations of aldosterone, corticosterone and DHEA, as well as adrenal tissue corticosterone levels, were determined by immunoassay. When compared to the SO group, orchidectomy decreased the ZG and ZR cell volume by 43% and 29%, respectively (P < 0.05). Serum concentrations of aldosterone and DHEA were markedly lower [13% and 41%, respectively (P < 0.05)], while serum and adrenal tissue levels of corticosterone did not change after orchidectomy. Orchidectomy followed by genistein treatment increased the ZG, ZF and ZR cell volume by 54%, 34% and 77%, respectively (P < 0.05), compared to the untreated orchidectomized group. Histological analysis revealed noticeable vacuolization of the ZG and ZF cells in the Orx+G group. Serum aldosterone and corticosterone concentrations together with adrenal tissue corticosterone were 47%, 31% and 44% lower, respectively (P < 0.05), whereas serum DHEA concentration was 342% higher (P < 0.05) in this group in comparison with the Orx group. This study shows that in orchidectomized middle-aged rats, genistein can cause the shunting of metabolic pathways in the adrenals, supporting DHEA secretion and inhibiting corticosterone and aldosterone secretion.

Introduction

In males the decline of serum testosterone with ageing, accompanied by decreasing DHEA, growth hormone, thyroxine and melatonin concentrations, cause a set of symptoms known as the andropause (1). During this period the risk of cardiovascular diseases, cancer and osteoporosis increases, together with the incidence of stress-related psychiatric disorders i.e., depression, irritability and sleep disturbance (2). Amplified activity of the hypothalamic-pituitary-adrenal (HPA) axis is associated with a high incidence of stress-related psychiatric disorders with advancing age (3).

Genistein, a soybean isoflavone that is structurally similar to estradiol 17β (4, 5), acts as a phytoestrogen. It competes with endogenous estradiol for binding to estrogen receptors and has significantly higher affinity for estrogen receptor β (Er β) than for estrogen receptor α (Er α) (6). The polyphenolic nature of genistein enables it to act as an antioxidant (7), performing either a free radical-scavenging (8, 9) or an antioxidant enzyme-activating role (10). Besides the mild estrogenic/antiestrogenic and antioxidant activities, genistein is a strong inhibitor of the tyrosine kinase family (11), which is certainly the cause of its antiproliferative effect as demonstrated both in vitro and in vivo (12). The reported mechanisms positively contribute to the supposed beneficial influence of genistein in the prevention and treatment of cancer, cardiovascular and osteoporosis (12), so nutritional supplements containing genistein are widely used as alternative therapy for these age-related diseases.

Increased activity of the hypothalamic-pituitary-adrenal (HPA) axis is a frequent concomitant of ageing (3), and a probable cause of the higher incidence of stress-related psychiatric disorders in older people. Data describing the potential effects of genistein on stress-related and other corticosteroid hormone production and secretion are expanding. Within the adrenal, steroids are produced through the action of several forms of cytochrome P450 enzymes and 3β-hydroxysteroid dehydrogenase (3βHSD) (13). It is the differential expression of these enzymes in the three adrenocortical zones that leads to the production of specific steroids within each zone (14). Newly produced pregnenolone, as a precursor for steroidogenesis, can be metabolized through different modes. Namely, inhibition of some steroidogenic enzymes may support alternative metabolic pathways leading to the production of either aldosterone and corticosterone or adrenal androgens (15). Increased presence of 17α-hydroxylase/17,20-lyase (CYP17), caused by src tyrosine kinase family inhibition, effectively moves the precursor towards the route leading to adrenal androgens (15). Additionally, it was reported that estrogen application, which causes a decline in 3βHSD activity, blocks steroido-genesis in rat adrenals (16). Genistein also inhibits 3βHSD activity in vitro (17) and is well known as a potent competitive inhibitor of adrenocortical cytochrome P450 21-hydroxylase activity in primary cultured adrenocortical cells (18). Thus, genistein, a potent inhibitor of the main steroidogenic enzymes and tyrosine kinases family, may seriously affect the network of multiple factors regulating adrenal steroid hormone synthesis.

The present study was undertaken to provide additional insight into the actions of genistein (subcutaneously applied) on adrenal cortex histomorphometric features and corticosteroid hormone production and secretion in the andropause, mimicked by using orchidectomized middle-aged rats. Despite near-cessation of production of testosterone in middle-aged rats, low levels of testosterone and estradiol synthesis do continue to persist in the testes (19). In addition, some authors reported a decline in serum testosterone to estradiol ratio with aging in brown Norway rats (20). Orchidectomy should eliminate both testosterone and estradiol from the hormone milieu and therefore eliminate their residual effects on the adrenals. This is an established approach for examining the potential effects of sex hormone-like compounds on hormonal homeostasis. The dose of genistein employed was chosen to mimic human exposure to elevated concentrations of isoflavones when nutritional supplements are used for therapeutic purposes (21). Orally consumed soybean-based therapeuticals contain genistin being hydrolyzed to unconjugated active form genistein, due to digestive enzymes. Some new data (22) suggest that subcutaneous treatment is legitimate, considering that the vast majority of the subcutaneously injected genistein enters the circulation (about 80% is absorbed into blood when genistin is orally applied), so biological effects caused by either oral or subcutaneous application are very similar. To that end, using an injection strategy, we have operated with the form of soybean isoflavone available after digestive transformation.

Materials and Methods

Animals and Diets.

The experiments involved 16-month-old male Wistar rats. They were bred in the Institute for Biological Research, Belgrade, Serbia, housed one per cage, exposed to a 12–12 hrs light-dark cycle and kept at 22 ± 2°C. Two weeks before the experiment, the rats were fed a soy-free diet prepared in cooperation with the Department of Food, School of Veterinary Medicine, Belgrade, Serbia, and INSHRA PKB, Belgrade, Serbia, according to Picherit et al. (23), with corn oil as the fat source.

The diet contained, per 100 g: 20.3 g casein; 65 g carbohydrate (45 g cornstarch + 20 g sucrose); 5.2 g corn oil; 3.7 g fiber (crystalline cellulose); 1.5 g vitamin/mineral mix (Ca-phosphate deficient); 1.8 g calcium-phosphate dibasic; 1 g calcium carbonate; and 1.5 g DL-methionine. Casein and crystalline cellulose originated from Alfa Aesar, Johnson Matthey Gmbh & Co. KG, Karlsruhe, Germany; carbohydrate, oil, vitamin/mineral mix, calcium carbonate and calcium phosphate from INSHRA PKB, Belgrade, Serbia; and DL-methionine from Sigma Chemical Company, St. Louis, MO, USA. Food and water were available ad libitum.

Animal Treatments.

Sham surgery and orchidectomy were performed under ketamine anaesthesia (15 mg/kg body wt). Ketamine hydrochloride was obtained from Richter Pharma, Wels, Austria. The sham-operated (SO) group, consisting of eight animals, and the orchidectomized rats were allowed to recover for two weeks. The orchidectomized rats were then divided into two groups of eight animals each. One group (Orx+G) was then given genistein subcutaneously (30 mg/kg body wt) every day, except on Sundays, for three weeks. Genistein (Nutraceutica, Monterenzio, Italy) was dissolved in a minimal volume of absolute ethanol (approx. 0.17 ml) and then mixed with sterile olive oil (approx. 0.33 ml). Each animal was injected with the final volume of 0.5 ml of mixture. Final concentration of genistein was 36 mg/ml of the mixture, considering the dose, the mean animal body weight and volume of injection. The other group (Orx) and the SO group were treated with a mixture of the same volume of absolute ethanol dissolved in sterile olive oil (0.5 ml of the mixture per animal). All animals were killed by decapitation under ether anesthesia (ether ad narcosis Ph. Iug. III., Lek, Ljubljana, Slovenia) 24 hrs after the last injection.

All experimental protocols were approved by the local Animal Care Committee in conformity with the recommendations in the Guide for the Care and Use of Laboratory Animals (Washington, DC: National Academy Press, 1996).

Morphometric Measurements in the Adrenal Gland.

Left adrenal glands were excised, weighted, fixed in Bouin’s solution for 48 hrs, embedded in paraffin and serially cut into 5 μm thick sections. These were stained with hematoxylineosin and examined under a light microscope (Carl-Zeiss, Jena). The relative weight of the adrenal to the body weight was determined. The volume of the glands was calculated from their weight, assuming an average specific gravity of 1.039 gcm⁻³ (24). In order to estimate the volumes of the adrenocortical zones, every tenth section (5 μm thick) of the gland was analysed at ×125 magnification with the multipurpose test system M₄₂ (25).

The same system was used to estimate the nuclear and cytoplasmic volumes of parenchymal cells on 5 μm thick sections under the light microscope at ×1000 magnification (25). For each adrenal gland, a single paraffin section containing the zona medullaris was chosen and 30 test areas of the ZG and 50 test areas of both the ZF and ZR were analysed. On the basis of earlier karyometric studies (26), the shape coefficient β was assumed to be 1.382 for the ZF and 1.500 for the ZG. It relates N_v (number of cells counted per unit volume) to N_a (number of cells counted per mm²) and V_v (volume density) and depends on the axial ratio of the estimated nuclei.

Serum Hormone Assays.

Blood samples were collected from the trunk after decapitation. The separated serum samples were stored at −20°C until assayed for hormone concentrations in duplicate within single assays, respectively, as follows: aldosterone concentration by enzyme immunoassay for direct quantitative determination (Aldosterone ELISA, IBL Hamburg, Germany) with an intra-assay CV of 4.1% (128.61 pg/ml); corticosterone concentration by immunoassay (R&D Systems Inc., Minneapolis, USA) with an intra-assay CV of 8.0% (171 ng/ml); DHEA concentration by enzyme immunoassay for quantitative determination (DHEA ELISA, IBL Hamburg, Germany) with an intra-assay CV of 6.92% (0.58 ng/ml).

Adrenal Tissue Corticosterone Assay.

The right adrenal glands were excised, weighted and immediately shredded on ice. Shredded tissue was then homogenized in TRIS-saccharose buffer (pH 7.9; 1 mg of tissue: 1 μl of buffer) using a dispersion system (Ultra-Turax T25, Janke & Kunkel, IKA-Labortechnik) at 8000 rpm. The homogenate was centrifuged at 35,000 rpm (105,000 g) for 1 hr (BECKMAN ultracentrifuge, L7–55) and corticosterone concentration in the supernatant determined by immunoassay (R&D Systems Inc., Minneapolis, USA).

Statistical Analyses.

Morphometric and biochemical data obtained for the experimental groups were subjected to one-way analyses of variance (ANOVA). Duncan’s multiple range test was used for post hoc comparisons between groups. A confidence level of P < 0.05 was considered statistically significant. The data are presented as means ± SD.

Results

Body and Adrenal Weights.

Data for body weight, absolute and relative adrenal weights are summarized in Table 1. Mean body weight in the Orx group decreased by 10% (P < 0.05) in comparison to the SO group. After orchidectomy the absolute adrenal weight increased by 14% (P < 0.05). Orchidectomy and subsequent genistein treatment led to a greater increase (P < 0.05) of adrenal weight, by 13% in comparison with the Orx group and 29% more than the SO group. The relative adrenal weight was 22% higher (P < 0.05) for the Orx group and 31% higher (P < 0.05) for the Orx+G group in comparison with the SO group.

Morphometric and Histological Findings in Adrenal Cortex.

Zona Glomerulosa (ZG).

The ZG was arranged in closely packed cell clusters of small, columnar or pyramidal, cells with oval nuclei (Fig. 1a ). The overall shape of ZG cells did not change either in the Orx (Fig. 1d ) or in the Orx+G group (Fig. 1g ), but a noticeable feature of these cells in the latter group was vacuolisation as a result of lipid-droplet pull down during fixation (Fig. 1g ).

The absolute and relative volumes of the ZG remained unaltered in the Orx and Orx+G groups when compared to the SO group (Figs. 2 and 3 ). However, in the Orx group, stereological analysis showed that the volumes of ZG cells and their nuclei decreased by 43% and 37% (P < 0.05), respectively, in comparison with values for the SO group (Table 2 ). In the Orx+G group, ZG cell volume was 13% smaller (P < 0.05) but nuclear volume 30% larger (P < 0.05) than in the SO group. In comparison with the Orx group, the volume of ZG cells and their nuclei in the Orx+G group was increased by 54% and 107% (P < 0.05), respectively (Table 2 ).

Zona Fasciculata (ZF).

The ZF cells were large, with clearly visible single oval nuclei and were arranged in long, straight cords separated by sinusoidal capillaries (Fig. 1b ). The shapes and positions of the cells did not change either after orchidectomy (Fig. 1e ) or orchidectomy followed with genistein treatment (Fig. 1h ), although noticeable vacuolisation characterized the latter (Fig. 1h ).

The absolute volume of the ZF tended to increase (P > 0.05) in the Orx and Orx+G groups (Fig. 2 ), but the relative volume remained unchanged when compared to the SO group (Fig. 3 ). While the volume of ZF cells in the Orx group increased slightly (P > 0.05), their nuclei volume increased significantly (P < 0.05) by 35%, when compared to the SO group (Table 2 ). In the Orx+G group, the volume of ZF cells was 51% and 34% greater (P < 0.05) than in the SO and Orx groups respectively, while the volume of their nuclei was 49% greater (P < 0.05) than in the SO group (Table 2 ).

Zona Reticularis (ZR).

Cells of the ZR were dark and arranged net-like around the blood vessels (Fig. 1c ). In the Orx group (Fig. 1f ), these cells were small and around dilated blood vessels. The ZR cells in the Orx+G group (Fig. 1i ) had dark nuclei and the blood vessels were dilated.

In comparison with the SO group, the absolute volume of ZR increased by 37% after orchidectomy and by 35% after orchidectomy followed with genistein treatment (P < 0.05) (Fig. 2 ). The relative volume of ZR after these treatments was also slightly greater than in the SO group, but not statistically significant (P > 0.05; Fig. 3 ). In the Orx group, the volume of ZR cells and their nuclei decreased by 29% and 25% (P < 0.05), respectively, in comparison with the SO group (Table 2 ). However, in the Orx+G group ZR cell volume was 25% greater (P < 0.05) than in the SO group. Thus, in comparison with the Orx group, the volumes of ZR cells and their nuclei in the Orx+G group showed 77% and 44% increases, respectively (P < 0.05) (Table 2 ).

Absolute and Relative Volumes of the Adrenal Cortex.

In the Orx+G group, the absolute volume of the adrenal cortex increased by 15% (P < 0.05) compared to the SO group (Fig. 2 ). The relative volumes of the adrenal cortices remained unchanged in both the Orx and Orx+G groups (Fig. 3 ). The absolute and relative volumes of the adrenal cortices did not differ significantly between the Orx and Orx+G groups (Figs. 2 and 3 ).

Serum Aldosterone Concentrations.

The serum concentrations of aldosterone declined by 13% in the Orx group and by 54% in the Orx+G group (P < 0.05), when compared to the SO group (Fig. 4a ). In the Orx+G group, serum aldosterone concentration was 47% lower (P < 0.05) than in the Orx group (Fig. 4a ).

Serum Corticosterone Concentrations.

The mean serum concentrations of corticosterone are depicted in Figure 4b . Orchidectomy alone did not affect the serum concentration of this hormone, but in the Orx+G group serum corticosterone concentration was reduced by 31% (P < 0.05; Fig. 4b ) in comparison with the vehicle-treated Orx group and by 34% compared to the SO group (P < 0.05; Fig. 4b ).

Serum DHEA Concentrations.

Changes in the mean serum DHEA concentrations after orchidectomy and orchidectomy followed with genistein treatment are displayed in Figure 4c . Orchidectomy led to a decrease of serum DHEA concentration by 41% (P < 0.05) in comparison with the SO group. In the Orx+G group, serum DHEA concentration was much higher (P < 0.05) than in the Orx and SO groups (342% and 163%, respectively) (Fig. 4c ).

Adrenal Tissue Corticosterone Levels.

In the Orx+G group the adrenal tissue level of corticosterone decreased by 44% (P < 0.05) compared to the Orx group(Fig. 4d ) and by 46% in comparison with the SO group (P < 0.05; Fig. 4d ).

Discussion

Under our experimental conditions, a significant decrease of mean body weight was detected five weeks after orchidectomy. This confirms previous reports (27) and may be due to atrophy of skeletal muscles induced by testosterone deprivation (28). Subcutaneous genistein treatment of orchidectomized rats did not affect body weight in our animals. Described effects of genistein on body weight in males are contradictory, including decreases (29), increases (30) or, as in this study, no effect (31), probably due to different treatment protocols and experimental conditions.

Increases of absolute and relative adrenal weight after orchidectomy and orchidectomy followed with genistein treatment were observed. Orchidectomy was earlier reported to increase adrenal weight, an effect reversed by testosterone (26). However, the detected elevation of absolute and relative adrenal weight was higher in the Orx+G group and may partly be the result of precursor accumulation due to blockade of corticosterone biosynthesis by genistein. Namely, continuous administration of genistein to weaning rats led to a greater total protein content in the adrenal gland and to cell expansion in the ZF and ZR areas (32).

Orchidectomy decreased the ZG cell and nuclear volume as well as serum aldosterone concentration in comparison with the SO group. Testosterone exerts an inhibitory action on the activity of steroid 5α-reductase, an intraadrenal enzyme system catalyzing the reduction of corticosterone to dihydro- and tetrahydrocorticosterone (33). Its activity markedly increases in orchidectomized rats and favors utilization of corticosterone as a substrate for reduced metabolites instead of aldosterone synthesis. Furthermore, Malendowicz (26) reported considerable diminution of the ZG volume fraction and slight decreases in ZG cell cytoplasmic and nucleari volume, after orchidectomy. The decline in ZG cell volume, also observed here, can be explained by continuous discharge of efficiently produced dihydro- and tetrahydrocorticosterone. Orchidectomy followed with genistein treatment also caused a reduction of both ZG cell volume and aldosterone concentration when compared to the SO group. However, although aldosterone concentration was significantly lower than that for the group where only orchidectomy was performed, ZG cell volume was noticeably larger. In addition, the ZG nuclei volume was greater compared to the same parameter in both of the SO and Orx groups. Genistein was reported to block aldosterone production as a result of a non-specific effect on 3βHSD (34). Some studies have suggested that inhibition of the src family of tyrosine kinases blocks aldosterone synthesis (15). Namely, inhibition of src increases CYP17 expression, which effectively removes substrate away from the pathway leading to aldosterone. Here, we confirm that genistein, a potent inhibitor of the tyrosine kinase family and 3βHSD activity, decreases aldosterone production. Enhancement of the ZG nuclear and cell volume in the Orx+G group is probably a consequence of CYP17 mRNA levels increasing and pregnenolone accumulation due to src tyrosine kinase family and 3βHSD blockade. Further investigations are necessary to confirm this hypothesis.

After orchidectomy and genistein treatment, the ZF cell and nuclei volumes increased, but both serum and adrenal tissue corticosterone levels markedly decreased when compared to the SO group. The ZF cell volume was also greater and the serum and adrenal tissue corticosterone levels were lower in the Orx+G group than for the orchidectomized group. In the inhibition of corticosterone production by genistein, the central effects play a very important role as well as direct inhibition of enzymes in the corticosteroidogenesis pathway(s). Estrogen replacement was reported to lower the POMC mRNA level and the ACTH response to repeated stressful stimuli in ovariectomized rats (35). Besides the estrogenic mechanism, genistein may also reduce the level of ACTH through inhibition of tyrosine kinase phosphorylation cascades (36). We previously noticed that genistein lowers pituitary ACTH release in orchidectomized middle-aged rats (37). Therefore, it seems logical to assume that serum corticosterone concentration was reduced through central effects. On the other hand, direct action of genistein on the adrenal cortex cannot be overlooked. Genistein was shown to suppress cortisol secretion in the H925 human adrenocortical cell line and a porcine adrenocortical cell line (18, 38). It was shown that subcutaneous administration of genistein at 3 nmol/100 g significantly lowered the blood corticosterone level in ovariectomized rats unrelated to its estrogen-like activity (39). On the other hand, genistein was found to strongly inhibit human adrenocortical 3βHSD in vitro (17), similarly to estrogen action (16), and to reduce the activity of 21-hydroxylase enzyme (P450c21) (18). A possible basis for the ZF cell volume increase is precursor accumulation due to blockade of corticosterone biosynthesis by genistein. Histological analysis showed noticeable vacuolization in the ZF and ZG, after orchidectomy and genistein treatment caused by pulling down of lipid droplets during fixation. Lipid droplets are the intracellular stores of the obligate precursors of steroid hormones in rats (40).

In the Orx group, we observed a significant decrease in the ZR cell and nuclei volume but a significant increase of the whole ZR volume (including cell volumes + intercellular spaces), together with a considerable fall in serum DHEA concentration when compared to SO group. In addition, histological analysis of the ZR in the Orx group revealed massive blood vessel dilatation, which was possibly the basis for the ZR volume increase. Production of C₁₉ steroids, often called adrenal androgens, is a unique characteristic of the adrenal glands but the mechanisms regulating production of the major androgens, DHEA and DHEA sulfate (DHEAS), are poorly understood (41). Most circulating DHEA comes from the conversion of DHEAS in peripheral tissues (42). It was reported that the level of serum DHEA and DHEAS in 10-weeks-old castrated hamsters was at least as high as for intact animals, suggesting that the testes did not contribute to a large amount of these hormones in the circulation (43). On the other hand, in prostate cancer patients DHEAS concentration decreased after orchidectomy (44). Finally, some authors have suggested stimulation of DHEA and DHEAS secretion by ACTH (45, 46), as well as increased (33) or decreased (47) ACTH secretion after orchidectomy. We believe that some central mechanisms related to gonadothropin (FSH and LH) secretion may also be responsible for the reduction in ZR cell and nuclei volume, which accompanies the decline in serum DHEA concentration observed in the Orx group. Namely, LH and FSH levels are extremely high after orchidectomy (48) and adult rats exhibit distinct atrophy of the ZR after gonadothropin administration (49). Therefore, consequences of increased gonadothropin secretion after orchidectomy on the ZR should be seriously considered. Orchidectomy followed with genistein treatment led to significant enhancement of the ZR volume, ZR cell volume and DHEA serum concentration in comparison with the SO group and with the group where only orchidectomy was performed. It was mentioned that differential expression of five forms of cytochrome P450 and 3βHSD in the three adrenocortical zones leads to the production of specific steroids within each zone (13, 14). All steroidogenic cells use pregnenolone as a precursor for steroidogenesis. Pregnenolone can be metabolized by either 3βHSD or CYP17, whereas a high CYP17/3βHSB ratio supports DHEA synthesis (15). Genistein, most likely, stimulates CYP17 overexpression via inhibition of the src tyrosine kinases family and, as observed, directly inhibits 3βHSD. That way, genistein shunts steroid metabolic pathways towards the synthesis of DHEA and opposes corticosterone and aldosterone synthesis. We found inhibition of ACTH release after orchidectomy and genistein treatment (37), and histomorphometric analysis revealed strong stimulation in the ZR, despite orchidectomy and its inhibiting effects on the same zone. We, therefore, believe that the direct effects of genistein on steroid metabolic pathways are predominant.

In conclusion, this study shows that subcutaneous genistein administration in orchidectomized middle-aged rats inhibits aldosterone secretion, as well as corticosterone production and secretion, while DHEA secretion was stimulated. In the same group, histological analysis showed noticeable vacuolisation of the ZG and ZF cells, while morphological features of the adrenal ZG, ZF and ZR were affected by increasing the cell and nuclei volumes, compared to the group where only orchidectomy was performed. The presented biochemical and histomorphometric data suggest that genistein caused shunting of steroid metabolic pathways in the adrenals, supporting DHEA production and inhibiting corticosterone and aldosterone production in orchidectomized middle-aged rats.

Table 1.
The Effects of Orchidectomy and Orchidectomy Followed with Genistein Treatment on Body Weight, Absolute and Relative Adrenal Weights in Middle Aged Male Rats^a

Groups Body weight (g) Absolute adrenal weight (mg) Relative adrenal weight (mg%)

^aMean ± SD, n = 8.

^a P < 0.05 vs. sham operated (SO).

^b P < 0.05 vs. orchidectomized (Orx).

SO 650.2 ± 28.9 32.5 ± 1.3 5.1 ± 0.4

Orx 552 ± 45.5^a 37 ± 3^a 6.2 ± 0.5^a

Orx+G 598.6 ± 57.2 41.8 ± 1.9^a,b 6.7 ± 0.4^a

Table 2.
The Effects of Orchidectomy and Orchidectomy Followed with Genistein Treatment on the Morphometric Parameters of Middle Age Male Rat Zona Glomerulosa (ZG), Zona Fasciculata (ZF) and Zona Reticularis (ZR)^a

Volume of cells (μm³) Volume of nuclei (μm³)

Groups ZG ZF ZR ZG ZF ZR

^aMean ± SD, n = 8.

^a P < 0.05 vs. sham operated (SO).

^b P < 0.05 vs. orchidectomized (Orx).

SO 1454 ± 55 1892 ± 23 941 ± 81 220 ± 4 237 ± 7 225 ± 16

Orx 825 ± 29^a 2120 ± 102 665 ± 6^a 138 ± 8^a 319 ± 7^a 169 ± 10^a

Orx+G 1268 ± 2^a,b 2849 ± 202^a,b 1178 ± 36^a,b 286 ± 5^a,b 353 ± 21^a 243 ± 2^b

Figure 1.
Adrenal cortex in: a.–c. sham operated rats; d.–f. orchidectomized rats; and g.–i. orchidectomized and genistein treated rats. Capsula (C), zona glomerulosa (ZG), zona fasciculata (ZF), zona reticularis (ZR), (H&E, bar 25 μm).

Figure 2.
The absolute volume (cm³) of the adrenal cortex, zona glomerulosa (ZG), zona fasciculata (ZF) and zona reticularis (ZR) after orchidectomy and orchidectomy followed with genistein treatment. All values are the means ± standard deviation, n = 8 animals per group, ^a P < 0.05 vs. sham operated (SO).

Figure 3.
The relative volume (%) of the adrenal cortex, zona glomerulosa (ZG), zona fasciculata (ZF) and zona reticularis (ZR) after orchidectomy and orchidectomy followed with genistein treatement. All values are the means ± standard deviation, n = 8 animals per group.

Figure 4.
a. Serum concentration of aldosterone; b. serum concentration of corticosterone; c. serum concentration of DHEA; d. tissue level of corticosterone, in middle aged male rats. The values are the means ± standard deviation, n = 8 animals per group, ^a P < 0.05 vs. sham operated (SO), ^b P < 0.05 vs. orchidectomized (Orx).

Groups	Body weight (g)	Absolute adrenal weight (mg)	Relative adrenal weight (mg%)
^aMean ± SD, n = 8.
^a P < 0.05 vs. sham operated (SO).
^b P < 0.05 vs. orchidectomized (Orx).
SO	650.2 ± 28.9	32.5 ± 1.3	5.1 ± 0.4
Orx	552 ± 45.5^a	37 ± 3^a	6.2 ± 0.5^a
Orx+G	598.6 ± 57.2	41.8 ± 1.9^a,b	6.7 ± 0.4^a

	Volume of cells (μm³)	Volume of nuclei (μm³)
^aMean ± SD, n = 8.
^a P < 0.05 vs. sham operated (SO).
^b P < 0.05 vs. orchidectomized (Orx).
SO	1454 ± 55	1892 ± 23	941 ± 81	220 ± 4	237 ± 7	225 ± 16
Orx	825 ± 29^a	2120 ± 102	665 ± 6^a	138 ± 8^a	319 ± 7^a	169 ± 10^a
Orx+G	1268 ± 2^a,b	2849 ± 202^a,b	1178 ± 36^a,b	286 ± 5^a,b	353 ± 21^a	243 ± 2^b

Footnotes

This work was supported by the Ministry of Science and Technological Development of Serbia, Grant number 143007B.

Acknowledgements

The authors are especially grateful to Vladan Milosavljević (Carl Zeiss, Belgrade, Serbia) and Goran Rakić (Alfatrade Enterprise D. O. O., Belgrade, Serbia) for help in this work.

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