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Effect of adrenalectomy and hydrocortisone on ventral prostate of rats

Neena Nair, R.S. Bedwal, R.S. Mathur

Cell Biology Laboratory, Department of Zoology, University of Rajasthan, Jaipur  302004,  India

Asian J Androl  2001 Dec; 3: 289-300

Keywords: adrenalectomy; ventral prostate; hydrocortisone; testosterone; FSH; LH; cholesterol; zinc; copper; acid phosphatase; alkaline phosphatase; aryl sulphatase; lactic dehydrogenase; leucine aminopeptidase

Abstract

Aim:  To study the effects of adrenalectomy and hydrocortisone on the ventral prostate of SD rats. Methods: In adrenalectomised (ADX) and ADX+hydrocortisone (1, 2, or 4 mg) treated rats, the prostatic histology and the cholesterol, protein, zinc, and copper levels and the enzymic profile (acid phosphatase, alkaline phosphatase, aryl sulphatase, lactic dehydrogenase, and leucine aminopeptidase) in the prostatic tissue were determined; the serum hormonal profile (testosterone, FSH and LH) was also assayed. Results:   Adrenalectomy caused a progressive degeneration in prostatic structure that was not reversed by hydrocortisone treatment. The serum testosterone were significantly lower in ADX than in sham operated rats and lower in ADX+hydrocortisone than in ADX-C rats (P<0.01). The serum FSH and LH were below the detection limit of 1 mIU/mL. The enzymatic activity was higher in ADX than in sham operated rats and higher in ADX+hydrocortisone than in ADX-C rats (P<0.05-0.01). The prostatic zinc levels were significantly higher in sham operated and higher in ADX-C than in ADX+hydrocortisone rats (P<0.05-0.01). Prostatic copper level was significantly lower in sham operated and lower in ADX-C than in the ADX+hydrocortisone rats (P<0.01). Conclusion:  In rats, adrenalectomy leads to pathological and functional changes of the prostate. Hydrocortisone treatment at the doses employed did not reverse these changes.

1 Introduction

Adrenalectomy lowers the serum dihydrotestosterone to non-detectable levels, but does not impede the prostatic growth[1]. The ventral prostate, an androgen dependent tissue, requires both epididymis and testis for a maximal androgen effect[2]. Blok et al[3] reported that the androgen receptor (AR) mRNA expression in the ventral prostate and epididymis was regulated in a manner by testosterone different from that in the testis. Androgen receptors have been found in the epithelial as well as the stromal cells[4,5]. Oestrogen and glucocorticoids receptors have also been observed in the prostate[6,7].  Moreover, certain prostatic proteins have been found to be androgen dependent[8,9]. Prostatic acid phosphatase, a sialoglycoprotein (mol. wt. 100 kDa), composed of 2 identical subunits[10] and synthesized in the secretory epithelium of the prostate gland, is secreted into the acinar lumen by merocrine and apocrine processes[11]. Leucine aminopeptidase (exists in 2 forms in the human prostate with mol. wt. of 107 and 305 kDa) is a product of the epithelial cells of the prostate[12-14].

It has been postulated that androgen metabolism can be inhibited by the zinc ion[15]. Radioautography and histochemical studies on administered 65Zn revealed the presence of zinc mainly in the glandular epithelial cells[16]. Glucocorticoid has been shown to regulate copper homeostasis[17]. Whole tissue analysis  by atomic absorption spectrophotometer indicated that zinc and copper levels were afgected after adrenalectomy and hydrocortisone treatment, leading to pathological changes in the testis[18,19] and epididymis[20]. The aim of the present study was to examine the histology and levels of various biochemical parameters in the ventral prostate after adrenalectomy and hydrocortisone treatment.
2 Subjects  and  methods
The investigation was carried out with 2 experiments. Experiment 1 studied the effect of adrenalectomy on the rats and Experiment 2, the effect of hydrocortisone treatment on the adrenalectomized rats.

2.1 Animals and treatment  

2.1.1 Experiment 1

Colony bred Sprague-Dawley rats  (290-350 g) were provided by the Animal House of the University and maintained in a well ventilated animal room (temperature 282, 12:12 h light: darkness) in polypropylene cages with stainless steel grills. Sixty animals were randomly divided into 2 groups of 30 animals each: the sham operated (SO) and the adrenalectomized (ADX). The sham operation and bilateral adrenalectomy were performed under light ether anaesthesia through a dorsal approach. The peritoneum and subcutaneous tissues were sutured by cat gut and the skin, ethicon silk thread ( 30). Rats had free access to standard food (Lipton India Ltd.) and 0.9% NaCl solution ad libitum.

Six animals were sacrificed on each of the following days: 2, 4, 8, 12 and 16.  Blood samples were collected through cardiac puncture. Serum was prepared and stored at -20 until hormonal assay. The ventral prostate was then excised, trimmed off extraneous tissues, weighed and stored at -20 for the analysis of the biochemical parameters, the enzymatic profile and the zinc and copper levels.

2.1.2 Experiment 2

One hundred and twenty similar rats were randomly divided into 4 groups of 30 animals each: Group 1: Adrenalectomized control (ADX-C), Group 2: Adrenalectomy+hydrocortisone 1 mg, Group 3: Adrenalectomy+hydrocortisone 2 mg, Group 4: Adrenalectomy+hydrocortisone 4 mg. Bilatral adrenalectomy was performed as in Experiment 1. A single dose of hydrocortisone at the dose levels indicated above was  injected subcutaneously in 1 mL of 0.9% NaCl 7 days postoperatively. To the ADX-C rats only the vehicle was injected. The animals were sacrificed 2, 4, 8, 12 and 16 days after drug administration, 6 at a time, and the blood and ventral prostate were collected in the same manner as in Experiment 1.

2.2 Histological study

A portion of the ventral prostate from each animal was fixed in Bouin's fixative. Sections were cut at 5 m and stained with Erhlich haematoxylin and eosin. Histometric studies were carried out using occulometer and expressed in m.

2.3 Biochemical analysis

Cholesterol was assayed by the Leiberman Buchard method described by King and Wolten[21]. The quantification of protein was performed according to the method of Bradford[22]. The alkaline phosphatase (AKPase) activity was estimated by the method of Besay et al (1946) as given in Glick[23]. The acid phosphatase (ACPase) activity was assayed according to Andersch and Szcypinski (1947) and Besay et al(1946) as given in Bergmeyer[24]. The aryl sulphatase activity was assayed according to the method of Dodgson and Spencer as given by Glick[25]. The Lactic dehydrogenase (LDH) was determined by the method of Wroblewski as given in Natelson[26]. Leucine aminopeptidase (LAP) was assayed according to the Louis Berger and Dan Broida method as given in Sigma Technical Bulletin 251[27]. Absorbance of the aforesaid biochemical parameters was measured on Carl-Zeiss Spekol ZV spectrophotometer  (Jena, Germany).

2.4 Zinc and copper determination

After digesting the ventral prostate in a di-acidic (HClO4:HNO3=1:5) solution, zinc and copper were estimated at 213.9 and 324.8 nm, respectively, in an air-acetylene flame with 0.5 nm slit width, background correction and an integration time of 3 sec on a GBC-902 double beam atomic absorption spectrophotometer (Australia).

2.5 Hormone assay

Serum LH, FSH and testosterone were determined by radioimmunoassay (RIA) using coat-A-count kit (Diagnostic Products Corporation, Los Angeles).

2.6 Data processing

The data were expressed in meanSEM, if applicable. The Student's t test was used for statistical analysis and P<0.05 was considered significant.

3 Results  

3.1 Experiment 1

3.1.1 Prostatic weight

The wet weights of the ventral prostate exhibited a significant and progressive decrease in the ADX, but not in the SO animals (Table 1).

Table 1.  Effect of adrenalectomy on prostatic  weight (mg).

3.1.2 Histological observation

The prostate of SO rats all along revealed normal histological structures. With the ADX rats, a decrease in prostatic secretion, cellular cytoplasmolysis and nuclear degeneration were observed at Day 2 after adrenalectomy (Figure 1). At Days 4 (Figure 2) and 8 (Figure 3, 4) the degeneration was more pronounced. However at Day 8, the most prominent feature was the exfoliation of the necrotic nuclei both in the lumina of follicles and in the interfollicular space (Figure 3). At Day 12, the prostatic secretion was more or less absent with microvilli and nuclei at various stages of degeneration (Figure 5). At Day 16 after adrenalectomy, a further decrease in the secretion, loss of epithelial cytoplasmic zone and numerous pycknotic nuclei were the characteristic features of the prostate (Figure 6). These pathological changes were supported by morphometric measurements. The cell height was increased significantly (P<0.01) at Days 8, 12 and 16 (Table 2).

Figure 1.    Microphotograph of prostate, ADX rats on day 2,  showing microvilli projection,  cytoplasmolysis,  few necrotic nuclei. HE, 100.
Figure 2.      Microphotograph of prostate,  ADX rats on day 4,  showing conspicuous microvilli  projection and some epithelial cells devoid of nuclei. HE, 100.
Figure 3.      Microphotograph of prostate of ADX rats on day 8,  showing cytoplasmolysis,  necrotic nuclei and loss of epithelial cytoplasmic zone. HE,  50.
Figure 4.      Microphotograph of prostate,  ADX rats on day 8,  showing cytoplasmolysis,  pycknotic nuclei and decreased secretion. HE, 100.
Figure 5.      Microphotograph of prostate,  ADX rats on day 12,  showing cytoplasmolysis,  necrotic nuclei,  conspicuous microvilli projections in the follicles,  and exfoliation of necrotic nuclei in the interfollicular region. HE. 100x.
Figure 6.      Microphotograph of prostate,  ADX rats on day 16 showing hypertrophied epithelium,  necrotic nuclei and edematous fluid in interfollicular space. HE, 100.
Table 2.  Effect of adrenalectomy on morphometric data (mol/L). 

3.1.3 Bichemical data

Prostatic zinc exhibited a significant (P<0.01) increase while copper, a significant decrease (P<0.01) at Day 16; the cholesterol concentration was significantly increased and protein significantly decreased  (P<0.01, in both cases) in most occasions (Table 3). The activities of various enzymes (AKPase, ACPase, aryl sulphatase, LDH and LAP) were increased (Table 4).

Table 3.  Effect of adrenalectomy on prostatic biochemistry. 
Table 4.  Effect of adrealectomy on enzymatic profile of  prostate.

3.1.4 Hormone assay

The serum testosterone level exhibited significant decrease at all days (Table 5). The serum FSH and LH were all below the detectable limit of 1 mIU/mL.

Table 5. Effect of adrenalectomy on serum testosterone (ng/mL).

3.2 Experiment 2

3.2.1 Prostatic weight

The wet weights of the ventral prostate exhibited a significant and progressive decrease both in the ADX-C and the ADX+hydrocortisone rats (Table 6).

Table 6. Effect of hydrocortisone on prostatic weight  (mg)  in adrenaltom

3.2.2 Histological observation

(a) ADX+1 mg hydrocortisone

Decreased secretion, cell loss, and pycknotic and crenated nuclei were found at Day 2, along with degeneration of the interfollicular connective tissue (Figure 7). The degeneration became more pronounced at Days 4, 8 (Figure 8), 12 and 16 (Figure 9). The cell height and the nuclear diameter were also changed significantly (Table 7). 

Figure 7.      Microphotograph of prostate,  ADX+1 mg,  day 2,  showing loss of cells,  pycknotic nuclei and degeneration of the interfollicular connective tissue. HE, 100.
Figure 8.      Microphotograph of prostate,  ADX+1 mg,  day 8,  showing acini formation,  degenerated nuclei,  exfoliation of nuclei into interfollicular space. HE, 100.
Figure 9.    Microphotograph of prostate,  ADX+mg,  day 16,  showing edematous fluid in  interfollicular space,  exfoliation of nuclei and karyorhexis nuclei. HE, 100.
Table 7.  Effect of hydrocortisone on morphometric data (mol/L) in adrenalectomized rats.

(b) ADX+2 mg hydrocortisone  

The pathological changes observed at Day 2 included (1) scarce secretion,  (2) cytoplasmolysis, (3) nuclei at various stages of degeneration and (4) interfollicular spaces completely devoid of fibrous matrix (Figure 10).At Day 4, there were a further decrease in secretion, in acini formation (a feature more prominent in the peripheral than the central tubules), loss of cellularity, gran ulated and vacuolated cytoplasm and degenerated nuclei (Figure 11). The deterioration of prostate was more pronounced at Days 8, 12 and 16 (Figure 12). The cell height and the nuclear diameter were also changed significantly (Table 7).   

Figure 10.     Microphotograph of prostate,  ADX+2 mg,  day 2,  showing cytoplasmolysis,  degenerated nuclei and interfollicular space devoid of fibrous matrix. HE, 100.
Figure 11.     Microphotograph of prostate,  ADX+2 mg,  day 4,  showing acini formation,  necrotic nuclei,  follicles devoid of secretion loss of cellularity. HE,  100.
Figure 12.     Microphotograph of prostate,  ADX+2 mg,  day 16,  showing loss of cellularity,  pycknotic nuclei and edematous fluid in interfollicular space. HE, 100.

(c) ADX+4 mg hydrocortisone  

Similar observations were seen on Day 2 (Figure 13). The degenerations were more pronounced at Days 4 (Figure 14), 8 (Figure 15), 12 (Figure 16) and 16 (Figure 17). The cell height and the nuclear diameter were also changed significantly (Table 7).   

Figure 13.     Microphotograph of prostate of ADX+4 mg,  day 2,  showing cytoplasmolysis,   necrotic nuclei and increase in acini formation. HE, 100.
Figure 14.     Microphotograph of prostate,  ADX+4 mg,  day 4,  showing cytoplasmolysis and necrotic nuclei. HE, 100.
Figure 15.     Microphotograph of prostate,  ADX+4 mg,  day 8,  showing acini formation,  degenerated nuclei,  decrease in secretion and degeneration of  interfollicular fibrous matrix. HE, 100.
Figure 16.     Microphotograph of prostate,  ADX+4 mg,  day 12,  showing cytoplasmolysis,  pycknotic nuclei and degenerated interfollicular fibrous matrix. HE, 100.
Figure 17.     Microphotograph of prostate,  ADX+4 mg,  day 16,  showing degenerated epithelium and pycknotic nuclei. HE, 100.

(d) ADX-C  

It can be seen that the histological pictures of the control animals at Day 2 (Figure 18), Day 4 (Figure 19), Day 8 (Figure 20), Day 12 (Figure 21) and Day 16 (Figure 22) were not significantly different from those of the respective days of  thehydrocortisone-treated animals.

Figure 18.     Microphotograph of prostate,  ADX-C rats on day 2,   showing loss of cells,  vacuolisation of the secretory material. HE, 100.
Figure 19.     Microphotograph of prostate,  ADX-C rats,  day 4,  showing cytoplasmolysis,  necrotic nuclei and edematous fluid in the interfollicular space. HE, 100.
Figure 20.     Microphotograph of prostate,  ADX-C rat,  day 8,  showing autophagic vacuolisation,  acini formation and karyolytic nuclei. HE, 100.
Figure 21.     Microphotograph of prostate,  ADX-C rats,  day 12,  showing microvilli projections,  degenerated cytoplasm and nuclei. HE, 100.
Figure 22.     Microphotograph of prostate,  ADX-C rats,  day 16,  showing microvilli projections and nuclei at various stages of degeneration. HE, 100.

3.2.3 Biochemical data

The zinc concentration was significantly decreased and the copper significantly increased (P<0.01) in most occasions. The cholesterol concentrations were in creased at all dose levels while the protein, decreased significantly (P<0.01) at Days 4, 12 and 16 (1 mg), Days 2, 4, 8, and 12 (2 and 4 mg) (Table 8). The AKPase, ACPase, aryl sulphatase, LDH and LAP activities were all increased irrespective of the dose administered (Table 9).

Table 8. Effect of hydrocortisone on biochemical data of ventral prostate in adrenalectomized rats.
Table 9. Effect of hydrocortisone on prostatic biochemistry in adrenalectomized rats.

3.2.4 Hormone assay

At all dose levels the serum testosterone concentra-tions were low as compared with their respective controls  (Table 10). The serum FSH and LH were below the detectable limit.

Table 10.  Effect of hydrocortisone on serum testosterone (ng/mL) in adrenalectomized rats.

4 Discussion

The data obtained in the present study provide evidence for the role of adrenal gland on the ventral prostate. Following adrenalectomy, there is a significant decrease in prostatic weight, cellularity and secretion, with increased cytoplasmolysis and nuclear degeneration. This is supported by morphometric measurement viz. an increase in cell height and a decrease in nuclear diameter. Previous studies have shown that adrenalectomy caused testicular and epididymal regression[18,19]. This would explain a significant decline in the serum testosterone level in the present study. The causal relationship between prostate cell death and deficient serum testosterone has been verified[28-30].

Adrenalectomy increased the cholesterol levels and decreased the protein levels as a result of tissue necrosis. In accordance with the low testosterone level, it can be postulated that protein synthesis has been adversely affected, as the majority of prostatic proteins are androgen dependent[31]. Several workers have demonstrated the hormonal regulation of lysosomal hydrolases in the reproductive system[32,33]. Increased level of alkaline phosphatase in the present study may be caused by prostatic regression and low level of circulating testosterone. Acid phosphatase, an androgen dependent marker enzyme for glandular epithelium[34], increased significantly, which may be attributed to the release of lysosomal enzymes due to cellular breakdown.  Aryl sulphatases (lysosomal enzymes) are increased in malignant tissue and an increase in aryl sulphatase in ADX and ADX+hydrocortisone rats can be correlated to decreased testosterone levels leading to changes in the lysosomal membrane permeability and release of these enzymes. The increase in LDH and LAP in the present study may reflect prostatic damage.

Chandler et al[35] suggested that zinc was uptaken by the prostatic nuclei from the stroma and was then condensed at the Golgi apparatus, transformed into secretory vesicles and secreted to the prostatic lumen. A positive correlation exists between zinc and prostate function[36]. The high level of zinc content in the present study can be related to the histopathological state and edematous fluid. Copper concentration was decreased in the present study, which may lead to impairment in reproduction, as physiological copper imbalance is related to alteration of cell membrane integrity, enzyme inhibition and reduced stability of DNA[37].

Previous studies have shown that glucocorticoid treatment failed to maintain the normal architecture of testis and epididymis[18-20], which may be due to  decreased steroidogenesis either directly by affecting Leydig cells function or indirectly by affecting LH release[18,19]. Decreased serum testosterone level was observed in the present study and since prostate is an androgen dependent organ, its degeneration was comprehensible. Hydrocortisone at the doses employed did not reverse the serum testosterone level and the prostatic weight. 

Hydrocortisone did not reverse the Zn and Cu levels, the cholesterol level, the protein concentration and the enzymic activities of the prostate. Some of the enzymes, as alkaline phosphatase, lactic dehydrogenase, are zinc metalloproteins where zinc occupies key positions in their structure. Moreover, seminal plasma zinc (secreted by prostate) has antibacterial activity[38]. Increased copper levels have been correlated to the production of free radicals (OH¯) by Haber-Weiss reaction. Thus, the pathological status of the gland in the present study could be a result of free radical generation. In conclusion, adrenalectomy in rats leads to pathological and functional changes of the prostate. Hydrocortisone treatment at the doses employed did not reverse these changes.

Acknowledgements

Dr. Neena Nair thanks the Council of Scientific and Industrial Research, New Delhi for award of Research Associateship and financial assistance.

References

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Correspondence to:  Dr. Neena Nair, Cell Biology Laboratory, Department of Zoology, University of Rajasthan, Jaipur 302004,  India.
E-mail: priyankasharma@id.eth.net
Received 2001-05-15                                          Accepted 2001-12-06