Effect of adrenalectomy on rat epididymidis

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

Cell Biology Laboratory, Department of Zoology, University of Rajasthan, Jaipur - 302 004, India

Asian J Androl 2002 Dec; 4: 273-279           

Keywords: adrenalectomy; epididymis; cholesterol; protein; alkaline phosphatase; acid phosphatase; leucine aminopeptidase; lactic dehydrogenase; zinc; copper; testosterone; FSH; LH

Abstract

Aim: To investigate the effect of adrenalectomy (ADX) on the epididymidis of Sprague-Dawley rats. Methods: The histological, biochemical (cholesterol protein, zinc, copper, alkaline and acid phosphatase aryl sulphatase, lactic dehydrogenase and leucine amino peptidase) and hormonal (FSH, LH and testosterone) changes of caput and cauda epididymis in ADX rats were observed. Results: Organ wet weight, histological studies and morphometric measurements indicated a cellular degeneration in caput and cauda epididymis of ADX rats. Serum testosterone level was significantly lower in ADX than in sham-operated rats, while 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. Epididymal zinc level increased whereas copper level decreased in ADX rats compared to the sham-operated. Conclusion: Adrenalectomy leads to degeneration of caput and cauda epididymidis epithelial cells as a result of decreased supply of testosterone.

1 Introduction

Chronically elevated glucocorticoids either because of increased endogenous secretion (such as in psychogenic amenorrhoea, anorexia nervosa, Cushing's syndrome and marathon runners) or exogenous administration have adverse effects on reproductive system [1, 2]. Adrenalectomy has been shown to abolish hypothalamic - pituitary - adrenal (HPA) axis induced suppression of gonadotropin secretion, hence glucocorticoid in excess per se may be responsible for hypogonadism [3]. However, Lemaire et al [4] reported that the influence on HPA axis is not always inhibitory but depends upon interaction of the two neuroendocrine axis. There are reports indicating that glucocorticoids affect both steroidogenesis and spermatogenesis, as glucocorticoid receptor-like immunoreactivity (GR-LI) has been found on Leydig cells, early pachytene and zygotene primary spermatocytes [5], peritubular myoid cells, fibroblast and basal cells of epididymis [6]. Androgen receptor (AR) and androgen binding protein (ABP) have been reported to be present in cauda [7]. In the rete testicular fluid, there is a fairly high aryl sulphatase activity [8]. However, there is not much information about epididymal lactic dehydrogenase and leucine aminopeptidase.

Reports indicate that zinc and copper seem to be regulated by glucocorticoids [9, 10]. Various studies have indicated that high zinc levels are responsible for maintaining spermatozoa in metabolically quiescent stage [11] as well as for stabilizing spermatozoa membrane as it is associated with phospholipids, lipoproteins and membrane bound metalloenzymes during caudal storage and/or ejaculation [12]. Chromatin contains 0.4 mmol of tightly bound copper per gm of DNA [13], which is homeosta-tically regulated by glucocorticoids [9]. Thus, the present study was undertaken to evaluate the effect of adrenalectomy on epididymal histology, biochemistry, hormones and trace elements - zinc and copper.

2 Materials and methods

2.1 Animals and experiment

Colony bred Sprague-Dawley rats (290 g~350 g) were provided by the Animal House of the University and maintained in a well ventilated animal room (28 2 , 12 h :12 h light : darkness) in polypropylene cages with stainless steel grills. Sixty animals were randomly divided into two 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 by ethicon silk thread (No 30). Rats had free access to standard food (Lipton India Ltd.) and 0.9 % NaCl solution ad libitum.

Six animals were sacrificed on day 2, 4, 8, 12 and 16. Blood samples were collected through cardiac puncture. Serum was prepared and stored at -20 until hormonal assay. The caput and cauda epididymides was then excised, trimmed off extraneous tissues, weighed and stored at -20 for various biochemical parameters.

2.2 Histological study

One of the caput and cauda epididymidis from each animal was fixed in Bouin's fixative. Sections were cut at 5 mm and stained with Erhlich haematoxylin and alcoholic eosin. Histometric studies were carried out using occulometer and expressed in mm.

2.3 Biochemical analysis

Cholesterol was assayed by the Leiberman Buchard method described by King and Wolten [14]. The quantification of protein was performed according to the method of Bradford [15]. The alkaline phosphatase (AKPase) activity was estimated by the method of Besay et al (1946) as given in Glick [16]. The acid phosphatase (ACPase) activity was assayed according to Andersch and Szcypinski (1947) and Besay et al (1946) as given in Bergmeyer [17]. The aryl sulphatase activity was assayed according to the method of Dodgson and Spencer as given by Glick [18]. The lactic dehydrogenase (LDH) was determined by the method of Wroblewski as given in Natelson [19]. Leucine aminopeptidase (LAP) was assayed according to the Louis Berger and Dan Broida method as given in Sigma Technical Bulletin No. 251 [20]. 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 caput and cauda epididymidis 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 mm 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, USA).

2.6 Statistical analysis

The data were expressed in mean SE. The Student's-t test was used for statistical analysis and P<0.05 was considered significant.

3 Results

3.1 Epididymal weight

The wet weights of caput and cauda epididymidis exhibited a decrease in ADX rats compared to the sham operated ones (Tables 1a, b).

Table 1a. Effect of adrenalectomy on caput epididydimis weight (mg). ^bP<0.05, ^cP<0.01, compared with sham-operated.

Table 1b : Effect of adrenalectomy on cauda epididydimis weight (mg). ^cP<0.01, compared with sham-operated.

3.2 Histological and histometric observation

3.2.1 Caput epididymidis

In sham operated rats, the histology was normal on day 2, 4 (Figure 1), 8 and 16. In the ADX rats, a decrease in stereocilia and spermatozoa was observed and a few pycknotic nuclei were seen on day 2; on day 4, the principal cells displayed pycknotic nuclei and vacuolization at the adluminal surface (Figure 2), whereas on days 8 and 12, further degeneration was evident; on day 16, the epithelial cells were detached from the basement membrane, the epithelial lining had completely lost its normal architecture and the lumen was obliterated with exfoliated epithelial cells. Spermatozoa were rarely seen. These pathological changes were supported by morphometric measurements (Table 2a).

Table 2a : Effect of adrenalectomy on caput morphometric data (mm). ^bP< 0.05, ^cP<0.01, compared with sham-operated.

Figure 1. Caput epididymis of sham operated rat on day 4 showing normal architecture. H & E 100.
Figure 2. Caput epididymis 4th day of ADX rat exhibiting decreased density of stereocilia and sperms and degeneration of chromatin and intertubular connective tissue. H & E 100.

3.2.2 Cauda epididymidis

The sham operated rats showed normal histology on days 2, 4, 8, 12 (Figure 3) and 16. In ADX rats on day 2 and 4, there were centrally located small masses of spermatozoa and epididymal secretion and a few necrotic epithelial cells; the basement membrane around the tubules and intertubular connective tissue were disrupted at some places. On day 8 there were decreased sperm density, necrotic epithelial cells with deterioration of intertubular connective tissue. Sparse masses of spermatozoa in the lumen, several necrotic epithelial cells, uneven surfaces of epithelial cells with secretory vesicles at adluminal surfaces and disorganised intertubular connective tissue were evident on day 12 (Figure 4). On day 16, cauda tubules illustrated disorganized epithelial cell lining, the epithelial cells were necrotic and the lumen filled with oedematous fluid intermingled with a few spermatozoa (Figure 5). Histometric measurement revealed significant decreases in the diameters of cauda epididymal tubules (on day 12 and 16), nuclei and the cell height (Table 2b).

Table 2b : Effect of adrenalectomy on cauda morphometric data (mm). ^b P < 0.05, ^c P<0.01, compared with sham-operated.

Figure 3. Cauda epididymis of sham operated rat on day 12 showing normal architecture. H & E100.
Figure 4. Cauda epididymis of adrenalectomised rat on day 12 illustrating spermatozoa, necrotic nuclei and disorganised basement membrane. H & E 100.
Figure 5. Cauda epididymis of adrenalectomised rat on day 16 showing fibrosis in the intertubular space, condensation of sperms and epithelial cells reduced to necrotic masses. H & E100.

3.3 Biochemical data

In the ADX rats, the epididymal cholesterol was increased (P<0.05, P< 0.01), while the protein level decreased (P<0.01) significantly (Tables 3a, b); Various enzymes (AKPase, ACPase, arylsulphatase, LDH and LAP) were increased (Table 4a, b), while the zinc level was increased and copper, significantly decreased (P<0.01) (Table 3a, b).

Table 3a : Effect of adrenalectomy on caput biochemistry. ^bP< 0.05, ^cP<0.01 compared with sham operated. Units: Cholesterol and Protein (mg/g), Zinc and Copper (mg/g).

Table 3b : Effect of adrenalectomy on cauda biochemistry. ^cP<0.01, compared with sham operated. Units: Cholesterol and Protein (mg/g), Zinc and Copper (mg/g).

Table 4a: Effect of adrenalectomy on enzymatic profile of caput. ^cP<0.01 compared with sham operated. Units: Alkaline phosphatase, Acid phosphatase, Aryl sulphatase (mmol of p-nitrophenol liberated /mg/ protein/h), Lactic dehydrogenase (LDH units / mg protein/ h), Leucine aminopeptidase (mmol of b-napthylamine liberated/ mg protein / h).

Table 4b: Effect of adrenalectomy on enzymatic profile of cauda. ^bP< 0.05, ^cP<0.01, compared with sham operated. Units: Alkaline phosphatase, Acid phosphatase, Aryl sulphatase (mmol of p-nitrophenol liberated /mg/ protein/h), Lactic dehydrogenase (LDH units/mg protein/h), Leucine aminopeptidase (mmol of b-napthylamine liberated/ mg protein/h).

3.4 Hormone assay

In the ADX rats, the serum testosterone was decreased significantly at all days with FSH and LH below the detectable limit 1 mIU/mL (Table 5).

Table 5: Effect of adrenalectomy on serum testosterone level (nmol/L). ^bP< 0.05, ^cP< 0.01, compared with sham operated. Serum FSH & LH < 1 mlU/mL.

4 Discussion

Cauda epididymal activities are androgen dependent [21]. The fact that adrenalectomy in male rats causes a degeneration of testicular structures, delays the onset of spermatogenesis and reduces the spermatogonia A cell count seems to suggest a possible role of adrenal cortical hormones in spermatogonial proliferation and subsequent differentiation [22]. Degenerative changes in testis and atrophy with decreased nuclear diameters of Leydig cells of adrenalectomised rats have been correlated to perturbation in testosterone production [23]. The deleterious changes in caput and cauda epididymal principal cells, decreased tubular and lumen diameters, cell heights and nuclear diameters and decreased sperm density in the tubules observed in ADX rats in the present investigation indicate that metabolic stress decreases serum testosterone with non detectable FSH and LH level, suggesting that stress related changes are mediated through the HPA axis. Further, the degenerative changes may increase the harmful effects of oxyradicals generated in the epididymis following ADX, which can be toxic to spermatozoa [24].

Adrenalectomy increased the cholesterol and decreased the total protein level in caput and cauda epidi-dymis. Alteration in the internal milieu due to degenerative process and decreased sperm density must have inhibited protein synthesis as the synthesis and secretion of a number of proteins are to a large extent mediated by androgens [25]. This was further confirmed by Syntin et al [26] who reported that approximately 48 % of all the proteins secreted in the adult boar epididymis are androgen-dependent.

AKPase activity increased significantly in caput and cauda epididymis of ADX rats, which may be due to resorption of non-ejaculated spermatozoa. The increase in ACPase activity is not androgen-dependent. Moreover, macrophage-like cells that exhibit strong ACPase activity have been shown among the epithelial cells of the epididymis and have been associated with the removal of old and/or dead cells by phagocytosis and lysosomal digestion (Spermiophagy) [27]. Hence, it is suggested that the increased ACPase activity in caput and cauda epididymidis of ADX rats may be due to: (1) lysosomal activity of epithelial cells and (2) increased number of ACPase positive macrophage-like cells needed for phagocytosis and lysosomal digestion of dead and old epithelial cells and resorption of non-ejaculated surplus spermatozoa. Adrenalectomy is characterized by asper-matogenic condition and enormous oedematous fluid formation in the testes [23]. The increased 'testicular fluid' or rete testis fluid will lead to increased aryl sulphatase activity. Further, the degenerative process can be related to the increase in leucine aminopeptidase and lactic dehydrogenase activity [23, 28].

Epididymal zinc does not depend upon testicular secretions especially androgens [29], but is regulated by glucocorticoids [10] and it suppresses androgen binding activity at the receptor and acceptor levels at high concentrations [30]. Increased caput and cauda epididymal zinc in ADX rats can be correlated to oedematous fluid formed by degeneration of testicular germinal epithelium[23, 28] and epithelial cells and intertubular tissues of caput and corpus. Adrenalectomy or reduction in glucocorticoids results in an increase and accumulation in hepatic copper in the nucleus, which is apparently injurious to the nucleus and is ultimately responsible for the cellular death [31]. Besides epididymal degeneration, the decreased epididymal copper may be one of the factors related to reproductive dysfunction. Most probably it could account for the lack of copper-containing enzymes, a probability yet to be verified. This impact appears to be critical during stress as inflammation also alters copper metabolism [32].

In conclusion, adrenalectomy leads to degeneration of caput and cauda epididymidis epithelial cells as a result of decreased supply of testosterone.

Acknowledgments

One of the authors (N. Nair) thanks the Council of Scientific and Industrial Research, New Delhi for the award of research associateship and financial assistance.

A part of the data was presented at the 6th Nordic Symposium on Trace Elements in Human Health and Disease, Denmark, 1997.

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Correspondence to: Dr. Neena Nair, Cell Biology Laboratory, Department of Zoology, University of Rajasthan, Jaipur - 302 004, India.

Email: fasilec@eth.net

Received 2002-01-08     Accepted 2002-11-07