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Prof. Yi-Fei WANG,

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.