Effect of experimental varicocele on structure and function of epididymis in adolescent rats

Qiu-Yang ZHANG¹, Shu-Dong QIU¹, Xiao-Nian MA², He-Ming YU³, Yan-Wan WU³

Asian J Androl 2003 Jun; 5:  108-112             

Keywords: experimental varicocele; epididymis; ultrastructure; L-carnitine; alpha-glucosidase; adolescent rat

Abstract

Aim: To study the effect of experimental left varicocele (ELV) on epididymal structure and function in adolescent Sprague-Dawley rats. Methods: ELV was induced by partial ligation of the left renal vein. Sham-operated animals served as the controls. Four and 8 weeks after the operation, the histological, ultrastructural and biochemical (alpha-glucosidase activity and carnitine content) changes in different segments of the epididymis were observed. Results: In the treated animals, there were degeneration of the epididymal epithelium and edema of the interstitial tissue; numerous shedding cells, residual bodies, deformed sperm and macrophages appeared in the epididymal lumen. Morphometric measurement indicated a significant reduction in the epididymal tubular diameter (P<0.05) and a significant increase in the epididymal interstitial area (P<0.05) compared with the controls. Ultrastructural study showed sparse microvilli of the columnar epithelium, increased and enlarged lysosomes in the principal cells with defected organelles and the presence of large cytoplasmic vacuoles. The protein and carnitine contents and the alpha-glucosidase activity in the caput, corpus and cauda epididymis of the ELV rats were lower than those of the controls (P< 0.05). Conclusion: There were structural and functional changes in the epididymis of adolescent ELV rats, which may contribute to the infertility caused by varicocele.

1 Introduction

Varicocele is one of the main causes of male infertility. In a World Health Organization multicentre study on 9043 infertile couples, varicocele was found in 25.4 % of men with abnormal semen, compared with 11.7 % of normal semen [1]. Recently, it was shown that the presence of grade I varicocele in adolescence appears to have no effect on normal testicular growth, but patients with grade II varicocele are at risk of unilateral testicular volume loss and grade III varicocele, of bilateral testicular volume loss [2]. The detailed pathophysiology of the disease is still unknown, nevertheless increased blood flow causing an elevated intratesticular temperature may contribute to the testicular damage. However, varicocele repair reverted the intratesticular temperature, but fertility was improved only in about one half of the patients [3]. Thus the mechanism of varicocele-induced infertility remained to be elucidated.

Various studies had focused on the testicular damages caused by varicocele [4-7], but only a few on the epididymis. Although the epididymal Na⁺ and K⁺ concentrations, epididymal temperatures and the blood-epididymal barrier did not change 30 days after ELV surgery in rats [8], the sperm concentration and motility in the cauda epididymis were decreased [9] and the epididymal fluids acidified [10]. The above changes could be the results of epididymal disturbance or merely changes in the seminiferous tubular fluid transmitted to the epididymis. Up to date, nothing has yet been reported about the structural and biochemical lesions of the epididymis in adolescent varicocele rats. The present study was designed to help clarify these points.

2 Materials and methods

2.1 Animals

Adolescent (50 days old) male Sprague-Dawley rats were obtained from the Laboratory Animal Unit of the National Research Institute for Family Planning. They were maintained on a 12 h light and 12 h dark cycle and fed a standard diet and tap water ad libitum for a week before operation. Animals were divided at random into two groups: the experimental varicocele group (n=48) and the sham-operated control group (n=20). They were sacrificed 4 and 8 weeks after the operation for various observations.

2.2 Experimental varicocele

Partial left renal vein ligation: the rat was anesthetized with sodium pentobarbital (40 mg/kg) i.p. and a midline incision was made to expose the left renal vein. A 4-zero silk ligature was made around the vein with a metal probe interposed at the point medial to the entrance of the adrenal and spermatic vein. The diameter of the metal probe was selected in order to reduce the renal vein diameter approximately 50 %. The probe was then removed and the vein was allowed to expand within the boundary of the ligature. The incision was then closed. Sham-operated animals were subjected to the same anesthesia and sham-surgery. Animals of the two groups were then equally divided into 4 subgroups separately for the morphological and biochemical observation 4 and 8 weeks after the operation. The diameter of the left spermatic vein was measured under a dissecting microscope before and 4 and 8 weeks post-operation.

Four and eight weeks post-operation, animals of the relevant subgroups were anesthetized with sodium pentobarbital i.p.(4 mg/100 g; Simnitol, MTC Pharma-ceuticals, Hamilton, Canada) and their reproductive tracts were fixed with 2.5 % glutaraldehyde buffered in phosphate (0.1 mol/L, pH 7.4) by perfusion through the abdominal aorta for 10 minutes. After perfusion, the epididymides were removed and sectioned into 4 segments, i.e., the initial segment, caput, corpus and cauda. Parts of each segment were then cut into 1 mm³ pieces and immediately placed in 2.5 % glutaraldehyde buffered in phosphate (0.1 mol/L, pH 7.4) for 2 h at 4 . The tissues were then post-fixed in 1 % osmium tetroxide, dehydrated in gradient acetone and embedded in Epon 812-Aradite. Thin (75-nm) sections were cut on an ultra-microtome, counterstained with uranyl acetate and lead citrate and examined with a Philips 400 electron microscope (Philips, Eindhoven, The Netherlands).

The remains of the epididymis were immersed in Bouins fixative for 24 h and then dehydrated and embedded in paraffin. Sections were prepared, stained with haematoxylin and eosin and observed under a light microscope. The epididymal tubular diameter and interstitial area were analyzed using MCID/M4 image analyzer (Imaging Research Inc., Toronto, Canada); 15 smallest, roundest and separated epididymal tubules and 5 random fields in each rat were selected for epididymal tubular diameter and interstitial area measurement, respectively.

Four and eight weeks post-operation, the caput, corpus and cauda epididymis of the relevant subgroups were removed and weighed. They were minced with scissors followed by homogenization in 10 volumes of sodium phosphate buffer (pH 6.8). The homogenate was then centrifuged at 10,000 g for 25 min at 4 . The supernatant was used for the estimation of protein [11], alpha-glucosidase activity [12] and carnitine [13].

Data were expressed in meanSD. Statistical analysis was performed using the independent t-test and P< 0.05 was considered significant.

3 Results

In the 48 experimental rats, 6 were excluded because of death and in the remaining, the spermatic vein was not dilated and the weights of the left and right kidneys were significantly different; the left spermatic vein was significantly dilated than that in the controls (data not shown).

Table 1 shows the mean epididymal tubular diameter of different epididymal segments. In the 4-week animals, the epididymal tubular diameters of both the left and right initial and the right cauda segments were smaller (insigni-ficant) than those of the corresponding controls. However, the epididymal tubular diameters of the left and right caput and the left cauda segments were significantly smaller than those of the corresponding controls (P<0.05). In the 8-week animals, the epididymal tubular diameters of the left and right initial, caput and cauda segments were significanltly smaller than those of the corresponding controls (P<0.05~0.01). In the 4- and 8- week animals, the epididymal tubular diameters between their right and left segments were not significantly different.

Table 1. Effect of varicocele on epididymal tubular diameter. ^bP< 0.05, ^cP<0.01, compared with the corresponding controls.

Table 2 shows the results of the epididymal interstitial area of different segments. In the 4-week animals, the interstitial area of the right caput and the left and right initial segments were larger (insignificant) than those of the controls. However, the interstitial area of the left caput segment was significantly larger than that of the controls (P<0.05). In the 8-week animals, the interstitial areas of the left and right initial and the right cauda segments were not significantly increased, but the interstitial areas of the left and right caput and the left cauda segments were significantly larger than those of the corresponding controls (P<0.05~0.01).

Table 2. Effect of varicocele on epididymal interstitial area. ^bP< 0.05, ^cP<0.01, compared with the corresponding controls.

Figure 1 is the light micrographs of the epididymal specimens. Compared with the corresponding control groups (panel A), there were significant histological changes in the different segments of the epididymis in the ELV rats and more severe damages were seen in the 8-week than in the 4-week rats. The main pathological changes included epithelial disorganization and even vacuolar-denaturalization (panel B). Halo cells were increased in number (panel C). The interstitial tissue was edematous with vascular dilatation and hyperemia. Lymphocytes and macrophages were accumulated and spermatic granuloma developed in the interstitial tissue, mainly in the corpus and cauda epididymis (panel D). Numerous shedding cells, residual bodies and macrophages were found in the epididymal lumen. The lumina were azoospermic (panels B & C).

Figure 2 is the electron micrographs of epididymal specimens. Compared with the controls (panels A & B), the microvilli of some principal cells were scarce, deciduous or damaged and the basement membrane thickened. There was a remarkable increase in the size and number of lysosomes in the principal cells (panel C). Some principal cells showed damaged organelles, such as mitochondria swelling, cristae clouding, rough endoplasmic reticulum dilatation and Golgi stacks of saccules dilatation and interrupted by large vacuoles. A few principal cells contained large vacuoles. Clear cells were filled with lysosomes and the dense lysosome and lipid droplet compartments were greatly enlarged and not clearly separated (panel D). Halo cells were increased in number and size and were also filled with lysosomes. In the epididymal lumen, the sperm residual bodies were increased.

Figure 2. Electron micrographs of epididymal sections. (A) Epithelium of caput epididymis in a control rat (8,000). (B) A principal cell of corpus epididymal epithelium in an ELV rat, showing numerous enlarged lysosomes (lys) (15,000). (C) A principal cell of corpus epididymal epithelium in an ELV rat, showing a heterophagosome in apical region (8,000). (D) Epithelium of caput epididymis in an ELV rat, showing clear cells bulging into lumen (2,500).
p: principal cell; b: basal cell; C: clear cell; my: myoid cell; N: nucleus; bm: basement membrane; mv: microvilli; lys: lysosome; cj: cell junction.

Table 3 summarizes the effect of varicocele on the alpha-glucosidase activity in different segments of the epididymis. In 4-week rats, the alpha-glucosidase activity in the right caput and cauda segments was lower (insignificant) than that of the controls. However, in the left caput and cauda segments of the 4-week rats and in both the left and right caput and cauda segments of the 8-week rats, the alpha-glucosidase activity was significantly lower than that of the corresponding controls (P<0.05~0.01).

Table 3. Effect of varicocele on alpha-glucosidase activity. ^bP < 0.05, ^cP<0.01, compared with the corresponding controls.

3.3 Effect on carnitine content

The effects of varicocele on carnitine content of the cauda epididymis in rats are shown in Table 4. When expressed as per g tissue weight or per mg protein, the carnitine content of the cauda epididymis was significantly lower in both the 4-week and 8-week rats than that in the corresponding controls (P<0.05).

Table 4. Effect of varicocele on cauda epididymal carnitine content. ^bP<0.05, ^cP<0.01, compared with the corresponding controls.

4 Discussion

Varicocele is often associated with male infertility. Clinical studies in adolescents had shown that varicocele early in puberty will often lead to testicular volume loss [2]. Choi et al. [5] and our previous study [7] indicated that experimental varicocele in adolescent rats led to more severe testicular abnormality than those in adult rats. Recently, it was shown that AZF microdeletions were seen in both idiopathic and non-idiopathic varicoceles [14], suggesting a genetic factor in the effect of varicocele on spermatogenesis.

Wang et al. [4] reported that experimental varicocele brought about defects in the microvilli of epididymal epithelium in adult rats. The present study showed that varicocele caused morphological abnormality in the epididymis. The ultrastructural changes were region-specific, which were in consistent with the region-specific structure of the epididymis. The present study also showed that the epididymal changes in adolescent rats were more severe than those in adult rats [5]. The results were coincident with the clinical study conducted by Zeng et al. [15] that epididymal damages, including epithelial degeneration and interstitial tissue edema, were present in infertile men with varicocele. Epididymal change may be an important contributory factor in infertility caused by varicocele. To the best of our knowledge, quantitative study on the alterations of the epididymal tubular diameter and interstitial area in varicocele had not previously been reported. The present study may provide important information for the clarification of its mechanism of development.

Epididymal alpha-glucosidase and carnitine are indicators for epididymal sperm content and epididymal function [16, 17]. Changes in these 2 parameters in ELV rats presented in this study were coincident with previous clinical research, indicating that the seminal carnitine content and alpha-glucosidase activity were distinctly lowered in varicocele patients with sperm maturation disturbance[18].

The present study supported the notion that varicocele-related male infertility was associated with impaired disposal of residual sperm cytoplasm by the testis and/or epididymis [19], as the abnormal spermatozoa and residual bodies in the epididymal lumen of adolescent ELV rats were significantly increased. All the abnormalities presented in this study were found to be bilateral, i.e., a unilateral varicocele will cause bilateral damages of the epididymis, which was also the case with the testis [2].

In conclusion, we believe that the observed abnormalities and reduced fertility potential associated with varicocele may be the result of both testicular and epididymal involvement. However, the mechanism of the alterations awaits farther study.

We thank Professor Tian-Bao SONG, Department of Histology & Embryology, Medical College, Xian Jiaotong University for reviewing the manuscript.

The project was supported by grants from the Natural Science Foundation of Shanxi Province, China (2002C278) and the Science Research Foundation of Xian Jiaotong University (PZ075).

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Correspondence to: Prof. Shu-Dong QIU, Department of Histology & Embryology medical college, Xi'an Jiaotong University, Xi'an 710061, China
Tel: +86-29-5275180
Email: qiusdxa@mail.xjtu.edu.cn
Received 2002-08-26      Accepted 2003-04-29