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Effect of testicular capsulotomy on lipid droplets in the seminiferous tubules  of rats

 Da-Nian QIN, Mary A. Lung1

Department of Physiology, Shantou University School of Medicine, Shantou  515031, China
1Department of Physiology, The University of Hong Kong, HKSAR, China

Asian J Androl  2001 Jun; 3:  121-124

Keywords: testis; testicular capsulotomy; lipid droplets
Abstract

Aim: In order to reveal the histochemical alteration that might occur during the processes of the spermatogenic disruption induced by testicular capsulotomy, the location and alteration of lipid droplets in the seminiferous tubules were observed in the present study. Methods: Osmium tetroxide was used to demonstrate the lipid droplets in the seminiferous tubules of capsulotomized and sham-operated control testes. Results: In the seminiferous tubules of the sham-operated rat testes, many small lipid droplets were located close to the basement membrane of the seminiferous tubules. But for the capsulotomized testes, the lipid droplets in the seminiferous tubules had increased in size and number, with many lipid droplets migrated towards the lumen of the tubules. Conclusion: The results indicated that a progressive fatty degeneration occurred in the seminiferous tubules after testicular capsulotomy.

1 Introduction

It was found in our previous study that a progressive degeneration of seminiferous tubules could be induced by testicular capsulotomy in adult male rats with a progressive reduction in the testosterone concentration in the testicular venous blood and a gradual increase in the LH and FSH levels in the peripheral blood from approximately 30 days post-capsulotomy. At that time, the fertility of capsulotomized rats was gradually depressed[1-3]. However, the underlying mechanism(s) are not clearly defined.

In order to reveal the histochemical alteration that might occur in the testis during processes of the spermatogenic disruption induced by testicular capsulotomy, osmium tetroxide was used in present study to demonstrate the change of lipid droplets in the seminiferous tubules after testicular capsulotomy in rats.
2 Materials and methods

2.1  Experimental animals and testicular capsulotomy

Mature Sprague-Dawley male rats (60 days of age) were obtained from the Laboratory Animal Unit of The University of Hong Kong and divided into sham-operated group and capsulotomized group. They were raised in condition with free access to food and water. Five rats were used in each group. Surgical intervention was performed in the Minimal Disease Operation Theatre of the University. The animals were anaesthetized intraperitoneally with sodium pentobarbitone (Sigma, USA) at the priming dose 60 mg/kg and thereafter a maintenance dose of 10 mg per kg per h was given. Testicular capsulotomy was carried out in the rats as previously described[1]. Briefly, it was performed with the aid of a dissecting microscope (Wild M60, Switzerland), starting halfway down the rostral half of the testis along the two lateral borders down to the middle of the caudal half of the testis. Only the two outer layers of the capsule, i.e., tunica vaginalis and tunica albuginea, were carefully incised.

2.2 Lipid droplet staining and measurement

The following procedure as previously described[4] was adopted for demonstration of lipid in the rat testes. (1) Fix thin pieces of fresh testicular tissue in Flemming's fluid which comprises of 1% aqueous chromic acid (15 mL), 2% aqueous osmium tetroxide (4 mL, Sigma, USA) and glacial acetic acid (1 mL, E. Merck), 12 hours;  (2) Rinse in distilled water;  (3) Rinse in running tap water, 3-4 hours; (4) Dehydrate by successive treatment with 50%, 70%, and 90% alcohol and finally undiluted alcohol (3 times);  (5) Clear in chloroform overnight; (6) Impregnate three times with paraffin wax (2 hours each);  (7) Embed in fresh wax;  (8) Cut sections, mount on slides, dry, dewax in chloroform and mount.

The slides were observed under microscope for lipid droplets. It is well recognized that in the presence of lipid, osmium tetroxide is reduced to insoluble lower oxides, which are deposited in the tissue at the site of the lipid. The lipid material will appear black while other structures show shade of dull yellow. The diameter of the lipid droplets was measured using the Vidas Image Processing System (Kontron, Germany). For each rat, twenty independent microscopic fields from sections of the polar and equatorial portions of the testis were analyzed and an average value was calculated.

2.3 Statistical analysis

Results were expressed as meanSEM. All two-group comparisons were made by student's t-test. All multiple comparisons were made by two-way analysis of variance (ANOVA) followed by the Student-Newman Keuls test. P<0.05 was assumed to denote a significant difference.

3 Results

In our histochemical studies, we found that in the seminiferous tubules of a sham-operated rat at the age of Day 60, many small lipid droplets were located close to the basement membrane of the seminiferous tubules. These small lipid droplets formed almost a ring adjacent to the tubular walls (Figures 1A, 2A, 3A). But for the capsulotomized testes, the lipid droplets in the seminiferous tubules had increased in their size and number (Figures 1B, 2B, 3B), with many lipid droplets, including large and small ones migrated towards the lumen of the tubules (Figures 2B, 3B).

The average diameter of the lipid droplets was measured and given in Figure 4. For the capsulotomized testes at 5 days after operation, the average diameter of lipid droplets was not significantly different from that of sham-operated controls. However, the average diameter of the lipid droplets in the capsulotomized rats was found to increase significantly (P<0.05) in comparison to that of sham-operated rats from 10 days to 60 days after operation. For the capsulotomized rats, the average diameter of the lipid droplets were significantly (P<0.05) increased by 36% at 20 days, 54% at 40 days and 88% at 60 days respectively when compared with that of capsulotomized rats at 5 days.

Figure 1.  Light micrographs of the testis at 20 days postoperation from rats which were operated at the age of Day 60. (A) Sham-operated control rat. Small lipid droplets (arrows) were located mainly in the basal aspect of the seminiferous epithelium. (B) Capsulotomized rat. Lipid droplets increased in size (big arrow). (A) 320; (B) 300. Osmium tetroxide.
Figure  2.   Light micrographs of the testis at 40 days post-operation from rats which were operated at the age of Day 60. (A) Sham-operated control rat. Small lipid droplets (arrows) were located mainly in the basal aspect of the seminiferous epithelium. (B) Capsulotomized rat. Lipid droplets increased in size and number (big arrow). Some lipid droplets had migrated towards the lumen of the seminiferous tubules (small arrows). (A) 320; (B) 300. Osmium tetroxide.
Figure 3.   Light micrographs of the testis at 60 days post-operation from rats that were operated at the age of Day 60. (A) Sham-operated control rat. Small lipid droplets (arrows) were located mainly in the basal aspect of the seminiferous epithelium. (B) Capsulotomized rat. Lipid droplets markedly increased in size and number (big arrow). Many lipid droplets had migrated into the lumen of the seminiferous tubules (small arrows). (A) 320; (B) 300. Osmium tetroxide.
Figure 4.  Average diameter of lipid droplets in the seminiferous tubules of sham-operated and testicular capsulotomized rats at various intervals post-operation. Sham-operation and testicular capsulotomy were performed in rats at the age of Day 60. Open histograms, sham-operated controls. Filled histograms, capsulotomized rats. Values are meanSEM from a group of 5 rats. bP<0.05, when compared with corresponding sham-operated control, Student's t-test.

4 Discussion

In the normal rat testis, it had been demonstrated that there was a cyclic change in the amount of lipid droplets in the cytoplasm of Sertoli cells during the spermatogenic cycle. The lipid droplets were abundant at stages IX-XIV, declined at stages I-III and remained low at stages IV-VIII[5-7]. Kerr et al[5] suggested that in normal physiological condition the accumulation of lipid droplets after spermiation was caused by resynthesis of lipids by Sertoli cells after phagocytosis of the residual bodies of the spermatids. But Chemes[6] claimed that the lipid droplets were the last remnants of digested residual bodies. However, it is generally agreed that there is a balance between lipolysis and synthesis of lipid in the Sertoli cells according to the stages of the spermatogenic cycle so as to provide local co-ordination for the proliferation and maturation of germ cells[5].

Results of our histochemical studies clearly demonstrated that lipid droplets were located inside the seminiferous tubules in both sham-operated (Figures 1A, 2A, 3A) and capsulotomized testes (Figures 1B, 2B, 3B). Lipid droplets in the sham-operated controls were seen mostly localized close to the basement membrane of the seminiferous tubule, whereas those in the capsulotomized testis appeared to migrate into the lumen of the seminiferous tubules and scattered inside the tubules (Figures 2B, 3B). In the sham-operated control rats, the average diameter of the lipid droplets did not vary significantly throughout the post-operation period of 60 days whereas the size of the lipid droplets in the seminiferous tubules of capsulotomized testis increased progressively after the treatment (Figure 4). The changes in the size and amount of the lipid droplets seemed to parallel the degeneration of the germ cells in the seminiferous tubules[1], i.e., the more the germ cells degenerated, the more the lipid droplets produced. 

We found in our sham-operated control that the number and average diameter of lipid inclusions remained relatively unchanged throughout the post-operation period of 60 days. Hence, despite the existence of a cyclic variation in Sertoli cell lipid droplets as found by previous workers, our results indicate that the average amount of lipid in a normal adult testis stays fairly constant and does not significantly fluctuate with time. But in the capsulotomized testis, we found an apparent progressive increase in the amount of lipid with time after the treatment, suggesting that lipolysis and synthesis of lipid in the Sertoli cells were not in balance in capsulotomized testes.

Several studies have shown that during periods when spermatogenesis was impaired large lipid droplets in various forms were seen to accumulate in the Sertoli cells[7,8]. Dramatic increases in lipid content have been found in situations of spermatogenic arrest or under conditions that cause germ cell damage[8-10]. It was proposed that the increases in the Sertoli cells lipid droplets in these situations were contributed by the degradation products of the affected germ cells that were phagocytosed by the Sertoli cells. In the capsulotomized testis, there was a progressive degeneration and exfoliation of spermatocytes and spermatids[1]. Hence, Sertoli cells would phagocytose a large number of degenerating cells, enhancing the accumulation of lipid droplets in the cytoplasm of these cells. However, we could not rule out the possibility that the increase in the lipid droplets in capsulotomized testis can result from the dysfunction of Sertoli cells themselves.

Acknowledgements

We wish to thank Mr. K. K. Tsang (The University of Hong Kong) for his skillful technical assistance.

References

[1] Qin DN, Lung MA. Studies on the relationship between testicular capsule and sperm transport in the rat testis. Asian J Androl 2000; 2: 191-8.
[2] Qin DN, Lung MA. Effect of testicular capsulotomy on secretion of testosterone and gonadotrophins in rats. Asian J Androl 2000; 2: 257-61.
[3] Qin DN, Lung MA. Effect of testicular capsulotomy on fertiltiy of rats. Asian J Androl 2001; 3: 21-5.
[4] Mai ZH. Histopathological technique. Beijing: People's Medical Publishing House; 1964. p 118-22.
[5] Kerr JB, Mayberry RA, Irby DC. Morphometric studies on the lipid inclusions in Sertoli cells during the spermatogenic cycle in the rat. Cell Tiss Res 1984; 236: 699-706.
[6] Chemes H. The phagocytic function of Sertoli cells: a phorphological, biochemical, and endocrinological studies of lysosomes and acid phosphatase localization in the rat testis. Endocrinology 1986; 119: 1673-81.
[7] Ueno H, Mori H. Morphometrical analysis of Sertoli cell ultrastructure during the seminiferous epithelial cyclic in rats. Biol Repod 1990; 43: 769-76.
[8] Abreu I, David-Ferrira JF. Fine structure of seminiferous tubules from prenatally irradiated rats. Cell Tiss Res 1982; 222: 143-52.
[9] Paniagua R, Nistal M, Saez FJ, Fraile B. Ultrastructure of the aging human testis. J Electron Microsc Tech 1991; 19: 241-60.
[10] Bensoussan K, Morales CR, Hermo L. Vitamin E deficiency cause incomplete spermatogenesis and affects the structural differentiation of epithelial cells of the epididymis in the rat. J Androl 1998; 19: 266-88.

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Correspondence to:  Dr Da-Nian QIN, Shantou University School of Medicine, Shantou 515031, China.  
Tel: +86-754-856 6776   Fax: +86-754-855 7562
E-mail: lqchen@mailserv.stu.edu.cn
Received 2001-04-17     Accepted 2001-04-30