1 Introduction

It is well known that sperm produced in the seminiferous tubules are immotile and they attain their motility while passing through the epididymis^[1]. Hence, it seems to be indisputable that the sperm produced in the seminiferous tubules are passively transported from the testis into the epididymis. But the mean pressure in the caput epididymidis is significantly greater than that in the seminiferous tubules^[2]. A force must thereby be exerted onto the luminal contents to propel them from the seminiferous tubules into the caput epididymidis. It is generally believed that three factors may be involved: the active secretion of fluid by the Sertoli cells (and its absorption in the efferent ducts), the contractility of the lamina propria of the seminiferous tubules, and the contractile activity of the testicular capsule.

It was found that acetylcholine, noradrenaline, and adrenaline can produce marked contraction whereas isoproterenol causes relaxation of the testicular capsule, and the response induced by each agent is dose-dependent^[3,4]. In rats, the response to norepinephrine is increased markedly after 45 days of age, which corresponds to the end of puberty. Histamine has also been found to cause contraction of isolated testicular capsules; this stimulatory effect is believed to be mediated via H₁ receptors in the capsule^[5]. Specific receptors for oxytocin have been suggested to be present in the tunica albuginea and oxytocin has been found to produce contraction of the rat testicular capsule in a dose-dependent manner^[6]. It has been found that prostaglandins, such as PGE₂ and PGF₂, are present in the isolated rat testicular capsule^[7] and both prostaglandin synthetase and dehydrogenase activities have also been demonstrated in the capsule^[8]. PGE₁ has been shown to inhibit capsular contraction^[9] whereas PGF₂, stimulates capsular contraction in vitro. In isolated rat capsules, a sudden increase in the contractile response to PGA₂ occurs at 50 days of age^[10].

2 Materials and methods

2.1 Testicular capsulotomy

Mature (60 days of age) male Sprague-Dawley rats were obtained from the laboratory animal unit of The University of Hong Kong and divided into sham-operated group and capsulotomized group. Five to eight rats were used in each of the groups. Surgical intervention was performed in the Minimal Disease Operation Theatre of the University. The animals were anaesthetized intraperitoneally with sodium pentobarbitone (Sigma USA) at a priming dose of 60 mg/kg and thereafter a maintenance dose of 10 mg per kg per h was given. Testicular capsulotomy was done on both testes via a mid-abdominal incision.

Capsolutomy was performed with the aid of  a dissecting microscope (Wild M60, Switzerland), starting half-way 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 as illustrated in Figure 1. The cutting size was about 1 centimeter in length. Since in the rat the tunica vasculosa and the tunica albuginea are separated by very loose connective tissue, when the outer two layers of the capsule were longitudinally cut open, the seminiferous tubules were still well protected and enclosed by the tunica vasculosa. Hence, bilateral capsulotomy will cause minimal disturbances to the blood vessels, lymphatic drainage and innervations, and the rete testis.

After capsulotomy, the testes were placed back into the scrotum. This was done simply by holding the animal in a vertical position and allowing gravity to cause the testes to descend from the abdomen to the scrotum via the inguinal canal. The muscle and skin layers of the abdomen were closed separately with interrupted silk sutures.  Dressing (Opsite spray, Smith & Nephew Co., Ltd, UK) was sprayed thinly over the site of operation. Sham operations were performed in control rats.

Figure 1. Light micrograph of testes on day 20 of sham-operated (left) and capsulotomized rat (right). The distribution of the testicular artery (broad arrows) and testicular vein (narrow arrows) in the capsulotomized testis was the same as in the control. The surface of the testes was smooth, without apparent changes in their appearance. Degenerated seminiferous tubules (arrowhead) can be seen under the tunica vasculosa at the capsulotomized area (asterisk).

2.2 Gross examination

With the rats under sodium pentobarbitone anaesthesia (Sigma, USA, at a dose of 60 mg/kg), the testes and the epididymides were exposed via a scrotal incision. The appearance and colour of both organs were examined with the naked eye and the arrangement and distribution of the seminiferous tubules and blood vessels in the testis were observed with a dissecting microscope (Wild M60, Switzerland).

2.3 Histological preparation

With the rats under sodium pentobarbitone anaesthesia (Sigma, USA, at a dose of 60 mg/kg), the testes were exposed through a scrotal incision and the spermatic blood vessels were ligated close to the surface of the testis. The testes and epididymides were then removed by cutting off their connections with the spermatic cords, pampiniform plexuses and vasa deferentia.

The testes were fixed in Helly's fluid^[11] overnight, then cut into two parts and placed again in the Helly's solution for another 32 hours.

3 Results

3.1 Gross examination

In both the experimental and control groups, the testes were located normally in the scrota without any adhesion. Under the dissecting microscope, it was seen that the general arrangement of the seminiferous tubules and the distribution of the blood vessels in the capsulotomized testis were the same as those of the controls. The surface of the capsulotomized testes was smooth, without any notable change in its appearance. In the capsulotomized area, degenerated seminiferous tubules could be seen underneath the tunica vasculosa (Figure 1).

3.2 Changes in transitional distal seminiferous segment

On day 10 post-operation, light micrographs showed a normal appearance in the controls (Figure 2A). However, in the experimental animals, a large number of sperm were seen aggregated in the lumen of this segment close to the rete testis. The luminal diameter of these tubules was progressively increased as more and more sperm were accumulated (Figure 2B). The epithelial cells of these tubules were highly compressed by the packed sperm and non-cellular debris. The nuclei of the epithelial cells were deeply stained and spindle-shaped (Figure 3A and 3B). The direction of sperm within the transitional distal segment was completely disordered, and their distribution was highly irregular.

Figure 2. Light micrographs of the testis on day 10 post-operation from (A): a sham-operated rat. The rete testis (big arrow) and the transitional distal seminiferous segments (small arrows) close to the rete testis shows normal structural appearance; (B): a capsulotomized rat. Aggregated spermatozoa (large arrows) are accumulated at the transitional distal seminiferous segments close to the rete testis (small arrows). H & E staining  156.  
Figure 3. Light micrographs of the testis on day 10 post-operation from a capsulotomized rat. (A): As more and more spermatozoa are detained at the transitional distal seminiferous segment (large arrows), the epithelial cells of these tubules are heavily compressed by the packed spermatozoa and matrix (small arrow). PAS staining  350. (B): The epithelial cells of these tubules are heavily compressed by the packed spermatozoa and non-cellular debris (small arrow). PAS staining 800.

3.3 Changes in seminiferous tubules

On day 10 after capsulotomy, the seminiferous tubules showed focal degenerative changes in areas close to the rete testis (Figure 4A). About 25% of the tubules in a section presented signs of degeneration. The major features of the degenerative changes included the following: a) Some spermatids and spermatocytes lost their contact to the  Sertoli cells with sloughing of spermatids (Figures 5A and 6A); b) vacuolation of variable configuration and size appeared in the basal aspect of the epithelium. The vacuoles were located in both the cytoplasm of Sertoli cells and in the intercellular spaces between Sertoli cells and neighbouring germ cells (Figure 6A). The severity of these changes was different in different seminiferous tubules.

On day 20 after capsulotomy, degeneration of the seminiferous tubules became somewhat diffuse. Epithelial alterations reached 50 to 60% of the tubules, but to different degrees. In addition to those changes that occurred at day 10, the nuclei of many round spermatids underwent ring-like chromatin condensation, i.e., with vacuolation of the nucleus, in which the chromatin was pushed to the periphery. Binucleated or even multinucleated cells could sometimes be seen in some tubules. Retention of step 19 spermatids in the basal region of the epithelium was also be observed (Figure 7A).

By day 30, considerable disorganization of the germinal epithelium was noted in about 60 to 70% of the tubules close to the rete. Many affected tubules had further degeneration. More and more multinucleated cells, which contained quite a number of spermatids with ring-like chromatin condensation, were observed. Many tubular lumina were markedly distended. Retention of step 19 spermatids could also be observed (Figure 7B).

On day 40 after capsulotomy, the degenerative changes had progressed to most tubules. The majority of germ cells appeared to have been shed, leaving only Sertoli cells, some spermatogonia, a few spermatocytes and spermatids with ring-like chromatin condensation. The cytoplasmic extensions of the Sertoli cells were seen protruding into the tubular lumen (Figure 7C).

On day 60 following capsulotomy, almost all seminiferous tubules were atrophied, containing only degenerated Sertoli cells surrounded by a thickened basement membrane. The tubular lumina were seen fully occupied by the protruded cytoplasmic extensions of the Sertoli cells. The contour of the tubules became irregular (Figure 7D).

References

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