Assessment of germ cell apoptosis in cryptorchid rats

Izzet Koçak¹, Mehmet Dndar¹, Mine Hekimgil², Pınar Okyay³

Asian J Androl 2002 Sep; 4: 183-186           

Keywords: cryptorchidism; germ cells; apoptosis; TUNEL method

Abstract

Aim: To investigate the relationship between germ cell degeneration and apoptosis in cryptorchid rats. Methods: Thirteen 21-day-old Wistar rats were made unilaterally cryptorchid by closing the left inguinal canal. At day 30 (Group 1, n=6) and day 60 (Group 2, n=7) after operation, the testes were removed for histopathological examination. The controls (n=8) were sham operated and were sacrificed at day 60. Germ cell apoptosis was assessed by means of the TUNEL method. Results: Spermatogenesis was arrested and the testicular and seminiferous tubular diameters were significantly reduced In the unilateral undescended testes (UUTs) compared with the contralateral descended testes (CDTs) and the control rats. However, atrophic changes, pathological calcification, necrosis of seminiferous tubule, and absence or sloughing of germ cells were not found in all the animals. The spermatocytes were the main type of germ cells undergoing apoptosis in all the groups. In the UUTs, there was a significant and time-dependent increase in the mean apoptotic index. By 60 days after surgery, increased apoptosis in germ cells was also observed in the CDTs. Conclusion: Apoptosis is the predominant mechanism of germ cell death rather than atrophy and necrosis in cryptorchidism.

Cryptorchidism is a primary congenital illness affecting around 3 % of boys [1,2]. The histopathological changes of the cryptorchid testis have been precisely described in both experimental animals and human. Most authors suggest that morphological alterations in the cryptorchid testis can be detected by 1 year of age and then advance progressively with time, including reduction in the testicular sizes and impairment of spermatogenesis [3-5]. Fertility can hardly revive after bilateral orchiopexy. However, the pathogenesis of reduced fertility in cases of unilateral undescended testis (UUT) remains controver-sial.

Recent studies have demonstrated that germ cell degeneration in rat testis involved the apoptotic mecha-nism [6,7]. Unilateral cryptorchidism has also been shown to induce germ cell apoptosis in the ipsilateral testis [8-11]; attention is now focused on changes in the contralateral descended testis (CDT).

In the present paper, the quantitative testicular histology as well as the effect of cryptorchidism on germ cell apoptosis was studied in the UUT as well as the CDT by using the in situ TUNEL method.

2 Materials and methods

2.1 Animals and treatment

Twenty-one immature Wistar rats weighing 70-80 g were raised in a light-controlled (12 hour light/darkness) environment with free access to pellet diet and tap water. On day 21 after birth, before complete descent of the testis, the animals were weaned and after an overnight fast, the rats were anaesthetised with an intraperitoneal injection of 2 mg/kg xylazine hydrochloride (Rompun, Bayer, Germany) and 10 mg/kg ketamine hydrochloride (Ketalar, Parke Davis, Eczac@bas@, Turkey). In animals of Groups 1 (n=6) and 2 (n=7), cryptorchid procedure was carried out by performing left inguinal canal closure through the inguinoscrotal approach [12]. Sham-operated animals served as the controls (Group 3, n=8). The experimental procedures were approved by the local ethics committee. On day 30 (Group 1) and day 60 (Groups 2 and 3) after operation, the animals were sacri-ficed and the testes were removed.

2.2 Histological examination

After determining the testicular diameter, the testis was fixed in 10 % formalin, cut vertically into two and embedded in paraffin. Five-micron thick sections were prepared and stained with hematoxylin and eosin (HE). The specimens were examined under a light microscope for routine observation, including atrophy, tubular calcification, tubular necrosis, interstitial oedema, apical vacuolation of Sertoli cells, tubular ectasis and absence or sloughing of germ cells. Tubular ectasis was identified when the tubular lumen was larger than the germ cell layer.

2.3 Quantitative analysis

The mean seminiferous tubular diameter was evaluated by measuring the smallest diameters of ten tubules on ten randomly selected fields (100 tubules in total), using an ocular micrometer at 400. The mean numbers of Leydig cells and germ cells (spermatogonia, primary spermatocytes, round spermatids and elongating sperma-tids) per tubular section were also calculated.

2.4 TUNEL method

The in-situ DNA fragmentation was visualised by a modification of the TUNEL method [13]. Briefly, dewaxed tissue sections were predigested with 20 mg/ml proteinase K for 20 min and incubated in phosphate buffered saline solution (PBS) containing 3 % H₂O₂ for 10 min to block the endogenous peroxidase activity. After incubating in 0.1 % Triton X-100 in 0.1 % sodium citrate for 2 min on ice (4), the sections were incubated with the TUNEL reaction mixture, fluorescein-dUTP (In Situ Cell Death Detection, POD kit, Roche, Germany), for 60 min at 37. The slides were then rinsed three times with PBS and incubated with secondary antifluorescein-POD-conjugate for 30 min. After washing three times in PBS, diaminobenzidine-H₂O₂ (DAB, Dako, Denmark) chromogenic reaction was added on sections and counterstained with hematoxylin. As a control for method specificity, the step using the TUNEL reaction mixture was omitted in negative control serial sections, and nucleotide mixture in reaction buffer was used instead.

A cell was considered TUNEL-positive when the nuclear staining was intense, dark brown and homo-genous. The apoptotic index (AI) was calculated by dividing the number of TUNEL-positive germ cells in a randomly focused seminiferous tubule by the total number of germ cells in that tubule and the result was multiplied by 100. The AIs of ten randomly selected tubules were evaluated and the mean AI of each case was calculated.

2.5 Data analysis

The data were expressed as meanSD. The Kruskal-Wallis one-way analysis of variance test, the Mann-Whitney U-test, and the Wilcoxon signed rank test were used for the statistical analysis. Results were considered statistically significant at P< 0.05.

3 Results

From Table 1, it can be seen that cryptorchidism exerts significant harmful effect on the UUTs compared with the CDTs and the sham-operated rats (P< 0.05). These effects included significant decreases in the numbers of germ cells and the diameters of the testis and seminiferous tubule. The histopathologic changes were most prominent in UUTs of Group 2. However, the number of germ cells and the diameter of the seminiferous tubules were significantly higher in CDTs of Group 2 than in the controls (P= 0.01 and P<0.05, respectively). Tubular ectasis in the CDTs was also more prominent in Group 2 than in Group 1 (P<0.05). In all the animals, atrophic changes, apical vacuolation of Sertoli cells, calcification or necrosis of the seminiferous tubule, absence or sloughing of germ cells and interstitial edema were not seen. The number of Leydig cells was found to be similar in all the groups.

Table 1. Effect of cryptochidism on testicular histology. ^bP< 0.05, compared with right testis; ^eP< 0.05, ^fP=0.01, compared with controls; ^cP< 0.05, compared with controls.

There were significant variations in labelled apoptotic germ cells per seminiferous tubule in different groups (Figure 1). The spermatocytes (most frequent) and spermatogonia were the main germ cells undergoing apoptosis. Although in low numbers, apoptotic germ cells were also observed in the control rats (Figure 2A and B). The mean AIs of UUTs in both study groups were signifi-cantly different from that in CDTs (P<0.05; Table 1). There was a time-dependent increase in the mean AIs of UUTs. By 60 days after surgery, apoptosis of germ cells was significantly increased not only in the UUTs but also in the CDTs (P<0.05; Figure 3 A and B).

Figure 1. Variations of number of TUNEL-positive germ cells in neighbouring seminiferous tubules at d 30. Seminiferous tubule in the middle has a high percentage of apoptotic cells (TUNEL method, 10).

Figure 2. Apoptotic changes localised to a single cell (arrow) of control rat testis. (A) HE, 40); (B) TUNEL method, 40).

Figure 3. Chromatin condensation, nuclear pyknosis and increased eosinophilia of three apoptotic cells (arrows) in UUT at d 60. (A)HE, 40); (B) TUNEL method, 40).

Many studies have shown that surgical induction of cryptorchidism in rats resulted in increased death of germ cells [14-16]. Although developmental alterations of the cryptorchid testis have been studied in depth, little attention has been paid to the effect on the CDT and the results remained controversial.

In the current study, the cryptorchid procedure resulted in detrimental effects on UUT which is consistent with previous reports [15,16]. These effects included significant decreases in the number of germ cells and the diameters of testis and seminiferous tubule. However, atrophic changes, pathological calcification or necrosis of the seminiferous tubule and absence or sloughing of germ cells were not seen in all the animals.

In unilaterally cryptorchid rats, the UUT showed an increase in apoptotic germ cells but not with the CDT [8]. A decrease in the number of germ cells was further confirmed by our data showing the markedly increased AIs in the UUTs. These observations are in agreement with recent reports demonstrating similar findings in different types of mechanically-induced cryptorchid rat testes [9-11]. In keeping with the work of Wang et al [9], we also observed a significant increase in the AI in CDTs of Group 2 compared with the controls. Further, we found the increases in germ cell and tubular diameter in CDTs at 60 days. These results led us to assume that unilateral cryptorchidism may have compounding effect on the CDT. This effect might be mediated by the local control mechanisms or by an immune reaction. Additional studies are required to elucidate this problem.

The reason responsible for the increased apoptosis in germ cells is not known. Although the primacy of the endocrine control in testicular function is clear, the complexity of the intratesticular events highlights the importance of local regulatory mechanisms and interactions. Growth factors and cytokines are also involved in local control mechanisms influencing testicular apoptosis through paracrine and autocrine mechanisms [17]. Intratesticular androgens, secreted by Leydig cells, also play an important paracrine role in preventing germ cell degeneration [17,18]. As the number of Leydig cells was similar in all groups, we thought that the increase in apoptosis may not be due to hormonal influence.The elevation of testicular temperature or other changes associated with cryptorchidism may cause germ cell apoptosis [19]. However, the mechanism by which testicular hyperthermia induces germ cell apoptosis is not known.

In conclusion, the lack of atrophic or necrotic changes support the argument that apoptosis may be the major cause of germ cell death in cryptorchidism.

References

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Correspondence to: Dr. Izzet Koçak, Asistant Professor, Adnan Menderes University, Tip Fakltesi, Üroloji Anabilim Dali, 09100 Aydın-TURKEY.
Tel: +90-542-232 7032, Fax: +90-256-212 0146
E-mail: ikocak@excite.com
Received 2002-03-04      Accepted 2002-06-27