Department of Urology, Yamagata University School of Medicine, 2-2-2 Iidanishi, Yamagata 990-9585, Japan
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Abstract |
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Key words: apoptosis/germ cell/glucocorticoid/rat
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Introduction |
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Reproductive function in male primates or rats is suppressed by psychogenetic or somatic stress (Sapolsky, 1985; Orr and Mann, 1992
). Stress provokes elevation of glucocorticoid concentration which precedes a decline in testosterone concentration in the male. Glucocorticoids act at the level of the pituitary and testes to suppress testosterone secretion (Sapolsky, 1985
). Thus, glucocorticoids are considered to be stress-induced hormones. To our knowledge, however, there is no published report detailing the effect of exogenous glucocorticoids on testicular germ cell apoptosis.
In the present study, we investigated the role of exogenous glucocorticoids on the apoptosis of testicular germ cells in male rats and the effect of in-vivo treatment with a glucocorticoid receptor agonist (GR-A) on testicular germ cell apoptosis during glucocorticoid-induced stress.
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Materials and methods |
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Treatment protocols
The animals were allocated to four groups. Dexamethasone (Dex)-treated rats (n = 10) were given 7 mg/kg of Dex (Wako Junyakukogyo, Osaka, Japan) daily by i.p. injection at 10:00 h for 7 days. GR-A-treated rats (n = 11) were given 10 mg/kg mifepristone (Exelgyn, Paris, France) daily by i.p. injection at 10:00 h for 7 days. Dex + GR-A-treated rats (n = 11) were given 7 mg/kg Dex and 10 mg/kg mifepristone daily by i.p. injection at 10:00 h for 7 days. Control rats (n = 10) were treated with saline (i.p. injection) daily for 7 days.
At 1 day after the final injection, the rats were killed by decapitation, the body weight was recorded, and the bilateral testes were removed and weighed.
DNA nick-end-labelling of tissue sections (TUNEL method)
Tissue sections were excised from the testes, embedded in Tissue-tek (Miles Inc., Elkhart, IN, USA) and stored at 70°C. Frozen 5 µm thick sections were mounted on silane-coated glass slides (Dako Japan, Tokyo, Japan) and fixed for 1530 min at room temperature in freshly prepared 4% paraformaldehyde buffered with 0.1 mol/l sodium phosphate (pH 7.4). Endogenous peroxidase was inactivated by 0.3% H2O2 for 1530 min at room temperature. Permeabilization of the sections was achieved by incubation with a permeabilization buffer for 5 min at 4°C. In-situ end-labelling was performed using an in-situ Apoptosis detection kit (Takara Biomedicals, Tokyo, Japan), which comprised non-radioactive fluorescein-dideoxyuridine triphosphate (dUTP). The sections were incubated with terminal deoxynucleotidyl transferase (TdT) and fluorescein-dUTP at 37°C for 6090 min in a dark humidified chamber, and 3'-OH ends of the DNA fragments were tailed with fluorescein. The sections were then washed three times in phosphate-buffered saline (PBS). After incubation with anti-fluorescein isothiocyanatehorseradish peroxidase (FITCHRP) conjugate for 30 min at 37°C, the slides were washed three times in PBS, developed with 0.05% diaminobenzidine (DAB), and stained for 1015 min at room temperature. The specimens were then washed three times in distilled water, counterstained in Mayer's haematoxylin solution for 510 min, dehydrated and then mounted. For evaluation of apoptosis, an Olympus BX50 microscope with a x20 objective (Tokyo, Japan) was used. The percentage of the apoptotic germ cells was determined by counting a total of 1000 germ cells from apoptotic tubule cross-sections (TCS) of each specimen. The percentage of apoptotic tubules was determined by counting 100 TCS from each specimen (Yin et al., 1998).
Statistical analysis
Results were analysed for statistical significance using Student's unpaired t-test. Differences were considered to be statistically significant if P was < 0.05.
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Results |
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Discussion |
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It is well known that glucocorticoids induce apoptosis in immature thymocytes (McConkey et al., 1990). It has also been shown (Compton and Cidlowski, 1987
) that in-vivo treatment of rats results in rapid degradation of the thymocyte genome and, ultimately, cell death. Similar results were obtained in an avian species by treating chickens with Dex (Compton et al., 1990
). The present study demonstrated that exogenously administered Dex induced testicular germ cell apoptosis in rats. To our knowledge, this is the first report of any relationship between glucocorticoids and testicular germ cell apoptosis.
Although the mechanisms involved in the results observed in our study remain unclear, the following possibilities can be considered. Glucocorticoid hormone binds to the glucocorticoid receptor in the cytoplasm of target cells, and the formation of a complex between the ligand and the receptor regulates gene expression of the glucocorticoid response element, a specific DNA sequence in the chromosome (DeGroot, 1989). Mifepristonea potent glucocorticoid receptor agonistis related to a strong interaction with the cytosolic glucocorticoid receptor. The binding affinity of mifepristone for the rat thymus glucocorticoid receptor is about three times higher than that of Dex at 0°C (Moguilewsky and Philibert, 1984
). The present study showed that a potent glucocorticoid receptor agonist completely suppressed Dex induction of testicular germ cell apoptosis. Therefore, the regulation of transcription of genes mediated by the glucocorticoid receptor may also enhance apoptosis of testicular germ cells.
The regulation of apoptosis is dependent upon specific gene products. Bcl-2 is only one gene among a growing multigene family whose members are thought to regulate apoptosis by the formation of hetero- and homodimers. Bcl-2, as well as its structural homologue, the long form of Bcl-x (Bcl-xl), promotes cell survival by inhibiting apoptosis (Vaux et al., 1988; Boise et al., 1993
). Other members of the Bcl-2 family, including Bax, Bak and Bad, can block the ability of Bcl-2 to inhibit apoptosis and subsequently promote cell death (Yang et al., 1995
). After androgen withdrawal using EDS, the concentrations of the apoptosis-related genes, such as Bcl-xl, Bak and Bad do not change. However, the expression of Bcl-2 and Bax is up-regulated (Woolveridge et al., 1999
). Thus, the induction of Bax may play a role in germ cell apoptosis following androgen withdrawal. In experimental cryptorchidism, abdominal stress induces germ cell apoptosis by both p53-dependent and p53-independent pathways (Yin et al., 1998
). The initial phase of apoptosis needs p53, whereas other biochemical triggers of apoptosis are involved in a later phase of cell death. Furthermore, the Fas system has recently been implicated as a possible key regulator of germ cell apoptosis in the mammalian testis (Lee et al., 1997
; Pentikainen et al., 1999
). Fas is a type I transmembrane receptor protein that belongs to the tumour necrosis factor (TNF)/nerve growth factor receptor family, and Fas ligand (FasL) is a type II transmembrane protein of the TNF family (Nagata and Golstein, 1995
). Binding of FasL to Fas activates the cytoplasmic death domain of Fas, which initiates a cascade of interleukin-1ß-converting enzyme family protease (caspase) activity (Nagata, 1997
). The activated caspases cleave various cellular substances, such as actin, fodrin, lamin, poly(ADP-ribose) polymerase and DNA-dependent protein kinase, resulting in apoptosis. Radiation exposure to the testis mainly causes DNA damage in spermatogonia. Up-regulation of Fas is seen corresponding with an increasing incidence of apoptosis (Lee et al., 1999
). Thus, it appears that p53 and Fas are other factors for germ cell apoptosis induced by treatment with Dex. Since the apoptosis of testicular germ cells would involve certain specific gene activity and proteins, molecular investigations may help to determine whether the different pathways possess mechanisms of germ cell apoptosis induced by Dex.
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Acknowledgments |
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Notes |
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References |
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Submitted on March 17, 2000; accepted on June 15, 2000.