Apoptosis of testicular germ cells induced by exogenous glucocorticoid in rats

H. Yazawa, I. Sasagawa1 and T. Nakada

Department of Urology, Yamagata University School of Medicine, 2-2-2 Iidanishi, Yamagata 990-9585, Japan


    Abstract
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 Abstract
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 Materials and methods
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The influence of exogenous glucocorticoid, dexamethasone (Dex), on testicular germ cell apoptosis was investigated in rats. The percentages of apoptotic tubules and apoptotic germ cells in the Dex-treated group of rats were about seven-fold and 10-fold higher respectively than in either the control group, or in rats treated with glucocorticoid receptor agonist (GR-A), or in rats treated with both Dex and GR-A. These results suggest that, in rats, apoptosis of testicular germ cells is mediated by glucocorticoid receptors.

Key words: apoptosis/germ cell/glucocorticoid/rat


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Apoptosis—programmed cell death—is characterized morphologically by a shrinkage of total cell volume, increased cell densities and compaction of cell organelles (Wyllie et al., 1980Go; Arends et al., 1990Go). During apoptosis there is often a double-strand, endonuclease-specific cleavage of nuclear DNA at the linker regions between nucleosomes, leading to the production of oligonucleosomal fragments that are multiples of 180 DNA base pairs (Wyllie, 1980Go).

Reproductive function in male primates or rats is suppressed by psychogenetic or somatic stress (Sapolsky, 1985Go; Orr and Mann, 1992Go). 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, 1985Go). 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.


    Materials and methods
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Animals
The following experiments were conducted in accordance with the principles and procedures of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (1984). In addition, all regulations on experimental animal studies recommended by Japanese law were adhered to. Young adult male Sprague-Dawley rats of body weight 250–300 g (Cea Japan Inc., Tokyo, Japan) were housed (two to three animals per cage) in hanging wire mesh cages under controlled lighting conditions [14 h light (commencing 06:00) and 10 h dark] at a temperature of 20–24°C. The animals were handled daily for 1 week before the experiments were performed.

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 15–30 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 15–30 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 60–90 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 isothiocyanate–horseradish peroxidase (FITC–HRP) conjugate for 30 min at 37°C, the slides were washed three times in PBS, developed with 0.05% diaminobenzidine (DAB), and stained for 10–15 min at room temperature. The specimens were then washed three times in distilled water, counterstained in Mayer's haematoxylin solution for 5–10 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., 1998Go).

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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There was no significant difference in testicular weight (expressed per 100 g body weight) among the four groups (Figure 1Go). The testes from Dex-treated rats showed strong positive staining when they were TUNEL labelled (Figure 2BGo). Positive staining was mostly observed in the spermatogonia (Figure 2CGo). However, neither interstitial cells nor Sertoli cells showed apoptosis. The testes from control, GR-A-treated and Dex + GR-A-treated rats showed a normal architecture of seminiferous epithelium with only slight signs of apoptosis (Figure 2A, D and EGo). The percentages of apoptotic tubules and apoptotic cells in Dex-treated rats were about seven-fold and 10-fold higher respectively than those in control, Dex + GR-A-treated and GR-A-treated animals (P < 0.001) (Table IGo).



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Figure 1. Testicular weight per 100 g body weight in control rats, and in rats treated with dexamethasone (DEX), glucocorticoid receptor agonist (GR-A) or DEX + GR-A. Vertical bars represent the SD.

 



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Figure 2. In-situ end labelling of DNA fragmentation on testis sections. (A) Testis from control rat. Few in-situ end-labelled germ cells are observed in the seminiferous epithelium. (B) Testis from dexamethasone (Dex)-treated rat. Labelled cells are frequently found in the seminiferous epithelium. (C) Positivity is mainly observed in spermatogonia of testis from Dex-treated rats. (D) Testis from glucocorticoid receptor agonist (GR-A)-treated rat. In-situ end-labelled germ cells are rarely observed in the seminiferous epithelium. (E) Testis from Dex + GR-A-treated rat. Few labelled cells are found in the seminiferous epithelium. Scale bars: (A), (B), (D), (E) = 10 µm; (C) = 2.5 µm.

 

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Table I. Percentages of apoptotic tubules and apoptotic germ cells in control rats, and in rats treated with dexamethasone (Dex), glucocorticoid receptor agonist (GR-A) or Dex + GR-A
 

    Discussion
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 Materials and methods
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 Discussion
 References
 
A transient increase in the concentration of serum corticosterone and a transient decline in that of serum testosterone during immobilization stress is associated with apoptosis of testicular germ cells in rats (Yazawa et al., 1999Go). When adult male rats are treated with ethane dimethane sulphonate (EDS)—an agent which leads to testosterone withdrawal by the selective destruction of Leydig cells—the incidence of germ cell loss by apoptosis increased remarkably (Troiano et al., 1994Go). In addition, androgen withdrawal as a result of hypophysectomy (Tapanainen et al., 1993Go), administration of gonadotrophin-releasing hormone (GnRH) antagonists (Brinkworth et al., 1995Go; Sinha Hikim et al., 1995Go), or of anti-LH antibodies (Marathe et al., 1995Go) resulted in testicular germ cell apoptosis in rats. Thus, programmed cell death in the seminiferous epithelium is thought to be mediated by androgens. In response to androgen withdrawal, the cell types that have been shown to undergo apoptosis are primary spermatocytes and spermatids (Tapanainen et al., 1993Go; Brinkworth et al., 1995Go; Marathe et al., 1995Go). In the present study, the predominant germ cell types undergoing apoptosis included spermatogonia. Therefore, the germ cell apoptosis arising from androgen withdrawal and Dex administration presumably result from different mechanisms. According to a previous report however (Billing et al., 1995Go), suppression of gonadotrophin secretion by GnRH antagonist treatment increased germ cell apoptosis in 16- to 32-day-old rats, but not in younger or adult rats, and showed age-dependent changes of the predominant germ cell types undergoing apoptosis. The principal types of cells undergoing apoptosis as a result of Dex administration may differ according to age.

It is well known that glucocorticoids induce apoptosis in immature thymocytes (McConkey et al., 1990Go). It has also been shown (Compton and Cidlowski, 1987Go) 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., 1990Go). 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, 1989Go). Mifepristone—a potent glucocorticoid receptor agonist—is 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, 1984Go). 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., 1988Go; Boise et al., 1993Go). 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., 1995Go). 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., 1999Go). 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., 1998Go). 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., 1997Go; Pentikainen et al., 1999Go). 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, 1995Go). 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, 1997Go). 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., 1999Go). 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.


    Acknowledgments
 
We are grateful to Dr R.Sitruk-Ware (Exelgyn) for a gift of mifepristone (RU486).


    Notes
 
1 To whom correspondence should be addressed at: Department of Urology, Yamagata University, School of Medicine, 2-2-2 Iidanishi, Yamagata-shi, Yamagata 990-9585, Japan.E-mail: isasaga{at}med.id.yamagata-u.ac.jp Back


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 Introduction
 Materials and methods
 Results
 Discussion
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Submitted on March 17, 2000; accepted on June 15, 2000.