Apoptosis related gene products in differentiated and tumorigenic rat Leydig cells and following regression induced by the cytotoxin ethane dimethanesulphonate.

Androgen secreting Leydig cells in the adult are differentiated with a very low turnover, however, Leydig cell tumours can arise spontaneously or after treatment with toxins. This study in the rat investigated whether changes in components of programmed cell death could be involved. In contrast to their absence in differentiated Leydig cells, antiapoptotic Bcl-2 and proapoptotic Bax were expressed in tumours. Bak and Bcl-xl were found in both tumour and normal Leydig cells. Apoptosis was induced in subcutaneous implants of Leydig cell tumour by ethane dimethanesulphonate (EDS) which is known to kill differentiated Leydig cells. The marked regression of the tumour following EDS treatment was transient and re-growth occurred between 6 and 14 days later. Tumour regression and growth was associated with a similar weight pattern in the seminal vesicles caused by changes in serum testosterone. During tumour regression, clusterin and Bax proteins were elevated but Bak, Bcl-xl and Bcl-2 were unchanged. Fas-R, Fas-L and Bax were upregulated after tumour regression had taken place. These data show that Leydig cell tumours possess many of the apoptosis related gene products and can die by apoptosis, however, regulation is clearly different in differentiated and mitotic Leydig cells.

Leydig cell apoptosis after the administration of ethane dimethanesulfonate to the adult male rat is a Fas-mediated process.

Leydig cells undergo apoptosis in response to the cytotoxin ethane dimethanesulfonate (EDS), with numbers declining at 12-18 h and maximal apoptosis at 24 h postinjection. The Bcl-2 family members, Bcl-2, Bcl-xl, and Bax, appear not to be involved in this process. To further investigate this phenomena, a single dose of EDS was administered to adult rats to induce the killing of Leydig cells. The interstitial cells were examined up to 3 days after EDS administration by Western blot analysis for the Bcl-2 family members (Bak and Bcl-w). Western blotting showed that Bak expression in the interstitial cell preparations was unchanged after EDS, and immunohistochemistry showed that it was not up-regulated in Leydig cells in response to EDS. Bcl-w expression in the Leydig cells and interstitial cell preparations was unchanged until 48 h when it became undetectable, suggesting that Leydig cell-associated Bcl-w is not involved in initiating apoptosis. We also investigated the role of the Fas system in Leydig cell apoptosis. Both Fas receptor and Fas ligand protein levels increased after EDS, peaking at 12-18 h and declining thereafter. Fas receptor and ligand were shown by immunohistochemistry to be present in Leydig cells, and after EDS all Leydig cells became strongly positive for both proteins. The intensity of staining increased in the early stages of apoptosis and decreased as the nuclear morphology became more fragmented. These data suggest that Bcl-2 family members are not involved in Leydig cell apoptosis after EDS administration. However, up-regulation of the Fas system does occur, implicating activation of Fas receptor in the induction of Leydig cell apoptosis.

Apoptosis in the rat spermatogenic epithelium following androgen withdrawal: changes in apoptosis-related genes.

Programmed cell death is an important regulatory event in spermatogenesis. However, the molecular events governing apoptosis have not been characterized. Using the Leydig cell-specific toxin ethane dimethanesulfonate (EDS) to withdraw androgen support, we have investigated the relationship between apoptosis and apoptosis-related genes. Adult male Sprague-Dawley rats were injected (i.p.) with 100 mg/kg EDS and killed at times of androgen depletion 2, 5, and 8 days postinjection. A 24-fold increase in the apoptotic index 8 days after EDS administration was demonstrated in tissue sections by in situ end-labeling of fragmented DNA. Leydig cell death and androgen withdrawal were confirmed by the absence of 3beta-hydroxysteroid dehydrogenase in testes from animals treated with EDS for 2 days. After androgen withdrawal, there were no significant changes in the levels of clusterin, Bcl-xl, Bak, and Bad. However, the expression of Bcl-2 and Bax was up-regulated at 8 days after EDS administration. The induction of Bax at this time suggests that it may play a role in germ cell apoptosis following androgen withdrawal. The concomitant elevation in Bcl-2 expression may represent a survival mechanism for the remaining germ cells. There was also a decline in the expression of Fas-L and Fas-R in the pachytene spermatocytes and spermatids. Fas-R was also present in Sertoli cells, although Fas-L staining was minimal. As the colocalization of Fas-L and Fas-R correlates with the germ cell types that die in response to androgen withdrawal, the potential exists for apoptosis in the rat spermatogenic epithelium to be regulated by the Fas pathway.

Apoptosis and expression of apoptotic regulators in the human testis following short- and long-term anti-androgen treatment.

Apoptosis and its augmentation by androgen withdrawal is an important event in the testis. In other tissues apoptosis is regulated by genes belonging to the bcl-2 family. However, little is known about these pathways in the human testes. Human testes were obtained from patients with prostate cancer, undergoing orchidectomy for permanent androgen ablative treatment. The patients were either untreated or had previously received short- or long-term anti-androgen therapy by cyproterone acetate or GnRH agonist (goserelin). In comparison with untreated patients, testicular testosterone concentrations were reduced by 83% in patients treated with cyproterone acetate and by 99% in patients treated with goserelin. Apoptotic cells were identified in tissue sections by in-situ end labelling of fragmented DNA. The expression of Bcl-2, Bcl-xl, Bax, p53 and poly(ADP) ribose polymerase (PARP) was demonstrated in tissue extracts by Western blotting. Apoptotic germ cells were present in the spermatogenic epithelium of untreated patients and patients who received short-term anti-androgen treatment. There were few or no apoptotic cells in the seminiferous tubules following long-term anti-androgen treatment. Following short-term treatment, the concentrations of the apoptosis-related proteins examined did not change. However, in the long-term treated testes, Bcl-xl and PARP expression declined, Bax and p53 protein concentrations were unchanged, and Bcl-2 was up-regulated. In conclusion, apoptosis occurs in spermatogenic cells of the human testis and may contribute to the regulation of germ cell populations. The apoptosis-related gene products which have been described in other tissues are present in the human testis and are modulated by androgenic stimuli.

Leydig cell apoptosis in the rat testes after administration of the cytotoxin ethane dimethanesulphonate: role of the Bcl-2 family members.

Ethane dimethanesulphonate (EDS) is cytotoxic to Leydig cells in the adult rat. To investigate the role and regulation of apoptosis in the Leydig cell, EDS (100 mg/kg i.p.) was administered to adult male rats and the testes examined 6, 12, 18, 24, 48 and 72 h later. Numbers of Leydig cells, identified by 3 beta-hydroxysteroid dehydrogenase immuno-histochemistry started to fall by 12 h after EDS injection and were almost undetectable by 72 h. Apoptotic cells in the interstitium, visualised by in situ end labelling of DNA, increased in number to reach a maximum 24 h after injection of EDS, and were undetectable by 72 h. In many tissues the apoptosis-related gene products act in cohort: Bcl-2 and Bcl-xl promoting survival of a cell, whilst Bax promotes cell death often positively regulated by the tumour-suppressor gene p53. Western blot analysis showed that: (1) Bcl-2 and p53 were absent from interstitial Leydig cells but were expressed in the seminiferous tubules. (2) Bax protein although expressed in the interstitium was not present in the Leydig cells. (3) Bcl-xl in Leydig cells was transiently increased after EDS. In conclusion, EDS kills Leydig cells by apoptosis; however the control of Leydig cell death does not involve p53 or the Bcl-2 family members but may require other gene products yet to be identified.

Apoptosis and related genes in the rat ventral prostate following androgen ablation in response to ethane dimethanesulfonate.

BACKGROUND: Following androgen withdrawal, regression of the prostate is characterized by apoptotic cell death. The molecular events governing this process have not been fully characterized. METHODS: Using ethane-1,2-dimethanesulfonate (EDS) to induce androgen ablation, we investigated the role of the Bcl-2 family members and Fas pathway in this phenomenon. Prostates were examined from adult male rats injected with 100 mg/kg EDS and killed 2, 5, and 8 days later. RESULTS: Regression of the prostate was evident as a time-dependent decrease in weight. The number of apoptotic cells identified by in situ end labeling was maximal after 5 days of treatment. There was no statistically significant change in the expression of Bax, Bcl-xl, Bcl-2, or p53 following androgen withdrawal. In contrast, 5 days post-EDS treatment, testosterone-repressed prostate message (TRPM-2) and Fas-R expression were induced. There was a decline in Fas-L levels 8 days after EDS administration. CONCLUSIONS: This study extends previous work which has shown that androgen withdrawal induces apoptosis in the prostate. We have shown that although p53 and the Bcl-2 family members examined in this study do not seem to be important in this process, the Fas pathway may play a role in apoptosis of the ventral prostate in response to androgen ablation.

The inhibition of androstenedione production in mature thecal cells from the ovary of the domestic hen (Gallus domesticus): evidence for the involvement of progestins.

In the 24 hours before ovulation, there is an abrupt decline in the ability of theca cells from the largest chicken preovulatory follicle to produce androstenedione from all substrates except dehydroepiandrosterone. In this study, we tested the hypothesis that progesterone from granulosa cells might inhibit andostenedione production by the adjacent theca cells. Physiological concentrations of progesterone inhibited andostenedione production by dispersed thecal cells from the substrate 17 alpha-hydroxyprogesterone but not dehydroepiandrosterone in both a dose- and time-dependent manner. In contrast, the metabolites of progesterone, 17 alpha-hydroxyprogesterone, and androstenedione at a high concentration (100 nM) failed to produce such an inhibitory effect. In addition, this inhibitory effect of progesterone was reversed by the protein synthesis inhibitor cycloheximide. The results of this study seem to suggest that progesterone acts indirectly through its nuclear receptor to induce the synthesis of a protein that possibly inhibits C17,20 lyase activity and/or C17,20 lyase gene expression.

Characterization of transforming growth factor-alpha receptors in the avian ovary: alterations in ligand binding to granulosa cells during follicular maturation.

The presence of epidermal growth factor receptors (EGF-R) and the ligands epidermal growth factor/transforming growth factor-alpha (EGF/TGF alpha) have been reported in mammalian ovaries where they are implicated in folliculogenesis and steroidogenesis. Evidence is presented to show that authentic EGF/TGF alpha receptors are expressed by the avian granulosa cells. The TGF alpha receptors (TGF alpha-R) from chicken granulosa cells were characterized by specific binding of 125I-human TGF alpha. In this study, competition with human EGF, human TGF alpha, human IGF-I, human basic fibroblast growth factor (bFGF) and insulin for 125I-human TGF alpha binding demonstrated that the avian granulosa cell TGF alpha-R binds human EGF with 300-fold lower affinity than human TGF alpha. IGF-I, bFGF and insulin did not displace bound 125I-TGF alpha. Scatchard analysis showed that a single class of high-affinity binding sites is present on the granulosa cells (Kd 0.23 +/- 0.009 nM). However, the number of binding sites altered during follicular maturation with a significant decline in the most mature follicle. These results go some way to explaining the basis for the changing sensitivity of avian granulosa cells to EGF/TGF alpha stimulation as they mature. In addition, the gonadotrophins, LH and FSH, increased the number of receptors in cultured granulosa cells and may therefore partially influence folliculogenesis and steroidogenesis through this route.

Immunohistochemical localization of epidermal growth factor receptors, epidermal-growth-factor-like and transforming-growth-factor-alpha-like peptides in chicken ovarian follicles.

The purpose of this study was to determine the presence of epidermal growth factor receptor and its potential ligands epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) in the tissues of the maturing follicles in the ovary of laying ISA-Brown hens using peptide-specific immunohistochemical methods. Cryostat sections, 6-8 microns thick, were made from fresh-frozen tissues of F1-F4 (largest to fourth largest) and large white follicles and they were immunostained for epidermal growth factor receptor, epidermal growth factor or transforming growth factor alpha using specific polyclonal antibodies. The EGF receptor and both ligands were detected in the granulosa, theca interna and theca externa layers of the follicles. The EGF receptor was localized both in the plasma membrane and cytoplasm of all cell types. EGF was predominantly cytosolic, whereas TGF-alpha was found in the plasma membranes and perinuclear areas of all cell types. The concentration of the receptor and both ligands decreased with follicular maturation. This observation is consistent with our previous observation that the response to EGF and TGF-alpha decreases as follicles mature, and thus provides further evidence that the receptor or the ligands may have a regulatory role in avian ovarian function.