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.

Ethane dimethanesulphonate (EDS) is cytotoxic to Leydig cells in the adult rat.

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 longterm 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 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.

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 \g=a\ (TGF-\g=a\) in the tissues of the maturing follicles in the ovary of laying ISA-Brown hens using peptide-specific immunohistochemical methods. Cryostat sections, 6\p=n-\8\g=m\mthick, were made from fresh\p=n-\frozen tissues of F1\p=n-\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 \g=a\ 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