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a Division of Physiology, Pharmacology and Toxicology, School of Biological Sciences, University of Manchester, Oxford Road, Manchester, M13 9PT, United Kingdom
b Department of Cell Biology and Histology, Faculty of Veterinary Medicine, Utrecht University, 3508 TD Utrecht,The Netherlands
c Department of Medicine, University of Manchester, Manchester, M13 9PT, United Kingdom
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 3ß-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.
2 Correspondence: Ian Woolveridge, G38 Stopford Building, Manchester University, Oxford Road, Manchester, M13 9PT, UK. FAX: 0161 275 5600; ian.woolveridge{at}man.ac.uk
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