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Long-Term Apoptotic Cell Death Process with Increased Expression and Activation of Caspase-3 and -6 in Adult Rat Germ Cells Exposed in Utero to Flutamide

Institut National de la Santé et de la Recherche Médicale (Unité 407; A.O., S.C., C.M., A.F., E.T., M.B.), Faculté de Médecine Lyon-Sud, 69921 Oullins, France; and BayerCropScience (F.C., R.B.) 06903 Sophia-Antipolis, France

Address all correspondence and requests for reprints to: Mohamed Benahmed, Institut National de la Santé et de la Recherche Médicale Unité 407, Faculté de Médecine Lyon-Sud, BP 12, 69921 Oullins Cedex-France. E-mail: benahmed{at}grisn.univ-lyon1.fr.

Although it is established that in utero exposure to antiandrogenic compounds such as flutamide induces hypospermatogenesis in adult male rat offspring, the cellular and molecular mechanisms remain to be investigated. By using adult rats exposed in utero to flutamide (0.4, 2, 10 mg/kg·d) as a model, we show that the hypospermatogenesis could be related to a chronic apoptotic cell death process associated with a long-term increase in caspase-3 and -6 expression and activation in germ cells. The number of apoptotic (terminal deoxynucleotidyl transferase-mediated deoxyuridine positive) adult germ cells was dependent on the dose of flutamide. The apoptotic germ cell death process could be related to an increased expression and activation of effector caspases-3 and -6. Procaspases-3 and -6 were immunodetected in germ cells from both untreated or flutamide-treated rats, whereas cleaved active caspase-3 was detected exclusively in germ cells from adult rat exposed in utero to flutamide. Exposure to the antiandrogen increased in a dose-dependent manner as caspase-3 and -6 mRNA (in RT-PCR approaches) as well as procaspase-3 and -6 protein (in Western blotting analyses) levels in the adult rat testis. Flutamide also activates procaspases. Indeed, whereas cleaved active caspase-3 and -6 proteins were absent in control animals, they were detected in adult rat testes exposed in utero to flutamide. Our results show that whereas the apoptotic germ cell death process associated with the increased caspase expression and activation in adult rat germ cells was chronic and nonreversible when exposure to flutamide occurred in utero, it was transient when such an exposure occurred during adulthood. Indeed, although an increase in caspase-3 and -6 mRNA and procaspase-3 and -6 protein levels was observed in germ cells after 3 d of exposure to flutamide, 1–2 wk after the cessation of the antiandrogen exposure, the caspase mRNA and procaspase protein levels were back to control. Active cleaved caspase-3 and -6 protein appeared following the exposure to the antiandrogen, whereas they disappeared at cessation of exposure to flutamide. In summary, the present findings indicate that in utero exposure to the antiandrogen induced in the adult rat testes a chronic apoptotic germ cell death associated with a long-term increase in the expression and activation in germ cells of caspases-3 and -6, two key components in the death machinery.

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