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

Abstract

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.

Alterations of Sertoli cell activity in the long-term testicular germ cell death process induced by fetal androgen disruption

“…Effects of in utero exposure to flutamide on Sertoli cell activity in adult germ cell survival As fetal androgen disruption induced a long-term apoptosis in adult germ cell characterized by caspase-3 activation in spermatocytes and spermatids (Omezzine et al 2003) and Sertoli cells are targeted by androgens in the adult seminiferous tubules, we addressed the question as to whether the increased adult germ cell death might be related to altered Sertoli cell activity. For this purpose, purified post-natal (PND 15) Sertoli cells from rat testes exposed in utero to 10 mg/kg d of flutamide were co-cultured with adult purified germ cells (i.e., spermatocytes or spermatids) not treated with flutamide.…”

“…(Bozec et al 2004), reduced ventral prostate weight, and increased malformations of vas deferens. Fetal exposure to flutamide induces a long-term apoptosis in testicular germ cells in adult male rat offspring (Omezzine et al 2003, Bozec et al 2004, particularly in spermatocytes and spermatids at the level of stages VII-VIII of the seminiferous epithelium androgen-dependent seminiferous tubules (Bozec et al 2004). As in the adult seminiferous tubules, it is Sertoli cells that are primarily targeted by the androgens; we have further addressed the question as to whether the long-term germ cell death process might be related to altered Sertoli cell functions.…”

“…It has been reported that the fetal hormonal disruption induced either by chemical anti-androgens such as flutamide or by different environmental anti-androgen compounds (e.g., vinclozolin, phthalates) gives rise to a wide range of reproductive system abnormalities such as hypospadias, undescended testes, developmental testicular alterations (Fisher 2004, Crews & McLachlan 2006, Gray et al 2006, Newbold et al 2006, and particularly to a long-term apoptosis of germ cells in the adult testis (Omezzine et al 2003, Bozec et al 2004, Uzumcu et al 2004 transmitted by male through at least four generations (Anway et al 2005). These observations support the concept of a fetal and transgenerational programming of adult testicular germ cell apoptosis.…”

“…These observations support the concept of a fetal and transgenerational programming of adult testicular germ cell apoptosis. Although it was shown that this apoptotic process was related to a long-term increase in the expression and activation of effector caspases-3 and -6 (Omezzine et al 2003), resulting from changes in the ratio of Bcl-2 family peptides in favor of the pro-apoptotic members (Bozec et al 2004), the upstream cellular and molecular mechanisms still remain to be investigated. Indeed, one of the major questions raised by these observations is related to the potential role of the Sertoli cells in the apoptotic process that affects germ cells in the adult rat testes exposed in utero to antiandrogens.…”

Origine développementale et environnementale de l’infertilité masculine

“…La projection de ces données chez l'être humain indique qu'à partir de la 7 e semaine de gestation et jusqu'à la puberté, l'individu est très sensible aux PEE. Les modèles expérimentaux ont égale-ment permis d'établir que l'exposition périnatale aux PEE induit des lésions irréversibles de la spermatogenèse (diminution du nombre de spermatozoïdes), alors que ces mêmes altérations sont réversibles (pour des doses faibles de PEE) chez l'adulte [19]. En somme, l'exposition périnatale (in utero et/ou néonatale) chez le rongeur à des substances susceptibles d'être toxiques, est un bon outil pour identifier leur activité perturbatrice endocrinienne, le développement testiculaire du rat mâle étant très proche de celui de l'homme.…”