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Glutathione S-transferases (GSTs) are a family of soluble enzymes of detoxification that use reduced glutathione in conjugation and reduction reactions. Toxic electrophiles are substrates for the GSTs. GSTalpha is expressed at high levels in different tIssues such as the testis. Among the different GSTs present in the testis, GSTalpha is specifically expressed in Leydig and Sertoli cells known to be under the control of hormonal and local regulatory factors. The present study investigated the regulatory action of tumor necrosis factor-alpha (TNFalpha) on basal and hormone (FSH and testosterone)-stimulated GSTalpha expression in cultured Sertoli cells. Treatment with TNFalpha (0-20 ng/ml, 48 h) induced a decrease in basal GSTalpha mRNA levels in a dose-dependent manner (fivefold decrease; P<0.001). The maximal and half maximal effects were observed at 20 ng/ml and 7 ng/ml respectively. The inhibitory effect of TNFalpha was also time-dependent with a maximal inhibitory effect (threefold decrease; P<0.001) observed at 48 h. The inhibitory effect of the cytokine was also observed on basal GSTalpha protein (28 kDa) levels. TNFalpha also inhibited the hormone-stimulated GSTalpha expression in Sertoli cells. The treatment of cultured Sertoli cells with both FSH and TNFalpha (100 ng/ml and 10 ng/ml respectively, 48 h) resulted in a complete suppression of the stimulatory action of FSH on GSTalpha mRNA levels. Similarly, in Sertoli cells treated with testosterone or its non-aromatizable metabolite dihydrotestosterone (100 ng/ml, 24 h), TNFalpha reduced the hormone-stimulated GSTalpha mRNA and protein levels. TNFalpha inhibited basal GSTalpha expression without affecting mRNA stability. Indeed, the decay curves (mRNA half-life time=18 h) for the GSTalpha basal mRNA levels in Sertoli cells was similar in the absence or presence of TNFalpha (10 ng/ml, 48 h). Testosterone increased GSTalpha mRNA without affecting the enzyme mRNA stability. TNFalpha antagonized the androgen-stimulated GSTalpha mRNA levels without affecting the enzyme mRNA stability, suggesting that the interaction between the androgen and the cytokine is mostly exerted at a transcriptional level. FSH increased GSTalpha mRNA levels through an increase in mRNA stability (increased mRNA half-life times to 119 h). TNFalpha antagonized the stimulatory effect of FSH on GSTalpha mRNA levels by antagonizing the stabilizing effect exerted by the hormone on GSTalpha mRNA. Together, these results suggest that the increase in the cytokine levels within the testis would alter the detoxification processes against genotoxic products during spermatogenesis.

In utero exposure to exogenous anti-androgenic compounds induces a wide range of abnormalities of the reproductive system, including hypospermatogenesis, cryptorchidism and hypospadias. By using rats exposed in utero to the anti-androgenic compound flutamide (0.4, 2 or 10 mg/kg per day), it has been shown that hypospermatogenesis in adult testes could be related to (i) a long-term apoptosis in germ cells but not in somatic Leydig and Sertoli cells as evidenced by the TUNEL approach and (ii) alterations in the mRNA and protein expression of pro- (Bax, Bak, Bid) and anti-apoptotic (Bcl-2, Bcl-w) members of the Bcl-2 family. Indeed, the number of apoptotic germ cells increased with the dose of flutamide administered and the apoptotic germ cells were mainly detected at androgen-dependent stages VII–VIII. Moreover, for the Bcl-2-related proteins that were expressed mainly in the germ cells, a decrease in the levels of anti-apoptotic peptides Bcl-w (60%, P=0.003) and Bcl-2 (90%, P=0.0001) was observed at 2 mg/kg per day flutamide and an increase in levels of the pro-apoptotic Bax (2.3-fold, P=0.0004) was detected at 10 mg/kg per day. In contrast, the levels of pro-apoptotic peptide Bak that was mainly expressed in somatic cells decreased (70%, P=0.0008) at 10 mg/kg per day. Such alterations in Bcl-2-related peptides occurred mainly at the protein level except for Bcl-2 (72%, P=0.0001) and Bak (43%, P=00002) transcripts. Together, these results showed that the apoptosis observed in adult germ cells from rats exposed in utero to flutamide may result from a long-term alteration in the balance between pro- and anti-apoptotic Bcl-2-related molecules in favour of pro-apoptotic proteins. These data further supported the concept of an androgen-dependent fetal programming that is in relation with an alteration of the expression of Bcl-2-related genes/proteins promoting apoptosis in testicular germ cells of adult rats with fetal androgen disruption.