Alteration of transforming growth factor-beta signaling system expression in adult rat germ cells with a chronic apoptotic cell death process after fetal androgen disruption.

In utero exposure to chemicals with antiandrogen activity induces undescended testis, hypospadias, and sub- or infertility. The hypospermatogenesis observed in the adult rat testis exposed in utero to the antiandrogen flutamide has been reported to be a result of a long-term apoptotic cell death process in mature germ cells. However, little if anything is known about the upstream signaling mechanisms controlling this apoptosis. In the present study, we have investigated the possibility that the TGF-beta signaling pathway may be at play in this control of the apoptotic germ cell death process. By using a model of adult rat exposed in utero to 0, 0.4, 2, or 10 mg/kg.d flutamide, we observed that pro-TGF-beta signaling members, such as the three isoforms of TGF-beta ligands (TGF-beta1-3), the two TGF-beta receptors (TGF-betaRI and -RII) and the R-Smads Smad 1, Smad 2, Smad 3, and Smad 5 were inhibited at the mRNA and protein levels, whereas the anti-TGF-beta signaling member Smad 7 was overexpressed. Furthermore, we report that the overexpression of Smad 7 mRNA could induce an activation of c-Jun N-terminal kinase, because of the observed c-Jun overexpression, activation, and nuclear translocation leading to an increase in the transcription of the proapoptotic factor Fas-L. Together, the alterations of TGF-beta signaling may represent upstream mechanisms underlying the adult germ cell apoptotic process evidenced in adult rat testis exposed in utero to antiandrogenic compounds such as flutamide.

Alteration of lactate production and transport in the adult rat testis exposed in utero to flutamide.

Although it is established that in utero exposure to the antiandrogen flutamide induces alteration of spermatogenesis in the adult rat testis offspring, the cellular and molecular mechanisms involved in such an effect remain to be investigated. In the present paper, by using as model adult rats exposed in utero to flutamide (0, 2, 10 mg/kg per day), we have investigated the hypothesis that germ cell alterations could be related to defects of energy metabolism and particularly to defects of the production and transport of lactate. Lactate is a preferential energy substrate produced by Sertoli cells and transported to germ cells by monocarboxylate transporters (MCT). A significant decrease (60%, P<0.001) in lactate production was observed in cultured Sertoli cells from rat testes exposed in utero to flutamide from the dose of 2 mg/kg per day. Such a decrease is concurrent to a decrease in lactate dehydrogenase A (LDHA) mRNA levels (evaluated through semiquantitative RT-PCR) and LDHA4 activity. The decrease in LDHA mRNA levels (to 64 +/- 9% of the control, P<0.05) was observed with the lowest dose (2 mg/kg per day) of flutamide tested. The decrease in LDHA mRNA levels was observed in both the whole testis and in isolated Sertoli cells, suggesting that such a decrease in LDHA expression occurred also in the (Sertoli) cells producing lactate. Lactate is transported from Sertoli cells to germ cells via MCT1 and MCT2. We immunolocalized MCT1 to all the different germ cell types and MCT2 exclusively to elongated spermatids. In the adult testis exposed in utero to flutamide, MCT1 (53 +/- 8%, P<0.02) and MCT2 (52 +/- 9%, P<0.02) mRNA levels were significantly reduced indicating that lactate transport to germ cells could be also altered. Together, these data support (i) the existence of a relationship between the antiandrogen activity and the energy metabolism in the testis and (ii) the concept of an androgen-dependent programming, occurring early in the fetal life in relation to the expression of some of the key genes involved in the production and transport of lactate in the seminiferous tubules.