The proto-oncoprotein Cbl is known to control several signaling processes. It is highly expressed in the testis, and because spermatogenesis is androgen dependent, we investigated the androgen dependency expression of Cbl through its testicular sublocalization and its expression levels in rats that were exposed to the antiandrogen flutamide or were hypophysectomized. We report the androgen dependency of Cbl as it localizes in pachytene spermatocytes during androgen-dependent stages, is down-regulated upon flutamide exposure, and is up-regulated with testosterone in hypophysectomized rats. Coculture experiments showed the key control exerted by the Sertoli cell on Cbl activity. As flutamide induces germ cell apoptosis, we investigate members of the Bcl-2 family upon flutamide exposure. We show that the proapoptotic Bcl-2 family member Bim mirrored Cbl expression through a posttranscriptional process. We also show that in Cbl knockout mouse testes, the imbalance between the high expression of Bim and Smac/Diablo and antiapoptotic factors such as cellular inhibitor of apoptosis 2 favors a survival process, which makes these mice unresponsive to androgen withdrawal and could explain their hypofertility.

In mammalian and fish species, P450c17 mediates both 17 alpha-hydroxylase and 17,20-lyase activities in the synthesis of steroid hormones. Previous results have shown that among the adrenal steroid hydroxylase enzymes involved in adrenal C19 steroid and glucocorticoid synthesis, regulation of cytochrome P450c17 is of primary importance because it is localized at the key branch between glucocorticoid and C19 steroid synthesis. A cDNA library from guinea pig adrenal was constructed, and the complete 17 alpha-hydroxylase cytochrome P450 cDNA was isolated. The guinea pig P450c17 cDNA includes the full-length coding region (1,524 nucleotide), the complete 3' untranslated region (169 nucleotide), and 39 bases of the 5' untranslated region. Our clone shares most of the features of the other P450c17 cDNAs; however, in addition, we identified a novel conserved region of 18 amino acids located in exon I between residues 80 and 97. This region presents the highest percentage of identity among the other P450c17 enzymes and is positioned one helixturn upstream of the important Ser106 on the corresponding human form. On Northern blot, the cDNA hybridizes with a major 1.8-kb mRNA and with two other related P450c17 mRNA of about 3 and 4 kb. P450c17 mRNA is equally distributed in male and female gonads and adrenals. Characterization of the enzymatic activity shows that 17 alpha-hydroxylase and 17,20-lyase are carried by a single protein, but in homogenates 17,20-lyase activity is barely detectable. Moreover, we demonstrate in vitro and in vivo that the guinea pig enzyme preferentially has very high levels of 17 alpha-hydroxylase and 17,20-lyase activities only toward delta 4 steroids. Second-messenger cyclic adenosine monophosphate and adrenocorticotropin specifically increased the abundance of P450c17 mRNA levels in guinea pig adrenal cells.

3 beta-Hydroxysteroid dehydrogenase (3 beta HSD) in human placenta converts 3 beta-hydroxy-5-ene steroids producing progesterone, whereas 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) mediates the interconversion of estrone and estradiol. We first showed that the expression of type I 17 beta HSD (17 beta HSD-I) gene was undetectable in human JEG-3 cells. We then studied the effects of cAMP- and protein kinase-C-dependent pathways on the expression of 3 beta HSD-I and 17 beta HSD-II genes using an analog of cAMP [8-(4-chlorophenylthio)cAMP (8CPTcAMP)] and a protein kinase-C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), in JEG-3 cells. Novel inhibitors of protein kinase-A (PKA) and PKC were also used. The 3 beta HSD cDNA probe hybridized to a single 1.7-kilobase (kb) 3 beta HSD mRNA species corresponding to the transcript of the 3 beta HSD-I gene. The 17 beta HSD cDNA probe hybridized to two 17 beta HSD transcripts of 1.3 and 2.2 kb. The 1.3-kb 17 beta HSD mRNA species was regulated, whereas the 2.2-kb species was constitutively expressed in JEG-3 cells. When JEG-3 cells were exposed to 8CPTcAMP or PMA, 3 beta HSD-I and 17 beta HSD-II gene transcriptions were increased in a dose- and time-dependent manner. Moreover, the combined effects of PMA and 8CPTcAMP on 3 beta HSD-I mRNA levels was additive and synergistic on 17 beta HSD-II mRNA levels. The mechanism by which cAMP activated accumulation of 3 beta HSD-I and 17 beta HSD-II mRNAs involved an activation of the cyclase. The effects of a cAMP-dependent kinase inhibitor and a diacylglycerol-dependent kinase inhibitor in JEG-3 cells indicated that cAMP acts on 3 beta HSD-I mRNA via a PKA-dependent mechanism, but on 17 beta HSD-II mRNA via another nonclassical cAMP-dependent mechanism. Finally, the effect of activation of both signaling pathways on expression of the 17 beta HSD-II gene as well as the effect of PMA on the 3 beta HSD-I gene did not require protein synthesis. These data provide strong evidence for the regulation of the 3 beta HSD-I and 17 beta HSD-II genes by cAMP and PKC and, thus, indicate an important endocrine and/or paracrine regulation of steroid hormone production in human placenta.

Two 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) genes, types 1 and 2, have been cloned. The two isozymes show a 30% sequence homology but differ markedly in their kinetic properties. To date, the steroidogenic capacity of the placenta has been associated with syncytium formation. In this study, we have investigated 17 beta-HSD type 1 and type 2 gene expression during trophoblast differentiation in culture. We observed that term placenta and fetal cotyledons contain large amounts of both messenger RNAs (mRNAs). In culture, the type 1 gene is expressed concurrent with syncytium formation. However, type 2 expression is barely detectable in freshly isolated cytotrophoblasts and undetectable in syncytiotrophoblasts. Incubation of trophoblasts with progesterone and estradiol increased type 1 mRNA but did not restore 17 beta-HSD type 2 expression. 17 beta-HSD activities with substrates that differentiate the type 1 and type 2 enzymes correlated with the gene expression results. Type 1 activity decreased in freshly isolated trophoblasts by 2-fold and remained at these levels throughout the culture period. However, when compared with levels measured in term microsomes, type 2 activity decreased by 20-fold in freshly isolated cells and decreased again in culture by 5-fold. The expression pattern of 17 beta-HSD type 1 and type 2 activity in trophoblasts in culture suggests differing mechanisms regulate type 1 and type 2 mRNA levels.

We have investigated the expression of cholesterol side-chain cleavage cytochrome P450 (P450scc) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) type 1 genes during human trophoblast differentiation in culture and the modulation of their steady-state mRNA levels by steroids. During the first 24 to 48 h after plating, mononucleated cells aggregated, forming colonies. After 60 h in culture, cell diameters were increased and nuclei appeared centrally distributed within large cells, consistent with syncytiotrophoblast formation. During these striking morphological changes in culture the expression and activity levels of 3 beta-HSD type 1 and P450scc increased significantly as isolated cytotrophoblasts progressed to a differentiated state, with P450scc and 3 beta-HSD type 1 mRNAs activities being more abundant in cells cultured for 48 to 72 h. In the same culture, however, the amount of 3 beta-HSD protein decreased during the first 12 to 24 h by 50% compared with freshly isolated trophoblasts but remained at these levels throughout the culture period. The specific activity of the 3 beta-HSD as determined with pregnenolone or dehydroepiandrosterone was similar but increased with time as syncytiotrophoblast was formed in vitro. These observations provide additional evidence that the expression of these two progesterone-synthesizing enzymes is coincident and that they reach their maximum steady-state mRNA levels at a time when syncytium formation occurs in vitro. Incubation of trophoblast cells with progesterone or estradiol increased the abundance of P450scc and 3 beta-HSD type 1 mRNAs but had no significant effect on the amount of 3 beta-HSD protein. These observations of the regulation of 3 beta-HSD type 1 mRNA levels by steroids suggest a complex relationship of the mechanisms regulating transcription/mRNA processing and transduction of the 3 beta-HSD type 1 gene.