No abstract

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.

The 17 -hydroxysteroid dehydrogenases (17 -HSDs) play a key role in the synthesis of sex steroids. The hallmark of this family of enzymes is the interconversion, through their oxydoreductive reactivity at position C17, of 17-keto-and 17 -hydroxy-steroids. Because this reaction essentially transforms steroids having low binding activity for the steroid receptor to their more potent 17 -hydroxysteroids isoforms, it is crucial to the control of the physiological activities of both estrogens and androgens. The human placenta produces large amounts of progesterone and estrogens throughout pregnancy. The placental type 1 17 -HSD enzyme (E17 -HSD) catalyzes the reduction of the low activity estrogen, estrone, into the potent estrogen, estradiol. We studied the cell-specific expression of type 1 17 -HSD in human term placental villous tissue by combining in situ hybridization to localize type 1 17 -HSD mRNA with immunohistochemistry using an antibody against human placental lactogen, a trophoblast marker. Immunolocalization of E17 -HSD was also performed. To ascertain whether other steroidogenic enzymes are present in the same cell type, cytochrome P450 cholesterol side-chain cleavage (P450scc), P450 aromatase, and type 1 3 -hydroxysteroid dehydrogenase (3 -HSD) were also localized by immunostaining. Our results showed that the syncytium is the major steroidogenic unit of the fetal term villi. In fact, type 1 17 -HSD mRNA and protein, as well as P450scc, P450 aromatase, and 3 -HSD immunoreactivities were found in these cells. In addition, our results revealed undoubtedly that extravillous cytotrophoblasts (CTBs), e.g. those from which cell columns of anchoring villous originate, also express the type 1 17 -HSD gene. However, CTBs lying beneath the syncytial layer, e.g. those from which syncytiotrophoblasts develop, contained barely detectable amounts of type 1 17 -HSD mRNA as determined by in situ hybridization. These findings, along with those from other laboratories confirm the primordial role of the syncytium in the synthesis of steroids during pregnancy. In addition, our results indicate for the first time that CTBs differentiating along the invasive pathway contain type 1 17 -HSD mRNA.

No abstract

Progesterone and estradiol are the most potent human sex steroid hormones of placental origin and are essential to the maintenance of pregnancy, the timing of parturition, the maturation of many fetal organs, and the preparation of the maternal reproductive system. Naturally, regulatory mechanisms must be in place to coordinate the synthesis and inactivation of these two hormones. We have previously shown that the highest levels of type 1 and type 2 17beta-hydroxysteroid dehydrogenase (17betaHSD) messenger ribonucleic acids (mRNAs) occur in the placenta, particularly in the villi. However, in contrast to type 1 17betaHSD mRNA, type 2 17betaHSD mRNA was not detectable in cell cultures of human cytotrophoblasts or syncytiotrophoblasts. Using in situ hybridization, we unequivocally identified endothelial cells as the only cell type expressing the type 2 17betaHSD gene in fetal villi. Moreover, type 2 17betaHSD mRNA was specifically detected in the endothelial cells of the arterial system, and at higher levels in the villi compared with endothelial cells of the cord arteries when the two tissue sections were cohybridized. In fact, both mRNA levels and enzymatic activity are at their highest levels in arterial endothelial cells. In conclusion, the endothelial cells of the villous arterioles are the primary site of type 2 17betaHSD gene expression. This suggests a regulatory role for these cells in the control of progestin, androgen, and estrogen levels during pregnancy, thus opening a whole new way of viewing regionalization and localization of steroidogenesis in the human villi.