Steroidogenesis by cells cultured from second trimester human amniotic fluids
Abstract (Summary)Restricted Item. Print thesis available in the University of Auckland Library or available through Inter-Library Loan. Three major cell types, termed E, F and AF, constitute cultures of second trimester human amniotic fluids and are widely used for the prenatal diagnosis of inherited disorders of metabolism. Current evidence suggests that the predominant cell type, the AF cells, retain properties of trophoblast and it has been proposed that they are derived from the trophoblast of the placenta. Since the trophoblast is thought to be the site of the aromatization of C19 -steroids in the placenta, it was considered that AF type amniotic fluid cell cultures should possess this steroidogenic activity. Investigations were therefore undertaken to explore this hypothesis. Initial experiments involved culturing AF and F type cells in the presence and absence of dehydroepiandrosterone sulphate and subsequently incubating the cell layers with [4-14C] androstenedione. Isolation and identification of the steroidal metabolites showed that neither cell type aromatized [14-14C] androstenedione under the conditions of the assay. However, a qualitative similarity was shown for the pattern of metabolites isolated from incubations with F type amniotic fluid cells and DF type adult dermal fibroblasts suggesting that F type cells are typical fibroblasts from dermis or other connective tissues. Results of radioimmunoassays of media extracts taken sequentially over a 7 day culture period for all cell types, indicated the absence of de novo estrogen production or conversion of dehydroepiandrosterone sulphate to estrogens under the culture conditions described. It was subsequently shown that culture of AF, F or DF type cells in the presence of various estrogen precursors including testosterone, androstenedione and dehydroepiandrosterone, did not result in the production of estrogens by any of the cell types when extracts of media were analysed by radioimmunoassay. However, culture of choriocarcinoma, a trophoblastic neoplasm, under identical conditions, showed that this cell type exhibits similar steroidogenic activities to those of the normal term human placenta. Variations of the composition of the medium and addition of exogenous agents known to stimulate aromatase activity of various systems were then investigated with the aim of maintaining/inducing aromatase activity of primary cultures of amniotic fluid cells. The composition of the medium had no apparent effect on this activity. Addition of a hormone mixture which maintains cytochrome P450 concentrations in primary cultures of rat liver parenchymal cells, was without effect on either the cytochrome P450 concentration or the aromatase activity of primary cultures of amniotic fluid cells. The gonadotropins hCG and FSH, prostaglandins E2and F2? and dibutyryl CAMP, were all shown to be unable to stimulate aromatase activity of these cultures when grown under a variety of conditions. These findings, obtained by radioimmunoassay of extracts of media, were subsequently confirmed by use of a tritium release assay. Cultures used in these studies were shown to be viable by demonstration of mitotic events, incorporation of L-[4,5-3H] leucine into cellular protein and by the production of hCG. These results suggest that the inability of primary cultures of amniotic fluid cells to aromatize C19 -steroids was not due to their being metabolically inactive. It can be concluded that either amniotic fluid cell cultures do not possess aromatase activity under the experimental conditions described, or that the analytical methods employed were not sensitive enough to detect this activity. However, if the latter were true, it can be assumed that such activity would not have any physiological significance.
School Location:New Zealand
Source Type:Master's Thesis
Date of Publication:01/01/1983