STUDIES INTO AROMATASE-ACTIVITY ASSOCIATED WITH FETAL ALLANTOCHORIONIC AND MATERNAL ENDOMETRIAL TISSUES OF EQUINE PLACENTA - IDENTIFICATIONOF METABOLITES BY GAS-CHROMATOGRAPHY MASS-SPECTROMETRY

Maternal endometrial and fetal allantochorionic tissues were separated manually from the placentae of seven healthy thoroughbred and three p ony mares, ranging in gestational age from 100 to 318 days. The homoge neity of subcellular fractions prepared from these tissues was assesse d initially using the marker enzymes, succinate dehydrogenase, NADPH c ytochrome C reductase and lactate dehydrogenase for the mitochondrial, microsomal and cytosolic fractions, respectively. Light microscopy an d histochemical analysis demonstrated that the separated fetal allanto chorionic membrane, which is made up of allantoic and chorionic epithe lia, contained no significant contamination of maternal tissues. The m aternal endometrium, however, was found to contain appreciable amounts of fetal chorion torn off during the separation process. Tissue homog enates and subcellular fractions were incubated with testosterone toge ther with [4-C-14] and [H-2(5) or H-2(3)] labelled analogues in either an NADPH (1 mM) or a NADPH-regenerating environment; control experime nts (without additional cofactor) were also performed. After extractio n of the tissue homogenates, neutral and phenolic (oestrogen) unconjug ated steroids were separated by column chromatography. Radiolabelled s tudies revealed that in allantochorionic tissue incubations 67-77% of testosterone was converted to oestrogenic material, subcellular fracti onation indicating that oestrogen production was largely confined to t he microsomal fraction and time-course studies showing that the rate o f formation appeared to be linear up to 90 min. In contrast, only 5-25 % conversion occurred using maternal endometrial tissues, which could be accounted for by the contaminating presence of fetal chorion. No oe strogen production was detected in control incubations. These radiolab elled studies demonstrate that aromatase activity is located on the fe tal allantochorionic surface and, together with the histochemical data , further delineate this activity to the chorion in mature equine plac enta. Gas chromatographic-mass spectrometric (GC-MS) analysis of the p henolic extracts from allantochorionic tissue homogenate incubations i ndicated the presence of substrate-derived oestradiol-17 beta (E(2)), 6-oxo-oestradiol-17 beta (6-oxo-E(2)) and 6 beta-hydroxyoestradiol-17 beta (6 beta-OH-E(2)). Whereas all three oestrogens were identified as metabolites from testosterone in incubations performed using allantoc horionic tissue homogenates and post-mitochondrial suspensions (PMS), only E(2) was identified from incubations performed using microsomal f ractions prepared from this tissue. We conclude that both the microsom al and cytosol fractions are required for the conversion of E(2) to th e 6-oxygenated species in vitro. Using stable isotope-labelled substra tes and GC-MS analysis the mechanism of formation of these metabolites from these in vitro incubation studies may be inferred. GC-MS analysi s of the neutral extracts from allantochorionic tissue homogenate incu bations confirmed the presence of small quantities of substrate-derive d 5(10)-oestrenediols. No substrate-derived 5(10)-oestrene-3,17-diols were detected in extracts from microsomal preparations incubated in th e absence of cytosol. These data suggest that demethylation of C-19 st eroids to produce C-18 neutral steroids may require the synergistic ac tion of enzymic activities that appear to reside both in the microsoma l and cytosolic fractions of equine allantochorionic tissues. Copyrigh t (C) 1996 Published by Elsevier Science Ltd.