In vitro evaluation of aromatase enzyme in granulosa cells using a [C-11]vorozole binding assay

An in vitro method for measuring aromatase cytochrome P450 enzyme (P450(ARO M)) in human granulosa cells (GC) has been developed, based on binding of t he C-11-labeled aromatase inhibitor vorozole. GC were obtained following su perstimulation during in vitro fertilisation. The method revealed a binding affinity (K-D) of 0.4 nM and a maximum binding (B-max) at 11 fmol/4000 cel ls which is equal to 1.6 million binding sites per cell. Linear Scatchard p lots indicated a single type of binding site, P450(AROM) concentrations mea sured by [C-11]vorozole binding correlated positively with aromatisation of [1 beta-H-3]androst-4-ene-3,17-dione measured as [H-3]water release, and a positive association was also found with the ovarian in vivo response to f ollicle-stimulating hormone (FSH) stimulation expressed as 1000 times the r atio of the number of oocytes recovered from a patient and the total dose o f recombinant FSH administered. Frozen cells could be used for P450(AROM) q uantitation, provided the correct freezing procedure was used. Quantitation of P450(AROM), based on binding of [C-11]vorozole is an accurate and sensi tive in vitro method, which might be extended to the measurement of aromata se expression by a noninvasive technique in the intact ovary in vivo using positron emission tomography. (C) 1999 Elsevier Science Inc. All rights res erved.