Molecular Human Reproduction, Vol. 6, No. 8, 677-680,
August 2000
© 2000 European Society of Human Reproduction and Embryology
Endocrinology |
Improved FSH sensitisation and aromatase assay in human granulosa–lutein cells
University of Manchester, Department of Medicine (Clinical Biochemistry), Clinical Sciences Building, Hope Hospital, Stott Lane, Salford M6 8HD, UK
Abstract
Granulosa–lutein (GL) cells from follicular aspirates from women undergoing in-vitro fertilization (IVF) treatment are usually refractory to follicle stimulating hormone (FSH) regarding the induction and/or maintenance of aromatase activity which converts androgens (e.g. testosterone) to oestrogens. The normal method of assaying FSH-stimulated aromatase activity in GL cell cultures is to add exogenous testosterone throughout the cell culture period and measure the secreted oestradiol. Thus under the conditions usually employed for studying FSH-stimulated oestradiol secretion in GL cells, the `total' FSH effect is dependent both on the decay of the aromatase concentration in culture relative to its induction/maintenance by FSH and on changes in its activity in the face of a declining substrate concentration as the exogenous testosterone is converted over several days to oestradiol. We have therefore used a technique for challenging the cells with testosterone (10 µmol/l) for just 2 h at the end of the normal longer-term culture period such that its concentration was essentially unchanged, thus ensuring that there was no depletion of the aromatase substrate and that the FSH stimulation phase could be performed independently of exogenous testosterone. Consequently, GL cells were incubated for 0, 24 and 48 h prior to stimulation with FSH (100 IU/l) for 24 h after which they were washed and challenged with testosterone for 2 h and the secreted oestradiol was assayed. Freshly isolated GL cells from women undergoing IVF were refractory to FSH but after preincubation were responsive such that there was a 3–14-fold increase over basal activity depending on the cell preparation. In conclusion, we have developed a simple 48 h procedure for sensitizing GL cells to FSH and established the conditions for optimizing the assay of aromatase activity independently from the effect of FSH on its induction.
aromatase/FSH/granulosa cells/granulosa/lutein cells
Notes
1,4 Present address: Nuffield Department of Obstetrics and Gynaecology, Women's Centre, John Radcliffe Hospital, Headley Way, Headington, Oxford OX3 9DU, UK
2 Present address: Willink Biochemistry Genetics Unit, Royal Manchester Children's Hospital, Pendlebury, Salford M27 4HA, UK
3 Present address: Nurture, Queen's Medical Centre, Nottingham NG7 2UH, UK
5 To whom correspondence should be addressed. E-mail: ann.lambert{at}obs-gyn.ox.ac.uk
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