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Department of Veterinary Basic Sciences, Royal Veterinary College, Royal College Street, London NW1 0TU, UK
¹ Division of Clinical Developmental Sciences, Academic Section of Obstetrics and Gynaecology, Centre for Developmental and Endocrine Signalling, St George’s University of London, Cranmer Terrace, London SW17 0RE, UK

(Requests for offprints should be addressed to A E Michael; Email: tony.michael{at}sgul.ac.uk)

Cortisol–cortisone metabolism is catalysed by the bi-directional NADP(H)-dependent type 1 11ß-hydroxysteroid dehydrogenase (11ßHSD1) enzyme and the oxidative NAD⁺-dependent type 2 11ßHSD (11ßHSD2). This study related the expression of 11ßHSD1 and 11ßHSD2 enzymes (mRNA and protein) to net 11-ketosteroid reductase and 11ß-dehydrogenase (11ß-DH) activities in bovine follicular granulosa and luteal cells. Granulosa cells were isolated from follicles of < 4, 4–8, > 8 and > 12 mm in diameter in either the follicular or luteal phase of the ovarian cycle. Luteal cells were obtained from corpora lutea (CL) in the early non-pregnant luteal phase. Enzyme expression was assessed by reverse transcription-PCR and western blotting, while enzyme activities were measured over 1 h in cell homogenates using radiometric conversion assays with 100 nM [³H]cortisone or [³H]cortisol and pyridine dinucleotide cofactors. Irrespective of follicle diameter, the expression of 11ßHSD2 and NAD⁺-dependent oxidation of cortisol predominated in granulosa cells harvested in the follicular phase. In contrast, in granulosa cells obtained from luteal phase follicles and in bovine luteal cells, expression of 11ßHSD1 exceeded that of 11ßHSD2 and the major enzyme activity was NADP⁺-dependent cortisol oxidation. Increasing follicular diameter was associated with progressive increases in expression and activities of 11ßHSD2 and 11ßHSD1 in follicular and luteal phase granulosa cells respectively. In follicular phase granulosa cells from antral follicles < 12 mm, 11ßHSD1 migrated with a molecular mass of 34 kDa, whereas in the dominant follicle, CL and all luteal phase granulosa cells, a second protein band of 68 kDa was consistently detected. In all samples, 11ßHSD2 had a molecular mass of 48 kDa, but in large antral follicles (> 8 mm), there was an additional immunoreactive band at 50 kDa. We conclude that 11ßHSD2 is the predominant functional 11ßHSD enzyme expressed in follicular phase granulosa cells from growing bovine antral follicles. In contrast, in bovine granulosa cells from dominant or luteal phase follicles, and in bovine luteal cells from early non-pregnant CL, 11ßHSD1 is the major glucocorticoid-metabolising enzyme. The increasing levels of cortisol inactivation by the combined NADP⁺- and NAD⁺-dependent 11ß-DH activities suggest a need to restrict cortisol access to corticosteroid receptors in the final stages of follicle development.

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