11β-Hydroxysteroid dehydrogenase expression and activities in bovine granulosa cells and corpora lutea implicate corticosteroids in bovine ovarian physiology

D R E Abayasekara,L M Thurston,A E Michael

doi:10.1677/joe.1.07025

D R E Abayasekara, L M Thurston + Show 1 more

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https://doi.org/10.1677/joe.1.07025

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Abstract

Cortisol-cortisone metabolism is catalysed by the bi-directional NADP(H)-dependent type 1 11beta-hydroxysteroid dehydrogenase (11betaHSD1) enzyme and the oxidative NAD(+)-dependent type 2 11betaHSD (11betaHSD2). This study related the expression of 11betaHSD1 and 11betaHSD2 enzymes (mRNA and protein) to net 11-ketosteroid reductase and 11beta-dehydrogenase (11beta-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 [(3)H]cortisone or [(3)H]cortisol and pyridine dinucleotide cofactors. Irrespective of follicle diameter, the expression of 11betaHSD2 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 11betaHSD1 exceeded that of 11betaHSD2 and the major enzyme activity was NADP(+)-dependent cortisol oxidation. Increasing follicular diameter was associated with progressive increases in expression and activities of 11betaHSD2 and 11betaHSD1 in follicular and luteal phase granulosa cells respectively. In follicular phase granulosa cells from antral follicles < 12 mm, 11betaHSD1 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, 11betaHSD2 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 11betaHSD2 is the predominant functional 11betaHSD 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, 11betaHSD1 is the major glucocorticoid-metabolising enzyme. The increasing levels of cortisol inactivation by the combined NADP(+)- and NAD(+)-dependent 11beta-DH activities suggest a need to restrict cortisol access to corticosteroid receptors in the final stages of follicle development.

Highlights

The current decline in the fertility of domestic livestock has major financial implications for the farming industry (Lamming et al 1998), resulting in a need to investigate aspects of the reproductive system that could be manipulated to increase productivity
The follicles were flushed with 1:1 Dulbecco’s modified Eagle’s medium (DMEM):Ham’s F12 medium supplemented with penicillin (87 000 IU/l) and streptomycin (87 mg/l), and granulosa cells were extracted by lightly scraping the superficial interior surface of the follicle wall with a sterile plastic inoculation loop taking care not to rupture the basement membrane. (Culture media and supplements were purchased from Invitrogen Life Technologies.) Media containing these granulosa cells was subsequently aspirated from the follicle
Previous studies of 11b-hydroxysteroid dehydrogenase (11bHSD) in the bovine ovary have been confined to studies of mRNA expression (Tetsuka et al 2003)

Summary

Introduction

The current decline in the fertility of domestic livestock has major financial implications for the farming industry (Lamming et al 1998), resulting in a need to investigate aspects of the reproductive system that could be manipulated to increase productivity. It has been recognised that increased output of the glucocorticoid cortisol from the cortex of the adrenal gland is associated with impaired gonadal function and decreased fertility. This endocrine interaction between the adrenal glands and the gonads is well documented in conditions such as stress-related infertility (Michael & Cooke 1994). It has been proposed that this periovulatory rise in ovarian glucocorticoids may directly influence the oocyte quality and control the inflammatory process of ovulation (Hillier & Tetsuka 1998, Andersen et al 1999).

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