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

Abstract

Cortisol-cortisone metabolism is catalysed by the bi-directional NADP(H)-dependent type 1 11b-hydroxysteroid dehydrogenase (11bHSD1) enzyme and the oxidative NAD C -dependent type 2 11bHSD (11bHSD2). This study related the expression of 11bHSD1 and 11bHSD2 enzymes (mRNA and protein) to net 11-ketosteroid reductase and 11b-dehydrogenase (11b-DH) activities in bovine follicular granulosa and luteal cells. Granulosa cells were isolated from follicles of !4, 4-8, O8 and O12 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 11bHSD2 and NAD C -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 11bHSD1 exceeded that of 11bHSD2 and the major enzyme activity was NADP C -dependent cortisol oxidation. Increasing follicular diameter was associated with progressive increases in expression and activities of 11bHSD2 and 11bHSD1 in follicular and luteal phase granulosa cells respectively. In follicular phase granulosa cells from antral follicles !12 mm, 11bHSD1 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, 11bHSD2 had a molecular mass of 48 kDa, but in large antral follicles (O8 mm), there was an additional immunoreactive band at 50 kDa. We conclude that 11bHSD2 is the predominant functional 11bHSD 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, 11bHSD1 is the major glucocorticoid-metabolising enzyme. The increasing levels of cortisol inactivation by the combined NADP C -and NAD C -dependent 11b-DH activities suggest a need to restrict cortisol access to corticosteroid receptors in the final stages of follicle development.

Ovarian 11β-Hydroxysteroid Dehydrogenase (11βHSD) Activity Is Suppressed in Women With Anovulatory Polycystic Ovary Syndrome (PCOS): Apparent Role for Ovarian Androgens

“…More importantly, across all 51 FF samples, there was no significant association between follicle diameter and any of the measured steroid concentrations/ratios. The elevated cortisol to cortisone ratio observed in follicles from anovulatory PCOs parallels the decreased oxidation of cortisol to cortisone reported previously for small antral follicles and spontaneous ovarian cysts from bovine and porcine ovaries (12,13), consistent with suppressed inactivation of cortisol by 11␤HSD1 in hyperandrogenic follicular environments.…”

“…After initial findings linking ovarian cortisol metabolism to the clinical outcome of assisted conception cycles (9 -11), we established that the net oxidation of cortisol in follicular granulosa cells increases progressively during the growth of antral ovarian follicles in pig and cow ovaries (12,13). Moreover, the net 11␤-dehydrogenase activity in porcine granulosa cells isolated from spontaneous ovarian cysts is decreased dramatically relative to preovulatory follicles (12).…”

Gene Expression of 11.BETA.-HSD and Glucocorticoid Receptor in the Bovine (Bos taurus) Follicle During Follicular Maturation and Atresia: The Role of Follicular Stimulating Hormone

“…However, the11HSD2 expression in GCs, albeit low, might still play a physiological role in these follicles. It has been shown that bovine GCs express both 11HSD2 mRNA and protein, and NAD(+)-dependent dehydrogenase activity (i.e., 11HSD2 activity) was evident in GCS homogenate prepared from preovulatory follicles [38]. In rat Leydig cells, the gene expression of 11HSD2 was shown to be about 1000-fold lower than that of 11HSD1, yet significant NAD(+)-dependent 11HSD2 dehydrogenase activity was observed [32].…”

Immunohistochemical Demonstration of the Mineralocorticoid Receptor, 11β-Hydroxysteroid Dehydrogenase-1 and −2, and Hexose-6-phosphate Dehydrogenase in Rat Ovary

“…In our present studies, IHC did not detect H6PDH in rat granulosa cells, which would further decrease the availability of NADPH. The resulting decrease in local cortisol concentrations was thought to be important in the late follicular phase (Thurston et al 2003a(Thurston et al ,2007). An endogenous inhibitor of 11b-HSD reductase activity produced by granulosalutein cells has been proposed (Thurston et al 2003b), and inappropriate production of this inhibitor has been postulated to be the cause of infertility in some women (Michael et al 1996).…”