Background: The local actions of glucocorticoids in the placenta can be modulated by 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) enzymes, which catalyse inter-conversion of cortisol with its inert metabolite, cortisone, and are known to be expressed in the term placenta and decidua. However, the expression and activity of these enzymes have not been well characterised in the first trimester placenta. The aim of this study was to compare 11 beta HSD2 expression and activity in first trimester placental tissue from pregnancies at either relatively low or high risk of developing pre-eclampsia as determined by Doppler ultrasound.Methods: Enzyme expression was assessed by western blot analysis and immunohistochemistry while 11 beta HSD enzyme activities were quantified using radiometric conversion of [3H]-cortisol in the presence of NADP(+) or NAD(+).Results: 11 beta HSD2 was expressed in syncytiotrophoblast of first trimester placenta, and there was no difference in the level of expression of placental 11 beta HSD2 protein between 9 high pre-eclampsia risk and 14 low pre-eclampsia risk pregnancies. NAD(+)-dependent cortisol oxidation was elevated 3-fold in placental tissue from pregnancies at higher risk of pre-eclampsia than in normal pregnancies (50.9 +/- 15.9 versus 18.3 +/- 1.9 pmol cortisone/mg protein.10 min, n = 11 & 12, respectively; P < 0.05).Conclusions: Expression of 11 beta HSD2 is thought to protect the fetus from exposure to maternal cortisol. While other studies have suggested that 11 beta HSD2 is down regulated in term pre-eclamptic placentae, our study suggests that there is increased cortisol inactivation in first trimester placenta prior to week 10 of gestation, from pregnancies at higher risk of developing pre-eclampsia. (C) 2011 Elsevier Ltd. All rights reserved.

To address the potential luteolytic role for prostaglandin F(2 alpha) (PGF(2 alpha)) in the corpus luteum of the common marmoset monkey (Callithrix jacchus), the ability of marmoset luteal cells, maintained in monolayer culture, to produce PGF(2 alpha) was determined in vitro in the presence and absence of human chorionic gonadotrophin (hCG) and other established pharmacological modulators of PGF(2 alpha) synthesis. We also assessed the effects of the PGF(2 alpha) analogue, cloprostenol, on progesterone output from luteal cells isolated in the early luteal phase versus the mid-luteal phase (days 3 and 14 post ovulation, respectively). Cloprostenol had no effect on progesterone output from luteal cells isolated on day 3 of the luteal phase, whereas it significantly inhibited both basal and hCG-stimulated progesterone synthesis by day 14 luteal cells during the culture period 48-72 h (P<0.001). Intra-luteal PGF(2 alpha) concentrations were 5-fold higher in luteal cells isolated in the early luteal phase than in mid-luteal phase cells (16.5+/-3.5 versus 3.5+/-0.6 pmol/10(5) cells). While PGF(2 alpha) production was unaffected by hCG in vitro, it was decreased by indomethacin (1000 ng/ml) (P<0.05) and stimulated by the calcium ionophore A23187 (10 micromol/l) (P<0.05) in luteal cells from both stages of the luteal phase. Phospholipase A(2) did not influence PGF(2 alpha) production by day 3 luteal cells whereas at 10 IU/ml, it significantly stimulated PGF(2 alpha) production by day 14 luteal cells (P<0.05). Hence, the timing of luteolysis in the common marmoset monkey appears to involve changes in both the luteal cell response to and production of PGF(2 alpha).

In luteinizing granulosa cells, prostaglandin E(2) (PGE(2)) can exert luteotrophic actions, apparently via the cAMP signalling pathway. In addition to stimulating progesterone synthesis, PGE(2) can also stimulate oxidation of the physiological glucocorticoid, cortisol, to its inactive metabolite, cortisone, by the type 1 11beta-hydroxysteroid dehydrogenase (11betaHSD1) enzyme in human granulosa-lutein cells. Having previously shown these human ovarian cells to express functional G-protein coupled, E-series prostaglandin (PTGER)1, PTGER2 and PTGER4 receptors, the aim of this study was to delineate the roles of PTGER1 and PTGER2 receptors in mediating the effects of PGE(2) on steroidogenesis and cortisol metabolism in human granulosa-lutein cells. PGE(2)-stimulated concentration-dependent increases in both progesterone production and cAMP accumulation (by 1.9 +/- 0.1- and 18.7 +/- 6.8-fold respectively at 3000 nM PGE(2)). While a selective PTGER1 antagonist, SC19220, could partially inhibit the steroidogenic response to PGE(2) (by 55.9 +/- 4.1% at 1000 nM PGE(2)), co-treatment with AH6809, a mixed PTGER1/PTGER2 receptor antagonist, completely abolished the stimulation of progesterone synthesis at all tested concentrations of PGE(2) and suppressed the stimulation of cAMP accumulation. Both PGE(2) and butaprost (a preferential PTGER2 receptor agonist) stimulated concentration-dependent increases in cortisol oxidation by 11betaHSD1 (by 42.5 +/- 3.1 and 40.0 +/- 3.0% respectively, at PGE(2) and butaprost concentrations of 1000 nM). Co-treatment with SC19220 enhanced the ability of both PGE(2) and butaprost to stimulate 11betaHSD1 activity (by 30.2 +/- 0.2 and 30.5 +/- 0.6% respectively), whereas co-treatment with AH6809 completely abolished the 11betaHSD1 responses to PGE(2) and butaprost. These findings implicate the PTGER2 receptor-cAMP signalling pathway in the stimulation of progesterone production and 11betaHSD1 activity by PGE(2) in human granulosa-lutein cells.

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.

P151 Protein Expression of Cortisol Metabolising Enzymes in the Boar Reproductive Tract V Sharp, AE Michael and LM Thurston Royal Veterinary College, University College London, London, UK The stress hormone cortisol is implicated in a wide range of reproductive processes including semen quality. Cortisol is interconverted with cortisone by isoforms of the enzyme 11beta hydroxysteroid dehydrogenase (11bHSD). Investigations in our laboratory have demonstrated that the boar reproductive tract secretes modulators of 11bHSD activity. We hypothesise that modulation of 11bHSD in the boar tract alters the environment in which the sperm mature, thus modifying sperm quality and the ability to withstand cold temperature storage. The aims of this study were to establish whether 11bHSD1 and 11bHSD2 proteins are expressed in the boar reproductive tract. Boar testis, epididymis, vas deferens, bulbourethral glands and penis were obtained from slaughterhouse material from 7 animals. Tissues were homogenised in a Tris-glycerol-SDS lysis buffer containing a protease inhibitor cocktail. Proteins were resolved by SDS-PAGE and 11bHSD protein expression was assessed using Western blotting. The immunopurified 11bHSD1 antibody recognised a protein band of 32 kDa in porcine liver (positive control) and in boar testis, epididymis and vas deferens, but not in porcine kidney (negative control), bulbourethral glands or penis. Alternatively, the immunopurified 11bHSD2 antibody recognised a protein band of 57 kDa in all reproductive tissues assessed. In conclusion, 11bHSD protein was expressed at several sites in the boar reproductive tract. Cortisol generation by 11bHSD is known to influence both sodium concentration and pH, thus modulation of this protein may result in changes to the luminal environment ultimately affecting sperm quality.

In the placenta, cortisol is inactivated by NADP(+)- and NAD(+)-dependent isoforms of 11beta-hydroxysteroid dehydrogenase (11betaHSD). Decreased placental 11betaHSD activities have been implicated in intrauterine growth restriction (IUGR) and fetal programming of adult diseases. The objective of this study was to investigate whether placental 11betaHSD activities and fetal plasma cortisol: cortisone ratios could be affected by nutritional restriction of ewes (70% maintenance diet) throughout gestation, for specific stages of gestation, or prior to mating. Chronic nutritional restriction from day 26 of gestation onwards decreased NAD(+)-dependent 11betaHSD activities by 52 +/- 4% and 45 +/- 6% on days 90 and 135 of gestation respectively. Although the decreases in enzyme activities were associated with fetal IUGR, the cortisol:cortisone ratio in fetal plasma was unaffected by chronic nutritional restriction throughout pregnancy. Nutritional restriction confined to early (days 26-45), mid- (days 46-90) and late gestation (days 91-135), or the 30 days prior to mating, had no significant effect on NAD(+)-dependent, placental 11betaHSD activities, nor was there evidence of IUGR. However, nutritional restriction at each stage of pregnancy and prior to mating was associated with significant decreases in the fetal plasma cortisol:cortisone ratio (3.2 +/- 0.7 in control fetuses; 1.0 to 1.6 in fetuses carried by nutritionally restricted ewes). We conclude that nutritional restriction of pregnant ewes for more than 45 consecutive days can significantly decrease NAD+-dependent placental 11betaHSD activities in association with IUGR. While the cortisol:cortisone ratio in fetal plasma is sensitive to relatively acute restriction of nutrient intake, even prior to mating, this ratio does not reflect direct ex vivo measurements of placental 11betaHSD activities.

In human granulosa-lutein cells, high-density lipoproteins (HDL) can stimulate progesterone synthesis. The objective of the present study was to establish whether prostaglandins (PGs) participate in the steroidogenic response to HDL. Both HDL and apolipoprotein AI (ApoAI) stimulated concentration-dependent increases in PGE2, cAMP and progesterone accumulation. The minimum concentrations of HDL and ApoAI required to elevate PGE2 production were the same as those required to stimulate cAMP accumulation and progesterone synthesis. Concentrations of PGE2 were elevated within 10 min in cells exposed to HDL and rose progressively over 24 h, whereas cAMP and progesterone were only increased significantly after 24 h of treatment with HDL. Co-treatment with prostaglandin H synthase inhibitors (meclofenamic acid and indomethacin) abolished the cAMP and progesterone responses to both HDL and ApoAI. Hence, the ability of HDL to stimulate progesterone synthesis can be mimicked by ApoAI and appears to involve increased generation of one or more luteotrophic PGs, possibly acting via cAMP.