ABSTRACT

The antigonadotrophic action of a prostaglandin F_2α analogue, cloprostenol, has been investigated in human granulosa cells obtained from cycles stimulated for in-vitro fertilization and induced to secrete luteal quantities of progesterone by culture in serum-supplemented medium. Cells were exposed to conditions which may mimic those occurring in early pregnancy to establish the roles of human chorionic gonadotrophin (hCG) versus LH and that of cyclic AMP (cAMP) in the anti-gonadotrophic action of cloprostenol. When human granulosa cells were cultured in the absence of treatment for 3 days, exposure to cloprostenol had no effect on basal progesterone production but inhibited hCG-stimulated progesterone (60% decrease; P<0·01), hCG-stimulated cAMP (40% decrease; P < 0·05) and the progesterone response to dibutyryl cAMP (dbcAMP; 70% decrease; P < 0·01), suggesting pre- and post-cAMP sites of cloprostenol action. The inhibitory actions of cloprostenol were prevented when the granulosa cells were either continuously exposed to treatment from the start of culture or pre-exposed for 3 days to maximum concentrations of LH, hCG, dbcAMP or 8-bromo-cAMP. We conclude that prior exposure either in vivo or in vitro to LH or hCG prevents the subsequent antigonadotrophic action of cloprostenol via a cAMP-dependent mechanism. Prevention of the antigonadotrophic action of cloprostenol after exposure to hCG may be a mechanism through which CG prevents regression of the corpus luteum in early pregnancy, while the suppressive effect of LH pretreatment may account for the refractory response of the early corpus luteum to cloprostenol following the midcycle LH surge.

Journal of Endocrinology (1991) 131, 319–325

ABSTRACT

Progesterone production by dispersed luteal cells obtained from the marmoset monkey on day 14 after ovulation can be stimulated by both prostaglandin F_2α (PGF_2α) and its structural analogue, cloprostenol. To establish whether these responses can be attributed to cross-reaction with the prostaglandin E₂ (PGE₂) receptor, this study compared the involvement of cyclic adenosine-3′,5′-monophosphate (cAMP) and protein kinase C (PKC) in the luteotrophic responses to PGE₂, PGF_2α and cloprostenol. While all three prostaglandins stimulated similar increases in progesterone production (239·5 ± 7·9% of control; P <0·01), only PGE₂ stimulated a significant increase in cAMP accumulation (373·2 ± 28·4% of control; P <0·01). This study is the first to demonstrate PKC activity in the marmoset ovary. Following down-regulation of PKC with a tumour-promoting phorbol ester, 4β-phorbol 12-myristate 13-acetate (4β-PMA), basal progesterone production was significantly increased (150·9 ± 8·2% of control; P <0·05) and the luteotrophic effects of PGF_2α and cloprostenol were no longer evident, whereas the response to PGE₂ was unaffected. These observations are consistent with the differential involvement of cAMP and PKC in the luteotrophic responses to PGE₂ and PGF_2α/cloprostenol respectively. Hence, we conclude that the luteotrophic actions of prostaglandins E₂ and F_2α on dispersed marmoset luteal cells are mediated via different receptors and signal transduction pathways.

Journal of Endocrinology (1993) 138, 291–298

Cortisol–cortisone metabolism is catalysed by the bi-directional NADP(H)-dependent type 1 11β-hydroxysteroid dehydrogenase (11βHSD1) enzyme and the oxidative NAD⁺-dependent type 2 11βHSD (11βHSD2). This study related the expression of 11βHSD1 and 11βHSD2 enzymes (mRNA and protein) to net 11-ketosteroid reductase and 11β-dehydrogenase (11β-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 11βHSD2 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 11βHSD1 exceeded that of 11βHSD2 and the major enzyme activity was NADP⁺-dependent cortisol oxidation. Increasing follicular diameter was associated with progressive increases in expression and activities of 11βHSD2 and 11βHSD1 in follicular and luteal phase granulosa cells respectively. In follicular phase granulosa cells from antral follicles < 12 mm, 11βHSD1 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, 11βHSD2 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 11βHSD2 is the predominant functional 11βHSD 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, 11βHSD1 is the major glucocorticoid-metabolising enzyme. The increasing levels of cortisol inactivation by the combined NADP⁺- and NAD⁺-dependent 11β-DH activities suggest a need to restrict cortisol access to corticosteroid receptors in the final stages of follicle development.

Abstract

We have previously shown that detectable metabolism of cortisol to cortisone by 11β-hydroxysteroid dehydrogenase (11βHSD) in human granulosa-lutein cells, pooled for each patient from all aspirated ovarian follicles, is associated with failure to conceive by in vitro fertilization and embryo transfer. The aims of the present study were to assess: (1) the variation in the 11β-HSD activities of granulosa-lutein cells obtained from individual follicles in relation to oocyte maturity and (2) whether the 11βHSD activity of pooled granulosa-lutein cells reflects the 11βHSD activities of the individual follicles for a given patient. 11βHSD activities were measured in intact cells in serum-free medium by a radiometric conversion assay (100 nmol/l [ ³H]cortisol to [³H]cortisone). Follicular 11βHSD activities ranged from <10 (undetectable) to 514 pmol/mg protein per 4 h (n=105 follicles from 12 patients) and did not correlate with oocyte maturity. In three separate patients, the follicular 11βHSD activities ranged from <10 to 117 pmol/mg protein per 4 h (n=8 follicles), 19 to 514 pmol/mg per 4 h (n=9) and 60 to 390 pmol/mg per 4 h (n=8). The 11βHSD activities of the corresponding multi-follicular pools of cells were <10, <10 and 44 pmol/mg per 4 h respectively, all of which were significantly lower (P<0·05) than the arithmetic means for the activities in the individual follicles (52, 132 and 215 pmol/mg per 4 h respectively). Likewise, the 11βHSD activities of two independent multi-patient pools of cells were significantly lower than the mean values of the 11βHSD activities of the appropriate individual patients. We conclude that ovarian 11βHSD activity varies between follicles and that co-culture of granulosa-lutein cells with low enzyme activity can suppress the ovarian 11βHSD activity in cells from different follicles (or patients) with high rates of cortisol metabolism. Hence, these data indicate the potential for paracrine inhibition of ovarian 11βHSD activity in human granulosa-lutein cells.

Journal of Endocrinology (1996) 148, 419–425

In luteinizing granulosa cells, prostaglandin E₂ (PGE₂) can exert luteotrophic actions, apparently via the cAMP signalling pathway. In addition to stimulating progesterone synthesis, PGE₂ can also stimulate oxidation of the physiological glucocorticoid, cortisol, to its inactive metabolite, cortisone, by the type 1 11β-hydroxysteroid dehydrogenase (11βHSD1) 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 PGE₂ on steroidogenesis and cortisol metabolism in human granulosa–lutein cells. PGE₂-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 PGE₂). While a selective PTGER1 antagonist, SC19220, could partially inhibit the steroidogenic response to PGE₂ (by 55.9 ± 4.1% at 1000 nM PGE₂), co-treatment with AH6809, a mixed PTGER1/PTGER2 receptor antagonist, completely abolished the stimulation of progesterone synthesis at all tested concentrations of PGE₂ and suppressed the stimulation of cAMP accumulation. Both PGE₂ and butaprost (a preferential PTGER2 receptor agonist) stimulated concentration-dependent increases in cortisol oxidation by 11βHSD1 (by 42.5 ± 3.1 and 40.0 ± 3.0% respectively, at PGE₂ and butaprost concentrations of 1000 nM). Co-treatment with SC19220 enhanced the ability of both PGE₂ and butaprost to stimulate 11βHSD1 activity (by 30.2 ± 0.2 and 30.5 ± 0.6% respectively), whereas co-treatment with AH6809 completely abolished the 11βHSD1 responses to PGE₂ and butaprost. These findings implicate the PTGER2 receptor–cAMP signalling pathway in the stimulation of progesterone production and 11βHSD1 activity by PGE₂ in human granulosa–lutein cells.

The adrenal glands are the primary source of mineralocorticoids, glucocorticoids, and the so-called adrenal androgens. Under physiological conditions, cortisol and adrenal androgen synthesis are controlled primarily by ACTH. Although it is well established that ACTH can stimulate steroidogenesis in the human adrenal gland, the effect of ACTH on overall production of different classes of steroid hormones has not been defined. In this study, we examined the effect of ACTH on the production of 23 steroid hormones in adult adrenal primary cultures and 20 steroids in the adrenal cell line, H295R. Liquid chromatography/tandem mass spectrometry analysis revealed that, in primary adrenal cell cultures, cortisol and corticosterone were the two most abundant steroid hormones produced with or without ACTH treatment (48 h). Cortisol production responded the most to ACTH treatment, with a 64-fold increase. Interestingly, the production of two androgens, androstenedione and 11β-hydroxyandrostenedione (11OHA), that were also produced in large amounts under basal conditions significantly increased after ACTH incubation. In H295R cells, 11-deoxycortisol and androstenedione were the major products under basal conditions. Treatment with forskolin increased the percentage of 11β-hydroxylated products, including cortisol and 11OHA. This study illustrates that adrenal cells respond to ACTH through the secretion of a variety of steroid hormones, thus supporting the role of adrenal cells as a source of both corticosteroids and androgens.

Germline mutations of the multiple endocrine neoplasia type 1 (MEN1) gene cause parathyroid, pancreatic and pituitary tumours in man. MEN1 mutations also cause familial isolated primary hyperparathyroidism (FIHP) and the same MEN1 mutations, in different families, can cause either FIHP or MEN1. This suggests a role for genetic background and modifier genes in altering the expression of a mutation. We investigated the effects of genetic background on the phenotype of embryonic lethality that occurs in a mouse model for MEN1. Men1 ^{+ /−} mice were backcrossed to generate C57BL/6 and 129S6/SvEv incipient congenic strains, and used to obtain homozygous Men1 ^−/− mice. No viable Men1 ^−/− mice were obtained. The analysis of 411 live embryos obtained at 9.5–16.5 days post-coitum (dpc) revealed that significant deviations from the expected Mendelian 1:2:1 genotype ratio were first observed at 12.5 and 14.5 dpc in the 129S6/SvEv and C57BL/6 strains respectively (P<0.05). Moreover, live Men1 ^−/− embryos were absent by 13.5 and 15.5 dpc in the 129S6/SvEv and C57BL/6 strains respectively thereby indicating an earlier lethality by 2 days in the 129S6/SvEv strain (P<0.01). Men1 ^−/− embryos had macroscopic haemorrhages, and histology and optical projection tomography revealed them to have internal haemorrhages, myocardial hypotrophy, pericardial effusion, hepatic abnormalities and neural tube defects. The neural tube defects occurred exclusively in 129S6/SvEv embryos (21 vs 0%, P<0.01). Thus, our findings demonstrate the importance of genetic background in influencing the phenotypes of embryonic lethality and neural tube defects in Men1 ^−/− mice, and implicate a role for genetic modifiers.

Glucagon plays a central role in amino acid (AA) homeostasis. The dog is an established model of glucagon biology, and recently, metabolomic changes in people associated with glucagon infusions have been reported. Glucagon also has effects on the kidney; however, changes in urinary AA concentrations associated with glucagon remain under investigation. Therefore, we aimed to fill these gaps in the canine model by determining the effects of glucagon on the canine plasma metabolome and measuring urine AA concentrations. Employing two constant rate glucagon infusions (CRI) – low-dose (CRI-LO: 3 ng/kg/min) and high-dose (CRI-HI: 50 ng/kg/min) on five research beagles, we monitored interstitial glucose and conducted untargeted liquid chromatography–tandem mass spectrometry (LC-MS/MS) on plasma samples and urine AA concentrations collected pre- and post-infusion. The CRI-HI induced a transient glucose peak (90–120 min), returning near baseline by infusion end, while only the CRI-LO resulted in 372 significantly altered plasma metabolites, primarily reductions (333). Similarly, CRI-HI affected 414 metabolites, with 369 reductions, evidenced by distinct clustering post-infusion via data reduction (PCA and sPLS-DA). CRI-HI notably decreased circulating AA levels, impacting various AA-related and energy-generating metabolic pathways. Urine analysis revealed increased 3-methyl-l-histidine and glutamine, and decreased alanine concentrations post-infusion. These findings demonstrate glucagon’s glucose-independent modulation of the canine plasma metabolome and highlight the dog’s relevance as a translational model for glucagon biology. Understanding these effects contributes to managing dysregulated glucagon conditions and informs treatments impacting glucagon homeostasis.