Glucocorticoids inhibit the transcriptional response of the uncoupling protein-1 gene to adrenergic stimulation in a brown adipose cell line.

Glucocorticoids may inhibit brown adipose tissue (BAT) thermogenesis acting at a central level as well as reducing the responses of the tissue to adrenergic stimulation in vivo. This latter effect is not well understood. We investigated whether or not glucocorticoids directly reduce the expression of the key molecule for BAT thermogenesis, uncoupling protein-1 (UCP1), and if so, to what extent and by what mechanisms. We used HIB-1B brown adipose cells obtained from a hibernoma. The response of UCP1 mRNA to adrenergic stimulation in these cells is qualitatively and quantitatively similar to that seen in vivo. Dexamethasone and other glucocorticoids, given simultaneously with NE, nearly abolish the ensuing UCP1 mRNA accumulation. This effect was negated by the glucocorticoid receptor antagonist RU-486. Significant inhibition is seen within the physiological range of concentrations, with ID(50)s for dexamethasone and corticosterone of 1 and 75 nM, respectively. Within the time span of the experiments, glucocorticoids did not reduce the strength of the NE signal nor did they necessitate ongoing protein synthesis or reduce the stability of mature UCP1 mRNA, but they significantly inhibited the stimulation of transcription by NE in a run-on in vitro transcription assay. These observations indicate that glucocorticoids are powerful inhibitors of the UCP1 gene response to adrenergic stimulation acting at transcriptional level, and provide further evidence for a global inhibitory effect of glucocorticoids on BAT thermogenesis.

Levels of messenger RNA encoding ovarian receptors for FSH and LH in cattle during superovulation with equine chorionic gonadotrophin versus FSH.

This study tested the hypothesis that luteal LH receptor (LHr) and follicular LHr and FSH receptor (FSHr) steady-state mRNA levels are greater during superovulation with equine chorionic gonadotrophin (eCG) compared with that with FSH. Heifers were stimulated with eCG (n = 10) or FSH (n = 10), and ovaries were recovered the day before and at 12 and 24 h after luteolysis was induced with prostaglandin F2 alpha (PGF2 alpha). Total RNA was purified from individual follicles and corpora lutea. Steady-state levels of LHr and FSHr mRNA were assessed by slot blot analysis employing homologous cDNA probes. There were no differences in luteal LHr between FSH- and eCG-stimulated animals before luteolysis, and hybridization signals were detected in only one of six animals by 12 h after injection of PGF2 alpha. After PGF2 alpha injection, steady-state levels of follicular LHr were 4-fold lower (P < 0.05) and follicular FSHr mRNA levels were 2.4-fold lower (P < 0.05) in eCG- compared with FSH-treated cattle. In eCG-treated animals, induction of luteolysis led to a significant increase in follicular LHr mRNA levels (P < 0.01) and a significant decrease in follicular FSHr mRNA levels (P < 0.01). There was no such effect of luteolysis in FSH-treated animals. We conclude that superovulation with eCG, compared with FSH, results in lower follicular levels of LHr and FSHr mRNA but does not affect luteal LHr mRNA levels.

Ovarian follicular steroidogenic acute regulatory protein, low-density lipoprotein receptor, and cytochrome P450 side-chain cleavage messenger ribonucleic acids in cattle undergoing superovulation.

Ovarian hyperstimulation with eCG in cattle results in increased plasma estradiol and progesterone concentrations, whereas hyperstimulation with FSH increases only estradiol concentrations. This study tested the hypothesis that eCG, compared to FSH, increases mRNA abundance for steroidogenic acute regulatory protein (StAR), low-density lipoprotein receptor (LDL-R), and/or cytochrome P450 cholesterol side-chain cleavage (P450scc), the main elements of the progesterone biosynthetic pathway. Heifers were stimulated with eCG (n = 10) or commercial FSH (n = 10), and ovaries were removed by colpotomy the day before and at 12 and 24 h after luteolysis was induced with prostaglandin (PG) F2alpha. RNA was extracted from individual follicles, and relative abundance of StAR, LDL-R, and P450scc mRNA was assessed by slot blots. In ovaries of abattoir origin, StAR mRNA was detected in all follicles and was present in the theca but not the granulosa cell layer, as shown by Northern blotting. Levels of StAR mRNA increased 2-fold (p < 0.05) after PGF2alpha injection in small (< 6 mm) follicles from eCG-treated but not from FSH-treated animals. After PGF2alpha, injection, StAR mRNA levels were 2- to 3-fold higher (p < 0.01) in large (> 9 mm) and medium (6-9 mm) follicles in eCG- compared with FSH-treated heifers. In contrast, P450scc and LDL-R mRNA levels did not consistently differ according to treatments. We show that StAR is expressed in the theca cells of bovine follicles and that stimulation with eCG increases follicular accumulation of StAR mRNA in comparison to stimulation with FSH.

Follicular 3 beta-hydroxysteroid dehydrogenase and cytochromes P450 17 alpha-hydroxylase and aromatase messenger ribonucleic acids in cattle undergoing superovulation.

In this study, we tested the hypothesis that there is altered abundance of transcripts of genes coding for the enzymes cytochrome P450 17 alpha-hydroxylase (P450(17 alpha)), cytochrome P450 aromatase (P450arom), and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in follicles of cattle hyperstimulated with eCG compared to FSH. Treatments were initiated on Day 10 of the cycle, and all cows received prostaglandin (PG) on Day 12. In experiment 1, blood samples were taken to determine plasma progesterone and estradiol concentrations during ovarian stimulation. In experiment 2, both ovaries were removed from stimulated cows by colpotomy before (n = 4 cows/treatment) and at 12 (n = 3/treatment) and 24 h (n = 3/treatment) after PG injection, and from nonstimulated controls (n = 4) 72 h after PG. The preovulatory follicle from nonstimulated heifers, and all follicles greater than 3 mm in diameter from superovulated heifers, were isolated and classified as small (3-5 mm), medium (6-9 mm), or large (> 9 mm). Steady-state levels of RNA for 3 beta-HSD, P450(17 alpha), and P450arom genes were determined by Northern analysis in the individual follicles. In experiment 1, stimulation with eCG significantly (p < 0.01) increased plasma progesterone concentrations compared to FSH-stimulated and nonstimulated controls, and increased (p < 0.05) plasma estradiol concentrations compared to FSH-stimulated controls. Stimulation with FSH did not alter progesterone concentrations, but significantly increased plasma estradiol concentrations compared to those of controls. In experiment 2, the number of large follicles increased significantly with time (p < 0.01), but there were no differences between eCG and FSH treatments in size distribution of follicles (p > 0.05). Relative abundance of P450(17 alpha) message (per 20 micrograms RNA) was significantly higher in large and small follicles (p < 0.05) in eCG-treated compared to FSH-treated heifers after PG injection. Analysis within this period revealed significant treatment effects at 12 h but not 24 h after PG injection. The bovine P450arom cDNA hybridized to 3 transcripts: a 6.5-kilobase (kb) polyadenylated transcript, and non-polyadenylated messages of 3.4 and 1.8 kilobases (kb), all of which hybridized with an oligonucleotide probe specific for the heme-binding region. In medium and small follicles, the 6.5-kb and 3.4-kb transcripts were present in similar quantities, and the 1.8-kb transcript was 25% less abundant. In large follicles recovered after luteolysis, the 3.4 and 1.8-kb transcripts were 3- to 4-fold more abundant in eCG-treated compared with FSH-treated and nonstimulated animals (p < 0.05). There were no significant differences between eCG and FSH treatments on steady-state 3 beta-HSD mRNA levels. Levels of 3 beta-HSD and P450(17 alpha) mRNA in large follicles in hyperstimulated heifers were not different from those in preovulatory follicles in nonstimulated cows. We conclude that hyperstimulation with eCG results in greater stimulation of follicular P450(17 alpha) message abundance compared to hyperstimulation with FSH, and that this may contribute to increased follicular estradiol secretion.

Steroidogenic acute regulatory protein in bovine corpora lutea.

Steroidogenic acute regulatory protein (StAR) transfers cholesterol from the outer to the inner mitochondrial membrane to initiate steroidogenesis. Our purpose was to determine the tissue distribution of StAR mRNA and the occurrence of StAR gene products in the bovine corpus luteum (CL). Tissues were taken from the slaughterhouse or by ovariectomy of cattle at specific times after estrus or ovulation. StAR mRNA was identified by Northern analysis employing a 1.6-kb cDNA mouse StAR probe, and polyclonal antiserum against mouse StAR was used in Western analysis of StAR protein in bovine luteal tissue. The mRNA for cytochrome P450 side-chain cleavage enzyme (P450scc) was also evaluated by means of a homologous cDNA probe. Two isoforms of StAR mRNA, approximately 2.9 and 1.8 kb, were present in bovine CL, adrenal, theca, and granulosa cells and caruncles and cotyledons. One, or sometimes two, protein bands were recognized by the mouse StAR antiserum. P450scc mRNA colocalized in all sites where StAR mRNA was found, and in bovine liver. StAR mRNA was low in developing CL, increased 9- to 15-fold during the mid- to late-luteal phase, and disappeared in CL that had regressed. StAR protein concentrations were highly correlated with StAR mRNA throughout the estrous cycle (r = 0.93, p < 0.05). P450scc mRNA abundance did not vary through the luteal phase except for its disappearance in regressed CL. Corpora lutea from intact animals treated with prostaglandin F2 alpha displayed a 50% decline in StAR mRNA over 12 h while P450scc mRNA remained unchanged. At 24 h StAr mRNA was undetectable, while P450scc mRNA had declined to 50% of pretreatment values. We conclude that StAR mRNA and protein are tightly coupled in the bovine CL, being present at low levels during CL development and in elevated concentrations during the midluteal phase, and disappearing in regressed CL within 24 h of prostaglandin-induced luteolysis. We have further shown, for the first time, that StAR mRNA is present in the mammalian placenta.