Rates Of Steroidogenesis Research Articles

SCAP/SREBP pathway is required for the full steroidogenic response to cyclic AMP

Luteinizing hormone (LH) stimulates steroidogenesis largely through a surge in cyclic AMP (cAMP). Steroidogenic rates are also critically dependent on the availability of cholesterol at mitochondrial sites of synthesis. This cholesterol is provided by cellular uptake of lipoproteins, mobilization of intracellular lipid, and de novo synthesis. Whether and how these pathways are coordinated by cAMP are poorly understood. Recent phosphoproteomic analyses of cAMP-dependent phosphorylation sites in MA10 Leydig cells suggested that cAMP regulates multiple steps in these processes, including activation of the SCAP/SREBP pathway. SCAP [sterol-regulatory element-binding protein (SREBP) cleavage-activating protein] acts as a cholesterol sensor responsible for regulating intracellular cholesterol balance. Its role in cAMP-mediated control of steroidogenesis has not been explored. We used two CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR associated protein 9) knockout approaches to test the role of SCAP in steroidogenesis. Our results demonstrate that SCAP is required for progesterone production induced by concurrent inhibition of the cAMP phosphodiesterases PDE4 and PDE8. These inhibitors increased SCAP phosphorylation, SREBP2 activation, and subsequent expression of cholesterol biosynthetic genes, whereas SCAP deficiency largely prevented these effects. Reexpression of SCAP in SCAP-deficient cells restored SREBP2 protein expression and partially restored steroidogenic responses, confirming the requirement of SCAP-SREBP2 in steroidogenesis. Inhibitors of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase and isoprenylation attenuated, whereas exogenously provided cholesterol augmented, PDE inhibitor-induced steroidogenesis, suggesting that the cholesterol substrate needed for steroidogenesis is provided by both de novo synthesis and isoprenylation-dependent mechanisms. Overall, these results demonstrate a novel role for LH/cAMP in SCAP/SREBP activation and subsequent regulation of steroidogenesis.

Effects of food restriction on steroidogenesis in dispersed adrenocortical cells from Yarrow’s Spiny Lizard (Sceloporus jarrovii)

Changes in energy balance can lead to functional alterations at all levels of the hypothalamic–pituitary–adrenal (HPA) axis. However, relatively little is known about how energy balance affects functional properties of adrenocortical cells themselves. We investigated effects of restricted food intake on sensitivity to ACTH and rates of steroidogenesis in adrenocortical cells isolated from growing female and male Yarrow’s Spiny Lizards (Sceloporus jarrovii). At the end of the feeding regimen, we assayed acute (3h) progesterone (P4), corticosterone (B), and aldosterone (ALDO) production in response to ACTH in dispersed adrenocortical cells. Food restriction depressed growth rate by about 50% in both males and females but did not alter baseline plasma B measured at 10weeks in either sex. At the cellular level, food restriction had the following effects: (1) increased basal B production in both sexes and basal ALDO production in males, (2) increased net maximal rates of production of P4, B, and ALDO in response to ACTH, and (3) no overall effect on adrenocortical cellular sensitivity to ACTH. There were modest sex differences: overall rates of P4 production were 46% greater in cells from females than from males, and in response to food restriction, the net maximal rate of ALDO production was 50% greater in cells from males than from females. Our results demonstrate that food restriction in S. jarrovii increases adrenocortical cellular rates of steroid production without affecting overall cellular sensitivity to ACTH.

Altered bioavailability of testosterone in androgen-binding protein-transgenic mice

Serum and intra-testicular total and free testosterone levels in different age groups of mice (7–360-day-old) were analyzed by radioimmunoassay (RIA) in age-matched wild type (WT)-control and in transgenic mice homozygous to rat androgen-binding protein (ABP-TG), in order to identify possible causes of increased pre-pubertal germ cell apoptosis, spermatogenetic defect and reduced fertility seen in ABP-TG mice. Total intra-testicular testosterone levels in the pre-pubertal ABP-TG (7, 14, 21 and 30-day-old) mice were significantly lower than those in age-matched WT-controls. After puberty (60 days and older) the total intra-testicular testosterone levels were higher than those in age-matched WT-controls and increased gradually, peaking on day 180. Serum total testosterone levels in ABP-TG mice did not differ from those in WT-control until day 30. However, a significant increase in the level of serum total testosterone was observed from day 60. Serum and intra-testicular free testosterone levels were significantly lower in 30, 120, 180 and 360-day-old ABP-TG mice than in age-matched WT-controls. Immunohistochemistry for the cholesterol side-chain cleavage (cytochrome P450) enzyme and quantitative real-time RT-PCR analysis of mRNAs for androgen receptor and for enzymes related to steroidogenesis did not show any changes in 30-day-old ABP-TG mice, indicating that the rates of steroidogenesis and utilization were not altered. Human chorionic gonadotrophin (hCG) administration to adult ABP-TG mice increased the intra-testicular total and free testosterone as well as total germ cell counts. We conclude that the presence of greater than physiological concentration of ABP in the mouse testis alters the ratio of free/bound testosterone, and thereby decreases the availability of free testosterone. As a result, a heightened wave of germ cell apoptosis during the pre-pubertal period followed by a reduction in germ cell numbers and reduced fertility is seen in these mice.

Facilitative Interactions between Estradiol and Luteinizing Hormone in the Regulation of Progesterone Production by Cultured Swine Granulosa Cells: Relation to Cellular Cholesterol Metabolism*

Well differentiated swine granulosa cells in monolayer culture were employed to investigate the mechanisms by which estradiol amplifies the stimulatory actions of LH in the later stages of follicular maturation. The facilitative interaction between estradiol and LH could not be attributed to altered rates of catabolism of progesterone to 20 alpha-hydroxypregn-4-en-3-one. Moreover, estradiol, LH, and estradiol combined with LH clearly stimulated pregnenolone production, measured in the presence of trilostane, to inhibit 3 beta-hydroxysteroid dehydrogenase-delta-5-4-isomerase activity. Thus, at least one component of the synergism between estradiol and LH must reside at or proximal to the cholesterol side-chain cleavage system. In the absence of lipoproteins, the magnitude of the synergism between estradiol and LH was significantly reduced. However, the facilitative interaction between estradiol and LH could still be observed in lipoprotein-deficient and serum-free medium and after the administration of ML-236B to suppress the de novo biosynthesis of cholesterol. In contrast, estradiol alone and LH alone significantly augmented progesterone production in the presence of the oxygenated sterol 5-cholesten-3 beta,25-diol, which can serve as an effective substrate for cholesterol side-chain cleavage. In addition, in the presence of 5-cholesten-3 beta,25-diol, the magnitude of the synergism between estradiol and LH was increased markedly. Thus, the present studies demonstrate that estradiol and LH significantly stimulate progesterone production, at least in part, by augmenting pregnenolone biosynthesis, but not by inhibiting progesterone catabolism. Studies with 5-cholesten-3 beta-25-diol further suggest that estradiol and LH increase cholesterol side-chain cleavage activity. These observations delineate important mechanisms by which estradiol and LH prepare well differentiated granulosa cells for the high rates of steroidogenesis that are ultimately required in the corpus luteum.