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Title: The regulation of steroidogenic enzymes in rat adrenal gland
Author: Lo, Yi-Chen
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2003
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Functional zonation of the rat adrenal cortex may be defined by the expression of the mitochondrial enzymes aldosterone synthase (CYP11B2) and 11 (3-hydroxylase (CYP11B1) that regulate the secretion of the steroids aldosterone in the zona glomerulosa (ZG) and corticosterone in the zona fasciculata (ZF), respectively. Synthesis of these steroids is also dependent on steroidogenic acute regulatory (StAR) protein which limits the transport of cholesterol into the mitochondria. The main aims of this thesis were to study the distribution and expression of CYP11B2 and CYP11B1 in the adrenal cortex of Wistar adult female rats, pregnant rats, their fetuses and offspring, after maternal manipulation of (i) the renin-angiotensin system (RAS) by changing dietary sodium, and (ii) the hypothalamo-pituitary-adrenal (HPA) axis by chronic administration of dexamethasone (DEX). Thirdly, an antibody against a bovine StAR peptide sequence was characterised for future studies on StAR protein expression in the rat adrenal paradigms. Immunohistochemistiy, immunoblotting and competitive reverse transcriptase-polymerase chain reaction (cRT-PCR) methods were developed to examine protein and mRNA expression for CYP11B2 and CYP11B1. Radioimmunoassays were used for plasma steroid levels. In the first experimental series, fetal adrenocortical functions were compared at embryonic day 20 (E20) after feeding high (HS; 3% Na), normal (NS; 0.3%) and low (LS; 0.03%) sodium diets throughout pregnancy. The effects of dietary sodium on fetal adrenal histology were compared with those of adult adrenals of non-pregnant female and pregnant rats. As expected, compared with tissues of rats fed a NS-diet, LS-diet caused significant increases of ZG width in non-pregnant and pregnant rats whereas HS-diet markedly reduced ZG width in both non-pregnant and pregnant rats. Cell hypertrophy was seen in the ZG of the adrenals from both the non-pregnant and pregnant LS-treated rats. In pregnant rats, the adrenal CYP11B2 level was 2.8-fold higher in LS-treated rats and 50% lower in HS-treated rats compared with NS-treated rats. Similarly, levels of CYP11B2 mRNA were significantly increased and decreased by LS- and HS-treatment, respectively. A HS-diet also significantly reduced the ZG staining for CYP11B2. There were no effects of sodium diets on either adrenal CYP11B1 protein or mRNA expression in pregnant rats. In fetal rats, plasma aldosterone levels were significant increased in the LS- compared to those in the NS- and the HS-groups (LS; 1005.0 ± 139.6 pmol/L, NS; 436.0 ± 46.4 pmol/L, HS; 520.9 ± 69.5 pmol/L). The staining intensity of CYP11B2 was markedly increased (P < 0.005) in the LS- compared to the NS- and HS-groups. However, there were no significant differences of protein or mRNA expression of CYP11B2 among the groups and no differences of any measured variable between NS- and HS-groups. Plasma corticosterone levels and the protein and mRNA expression levels of the fetal adrenal CYP11B1 were not affected by maternal sodium intake. These results suggest that dietary sodium restriction may activate the RAS to induce the expression of CYP11B2 in fetal adrenals but that responses of protein and mRNA are muted compared with changes in maternal adrenal expression. These modest fetal effects are compatible with published data suggesting that aldosterone biosythesis is initiated only in the final few days of gestation. In the second experimental series, chronic daily injections of DEX (100 pg/kg body weight/day) throughout gestation significantly reduced the protein and mRNA expression of the CYP11B1 and caused modest increases in CYP11B2 mRNA and protein in adrenals of DEX-treated pregnant rats. Regulation of protein and mRNA levels of CYP11B2 and CYP11B1 in these E20 adrenals appeared to be similar to those in maternal adrenals. These results suggest that DEX readily crosses the placenta and down-regulates fetal HPA axis activity, and are compatible with previous in vitro studies demonstrating that fetal adrenal CYP11B1 activity is regulated from El5. Intrauterine DEX-treated rats exhibited fetal growth retardation with differential effects on heart, kidney and adrenal weights. Some of these effects persisted after birth when DEX-treatment had been stopped. Postnatal body weights continued to be lower at age 7-, 28-day and 4-month-old whereas heart weight was proportionately greater after DEX-treatment in utero, particularly at days 7 and 28. In addition to CYP11B1 and CYP11B2, transcripts of CYP11B3 a third gene encoding an 11 P-hydroxylase enzyme were found in 7- and 28-day-old rat adrenals, but not in E20, 4-month-old and pregnant rat adrenals. The relative amount of CYP11B3 mRNA was higher in 28-day-old than in 7-day-old rat adrenals. No obvious effect of the prenatal DEX-treatment on the amount of the CYP11B3 transcripts was observed. Long-term DEX exposure throughout gestation causes significant changes of the expression of the CYP11B1 in the adrenals of pregnant rats. In the adrenals of the 7-, 28-day- and 4-month-old rat offspring, gene and protein expression of CYP11B2 and CYP11B1 was regulated in a different manner from those in E20 and pregnant rats. Intrauterine DEX-treatment slightly increased the CYP11B1 and CYP11B2 mRNA and protein expression, especially in the 4-month-old offspring. The results suggest that maternal DEX-treatment may programme the physiological state of the adult offspring via changes in the activity of the HPA axis or possibly the RAS system. Immunohistochemical studies in adult rat adrenal glands, using a peptide StAR antibody, showed the distribution of StAR protein in the ZG and ZF with less intense staining in the zona reticularis (ZR). In addition, intense immunoreactivity was seen in the adrenal medulla. The specificity of the peptide StAR antibody was examined by immunoblotting. Consistent with other studies, a 30 kDa band, corresponding to StAR, was detected mainly in the total protein homogenate of the adrenal and ovary and the mitochondria of the ZG and ZF. These results suggest that StAR protein may have some other as yet unidentified functions in the adrenal gland, especially in the medulla. Alternatively, the adrenal medulla may contain a protein closely related to StAR, sharing common antigenicity. These preliminary studies emphasise the need for further studies on the role of StAR (and related) proteins in the adrenal gland and in the other endocrine organs in different physiological situations.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available