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Development of the adrenal gland and brain of the spiny mouse in relation to dehydroepiandrosterone (dhea) biosynthesis
thesisposted on 31.01.2017, 03:49 by Quinn, Tracey Anne
Appropriate growth of the fetal adrenal glands is important for fetal maturation, as well as to facilitate the synthesis and release of steroid hormones needed for pre- and post-natal survival. The innermost zone of the human fetal adrenal cortex, the fetal zone, is the primary site of growth and steroidogenesis in the developing fetus. This important zone is responsible for the massive quantities of DHEA synthesised and secreted into the fetal circulation after mid-gestation. A smaller definitive zone in the outer human neo-cortex is comprised of a narrow band of cells exhibiting ultrastructural characteristics typical of cellular proliferation. A transitional zone lies between these two zones, and is the primary site of cortisol synthesis in the human fetus. Comparable to the human and non-human primate, the spiny mouse (Acomys cahirinus) has been shown to synthesise cortisol from mid-gestation (Lamers et al 1986), and the overall aim of this thesis was to determine if the spiny mouse adrenal gland is also capable of DHEA synthesis during fetal and postnatal life. Results presented in Chapter 2 show that there is a distinct inner-most zone of the spiny mouse fetal adrenal cortex characterised by cells that show eosinophilia of cytoplasm and large nuclei - phenotypical markers of fetal zone cells. This zone expanded in size from 30-35 days (0.75-0.8) of gestation (term is 39 days). Subsequent involution/regression of this inner zone was evident between 35 and 38 days of gestation, a period also associated with an increase in programmed cell death in the inner zone of the cortex. At the same time, there was increased proliferation in the outermost zone of the fetal adrenal, which also became the dominant zone of the neo-cortex. Together, these results suggest the innermost zone of the spiny mouse fetal adrenal cortex is analogous to that of the ‘fetal zone’ in humans. The aim of Chapter 3 was therefore to determine if the adrenal gland of the spiny mouse synthesized DHEA (in addition to cortisol) from the innermost zone of the adrenal cortex. Synthesis of DHEA by the fetal adrenal gland is important for placental estrogen production, and may also be important for modulating the effects of glucocorticoids on the developing brain. The results presented in Chapter 3 show that the enzymes and accessory proteins needed for DHEA synthesis - cytochrome P450 enzyme 17α-hydroxylase /17,20 lyase (P450c17), cytochrome-b5 (Cytb5), 3β-hydroxysteroid dehydrogenase (3βHSD) – are indeed expressed in the adrenal gland from 30 days gestation, and DHEA, cortisol and aldosterone are present in fetal plasma from this time. Plasma DHEA concentrations increased 4-fold, whereas cortisol concentrations decreased from day 30 of gestation until the day of birth. Explant culture of fetal adrenal tissue showed that DHEA was produced from the precursor steroid pregnenolone, and thus the DHEA in the fetal circulation was likely to be of fetal origin. Clear zonation of the fetal adrenal cortex was evident by 38 days gestation when expression of Cytb5 was present throughout the cortex, and co-expression of P450c17 and Cytb5 occurred in the zona reticularis and fasciculata. 3βHSD was expressed in the cortex from at least 30 days gestation and decreased as term approached, consistent with the fall of cortisol in late gestation in this species. Together, these results show that the spiny mouse adrenal gland, like that of the human fetus, can synthesize and secrete DHEA from at least 0.75 of gestation, and suggest that DHEA may have an important role(s) in placental biosynthesis of estrogens and in modulating the actions of glucocorticoids in the developing brain in this species. Chapter 4 presents data showing that the secretory pattern of DHEA changes markedly in postnatal and adult life in the spiny mouse. In humans, there is an increase in adrenal synthesis of C19 androgens during adolescence, which is characterized by a pre-pubertal, but gonadally independent, rise in circulating concentrations of DHEA, DHEA sulfate (DHEA-S), and androstenedione. This physiological process is known as adrenarche, a complex morphological, biochemical and endocrinological event thought to occur only in humans and old world primates. Conversely, the process of adrenopause is the corresponding diminution in DHEA and DHEA-S concentrations in later life. The mechanisms by which adrenarche and adrenopause are induced and regulated are unknown, however emerging evidence suggests that intra-adrenal events, particularly those concerning P450c17, Cytb5, DHEA sulfo-transferase (SULT2A1) and 3βHSD are likely to be involved. To further examine the nature of postnatal adrenal development in the spiny mouse, and the possible role of the gonads and the regulatory hormones corticotropin releasing hormone (CRH)/adrenocorticotropic hormone (ACTH) on adrenal androgen synthesis, plasma samples and adrenal tissue were taken from animals at key developmental stages of life. Incubation of explant adrenal glands with CRH and ACTH showed that the fetal and neonatal spiny mouse adrenal gland was more sensitive to CRH and ACTH when compared with the adult. There was a significant increase in the synthesis and secretion of DHEA in plasma from 8 to 20 days of age in the spiny mouse, just prior to puberty. During this time, there was increased immuno-expression of P450c17 and Cytb5 expression in the zona reticularis of the adrenal gland, whilst 3βHSD was found predominantly in the zona fasciculata and zona glomerulosa. In addition, Chapter 4 shows the high circulating DHEA levels seen in the spiny mouse remain stable for at least one month in both males and females following gonadectomy. Consistent with human studies, these results suggest that the zona reticularis is largely responsible for the increase in DHEA levels seen from 8- 20 days of age, a period of high adrenal androgen production consistent with the human phenomenon of adrenarche. Previous studies have shown that antenatal stress can disturb the development of the fetal hypothalamic-pituitary-adrenal axis, with long-lasting and permanent implications for steroid synthesis in both pre and post-natal life. Results presented in Chapter 5 describe the effect of a brief maternal exposure to high glucocorticoids (dexamethasone, DEX) at mid- and late-pregnancy on adrenal structure and production of DHEA, cortisol and testosterone in the spiny mouse. Pregnant spiny mice were treated for 60 h with either 125µg/kg DEX or saline delivered subcutaneously from an implanted osmotic minipump from day 20 (0.5 term) or day 30 (0.75 term) of gestation. Protein expression of steroidogenic acute regulatory-protein (StAR), 3βHSD, P450c17, and Cytb5 were determined by immunohistochemistry in adrenal glands at 21-27 weeks of age. DHEA, testosterone, and cortisol were measured by radioimmunoassay. Maternal DEX at 20 days gestation did not affect adrenal structure, but expression of StAR, P450c17, and Cytb5 were significantly reduced in the adult adrenal zona reticularis (ZR), with greater change in male compared to female offspring (p<0.05). Plasma DHEA was significantly decreased in male offspring from DEX-treated (6.84±1.24 ng/ml) vs saline-treated (13±0.06 ng/ml; p=0.01) dams, and the DHEA:cortisol ratio was significantly lower in only the males (p<0.05). Testosterone levels significantly increased in male offspring from DEX- (266.03±50.75 pg/ml) vs saline- (83.47±32.3pg/ml, p<0.05) treated dams. DEX treatment at 0.75 gestation had no significant effect on adrenal enzyme expression or plasma steroids in either sex at 21+ weeks of adult age. As DHEA has been shown to be important for brain development, it is further possible that DHEA suppression in adult life might also contribute to the neurological pathologies that can arise after illness and stress during pregnancy. During development, DHEA has indeed been shown to have trophic effects on brain growth, and to have anti-glucocorticoid actions that diminish neurotoxicity and oxidative stress. The studies summarised in Chapter 6 determined for the first time in this species, if DHEA is produced de novo in the developing brain of the spiny mouse. Expression of P450c17 and cytochrome-b5 (Cytb5) - the enzyme and accessory protein responsible for the synthesis of DHEA – together with expression of the glucocorticoid receptor (GR), were determined in the fetal brain at 25, 30, and 35 days of gestation, in the neonatal brain on the day of birth, and 80 days old adult brains by immunocytochemistry. Double-label immunofluorescence was used to determine co-localisation in neurons, astrocytes or oligodendrocytes. P450c17 bioactivity was determined using radioimmunoassay of conversion of pregnenolone to DHEA by explants of fetal, neonatal and adult brain. The fetal brain produced significantly more DHEA after 48 h in culture (22.46±2.0 ng/mg) than the adult brain (5.04±2.0 ng/mg; p<0.0001). P450c17 and Cytb5 were found to be expressed together in neurons in the fetus, but were detected together only in oligodendrocytes and associated white matter tracts in the adult brain. GR expression changed from predominantly neurons in the fetal brain, to white matter tracts and oligodendrocytes in the adult brain. These results showed that the spiny mouse brain can indeed produce DHEA from pregnenolone in a time-dependant manner, and coupled with the identification of P450c17 and Cytb5 protein in several regions of the brain, support the idea that DHEA is an endogenous neuro-active steroid in this species. Together, the studies outlined in this thesis indicate that the androgen DHEA is an important hormone of adrenal and CNS origin in the fetal and postnatal spiny mouse.