The human fetal adrenal gland and the influence of maternal smoking

Johnston, Zoë Claire (2018) The human fetal adrenal gland and the influence of maternal smoking. PhD thesis, University of Glasgow.

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The human fetal adrenal cortex is highly active, producing large amounts of Δ5 androgens. Together with the placenta, the fetal adrenal cortex regulates circulating progesterone, estrogen and corticosteroids in the fetus and has a major effect on maternal steroid levels. At birth, production of adequate aldosterone and cortisol are essential for survival, and shortly after birth in the human the cortex undergoes rapid remodelling. Catecholamines, produced by the adrenal medulla are also vital to the body’s ability to respond to stress, and regulate metabolic processes and blood pressure. Normal development of both the cortex and medulla of the human fetal adrenal are therefore critical for post-natal human endocrinology and health. Despite this, adrenal development and function during fetal life is poorly understood due both to the inherent difficulties in obtaining and studying human samples and to the unique nature of human adrenal/endocrine developmental which means animal models are of limited value. While poorly understood, the processes involved in fetal development are highly regulated although they can be disrupted by exposure to environmental chemicals or endocrine disruptors. Exposure to maternal smoking, for example, can disrupt normal fetal programming and has been linked with an altered postnatal stress response in the offspring, as well as a higher risk of developing cardiovascular disease, diabetes, and metabolic syndrome in adult life. These programming effects point to the possible involvement of the human fetal adrenal gland in the post-natal pathologies associated with maternal smoking.
The aims of the studies described here were to develop a method to measure 26 steroids by liquid chromatography mass spectrometry (LC/MS) from samples, from which RNA and protein were also extracted, in order to make optimal use of small amounts of human tissue and use this method, and others, to examine the development and steroidogenic capacity of the human fetal adrenal cortex as well as the migration and development of the pheochromoblast cells which will go on to form the adrenal medulla. The influence of maternal smoke-exposure was also examined in these tissues. Additional aims of a set of in vitro studies were to examine the effects of cigarette smoke extract (CSE) and its components on the steroidogenic capacity of a human pluripotent adrenocortical cell line, as well as the influence of placental steroids and their withdrawal.
109 human fetal adrenals were obtained from elective terminations (REC 04/S0802/21) of second trimester fetuses between 11-21 weeks of gestation. Fetuses were grouped according to sex, gestational age and maternal smoking. Cortical steroids extracted from these adrenals were quantified by LC/MS. Cortical and medulla development was examined by measurement of key elements of the steroidogenic, catecholaminergic, and angiogenic pathways using real-time PCR (RT-qPCR), western blot and immunohistochemistry. Statistical analysis of the data was carried out using generalised linear models with age, sex and maternal smoking status as covariates. For in vitro smoke-exposure experiments, H295R cells were cultured for 3 full days in the presence of cotinine, nicotine, or CSE, and stimulated with forskolin. For examinations of the effects of placental steroids, cells were cultured in the presence of estrogen and/or progesterone for 4 days followed by an additional 4 days of culture without added steroids. Steroids and mRNA transcript levels from cells were measured by ELISA, LC/MS and RT-qPCR. Data was analysed using mixed-effects general linear models.
The most abundant steroid (ng/mg of tissue) in the human fetal adrenal was pregnenolone, followed by dehydroepiandrosterone-sulphate and 17-hydroxyprogesterone and levels of these steroids were similar between male and female fetuses. Cortisol was present in all adrenals examined although aldosterone was undetected. This data suggests adrenal regulation of circulating fetal ACTH remains a factor in development of congenital adrenal hyperplasia during the 2nd trimester while lack of aldosterone probably explains salt-wasting disorders frequently seen in extreme preterm neonates. Fetal plasma levels of ACTH, intra-adrenal levels of progesterone, 17α-hydroxyprogesterone and 16α-hydroxyprogesterone and adrenal transcript levels of the transcription factors GATA-6 and NR5A1 were increased by maternal smoking. However, plasma and intra-adrenal cortisol, and intra-adrenal DHEAS were unaffected.
The enzymes involved in catecholamine biosynthesis were all highly expressed in the developing medulla. Migrating prechromoblast cells were clearly visible in the adrenal fetal zone through H&E staining and could be categorised into noradrenaline- and adrenaline-producing cells by immunohistochemistry for tyrosine hydroxylase and phenylethanolamine-N-methyl transferase. Maternal smoking was also associated with increased levels of PHOX2B transcript, a transcription factor involved in the maturation of chromaffin cells.
In cell culture experiments, levels of CYP11A1, CYP17A1, CYP21A2, HSD3B, PGR and ESR2 transcripts were all significantly reduced in cells exposed to CSE. The effects of CSE-exposure on steroid production was variable but pregnenolone and progesterone production was highly stimulated under basal conditions, indicating that CSE has both cell-stimulatory and cell-inhibitory effects. Estrogen and progesterone exposure also had variable effects on steroid production by the cells, depending on other stimulatory factors, and results indicate that other placental factors, in addition to placental steroids, are likely to play a role in cortical remodelling after birth.
In conclusion, the studies described here have comprehensively examined the steroidogenic capacity of the human fetal adrenal cortex during the second trimester and have begun to characterise the development and migration of the pheochromoblasts. The results described here provide an understanding of normal development of the steroidogenic capacity of the human fetal adrenal during the second trimester, as well as some of the pathologies associated with abnormal adrenal development. The effects of maternal smoking on the processes examined were not marked, however the disruption of steroid production or dysregulation of transcription factors may lead to changes in adrenal function in postnatal life.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Human, fetal, adrenal, maternal smoking, adrenal steroidogenesis, human development, endocrine disruptors.
Subjects: Q Science > QH Natural history > QH301 Biology
Q Science > QH Natural history > QH345 Biochemistry
Q Science > QM Human anatomy
Q Science > QP Physiology
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Biodiversity Animal Health and Comparative Medicine
Funder's Name: Medical Research Council (MRC)
Supervisor's Name: Bellingham, Dr. Michelle and O'Shaughnessy, Professor Peter
Date of Award: 2018
Embargo Date: 28 November 2021
Depositing User: Dr Zoe C Johnston
Unique ID: glathesis:2018-38978
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 06 Dec 2018 09:35
Last Modified: 30 Jan 2019 09:55
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