Schwarz, Tamar Imogen (2016) The origin of vertebrate steroids in molluscs: uptake, metabolism and depuration studies in the common mussel. PhD thesis. https://theses.gla.ac.uk/7436/ Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Enlighten: Theses https://theses.gla.ac.uk/ [email protected] THE ORIGIN OF VERTEBRATE STEROIDS IN MOLLUSCS: UPTAKE, METABOLISM AND DEPURATION STUDIES IN THE COMMON MUSSEL TAMAR IMOGEN SCHWARZ Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular and Cellular Biology Institute of Molecular, Cell and Systems Biology College of Medical, Veterinary and Life Sciences University of Glasgow In collaboration with the Centre for the Environment, Fisheries and Aquaculture Science (Cefas) April 2016 ©Tamar I. Schwarz, 2016 1 Abstract Many studies have found vertebrate sex steroids, such as testosterone (T), 17 β-oestradiol (E 2) and progesterone (P) to be present in molluscan tissues. The underlying assumption of most, if not all, these studies has been that these steroids are formed endogenously and furthermore, act as reproductive hormones in the same way that they do in vertebrates (i.e. they bind to receptors and induce physiological processes such as egg yolk protein production in females, sex reversal in bivalves and penis formation in neogastropods). However, an in depth evaluation of the literature indicates that the evidence for endogenous formation of vertebrate steroids is rather weak (for example, mollusc genomes do not contain sequences for critical P450 enzymes such as 17-hydroxylase and aromatase nor for functional steroid nuclear receptors). The evidence for the uptake of at least two of these steroids, 17β-oestradiol (E 2) and testosterone (T) from the environment appears, on the other hand, to be very strong and the aim of this thesis was to study this in more detail in common mussels (Mytilus edulis and a mixed population of M. edulis / M ytilus galloprovincialis ). This involved exposing mussels to tritiated E2, T, progesterone (P), 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P), 17α- ethinyl-oestradiol (EE 2) and cortisol (F) and then determining the rate and capacity of uptake, how much was esterified (i.e. conjugated to fatty acids), how much if any was sulphated and finally, how rapidly it was depurated. The study also required novel procedures to be developed for optimising in vivo radiolabel uptake and for separating free (i.e. non- conjugated), esterified and sulphated steroids. It was discovered that all steroids except, intriguingly, F, were rapidly absorbed by mussels from the water. It was discovered that the rate of uptake of radioactive steroids could not be saturated by the addition of non-radioactive steroid (even at 25µg L-1), i.e. the uptake capacity was far higher than amounts found in nature . It was found that the largest proportion (> 70 %) of radioactive E 2 and T in the tissues was present as fatty acid esters. These depurated very slowly (E2 had a half-life of ca. 12 days, while T showed no signs of depletion after 10 days). It was found that the level of esterification of EE 2, P and 17,20β-P was noticeably lower than that of E 2 and T. Following saponification of esterified E 2, a single steroid (E 2 itself) was recovered. 2 However, following saponification of esterified P, two steroids, neither of them P and not yet fully identified, were recovered. Following saponification of T, four steroids were recovered. The least abundant (< 10 %) was T itself and the most abundant was 5α-dihydrotestosterone (47 %). It was discovered that sulphation played an important role in the metabolism of E 2. While none of these results disprove endogenous synthesis of vertebrate steroids, they do call into question those studies that have claimed a cause and effect relationship between vertebrate steroid concentrations in mollusc tissues and factors such as the stage of the reproductive cycle, gender or exposure to presumed ‘endocrine’ disrupters (i.e. it is impossible to determine in any of these studies whether any of the steroids were of endogenous origin). 3 Table of Contents Abstract ............................................................................... 2 List of Tables ......................................................................... 12 List of Figures ........................................................................ 13 Acknowledgement .................................................................. 17 Author’s Declaration ................................................................ 19 Definitions/Abbreviations .......................................................... 20 1 Chapter 1 ....................................................................... 22 General introduction ............................................................... 22 Are vertebrate steroids present in molluscs and, if so, are they of endogenous or exogenous origin? ................................................. 22 1.1 Bivalve Physiology ........................................................ 23 1.2 Evidence of the presence of steroids in molluscs ................... 24 1.2.1 Why is measurement of free steroids inadequate? .............. 24 1.2.2 Is choice of method important? .................................... 25 1.2.3 Evidence for bioaccumulation of steroids ........................ 27 1.2.4 Do steroid levels change with maturation cycles? ............... 28 1.3 Evidence for biosynthesis of vertebrate-like steroids .............. 33 1.3.1 The strength of the evidence for steroid interconversions .... 36 1.3.1.1 Could esterification explain low yields in some biosynthesis experiments? ................................................................ 39 1.3.1.2 Molecular biological evidence to support steroid inter- conversions .................................................................. 40 1.4 Evidence of steroid receptor presence and activity in molluscs .. 44 1.4.1 Do molluscs have steroid receptors? .............................. 45 1.4.2 Are these receptors functional? .................................... 46 1.5 Evidence for biological activity ........................................ 48 1.5.1 Does exposure to oestrogens affect transcription of mER-like?48 1.5.1.1 Does VTG serve as an oestrogen exposure biomarker in molluscs? ..................................................................... 50 1.6 Objectives of this thesis ................................................. 53 2 Chapter 2 ....................................................................... 70 Method Development ............................................................... 70 2.1 Introduction ............................................................... 70 2.2 Materials and methods .................................................. 74 2.2.1 Chemicals ............................................................. 74 2.2.2 Laboratory exposures of Mytilus spp. to radiolabelled 17β- oestradiol ....................................................................... 74 2.2.2.1 Study 1 ............................................................ 74 2.2.2.1.1 Collection and acclimation ................................ 74 4 2.2.2.1.2 Exposure ..................................................... 75 2.2.2.2 Study 2 and 3 .................................................... 75 2.2.2.2.1 Collection and acclimation ................................ 75 2.2.2.2.2 Exposure ..................................................... 76 2.2.2.3 Study 4 ............................................................ 77 2.2.2.3.1 Collection and acclimation ................................ 77 2.2.2.3.2 Exposure ..................................................... 77 2.2.2.4 Study 5 ............................................................ 78 2.2.2.4.1 Collection and acclimation ................................ 78 2.2.2.4.2 Exposure ..................................................... 78 2.2.3 Condition index analysis ............................................ 79 2.2.4 Clearance rates ....................................................... 79 2.2.5 Steroid extraction methods ......................................... 80 2.2.5.1 Water extraction and extract clean-up ...................... 80 2.2.5.2 Tissue extraction ................................................ 80 2.2.5.3 Procedure for separating free, esterified and sulphated steroids in extracts ......................................................... 81 2.2.6 Chromatography ...................................................... 82 2.2.6.1 Normal-phase High Performance Liquid Chromatography . 82 2.2.6.2 Reverse-phase High Performance Liquid Chromatography 82 2.2.7 Thin layer chromatography ......................................... 83 2.2.7.1 Separation of free steroid and ester ......................... 83 2.2.7.2 Separation of free steroid and water soluble metabolites 83 2.2.8 Statistics ............................................................... 84 2.2.9 Overall strategy