Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology THE ESTRADIOL SIGNALLING PATHWAY IN CAPITELLA TELETA THE ESTRADIOL SIGNALLING PATHWAY IN THE MARINE POLYCHAETE, CAPITELLA TELETA ANDREA MICHELLE MURILLO RAMOS, B.Sc., M.Sc. A Thesis Submitted to the School of Graduate Studies in Partial Fulfillment of the Requirements For the Degree Doctor of Philosophy McMaster University © Andrea M. Murillo Ramos, April 2024 Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology ii McMaster University DOCTOR OF PHILOSOPHY (2024) Hamilton, Ontario (Biology) TITLE: The estradiol signalling pathway in the marine polychaete, Capitella teleta AUTHOR: Andrea M. Murillo Ramos, B.Sc., M.Sc. (McMaster University) SUPERVISOR: Dr. Joanna Y. Wilson NUMBER OF PAGES: i-xvi, 1-201 Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology iii Lay abstract Estrogen receptors are present in many metazoan species, but their physiological roles and whether those roles are mediated by estradiol remain elusive. Capitella teleta is a marine polychaete worm and in vitro studies suggest their estrogen receptor is activated by estradiol. This thesis investigated the estradiol signalling pathway in this species and its roles in development, sexual maturation, and behaviour. I found that estrogen receptor is highly expressed in the larval brain and foregut, suggesting a role in development; gene expression patterns do not suggest a role in sexual maturation or reproduction. Pharmacological manipulations demonstrate a clear role in male locomotory behaviour. I discuss possible avenues for future research in both estradiol signalling in Capitella teleta and its role as a model for labile sexual expression. Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology iv Abstract The use of estrogen as an endocrine regulator was thought to be restricted to vertebrates until estrogen receptors were discovered in a wide range of invertebrates, raising intriguing questions about the function of estrogen receptors across metazoans. Capitella teleta is a marine lophotrochozoan used as an indicator species for pollution and an emerging model for regeneration and developmental evolutionary studies. In vitro studies using C. teleta have revealed that their estrogen receptor is ligand-activated, specifically by estrogens and not other steroids. This thesis investigated whether the estradiol signalling pathway was present, and its role in development, sexual maturation, and behaviour. I found that estrogen receptors are present at various life stages and sexes in Capitella teleta; but patterns of expression and estrogen levels do not suggest a role in sexual maturation or reproduction. Based on the expression of receptors in the brain, I developed a locomotory behavioural assay in Capitella teleta and identified that estradiol exposure causes hypoactivity in males but not females. I used RNA-seq to investigate gene expression in juveniles and adult males, females, and hermaphrodites to identify genes that may be important in maturation and reproduction. Male-transitioned hermaphrodites were more transcriptionally similar to females than males. I discuss the advantages of using Capitella teleta as a model for labile sexual expression. Overall, the data in this thesis suggest a role for the estradiol signalling in development and behaviour, but not sexual maturation or reproduction. This thesis explores the estradiol signalling pathway and further advances knowledge of the understudied juvenile and adult stages of the emerging model polychaete Capitella teleta. Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology v Acknowledgements I would like to thank first and foremost my supervisor, Dr. Joanna Wilson. Thank you for your mentorship and your support. I grew so much as a scientist and as a person under your tutelage. Thank you for your patience and the reassurance when I needed it. Thank you also for working with me to find the best ways for me to be a scientist. I appreciate that you don’t think that one size fits all in graduate school and that helped me more than you’ll know. Thank you for sharing my ups and my downs in a way that always made me feel supported. Thanks to all Wilson members, present and past. When I first came to the lab, Joanna, Lana, James, Derek, and Lisa, made me feel so welcome and made it easy for me to create a second family away from home. So many wonderful friendships began for me in this lab, and I will treasure them forever. Thank you to Andrew for being an absolute mad scientist and for pushing me to think outside the box. Thank you for helping my journey as a writer and letting me find my voice. Thank you to Derek for always lending an ear and having unbridled enthusiasm for my project, especially when you asked really hard questions I could not possibly answer. Thank you to Lana for being my paranoid twin in the lab and for always being there for me. Thank you to Mellissa, Jack, and Shemar for always listening and troubleshooting with me. Thank you to Lisa for always being there to help no matter what. Thank you to Oana Birceanu for introducing me to science writing during my Ph.D. and helping me find the next step in my career. Importantly, thank you to the undergraduate students that I had the privilege to mentor as part of Team Capitella. Each one of you contributed so much and this project could not have happened without you. Thank you to my colleagues and friends in the department who made it a safe place for me to learn and grow. Thank you to Team 2023/2024 for the amazing support in writing and for celebrating my highs and helping me through my lows. You motivated me even when I could not motivate myself. Every single friend in the department made especially challenging moments bearable. Special thanks to Katie, Jess, and Zach for motivating me and helping me with code when I desperately needed it, that last chapter was brutal for me and you helped me so much. Rachel, thank you so much for all your indispensable help as well, this thesis could not have been submitted without you. Thank you to my parents, Teresa and Juan, and siblings, Mari and Juan, for all their sacrifices (which were many!!!) and their unrelenting love and faith in me. Their hard work, ethic, love, and kindness are inspiring to me. Thank you to every Murillo and Ramos family member for your love, support, and financial contributions. In particular, my Tia Estela, Tio Cesar, and Tia Margarita. To my favourite human, my grandfather Anibal, thank you for inspiring me always and sharing my love for biology. Thank you to those who couldn’t be here with me to finish the journey, my grandmothers Estela and Edelmira, my grandfather Carlos, and Peter, for always believing in me and knowing I could do it. I dedicate this thesis to the four of you. I love you and rest in peace. To my chosen family, thank you for always believing in me even when I didn’t believe in myself. It takes a village to go through this journey and I could not have asked for a better support system. Paula, Jackie, Rachel, Lindy, Allison, Alex, Nicole, and Alexis, thank you for listening to me and always giving me support from and for inspiring me to be better. Thank you to Mazin for being a true partner with every step of this journey, I know it was hard for us both, but your belief and your love made all the difference. Thanks for always readily listening and providing love and support for me. I love you and will always be thankful to you. Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology vi Table of Contents Title page………….……………………………………………………………….…. i Descriptive note……………………………………………………………………..... ii Lay abstract………….……………………………………………………………....... iii Abstract……………………………………………………………………………….. iv Acknowledgements………………………………………………..………………….. v Table of Contents………..……………………………………………………………. vi List of Figures…………………………………………………………………..…...... ix List of Tables……………………………………….……………………………........ x List of Abbreviations…………………………………………………………….....… xii Declaration of Academic Achievement……………………………..…………...…… xv COVID-19 impacts…………...………………………………………………...…..… xvi 1 Introduction Abstract …………………………………………………………………………... 1 1 Introduction……………………………………………………………...…....... 2 The de novo synthesis of estrogens…………………............................………..... 6 STAR…………………………………………………………………………. 6 CYP11A1………………………………………………………………….… 8 3β-HSD ………………………………..……………….…………..………... CYP17A1…………………………………………………………………..… 17β-HSD………………………………………………………………..….… CYP19………………………………………………………………..………. Estrogen receptors and their specific activation by ligands such as 17β – estradiol…………………………..………………………………..…………….. 10 12 14 15 18 Evolution of the estrogen receptor ….………………….……………….…... Estrogen Receptors in Lophotrochozoa……………………………...........… Endogenous Expression of Estrogen Receptors …………….……………..... 18 21 23 Physiological response to estrogens………….………………………………....... 24 Conclusions…………………………………………………………………......... 27 Thesis Goals……………………………………………………………................ 30 2 Life stage specific expression of the estrogen receptor gene in the polychaete, Capitella teleta …………………………………………………………………… 50 Abstract………………………………………………………………………….... 50 Introduction……………………………………………………………………….. 51 Methods…………………………………………………………………………… 55 Culture Conditions………………………………………………………...... 55 Localization of ER expression across larval stages………………………… 55 Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology vii Collection of larval stages ….…………………………………………. 55 ER probe design and optimization………………….………..………... 56 Colorimetric whole mount in situ hybridization………………………. 56 Microscopy and Scoring……..…………………………………………. 57 Relative ER expression across life stages………………………………….. Collection of whole-body juveniles and adults……………..................... 57 57 Collection of anterior portions and guts of sexually mature adults……... 58 RT-qPCR…………………………………………..…………………...... 58 Statistics………………..…………………………..…………………..... 60 Results…………………………………………………………………………….. 61 Localization of ER expression across larval stages……………………...…. 61 Relative ER expressions across life stages……………………………….. 62 Discussion………………………………………………………………………. 63 References………………………………………………………………………. 75 3 Estrogen receptor activation modulates locomotory behaviour in the marine polychaete, Capitella teleta, in a sex-specific manner………….…..................... 82 Abstract…………………………………………………………………………….. 82 Introduction………………………………………………………………………… 83 Methods……………………………………………………………………………. 88 Culturing Conditions……………………………………………………........ 88 Tissue 17β-estradiol concentrations …………………………..…………….. 88 Chemical Exposures………………………………………………………… Nicotine Exposure…………………...……………………....................... 89 89 17β-estradiol Exposure……..…………….…………...…………….……. 90 Fulvestrant and 17β-estradiol co-incubation exposure …….……….……. 91 Behavioural Assay for Locomotion in Novel Arena ……………..…........ 92 AKR and SDR Gene Identification, Annotation, and Phylogenetic Analyses 93 Statistical Analyses………………………………...………………………... 95 Results…………………………………………………………………………....... 96 Nicotine exposures alter C. teleta locomotion………………………………. 96 17β-estradiol alters locomotory behaviour in male, but not female, C. teleta 96 Fulvestrant co-exposure blocks the effect of estradiol on male C. teleta locomotory behaviour……………………………………………………….. 97 Steroid biosynthetic genes and tissue levels of 17β-estradiol……………….. 98 Discussion……...………………………………………………………………… 100 References…………………...………………………………………………….….. 123 4 Capitella teleta simultaneous hermaphrodites have transcriptionally female signatures. ………………..………………………………………….... 130 Abstract……………………………………………………………………….... 130 Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology viii Introduction………………………………………………………………….…. 131 Methods……………………………..………………………………………..… 134 Culture conditions and sampling…………………………….…………….. 134 RNA extraction and RNA Sequencing…………………………………..... 135 Differential expression analyses and statistics……..……………………… 136 Gene Ontology Analyses……...…………………………………………… 137 Results………………………………………………………………………..…. 138 Principal Component Analysis (PCA)…………………………………….. 138 Enriched GO analyses……………………………………………………. 139 Differentially expressed cluster in juveniles.……………..……....……….. 139 Differentially expressed gene clusters in hermaphrodites……….……….. 140 Genes upregulated in males……………………………………………..... 141 Discussion………………………………………………………………………. 141 References……………………………………………………………………… 175 5 General Discussion……….……………………………………………… 180 References ………………………………………………………………. 189 Appendix……….……………………………………………………… 192 Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology ix List of Figures Introduction Page 1.1 Vertebrate Estrogen Biosynthesis Pathway 33 1.2 Estrogen receptors in bilaterian phylogeny 34 Chapter 2 Page 2.1 The estrogen receptor (ER) of Capitella teleta is expressed throughout larval stages (4-8). 70 2.2 ER mRNA levels across different ages of Capitella teleta juveniles. 71 2.3 ER mRNA levels in whole bodies, anterior head regions, and guts of adult Capitella teleta 72 2.4 Percentage of C. teleta juveniles at different ages post-emergence. 74 Chapter 3 Page 3.1 Experimental design for co-exposure of ER antagonist prior to behavioral assay. 108 3.2 Nicotine exposure modulates C. teleta adult locomotory behaviour. 109 3.3 Estradiol exposure does not impact C. teleta adult female locomotory behaviour. 111 3.4 Exposure to estradiol induced a hypoactive phenotype in C. teleta adult male locomotory behavior. 112 3.5 Co-incubation of 17𝛃 -estradiol and fulvestrant suppressed hypoactive phenotype in C. teleta adult male locomotory behavior. 114 3.6 Phylogeny of metazoan 3𝛃HSD genes. 116 3.7 Phylogeny of metazoan AKR genes. 118 3.8 Phylogeny of metazoan 17𝛃HSD. 120 3.9 17𝛃 -estradiol levels are similar across different (A) ages of juveniles and (B) and age and sex of adult Capitella teleta. 121 Chapter 4 Page 4.1 Principal component analysis (PCA) plots for the top (A) 500 and (B) 5,487 differentially expressed Capitella teleta show unique clustering by group. 154 4.2 Heat map schematic of five clusters of differentially expressed genes. 156 4.3 Heat map of genes upregulated in 2-week-old Capitella teleta juveniles compared to 12-week-old females, males, and hermaphrodites (Cluster 1). 157 4.4 Farnesyl pyrophosphate synthase (FPPS) counts for juveniles, females, hermaphrodites, and males. 159 Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology x 4.5 Farnesyl pyrophosphatase (FP; ortholog 1) counts for juveniles, females, hermaphrodites, and males. 160 4.6 Farnesyl pyrophosphatase (FP; ortholog 2) counts for Capitella teleta juveniles, females, hermaphrodites, and males. 161 4.7 Farnesol oxidase/dehydrogenase (SDR11) counts for Capitella teleta juveniles, females, hermaphrodites, and males. 163 4.8 Aldehyde dehydrogenase 3 (ALDH3) counts for Capitella teleta juveniles, females, hermaphrodites, and males. 165 4.9 Putative methyltransferase counts for Capitella teleta juveniles, females, hermaphrodites, and males. 166 4.10 Heat map of genes downregulated in 2-week-old Capitella teleta juveniles compared to 12-week-old females, males, and hermaphrodites (Cluster 3) 167 4.11 Heat map of genes upregulated in 12-week-old Capitella teleta males and hermaphrodites compared to 2-week-old juveniles and 12-week-old females (Cluster 2). 169 4.12 Heat map of genes upregulated in 12-week-old Capitella teleta females and hermaphrodites compared to 2-week-old juveniles and 12-week-old males (Cluster 5). 170 4.13 Heat map of genes upregulated in 12-week-old Capitella teleta males compared to 2-week-old juveniles and 12-week-old females and hermaphrodites (Cluster 4). 173 S1 Bioanalyzer 2100 outputs for Capitella teleta samples (D1-D4) used in current transcriptomic studies. 174 S2 Bioanalyzer 2100 outputs for Capitella teleta samples (8 replicates) used in current transcriptomic studies 175 List of Tables Chapter 1 Page 1.1 Evidence for the presence and function of steroidogenic enzymes in polychaetes, rotifers, and platyhelminthes. 32 Chapter 2 Page 2.1 Primer sequences, annealing temperature, amplicon size, and primer efficiency for the estrogen receptor (ER), elongation factor 1 alpha, (elfa) and 18s RNA (18s) genes used for RT-qPCR analysis. 69 Chapter 4 Page 4.1 Cluster 1 Gene Ontology (GO) analyses for enriched GO terms for biological process (BP), molecular function (MF), and cellular component (CC) ontologies. 150 Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology xi 4.2 Cluster 3 Gene Ontology (GO) analysis for enriched GO terms for biological process (BP), molecular function (MF), and cellular component (CC) ontologies. 151 4.3 Cluster 2 and Cluster 5 Gene Ontology (GO) Analysis for enriched GO terms for biological process (BP), molecular function (MF), and cellular component (CC) ontologies. 153 4.4 Cluster 4 Gene Ontology (GO) Analysis for enriched GO terms for biological process (BP), molecular function (MF), and cellular component (CC) ontologies. 154 Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology xii List of Abbreviations AncSR1 – ancestral steroid receptor AKR – aldoketo reductase ALDH3 – aldehyde dehydrogenase AMI – arylideneimidazolidinone anti-digoxigenin-AP – anti-digoxigenin-alkaline phosphatase ANOVA – analysis of variance AR– androgen receptor BLASTP – search protein databases using protein query BMP – Bone morphogenetic protein BP – Biological processes CC – cellular component cDNA – complementary DNA CAS – Chemical Abstract Service CYP – cytochrome DBD – DNA binding domain DEG – Differentially Expressed Genes DHEA– Dehydroepiandrosterone DIC – Differential Interference Contrast DMSO – dimethyl sulfoxide E2 – 17β-estradiol EBI – European Bioinformatics Institute EE2 – 17α-ethinylestradiol EC50 – half maximal effective concentration ELISA – enzyme-linked immunosorbent assay ER – estrogen receptor ERE – estrogen-responsive element ERR – estrogen-related receptor EST – expressed sequence tag Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology xiii ExPASy – Expert Protein Analysis System FP– farnesyl pyrophosphate FPPS – farnesyl pyrophosphate synthase GABA – Gamma Amino Butyric Acid GO – Gene Ontology GPR30 – E2-activated G-protein coupled receptors GNRH – gonadotropin-releasing hormone GR – glucocorticoid receptor HSD – hydroxysteroid dehydrogenase JGI – Joint Genome Institute IC50– half-maximal inhibitory concentration Kd – dissociation constant LBD – ligand-binding domain LC-MS/MS-ESI – Liquid chromatography-mass spectrometry/ mass spectrometry- electron spray ionization LRT – likelihood ratio test MAPK – Mitogen-activated protein kinase MCMC – Markov Chain Monte Carlo MF – Methyl farnesoate MF – Molecular Function MgCl2 – Magnesium chloride MPC – Multipotent Progenitor Cells ML– Maximum Likelihood MR– mineralocorticoid receptor mRNA– messenger RNA NBT/BCIP – nitroblue tetrazolium / 5-bromo-4-chloro-3-indolyl-1-phosphate NCBI – National Center for Biotechnology Information NR– nuclear receptor PBS– phosphate-buffered saline Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology xiv PBT– phosphate-buffered saline with Triton-X PC– principal component PCA– principal component analysis PCR– polymerase chain reaction PFA– paraformaldehyde PR– progesterone receptor PGC– primordial germ cells RIA – radioimmunoassays RIN – RNA Integrity Number rRNA – ribosomal RNA RT-qPCR – quantitative reverse transcription polymerase chain reaction SDR – short-chain dehydrogenase SDR11 – farnesol oxidase/dehydrogenase SEM – standard error of the mean STAR – steroid acute regulatory protein TNPO2 – Transportin-2 tRNA – transfer RNA tBLASTn – Search translated nucleotide databases using a protein query VMHvl – ventrolateral area of the ventromedial hypothalamus Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology xv Declaration of Academic Achievement This thesis is organized in a sandwich format and has 5 chapters. Chapter 1 is an introduction to the estrogen signalling pathway in lophotrochozoan and discusses the concordant and discordant data for an estrogen signalling pathway in this area of the tree of life. Chapter 2 to 4 are written as manuscripts that are ready to be submitted into peer- reviewed journals. Last, Chapter 5 is a general discussion of the major findings in this thesis. I wrote all 5 chapters of this thesis and received editorial feedback from Dr. Joanna Wilson and Dr. Andrew Thompson. Chapter 1 of this thesis (sections 1-4 only) has been accepted to General and Comparative Endocrinology. Manuscript number= GCE-D-24-00056. DOI= https://doi.org/10.1016/j.ygcen.2024.114519 Authors: Murillo, A.M.1, Wilson, J.Y.1 Chapter 2: Authors: Murillo, A.M.1, Lanza, A.R.2, Hendershot, M.1, Seaver, E.C.2, Wilson, J.Y.1 I planned and conducted most of the study and wrote the manuscript under the supervision of J.Y.W. A.R.L. and E.C.S. supported the optimization and experimental design of the in-situ hybridizations in larval stages. A.R.L. trained me on the technique and independently assessed some of the in-situ slides to determine that the scoring was reliable. I carried out the in-situ hybridizations in E.C.S.’s laboratory. M.H. sampled the gastrointestinal tract samples of adult male and female worms, extracted RNA and determined gene expression in this tissue under the supervision of A.M. and J.Y.W. Chapter 3: Authors: Murillo, A.M.1, Thompson, W.A.1, Fasih, B.1, Liu, A.1, Laframboise, L. 1, Wilson, J.Y.1 I planned and conducted most of the study and wrote the manuscript under the supervision of J.Y.W. The behavioural assay was designed with W.A.T. W.A.T. and L.L. helped with the data collection for the behavioural assay. W.A.T. blindly assessed and performed statistics on the behavioural assay. B.F. and A.L. carried out HSD annotations under the supervision of J.Y.W. B.F and J.Y.W. completed the phylogenetic trees for the HSD families. Chapter 4: Authors: Murillo, A.M.1, Wilson, J.Y.1 I planned and conducted all of the study and wrote the manuscript under the supervision of J.Y.W. Affiliations: 1 Department of Biology, McMaster University, 1280 Main St. West, Hamilton, ON, L8S 4K1, Canada 2 Whitney Laboratory for Marine Bioscience, University of Florida, 9505 Ocean Shore Blvd., St. Augustine, FL 32080, USA https://doi.org/10.1016/j.ygcen.2024.114519 Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology xvi COVID-19 Impacts During my PhD, my project was deeply impacted by the COVID-19 pandemic. I had to stop an experiment that was 6 months in process for the shutdown. I had 4 months of no data collection during the shutdown, and I had to cut down the number of animals in my colony to ensure that I could properly maintain the colony during the shutdown, when I could only go in twice a week for maintenance of the colony. Capitella teleta is not a species that can be frozen and resumed in work so during the shutdown, I was only allowed to maintain a small colony to limit the work hours each week. When Phase 1 of reopening occurred, the focus was on increasing colony size, however, this species takes about 2 months to reach sexual maturity, so the increase in colony size took some time to get back to a size to support experiments. Furthermore, my time points chosen take a long time to collect 2-12 weeks. At the height of the pandemic, it was also hard to get consumables needed for my first data chapter, due to its molecular biology nature. My last chapter completely changed due to the nature of shutting down the experiment, so I had to learn bioinformatics and coding skills to do my Chapter 3. Though there was power in the sampling occurring in the way it did during COVID, it added significant time to my degree overall. Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 1 Chapter 1: Is there potential for estradiol receptor signalling in lophotrochozoans? Murillo, A.M.1, Wilson, J.Y.1 1 Department of Biology, McMaster University, 1280 Main St. West, Hamilton, ON, L8S 4K1, Canada Abstract Estrogen receptors (ERs) are thought to be the ancestor of all steroid receptors and are present in most lophotrochozoans studied to date, including molluscs, annelids, and rotifers. A number of studies have investigated the functional role of estrogen receptors in invertebrate species, although most are in molluscs, where the receptor is constitutively active. In vitro experiments provided evidence for ligand-activated estrogen receptors in annelids, raising important questions about the role of estrogen signalling in lophotrochozoan lineages. Here, we review the concordant and discordant evidence of estradiol receptor signalling in lophotrochozoans, with a focus on annelids and rotifers. We explore the de novo synthesis of estrogens, the evolution and expression of estrogen receptors, and physiological responses to activation of estrogen receptors in the lophotrochozoan phyla Annelida and Rotifera. Key data are missing to determine if de novo biosynthesis of estradiol in non-molluscan lophotrochozoans is likely. For example, an ortholog for the CYP11 gene is present, but confirmation of substrate conversion and measured tissue products is lacking. Orthologs CYP17 and CYP19 are lacking, yet intermediates or products (e.g. estradiol) in tissues have been measured. Estrogen receptors are present in multiple species, and for a limited number, in vitro data show agonist binding of estradiol and/or transcriptional activation. The expression patterns of Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 2 the lophotrochozoan ERs suggest developmental, reproductive, and gastrointestinal roles but are highly species dependent. E2 exposures suggest that lophotrochozoan ERs may play a role in reproduction, but no strong dose-response relationship has been established. Therefore, we expect most lophotrochozoan species, outside of perhaps platyhelminths, to have an ER but their physiological role remains elusive. Mining genomes for orthologs gene families responsible for steroidogenesis, coupled with in vitro and in vivo studies of the steroid pathway are needed to better assess whether lophotrochozoans are capable of estradiol biosynthesis. One major challenge is that much of the data are divided across a diversity of species. We propose that the polychaetes Capitella teleta or Platynereis dumerilii, and rotifer Brachionus manjavacas may be strong species choices for studies of estrogen receptor signalling, because of available genomic data, established laboratory culture techniques, and gene knockout potential. Introduction Steroid hormones and their receptors play important physiological roles in biological pathways that control reproduction, development, and immune responses (Baker, 2019). The steroid hormones with established roles in vertebrate reproduction include progestins (e.g., progesterone), androgens (e.g., testosterone), and estrogens (e.g., 17β-estradiol). Cholesterol is the initial backbone molecule for the vertebrate steroid biosynthesis pathway; a pathway executed by enzymes from the cytochrome P450 (CYP11A, CYP17, CYP19), short-chain dehydrogenase (3β-hydroxy steroid dehydrogenase; HSD), and aldo-keto reductase (17β-HSD) enzyme superfamilies. The key biochemical step in the production of estrogens in vertebrates is the conversion of Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 3 androgens through aromatase (CYP19). The steroids produced by this pathway act as paracrine signals to bind and activate specific membrane and/or cellular receptors, for which they have high specificity and selectivity (Beato & Klug, 2000). Cellular steroid hormone receptors are transcription factors, which control gene expression (Wierman, 2007). The biosynthetic pathway, critical genes for biosynthesis and effects, and physiological roles of the signalling systems for each steroid hormone are well established in vertebrates, but it is clear that at least some components of this system developed prior to the vertebrate lineage, raising questions about whether these signalling pathways have biological roles in invertebrates. Vertebrate steroid hormone receptors, which are part of the nuclear receptor 3 (NR3) subfamily can be categorized into two groups: the estrogen receptors (ER; NR3A), which in vertebrates are ligand-activated transcription factors that bind to estrogen- responsive element (ERE), and the 3-ketosteroid receptors (NR3C). There are other NR3 groups in metazoans including NR3D, which contain genes from protostomes (Markov & Laudet, 2011; Khalturin et al., 2018). In many phylogenetic analyses, NR3D and NR3A genes are sister clades (Keay & Thornton, 2009; Bridgham et al., 2014; Vogeler et al., 2016; Kaur et al., 2015; Huang et al., 2015; Cheng et al., 2021) and throughout the scientific literature, most studies refer to the NR3D genes as ERs or ER-like. We use ER here to avoid confusion with this literature, although others have recommended using the formal gene nomenclature (Markov & Laudet, 2011). Since it is hypothesized that the ER is the ancestral steroid receptor, it is likely to have the widest taxonomic distribution (Keay & Thornton, 2009). Within the protostome Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 4 clade, there are two superphyla: ecdysozoans (including insects, nematodes, and crustaceans) and lophotrochozoans (also called spiralians, including molluscs, annelids, rotifers, and platyhelminthes; Dunn et al., 2014). The first invertebrate genomes sequenced were ecdysozoans Caenorhabditis elegans and Drosophila melanogaster, and the lack of ERs in these genomes suggested that ERs were absent in protostomes (J. Thornton et al., 2003). Since more taxonomically diverse genome sequencing has provided better coverage of the tree of life, estrogen-like receptors have been reported in some invertebrate taxa. Baker (2008) iteratively searched the genomes of Trichoplax adhaerans (Placozoan, a sister group to bilaterians; Srivastava et al., 2008), and three species of lophotrochozoans. No estrogen receptor was found in Trichoplax, but ERs were found in two of the lophotrochozoans (polychaete Capitella teleta and mollusc Lottia gigantea; Baker, 2008). The superphylum Lophotrochozoa thus represent important phyla to examine the role of estrogen receptors. Molluscs have been perhaps the most studied of the lophotrochozoans, partly because of reports of endocrine disruption associated with altered aromatase activity (e.g., Morcillo et al., 1999). While molluscs have estrogen receptors, these receptors are constitutively active (Bridgham et al., 2014). This raised questions about whether molluscs are capable of estrogen synthesis and the biological role of ER signalling in molluscs. Several reviews have focused on steroid synthesis and signalling pathways in molluscs, arguing that although the ER is present, testosterone, progesterone, and 17β- estradiol signalling is not active (Scott, 2012, 2013, 2018). While estrogen receptors, but not androgen or progesterone receptors, are present, molluscs do not have some orthologs Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 5 for enzymes involved in synthesizing vertebrate steroids from cholesterol, including CYP11A1 and CYP19 (Scott, 2012). Furthermore, enzyme activities have not been found for all steps in the biosynthetic pathway for a single mollusc species (Scott, 2012). While 17β-estradiol has been measured in mollusc tissues, it was argued that it was not endogenously produced (Scott, 2018). Molluscs can uptake steroids from their environment, and tissue estrogens were suggested to reflect environmental uptake rather than de novo synthesis (Scott, 2012). Studies documenting endocrine disruption in molluscs have used environmental compounds (e.g., bisphenol A and nonylphenol), but not 17β-estradiol, to stimulate specific responses in molluscs (Scott, 2013). Although these compounds activate vertebrate ERs, direct activation of ER and responses to 17β-estradiol showing dose- dependent effects are lacking (Scott, 2013). Regardless, the need for the synthesis of 17β- estradiol and the likelihood of a dose-response relationship is low since they have a constitutively active ER with an occluded ligand-binding pocket (Bridgham et al., 2014). Ligand-activated ERs have been reported in other lophotrochozoan phyla, particularly in polychaetes and rotifers. Capitella teleta, a marine polychaete, has an estrogen receptor gene with moderate phylogenetic support for placement with vertebrate and molluscan ERs (Baker, 2008; Keay & Thornton, 2009). In vitro reporter assays of the Capitella ER and an ER from the polychaete Platynereis dumerilii, showed that both receptors could bind 17β-estradiol with high affinity (Keay & Thornton, 2009). These intriguing data suggest that some lophotrochozoan species may have ligand-activated ERs, and thus an estrogen signalling pathway is more likely. The goals of this review are Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 6 to investigate the weight of the evidence for an estrogen signalling pathway in the Lophotrochozoa superphylum, emphasizing annelids and rotifers where possible (and minimizing molluscs which are well explored elsewhere). In particular, we examine: (1) The de novo synthesis of estrogens, based on the measurement of tissue steroids and the presence and function of steroid biosynthesis enzymes. (2) The presence of estrogen receptors and their specific activation by ligands known to activate vertebrate ERs, particularly 17β-estradiol. (3) A physiological response to estrogens, preferably in tissues that express the estrogen receptor or that involve key target genes or responses linked to estrogen signalling. The de novo synthesis of estrogens STAR (steroidogenic acute regulatory protein) The de novo synthesis of estrogen has been well established for vertebrates as a complex biochemical pathway executed by cytochrome P450 and hydroxysteroid dehydrogenase enzymes on a cholesterol backbone (Figure 1.1). The first step of steroid synthesis involves the translocation of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane (Miller & Auchus, 2011) by steroid acute regulatory protein (STAR; Step 1 in Figure 1.1). STAR has a cholesterol-binding domain and facilitates transport; conformational changes at the outer mitochondrial membrane regulate the acceptance and discharge of the cholesterol molecule (Miller & Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 7 Auchus, 2011). STAR translocation of cholesterol is the rate-limiting step for steroid biosynthesis, providing the critical first substrate. Early studies showed a strong relationship between STAR synthesis and steroid hormone biosynthesis using rat corpus luteum cells, mouse Leydig cells, and MA-1- mouse Leydig tumour cells (reviewed in Stocco et al., 2017). STAR protein is encoded by the STARDI gene in mammals and belongs to the StAR-related lipid transfer domain family. STAR proteins are conserved in chicken, Xenopus, and zebrafish species (Bauer et al., 2000), and STAR expression patterns were as expected for a role in steroidogenesis in several non-mammalian vertebrates (Bauer et al., 2000; Teng et al., 2013). STAR protein orthologs have been found in some (e.g., nematode C. elegans) but not all invertebrates (e.g., fruit fly D. melanogaster and sea squirt Ciona intestinalis; Soccio & Breslow, 2003; Campbell et al., 2004). STAR proteins from nematodes and insects phylogenetically clustered with the vertebrate STARD3 and STARD1 genes, although functional divergence analysis suggests a significant difference between vertebrate and invertebrate STAR genes (Fan & Papadopoulos, 2013). A recent study in metazoans examined STAR sequences from invertebrate species, including three echinoderms, a hemichordate, five arthropods, and four molluscs (octopus and bivalves; Thongbuakaew et al., 2021). Domains for interacting with the cholesterol-rich domains of cell membranes and the motifs critical for proper protein folding were well conserved (Thongbuakaew et al., 2021). A STAR-related lipid transfer protein has also been identified in freshwater prawn M. Rosenbergii (Thongbuakaew et al., 2016) and scallop (Thitiphuree et al., 2019). However, whether these are STAR orthologs or related Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 8 proteins is not yet clear. The sea cucumber STAR and other steroidogenic genes were expressed in gonadal tissues and upregulated in ovaries and testes during reproductive development (Thongbuakaew et al., 2021). Mutations of residues conserved in human STAR protein reduced the steroidogenic activity of the C. elegans STAR protein (Watari et al., 1997). Collectively, this suggests that STAR orthologs are present in multiple metazoan species, including lophotrochozoan molluscs, and implicated in steroidogenesis for at least a few species. A more robust analysis of the STAR protein family in metazoans would provide a better evolutionary context for understanding STAR orthologs in invertebrate species, particularly in annelid lophotrochozoan phyla that contain ligand-depended ERs. Likewise, experimental data that demonstrates invertebrate STAR proteins move cholesterol across mitochondrial membranes is needed. CYP11A1 After STAR transports cholesterol across the outer mitochondrial membrane, cholesterol is converted into pregnenolone by CYP11A1 in vertebrates (P450scc; Step 2 in Figure 1.1). CYP11A1 is a side-chain cleavage enzyme that catalyzes cholesterol 20- and 22- hydroxylation, and C22-C20 bond cleavage (Pikuleva, 2006). A CYP11A gene was cloned from Branchiostoma belcheri (amphioxus; Mizuta & Kubokawa, 2007). By sequence similarity, two CYP11 genes were identified in B. floridae and the hemichordate acorn worm Saccoglossus kowalevskii, while a single CYP11 gene was identified in the sea urchin Stronglyocentrotus purpuratus (Goldstone et al., 2016). Overall sequence similarity was low between vertebrate and invertebrate CYP11 sequences, prompting one study to place the B. floridae CYP11 orthologs in different Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 9 subfamilies (CYP11D and CYP11E) than vertebrate (CYP11A; Baker et al., 2015). Yet, amino acid residues that surround the substrate and are critical for substrate binding in human CYP11A1 were conserved in the invertebrate CYP11 sequences (Goldstone et al., 2016). In amphioxus, the CYP11 shared synteny with an opossum syntenic gene block that included the gene TNPO2, providing evidence that this gene is orthologous to the vertebrate CYP11A, CYP11B, and CYP11C genes (Nelson & Habibi, 2013). In the Capitella teleta genome, a single gene (CYP376A1) was orthologous to vertebrate CYP11 genes (Dejong & Wilson, 2014). In some studies, the placement of candidate CYP11 genes from invertebrates were within an unresolved cluster of vertebrate CYP11, CYP27, and CYP24 sequences, preventing an assessment of whether these genes are orthologs to vertebrate CYP11s (e.g., see Figure 3 in Markov et al., 2009). Markov et al. (2009) rightly pointed out that caution is needed to attribute vertebrate-like steroidogenic activities to genes solely based on sequence similarity and phylogenetic placement. A combination of ortholog identification coupled with multiple lines of evidence demonstrating function are needed. Evidence for function could include appropriate gene or protein expression data (e.g. tissues relevant for steroid production), presence of substrate and product in tissues that express putative steroidogenic genes/proteins, in vitro functional data showing conversion of substrate to product, or loss of product with in vivo knockout lines. CYP11A has been measured in amphioxus in female ovaries (transcript; Mizuta et al., 2008) and in the nervous system (protein; Takeda et al., 2003). In vitro C20, 22-lyase activity, which converts cholesterol to pregnenolone, was detected in the gonads of the cuttlefish with a rate of 0.01% (Carreau Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 10 & Drosdowsky, 1977). Pregnenolone has been measured in amphioxus (Chang et al., 1985), crustaceans (e.g., Artemia sp. shrimp, (Novak & Lambert, 1989); lobster Nephrops norvegicus (Fairs et al., 1989); crayfish Astacus leptodactylus (Ollevier et al., 1986), and insects (e.g., locust Locusta migratoria, Novak & Lambert, 1989). Pregnenolone has been measured in the ovaries and testes of amphioxus and insects (Chang et al., 1985; Novak & Lambert, 1989). Collectively, this suggests that pregnenolone production may be possible in multiple invertebrate species; but only in amphioxus are there multiple lines of evidence supporting CYP11 function in the same tissue (both gene expression and product measurements in ovaries) and data from key lophotrochozoans are largely absent, particularly for in vitro substrate conversion in tissues or with expressed proteins (Table 1.1). 3β Hydroxysteroid dehydrogenase (3β-HSD) Pregnenolone is the final common steroid in the vertebrate biosynthesis pathway, from which all sex steroids are formed (Fuentes & Silveyra, 2019). Pregnenolone is the precursor to progesterone (Step 3 on Figure 1.1) and 17α-hydroxy-pregnenolone (Step 4 on Figure 1.1), creating bifurcating routes toward the generation of androgens and estrogens. Progesterone has been measured in tissues of a variety of invertebrate species, including the ovaries of sea urchin (Botticelli et al., 1961; Hines et al., 1992), ovaries and pyloric ceca of starfish (Dieleman & Schoenmakers, 1979), gonads of amphioxus (Mizuta & Kubokawa, 2007), hemolymph of prawn (Quinitio et al., 1991), hemolymph and ovary of mud crab (Ye et al., 2010), whole polychaetes (Meunpol et al., 2007), and gonads and whole-body homogenates of rotifers (Stout et al., 2010). Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 11 Vertebrate progesterone synthesis is catalyzed by 3β-hydroxysteroid dehydrogenase (3β-HSD) enzymes, which belong to the short-chain dehydrogenase superfamily (Miller & Auchus, 2011). 3β-HSD is required for additional steps in steroid biosynthesis, converting 17α-hydroxy-pregnenolone into 17α-hydroxyprogesterone (Step 6 in Figure 1.1) and dehydroepiandrosterone into androstenedione (Step 9 in Figure 1.1). In each case, the enzymatic step catalyzed by 3β-HSD is the conversion of the hydroxyl group on carbon 3 into a ketone and the conversion of the B ring into the A ring through the isomerization of the double bond (Miller & Auchus, 2011). 3β-HSD genes have been identified in tunicates (reviewed in Campbell et al., 2004) and most recently in amphioxus (reviewed in Baker et al., 2015; Holland & Li, 2021). 3β-HSD transcripts were identified in the gonads of sea cucumber, and expression increased with reproductive maturation (Thongbuakaew et al., 2021). The sea cucumber 3β-HSD gene had the motif (GxxGxxG), important for binding with NAD, NADP, and other cofactors for the short-chain dehydrogenase/reductase superfamily (Thongbuakaew et al., 2021). Overall sequence similarity of the sea cucumber 3β-HSD was low to vertebrate sequences, and it clustered with crustacean sequences (Thongbuakaew et al., 2021). 3β-HSD was identified in the freshwater prawn, contained the expected conserved motifs, and clustered with vertebrate sequences (Thongbuakaew et al., 2016). Phylogenetic analyses of the short-chain dehydrogenase family strongly cluster sequences from a variety of cnidarians (Nematostella vectensis), lophotrochozoans (Lottia gigantea, Crassostrea gigas, Helobdella robusta, Capitella sp.), ecdysozoans (Daphnia pulex), deuterostomes (Ciona intestinalis), echinoderms (Stroglyocentrotus purpuratus), Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 12 hemichordates (Saccoglossus koweleski) and cephalochordates (Branchiostoma floridae) with vertebrate 3β-HSD genes (See Figure S5 of Markov et al., 2009; see Figure S1A of Thongbuakaew et al., 2016). 3β-HSD activity had been inferred in microsomal and cytosolic fractions isolated from the gonads of the echinoderm species Paracentrotus lividus (Janer et al., 2005) and Asterias rubens (Dieleman & Schoenmakers, 1979). 3β- HSD activity has been detected in lophotrochozoans (tapeworm Taenia solium, Fernández Presas et al., 2008; polychaete Nereis virens, Garcia-Alonso & Rebscher, 2005), specifically in the gut epithelium of Nereis virens, and in the neck (immature) and testes (mature) of tapeworm (Garcia-Alonso & Rebscher, 2005; Fernández Presas et al., 2008). While there are multiple lines of evidence for functional 3β-HSD genes in polychaetes, one challenge is that each line of evidence is from a different species (e.g. gene from C. teleta, progesterone from Perinereis sp, substrate conversion in N. virens). CYP17A1 Vertebrate CYP17A1 is responsible for the conversion of pregnenolone (Step 4 in Figure 1) and progesterone (Step 5 in Figure 1.1) into 17α-hydroxy-pregnenolone and 17α-hydroxy-progesterone, respectively (Alex et al., 2016). In these reactions, CYP17 functions as a 17α -hydroxylase. 17α-hydroxy-pregnenolone and 17α-hydroxy- progesterone were detected after in vitro incubation of pyloric caeca or homogenates from Asterias sea star with pregnenolone and progesterone, suggesting CYP17 activity (den Besten et al., 1991; Meunpol et al., 2007). 17α-hydroxy-progesterone has been identified in the polychaete Perinereis sp. (Meunpol et al., 2007), while 17α-hydroxy- pregnenolone was found in the hemolymph of the crustacean Astacus leptodactylus Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 13 (Ollevier et al., 1986). CYP17A1 also functions as a C17-20 lyase, catalyzing 17α- hydroxy-pregnenolone (Step 7 on Figure 1.1) and 17α-hydroxy-progesterone (Step 8 on Figure 1.1) into dehydroepiandrosterone and androstenedione, respectively. Reports of these steroids in invertebrate tissues appear to be mostly lacking; dehydroepiandrosterone pathways (production and metabolism) are thought to be missing in amphioxus (Mizuta et al., 2008). Androstenedione has been reported in crustaceans (e.g., shrimp, Verslycke et al., 2002; crayfish, Ollevier et al., 1986). A partial CYP17 transcript and a motif associated with a heme-binding region common to the CYP superfamily and the PEHF motif common to CYP17 was identified in the sea cucumber (Thongbuakaew et al., 2021). The sea cucumber CYP17 was expressed in several tissues, including gonads; gonadal expression differed between males and females and across maturation stages (Thongbuakaew et al., 2021). CYP17 was cloned from amphioxus and phylogenetically clustered with vertebrate CYP17 sequences (Campbell et al., 2004; Mizuta & Kubokawa, 2007). Yet, CYP17 is lacking in tunicates (Campbell et al., 2004). The Daphnia magna (Baldwin et al., 2009) and Capitella teleta (Dejong & Wilson, 2014) genomes did not contain an ortholog to CYP17. Seven CYP genes have been identified in three marine and one freshwater species of Brachionus rotifers that may be related to vertebrate CYP17 genes (all Clan 2 CYP genes), but phylogenetic analyses did not include vertebrate sequences (Han et al., 2018; Byeon et al., 2021) to assess whether these are orthologs. Studies on the rotifer CYP17 are needed to investigate whether they are orthologs as well as the mining of additional genomes of lophotrochozoans for CYP17 would be helpful in resolving Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 14 whether CYP17 orthologs are present. Furthermore, substrate conversion and tissue concentrations of products expected from CYP17-like activity are entirely lacking in annelid and rotifer lophotrochozoans. 17β-HSD 17β-hydroxysteroid dehydrogenase (17β-HSD), a short-chain dehydrogenase superfamily member, converts androstenedione into testosterone (Step 10 in Figure 1.1), the precursor to 17β-estradiol. There are different types of 17β-HSD genes in mammals; individual genes have different sites of expression and are responsible for different activities. The mammalian type 3 enzyme is responsible for testosterone production from androstenedione, and we focus here since it is one of the intermediate steps in making 17β-estradiol in vertebrates. In the mature gonads of the amphioxus Branchiostoma belcheri, a 17β-HSD gene was identified using RT-PCR (Mizuta & Kubokawa, 2007). The amphioxus 17β-HSD gene has similar primary structural characteristics to vertebrate 17β-HSD8 orthologs (Mizuta & Kubokawa, 2007). 17β-HSD was identified in tunicates using EST profiles of the testis (Inaba et al., 2002; Kho, 2004), and its expression was localized to sterol-producing cells (Leydig cells equivalents) using a custom antibody (Kho, 2004). 17β-HSD transcripts have been identified in the sea cucumber (Thongbuakaew et al., 2021), urchin (Campbell et al., 2004), and amphioxus (Baker et al., 2015). Transcriptomics identified multiple 17β-HSD genes in the giant freshwater prawn; one of these sequences had the expected conserved motifs and catalytic triad and was widely expressed in tissues and across ovarian maturation stages (Thongbuakaew et al., 2016). 17β-HSD activity has been identified in multiple species of shrimp (Ghosh & Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 15 Arun, 1993; Summavielle et al., 2003) and in amphipods (Janer et al., 2005). 17β-HSD genes and activity were confirmed in C. elegans (Desnoyers et al., 2007). Likewise, the gene was isolated from the tapeworm T. solium contained the expected conserved motifs for the SDR family and the catalytic tetrad, and phylogenetically clustered with other sequences from platyhelminthes (Aceves-Ramos et al., 2014). The expressed protein was functionally capable of testosterone production in vitro from androstenedione (Aceves- Ramos et al., 2014); in vitro steroid synthesis has been demonstrated in this species (Gomez et al., 2000, Romano et al., 2003, Valdéz et al., 2006). Therefore, there are significant data in platyhelminthes for a functional 17β-HSD. CYP19 The final step of estrogen biosynthesis is the production of estrogens (17β- estradiol or estrone) from androgen precursors via CYP19, the aromatase enzyme (Corbin et al., 1988). This gene was thought to evolve at the origin of the vertebrates, although the genomic environment has an invertebrate origin, and a CYP19 gene (L. F. C. Castro et al., 2005) and the syntenic region (Nelson & Habibi, 2013) is present in amphioxus. There are three CYP19 sequences representing two different subfamilies (CYP19B and CYP19C) identified from two species of amphioxus (Goldstone et al., 2016). One of the Branchiostoma CYP19 genes was originally cloned from ovarian tissue (Mizuta & Kubokawa, 2007), had a similar number of exons, a 40% amino acid residue similarity, and 60% amino acid similarity to the human CYP19 gene sequence, and contained at least six expected transcription factor binding motifs in the 5’ flanking region (Mizuta & Kubokawa, 2007; Callard et al., 2011). The Branchiostoma aromatase was modelled, and Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 16 docking studies suggest androstenedione has a similar affinity for the human and amphioxus aromatase catalytic site (Callard et al., 2011). However, CYP19 is not expected in other invertebrates because this gene was not found in the tunicate (Campbell et al., 2004), urchin, acorn worm (Goldstone et al., 2016), polychaete Capitella teleta (Dejong & Wilson, 2014), crustacean Daphnia pulex (Baldwin et al., 2009), or 21 arthropodae (Feyereisen, 2011) genomes. Neither was a CYP19 identified in transcriptomes from rotifers (Kim et al., 2013; Kim et al., 2017; Han et al., 2018), sea cucumber (Thongbuakaew et al., 2021), or giant freshwater prawn (Thongbuakaew et al., 2016). Thus, the aromatase function, if present, must be completed by another enzyme. Tissue estradiol measurements have been reported in various species, using radioimmunoassays (RIA), enzyme immunoassays (EIA or ELISA), liquid chromatography, and different types of mass spectrometry. Some have raised concerns regarding the potential for environmental or dietary uptake of estrogens (Scott, 2012, 2018), which would be indistinguishable from the endogenously synthesized hormone. In fact, uptake and elimination of estradiol and active endocrine contaminants were found in the polychaete Perinereis nuntia, and estradiol was not detected in tissues without dietary exposure (Nurulnadia et al., 2013). Yet, estradiol levels have been detected in coelomic fluids from the polychaete Nereis virens (20 pg/ml, EIA; (Garcia-Alonso & Rebscher, 2005) and whole-body homogenates from N. diversicolor (2-10 ng/g, EIA; Mouneyrac et al., 2006, 0.5-2 ng/g, EIA; Durou & Mouneyrac, 2007) and Paraprionospio sp. (1-4 ng/g; LC-MS/MS-ESI, Nurulnadia et al., 2014). Estradiol concentrations were higher in immature N. virens (Garcia-Alonso & Rebscher, 2005) and the smallest weight classes in Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 17 N. diversicolor (Mouneyrac et al., 2006). Estradiol concentrations varied seasonally, but not with sexual maturity, in N. diversicolor (Durou & Mouneyrac, 2007). While seasonal estradiol concentrations of Paraprionospio sp. were reported, replication was insufficient to determine statistical differences across time (Nurulnadia et al., 2014). The assumption in the Nurulnadia et al. study (2014) was that estradiol was environmentally sourced, yet two of the three sampling sites did not have detectable estradiol. It was unclear which site(s) the animals were sampled from, making it difficult to determine the potential for environmentally sourced estrogens in this study. Therefore, the potential for environmental uptake of estradiol in polychaetes is clear, and careful consideration of this is needed to determine if tissue estradiol levels are a result of uptake, biosynthesis, or both. Beyond polychaetes, estradiol has been reported in a broad range of crustaceans, a few insects, and one urchin species. Estradiol was found in the hemolymph of red king crab (1249 pg/ml using RIA), but levels did not appear to differ with sex (male versus female), maturity (immature versus mature), or seasonally (by month; Dvoretsky et al., 2021); similar concentrations of estradiol were reported for males and females in three amphipod crustaceans (Lewis et al., 2015). Estradiol has been measured in hemolymph, eggs, ovary, hepatopancreas, and testes. Estradiol concentrations are typically in the pg- ng/ml or pg-ng/mg range but with higher levels reported in gonads, eggs, and hepatopancreas (see data for 15 crustacean species in Table 2 from Dvoretsky et al., 2021; Wijaya et al., 2020). Age-dependent estradiol concentrations had sharp changes with instar age (minimum 121 and maximum 1066 pg/ml for hemolymph; RIA) in the Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 18 hemolymph and posterior silk gland of the insect Bombyx mori (Keshan & Ray, 2000, 2001); hemolymph levels were similar to Locusta migratoria (Novak & Lambert, 1989). The rate of change was large and coupled with very different patterns of estradiol concentrations in hemolymph and posterior silk gland over time; it is most likely that such differences are driven by biosynthesis (Keshan & Ray, 2000; Keshan & Ray, 2001). Daily estradiol concentrations rose >3 fold in hemolymph but fell two-fold in the silk gland between days 9 and 10 days of the fifth instar; a three-fold increase in silk gland estradiol was between the 5 and 7 days of the fifth instar (Keshan & Ray, 2000, Keshan & Ray, 2001). Lastly, estradiol was 100 pg/g in the gonads of the urchin Paracentrotus lividus, suggesting that gonads could be a site of estrogen biosynthesis for the urchin if they are indeed endogenously produced (HPLC; Lavado et al., 2006). Collectively, these studies show measurable estradiol levels in a wide variety of invertebrate species, but strong evidence for endogenous production is lacking. Estrogen receptors and their specific activation by ligands such as 17β-estradiol Evolution of the Estrogen Receptor To discuss the presence of the estrogen receptor in lophotrochozoans, a brief discussion of the evolutionary history of the ER and the NR3 gene subfamily is warranted; several excellent reviews are available with more detail (see Baker, 2011; Callard et al., 2011; Markov et al., 2017; Holzer et al., 2017). The estrogen receptor (ER) is part of the NR3 subfamily, which contains the steroid and adrenal hormone-binding receptors. The NR3 subfamily is further divided into 5 groups: NR3A (vertebrate ER), Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 19 NR3B (estrogen-related receptor or ERR), NR3C (receptors that bind to androgens, progesterone, glucocorticoids, and mineralocorticoids; AR/PR/GR/MR), NR3D (protostome ER), NR3E (cnidarian steroid-related receptor), and NR3F (placozoan ERR) (Markov & Laudet, 2011; Khalturin et al., 2018). The diversification of the NR3 family likely occurred due to a duplication in bilaterians, resulting in the ERR and the ancestral steroid receptor (AncSR1), an ER-like receptor from which all other steroid receptors descended (Markov & Laudet, 2011; Thornton et al., 2003). AncSR1 is thus the ancestral receptor to NR3A, NR3D, and NR3C proteins; this reconstructed protein was based on a phylogeny that had NR3A and NR3D as sister clades (Thornton et al., 2003; Keay & Thornton, 2009). The predicted ligand binding (LBD) and the DNA binding (DBD) domains of AncSR1 had the highest sequence similarity to the extant NR3A subfamily, making the ER “the most ancient steroid receptor” (Eick et al., 2012; Holzer et al., 2017; Baker & Lathe, 2018). There is much debate about whether AncSR1 was estrogen- activated and the identity of the physiological ligand for this receptor. The placement of NR3D as a sister clade to NR3A is found in multiple phylogenetic studies with strong support (Vogeler et al., 2014; Cheng et al., 2021; Keay & Thornton, 2009; Bridgham et al., 2010; Kaur et al., 2015). Alternatively, NR3A and NR3C are sister clades, meaning that all vertebrate steroid receptors are more closely related, and the NR3D genes are not ER orthologs (Bridgham et al., 2014; Katsu et al., 2016). Other studies have failed to resolve this relationship with strong support (Huang et al., 2015; Kim et al., 2017; Khalturin et al., 2018; Beinsteiner et al., 2022; Eick et al., 2012). An important point about these different phylogenetic analyses is that they are Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 20 heavily biased towards vertebrate sequences, with low taxon sampling of lophotrochozoans overall (Keay & Thornton, 2009; Eick et al., 2012; Bridgham et al., 2014; Katsu et al., 2016). These studies included lophotrochozoan sequences biased towards molluscan ERs and 1-2 sequences for annelids at most (Platynereis dumerilii and/or Capitella teleta). With an increase in lophotrochozoan genomes sequenced, it is essential to mine for ER orthologs and incorporate sequences beyond Mollusca and Annelida to resolve the evolutionary uncertainties within the NR3 subfamily. The interpretation of NR3D genes as ER orthologs remains somewhat controversial because of the conflicts across phylogenetic studies. Estradiol-activated estrogen receptors are in all vertebrate classes, including in the basal vertebrates such as sea lamprey and hagfish (Callard et al., 2011; Baker et al., 2015). In basal chordates, the tunicate Ciona intestinalis does not contain an ER (Yagi et al., 2003; Campbell et al., 2004), while amphioxus contains an ER but differs in key residues in the ligand binding domain, does not bind E2, and was not ligand-activated by E2 and other ER agonists in a reporter assay (Paris et al., 2008; Callard et al., 2011). In basal deuterostomes, Baker et al. (2015) did not find estrogen receptors in sea urchins. However, estradiol functions as a ligand to activate NR3D receptors in some (polychaetes and rotifers; Keay & Thornton, 2009; Jones et al., 2017), but not all (molluscs, Baker & Chandsawangbhuwana, 2007; Bridgham et al., 2014), lophotrochozoans. Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 21 Estrogen receptors in Lophotrochozoa ER genes have been identified in three polychaetes, C. teleta, P. dumerilii, and Perinereis aibuhitensis (Keay & Thornton, 2009; Lv et al., 2017), and in five rotifers, B. calyciflorus, B. koreanus, B. plicatilis, B. rotundiformis, and B.manjavacas (D. H. Kim et al., 2017; Jones et al., 2017). Importantly, an ER gene is absent from the genome of the leech Helobdella robusta (Baker, 2008). ER genes were absent from 34 different species of platyhelminthes, identifying a possible gene loss for this phylum (e.g., Schistosoma mansoni, Wu et al., 2006; 33 different platyhelminth species, Wu & LoVerde, 2021). However, only two species were free-living, Macrostomum lignano and Schmidtea mediterranea, and the remaining parasitic to vertebrate hosts (e.g., birds, cattle, sheep, and humans; Wu et al., 2006; Wu & LoVerde, 2021). Therefore, among the lophotrochozoan lineages, we expect ER genes in rotifers and polychaetes, but may not in (vertebrate parasitic) platyhelminthes. ER genes have two critical functional domains: the ligand (LBD) and DNA binding (DBD) domains. The DBD sequences in polychaetes C. teleta (Keay & Thornton, 2009a), P. dumerilii (Keay & Thornton, 2009a), and P. aibuhitensis (Lv et al., 2017) had 74, 80, and 82% protein sequence identity compared to the human ERα, respectively; similar data for rotifer DBD sequences were not reported (Jones et al., 2017). Both rotifer and polychaete ERs contain the characteristic P-box motif (CEGCKA) in the DBD (Jones et al., 2017; Lv et al., 2017; Keay & Thornton, 2009). The percent sequence identity of the LBDs between lophotrochozoan ERs and human ERα LBD is 25-35% (25% in the rotifer Brachionus manjavacas; Jones et al., 2017, 28-29% in polychaetes Platynereis Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 22 dumerilii and Capitella teleta; Keay & Thornton, 2009, and 35% in polychaete Perinereis aibuhitensis; Lv et al., 2017). The polychaete and rotifer LBDs had an unobstructed ER ligand pocket, necessary for ligand binding, with conserved residues essential for contact with 17β-estradiol (Jones et al., 2017; Lv et al., 2017; Keay & Thornton, 2009), which suggests that these ERs bind and can be activated by estradiol. Direct testing of the binding and activation of lophotrochozoan ERs has been assessed in two polychaetes (Keay & Thornton, 2009a) and one rotifer (B. L. Jones et al., 2017). The P. dumerilii LBD binds radiolabeled estradiol with high affinity (Kd=5.3 nM; Keay & Thornton, 2009). Furthermore, the ability of 17β-estradiol to activate transcription via the C. teleta and P. dumerilii LBD and full receptor (P. dumerilii only) in a luciferase reporter assay was tested (Keay & Thornton, 2009a). The C. teleta LBD, P. dumerilii LBD, and P. dumerilii full receptor, activated transcription in response to physiologically relevant levels of estradiol (PdER EC50=8.5 nM; CtEC50 = 9.2 nM) but not in response to other steroid hormones (Keay & Thornton, 2009a). The P. dumerilii full ER had 10-fold lower activity than the human ERα, which could be due to the testing of the reporter activation in a mammalian cell line (Keay & Thornton, 2009a). A yeast genetic selection assay of the B. manjavacas LBD showed activation of ER LBD upon exposure to ER ligands (17β-estradiol and AMI fluorophore ligands; no other hormones tested; Jones et al., 2017). This assay measured activation through yeast growth after ER ligand-dependent histidine production (Jones et al., 2017). Collectively, these data support the binding and activation of ERs by 17β-estradiol in polychaetes and rotifers. Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 23 Endogenous expression of estrogen receptors in Lophotrochozoa The expression of the receptor has been examined in two polychaete and one rotifer species. Endogenous ER expression in P. dumerilii embryos was localized in the micromeres after cleavage (Lidke et al., 2014). In larval stages of P. dumerilii, ER expression localized in the brain at 72 hours post-fertilization (Lidke et al., 2014). In the adult polychaete Perinereis aibuhitensis, ER was broadly expressed in the head, body wall, esophageal gland, esophagus, and stomach, with the highest expression in the intestines compared to all other organs (Lv et al., 2017). In adult rotifers, ERs localized to the pharynx, ovaries, and yolk gland of female B. manjavacas (Chen et al., 2017; Jones et al., 2017). ERs localized to testes, ovaries, and eggs in the platyhelminthes, Raillietina echinobothrida and Raillietina tetragona (Chen et al., 2017). ER localized to eleocytes, coelomic cells that secrete vitellogenin, in female polychaete Nereis virens at the start of vitellogenesis but not in the gut, oocytes, or muscle tissues (Garcia-Alonso & Rebscher, 2005). These expression patterns suggest possible developmental, reproductive, and digestive roles, depending on the species. To better understand expression patterns, data are needed from multiple life stages, e.g., embryonic, larval, juvenile, and adult stages for a single lophotrochozoan species. Data are likewise needed at the same life stage from multiple species to determine if there is a conserved pattern of expression, such as has been completed for the estrogen-related receptor expression (Papadogiannis et al., 2023). Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 24 Physiological response to estrogens Studies with ER agonist exposures in lophotrochozoans may provide important links between the activation of the estrogen receptor and biological processes. Here, we prioritize 17β-estradiol (E2) and the synthetic estrogen, 17α-ethinylestradiol (EE2), potent agonists of the vertebrate estrogen receptors (pM- nM EC50, Van Den Belt et al., 2004). The effects of chronic E2 and EE2 exposures on lophotrochozoan survival, growth, and life-stage progression have been studied in two rotifers (Gallardo et al., 1997; Huang et al., 2012), one polychaete (Lv et al., 2016), and ten leech (Heger et al., 2015; Kidd et al., 2020) species. In the rotifer Brachionus calyciflorus, chronic exposures (up to 10 days, from 2-h post hatch) to E2 (0.003671-3671 nM) decreased the duration of the embryonic period (≦36.71 nM) and increased the duration of the juvenile (0.03671-36.71 nM), reproductive (≦3671 nM), and post-reproductive (367.1 and 3671 nM) periods, generation time (0.03671-3671 nM), and life expectancy at hatching (0.03671-3671 nM; Huang et al., 2012). However, these effects did not have a strong dose-dependent relationship despite the six orders of magnitude in dose. Exposure (48 h) of E2 (183 nM- 183 µM) in the rotifer B. plicatilis did not affect body size (Gallardo et al., 1997). Chronic exposure (205 days) of the polychaete Perinereis aibuhitensis to E2 (0.3671-3671 nM) had no effect on weight gain or specific growth rate (Lv et al., 2016). A 2-week E2 exposure (10 – 100 µg/kg) to the earthworm Eisenia fetida did not affect growth or mortality (Heger et al., 2015). In a whole lake exposure to EE2 (16.2-20.6 pM), there were no effects of EE2 exposure growth rates for 9 leech species (Kidd et al., 2020). Thus, in B. calyciflorus rotifers, estradiol appears important for life-stage progression. Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 25 Survival and growth data are limited but, to date, there are no reported effects of E2 in any lophotrochozoan species. In vertebrates, E2 increases the number of primordial germ cells (PGC; in the primitive gonads; Ge et al., 2012), and this effect has been examined in polychaetes (Lidke et al., 2014). E2 exposure (0.367- 367 nM) during PGC formation increased the number of larvae with more than four PGCs in P. dumerilii in a dose-dependent manner after 36.71 nM (Lidke et al., 2014). Interestingly, the effect of E2 exposure was highest during PGC formation (28.5% vs. 0-4.3% of larvae), a stage that had increased ER expression (Lidke et al., 2014). This effect was reversed with the co-exposure of E2 and an ER antagonist, supporting a mechanism via interactions with the ER (Lidke et al., 2014). Vitellogenesis is an important process in egg-bearing vertebrates and is regulated by E2 via the ER (Nelson & Habibi, 2013; Levi et al., 2009). Vitellogenin is a yolk precursor protein important for egg development in vertebrates (Biscotti et al., 2018). In polychaetes, platyhelminthes, and leeches, exposures to E2 and EE2 show possible roles for E2 in the regulation of vitellogenesis and sexual maturation. In the polychaete Nereis virens, eleocytes from females starting vitellogenesis increased the amount of vitellogenin secreted in vitro after exposure to 3.67 nM E2 for 48 and 72 hrs, but vitellogenin secretion was not induced by E2 in eleocytes from immature worms (Garcia- Alonso & Rebscher, 2005). Exogenous E2 exposure (3.67-367 nM) inhibited the regeneration of sexual organs (yolk gland) in the hermaphroditic flatworm Dugesia Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 26 ryukyuensis seven weeks post-ablation, compared to the four weeks it takes for regeneration to occur in control conditions. E2 exposures have effects on rotifer reproduction and population-level effects, but the direction of the effect (positive or negative) varies even in the same species tested. Net reproductive rate increased in B. calyciflorus after exposure to higher doses of E2 (367 and 3671 nM); E2 prolonged the reproductive period in most doses (Huang et al., 2012) and increased the intrinsic rate of population growth only at one lower dose (3.67 nM E2; Huang et al., 2012). Yet, E2 exposure (3671 nM) decreased the total number of resting eggs in B. calyciflorus and decreased the asexual population growth rate (Yang & Snell, 2010). Lastly, E2 exposure did not alter the percent of ovigerous females fertilized in B. calcyciflorus (36.7 nM; Preston et al., 2000). In these latter studies (Yang & Snell, 2010; Preston et al., 2000), only a single concentration was used which limits their interpretation. Lastly, EE2 (0.27 and 1.72-27.1 µM) decreased the ratio of mictic (sexual) females/total number of amictic (asexual) females and the intrinsic rate of population growth at concentrations higher than 1.72 µM (EC50= 4.15 µM) in B. calyciflorus (Radix et al., 2002). In B. plicatilis, mictic (sexual) female production increased at the highest concentration (18.4 µM E 2) only, and this result was time-dependent (day 6, but not day 2, 4, or 8) but did not alter population density (Gallardo et al., 1997). Finally, the reproductive output of B. manjacavas was not affected by 10 µM E2 (B. L. Jones et al., 2017). Beyond rotifers, exposure of adult leeches E. fetida to E2 (0-100 µg/kg) for 4 weeks stimulated reproduction in a dose-dependent manner at lower doses (10, 30, and 50 Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 27 µg/kg), while higher doses (80 and 100 µg/kg) inhibited reproduction (Heger et al., 2015). In a whole lake exposure to EE2 (16.2-20.6 pM), there were no effects of EE2 exposure on leech community composition or abundance or leech egg production for 9 leech species and there were no treatment effects on the total gonadosomatic index in a subset of species (5 species; Kidd et al., 2020; Simakov et al., 2013). However, low concentrations of EE2 (6.2-20.6 pM) for the leech Haemopis marmorata, altered growth of reproductive organs (increased length of relative sperm sac, relative vaginal bulb, ovisac plus albumen gland; and decreased relative epididymitis weight; Kidd et al., 2020). Overall, these data are weak support for a role for E2 in the reproduction of lophotrochozoans because there is clear ambiguity in the direction of the effects, whether this impacts population dynamics, and no strong dose-response relationships have been established. Conclusions Based on current data, we might expect that most lophotrochozoan species, outside of perhaps platyhelminths, have an ER gene. Yet, the resolution of the evolutionary relationships within the NR3 subfamily is greatly needed to clearly resolve whether these are orthologs to the vertebrate ERs or have evolved from the ancestor to all vertebrate steroid hormone receptors. While there is evidence of either putative or obvious orthologs of several steroid biosynthetic genes, notable exceptions are for CYP17 and CYP19, which raise significant questions regarding endogenous estradiol synthesis in lophotrochozoans. Currently, lophotrochozoan species whose genomes have been sequenced include, but are not limited to: Capitella teleta (Simakov, Marletaz, Cho, Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 28 Edsinger-Gonzales, Havlak, Hellsten, Kuo, Larsson, Lv, Arendt, Savage, Osoegawa, De Jong, et al., 2013), Platynereis dumerilii (Raible et al., 2005), Hirudo medicinalis (Kvist et al., 2020), Helobdella robusta (Simakov, Marletaz, Cho, Edsinger-Gonzales, Havlak, Hellsten, Kuo, Larsson, Lv, Arendt, Savage, Osoegawa, De Jong, et al., 2013), Spirobranclus lamarcki (Kenny et al., 2015), and Ridgeia piscesae (M. Wang et al., 2023), Aditena vaga (Vakhrusheva et al., 2020), and Brachionus calyciflorus (H. Kim et al., 2018). As more genomes become available, a substantial effort to mine lophotrochozoan genomes for NR3, STAR, CYP, SDR, and AKR gene families to identify and resolve phylogenetic relationships among genes encoding putative steroid receptors and steroid biosynthesis proteins are crucial. To determine whether lophotrochozoans can endogenously produce 17β-estradiol, more functional data are clearly required. Considering that the current data comes from a patchwork of many different species, it would be most valuable to focus on lophotrochozoans whose genomes have been sequenced and complete phylogenetic and functional work in the same species. Of these, species that can be coupled with gene knockout techniques, such as CRISPR/Cas9 mutagenesis, like B. manjavacas (H. Feng et al., 2023), P. dumerilii (Bezares-Calderó et al., 2018) and C. teleta (Neal et al., 2019) would provide a significant advantage to determining function in vivo. A major and exciting advantage is that ligand-activated ERs have already been established in these species of lophotrochozoans. In C. teleta, gene knockouts of rhopsin have been performed at a high mutation rate (94%-100%) in the F0 generation (Neal et al., 2019). This, coupled with a short generation time (about 6 weeks at room temperature and 8 weeks at 19°C), Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 29 iteroparity, and availability year-round, makes it a powerful candidate species where you can combine studies of phylogenetics, gene expression, protein function, and assess phenotypes across life stages (Seaver, 2016; Neal et al., 2019). In comparison, P. dumerilii and B. manjavacas have a significantly longer (3–4-months) and shorter (4 day) generation time, respectively. Gene knockdowns in P. dumerilii are possible in the F2 generation (Bezares-Calderó et al., 2018) and the F1 generation in B. manjavacas, although at a 40% mutation rate (H. Feng et al., 2023). One important consideration is that estradiol may not be the natural ligand of invertebrate ERs. One of the limitations of ancient protein reconstruction is that it omits the evolution of small molecules (e.g., hormones) and assumes that the extant ligands are the same from millions of years ago (Markov & Laudet, 2022). The approach from their analysis in 2017 compared chemical pathways rather than enzymes and examined the potential for synthesis of paraestrol A and its capacity for transcriptional activation of ancestral steroid receptors (Markov et al., 2017). In fact, multiple steroids have been proposed as the ancient steroid ligand, including 27-hydroxycholesterol, Δ5- androstenediol, and 5(alpha)-androstanediol (Baker & Lathe, 2018). Assessing putative ancestral ligands (and their potential biosynthesis) may provide useful avenues to understanding the function of the ER genes in extant taxa. One limitation in current studies is the major focus on the role of estrogens in reproductive functions. However, vertebrate estrogens are pleiotropic, with many non- reproductive functions. We suggest investigating non-reproductive endpoints may be Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 30 fruitful, especially if that research is driven from established patterns of ER expression. Thesis Goals Estradiol signalling has been characterized in vertebrates and was previously thought to be restricted to chordates. However, multiple lines of evidence suggest that E2 signalling may be more broadly found in the tree of life. Lophotrochozoans are important taxa to investigate estradiol signalling because they contain phyla with E2-dependent and E2-independent ERs (Jones et al., 2017; Figure 1.2). Questions remain about whether vertebrate steroids are important for endocrine regulation in lophotrochozoans (Markov et al., 2017). This is in part due to a lack of in-depth analysis in a single lophotrochozoan species on whether E2 signalling pathway components are present. The overall goal of this thesis is to determine whether estradiol signalling is present in C. teleta. A signalling pathway requires a receptor that processes a signal (ER; Aim 1), a signalling molecule (estradiol; Aim 2), and a response to the signal (behavioural in Aim 2, definitions from Callard et al., 2012). I hypothesized that an estradiol signalling pathway was present in Capitella teleta and had roles in development, reproduction, and behaviour. In Chapter 2, I characterized estrogen receptor gene expression during the larval, juvenile, and adult life stages of Capitella teleta. In Chapter 3, I annotated and determined the phylogeny of hydroxysteroid dehydrogenase genes from the aldo-keto reductases and the short chain dehydrogenases reductases to identify orthologs of vertebrate steroid biosynthesis genes. Further, I measured 17β-estradiol in juvenile and adult worms and developed and applied a novel behavioural assay to determine the responsiveness of Capitella teleta adults to exogenous estradiol. Lastly, in Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 31 Chapter 4, I performed transcriptomics in C. teleta to determine changes in gene expression with age and sex. This study will aid in understanding of evolution of the estrogen receptor and its physiological function in polychaete lophotrochozoans. The knowledge of the basic biology of endocrine systems, and transcriptomics across major life stages, can help inform future research, possibly including toxicological studies considering the widespread and persistent distribution of endocrine-disrupting compounds. This may be important considering the established tolerance of this species to disturbed and contaminated marine environments. Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 32 Table 1.1. Evidence for the presence and function of steroidogenic enzymes in polychaetes, rotifers, and platyhelminthes. Taxa are chosen to represent three major phyla of lophotrochozoans. Evidence for the presence of gene orthologs, tissue products of the enzyme, and in vitro substrate conversion with tissues or expressed proteins are listed. When there is evidence in at least one species, the phylum is in black text. Blank spaces are when data are absence. If evidence is lacking (e.g., has been investigated in at least one species and was not found) the taxa is in gray text and struck out. Those in grey with a question mark indicate a possible gene ortholog that lacks strong phylogenetic support. Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 33 Figure 1.1. Vertebrate Estrogen Biosynthesis Pathway. Vertebrate steroid synthesis is shown with an emphasis on estradiol production; corticosteroids are not shown and androgens are limited to those used as precursors to 17ß-estradiol, the most potent estrogen. The steps of this pathway are numbered. Step 1 is the movement of cholesterol across the outer mitochondrial membrane by steroidogenic acute regulatory (STAR) protein. Steps 2-11 are enzymatic reactions mediated by proteins from the cytochrome P450 (CYP) and hydroxysteroid dehydrogenase (HSD) enzymes. The specific enzymes responsible for each step are listed below or to the right of the arrow. Ph.D. Thesis – A. Murillo Ramos; McMaster University – Department of Biology 34 Figure 1.2. Estrogen receptors in Bilateria. Phylogeny of bilaterian clade showing whether species corresponding to different bilaterian phyla have the presence of an estrogen receptor (ER) and whether there is a ligand-activated estrogen receptor. Modified from Kim et al. 2017. Ph.D. Thesis – A. 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