Medicine, Faculty of
DSpace
UBCV
Adams, Arlo Cordell
Doctor of Philosophy - PhD
University of British Columbia
The focus of this thesis is the characterization of the functions of the membrane contact sites (MCSs) between tubulobulbar complexes (TBCs) and the endoplasmic reticulum (ER). TBCs are double membrane endocytic invaginations that are endemic to Sertoli cells of the mammalian testis. They have been proposed to internalize intact intercellular junctions between late spermatids and Sertoli cells, allowing late spermatids to be released into the seminiferous tubule lumen. However, there is little data showing how TBCs may be regulated temporally and spatially to initiate junction turnover and subsequent spermatid release (spermiation). One of the most striking features of TBCs is the close association of ER to the ‘bulb’ portion, which is present for much of the time a TBC is developing and up until the moment of its vesiculation, all while it is still continuous with the plasma membrane. Although ER contacts are known to help regulate the form and function of other organelles such as endosomes, there have never been an investigation into what the functions of the TBC-ER contact sites are. It is my overall hypothesis that the TBC-ER contact sites are necessary for the normal development of TBCs into mature endocytic structures. Some of the well-established functions of ER contacts are both calcium and lipid exchange. Both processes have been shown to be fundamental to the progression of endosomes through the endosomal-lysosomal system. In this thesis, I begin by using qualitative approaches to establish that proteins known to be involved in both calcium and lipid exchange are present at TBC-ER contacts. I follow-up on my qualitative works with functional studies where I explore the role some of these proteins play in TBC development and in spermiation. Using in vivo knockdown experiments in rat testes to target two of these proteins (IP3R1 and ORP9), I demonstrate that both are necessary for the normal maturation of TBC bulbs and for sperm release to occur. These data support that conclusion that both calcium and lipid exchange are functions of the TBC-ER contact sites and provide evidence that the contact sites are necessary components of the sperm release mechanism.
THE ROLE OF TUBULOBULBAR COMPLEX-ENDOPLASMIC RETICULUM CONTACT SITES DURING JUNCTION TURNOVER IN THE SEMINIFEROUS EPITHELIUM by Arlo Cordell Adams B.Sc. (Hons), The University of British Columbia, 2015 A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate and Postdoctoral Studies (Cell and Developmental Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April, 2021 © Arlo Cordell Adams, 2021 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: The role of tubulobulbar complex-endoplasmic reticulum contact sites during junction turnover in the seminiferous epithelium submitted by Arlo Cordell Adams in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Cell and Developmental Biology Examining Committee: Dr. A Wayne Vogl, Cell and Developmental Biology, UBC Supervisor Dr. A Lacey Samuels, Botany, UBC Supervisory Committee Member Dr. Joy Richman, Craniofacial Science, UBC University Examiner Dr. Jeffrey Richards, Zoology, UBC University Examiner Additional Supervisory Committee Members: Dr. Edwin Moore, Cell and Developmental Biology, UBC Supervisory Committee Member Dr. Christopher Loewen, Cell and Developmental Biology, UBC Supervisory Committee Member Dr. Julian Guttman, Molecular Biology and Biochemistry, SFU Supervisory Committee Member iii Abstract The focus of this thesis is the characterization of the functions of the membrane contact sites (MCSs) between tubulobulbar complexes (TBCs) and the endoplasmic reticulum (ER). TBCs are double membrane endocytic invaginations that are endemic to Sertoli cells of the mammalian testis. They have been proposed to internalize intact intercellular junctions between late spermatids and Sertoli cells, allowing late spermatids to be released into the seminiferous tubule lumen. However, there is little data showing how TBCs may be regulated temporally and spatially to initiate junction turnover and subsequent spermatid release (spermiation). One of the most striking features of TBCs is the close association of ER to the ‘bulb’ portion, which is present for much of the time a TBC is developing and up until the moment of its vesiculation, all while it is still continuous with the plasma membrane. Although ER contacts are known to help regulate the form and function of other organelles such as endosomes, there have never been an investigation into what the functions of the TBC-ER contact sites are. It is my overall hypothesis that the TBC-ER contact sites are necessary for the normal development of TBCs into mature endocytic structures. Some of the well-established functions of ER contacts are both calcium and lipid exchange. Both processes have been shown to be fundamental to the progression of endosomes through the endosomal-lysosomal system. In this thesis, I begin by using qualitative approaches to establish that proteins known to be involved in both calcium and lipid exchange are present at TBC-ER contacts. I follow-up on my qualitative works with functional studies where I explore the role some of these proteins play in TBC development and in spermiation. Using in vivo knockdown experiments in rat testes to iv target two of these proteins (IP3R1 and ORP9), I demonstrate that both are necessary for the normal maturation of TBC bulbs and for sperm release to occur. These data support that conclusion that both calcium and lipid exchange are functions of the TBC-ER contact sites and provide evidence that the contact sites are necessary components of the sperm release mechanism. v Lay Summary The release of sperm cells is fundamental to male fertility. In Sertoli cells of the mammalian testis, sperm release occurs when the attachments between Sertoli cells and sperm cells are removed by subcellular machines termed ‘tubulobulbar complexes’ (TBCs). Another cellular structure, the endoplasmic reticulum (ER), forms a close connection with TBCs. This connection may serve to regulate the maturation of TBCs, and facilitate sperm release. Here I provide evidence that proteins involved in both calcium and lipid exchange in other parts of cells generally, are also present at the TBC-ER connections. I further demonstrate that of these proteins, the calcium exchanger IP3R1, and the lipid exchanger ORP9, are both necessary for the normal maturation of TBCs and the subsequent release of spermatids. These data provide evidence that both calcium and lipid exchange occur at these contact sites and that these processes are necessary for TBC maturation and subsequent spermatid release. vi Preface All of the work presented in this thesis was conducted in the laboratory of Dr. A Wayne Vogl at the University of British Columbia. I was the lead author for the majority of the work presented here and have indicated sections where I was a co-author. All of the animal work performed in this thesis was done under approval by the Animal Care Committee at the University of British Columbia (A16-0069, A20-0076). Chapter 1: A version of parts of this chapter is a published review article where I am the first author. Published work: Adams, A., Sriram, A. and Wayne Vogl, A. (2018). Internalization of Intact Intercellular Junctions in the Testis by Clathrin/Actin‐Mediated Endocytic Structures: Tubulobulbar Complexes. Anat. Rec. 301, 2080-2085. Author contributions: I wrote the sections on comparisons of tubulobulbar complexes to other endocytic structures, and reviewed the manuscript prior to publication. Aarati Sriram reviewed the manuscript prior to publication. A. Wayne Vogl wrote the outline for the first draft of the manuscript and reviewed the manuscript prior to publication. Chapter 2: A version of this chapter is a published paper and is based on experiments that I began as an undergraduate and completed as a graduate student in the laboratory of Dr. A Wayne Vogl at UBC. vii Published work: Adams, A. and Vogl, AW. (2017). High Resolution Localization of Rab5, EEA1, and Nectin‐3 to Tubulobulbar Complexes in the Rat Testis. Anat. Rec. 300, 1160-1170. Author contributions: I performed all the experiments, data analysis, and prepared the manuscript for publication. A. Wayne Vogl contributed to experimental design, data analysis, and editing of the manuscript prior to publication. Chapter 3: A version of this chapter is a published paper where I am co-first author. Published work: Lyon, K., Adams, A., Piva, M., Asghari, P., Moore, E. D. and Vogl, A. W. (2017). Ca2+ signaling machinery is present at intercellular junctions and structures associated with junction turnover in rat Sertoli cells. Biol. Reprod. 96, 1288-1302. Author contributions: The immunofluorescence experiments for the ORAI, and STIM1 localization were performed by Kevin Lyon, as were the initial immunofluorescence experiments for IP3R. I performed all of the other experiments (immunofluorescence and immunoelectron microscopy) for IP3R, TRPM6 and HOMER1. Matthew Piva performed the tomography experiments. Parisa Asghari and Edwin Moore contributed to experiments not described in this thesis and reviewed the final manuscript prior to publication. A. Wayne Vogl viii contributed to experimental design, data analysis, and editing of the manuscript prior to publication. Chapter 4: A version of this chapter is a published paper where I am the first author. Published work: Adams, A., Yoo, K. M. and Vogl, W. (2019). Lipid transfer machinery is present at membrane contact sites associated with the internalization of junctions in Sertoli cells. Biol. Reprod. 101, 662-663. Author contributions: I performed all the experiments, data analysis, and prepared the manuscript for publication. Kang-Mu Yoo assisted with all the experiments and reviewed the final manuscript. A. Wayne Vogl contributed to experimental design, data analysis, and editing of the manuscript prior to publication. Chapter 5: A version of this chapter is a published paper where I am the first author. Published work: Adams, A. and Vogl, W. (2020a). Knockdown of IP3R1 disrupts tubulobulbar complex-endoplasmic reticulum contact sites and the morphology of apical processes encapsulating late spermatids. Biol. Reprod. 103(3), 669-680. Author contributions: I performed all the experiments, data analysis, and prepared the manuscript for publication. A. Wayne Vogl contributed to experimental design, data analysis, and editing of the manuscript prior to publication. ix Chapter 6: A version of this chapter is a published paper where I am the first author. Published work: Adams, A. and Vogl, W. (2020b). ORP9 Knockdown Delays the Maturation of Junction Related Endocytic Structures in the Testis and Leads to Impaired Sperm Release. Biol. Reprod. 103(6), 1314-1323. Author contributions: I performed all the experiments, data analysis, and prepared the manuscript for publication. A. Wayne Vogl contributed to experimental design, data analysis, and editing of the manuscript prior to publication. x Table of Contents Abstract ............................................................................................................................... iii Lay Summary ....................................................................................................................... v Preface ................................................................................................................................ vi Table of Contents ................................................................................................................ x List of Figures .................................................................................................................. xvii List of Abbreviations ......................................................................................................... xxi Acknowledgments .............................................................................................................. iii Dedication ........................................................................................................................... iv 1 Introduction .................................................................................................................. 1 1.1 Spermatogenesis ...................................................................................................... 1 1.2 Sertoli cells of the seminiferous epithelium .............................................................. 2 1.3 Turnover of intact junctions in cells .......................................................................... 3 1.4 Tubulobulbar complexes (TBCs) .............................................................................. 4 1.5 Internalization of intact junctions by TBCs ............................................................... 6 1.6 TBC domains ............................................................................................................ 7 1.7 Comparison of TBCs to other endocytosis mechanisms ........................................ 7 1.8 TBC formation and scission ..................................................................................... 8 1.9 The role of actin in TBC structure ............................................................................ 9 1.10 Regulation of TBCs .............................................................................................. 10 xi 1.11 ER-endosome contact sites ................................................................................. 10 1.12 Overall hypothesis and chapter aims ................................................................... 12 1.13 Introduction figures ............................................................................................... 16 2 High Resolution Localization of Rab5, EEA1, and Nectin-3 to Tubulobulbar Complexes in the Rat Testis ............................................................................................ 23 2.1 Brief Synopsis ......................................................................................................... 23 2.2 Results .................................................................................................................... 25 2.2.1 Immunofluorescence ....................................................................................... 25 2.2.2 Immunoelectron Microscopy ............................................................................ 25 2.2.3 Western Blots ................................................................................................... 26 2.3 Discussion ............................................................................................................... 27 2.4 Materials and Methods ........................................................................................... 30 2.4.1 Animals ............................................................................................................. 30 2.4.2 Reagents .......................................................................................................... 30 2.4.3 Immunofluorescence ....................................................................................... 30 2.4.4 Immunoelectron Microscopy ............................................................................ 32 2.4.5 Electron microscopy......................................................................................... 33 2.4.6 Western Blotting ............................................................................................... 34 2.5 Figures .................................................................................................................... 35 xii 3 Ca2+ Signaling Machinery is Present at Intercellular Junctions and Structures Associated with Junction Turnover in Rat Sertoli Cells ................................................... 43 3.1 Brief Synopsis ......................................................................................................... 43 3.2 Results .................................................................................................................... 46 3.2.1 Characterization of the endoplasmic reticulum at ectoplasmic specializations and tubulobulbar complexes ..................................................................................... 46 3.2.2 The Ca2+ release channel, IP3R, localizes to the bulb regions of tubulobulbar complexes ................................................................................................................. 47 3.2.3 TRPM6 and HOMER1 localize to the bulb regions of tubulobulbar complexes ................................................................................................................................... 48 3.3 Discussion ............................................................................................................... 49 3.4 Materials and Methods ........................................................................................... 52 3.4.1 Animals ............................................................................................................. 52 3.4.2 Anesthesia and euthanasia ............................................................................. 52 3.4.3 Reagents .......................................................................................................... 52 3.4.4 Electron microscopy......................................................................................... 52 3.4.5 Electron tomography ........................................................................................ 54 3.4.6 Distance measurements and statistical analysis ............................................ 54 3.4.7 Pre-embedding immunoelectron microscopy.................................................. 55 3.4.8 Immunofluorescence microscopy .................................................................... 56 3.4.9 Western blotting ............................................................................................... 58 xiii 3.5 Figures .................................................................................................................... 61 4 Lipid transfer machinery is present at membrane contact sites associated with the internalization of junctions in Sertoli cells ........................................................................ 67 4.1 Brief Synopsis ......................................................................................................... 67 4.2 Results .................................................................................................................... 70 4.2.1 Lipid transfer protein ORP9 and its integral membrane anchor, VAPA, specifically label TBC-ER contacts ........................................................................... 70 4.3 Discussion ............................................................................................................... 71 4.4 Materials and Methods ........................................................................................... 74 4.4.1 Animals ............................................................................................................. 74 4.4.2 Tissue collection .............................................................................................. 74 4.4.3 Antibodies......................................................................................................... 74 4.4.4 Electron microscopy......................................................................................... 75 4.4.5 Immunofluorescence microscopy .................................................................... 75 4.4.6 STED Microscopy ............................................................................................ 76 4.4.7 Western blotting ............................................................................................... 77 4.4.8 Reagents .......................................................................................................... 77 4.5 Figures .................................................................................................................... 78 5 Knockdown of IP3R1 Disrupts TBC-ER Contact Sites and the Morphology of Apical Processes Encapsulating Late Spermatids ..................................................................... 80 xiv 5.1 Brief Synopsis ......................................................................................................... 80 5.2 Results .................................................................................................................... 82 5.2.1 Knockdown of IP3R1 by intratesticular siRNA injection ................................. 82 5.2.2 IP3R1 Knockdown Changes the Actin Structure in Sertoli cells and Late Spermatids ................................................................................................................ 83 5.2.3 The Abundance and Distribution of Intact and Vesiculated TBC bulbs Are Altered in IP3R1 Knockdown Testes ........................................................................ 84 5.2.4 IP3R1 Knockdown Causes a Delay in Spermiation ........................................ 85 5.3 Discussion ............................................................................................................... 87 5.4 Materials and Methods ........................................................................................... 90 5.4.1 Animals ............................................................................................................. 90 5.4.2 Reagents .......................................................................................................... 90 5.4.3 Intratesticular injections ................................................................................... 91 5.4.4 Tissue Collection .............................................................................................. 91 5.4.5 Antibodies......................................................................................................... 92 5.4.6 Electron Microscopy and Bright Field Microscopy .......................................... 92 5.4.7 Immunofluorescence ....................................................................................... 93 5.4.8 STED Microscopy ............................................................................................ 94 5.4.9 Western blotting and Densitometry ................................................................. 95 5.5 Figures .................................................................................................................... 97 xv 6 ORP9 Knockdown Delays the Maturation of Junction Related Endocytic Structures in the Testis and Leads to Impaired Sperm Release .................................................... 108 6.1 Brief Synopsis ....................................................................................................... 108 6.2 Results .................................................................................................................. 110 6.2.1 Knockdown of ORP9 by Intratesticular siRNA Injection ............................... 110 6.2.2 The Length of TBCs is Increased by ORP9 Knockdown .............................. 110 6.2.3 ORP9 Knockdown Prevents the Maturation and Fusion of TBC Bulbs ....... 111 6.2.4 TBC Length is Increased and TBC Bulbs Accumulate at the Ultrastructural Level in ORP9 Knockdown Tissue ......................................................................... 112 6.2.5 ORP9 Knockdown Causes a Delay in Spermiation ...................................... 113 6.2.6 PI(4,5)P2 Localizes to the Area Containing TBCs ........................................ 114 6.3 Discussion ............................................................................................................. 115 6.4 Materials and Methods ......................................................................................... 121 6.4.1 Animals ........................................................................................................... 121 6.4.2 Reagents ........................................................................................................ 121 6.4.3 SiRNAs ........................................................................................................... 121 6.4.4 Intratesticular injections ................................................................................. 122 6.4.5 Tissue Collection ............................................................................................ 122 6.4.6 Antibodies and Control Reagents.................................................................. 123 6.4.7 Western blotting and Densitometry ............................................................... 123 xvi 6.4.8 Immunofluorescence ..................................................................................... 125 6.4.9 STED Microscopy and TBC Measurements ................................................. 126 6.4.10 Electron Microscopy and Bright Field Microscopy ...................................... 126 6.5 Figures .................................................................................................................. 128 7 Conclusion ............................................................................................................... 136 7.1 Overall Analysis .................................................................................................... 136 7.1.1 Structure ......................................................................................................... 136 7.1.2 Function .......................................................................................................... 138 7.2 Conclusions of Hypotheses .................................................................................. 142 7.3 Specific Contributions ........................................................................................... 144 7.4 Strengths and Limitations ..................................................................................... 146 7.5 Potential Research Applications .......................................................................... 148 7.6 Future Directions .................................................................................................. 149 Bibliography .................................................................................................................... 153 xvii List of Figures Figure 1.1 Schematic diagram showing the position of major intercellular junction in Sertoli cells. ............................................................................................................... 16 Figure 1.2 Fixed frozen sections of perfusion fixed rat testis, stained with fluorescent phallotoxin (green) for filamentous actin. ................................................................. 17 Figure 1.3 TBCs associated with the heads of mature spermatids at the apex of the seminiferous epithelium of the rat. ............................................................................ 18 Figure 1.4 Schematic diagram showing the different parts of a TBC. ............................ 19 Figure 1.5 Schematic diagram illustrating the current model of the life cycle of a TBC. 20 Figure 1.6 Schematic diagram of an endoplasmic reticulum-endosome membrane contact site (MCS) and some of its common functions............................................ 21 Figure 1.7 Schematic diagram of apical TBCs with high-magnification views of the TBC-ER MCS and potential functions modelled at these sites. ....................................... 22 Figure 2.1 Diagram showing a late stage spermatid with a surrounding apical process from a related Sertoli cell. ......................................................................................... 35 Figure 2.2 Immunofluorescence micrographs of stage VII spermatids and related Sertoli cell cytoplasm (apical processes) isolated from rat seminiferous epithelium. ........ 36 Figure 2.3 Immunofluorescence micrographs of stage VII spermatids stained with Nectin-3 (green) and DAPI for DNA (blue). .............................................................. 37 Figure 2.4 Electron micrographs of stage VII seminiferous tubules probed with a nanogold conjugated anti-Rab5 antibody enhanced with silver particles. .............. 38 Figure 2.5 Electron micrographs of stage VII seminiferous tubules probed with a nanogold conjugated anti-EEA1 antibody enhanced with silver particles. .............. 39 xviii Figure 2.6 Electron micrographs of stage VII seminiferous tubules probed with a nanogold conjugated anti-nectin-3 antibody enhanced with silver particles. .......... 40 Figure 2.7 Electron micrographs of stage VII seminiferous tubules probed with normal rabbit IgG (NRIgG) and buffer lacking primary antibody as controls for Rab5, EEA1, and nectin-3 stained tissue. Western blots of Rab5, EEA1, and nectin-3 antibodies used in the immunoelectron and immunofluorescence studies are also shown alongside their respective NRIgG antibody and calnexin loading controls. ............ 41 Figure 2.8 Schematic diagram showing the progression of a TBC as it forms. ............. 42 Figure 3.1 Endoplasmic reticulum associated with ES and TBCs. ................................. 61 Figure 3.2 IP3R localizes to the bulb regions of TBCs.................................................... 62 Figure 3.3 HOMER1 and TRPM6 localize predominately to the bulbs of TBCs. ........... 64 Figure 3.4 Diagrammatic summary of ER distribution in apical processes of Sertoli cells and the location of Ca2+ regulatory proteins at the various sites. ............................ 66 Figure 4.1 STED micrographs of perfusion fixed cryosections of rat testis at stage VII of spermatogenesis. ...................................................................................................... 78 Figure 4.2 Visualization of ORP9 and VAPA localization to the TBC-ER contact and their Western blot specificity controls. ...................................................................... 79 Figure 5.1 IP3R1 protein levels are significantly reduced by in vivo IP3R1 siRNA injections, while IP3R2 is unaffected. ....................................................................... 97 Figure 5.2 Reduction of IP3R1 levels causes a loss of IP3R staining only in the apical cytoplasm around late spermatids. ........................................................................... 98 xix Figure 5.3 Reduction of IP3R1 levels causes a loss of TBC actin, an apparent increase in ES actin, and the appearance of ectopic actin ‘stalks’ throughout the Sertoli cells. ................................................................................................................................. 101 Figure 5.4 Ectopic actin bundles that are present in the IP3R1 siRNA treated tissue have an ‘ES-like’ appearance at the ultrastructural level. ...................................... 102 Figure 5.5 Reduction of IP3R1 levels causes a loss in ORP9 and EEA1 staining around late spermatids. ORP9 and EEA1 staining demonstrate the TBC-ER contact sites and the vesiculated/fused TBC bulbs respectively. ............................................... 103 Figure 5.6 Reduction in IP3R1 levels causes the absence of TBCs and TBC bulbs, a disruption to ER morphology, and the presence of ectopic actin patches. ........... 104 Figure 5.7 Loss of IP3R1 causes late spermatids to be retained in the epithelium at late stage VIII of spermatogenesis. ............................................................................... 106 Figure 5.8 Summary diagram of the observed IP3R1 knockdown phenotypes. .......... 107 Figure 6.1 ORP9 knockdown is achieved in rat testes for periods of 2 and 3 days with daily injections of siRNA. ........................................................................................ 128 Figure 6.2 Tubulobulbar complexes (TBCs) are longer relative to controls in ORP9 knockdown testes. ................................................................................................... 129 Figure 6.3 EEA1 positive (fused) TBC bulbs are largely absent in ORP9 knockdown testes. ...................................................................................................................... 130 Figure 6.4 TBC tubes are longer and coalesced/fused TBC bulbs are largely absent at the ultrastructural level. ........................................................................................... 131 Figure 6.5 Knockdown of ORP9 prevents late spermatids from being released from the epithelium at late stage VIII of spermatogenesis. .................................................. 133 xx Figure 6.6 PI(4,5)P2 is localized to the apical cytoplasm of late spermatids in the area containing TBCs. ..................................................................................................... 134 Figure 6.7 Summary diagram of the observed ORP9 knockdown phenotypes. .......... 135 xxi List of Abbreviations AR Androgen receptor BSA Bovine serum albumin BTB Blood-testis barrier CME Clathrin-mediated endocytosis ER Endoplasmic reticulum ES Ectoplasmic specialization FSH Follicle-stimulating hormone GEL Gelatin IgG Immunoglobulin G LH Luteinizing hormone LTP Lipid-transfer protein MCS Membrane contact site NGS Normal-goat serum NMIgG Normal-mouse IgG NRIgG Normal-rabbit IgG ORP Oxysterol-binding protein (OSBP)-related protein OSBP Oxysterol-binding protein PBS Phosphate-buffered saline PI Phosphoinositide PIP Phosphatidylinositol phosphate PM Plasma membrane PS Phosphatidylserine PVDF Polyvinylidene fluoride RT Room temperature STED Stimulated emission depletion TBC Tubulobulbar complex TBS Tris-buffered saline TBST Tris-buffered saline, Tween-20 TEM Transmission electron microscopy iii Acknowledgments The greatest gift to my life has been the abundance of friends and family who have contributed to my story, and allowed me the privilege to be a character in theirs. So it is no overstatement that I could not thank them all adequately in a single page. But here is my best attempt. Thank you to my mentor Dr. Wayne Vogl, without whom none of this work would be possible. His ability to perform science with rigour and precision while doing so within the framework of the most admirable social conscience is beyond inspiring, and something exceedingly rare in our area of academia. I hope to be at least half the anatomist and human being that he is. I would like to thank some of my chosen family – Kyle, Jacob, Derek, Monika, Pete, Joe, Arshia, Rob, and countless others for being there for me when family was hard to come by. I would also like to thank Dr. Bradley Little and Dr. Stephen van Gaal for their courage as we worked towards breaking down social barriers in order to build the life we have together. Nothing worth doing is easy, or has been, but I would do it all again with those two. Finally, I owe a special thanks to my husband, Jake Stumph, whom I miss more than I can possibly say. Being separated from him by a global pandemic has challenged me more than any dissertation ever could. But he has always managed to show me his love from a distance and has kept me afloat. Thank you for making this whole adventure worth it. We will meet again. iv Dedication To my mother, Jacqueline, for giving me both grit and humour. These were essential to me sticking with this work. To my adopted parents, Diane and Scotty, whose compassion and care quite literally saved my life. 1 1 Introduction 1.1 Spermatogenesis The production of sperm cells (spermatozoa) by spermatogenesis is the cellular basis for male fertility. This process has three steps and is coordinated by the nurse-like Sertoli cells in the seminiferous tubules of the testes, which are responsible for providing nourishment and protection to developing spermatogenic cells (Griswold, 1995; Franca et al., 2016) (Figure 1.1). Spermatocytogenesis is the first step in this process, where primary spermatocytes are produced. This occurs when diploid spermatogonia in the basal compartment (the spermatogonial stem cell niche of the epithelium) undergo mitosis to produce diploid primary spermatocytes. These primary spermatocytes migrate into the adluminal compartment of the tubules, moving past a junction complex containing the blood-testis barrier (BTB) (O’Donnell et al, 2011; Russell, 1977; Smith and Braun, 2012; Dym and Fawcett, 1970). After traversing the BTB, these diploid cells complete meiosis-I to produce haploid secondary spermatocytes in the adluminal compartment. Secondary spermatocytes undergo meiosis-II and differentiate into spermatids, finishing the meiosis stage. Subsequently, these spermatids gain a tail, an acrosome, and undergo extensive DNA packaging in the last step of this process, spermiogenesis. As the spermatids polarize during spermiogenesis, they become attached to Sertoli cells by actin-related adhesion junctions called ‘ectoplasmic specializations’ (ESs), which are subsequently removed as spermatids mature and are prepared for release into the tubule lumen (O’Donnell et al, 2011). 2 The movement of developing sperm cells through the epithelium during spermatogenesis requires temporal and spatial coordination. Leydig cells in the testis produce testosterone when stimulated by luteinizing hormone (LH) from the pituitary gland. Sertoli cells are able to coordinate the timing of spermatogenesis in response to testosterone binding their androgen receptors (ARs) and from receiving follicle stimulating hormone (FSH) from the pituitary gland. 1.2 Sertoli cells of the seminiferous epithelium Sertoli cells are the major architectural and regulatory units of the seminiferous epithelium of the testis (Franca et al., 2016). Beyond their foundational role in spermatogenesis, they are also an ideal model system for exploring cytoskeleton/junction interrelationships in cells within the context of tissues. All major junction types are present in Sertoli cells (Cheng and Mruk, 2015), and elements of the cytoskeleton, particularly actin, are abundant (Hess and Vogl, 2015). Moreover, both the cytoskeleton and the associated junctions are dynamic and change in a regular and cyclical fashion during spermatogenesis. At the apex of the epithelium, unique actin/endoplasmic reticulum associated adhesion junctions, known as ESs, adhere Sertoli cells to spermatids. At the base of the epithelium, Sertoli cells are joined to neighboring Sertoli cells by enormous junction complexes that include ectoplasmic specializations, tight junctions, gap junctions, and desmosome-like junctions (Figure 1.1). Tight junctions within this complex contribute to formation of the blood–testis barrier (Dym and Fawcett, 1970). 3 Junction turnover is associated with two major morphogenic events that are fundamental to male fertility — (1) the release of spermatozoa into the duct system of the male reproductive tract, and (2) the movement of the next generation of spermatogenic cells from the spermatogonial stem cell niche at the base of the seminiferous epithelium into more apical regions where these cells complete meiosis and morphologically differentiate. At the apex of the epithelium, junctions between Sertoli cells and mature spermatids are disassembled as part of the process of sperm release, and new junctions are formed with early spermatids that are beginning to polarize deeper in the epithelium. At the base of the epithelium, the large junction complexes between neighboring Sertoli cells are disassembled above and assembled below spermatocytes as they translocate from basal to adluminal compartments of the epithelium (Russell, 1977; Smith and Braun, 2012). In both cases, the intercellular junctions are proposed to be internalized ‘intact’ as double-membraned invaginations into the Sertoli cells. 1.3 Turnover of intact junctions in cells In cells generally, internalization of intact intercellular junctions by double membrane vesicles into one or the other of two attached cells may be more common than currently appreciated. Gap junctions are routinely internalized by double membrane vesicles (Gaietta, 2002; Berthoud, 2004; Norris and Terasaki, 2020) and the process is clathrin dependent (Piehl et al, 2007). Claudins from adjacent cells also have been reported to be internalized into one or other attached cells in cultured epithelial cell lines where junction turnover is occurring rapidly (Matsuda et al, 2004). Tight junction 4 strands have been reported in large intracellular vesicles in cultured human colon adenocarcinoma cells when processed for freeze-fracture electron microscopic analysis (Polak-Charcon and Ben-Shaul, 1979). Presumably these latter structures would have been internalized as double membrane vesicles containing intact junctions. Internalization of intact nectin-based adhesion junctions, together with enclosed cytoplasmic contents of the adjacent cell, have been recently reported in a cultured human lung cell line (Generous et al, 2019). There are hints in the literature that similar processes observed in culture cells may also occur at the tissue level in animals. Similar to cultured cells, tight junction strands have been reported in large intracellular vesicles of mammalian intestinal epithelial cells processed for freeze-fracture analysis (Staehelin, 1973; Madara, 1990). In the seminiferous epithelium, the internalization of the attached membranes of two adjacent cells via double membrane endosomes is common and is mediated by unique double-membraned and actin-related structures termed “tubulobulbar complexes” (TBCs). TBCs form in Sertoli cells both at apical and at basal sites of intercellular attachment (Figure 1.1), and have been proposed to be “subcellular machines” that internalize intact junctions. 1.4 Tubulobulbar complexes (TBCs) TBCs are long tubular extensions of one cell that project into corresponding invaginations of the adjacent Sertoli cell (Russell and Clermont, 1976) (Figure 1.2 and Figure 1.3). At apical sites (Figure 1.2A and Figure 1.3) extensions of spermatids project into Sertoli cells. At basal sites (Figure 1.2B) projections of one Sertoli cell extend into the adjacent Sertoli cell. The structures have best been studied at apical 5 sites of attachment between Sertoli cells and spermatids where they appear prior to sperm release. Here, multiple generations of complexes develop in Sertoli cell regions associated with each maturing spermatid head. TBCs have been documented in all mammals studied, but are best morphologically characterized in the rat (Russell and Malone, 1980), making them the ideal model system to study TBCs. Although TBCs exist in humans (Vogl et al, 2013), comparatively little work has been done on human TBCs because of the lack of a human Sertoli cell culture system that can produce TBCs consistently. In the rat, a cluster of as many as 24 complexes form in two rows adjacent to the concave margin of each hook-shaped spermatid head (Russell and Clermont, 1976). At basal sites, the complexes occur at sites of intercellular attachment between neighboring Sertoli cells and are most numerous at stages of spermatogenesis well before spermatocyte translocation (Russell, 1979). Their early appearance has been suggested to gradually reduce the size of the massive intercellular junction complexes prior to the translocation event itself (Du et al., 2013; Vogl et al., 2014). TBCs are classically described as consisting of a long proximal tubule, a swollen bulbous (‘bulb’) region, and a short distal tubule ending in a 'bristle-coated” pit (Russell and Clermont, 1976). A network of microfilaments cuffs the tubular elements and cisternae of endoplasmic reticulum (ER) are closely associated with the bulb. The structures in most animals are approximately 1–2 μm in length, but they can reach up to 8 μm in some species, and although they appear to be general features of mammalian Sertoli cells (Russell and Malone, 1980), including human (Vogl et al., 2013), they have not been described in nonmammalian vertebrates. As the structures mature, the bulbs 6 separate from the structures, coalesce with other internalized bulbs and continue through endosomal compartments of the Sertoli cells. 1.5 Internalization of intact junctions by TBCs Results, mainly from immunofluorescence, show that TBCs contain junction proteins, and electron microscopic images demonstrate that “intact” junctions occur in the structures; that is, adjacent membranes do not separate, but remain attached. At apical sites, the structures have been shown to contain the adhesion junction proteins nectin-2 and α6β1 integrin (in the Sertoli cell membrane) and nectin-3 (in the spermatid membrane) (Guttman et al., 2004a; Young et al., 2009b, 2012b; Adams and Vogl, 2017). At basal sites, TBCs have been shown to contain the tight junction protein claudin-11 and the gap junction protein connexin-43 (Du et al., 2013), and both tight and gap junctions have been morphologically identified in electron micrographs of the structures (Russell, 1979; Du et al., 2013). Importantly, TBCs are associated with the endocytosis markers Rab5, EEA1, and LAMP1 (Guttman et al., 2004b; Young et al., 2009a, 2012a; Du et al., 2013). Together these results are consistent with the conclusion that TBCs internalize intercellular junctions (Figure 1.4). At apical sites, the absence or disruption of TBCs is correlated with a failure or a delay in sperm release (Kusumi et al., 2007; D’Souza et al., 2009; Young et al., 2012a). At basal sites, TBCs have yet to be linked directly to the mechanism of spermatocyte translocation. 7 1.6 TBC domains We now have a reasonably good understanding of the general molecular domain structure of TBCs (Figure 1.4). The coated pit contains clathrin and related proteins (Young et al., 2009a; Nicholls et al., 2011; Du et al., 2013). The tubular membrane cores are associated with the membrane curvature and scission related proteins, amphiphysin, and dynamin (Kusumi et al., 2007; Vaid et al., 2007), and are cuffed by a three-dimensional network of actin filaments (Vogl, 1989) together with a multitude of actin-related proteins (Mulholland et al., 2001; Guttman et al., 2004a; D’Souza et al., 2009; Lie et al., 2011; Young et al., 2009a; Young and Vogl, 2012; Chojnacka et al., 2017). The actin cuffs are in turn surrounded by an outer shell consisting of spectrin and plectin (Upadhyay et al., 2011; De Asis et al., 2013). ER-plasma membrane contact sites occur at the bulbs, and although there is some data suggesting that endosomal markers like Rab5 and EEA1 may be associated with these sites (Young et al, 2012) there is currently no high-resolution data to support the localization of specific proteins to the membrane contact sites at the bulbs. Other endosomal proteins reported to be present at TBCs include endophilin, syntaxin-7, and Vamp-2, and the lysosomal marker Lamp-2 (Upadhyay et al., 2014). The morphology and molecular composition of TBCs support the conclusion that the structures are clathrin and actin-network related, and are endocytic in nature. 1.7 Comparison of TBCs to other endocytosis mechanisms TBCs have similarities to tubular membrane invaginations reported in other cell systems. The morphology and composition of the tubular elements of the complexes 8 resemble podosomes in osteoclasts (Ochoa et al., 2000). Although the later structures do not appear associated with clathrin-coated pits or other endosomal structures and vesicles, they do have a tubular plasma membrane core that is cuffed by a network of actin filaments. Interestingly, TBCs bear a resemblance to the clathrin-related and actin-cuffed tubular invaginations observed in dynamin-1 & -2 knockout cells in a culture system (Ferguson et al., 2009), as well as to actin-cuffed tubular extensions seen on endosomes in VAP knockout cells (Dong et al., 2016). The most striking similarity of TBCs to other systems is to the basic architecture of clathrin-mediated endocytosis structures in cells generally (Taylor et al., 2011). In fact, as TBCs form, they resemble clathrin-coated pits with extremely long necks. Moreover, as the complexes mature, the bulbs begin to resemble early endosomes, which are also known to form contact sites with the ER (Phillips and Voeltz, 2016). TBCs differ from general clathrin-mediated endocytosis pathways in at least three major ways; (1) the membrane cores are formed by the plasma membranes of two adjacent cells that remain attached, (2) ‘bulbs’ form in the elongate necks, and (3) the bulbs appear to establish endosome-like features even before scission. 1.8 TBC formation and scission TBCs are initiated as clathrin-coated pits at intercellular junctions; however, the pits do not undergo scission (Figure 1.5). Rather, the actin related necks continue to elongate into the Sertoli cell. As each structure matures, a bulb begins to form near the end of the complex through the local loss of the actin network and the formation of extensive membrane contact sites with cisternae of ER. The bulb expands proximally 9 and eventually undergoes scission near the base of the structure (Lyon et al., 2015). It is not entirely clear what happens to the distal tubules and clathrin-coated pits at the end of the structures, but I suspect they likely are resorbed back into the bulbs prior to or during scission. 1.9 The role of actin in TBC structure Actin filament networks are clearly major components of TBCs. Perturbation of the networks with the actin destabilizing drug cytochalasin either prevents the tubular elements of TBCs from forming (Russell et al., 1988), or results in short, dilated, and abnormally shaped TBCs (Lyon et al., 2015; Sriram et al., 2016). The results of these studies are consistent with the conclusion that actin is involved with the elongation and maintenance of the tubular components of TBCs. These results also are in agreement with the proposal that the actin filaments associated with clathrin-mediated endocytosis generally in cells function “to constrict and elongate the bud neck and drive the endocytosed vesicles from the plasma membrane” (Collins et al., 2011). In yeast, the formation of dendritic actin networks is more necessary than even clathrin for endocytosis to occur, because it serves as the propulsive force to create invaginations in their dense, ergosterol rich membranes (Goode et al., 2015). While not as ubiquitous at sites of clathrin-mediated endocytosis in mammalian cells as in yeast, dendritic actin networks have been shown to be associated with and necessary for endocytosis that occurs at membranes under high tension such as those at intracellular junctions or those layered by associated cortical actin (Boulant et al., 2011; Skau and Waterman, 2015). The sheer amount of both junction proteins and para-crystalline actin layering the 10 plasma membrane at junctions in Sertoli cells likely provides similar membrane tension and rigidity to that seen in other systems. Thus, it is possible that the long actin-related regions of TBCs may have evolved in Sertoli cells as a substitute for conventional clathrin-mediated endocytosis at ESs in order to endocytose large “intact junctions” at membranes under high-tension. 1.10 Regulation of TBCs Although we now have a reasonably good understanding of the basic structure and function of TBCs, there are a number of key issues left to resolve - many of which involve regulation. What initiates the formation of TBCs is not known, nor is the signal for establishing formation of the bulb. The role of the endoplasmic reticulum membrane contact sites at TBCs is an important area to explore, particularly with respect to possibly establishing fluctuations in calcium levels, as well as having a potential role in establishing lipid domains in the plasma membrane at TBCs. 1.11 ER-endosome contact sites The ER has a multitude of important functions at membrane contacts, with some of these being to act as the primary vehicle for calcium dynamics in both excitable and non-excitable cells, initiating lipid modification and recycling, and facilitating endosome maturation, trafficking, and fission (Salvador-Gallego, 2017) (Figure 1.6). The ER forms extensive membrane contacts with other organelles, including those in the endocytic pathway such as endosomes (Friedman et al, 2013). The ER and endosomes are the two largest calcium stores in the cell and the close association of the two via MCSs has 11 been proposed to allow for calcium exchange between the two organelles (Sillence et al, 2008). This is reinforced by the fact that the main ER calcium release channel, IP3R, tends to be clustered and active at ER-endosome contact sites (Atakpa et al, 2018) and that calcium release from endosomes at ER contacts can stimulate further calcium release from the ER (Kilpatrick et al., 2017). The function of calcium exchange at ER-endosome contact sites is still not fully understood although it has been shown that ER calcium release at these sites is necessary for endosome acidification and maturation, indicating bi-directional calcium exchange is at least one crucial function of ER-endosome contacts (Morgan et al., 2013) (Figure 1.6B). Interestingly, depletion of the endosome calcium release channel TPC1 has been shown to disrupt the structure of ER-endosome contact sites (Kilpatrick et al, 2017), which may suggest that tethering between calcium channels at ER-endosome contact sites can contribute to the contact site’s stability. Tethering between the ER and endosomes is also thought to be partially established by VAP proteins (VAMP-associated protein) in the ER binding with various integral endosome proteins via their FFAT motifs (Loewen et al, 2003; Loewen & Levine, 2005). Some of the endosome associated proteins containing FFAT motifs include members of the StARkin superfamily (eg. STARD [START domain-containing] proteins) as well as the ORP (Oxysterol-binding protein [OSBP]-related proteins) family (Di Mattia et al, 2019). Additionally, both STARDs and ORPs are thought to mainly act as lipid transfer proteins (LTPs) (Di Mattia et al, 2019). LTPs like ORPs have been shown to contribute to non-vesicular lipid transfer between organelles at MCSs, making lipid transfer/exchange a principle feature of ER 12 membrane contacts (Holthuis & Levine, 2005). Some of these lipid exchange/modifying events include phosphoinositide (PI) replenishment and recycling (Kim et al., 2015) and phosphotidylserine (PS)/sterol counter transport driven by PI hydrolysis (Chung et al., 2015) (Figure 1.6C). One of the main proposed functions of ORPs generally is counter exchange between phosphoinositide phosphates (PIPs) in target organelle membranes (eg. Endosomes) with sterols from the ER (Mesmin et al, 2013). ORPs are able to bind sterols through their oxysterol-binding domain (Ridgway et al, 1992) and bind PIs (specifically PIPs, usually PI(4)P) through a pleckstrin homology (PH) domain (de Saint-Jean et al, 2011). The PIP and sterol are then exchanged between the two membranes in a process that is thought to be driven by the hydrolysis of the PIP once it is in the ER membrane, providing the energy to move the sterols across the hydrophilic cytosol of the contact site (Mesmin et al, 2017). This allows the ER to exert control over cellular events related to the target organelle by changing its lipid profile. At endosomes for example, the removal of PI(4)P from the membrane by ORPs allows the ER to control the nucleation of actin around the retromer complex, thus determining if the complex can undergo tubulation and budding (Dong et al, 2016). It is clear that ER membrane contacts are emerging as an important factor in regulating the composition and function of both the ER and its target organelles like endosomes, thus influencing a wide range of cellular events (Gallo et al., 2016). 1.12 Overall hypothesis and chapter aims My overall hypothesis is that the TBC-ER contact sites are partially responsible for the maturation of TBCs from the point of their initiation at the plasma membrane to 13 their eventual vesiculation. It is likely that processes such as calcium and lipid exchange, which occur at other ER contact sites, play a role in this regulatory function (Figure 1.7). There are five main predictions I have made and tested to support this overall hypothesis, as outlined in the following chapters of this dissertation. To begin, it would be crucial to understand the endocytic identity of the ‘bulb’ portion of the TBC. Previous studies provided evidence that TBCs were endocytic in nature due to their association with many known endocytic protein markers (Young, 2013). Of these, the GTPase Rab5 and its effector tethering protein EEA1 were found to localize to the area of TBCs using standard immunofluorescence and confocal microscopy (Young et al, 2012). Both proteins are known markers of endosomes generally in cells. However, confocal microscopy on its own did not have the resolving power to determine exactly which domains of the TBC Rab5 and EEA1 were associated with. In Chapter 2, I test the prediction that Rab5 and EEA1 are localized to the ‘bulb’ portion of the TBC by using an immunoelectron microscopy technique I adapted for use in whole tissues. Because their TBC morphology is well-defined, rat testes are used for all the experiments in this thesis. Localization of these proteins to the TBC ‘bulb’ will clarify its endocytic identity, and support the direction of the subsequent chapters of my dissertation, by allowing me to make appropriate analogies between TBC-ER contact sites and other ER MCSs. In Chapter 3, I explore whether or not the proteins known to be involved in common processes at ER MCSs are present at TBC-ER contact sites. Specifically, I test the prediction that calcium exchange machinery is localized to TBC-ER contact sites (Figure 1.7B). In these experiments, I use immunolocalization techniques 14 (immunofluorescence and immunoelectron microscopy) to provide evidence that calcium exchange proteins are enriched at these sites. This machinery would need to be present at the contact sites in order to enable calcium exchange as a possible mechanism used by the ER to regulate the maturation of TBCs. After providing evidence for my hypotheses related to calcium exchange, I then explore the prediction that lipid exchange may be a fundamental function of the TBC-ER contact sites as well. In Chapter 4, I test the prediction that common lipid exchange machinery is also localized to the TBC-ER contact sites (Figure 1.7C). Similar to my approach in Chapter 3, I use high-resolution immunolocalization techniques to provide evidence that common lipid exchange proteins are enriched at the contact sites. Chapter 5 represents a functional sequel to Chapter 3, where I test the prediction that the calcium exchange protein, IP3R (Itpr), that I localized to the TBC-ER contact sites previously, is functionally necessary for TBC maturation. Here I predict that if IP3R levels are reduced, then the maturation of TBCs and their ‘bulbs’ will be altered. Secondary to this phenotype, I also test the prediction that sperm release will be delayed if TBC maturation is affected by reduced IP3R levels. In these experiments, I use in vivo grade siRNA to target and degrade the mRNA of the IP3R1 isoform in rat testis, thus reducing the IP3R1 protein levels. I then use stimulated emission depletion (STED) and electron microscopies to make high-resolution observations of any cellular phenotypes. Finally, in Chapter 6 I explore the prediction that the lipid exchange protein ORP9, which I localized to the TBC-ER contact sites in Chapter 4, is necessary for TBC maturation. This is done by again using an in vivo approach, where I inject siRNA 15 targeting ORP9 mRNA into rat testes to reduce ORP9 protein levels. I then use high-resolution microscopy techniques to evaluate TBCs for changes to their structure due to ORP9 knockdown. 16 1.13 Introduction figures Figure 1.1 Schematic diagram showing the position of major intercellular junction in Sertoli cells. TBCs appear at intercellular junctions prior to sperm release at the apex of the epithelium and spermatocyte translocation at the base. Actin filaments are shown in red and endoplasmic reticulum is shown in yellow. 17 Figure 1.2 Fixed frozen sections of perfusion fixed rat testis, stained with fluorescent phallotoxin (green) for filamentous actin. Deconvolved super-resolution images taken with a STED (stimulated emission depletion) microscope (Leica TCS SP8). TBCs are visualized associated both at apical and at basal sites in the seminiferous epithelium. (A) At apical sites, clusters of TBCs are associated with each spermatid head. (B) At basal junction complexes, TBCs are clustered at sites where junction-related actin in ectoplasmic specializations is less apparent. In this image, the intercellular junction complexes are sectioned perpendicular to the long axis of the Sertoli cells and actin filament bundles associated with the junctions circle the cells like belts. 18 Figure 1.3 TBCs associated with the heads of mature spermatids at the apex of the seminiferous epithelium of the rat. (A) Deconvolved STED image of an apical process of a Sertoli cell surrounding the head of a late spermatid. Actin filament bundles associated with the junction and TBCs are labeled with a fluorescent phallotoxin. (B) Electron micrograph of an apical TBC. (C) Electron micrograph of the distal end of a TBC showing different regions (ER, endoplasmic reticulum). (D) Electron micrograph of a cross section through a TBC showing the two plasma membranes that make up the membrane core of the structure. 19 Figure 1.4 Schematic diagram showing the different parts of a TBC. The proteins that have been reported as being associated with each region (ER, endoplasmic reticulum; PM, plasma membrane) are shown. 20 Figure 1.5 Schematic diagram illustrating the current model of the life cycle of a TBC. The process begins from initiation as a clathrin-coated pit at an intercellular junction until scission and internalization into the Sertoli cell. Actin filaments are shown in red and endoplasmic reticulum is shown in yellow. 21 Figure 1.6 Schematic diagram of an endoplasmic reticulum-endosome membrane contact site (MCS) and some of its common functions. (A) An endosome (white) is shown with a small vesicle fusing at its surface. The ER (blue) makes contact with much of the endosome surface. (B) A high-magnification diagram of the ER-endosome MCS modelling calcium exchange. Calcium ions in high-concentrations in the ER move through IP3R channels (green) towards the endosome and are hypothesized to move into the endosome through yet to be identified uptake channels (yellow). (C) A high-magnification diagram of the ER-endosome MCS modelling lipid exchange. A lipid transfer protein (LTP) (purple) is attached to the ER via a dedicated membrane anchoring protein (orange). Step 1: the LTP grabs onto a phosphoinositide (PI, Red) in the endosome membrane. Step 2: the LTP brings the PI into the ER membrane and exchanges it for a sterol (white oval). Step 3: the sterol is inserted into the endosome membrane and the cycle repeats. 22 Figure 1.7 Schematic diagram of apical TBCs with high-magnification views of the TBC-ER MCS and potential functions modelled at these sites. (A) Schematic diagram of a late spermatid surrounded by a Sertoli cell apical process. TBCs are shown projecting into the apical process and have established ER contact sites at the bulbs (blue). A high-magnification insert shows the membrane contact site. We currently have no high-resolution data showing specific proteins at these sites. (B) One possible model for how calcium exchange may occur at the TBC-ER contact sites. Calcium is seen flowing through calcium channels from the ER and through the TBC outer membrane. (C) One possible model for how lipid exchange may occur at the TBC-ER contact sites. A phosphoinositide (red) in the TBC bulb outer membrane is shown being counter-exchanged for a sterol (white) from the ER membrane. 23 2 High Resolution Localization of Rab5, EEA1, and Nectin-3 to Tubulobulbar Complexes in the Rat Testis 2.1 Brief Synopsis While there is a growing body of work that supports the hypothesis that TBCs are endocytic devices (Young, 2013), there is still a lack of evidence for what the exact identity of the TBC ‘bulb’ is as it develops, vesiculates, and joins the endosomal-lysosomal system. TBCs are the mechanism by which ‘intact’ junctions are internalized into Sertoli cells (Figure 2.1). Two of the endocytosis markers that have been localized to TBCs are the GTPase Rab5 and its effector tethering protein EEA1. Rab proteins are GTPases considered to be master regulators of the endosomal system (McBride and Zerial, 2001). Their primary function is to activate effector proteins that control the distribution and fusion of vesicles and endosomes. Rab5 has been localized to the plasma membrane, vesicles, and early endosomes (Chavrier et al., 1990; Von Mollard et al., 1994). EEA1 is a tethering protein that mediates vesicle–endosome and endosome–endosome fusion and is the most widely known effector protein acted on by Rab5 (Mu et al., 1995). Recently, EEA1 has been shown to undergo a conformational change and provide the mechanical force necessary to bring vesicles close together when it is bound by Rab5-GTP on an incoming vesicle (Murray et al., 2016). Together, Rab5 and EEA1 are essential to the progression of internalized extracellular materials into the endosomal pathway. Although both Rab5 and EEA1 are enriched at or near the ends of TBCs when localized by immunofluorescence, their exact locations with respect to the 24 bulbs and to double-membrane vesicles that have been internalized is unclear. In fact, in many images from immunofluorescence studies, Rab5 appears discretely localized at the end of each TBC, raising the possibility that it is associated with the bulbs prior to scission. While nectin-3 has been localized to TBC bulbs with immunofluorescence, it has not been seen at TBC tubes and ‘budded’ bulbs as would be predicted by the junction internalization hypothesis. In this Chapter, I use a recently developed immunoelectron microscopy protocol (Melo et al., 2014) to precisely localize Rab5, EEA1 and nectin-3 to apical TBCs at the ultrastructural level. The fixation and staining protocol I use in the study preserves membranes much better than more traditional methods. My results indicate that Rab5 associates with nectin-3 containing bulbs prior to scission and that EEA1 binds later. The data are consistent with the hypothesis that TBC junction-containing bulbs are internalized into Sertoli cells and enter endocytic pathways, and that the TBC bulbs have an ‘endosome-like’ identity. 25 2.2 Results 2.2.1 Immunofluorescence Staining for Rab5 GTPase was concentrated near the ends of TBCs when processed for immunofluorescence (Figure 2.2A-C). Immunofluorescence with an EEA1 antibody showed staining of large vesicles in Sertoli cell apical processes surrounding late spermatids and clusters of TBCs (Figure 2.2D-F). Antibodies to nectin-3 adhesion proteins were seen, as predicted, around stage VII spermatid heads where they are known to be components of intercellular adhesion junctions (ectoplasmic specializations) between spermatids and Sertoli cells, and were also seen in the area of intact TBC tubes and bulbs (Figure 2.2G-I and Figure 2.3A-C). 2.2.2 Immunoelectron Microscopy At the ultrastructural level, staining for Rab5 occurred around intact bulbs that were identified by their double-membrane structure and close association with cisternae of ER (Figure 2.4A-D). Labeling was seldom seen at large vesicles nearby in the related Sertoli cell cytoplasm. Labeling with the anti-EEA1 antibody showed heavy reactivity with large complex vesicles near the ends of clusters of TBCs (Figure 2.5A-D). These are characterized by their large size, the presence of ‘double membrane’ profiles, and lack of association with ER cisternae. The large vesicles previously have been described as resulting from the aggregation of bulbs from TBCs that have been internalized by Sertoli cells (Russell, 1979). There was no antibody reactivity associated with TBC bulbs, which were identified by the presence of a double plasma membrane, the close association with cisternae of ER, their continuity with TBC tubular regions, and 26 the presence of a terminal coated pit (asterisk in Figure 2.5B). Staining for nectin-3 was seen at the ultrastructural level around spermatid heads, in TBC bulbs, tubes, and in the related large endosomes (Figure 2.6A-E). Small “specks” of nonspecific silver particles from the enhancement step were often seen in the lumen of TBC bulbs and small vesicles (Figure 2.6C); however, control tissue also contains these nonspecific particles. Controls for the primary antibodies consisted of replacing the antibodies with the same concentration of normal rabbit IgG (NRIgG) while controls for the secondary antibody involved omitting any primary antibody. Both controls showed no specific staining at TBC bulbs, tubes, or related large vesicles (Figure 2.7A-C). 2.2.3 Western Blots Specificity of the antibodies used for immunofluorescence and immunoelectron microscopy was determined with Western blots of both whole testis (WT) and seminiferous epithelium (SE) lysates. A band close to 24 kDa was detected corresponding to the molecular weight of Rab5 when stained with the anti-Rab5 antibody with two faint, nonspecific bands seen at 40 and 70 kDa (Figure 2.7D) (Chavrier et al., 1990). Western blots with the anti-EEA1 antibody showed a band around 180 kDa corresponding to previously published values for the EEA1 (Figure 2.7E) (Mu et al., 1995). Blots with the anti-nectin-3 antibody also showed a strong band around 67 kDa corresponding to the molecular weight of nectin-3 (Figure 2.7F) (Takahashi et al., 1999). 27 2.3 Discussion In this chapter, I demonstrate at the ultrastructural level that Rab5, EEA1 and nectin-3 are present in TBCs and related endosomes that form at sites of apical attachment of Sertoli cells to spermatids. The results are consistent with the hypothesis that TBCs internalize intercellular junctions. A summary of these results are diagrammed in Figure 2.8. Moreover, the results indicate that Rab5 initially associates with bulbs prior to their separation from TBCs and internalization by Sertoli cells. TBCs are double-membrane structures that form only at sites of intercellular attachment in the testis and are proposed to internalize junctions during sperm release and during spermatocyte translocation in the seminiferous epithelium (Vogl et al., 2014). Previous studies using immunofluorescence techniques have indicated that markers for endocytosis (Rab5 and EEA1) are present at apical TBCs (Young et al., 2012); however, it has been unclear exactly where these proteins localize in relation to TBCs and their related vesicles. Here I show that antibodies to Rab5 were localized mainly to the bulbs of intact TBCs. Rab5 GTPase is primarily responsible for activating EEA1 tethers on vesicles and early endosomes. Although Rab5 is understood to associate primarily with vesicles and early endosomes, it has also been reported to be involved in ligand sequestration at budding vesicles in vitro (McLauchlan et al., 1998). As Rab5 antibodies were localized to intact TBC bulbs, these results show the in vivo association of Rab5 to structures that are still continuous with the plasma membranes and have not yet undergone scission. Interestingly, this suggests that the identity of TBC bulbs as endosomes may begin prior to their actual separation from TBCs and their internalization by the Sertoli cell. 28 Unlike probes for Rab5, antibodies to EEA1 were localized to large endosomes near the ends of TBCs. These structures are presumed to result from the aggregation of bulbs that have budded from TBCs. In endocytosis pathways generally, EEA1 is a tethering protein that binds to phosphatidylinositol-3-phosphate (PI(3)P) in the membrane of early endosomes mediated by Rab5-GTP (Simonsen et al. 1999). EEA1 tethers on opposing vesicles facilitate early endosome fusion. Data presented here clearly indicates that EEA1 does not associate with TBCs themselves. Rather, it associates with vesicles formed by bulbs after they have undergone scission from TBCs. The association of these vesicles with EEA1 indicates that they can be classified as early endosomes. Antibodies to nectin-3, an adhesion protein present in the plasma membrane of spermatids, labeled TBC tubes and bulbs by immunofluorescence, as predicted if TBCs internalize intercellular junctions. Previous reports using fluorescent microscopy described nectin-3 as concentrated at the ends of TBCs at the bulbs (Young et al. 2009). However, here I used a higher resolution confocal microscope to resolve the localization of nectin-3 to TBC tubes as well as bulbs. At the ultrastructural level, antibodies to nectin-3 labeled TBC tubes, bulbs, and the related endosomes. In this chapter, I’ve shown that Rab5 appears largely localized to the bulbs of TBCs prior to their separation and internalization by the Sertoli cell, and that EEA1 is related to the bulbs somewhat later after they have undergone scission from TBCs and are aggregating into large endosomes. The observation that nectin-3 is present in TBCs and in associated endosomes clearly demonstrates that this spermatid junction molecule is internalized by adjacent Sertoli cells. These data support the hypothesis 29 that TBCs internalize intercellular junctions and clarifies the relationship between markers of endocytosis at the various stages of the TBC internalization process. 30 2.4 Materials and Methods 2.4.1 Animals All animals used in this chapter were reproductively active male Sprague Dawley rats (Rattus norvegicus) obtained from Charles River. Three animals were used for the pre-embedding immunoelectron microscopy, two for immunofluorescence, and three for lysates for Western Blot analysis. The animals were maintained according to the guidelines established by the Canadian Council on Animal Care. They were housed in OptiRAT cages, had ad libitum access to food (Teklad global protein 18%—diet 2918) and water, and were maintained in a 12/12 light cycle with lights on at 0700 h and off at 1900 h. The Animal Care Committee of the University of British Columbia approved all the protocols used in this study. 2.4.2 Reagents Unless otherwise indicated, all the reagents used in the chapter were obtained from Sigma-Aldrich. 2.4.3 Immunofluorescence Tissue preparation: Testes were removed from rats under deep anesthesia induced with isoflurane inhalation. Blood was cleared from the testes by perfusing the spermatic artery with PBS (150 mM NaCl, 5 mM KCl, 0.8 mM KH2PO4, 3.2 mM Na2HPO4, pH 7.3) for 30 min. Perfusion medium was then changed to 3% paraformaldehyde in PBS for 30 min followed by perfusion with PBS for another 30 min. Perfusion-fixed testes were decapsulated in PBS and the seminiferous tubules were 31 diced with razor blades. Tissue was transferred into a 50 mL plastic Falcon tube in PBS and gently passed into a syringe with an 18-gauge needle multiple times to further fragment the tissue. The precipitate was allowed to settle at the bottom of the tube for 5 min at room temperature and then the supernatant was transferred to a 14 mL falcon tube. The fragmented material was centrifuged for 30 s. The supernatant was removed and the pellet containing the spermatogenic cells was then suspended in PBS. The cells were added to poly-L-lysine-coated slides and allowed to incubate in a humidity chamber for 10 min. Immunostaining: Slides with attached tissue fragments were blocked with 1% bovine serum albumin (BSA) PBS for 1 h at room temperature. Primary antibodies were diluted in 0.1% BSA, 0.1% Saponin-PBS and consisted of a rabbit anti-EEA1 antibody used at 4.4 ug/ mL (New England Biolabs, Whitby, ON), rabbit anti- Rab5 antibody used at 10 ug/mL (Abcam, Cambridge, MA) and rabbit anti-nectin-3 antibody used at 5 ug/mL (Abcam Inc., Cambridge, MA). Antibodies were added to the tissue and incubated overnight at 4o C in a humidity chamber. The following day, the material was washed three times with PBS then incubated for 60 min at 37o C with goat anti-rabbit antibodies conjugated to Alexa Fluor 488 in 0.1% saponin–PBS. Alexa Fluor 488 goat-anti-rabbit antibodies were used to label the primary antibodies EEA1, Rab5, and nectin-3 and DAPI was used to stain the spermatid heads. The slides were again washed and coverslips mounted using Vectashield (Vector Labs, Burlington, ON) and the tissue was immediately visualized using a Leica DMI6000 Confocal microscope. Controls for immunofluorescent localization consisted of replacing the primary antibodies with normal rabbit immunoglobulin G (NRIgG) at an identical concentration to the highest 32 concentrated primary antibody (10 ug/mL), and replacing the primary antibodies with buffer alone to control for the Alexa Fluor 488 goat–anti-rabbit antibody. 2.4.4 Immunoelectron Microscopy Tissue preparation: The fixation protocol used was modified after that described elsewhere (Young et al., 2009). Rat testes were removed from animals deeply anesthetised with isoflurane and were perfusion fixed for 30 min at room temperature with 3% paraformaldehyde in PBS (150 mM NaCl, 5 mM KCl, 0.8 mM KH2PO4, 3.2 mM Na2HPO4 pH 7.3). Then, the testes were cut into 1– 2 mm sections with a razor blade and washed with PBS. The samples were processed further generally as described by Melo et al. (2014). After washing three times with PBS testes slices were immersed in 30% sucrose in PBS for 48 h at 4o C. The slices were immersed in Tissue-Tek O.C.T in aluminum foil molds and frozen and stored in liquid nitrogen. Sections of 10 uM thickness were cut on a cryostat at -20o C and transferred to poly-L-lysine coated slides. Sections were allowed to adhere to the slides for 8 min in humidity chambers before adding PBS for 5 min and then quenched of free aldehyde groups with 0.05M Glycine in PBS. Tissue was blocked with 1% bovine serum albumin (BSA) and 0.1% gelatin (GEL) in PBS for 30 min and further blocked with BSA–GEL–PBS with 1% normal-goat serum (NGS) for another 30 min. Immunolabeling and silver enhancement: Immunolabeling and silver enhancement were done as described by Melo et al. (2014). Primary antibodies were used at a dilution of 1:10 made up in BSA–GEL–PBS for 1 h. Tissue was blocked again with BSA–GEL–NGS– PBS for 30 min. The secondary antibody was a nanogold 33 conjugated goat-anti-rabbit fab’ fragment (Nanoprobes, Yaphank, NY) used at a 1:100 dilution in BSA–GEL– NGS–PBS for 1 h. Immunogold labeling was then enhanced using a HQ Silver Enhancement kit (Nanoprobes, Yaphank, NY) in the darkroom for 8 min before being immersed in 5% sodium thiosulfate. Silver enhancement is necessary to visualize the otherwise small gold particles; however, because the resulting metal precipitates vary in size, double labeling is not possible using this methodology. In addition, all antibodies used here were raised in rabbit, which also makes double labeling experiments problematic. 2.4.5 Electron microscopy Sections on slides were postfixed with 1% osmium tetroxide for 10 min followed by en bloc staining with 1% uranyl acetate for 5 min. All slides then underwent serial dehydration with increasing concentrations of ethanol solutions of 30, 50, 70, 95, and 100% for 10 min each before being infiltrated with a 1:1 mixture of propylene oxide and EMbed 812 (Electron Microscopy Sciences, Hatfield, PA) for 1 h. Custom made silicone capsules were filled with 100% EMbed 812 and inverted over the slides containing the tissue and carefully transferred to an oven to polymerize at 60o C for 48 h. After polymerization of the resin, the slides with attached polymerized blocks were placed in liquid nitrogen for ~2–3 min and then removed. This allowed the glass slides to be separated from the tissue which remained in the polymerized Embed 812 blocks. Stage VII seminiferous tubules were selected based on their characteristic morphology by viewing the blocks under a dissecting microscope and the blocks trimmed accordingly. 34 The blocks were sectioned on a Leica Ultramicrotome and sections imaged using a FEI Tecnai G2 Spirit Electron Microscope operated at 120 kV. 2.4.6 Western Blotting Western blots were performed on both whole testis and seminiferous epithelium lysates that were prepared as described by Vogl (1996). Proteins were transferred onto Immobilon-P transfer membrane (Millipore, Billerica, MA) and then washed for 15 min at room temperature with TBST (500 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween-20). The blots were blocked overnight at 4-80 C using 4% non-fat milk. Membranes were then incubated for 1 hr at room temperature with a rabbit anti-EEA1, rabbit anti-Rab5, or rabbit anti-nectin-3 antibody, and then washed three times with 4% milk/TBST. Controls consisted of replacing the primary antibodies with normal rabbit IgG or normal mouse IgG at the identical primary antibody concentration. Membranes were then incubated at room temperature with a goat anti-rabbit horseradish peroxidase-conjugated antibody for 1 h at room temperature and then washed three times with TBST. Blots were reacted with ECL (Millipore, Etobicoke, ON) and bands visualized with an ImageQuant LAS4000 Imager (GE Healthcare Lifesciences, Mississauga, ON). 35 2.5 Figures Figure 2.1 Diagram showing a late stage spermatid with a surrounding apical process from a related Sertoli cell. Tubulobulbar complexes (TBCs) can be seen growing into the Sertoli cell cytoplasm away from the concave surface of the late spermatid. Apical TBCs are proposed to internalize junction proteins allowing late spermatids to be released from the seminiferous epithelium. The following features are color coded: actin (red), endoplasmic reticulum (blue), and clathrin-coated pits (green). 36 Figure 2.2 Immunofluorescence micrographs of stage VII spermatids and related Sertoli cell cytoplasm (apical processes) isolated from rat seminiferous epithelium. Respective antibody labeling is shown in green and spermatid nuclei are stained with DAPI for DNA. Controls consisted of substituting primary antibody for NRIgG, and omitting primary antibody (2o control). (a) Staining with an anti-Rab5 antibody shows a signal in the Sertoli cell apical process where TBC bulbs are known to occur. (b,c) Controls for Rab5 show no staining in the area of TBC bulbs. (d) EEA1 is seen in a more central position in the apical Sertoli cell process. (e,f) Controls for EEA1 showed no apical process staining. (g) Nectin-3 staining appeared around the spermatid head, and also appeared to line TBC tubes extending into the Sertoli cell cytoplasm. (h,i) Controls for nectin-3 were negative for antibody signal. 37 Figure 2.3 Immunofluorescence micrographs of stage VII spermatids stained with Nectin-3 (green) and DAPI for DNA (blue). (a) Nectin-3 staining is seen in TBC tubes (white arrow) and around the spermatid head. (b) Nectin-3 staining is seen largely along the entire length of TBC tubes and around the spermatid head. (c) Nectin-3 staining is shown to be more concentrated at the ends of TBCs and around the spermatid head. 38 Figure 2.4 Electron micrographs of stage VII seminiferous tubules probed with a nanogold conjugated anti-Rab5 antibody enhanced with silver particles. White arrows are used to indicate prominent particles corresponding to anti-Rab5 antibodies and asterisks are used to indicate ER cisternae. (a) Cross section through a late spermatid with anti-Rab5 antibody conjugated gold particles concentrated in the surrounding Sertoli cell cytoplasm. (b) Intact TBC bulb identified by its double membrane and association with cisternae of ER. Particles are seen both between the ER and the outer membrane of the bulb as well as at an area of the bulb with no associated ER. (c) A gold particle is seen localized to an intact TBC bulb identified by its double membrane and clathrin-coated pit. (d) High-magnification of (a) showing particles bound to an intact TBC bulb and to vesicles in the surrounding cytoplasm. Small, nonspecific “specks” of silver can be seen in the lumen of some of the surrounding vesicles. 39 Figure 2.5 Electron micrographs of stage VII seminiferous tubules probed with a nanogold conjugated anti-EEA1 antibody enhanced with silver particles. White arrows are used to indicate prominent particles corresponding to anti-EEA1 antibodies. (a) Cross section through a late spermatid with anti-EEA1 antibody conjugated gold particles concentrated in the surrounding Sertoli cell cytoplasm around TBC bulbs. (b) Vesiculated bulb with heavy anti-EEA1 antibody staining next to an intact TBC bulb (ER labeled with asterisks) that lacks localized gold particles. (c) High-magnification of (a) showing gold particles bound to a vesiculated TBC bulb. (d) Vesiculated bulb with heavy anti-EEA1 antibody staining. 40 Figure 2.6 Electron micrographs of stage VII seminiferous tubules probed with a nanogold conjugated anti-nectin-3 antibody enhanced with silver particles. White arrows are used to indicate prominent particles corresponding to anti-nectin-3 antibodies. (a) Cross section through a late spermatid with anti-nectin-3 antibody conjugated gold particles concentrated in various regions of the surrounding Sertoli cell cytoplasm. (b) Cross section through the proximal end of a late spermatid head with antinectin-3 antibody conjugated gold particles lining the spermatid-Sertoli cell adhesion junction. (c) Anti-nectin-3 antibody staining shown at a TBC tube. (d) Gold particle staining is seen at coalescing and fusing TBC bulbs. (e) Gold particles conjugated to nectin-3 antibodies are seen at an intact bulb as well as various small vesicles and vesiculated TBC bulbs. 41 Figure 2.7 Electron micrographs of stage VII seminiferous tubules probed with normal rabbit IgG (NRIgG) and buffer lacking primary antibody as controls for Rab5, EEA1, and nectin-3 stained tissue. Western blots of Rab5, EEA1, and nectin-3 antibodies used in the immunoelectron and immunofluorescence studies are also shown alongside their respective NRIgG antibody and calnexin loading controls. (a) Cross section through a late spermatid probed with NRIgG with no substantial gold particle staining seen. (b) TBC bulb from tissue probed with NRIgG showing no gold particle staining around the bulb and only minimal, nonspecific staining in the surrounding cytoplasm. (c) Cross section through a late spermatid probed only with secondary antibody with no substantial gold particle staining seen. (d-f) Western blots of whole testis (WT) and seminiferous epithelial (SE) lysates from normal rats. (d) Western blot with the anti-Rab5 antibody showing a band close to 24 KDa corresponding to the molecular weight of Rab5, and two nonspecific bands appearing at 40 and 70 KDa. (e) Western blot with the anti-EEA1 antibody showing at 180 KDa corresponding to the molecular weight of EEA1. (f) Western blot with the anti-nectin-3 antibody showing a band at 70 KDa corresponding to the molecular weight of nectin-3. 42 Figure 2.8 Schematic diagram showing the progression of a TBC as it forms. (a,b) A TBC undergoes scission, and fuses with other bulbs (c). Nectin-3 is associated with the spermatid plasma membrane and is internalized with the TBC as it grows into the Sertoli cell cytoplasm. Rab5 begins to associate with the bulb region of the TBC while it is continuous with the spermatid and Sertoli cell plasma membranes, and while it is in contact with cisternae of ER (navy blue). EEA1 becomes associated with the bulb after its vesiculation and remains associated with these and other endosomes, helping to facilitate their fusion. 43 3 Ca2+ Signaling Machinery is Present at Intercellular Junctions and Structures Associated with Junction Turnover in Rat Sertoli Cells 3.1 Brief Synopsis TBCs are proposed to be subcellular machines that internalize intercellular junctions during the process of junction turnover in the seminiferous epithelium (Vogl et al, 2013). Previous work by Clermont and coworkers (Clermont et al, 1980) has shown that the ER forms a continuous network in the apical Sertoli cell processes. The function of the ER at ESs and at TBCs is not entirely clear. One function that has been postulated for the ER at ESs is that it is somehow involved with Ca2+ signaling, and that this regulates either the junctions themselves or the actin cytoskeleton (Means et al, 1980; Franchi et al, 1985; Guttman et al, 2002). A role in Ca2+ signaling also has been suggested for the ER associated with TBC bulbs (Vogl et al, 2013). In cells generally, one of the major functions of the ER, partly through the establishment of close contacts with the PM and with other organelles, is maintaining Ca2+ homeostasis and generating Ca2+ fluxes that serve as signals (Burgoyne et al, 2015; Gallo et al, 2016; Phillips and Voeltz, 2016). If Ca2+ signaling/exchange is one of the functions of the ER at ESs and at TBCs, then the molecular machinery known to orchestrate changes in local Ca2+ levels in other systems should be present at these sites. One of the proteins that may be present in the ER of Sertoli cells is the Ca2+ release channel, inositol 1,4,5-trisphosphate receptor (IP3R, Itpr). 44 As there have been no previous studies evaluating IP3R at ESs or elsewhere in the Sertoli cell, we sought to determine whether or not IP3R is indeed present. If IP3R is present at TBC bulbs, then there may be binding partners present that couple IP3R in the ER to the Sertoli cell PM. One well-established binding partner for IP3R is HOMER1. HOMER1 is a scaffolding protein that interacts with other proteins through its EHV1 domain (Tu et al, 1998). The HOMER1 EHV1 domain binds to a proline-rich region in other proteins characterized by proline-proline-x-x-phenylalanine (PPXXF) sequence (Hayashi et al, 2006). The PPXXF motif appears in a variety of other receptors and channels such as transient receptor potential (TRP) channels, allowing them to be coupled with IP3R (Yuan et al, 2003). A candidate for coupling to IP3R via HOMER1 in Sertoli cells is TRP channel subfamily M member 6 (TRPM6). The Ca2+ permeable TRPM6 channel is part of the melastatin-related TRP channels that localize to the PM (Runnels, 2001). There have been no reports of TRPM6 functioning in a complex with HOMER1 and IP3R; however, TRPM6 contains a Homer-binding ligand, PPXXF, at residues 1367–1371 (accession no. F1M7G0). Combined with the fact that TRPM6 is upregulated in the testis (Uhlen et al, 2015), these observations suggested to us that TRPM6 may be present at TBC bulbs. Here we use conventional electron microscopy to explore the relationship of the ER to adjacent membranes in apical processes, and use immunofluorescence and immunoelectron microscopy to probe the system for those proteins known to be involved in generating ER-related Ca2+ fluxes in other systems. We demonstrate that the transient receptor potential channel subfamily M member 6 (TRPM6), Homer1 (HOMER1), and inositol 1,4,5-trisphosphate receptor (ITPR, IP3R) are present at ER- 45 PM contacts associated with TBC bulbs. In Sertoli cells, the molecular machinery associated with ER generated Ca2+ fluxes is present at structures directly related to junction remodelling - a process necessary for sperm release. 46 3.2 Results 3.2.1 Characterization of the endoplasmic reticulum at ectoplasmic specializations and tubulobulbar complexes We first attempted to characterize the ER both at ESs (Figure 3.1A) and at TBCs (Figure 3.1B) by measuring the distance between the ER cisternae and Sertoli cell PM in transmission electron micrographs of normal tissue from two different animals. The averaged closest distances between the ER and the PM at ESs were determined to be 72.5 ± 3.5 nm (95% CI, n = 21) and 73.0 ± 2.1 nm (95% CI, n = 19) (Figure 3.1, inset). An area of dense hexagonally-packed actin was consistently observed to separate the ER from close contact with the Sertoli cell PM (Figure 3.1A). In contrast, the average closest distance between the ER and the Sertoli cell PM at the bulbs of TBCs was 12.2 ± 0.7 nm (95% CI, n = 24) and 12.7 ± 0.6 nm (95% CI, n = 21) (Figure 3.1, inset). When compared statistically, membrane distances were found to be significantly affected by subcellular location (ESs versus TBCs) (p < 0.0001, two-way ANOVA). Membrane distance was not affected by animal (p = 0.81, two-way ANOVA), and subcellular location and animal did not significantly interact (p = 0.88, two-way ANOVA). The data indicate that any variations in fixation quality between the two utilized animals did not significantly affect membrane distance. In both animals, the ER was closer to the PM at the bulbs of TBCs than at ESs (Figure 3.1, inset). The average measured intermembrane gap between the ER and PM at the bulbs was well within the range of that expected for an ER-PM contact of 10–30 nm (Stefan et al, 2013). When evaluated in tomograms, the ER related to bulbs form flattened cisternae that encapsulate each 47 bulb and interconnect, either as sheets or tubes, with the ER of neighboring bulbs (Figure 3.1C and D). Filamentous linkages connecting the ER and the Sertoli cell PM were clearly visible in the gap between the two adjacent membranes (Figure 3.1B, E, and F). 3.2.2 The Ca2+ release channel, IP3R, localizes to the bulb regions of tubulobulbar complexes In cryosections of stage VII seminiferous tubules, the IP3R antibody most strongly labeled regions within Sertoli cell apical processes where TBCs are known to occur and did not convincingly label ESs (Figure 3.2A). To evaluate the position of the IP3R labeling with respect to TBCs, we used phalloidin staining of the actin networks present at tubular regions of TBCs or an antibody labeling cortactin (CTTN), a component of these actin networks. In cryosections of stage VII seminiferous tubules, the IP3R antibody labeling is seen near the ends of labeled TBCs (Figure 3.2B and C). The antibody also labeled the acrosomes of round spermatids in stage VII seminiferous tubules (Figure 3.2A and B). At TBCs, the IP3R antibody labeled TBC bulbs in regions corresponding to the gap between the actin networks of proximal and distal tubules (Figure 3.2D). The pattern of labeling at TBCs with the primary antibody was not observed in the NMIgG specificity controls for sections and fragments (Figure 3.2E and F), and the antibody reacted with a single band in the range expected for IP3R on immunoblots (Figure 3.2G). We further investigated the spatial location of IP3R at TBCs using immunoelectron microscopy. At bulb regions of TBCs, the IP3R antibody labels the ER that is closely associated with the Sertoli cell PM (Figure 3.2H and I). The same 48 labeling pattern was not seen in equivalent electron micrographs of primary antibody controls (Figure 3.2J). 3.2.3 TRPM6 and HOMER1 localize to the bulb regions of tubulobulbar complexes Using immunofluorescence and pre-embedding immunoelectron microscopy, we found that both HOMER1 and TRPM6 localized to TBC bulbs (Figure 3.3). At the fluorescent level, antibodies to both proteins localized to regions where TBC bulbs are known to occur (Figure 3.3A and B). Additionally, the HOMER1 antibody labeled regions where we would expect ESs to be (Figure 3.3A). Using pre-embedding immunoelectron microscopy, we were more accurately able to see labeling at TBC bulbs for HOMER1 (Figure 3.3D and E), and TRPM6 (Figure 3.3G and H). Normal rabbit IgG (NRIgG) controls were negative for staining at TBC bulbs (Figure 3.3F and I). Western blots were also performed as specificity controls for both HOMER1 and TRPM6 antibodies (Figure 3.3C). A band for HOMER1 appears around 40 kDa corresponding to its predicted molecular weight. A prominent band also appears around 60 kDa, which may be due to interactions between HOMER1 and one or more of its binding partners. Western blots for TRPM6 show a band at ∼230 kDa that also corresponds to its molecular weight with additional bands that also appear in the NMIgG control blot (Figure 3.3C). 49 3.3 Discussion The ER is a prominent component of Sertoli cells (Clermont et al, 1980), and elements of the system have for many years been recognized as characteristic features both of unique actin filament-related junctions (ESs) in this cell type (Russell, 1977; Brokelmann 1963; Nicander, 1967) and of structures termed tubulobulbar complexes responsible for junction internalization (Russell and Clermont, 1976; Russell et al, 1988; Guttman et al, 2004). Here we show that cisternae of ER associated with the bulb regions of TBCs meet the morphological criteria for being membrane contact sites (MCSs). Proteins involved with Ca2+ regulation are found at these contact sites. A diagrammatic summary of this localization pattern in apical processes of rat Sertoli cells is shown in Figure 3.4. It has long been suspected that the ER component of the unique adhesion junctions (ESs) in Sertoli cells functions to generate local changes in Ca2+ levels which in turn regulate either the junctions themselves or the related actin bundles (Franchi et al, 1985; Pelletier et al, 1999). A model previously proposed by Guttman et al. (Guttman et al, 2002) predicted that IP3R was present in the ER at ESs and releases Ca2+ to initiate disassembly of the actin bundles at sites during junction remodeling that occurs at sperm release and turnover of the blood–testis barrier. Although this disassembly was thought to be due to the Ca2+-dependent actin-severing protein, gelsolin, this was later shown not to be the case (Guttman et al, 2007). A more recent study has shown that plastin-3 (PLS3), an actin-bundling protein that is negatively regulated by Ca2+, is present at ESs (Li et al, 2015) and a release of Ca2+ may function to inhibit the bundling activity of PLS3. 50 At TBC bulbs, our ultrastructural analysis shows the presence of extensive ER-PM contact sites where the gap between the two adjacent membranes is approximately 12 nm. At these sites, our immunolocalization results indicate that IP3R, TRPM6, and HOMER1 are present. In HEK cells, a different TRP channel (TRPC1) has been shown to form a complex with IP3R and HOMER1 and is involved in store-operated calcium entry (SOCE) (Yuan et al, 2003). HOMER1 binding blocks DD/E residues on TRPC1 (Yuan et al, 2012). The stimulation of IP3R leads to dissociation of the IP3R-HOMER1-TRPC1 complexes (Kim et al, 2006) revealing the DD/E residues. Our observation that TRPM6 is in the same regions as IP3R and HOMER1 makes this potential mechanism attractive. The function of any potential ER-mediated Ca2+ oscillations at TBCs is unknown; however, Ca2+ signaling could be related either to regulation of the actin cytoskeleton in neighboring tubular regions or to the eventual identification and function of the bulbs as putative endosomes. It has previously been shown that TBC bulbs are associated with early endosome markers before and after budding, providing evidence for their identity as putative endosomes (Adams & Vogl, 2017). Interestingly, the maturation and acidification of endosomes has been linked to their release of luminal Ca2+, a process that is inhibited when endosome Ca2+ levels are artificially reduced (Gerasimenko et al, 1998). At the same time, TRP channels have been found to enable regulated release of Ca2+ from both endosomes and lysosomes (Abe and Puertollano, 2011). Because TBC bulbs are putative endosomes, it is perhaps not surprising then that TBC bulbs would contain TRP channels like TRPM6 to potentially release Ca2+ for maturation and acidification. While TRPM6 was selected as a Ca2+ release candidate at TBCs based on 51 bioinformatics data, it is likely that other TRP channels could also be involved in this process such as in endosomes generally. Furthermore, the possibility remains that Ca2+ movement between TBCs and the ER, if occurring, could be bidirectional. Endoplasmic reticulum Ca2+ is known to be sequestered into lysosomes and possibly endosomes via currently unidentified uptake channels (Lopez-Sanjurjo et al, 2013). Further experimentation making use of Ca2+ imaging of TBC bulbs as they develop and mature will be crucial for elucidating any Ca2+ fluxes between the ER and TBCs. The presence of IP3R, TRPM6, and HOMER1 at the bulbs certainly indicates that the machinery necessary to generate Ca2+ fluxes occurs at these MCSs. In this study, we identify a new ER-PM membrane contact site in Sertoli cells at the bulbs of TBCs in the apical processes. Our results also show that proteins known to be involved with regulating intracellular Ca2+ levels in other systems are present at TBCs. Importantly, this work provides the first solid support for the hypothesis that local fluctuations in Ca2+ generated by subdomains of the ER in Sertoli cells may play a significant role in events that occur during spermatogenesis, particularly those related to junction remodeling. 52 3.4 Materials and Methods 3.4.1 Animals All animals utilized in this study were reproductively mature male Sprague-Dawley rats (Rattus norvegicus) obtained from Charles River Laboratories. They were housed in accordance with guidelines established by the Canadian Council on Animal Care and with protocols approved by the Animal Care Committee of the University of British Columbia. These guidelines were also in accordance with the Ethical Guidelines for Publication of Research Results in Biology of Reproduction. All rats were healthy at the time of use and ranged from 300 to 500 g in weight. 3.4.2 Anesthesia and euthanasia All animals used in this study were anesthetized by isoflurane inhalation. While under deep anesthesia, the testes were removed and then processed further using protocols specific to each experiment. After testes were removed, animals were euthanized while under deep anesthesia by opening the thorax and cutting the heart. 3.4.3 Reagents Unless otherwise noted, reagents were obtained from Sigma-Aldrich. 3.4.4 Electron microscopy Fixation and embedding: The spermatic artery on the dorsal surface of each testis was canulated with a 26-G needle and then the organs were gravity perfused briefly with phosphate buffered saline (PBS) (150 mM NaCl, 5 mM KCl, 0.8 mM 53 KH2PO4, 3.2 mM Na2HPO4, pH 7.3, 33o C) to remove blood, and then for 30 min with fixative [0.1 M sodium cacodylate (EM Sciences), 1.5% paraformaldehyde (Fisher), and 1.5% glutaraldehyde (EM Sciences), pH 7.3, 33o C]. The testes were cut into small (1 cm3) blocks, and the blocks fixed further by immersion for an additional 2 h in the same fixative at room temperature. Samples were washed three times in 0.1 M sodium cacodylate for 10 min and then post-fixed on ice for 1 h in 1% OsO4 (EM Sciences) in 0.1M sodium cacodylate buffer. Samples were washed three times (10 min each wash) with ddH2O and then stained en bloc with 1% uranyl acetate (EM Sciences) for 1 h at room temperature. Samples were washed three times with ddH2O (10 min each wash), dehydrated through an ascending alcohol series (30%, 50%, 70%, 95%, 100%), passed through two incubations in 100% propylene oxide (Fisher), left overnight in 1:1 solution of propylene oxide: EM-bed 812 (EM Sciences), passed through two 2-h incubations in 100% EM-bed 812, and then embedded in 100% EM-bed 812 and incubated at 60o C for approximately 48 h. Sectioning and imaging: Thick (1 μm) sections for light microscopy were obtained using a Leica EM UC6 Ultramicrotome (Leica Microsystems) fitted with glass knives. Sections were stained with toluidine blue and photographed using a Zeiss Imager A1 Microscope (Carl Zeiss) utilizing bright-field optics. For analysis by conventional electron microscopy, thin sections (900 Å) were obtained using the same ultramicrotome as indicated above, but fitted with an Ultra-AFM 3.0 mm diamond knife (DiATOME). Sections were collected on naked 200 mesh copper grids and then stained first with saturated uranyl acetate in 75% methanol and then with standard Reynolds’ 54 lead citrate. The material was imaged using a Tecnai G2 Spirit Transmission Electron Microscope (FEI) operated at 120 kV. 3.4.5 Electron tomography For electron microscopic tomography studies, material from three animals was used. Sections were cut at around 2000 Å and collected on formvar-coated copper slot grids (EM Sciences). Sections were stained with 2% aqueous uranyl acetate for 30 min and then Reynolds’ lead citrate for 15min. Grids were stored in a desiccator at room temperature until they were analyzed. Images for tomography were collected using a Tecnai G20 LaB6 Transmission Electron Microscope (FEI) operated at 200 kV. Data were extracted using TIA (FEI), an automated acquisition program. The 3D volumes were reconstructed using real-space back-weighted projection implemented in Inspect 3D (FEI). Tilt series alignment and modeling was done using Amira 5.50 (FEI). 3.4.6 Distance measurements and statistical analysis Transmission electron microscopy (TEM) images from two animals were used to measure the distance between the PM and ER both at the bulbs of apical TBCs, at ESs, and at the periphery of the apical process. Each associated cistern of ER was measured at the closest distance between the two membranes using ImageJ software. Average values for distances were calculated for each animal and expressed as means ± 95% confidence interval. Because the data were distributed along a continuous variable (distance) and defined by two independent, categorical variables (animal and subcellular location), two-way analysis of variance (ANOVA) was utilized for statistical 55 comparison between ER at TBC bulbs and at ESs. Significance was defined as P < 0.05. Measurements of the distance between the ER and the periphery of apical processes were obtained identically but were not compared statistically to bulbs or ESs. 3.4.7 Pre-embedding immunoelectron microscopy The immunoelectron microscopy fixation protocol used was modified after that described by Young et al. (2009). Rats were anesthetized with isoflurane before testes were removed and perfusion fixed for 30 min at room temperature with 3% paraformaldehyde in PBS. Sections of fixed testis (1–2 mm) were cut with a razor blade and then washed three times with PBS. Testis slices were then immersed in 30% sucrose in PBS as a cryoprotectant for 48 h at 4o C. Cryoprotected slices were then covered in Tissue-Tek O.C.T in aluminum foil holders then frozen and stored in liquid nitrogen. Frozen tissue was sectioned to 10-μm thickness with a cryostat at –200 C and transferred to poly-L-lysine-coated slides. Tissue sections were allowed to adhere to the slides for 8 min, washed with PBS for 5 min, and incubated in 0.05 M glycine in PBS for 10 min. Blocking was then done with 1% bovine serum albumin (BSA) and 0.1% gelatin (GEL) in PBS for 30 min and BSA–GEL–PBS with 1% normal goat serum (NGS) for another 30 min. Immunolabeling and silver enhancement were done using an original protocol by Melo et al. (2016) with several modifications to this and the electron microscope done as described in detail by Adams and Vogl (2017). Primary antibodies were used at a dilution of 1:10 made up in BSA–GEL–PBS for 1 h. Blocking was then done with BSA–GEL–NGS–PBS for 30 min. The secondary antibodies were diluted in blocking buffer and samples were incubated with secondary for 1 h. Gold particles were 56 then enhanced using an HQ Silver Enhancement kit from Nanoprobes in the darkroom for 8 min before being incubated in 5% sodium thiosulfate. Sections were post-fixed with 1% OsO4 for 10 min followed by en bloc staining with 1% uranyl acetate for 5 min. Serial dehydration was performed with ethanol solutions (30%, 50%, 70%, 95%, and 100%) for 10 min each before being infiltrated with a 1:1 mixture of propylene oxide and EMbed 812 for 1 h. Silicone capsules containing EMbed 812 were inverted over tissue and polymerized at 60o C for 48 h. Polymerized blocks were placed in liquid nitrogen for 2–3 min to allow the glass slides to be separated from the tissue in the blocks. Stage VII tubules were selected using a dissecting microscope and the blocks were trimmed to isolate these tubules. Blocks containing tissue were sectioned on a Leica Ultramicrotome, and imaging was done using an FEI Tecnai G2 Spirit Electron Microscope operated at 120 kV. 3.4.8 Immunofluorescence microscopy Fixation: Testes were fixed as described for EM using fixative for fluorescence (PBS, 3% paraformaldehyde, pH 7.3). After 30 min, the organs were washed by perfusion with 0.22-μm-filtered PBS warmed to 33o C. One half of the testis was used to obtain cryosections, while the other was used to obtain epithelial fragments. Cryosections: The half of each testis dedicated to cryosections was mounted onto an aluminum stub using optimal cutting temperature compound (Sakura Finetek). The testis was flash-frozen using liquid nitrogen, and 10-μmthick sections were obtained using a cryomicrotome. Sections were collected on poly-L-lysine-coated slides and 57 immediately plunged into cold acetone for 5 min and subsequently placed into a PBS/0.1% BSA bath for 5 min. Epithelial fragments: The half of each testis dedicated to epithelial fragments was decapsulated, and the seminiferous tubules were cut into small pieces using scalpels in a petri dish containing PBS. The pieces were transferred into 50 mL falcon tubes and fragmented by aspirating them first through an 18-G needle followed by a 21-G needle. The solution was allowed to stand for 10 min to allow larger fragments to sink to the bottom. The supernatant was transferred to a 14 mL falcon tube and centrifuged to precipitate fragments that contained late spermatids with surrounding Sertoli cell apical processes. The supernatant was removed and the pellet was resuspended in 1 mL of PBS. These fragments were transferred to poly-L-lysine-coated slides. The fragments were allowed to adhere to the slide for 10 min inside a humidity chamber. Antibody labeling and imaging: Both sections and fragments had excess liquid drained off, and a hydrophobic marker was used to draw a well around the samples to keep solutions over the tissue. Sections were blocked with 5% BSA/PBS for 1 h, while fragments were blocked with 5% BSA/0.1% saponin/PBS for 1 h. Primary antibodies for sections were diluted in 0.1% BSA/PBS, while primary antibodies for fragments were diluted in 0.1% BSA/0.1% saponin/PBS. Once added to the samples, antibodies were left on overnight at 4o C in a humidity chamber. The samples were washed three times for 10 min each by replacing the solution on the tissue with fresh 0.1% BSA/PBS each time. Secondary antibody was diluted in 0.1% BSA/PBS for sections and 0.1% BSA/0.1% saponin/PBS for fragments. The secondary antibody was added to the samples and incubated at 37o C for 1 h. They were then washed for 10 min by replacing 58 the solution with fresh 0.1% BSA/PBS and stained with 300 nM DAPI in 0.1% BSA/PBS for 10 min. The samples were washed again with fresh 0.1% BSA/PBS and then mounted using Vectashield antifade mounting medium (Vector Laboratories). Samples were imaged using a Leica TCS SP5 confocal microscope. Select images were further processed using a Lucy-Richardson deconvolution algorithm in the ImageJ plugin, Deconvolution Lab 3.4.9 Western blotting Whole testis lysates: Testes from several animals were decapsulated, and the tissue was pooled. The tissue was homogenized in radioimmunoprecipitation assay (RIPA) buffer (150mMNaCl, 50mMTris, 5 mM, EDTA, 1% Nonidet P-40, 1% deoxychloic acid, 0.1% SDS, pH 7.3) containing mini-EDTA-free protease inhibitor (Roche). Seminiferous epithelium lysates: This lysate was prepared using the method developed by Guttman and colleagues. (Guttman et al, 2004) and is described here briefly. Testes were decapsulated and cut into smaller pieces while in cold PEM/250 buffer (0.2 M Pipes, 0.1 M EGTA, 0.1 M MgCl2, 250 mM sucrose). The seminiferous epithelium was removed from the tubule walls using two probes. Tissue was concentrated by centrifugation and homogenized using RIPA lysis buffer containing mini-EDTA-free protease inhibitor (Roche). Protein concentration of both lysates was determined using Pierce BCA protein assay kit (Thermo Fisher Scientific) and then diluted down to 2mg/mL using Laemmli sample buffer. Aliquots were stored at –20o C until needed. 59 Sodium dodecyl sulfate polyacrylamide gel electrophoresis and membrane transfer: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Laemmli and is briefly described here. Lysates were loaded into 1-mm-thick SDS-PAGE gels and run at 100 V for 1 h. The gel was allowed to equilibrate in wet transfer buffer (25 mM Tris, 192 mM glycine, pH 8.3) for 10 min. Proteins were transferred to polyvinylidene fluoride membranes using wet transfer for either 1 h at constant 350 mA or overnight at 4o C and constant 90 mA. Immunolabeling and detection: We used our standard blocking buffer, 4% non-fat milk in TBST (50 mM Tris, 150 mM NaCl, 0.1% Tween, pH 7.5), for washing the membrane throughout the experiment as well and refer to it as blot buffer. The membrane was blocked with blot buffer either overnight at 4o C or for 1 h at room temperature. Primary antibody was diluted in blot buffer and added to the membrane for 1 h at room temperature. This was followed by another three washes with blot buffer for 5 min each wash. Secondary antibody was diluted in blot buffer and added to the membrane for 1 h at room temperature. The membrane was washed three times for 5 min each wash with TBST and transferred to ECL solution for 3 min. Membranes were imaged on an ImageQuant LAS 4000 system (GE Life Sciences). Optimizations: The ITPR blot had 20 μg of protein loaded into a 6% polyacrylamide gel and was transferred overnight at 4o C. The STIM1 and ORAI1 blot had 30 μg of protein loaded into a 10% polyacrylamide gel and was transferred for 1 h at room temperature. The ATP2A2 blot had 60 μg of protein loaded into a 10% polyacrylamide gel and was transferred for 1 h at room temperature. The TRPM6 blot had 20 μg protein loaded into a 6% polyacrylamide gel and was transferred overnight at 60 4o C. Following transfer, the TRPM6 blot was incubated in stripping buffer (62.5 mM Tris-HCl, 2% SDS, 0.7% β-mercaptoethanol, pH 6.8) prior to blocking in order to facilitate the exposure of protein epitopes that is often necessary with transmembrane proteins. The HOMER1 blot had 20 μg protein loaded into a 10% polyacrylamide gel and was transferred for 1 h at room temperature. 61 3.5 Figures Figure 3.1 Endoplasmic reticulum associated with ES and TBCs. (A) Electron micrograph of a typical ES comprised of the Sertoli cell PM, associated bundles of actin filaments, and an adjacent cisternae of ER. (B) Electron micrograph of a typical bulb region of a TBC showing filamentous linkages between the ER and PM of the Sertoli cell (arrowheads). (A/B, inset) The distance between the ER and the Sertoli cell PM was measured at both ESs (arrowhead in A) and TBCs in two animals. Distance of the ER from the PM at ESs was found to be significantly different than the ER at TBC bulbs (P < 0.0001, two-way ANOVA). Error bars represent 95% CI. (C) One angle of view from a tomogram showing multiple TBC bulbs (asterisks). (D) Three-dimensional reconstruction of the tomogram in panel C showing that the ER at the bulb region of TBCs is continuous with the ER of neighboring bulbs. (E) A single angle view of a TBC bulb tomogram showing a filamentous connection between the ER and the Sertoli cell PM (arrowhead). (F) The 3D reconstruction of the tomogram in panel E shows multiple filamentous connections between the ER and Sertoli cell PM (red). It also clearly shows that the ER is a lamellar. 62 Figure 3.2 IP3R localizes to the bulb regions of TBCs. 63 Cryosections of rat testes were probed with antibodies against IP3R (ITPR) and analyzed by confocal microscopy. (A) Maximum projection of stage VII seminiferous epithelium showing the IP3R antibody labeling regions within the apical processes of Sertoli cells (arrowheads) in regions known to contain the bulbs of TBCs. (B) Phalloidin-labeled actin merged with IP3R antibody labeling shows that the latter probe labels regions near the ends of TBCS. (C) Maximum projection of a deconvolved Z stack better illustrating the labeling of the IP3R antibody (arrowheads) at the ends of TBCs (arrows). (D) Maximum projection of a deconvolved Z stack of isolated epithelial fragments showing that the IP3R antibody binding (arrowheads) is located between the proximal (PT) and distal tubules (DT—arrows). (E, F) Maximum projection of an epithelial fragment and cryosection treated with normal mouse IgG (NMIgG) as a control for the specificity of the IP3R antibody which was acquired using the same settings as those used to acquire panels A and B. (G) Western blot analysis of the specificity of the IP3R antibody shows a single band above 250 kDa in both whole testis lysate (WT) and seminiferous epithelium lysate (SE). Lysates probed with the same concentration of NMIgG show no bands. An antibody against calnexin (CANX) was used as a loading control. Expected molecular weight of IP3R I/II/III = 316, 260, 250 kDa. (H, I) Immunoelectron micrographs showing IP3R antibody labeling (arrowheads) at the ER (false colored blue) associated with the bulb regions of TBCs (false colored green). (J) A bulb region of a TBC in tissue probed with NMIgG instead of the IP3R antibody as a specificity control. 64 Figure 3.3 HOMER1 and TRPM6 localize predominately to the bulbs of TBCs. 65 Cryosections of rat testes were probed with antibodies against either HOMER1 or TRPM6. (A) Confocal maximum projection of stage VII seminiferous epithelium with HOMER1 antibody largely labeling the area of TBC bulbs as well as the dorsal aspect of the spermatid head where we would expect ESs to occur (arrowheads). The NRIgG control is also shown and is absent for stain in both the area of TBCs and the PM. (B) Confocal maximum projection of stage VII seminiferous epithelium with TRPM6 antibody showing concentrated stain in the area of TBCs as well as at the PM. The NRIgG control has no such staining pattern in the area of TBCs. (C) Western blots for the specificity of the HOMER1 and TRPM6 antibodies. The membrane stained with HOMER1 antibody shows a band around 40 kDa corresponding to the molecular weight of HOMER1 with an additional band around 60 kDa that may be due to aggregation of HOMER1 with one or more of its binding partners. The normal rabbit serum (NRS) control is absent for the 40 kDa HOMER1 band. A band for TRPM6 appears around 230 kDa that corresponds to its molecular weight as well as numerous over small bands. The NRIgG control shows many of the smaller bands in the TRPM6 stained membrane, and is absent for the 230 kDa TRPM6 band. (D, E) Electron micrographs showing TBC bulbs (green), associated ER (blue), and gold particles indicating HOMER1 antibodies (arrowheads). Gold particles are shown localized to TBC bulbs adjacent to the ER. (F) NRIgG control for HOMER1 shows no gold particles at bulbs. (G, H) Electron micrographs showing TBC bulbs (green), associated ER (blue), and gold particles indicating TRPM6 antibodies (arrowheads). Binding of gold particles is concentrated at TBC bulbs both associated with ER (G) and those already vesiculated (H). (I) NRIgG control for TRPM6 is absent for gold particles at TBC bulbs. 66 Figure 3.4 Diagrammatic summary of ER distribution in apical processes of Sertoli cells and the location of Ca2+ regulatory proteins at the various sites. Within the apical process of Sertoli cells, the ER associates with the PM of the Sertoli cell at the bulbs of TBCs. At these locations, we have identified IP3R, TRPM6, and HOMER1 where they may act in a complex where HOMER1 is the cytosolic factor bridging TRMP6 in the PM with IP3R in the ER. The ER is also a component of the ESs where we have identified both IP3R and ATP2A2 which may function to regulate Ca2+ levels within ESs. At the periphery of the apical process where ER is closely associated with the PM, we have identified STIM1, ORAI1, and ATP2A2 which may function to replenish depleted Ca2+ stores by SOCE. STIM1 and ORAI1 were also observed in the general ER of Sertoli cells. 67 4 Lipid transfer machinery is present at membrane contact sites associated with the internalization of junctions in Sertoli cells 4.1 Brief Synopsis TBCs have many features in common with clathrin-mediated endocytosis (CME) generally in cells (Young et al, 2009; Adams and Vogl, 2017). In fact, TBCs likely evolved from conventional CME machinery because their extremely long tubular parts resemble the much shorter necks of coated pits, and the structures are actually capped by a clathrin-coated pit at their ends (Young et al, 2009). The ‘neck’ of a TBC may reach microns in length and is cuffed by a dendritic actin network. During the elongation phase, a MCS is established with the endoplasmic reticulum at a site just proximal to the ends of the TBC (Lyon and Adams et al, 2017). Unlike the long tubular neck, the membrane contact site is devoid of actin and the contacted area progressively swells into a large ‘double membrane’ endocytic vesicle or bulb. The entire process from TBC initiation to the swelling of the bulb occurs prior to scission, creating ambiguity about whether or not the MCS is more like an ER-PM or an ER-Endosome contact, or something entirely different. Identifying the proteins that populate the contact site would not only provide insight into how the contact may function, but also how it might relate to the overall mechanism of junction turnover in the seminiferous epithelium. Among the numerous functions attributed to MCSs generally in cells is lipid transfer/exchange (Raiborg et al, 2015). The movement of lipids between the ER and opposing membranes allows for the initiation of important cellular events, establishing 68 and maintaining membrane identity, and lipid storage. In cells generally, the ER forms extensive membrane contact sites with endosomes in order to exchange and store calcium, mark positions where they will undergo scission, and store sterols (Phillips and Voeltz, 2016). I’ve previously proposed that TBC bulbs are likely putative endosomes, due to the presence of known endosomal proteins at the bulb PM (Adams and Vogl, 2017) and the presence of calcium exchange proteins at TBC-ER MCSs (Lyon and Adams et al, 2017). If TBC bulbs have an endosome-like identity, then it is likely that in addition to proteins involved with Ca2+ exchange, lipid transfer machinery also may be present at the contacts. To determine if elements involved in lipid transport occur, I screened for the presence of known lipid transfer proteins at ER-TBC contacts. I focused on the Oxysterol-Binding Protein (OSBP)-Related Protein (ORPs) family and their integral ER membrane anchor, VAPA, as possible indicators of lipid exchange mechanisms, because of their extensive involvement at other ER-MCSs (Jackson et al, 2016). ORPs are proposed to be able to remove PIs from the target organelle membrane and exchange them for sterols from the ER. This ‘counter-exchange’ model is proposed to be driven by hydrolysis of the PI once it is brought into the ER (de Saint-Jean et al, 2011). Because PIs and sterols both act as signalling molecules, ORPs are able to have a substantial impact on cellular events such as trafficking related to the target organelle (Lipp et al, 2020). The idea that ORPs may be present at TBC-ER contacts is attractive, as it presents one possible mechanism for the regulation of TBC bulb maturation and progression. In order explore the prediction that lipid transfer machinery is present at TBC MCSs, I immunofluorescently labelled perfusion fixed cryo-sections of rat testis with probes specific for each of the lipid 69 exchange proteins ORPs 1,3,5,7,8,9,10,11 and VAPA, and imaged the sections using stimulated emission depletion (STED) microscopy. Antibodies to ORP9 (Osbpl9) and VAPA were the only probes positive for signal and were found to be localized to the TBC-ER contacts. I propose that lipid exchange is one of the functions of this MCS and that lipid exchange between the ER and Sertoli cell plasma membrane at TBCs may play a significant role in the mechanism of junction turnover in the seminiferous epithelium. 70 4.2 Results 4.2.1 Lipid transfer protein ORP9 and its integral membrane anchor, VAPA, specifically label TBC-ER contacts In Stage VII seminiferous tubules just prior to spermatid release, ORP9 was localized to the area of the TBC-ER contacts as determined by its specific, periodic distribution in the concavity of the late spermatid ends where I would predict to see TBC bulbs and is not concentrated elsewhere in regions surrounding the spermatid heads (Figure 4.1A). Antibodies for all other ORPs (1,3,5,7,8,10,11) did not produce a positive immunofluorescent signal in my hands (data not shown). Co-labelling with phalloidin to mark for the TBC actin network reinforced the localization by demonstrating that ORP9 was present near the ends of the actin-related tubular regions (necks) of the TBCs - regions consistent with the localization of the TBC-ER contacts (Figure 4.1B and C). Normal mouse IgG and secondary antibody controls were negative for any similar staining patterns (Figure 4.1D and E). The VAMP-associated Protein A (VAPA; Vapa) was found to label the ER generally in Sertoli cell cytoplasm encapsulating spermatid heads, but appeared to be more specifically concentrated in the area of TBC bulbs (arrow heads) (Figure 4.1F). This is particularly evident in sections co-labelled for actin (Figure 4.1G and H). There was little to no staining seen in the normal mouse IgG and secondary antibody controls (Figure 4.1I and J). A high-resolution electron micrograph of the TBC-ER contact is shown (Figure 4.2A) to better demonstrate the area of ER that ORP9 and VAPA localize to. Western blots of rat seminiferous epithelium lysate showed single bands at the appropriate 71 molecular weights for ORP9 and VAPA (Figure 4.2B). This result together with the staining controls indicate that the antibodies are likely specific for ORP9 and VAPA. 4.3 Discussion Here I show that lipid exchange machinery localizes to ER MCSs at those TBCs associated with junctions between Sertoli cells and late spermatids. I chose to screen for Oxysterol-Binding Protein (OSBP)-Related Proteins (ORPs) because of their ability to counter exchange specific PIs, phosphatidylinositol phosphates (PIPs), with sterols (Antonny et al, 2018). PIPs have been identified as serving as both crucial markers for the initiation of CME and also as key determinates of membrane identity throughout the endosomal system (Kaksonen and Roux, 2018; Jackson et al, 2016). Because I have previously established that the TBC bulb has an endosome-like identity even prior to scission (Adams and Vogl, 2017), the idea that the insertion of certain PIPs in the Sertoli cell plasma membrane may be important for establishing the TBC bulb’s identity as a putative endosome prior to scission is attractive. I used available antibodies to screen for ORPs that are known to be associated with ER-endosome contacts, ORP-5 & -8 (Phillips and Voeltz, 2016). There was no evidence for staining of ORP-5 & -8 at TBCs (data not shown), and so I proceeded to test other members of the ORP family – ORPs 1,3,7,9,10,11. Of these antibodies, only the antibody to ORP9 reacted with regions known to contain TBCs bulbs. The ORP family all have several domains in common that make them suitable for PI and sterol counter exchange: an N-terminal PH domain that recognizes PIs and allows binding to the target membrane (Levine and Munro, 2002), an FFAT domain in the middle of the protein that enables them to associate with VAPs (Loewen and Levine, 72 2003), and an ORD domain near the C-terminal end that binds target sterols (Mesmin et al, 2013). ORP9 is known to have both long (ORP9L) and short forms (ORP9S), and the long form is known to localize to ER-Trans-Golgi contacts and has PH, FFAT, and ORD domains (Ngo and Ridgway, 2009). Given my observation here of the molecular weight of ORP9 in Western blots, it is likely that the form localized to the TBC-ER contacts is the long form. Furthermore, there is in vitro evidence that ORP9 functions in sterol transport between the trans-Golgi and the ER in a PI(4)P dependent manner (Liu and Ridgway, 2014). The exchange of PI(4)P with sterols is known to be driven by a phosphatidylinositide phosphatase (Sac1/Sacm1L), which hydrolyzes PI(4)P at the ER membrane (Mesmin et al, 2013). I have yet to find a suitable antibody to localize Sacm1L in rat testis, though I would predict it to have a similar localization to ORP9 and VAPA at TBC-ER contacts if PI(4)P is being transported at these sites. It is also possible that other PIs are being transported and perhaps acted upon by different phosphatidylinositide phosphatases. My finding that ORP9 is associated with MCSs that are being identified at putative endosomes prior to scission from the plasma membrane is new. My Western blot data using rat seminiferous epithelium lysate showed a single, specific band at the corresponding molecular weight for ORP9. This result together with the staining controls indicate that antibody is likely specific for ORP9 in the seminiferous epithelium. Because ORPs are known to require VAPA or VAPB as an integral membrane anchor that binds them via their FFAT domains (Loewen and Levine, 2003), I also screened for VAPA and VAPB in the same manner as the ORPs. VAPA was found to distribute widely throughout the ER of Sertoli cells, though appeared more heavily 73 concentrated at the apex of the epithelium around the late spermatid heads. While VAPA is known to localize widely throughout the ER, it is thought to be only functionally active at ER contacts with the Golgi and endosomes, where one of its functions is to anchor lipid transfer proteins to the ER (Wu et al, 2018). My data is the first evidence that lipid transfer proteins are localized to MCSs at TBCs and that lipid transfer may be occurring at these sites. From a general cell biology perspective, it is the first time to my knowledge that lipid transfer machinery has been seen at an endocytic structure while it is still attached to the plasma membrane. TBCs are known to be crucial for junction turnover, and at apical junctions with spermatids their disruption results in delayed sperm release or spermiation failure (Young et al, 2012). It is interesting that at TBCs, ER contacts are established prior to scission, unlike at other endocytic structures. The presence of lipid transfer machinery at these contacts while the TBCs are still continuous with the plasma membrane suggests that lipid transfer may be necessary for TBC maturation and/or involved with establishing the identity of the bulbs as putative endosomes. The presence of lipid transfer machinery at ER MCSs associated with TBCs opens a new field of inquiry into the function of lipid transfer at MCSs during junction turnover in the seminiferous epithelium and the impact of this function on the mechanisms of sperm release and spermatocyte translocation. Future works should focus on further characterization of lipid transfer proteins at the TBC-ER contacts, as well as knockdown experiments to observe any effects on junction turnover and sperm release. 74 4.4 Materials and Methods 4.4.1 Animals Animals used in this study were Sprague-Dawley rats (Rattus norvegicus) and were obtained from Charles River Laboratories. Rats were maintained and housed in conditions that follow the guidelines established by the Canadian Council on Animal Care and with approval from the Animal Care Committee of the University of British Columbia. These guidelines were also in accordance with the Ethical Guidelines for Publication of Research Results in Biology of Reproduction. All rats 300 to 500 g in weight when sacrificed. 4.4.2 Tissue collection All animals were anesthetized using isoflurane and their testes were removed and processed using protocols specific to each experiments, which are detailed below. After collecting tissues, animals were euthanized while under anesthesia via thoracotomy and cutting of the heart. 4.4.3 Antibodies Anti-ORP9 (Cat #: sc-398961) and Anti-VAPA (Cat #: sc-293278) antibodies were obtained from Santa Cruz Antibodies (Dallas, Texas). The ORP9 antibody was used at a 1:100 dilution in 0.1% BSA/PBS and the VAPA antibody was used at 1:20 dilution in 0.1% BSA/PBS. 75 4.4.4 Electron microscopy Testis were gravity perfused briefly with phosphate buffered saline (PBS) (150 mM NaCl, 5 mM KCl, 0.8 mM KH2PO4, 3.2 mM Na2HPO4, pH 7.3, 33° C) wash and then fixed for 30 min with [0.1 M sodium cacodylate (EM Sciences), 1.5% paraformaldehyde (Fisher), and 1.5% glutaraldehyde (EM Sciences), pH 7.3, 33° C]. The testes were cut into smaller blocks and were immersed overnight in 0.1 M sodium cacodylate. Samples were then post-fixed on ice for 1 h in 1% OsO4 (EM Sciences). Samples were washed three times with ddH2O (10 min each wash), dehydrated through an ascending alcohol series (30%, 50%, 70%, 95%, 100%), passed through two incubations in 100% propylene oxide (Fisher), left overnight in 1:1 solution of propylene oxide : EM-bed 812 (EM Sciences), passed through two 2 h incubations in 100% EM-bed 812, and then embedded in 100% EM-bed 812 and incubated at 60° C for approximately 48 h. Thin sections of ~85 nm were obtained using a Leica EM UC6 Ultramicrotome with an Ultra-AFM 3.0 mm diamond knife (DiATOME). Sections were collected on 200 mesh copper grids and then sequentially stained first with uranyl acetate in 75% methanol and then with standard Reynolds’ lead citrate. Grids were imaged using a Tecnai G2 Spirit Transmission Electron Microscope (FEI) operated at 120 kV. 4.4.5 Immunofluorescence microscopy Testes were fixed using a similar mechanical setup as above in Electron Microscopy, but with PBS, 3% paraformaldehyde, pH 7.3 for 30 min. Testes were then washed by perfusion with PBS warmed to 33° C. Testes were mounted onto an aluminum stub using Tissue-Plus O.C.T compound (Fisher Scientific). The testes were flash-frozen 76 using liquid nitrogen, and 6-μm thick sections were obtained using a Cryostat (Microm HM 525, Thermo Scientific). Sections were collected on poly-L-lysine-coated slides and immediately plunged into cold acetone for 5 min and subsequently placed into a PBS/0.1% BSA bath for 5 min. Sections were blocked with 1% BSA/PBS for 1 h and then primary antibodies diluted in 0.1% BSA/PBS were added for 1 h. The samples were washed three times for 10 min each with 0.1% BSA/PBS. Secondary antibodies were diluted in 0.1% BSA/PBS, added to the samples, and incubated at 37° C for 1 h. They were then washed for 10 min in PBS and stained before being stained with 5uM Yoyo-3 Iodide (Thermofisher) for 20 min. The samples were washed again with PBS and then mounted using ProLong Diamond Antifade mountant (Thermofisher). 4.4.6 STED Microscopy Imaging was performed with a Leica TCS SP8 STED Microscope. Alexa Fluor 488 was excited at 499 nm and depleted using the 592 nm depletion laser. Alexa Fluor 594 was excited at 592 nm and depleted at 775 nm, Yoyo-3 Iodide was excited at 638 nm. For more detailed information on the microscope setup used here, see Gao et al, 2019. 77 4.4.7 Western blotting Testes were decapsulated and cut into smaller pieces while in cold PEM/250 buffer (0.2 M Pipes, 0.1 M EGTA, 0.1 M MgCl2, 250 mM sucrose). The epithelium was ‘stripped’ from the seminiferous tubules using microsurgical forceps. Tissue was concentrated by centrifugation and homogenized using RIPA lysis buffer containing mini-EDTA-free protease inhibitor (Roche). Protein concentrations were determined using Pierce BCA protein assay kit (ThermoFisher Scientific) and then diluted down to 2 mg/mL using Laemmli sample buffer. Aliquots were stored at –20° C until needed. For more detailed information, see Guttman et al (2004). Lysates were loaded into 1-mm-thick SDS-PAGE, 10% gels and run at 120 V for 1-2 h. The gels were allowed to equilibrate in wet transfer buffer (25 mM Tris, 192 mM glycine, pH 8.3) for 10 min. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes using wet transfer for 1 h at constant 350 mA. Membranes were blocked in 4% milk in TBST (50 mM Tris, 150 mM NaCl, 0.1% Tween, pH 7.5) for 1h at RT. Primary antibody was diluted in 4% Milk/TBST and added to the membrane for 1 h at RT. Membranes were then washed with 4% Milk/TBST for 5 min, three times. Secondary antibody was diluted in 4% Milk/TBST and added to the membrane for 1 h at RT. The membrane was washed three times for 5 min each with TBST and transferred to ECL solution for 4 min. Membranes were imaged on an ImageQuant LAS 4000 system (GE Life Sciences). 4.4.8 Reagents Unless stated otherwise, all reagents were purchased from Sigma-Aldrich. 78 4.5 Figures Figure 4.1 STED micrographs of perfusion fixed cryosections of rat testis at stage VII of spermatogenesis. (a-c) Anti-ORP9 antibody (green). (b & c) ORP9 (green), actin (red). (d) NMIgG control and secondary antibody control (e). (f-g) Anti-VAPA antibody (green) is concentrated at TBC bulbs (arrow heads). (f-h) VAPA (green), actin (red). (i) NMIgG control and secondary antibody control (j). 79 Figure 4.2 Visualization of ORP9 and VAPA localization to the TBC-ER contact and their Western blot specificity controls. (a) Electron micrograph of a TBC. The arrow head indicates ER at the TBC bulb while the arrow indicates the Sertoli cell PM of the TBC. Asterisks indicate the actin cuff around the TBC neck. (b) Western blots stained with the anti-ORP9 and anti-VAPA antibodies. (c) Schematic diagram illustrating the localization of ORP9 and VAPA at TBC-ER contacts and their possible function in lipid exchange. 80 5 Knockdown of IP3R1 Disrupts TBC-ER Contact Sites and the Morphology of Apical Processes Encapsulating Late Spermatids 5.1 Brief Synopsis One of the characteristic features of TBCs is that they form ‘bulbs’ or swollen regions that have well defined membrane contact sites (MCS) with adjacent cisternae of endoplasmic reticulum. In Chapter 3, I localized the IP3R calcium channel to the TBC-ER contacts and have hypothesized that fluctuations in local calcium levels may alter local actin networks that cuff adjacent tubular regions of the TBCs, or facilitate the maturation of TBC bulbs into putative endosomes. Changes in intracellular calcium levels are known to be correlated with changes in actin networks (Veksler and Gov, 2009). As well, we know that early endosomes release calcium in order to mature and fuse into late endosomes (Saito et al, 2007). Thus, it is also possible that TBC bulbs, which are putative endosomes, must receive calcium from the ER in order to later release it and mature. To test these predictions, I injected the testes of Sprague Dawley rats with siRNAs against IP3R1 and processed the tissues for either western blot, immunofluorescence, or electron microscopy. If calcium exchange is responsible for regulation of the TBC actin network and/or the maturation of TBC bulbs into endosomes, then knocking down the IP3R calcium channel would be predicted to result in changes to the TBC actin network and/or to impede the maturation of TBC bulbs. Here, I target and knockdown IP3R1, and subsequently evaluated the tissue for protein knockdown using Western blot. Of the three 81 isoforms of IP3R, IP3R1 is the most highly expressed in testis (Tash et al, 2008). Previously, I’ve established that the lipid transfer protein, ORP9 (OSBPL9), is a specific marker for TBC-ER contacts (Adams et al, 2019) and EEA1 marks endocytosed bulbs that have coalesced and fused (Adams and Vogl, 2017). I used these markers to examine IP3R1 knockdown testis for morphological changes to TBC bulbs using stimulated emission depletion (STED) microscopy as well as electron microscopy (EM) to more precisely determine any changes to TBC bulbs. Using the same techniques, I also evaluated whether or not the knockdown of IP3R1 affected related actin networks. When compared to control testes injected with non-targeting siRNAs, Sertoli cells in knocked-down testes showed significant morphological alterations to the actin networks including a loss of TBC actin and the appearance of ectopic para-crystalline actin bundles in Sertoli cell stalks. There also was a change in the abundance and distribution of TBC-ER contact sites and large internalized endosomes. This disruption of TBCs resulted in delay of the withdrawal of apical processes away from spermatids and in spermiation. Together, these findings are consistent with the hypothesis that calcium exchange at TBC-ER contacts is involved both in regulating actin dynamics at TBCs and in the maturing of TBC bulbs into endosomes. The results are also consistent with the hypothesis that TBCs are part of the sperm release mechanism. 82 5.2 Results 5.2.1 Knockdown of IP3R1 by intratesticular siRNA injection There were noticeable decreases in the protein levels of IP3R1 between experimental and control testes as analyzed by Western blot and densitometry analysis (Figure 5.1A). All three animals used for Western blot showed thicker bands in the lanes from the control testes relative to their paired experimental testes. Densitometry was used to quantify the bands, and samples were compared using Student’s paired t-test. The IP3R1 knockdown was found to be statistically significant (p = 0.0152) (Figure 5.1A). To confirm that there was no effect on the other major isoform in testis, IP3R2, I analyzed lysates from the same three animals using Western blots probed with an anti-IP3R2 antibody and found no statistically significant difference in the IP3R2 levels as analyzed by densitometry and compared using Student’s paired t-test (p = 0.423) (Figure 5.1B). Calnexin loading controls confirm that the total amount of protein loaded was similar in control and experimental lanes (Figure 5.1A and B). After confirming the knockdown by Western blot, I evaluated siRNA treated testes using STED microscopy to look for changes in IP3R1 staining. I began by replicating our previously reported localization of IP3R on untreated normal tissues using an IP3R1/2/3 antibody (Lyon and Adams et al, 2017). As previously reported, IP3R labelling in normal rat testis localized to the ends of the actin networks of TBCs, in the area occupied by the TBC-ER contact sites (Figure 5.2A). Similarly, tissue from control non-targeting siRNA injected testes stained with an antibody to IP3R1/2/3 showed staining at stage VII of spermatogenesis both at the apex of epithelium in Sertoli cell regions adjacent to the 83 concave contour of the ‘hook-shaped’ spermatid heads where TBCs are found as well as around the early germ cell nuclei as ‘halo’ like structures (Figure 5.2B). Higher magnification of the late spermatids in epithelium show a similar staining pattern (Figure 5.2C). In contrast, tissue treated with siRNA against IP3R1 showed reduced staining at the apex of the epithelium in the area of TBCs, but not in the ‘halo’ structures around the early germ cell nuclei (Figure 5.2D and E). I interpret the loss of staining in the knockdown tissue to mean that the majority isoform of IP3R at TBCs is IP3R1, while the ‘halos’ around the early germ cell nuclei may be largely composed of either IP3R2, or less likely, IP3R3. 5.2.2 IP3R1 Knockdown Changes the Actin Structure in Sertoli cells and Late Spermatids Using STED and electron microscopy I compared IP3R1 siRNA treated and control testes for any evidence of differences in actin distribution, TBC-ER contacts, and endocytosed bulbs. In control tissues, ORP9 had a periodic distribution that corresponds to the TBC-ER contact sites at the ends of actin-labeled TBC proximal tubules (Figure 5.3A). ORP9 and actin staining in control testes showed normal distributions and arrangements at stage VII tubules similar to those I have previously reported (Adams et al, 2019). Actin staining in ESs adjacent to the late spermatid heads mirrored the ‘hook-shaped’ contours of the maturing spermatid heads at the apex of the epithelium. There also was little actin staining in columnar regions and apical stalks of Sertoli cells at this stage of spermatogenesis (Figure 5.3A). In contrast to control samples, IP3R1 knockdown tissue had significant changes to the distribution and apparent abundance of ORP9 as well as the arrangement and 84 location of actin in the Sertoli cells (Figure 5.3B). ORP9 staining either was lost at the apex of the epithelium or the staining was clumped and displaced from central regions of the apical processes of Sertoli cell processes surrounding the spermatid heads. Actin associated with TBCs was dramatically reduced or absent. Also, the actin in ectoplasmic specializations (ESs) adjacent to late spermatids appeared intensely labelled and more concentrated than in controls. Another consistent phenotype was the presence of large ectopic linear actin arrays in columnar regions of Sertoli cells and in the stalks supporting apical processes at stage VII of spermatogenesis. The presence of ectopic actin arrays was confirmed at the ultrastructural level (Figure 5.4A). Many of the actin arrays were flanked on either side by cisternae of ER and are reminiscent of an ‘ES-like’ arrangement (Figure 5.4B and C). Lone bundles of actin were present throughout the Sertoli cells, running parallel to the orientation of the Sertoli cells (Figure 5.4D and E). In some cases, fully formed, ‘ectopic’ ESs could be seen between Sertoli cells and early germ cells (Figure 5.4F). 5.2.3 The Abundance and Distribution of Intact and Vesiculated TBC bulbs Are Altered in IP3R1 Knockdown Testes To evaluate samples by STED microscopy, ORP9 and EEA1 were used as proxy markers for intact TBCs and vesiculated TBC ‘bulbs’, respectively. The distribution of ORP9 and EEA1 in control tissues showed a regular distribution similar to what I’ve previously reported (Adams and Vogl, 2019; Adams and Vogl 2017). ORP9 appeared in a periodic distribution along the TBC-ER contact sites, while EEA1 appeared in small clumps representing the vesiculated bulbs just distal to the ORP9 staining (Figure 5.5A 85 and B). This staining demonstrates the normal transition from intact TBCs that are continuous with the plasma membrane, to vesiculated TBC bulbs that are coalesced and fused. In contrast, the ORP9 and EEA1 staining are both largely absent from the stage VII spermatids from IP3R1 siRNA treated testis (Figure 5.5C and D). At the ultrastructural level, late spermatids from control testes showed a typical number and arrangement of TBCs as seen by the arrangement of TBC proximal tubules (Figure 5.6A). In addition, TBC bulbs were a consistent feature of control tissue (Figure 5.6B and C). The ER and actin of ESs are intact around the spermatid heads as is the ER associated with the plasma membrane (Figure 5.6A-D). TBC bulbs can be seen coalescing and fusing into larger endocytic structures (Figure 5.6E). Comparatively, TBCs were frequently absent from the apical cytoplasm around late spermatids in knockdown tissues (Figure 5.6F-J). Much of the ER around the late spermatids was pulled away from the plasma membrane and appeared to form large membrane swirls in the apical cytoplasm (Figure 5.6G and H). Many of the late spermatids had ectopic patches of actin scattered throughout the apical cytoplasm and TBCs were rare relative to control samples (Figure 5.6I and J). When present, TBC bulbs appeared small and lacked ER contacts. Also, large endosomes were rarely observed (Figure 5.6K). 5.2.4 IP3R1 Knockdown Causes a Delay in Spermiation To determine if the disruption of the TBC-ER contacts by IP3R1 knockdown resulted in delayed spermiation, I examined sections from whole testis prepared for bright field microscopy and looked for abnormalities in the release of late spermatids. Control testes showed a normal distribution of late spermatids at the apex of the epithelium during 86 stage VII of spermatogenesis (Figure 5.7A). This stage is identifiable by the presence of the ‘hook-shaped’ late spermatids at the apex of the epithelium and earlier stage spermatids deeper in the epithelium have a rounded cell shape and a nucleus located centrally in the cytoplasm. Similarly, stage VII tubules from IP3R1 knockdown testes also had late spermatids attached at the apex (Figure 5.7B). By late stage VIII of spermatogenesis, the vast majority of late spermatids should be released, and this appears consistently in sections from control testes (Figure 5.7C). Stage VIII is differentiated from stage VII by the polarization of the next generation of spermatids. These cells elongate and the nucleus and attached acrosome become oriented towards the base of the epithelium. In contrast to control tissue, stage VIII epithelium from IP3R1 knockdown tissues consistently showed the presence of late spermatids that had retained an association with apical processes of Sertoli cells (Figure 5.7D). The presence and abundance of abnormal late spermatids at stage VIII of spermatogenesis is indicative of delayed spermiation. 87 5.3 Discussion Calcium release from the ER has been implicated in changes in actin dynamics in cells generally as well as in the maturation of endosomes (Wales et al, 2016, Luzio et al, 2007). Here I show that the knockdown of the ER calcium release channel, IP3R1, causes marked changes in actin throughout Sertoli cells as well as changes to TBC bulbs, which have an endosome-like identity. The observed phenotypes are diagrammed in Figure 5.8. In Chapter 3, I showed that IP3R is localized to the membrane contact sites at TBC bulbs using an antibody that recognizes all three isoforms of the protein (Lyon and Adams et al, 2017). I chose to knockdown the type-1 isoform since it is known to be highly expressed in testis relative to the other two isoforms (Tash et al, 2008). Testes injected with IP3R1 specific siRNA showed changes in staining with the pan-specific IP3R antibody only in the area of TBC-ER contacts in apical Sertoli cell cytoplasm. Specific staining in other areas of the epithelium, was unaltered. In immunoblots, samples from testes injected with IP3R1 siRNA showed a significant knockdown of IP3R1 bands relative to controls. There was no knockdown of IP3R2 bands when blots were labelled with IP3R2 antibodies. RNAseq data suggest that IP3R3 is negligibly expressed in testis (Tash et al, 2008). Our data are consistent with the conclusion that the distribution of IP3R at TBC-ER contacts is largely composed of the type-1 isoform (IP3R1), while the other two isoforms may be more concentrated elsewhere in the epithelium. After confirming the knockdown of IP3R1, I examined the seminiferous epithelium from IP3R1 siRNA treated testes for changes to actin-based structures such as TBCs and ESs. Intracellular calcium fluctuations generated by the ER are known to be correlated with changes in adjacent actin filaments (Chakrabarti et al, 2018) as well as 88 changes in more global actin dynamics throughout the cell (Wales et al, 2016). The actin networks associated with TBCs were noticeably reduced or absent from apical processes. Also, those regions that lacked TBCs appeared to have ‘denser’ actin in ESs. These observations are consistent with the hypotheses that (1) changes in intracellular calcium in the apical processes of Sertoli cells affects the dynamics of actin associated with TBCs and that (2) the appearance of TBCs is correlated with the disassembly of ESs. In addition to changes in the actin filament distribution in apical processes associated with late spermatids, there were equally striking changes to actin in other areas of the Sertoli cells. Affected tubules appeared to have ectopic linear arrays of actin filaments in apical stalks and columnar regions of the Sertoli cells. The presence of these actin arrays was confirmed at the ultrastructural level, which also revealed that a large amount of this actin was associated with parallel running leaflets of ER. These ER-actin complexes appear morphologically similar to ESs, though many of these ‘ES-like’ structures were either not associated with the Sertoli cell PM or were juxtaposed to the PM adjacent to regions of early germ cells not normally associated with ESs. I suspect that the loss of IP3R1 at membrane contact sites associated with apical TBC bulbs not only altered calcium fluxes at these sites leading to a loss of TBCs, but may also have affected calcium signaling more globally in the cell promoting the formation of ectopic ‘ES-like’ actin arrays elsewhere in Sertoli cells. In cells that did contain TBC bulbs, the bulbs were often small in diameter with very few noticeable ER contacts and there was a general lack of ORP9 and EEA1 staining in treated tissue evaluated by STED microscopy. In cells generally, calcium is known to be dynamically exchanged at ER contact sites associated with multiple organelles (Wu et al, 89 2018). At endosomes specifically, calcium from the ER is required for endosomes to mature and acidify into lysosomes (Luzio et al, 2007) and is also known to be required for maintenance of ER-endosome contact sites (Kilpatrick et al, 2017). The identity of TBC bulbs as putative endosomes and their association with the ER while still continuous with the Sertoli cell PM suggests that calcium exchange by IP3R1 at the TBC-ER contact sites may be necessary for maintaining TBCs, and for enabling TBC bulbs to mature, undergo scission and fuse. Knockdown of IP3R1 may cause a failure of TBC bulbs to fuse and mature into larger endocytic structures. The observed delay in spermiation also further enforces the hypothesis that TBCs are an endocytic mechanisms necessary for sperm release. Here, I show that the knockdown of IP3R1 causes alterations to actin dynamics not only at TBCs in Sertoli cells, but also to actin dynamics elsewhere in the cell. In addition, reduced levels of IP3R1 may prevent the maturation of TBC bulbs through the endocytic pathway. This is likely due to impeded calcium exchange at the TBC-ER contact sites, and provides support for the hypothesis that calcium fluxes may be a primary function of TBC membrane contact sites. My data is also the first to link TBC-ER contact sites to TBC development and normal spermiation. Overall, these observations provide insight into the function of TBC-ER membrane contacts and their role during junction turnover at sperm release. 90 5.4 Materials and Methods 5.4.1 Animals All animals used in this study were Sprague-Dawley rats (Rattus norvegicus) between 300-600g in weight, and were purchased from Charles River Laboratories. Rats were housed and maintained according to guidelines established by the Canadian Council on Animal Care and all experimental protocols were approved by the Animal Care Committee of the University of British Columbia. All rats were weighed each day during the experiment. After handling and injections, the animals were given small food treats. A total of seven animals were used for these experiments. Three animals were used for Western blot, two for immunofluorescence, and two for electron microscopy. An additional untreated animal was used for immunofluorescence to confirm the normal distribution of IP3R in Sertoli cell apical processes. 5.4.2 Reagents Unless stated otherwise, all reagents were purchased from Sigma-Aldrich. 5.4.3 SiRNAs All siRNAs were in vivo grade Accell siRNA pools purchased from GE Healthcare Dharmacon (Lafayette, CO). Accell rat IP3R1 SMART pools (E-095360-00) and Accell Non-targeting pools (D-001910-10) were made to a final injection concentration of 0.5 ug/uL in 5% sterile glucose injection solution. 91 5.4.3 Intratesticular injections For intratesticular injections, anesthesia was induced and maintained with isolfuorane administered by inhalation. Animals were placed on a warming pad and once animals were under deep anesthesia, eye protection was applied and the rats were injected subcutaneously with saline (10 mL/Kg) to maintain hydration and Metacam (2mg/Kg on the first day of procedures and 1 mg/Kg for subsequent days) for pre-emptive pain management. The scrotum was shaved and then washed with chlorohexidine soap (Partnar) and swabbed with isopropanol. Each testis was manually localized and then the caudal third of the organ was transcutaneously injected either with 100 ul targeted IP3R1 siRNA reagents (right testis) or with 100 ul of control non-targeted siRNA (left testis). A maximum volume of 100 uL was used because of reports that volumes much greater than 50 uL can create turgidity in rat testis and restrict the dispersion of injectable agents (Russell et al, 1987). Only the caudal third of the testis was injected to help localize the small amount of injected volume to an isolatable area, in order to maximize the chances of inducing a phenotype. The entire procedure from induction of anesthesia to recovery was approximately 10 min for each animal. Tissues were collected and processed 2 days after the siRNA injections. 5.4.4 Tissue Collection All animals were placed under deep anesthesia using isofluorane administered by inhalation and then their testes were removed and processed using protocols that are detailed below. Animals were euthanized while under deep anesthesia by opening the thorax and incising the heart. 92 5.4.5 Antibodies Anti-IP3R1 (Cat #: SAB5200080) was obtained from Millipore Sigma (Darmstadt, Germany). Anti-IP3R1/2/3 (Cat #sc-377518), Anti-IP3R2 (Cat #sc-398434), and Anti-ORP9 (Cat #: sc-398961) antibodies were obtained from Santa Cruz Antibodies (Dallas, Texas). Anti-EEA1 (Cat #: 3288S) antibody was obtained from New England Biolabs (Whitby, Ontario, Canada). Goat anti-mouse IgG Alexa Fluor 488 (Cat #: A-21121) and Goat anti-rabbit IgG Alexa Fluor 568 (Cat #: A-11036) were obtained from Thermo Fisher Scientific (Waltham, Massachusetts, USA). 5.4.6 Electron Microscopy and Bright Field Microscopy Each testis was gravity perfused first with a brief wash of phosphate buffered saline (PBS) (150 mM NaCl, 5 mM KCl, 0.8 mM KH2PO4, 3.2 mM Na2HPO4, pH 7.3, 33° C) to clear the organs of blood and then perfused with fixative [0.1 M sodium cacodylate (EM Sciences), 1.5% paraformaldehyde (Fisher), and 1.5% glutaraldehyde (EM Sciences), pH 7.3, 33° C]. After 30 min, the caudal third of each testis was cut into roughly 0.1 cm2 blocks and the samples immersion fixed for an additional 2-3 h. Following this, the fixative was removed and replaced with 0.1 M sodium cacodylate buffer (pH 7.3). The next day, the samples were washed twice with buffer and then post-fixed with 1% OsO4 (EM Sciences) in 0.1 M sodium cacodylate (pH 7.3) on ice for 1 h. The samples were washed 3 times in ddH2O for 10 min each wash, stained ‘en bloc’ for 1 h with 1% aqueous uranyl acetate, and then washed 3 times with ddH2O. This was followed by dehydration through an increasing concentration series of ethyl 93 alcohol (30%, 50%, 70%, 95%, 100%) and incubation in two changes of 100% propylene oxide (Fisher). Samples were then left overnight in a 1:1 solution of propylene oxide:EM-bed 812 (EM Sciences). The following day, the tissue was put through two, 2 h incubations in 100% EM-bed 812, followed by embedding in 100% EM-bed 812 and polymerization at 60°C for 2 days. Using a Leica EM UC6 Ultramicrotome with an Ultra-AFM 3.0 mm diamond knife (DiATOME), thin sections of ~85 nm were collected on 200 mesh copper grids and then subsequently stained with both uranyl acetate and with Reynolds’ lead citrate. After staining, the sections were imaged using a Talos L120C Transmission Electron Microscope (ThermoFisher Scientific) operated at 120 kV. For bright field microscopy, EM-bed 812 embedded tissue was sectioned at a thickness of ~0.8um using a glass knife and stained with toluidine blue for ~2-3 min. Images of whole seminiferous epithelium were taken on a Zeiss Axioscope A1 Imager with 40x APOCHROMAT objective, mounted with a Hitachi HV-F22 digital camera. 5.4.7 Immunofluorescence Rat testes were gravity perfused first with PBS (33o C) to clear the organs of blood, and then with warm PBS containing 3% paraformaldehyde (pH 7.3, 33o C) for 30 min. After fixation, the testes were again perfused with PBS for an additional 30 min. The caudal third of each testis was excised, placed in Tissue-Plus O.C.T (Fisher Scientific), frozen with liquid nitrogen, and then stored at -80o C until the blocks were sectioned. For sectioning, each frozen block was secured onto a cryotome sectioning stub using Tissue-Plus O.C.T compound. Sections of 10-μm thickness were cut using a Cryostat (Microm 94 HM 525, Thermo Scientific) and collected on poly-L-lysine-coated slides. After collecting the sections, the slides were placed in a humidity chamber for ~1 min so sections could adhere to the slides without drying. This was either followed by plunging the slides into cold acetone for 5 min or placing the slides in a bath of PBS/0.1%BSA/0.1% saponin. Most antibodies used in this study reacted best with acetone permeabilized tissue, but the Anti-IP3R1/2/3 antibody reacted better with saponin permeabilization. Acetone permeabilized slides were subsequently placed into a PBS/0.1% BSA bath for 5 min. Sections were treated with either PBS/1% BSA or PBS/1% BSA/0.1% saponin for 1 h before incubating with primary antibodies diluted in either PBS/0.1% BSA or PBS/0.1% BSA/0.1% saponin for an additional 1 h at 37o C. Following primary antibody incubation, the sections were washed three times for 10 min each with PBS/0.1% BSA or PBS/0.1% BSA/0.1% saponin. Secondary antibodies were diluted in PBS/0.1% BSA or PBS/0.1% BSA/0.1% saponin before being added to the section for 1 h at 37o C. Sections were then washed for 10 min in PBS and stained with either 5uM Yoyo-3 Iodide (Thermofisher) for 20 min or with Alexa Fluor 568 Phalloidin (Cat #: A12380) diluted 1:40 in PBS for 20 min. All sections were then washed a final time in PBS for 10 min followed by mounting using ProLong Diamond Antifade mountant (Thermofisher). 5.4.8 STED Microscopy All images were taken using a 100×/1.4 Oil HC PL APO CS2 STED White objective on a Leica TCS SP8 3× STED microscope (Leica, Wetzlar, Germany) equipped with a white light laser, HyD detectors, and Leica Application Suite X (LAS X). All acquisitions were done at a scan speed of 600 Hz with a line average of 6, and were sequentially 95 acquired between frames in the sequence of Yoyo-3 Iodide, Alexa Fluor 568, and Alexa Fluor 488, with a step size of 100 nm. Alexa Fluor 488 was excited at 499 nm and depleted using a 592 nm depletion laser, Alexa Fluor 568 was excited at 577 nm and depleted with a 660 nm depletion laser, and Yoyo-3 Iodide was excited at 612 nm and depleted with a 775 nm depletion laser. After acquisitions, all images were deconvolved using Huygens Professional software (Scientific Volume Imaging, Hilversum, the Netherlands) using the same algorithms, and subsequently processed into publication formats using ImageJ software. 5.4.9 Western blotting and Densitometry The caudal third of both the right (IP3R1 siRNA treated) testis and the left (control) testes from each animal were decapsulated and homogenized in radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 50 mM Tris, 5 mM, EDTA, 1% Nonidet P-40, 1% deoxychloic acid, 0.1% SDS, pH 7.3) containing mini-EDTA-free protease inhibitor (Roche). The protein concentrations of each of the testis lysates were determined using Pierce BCA protein assay kit (ThermoFisher Scientific) before being diluted to 1 mg/mL using RIPA and 6X Laemmli sample buffer, aliquoted, and stored at –20° C. Testis lysates were loaded into Criterion XT 1.0-mm-thick 3-8% gradient tris-acetate gels (BioRad) and run at 120 V for 1 h in XT Tricine Running Buffer (BioRad). Gels were immersed in Tris/Glycine Transfer Buffer (BioRad) for 10 min and then were transferred for 1 h at constant 350 mA to polyvinylidene fluoride (PVDF) membranes transfer buffer. After transfer, PVDF membranes were blocked in 4% milk in TBST (50 mM Tris, 150 96 mM NaCl, 0.1% Tween, pH 7.5) for 1 h at RT before adding primary antibody, diluted in 4% Milk/TBST, for an additional 1 h at RT. Excess primary antibody was washed off with 4% Milk/TBST for 10 min, three times each wash, before adding secondary antibody, diluted in 4% Milk/TBST, for 1 h at RT. Excess secondary antibody was washed off with three washes for 5 min each with TBST, and then incubated in ECL solution for 5 min. Membranes were imaged on an ImageQuant LAS 4000 system (GE Life Sciences). The band intensities for each protein were determined using ImageQuant TL software (GE Life Sciences). Three technical replicates were analyzed from the same blot for each individual animal (n = 3) and samples from each animal were run separately. IP3R1 and IP3R2 protein levels were normalized to Calnexin loading controls and the technical replicates were averaged together to produce a measure for each animal. The relative measures for knockdown testes were compared to their paired control testes using Student’s paired t-test. 97 5.5 Figures Figure 5.1 IP3R1 protein levels are significantly reduced by in vivo IP3R1 siRNA injections, while IP3R2 is unaffected. (A) Western blot of whole testis lysate stained with an anti-IP3R1 antibody showing noticeable decreases in the amount of IP3R1 in the treated testis compared to the control testis. Calnexin loading controls are similar sizes between lanes. Densitometry data of western blots for n = 3 independent animals showing a significant reduction in IP3R1 in knockdown testes compared to paired control testes. *P<0.05 when knockdown testes are compared to their paired controls using Student’s paired t-test. (B) Western blot of whole testis lysate stained with an anti-IP3R2 antibody showing no change in the amount of IP3R2 in the treated testis compared to the control testis. Calnexin loading controls are similar sizes between lanes. Densitometry data of western blots for n = 3 independent animals showing no significant change in IP3R2 in knockdown testes compared to paired control testes. **P>0.05 when knockdown testes are compared to their paired controls using Student’s paired t-test. Bars indicate standard deviation. 98 Figure 5.2 Reduction of IP3R1 levels causes a loss of IP3R staining only in the apical cytoplasm around late spermatids. 99 (A) IP3R is localized to the ends of TBC tubes in STED microscopy, confirming our previous findings with confocal microscopy [5]. IP3R1/2/3 is shown to primarily label the ends of the actin (red) coated tubulobulbar complexes that project into the concavity of the ‘sickle’ shaped spermatid head, in the area occupied by the TBC-ER contact sites. (B) Late spermatids from control siRNA treated testes are shown with IP3R1/2/3 (green) showing normal labelling of the apical cytoplasm in the area of the late spermatids heads, as well as labeling of ‘halo’ shaped structures in germ cells located more basally in the epithelium. (C) Higher magnification micrograph of late stage spermatids from control tissues further demonstrating the staining pattern of IP3R1/2/3 in the area of TBC-ER contact sites. (D) Late spermatids from IP3R1 siRNA treated testes are shown with IP3R1/2/3 (green) being largely absent from the apical processes of the late spermatids. The ‘halo’ shaped structures in the epithelium are not affected by IP3R1 siRNA treatment. (E) Higher magnification micrograph of late stage spermatids from IP3R1 siRNA treated tissues which also lack significant staining for IP3R1/2/3 in the apical processes of late spermatids. 100 101 Figure 5.3 Reduction of IP3R1 levels causes a loss of TBC actin, an apparent increase in ES actin, and the appearance of ectopic actin ‘stalks’ throughout the Sertoli cells. (A) STED micrograph of stage VII seminiferous epithelium from control siRNA treated whole testis. The actin (red) is largely confined to the curvature of the late spermatid heads and to tubulobulbar complexes, while the ORP9 (green) specifically labels regions of TBC-ER contacts at the ends of the actin labeled TBCs as previously reported [9]. A high mag insert shows a late spermatid with TBC actin labelled (white arrow). (B - 3 panels) Sections of stage VII seminiferous epithelium from IP3R1 siRNA treated whole testis. High mag insert in the first panel shows a late spermatid with the area normally occupied by TBC actin labelled (white arrow), indicating a general loss of TBC actin around the late spermatids. The actin around the late spermatids appears more concentrated at the ESs and the overall shape and arrangement of the actin is disorganized compared to controls. There also appears to be an increase in large actin ‘stalks’ running parallel throughout the height of the epithelium not seen in controls. ORP9 staining is largely absent from the apex of the epithelium. Late spermatid actin morphology is disrupted and prominent actin ‘stalks’ are seen in the epithelium. ORP9 staining is somewhat present near the late spermatids, but it appears ‘clumped’ and its localization is displaced from within the curvature of the late spermatid heads. 102 Figure 5.4 Ectopic actin bundles that are present in the IP3R1 siRNA treated tissue have an ‘ES-like’ appearance at the ultrastructural level. (A) Low-magnification electron micrograph images of a Sertoli cells where ‘ectopic’ actin structures can be seen lining the plasma membrane on the lateral edges of the cells. (B&C) Magnified views of regions outlined by the white boxes in (A) demonstrating that the parallel actin arrays are flanked by leaflets of ER, giving an ‘ES-like’ arrangement, though there are no corresponding arrangements of actin and ER on the opposing cell and the complexes are seen in areas far from where ESs are normally found. (D) Actin bundles without opposing ER leaflets. (E) Actin bundles between two cisternae of ER. (F) An ‘ES-like’ arrangement of actin and ER at the Sertoli cell plasma membrane opposing the plasma membrane of an early germ cell. 103 Figure 5.5 Reduction of IP3R1 levels causes a loss in ORP9 and EEA1 staining around late spermatids. ORP9 and EEA1 staining demonstrate the TBC-ER contact sites and the vesiculated/fused TBC bulbs respectively. (A&B) Sections of stage VII seminiferous epithelium from control treated testis. (A) STED micrographs of whole rat testis treated with control siRNA showing that ORP9 (green) and EEA1 (red) staining is largely localized to the apical processes of late spermatids. (B) High-mag image showing the periodic distribution of ORP9 staining in the area of the TBC-ER contacts, while the EEA1 staining is shown to label discrete puncta just distal to the ORP9 staining, representing the vesiculated TBC bulbs that lack ER contacts. (C&D) Sections of stage VII seminiferous epithelium from IP3R1 siRNA treated whole testis. Both ORP9 and EEA1 labeling is shown to be largely absent from the apical processes surrounding late stage spermatids. 104 Figure 5.6 Reduction in IP3R1 levels causes the absence of TBCs and TBC bulbs, a disruption to ER morphology, and the presence of ectopic actin patches. 105 (A-E) Electron micrographs of control tissue showing stage VII late spermatids. (A) TBC tubes (arrowheads) are seen arranged in two rows projecting into the concavity of the spermatid head. (B-C) Bulbs are present (arrowheads), along with coalesced and fused internalized bulbs (arrow), in an area of the apical process not occupied by any TBC tubes. (D) Cross section through one side of a spermatid head showing a normal ES (bracket), with parallel actin arrays and an associated leaflet of ER. (E) Large, endocytosed bulbs are seen and are a consistent feature of the control tissue (arrowheads). (F-K) Images of stage VII late spermatids from IP3R1 siRNA treated testes. (F) Late stage spermatid surrounded by an apical process with disrupted morphology. TBCs and TBC bulbs are noticeably absent. (G) The ER in the apical process has pulled away from the plasma membrane and is expanded in the cytoplasm, and TBCs are absent. (H) The ER has again pulled away from the plasma membrane of the cell and formed an ER ‘swirl’ in the center of the apical process. (I&J) Scattered actin patches can be seen in the apical processes of some cells and TBCs or TBC bulbs are reduced or not present. (K) When present, TBC bulbs appear smaller than controls and coalesced bulb structures are not apparent in knockdown tissues. 106 Figure 5.7 Loss of IP3R1 causes late spermatids to be retained in the epithelium at late stage VIII of spermatogenesis. Light micrographs of stage VII and VIII from whole testis treated with either control or IP3R1 siRNA. (A) Stage VII of spermatogenesis from control tissue showing the normal morphology of late spermatids and the surrounding apical processes of Sertoli cells. (B) Stage VII of spermatogenesis from IP3R1 siRNA treated tissue . (C) Stage VIII of spermatogenesis from control tissue showing that absence of late spermatids from the epithelium. (D) Stage VIII of spermatogenesis from IP3R1 siRNA treated tissue showing delayed withdrawal of apical Sertoli cell cytoplasm (arrowheads) from the heads of late spermatids, and a delay in sperm release. 107 Figure 5.8 Summary diagram of the observed IP3R1 knockdown phenotypes. In control tissue, TBCs extend to their regular length before vesiculating and fusing into a large, coalesced structure just distal to the site of the TBCs. In the IP3R1 knockdown tissue TBCs are mostly non-existent, and any observable TBC bulbs that have vesiculated have failed to ‘swell’ and have accumulated, and not fused into a coalesced structure. This also results in spermiation delay where the late spermatids are retained in the epithelium beyond stage VII of spermatogenesis. 108 6 ORP9 Knockdown Delays the Maturation of Junction Related Endocytic Structures in the Testis and Leads to Impaired Sperm Release 6.1 Brief Synopsis One of the well-established functions of ER MCSs is lipid exchange (Jackson et al, 2016). In Chapter 4, I established that the ORP9 lipid exchange protein is localized to the TBC-ER MCS; however, the function of ORP9 and lipid exchange at the sites is not known. I previously established that the ‘bulb’ region of TBCs has an early endosome-like identity (Adams and Vogl, 2017), and it is known that endosomes generally have a robust association with the ER (Raiborg et al, 2015). It perhaps is not surprising then that the bulbs of TBCs, which are putative endosomes, establish MCSs. However, the association of the ER with an endocytic structure prior to scission is a unique phenomenon among cells generally. In Chapter 5, I provided evidence that the TBC-ER contacts may be involved in calcium exchange, although it is likely that the TBC-ER MCS is multi-functional similar to other MCSs. One of the other well-established functions of MCSs generally, including those at early endosomes, is lipid exchange/lipid transfer (Phillips and Voeltz, 2016). If lipid exchange is involved with regulating the structure/function of TBCs, then perturbing the lipid exchange machinery at TBCs could be predicted to alter the bulbs and progression through endosomal compartments. 109 Here I use an in vivo knockdown approach to probe the function of ORP9 at TBC-ER MCSs. The testes of Sprague-Dawley rats were injected with ORP9 targeted siRNA or non-targeted reagents, and the tissues examined by bright field, super-resolution (STED), and electron microscopy. Knockdown of ORP9 was achieved and maintained with daily injections of siRNA for 2-3 day intervals. Compared to controls, sections from ORP9 siRNA injected testes had longer TBC tubes and fewer fused TBC bulbs. Late spermatids were also abnormally retained in the epithelium of knockdown tissue. These results suggest that ORP9 is necessary for normal TBC bulb vesiculation and fusion, most likely by changing the plasma membrane lipid profile of the TBC. These data also further support the conclusion that TBCs are part of the normal mechanism of sperm release. 110 6.2 Results 6.2.1 Knockdown of ORP9 by Intratesticular siRNA Injection There were significant differences in the protein levels between the ORP9 siRNA (siORP9) injected testes (right testis) and non-targeting control siRNA (NT-control) injected testes (left testis) for all animals both of 2 day and of 3 day injection courses as determined by Western blot. Densitometry was done on the blots and then the data was analyzed using a Student’s paired t-test (Figure 6.1). ORP9 levels in testes of animals (n = 3) from the 2 day course of siRNA injections were found to be significantly different (p = 0.0447) between right and left testes, with average ORP9 levels in the siORP9 injected testes being ~40% reduced compared to controls (Figure 6.1A). Similarly, ORP9 levels in testes of animals (n = 3) from the 3 day course of siRNA injections were significantly different (p = 0.0262) between right and left testes, with the average ORP9 levels in the siORP9 injected testes being ~30% reduced compared to controls (Figure 6.1B). These results suggest that modest knockdowns of ORP9 was achieved with daily siRNA injections, and that the knockdown was maintainable and consistent between 2 and 3-day courses. 6.2.2 The Length of TBCs is Increased by ORP9 Knockdown Using standard bright field microscopy, I examined resin-embedded tissues from siORP9 and NT-control treated testes to locate stage VII seminiferous tubules where late spermatids are present. Relative to controls (Figure 6.2A), TBCs were noticeably longer in siORP9 treated tissues (Figure 6.2B). To explore this phenotype at higher 111 resolution, I processed siRNA treated tissues for stimulated emission depletion (STED) microscopy and stained for ORP9 as well as cortactin, a protein known to be a component of the actin networks of TBCs (Young et al, 2009). I then measured the length of the cortactin staining as a proxy for TBC proximal tubule length, and compared the means between control testes and their paired siORP9 injected testes using Student’s paired t-test. TBCs associated with late spermatids from control testes of the 2-day course of injections had cortactin labeling outlining the tubular regions of TBCs with ORP9 labeling at their distal ends (Figure 6.2C). Controls for the 2-day course of siORP9 injected testes had a mean TBC cortactin length of 0.698 uM while the cortactin length on the TBCs from the 2-day course of siORP9 injected testes were longer, with a mean length of 1.98 uM. The means were found to be significantly different (p = 0.0183, n = 2). The ORP9 staining at the ends of the TBCs also appeared less intense (Figure 6.2D). The same difference in cortactin and ORP9 staining was seen between the control and siORP9 injected testes in the 3-day post siRNA injected animals (Figure 6.2E and F), with the controls having a mean TBC cortactin length of 0.887 uM and the siORP9 injected testes having a mean TBC cortactin length of 2.00 uM. The means were found to be significantly different (p = 0.0428, n = 2). These observations together indicate that a reduction in ORP9 at TBC-ER contacts caused an increase in TBC length as indicated by the cortactin labeling in the associated actin cuffs. 6.2.3 ORP9 Knockdown Prevents the Maturation and Fusion of TBC Bulbs Because PIs can recruit proteins to endosomes to allow their maturation in the endocytic pathway, I looked to see if the ORP9 knockdown prevented TBC bulbs from 112 maturing into fused, EEA1 (Early endosome antigen 1) positive structures. Using STED microscopy, I labelled sections from treated testes with probes for EEA1 and ORP9. Control tissues from the 3-day course of injections showed a distribution of EEA1 and ORP9 as has previously been reported (Adams and Vogl, 2017; Adams and Vogl, 2019) (Figure 6.3A and B); that is, ORP9 labelling was localized in areas known to contain TBC bulbs and the associated MCSs, while the EEA1 staining occurred just distal to the ORP9 in the area known to contain the large coalesced/fused vesicles from TBCs that have undergone scission (Figure 6.3A and B). In contrast, siORP9 injected testes from the 3-day treatment showed a pronounced loss in EEA1 staining. In addition, the ORP9 staining also was consistently reduced compared to controls (Figure 6.3C and D). These data suggest that a reduction in ORP9 levels leads to a failure of TBC bulbs to progress into EEA1 positive, coalesced structures. 6.2.4 TBC Length is Increased and TBC Bulbs Accumulate at the Ultrastructural Level in ORP9 Knockdown Tissue At the ultrastructural level, I observed phenotypes consistent with those seen with STED microscopy. Apical cytoplasm surrounding late spermatids in the control tissue from the 2-day course of injections contained longitudinal profiles of TBC tubular regions (Figure 6.4A), TBC bulbs (Figure 6.4B), cross sectional profiles of TBCs (Figure 6.4C) and large vesicles resulting from bulbs that have undergone scission and fused (Figure 6.4D). In contrast, TBCs from the 2-day course of siORP9 injected testes were often abnormally long (Figure 6.4E) with extended narrow bulbs (Figure 6.4F). 113 Accumulations of extended bulbs were obvious in many sections in regions where larger vesicles also were present (Figure 6.4G and H). Control (Figure 6.4I-J) and siORP9 (Figure 6.4M-P) injected 3-day samples were morphologically similar to the 2-day samples. These data provide an ultrastructural reinforcement to those observations of phenotypes with STED microscopy; that is, ORP9 knockdown increased the length of TBCs and prevented TBC bulbs from maturing and progressing through the endosomal pathway. 6.2.5 ORP9 Knockdown Causes a Delay in Spermiation To explore whether or not the observed disruption of TBC structure/function was correlated with the secondary phenotype of delayed spermiation, I examined resin embedded testicular tissue from the 3-day siRNA treatment course with bright-field microscopy. Prior to spermiation, which occurs at Stage VIII of spermatogenesis in the rat, late spermatids are anchored in the apex of the epithelium at Stage VII. At this stage, the cytoplasm of apical Sertoli cell processes encapsulates each spermatid head. Stage VII seminiferous tubules from control testes had late spermatids anchored to the apex of the epithelium (Figure 6.5A). At Stage VIII of spermatogenesis in control samples, late spermatids generally had been released and were mostly absent from the epithelium. For the few that remained attached, the apical processes of Sertoli cells were being withdrawn away from the heads (arrowheads in Figure 6.5B). Similar to the control tissues, stage VII tubules from siORP9 treated testes had late spermatids anchored at the apex of the epithelium with surrounding apical processes (Figure 6.5C). 114 However, in Stage VIII tubules from siORP9 treated testes, late spermatids remained anchored to the epithelium and were still surrounded by apical processes of the associated Sertoli cells (arrowheads in Figure 6.5D). These results are consistent with a delay in sperm release from the epithelium and represent a secondary phenotype to those effects seen at TBCs, reinforcing our primary findings. 6.2.6 PI(4,5)P2 Localizes to the Area Containing TBCs Because ORPs are known to bind PIs generally, I chose to screen for all common PIs at TBCs using immunofluorescence with STED microscopy: PI(3)P, PI(3,4)P2, PI(3,4,5)P3, PI(4)P, and PI(4,5)P2. Other than PI(4,5)P2 (Figure 6.6), most of these antibodies had no significant fluorescent signal at TBCs (data not shown). PI(4,5)P2 was localized in discrete ‘donut- shaped’ structures close to the inner concavity of the spermatid head where TBCs are located (Figure 6.6A-C). A similar pattern of staining was absent in sections stained with anti-PI(4,5)P2 and pre-absorbed with purified PI(4,5)P2, indicating that the PI(4,5)P2 antibody was specific (Figure 6.6D). Both normal mouse IgM and secondary antibody controls also lacked any staining (Figure 6.6E and F). These data indicate that PI(4,5)P2 localizes to the area of TBCs and may be a target for the lipid transfer ability of ORP9. 115 6.3 Discussion Lipid transfer/exchange by lipid transfer proteins (LTPs) at MCSs plays a vital role in shaping membrane identity and organelle function (Jackson et al, 2016), and can affect actin polymerization both in association with CME vesicles (Encinar del Dedo et al, 2017) and with endosomes (Dong et al, 2016). Here I show that knockdown of a known LTP at MCSs at TBCs in Sertoli cells of the testis causes alternations to the structure of TBCs, prevents their progression through the endocytic pathway, and ultimately results in a delay in spermiation. These phenotypes are diagramed in Figure 6.7. This work is the first to suggest a role for lipid transfer in regulating the TBC mediated internalization of intercellular junctions in the testis. The intratesticular injection of siORP9 resulted in a significant mean reduction of ORP9 protein levels by about 40% and 30% respectively in the 2-day and 3-day courses of injections, and a reduced signal at TBC bulbs relative to controls in tissue sections probed for ORP9 and imaged by STED. Although knockdown of ORP9 resulted in diminished fluorescent staining, it did not result in a complete loss of staining in all tissue sections or a loss in the periodic distribution of ORP9 at the distal ends of the TBC tubes, indicating that the MCS were still intact – a result confirmed at the ultrastructural level. While large knockdowns are difficult to achieve in vivo due to siRNA degradation by endogenous nucleases and barriers to cellular uptake (Hoerter and Walter, 2007) these results show marked phenotypic changes associated with TBCs and other Sertoli cell structures that are in line with previously reported works with these methods (Adams and Vogl, 2020a). In in vivo studies in the testis, it appears that modest degrees of target protein knockdown, indicated by immunoblots of tissue 116 lysates, can provide interpretable phenotypes when tissues are examined morphologically. One of the more striking morphological phenotypes observed in the siORP9 injected tissues was an increase in the overall length of TBCs relative to controls. This phenotype was first observed in thick (0.08 m) toluidine blue stained resin sections, and was then confirmed both by STED analysis of fixed frozen testis sections probed with cortactin antibodies to label the actin networks of TBCs, and by electron microscopy of thin resin sections. Though the difference in TBC length as determined by measurement of the cortactin staining in STED images was found to be significant, the power of this statistical analysis would benefit from higher n-values. However, the observation that the TBCs appear longer in the resin embedded tissues for both 2-day and 3-day animals (n = 2, respectively) as well as for the 2-day and 3-day animals (n = 2, respectively) processed for STED, increases my confidence in the observed length differences. My interpretation of this increase in length of TBCs is that reduced ORP9 levels at TBC-ER contacts results in the continued growth or extension of dendritic actin networks ‘cuffing’ the necks of TBCs, and that the exchange of lipids by ORP9 at the TBC-ER contact somehow influences actin dynamics at the bulb/neck interface. One possibility is that ORP9 is responsible for removing a specific lipid that recruits and promotes actin polymerizing proteins at the distal end of the TBC proximal tube adjacent to the MCS. With reduced removal of a lipid that recruits actin polymerizing machinery, the actin network would continue to polymerize and the TBC tubular region or neck would continue to elongate. Some of the proteins known to polymerize actin networks are associated with TBCs, including Arp2/3 and its activator 117 N-WASP (Young et al, 2009; Vaid et al, 2007), both of which are recruited by PI(4,5)P2 (Rohatgi et al, 2001). Previous works have shown that ORP9 is able to bind several PIs including PI(4,5)P2 (Fairn and McMaster, 2005). If ORP9 is responsible for removing PI(4,5)P2 from the TBC membrane, then knockdown of ORP9 may cause a failure to disengage actin polymerizing machinery like Arp2/3 and N-WASP, leading to the observed phenotype. Our observation here that antibodies to PI(4,5)P2 are reactive in areas containing TBCs coupled with the observation that ORP9 leads to longer TBC actin networks suggests that PI(4,5)P2 could be a target for ORP9 at TBCs. Alternatively, others have suggested that the transfer of sterols from the ER to the plasma membrane can promote actin polymerization during canonical endocytosis (Encinar del Dedo et al, 2017). Rather than the removal of a PI, it may be that the addition of a sterol to the TBC membrane by ORP9 could also be regulating the actin polymerization machinery associated with TBCs. Interestingly, I also observed a phenotype related to TBC bulbs and their progression through the endosomal pathway. The presence of large coalesced and fused ‘bulb’ structures that were EEA1 positive, were reduced relative to controls when analyzed by STED microscopy. Further, there appeared to be less large vesicles in knockdown sections than in controls at the ultrastructural level, and a striking accumulation of narrow elongate bulbs that had apparently not undergone scission and coalesced to form the larger vesicles. These observations indicate that a reduction in ORP9 levels prevents TBC bulbs from maturing through the endosomal pathway. Because ER contacts are known to have a crucial role in endosome maturation (Rowland et al, 2014), it perhaps is not surprising that reducing the abundance of a 118 TBC-ER contact protein delays TBC maturation. On early endosomes, PI(3)P recruits Rab5 GTPase, allowing it to activate effectors like the tethering protein EEA1 (Mills et al, 1998). The conversion of PI(3)P into PI(3,5)P2 causes dissociation of Rab5 and recruitment of Rab7, and is a necessary step in endosome maturation (Ikonomov et al, 2006). I’ve previously shown that pre-scission, TBC bulbs are Rab5 positive, while they only become EEA1 positive after undergoing scission and fusing (Adams and Vogl, 2017). It also has been shown that PI(3)P is one of the PIs that ORP9 is able to bind (Fairn and McMaster, 2005). It is possible that ORP9 plays a role in regulating the PI(3)P levels on TBC bulbs, which in turn would recruit proteins like Rab5 that contribute to TBC bulb maturation and progression through the endosomal pathway. Though I did not observe PI(3)P staining at TBCs (data not shown), this does not exclude it being present in the TBC membrane or from being a target for ORP9 at TBCs. The increase in TBC length and the accumulation of elongate TBC bulbs that have not undergone scission in ORP9 knockdown tissue, indicate that ORP9 may function to change the lipid profile of the TBC bulb membrane, marking it for both vesiculation and for later fusion into large, coalesced structures. The same lipid change in the membrane of the TBC bulbs may both contribute to the dissociation of actin polymerizing machinery and also the recruitment of proteins responsible for TBC bulb maturation. The major function of TBCs at apical sites in the seminiferous epithelium has been proposed to be internalization of adhesion junctions between Sertoli cells and late spermatids (Vogl et al, 2014). Perturbation of the structure/function of TBCs would be 119 predicted to delay or prevent spermiation. In the ORP9 knockdown tissue, spermatids remained attached to the apex of the epithelium at Stage VIII of spermatogenesis compared with control tissues in which spermatids were released. Moreover, the apical processes of the Sertoli cells in which clusters of TBCs reside remained associated with each spermatid head in the knockdown tissues, but were withdrawn away from the heads in control samples. ORP9 would appear to be integral to the normal function of TBCs and to the mechanism of sperm release. There is still much we do not know about ORP9’s function at TBC-ER contacts. Because ORPs can vary between which PIs/Sterols they bind generally (Fairn and McMaster, 2005; Pietrangelo and Ridgway, 2018) I cannot predict which lipids ORP9 may be interacting with at TBCs. Though I have observed PI(4,5)P2 at TBCs, I do not have direct evidence of it being bound by ORP9. Similarly, while I did not observe staining at TBCs with other PI antibodies, it is possible that the antibodies I used were not able to detect their respective targets in my hands. These data would not exclude PIs other than PI(4,5)P2 from being present in the membrane of TBCs. Without this information, I also cannot narrow down which proteins are possibly affected by the knockdown of ORP9. Moreover, the observed phenotypes do not preclude the possibility that the reduction in ORP9 levels may be affecting the functionality of other proteins at the TBC-ER contact that may be more directly involved in lipid transfer or other processes. For instance, it may be that ORP9 is not itself acting to transfer lipids, but is an accessory lipid sensor aiding the overall lipid transfer process. This has previously been suggested as an alternative function for ORPs (Olkkonen and Li, 2013). It is also possible that my knockdown of ORP9 resulted in loss of integrity of the TBC- 120 ER contact generally, or disrupted other cellular functions at the MCS, such as calcium exchange by the IP3R1, as I’ve previously suggested may occur (Adams and Vogl, 2020a). Many works have linked lipid exchange at MCSs to calcium exchange and subsequent actin dynamics on target organelles (Balla, 2018). The knockdown of IP3R1 (Adams and Vogl, 2020a) and of ORP9 (this chapter) result in the same secondary phenotype of delayed spermiation; however, there are crucial differences in the observed primary TBC phenotypes. Knockdown of IP3R1 results in stunted TBCs whereas knockdown of ORP9 results in longer TBCs, presumably both through effects on actin dynamics. Also, bulbs observed in the IP3R1 knockdown were small and failed to swell (Adams and Vogl, 2020a). Bulbs seen in ORP9 knockdown tissues were elongate and narrow. In knockdowns of each protein, the ultimate effect was a loss of material progressing through the endocytic pathway. Here I’ve shown that reduction of a known LTP inhibits the maturation of TBCs through the endosomal pathway. This inhibition impedes their primary function of internalizing intercellular attachments of Sertoli cells with spermatids, which in turn leads to a spermiation defect. This is the first data to suggest that a lipid transfer mechanism is functionally active at TBC-ER contacts and plays a role in sperm release. 121 6.4 Materials and Methods 6.4.1 Animals Sprague-Dawley rats (Rattus norvegicus) of reproductive age between 300-600 g in weight were used for this study. All rats were purchased from Charles River Laboratories. Rats were housed and maintained according to guidelines established by the Canadian Council on Animal Care. The Animal Care Committee of the University of British Columbia reviewed and approved all experimental protocols used in this study and all protocols used here fall within the Ethical Guidelines for publication in Biology of Reproduction. Rats were handled and weighed in the days leading up to and including the days of the experiments to acclimatize them to the procedures and were given small food treats both after handling and after injections. For these experiments, a total of fourteen animals were used: six animals were used for Western blot, four for immunofluorescence, and four for bright field & electron microscopy. 6.4.2 Reagents Unless stated otherwise, all reagents were purchased from Sigma-Aldrich. 6.4.3 SiRNAs All siRNAs used in this study were in vivo grade Accell siRNA pools purchased from GE Healthcare Dharmacon (Lafayette, CO). Accell rat ORP9 SMART pools (E-098474-00) and Accell Non-targeting pools (D-001910-10) were made to a final injection concentration of 0.5 ug/uL in 5% sterile glucose injection solution. 122 6.4.4 Intratesticular injections Rats were anesthetized with isolfuorane administered by inhalation and placed on a warming pad. Lacri-lube (Refresh) eye protection was applied and a hydrating solution of saline (10 mL/Kg) was injected subcutaneously. For pain management, Metacam dosed at 2 mg/Kg was injected on the first day of procedures, and this dose was reduced to 1 mg/Kg on subsequent days of the procedure. For the first day of the procedure only, the scrotum was shaved to allow better visualization and positioning of the testes. The scrotum was washed with chlorohexidine soap (Partnar) and swabbed with isopropanol before any siRNA injections into the testes. The testes were anchored between the thumb and index finger and then the caudal third of each testis was transcutaneously injected. Injections consisted of either 100 ul of ORP9 siRNA (siORP9) reagents (right testis) or 100 ul of non-targeted control (NT-control) siRNA (left testis). SiRNA injections were performed each day for either 2 or 3 days and the testes were collected and processed one day after the last injection. 6.4.5 Tissue Collection Rats were anesthetized using isofluorane and tested for reflexive responses (toe-pinch) before their testes were removed and the caudal third of each testis was processed using the various protocols used in this study. All subsequent protocols are detailed below. After collecting the testes, rats were euthanized while still under deep anesthesia by thoracotomy and incising the heart. 123 6.4.6 Antibodies and Control Reagents Anti-ORP9 (Cat #: sc-398961) antibody was obtained from Santa Cruz Antibodies (Dallas, Texas). Anti-Cortactin antibody was obtained from Sigma-Aldrich (Cat #: C7112). Anti-EEA1 (Cat #: 3288S) antibody was obtained from New England Biolabs (Whitby, Ontario, Canada). Goat anti-mouse IgG Alexa Fluor 488 (Cat #: A-21121) and Goat anti-rabbit IgG Alexa Fluor 568 (Cat #: A-11036) were obtained from Thermo Fisher Scientific (Waltham, Massachusetts, USA). Anti-PI(4,5)P2 (Z-P045) antibody was obtained from Echelon Biosciences (Salt Lake City, Utah, USA). All antibodies used to screen for PIs and purified PI(4,5)P2 (P-4504) for pre-absorption control were also obtained from Echelon Biosciences. 6.4.7 Western blotting and Densitometry Testes were collected for Western blot analysis after either 2 or 3 days of daily siRNA injections. Each testis was decapsulated and homogenized in radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 50 mM Tris, 5 mM, EDTA, 1% Nonidet P-40, 1% deoxychloic acid, 0.1% SDS, pH 7.3) containing mini-EDTA-free protease inhibitor (Roche). Testis lysates had their protein concentrations determined using Pierce BCA protein assay kit (ThermoFisher Scientific) before being diluted to 1 mg/mL using RIPA and 6X Laemmli sample buffer. Samples were loaded into Criterion XT 1.0-mm-thick 3-8% gradient tris-acetate gels (BioRad) and run at 120 V for 1 h in XT Tricine Running Buffer (BioRad). Gels were washed for 10 min in Tris/Glycine Transfer Buffer (BioRad) before the proteins were transferred in fresh transfer buffer for 1 h at constant 350 mA to polyvinylidene fluoride (PVDF) membranes. PVDF membranes were blocked in 4% 124 milk in TBST (50 mM Tris, 150 mM NaCl, 0.1% Tween, pH 7.5) for 1 h at RT and then incubated for 1 h at RT in primary antibody diluted in 4% Milk/TBST. The membranes were then washed three times with 4% Milk/TBST for 10 min. Secondary HRP antibodies were diluted in 4% Milk/TBST and added to the membranes for 1 h at RT and subsequently washed three times for 5 min each with TBST. For chemiluminescence, the membranes were then incubated in ECL Select (GE Life Sciences) solution for 5 min and imaged immediately on an ImageQuant LAS 4000 system (GE Life Sciences). Each gel was first incubated with ORP9 antibody and processed as above before being stripped with Restore Western Blot Stripping Buffer (ThermoFisher) and incubated with Calnexin antibody (loading control) and also processed as stated above. Band intensities for each protein were determined using ImageQuant TL software (GE Life Sciences). Three technical replicates were analyzed from the same blot for each individual animal from both the 2 day (n = 3) and 3 day (n = 3) courses of injections. Samples from each animal were run on their own gels. Technical replicates of the ORP9 bands on each gel were normalized to the corresponding Calnexin loading control bands from the same lane. The normalized technical replicates for both the siORP9 injected testis and the paired control testis were averaged for each gel to produce a relative measure of the knockdown for each animal. The relative measures the siORP9 injected testis and NT-control injected testis from the 2 day and 3 day courses of injections were compared using Student’s paired t-test. 125 6.4.8 Immunofluorescence Testes were briefly gravity perfused with phosphate buffered saline (PBS) (150 mM NaCl, 5 mM KCl, 0.8 mM KH2PO4, 3.2 mM Na2HPO4, pH 7.3, 33° C) via the dorsal testicular artery to clear the organs of blood. This was followed by a 30 min perfusion with warm PBS containing 3% paraformaldehyde (pH 7.3, 33oC) for 30 min followed by a subsequent wash with PBS for an additional 30 min to clear any residual fixative. The caudal third of each testis was collected using a scalpel and then placed in Tissue-Plus O.C.T (Fisher Scientific) and frozen in liquid nitrogen. Frozen testes were stored at -80o C until further processed for immunofluorescence. For sectioning, frozen testes were mounted onto a stub using Tissue-Plus O.C.T compound and then the stubs placed in a Cryostat (Microm HM 525, Thermo Scientific). Sections (10-μm thick) were obtained and then collected on poly-L-lysine-coated slides and placed immediately into in a humidity chamber for ~1 min to allow sections to adhere and avoid tissue dehydration. Slides were then plunged into cold acetone for 5 min and then placed into a wash of PBS/0.1% BSA bath for an additional 5 min. Sections were blocked with PBS/1% BSA for 1 h before incubating with primary antibodies diluted in PBS/0.1% BSA 1 h at 37o C. Sections were then washed three times (10 min each wash) with PBS/0.1% BSA and then secondary antibodies diluted in PBS/0.1% BSA were added to the sections for 1 h at 37o C. Sections were then washed for 10 min in PBS and optionally stained with 5uM Yoyo-3 Iodide (Thermofisher) to visualize the nuclei. All sections were then washed a final time in PBS for 10 min before mounting using ProLong Diamond Antifade mountant (Thermofisher). 126 6.4.9 STED Microscopy and TBC Measurements All images were taken with a Leica TCS SP8 3× STED microscope (Leica, Wetzlar, Germany) mounted with a 100×/1.4 Oil HC PL APO CS2 STED White objective, white light laser, and HyD detectors. Leica Application Suite X (LAS X) was used to acquire images at a scan speed of 600 Hz with a line average of 6, and a step size of 100 nm. Frames were acquired sequentially in the order of Yoyo-3 Iodide, Alexa Fluor 568, and Alexa Fluor 488. Alexa Fluor 488 was excited at 499 nm and depleted using a 592 nm depletion laser, Alexa Fluor 568 was excited at 577 nm and depleted with a 660 nm depletion laser, and Yoyo-3 Iodide was excited at 612 nm and depleted with a 775 nm depletion laser. All STED images were deconvolved using the same algorithms with Huygens Professional software (Scientific Volume Imaging, Hilversum, the Netherlands) and compiled into final images using ImageJ software. The TBCs in all STED images with cortactin staining were measured to get a relative measure of any length differences between control and experimental samples. Cortactin stained TBCs were measured from the base (spermatid head) to the distal end using ImageJ software. 6.4.10 Electron Microscopy and Bright Field Microscopy Testes were briefly gravity perfused with phosphate buffered saline (PBS) (150 mM NaCl, 5 mM KCl, 0.8 mM KH2PO4, 3.2 mM Na2HPO4, pH 7.3, 33°C) via the dorsal testicular artery to clear the organs of blood. This was followed by a 30 min perfusion with fixative [0.1 M sodium cacodylate (EM Sciences), 1.5% paraformaldehyde (Fisher), and 1.5% glutaraldehyde (EM Sciences), pH 7.3, 33° C]. Tissue from the caudal third of 127 each fixed testis was collected and cut into roughly 0.1 cm2 blocks, and further immersed in PFA/glutaraldehyde fixative for an additional 2-3 h. The fixative was replaced with 0.1 M sodium cacodylate buffer (pH 7.3) and left overnight at room temperature. Samples were washed another two times with sodium cacodylate buffer before being fixed with 1% OsO4 (EM Sciences) in 0.1 M sodium cacodylate (pH 7.3) on ice for 1 h. Tissues were then washed 3 times in ddH2O for 10 min each wash followed by ‘en bloc’ staining with 1% aqueous uranyl acetate for 1 h followed by 3 washes with ddH2O. Tissue dehydration was done with a series of ethyl alcohol washes (30%, 50%, 70%, 95%, 100%) before infiltration with two changes of 100% propylene oxide (Fisher). Samples were further infiltrated overnight in a 1:1 solution of propylene oxide:EMbed 812 (EM Sciences) followed by resin infiltration via two, 2 h incubations in 100% EMbed 812. Tissues were then embedded in resin and polymerized in an oven at 60° C for 2 days. Thin sections of the polymerized tissues were cut using a Leica EM UC6 Ultramicrotome with an Ultra-AFM 3.0 mm diamond knife (DiATOME). Sections were collected with 200 mesh copper grids before being stained both with uranyl acetate and with Reynolds’ lead citrate. Stained sections were imaged using a Talos L120C Transmission Electron Microscope (ThermoFisher Scientific) operated at 120 kV. EM-bed 812 embedded tissues also were used to obtain thick sections for bright field microscopy. Sections of 0.8um thickness were cut using glass knives and collected on glass slides. Sections were then stained with toluidine blue for 2-3 min. A Zeiss Axioscope A1 Imager photomicroscope fitted with a 40x APOCHROMAT objective and a Hitachi HV-F22 digital camera was used to collect bright field images. 128 6.5 Figures Figure 6.1 ORP9 knockdown is achieved in rat testes for periods of 2 and 3 days with daily injections of siRNA. (A) Western blot densitometry data and a representative blot shows a significant difference (p < 0.05) in ORP9 protein levels between the NT-control and siORP9 injected testes after 2 days of daily injections with siRNA. The mean reduction for all animals (n = 3) is ~40%. (B) Western blot densitometry data and a representative blot showing a significant difference (p < 0.05) in ORP9 protein levels between the control and siORP9 injected testes after 3 days of daily injections with siRNA. The mean reduction for all animals (n = 3) is ~30%. Bars indicate standard deviation. 129 Figure 6.2 Tubulobulbar complexes (TBCs) are longer relative to controls in ORP9 knockdown testes. (A) Bright field micrograph of a late spermatid from testes injected with NT-control over a 2-day period. TBCs can be seen projecting from the concavity of the spermatid head into the apical cytoplasm. (B) Late spermatid from testes injected with siORP9 over a 2-day period showing much longer TBCs compared to controls. (C) STED micrograph of a late spermatid from testes injected with NT-control over a 2-day period stained for cortactin (red) and ORP9 (green). TBCs proximal tubes can be seen outlined by cortactin in the concavity of the spermatid head with ORP9 labelling the TBC-ER contacts at their distal ends. (D) Late spermatid from testes injected with siORP9 over a 2-day period showing much longer cortactin labelled TBC tubes and a reduced intensity of ORP9 compared to controls. (E) Late spermatid from testes injected with NT-control over a 3-day period showing a similar length of cortactin labelled TBC tubes to the 2-day control. (F) Late spermatid from testes injected with siORP9 over a 3-day period showing much longer cortactin labelled TBC tubes and a reduced intensity of ORP9 compared to controls, similar to the 2-day period of siORP9 injections. 130 Figure 6.3 EEA1 positive (fused) TBC bulbs are largely absent in ORP9 knockdown testes. (A&B) STED micrographs of late spermatids from testes injected with NT-control over a 3-day period stained for EEA1 (red), ORP9 (green) and YOYO-3 iodide (cyan) to show the spermatid nuclei. TBC-ER contacts can be seen labelled with ORP9, and coalesced/fused TBC bulbs are shown distal to the TBC-ER contacts as EEA1 positive structures (white arrow heads). (C-D) Late spermatid from testes injected with siORP9 over a 3-day period showing a pronounced loss of ORP9 and EEA1 staining. 131 Figure 6.4 TBC tubes are longer and coalesced/fused TBC bulbs are largely absent at the ultrastructural level. 132 (A-D) Electron micrographs of late spermatids in the control tissue from the 2-day course of injections. (A) The Sertoli cell apical cytoplasm surrounding late spermatids contains longitudinal profiles of TBC tubular regions. (B) TBC bulbs are present in the apical cytoplasm as well. (C) Cross sectional profiles of TBCs in the apical cytoplasm are shown where no TBC bulbs are present. (D) Large vesicles resulting from TBC bulbs that have undergone scission and fused are present just distal to the intact TBC bulbs. (E-H) Electron micrographs of late spermatids in the siORP9 injected tissue from the 2-day course of treatments. (E) Abnormally long TBCs are seen projecting into the apical cytoplasm. (E-H) TBC bulbs also appear extended and narrow relative to controls (white arrow heads). (G&H) Accumulations of extended bulbs are shown in in regions where some larger vesicles are also present. (I-J) Late spermatids in the control tissue from the 3-day course of injections appear morphologically similar to those from the 2-day course of injection. (M-P) Late spermatids in the siORP9 injected tissue from the 3-day course of treatments are also morphologically similar to the 2-day siORP9 injected tissue. (M&N) TBCs appear longer relative to controls. (O&P) TBC bulbs appear narrow (white arrow heads) and there are less obvious large vesicles resulting from TBC bulb fusion. 133 Figure 6.5 Knockdown of ORP9 prevents late spermatids from being released from the epithelium at late stage VIII of spermatogenesis. (A) Bright field micrographs of stage VII seminiferous epithelium from 3-day NT-control injected tissue showing late spermatids in the apex of the epithelium with surrounding apical processes of Sertoli cells. (B) Stage VIII seminiferous epithelium from control tissue showing that absence of late spermatids from the apex of the Sertoli cells. (C) Stage VII seminiferous epithelium from the 3-day course of siORP9 injected tissue showing late spermatids at the apex of the Sertoli cells similar to the control tissue. (D) Stage VIII seminiferous epithelium from the 3-day course of siORP9 injected tissue showing retained late spermatids with delayed withdrawal of apical Sertoli cell cytoplasm (arrowheads). 134 Figure 6.6 PI(4,5)P2 is localized to the apical cytoplasm of late spermatids in the area containing TBCs. (A-C) STED micrographs of late spermatids from normal testes showing PI(4,5)P2 (green) and YOYO-3 iodide (cyan) to show the spermatid nuclei. PI(4,5)P2 appears in the area containing TBCs and often has a ‘donut- like’ shape. Arrowheads indicate some of the examples of this staining. (D) Pre-absorption control showing late spermatids without any staining in the apical cytoplasm. (E) Normal mouse IgM (NMIgM) and (F) secondary antibody controls are also absent of any staining. 135 Figure 6.7 Summary diagram of the observed ORP9 knockdown phenotypes. In control tissue, TBCs extend to their regular length before vesiculating and fusing into a large, coalesced structure just distal to the site of the TBCs. In the ORP9 knockdown tissue TBCs are extended, and those TBC bulbs that have vesiculated have accumulated, and not fused into a coalesced structure. This also results in spermiation delay where the late spermatids are retained in the epithelium beyond stage VII of spermatogenesis. 136 7 Conclusion In this thesis, I present morphological and functional data that are consistent with the hypothesis that membrane contact sites (MCSs) formed between tubulobulbar complexes (TBCs) and the endoplasmic reticulum (ER) serve to regulate the maturation and development of the TBC via calcium and lipid exchange mechanisms. These data further reinforce the broader hypothesis, that TBCs in the apical processes of Sertoli cells are necessary for the normal release of late spermatids. 7.1 Overall Analysis 7.1.1 Structure TBCs have a protein composition and internalization process that is most similar to actin-related CME machinery (Young et al, 2009; Zakrzewski et al, 2020; Kusumi et al, 2007; Vaid et al, 2007). TBC and CME vesicles are initiated by clathrin-coated pits (Young et al, 2009) and have a network of dendritic actin along their ‘necks’. Moreover, in both TBC and CME, internalized vesicles eventually join the endosomal-lysosomal system (Vogl et al, 2014). However, there are a number of significant differences between the two types of machinery. TBCs are double membraned invaginations of either two Sertoli cell plasma membranes (PMs) at the base of the epithelium or of Sertoli cell and late spermatid PMs at the apex of the epithelium, whereas CME vesicles are formed by the invagination of the PM of a single cell. Unlike clathrin-coated pits of CME that have short necks and undergo scission soon after formation, the double membrane coated pits of TBCs do not undergo scission and the necks continue to 137 elongate into actin cuffed tubes that are microns in length (Russell and Malone, 1980; Vogl et al, 2014). The most striking structural feature of TBCs is that the neck of each structure swells near the distal end of the tube creating a ‘bulb’ that loses its actin cuff and forms a membrane contact site (MCS) with the endoplasmic reticulum (ER), all occurring while the TBC is still continuous with the PM (Russell, 1977; Russell and Clermont, 1976). The association of the ER with an endocytic structure prior to scission is a unique phenomenon among cells generally. In this thesis, I provide structural data using a high-resolution immunoelectron and STED microscopy techniques to show that TBC bulbs have an endosome-like identity. I begin by showing that the endocytic markers Rab5 and EEA1, are localized to the bulb region of TBCs (Adams & Vogl, 2017). These data provide evidence that the bulb region of the TBC is a putative endosome, a conclusion that is further reinforced by the fact that TBC bulbs go on to join the endosomal-lysosomal system in a manner similar to endosomes (Young et al, 2012). Calcium exchange machinery (Atakpa et al, 2018) and lipid exchange machinery (Raiborg et al, 2015) are known components of ER-endosomes MCSs. Given that TBC bulbs are similar in structure to endosomes, it is likely that proteins involved in both calcium and lipid exchange processes are also present at TBC-ER contact sites. I have shown that the main ER calcium release channel IP3R, the TRPM6 PM calcium channel, and their shared tethering protein HOMER1, are all localized to TBC-ER contact sites. These data support the conclusion that calcium exchange is a function of the TBC-ER contact site. I further demonstrated that the lipid exchange protein ORP9, and its ER membrane anchor VAPA, are also 138 localized to TBC-ER contact sites, providing evidence that lipid exchange is also likely a function of these sites. The presence of proteins like Rab5, EEA1, IP3R, VAPA, and ORP9 at the TBC-ER contact site provide structural evidence that the TBC bulb has an endosome-like identity at that the TBC MCS may function similarly to ER-endosome contact sites. 7.1.2 Function The data I’ve presented in this thesis support the hypotheses that TBC-ER membrane contact sites (MCSs) regulate the development and maturation of TBCs via calcium and lipid exchange proteins. In general, MCSs are now understood to be crucial regulators of organelle function. ER MCSs exist between many organelles including mitochondria, the plasma membrane, the Golgi, and endosomes (Raiborg et al, 2015). At endosomes, they facilitate functions that include calcium exchange, lipid transfer, and organelle positioning (Phillips and Voeltz, 2016). Because I have demonstrated that TBC bulbs are structurally similar to endosomes, the idea that similar processes may occur at the TBC-ER contact sites would be predicted. As mentioned previously, I have localized both calcium exchange machinery (Lyon and Adams et al, 2017) and lipid exchange machinery (Adams et al, 2019) to the TBC-ER contact sites, which suggests that these functions could occur at these sites and that they may be necessary for regulating TBC development and maturation. Calcium exchange at TBC-ER contact sites could be responsible for regulating the TBC actin network and/or for the maturation of TBC bulbs into endosomes. Of the 139 proteins I’ve localized to the TBC-ER contact site, inositol 1,4,5-triphosphate receptor (IP3R) would be an ideal candidate for functional experiments because it is the main calcium release channel of the ER. Using an in vivo approach, I have demonstrated that knockdown of the IP3R1 isoform in rat testis results in a loss of TBCs generally, as evidenced by a lack of actin staining in the regions associated with the proximal tubules of TBCs. This phenotype may be expected because changes in intracellular calcium levels are known to be correlated with changes in actin networks (Veksler and Gov, 2009). Moreover, actin-regulating proteins such as cortactin, which helps polymerize actin, as well as cofilin, which disassembles and rearranges actin networks, can be regulated by the release of calcium locally (Gonzalez-Jamett et al, 2017; Wang et al, 2005). Both of these proteins have been localized to TBCs (Guttman et al, 2004; Young et al, 2009), so it is possible that calcium release from TBC-ER contacts promotes remodelling of the TBC dendritic actin network, promoting their growth. This would explain the absence of the dendritic actin networks normally associated with TBCs in the IP3R1 knockdown tissue (Adams et al, 2020a). Another possible function of calcium release at TBC MCSs is the direct loading of calcium into the bulbs as they swell. Because apical TBC bulbs contain cytoplasm from adjacent sperm cells, which do not have the same high concentration of calcium that early endosomes do (Costello et al, 2009), it is possible they must receive calcium from the ER in order to facilitate their budding and maturation through the endocytic pathway. Later, early endosomes must then release calcium in order to mature and fuse into late endosomes (Saito et al, 2007). This conclusion would also be supported by the lack of ‘swollen’ bulbs seen in the IP3R1 knockdown tissue as examined by electron 140 microscopy (Adams and Vogl, 2020a). It is also supported by the observation that TBC bulbs were unable to fuse into larger endosome-like structures (Adams and Vogl, 2020a), which normally is the next step in their process through the endosomal-lysosomal system (Young et al, 2009). The data I have presented on the knockdown of IP3R in rat testis suggest that the function of calcium exchange at the TBC-ER contact sites is either to modulate the polymerisation of the actin network around TBCs and/or to participate in calcium exchange with the TBC bulb to influence its maturation. I have also provided evidence that the TBC-ER contact sites function in lipid transfer/exchange. In cells generally, non-vesicular lipid transfer at MCSs is a crucial avenue for lipid trafficking in cells. Lipids are first manufactured in the ER and must be transported to various organelles throughout the cell, which all have unique lipid membrane profiles. Lipid transfer occurs in cells both by vesicular means via the endocytic and secretory pathways, and by non-vesicular transport at MCSs (Jackson et al, 2016). While vesicular transport allows for bulk lipid transport, it also results in a progressive homogenization of the lipid makeup of organelles. Lipid transfer proteins (LTPs) present at MCSs allow the selective exchange of lipids between ER and organelle membranes (Jackson et al, 2016) as a mechanism of maintaining membrane identity. It is important for organelles to maintain their unique lipid profiles as many of these lipids serve as the basis for recruiting the proteins specific to those organelles and enabling their function. An example of these includes phosphoinositides (PIs), which are major lipid signaling molecules and protein recruitment markers in cells (Balla et al, 2013), and play a major role in establishing membrane identity. 141 The specific functions of PIs in membranes can depend on their localization and phosphorylation state. For example PI(4,5)P2 can recruit actin proteins associated with endocytosis, and conversion of PI(4,5)P2 into other PIs can alter and disassemble the actin associated with vesicles (Senju and Lappalainen, 2019). Similarly, PI(4)P is implicated in the recruitment of actin-polymerizing machinery to generate actin ‘comets’ on early endosomes (Dong et al, 2016). Thus, the type and control of PIs on endocytic and endosomal structures establishes function. One of the main LTP families known to act on PIs is the OSBP (oxysterol-binding protein) related (ORP) family of proteins, which are known to generally interact with phosphoinositides like PI(4)P and PI(4,5)P2, and sterols like cholesterol (Olkkonen and Li, 2013; Ghai et al, 2017). One of the proposed mechanisms for ORP mediated PI/Sterol transport is a counter-exchange model where an ORP is able to transport cholesterol out of the ER into the opposing membrane, and bring a PI from the opposing organelle’s membrane back into the ER. The driving force for this exchange is thought to be the creation of a favorable concentration gradient via hydrolysis of the PI by specific phosphatases on the ER membrane once the PI is brought to the ER from the opposing organelle (Antonny et al, 2018). Because ORPs can remove PIs from an organelle’s membrane, they can have a substantial impact on the identity and function of that organelle. This allows the ER to act as a crucial regulator of the progression and development of other organelles, including endocytic structures (Encinar del Dedo et al, 2017). I have provided evidence that ORP9 is localized to the TBC-ER contact sites (Adams et al, 2019), as well as provided evidence that PI(4,5)P2 is a component of the 142 TBC membrane (Adams & Vogl 2020b), suggesting that the exchange of PIs like PI(4,5)P2 and/or sterols may be a function of the TBC-ER contact. I have further demonstrated through in vivo knockdown of ORP9 in rat testis that TBCs were longer and their vesiculated bulbs failed to fuse (Adams & Vogl, 2020b). In ORP9 knockdown tissue, it is possible that failure to remove a PI such as PI(4,5)P2 from the TBC plasma membrane allows it to remain in the TBC outer membrane and continuously recruit actin-polymerizing machinery, causing TBC tubes to grow much longer than normal. The additional observation in ORP9 knockdown tissue that vesiculated TBC bulbs swell, but fail to fuse into larger endosome-like structures, suggests that ORP9 may function to change the TBC bulb membrane lipid profile to mark it for fusion with other bulbs. The function of ORP9 at TBC-ER contacts may be to either disengage actin polymerization machinery by removing the PI responsible for recruiting them and/or to change the lipid profile of the TBC bulb so it can acted upon by other endosomal proteins (eg. Rabs) in order to mature. 7.2 Conclusions of Hypotheses The data provided in each chapter of this thesis are consistent with my overall hypothesis is that TBC-ER contact sites are involved with the maturation of TBCs into mature endocytic structures, and as a consequence, also support the argument that the contact sites are necessary for the overall mechanism of sperm release. Support for this hypothesis was provided by way of five main predictions that are explored in the chapters of this thesis. Firstly, based on the general localization of endosomal markers 143 to TBCs using immunofluorescence (Young et al, 2012), I predicted that immuno-electron microscopy with similar antibodies would reveal the precise membrane association of these markers on TBCs, providing support for their identity as a putative endosome. I probed rat testis for these endosomal markers, Rab5 and EEA1, and found that they were associated with the TBC bulbs before and after vesiculation, respectively (Adams & Vogl, 2017). This lead to my conclusion that TBC bulbs were indeed endosome-like in identity, suggesting that the associated ER contact sites on TBC bulbs may function similarly to the way in which they do on endosomes. With evidence that TBC bulbs are putative endosomes, I made my second prediction that calcium exchange proteins would be localized to the TBC-ER contact sites as has been shown by others to be the case at ER-endosome contacts. Using immunofluorescence and immunoelectron microscopy, I demonstrated that the main ER calcium release channel IP3R, the calcium channel TRPM6, and their shared tethering protein HOMER1, are localized to the TBC-ER contact sites (Lyon and Adams et al, 2017). This provided structural evidence that calcium exchange could be a function of the TBC-ER contact sites, as is seen at ER-endosome contact sites. Following this, I present data to support my third prediction, that lipid exchange machinery is also localized to TBC-ER contact sites. I show that the lipid exchange protein ORP9, and it’s ER membrane anchor VAPA, are localized to TBC-ER contact sites (Adams et al, 2019). These data provide further structural evidence that another known ER-endosome contact site function, lipid exchange, could also be occurring at TBC-ER contact sites. My fourth prediction was that if calcium exchange was a function of TBC-ER contact sites, then knocking down the previously localized IP3R channel would interfere 144 with the normal maturation of TBCs. Using in vivo injection into rat testis of siRNA targeting IP3R1 mRNA, I demonstrated that a reduction in IP3R1 levels lead to a loss of TBC actin, and a failure of TBC bulbs to ‘swell’ and fuse (Adams & Vogl, 2020a). I also demonstrated that this phenotype lead to impaired sperm release. These data provided evidence that calcium exchange via IP3R at TBC-ER contact sites is necessary for TBCs to develop and mature, and further supports the importance of TBCs to spermatid release generally. Finally, my fifth prediction was that if lipid exchange was also occurring at TBC-ER contact sites, then knocking down the lipid exchange protein ORP9, which I had earlier localized to the contact sites, would affect the normal development and maturation of TBCs. Using in vivo injection into rat testis of siRNA targeting ORP9 mRNA, I showed that reduced ORP9 levels lead to elongated TBC actin networks, prevented vesiculated bulbs from fusing, and led to impaired sperm release (Adams & Vogl, 2020b). These results support the conclusion that ORP9 at the TBC-ER contacts is necessary for the vesiculation of the TBCs, perhaps by changing the lipid profile and disengaging actin polymerizing machinery, and for the fusion of TBC bulbs into large endosome-like structures. Crucially, these data further support the conclusion that TBCs are necessary components of the sperm release mechanism. 7.3 Specific Contributions A number of important contributions to the fields of cell biology and reproductive biology are made in this thesis. Firstly, never before has anyone localized proteins specifically to TBC-ER contact sites. The associated of the ER to the TBC bulbs was 145 characterized over forty years ago (Russell and Clermont, 1976), but the function of these contact sites has never been established. The data presented here are the first to provide evidence for specific proteins being present and functional at the TBC-ER contacts. Interestingly, TBC-ER contact sites are the only known membrane contact sites established with endocytosis derived structures while they are still continuous with the plasma membrane. Because apical TBCs are both easy to visualize with even standard light microscopes and are long lived compared to clathrin-mediated endocytosis derived vesicles, they are ideal for studying membrane contacts in an in vivo system. In other words, TBC-ER contacts are an ideal in vivo model for studying membrane contacts generally in cells. This work significantly contributes to our understanding of how Sertoli cells regulate the development of TBCs and thus the release of late spermatids. The in vivo observations made here that late spermatids are abnormally retained in the epithelium beyond their normal stage of release when interrupting either calcium or lipid exchange functions at TBC-ER contact sites, demonstrates that the TBC-ER contact sites have a regulatory effect on TBCs. These observations thus also add substantial weight to the hypothesis that TBCs are responsible for allowing late spermatids to be released as spermatozoa into the male reproductive tract, likely by internalizing intact intercellularly junctions between Sertoli cells and late spermatids. This new understanding of a major regulatory feature in sperm release adds substantially to our understanding of male fertility in mammals. 146 7.4 Strengths and Limitations The data provided in this thesis establishes a regulatory role of TBC-ER contacts in TBC development and late spermatid release. Both the biggest strength and limitation of this thesis is its reliance on whole tissue and in vivo experiments. The immediate advantages of these approaches are that the cells are observed and manipulated in their native environments, giving the results an unparalleled scope of accuracy into the workings of the male reproductive system that would not be obtainable with in vitro approaches such as cell culture. In vivo work is more cumbersome and involved than in vitro, but there exists an advantage in the applicability to the native system of the results obtained through in vivo work. Though many others have tried with some limited success (Du et al, 2013; Sriram, 2019), there are currently no established primary Sertoli cell culture techniques that produce typical looking TBCs to study in vitro, leaving in vivo approaches as the only option for studying TBCs. Substantial advancements in microscopy techniques in the last decade have allowed biologists to explore exciting new avenues of cell biology, and many of those techniques have been utilized in this thesis. The pre-embedding immunoelectron microscopy used throughout this work to accurately localize protein to TBC-ER contact sites is one that I adapted for use in whole testis tissues from protocols meant for cell isolates, that I began as part of my undergraduate thesis work in the Vogl lab (Adams, 2015). This retooling of an established microscopy technique is an approach that is unique to our lab and has strengthened the conclusions of the morphological data presented throughout this thesis. Another microscopy technique introduced for the first time to the field of Sertoli cell biology by the work in this thesis is the use of stimulated 147 emission depletion (STED) microscopy. This work is the first to use STED microscopy in whole testis tissue, and has allowed me to localize proteins with antibodies at resolutions exceeding our standard confocal microscopy techniques. Both the use of pre-embedding immunoelectron microscopy and STED microscopy in testis are unique to my thesis work and contribute substantially to the strength of the morphological conclusions drawn in this work. Because we are limited to in vivo approaches to study TBCs, functional experiments can be challenging as it is much more difficult to knockdown or knockout genes in whole animal models versus in cell culture. However, the use of siRNA to knockdown gene expression for TBC associated proteins in vivo has been performed successfully in our lab by others (Young et al, 2012) and has been used similarly throughout this thesis (Adams & Vogl, 2020a; Adams & Vogl, 2020b). There are several limitations to this approach versus using siRNA in cell culture and these include: the need to inject substantial quantities of siRNA to affect large areas of the testis, the need to use in vivo grade siRNAs that are degradation resistant but more expensive, and the need to inject siRNA repeatedly over many days to maintain the knockdown. Until the time that there is a well-established Sertoli cell culture system that can produce TBCs, these limitations are acceptable in order to be able to study the function of TBC-associated proteins. While whole animal gene knockouts are a potential alternative to in vivo knockdowns with siRNA, there are major obstacles with this approach that make it less than ideal. Firstly, the cost of producing knockout animals, even with the advent of CRISPR-cas9 gene editing technology, is substantially greater than using in vivo grade siRNA. Even more challenging to overcome than cost is the fact that many proteins one 148 suspects of being necessary for TBC function are also expected to be necessary for male fertility generally. Thus, knockout animal lines for TBC proteins run the risk of being unmaintainable as the animals would theoretically not be able to successfully reproduce. Further, even if these animals were able to reproduce, a complete knockout of a protein is likely to prevent most TBC formation all together. Though, this would allow us to infer the necessity of the protein to TBCs, it would not enable us to draw conclusions on which specific part of the TBC maturation process were interrupted by the loss of the protein. These limitations make in vivo knockdown with siRNA advantageous, as spermatogenesis can be safely affected for temporary periods, and the immediate effects on TBCs can be characterized. Crucially, in vivo grade siRNA enables us to evaluate the effect of reduced protein levels on TBCs at various time points, allowing us to draw conclusions on which TBC maturations steps are affected by the knockdown. 7.5 Potential Research Applications The observations that knockdown of TBC-ER contact associated proteins resulted in spermiation delay, are consistent with the conclusion that TBCs are a vital component of normal sperm release. Nearly half of male infertility disorders in men have unknown causes (Rowe, 2000), and so an understanding of the basic cell biology of late spermatid release by Sertoli cells would likely be prerequisite to explaining many of these pathologies. Alternatively, it would also be possible to design pharmaceutical agents to target proteins known to be specific to TBC-ER contact sites as a form of male contraception. 149 One potential example of this may be ORP9, which in our hands, was shown to concentrate at TBC-ER contact sites with only low amounts at the Sertoli cell Golgi apparatus (Adams et al, 2019). In whole testis sections, ORP9 is so specific to the TBC-ER contact sites that we now use it as our gold standard in the Vogl lab for marking these sites when doing immunolabelling studies. Proteins like ORP9 that are nearly exclusively localized to TBC-ER contact sites could be chemically targeted and interfered with in vivo, potentially having contraceptive outcomes. However, prior to any clinical studies, further understanding of the protein architecture of the TBC-ER contact sites would be ideal in order to select the most promising targets for any potential contraceptive agent. 7.6 Future Directions As previously indicated as a limitation, the lack of a functional Sertoli cell culture system that can support spermatogenic cells and subsequently produce TBCs is a major drawback for studying these structures. The introduction of a reproducible, available, and morphologically mature Sertoli cell line culture system would vastly increase our ability to perform a range of knockdown and knockout experiments that are cumbersome or expensive to do in vivo. Such a system would add significantly to the field, and would likely attract a broader range of cell biologists to what is otherwise considered a ‘niche’ area of research. By extension of this, an alternative to cell culture systems could be the use of whole organ culture of seminiferous tubules. This type of experimental approach would provide a half-way point between cell culture and in vivo work, keeping the epithelial architecture mostly intact while also enabling greater 150 experimental manipulation by means of knockdowns, knockout, or imaging approaches. Currently, there are at least a few seminiferous tubule organ culture protocols in existence that vary only slightly in design, but which are reportedly able to maintain spermatogenesis in culture for several weeks (Gholami et al, 2018; Sato et al, 2011). The introduction of either a TBC producing primary Sertoli cell culture or an accessible seminiferous tubule organ culture would enhance the scope of work that can currently be done on TBCs. Once a Sertoli cell or seminiferous tubule culture system is established, this would open the door for live cell imaging of spermatogenic processes, something which is currently infeasible in an in vivo system. For example, the two processes I have proposed to likely occur at TBC-ER contact sites are calcium and lipid exchange, which could both be visualized in an in vitro system. Live-cell calcium imaging of a seminiferous tubule organ culture would be feasible using multi-photon microscopy. Tubules could be transfected with genetically encoded calcium indicators (GECIs) such as a GCaMP series indicator and then imaged through the intact tubule wall and into the tubule lumen where the late spermatids and surrounding Sertoli cell apical processes occur. Similarly, an organ culture system could be transfected with plasmids coding for fluorescently labelled PI-binding proteins specific to different phosphoinositides, and imaging could be again done with multi-photon microscopy. Results from both approaches could add significant support to the hypotheses that calcium and lipid exchange are functions of TBC-ER contact sites. Until live-cell imaging of TBCs is possible, it will be important to continue to characterize the protein architecture of TBC-ER contact sites in order to elucidate their 151 function in TBC development and overall spermatid release. While I have shown examples of both calcium and lipid exchange proteins being both present and functional at these sites, it is likely that other proteins involved in these processes are also localized to the contact sites. For example, while I have focused largely on the ORP family of lipid exchange protein in this thesis, it would be reasonable to expect that examples of other families of lipid exchange proteins could be localized to these sites. There are at least 27 families of lipid exchange proteins and at least two of these, the ORP family and StARkin superfamily proteins, are commonly found at ER membrane contact sites (Wong, et al 2018). Thus there remains many possibilities of proteins in the lipid exchange category that may be present at TBC-ER contacts. However, this does not exclude the presence of proteins involved in functions outside calcium and lipid exchange also being components of the contact sites. Ultimately, a ‘shotgun’ approach wherein we localize proteins to the contact sites using antibodies and our best ‘guesses’ will not be adequate in the long-term to fully understand the breadth of their function. The best approach to solving this issue would be to develop an isolation protocol that can largely concentrate and purify TBCs or the entire Sertoli cell apical processes so that they can be analyzed by mass spectrometry. This would substantially narrow down the list of candidate proteins that may be functional at the contact sites, allowing for more targeted searches. 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Thesis/Dissertation
eng
Cell and Developmental Biology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
The role of tubulobulbar complex-endoplasmic reticulum contact sites during junction turnover in the seminiferous epithelium
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