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Implicating calcium signaling at intercellular junctions and structures associated with junction turnover… Lyon, Kevin 2017

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 IMPLICATING CALCIUM SIGNALING AT INTERCELLULAR JUNCTIONS AND STRUCTURES ASSOCIATED WITH JUNCTION TURNOVER IN RAT SERTOLI CELLS by  Kevin Lyon  B.Sc., The University of British Columbia, 2015  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Reproductive and Developmental Sciences)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2017  © Kevin Lyon, 2017 ii  Abstract The endoplasmic reticulum (ER) is a prominent organelle in Sertoli cells. It is an integral component of unique adhesion junctions (ectoplasmic specializations - ESs) in this cell type, and is closely associated with structures termed tubulobulbar complexes (TBCs) that internalize intercellular junctions during sperm release and during the translocation of spermatocytes through the blood-testis barrier. A role for the ER in Ca2+ regulation at ESs and TBCs has been suspected, but evidence for this function has proved elusive. The focus of this thesis is identification of molecular machinery involved in Ca2+ signaling and obtaining functional evidence of Ca2+ regulation of the actin networks at TBCs. Functional experiments using EGTA and thapsigargin to lower and raise Ca2+ levels did not provide evidence that TBC actin networks are regulated by Ca2+. Using immunofluorescence, I demonstrated that Ca2+ regulatory machinery is present at the ESs attached to spermatid heads, and at ER-PM contacts. SERCA2 is present at ESs, IP3R is present at ER-PM contacts associated with TBC bulbs, and STIM1, ORAI1 and SERCA2 are present at the ER-PM contacts around the margins of Sertoli cell apical processes. The results support the conclusion that the molecular machinery necessary for ER generated Ca2+ fluxes is present in regions and structures directly related to junction remodeling in Sertoli cells, a process necessary for sperm release. iii  Preface I designed, carried out the experiments and analyzed data in chapter 3. The figures were prepared by me with the exception of the schematic diagrams that appear in the introduction and in chapter 3 which were prepared by Dr. Wayne Vogl. Clement Ho was involved in the execution of experiments related to Src and PTP1B. Julien Lima was involved in execution of preliminary experiments relating to STIM1 and ORAI1. Dr. Wayne Vogl was involved in the execution of experiments. Both Dr. Wayne Vogl and I collected electron micrographs for analysis of ER-PM contacts. The EGTA and thapsigargin studies in chapter 4 were designed and executed by myself. Dr.  Wayne Vogl was involved in the design and execution of these experiments. The live Ca2+ imaging experiments were designed by myself, Dr. Wayne Vogl, Dr. Edwin Moore and Dr. Parisa Asghari. This experiment was carried out by a number of individuals. Dr. Wayne Vogl and Arlo Adams handled the animals and isolated seminiferous tubules. Aarati Sriram prepared fresh media specifically designed to culture Sertoli cells to be used for incubating the isolated tubules. Dr. Parisa Asghari performed the live Ca2+ imaging and helped in the analysis of data. Dr. David Scriven developed software for data analysis as well as provided insights during data analysis. I was involved throughout all of these processes and analyzed the data obtained from these experiments. Dr. Edwin Moore, Dr. Parisa Asghari and Dr.  David Scriven contributed to the analysis of the data.  The majority of the data from Chapter 3 has been submitted for publication in “Biology of Reproduction” under the title “Ca2+ signaling machinery is present at intercellular junctions and structures associated with junction turnover in rat Sertoli cells”. This manuscript was written by myself, Dr. Wayne Vogl and Arlo Adams with editing by Dr. Parisa Asghari, Dr. Edwin Moore iv  and Matthew Piva. Parts of this manuscript were adapted for use in this thesis. The Src and PTP1B data in this chapter is intended to be submitted as a separate manuscript to “The Anatomical Record”. v  Table of Contents Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iii Table of Contents ...........................................................................................................................v List of Tables ............................................................................................................................... vii List of Figures ............................................................................................................................. viii List of Abbreviations .....................................................................................................................x Acknowledgements ...................................................................................................................... xi Chapter 1: Introduction ............................................................................................................... 1 1.1  Spermatogenesis and mammalian seminiferous epithelium ....................................... 1 1.2  Stages of spermatogenesis .......................................................................................... 2 1.3  Turnover of ectoplasmic specializations is critical for spermatogenesis .................... 3 1.4  Tubulobulbar complexes: the endocytic machines of junction turnover in spermatogenesis ...................................................................................................................... 4 1.5  Endoplasmic reticulum: calcium regulation of tubulobulbar complexes? .................. 6 1.6  Hypothesis and Objectives .......................................................................................... 6 Chapter 2: Methods ...................................................................................................................... 9 2.1  Animals ....................................................................................................................... 9 2.2  Anesthesia and euthanasia .......................................................................................... 9 2.3  Reagents ...................................................................................................................... 9 2.4  Electron microscopy ................................................................................................... 9 2.5  Immunofluorescence microscopy ............................................................................. 11 2.6  Ca2+  imaging ............................................................................................................ 14 vi  2.7  Immunoblotting......................................................................................................... 15 Chapter 3: Evidence for Ca2+ signaling machinery at ectoplasmic specializations and tubulobulbar complexes in Sertoli cell apical processes .......................................................... 18 3.1  Introduction ............................................................................................................... 18 3.2  Results ....................................................................................................................... 20 3.3  Discussion ................................................................................................................. 25 Chapter 4: Studies on obtaining functional evidence of calcium fluxes in Sertoli cell apical processes....................................................................................................................................... 45 4.1  Introduction ............................................................................................................... 45 4.2  Results ....................................................................................................................... 46 4.3  Discussion ................................................................................................................. 47 Chapter 5: Conclusion ................................................................................................................ 53 5.1  Overall analysis and conclusions in light of current research ................................... 53 5.2  Strengths and limitations of thesis ............................................................................ 54 5.3  Future directions ....................................................................................................... 55 References .....................................................................................................................................58  vii  List of Tables Table 2.1 Antibodies ..................................................................................................................... 17  viii  List of Figures Figure 1.1 Schematic diagram of the seminiferous epithelium. ..................................................... 8 Figure 3.1 Cryosections of stage VII seminiferous tubules show IP3R labeling near the distal ends of tubulobulbar complexes. .................................................................................................. 30 Figure 3.2 Epithelial fragments show IP3R labeling at the bulb regions of tubulobulbar complexes. .................................................................................................................................... 31 Figure 3.3 Cryosections of stage VII seminiferous tubules show STIM1 labeling Sertoli cells and their apical processes. ................................................................................................................... 32 Figure 3.4 Epithelial fragments show STIM1 labeling throughout apical processes. .................. 33 Figure 3.5 Cryosections of stage VII seminiferous tubules show ORAI1 labeling Sertoli cells and their apical processes. ................................................................................................................... 34 Figure 3.6 Epithelial fragments show ORAI1 throughout apical processes. ................................ 35 Figure 3.7 STIM1 and ORAI1 colocalize at the periphery of apical processes. .......................... 36 Figure 3.8 ER-PM contacts at the periphery of apical processes. ................................................ 37 Figure 3.9 Cryosections of stage VII seminiferous tubules show SERCA2 labeling the ER throughout Sertoli cells and their apical processes. ...................................................................... 38 Figure 3.10 Epithelial fragments show SERCA2 labeling ectoplasmic specializations. .............. 39 Figure 3.11 Cryosections of stage VII seminiferous tubules show Src labeling near the distal ends of tubulobulbar complexes. .................................................................................................. 40 Figure 3.12 Epithelial fragments show Src labeling at the bulb regions of tubulobulbar complexes. .................................................................................................................................... 41 Figure 3.13 Cryosections of stage VII seminiferous tubules show PTP1B labeling of Sertoli cells and their apical processes.............................................................................................................. 42 ix  Figure 3.14 Epithelial fragments show PTP1B labeling throughout Sertoli cell apical processes........................................................................................................................................................ 43 Figure 3.15 Summary of Ca2+ signaling machinery localized within apical processes of Sertoli cells. .............................................................................................................................................. 44 Figure 4.1 Perfusion of 1 mM EGTA for 1 hr has no effect on length of tubulobulbar complexes........................................................................................................................................................ 50 Figure 4.2 Perfusion of thapsigargin for 1 hr has no effect on length of tubulobulbar complexes........................................................................................................................................................ 51 Figure 4.3 Live imaging of Ca2+ in isolated apical processes. ...................................................... 52  x  List of Abbreviations ATP – adenosine triphosphate BTB – blood-testis barrier DMSO – dimethyl sulphoxide EGTA - ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid ER – endoplasmic reticulum ES – ectoplasmic specialization GECI – Genetically encoded calcium indicators NMIgG – Normal mouse immunoglobulin gamma NRIgG – Normal rabbit immunoglobulin gamma IP3R – inositol 1,4,5-trisphosphate receptor PIP2 – phosphatidylinositol 4,5-bisphosphate PLCγ – phospholipase C-γ PM – plasma membrane PMCA – plasma membrane Ca2+ ATPase PTP1B – protein tyrosine phosphatase SERCA2 – sarco/endoplasmic reticulum Ca2+-ATPase 2 SOCE – store-operated Ca2+ entry STIM1 – stromal interacting molecule 1 TBC – tubulobulbar complex TRPM6 – transient receptor potential channel subfamily M member 6  xi  Acknowledgements To my greatest role model and mentor, Dr. Wayne Vogl. It has been my pleasure to have been a part of your lab over the past 5 years. I am ever grateful to Dr. Lacey Samuels for having connected me with you early in my undergraduate degree. Your seemingly never ending patience, encouragement and support over the years has made all the difference in my university degrees.  I would like to thank Dr. Edwin Moore, Dr. Parisa Asghari and Dr. David Scriven for their help in designing the Ca2+ imaging experiment and in the analysis of the data. Special thanks to Dr. Maxence Le Vasseur for taking the time to train me on the confocal microscope and how to perform deconvolution. Your advice was helpful in optimizing my immunoblotting and immunofluorescence protocols. Thank you Warren Meyers for the fruitful discussions of my work and for the helpful tips regarding my immunoblotting protocols. I am ever grateful for providing access to your lab’s blot imager. To my lab mates, Arlo Adams and Aarati Sriram, thank you both for your help and entertaining conversations. Our adventures have definitely been a highlight throughout this thesis.   Last but certainly not least, thank you to my partner, Trevor Leggat, for putting up with my late nights at the lab and supporting me throughout my degree.1  Chapter 1: Introduction 1.1 Spermatogenesis and mammalian seminiferous epithelium The process of mammalian spermatozoa production, spermatogenesis, takes place in the seminiferous epithelium that lines seminiferous tubules of the testes. There are two groups of cells within the seminiferous epithelium: Sertoli cells and developing spermatogenic cells. Sertoli cells are the architectural units of the seminiferous epithelium. They are columnar in shape and extend from the base to apex of the epithelium. Their nuclei are typically located basally in the cells adjacent to the basement membrane which is secreted both by Sertoli cells and by peritubular myoid cells that form part of  the walls of the seminiferous tubules [1]. Sertoli cells are located adjacent to each other and embedded between them are the spermatogenic cell line.  The blood-testis barrier (BTB) is formed predominantly by tight junctions between adjacent Sertoli cells and divides the epithelium into an adluminal compartment and a basal compartment. The basal compartment is continuous with the interstitial space between adjacent tubules [2] which contains Leydig cells and blood vessels. In addition to providing an immune-privileged environment in the adluminal compartment, the blood-testis barrier also enables Sertoli cells and germ cells to produce and maintain a unique physiological environment in the this compartment [3].  Spermatogonial stem cells are located ‘beneath’ Sertoli cell tight junctions and on the basement membrane where they undergo a series of mitotic divisions to give rise to primary spermatocytes. These primary spermatocytes then lift off of the basement membrane and translocate through the BTB into the adluminal compartment [4] where they complete meiosis giving rise to the haploid round spermatids. These round spermatids undergo morphological 2  differentiation into spermatids which are eventually released as spermatozoa into the lumen of the tubules in a process referred to as spermiation.  1.2 Stages of spermatogenesis Leblond and Clermont staged the rat spermatogenic cycle based on the cellular associations seen in cross sections of seminiferous tubules [5]. The number of stages vary between species with one spermatogenic cycle consisting of 14 stages in rats [5], 12 in mice [6], and 6 in humans [7]. By convention, the stages of the spermatogenic cycle are indicated with Roman numerals to avoid confusion with ‘steps’ of the spermatid differentiation. Although within a single spermatogenic cycle spermatozoa will be released into the lumen, the time it takes for a spermatogonium to develop into a spermatozoa takes multiple spermatogenic cycles. This is due to the presence of multiple cohorts of germ cells at different steps of development for any given stage of spermatogenesis. For example, in a stage I seminiferous tubule of a rat there will be spermatogonia, primary spermatocytes originating from spermatogonia of the previous spermatogenic cycle, and two groups of spermatids originating from spermatogonia of two and three cycles previously.   A single spermatogenic cycle takes 12.9 days in rats [8], 8.6 days in mice [9], and ~16 days in human [7]. It takes approximately 4.5 spermatogenic cycles in mammals for the entire process of spermatogenesis to occur [10,11]– that is, it takes roughly 57 days in rats and 73 days in humans for spermatogonium to differentiate into a mature spermatozoa. Although spermatozoa are released only at one specific stage during each cycle, spermatozoa are released continuously from any given seminiferous tubule.  This is due to the phenomenon known as the spermatogenic wave where segments along a seminiferous tubule will be at different stages of the spermatogenic cycle in succession [12]. For example,  a segment of a seminiferous tubule at Stage I will have a 3  following segment at Stage II. This allows for the continuous production of spermatozoa by the testis throughout adulthood.  1.3 Turnover of ectoplasmic specializations is critical for spermatogenesis  The seminiferous epithelium contains unique plaques of intercellular junctions termed ectoplasmic specializations (ESs) [13]. These are tripartite structures characterized by three morphological characteristics: a region of plasma membrane (PM) containing junction proteins, attached bundles of hexagonally packed actin filaments, and an associated cisterna of endoplasmic reticulum (ER). These structures are found in two regions within the seminiferous epithelium. The first is located basally between adjacent Sertoli cells where PM contains the tight junctions forming the BTB occur [2] in addition to adhesion junctions [14,15] and gap junctions [16]. The associated actin bundles and ER cisterna are found in both Sertoli cells participating in attachment as such these sites are often referred to as homotypic junction complexes to differentiate it from the second region where ESs occur (Fig. 1.1). The second location where ESs are found is apically where projections of Sertoli cells, termed apical processes, surround the heads of late spermatids to anchor them in the epithelium prior to their release (Fig. 1.1). These apical ESs are comprised of nectin-based and integrin-based adhesion junctions [14,15,17,18]. However, the tripartite structure of an ES is only seen on the Sertoli cell side of the intercellular junction where the spermatid side of the intercellular junction lacks associated bundles of actin and cisterna of ER. Thus apical ESs are referred to as heterotypic junction complexes to distinguish them from the basal homotypic junction complexes.   The turnover of the junctions within the ESs both at basal and at apical sites is crucial for spermatogenesis. During spermatocyte translocation from the basal to adluminal compartments of the seminiferous epithelium, junctions in basal ESs must disassemble above spermatocytes and 4  reassemble below to maintain compartmentalization of the epithelium. At apical sites, junctions in ESs anchoring the spermatid must be disassembled in the final step of spermiogenesis where the spermatid is released into the lumen. The process of release is referred to as spermiation and the free germ cell is now referred to as a spermatozoan. 1.4 Tubulobulbar complexes: the endocytic machines of junction turnover in spermatogenesis Clathrin-based endocytic structures known as tubulobulbar complexes (TBCs) internalize and remove intact intercellular junction proteins both at apical and at basal ESs [19–21]. These structures form initially as clathrin-coated pits which eventually grows into elongated structures of up to 1-2 µm in eutherian mammals. In electron micrographs, cross-sections of these structures reveal that they consist of a double membrane core. The outer membrane is formed from the invagination of the Sertoli cell PM while the inner membrane consists of an evaginated adjacent Sertoli cell PM or spermatid membrane [22] that has been pulled along through its intercellular junctions. These elongated structures are comprised of three distinct morphological regions: a long proximal tubule, a bulb region and a short distal tubule [22]. The proximal and distal tubule regions contain an associated dendritic actin network that cuffs the membrane core with a clathrin-coated pit at the end of the distal tubule (Fig. 1.1) [23,24]. A swelling develops in the distal end of the structure which separates the proximal and distal tubules and is referred to as the bulb. The bulb region lacks the actin cuffs associated with the tubule regions, but is closely related to a cisterna of ER [22].  The actin networks of the tubule regions eventually disassemble resulting in the swelling of both the distal and proximal tubules effectively being absorbed into the bulb [25]. The structure undergoes scission at its base and enters the endosomal pathway of the Sertoli cell resulting in the 5  fusion with a lysosome [22,23]. This process effectively removes intact intercellular junctions at basal and apical sites to allow for spermatocyte translocation and spermiation respectively. The disruption of these structures has been shown to delay the release of spermatogenic cells from the seminiferous epithelium. When the actin network stabilizing protein present at TBC actin cuffs, cortactin, was knocked down using siRNA injections, TBCs appeared shorter and spermatids were retained in the epithelium [26] The protein components of the TBCs have been characterized extensively. The coated pits contain clathrin [27], the protein adaptor AP-2 [28], and the AP-2 binding protein, Eps15 [29]. Amphiphysin [30] and dynamin [30,31] line the tubular regions of TBCs. Amphiphysin contains a BAR domain that associates with membrane curvature and is thought to aid in tubulation of membrane structures [32]. Although dynamin does not contain this BAR domain, they are also known to be able to induce membrane tubulation [33]. The finding of dynamin along the entire length of the tubular regions suggested that these structures undergo scission at multiple sites [31], however, a recent study suggests that the tubular regions eventually swell into the bulb and the structures undergoes scission at its base [25]. The dendritic actin networks that cuff the tubular regions contain a protein profile similar to that of dendritic actin networks in general. N-WASP, Arp2/3 and cortactin have all be localized to TBC actin cuffs [27,31]. N-WASP binds to the ends of an existing actin filament and regulates the activity of Arp2/3 complex which nucleates new actin filaments at a 70° angle. Cortactin is another known promoter of the Arp2/3 complex [34,35] and its activity is regulated by post-translational modifications such as phosphorylation [36]. The actin cuffs are essential for the formation of TBCs as demonstrated by elimination of the structures except for coated pits after intratesticular injection of high doses of cytochalasin D [37]. 6  1.5 Endoplasmic reticulum: calcium regulation of tubulobulbar complexes?  Little is known about the function of the ER associated with the bulb regions of TBCs. Morphologically similar structures, podosomes, are cell protrusions with an actin network core that functions in motility and adhesion. It has been reported that Ca2+ oscillations caused by store-operated Ca2+ entry (SOCE) drive the polymerization of the actin network elongating the podosomes in melanoma cells [38]. Perhaps the ER at TBC bulbs functions in a similar fashion, to drive the polymerization of the actin cuffs resulting in the elongation the structure. Alternatively, it may function in the disassembly of the structure rather than its formation. When rat testes were perfused ex vivo with cytochalasin D, actin cuffs were disrupted and the resulting TBCs resemble early TBCs that contain massive bulbs [25]. Perhaps Ca2+ signaling at the bulb region functions to progressively remove the actin cuffs prior to vesiculation of the structures. 1.6 Hypothesis and Objectives In this thesis, I hypothesize that Ca2+ signaling occurs at TBCs to orchestrate changes in their actin networks. To investigate this hypothesis, I had two major objectives.  My first objective was to determine if the molecular machinery for Ca2+ signaling is present at TBCs. If Ca2+ signaling occurs at these structures then they should possess molecular machinery known in other systems to orchestrate local changes in Ca2+ levels. To evaluate whether this machinery is present at both ESs and TBCs, I used immunofluorescence to probe tissue from Rattus norvegicus for Ca2+ machinery.   My second objective was to obtain functional evidence of Ca2+ signaling altering the actin networks at TBCs. I evaluated the effects of the Ca2+ chelator, EGTA, to investigate the effect of lowering Ca2+ concentration on TBCs. To investigate the effect of increasing Ca2+ concentration on TBCs, I used the ER Ca2+ pump blocker, thapsigargin, which would prevent the pumping of 7  Ca2+ from the cytoplasm into the ER resulting in a rise in cytosolic Ca2+. Additionally, I attempted to visualize Ca2+ fluxes at apical TBCs using live Ca2+ imaging.    8   Figure 1.1 Schematic diagram of the seminiferous epithelium.  Positions of ectoplasmic specializations (ESs) and tubulobulbar complexes (TBCs) in the seminiferous epithelium are shown. Germ cells are shaded with grey and are embedded between adjacent Sertoli cells which are shaded with yellow. The ER is highlighted in blue. Actin filaments are highlighted in red. TBCs occur at basal ESs to internalize junctions between adjacent Sertoli cells to allow for spermatocyte translocation. Within the apical processes of Sertoli cells, ESs occur at sites of attachment with spermatid heads. Removal of junctions by TBCs at apical sites is a crucial step of spermiation. 9  Chapter 2: Methods 2.1 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 (#A16-0069) approved by the Animal Care Committee of the University of British Columbia. All rats were healthy at the time of use and ranged from 300 to 500 g in weight.  2.2 Anesthesia and euthanasia 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.  2.3 Reagents Unless otherwise noted, reagents were obtained from Sigma-Aldrich. See Table 2.1 for details on antibodies used. 2.4 Electron microscopy  2.4.1 Fixation and embedding The spermatic artery on the dorsal surface of each testis was canulated with a 26-gauge needle and then the organs were gravity perfused briefly with PBS (150 mM NaCl, 5 mM KCl, 0.8 mM KH2PO4, 3.2 mM Na2HPO4, pH 7.3, 33°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, 33°C]. The testes were cut into small (1 cm3) blocks, and the blocks fixed further by immersion for an additional 2 hrs in the same fixative at room 10  temperature. Samples were washed three times in 0.1 M sodium cacodylate for 10 min and then post-fixed on ice for 1 hr in 1% OsO4 (EM Sciences) in 0.1 M sodium cacodylate buffer. Samples were washed three times (10 min each wash) with ddH2O then stained en bloc with 1% uranyl acetate (EM Sciences) for 1 hr 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 hr incubations in 100% EM-bed 812, and then embedded in 100% EM-bed 812 and incubated at 60°C for approximately 48 hrs.  2.4.2 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 (90 nm) 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’ lead citrate.  The material was imaged using a Tecnai G2 Spirit Transmission Electron Microscope (FEI) operated at 120 kV. 2.4.3 Distance measurements and statistical analysis TEM images from two animals were used to measure the distance between the PM and ER both at the periphery of the apical process. Each associated cistern of ER was measured at the 11  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. 2.5 Immunofluorescence microscopy 2.5.1 Drug perfusion For ex vivo drug perfusions, a pair of testes were removed from the same animal (when possible) and each testis was perfused with Krebs-Henseleit buffer (with 4% bovine serum albumin) containing either the drug of study (EGTA or thapsigargin) or the control buffer at 33°C using 2 channel peristaltic pump set to 1 mL/min. Then the testes were gravity-perfusion fixed for 30 minutes as described below.  For EGTA studies, 1 mM of EGTA was perfused for 10 min with a control testis was perfused with buffer containing 3 mM Ca2+ for the same length of time. For the thapsigargin studies, 1 µM of thapsigargin was perfused for 1 hr with a control testis being perfused with buffer containing DMSO at the same concentration.  2.5.2 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 33°C. One half of the testis was used to obtain cryosections while the other was used to obtain epithelial fragments.  2.5.3 Cryosections  The half of each testis dedicated to cryosections was mounted onto an aluminum stub using Optimal Cutting Temperature (OCT) compound (Sakura Finetek). The testis was flash-frozen using liquid nitrogen and 10 µm thick sections were obtained using a cryomicrotome. 12  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.  In the drug perfusion experiments, the acetone step was replaced with a 10 min incubation to attach the sections to the slides. Tissue was subsequently washed with PBS/0.1% BSA for 10 minutes  and stained with fluorescent phallotoxins followed by mounting with Vecta Shield Mounting medium (Vector Laboratories). 2.5.4 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-gauge needle followed by a 21-gauge needle. The solution was allowed to stand for 10 minutes 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 minutes inside a humidity chamber.  2.5.5 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 hr while fragments were blocked with 5% BSA/0.1% saponin/PBS for 1 hr. 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 4°C in a humidity chamber. The samples were washed three times for 10 13  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 37°C for 1 hr. They were then washed for 10 min by replacing 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 [39]. 2.5.6 Optimizations  Sections labeled with mouse anti-SERCA2 were not treated with acetone immediately after sectioning, instead they were treated identically to epithelial fragments with the inclusion of 0.1% saponin in buffers for permeabilization.  2.5.7 Colocalization analysis  Deconvolved Z stacks of epithelial fragments were used for colocalization analysis of STIM1 and ORAI1 antibody labeling. The imageJ plugin, JACoP [40], was used to calculate Manders’ coefficients for STIM1 and ORAI1 labeling. Due to a limitation with the plugin not being able to measure colocalization within a region of interest in a Z stack, all labeling outside of the fragment consisting of debris and artifact was removed using functions contained in the imageJ software prior to colocalization analysis. This enabled a measure of colocalization of the fragment without artificial inflation caused by debris and artifacts. The thresholds for both STIM1 and ORAI1 channels were determined visually and applied identically across all experiments.  14  2.5.8 Statistical analysis of tubulobulbar complex actin cuff measurements  Micrographs of apical TBCs were used to measure the length of TBC actin cuffs. All groups of data followed a Gaussian distribution as verified by a Shapiro-Wilk normality test. Statistically significant differences between groups was evaluated using an unpaired two-tailed t-test. The variances between groups that were compared were evaluated with an F test to determine if their variances were significantly different – no significant differences were found. 2.6 Ca2+  imaging 2.6.1 Serum-free defined medium (SFDM) The media used to isolate and image was prepared using DMEM/F12 cell culture medium as a base. Reagents were added to give a final composition of: 100 U/ml penicillin, 0.1 mg/ml streptomycin, 6.7 ng/ml sodium selenite (Invitrogen), 10 µg/ml insulin (Invitrogen), 5.5 µg/ml transferrin (Invitrogen), 250 ng/ml Fungizone, 100 ng/ml rat follicle-stimulating hormone (FSH) (National Hormone and Peptide Program), 10 ng/ml recombinant human epidermal growth factor, 50 ng/ml vitamin A, 200 ng/ml vitamin E, 1 nM hydrocortisone (Sigma-Aldrich), 0.1 µM testosterone, 10 nM estradiol, 2 mM L-glutamine (Invitrogen), 3 µg/ml cytosine beta-D-arabinofuranoside.  2.6.2 Epithelial isolation Animals were put under deep anesthesia using an intraperitoneal injection of pentobarbital rather than isoflurane which is known to induce IP3R calcium release. Testes were removed and decapsulated in SFDM media. The seminiferous tubules were cut into pieces using scalpels. The epithelium from stage VII seminiferous tubules was isolated using a technique developed by Guttman and colleagues [41]. After the epithelium has been stripped from the tubules, they were 15  transferred to 4-chambered #1.0 borosilicate coverslips (Lab-Tek) coated with poly-L-lysine and allowed to attach for 10 min.   2.6.3 Ca2+ monitoring Chambers were washed with fresh media to remove unattached material. The isolated epithelium was then permeabilized for 4 min using modified media containing 30 µg/mL saponin, 5 mM ATP and buffered to 100 µM Ca2+. EGTA was used to buffer Ca2+ to 100 µM as calculated by the online resource MAXchelator Ca-Mg-ATP-EGTA Calculator V1.0 using constants from NIST database #46 v8. Saponin was washed away using the above modified media without saponin and with 30 mM Fluo-4. The chamber was then placed on a widefield microscope fitted with a CCD camera and recording initiated. The media was then replaced with media containing 3.5 µM Adenophostin A, 30 mM Fluo-4, 5 mM ATP, 100 µM Ca2+.   2.7 Immunoblotting 2.7.1 Whole testis lysates  Testes from several animals were decapsulated and the tissue was pooled. The tissue was 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).  2.7.2 Seminiferous epithelium lysates This lysate was prepared using the method developed by Guttman et al. (2007) 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 16  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 2 mg/mL using Laemmli sample buffer. Aliquots were stored at -20°C until needed. 2.7.3 SDS-PAGE and membrane transfer  SDS-PAGE was performed as described by Laemmli (1970) and is briefly described here. Unless otherwise stated, 20 µg of lysates were loaded into 1-mm-thick 10% SDS-PAGE gels and run at 100 V for 1 hour. The gel was allowed to equilibriate in wet transfer buffer (25 mM Tris, 192 mM glycine, pH 8.3) for 10 minutes. Proteins were transferred to PVDF membranes using wet transfer for either 1 hr at constant 350 mA or overnight at constant 90 mA in 4°C. 2.7.4 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 4°C or for 1 hr at room temperature. Primary antibody was diluted in blot buffer and added to the membrane for 1 hr 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 hr at room temperature. The membrane was washed three times for 5 min each wash with TBST and transferred to ECL solution for 3 minutes. Membranes were imaged on an ImageQuant LAS 4000 system (GE Life Sciences). 17  2.7.5 Optimizations The IP3R blot had 20 µg of protein loaded into a 6% polyacrylamide gel and was transferred overnight at 90 mA 4°C. The STIM1 and ORAI1 blot had 30 µg of protein loaded into a 10% polyacrylamide gel and was transferred for 1 hr at room temperature. The SERCA2 blot had 60 µg of protein loaded into a 10% polyacrylamide gel and was transferred for 1 hr at room temperature.  Table 2.1 Antibodies Antibody Manufacturer Catalog Number Lot # IF (µg/mL) Blotting (µg/mL) Rabbit anti-Calnexin  Sigma-Aldrich C4731 054M4870V - 1:40,000 dilution Rabbit anti-Cortactin  Sigma-Aldrich C7112 076M4790V 90 - Mouse anti-IP3R Santa Cruz Biotechnology sc-377518 D1514 2 0.25 NMIgG Sigma-Aldrich I5381 SLBK0107V variable variable NRIgG Sigma-Aldrich I5006 SLBM2617V variable variable Mouse anti-ORAI1  ProSci Inc. PM-5205 0902 10 0.5 Rabbit anti-PTP1B  Abcam Ab201974 GR234215-1 2 0.5 Mouse anti-SERCA2 Abcam Ab2861 GR283954-1 10 0.5 Rabbit anti-Src Abcam Ab47405 GR104503-6 1 1 Rabbit anti-STIM1 Santa Cruz Biotechnology sc-68897 F0409 2 0.1 Goat anti-Mouse Alexa Fluor 488 Invitrogen A11029 1745855 20 - Goat anti-Rabbit Alexa Fluor 488 Invitrogen A11034 1531670 20 - Goat anti-Rabbit CF 633 Biotium 20122 13c1015 20 - Goat anti-Mouse Alexa Fluor 488 Invitrogen A11029 1745855 20 - Goat anti-Rabbit Alexa Fluor 488 Invitrogen A11034 1531670 20 - Goat anti-Rabbit CF 633 Biotium 20122 13c1015 20 - Goat anti-Mouse HRP Bio-Rad 170-6516 L005680 A - 1:40,000 dilution Goat anti-Rabbit HRP Bio-Rad 170-6515 L005679 A - 1:40,000 dilution  18  Chapter 3: Evidence for Ca2+ signaling machinery at ectoplasmic specializations and tubulobulbar complexes in Sertoli cell apical processes 3.1 Introduction This chapter details the experiments related to the first objective of this thesis. To determine if Ca2+ signaling machinery is present at TBCs, I performed immunofluorescence microscopy using testes from Rattus norvegicus. This species was chosen for study as apical TBCs are large and easy to visualize in rat compared to other species. In this study only apical TBCs were examined as basal TBCs are difficult to visualize at the light level. Apical TBCs occur in clusters of up to 24 [22] that are typically oriented in the same direction making it easy to obtain them in cross section whereas basal TBCs do not occur in clusters and their orientations in the epithelium are unpredictable [28]. Stage VII seminiferous tubules were chosen for evaluation because apical TBCs are well developed at this stage of spermatogenesis in rat [19,43].  Previous observations have shown that phosphatidylinositol 4,5-bisphosphate (PIP2) and phospholipase C-γ  (PLCγ) are present at ESs [44]. The finding of these components suggested that phosphoinositide signaling may be occurring at ESs which involves generation of IP3 from the cleavage of PIP2 by PLCγ. This led to a model proposed by Guttman and colleagues [44] where inositol 1,4,5-trisphosphate receptor (IP3R), a Ca2+ release channel in the ER, at ESs releases Ca2+ to trigger ES disassembly. Although this model proposed that IP3R is present at ESs, it is also possible that IP3R may occur at the ER associated with the bulbs of TBCs. As there have been no previous studies evaluating IP3R at ESs or TBCs, I used immunofluorescence to investigate whether IP3R is indeed present at ESs and TBCs.  19  In addition to IP3R, there are other candidate proteins that could be present at TBCs. The ER and PM at the bulbs of TBCs are in close apposition and suggest a potential ER-PM contact. Tomography studies [45, submitted] indicate there are filamentous connections between the ER and PM at these sites. The proteins stromal interaction molecule 1 (STIM1) and ORAI1 occur at ER-PM contact sites involved in Ca2+ signaling making them attractive candidates at TBC bulbs. These proteins are components of store-operated Ca2+ entry (SOCE) where depletion of intracellular Ca2+ stores triggers an influx of Ca2+ from the extracellular environment through channels in the PM. STIM1 is a Ca2+ sensor in the ER membrane which is activated upon depletion of ER Ca2+ stores [46,47]. Its activation causes the cytosolic domain to become positively charged which brings it into close apposition to the negatively charged phospholipids of the PM [48–50]. Once in close proximity to the PM, it can then activate ORAI1, a Ca2+ channel subunit in the PM [51,52]. Activation of ORAI1 subunits allows them to oligomerize to form a Ca2+ channel resulting in Ca2+ influx into the cytoplasm from the environment [53]. If this candidate pair is in fact present at TBCs, this mechanism may explain how the ER becomes closely associated with the PM at the bulbs of TBCs.  If regulation of Ca2+ is important in apical TBCs, then there may be Ca2+ pumps present in the ER membrane to refill ER stores in the apical process especially since there are multiple generations of TBCs that occur during Stages VI and VII. In addition to resetting cytosolic Ca2+ levels, reuptake of Ca2+ into the ER is important for Ca2+ oscillations. This is an important mechanism of Ca2+ signaling in cells such as in the elongation of actin networks in podosomes [38] and in the regulation of transcription factors such as NF-KB [54]. I hypothesized that sarco/endoplasmic reticulum Ca2-ATPase 2 (SERCA2), a Ca2+ pump in the ER that refills ER Ca2+ stores, is present in apical processes. 20  In addition to looking for Ca2+ signaling machinery at TBCs, I was particularly interested in whether or not the components involved in Ca2+ oscillation-induced actin network elongation of podosomes were also present at TBCs. The oscillations of Ca2+ created by STIM1 and ORAI1 at podosomes function to activate Src kinase [38] which will then phosphorylate cortactin to promote polymerization of the actin network [55]. More specifically, Src is activated by the activity of protein tyrosine phosphatase 1B (PTP1B) which removes an inhibitory phosphate from Src [56]. I hypothesized that Src and PTP1B may be present at apical TBCs and probed for their presence.  3.2 Results 3.2.1 The Ca2+ release channel, IP3R, localizes to the bulb regions of tubulobulbar complexes In cryosections of stage VII seminiferous tubules, the IP3R antibody strongly labelled the acrosomes of round spermatids and regions of the apical processes (Fig. 3.1A). Similar labeling patterns were not observed in primary antibody controls (Fig. 3.1B) where sections were probed with normal mouse IgG at the same concentration instead of the IP3R antibody. In controls where the primary antibody was replaced with buffer (secondary control) (Fig 3.1C) and in controls where both primary and secondary antibodies were replaced with buffer (blank control) (Fig. 3.1D), a similar pattern was not observed. Higher magnification of the epithelium that has been phallotoxin-labeled to show the actin networks of TBCs, showed that the IP3R labeling is located near the ends of TBCs (Fig. 3.1E). This was better observed in deconvolved micrographs using an antibody against cortactin to label TBCs (Fig 3.1I). Controls of similar magnification did not show similar labeling patterns (Fig. 3.1F-H).  To evaluate the position of the IP3R labeling with respect to TBCs, I used deconvolved micrographs of isolated apical processes. IP3R labeling was seen in the apical process distal to the 21  spermatid head in a region where the ends of TBCs are expected to occur (Fig. 3.2A, D). Interestingly, the antibody did not convincingly label regions where ESs occur. Labeling of the stalk of the spermatid head proximal to the midpiece was also seen (Fig. 3.2A, D). Cortactin was used to label the actin networks of TBCs (Fig. 3.2B, E) and the proximal and distal tubules could sometimes be distinguished (Fig. 3.2B). When these channels were merged, the IP3R labeling could be seen at the ends of TBCs (Fig. 3.2C, F) and when the proximal and distal tubules could be distinguished, the labeling of IP3R occurred in the gap between (Fig. 3.2C). Controls for the IP3R antibody did not show similar labeling patterns (Fig. 3.2G-I). The IP3R antibody reacted with a single band in the range expected for IP3R on immunoblots (Fig. 3.2G). The antibody is expected to interact with 3 isoforms of IP3R and their expected molecular weights are 250, 260, and 313 kDa. Calnexin was used as a loading control. 3.2.2 The ER Ca2+ sensor, STIM1, is present in Sertoli cells and their apical processes In cryosections of stage VII seminiferous tubules, the antibody against STIM1 labelled Sertoli cells extending from the basal region into apical processes surrounding late spermatids (Fig. 3.3A). This pattern was not seen in controls (Fig 3.3B-D). At higher magnification, the labeling pattern was seen as filamentous projections from the base to apex consistent with an ER labeling pattern (Fig. 3.3E). This pattern was not seen in controls at similar magnification (Fig. 3.3F-H).  In deconvolved micrographs of isolated apical processes, STIM1 labeling was seen throughout the apical process (Fig. 3.4A, B). This pattern was not seen in controls (Fig. 3.4C-E). In immunoblots, the STIM1 antibody detected a band between 70–100 kDa which is consistent with the predicted molecular weight of STIM1 (86 kDa) (Fig. 3.4F). The antibody also detected a band at a lower molecular weight between 55 – 70 kDa which was not seen in control lanes probed 22  with NRIgG. This band may represent STIM1 that has undergone cleavage as it has been reported to be regulated by calpain cleavage [57]. Calnexin was used as a loading control. 3.2.3 The plasma membrane Ca2+ subunit, ORAI1, is present in Sertoli cells and their apical processes The ORAI1 antibody showed a similar Sertoli cell pattern to that of STIM1 in stage VII seminiferous tubules, extending from the base of Sertoli cells into the apical process but the labeling was more diffuse (Fig. 3.5A) compared to the filamentous pattern seen with the STIM1 antibody. Similar patterns were not seen in controls (Fig. 3.5B-D). This Sertoli cell labeling pattern could better be seen at higher magnification (Fig. 3.5E), which was not seen in controls at similar magnification (Fig. 3.5F-H).  Deconvolved micrographs of isolated apical processes showed labeling throughout the apical process with a particular bias towards labeling the periphery (Fig. 3.6A, B). Controls did not show the same labeling pattern (Fig. 3.6C-E). Immunoblot analysis of the specificity of the ORAI1 antibody detected a band between 40–55 kDa which was consistent with glycosylated ORAI1 (50 kDa) (Fig. 3.6F). The ORAI1 antibody also detected a band just below 55 kDa in whole testis lysate only and another just below 25 kDa in both whole testis and seminiferous epithelium lysates that were also seen in the control lysates probed with NMIgG. Calnexin was used as a loading control. 3.2.4 STIM1 and ORAI1 colocalize at the periphery of apical processes  Colocalization of STIM1 and ORAI1 in apical processes of Sertoli cells was analyzed using deconvolved micrographs of isolated epithelial fragments labeled for both STIM1 and ORAI1 (Fig. 3.7A, B). When STIM1 and ORAI1 patterns were overlayed, regions of colocalization were seen near the periphery of the apical process (Fig. 3.7C), not at regions known to contain bulbs. 23  The colocalization of STIM1 and ORAI1 in apical processes was quantified in deconvolved Z stacks of epithelial fragments from three different animals. The fraction of STIM1 colocalized with ORAI1 at peripheral sites in apical processes in the three animals was 0.41 ± 0.07, 0.21 ± 0.05, and 0.31 ± 0.09 (95% c.i., n = 11, 11, 10) respectively (Fig. 3.7D). The fraction of ORAI1 in the same three animals was 0.43 ± 0.07, 0.33 ± 0.05, and 0.41 ± 0.06 (95% c.i., n = 11, 11, 10) respectively (Fig. 3.7D). 3.2.5 ER-PM contacts occur at the periphery of the apical processes The apparent co-localization of STIM1 and ORAI1 at the periphery of apical processes prompted me to evaluate these regions at the ultrastructural level and search for ER-PM contact sites. At the periphery of apical processes, concentric layers of ER surround more central regions that encase the heads of spermatids and the related cluster of TBCs. The outermost tubular cisternae were often swollen and form close contacts with the PM (Fig. 3.8 A, B). The distance between the PM and ER at these regions of close contact was measured to be 13.3 ± 1.1 nm (95% c.i., n = 47) and 14.2 ± 1.0 nm (95% c.i., n = 45) in two different animals. When these contact sites were viewed at high magnification, electron dense connections could be seen between the two closely apposed membranes (Fig. 6D-M).  3.2.6 The sarco/endoplasmic reticulum Ca2+ ATPase, SERCA2, is present in Sertoli cells and is associated with apical ectoplasmic specializations In cryosections probed with antibodies against SERCA2, a Sertoli cell pattern was seen with labeling extending from the base of the epithelium to the apex (Fig. 3.9A). At a higher magnification in cryosections, the labeling surrounded the nucleus and extended filamentous projections towards the apex of each cell where it labeled apical processes (Fig. 3.9E). This labeling pattern was not seen in controls (Fig. 3.9B-D, F-H).  24  In epithelial fragments, the antibody labeling could be seen throughout the apical process and in particular at the periphery proximal to the dorsal aspect of the spermatid head (Fig. 3.10A, D). The actin networks were labeled with phallotoxins to show the position of the ES and TBCs (Fig. 3.10B, E). When the antibody labeling was overlayed with the actin, the antibody label occurred in association with the ES along the dorsal aspect and the inner concavity of the spermatid head (Fig. 3.10C, F). This pattern was not observed in controls (Fig. 3.10G-I). The specificity of the antibody was further evaluated by immunoblot where it recognized a single band between 70-100 kDa in both whole testis lysate and seminiferous epithelium lysate (Fig. 3.10J). This was lower than the predicted molecular weight for SERCA2 of 110 kDa. Calnexin was used as a loading control. 3.2.7 Src kinase is associated with the ends of tubulobulbar complexes In cryosections probed with antibodies against Src, was seen strongly labeling apical process of Sertoli cells (Fig. 3.11A). At a higher magnification in cryosections that have been labeled with phallotoxins, the labeling was observed at the ends TBCs (Fig. 3.11E). This labeling pattern was not seen in controls (Fig. 3.11B-D, F-H).  In deconvolved micrographs of epithelial fragments, the antibody labeling was concentrated within the apical process distal to the spermatid head (Fig. 3.12A, D). The actin networks were labeled with phallotoxins to show the position of TBCs (Fig. 3.12B, E). When the antibody labeling was overlayed with the actin, the antibody label occurred at the ends of TBCs (Fig. 3.12C, F). This pattern was not observed in controls (Fig. 3.12G-I). The specificity of the antibody was further evaluated by immunoblotting where it recognized a single band between 55-70 kDa in both whole testis lysate and seminiferous epithelium lysate (Fig. 3.12J) which is consistent with the molecular weight of Src (60 kDa). Calnexin was used as a loading control. 25  3.2.8 The phosphatase regulator of Src, PTP1B, is present in Sertoli cells and their apical processes In cryosections of stage VII seminiferous tubules, PTP1B antibody had a typical Sertoli cell labeling pattern with staining extending from the base of the tubule to the apex (Fig. 3.13A). This pattern was not seen in controls (Fig. 3.13B-D). At higher magnification, labeling surrounded the nucleus and extended apically into Sertoli cell apical processes (Fig. 3.13E). This pattern was not seen in controls (Fig. 3.13F-H).   Deconvolved micrographs of isolated epithelial fragments showed that the labelling was distributed throughout the apical process (Fig. 3.14A, B). This labeling pattern was not seen in controls (Fig. 3.14C-E). Immunoblot analysis of the antibody specificity revealed a single band between 37-50 kDa which is consistent with the molecular weight of PTP1B (50 kDa).  3.3 Discussion Figure 3.15 is a diagrammatic summary of the immunolocalization data showing the location of proteins within the apical process of Sertoli cells with respect to TBCs and ESs. 3.3.1 Role of SERCA2 at ectoplasmic specializations: maintain microdomain of Ca2+? 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 as it has been shown to contain Ca2+ through precipitation and x-ray microanalysis of electron micrographs [58]. The effects of this potential signaling was first thought to result in a contraction of the actin bundles resulting in the expulsion of the spermatids into the lumen [59], however, it was later shown that the actin bundles are non-contractile [60]. Others hypothesized that it may be involved in regulating either the junctions themselves or the related actin bundles [61,62]. Chelation of Ca2+ has been shown to 26  interfere with the arrangement of actin filaments within ESs [61] suggesting that Ca2+ regulation may function to alter their organization.  A model previously proposed by Guttman et al. [44] 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 blo od-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 [63]. A more recent study has shown that plastin 3, an actin bundling protein that is negatively regulated by Ca2+, is present at ESs [64] and a release of Ca2+ may function to inhibit the bundling activity of plastin 3. The immunolocalization data did not convincingly demonstrate that IP3R is present at ESs; rather, it showed that SERCA2 was present at the sites. It is possible that one of the functions of SERCA2 at ESs may be to maintain a micro-domain of normally low Ca2+ within the ES prior to any junction remodeling to promote the bundling of actin by plastin 3. 3.3.2 Bulbs of tubulobulbar complexes: a site of store-operated Ca2+ entry? Although STIM1 and ORAI1 did not appear to localize to TBC bulbs, others have shown that TRPM6, a Ca2+ channel in the PM, and a cytosolic scaffolding protein, HOMER1, are present at the bulb regions of TBCs [45, submitted]. Additionally, the immunolocalization data of IP3R also indicates that it is present at the bulbs of TBCs. In HEK cells, a different TRP channel (TRPC1) has been shown to form a complex with IP3R and HOMER1 and is involved in SOCE [65]. HOMER1 binding blocks DD/E residues on TRPC1 [66] which is the site of STIM1 binding [67,68]. The stimulation of IP3R leads to activation of STIM1 and the dissociation of IP3R-HOMER1-TRPC1 complexes [69] revealing the DD/E residues. STIM1 then is able to bind to the DD/E residues on TRPC1 and stabilize the channel in its open state [67,68]. The observation that 27  TRPM6 is in the same regions as IP3R and HOMER1, together with the observation that STIM1 is generally distributed in the ER, makes this potential mechanism attractive. TRPM6 contains both a Homer-binding ligand (residues 1367-1371) and STIM1 binding residues (residues 1380-1381), though these two sequences are 8 residues apart in TRPM6 compared to 4 residues in TRPC channels. 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. As TBCs will eventually vesiculate to enter endosomal compartments of Sertoli cells, the markers associated with early endosomes are found at TBC bulbs such as Rab5 and EEA1 [20,21,70]. It is thought that Ca2+ release is crucial for early endosomes potentially to aid in their acidification [71–73]. Since the luminal environment of endosomes is initially composed from that of the extracellular space, the Ca2+ concentration is relatively high. The seminiferous tubule fluid contains 400 µM of Ca2+ [74] compared to 80-90 nM in the cytoplasm of Sertoli cells [75,76] resulting in a concentration in the bulbs four times higher compared to the Sertoli cell. The concentration of Ca2+ found in early endosomes characterized by Rab5 is 0.5 µM indicating that a massive release of Ca2+ has occurred [77] which may be an alternate explanation for why TRPM6 occurs at the bulb regions of TBCs. Although this is complicated by the portion of the spermatid cytoplasm that occurs within the bulbs which will also have a lower concentration of Ca2+ compared to the seminal tubule fluid. However, the data shows that IP3R, TRPM6, HOMER1 and STIM1 occur at TBC bulbs indicating that the machinery necessary to generate Ca2+ fluxes is present.  28  3.3.3 Peripheral ER-PM contacts in apical processes: a site of store-operated Ca2+ entry? The combination of STIM1 and ORAI1 colocalizing at the periphery and finding ~14 nm ER-PM contacts suggest that SOCE may occur at these sites. The distance between the ER and PM at these contacts is within the reported range of STIM1 contact sites 10-25 nm [78]. SOCE at the periphery may function to replenish the Ca2+ stores of the ER within the apical process which may be particularly important if Ca2+ is occurring both at ESs and TBCs. Loss of Ca2+ from these potential signaling sites may be due to plasma membrane Ca2+ATPases (PMCAs) that pump Ca2+ out of the cell intro the environment and function similarly to SERCAs to dissipate Ca2+ transients in the cell. Although they are known to be present in spermatids and are necessary for hyperactivated motility of mature sperm for fertilization [79], it is unknown whether they are also present in Sertoli cells or their apical processes. To investigate this potential function, preliminary studies could be conducted aimed at evaluation of PMCA distribution in Sertoli cells. Additionally, if these sites are important in refilling intracellular Ca2+ stores then inducing Ca2+ release using thapsigargin or ionomycin should result in an increased colocalization of STIM1 and ORAI1 at the periphery of Sertoli cell apical processes. 3.3.4 Potential role of Src in regulation of the actin cuffs of tubulobulbar complexes Currently the mechanism of TBC actin cuff elongation and subsequent depolymerization is unknown. Based on the mechanism of dendritic actin network polymerization-driven elongation of podosomes, I looked for Src kinase and its regulator PTP1B at TBCs. In podosomes, Ca2+ oscillations from STIM1/ORAI1 results in activation of Src which phosphorylates cortactin to drive actin network polymerization [38]. The mechanism of Src activation by Ca2+ oscillation is thought to be mediated by the Ca2+-dependent protease, calpain2 [55]. Activation of calpain 2 protease activity by Ca2+ results in cleavage of PTP1B producing a fragment with enhanced 29  phosphatase activity [55]. The PTP1B fragment removes an inhibitory phosphate from Src which can then phosphorylate cortactin to promote actin network assembly [55,80–82]. The finding of Src localized to the ends of TBCs may suggest these structures are elongated by a mechanism similar to podosomes. Although its regulator, PTP1B, is observed throughout the apical process, it may only exert its phosphatase activity in regions where there are Ca2+ fluxes to increase phosphatase activity through calpain 2-mediated cleavage.  The mechanism involving calpain 2, PTP1B and Src described above is further complicated by the fact that calpain 2 also recognizes cortactin as a substrate for cleavage and is involved in disassembly of podosomes [55]. In addition, Src phosphorylation of paxillin, which is also a component of TBC actin cuffs [83], can result in activation of calpain through Erk signaling to initiate podosome disassembly [84,85]. It is hypothesized that the spatiotemporal regulation of this potential feedback loop between Src and calpain 2 coordinates the assembly and disassembly of podosomes [86]. An alternative function of SOCE at peripheral sites may be to participate in spatiotemporal regulation of Src by potentially activating Src and/or calpain 2 that are proximal to the periphery. A role for Src in spermatogenesis is suggested by previous studies showing that incubation of isolated stage VII-VIII with Src inhibitor, PP2, results in a 45% reduction in sperm release [87]. It was postulated that this was a result of inhibiting the Src activation of the disengagement complex [87], the last Sertoli cell-spermatid attachments that remain after ES disassembly. Disruption of TBCs through siRNA knockdown of cortactin have been shown to inhibit sperm release [26]. Therefore it would not be surprising if the inhibition of Src caused a reduction in sperm release due to a disruption at the level of TBC regulation.    30   Figure 3.1 Cryosections of stage VII seminiferous tubules show IP3R labeling near the distal ends of tubulobulbar complexes. (A) Maximum projection showing the IP3R antibody labeling regions within the apical processes of Sertoli cells (arrowheads) known to contain the bulbs of TBCs. Labeling of round spermatid acrosomes is also seen. (B-D) Primary, secondary, and blank controls. (E) Maximum projection of phalloidin-labeled actin merged with IP3R antibody labeling shows that the latter probe labels regions near the ends of TBCS (arrowheads). (F-H) Primary, secondary, and blank controls. (I) Maximum projection of a deconvolved Z stack better illustrating the labeling of the IP3R antibody (arrowheads) at the distal ends of TBCs labeled by cortactin antibody. 31   Figure 3.2 Epithelial fragments show IP3R labeling at the bulb regions of tubulobulbar complexes. Maximum projections of deconvolved Z stacks of isolated epithelial fragments (A, D) showing IP3R antibody labeling in the apical process distal to spermatid heads (arrowheads). Labeling of the acrosome proximal to the tail regions is also seen (arrows). (B, E) Cortactin labeling actin networks at TBCs clearly showing the proximal (PT) and distal (DT – arrows) tubules of TBCs. Merged micrographs show IP3R labeling at the ends of TBCs (F – arrowhead) and between the distal and proximal tubules (C – arrowhead). (G-I) Primary, secondary, and blank controls. (J) Immunoblot analysis of the specificity of the IP3R antibody on whole testis (WT) and seminiferous epithelium (SE) lysates compared to lysates probed with normal mouse IgG (NMIgG). The antibody is expected to interact with 3 isoforms of IP3R and their expected molecular weights are 250, 260, and 313 kDa. Calnexin was used as a loading control.  32   Figure 3.3 Cryosections of stage VII seminiferous tubules show STIM1 labeling Sertoli cells and their apical processes. (A) Maximum projection showing STIM1 antibody labeling Sertoli cells which extend from the base of the epithelium towards the apex where their apical processes contact spermatids. (B-D) Primary, secondary and blank controls. (E) Maximum projection showing STIM1 antibody labeling of Sertoli cells at a higher magnification. STIM1 labeling is seen extending from the nucleus (arrow) at the base through filamentous projections to the apical processes (arrowheads). (F-H) Primary, secondary, and blank controls. Arrows indicate Sertoli cell nuclei. 33   Figure 3.4 Epithelial fragments show STIM1 labeling throughout apical processes. Maximum projections of deconvolved Z stacks of isolated epithelial fragments (A, B) showing STIM1 antibody labeling throughout the apical process. (C-E) Primary, secondary, and blank controls. (F) Immunoblot analysis of the specificity of the STIM1 antibody on whole testis (WT) and seminiferous epithelium (SE) lysates compared to lysates probed with normal rabbit IgG (NRIgG). Expected molecular weight of STIM1 is 84 kDa. Calnexin was used as a loading control.  34   Figure 3.5 Cryosections of stage VII seminiferous tubules show ORAI1 labeling Sertoli cells and their apical processes. (A) Maximum projection showing ORAI1 antibody labeling Sertoli cells which extend from the base of the epithelium towards the apex where their apical processes contact spermatids. (B-D) Primary, secondary and blank controls. (E) Higher magnification of the seminiferous epithelium showing ORAI1 antibody labeling of Sertoli cells at a higher magnification. STIM1 labeling is seen extending from the nucleus (arrow) at the base to the apical processes (arrowheads). (F-H) Primary, secondary, and blank controls. Arrows indicate Sertoli cell nuclei.   35   Figure 3.6 Epithelial fragments show ORAI1 throughout apical processes. Maximum projections of deconvolved Z stacks of isolated epithelial fragments (A, B) showing ORAI1 antibody labeling throughout the apical process. (C-E) Primary, secondary, and blank controls. (F) Immumoblot analysis of the specificity of the STIM1 antibody on whole testis (WT) and seminiferous epithelium (SE) lysates compared to lysates probed with normal mouse IgG (NMIgG). Expected molecular weight of ORAI1 is 33 kDa, expected weight of glycosylated ORAI1 is 50 kDa. Calnexin was used as a loading control.    36   Figure 3.7 STIM1 and ORAI1 colocalize at the periphery of apical processes. Maximum projections of a deconvolved Z stack of an isolated apical process showing STIM1 (A) and ORAI1 (B) antibody labeling. (C) When the two channels are combined, colocalization of STIM1 and ORAI1 is observed near the periphery of the apical process (arrowheads). (D) Colocalization of STIM1 and ORAI1 in deconvolved Z stacks of epithelial fragments from three animals was quantified. Error bars represent 95% confidence intervals.    37   Figure 3.8 ER-PM contacts at the periphery of apical processes. (A) An electron micrograph showing a cross-section through an apical process. The hook-shaped spermatid head is sectioned in two places (asterisks) and the proximal tubular regions of numerous TBCs are seen in cross-section (arrowheads). The ER in regions around the periphery of the apical process occurs in multiple layers. (B) An area similar to that outlined by the box in panel (A). Here, the outermost cisternae swell (arrows) into tubular ER (asterisks) that come into close contact with the PM of the Sertoli cell (arrowhead). (C) The distance between the ER and PM was 13.3 ± 1.1 nm in one animal and 14.2 ± 1.0 nm in another. (D-M) Electron micrographs showing these ER-PM contacts (arrowheads) in representative images from two animals. Scale bar in D applies to E-M. 38   Figure 3.9 Cryosections of stage VII seminiferous tubules show SERCA2 labeling the ER throughout Sertoli cells and their apical processes. (A) Maximum projection showing SERCA2 antibody labeling Sertoli cells extending from the base to the apical processes. (B-D) Primary, secondary and blank controls. (E) Higher magnification of the seminiferous epithelium showing SERCA2 labeling surrounding a Sertoli cell nucleus (arrow) and extending through filamentous projections to the apical processes (arrowheads). (F-H) Primary, secondary, and blank controls. Arrows indicate Sertoli cell nuclei.   39   Figure 3.10 Epithelial fragments show SERCA2 labeling ectoplasmic specializations. Maximum projections of deconvolved Z stacks of isolated epithelial fragments (A, D) showing SERCA2 antibody throughout the apical process (arrowheads). (B, E) Phallotoxin staining of actin networks at TBCs. (C-F) Merged micrographs show SERCA2 labeling (arrowheads) at ESs (arrows). (G-I) Primary, secondary, and blank controls. (J) Immunoblot analysis of the specificity of the SERCA2 antibody on whole testis (WT) and seminiferous epithelium (SE) lysates compared to lysates probed with normal mouse IgG (NMIgG). The expected molecular weight of SERCA2 is 110 kDa. Calnexin was used as a loading control.    40   Figure 3.11 Cryosections of stage VII seminiferous tubules show Src labeling near the distal ends of tubulobulbar complexes. (A) Maximum projection showing the Src antibody strongly labeling the apical processes of Sertoli cells. (B-D) Primary, secondary, and blank controls. (E) Maximum projection showing that Src labeling (arrowheads) is at the ends of phalloidin-labeled TBCs (arrows). (F-H) Primary, secondary, and blank controls.   41   Figure 3.12 Epithelial fragments show Src labeling at the bulb regions of tubulobulbar complexes. Maximum projections of deconvolved Z stacks of isolated epithelial fragments (A, D) showing Src antibody labeling in the apical process distal to spermatid heads (arrowheads). (B, E) Phallotoxin-labeling of actin shows location of TBCs (arrows). (C, F) Merged micrographs show Src labeling (arrowheads) at the ends of TBCs (arrows). (G-I) Primary, secondary, and blank controls. (J) Immunoblot analysis of the specificity of the Src antibody on whole testis (WT) and seminiferous epithelium (SE) lysates compared to lysates probed with normal rabbit IgG (NRIgG). The expected molecular weight of Src is 60 kDa. Calnexin was used as a loading control.   42   Figure 3.13 Cryosections of stage VII seminiferous tubules show PTP1B labeling of Sertoli cells and their apical processes. (A) Maximum projection showing the PTP1B antibody strongly labeling the apical processes of Sertoli cells. (B-D) Primary, secondary, and blank controls. (E) Maximum projection showing that PTP1B labeling at a higher magnification. Labeling is seen to surround the nucleus of a Sertoli cell (arrow) and extend to the apical processes (arrowheads). (F-H) Primary, secondary, and blank controls.   43   Figure 3.14 Epithelial fragments show PTP1B labeling throughout Sertoli cell apical processes. Maximum projections of deconvolved Z stacks of isolated epithelial fragments (A, B) showing PTP1B antibody labeling throughout the entire apical process. (C-E) Primary, secondary, and blank controls. (F) Immunoblot analysis of the specificity of the PTP1B antibody on whole testis (WT) and seminiferous epithelium (SE) lysates compared to lysates probed with normal mouse IgG (NMIgG). The expected molecular weight of PTP1B is 50 kDa. Calnexin was used as a loading control.   44   Figure 3.15 Summary of Ca2+ signaling machinery localized within apical processes of Sertoli cells. The Ca2+ release channel, IP3R, was localized to the bulb regions of tubulobulbar complexes (TBCs). STIM1 and ORAI1 were found to colocalize to the periphery of the apical process where ER-PM are found. SERCA2 was seen to associate with ectoplasmic specializations (ESs). SERCA2 and STIM1 were seen throughout the general ER of the Sertoli cell. ORAI1 was also generally distributed throughout the Sertoli cell (not pictured here).   45  Chapter 4: Studies on obtaining functional evidence of calcium fluxes in Sertoli cell apical processes  4.1 Introduction In chapter 3, I provide immunofluorescence data that indicates a number of proteins involved in Ca2+ signaling occur at ESs and TBCs. The studies in this chapter attempt to provide functional evidence to support the main hypothesis of this thesis that Ca2+ signaling at TBCs functions to alter the actin networks at TBCs. If Ca2+ signaling is a regulator of these actin networks then the manipulation of Ca2+ levels should elicit an observable phenotype. To test this prediction, I utilized the Ca2+ chelator, EGTA, to explore the effects of reduced levels of Ca2+ on TBC actin networks in rat. Conversely I used thapsigargin to explore the effects of increased Ca2+ levels on TBC actin networks.  Thapsigargin effectively increases the cytosolic levels of Ca2+ by blocking SERCA pumps which I have shown to be present at apical processes in chapter 3. I used an ex vivo testis perfusion technique that was developed by Hoffer and colleagues [88] to deliver EGTA or thapsigargin over a period time to the seminiferous tubules.  A previous study investigated the effect of Ca2+ depletion on basal ESs and found after 2 hours the junctions lost stability as Sertoli cells retracted from one another [61]. The investigators proposed this was due to contraction of the actin bundles associated with ESs but these actin bundles were shown by a later study to be non-contractile [60]. It has previously been shown that Ca2+ depletion causes rounding of cultured Sertoli cells due to changes in their cytoskeletal networks [89] which may explain the phenotype observed at basal ESs. The study involving EGTA in this chapter attempts to dissect the effects of Ca2+ depletion on the actin networks of TBCs rather 46  than ESs. Due to the severe phenotype observed from exposing tubules to 2 mM of EGTA for 2 hours, I decided to perfuse testes with the same concentration of EGTA but for 15 minutes. This was an attempt to detect any effects on the actin networks at TBCs without being obscured by the severe change in overall cell morphology seen at 2 hours of exposure.  4.2 Results 4.2.1 Ca2+ depletion by EGTA perfusion has no effect on length of actin networks at tubulobulbar complexes The quality of the actin networks was visualized using fluorescent phallotoxins. Quality of the actin networks at TBCs and ESs was comparable between control (Fig. 4.1A) and EGTA exposed groups (Fig. 4.1B). The mean length of TBCs in EGTA group was 1.6 ± 0.1 µm while the mean length in the control was 1.5 ± 0.1 µm (95% confidence intervals) (Fig. 4.2C). There was no statistically significant difference between the two groups (P = 0.06). 4.2.2 Thapsigargin has no effect on length of actin networks at tubulobulbar complexes Similar to the EGTA perfusion studies, thapsigargin had no visible effect on the quality of the actin networks at TBCs (Fig. 4.2B) when compared to controls (Fig. 4.2A). The mean length of TBCs in thapsigargin exposed group was 1.2 ± 0.1 µm while the mean length in the control was 1.3 ± 0.1 µm (95% confidence intervals) (Fig. 4.2C). There was no statistically significant difference between the two groups (P = 0.13). 4.2.3 Live imaging of Ca2+ in isolated apical processes  I attempted to visualize Ca2+ fluxes at TBCs using the fluorescent Ca2+ indicator fluo-4 in isolated apical processes. After initial studies were unsuccessful, I attempted to induce Ca2+ release from IP3R which I have shown in chapter 3 to be present at the bulbs of TBCs. Using the most potent agonist of IP3R, adenophostin A, I was unable to detect any events that could be classified 47  as a Ca2+ flux event. However, I did observe bright patches in regions of the apical process where the bulbs of TBCs would be expected (Fig. 4.3A, C). When looking at a line scans of this region (Fig. 4.3A, C insets) the change in fluorescence intensity at this region can be seen (Fig. 4.3B, D). The three micrographs in panels A and C represent three different time points and their position in the line scan is indicated by the arrows. Although minor fluxes in fluorescence intensity occur in these regions of interest (Fig. 4.3B, D), the change in intensity at these sites was at the same level of magnitude as background within the apical process. 4.3 Discussion 4.3.1 EGTA and thapsigargin studies  Attempts to deplete and inflate Ca2+ levels in Sertoli cells by perfusing with EGTA and thapsigargin respectively yielded no observable phenotypic changes in the actin networks at TBCs. The duration of EGTA perfusion of the testis was chosen to be 10 minutes as previous studies showed the retraction of Sertoli cells at even 30 minutes [61]. However, the reference study utilized isolated seminiferous tubules rather than perfusion methods used here which may impacted effectiveness of Ca2+ depletion in the seminiferous tubules. In particular, the use of isolated tubules allows for EGTA chelation of Ca2+ within the lumens of the seminiferous tubules which is directly in contact with the apical processes. With perfusion, however, chelation of Ca2+ is occurring via the vascular system. Thus perfusion may not be as efficient at depleting Ca2+ as bathing isolated seminiferous tubules in a Ca2+ chelating buffer. Future studies that extend the duration or increase the concentration of EGTA used in these perfusion experiments could be conducted to examine this further.   If the mechanism of Ca2+ regulation of TBCs is similar to that of podosomes then it may not be surprising that thapsigargin did not have any observable effects. Thapsigargin induces 48  constitutive Ca2+ influx rather than Ca2+ oscillations mediated by SOCE which was found to be sufficient for Src activation but not podosome formation [38]. This is an example of the importance of temporal Ca2+ oscillations orchestrating cellular processes which is also seen in mast cell exocytosis [90]. If the mechanism of TBC formation is similar to that of podosomes then it may explain why thapsigargin had no effect on its actin networks. Studies that manipulate Ca2+ oscillations may inform to what potential function if any they might have on TBC actin networks. Potential studies could involve overexpression or knockdown of the proteins involved in the oscillations which might include TRPM6, IP3R, STIM1 or ORAI1.  4.3.2 Live Ca2+ imaging Although I was unable to detect any significant flux of Ca2+ in isolated epithelial fragments, I did observe bright patches of fluorescence in regions where the ends of TBCs would be expected. The magnitude of change in the intensity of the region of interest did not suggest Ca2+ fluxes were occurring. It is important to note that the Ca2+ indicator was a non-ratiometric indicator and is not able to make a direct correlation to [Ca2+] as multiple factors contribute to the fluorescence intensity such as indicator concentration. The bright patch observed may be due to a higher concentration of probe accumulated inside of a membrane-bound structure such as a TBC bulb or post-vesiculation structures. However, changes in fluorescence intensity are indicative of a change in [Ca2+] but the fluctuations observed at these patches were not above background levels.  A number of factors may have made detection of fluxes difficult such as the buffer system used. In order to deliver the fluorophore and the agonist adenophostin A, fragments were permeabilized with saponin which meant that the level of Ca2+ within the cytosol would be controlled by the Ca2+ level of the buffer. It has been shown previously that Ca2+ is regulator of IP3R activity with no activity in the absence of Ca2+ but becomes increasingly active up to 300 nM 49  after which it becomes inhibitory [91–93]. Thus the levels of Ca2+ in the buffer was adjusted to ~100 nM to ensure it was not inhibitory and was close to values reported for Sertoli cell basal levels [75,76]. The use of EGTA to buffer the Ca2+ in the solution may have made detection of small Ca2+ fluxes impossible by buffering the Ca2+ immediately. Additionally, the structural integrity of the ER may have been compromised in the process of fragmentation. During isolation of these fragments, the apical processes are separated from the rest of the Sertoli cell by mechanical force which may have damaged the integrity of the ER network within the apical process. Future studies that investigate the possibility of Ca2+ fluxes at apical processes of Sertoli cells could utilize 2 photon microscopy of intact seminiferous tubules. This would allow for the depth penetration necessary to look through the wall of seminiferous tubule at the apical processes of Sertoli cells. This in combination with using genetically encoded calcium indicators (GECI) with peptide tags to localize the GECIs to endosomes could allow for visualization of Ca2+ fluxes at TBCs specifically. This approach would eliminate any potential compromise in the integrity of the intracellular morphology necessary for Ca2+ fluxes to occur.     50   Figure 4.1 Perfusion of 1 mM EGTA for 1 hr has no effect on length of tubulobulbar complexes. (A) Maximum projection of control tissue phallotoxin-labeled for actin to visualize TBCs. (B) Maximum projection of EGTA treated tissue phallotoxin-labeled for actin to visualize TBCs. (C) The mean lengths of TBCs between control and treated tissues were not significantly different (P = 0.06). Error bars = 95% confidence intervals.    51     Figure 4.2 Perfusion of thapsigargin for 1 hr has no effect on length of tubulobulbar complexes. (A) Maximum projection of control tissue phallotoxin-labeled for actin to visualize TBCs. (B) Maximum projection of thapsigargin (TG) treated tissue phallotoxin-labeled for actin to visualize TBCs. (C) The mean lengths of TBCs between control and treated tissues were not significantly different (P = 0.13). Error bars = 95% confidence intervals.    52   Figure 4.3 Live imaging of Ca2+ in isolated apical processes. Widefield micrographs showing a region in two different (A, C) apical process containing a bright patch where bulbs of TBCs occur (arrow) at three different time points. Insets are magnified regions of the bright patches with a green line indicating the pixels selected for line scan analysis. (B, D) Line scan analysis showing changes in fluorescence intensity over time. Each column represents the entire green line seen in the inset. Each square in a column represents a pixel 90 nm in width and height and thus the y-axis represents the x position on that line.   53  Chapter 5: Conclusion 5.1 Overall analysis and conclusions in light of current research The molecular components associated with actin cuffs of the tubular regions of TBCs have been well established. These networks contain hallmark proteins of dendritic actin networks found in other systems such as NWASP, Arp2/3 and cortactin [27]. To a lesser extent, molecular components of the bulbs regions have been reported including junction proteins being internalized and endosomal markers such as EEA1 and rab5 [20,21]. However, molecular components related to the ER at TBC bulbs have not been reported. This is similar to ESs, the molecular components that have been identified are associated with either the actin bundles or the intercellular junctions. Few molecular components associated with the ER of these structures have been reported.  The results of the immunolocalization studies show that proteins known to be involved with regulating intracellular Ca2+ levels in other systems are present at ESs, at TBCs and at peripheral PM contact sites. These results fulfill the first objective of this thesis which was to determine if the molecular machinery involved in Ca2+ was present at TBCs. The finding of IP3R at the bulbs of TBCs and SERCA2 at ESs 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. Additionally, the finding of STIM1 and ORAI1 colocalization at the periphery of Sertoli cells led to the identification of novel ER-PM contact sites in Sertoli cells – at the periphery of apical processes. However, the results of the functional studies were unable to fulfill the second objective of this thesis of obtaining evidence of Ca2+ regulation over actin networks at TBCs.  In addition to localizing Ca2+ signaling machinery to TBCs, the localization of Src at the ends of these structures was shown. Although distribution of Src in the seminiferous epithelium 54  has been previously reported [94], the data did not provide information on the precise localization with respect to TBCs and ESs due to low resolution. It has been previously hypothesized that Src functions as a molecular switch to trigger endocytosis of junctions at ESs [94] based on observations that it shuffles between the PM and late endosomes [95–97] and studies showing Src knockout mice are infertile [98]. I have shown that Src localizes to TBCs which suggests that TBCs may be the site at which Src regulates control over junction internalization. This regulation might act on actin networks as discussed in chapter 3.     5.2 Strengths and limitations of thesis A major strength of the work in this thesis involves the methods used in evaluating the distribution of molecular machinery in relation to ESs and TBCs. I used a technique that was developed in the Vogl lab that fragments fixed apical processes away from the rest of the Sertoli cell. Using these fragments in immunolocalization studies allows for a superior analysis of the spatial distribution of molecular machinery with respect to ESs and TBCs. This is nicely illustrated in cryosections and fragments probed for SERCA2. Cryosections nicely show the distribution of SERCA2 throughout Sertoli cells and at higher magnification show that SERCA2 is ubiquitously distributed throughout the apical process (Fig. 3.10). In isolated apical processes, the labeling occurs throughout the apical process but an association with ESs can be seen (Fig. 3.10). The differences in clarity between the two techniques may be due to overlapping apical processes in cryosections which can be reduced by using thinner sections. However, the use of thinner sections limit the ability to obtain an entire Sertoli cell in cross section. I opted to use both techniques in this thesis, using cryosections to show the overall distribution of specific proteins within the seminiferous epithelium and fragments to evaluate their distribution in relation to TBCs and ESs.  55  When designing the experiment to visualize Ca2+ fluxes in isolated apical processes, I made an assumption that the morphology crucial to facilitating Ca2+ fluxes would remain intact during the fragmentation process. Since the ER networks within the apical process are continuous with the rest of the Sertoli cell, fragmentation of the apical process away from the rest of the Sertoli cell may disrupt the distribution of the ER and associations with the PM that are necessary for Ca2+ fluxes to occur in apical processes. Additionally, the fragments were saponized to deliver the fluorescent Ca2+ indicator, Fluo-4, and the IP3R agonist, adenophostin A which may also have an impact on the integrity of the ER.   5.3 Future directions  The work presented here attempts to provide a foundation upon which future studies might further dissect the role of Ca2+ regulation of TBCs and ESs. As discussed in chapter 4, the use of two photon microscopy would allow for the depth penetration necessary to examine apical processes of intact seminiferous tubules. Coupled with the use of GECIs targeted to specific cellular domains to detect any Ca2+ fluxes. This could allow for detection of Ca2+ fluxes that were not visualized using methods in this thesis potentially due to several factors. The maintenance of the architecture of the seminiferous epithelium by using intact tubules would ensure that the integrity of the ER is not compromised. The use of GECIs targeted to specific domains will reduce background levels of fluorescence to make detection of Ca2+ fluxes easier. Visualizing apical processes in intact seminiferous tubules using two photon microscopy is an exciting prospect that has the potential to dissect the process of TBC formation and ES regulation. One experiment that would provide useful insight, would be the visualization of the actin dynamics throughout TBC formations. This could be accomplished by transfecting the seminiferous tubules with a plasmid encoding LifeAct, a small peptide that binds F-actin without interfering in cellular 56  dynamics. Since TBCs have never been visualized live before, this would inform on the length of time it takes for a TBC to form and eventually vesiculate which would be useful when designing drug perfusion studies similar to those performed in chapter 4. The visualization of the actin dynamics in the lifecycle of one TBC would inform whether current models of TBC formation are correct.  Studies aimed at evaluating the function of the Ca2+ signaling machinery identified at ESs and TBCs in this thesis would be productive. Knockdown studies using siRNA could inform on the function of these proteins on the regulation of ESs and TBCs. This approach has been used previously to show that disruption of TBCs by cortactin knockdown results in spermiation failure [26]. It would be interesting to knockdown IP3R and determine if it has a similar effect on spermiation which would provide functional evidence of Ca2+ regulation of TBCs. Similarly, knockdown studies on SERCA2 would provide insight into its role in regulating ESs. If the role of SERCA2 is to maintain low levels of Ca2+ to prevent disassembly of the actin bundles at ESs, then knockdown might result in disruption of these tripartite structures. Further investigation into potential Src regulation of TBCs is warranted from the immunolocalization data in chapter 3 showing it at the ends of TBCs. It has been reported previously that incubation of isolated seminiferous tubules with PP2, a Src inhibitor, results in a 45% decrease in sperm release [87]. Studies similar to the drug perfusion studies in chapter 4 could be used to deliver Src inhibitors to evaluate the function of Src in relation to TBCs. The same technique could be used to explore the potential spatiotemporal regulation of Src by Ca2+ oscillations by perfusing inhibitors to components of SOCE. Inhibitors could target STIM1/ORAI1 mediated SOCE as I have shown this pair to occur at peripheral ER-PM contacts in the apical 57  process. Alternatively, inhibitors to the TRPM6 channel could be used as it has recently been implicated at the bulb of TBCs [45]. This work contributes to our understanding of the role of calcium signaling in the unique process of junction remodeling in the testis. From a general cell biology perspective, it will lead to a new and alternative paradigm of junction internalization. An understanding of the basic biology of the testis is essential to understanding normal male fertility. Identifying major signaling regulators of TBC formation will help us identify the key control points of spermatocyte translocation and spermiation. Future studies can use this information to investigate the underlying causes of male infertility or to identify potential molecular targets for male contraceptives.  58  References [1] Tung PS, Skinner MK, Fritz IB. 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