Novel Tissue-Intrinsic Functions of SED1/MFG-E8 in the Epididymis Open Access

Raymond, Adam S (2009)

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Novel Tissue-Intrinsic Functions of SED1/MFG-E8 in the Epididymis

By Adam S. Raymond
SED1 is a bimotif cell adhesion molecule secreted by the initial segment of the epididymis where it binds sperm and facilitates adhesion to the egg coat. Herein, SED1-null animals are shown to harbor an unexpected increase in epididymal pathologies suggesting a previously unidentified tissue-intrinsic role for SED1 in the epididymis. Although the SED1-null epididymis develops and differentiates normally, >40% of SED1-null epididymides contain spermatic granulomas. Immunocytochemistry reveals that in addition to its apical secretion, SED1 is localized in basolateral domains of cells of the initial segment. In vitro assays show that SED1 supports epididymal cell adhesion via RGD binding to V integrins and epididymal cells from SED1-null males show reduced adhesion. These results suggest that SED1 facilitates epididymal cell adhesion that may be protective against spermatic granulomas, however the bulk of SED1 is expressed in the proximal epididymis while sperm granulomas most often manifest in the distal segments. In other models, disruption of fluid regulatory processes in proximal regions is accompanied by long-range effects including spermatic granulomas in the distal epididymis. In this regard, the SED1-null epididymis fails to properly acidify the luminal fluid, and the SED1-null epithelium exhibits histological hallmarks of disrupted luminal dynamics. Collectively, these data suggest that SED1 contributes to the regulation of epididymal fluid; however, a variety of proteins known to effect fluid transport and pH regulation are expressed normally. Since SED1 is secreted in exosomes that participate in fluid homoeostasis, it is feasible that the loss of SED1 leads to defective exosome contents, a possibility that will be explored in future studies.

Table of Contents

Table of Contents Chapter 1: Introduction 1 1.1 Introduction to the bimotif adhesion protein SED1 2 1.1.1 Abstract 2 1.1.2 SED1 is a mosaic protein 3 1.1.3 SED1 contributes to phagocytic removal of apoptotic cells in many 9 tissues and organs 1.1.4 SED1 facilitates a number of intercellular interactions 16 1.1.5 Concluding thoughts: SED1 as a therapeutic agent 27 1.2 Introduction to the male epididymis 29 1.2.1 Summary 29 1.2.2 Epididymal organization and anatomy 30 1.2.3 Epididymal function: protein secretion 31 1.2.4 Epididymal function: fluid reabsorption and pH modification 32 1.3 Focus of Dissertation 33 Chapter 2: Materials and Methods 35 2.1 Mice and recombinant SED1 36 2.2 Tissue preparation for histology 36 2.3 Epithelial morphometrics 37 2.4 Detection of endogenous b-galactosidase activity 37 2.5 Immunocytochemistry 38 2.5.1 In vivo 38 2.5.2 In vitro 38 2.6 Immunoblotting 39 2.6.1 MFG-E8 and a V (Chapter 4) 39 2.6.2 All other immunoblots (Chapter 5) 40 2.7 Preparation and culture of primary cells 40 2.8 Cell adhesion assays 41 2.9 BrdU proliferation assays 42 2.10 Determination of luminal pH ex vivo 43 2.11 Intracellular vesicle quantification 43 2.12 Preparation of epididymosomes 44 Chapter 3: The SED1-null Contains an Unexpected Epididymal 45 Phenotype 3.1 Introduction and experimental rationale 46 3.2 Results 47 3.2.1 SED1 localizes to numerous domains in principal, clear, and basal 47 cells of the epididymis 3.2.2 SED1-null epididymides exhibit a loss in tubule integrity and an 51 increase in spermatic granulomas 3.2.3 Reproductive pathologies associated with the loss of SED1 are 54 restricted to the epididymis 3.2.4 The SED1-null epididymis undergoes normal development and 57 differentiation 3.3 Discussion 61 Chapter 4: A Novel Role for SED1 in Maintaining the Integrity of 65 the Epididymal Epithelium 4.1 Introduction and experimental rationale 66 4.2 Results 68 4.2.1 SED1 is expressed in basal and basolateral domains of epididymal 68 epithelia in vivo 4.2.2 SED1 is deposited basally by polarized epithelial cells in vitro 72 4.2.3 rSED1 increases the initial adhesion of primary epididymal 76 epithelial cells 4.2.4 Epithelial cell adhesion to rSED1 is RGD-dependent 76 4.2.5 Epididymal epithelial cells express a V integrins 81 4.2.6 a V integrins are required for epididymal epithelial cell 84 adhesion to SED1 4.2.7 SED1-null epithelial cells show reduced adhesion in vitro 89 4.3 Discussion 92 Chapter 5: Loss of SED1 Results in Altered Luminal Physiology 100 in the Epididymis 5.1 Introduction and experimental rationale 101 5.2 Results 102 5.2.1 Loss of SED1 results in an increase in luminal pH 102 5.2.2 SED1-null epididymides exhibit morphological correlates of 105 defective fluid regulation 5.2.3 Many proteins that regulate the composition of the luminal fluid 111 are expressed and localized normally in the SED1-null epididymis 5.2.4 SED1 is associated with mouse epididymosomes 114 5.3 Discussion 132 Chapter 6: Conclusions and Future Directions 137 6.1 Conclusions 138 6.1.1 Overview 138 6.1.2 Maintenance of the epididymal epithelium 139 6.1.3 SED1 function in regulation of luminal fluid of the epididymis 139 6.1.4 Conclusions 140 6.2 Future directions 141 6.3 Use of SED1 in reproductive interventions 142

Chapter 7: References 146

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