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B1-integrin and cadherin mediated signaling molecules in the rat seminiferous epithelium 2000

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pl-INTEGRIN AND CADHERIN MEDIATED SIGNALING MOLECULES IN THE RAT SEMINIFEROUS EPITHELIUM by DAVID J. MULHOLLAND B.Sc. The University of British Columbia, 1996 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Anatomy) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December, 1999 © David J. Mulholland, 1999 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. DE-6 (2/88) II ABSTRACT The aim of this study was to assess whether pi-integrin mediated signaling molecules, known to be present at cell-matrix adhesion sites in other cell types, are present at unique cell/cell junctions in the seminiferous epithelium, called ectoplasmic specializations (ESs). To address this question several immunological techniques were used including Western blotting, indirect immunofluorescence and immunoprecipitation. Western blotting on whole epithelial lysates verified the specificity of each immunoprobe while immunofluorescence studies determined the spatial and temporal expression of each molecule during spermatogenesis and showed whether or not integrin related molecules were detectable at cell/cell junctions. Markers known to be specific for filamentous actin, which is concentrated at ESs, were used to counterstain immunological probes and serve as indicators for the location of the junctional plaques. Based on the specificity and codistribution of molecules observed in tissue sections and isolated cells, lysates of seminiferous epithelia were assessed for stable protein complexes using co-immunoprecipitation. Lysates used for immunoprecipitations contained only a population of seminiferous epithelium and did not contain myoid cells or the basement membrane component of the seminiferous tubule. (31-integrin and Integrin Linked Kinase (ILK) were shown, at the light level, to be spatially and temporally associated, throughout spermatogenesis, with sites known to contain ESs. These molecules co-distributed with the exception of some ILK staining associated with basal germ cells. Integrin staining appeared restricted to junctional areas known to contain ESs. ILK precipitates probed with (31 -integrin antibodies showed a specific band at 140 kD. pi-integrin precipitates showed the presence of a specific band at about 55 kD when non-conventional detection techniques were used. Pan- Cadherin antibodies showed a direct protein interaction with p-catenin and showed punctate immunofluorescence staining that was restricted to the Sertoli/Sertoli cell interface. Ultrastructural data indicates that these molecules are concentrated at desmosome-like plaques and not ESs. Staining for these molecules was not detected at the Sertoli cell/germ cell junction at any point during spermatogenesis. Focal adhesion Kinase (FAK), a known integrin associating molecule, did not codistribute with the integrin at the light level. Rather, it was restricted to the cytoplasm of early germ cells. Additionally, these molecules did not co- immunoprecipitate in whole epithelial lysates. The absence of detectable FAK at junctional sites prompted the question of whether another major tyrosine related protein is present. Polyclonal antibodies against phosphotyrosine containing proteins showed an abundance of expression throughout the epithelium at both ESs and other cell/cell borders. Paxillin was shown to be present abundantly throughout both the cytoplasm of germ cells and Sertoli cells. Additionally, it appeared to be concentrated at tubulobular complexes. FAK and vinculin, proteins both known to have binding domains with paxillin, co-immunoprecipitated with paxillin in whole epithelial lysates. Possible locations of a direct interaction for these molecules were determined by light level co-distribution. A paxillin/vinculin complex would likely be found at the tubulobulbar complexes, the only location where both molecules were observed to be concentrated. A FAK/paxillin complex, however, would likely be located in the basal germ cells, which is the only location where these molecules were shown to be co-distributed. Tyrosine phosphorylation studies suggest the presence of at least five major bands in blots of whole cell lysates. FAK is not phosphorylated at tyrosine while paxillin is highly phosphorylated. iv Together, these data provide support for the generation of a model showing the presence of molecules known to be fundamental to cell adhesion and signaling. Possible direct protein complexes occurring at junctional sites, and other locations within the epithelium, are based on the distribution of immunofluorescence, co-immunoprecipitations and known interactions that occur in other epithelial systems. Molecules that are detected at junctional sites likely have direct implications for sperm release and architectural changes in the blood- testes barrier. r TABLE OF CONTENTS ABSTRACT TABLE OF CONTENTS LIST OF FIGURES ' LIST OF ABBREVIATIONS ACKNOWLEDGEMENTS INTRODUCTION A. Organization of the seminiferous epithelium and spermatogenesis B. Cell-Cell junctions in the seminiferous epithelium C. Other organized concentrations of actin in the seminiferous epithelium.. D. Rationale for the presence of pi-integrin mediated signaling molecules in the rat seminiferous epithelium E. Statement of hypothesis MATERIALS AND METHODS A. Animals B. Reagents C. Preparation of tissue for immunostaining D. Immunofluorescence E. Tissue preparation for gel electrophoresis and immunoblotting F. Gel electrophoresis & Western blotting G. Electronmicroscopy H. Immunogold labelling /. Immunoprecipitation J. Controls 17 17 .17 .18 19 .20 .21 .21 .22 .23 VI RESULTS A. Immunofluorescence Localization of Signaling Molecules Actin 25 Pan-Cadherin/fi-Catenin 28 pi-integrin 31 Integrin Linked Kinase (ILK) 31 Focal Adhesion Kinase (FAK) 41 Vinculin 41 Paxillin 41 Phosphotyrosine 53 Controls for immunofluorescence. 53 Spatial and temporal distribution of signaling molecules 63 B. Western Blots and Immunoprecipitations Pan-Cadherin and p-catenin 67 p1-integrin and ILK 6 7 Paxillin and Vinculin 74 Paxillin and FAK 7 4 Phosphotyrosine 7 7 P1-integrin and FAK 7 7 Vinculin and Actin 7 7 C. Immunogold Labelling for pan-Cadherin 85 DISCUSSION 90 CONCLUSION 1 °5 FUTURE DIRECTIONS 106 BIBLIOGRAPHY 109 vii 114 APPENDIX A 116 APPENDIX B APPENDIX C 1 1 7 APPENDIX D 1 1 8 a " Vlll LIST OF FIGURES Fig. 1 Histological organization of the seminiferous epithelium Fig. 2 Schematic diagram of actin distribution in the seminiferous epithelium Fig. 3 Rational for the presence of (31-integrin mediated signaling molecules at ectoplasmic specializations Fig. 4 Phalloidin localization in tissue sections Fig. 5 p1-catenin and pan-Cadherin immunofluorescence localization in tissue sections Fig. 6 31-integrin immunofluorescence localization in tissue sections Fig. 7 pi-integrin immunofluorescence localization in isolated cells Fig. 8 ILK immunofluorescence localization in tissue sections Fig. 9 ILK immunofluorescence localization in isolated cells Fig. 10 FAK immunofluorescence localization in tissue sections Fig. 11 FAK immunofluorescence localization isolated cells Fig. 12 Vinculin immunofluorescence localization in tissue sections Fig. 13 Paxillin immunofluorescence localization in tissue sections Fig. 14 Paxillin immunofluorescence localization in isolated germ cells Fig. 15 Phosphotyrosine immunofluorescence localization in tissue sections Fig. 16 Phosphotyrosine immunofluorescence localization in isolated cells Fig. 17 Controls for immunohistochemistry Fig. 18 Controls for immunocytochemistry Fig. 19 Summary of staged immunofluorescence expression of molecules Fig. 20 Pan-Cadherin/p-catenin Western blot & co-immunoprecipitation Fig. 21 pi-integrin/ILK Western blot & co-immunoprecipitation Fig. 22 PaxillinA/inculin & Paxillin/FAK Western blot & co-immunoprecipitation ix Fig. 23 Phosphotyrosine Western blot & co-immunoprecipitation Fig. 24 p1-integrin/FAK Western blot & co-immunoprecipitation Fig. 25 Vinculin/Actin Western blot & co-immunoprecipitation Fig. 26 Immunoelectronmicroscopy localization of pan-Cadherin Fig. 27 Proposed model for the presence of integrin and cadherin related molecules at ectoplasmic specializations X LIST OF ABBREVIATIONS BSA Bovine Serum Albumin EDTA Ethylene Diamene Tetra Acetic Acid EGTA Ethylene Glycol-bis ((3-Amino Ethyl Ether)-N,N,N',N'-Tetra Acetic Acid ES Ectoplasmic Specialization FAK Focal Adhesion Kinase HRP Horse Radish Peroxidase IgG Immunoglobulin Ij Intercellular Bridge ILK Integrin Linked Kinase KCI Potassium Chloride KH2P04 Potassium Phosphate KOH Potassium Hydroxide MgCI2 Magnesium Chloride Na2HP04 Sodium Phosphate NaCI Sodium Chloride NP-40 Nonidet P-40 PBS Phosphate Buffered Saline PIPES Piperazine-N,N'-bis (2-Ethersulfonic Acid) PVDF Polyvinylidene Fluoride (PVDF) SDS Sodium Dodecyl Sulfate SDS-PAGE Sodium Dodecyl Sulfate-Poly Acrylamide Gel Electrophoresis S/Gj Sertoli Cell/Germ Cell junction S/Sj Sertoli Cell/Sertoli Cell junction TPBS Tween Phosphate Buffered Saline Tris-HCI (Tris[hydoxymethyl]aminomethane)-Hydro Chloride Tb Tubulobulbar Process Tween-20 Polyethylene Sorbitan Monolaurate xi ACKNOWLEDGEMENTS I would like to thank the members of my Supervisory Committee, Dr. Frank Jirik and Dr. Cal Roskelley. Special thanks go to Dr. Wayne Vogl for his guidance, encouragement and patience in allowing me to pursue my scientific endeavours throughout my degree. 1 INTRODUCTION A. Organization of the seminiferous epithelium and spermatogenesis The seminiferous tubules are lined by a complex stratified epithelium consist ing of two main cell types. Sertoli cel ls are a non-dividing cell population that extend from the basement membrane to the lumen of the seminiferous tubule. The spermatogenic cells are a proliferating population of cells that includes cells at different stages of differentiation (Fig. 1). Spermatogenes i s is often divided into three phases including: (1) the spermatogonia! p h a s e ; (2) the s p e r m a t o c y t e p h a s e (me io s i s ) a n d (3) the s p e r m a t i d p h a s e (spermiogenesis). The most immature cel l s of the semin i ferous ep i thel ium are located near the basement membrane and move apically as they proliferate and differentiate. T h e s e cel ls form four to five concentr ic layers which represent different generat ions of deve lop ing s pe rma togen i c ce l l s . S pe rma togen i c ce l l s are morpho log ica l l y d ist inct dur ing their d i f ferent iat ion and a re identif ied as spe rmatogon ia , s pe rmatocy te s or spe rmat id s . Spermatogonia are derived from stem cells found at the base of the epithelium and undergo severa l mitotic d iv is ions whi le chang ing slightly in morphology. T h e s e ce l l s d iv ide to produce the larger spermatocytes which then divide meiotical ly into round spermatids. T h e s e haplo id ce l l s subsequent ly undergo dramat ic morpho log ica l d i f ferent iat ion to ultimately produce mature spermatozoa (Fig. 1) (Fawcett, 1975a; Russel l , 1977). B. Cell-Cell junctions in the seminiferous epithelium During spermatogenes i s early round spermat ids t rans locate towards the tubule lumen and then become situated within Sertol i cel l crypts. A s germ cel l deve lopment 2 proceeds the crypts change in shape to accommodate the elongating spermatid which eventually undergoes spermiation, or release into the seminiferous tubule lumen (Russell, 1994). Throughout this process sites of actin based cellular adhesion exist both between Sertoli cells and elongating germ cells, Sertoli cell/germ cell junctions or S/Gj, and between the base of adjacent Sertoli cells, Sertoli cell/Sertoli cell junctions or S/Sj (Russell and Peterson, 1985) (Fig. 2). 3 Figure 1. Histological organization of the seminiferous epithelium in the rat. Germ cells undergo dramatic morphological change as they translocate from the basal compartment to the adluminal one and become situated in the Sertoli cell crypt. Morphologically mature spermatids undergo spermiation and are released into the tubule lumen (adapted from Vogl, 1989). ADLUMINAL COMPARTMENT BASAL COMPARTMENT 5 Figure 2. Major concentrations of filamentous actin in the rat seminiferous epithelium. Ectoplasmic Specializations (ESs) occur in two locations including the Sertoli/Germ cell junction (S/Gj) and the Sertoli/Sertoli (S/Sj) junction. Developing ESs also contain junctional actin in close association with the acrosome as seen in early round spermatids. Non-junctional sites of actin include the tubulobulbar processes, intercellular bridges and subacrosomal actin (adapted from Vogl, 1989). to Tubulobulbar Process Actin Sertoli/Germ Cell Junctional Actin Intercellular Bridge Actin Acrosomal Actin Adluminal Compartment Sertoli/Sertoli Junctional Actin Basement Membrane / Basal Compartment 7 The Sertoli cell junctional complex found at the base of the epithelium is morphologically distinct and is not found in any other type of epithelia. This complex functions to divide the epithelium into two compartments including a basal compartment and an adluminal compartment. The basal compartment contains the spermatogonia and younger spermatocytes while the adluminal compartments contains older spermatocytes and all spermatids. During the first meiotic division of spermatocytes, these cells pass through the "blood-testes" barrier and into the adluminal compartment while at the same time maintaining the structurally integrity of the barrier. The Sertoli-Sertoli junctional complex is composed of: (1) tight junctions, (2) gap junctions, (3) desmosome-like junctions, and (4) ectoplasmic specializations or ESs (Russell, 1976; Vogl , 1989). The tight junctions constitute what is called the "blood-testes" barrier, a structure with important immunological functions in the testes. ESs form at the interface between the Sertoli cell and germ cells and is morphologically similar to the ES which occurs at the Sertoli-Sertoli interface. Both the apical (Sertoli/germ cell) and basal (Sertoli/Sertoli) ectoplasmic specializations are thought to function primarily for cellular adhesion (Vogl, 1996). C. Other organized concentrations of actin filaments: intercellular bridges, tubulobular complexes and subacrosomal actin In addition to actin associated with ectoplasmic specializations, actin is also concentrated at other major sites in the seminiferous epithelium (Figure 2). Intercellular bridges appear as distinct tubular structures between spermatogenic cells which form as a result of incomplete cytokinesis, ultimately linking the cytoplasm of many spermatids. The function of these structures is not certain but it has been hypothesized that they play a role in maintaining synchrony of spermatogenesis (Fawcett, 1975a). 8 Tubulobulbar complexes are composed of many finger-like processes which project into the Sertoli cell cytoplasm from the spermatid. Actin filaments occur in the Sertoli cell cytoplasm surrounding the processes and are thought to function as anchoring devices for the spermatids to the Sertoli cells (Russell and Clermont, 1976). They are also thought to be involved in the recycling of junctions at locations of cellular adhesion during spermiation (sperm release). In the rat tubulobulbar processes are particularly prominent in late stage spermatids (Russell and Clermont, 1976. Actin found in the subacrosomal space changes in distribution during spermatogenesis and tends to spread over the nucleus as the acrosome does during germ cell differentiation (Fig. 2). Interestingly, subacrosomal filamentous actin is more prominent in the rat during early germ cells stages and eventually disappears in later ones. The function of subacrosomal actin has been thought to be involved in the capping process (Russell etal., 1986). D. Rationale for presence of pi-integrin mediated signaling molecules in rat seminiferous epithelium As developing spermatids elongate ectoplasmic specializations are formed in Sertoli cells adjacent to spermatid heads. This site of adhesion between the germ cell and Sertoli cell is disassembled as part of the process by which spermatids are released into the tubule lumen (Russell, 1977; Vogl, 1989). Ectoplasmic specializations are also thought to be important in the maintenance of adhesion between neighbouring Sertoli cells (Vogl 1989; Vogl et al., 1991) at the base of the epithelium. Like apical ectoplasmic specializations the structural components of the "blood-testes barrier" are structurally dynamic and undergo cyclic disassembly and reassembly as primary spermatocytes pass into the adluminal compartment (Russell, 1977). 9 Although much is known about the ultrastructure of ESs in the seminiferous epithelium, comparatively little is known about their molecular composition and their control. It is known, however, that ectoplasmic specializations are dissimilar to other intercellular junctions in that they do not contain the usually composition of adhesion molecules but, nevertheless, contain several known adhesion associated proteins including a-actinin (Franke et al., 1978, Russell and Goh, 1988), and vinculin (Grove et al., 1990; Pfeiffer et al., 1991). While proteins such as these are present, reports of other adhesion molecules have been unclear. For instance, some investigators have suggested the presence of N-CAM at junctional complexes in the seminiferous epithelium grown in culture but their absence in vivo by postnatal day 15 (Li et al., 1998). 10 Figure 3. (a) Rationale for the presence of integrin related molecules at cell adhesion sites in the seminiferous epithelium. The presence of integrins at cell-cell borders in the seminiferous epithelium, and the absence of other cell-cell adhesion related molecules, suggests that integrin related molecules, such as those found in well-defined cell-matrix attachment sites, may be present at ectoplasmic specializations. Integrin activation, or clustering, at a cell/matrix attachment sites is shown with molecules that associate with the integrin cytoplasmic domain and include Src Kinase, Focal Adhesion Kinase (FAK), talin, paxillin, vinculin and p130CAS (Giancotti and Ruoslahti, 1999). (b) A speculative model for the presence of p1-integrin mediated signaling molecules at ectoplasmic specializations in the rat seminiferous epithelium. p1-integrins have been shown to be present in the seminiferous epithelium and concentrated at ESs, but few other integrin related molecules have shown to be present. In other systems protein-protein interactions have been shown between the pi-integrin and ILK. FAK has been shown to associate with many molecules capable of signaling events including paxillin, a vinculin binding protein. These interactions are thought to be mediated by motifs containing phosphotyrosine which are critical to intermolecular interactions. i i a) Matrix RGO RGO RGD RGO Actin Cytoskeleton b) 12 Surprisingly, the only transmembrane adhesion molecule to be identified in vivo both at apical and basal attachment sites is p1 integrin (Pfeiffer et al., 1991; Palombi et al., 1992, i Salanova et al., 1995 Salanova et al., 1998) and an associated a6 integrin subunit (Salanova et al., 1995, Salanova et al., 1998). Although the a6p1 integrin complex has been previously thought to be mainly a laminin receptor (Sonnenberg et al., 1988), reports tiave suggested that it may function on the egg surface as a sperm receptor and therefore be involved in cell-cell interactions (Almeida et al., 1995). B1-integrins have also been implicated with white blood cells and their migration. Specifically, integrins on the cell surface of eosinophils are thought to interact with ligands on endothelial and epithelial cells (Seminario & Bochner, 1997). Further support for a functional role for integrins in the seminiferous epithelium is based on the disappearance of integrin expression concurrent with the turnover of ESs (Salanova et al., 1995). As spermatocytes translocate from the basal to the adluminal compartment, integrin expression disappears and then subsequently reappears (Salanova et al., 1995). Also, the disappearance of integrin expression has been shown to coincide with disassembly of the apical ESs and spermatid release into the tubule lumen (Palombi et al., 1992). With reports of pi integrin expression coinciding with key spermatogenic events it is possible that integrin-related molecules could also show changing patterns of expression in the seminiferous epithelium. Further motivation for investigating potential signaling pathways associated with ESs comes from preliminary immunoflourescence and biochemistry work, using a pan-antibody for ERK, a serine/threonine extracellular signal regulated kinase. These experiments showed that ERK is localized around the sperm head and is expressed in protein samples that are enriched for ESs. 13 Integrin mediated signaling molecules (Fig. 3) are often associated with cytoskeletal changes and have been found to be vital for development and maintenance of normal cellular structure and function (Hynes, 1992). Many researchers have shown that integrins are not only able to mediate signals between the extracellular matrix and cells (Clark and Brugge, 1995; Yamada and Miyamoto, 1995; Geiger et al., 1995; Schwartz et al., 1995; Parsons, 1996), but also between adjacent cells (Geiger et al., 1995; Kirkpatrik and Peifer, 1995; Gumbiner, 1996). Although phosphorylation is one of the earliest events associated with integrin activation or clustering (Burridge et al., 1992), there is growing evidence to suggest that the relationship between cell adhesion formation and phosphorylation events may occur independent of each other. Nevertheless there is also increasing evidence that pi-integrin subunits, a structural element found at focal adhesions, can associate, either directly or indirectly via their cytoplasmic domains, with molecules capable of phosphorylation (Burridge et al., 1992; Schoenwaelder SM and Burridge K, 1999). Phosphorylation events have also been shown to be p1 -integrin mediated and have been suggested to occur in either an "outside-in" or an "inside-out" manner and are thought to be closely associated with changes in the actin cytoskeleton (Dedhar and Yamada, 1996), This suggests that initiation of signaling events can come from an extracellular ligand or a cytoplasmic protein. A number of kinases have been shown to associate with integrins in vitro of which include Focal Adhesion Kinase (FAK) (Schaller and Parsons, 1994; Schaller et al., 1995) and Integrin Linked Kinase (ILK) (Hannigan et al., 1996). These submembranous molecules are thought to be closely associated with integrin cytoplasmic tails and to mediate signals to the actin cytoskeleton. FAK, a tyrosine kinase, has been shown to interact, in vitro, with p1. Specifically, it becomes phosphorylated, at tyrosine residues, during integrin activation, and has been shown to induce changes in other signaling 14 proteins (Schal ler and Parsons, 1994, Schal ler et al., 1995). Interestingly, F A K has never been shown to assoc iated with pi-integrin cytoplasmic tails in vivo or in non-cultured cells. This is unlike the situation with ILK, a serine-threonine kinase, which has been shown to have a strong in vivo a s soc ia t ion with f31, p2 and p3 integrin cy top la smic doma in s (Hannigan et al., 1996). C l a s s i c s ignal ing molecules downstream of ILK and F A K include v incul in and paxil l in. The se proteins are thought to have roles in regulat ion of both adhes ion and downst ream signal ing events and have, therefore, been termed linking or dock ing proteins to which other component s of the actin cytoske leton can a s soc i a te (Yamada and Geiger, 1997). Even though the as semb ly of many proteins a s soc ia ted with adhes ion p laques depend on the ability of F A K to form phosphorylation sites on other proteins, it is very interesting that tyros ine phosphorylat ion has been shown not to be neces sa ry for the gathering of structural components of focal adhes ions (Gi lmore and Romer, 1996). When leve l s of phosphotyros ine levels are enhanced the formation of foca l adhes i on s and adhes ion p laques is enhanced whi le dec rea sed levels of tyrosine phosphory lat ion can inhibit focal adhes ion formation (Burridge and Chrzanowska-Wodn icka, 1996). It appears that despite the importance of tyrosine phosphorylation in the formation of adhes ion sites the structural e lements of focal adhesions, the phosphorylation of their structural e lements, including vinculin and paxillin, is not necessary for adhes ion plaque formation. Ev idence suggests that molecular regulation by tyrosine phosphorylation is upstream of such focal adhes ion elements. Poss ib le protein tyrosine kinases upstream of Rho A, a smal l G T P a s e , could have the ability to regulate its activity and the subsequent regulation of Rho guanine nuc leo t i de e x c h a n g e factor (Nobe s et al . , 1995). T h e impo r t ance of t y ro s i ne phosphory lat ion, however, is under scored when cons ider ing the number of condit ions which can promote dephosphorylation of adhesion sites including many growth factors such 15 as insulin and platelet derived growth factor. Nevertheless, in either instance of phosphorylation or dephosphorylation it appears that the fundamental phosphorylation event is upstream of Rho A. Both stress fibers and focal adhesions are regulated by Rho- A, a GTP-binding protein (Knight et al., 1995). Further interest in the Rho family stems from the fact that of the molecular composition induced by activation of the small GTPases, focal adhesions, appear to resemble ectoplasmic specializations the closest of any cultured form of cellular adhesion. Although the tyrosine phosphorylation of the structural elements of adhesion plaques does not appear to be necessary for the formation of these sites, increased levels of tyrosine phosphorylation, especially by non-receptor tyrosine kinases such as Src and FAK, have been implemented with turnover with adhesion plaques (Schoenwaelder and Burridge, 1999). For example, cells which are transformed with v-src show disrupted focal adhesions and elevated tyrosine phosphorylation levels of many structural elements of focal adhesions including integrins, vinculin and also regulatory components including FAK and paxillin (Burridge and Chrzanowska-Wodnicka, 1996). Besides protein interactions which have been shown between FAK and many focal adhesion elements its importance in adhesion turnover is further supported when considering FAK deficient cells. Interestingly, in FAK deficient cells there is increased formation of focal adhesions but reduced motility (llic et al., 1995). Similar results have been observed in dominant-negative constructs where FAK expression is repressed (Gilmore and Romer, 1996). Conversely, over expression of FAK results in increased cell migration and decreased formation of adhesion plaques. FAK knock-out mice are lethal during embryonic development but show a phenotype which is similar to that of FAK deficient cells grown in culture (llic et al., 1995). 16 To date, very little is known about the presence and distribution of molecules capable of signaling in the seminiferous epithelium. In this study, using Western blotting, conventional immunofluorescence, immunoprecipitation and immunoelectronmicroscopy we explore the possibility that key members of classically defined (31-integrin signaling pathways are present at cell-cell junctions (ectoplasmic specializations) in the seminiferous epithelium as a prerequisite to determining their role in events such as sperm release and turnover of the basal junctional complex. In addition, we conjecture whether or not cadherins are components of ectoplasmic specializations and therefore a defining feature of their turnover. Together our results have implications for signaling events involved in sperm release and turnover of the "blood-testes" barrier. E. Statement of hypothesis The aim of this study was to determine whether classical pi-integrin mediated signaling molecules and complexes found at sites of cellular adhesion in other epithelial systems are present at ectoplasmic specializations in the adult rat seminiferous epithelium. If ectoplasmic specializations are similar to other forms of cellular adhesion, then 111- integrins and related signaling molecules should be spatially and temporally co-distributed with markers that are specific for the actin component of these junctions. 17 METHODS AND MATERIALS A. Animals Tis sue for all experiments was obtained from adult Sprague-Dawley rats that ranged in weight from 250 g to 550 g. An imal s were anaesthet ised using halothane administered via the respiratory tract. Testes were removed and rats were euthanized while under deep anaesthesia. B. Reagents Mos t reagent s we re obta ined from S i gma -A ld r i ch (Oakv i l le , O N , C a n a d a ) . F l uo re s cen t pha l lotox ins we re obta ined from Mo lecu l a r P r o b e s (Eugene, O R ) and paraformaldehyde was obtained from Fisher Scientific (Vancouver, BC, Canada). C. Preparation of Tissue for Immunostaining Testes were perfusion fixed, via the spermatic artery, with 3 % paraformaldehyde in phosphate buffered sal ine (PBS ) (150 mM NaCI, 5 mM KCI, 3.2 m M N a 2 H P 0 4 , 0.8 m M K H 2 P 0 4 , pH 7.3, 33 °C) for 30 min. Following fixation, testes were perfusion washed for an additional 30 min. with P B S . Both fixative and wash solutions were filtered using 0.22 um Millipore filters and warmed to 33 °C prior to perfusion. After fixing, the testes were frozen in O C T compound (Fisher Scientific) and then e q u i l i b r a t e d to c h a m b e r t e m p e r a t u r e in a c r yo s ta t (B r i ght s 5010 ) . F o r immunof luorescence studies, sect ions were cut to a th ickness of 10 um, co l lected on polylys ine coated sl ides, and then immediately treated with - 2 0 °C acetone for 10 min. Sect ions were then air-dried for 10 min. For immunostaining with the probes for p1-integrin 18 and ILK, testes were immediately frozen after excis ion from the animal; that is, the t i ssue was fresh frozen without prior fixation. D. Immunofluorescence Prior to immunostaining, t issue was treated for 30 min. with 5 % normal goat serum in T P B S ( 0 . 1 % BSA, 0 . 1 % Tween-20, PB S ) to minimize nonspecif ic binding of secondary ant ibodies. Incubation t imes with primary antibodies were either 1-2 hours at 3 7°C or overnight at 4 °C. Primary antibodies were used at the following dilutions: polyclonal anti- |3-catenin (Sigma, cat. C2206) at 1:1000; monoclonal anti-p-catenin (Sigma, cat. C7207) at 1:400; po lyc lona l ant i -Pan Cadhe r i n (S igma, C3678 ) at 1:1000; monoc l ona l an t i -Pan Cadher in (S igma, C1821) at 1:400; serum anti-B1 integrin (Chemicon International, cat. AB1952 ) at 1:200, polyclonal anti-ILK (gift of Dr. Shoukat Dedhar, Univers ity of British Columbia) at 1:100, monoclonal ant i -FAK (Transduction Laboratories, cat. F15020) at 1:50, monoc lona l anti-Paxi l l in (Transduction Laboratories, cat. F15020) at 1:400, monoc lona l ant i -V incu l in ( S i gma, cat. V9131 ) at 1:400 and po l yc lona l an t i - pho spho ty ro s i ne (Transduction Laboratories, cat. P11230) at 1:100. After primary antibody staining, t issue sect ions were washed 3 t imes (10 min. each wash) with T P B S and then incubated with secondary antibodies (Jackson Immunoresearch Laboratories Inc., Wes t Grove, PA) for 1 hour at 37°C at a 1:100 dilution. After secondary antibody staining, sections were washed 3 t imes (10 min e a c h wash) with T P B S and then mounted with Vec ta sh i e l d (Vector Laborator ies , Bur l inkgamem, C A ) and a covers l ip. In s o m e c a s e s the s e cond w a s h included rhodamine or f luorescein Phal loidin to specif ical ly label f-actin structures. S l ides were observed and photographed using a Ze i s s Ax iophot mic roscope (Carl Ze i s s , Inc., Thornwood, NJ). 19 E. Tissue preparation for gel electrophoresis and immunoblotting Tes te s were decapsu lated (see appendix II) and the semini ferous tubules put in a g lass petri dish containing cold P E M (80.0 m M P IPES , 1.0 m M E G T A , 0.1 m M MgCI 2 , 250 m M sucrose, pH 6.8 with KOH). T i s sue was minced with two scape l b lades decapsu lated in cold P E M containing a cocktai l of protease inhibitors (0.5 ug/ml Leupept in, 0.5 ug/ml Pepstatin, 0.5 jag/ml aprotinin, 0.1 m M PMSF ) , for a maximum of 60 min. at 4 °C. Using fine d issect ing tools and a Ze i s s Dissect ing M ic roscope fitted with dark-field condense r (Don Mills, Ontario, Canada ) epithelia were separated from tubule walls by secur ing one end of the tubule and using another probe to squeeze the tubule contents out towards the other end. Ejected epithelia were collected with a micropipette and placed in fresh buffer on ice. Epithelia were lysed both under denaturing (Western blots of whole cell lysates) and native (all co-immunoprecipitations) conditions. Denaturing conditions included 30 min. of NP -40 lysis buffer (30 m M Tris, pH 7.4, 150 m M NaCI, 1 % NP-40, 0 . 5 % deoxylacholate, 0 . 1 % SDS , 2 m M EDTA) fol lowed by boiling and 2 sec. of sonication at 60 MHz. Native conditions was with NP-40 lysis buffer (30 m M Tris, pH 8, 150 m M NaCI; 1 % NP-40, 1 0 % glycerol, 2 m M EDTA ) without sonicat ion or boiling. Lys is buffers inc luded a cocktai l of protease inhibitors (0.5 ug/ml Leupeptin, 0.5 ug/ml Pepstatin, 0.5 ug/ml aprotinin, 0.1 m M P M S F and 0.1 m M Na Vanadate ) . Who l e ce l l s lysates, but not immunoprecipitated samples , were a s s e s s ed for total protein using a bic inchoninic protein assay. S amp l e s were standardized to approximately 1 mg/ml and a total of 15 ug of protein loaded per wel l during electrophoresis. 20 F. Gel Electrophoresis & Western Blotting Ti s sue samples were then mixed with 2x S D S buffer (0.125 M Tris-HCI, pH6.8, 4% SDS , 2 0 % glycerol, 1 0 % 2-mercaptoethanol), boiled at 100 °C for 5 min., put on ice and then loaded into gels. T i s sue samples were e lectrophoresed on 7.5 % , 1 0 % and 12 % S D S - P A G E (Laemmli, 1970) minigels and transferred to P V D F membranes for 4 hours or overnight. After transfer, membranes were washed twice in T B S T (100 mM-Tris-HCI, pH 7.5, 0 . 9 % NaCI, 0 . 1 % Tween-20) (5 min each wash) and then b locked with 1 0 % dried sk im milk in T B S T for either 1 hour at room temperature or overnight at 4 °C. Pr imary antibodies for Western blotting were used at the following dilutions: monoclonal anti-actin (1:1000), polyclonal anti-actin (1:1000), monoclonal anti-|3-catenin (1:1000), polyclonal anti- p-catenin (1:5000); monoclonal ant i -Pan-Cadher in (1:1000); polyclonal ant i -Pan-Cadher in (1:5000); polyclonal anti-p1-integrin (Chemicon A B 1952) (1:1000), po lyc lona l anti-B1- integrin (G. Tarone, Italy) (1:10,000); monoc lona l anti-ILK (1:500): po lyc lona l anti- ILK (1:1000), monoc lona l ant i -FAK (1:500), monoc lona l anti-Paxi l l in (1:10,000), monoc lona l ant i -Vincul in (1:2000) and polyclonal ant i -phosphotyros ine (1:1000). Fo l lowing primary incubations membranes were washed 2 x 5 min. and 3 x 1 0 min. with T B S T followed by 2 x 5 min. and 3 x 1 0 min. with TBS . Membranes were incubated either with ant i -mouse-HRP (Santa C ruz Biotechnology, cat # sc-2031) or anti-rabbit-HRP (Santa C ruz Biotechnology, cat # sc-2030) at a dilution of 1:5000. Membrane bound antibodies were detected using chemi luminescence with E C L Western blotting detection agents (Amersham Life Sc ience, cat. R P N 2106) for incubat ion per iods of 1 min and then e x p o s e d immed ia te ly to radiographic film. 21 G. Electron Microscopy Testes were perfused with warm (33 °C) fixative (80 mM PIPES, 1.0 mM EGTA, 1.0 mM MgCI2, 0.5% gluteraldehyde, 3.0% paraformaldehyde adjusted to pH 6.8 with KOH) for 30 min. After perfusion, tissue was mixed by inverting the test tube and left at room temperature for 1 h. Cells were sedimented by centrifugation at 11,000 rpm in an Epindorf centrifuge and the supernatant was replaced with 0.1 M sodium cacodylate (pH 6.9). Samples were washed twice with fresh buffer and post-fixed for 1 h on ice in 0.1 M sodium cacodylate containing 1% osmium tetroxide. Samples were washed three times with dH20 (10 min. each wash) and then dehydrated through a series of increasing concentrations of ethanol. Tissue was then embedded in resin (J.B. EM Services Inc.), sectioned followed by staining with uranyl acetate and lead citrate. Photography was done on a Philips 300 electron microscope. H. Immunogold Labelling Testes were perfused for 30 min. by the spermatic artery briefly with PBS followed by fixative (3% paraformaldehyde and 20 mM ethylacetimidate in PBS, pH 7.3). After perfusion the testes was cut into small pieces and immersion fixed (3% paraformaldehyde and 0.1% gluteraldehyde in PBS, pH 7.3) for an additional 60 min. After fixation, the tissue was washed three times with PBS and dehydrated through a series of increasing concentrations of ethanol. Tissue was then embedded in lowicryl KM4. Tissue sections were collected on carbon/formvar coated nickel grids and treated for 5 min with a pre-block solution (0.01 M glycine, 0.1% BSA, 1:20 NGS and 0.05% Tween- 20). Sections were incubated with primary cadherin antibodies (Sigma, cat. 1821) in 22 antibody solutions (0.01 M glycine and 1% NGS) at a dilution of 1:50 for 2 h at 37 °C. After staining grids were washed three times (5 min each wash) with wash buffer (0.01 M glycine and 0.1% BSA) and then incubated for 60 min at 37 °C with the secondary antibody (Sigma, cat. G7652) diluted 1:20 in secondary antibody solution (0.1% BSA, 0.05% Tween- 20, and 5% fetal bovine serum). Sections were washed with PBS and fixed for 10 min with 2% gluteraldehyde in PBS followed by two washes with dH20. Grids were stained with 1 % uranyl acetate, washed and dried. /. Immunoprecipitation Whole cells lysates of seminiferous epithelia were pre-cleared for at least 60 min by adding 30-50 ul of a 50% slurry of protein A-sepharose beads (Sigma, cat. P9424) to 500- 750 ul of total cell lysate. After pre-clearing, the lysates were centrifuged 5-10 sec. at high speed to pellet beads. Supernatants were then transferred to new pre-chilled tubes. Primary antibodies were added to cell lysates and incubated overnight without agitation. During the last 60 min. of incubation 30-50 ul of 50% protein A or protein G-sepharose was added to the lysates and allowed to incubate while on a platform roller. After tubes were centrifuged at high speed for 15-20 sec. to sediment immune complexes. The supernatants were saved for western blotting to assess the extent of specific antigen precipitation. Immune complexes were then washed 4-5 times by sedimenting the sepharose beads and then resuspending them in lysis buffer at a volume of 10 times that of the beads. In the third wash beads were resuspended in 20 mM Tris, pH 7.5 to remove detergents contained in the wash buffer. After washing liquid was removed with a 23 G needle on a syringe. Immunoprecipitate complexes were then resuspended in 50-100 ul of 2x SDS-PAGE sample buffer and boiled for 5 minutes to elute bound proteins. Beads were 23 pelleted and the supernatants were removed and either used immediately for gel electrophoresis or frozen at -20 °C. J. Controls: Immunofluorescence, Western blotting, Immuno EM and Immunoprecipitation In immunofluorescence, immuno EM and immunoblotting studies, three types of controls were used. First, controls for nonspecific primary antibody binding included non- immune sera used at an equivalent IgG concentration to that of the primary antibody. Controls for non-specific binding of secondary antibodies included tissue samples treated with buffer instead of primary antibodies and then incubated with secondary antibody. Controls for autofluorescence or autochemiluminescence consisted of incubation with buffer alone without primary or secondary antibodies. To control for non-specific binding during immunoprecipitation, several controls were used. Controls for non-specific binding of the monoclonal and polyclonal antibodies primary antibody included using non-immune IgG or sera, respectively, at a concentration the same as that of the immune antibody. Specificity of the precipitating antibody was determined by running immunoprecipitates on SDS-PAGE and probing with the immunoprecipitating antibody while also comparing to specific bands seen with non-precipitated whole cell lysates with Western blots. To determine whether there was an antibody or antigen excess non-precipitated supernatants were examined on SDS-PAGE and immunoblotted for the presence of non-precipitated antigen. In addition, the remaining non-soluble pellet from cell lysates was assessed for the presence of specific antigen. To separate cross reaction of denatured IgG molecules from specific detection of ILK two additional methods to conventional immunoprecipitation were used. Rather than using a secondary IgG conjugated to HRP, protein A-HRP (Sigma, P-8651) was used for secondary detection. 24 Using this method cross-reaction with electrophoresed, and therefore denatured, IgG molecules was avoided and detection only of ILK primary antibodies would occur. Secondly, proteins were electrophoresed under nonreducing conditions achieved by not including (3-mercaptoethanol in the treatment buffer. Non-denatured IgG molecules were then separated from faster migrating prospective ILK precipitates. 25 RESULTS A. IMMUNOFLUORESCENCE Distribution of actin The distribution of ec top la smic spec ia l i zat ions w a s determined by sta in ing for f i lamentous actin using F ITC-phal lo idin in f ixed frozen sect ions of semin i ferous tubules. Figure 4 shows paired phase and f luorescence double exposures (FITC-phalloidin, green; DAPI, blue) illustrating the changing distribution of f i lamentous actin structures. Serto l i - ge rm cel l junct iona l (S/Gj) actin is apparent at different points of deve l opment of spermatogenes is . It is most obvious in regions surrounding spermatid heads during s tages l-IV (Fig. 4a, a') and Stages V-VI (Fig. 4b, b'). Subsequently, at stage VIIA/III (Fig. 4c, c'), S/G junct ional actin shows greater concentrat ion on the dorsal s ide of the elongat ing spermatid. In addition to well establ ished junctions, junctional actin is present in developing ec top l a sm ic spec ia l i za t ions (dj) and is observed to be in c l o se appos i t ion to round spermatids during stages IX (Fig. 4d, d'). Specifically, these developing sites of adhes ion are recognized by a circular sheath of actin forming in Sertoli cell regions adjacent to germ cel l acrosomes. Sertol i cell-Sertoli cel l junctional (S/Sj) actin is identified by a l inear array of actin between the bases of adjacent Sertoli cells (Fig. 4a). This junction is present during most spe rmatogen i c s tages but undergoes structural change as spe rmatocy te s pa s s through the blood-testes barrier during stages VIIA/III (Fig. 4c). Tubulobulbar comp lexe s (tb), transient structures found predominately at stages VIIA/III (Fig. 4c), are recognized by thin parallel arrays of actin that interdigitate between the Sertoli cel l and late spermatids. These structures can be seen to distribute along the ventral portion of a late spermatid and are particularly evident in a digitally reconstructed serial image obta ined using confoca l m i c ro s copy (Fig. 4c"). Intracellular br idges (ij) between adjacent spe rmat id s are recognized as punctate staining at the apex of the epithelium (Fig. 4b). 26 Figure 4. Pa i r ed f l uo re scence and phase micrographs of f i l amentous act in (F ITC- phalloidin) countersta ined with a blue nuclear stain (DAPI) in c ryosect ions of adult rat seminiferous epithelium during stages l-IV (a, a'), \M/I (b, b'), VIl-VIII (c, c'), IX (d, d'), and X-X IV (e, e') of spermatogenesis. Act in filaments in ectoplasmic special izat ions are clearly s e e n at ap ica l Sertol i -germ cel l junctions (S/Gj) and basa l Sertol i -Sertol i junct ions (S/Sj). Other major f-actin assoc iated structures are stained including intracellular br idges (ij, b) and tubulobulbar comp lexe s (tb, c). F igure 4 c " is a confoca l micrograph of a late spermatid sta ined with FITC-phalloidin showing S/Gj actin and actin f i laments as soc iated with tubulobulbar processes (tb) (a'-e' bar = 10 um; c " bar = 5 um). 27 28 Distribution pan-Cadherin & p-Catenin p-catenin was detected in both the seminiferous epithelium and in the interstitium of rat testes while Cadher ins appeared most highly expressed in the seminiferous epithelium. Epithelial staining for p-catenin (Fig. 5a ' , b\ arrow) and Pan-Cadher in (Fig. 5a " , b", arrow) appear s restricted to the Sertol i cel l -Sertol i cel l junct ional complexes . Over lay staining shows Cadher in to be codistributed with p-catenin throughout the epithel ium with nearly identical spatial and temporal distribution. In both double label experiments and s ingle label exper iments neither molecule wa s detectable at the Sertoli cell/germ cell interface during any stage of spermatogenes i s (Fig. 5a-a" ' , arrow head). Even with high concentrat ions of primary antibodies (1:10) and extended incubation periods (2-3 hours at 37 °C) junctional staining wa s not observed at these sites. At all stages the staining pattern for the cadherin and catenin molecu les appear punctate and local ized rather than l inear and continuous. S ingle labelled t issue sections counterstained with phalloidin (data not shown) revealed that neither molecule co-distributed with junctional actin found at Sertoli/Sertoli junctional sites. In addition to the intense staining observed at basal sites discrete punctate staining for the cadherin and catenin wa s observed in more apical positions (Fig. 5e ' -e " ' , arrow head). This staining pattern appeared to local ize to areas of contact between adjacent cel l s and w a s ob se r ved in a s soc i a t i on with most ear ly ge rm ce l l s inc lud ing spe rmatocy te s , round spermatids but not elongate spermatids. Specificity of the catenin probe was verified by a conven ient internal marker, a blood ve s se l in the interstitium, wh ich s howed intense staining coincident with the borders of cells likely to be smooth musc le cells (Fig. 17a, panel 4a) 29 Figure 5. Pa i red immunof luorescence and phase micrographs showing local ization of fJ1- caten in (a '-e ' ), pan -Cadher in (a " -e " ) , and over lay (a " ' - e " ' ) in adult rat semin i fe rous epithelium cryosections during stages l-IV (a-a'"), V-VI (b-b' M), VII-VIII (c-c'"), IX (d-d'"), and X -X IV (e-e'") of spermatogenes is . Staining both for p-catenin and pan-Cadher in appear highly expres sed in regions of the Sertoli-Sertoli junctional complex but appear not to co- local ize with junctional actin (not shown) nor is staining detectable at apical junctions. The staining pattern both for p-catenin and pan-Cadherin appears punctate and local ized rather than linear and continuous (scale bar = 10 um).  31 Distribution of /31-integrin The spatial distribution of the p-1 integrin subunit in unfixed cryosect ions (Fig. 6) and f ixed isolated cells is consistent with actin staining and the presence of junctional actin. A lthough preservation of morphology is poorer in these t issue sections, integrin express ion is clearly coinc ident with the presence of junctional actin as ec top la smic spec ia l i zat ion remodel ing during spermatid developed from round to elongate in shape. Integrin staining was most abundant on dorsal s ides of the spermatid (Fig. 6c) throughout development and is consistent with the presence of the greatest amounts of E S actin. T i s sue sect ions a l so show developing junctions (dj) to express the integrin (Fig. 6a, d) and is even more evident in i so lated ce l l s (Fig. 7, a " - c " ) . Interestingly, immunocytochemist ry s hows (31-integrin staining in s ome locations where phalloidin staining (a'-c') is not detected where integrin exp re s s i on appea r s including m id - (b") and late- s taged (c") spermat ids . Integrin express ion was not detected at other actin structures throughout the epithel ium including intracellular bridges and tubulobulbar complexes. Intense staining at the basa l lamina is non-spec i f ic and can be accounted for by normal IgG and secondary ant ibody a lone negative controls. Distribution of ILK Spec i f ic staining for ILK (Fig. 8) is highly expressed at Serto l i -Germ cell junct ions as recognized by a linear staining pattern running the length of spermatids heads (Fig. 8; b, c), (Fig 9; b', c'). ILK was detected both at newly forming junctions and in those which have undergone cons iderable morphologically change (Fig. 8; c, d; Fig 9; a'). A s with staining for the integrin and actin, ILK express ion was most prominent a long the dorsa l ridge of developing spermatids. This staining pattern is particularly evident in isolated cel l s (Fig. 9, c ' " ) . ILK express ion, although weaker, can also be observed in the basal compartments of the epithel ium outlining spermatogonia and spermatocytes. Although, at these sites, ILK 32 appeared to co-localize to areas of specific staining for pi-integrin, this molecule was also expressed at non-junctional areas. That is, areas where the p1-integrin subunit or actin is not detectable (Fig. 8, e) including the margins of spermatocytes and spermatogonia (Fig. 8, e). In general, ILK was more difficult to localize at basal sites as it does not appear to form a linear contour that follows the junctional complex as defined by actin staining. 33 Figure 6. Paired immunofluorescence and phase micrographs of (3-1 integrin localization in adult rat seminiferous epithelium cryosections during stages l-IV (a, a'), V-VI (b. b'), VII-VIII (c, c'), IX (d, d') and X-XIV (e, e') of spermatogenesis. Expression of the p-1 integrin complex is consistent with staining for actin both at Sertoli cell-Germ cell junctions (S/Gj, a, c) and Sertoli cell-Sertoli cell junctions (S-Sj, c). Expression of the integrin is also observed in developing junctions (dj, d) (scale bar =10 pm).  35 Figure 7. Immunofluorescence showing phase (a-c), f-actin (a'-c', green), pi-integrin (a"- c", red) and overlay (a"'-c"') in isolated germ cells during early (a), mid- (b) and late (c) stages of spermatid development. Integrin staining is expressed at junctional sites and is coincident with the development of the Sertoli cell-Germ cell junction (S/Gj) as verified by the distribution of FITC-phalloidin (scale bar = 10 um).  37 Figure 8. Paired immunofluorescence and phase micrographs of ILK localization in adult rat seminiferous epithelium cryosections during stages l-IV (a, a'), V-VI (b. b'), VII-VIII (c, c'), IX (d, d') and X-XIV (e, e') of spermatogenesis. Specific staining is seen at Sertoli cell- Germ cell junctions (S-Gj), (b, c), at Sertoli cell-Sertoli cell junctions (S-Sj), (a, c) junctions and at developing junctional sites (dj, d). Staining is also observed at non-junctional areas around spermatogonia and spermatocytes found in the most basal aspect of the epithelium (scale bar =10 pm). 38 39 Figure 9. Immunofluorescence showing phase (a-c), f-actin (a'-c\ green), ILK (a"-c", red) and overlay (a"'-c"') in isolated germ cells during early (a), mid- (b) and late (c) stages of development. ILK staining is coincident with development of the Sertoli cell- germ cell junction (S/Gj) as shown by counterstaining with FITC-phalloidin (scale bar = 10 um). U~0 41 Distribution of FAK Expres s i on of F A K was not detected at any cel l -cel l junctional s ites including Serto l i - Sertol i junct ions and Sertol i -germ cel l junct ions (Fig. 10, a, c, arrow head). To verify the ab sence of detectable F A K at basal junction sites t issue sect ions were counter-stained with phalloidin. In addition, by using components of the testis intersitium (Fig. 17, 5a) a s a positive control, a relat ive measu re of F A K expres s ion in the semin i ferous ep i thel ium cou ld be determined. The intense cytoplasmic staining observed in basal germ cells was not observed at any point in the cel l l ineage apical ly of primary spermatocytes (Fig. 11, b", c " ) . F A K exp re s s i on s e e m e d to be the highest in the cy top la sm of germ ce l l s jux tapos ing the extracel lu lar matrix, the spermatogonia, where staining intensity w a s comparab le to that of endothel ial cel l s (Fig. 11, d, arrowhead). Figure 11 (a") shows the localization of F A K to the cytoplasm of two adjoining spermatogonia. Distribution of Vinculin Immunostaining for vinculin (Fig. 12) local ized to Sertoli cel l -germ cell junct ions (S-Gj) and Sertoli cell-Sertoli cell junctions (S-Sj). Staining at apical junctions is observed around the entire a spect of elongate spermatid heads and becomes restricted to the dorsa l a spect of the spermat id head as it becomes more s ick le shaped (Fig. 12, b). V incul in staining is readily observed at developing junctions (dj) (Fig. 12, d'). V incul in a lso is expres sed at intercellular br idges (ij) (Fig. 12, b) and, just prior to spermiat ion, in a s soc ia t ion with tubu lobu lbar p roces se s (tb) (Fig. 12, c'). V incul in staining appears to be less abundant a long the dorsa l aspect of the elongate spermatids just prior to spermiation. Distribution of Paxillin Paxi l l in, has a very high express ion throughout the cytoplasm of both Sertol i cel l s and germ cel ls in almost all s tages of spermatogenes i s (Fig. 13, Fig. 14, a " -c " ) . Paxi l l in staining 42 does not concentrate at junctional sites (Fig. 14, a", arrow) but appears diffuse and cytoplasmic. Primary spermatocytes showed considerably less Paxillin staining than any other cell type (Fig. 13, a, arrow) while it appeared highly expressed in the tubulobulbar processes (tb, c') and the apical aspect of Sertoli cells just prior to spermiation (Fig. 13, d). At stage VIIA/III (Fig. 13, d), paxillin staining appears less intense in basal, columnar portions of the Sertoli cell and increased in apical expression. Apical staining of Sertoli cells is considerably less intense by stage X-XIV (Fig. 13, e). 43 Figure 10. Paired immunofluorescence and phase micrographs of FAK localization in cryosections of rat seminiferous epithelium during stages l-IV (a, a'), VA/I (b, b'), VII-VIII (c, c'), IX (d, d') and X-XIV (e, e') of spermatogenesis. Staining does not appear to localize to either Sertoli-germs cell junctions or Sertoli-Sertoli junctions but is highly diffuse throughout the cytoplasm of spermatogonia and spermatocytes. Specific staining is not observed at any point in the cell lineage past primary spermatocytes (scale bar = 10 pm).  45 Figure 11. Immunofluorescence showing phase (a-c), f-actin (a'-c', green), FAK (a"-c", red) and overlay (a'") in isolated germ cells during early (a), mid- (b) and late (c) stages of development, FAK staining is not detected at either the Sertoli cell-germ cell junction or Sertoli-Sertoli junction but is restricted to the cytoplasm of spermatogonia and early spermatocytes (scale bar = 10 pm).  4 7 Figure 12. Pa i red immunof luorescence and phase micrographs of V incul in local izat ion in cryosect ions of adult rat seminiferous epithelium during stages l-IV (a, a'), V-VI (b, b'), VII- VIII (c, c'), IX (d, d') and X-X IV (e, e') of spermatogenesis. V incul in is local ized to Sertoli c e l l - G e r m cel l junct ions (S/Gj) and Sertol i ce l l -Serto l i ce l l junct ions (S/Sj) in most spe rmatogen i c stages. V incu l in a l so appear s at other actin related structures in the epithelium including tubulobulbar processes (tb, c) and intracellular bridges (ij, b) (scale bar = 10 jam).  49 Figure 13. Paired immunofluorescence and phase micrographs of paxillin localization in cryosections of adult rat seminiferous epithelium during stages l-IV (a, a'), V-VI (b, b'), VII- VIII (c, c'), IX (d, d') and X-XIV (e, e') of spermatogenesis. Although staining is not concentrated at junctional sites, staining is abundant throughout both germ cell and Sertoli cell cytoplasm. The exception was that in primary spermatocytes which showed considerably less expression of paxillin throughout their cytoplasm (a, arrow). Tubulobulbar complexes show abundant levels of paxillin (c, arrow) (scale bar = 1 0 um). so 51 Figure 14. Immunofluorescence showing phase (a-c), f-actin (a'-c\ green), paxillin (a"-c", red) and overlay (a"'-c"') in isolated germ cells during early, mid- and late stages of development. Paxillin staining is not concentrated at either the Sertoli cell-germ cell junction (a", arrow) or Sertoli-Sertoli junction but is highly expressed throughout the cytoplasm of both germ cells (a", b") (scale bar = 10 um). sz 53 Distribution of Phosphotyrosine Immunosta in ing with a rabbit po lyc lona l ant ibody revea led pho spho ty ro s i ne - containing proteins to be widely expressed throughout the seminiferous epithelium (Fig. 15, 16) including non-junctional components of the epithelium. Express ion is concentrated at cell borders of most cells including germ cells (Fig. 15, c, arrow head) and adjacent Sertoli cel ls (Fig. 15, d, nj). Express ion, however, was most intense at S/S junctions (Fig. 15, a) and S/G junctions (Fig. 15, b) at sites where E S s are known to occur. Unl ike the other mo lecu les studied, phosphotyros ine showed staining around both the dorsa l and ventral components of the spermatid head (Fig. 16, c"). S tage VIIA/III staining at S/Sj appears distributed along only a smal l portion of the length of the junction (c', arrow) which differs from the more linear staining pattern (Fig. 15, a) that coincides with the majority of the basa l act in a s ob se r ved in s tages l-IV. Pho spho ty ro s i ne a l so appea r s concen t ra ted at tubulobulbar complexes (tb) (Fig15, c; Fig.16, c", arrow) but d isappears after sperm release (Fig. 15, d). Phosphotyrosine staining is not obvious in developing junctions (Fig. 15, c, d). Controls for Immunohistochemistry and Immunocytochemistry Staining with normal rabbit and mouse IgG, used at a protein concentration equal to that of the most concentrated primary antibody was used to control for non-specif ic staining of primary antibodies. In fixed t issue sect ions normal mouse IgG staining (Fig. 17a, 1a) typically showed some diffuse staining throughout the cytoplasm of round germ cel ls whi le normal rabbit IgG staining (Fig. 17, 2a) at concentrations equal to the IgG of the p1-integrin primary antibody showed intense staining at the basal lamina that could be accounted for, in part, by staining with the secondary rabbit antibody alone (Fig. 17, 2b). Fresh, or non- fixed, t issue sections treated with normal IgG (Fig. 17, 6a) showed strong reactivity with the basa l lamina and also reacted in a diffuse manner throughout the cytop lasm. Rabbit 54 secondary antibodies alone (Fig. 17, 7a) showed a similar reaction but was not as intense. Blank controls (Fig. 17, 3a) for tissue sections showed no non-specific staining. Controls for isolated cells (Fig. 18) show no non-specific staining with any control treatment. 55 Figure 15. Paired immunofluorescence and phase micrographs of phosphotyrosine localization in cryosections of adult rat seminiferous epithelium during stages l-IV (a, a'), V- VI b, b'), VII-VIII (c, c'), IX (d, d') and X-XIV (e, e') of spermatogenesis. Expression of phosphotyrosine residues appears at most cell-cell borders but is most highly expressed at apical (b) and basal junctions (a). Sertoli/Sertoli junctions (S/Sj) show prominent phosphotyrosine expression at stages l-IV (a) and VIIA/III (c) (scale bar = 10 jam).  57 Figure 16. Immunofluorescence showing phase (a-c), f-actin (a'-c\ green), phosphotyrosine (a"-c", red) and overlay (a"'-c"') in isolated germ cells during early (a), mid- (b) and late (c) stages of spermatogenesis. Phosphotyrosine is highly expressed at Sertoli cell-germ cell junctions (c", arrow) but is also associated with non-junctional areas of germ cells including germ cell borders (a", arrow). Staining also occurs in locations where tubulobulbar complexes (c", arrow head) are known to be present. Phosphotyrosine does not appear abundant in the germ cell cytoplasm (scale bar =10 um). Sf3 59 Figure 17. Paired phase and fluorescence controls for immunohistochemistry including staining with normal rabbit IgG (stage VII/VIII) (1a), Texas Red conjugated anti-rabbit IgG (1b) (stage V-VI), normal rabbit IgG (2a) (stage X-XIV) and Texas Red conjugated anti- rabbit IgG (2b) (Stage V-VI). A blank control was also included (3a) (stage VIIA/lll). Since many different antibodies at different protein concentrations were used for immunostudies fluorescence controls shown are at a protein concentration equal to that of the most concentrated primary antibody used. Internal positive controls, interstitial blood vessels, are included for p-catenin (4a), and FAK (5a) as a measure of antibody specificity and the relative abundance of antigens. Normal rabbit IgG (at a concentration equal to the working dilution of the p1-integrin) (6a) (stage IX) and secondary antibody alone (7a) (stage IX) controls for non-fixed tissue are also included to account for non-specific staining in experiments with p1-integrin and Integrin Linked Kinase (scale bar = 10 pm). I 1a' 61 Figure 18. Paired phase and fluorescence controls for immunocytochemistry including staining with normal mouse IgG (1a), Texas Red conjugated anti-mouse IgG (1b), normal rabbit IgG (2a) and Texas Red conjugated anti-rabbit IgG (2b). A blank control (3b) was also included (scale bar = 10 um).  63 Summary of Spatial & Temporal Distribution of Immunofluorescence During Spermatogenesis Temporal and spatial staining patterns of molecules used for immunolabelling studies are summarized in the form of a table (Figure 19). Expression of each molecule is seen as a function of the five spermatogenic stages used in immunofluoresence studies. Expression of each molecule is classified as either junctionally related or as being associated with the cytoplasm of either germ cells or Sertoli cells. Because it was not possible to definitively determine whether staining is associated with ESs or with other components of the basal junctional complex, apical junction can be inferred as being ES while basal junction cannot (even though ES staining at basal complexes shows a very particular staining pattern at the light level), p-catenin and Pan-Cadherin are indicated to be highly expressed but restricted to the basal junctional complex, pi-integrin and ILK are shown to be expressed at both apical and basal junction areas throughout all five stages with no detectable staining in the cytoplasm of either germ cells or Sertoli cells. Vinculin is indicated being concentrated at basal and apical stages throughout the stages except for stage VII/VIII spermatids where staining appears to be predominately associated with the tulbulobulbar complexes and not the ES. FAK expression is intense but restricted to spermatogonia and spermatocytes and is not detected at any junctional site. Similarly, we indicate that paxillin is not detected at junctional sites but is highly expressed throughout the cytoplasm both of germ cells and Sertoli cells except during stage IX where staining is less abundant but more apical within the columnar Sertoli cell. Of all the molecules studied phosphotyrosine seemed to show the greatest stage related changes in expression. Although phosphotyrosine was not observed in the cytoplasm of either germ cells or Sertoli cells staining was observed to be intense at apical junctions during most stages except just after spermiation. In addition, during architectural rearrangement of the blood-testes barrier (stage VIIA/III) phosphotyrosine staining is particularly prominent at basal junctions and 64 appears restricted to only a portion of the Sertoli/Sertoli junction which, at this stage, is co- distributed with the actin component of this interface. 65 Figure 19. Summary of staged immunofluorescence expression of signaling molecules in adult rat seminiferous epithelium. The relative amount of expression of each molecule at junctional and cytoplasmic locations is indicated. Expression was classified as either not detectable, relatively weak, or relatively high based on the intensity and distribution of immunofluorescence. a) Junctional Complex Concentration Stage of Spermatogenesis Basal Junction Apical Junction edict" 61 ILK FAK Pax Vine p-Tyr Cd/Ct 61 ILK FAK Pax Vine p-Tyr I-IV mm vz& ED cn mm mm CD • i • • CD CD V-VI mm m CD CD mm CD CD • • • H CD CD mm • VII-VII1 mm &i CD CD mm mm CD • • mm I I CD CD mm IX mm VM m ED CD mm CD CD mm mm CD CD mm CD X - X I V mm mcDCDmmcD CD • • mm CD CD m b) Cytoplasmic Concentration Germ Cells Sertoli Cell Cd/O 61 ILK FAK* Pax* Vine p-Tyr Cd/Ct 61 ILK FAK Pax Vine p-Tyr I-IV CD £53 mm CD CD • cn cn cn cn cn V-VI CD • v%k mm CD CD cn cn cn cn cn VII-VIII CD CD CD m mm CD CD cn cn cn cn mm i i cn IX CD CD CD m CD CD CD cn cn cn cn cn cn X - X I V CD CD CD m mm CD ED cn cn cn cn • i cn cn I—| Expression is not detectable E22 Expression is relatively weak WM Expression is relatively high "Staining is resticted to immature germ cells or spermatogonia +Staining is much less intense in spermatocytes "Puctate staining observed between adjacent round spermatids 67 B. WESTERN BLOTS & IMMUNOPRECIPITATIONS Western blotting of non-precipitated whole cell lysates (WCL) show the specificity for probes used throughout immunofluorescence studies while blotting of immunoprecipitates are used in corroboration with light level data to show possible protein complexes that could occur throughout the epithelium in vivo. Pan-Cadherin/Catenin Specificity of antibodies for pan-cadherin (130 kD) and catenin (92 kD) in WCL was shown by the presence of a single band at the appropriate molecular weight for each molecule. The presence of stable protein-protein interactions for pan-Cadherin and p- catenin (Fig. 20) are supported by immunoprecipitations with monoclonal pan-Cadherin, polyclonal pan-Cadherin and polyclonal p-catenin primary antibodies. Both monoclonal (Fig. 20i) and polyclonal Cadherin blots (Fig. 20ii) of p-catenin immunoprecipitations show a specific band at approximately 130 kD (arrow). Immunoprecipitations with the monoclonal pan-Caderin probed with antibodies against the C-terminus of the p-catenin molecule (Fig. 20iii) show a specific band at about 95 kD. Expression of both the cadherin and catenin bands appear prominent suggesting that these molecules are binding at roughly an equivalent ratio. p1-integrin & ILK Specificity of antibodies for p1-integrin (140 kD) and ILK (58 kD) in WCL was shown by the presence of a single band at the appropriate molecular weight for each molecule (Fig. 21). ILK immunoprecipitates show the presence of a band at approximately 140 kD when probed with polyclonal antibodies for pi-integrin (i). Precipitates of normal rabbit IgG used at an equivalent IgG concentration did not show a specific band at 140 kD. Both the 68 precipitating ILK antibody and the normal IgG showed an intense band at about 55 kD r e p r e s e n t i n g the heavy cha i n of the p rec ip i t a t i ng IgG m o l e c u l e . p 1-integrin immunprecipitates that were probed with polyclonal ILK antibodies, using convent ional IgG- H R P detection methods, proved to be problematic because of only a 3 kD dif ference in molecular weights between a ILK band and the heavy chain of the precipitating p 1-integrin antibody (Fig. 21 ii). Even though pi-integrin precipitates showed a more intense band at 55 kD than the normal IgG precipitates, it could not be confirmed 69 Figure 20. Whole cell lysate (WCL) and immunoprecipitates (IP) probed with monoclonal Pan-Cadherin (i),polyclonal Pan-cadherin (ii), and polyclonal p-catenin (iii) showing that each primary antibody reacts with a band at the appropriate molecular weight (arrow heads). Western blots of immunoprecipitates suggest that a stable in vivo cadherin/p- catenin complex occurs in whole cell lysates. For each primary antibody used for immunoprecipitation a non-immune IgG of equal protein concentration was used as a control. 7 0 i) BLOT: pan-Cadher in (monoclonal) P-Cad. Ms B-cat B-cat Rbt. NRS WCL IP: (mono.) IgG (N-t) (C-t) IgG ii) BLOT: pan-Cadher in (serum) W C L ip- P-Cad. NRS B ' C A T B - C A T R B T - (serum) (N-t) (C-t) IgG 1 3 5 - f 5 5 - 1 I 30 — 2*ro. 1 1** iii) BLOT: B-catenin WCL IP- B " C a t p _ C a d - M s - (C-t) (mono.) IgG 71 Figure 21. Whole cell lysates (WCL) and immunoprecipitates (IP) probed with polyclonal anti- p1-integrin (i), polyclonal ILK (ii-iv) and monoclonal ILK (v) antibodies. Since the IgG heavy chain of the precipitating antibody cross reacts at a molecular weight close to that of ILK several controls were used to verify the precipitation of ILK with pi-integrin antibodies. These include the use of protein A-peroxidase (iii), a non-reducing gel and a monoclonal ILK antibody (v). For each primary antibody used for immunoprecipitation a non-immune IgG of equal protein concentration was used as a control. 7 2 i) BLOT: B1-integrin ii) BLOT: ILK (anti-rbt IgG-HRP) iii) BLOT: ILK (protein A-peroxidase) WCL IP: PI Rbt ILK Rbt IgG IgG CPU (ILK) WCL IP: (il IgG WCL IP: p i ILK Rbt Pure IgG IgG - i 4 o H H 111 iv) BLOT: ILK (non-reducing gel) v ) B L 0 T : | L K ( m o n o c | o n a | j WCL IP: 73 that ILK w a s present in the integrin precipitates. A s a result, severa l additional controls were emp loyed to determine whether the p i - integr in was actually precipitating the ILK molecule. The first control w a s the use of protein A -pe rox ida se (Fig. 21 iii) rather than a conventional I gG-HRP secondary antibody as a means of secondary detection. A lthough reactivity occurred with control rabbit IgG the intensity of the band for the ILK precipitates was much greater. Similarly (31 -integrin preciptites showed a more intense ILK band than the IgG control. S ince the precipitating control was done at the s ame IgG concentration as the exper imental antibody this difference in band intensity is likely due to the p resence of specif ic ILK. The second control was the use of a non-reducing gel which was intended to separate the much larger heavy chain, which in a non-reduced state is about 155 kD, from ILK wh ich shou ld would still migrate to about 58 kD (Fig. 21 iv). A gel s ta ined with coomas s i e blue shows the presence of the heavy chains in both in a non-reducing (NR) and reducing gel (R) state. The presence of a band at about 58 kD in the non-reducing blot, and its absence in non-reducing normal IgG, suggests a specif ic reaction of ILK with p i - integr in precipitates. A reducing gel with normal IgG shows that the heavy chain IgG band migrates c lose to the specif ic ILK band. With the recent availability of a monoclonal ILK antibody (Fig. 21 v), raised in a different spec ies than the precipitating with pi- integr in antibody, prob lems of c ro s s reactivity with heavy cha in of the IgG mo lecu le cou ld be avoided. Despite the presence of several lower molecular weight bands, the monoc lona l ILK antibody showed a relatively intense band at the appropriate molecular weight that did not appear in the IgG precipitating control. 74 PaxillinA/inculin & Paxillin/FAK Figure 22 shows that paxillin and vinculin are able to form stable complexes in whole cell lysates. Pooled monoclonal vinculin antibodies show a strong signal at 70 kD when probed with Paxillin (Fig. 22i) . However, paxillin precipitates (Fig. 22ii) appear to contain relatively less vinculin. Also, mouse IgG precipitates, used at an equal protein concentration to that of the paxillin antibody, showed a faint band at a similar molecular weight. Paxillin was also shown to associate with polyclonal FAK precipitates (Fig. 22iii) as recognized by a band at 70 kD. When paxillin precipitates were assessed for specific FAK polyclonal FAK antibodies detected a band at the expected 120 kD eventhough FAK monoclonal antibodies did not. 75 Figure 22. Whole cell lysate (WCL) and immunoprecipitates (IP) probed with monoclonal anti-paxillin (i, iii), "pooled" monoclonal anti-vinculin (IP only) (ii) and monoclonal FAK (iv) showing that each primary antibody reacts as a single band at the appropriate molecular weight. 7b i) BLOT: Paxillin WCL IP: Pax. Vine. MsIgG j 1 68 -1 * ** I • mm iii) BLOT: Paxillin WCL IP: Pax FAK IgG ii) BLOT: Vinculin iv) BLOT: FAK WCL IP: Vine. Pax. MsIgG W C L IF Pax MsIgG 77 Phosphotyrosine Polyclonal antibodies against phosphotyrosine residues showed a broad array of bands in WCL. At lower WCL protein concentrations several prominent bands could be discerned and appeared at 55, 70, 120, 130 and 140 kD (Fig. 23i). With major phosphotyrosine bands in WCL being detected at 70 and 120 kD, the possibility that these bands were phosphophorylated forms of FAK and paxillin was considered. To investigate this paxillin and FAK immunoprecipitates, under denaturing conditions, were blotted for phosphotyrosine containing residues. Interestingly, immunoprecipitates for paxillin (Fig. 23ii) showed high level of reactivity while polyclonal FAK precipitates appear to have no detectable tyrosine phosphorylation (Fig. 23iii). When increasing amounts of paxillin precipitates was loaded into the gel increasing band intensity was observed when blotted with phosphotyrosine antibodies. pl-integrin/FAK Immunoprecipitates failed to show a direct interaction between pi-integrin and FAK (Fig. 24). Monoclonal and polyclonal integrin antibodies were blotted with monoclonal antibodies to FAK and failed to show any band at 120 kD (Fig. 24i). Similarly, polyclonal FAK precipitates did not show the presence of pi-integrin even after long periods of film exposure (Fig. 24ii). Vinculin/Actin With the close spatial and temporal distribution of actin and vinculin their direct in vivo binding was assessed. Pooled monoclonal vinculin precipitates (Fig. 25i) that were probed with a monoclonal actin antibody showed the presence of a high amount of actin indicated by the presence of a prominent band at 43 kD (arrow). Non-pooled vinculin antibodies failed to precipitate actin (data not shown). Polyclonal anti-actin antibodies 78 precipitated actin but in much smaller amounts than might be expected. Explanations for this include that the polyclonal actin antibody does not co-immunoprecipitate well (no data was available from the supplier) or that the amount of vinculin linked to actin relative to the total amount of actin precipitated is extremely small. 79 Figure 23. Whole cell lysates (WCL), FAK and paxillin immunoprecipitates (IP) probed for phosphosphotyrosine. In whole cell lysates 4 or 5 major bands appear with ones at 70 kD and 120 kD being candidates for phosphorylated paxillin and FAK respectively, (i). FAK (ii) and paxillin (iii) immunoprecipitates were probed with phosphotyrosine to assess this possibility. i ) B L O T : p T y r WCL 15 ul 3ul ip : pTyr Rbt. IgG (native) (native) - 1 4 0 — - 1 2 0 - - 7 0 • - 5 5 - ii) B L O T : Pax p T y r IP: Pax Rbt Pax IgG 3u l 10 ul 15 ul 15 ui 68 — i i i ) B L O T : F A K p T y r 120' IP: FAK FAK Ms I g G tat m — mm • ' : , ; : j | SasSs ;::M::: mm ! 81 Figure 24. Whole cell lysates (WCL) and immunoprecipitates (IP) probed with monoclonal FAK (i) and pi-integrin (ii) showing the specificity of each immunoprobe but the absence of their direct interaction in whole cell lysates. <?2_ i) BLOT: FAK ii) BLOT: B1-integrin WCL IP: pi FAK 83 Figure 25. Whole cell lysates (WCL) and immunoprecipitates (IP) probed with monoclonal vinculin (i) and monoclonal actin. Although both antibodies reacted specifically in whole cell lysates (WCL), actin immunoprecipitations probed for vinculin (i) failed to detect any specific antigen while actin was readily detected in vinculin immunoprecipitations (ii). i) BLOT: Vinculin mab pab Arb. Ms. WCL IP: Vine. Actin Actin IgG IgG 130 — t I 1 3 0 - m «4 • | 5 5 — Ip • Hi PI - • • ii) BLOT: mab Actin mab M s WCL |P; Actin Vine. | g Q 43— I • I 1 • 1 85 C. IMMUNOELECTRONMICROSCOPY Immunoelectronmicroscopy using monoclonal pan-Cadherin antibodies showed specific staining at desmosome-like junctions. These junctions which, in part, compose the junctional complex found at the base of adjacent Sertoli cells are found in close apposition to the ectoplasmic specialization component of the junctional complex. They are also recognized by direct attachment to the Sertoli cell intermediate filaments. Immunostaining, at the ultrastructural level, supports light level observations that Cadherins do not localize to the Sertoli cell/germ cell junction. Fig. 26 (ai & aii) show an elongate spermatid in cross section with no gold particles. Arrows show the location of the ectoplasmic specialization which includes a junctional actin. Fig. 26 (b-f) show lower magnification electronmicrographs of Cadherin staining indicating that Cadherin antibodies are specific to junctional sites and do not stain at other locations within the tissue section. Higher magnifications (b'-f) show localization of gold particles to Sertoli/Sertoli cell junctions and the radiating intermediate filaments. Gold particles appear to localize near to areas that are electron dense, which is likely, the submembranous plaque of the desmosome. Fig. 26g shows specific staining along an electron dense circular structure which when observed at higher magnification shows a cluster of intermediate filaments (Fig. 26g') leading to a group of gold particles. This structure is likely a desmosome that has been cut in an oblique section. In general, gold particles were observed in groups possibly as a result of discontinuous exposure of the antigen within the tissue section. Fig. 26 (g) shows a portion of a basal junctional complex with a desmosome-like junction on the left (arrow) and part of the ectoplasmic specialization on the right (arrowhead). The desmosome-like portion of the complex shows a linear array of gold particles while the actin portion shows almost no gold (Fig. 26g). Some gold appears to be present at the actin component of the junctional complex (Fig. 26g') but the presence of intermediate filaments 86 suggests that they could be associated with desmosome-like junctions in a different plane of section. Normal mouse IgG controls (Fig. 26h, h'), used at an equivalent protein concentration as the primary antibody, showed no gold particles at desmosome-like junctions. 87 Figure 26. Immunoelectronrnicroscopy localization of pan-Cadherin viewed at low and high magnification. Panel ai/aii shows a spermatid in cross section including the Sertoli cell membrane and the accompanying actin layer. The absence of gold is consistent with light level data and suggests that cadherin molecules are not concentrated at apical ESs. Panels b-g show the presence of specific p-Cadherin staining at Sertoli/Sertoli junctions. Gold particles (arrow heads) appear not to be concentrated at ectoplasmic specializations but, rather, to desmosome-like junctions which can be identified by the presence of radiating intermediate filaments (arrows). Panels b-e show the presence of clusters of gold localizing to desmosome-like junctions. Panel d' (big arrow head) shows a tight junction next to a desmosome-like junction. Panel f shows a desmosome-like junction cut in an oblique section with gold particles following the length of the plaque. Panel g shows that gold is concentrated at portions the Sertoli/Sertoli interface and is associated with intermediate filaments but absent from the ectoplasmic specialization. Normal mouse IgG controls (Panel h) show no gold particles at desmosome-like junctions.   90 DISCUSSION Overview By using the distribution of filamentous actin as a marker for sites of cellular adhesion between adjacent Sertoli cells (S/S junctions) and between Sertoli cells and germ cells (S/G junctions) I have confirmed the presence of (31-integrin and vinculin at these sites. I have further determined that Integrin Linked Kinase is associated with these sites while FAK and paxillin are not detectable. Pan-Cadherin and p-catenin were not observed at the Sertoli cell-germ cell ES but, rather, were highly expressed in areas associated with basal junctional complexes. Ultrastructural data support the absence of a cadherin molecule from the Sertoli/germ cell interface and indicates that that the cadherin complex does not appear to have an ES pattern of distribution at basal sites but rather is concentrated at desmosome-like junctions. Antibodies against phosphotyrosine containing proteins appear distributed abundantly throughout the epithelium but stain most intensely at junctional sites. Paxillin, but not FAK, appears to be present in a phosphorylated state of lysates of whole seminiferous epithelium. To corroborate immunofluorescence co- localization we have used co-immunoprecipitation to assess the possibility of direct protein- protein interactions in the epithelium. Previous experiments have shown that in the adult rat seminiferous epithelium there is a curious absence of several key adhesion molecules that have been implicated with signaling events associated with cellular adhesion. Some members of the cadherin family, such as E-cadherin, have been detected at cell-cell junctions in almost all types of epithelia (Hirano et al., 1987, Geiger et al., 1990) and have been shown to promote cellular adhesion, but appear not to be expressed in the rat seminiferous epithelium (Byers et al., 1994). Some investigators have suggested that N-cadherin may be present at adhesion 91 sites between Sertoli cells and early spermatids when grown in vitro (Newton et al., 1992, Byers et al., 1994). A recent study even reported the possibility of N-cadherin expression at sites of spermatid/Sertoli cell adhesion in whole seminiferous epithelium. The same study, however, was not able to detect the p 1-integrin at any site within the seminiferous epithelium (Wine and Chapmin, 1999), possibly because they used fixed tissue and harsh processing techniques. Further discrepancy arises when considering the presence of the a- and p-catenins, two cadherin- associated molecules, at basal S/S junctional complexes but their absence from apical S/G junctions (Byers et al., 1994). The only adhesion molecule found to be present both at apical and basal ESs is p 1-integrin (Pfeiffer et al., 1991; Palombi et al., 1992; Salanova et al., 1995 Salanova et al., 1998). The absence of a representative in vitro model of seminiferous epithelium has yielded a significant void in the literature pertaining to the control processes of cellular adhesion. As a prerequisite to understanding such events I have, for the first time, shown the spatial and temporal distribution of molecules likely to be involved in the regulation of turnover of cell-cell junctions in the seminiferous epithelium. In addition, this study is the first to indicate direct protein interactions which may exist in distinct locations in the epithelium as based on immunofluorescence localization. I have also shown the ultrastructural localization of p-Cadherin to a component of the blood-testes barrier, the desmosome-like junction. The changing morphology of actin in ectoplasmic specializations throughout spermatogenesis provides a basis for the notion that these adhesion sites are not static structures but ones that are constantly being remodeled as the germ cell changes from round, to elongate, to sickle-shaped. At basal sites, junction turnover allows the new 92 generation of spermatids to pass through the blood-testes barrier into the adluminal compartment. Fluorescent phallotoxins have been routinely used as markers for ectoplasmic specializations, which contain filamentous actin as a main structural components, at both S/G and S/S junctional sites. The staining pattern of phalloidin provides a sensitive and reliable marker for the presence and spatial distribution of these junctions throughout the spermatogenic stages. In addition to ectoplasmic specializations, other filamentous actin containing structures are readily labeled including tubulobulbar processes and intracellular bridges. P1-integrin & Integrin Linked Kinase are Present at ESs My immunofluorescence and immunoblotting results have confirmed earlier reports (Pfeiffer et al., 1991; Palombi et al., 1992; Salanova, et al., 1995, Salanova et al., 1998) that the pi-integrin subunit is expressed and concentrated at both S/S junctions and S/G junctions. My immunofluorescence has shown a high expression of the integrin at S/G junctions and moderate levels at S/G junctions that, at both sites, is co-distributed with actin filaments within ESs as indicated by phalloidin staining. ILK has a spatial and temporal distribution similar to that of p1-integrin. Maximum immunofluorescence staining of both molecules is observed adjacent to spermatids deep within the Sertoli cell crypts. Just prior to sperm release, staining occurs along the dorsal side of the spermatid head in the same location as actin staining in ESs. Staining for p i and ILK also appear co-distributed with newly forming ESs, which develop in close association with early spermatids during stage IX of spermatogenesis. The close spatial and temporal association of p1-integrin, ILK and ES actin filaments at ES sites indicates that the p1-integrin/ILK complex may participate in junction turnover during spermiation and 93 during turnover of the basal Sertoli cell junctional complex. Co-immunoprecipitation of these two molecules provides biochemical support for a direct B1 -integrin/ILK interaction in vivo. This interaction is likely to occur at junctional sites because the only instance where these two molecules are expressed together is at ES sites as defined by phalloidin staining. Because the expressions of the pi-integrin and ILK are not restricted to ESs only during events such as spermiation and turnover of the blood-testes barrier, it is possible that this complex is involved with continuous structural changes of the junction particularly at apical sites where the adhesion complex must conform to changes in shape of the associated spermatid head. As pi-integrin and ILK appear to be co-expressed throughout the spermatogenic stages it possible that the pi-integrin/ILK complex is a constitutive one rather than an induced one. The only location in the epithelium where pi-integrin and ILK is not co-distributed is in the early germ cells. Here, some ILK staining occurs at the periphery of primary spermatocytes. This staining could be attributed to ILK associating with other known substrates such as the p2- and p3- integrin cytoplasmic tails, two molecules which ILK has shown to have an in vivo association with (Hannigan et al., 1996) but have not been identified as being present in the adult rat seminiferous epithelium. Despite the fact that the tubule wall was removed during tissue collection and epithelium cells were washed prior to lysis the possibility remains that the detected pi-integrin/ILK interaction is occurring at sites between the base of epithelial cells and remaining portions of the limiting membrane even after dissection. Nevertheless, it is this investigators opinion that with the removal of the tubule wall, tissue sections controls accounting for intense staining at the basement membrane, absence of detectable ILK staining at the limiting membrane, and the intense spatial co-distribution of these two molecules at ESs throughout the spermatogenic stages that the detected pi-integrin/ILK interactions is occurring at ES sites. Additionally, during immunofluorescence studies of isolated cells integrin and ILK 94 expression was not detected at basal portions of Sertoli cells. If the p1-integrin is not expressed at these sites, then the question arises as to what is the adhesion molecule which is maintaining adherence between the base of the Sertoli cell and the limiting membrane. The known presence of hemidesmosome-like junctions at the base of Sertoli cells indicates that desmosome related molecules such as a6p4-integrin could be present at these sites, although this has not been investigated. Even thought previous studies have not detected desmosome related proteins, such as Plectin (Guttman et al., 1999) at these sites, intermediate filaments show contacts to these sites both at the light level and the ultrastuctural level. Focal Adhesion Kinase is not associated with ESs FAK is not detectable at junctional sites in the adult rat seminiferous epithelium as verified by the absence of staining at sites labelled with fluorescent phalloidin. FAK staining is restricted to basal germ cells and appears diffusely, but intensely, expressed throughout the cytoplasm of spermatogonia and spermatocytes. Expression of FAK in spermatogonia cytoplasm is more intense than that in spermatocytes and is comparable to FAK staining in endothelial cells, a convenient positive control found in the interstitial blood vessels. To further corroborate that FAK does not associate with the pi-integrin in vivo co- immunoprecipitation failed to show a direct interaction in whole cell lysates of these proteins. It is interesting that a direct in vivo association, in tissue or primary epithelia, between pi-integrin and FAK has never been shown despite their strong interaction in cultured cells (Schalleretal., 1995; Guan, 1997). By reason of the fact that FAK has been shown to be present at cell adhesion complexes in many other systems, the absence of this molecule at ectoplasmic 95 specia l izat ions (an actin related intracellular adhesion junction) is a significant finding. The spatial distribution of F A K staining is intriguing when considering that F A K is known to be one of the major mediators of integrin activated signaling events and is a lso a protein that has been shown to co-distribute with integrins, paxillin and vincul in at s i tes of cel lu lar adhes ion in cultured cells. A l so, the apparent lack of phosphorylated FAK, as suggested by the lack of a FAK/phosphoty ro s ine interact ion and co-d i s t r ibut ion of F A K and phosphotyros ine immunofluorescence, is intriguing. A poss ible explanation for the ab sence of F A K at adhes ion sites in the testes is that it may not be required for junction turnover. For instance, F A K has been shown to have many functions which are not all directly related to cell adhes ion and has been implicated for roles in cell mobility, migration and shape change (ll ic et al., 1997, Zachary, 1997). S tud ies have shown F A K expre s s i on to be more prominent in embryon ic epithel ia and developing sy s tems than adult t i s sue (Tani et al., 1996). In fact, s ome studies have shown that in deve lop ing t i s sues F A K is highly expres sed , but in the s a m e adult t i ssue express ion is faint, if not undetectable (Tani et al.,1996). The seminiferous epithelium is developmentally one of the most dynamic t i ssues known and unde rgoe s c omp lex morpho log ica l c hange s a s d ip lo id s pe rma togon i a proliferate, divide meiotically and differentiate into spermatozoa. Our data indicates strong F A K express ion in spermatogonia, the s tem cel ls of the seminiferous epithelia. Perhaps , F A K functions in the initial developmental changes of the spermatogenic cell l ineage or is assoc iated with s ignals from the extracellular matrix. If this is true, then perhaps F A K also shows greater levels of phosphorylation in embryonic t i ssue than in adult ones. S o m e authors have sugges ted that expres s ion of F A K in vitro is an adaptat ion to culture condit ions providing an explanation for the differences in staining patterns often observed between in vivo and in vitro conditions (Tani et al., 1996). 96 Phosphotyrosine Proteins are Concentrated at ESs The absence of FAK at ectoplasmic specializations suggests that another major tyrosine kinase may be downstream of the pi-integrin. The observation that a probe for phosphotyrosine intensely labels both S/S junctions and S/G junctions is consistent with this possibility. Significantly, phosphotyrosine staining dramatically highlights basal junctions coincident with movement of spermatocytes through the blood-testes barrier, an event known to occur during stage VIIA/III of spermatogenesis in the rat. This observation indicates that a major phosphotyrosine containing protein may be involved with the rearrangement of the S/S junctional complex. In addition, since phosphotyrosine was prominent at S/G junctions just prior to spermiation, the same or a similar phosphotyrosine containing protein could be functionally important for breakdown of the junction. When whole cell lysates were probed with polyclonal phosphotyrosine antibodies several prominent bands appeared at about 55, 70, 120, 130 and 140 kD. Although the presence of a band at about 120 kD seems to suggest the presence of the tyrosine kinase, FAK, this does not appear to be the case. FAK immunoprecipitates appear not to contain an detectable phosphotyrosine. Differently, is the level of phosphorylated paxillin which appears to be relatively high in cell lysates With the detection of a major phosphotyrosine band at about 70 kD and the abundance of paxillin detected at the light level, it is possible that paxillin is one of the major phosphotyrosine containing proteins in the adult rat seminiferous epithelium. It would be interesting to address which proteins could possibly account, completely or in part, for the bands observed at 55, 120, 130 and 140 kD. For instance, with the absence of junctional FAK it is possible that Src, a 60 kD protein, could be another major phosphotyrosine containing peptide with important roles at ESs. Additionally, with the apparent high expression of phosphotyrosine protein(s) at the ESs it would be interesting to evaluate whether or not tyrosine phosphorylation is a necessary 97 element for their formation. Primary seminiferous epithelia could be potentially incubated with a variety of growth factors or chemical inhibitors to evaluate this. Subsequent to incubation with phosphotyrosine junctions could be evaluated for the relative amounts of adhesion elements including actin and vinculin. Paxillin is Widely Distributed in the Seminiferous Epithelium Paxillin, a vinculin-binding protein, has a nearly ubiquitous spatial distribution throughout the seminiferous epithelium cytoplasm but is not concentrated at ectoplasmic specializations. The most outstanding observation regarding paxillin expression throughout spermatogenesis is its increase in abundance during stages VII/VIII in round spermatids and in the columnar portion of Sertoli cells. Curiously, paxillin does not appear to be present in the spermatocytes. The recruitment of paxillin to the tubulobulbar complexes, structures thought to have an anchoring function between the Sertoli and germ cell, just prior to spermiation suggests that Paxillin could be functionally involved with the turnover of the actin network and therefore a possible candidate for involvement with sperm release. The co-immunoprecipitation of Paxillin and FAK, but the absence of immunostaining for FAK in apical regions of the epithelia, suggests that a direct FAK/Paxillin interaction occurs in the basal portions of the epithelium. The localization of vinculin, a known paxillin binding protein, to both S/S junctions and S/G junctions occurs throughout the spermatogenic stages. Vinculin also occurs at other actin related structures such as the tubulobular complexes. These structures are the only site during spermatogenesis, where the codistribution of vinculin and paxillin is detectable by immunofluorescence, thus suggesting a location in the epithelium that accounts for their direct interaction. However, even though paxillin is not detectable at 98 junctional sites the abundance of this molecule throughout the epithelium indicates that a direct interactions may be occurring elsewhere in the epithelium. Since FAK and Paxillin are not concentrated at ESs, it is likely that a FAK/Paxillin protein complex is not directly associated with control of junctional actin architecture but may be associated with other developmental roles elsewhere in the epithelium. Vinculin, an actin linking protein, showed a close spatial and temporal distribution with actin throughout spermatogenesis. Expression of vinculin appeared to be particularly high at ectoplasmic specializations. Co- immunoprecipitations verified that vinculin binds to actin although the relative amount of vinculin detected in actin precipitates was much less than the amount of actin detected in vinculin precipitates. This could be explained by the fact that most vinculin precipitates contain actin while a much smaller portion of actin precipitates contain vinculin. Furthermore, total precipitated actin includes both globular and filamentous types while fluorescent phalloidin staining detects only the later form. Cadherin vs. Integrins at Junctional sites The apparent absence of a cadherin/catenin complex at apical ESs and its localization to desmosome-like junctions at S/S cell junctional complexes suggests that an integrin could be a key molecule involved in the control of ectoplasmic specialization turnover. Nevertheless, with the co-distribution of the Cadherin and B-catenin immunofluorescence, their co-immunoprecipitation and the ultrastructural localization of the pan-Cadherin to desmosome-like junctions it is likely that such a complex plays an turnover of the Sertoli/Sertoli junctional complex. The fact that the pan-Cadherin/catenin complex does not appear to be associated with ESs between adjacent Sertoli cells nor between Sertoli cells and germ cells indicates that a Cadherin mediated signaling pathway may not be a component of ES control. The absence of a Cadherin or catenin molecule between the 99 Sertoli cell and the germ cell at ESs is unusual, but the known association of catenins with desmosomes (Cowin et al., 1986) provides justification for their presence at desmosome- like junctions in the seminiferous epithelium. It is possible, however, that both integrin (directly) and cadherin (indirectly) related signaling pathways are required for control of the Sertoli cell/Sertoli ectoplasmic specialization. This could be explained by the cross-talk in signaling pathways that may occur between integrin and Cadherin associated molecules. With the presence of both molecules at the Sertoli cell junctional complex it seems almost certain that the coordinated involvement of both molecules are involved in its architectural change. The presence of pi-integrin, Integrin Linked Kinase and vinculin at both apical and basal ES sites suggests that integrin mediated signaling events have a prominent role in the architectural rearrangement of ES-related intracellular adhesion junctions. Possible Ligands for /31-integrin With the presence of a pi-integrin mediated signaling pathway at major sites of intercellular adhesion in the testes, it is interesting to contemplate what might initiate or activate such a sequence of signaling events in either an "outside-in" or an "inside-out" manner. This question seems particularly important since these sites are intimately related with sperm release and turnover of the basal junctional complex. Although integrins have been shown to be associated with a variety of ligands, one ligand stands out as being a good candidate in the seminiferous epithelium. Previous investigations have explored the relationship between an ADAM protein (fertilin) on the surface of the mammalian sperm acrosome, and the a6pi receptor on the surface of mouse eggs (Chen et al., 1999). Fertilin has a strong affinity for the a6pi receptor, and when blocked, disrupts sperm to egg adhesion suggesting that oc6B1 -fertilin binding is 100 important for adhesion and initiation signaling events (Almeida et al., 1995). Since it is known that the a6pi dimer is found at ectoplasmic specializations, it is possible that a member of the ADAM family is responsible for activation and or clustering of the integrins during ES structural modification. Activation of integrin-mediated signaling pathways may be initiated by submembranous molecules capable of changes in phosphorylation in an inside-out manner. Obvious candidates include ILK, which has been shown to affect the structural integrity of cell-cell junctions when up-regulated (Hannigan et al., 1996, Dedhar and Hannigan, 1996). It is possible that a submembranous event or internal signal could prompt ILK to phosphorylate the cytoplasmic tail of the (31 -integrin and thereby cause integrin activation. Since it is likely that an alternative tyrosine kinase, to FAK, exists in close association with the integrin cytoplasmic domain, it would be interesting to evaluate what the effects of inhibiting or up-regulation of such a kinase would be on sites of adhesion in the seminiferous epithelium. Some authors have speculated that elements that would contribute to inside-out cascades leading to junctional turnover may be hormones such as follicle-stimulating hormone. (Salanova et al., 1998). Other studies have linked testosterone with the fundamental spermatogenic events such as spermatid detachment (O'Donnell et al., 1996) and progression of round spermatids to elongate spermatids (McLachlan etal., 1994). Hypothetical Model for Junctional Adhesion and Control Molecules Based on the distribution of pi-integrin signaling molecules in the seminiferous epithelium and the hypothesis that ESs are structurally maintained and controlled by an integrin mediated pathway a hypothetical model of construction can be proposed. With the 101 consideration that the S/S and S/G interfaces are different in design I suggest independent models to represent adhesion molecules and junctional components likely to be involved in signaling events. Paired transmission electronmicographs with each model depict the ultrastructure of both S/G junctions (Fig. 27a) and S/S junctions (Fig. 27b). At both sites of adhesion ectoplasmic specializations consists of an endoplasmic reticulum (ER), a hexagonal array of actin and a submembranous space. The junctional complex found between adjacent Sertoli cells also contain desmosome-like junctions and tight junctions in close association with the ES actin. In both the S/G and S/S junctional model I have included the transmembrane a6pi dimer with integrin cytoplasmic tail extending into the Sertoli cytoplasm. I have also included the presence of a cadherin/p-catenin complex molecule at S/S junctional complexes but not at S/G junctional sites or in association with the actin component of the basal ectoplasmic specialization. I have shown the cytoplasmic tail of the integrin subunit to interact, directly or indirectly, with a putative tyrosine kinase which, 102 Figure 27. Proposed model for the presence of integrin and Cadherin related molecules at both apical (a) and basal (b) junctional complexes. The presence of the pi-integrin, but absence of a detectable Cadherin associated molecules, suggests that a potential downstream signaling cascade associated with changes in junctional actin could be integrin related. Upon interaction with spermatid surface proteins integrin activation or clustering could occur and a subsequent phosphorylation of its cytoplasmic domain by downstream kinases such as ILK. Alternatively, an initial signal could originate from a submembranous member of the cascade. The absence of detectable FAK, but prominent distribution of phosphotyrosine residues, suggests that other tyrosine kinases could be present and function as mediators to downstream molecules such as vinculin, which could directly interact with the actin cytoskeleton. 103 104 based on my data, is probably not FAK. Since vinculin typically does not directly interact with the pi-integrin, but is known to link directly to the actin cytoskeleton, I have diagrammed it downstream of a possible tyrosine kinase and make the speculation that it signals to a member of the small GTPases, possibly rho or rac. Molecules such as these could cause direct rearrangement of the actin cytoskeleton. At both the S/S junctions and S/G junctions, I have removed FAK from the ectoplasmic specialization to indicate that it does not concentrate at junctional sites but, rather, is found to be highly expressed throughout spermatogonia cytoplasm. Paxillin, but not FAK, appears with phosphotyrosine residues. It is also shown to be present in the cytoplasm of both the Sertoli cell and the germ cell but with question at S/S junctions. At S/G junctions paxillin and vinculin can be seen in close association with the actin network surrounding the tubulobulbar processes and are diagrammed to have a direct interaction. An unidentified transmembrane molecule is indicated opposite to the integrin dimer as a possible ligand of the p i - integrin. With the known association of the a6p4 integrin with desmosomes I have included the integrin dimer as a molecule that may be localized to the hemidesmosome-like junctions found at the base of the Sertoli cells. This molecule along with other unidentified transmembrane molecules may function in maintaining adhesion between the seminiferous epithelium and the limiting membrane. 105 CONCLUSION The presence and spatial co-distribution of p 1-integrin, ILK, vinculin, phosphotyrosine and actin-related cell-cell junctions in the rat seminiferous epithelium suggests the importance of integrin mediated molecules during the structural modification of basal and apical intracellular adhesion junctions. The absence of FAK, and concentration of paxillin, from junctional sites is intriguing and suggests that these molecules may not be directly associated with the control of junction turnover but could assume a different function during spermatogenesis. My data has provided the first evidence showing that ILK is present at an in vivo actin-related site of adhesion and the absence of a cadherin molecule from an in vivo epithelial cell/cell interface. The reported absence of E-cadherin and N-cadherin, but the positive reactivity of pan-Cadherin in the seminiferous epithelia indicates that another Cadherin molecule remains to be described at the basal junctional complex in the seminiferous epithelium. Based on the molecules detected at ectoplasmic specializations it appears that these junctions have properties both of focal adhesions and epithelial cell-cell junctions. The question of whether junction formation and control is integrin related, cadherin-related or both, during spermatogenesis is a complex issue and needs to be further addressed. 106 FUTURE DIRECTIONS The seminiferous epithelium provides a good system to test fundamental questions about signaling events associated with cellular adhesion and the actin cytoskeleton. The present study has addressed many issues but also raises many others requiring further study. The following are some of the possibilities that seem worthy of trial: Determining the Ligand of the pi-integrin in the seminiferous epithelium An important question to answer about a potential pi-integrin signaling pathway is to identify the ligand, or molecule with the potential to activate the integrin pathway. A good candidate is a member of the ADAM family. However, the distribution and expression of such a molecule has not been investigated in the seminiferous epithelium. If identified as being present in the seminiferous epithelium and co-distributed with the p1-integrin, blocking this molecule could provide further answers as to the function of the integrins and closely associated molecules. Cytoskeletal clustering of integrins and the small GTP-ases Rho family proteins functions as molecular switches that have the ability to cycle between a GDP-bound state and an active GTP-bound state. These molecules are also known to be involved in integrin clustering (Schoenwaelder and Burridge, 1999). It would be interesting to evaluate whether there is differential GTP-ase expression as integrin expression changes during development of the ectoplasmic specialization. Perhaps there is repetitive integrin clustering and cycling of Rho family members including Cdc42 and Rac. 107 Is PINCH present at Ectoplasmic Specializations? The N-terminal domain of ILK has been shown to interact with the recently described adapter protein, PINCH (Tu et al., 1999). Further studies have shown that PINCH may be necessary for localization of the PINCH/ILK complex to focal adhesions (Li et al., 1999, J. Cell Science, in press). Interestingly, this study has also provided evidence that ILK does not localize to E-cadherin mediated junctions. Since ectoplasmic specializations share features of both classical adherens junctions and focal adhesions, the question of whether PINCH is expressed at these sites and whether it directly interacts with ILK, and not pi -integrin, is an interesting one. FAK and pTyr: Levels of Expression in Embryonic vs. Adult Tissue In some tissues FAK has been shown to be expressed at higher levels in embryonic tissues than adult ones (Tani et al., 1996). It would be interesting to determine whether this holds true in the seminiferous epithelium. Although FAK does not appear to be directly associated with any type of adhesion structure in the adult seminiferous epithelium, it may in early developing tissues. The question of how the expression of FAK changes in development from embryonic to adult tissue needs to be addressed. It has also been reported that levels of phosphotyrosine are frequently greater in embryonic tissue than in adult (Maher and Pasquale, 1988). Since phosphotyrosine levels are already substantial in the adult seminiferous epithelium it would be interesting to evaluate them in embryonic and pre-pubescent tissue. 108 Chemical Inhibitors Phosphorylation, and in particular phosphorylation of tyrosine residues, clearly has important functions in many tissues. With the availability of several protein-tyrosine kinase inhibitors, such as herbimycin, specific events such as spermiation could be evaluated for their dependence upon tyrosine phosphorylation events. Blocking Peptides With the shown presence, distribution and possible interaction of several key signaling and adhesion molecules in the seminiferous epithelium associated functions of each molecule need to be explored. Despite the fact that a good representative in vitro model of the seminiferous epithelium does not exist, several methods could be used to ascertain what the functions of individual signaling molecules are. One approach could include the use of blocking peptides such as RGD-containing peptides. Primary epithelia maintained in culture for several days could be incubated with blocking peptides and then assessed for structural changes or changes in spermatogenic processes. Since a cycle in the rat takes about 12.9 days (about 53 days for total duration of spermatogenesis) it may be possible to maintain primary epithelia in culture long enough to allow a spermatogonia to go through many of the stages of spermatogenesis. Knock Outs Animals that are deficient for a certain molecule can be used to provide evidence for an in vivo function. 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Curr. Opin. Cell Biol. (7): 681-689. 113 Yamada KM and B Geiger (1997): Molecular interactions in cell adhesion complexes. Current Opinion Cell Biology (9): 76-85. Zachary I (1997): Molecules in Focus: Focal Adhesion Kinase. Int. J. Biochem. Cell Biol. (29): 939-934. 114 APPENDIX A Diagrammatic representation of how seminiferous epithelium is collected for Western Blotting and immunoprecipitations studies (adapted from Beach and Vogl, 1999). When the tubule wall is separated from epithelium components such as the basal lamina, basement membrane and surrounding myoid cells are removed. This dissection was done to decrease the possibility that tubule wall components could be accounting for false positive results when assessing direct protein interactions. U5 Tubulue Wall Components / Cells Lysed and Used for Western Blotting and Immunoprecipitation 116 APPENDIX B Table 1. Descriptive table of primary antibodies Antiserum M.W. Mono./Poly. Dilution (IF/IB) Stock (mg/ml) Source pi -integrin 140 polyclonal 1:200/1:1000 15 Chemicon (AB1952) pi-integrin 140 polyclonal 1:1000/1:10,000 15 G.Tarone ILK 58 polyclonal 1:100/1:1000 0.33 S. Dedhar ILK 58 monoclonal 1:500 (IB) 1.0 C. Wu, U.S. pan-Cad 135 monoclonal 1:400/1:1000 10 Sigma (C1821) pan-Cad 135 polyclonal 1:1000/1:5000 serum Sigma (C3678) P-catenin 92 monoclonal 1:400/1:1000 10 Sigma (C7207) p-catenin 92 polyclonal 1:1000/1:5000 serum Sigma (C2206) Actin 43 monoclonal 1:1000 (IB) 1.0 Chemicon (MAB1501) Actin 43 polyclonal 1:1000 (IB) 0.2 Santa Cruz (sc-1615) Vinculin 116 monoclonal 1:400/1:2000 8.4 Sigma (V9131) Paxillin Labor. 68 monoclonal 1:400/1:10,000 0.25 Transduction (P13520) FAK 120 monoclonal 1:50/1:500 0.25 Transduction (F15020) FAK 120 polyclonal 1:50/1:500 0.2 Santa Cruz (sc-557-G) pTyr polyclonal 1:100/1:1000 0.2 Transduction (P11230) 117 APPENDIX C (continued from page 51-52) Complete panel of controls used for immunohistochemistry. Paired fluorescence (a, b) and phase micrographs (a', b') include normal IgG (a) and secondary antibody alone (b). Normal IgG concentrations were used an equivalent IgG concentration to that of the working dilution of the primary antibody while secondary antibodies were used at a 1:100 dilution. Primaryantibody incubations were either at 1-2 hours for 37 °C or for overnight at 4 °C. Figure label Control for primary antibody 8 monoclonal pan-Cadherin 9 polyclonal p-catenin 10 polyclonal pTyr 11 monoclonal FAK 12 monoclonal Vinculin 13 monoclonal Paxillin 14 polyclonal ILK  1180L APPENDIX D Structural features of integrin receptors (adpated from Hynes, 1992) and Integrin Linked Kinase (adapted from Dedhar, 1999). Integrin space filling model (a) and schematic (b) show the locations of cysteine-rich repeats (crosshatched) and metal-binding sites in the a subunit (M++). Shaded areas represent the ligand binding domain. Dilsulfide loopes are seen in the schematic representation. Integrin Linked Kinase contain 4 ankyrin repeats at the N-terminus and the [31 cytoplasmic domain binding region at the carboxy terminus. The middle domains contain the PH-like domain and the kinase catalytic domain. I l l B1-Integrin Integrin Linked Kinase Kinase Catalytic Domain .452 4 Ankyrin Repeats 164 179 4 215 PH-Iike Domain 309 B l Cytoplasmic 452 Domain Binding Region


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