| Summary: | 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. β1-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 β1 -integrin
antibodies showed a specific band at 140 kD. Β1-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 coimmunoprecipitate
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.
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