of Work: The """"""""Mechanisms of Germ Cell Death"""""""" project aims to identify the mechanisms by which germ cells are killed in the testis. We earlier showed that the architecture of the normal seminiferous tubule was required for this apoptosis, which implicates a role for cell-cell adhesions and their related signals from the Sertoli cells. This had not been demonstrated previously. Working on glycol ether-induced apoptosis in pachytene spermatocytes, we showed that pharmaceuticals that inhibited calcium fluxes across membranes inhibited the apoptosis .Other labs have shown similar protection in thymus. However, recently, using fluorescent confocal laser microscopy, we demonstrated that such fluxes did not, in fact, occur, and we postulated alternative mechanisms, e.g., changes in membrane-bound kinases. Current studies have documented the recruitment of some PKC isoforms to around the dying cells, while other strategies are identifying the substrates that are phosphorylated during cell death, and which can be blocked when the lesion is also blocked. We plan to work backward to identify the kinases by first identifying the substrates. In the """"""""Inhibited Spermiation"""""""" project, we have found N-cadherin in Sertoli cells, and desmoglein on late spermatids. Both these cadherins are in a distribution that suggests that they act to mediate spermatid adhesion. Importantly, we have found a suite of proteins inside the Sertoli cells that are known, in other cells, to regulate cadherin adhesiveness by regulating protein phosphorylation. Using cultured everted tubule fragments, compounds that block protein phosphorylation also increase sperm release, showing the involvement of phosphorylation in this process. We are also using antibodies in a new in vitro spermatid-Sertoli binding culture to block spermatid adhesion, and thus to identify the adhesion molecules. Ongoing immunoprecipitation studies are being used to identify protein-protein interactions at these adhesions, which will in turn lead to new understandings about how these adhesions are controlled and interrupted by toxicants. Current work is focused on confirmation of several of the peripheral proteins involved in the multi-protein complexes involved in the process of spermiation, by co-immunoprecipitation and Western blotting. Additional ongoing experiments involve 2-D PAGE separation of proteins from seminiferous tubule lysates, before, during, and after spermiation for identification of serine and threonine phosphorylated proteins actively involved in sperm release. Serine and threonine phosphorylated proteins are identified by Western blotting, excised from corresponding 2-D PAGE gels, and analyzed by MALDI-TOF, MS/MS mass spectrometry. Identified proteins are localized in situ by immunohistochemistry to confirm their presence and involvement in the protein complex. The results of these experiments are to be published in the Journal of Andrology and acceptance of the manuscript will conclude this project.
