Project Grant R03HD110599

SOMATIC CONTROL OF GERMLINE DIFFERENTIATION IN SPERMATOGENESIS. - PROJECT SUMMARY THE BROAD, LONG-TERM OBJECTIVES OF THIS APPLICATION ARE TO CHARACTERIZE SOMA-GERMLINE INTERACTIONS DURING ADULT SPERMATOGENESIS. THE PROPOSAL WILL UTILIZE BIOCHEMISTRY, SITE-DIRECTED MUTAGENESIS IN CULTURED CELLS AND IMMUNOFLUORESCENCE, GENETICS, RNA INTERFERENCE, TARGETED PROTEIN DEGRADATION AND RESCUE ASSAYS IN THE ADULT DROSOPHILA TESTIS TO DETERMINE HOW A SECRETED, CONSERVED, IMMUNOGLOBULIN (IG) DOMAIN PROTEIN MAINTAINS THE BLOOD-TESTIS BARRIER (BTB) IN SOMATIC SUPPORT CELLS OF ADULT TESTES. WE WILL CAPITALIZE UPON THE POWERFUL GENETICS AVAILABLE IN DROSOPHILA, AS WELL AS THE ABILITY TO UNEQUIVOCALLY IDENTIFY THE NICHE, GERMLINE STEM CELLS (GSCS), SPERMATOGONIA AND SOMATIC SUPPORT CELLS IN THE DROSOPHILA TESTIS. THIS PROPOSAL IS SUPPORTED BY UNPUBLISHED RESULTS DEMONSTRATING THAT (1) THE SECRETED PROTEIN IS EXPRESSED IN SOMATIC CELLS OF THE TESTIS AND IS REQUIRED FOR SPERMATOGONIAL DIFFERENTIATION AND FOR ROBUST EXPRESSION OF A BTB DOMAIN PROTEIN; (2) THE ONLY KNOWN RECEPTOR FOR THE SECRETED PROTEIN IS A NEURONAL ADHESION PROTEIN THAT IS NOT EXPRESSED OR REQUIRED IN THE ADULT TESTIS, INDICATING THAT ANOTHER RECEPTOR IS INVOLVED; (3) ANOTHER RECEPTOR WITH HIGH HOMOLOGY TO KNOWN ONE IS EXPRESSED IN SOMATIC MEMBRANES IN THE ADULT TESTIS AND ITS DEPLETION LEADS TO PHENOTYPES SIMILAR TO LOSS OF THE SECRETED PROTEIN; (4) THIS OTHER RECEPTOR IS REQUIRED FOR MAINTENANCE OF THE BLOOD-BRAIN-BARRIER DURING DEVELOPMENT, SUGGESTING A CONSERVED BARRIER FUNCTION. AIM 1 CENTERS ON USING IN VITRO ASSAYS (CELL-CELL AGGREGATION, AFFINITY PURIFICATION FOLLOWED BY MASS SPECTROMETRY, STRUCTURE-FUNCTION ANALYSIS) TO DETERMINE HOW THE SECRETED LIGAND AND RECEPTOR INTERACT IN CULTURED CELLS AND ON EMPLOYING IN VIVO ASSAYS (SPLIT-GFP RECONSTITUTION AND DEGRADFP- DEPENDENT PROTEIN DEGRADATION WITH GENETIC "ADD BACK") TO TEST WHETHER THESE INTERACTIONS OCCUR IN THE ADULT TESTIS. THE AIM 2 IS FOCUSED ON DETERMINING WHETHER THE PERMEABILITY BARRIER FUNCTION OF THE BTB IS COMPROMISED IN TESTES LACKING THE LIGAND OR RECEPTOR. THESE EXPERIMENTS ARE DESIGNED TO REVEAL MECHANISTIC INSIGHTS INTO HOW THE BTB DOMAIN IS MAINTAINED IN ADULTS. THE STUDIES IN THIS PROPOSAL WILL INCREASE THE KNOWLEDGE BASE ABOUT SIGNALS THAT MAINTAIN SPERMATOGENESIS DURING ADULT STAGES AND WILL FOSTER NEW AVENUES OF RESEARCH INTO MECHANISMS AND TREATMENTS FOR AGE-RELATED MALE INFERTILITY.