SPERM MOTILITY AND ACQUISITION OF THE CAPACITY TO FERTILIZE ARE THE MOST FUNDAMENTAL COMPONENTS OF MALE REPRODUCTION NECESSARY FOR SPECIES SURVIVAL ON EARTH OR IN SPACE. PREVIOUS SPACE SHUTTLE FLIGHT EXPERIMENTS THAT WE PERFORMED DEMONSTRATED THAT THE SIGNAL TRANSDUCTION (I.E. PROTEIN PHOSPHORYLATION) REQUIRED FOR ACTIVATION OF SEA URCHIN SPERM OCCUR MORE RAPIDLY IN MICROGRAVITY THAN AT 1G. IN CONTRAST, WE FOUND THAT SIGNALING IN SPERM TRIGGERED BY EGG CHEMOTACTIC FACTORS IS DELAYED IN MICROGRAVITY COMPARED TO 1G. NO FLIGHT-CAPABLE MICROSCOPE WAS AVAILABLE DURING THOSE EXPERIMENTS TO DETERMINE WHETHER THE SIGNALING CHANGES WERE IN FACT COUPLED TO DIFFERENCES IN MOTILITY UNDER THE MICROGRAVITY VERSUS 1G CONDITIONS. OUR HYPOTHESIS IS 1) THAT SPERM MOTILITY (SEA URCHIN AND MAMMALIAN) IS ACTIVATED MORE RAPIDLY IN MICROGRAVITY THAN ON EARTH, AND 2) THAT THE RESPONSE OF SEA URCHIN AND MAMMALIAN SPERM (MOTILITY HYPERACTIVATION, AND MORPHOLOGICAL CHANGES) TO FERTILIZATION ACTIVATING CONDITIONS, AS WELL AS THE UNDERLYING METABOLIC AND REGULATORY MECHANISMS, ARE DELAYED OR BLOCKED IN MICROGRAVITY. IN THIS REGARD, IF THE CORRESPONDING ACTIVATION AND CHANGES IN SPERM MOTILITY DO NOT OCCUR, THEN FERTILIZATION CANNOT OCCUR AND REPRODUCTION WILL FAIL. THUS, IT IS ESSENTIAL TO ASSESS WHETHER THE CRITICAL COMPONENT OF SPERM FUNCTION, MOTILITY, AND THE UNDERLYING SIGNALLING AND METABOLIC SUPPORT ARE ALTERED IN SPACE FLIGHT CONDITIONS. WE PROPOSE TO USE EXISTING FLIGHT HARDWARE TO ACTIVATE SEA URCHIN AND RODENT SPERM IN MICROGRAVITY, AND QUANTIFY MULTIPLE PARAMETERS OF MOTILITY AND MORPHOLOGY, COMBINED WITH DETAILED METABOLOMIC PATHWAY ANALYSIS. PREVIOUSLY UTILIZED METHODS FOR PREPARING INACTIVE SEA URCHIN SPERM FOR SUBSEQUENT ACTIVATION IN SPACE FLIGHT WILL BE ADAPTED FOR MOUSE AND RAT SPERM. WE WILL ALSO TEST WHETHER ACTIVATING PEPTIDES FROM SEA URCHIN EGGS, OR MAMMALIAN CAPACITATING CONDITIONS TRIGGER TEMPORAL OR MAGNITUDE CHANGES IN SPERM MOTILITY, MORPHOLOGY, AS WELL AS ESSENTIAL REGULATORY GLYCOLYTIC AND ENERGY METABOLIC PATHWAY MARKERS (USING NOVEL METABOLOMIC ANALYSIS) IN MICROGRAVITY VERSUS 1G. MOST IMPORTANTLY, THIS COMPREHENSIVE PROJECT WILL LEAD TO THE FIRST DIRECT IN VITRO ASSESSMENT OF CANONICAL FERTILITY-CRITICAL FUNCTIONAL PROCESSES OF MAMMALIAN SPERM IN MICROGRAVITY THAT ARE IDENTICAL TO MANY HUMAN IN VITRO CLINICAL AND RESEARCH LABORATORY ASSESSMENTS. THESE EXPERIMENTS ARE 1) PART OF THE RAPID TURN-AROUND SPACE FLIGHT EXPERIMENTS RESEARCH EMPHASIS, AND 2) ADDRESS THE NRC 2011 DECADAL SURVEY TO DISCOVER AND CHARACTERIZE UNDERLYING MECHANISMS OF ADAPTATION TO THE SPACE FLIGHT ENVIRONMENT, AND TO DETERMINE CELLULAR AND ORGANISMAL MECHANISMS THAT REGULATE AND SUSTAIN REPRODUCTION AND DEVELOPMENT IN SPACE. IN THIS REGARD, THE RESULTS OF THESE EXPERIMENTS WILL PROVIDE IMPORTANT FUNDAMENTAL REPRODUCTIVE BIOLOGICAL DATA TO SUPPORT NASA S GOAL TO CONDUCT MULTIGENERATIONAL STUDIES OF MAMMALS IN SPACE.