Undergraduate Student Randolph-Macon College Henrico, Virginia, United States
Abstract Authors: A.C. Tetterton1 and Jim Foster1.
1. Biology Department, Randolph-Macon College, Ashland, VA, USA.
Abstract Text: Research into fertilization mechanisms informs infertility and the development of new contraceptives. Recently it was shown that mice lacking the GPR56 gene are infertile, suggesting that this protein is essential for fertility. Though it has been identified in germ cells and sperm, its location and function inside spermatozoa have never been characterized. Previous immunofluorescence data from our lab indicates that GPR56 colocalizes with a known acrosomal protein, ZP3R; however, the colocalization did not distinguish whether GPR56 was on the plasma membrane or beneath the cell surface on the acrosomal membrane. Since G-protein coupled receptors (GPCRs) are surface-active proteins and GPR56 was expected to be on the sperm surface, immunofluorescence microscopy was used to localize GPR56 in epididymal mouse sperm and determine when GPR56 is placed on the plasma membrane during maturation. The timing of surface exposure should coincide with receptor activation. Immunofluorescence data indicates that GPR56 localizes to the sperm acrosome in cauda epididymal sperm, and is not present in the rest of the sperm head or tail. GPR56 knockout sperm lacked the acrosomal label, indicating that the signal observed was specifically from GPR56. To test whether GPR56 was on the surface in epididymal sperm, permeabilized and unpermeabilized sperm were probed with anti-GPR56 antibodies. The data demonstrates that GPR56 is not on the plasma membrane but on the acrosomal membrane in cauda epididymal sperm. Anti-GPR56 antibodies were unable to stain GPR56 in unpermeabilized cells, presumably because GPR56 was sequestered on the acrosomal membrane and the antibodies could not breach the plasma membrane to reach it. Capacitation, a process where sperm prepare to fertilize the egg, often results in proteins concealed in the acrosome being released to the surface. To determine when GPR56 was exposed to the surface, permeabilized and unpermeabilized cells incubated in capacitating conditions were probed with anti-GPR56 antibodies. Anti-GPR56 antibodies were able to stain GPR56 in unpermeabilized cells once the cells were capacitated, indicating that GPR56 was exposed to the extracellular environment. Results in capacitated sperm suggest that once sperm enter the female reproductive tract, GPR56 is packaged and released to the plasma membrane. Based on these results, we propose that GPR56 is sequestered on the acrosomal membrane in epididymal sperm and is released to the plasma membrane during capacitation. The relocation of GPR56 during capacitation indicates that it may contribute to the signaling that begins acrosomal exocytosis.
