The developmental and functional characterization of glutathione-s-transferase omega 2 (GSTO2) within eutherian spermatozoa

Abstract

The eutherian sperm head has three main components: the nucleus, acrosome and perinuclear theca (PT). The PT is a dense cytosolic protein layer that lies between the nucleus and acrosome and houses proteins involved in spermiogenesis and fertilization. This thesis identifies two new PT constituents as isoforms of glutathione-s-transferase omega 2 (GSTO2) and characterizes their developmental and functional importance. We demonstrate that GSTO2 localizes to the postacrosomal sheath (PAS) of the PT and adheres to the developmental mechanisms of other PAS constituents, utilizing the microtubular manchette for protein transport during spermiogenesis. The PAS is of high functional significance, as the first region of the sperm head to solubilize after gametic fusion. The early release of GSTO2 from the sperm head and its ability to facilitate glutathione-centered reduction reactions suggests it may function in glutathione-dependent processes of early embryonic development. Past findings demonstrated that the sperm nuclear transition is centered on these reduction reactions but the enzymes involved remain elusive. Therefore, we hypothesized that PT-anchored GSTO2 facilitates the sperm nuclear transition, as ooplasmic constituents are being recruited. Through the use of intracytoplasmic sperm injections coupled with enzymatic inhibition, we have shown that blocking GSTO2’s functionality causes a delay in sperm nuclear decondensation in the mouse fertilization model. Additionally, we demonstrated that delays accrued persist through development leading to decreased embryo viability and blastocyst development. A secondary localization of GSTO2 was also discovered on the sperm plasmalemma. GSTO2’s high dehydroascrobate reductase activity coupled with an elevated concentration of ascorbic acid in seminal fluid suggested that GSTO2 may also function in a regulatory capacity on the plasmalemma during sperm capacitation. To test this hypothesis, we catalytically inhibited GSTO2 during in vitro capacitation and investigated the consequence on sperm performance parameters. GSTO2 inhibition resulted in a significant decrease of tyrosine phosphorylation activity and in turn negatively impacted acrosome exocytosis and motility, leading to the sperms inability to penetrate the oocyte. We conclude that surface-borne GSTO2 may function in a regulatory capacity during capacitation. Together, this thesis provides a comprehensive developmental and functional assessment of the importance of GSTO2 before, during and after fertilization.