Date

2013

Document Type

Dissertation

Degree

Doctor of Philosophy

Department

Molecular Biology

First Adviser

Lowe-Krentz, Linda J.

Other advisers/committee members

Falk, Matthias M.; Iovine, M Kathryn; Somkuti, Stephen G.

Abstract

The equatorial segment (the EqS) of mammalian sperm has been reported to be a site for sperm-egg fusion initiation. The subdomain, called the equatorial subsegment (the EqSS), within the EqS has been identified in sperm of many species; however, there is no clear report showing the existence of this subdoman in human sperm. The EqS has been hypothesized to be an organizing center for assembly of molecular complexes required for gamete interaction and fusion. Several sperm proteins have been reported to relocate to the EqS to make this region fusion competent. However, molecular mechanisms that regulate relocalization of sperm proteins have not been clearly elucidated. The current study aimed to understand mechanisms underlying the relocalization of sperm proteins during the acrosome reaction. Based on the previous studies in our laboratory, sperm-associated alpha-L-fucosidase was detected to relocalize from the anterior of the acrosome to the EqS of human sperm after the acrosome reaction. In conjunction with the study in our laboratory aimed to investigate roles of this enzyme during fertilization by using mice as a model of study, the current project first refined the subcellular localization of membrane-associated alpha-L-fucosidase in mouse sperm before and after the acrosome reaction using immunolocalization and fluorescence microscopy. The experiments revealed that membrane-associated alpha-L-fucosidase was originally localized throughout the anterior of the acrosomal region; however, the enzyme could be detected only when mouse sperm were permeabilized. These observations suggest that the enzyme may reside in the acrosomal membranes, and that alpha-L-fucosidase progressively migrates from the acrosomal membranes to the plasma membrane over the EqS by the influence of lateral diffusion. CRISP2 protein was another human sperm protein used as a marker to study mechanisms of protein relocalization during the acrosome reaction. We found that Latrunculin A could not reduce the presence of CRISP2 at the EqS of acrosome-reacted human sperm, indicating that actin polymerization is not involved in facilitating CRISP2 relocalization during the acrosome reaction. Results also revealed that CRISP2 is a component of the acrosomal matrix. During the acrosome reaction, but not capacitation, CRISP2 was released into the media and re-associated with the EqS in a dose-dependent manner. These results suggest that the released CRISP2 can reassociate with the EqS during the acrosome reaction. The stability of CRISP2 at the EqS of acrosome-reacted human sperm was also studied. The association stability was very strong since high ionic strength solution, mild sonication, reducing agents, and non-ionic detergent with low concentration could not dissociate CRISP2 from the EqS of acrosome-reacted human sperm. However, a low concentration with a short period of incubation in the anionic detergent, SDS, could extensively remove CRISP2 from the EqS, but could not extract the CRISP2 population at the neck of sperm cells. Interestingly, EGTA caused redistribution of CRISP2 over the sperm head. Data suggest that stabilization of CRISP2 at the EqS of human sperm depends on calcium ions. This project also investigated the oocyte binding ability of acrosomal CRISP2 by incubating zona-intact and zona-free hamster eggs with the purified biotinylated human acrosomal CRISP2. CRISP2 bound to the plasma membrane of the oocyte but not to the zona pellucida, suggesting the presence of conserved complementary sites of CRISP2 on hamster oocyte plasma membrane. These observations raise the possibility that CRISP2 at the EqS may help modify the EqS plasma membrane to make this domain fusion competent or act as an adhesion molecule for sperm-egg interaction and fusion. Taken together, the results revealed that human sperm use different mechanisms to facilitate the relocalization of sperm proteins from other regions to the EqS during the acrosome reaction. Membrane proteins like alpha-L-fucosidase seem to migrate laterally in the plasma membrane whereas soluble proteins stored in the acrosomal cap like CRISP2 are released and reassociate with the plasma membrane of the EqS. The reassociation of this protein may facilitate sperm-egg fusion since acrosomal CRISP2 can bind to the targets on the oolemma of hamster oocytes.

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