Date of Award

Spring 1993

Document Type

Thesis

Department

Life & Environmental Sciences

First Advisor

Rev. Joseph Harrington

Second Advisor

Marilyn Schendel

Third Advisor

John Downs

Abstract

The anterior end of the mammalian sperm nucleus is covered by a thin, double-layered membranous sac that contains hydrolytic enzymes including acrosin, hyaluronidase, esterases and acid hydrolases. These enzymes, which are involved in the initial phase of fertilization, are activated and released during the acrosome reaction. This exposure of the acrosomal contents occurs not only during fertilization, but also during sperm death.

Acrosome-reacted sperm cells were selected from thawed, cryopreserved samples of bovine spermatozoa using rabbit antibodies to bovine acrosine and goat anti-rabbit immunoglobulin-coated magnetic beads. The selection process was monitored by staining with fluorescence isothiocynate- beads coupled to anti-rabbit immunoglobins (FITC-IgG), Fura- red (FRED) and propidium iodide (PI). The spermatozoa were diluted in Tyrode salt solution and stained with FRED, anti- bovine acrosome, fluorescence labeled anti-rabbit IgG. An inhibitor, benzamidine, was used in all samples to control proteolysis, while ionophore A233187 was used in control samples to induce the acrosome reaction. Flow cytometric analysis revealed three distinct populations; dead cells stained only with PI and two acrosome-reacted populations stained with FITC-IgG and either FRED or PI. The acrosome- reacted spermatozoa were selected by exposing the samples to a magnet (5 min). Magnetic beads, when coupled to antibodies, were useful for selecting a specific sperm population. Magnetic beads, when coupled to specific antibodies, proved to be effective in selecting acrosome reacted spermatozoa.

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