Date of Award

Winter 2002

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program/Concentration

Biomedical Sciences

Committee Director

Sergio C. Oehninger

Committee Director

Mahmood S. Morshedi

Committee Member

R. James Swanson

Committee Member

Stephen J. Beebe

Abstract

Ejaculated sperm display markers that are indicative of apoptosis in somatic cells. The question remains as to whether sperm have operative apoptosis mechanisms. The aim of this research was to test the hypothesis that apoptosis markers in sperm and somatic cells are different.

Ejaculated human sperm from patients and donors were separated into high and low motility fractions using Percoll™ gradients. Contaminating cells were removed using anti-CD45 conjugated paramagnetic beads. Fractions were divided into groups: staurosporine, anti-Fas antibody, and hydrogen peroxide treated and control. Direct enzymatic measurement of caspase activity, flow cytometric evaluation of phosphatidylserine translocation, immunoblots, and immunocytochemistry were used in this investigation.

Of the pro-apoptosic treatments, only staurosporine resulted in elevated caspase activity in patients and donors. Differences in caspase activity were found between donor and patient sperm. No elevations low motility fraction caspase activities were detected.

In Annexin-V-FITC labeled high motility fractions, staurosporine, anti-Fas, and 25 micromolar hydrogen peroxide failed to stimulate phosphatidylserine translocation. However, 200 micromolar hydrogen peroxide resulted in translocation increases. No treatment stimulated translocation in low motility fractions.

In immunoblots, the presence of procaspase-3 was detected in high and low motility fractions from donors and patients. Active caspase-3 was detected in high and low motility fractions from donors and patients. Procaspase-7, but not active, was also detected. Procaspase-9 was detected in high and low motility fractions from donors and patients. Active caspase-9 was only detected in a low motility patient fraction. Neither intact Poly-ADP-ribose Polymerase nor its breakdown product was detected. Apoptosis Inducing Factor was present in immunoblots from all high and low motility fractions. BID was not detected in either high or low motility fractions.

Active caspase-3 was detected using immunofluorescent microscopy in severe oIigoasthenoteratozoospermic patients. Less than one percent of the sperm evaluated exhibited active caspase-3.

Sperm possess apoptosis markers. However, pro-apoptotic treatments indicated that apoptosis was not fully inducible in sperm. Sperm lack major signals associated with caspase dependent apoptosis in somatic cells. Since mitochondria are the only active organelle present in sperm, further work must be done to determine whether mitochondria dependent pathways are intact.

Comments

A Dissertation Submitted to the Faculty of Old Dominion University and Eastern Virginia Medical School in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in Biomedical Sciences.

DOI

10.25777/r0b7-wf73

ISBN

9780493976075

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