Date of Award

Winter 1997

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Committee Director

Mary C. Mahony

Committee Member

Sergio Oehninger

Committee Member

Peter F. Blackmore

Committee Member

Patricia A. Pleban

Abstract

The regional distribution of ChAT activity in human spermatozoa is altered during in vitro capacitation and it correlates with the fertilizing potential of sperm.

Regional immunoreactivity in human spermatozoa as assessed by fluorescent immunocytochemistry was compared with ChAT and CaAT activity determined by enzymatic methodologies. Increasing proportions of sperm exhibited ChAT immunoreactivity along the equatorial region with a concomitant decrease in ChAT reactivity in the midpiece. Also, competitive studies with unlabeled ChAT blocked the equatorial region labeling; the unlabeled CaAT blocked staining along the midpiece region of the tail, suggesting some cross-reactivity of the ChAT antiserum with the CaAT enzyme. A ChAT radioassay was used to compare regional immunoreactivity in human spermatozoa as assessed by fluorescent immunocytochemistry with ChAT and CaAT activity determined by enzymatic methodologies. Although a direct comparison was not made between the ChAT radioassay and the equatorial and midpiece/tail ChAT immunoreactivity, the two procedures appeared to not correlate. However, based on findings from the ChAT radioassay study, it was concluded that the ChAT enzyme is present within the sperm and not synthesized de novo.

The regional distribution of ChAT and CaAT immunoreactivity under sperm capacitating conditions was correlated with the physiological indicators of completed capacitation, including the physiological and pharmacological induction of the acrosome reaction. A significant increase in both equatorial and midpiece/tail immunoreactivity was observed when using both caffeine and pentoxifylline. The addition of caffeine significantly increased equatorial binding, midpiece/tail binding, and the acrosome reaction rate in both washed and post swim-up samples. Although the addition of caffeine increased both equatorial binding and the acrosome reaction rate, a correlation was not observed between equatorial binding and the acrosome reaction.

The potential fertility of human spermatozoa was correlated with ChAT and CaAT immunoreactivity by comparison of spermatozoa obtained from proven fertile males to spermatozoa incapable of attaining the physiological indicators of completed capacitation. A significant positive correlation was demonstrated between ChAT binding and the Hemizona Assay Index. A significant positive correlation was also demonstrated between CaAT binding and original sperm concentration, VAP, VSL, VCL, and both original and swim-up motility.

Comments

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

DOI

10.25777/q9cd-wf54

ISBN

9780591623314

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