STUDIES ON SPERM FERTILIZING ABILITY

拟议研究的总体目标是提供新的

The overall objective of the proposed research is to provide new information on the regulation of sperm:egg interaction in mammals by components of the normal oviductal milieu. This information is needed for a more complete understanding of the way that gamete interactions are facilitated and coordinated at the site of fertilization in vivo. Increased knowledge of these regulatory mechanisms could assist the development of improved methods for amelioration of infertility in humans and for fertility control. Specific aims are (i) to identify molecules modulating terminal events of sperm:zona interactions and acrosome reactions, and to differentiate their sites of production (follicle or oviduct); (ii) to isolate and identify a heat labile oviductal factor that facilitates sperm penetration into zonae pellucidae, and to determine its mechanism of action; and (iii) to isolate and identify a heat stable factor present in the ovulated cumulus oophorus that facilitates acrosome reactions in free-swimming (i.e., non-zona bound) sperm. The research will use well-defined in vitro fertilization techniques to examine details of the regulatory mechanisms with golden hamster gametes. Hamster epididymal spermatozoa will be capacitated in vitro, then incubated for 60 or 90 min with living or salt-stored follicular or ovulated eggs. These eggs and/or sperm will be treated with impure or (later) purified heat labile and/or heat stable factors and the effects on sperm penetration of the zonae pellucidae will be evaluated; differential responses with these preparations will provide detailed information about the functions and mechanisms of action of the factors. Sperm acrosome reactions will be monitored using fluorescent monoclonal antibodies. The sites of production and of functional activities of these factors will be localized using polyclonal and monoclonal antibodies. Factors will be isolated by standard biochemical procedures, including SDS-polyacrylamide gel electrophoresis followed by western blotting, and identified using immunoenzymatic assay. Tests will show if the factors' actions are enzyme-like or ligand-like. The hypotheses will be tested that the factor(s) act as co-acrosome reaction inducing factors, i.e., in synergism with zonae pellucidae, and that the factors alter the physical characteristics of zonae pellucidae. Possible homology between some of the oviductal factors and the oviductal protein 'oviductin' will be examined. It will be determined whether the acrosome reaction inducing activity of the heat-stable factor can be ascribed to a macromolecule or to a ligand, such as progesterone, bound to a macromolecule. The results of these experiments will provide deeper insights into the functional roles of these naturally-occurring factors, and into their mechanisms of action in regulating events immediately preceding egg penetration.