The long-term objectives of this work are to determine how sperm movement is regulated and how flagellar movement patterns affect the ability of sperm to reach the site of fertilization and penetrate the oocyte. We have focussed our efforts on a movement pattern termed hyperactivation, because it has been observed to occur in the oviduct near the site of fertilization. Our three specific aims at this point are as follows: (1) Investigate the functions of hyperactivation by comparing the ability of activated (the movement pattern of sperm in the ejaculate) and hyperactivated sperm to generate force for pulling away from the oviductal wall and for penetrating the zona pellucida. Pulling forces will be compared by determining the relative force required to hold activated and hyperactivated sperm onto suction micropipets. Zona penetration will be tested by comparing the rates at which acrosome- reacting activated and hyperactivated sperm penetrate zonae in vitro. (2) Characterize the mechanism that raises intracellular free Ca2+ ([Ca2+]in) to induce hyperactivation. Based on our observations, we hypothesize that a different mechanism regulates the calcium influx associated with hyperactivation than that associated with the acrosome reaction. Calcium channels can be characterized by their response to various organic agents and ions. We have the ability to estimate concentrations of [Ca2+]in in various regions of individual, moving sperm. These techniques can be used to determine whether an agent actually affects [Ca2+]in while it is affecting motility. (3) Determine whether levels of [Ca2+]in in sperm flagella may be regulated by the oviduct or zona pellucida. We have observed that hyperactivation is initiated in sperm that are attached to the oviductal wall and that only hyperactivated sperm flagella may be regulated by the oviduct or zona pellucida. We have observed that hyperactivation is initiated in sperm that are attached to the oviductal wall and that only hyperactivated sperm free themselves from the wall. It has also been reported that the pattern of flagellar beating alters when sperm bind to the zona pellucida. We hypothesize that elements in the oviductal wall and/or zona regulate sperm flagellar beating by affecting [Ca2+]in. [Ca2+]in will be measured in individual sperm as they attach to oviductal epithelium and to the zona pellucida. The information gained may be used to treat infertility, to monitor the effects of toxins and disease on fertility, and to develop methods of contraception that act prior to fertilization.
NIH ExPorter
NIH ExPorter