Very little is known about the molecular mechanisms controlling hyperactivation (HA) and how abnormal movements affect sperm transport through the female genital tract and penetration of the egg investments. The PI has shown that sperm from tw32/+ mice (tw32/+ sperm) have abnormal motility: HA occurs prematurely, and most of the nonhyperactivated motile sperm are abnormally slow. The PI will determine whether precocious HA or slow speed is also characteristic of other t haplotypes (Aim IA). Collaborative studies have shown that the axonemes of tw32/+ sperm are unusually sensitive to calcium (Ca). The PI will determine whether the very slow movements of sperm from mice carrying two t haplotypes are also sensitive, by (1) treating intact sperm with drugs that lower the intracellular free [Ca], and (2) measuring the [Ca] necessary to change flagellar curvature in demembranated sperm (Aim IB). Changes in invertebrate flagellar movement are controlled by cAMP, Ca-binding proteins, or both. Preliminary data suggest that tw32/+ sperm may contain elevated cAMP levels. The PI will determine whether there is a relationship between cAMP and premature HA by (1) measuring cAMP in tw32/+ sperm and (2) perturbing cAMP levels to affect the timing of HA (Aim IIA). Also the Ca-binding proteins of tw32/+ sperm flagella will be characterized by diagonal electrophoresis (Aim IIB). Since the PI has evidence that tw32/+ sperm have normal levels of capacitation, acrosome reaction and zona-binding, their only abnormality relevant to penetration of the investments is in motility. Therefore, the PI will determine whether tw32/+ sperm are abnormal in penetration of the investments by counting the numbers of sperm able to penetrate each investment (Aim II). Experiments from the PI's lab suggest that tw32/+ sperm that do not carry the tw32 haplotype (+ t sperm) are dysfunctional in fertilization in vivo. Although the mechanism of dysfunction is unknown, it must occur between the uterus and the site of gamete interaction and could involve abnormal sperm motility. The Pi will test the hypothesis that the dysfunction of + t sperm occurs in passage through the uterotubal junction by determining whether + t sperm can be recovered from oviductal populations, using the polymerase chain reaction to amplify a sequence which is different for + and t DNA (Aim IV). Determine the site of + t dysfunction will help to determine its nature. These experiments will increase our understanding of how Ca controls hyperactivation and how specific types of movements affect sperm fertilizing ability.