The Harry M. Zweig Memorial Fund for Equine Research

Media and Molecular Pathways Conducive to Stallion
Sperm Capacitation and Mobility Hyperactivation

Dr. Sylvia Bedford-Guaus

It has been known for long that freshly ejaculated sperm from all mammalian species are not immediately capable of fertilizing an oocyte (egg) but require to undergo a complex process that has been termed capacitation (Chang, 1951; Austin, 1952). Sperm capacitation typically occurs in the reproductive tract of the female, after sperm reach the oviduct (fallopian tube; Chang, 1951; Suarez et al., 1983) and shortly before fertilization. During capacitation, sperm undergo a series of membrane changes with consequent activation of signaling pathways that allows them to shed their acrosome, a membrane cap over the sperm head that contains enzymes which facilitate penetration of the oocyte at fertilization. This constitutes the acrosome reaction ( Austin , 1952). Additionally, sperm must also acquire a change in their movement pattern or hyperactivation conferring them with the impetus required to move through the viscous secretions of the oviduct and the outside layer of the oocyte (or zona pellucida; Suarez et al., 1991; DeMott and Suarez, 1992; Suarez and Ho, 2003).

In laboratory and livestock species, as well as in humans, all of the above processes can be induced by incubating sperm in the laboratory with conditions that mimic those found in the oviduct (Suarez et al., 1992, 1993; Galantino-Homer et al., 1997; Visconti et al., 2002; Sakkas et al., 2003). Additionally, recent research has begun to elucidate the signaling pathways that lead to the completion of these events in some species (Suarez and Ho, 2003; Visconti et al., 2003). This research is very important because in species where in vitro capacitation works successfully, assisted reproduction techniques such as in vitro fertilization (IVF) can be used to propagate the genetics of valuable animals as well as to obtain offspring from subfertile animals and humans. Another important application of in vitro technology includes the possibility of using it as a tool to evaluate the cause of certain infertility p rob lems, such as it is done commonly in men (Hortas et al., 2001; Munire et al., 2004). Overall, research in this area also helps advance the general knowledge of the molecular processes involved in mammalian fertilization.

Unfortunately, stallion sperm capacitation cannot be successfully achieved in the laboratory which is proven by the fact that only two foals have ever been born from IVF technology (Palmer et al., 1990; Bézard, 1992) and no one has since been able to repeat those results. There are numerous reports where stallion sperm capacitation has been attempted in the laboratory, but results have been unrewarding and techniques inconsistent (Farlin et al., 1993; Christensen et al., 1996; Meyers et al., 1996; Pommer et al., 2002; Rathi et al., 2003). Undoubtly, laboratory media and conditions to achieve in vitro capacitation and hyperactivation in the equine require precise and systematic evaluation. Since some of the important molecular pathways involved in capacitation in laboratory species have been recently published (Visconti et al., 2002; Suarez and Ho, 2003), we propose to test some of these findings in the horse. For instance, it has been shown that capacitation involves the removal of cholesterol from the sperm membrane, which allows the activation of certain ion ch anne ls, ultimately resulting in the activation of kinases (proteins that add phosphorus to other proteins) within the sperm head (Visconti et al., 1995ab, 2002). Conversely, hyperactivation, which requires calcium in the medium, results from the initiation of signaling pathways at the level of the sperm tail (Ho and Suarez, 2003; Suarez and Ho, 2003).

By applying some of the knowledge gained in other species, we will approach our research on the stallion by following two Specific Aims : 1) to study the media and incubation conditions, as well as the signaling pathways that support stallion sperm capacitation and the endpoint to this process, the acrosome reaction; and 2) to evaluate and characterize changes in motility compatible with hyperactivation and media requirements that facilitate this process. Worth noting is that our laboratory has already performed some preliminary experiments that show that in the process of capacitation stallion sperm undergo protein phosphorylation. Moreover, for the first time, we have been able to observe hyperactivated motility in stallion sperm. Preliminary results from these findings are described in page 6 of this proposal. Additional experiments are required to further define these processes in stallion sperm and it is expected that by completing the research outlined in this proposal we will be able to successfully compete for outside private and federal funding, with the purpose of continuing this line of work in the horse.

In summary, our ultimate goal is to develop an in vitro protocol that will support successful and consistent capacitation and hyperactivation of stallion sperm. This research will allow the application of IVF technology in the horse industry, as well as for the propagation of genetics from endangered wild equids (i.e. Catalonian ‘burro', African wild ass and zebras). Additionally, the ability of sperm to undergo capacitation in the laboratory will provide a functional test to evaluate the fertilizing ability of sperm from subfertile stallions. Finally, our research will expand the overall knowledge of fertilization in mammalian species.