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; DeMott and Suarez, 1992; Suarez et al., 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 (Visconti et al., 2002; Suarez and Ho, 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 problems, 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 channels, 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. 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). More importantly, the ability to perform IVF in the laboratory will allow us to use this as a functional assay to evaluate the fertilizing ability of sperm from stallions and thus serve as a potential fertility test. Finally, our research will expand the overall knowledge of fertilization in mammalian species.
Austin CR. The ‘Capacitation' of mammalian sperm. Nature 1952;170:326.
Bézard J . In vitro fertilization in the mare. Proc Int Sci Conf Biotech in Horse Reprod, Agricultural University of Krakow , Poland ; Abstr 1992;12.
Chang MC. Fertilizing capacity of spermatozoa deposited into the fallopian tubes. Nature 1951;168:697-698.
Christensen P, Whitfield CH, Parkinson TJ. In vitro induction of acrosome reactions in stallion spermatozoa by heparin and A23187. Theriogenology 1996;45:1201-1210.
DeMott RP, Suarez SS. Hyperactivated sperm progress in the mouse oviduct. Biol Reprod 1992:46;779-785.
Farlin ME , Jasko DJ, Graham JK, Squires EL. Heparin-induced capacitation: A comparison between the bull and the stallion. Equine Vet J 1993:15;49-52.
Galantino-Home r HL, Visconti PE , Kopf GS. Regulation of protein tyrosine phosphorylation during bovine sperm capacitation by a cyclic adenosine 3'5'-monophosphate-dependent pathway. Biol Reprod 1997:56;707.
Ho H-C, Suarez SS. Characterization of the intracellular calcium store at the base of the sperm flagellum that regulates hyperactivated motility. Biol Reprod 2003:68;1590-1596.
Hortas ML, Castilla JA, Gil MT, Samaniego F, Morell M, Redondo M. Alterations in sperm protein phosphorylation in male infertility. Andrologia 2001:33(5);282-286.
Meyers SA, Liu IKM, Overstreet JW, Vadas S, Drobnis EZ. Zona pellucida binding and zona-induced acrosome reactions in horse spermatozoa: Comparisons between fertile and subfertile stallions. Theriogenology 1996:46;1277-1288.
Munire M, Shimizu Y, Sakata Y, Minaguchi R, Aso T. Impaired hyperactivation of human sperm in patients with infertility. J Med Dent Sci 2004:51(1);99-104.
Palmer E , Magistrini M, Bézard J, Duchamp G. Gestation aprés fécondation in vitro dans l'espéce équine. CR Acad Sci Paris 1990:310;71-74.
Pommer AC, Rutllant J, Meyers SA. Phosphorylation of protein tyrosine residues in fresh and cryopreserved stallion spermatozoa under capacitating conditions. Biol Reprod 2002:58(7);1373-84.
Rathi R, Colenbrander B, Stout TAE, Bevers MM, Gadella BM. Progesterone induces acrosome reaction in stallion spermatozoa via a protein tyrosine kinase dependent pathway. Mol Reprod Dev 2003:64;120-128.
Sakkas D, Leppens-Luisier G, Lucas H, Chardonnens D, Campana A, Franken DR, Urner F. Localization of tyrosine phosphorylated proteins in human sperm and relation to capacitation and zona pellucida binding. Biol Reprod 2003:68;1463-1469.
Suarez SS , Katz DF, Overstreet JW. Movement characteristics and acrosomal status of rabbit spermatozoa recovered at the site and time of fertilization. Biol Reprod 1983:29;1277-1287.
Suarez SS , Dai XB, DeMott RP, Redfern K, Mirando MA. Movement characteristics of boar sperm obtained from the oviduct or hyperactivated in vitro . J Androl 1992:13;75-80.
Suarez SS , Varosi SM, Dai X. Intracellular calcium increases with hyperactivation in intact, moving hamster sperm and oscillates with the flagellar beat cycle. Proc Natl Acad Sci USA 1993:90;4660-4664.
Suarez SS , Ho H-C. Hyperactivated motility in sperm. Reprod Dom Anim 2003:38;119-124.
Visconti PE, Bailey JL, Moore GD, Pan D., Olds-Clarke P, Kopf G. Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation. Dev 1995a:121;1129-1137.
Visconti PE, Moore GD, Bailey JL, Leclerc P, Connors SA, Pan D, Olds-Clarke P, Kopf G. Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway. Dev 1995b:121;1139-1150.
Visconti PE , Galantino-Homer H, Moore GD, Bailey JL, Ning X, Fornes M, Kopf GS. The molecular basis of sperm capacitation. J Androl 1998:19(2);242-248.
Visconti PE , Westbrook VA , Chertihin O, Demarco I, Sleight S, Diekman AB. Novel signaling pathways involved in sperm acquisition of fertilizing capacity. J Reprod Immunol 2002:53;133-150.