Recent Developments in IVF & Oviduct Biology: New Experimental Tools & Biocontrol Targets
John Rodger 1, 2, Kuldip Sidhu 1, Karen Mate 1& Amanda Harman 2
Marsupial CRC at
1 Macquarie University, Sydney Australia
2 University of Newcastle, Newcastle Australia
Background
In vitro fertilization is a very powerful experimental tool widely used in eutherian gamete biology to identify gamete and fertilization targets and to test their potential as immuno-contraceptive agents. As well as allowing direct experimental access to events, which normally occur within the female tract, IVF is relatively quick and cost effective compared with in vivo experimentation. Indeed most of what we know about the cell and molecular mechanisms of eutherian fertilization is based on IVF.
A key process which sperm must complete to achieve fertilization in vivo and in vitro is capacitation. This sperm maturation process occurs within the female tract usually within the oviduct. In the possum it involves changes to the sperm conformation from streamlined to `T-shape' and the development of the ability to bind to the egg coat the zona pellucida (ZP). Using oviduct cell cultures and conditioned media we, and our CRC and AgResearch colleagues working at Landcare Research Lincoln have achieved capacitation in vitro and IVF for the possum.
This paper reports two recent developments in our understanding of possum capacitation and fertilization which increases the potential of IVF as an experimental tool and offers new, and possibly marsupial specific, targets for fertility control in the possum. The first is the finding of an unexpected critical role for Ca2+. In possum sperm low calcium facilitates change to `T shape' in a reversible manner. The second is observation of acrosome intact sperm heads within the cytoplasm of polyspermic eggs. Current ideas on the mechanism of fertilization in marsupials suggest that such a result is impossible. This observation, although almost certainly due to the unusual condition created in vitro which lead to polyspermy, raises major questions about the mechanism and site of sperm-egg membrane fusion in the possum.
Calcium & Capacitation
Ca2+ is important in the regulation of eutherian gamete function and fertilization but this is associated with high levels suggesting an unusual marsupial specific phenomenon may be involved. Our studies found that changes to T-shape and successful IVF were correlated with low concentrations of Ca 2+ in the media. The first indication of this result came in studies of wallaby sperm incubated in calcium-free media as part of other CRC supported experiments on the biochemistry of capacitation. When wallaby sperm were incubated for 2h in a calcium-free medium 70-75 percent of sperm became T-shape. This compared with 0 percent T-shape sperm in the calcium containing media routinely used for cell culture and IVF. Even more striking was the observation that the calcium free medium induced T-shape change was reversible. Addition of 1mM Ca2+ to the medium resulted in 100 percent of sperm displaying streamlined highly progressive motility. In earlier experiments it was found that although co-culture of wallaby sperm with oviduct cells resulted in a small percentage of sperm changing to T-shape (around 15 percent) there was no effect on sperm of conditioned medium from these cells. This was in contrast to the possum where oviduct conditioned media (CM) alone induced >75 percent of sperm to change to T-shape after 2h incubation. We then examined the effect on wallaby sperm of wallaby oviduct CM which had been depleted of Ca2+ by dialysis. This calcium-depleted oviduct CM induced 100 percent of sperm to become T-shape after 2h incubation. We then examined stored possum oviduct CM from earlier IVF trials for their Ca2+ concentration. In all cases calcium concentration was reduced by exposure to oviduct cells - pSOF (possum synthetic oviduct fluid 0.7mM > CM 0.4mM), EMEM (Eagles' modified essential medium 1.5mM > CM 0.7mM). These Ca2+ depleted CM had high T-shape inducing capacity (75-90 percent) and supported fertilization with a success rate of 10-15 percent (pSOF) and 30-45 percent (EMEM). These findings direct target research to oviduct calcium binding molecules or Ca2+ removal systems in the possum oviduct. In addition it will allow improved efficiency in IVF and in the quality of IVF produced embryos because it offers the promise of fully defined `off the shelf' IVF media for marsupials.
In Vitro Fertilization
IVF has been achieved for the possum, and depending on media components, has involved sperm-ZP binding, ZP penetration and sperm incorporation into egg. ZP penetration is via a large hole in the ZP, and under current IVF conditions is usually polyspermic. This is not observed in vivo and although not desirable for generation of viable embryos is a technical advantage for studies of the mechanism of fertilization and its disruption since the interactions of many sperm with one egg can be examined. In deed polyspermic fertilization may be a possible target in itself if the block to polyspermy in marsupials is dependent on the oviduct produced mucoid coat. Finally there is data on possum fertilization to suggest that the mode of sperm-egg fusion and incorporation does not fit accepted dogma. It has been accepted for some time that fertilization in Australian marsupials involves direct interaction and fusion between the inner membrane of the acrosome which is exposed at the acrosome reaction which occurs on the surface of the ZP prior to penetration of the ZP. This almost certainly happens for the primary sperm making any hole in the ZP in our IVF system. However it appears the subsequent supernumerary sperm which reach the egg membrane acrosome intact then fuse with the cell membrane of the egg by the sperm cell membrane itself not the supposedly specialised fusogenic inner acrosomal membrane. If this is so we will need to very critically reassess molecular targets on the sperm surface.
Conclusion
IVF for the possum is now nearing the stage of being a routine tool. Identification of a critical role for concentrations of extra cellular Ca2+in media and a role for oviduct cells in reducing available Ca2+ for the first time provides not only insight into the mechanism of capacitation but provides a potential marsupial-specific control target. In addition it opens a path to the development of a fully defined capacitation medium for the possum not dependent on oviduct cells. IVF system now in place, using oviduct CM, is however at a stage that it can will be used as a primary screening tool for assessment of new gamete and oviduct fertility control targets. This will allow more targets to be relatively rapidly examined to select those of promise to proceed to very expensive and timeconsuming animal experiments. Finally fertilization itself may offer new and unexpected marsupial-specific targets based on the sperm-egg fusion phenomena described above.
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