Sperm binding and penetration of the zona pellucida in vitro but not sperm–egg fusion in an Australian marsupial, the brushtail possum (Trichosurus vulpecula)

Abstract

Sperm capacitation and in vitro fertilisation (IVF) have been achieved in most eutherian mammals and American marsupials under relatively simple culture conditions. In contrast sperm capacitation in Australian marsupials has not been achieved in vitro and attempts at IVF have previously been characterised by a complete lack of sperm–zona pellucida (ZP) binding. Recently, co-culture of sperm with oviduct epithelial cell monolayers or with oviductal explant conditioned media has been shown to prolong the viability and motility of brushtail possum spermatozoa, as well as to induce capacitation-associated changes such as transformation of sperm to the T-shape orientation. In this study we report that these in vitro produced T-shaped sperm, and in vivo derived T-shaped sperm flushed from the oviduct of artificially inseminated possums as a control, are able to bind to and penetrate the ZP of approximately 25% of eggs recovered from PMSG/LH-superovulated possums in vitro. Development of ZP receptivity and penetrability towards sperm was also identified as a major factor affecting the outcome of IVF. Neither in vivo nor in vitro derived T-shaped sperm were able to bind to or penetrate the ZP if eggs were obtained from animals that were treated with pLH less than 76 h after PMSG. Thus this study provides preliminary evidence for the necessity of sperm–oviduct epithelial cell interactions for capacitation in Australian species and lends further support to the suggestion that the T-shape head orientation is indicative of sperm capacitation. Despite the occurrence of sperm–ZP binding and penetration, sperm–egg membrane fusion and egg activation were not observed. Although the factor(s) responsible for the lack of sperm–egg membrane fusion in the possum have not been identified it is possible that egg capacity for membrane fusion develops independently of zona receptivity and is defective in these eggs, or alternatively that membrane fusion requires strictly defined ionic conditions which are not provided by the IVF media used in this study.