Role of SPRASA in Sperm-Zona and Sperm-Oolemmal Binding.

Abstract

SPRASA (SPACA-3, SLLP-1) is a highly conserved protein located in the inner acrosome membrane of sperm. It was discovered based on its' reactivity with antisperm antibodies from some infertile men. SPRASA expression appears to be limited to the testis/sperm and others have shown that SPRASA-reactive antibodies inhibit human sperm binding to zona-free hamster oocytes. We have previously found that exposing gametes to SPRASA-reactive antibodies inhibits subsequent embryo development and have recently demonstrated that there are SPRASA binding sites on the oolemmal membrane of bovine oocytes. Using a bovine model, we investigated the effects of a SPRASA-reactive antiserum on sperm binding to oocytes at the zona pellucida and oolemmal membrane level in a bovine in vitro fertilisation model system. Bovine oocytes were obtained from Auckland Meat Processors and matured in vitro. To examine the effects of binding of sperm at the zona pellucida level, capacitated sperm and zona-intact oocytes were co-incubated with SPRASA-reactive antiserum, an irrelevant antiserum or in the absence of antiserum for 2 hours, followed by repeated pipetting with a Gilson pipette to remove loosely-bound sperm. Sperm and oocyte complexes were then stained with Hoechst and visualised on a fluorescent microscope. To examine the effects of binding of sperm and oocytes at the oolemmal membrane level sperm were capacitated then acrosome reacted using calcium ionophore then incubated with oocytes, from which the zona had been stripped using pronase, in the presence or absence of a SPRASA-reactive antiserum or an irrelevant rabbit antiserum for 2 hours. Following extensive washing, sperm/oocyte complexes were then stained with Hoechst and visualised on a fluorescent microscope. In both assays the number of sperm bound to each oocyte was counted. The significance of the difference between SPRASA-reactive antiserum and control groups was determined by the Student T-test. Analysis of sperm-zona binding showed that the number of sperm bound per zona-intact oocyte was not significantly different between the SPRASA-reactive antiserum treated group and the controls (P = 0.26). Similarly, SPRASA-reactive antiserum did not significantly alter the number of sperm binding to zona-free oocytes in comparison to the no antiserum control (P = 0.49). However, control rabbit antiserum appears to interfere with the binding assay, significantly increasing the number of sperm bound to the oolemmal membrane. In conclusion, this study shows that SPRASA-reactive antiserum had no effect on the binding of bovine sperm to bovine oocytes at either the level of the zona pellucida or the oolemmal membrane, and that the inhibitory effect of SPRASA-reactive antiserum on embryonic development that we have reported previously appears therefore to be downstream of sperm-oocyte binding. This study was supported by funding from the Marsden Fund of the Royal Society of New Zealand and the University of Auckland Staff Research Fund.