Human glucosamine-6-phosphate isomerase, a homologue of hamster oscillin, does not appear to be involved in Ca2+ release in mammalian oocytes.

Overview

abstract

Injections of cytosolic preparations from mammalian sperm into oocytes have been shown to trigger calcium [Ca2+]i oscillations and initiate activation of development. Recently, a protein isolated from hamster sperm has been suggested to be involved in the generation of these oscillations and it was named "oscillin." The human homologue of hamster oscillin is glucosamine 6-phosphate isomerase (GPI, EC no. 5.3.1.10), an enzyme so far described to be involved in hexose phosphate metabolism. To assess the role of GPI on Ca2+ signaling, a human recombinant protein was generated in a prokaryotic system and injected into fura-2-dextran-loaded metaphase II (MII) mouse oocytes. Injection of recombinant GPI failed to induce Ca2+ responses in 12/12 injected MII oocytes despite the fact that the recombinant GPI was active as assessed by an enzymatic assay. Injection of buffer (0/6 oocytes) or fructose-6-phosphate, a product of GPI enzymatic reaction (0/5 oocytes), also failed to initiate Ca2+ responses. Conversely, injections of sperm cytosolic factor induced [Ca2+]i oscillations in all 17/17 oocytes. In addition, injection of recombinant GPI or GPI mRNA failed to induce parthenogenetic activation (0/30 oocytes). Immunofluorescence studies using an anti-GPI polyclonal antibody (GK) resulted in localization of GPI to the sperm's equatorial region. Incubation of the GK antibody with sperm extracts failed to block the [Ca2+]i responses induced by these extracts. Moreover, near complete depletion of GPI from sperm fractions by immunoprecipitation did not impair the ability of these fractions to induce [Ca2+]i oscillations. In summary, our results support the role of a sperm cytosolic component(s) in the generation of [Ca2+]i oscillations during mammalian fertilization, although a protein other than GPI/oscillin is likely to be the active calcium releasing factor.