Immunolocalization of multiple Gα subunits in mammalian spermatozoa and additional evidence for Gαs

Abstract

Like somatic cells, mammalian spermatozoa appear to contain several different heterotrimeric G protein α‐subunits that could mediate specialized cell responses. However, the precise Gα subunits present, their subcellular location and their possible roles are still incompletely defined. In this study, using commercially available specific antibodies, we have shown by immunoblotting that Gα_s is present in human and mouse sperm lysates. Immunolocalization using intact spermatozoa from both species revealed this protein to be in the acrosomal cap region and the flagellum, particularly the principal piece. Treatment of permeabilized mouse spermatozoa with cholera toxin led to enhanced ADP‐ribosylation of a protein the same size as Gα_s, as well as an increase in cAMP, providing further proof for Gα_s. Evidence for the presence and distinct localizations of Gα_i2, Gα_i3, Gα_o, Gα_q/11, and Gα_olf was also obtained. Of particular interest was Gα_i2 which, like Gα_s, was present in the acrosomal cap region and flagellum, the same regions where stimulatory and inhibitory adenosine receptors are localized. These observations are consistent with our hypothesis that G proteins mediate adenosine receptor modulation of adenylyl cyclase, with consequent alterations in cAMP production, apparently crucial for the spermatozoon's acquisition and maintenance of fertilizing ability. Mol. Reprod. Dev. 65: 104–113, 2003.