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

Abstract

Like somatic cells, mammalian spermatozoa appear to contain several different heterotrimeric G protein alpha-subunits that could mediate specialized cell responses. However, the precise Galpha 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 Galpha(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 Galpha(s), as well as an increase in cAMP, providing further proof for Galpha(s). Evidence for the presence and distinct localizations of Galpha(i2), Galpha(i3), Galpha(o), Galpha(q/11), and Galpha(olf) was also obtained. Of particular interest was Galpha(i2) which, like Galpha(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.

Compartmentalization of Distinct cAMP Signaling Pathways in Mammalian Sperm

“…Similar to the case of G s , different results have been reported concerning the use of FK. Although several groups failed to detect cAMP elevation when the sperm were exposed to FK (8,13,22,27,28,68), others have shown that FK increased cAMP levels in sperm (15,17,34). Consistent with these last studies, Leclerc and Kopf (20,69) reported that FK significantly increased adenylyl cyclase activity in membrane fractions of capacitated mouse sperm.…”

“…the Kopf and Garbers laboratories) conclusively demonstrated the presence of G␣ i in mouse sperm (32,33). However, similar to the situation with tmACs, the presence of stimulatory G␣ s in mammalian sperm has been controversial with conflicting reports for (34) or against (26) its presence in these cells.…”

Mechanisms of action of angiotensin II on mammalian sperm function

“…Current evidence suggests that the binding of FPP, calcitonin and adenosine to their respective receptors results in a G protein-mediated regulation of mAC, with the stimulatory responses involving Ga s and the inhibitory responses involving Ga i (possibly Ga i2 ); both Ga s and Ga i2 are found in the same regions as those receptors (Baxendale & Fraser 2003a). Immunolocalization studies have identified the presence of several mACs, with mAC2, 3 and 8 being most abundant and mAC1 and 4 less so; mAC3 and 8 were located in both the acrosomal cap region and in the flagellum where calcitonin and adenosine receptors are also found (Baxendale & Fraser 2003b).…”

Phosphodiesterase 11 (PDE11) regulation of spermatozoa physiology

“…The recently identified human testicular olfactory receptor, hOR17-4, 28 and the cAMP-regulated Ca 2 þ ion channel expressed specifically in the sperm, termed CatSper, [29][30][31] are important components of the fertilization process and may play significant roles in cAMP-mediated regulation of capacitance. In spermatozoa, ligand-odorant activation of hOR17-4, with compounds such as bourgeonal, lilial and floralozone, stimulates mAC 32 and sAC 33 via neuronal and endothelial NO release. An intracellular rise in cAMP activates CatSper, leading to increased intracellular Ca 2 þ levels, membrane depolarization and the generation of action potentials essential for capacitance.…”