Extracellular Regulation of Sperm Transmembrane Adenylyl Cyclase by a Forward Motility Stimulating Protein

Abstract

Forward motility stimulating factor (FMSF), a glycoprotein isolated from buffalo serum, binds to the surface of the mature sperm cells to promote their progressive motility. This article reports the mode of signal transduction of this extracellular factor in goat sperm. The mechanism was investigated by assaying intracellular second messenger level and forward motility in presence of different pharmacological modulators. Mg++-dependent Forskolin responsive form of transmembrane adenylyl cyclase (tmAC) of goat spermatozoa was probed for its involvement in FMSF action. Dideoxyadenosine, a selective inhibitor of tmACs, was used to identify the role of this enzyme in the scheme of FMSF-signaling. Involvement of the α-subunit of G-protein in this regard has been inspected using GTPγS. Participation of protein kinase A (PKA) and tyrosine kinase was checked using IP20 and genistein, respectively. FMSF promotes tmAC activity in a dose-dependent manner through receptor/G-protein activation to enhance intracellular cAMP and forward motility. Motility boosting effects of this glycoprotein are almost lost in presence of dideoxyadenosine. But, FMSF displayed substantial motility promoting activity when movement of spermatozoa was inhibited with KH7, the specific inhibitor of soluble adenylyl cyclase indicating tmAC to be the primary target of FMSF action. Involvement of cAMP in mediating FMSF action was confirmed by the application of dibutyryl cAMP. Observed motility regulatory effects with IP20 and genistein indicate contribution of PKA and tyrosine kinase in FMSF activity; enhanced phosphorylation of a tyrosine containing ≈50 kDa protein was detected in this regard. FMSF initiates a novel signaling cascade to stimulate tmAC activity that augments intracellular cAMP, which through downstream crosstalk of phosphokinases leads to enhanced forward motility in mature spermatozoa. Thus, this article for the first time describes conventional tmAC-dependent profound activation of progressive motility by a physiologic extracellular factor in a mammalian species.

International Union of Basic and Clinical Pharmacology. CI. Structures and Small Molecule Modulators of Mammalian Adenylyl Cyclases

“…(6)-2-(1H-Benzimidazol-2-ylthio)propanoic acid 2-[(5-bromo-2-hydroxyphenyl)methylene]hydrazide (KH7) is the most widely used sAC inhibitor in intact-cell studies (Kumar et al, 2009;Schmid et al, 2010;Kolodecik et al, 2012;Bitterman et al, 2013;Dey et al, 2014;Hasan et al, 2014;Nickols et al, 2015;De Rasmo et al, 2015;Sosa et al, 2016). Caution must be exerted when Pharmacological Modulation of Mammalian Adenylyl Cyclases interpreting intact-cell studies with KH7 because of toxic effects of this compound in mitochondria (Di Benedetto et al, 2013) and b cells (Tian et al, 2011).…”

Major regulatory mechanisms involved in sperm motility

“…Both ACs are known to play an important role in male fertility. Transmembrane AC is involved in the basic mechanism for motility activation through cAMP-dependent protein phosphorylation and in progressive motility 56. Soluble AC is the predominant adenylyl cyclase responsible for the generation of most cAMP in spermatozoa and plays a critical role in cAMP signaling and is involved in the increase in beat frequency in spermatozoa 78.…”

Roles of glycogen synthase kinase 3 alpha and calcineurin in regulating the ability of sperm to fertilize eggs

“…30,31 The cells were washed twice with PBS and finally fluorescence was measured using BD FACSAria with FSC and SSC voltages being 220 and 350, respectively. Sperm populations incubated under capacitating medium with or without FK506 were used for IVF with oocytes collected from super-ovulated mice.…”