Cyclic AMP efflux through MRP4 regulates actin dynamics signalling pathway and sperm motility in bovines

Nicolás Chiarante,Nicolás Chiarante,Raquel Lottero-Leconte,Raquel Lottero-Leconte,Marcelo Miragaya,Carlos Davio,Carlos Davio,Claudia E Osycka-Salut,Claudia E Osycka-Salut,Camila Arroyo-Salvo,Agustín Yaneff,Agustín Yaneff,Jessica Plaza,Silvina Perez-Martinez,Carlos A I Alonso,Camila Arroyo-Salvo

doi:10.1038/s41598-020-72425-5

Abstract

Previously we demonstrated that multidrug resistance-associated protein 4 transporter (MRP4) mediates cAMP efflux in bovine spermatozoa and that extracellular cAMP (ecAMP) triggers events associated to capacitation. Here, we deepen the study of the role of MRP4 in bovine sperm function by using MK571, an MRP4 inhibitor. The incubation of spermatozoa with MK571 during 45 min inhibited capacitation-associated events. MRP4 was localized in post-acrosomal region and mid-piece at 15 min capacitation, while at 45 min it was mainly located in the acrosome. After 15 min, MK571 decreased total sperm motility (TM), progressive motility (PM) and several kinematic parameters. The addition of ecAMP rescued MK571 effect and ecAMP alone increased the percentage of motile sperm and kinematics parameters. Since actin cytoskeleton plays essential roles in the regulation of sperm motility, we investigated if MRP4 activity might affect actin polymerization. After 15 min capacitation, an increase in F-actin was observed, which was inhibited by MK571. This effect was reverted by the addition of ecAMP. Furthermore, ecAMP alone increased F-actin levels while no F-actin was detected with ecAMP in the presence of PKA inhibitors. Our results support the importance of cAMP efflux through MRP4 in sperm capacitation and suggest its involvement in the regulation of actin polymerization and motility.

Highlights

Homeostasis within the flagellar compartment, which is in contact with bicarbonate produced by the male’s seminal glands and the female’s reproductive fluids[9,10]
We have described that extracellular cyclic adenosine monophosphate (cAMP) is an inductor of bovine sperm capacitation[15] its role is not clear in the mouse g amete12. cAMP is a cyclic nucleotide involved in numerous signal transduction pathways in different reproductive processes such as spermatogenesis, sperm maturation and sperm capacitation[4]
A link between protein kinase A (PKA) activity and intracellular cAMP regulation through MRP4 has been suggested in fibroblasts, since it has been demonstrated that MRP4 blockade promotes focal PKA stimulation by punctual cAMP a ccumulation[32]

Summary

Introduction

Homeostasis within the flagellar compartment, which is in contact with bicarbonate produced by the male’s seminal glands and the female’s reproductive fluids[9,10]. We have investigated the role of cAMP efflux through MRP4 in sperm capacitation in these two species. The phosphorylation / dephosphorylation regulation of proteins is known to be essential for sperm capacitation and motility. PKA binds A-kinase anchor proteins (AKAP) and phosphorylates motility regulatory proteins in the principal piece of the tail. Under physiological conditions, PKA has been identified as the main regulator of actin polymerization[26]. The relevance of this pathway in reproduction has been proven as genetically modified mice not expressing the catalytic ( Cs) subunit of PKA were found to be infertile[27]. Actin polymerization in the sperm tail during capacitation is essential to modulate motility[30]

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Conclusion