Multidrug Resistance Protein 4 (MRP4/ABCC4) is present in mammalian sperm and is involved in the acquisition of fertilizing ability.

BUFFONE M; CASTELLANO L; ALONSO CAI; PEREZ MARTINEZ S; DI SIERVI N; PUGA-MOLINA L; DAVIO C; LOTTERO-LECONTE R; OSYCKA-SALUT C

Conferencia; Gordon Research Conference: 2017 Fertilization & Activation of Development; 2017

To fertilize the oocyte, mammalian spermatozoa must undergo a series of biochemical and structural changes in the female tract, known as capacitation. This process correlates with a HCO3- and Ca2+ influx, an activation of soluble adenylyl cyclase (sAC), a cAMP increase and PKA activation, and the increase in tyrosine phosphorylation (pTyr), among others. Although intracellular cAMP levels are mainly regulated by the nucleotide synthesis and degradation, in several tissues cAMP extrusion, through the multidrug resistance protein 4 (MRP4), is also involved in this modulation. In this sense, MRP4 has a dual role in the cell physiology, regulating intracellular cAMP levels, and providing this nucleotide to the extracellular space; this could trigger purinergic signalling events. Regardless of the broad importance of this transporter in other cell types, MRP4 has not been described in mammalian sperm yet.Therefore, the objective of the present work was to characterize cAMP efflux process by MRP4 and establish its role in sperm capacitation. To that end we performed in vitro sperm capacitation in three mammalian species: bovine (ejaculated/cryopreserved), human (ejaculated/fresh) and murine (epidydimal/fresh) spermatozoa, and the presence of MRP4 as well as capacitation-associated events were evaluated.Results showed that MRP4 is present in spermatozoa from the three species. At control (non-capacitating) conditions, MRP4 was mainly localized at the post-acrosomal region and the midpiece of bovine and human spermatozoa; whereas it was found in the principal piece of the flagellum in mouse spermatozoa. Interestingly, under capacitating conditions, most of the bovine spermatozoa presented MRP4 in the acrosomal region (in addition to the original protein location) and ∼ 40 % of human sperm showed MRP4 in the principal piece of the tail. However, a significant loss of label was observed in mice sperm, concomitantly with an augment in pTyr proteins.Regardless of MRP4 localization, cAMP was detected in the incubation media of spermatozoa suggesting that cAMP exclusion to the extracellular space occurred in the three species.To assess the role of MRP4 in sperm capacitation, we examined the effect of MK571 (a MRP4 inhibitor, 50 μM) on pTyr and phosphorylation of PKA substrates (pPKA) in sperm incubated under capacitating conditions. Results showed that bovine and human spermatozoashowed no change in the profile of total pTyr in the presence of MRP4 inhibitor. However, previous results indicated that MRP4 inhibitor blocks other capacitation related events, such as, LPC- induced acrosomal reaction and sperm release from oviductal cells in bovines. On the contrary, murine spermatozoa showed a significant decrease in the whole pTyr profile, with MRP4 inhibitor. Regarding pPKA, the incubation with MK571 produced an increase of these phosphorylated substrates in bovine and human spermatozoa; whereas a decrease of them was detected in murine spermatozoa.Altogether, these results indicate that MRP4 is present and is functional in mammalian spermatozoa, and that cAMP extrusion process might regulate sperm cAMP levels and play a significant role in the acquisition of fertilizing ability.