Extracellular cAMP activates molecular signalling pathways associated to sperm capacitation in bovines

ALONSO, CARLOS AGUSTÍN I.; OSYCKA-SALUT, CLAUDIA E.; CASTELLANO, LUCIANA; CESARI, ANDREINA; DI SIERVI, NICOLÁS; MUTTO, ADRIÁN; JOHANNISSON, ANDERS; MORRELL, JANE M.; DAVIO, CARLOS; PEREZ-MARTINEZ, SILVINA

MOLECULAR HUMAN REPRODUCTION.

OXFORD UNIV PRESS

Lugar: Oxford; Año: 2017 vol. 23 p. 521 - 534

1360-9947

STUDY QUESTION: Is extracellular cAMP involved in the regulation of signalling pathways in bovine sperm capacitation?SUMMARY ANSWER: Extracellular cAMP induces sperm capacitation through the activation of different signalling pathways that involvephospholipase C (PLC), PKC/ERK1-2 signalling and an increase in sperm Ca2+ levels, as well as soluble AC and cAMP/protein kinase A(PKA) signalling.WHAT IS KNOWN ALREADY: In order to fertilize the oocyte, ejaculated spermatozoa must undergo a series of changes in the femalereproductive tract, known as capacitation. This correlates with a number of membrane and metabolic modifications that include an increasedinflux of bicarbonate and Ca2+, activation of a soluble adenylyl cyclase (sAC) to produce cAMP, PKA activation, protein tyrosine phosphorylationand the development of hyperactivated motility. We previously reported that cAMP efflux by Multidrug Resistance Protein 4 (MRP4)occurs during sperm capacitation and the pharmacological blockade of this inhibits the process. Moreover, the supplementation of incubationmedia with cAMP abolishes the inhibition and leads to sperm capacitation, suggesting that extracellular cAMP regulates crucial signalling cascadesinvolved in this process.STUDY DESIGN, SIZE, DURATION: Bovine sperm were selected by the wool glass column method, and washed by centrifugation inBSA-Free Tyrode?s Albumin Lactate Pyruvate (sp-TALP). Pellets were resuspended then diluted for each treatment. For in vitro capacitation,10 to 15 × 106 SPZ/ml were incubated in 0.3% BSA sp-TALP at 38.5°C for 45 min under different experimental conditions. To evaluate therole of extracellular cAMP on different events associated with sperm capacitation, 10 nM cAMP was added to the incubation medium as wellas different inhibitors of enzymes associated with signalling transduction pathways: U73122 (PLC inhibitor, 10 μM), Gö6983 (PKC inhibitor,10 μM), PD98059 (ERK-1/2 inhibitor, 30 μM), H89 and KT (PKA inhibitors, 50 μM and 100 nM, respectively), KH7 (sAC inhibitor, 10 μM),BAPTA-AM (intracellular Ca2+ chelator, 50 μM), EGTA (10 μM) and Probenecid (MRPs general inhibitor, 500 μM). In addition, assays forbinding to oviductal epithelial cells and IVF were carried out to test the effect of cAMP compared with other known capacitant agents such asheparin (60 μg/ml) and bicarbonate (40 mM).PARTICIPANTS/MATERIALS, SETTING, METHODS: Straws of frozen bovine semen (20?25 × 106 spermatozoa/ml) were kindlyprovided by Las Lilas, CIALE and CIAVT Artificial Insemination Centers. The methods used in this work include western blot, immunohistochemistry,flow cytometry, computer-assisted semen analysis, live imaging of Ca2+ and fluorescence scanning. At least three independentassays with bull samples of proven fertility were carried.MAIN RESULTS AND THE ROLE OF CHANCE: In the present study, we elucidate the molecular events induced by extracellularcAMP. Our results showed that external cAMP induces sperm capacitation, depending upon the action of PLC. Downstream, this enzymeincreased ERK1-2 activation through PKC and elicited a rise in sperm Ca2+ levels (P < 0.01). Moreover, extracellular cAMP-induced capacitationalso depended on the activity of sAC and PKA, and increased tyrosine phosphorylation, indicating that the nucleotide exerts a broad rangeof responses. In addition, extracellular cAMP-induced sperm hyperactivation and concomitantly increased the proportion of spermatozoa withhigh mitochondrial activity (P < 0.01). Finally, cAMP increased the in vitro fertilization rate compared to control conditions (P < 0.001).LARGE SCALE DATA: None.LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study performed with bovine cryopreserved spermatozoa. Studies inother species and with fresh samples are needed to extrapolate these data.WIDER IMPLICATIONS OF THE FINDINGS: These findings strongly suggest an important role of extracellular cAMP in the regulationof the signalling pathways involved in the acquisition of bull sperm fertilizing capability. The data presented here indicate that not only a rise,but also a regulation of cAMP levels is necessary to ensure sperm fertilizing ability. Thus, exclusion of the nucleotide to the extracellular spacemight be essential to guarantee the achievement of a cAMP tone, needed for all capacitation-associated events to take place. Moreover, theability of cAMP to trigger such broad and complex signalling events allows us to hypothesize that cAMP is a self-produced autocrine/paracrinefactor, and supports the emerging paradigm that spermatozoa do not compete but, in fact, communicate with each other. A precise understandingof the functional competence of mammalian spermatozoa is essential to generate clinical advances in the treatment of infertility andthe development of novel contraceptive strategies.STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by Consejo Nacional de Investigaciones Científicas yTécnicas [PIP0 496 to S.P.-M.], Agencia Nacional de Promoción Científica y Tecológica [PICT 2012-1195 and PICT2014-2325 to S.P.-M.,and PICT 2013-2050 to C.D.], Boehringer Ingelheim Funds, and the Swedish Farmers Foundation [SLF-H13300339 to J.M.]. The authorsdeclare there are no conflicts of interests.