Fibronectin induces capacitation-associated events through the endocannabinoid system in bull sperm.

Mammalian ejaculated spermatozoa must undergo a series of changes in the female reproductive tract, collectively called capacitation, in order to fertilize the oocyte. We reported that fibronectin (Fn), a glycoprotein from the extracellular matrix, and anandamide (AEA), one of the major members of the endocannabinoid family, are present in the bovine oviductal fluid and regulate bull sperm function. Also, AEA induces bovine sperm capacitation, through CB1 and TRPV1 receptors. In this work, we investigated if Fn induces bovine sperm capacitation thought the activation of the endocannabinoid system in this process. We incubated sperm with Fn (100 µg/ml) and/or capsazepine, a TRPV1 antagonist (0.1 µM) and some events related to sperm capacitation such as LPC-induced acrosome reaction, sperm-release from the oviduct, induction of PKA phosphorylated substrates (pPKAs) and protein tyrosine phosphorylation (pY) and nitric oxide (NO) production were assessed. Also, we studied the activity of fatty acid amide hydrolase (FAAH), the enzyme that degrades AEA. We found that Fn, via α5ß1 integrin, induced capacitation-associated events. Also, Fn stimulated signaling pathways associated to capacitation as cAMP/PKA and NO/NO synthase. Moreover, Fn decreased the FAAH activity and this correlated with sperm capacitation. Capsazepine reversed fibronectin-induced capacitation, and pPKAs and NO levels. The incubation of spermatozoa with R-methanandamide (1.4 nM), a stable analogue of AEA, increased cAMP and pPKAs levels. The presence of H89 (50 µM) or KT5720 (100 nM) (PKA inhibitors) prevented AEA-induced capacitation. In addition, R-methanandamide and capsaicin (0.01 µM), a TRPV1 agonist, increased NO production via the PKA pathway. These results indicate that Fn, through α5ß1, supports capacitation in bovine spermatozoa. This effect is dependent on the activation of TRPV1 through cAMP/PKA and NO signaling pathways. We propose that Fn could be considered as a new agent that promotes sperm capacitation in bull sperm. Our findings contribute to better understand the significance of Fn signaling in the capacitating events that lead to successful fertilization and embryo development in mammals including humans.

MRP4-mediated cAMP efflux is essential for mouse spermatozoa capacitation.

Mammalian spermatozoa must undergo biochemical and structural changes to acquire the capacity for fertilization, in a process known as capacitation. Activation of PKA enzymes is essential for capacitation, and thus cAMP levels are tightly regulated during this process. Previously, we demonstrated that during capacitation, bovine spermatozoa extrude cAMP through multidrug resistance-associated protein 4 (MRP4, also known as ABCC4), which regulates intracellular levels of the nucleotide and provides cAMP to the extracellular space. Here, we report the presence of functional MRP4 in murine spermatozoa, since its pharmacological inhibition with MK571 decreased levels of extracellular cAMP. This also produced a sudden increase in PKA activity, with decreased tyrosine phosphorylation at the end of capacitation. Blockade of MRP4 inhibited induction of acrosome reaction, hyperactivation and in vitro fertilization. Moreover, MRP4 inhibition generated an increase in Ca2+ levels mediated by PKA, and depletion of Ca2+ salts from the medium prevented the loss of motility and phosphotyrosine inhibition produced by MK571. These results were supported using spermatozoa from CatSper Ca2+ channel knockout mice. Taken together, these results suggest that cAMP efflux via MRP4 plays an essential role in mouse sperm capacitation.This article has an associated First Person interview with the first author of the paper.

Molecular changes and signaling events occurring in spermatozoa during epididymal maturation.

After leaving the testis, spermatozoa have not yet acquired the ability to move progressively and are unable to fertilize oocytes. To become fertilization competent, they must go through an epididymal maturation process in the male, and capacitation in the female tract. Epididymal maturation can be defined as those changes occurring to spermatozoa in the epididymis that render the spermatozoa the ability to capacitate in the female tract. As part of this process, sperm cells undergo a series of biochemical and physiological changes that require incorporation of new molecules derived from the epididymal epithelium, as well as post-translational modifications of endogenous proteins synthesized during spermiogenesis in the testis. This review will focus on epididymal maturation events, with emphasis in recent advances in the understanding of the molecular basis of this process.

Sperm Release From the Oviductal Epithelium Depends on Ca(2+) Influx Upon Activation of CB1 and TRPV1 by Anandamide.

The oviduct acts as a functional sperm reservoir in many mammalian species. Both binding and release of spermatozoa from the oviductal epithelium are mainly modulated by sperm capacitation. Several molecules from oviductal fluid are involved in the regulation of sperm function. Anandamide is a lipid mediator involved in reproductive physiology. Previously, we demonstrated that anandamide, through activation of the cannabinoid receptor type 1 (CB1), promotes sperm release from bovine oviductal epithelial cells, and through CB1 and the transient receptor potential vanilloid 1 (TRPV1), induces sperm capacitation. Herein we investigate co-activation between CB1 and TRPV1, and Ca(2+) influx as part of the mechanism of action of anandamide during sperm release from oviductal cells. Our results indicate that in the absence of Ca(2+) anandamide failed to release spermatozoa from oviductal epithelial cells. Additionally, sperm release promoted by cannabinoid and vanilloid agonists was abolished when the spermatozoa were preloaded with BAPTA-AM, a Ca(2+) chelator. We also determined Ca(2+) levels in spermatozoa preloaded with FURA2-AM co-cultured with oviductal cells and incubated with different cannabinoid and vanilloid agonists. The incubation with different agonists induced Ca(2+) influx, which was abolished by CB1 or TRPV1 antagonists. Our results also suggest that a phospholypase C (PLC) might mediate the activation of CB1 and TRPV1 in sperm release from the bovine oviduct. Therefore, our findings indicate that anandamide, through CB1 and TRPV1 activation, is involved in sperm release from the oviductal reservoir. An increase of sperm Ca(2+) levels and the PLC activation might be involved in anandamide signaling pathway.