Fibronectin regulates sperm-oviduct interaction through endocannabinoid system in bovines

CLAUDIA ELENA OSYCKA SALUT

Woods Hole, Massachusetts

Simposio; Frontiers in Reproduction - Eighteenth Annual Symposium; 2015

Marine Biological Laboratory (MBL)

The mammalian oviduct acts as a functional sperm reservoir providing an environment that allows maintenance and competition for fertilization of the oocyte. In different species, spermatozoa attach to epithelial cells in the lower region of the oviduct until ovulation associated signals induce their release allowing the transit to the upper region of the oviduct where fertilization occurs. The binding and release of spermatozoa from the oviductal epithelium are modulated mainly by the sperm capacitation. Different molecules present in the oviductal fluid regulate this interaction. Fibronectin (Fn) is a glycoprotein from extracellular matrix produced from a single gene, but the alternative splicing of its mRNA lead to the formation of several isoforms: soluble (sFN) and cellular (cFn) forms. This glycoprotein binds to integrins. Fibronectin and integrins are involved in several reproductive functions. In other biological systems, Fn modulates endocannabinoid system.Anandamide (AEA), a major endocannabinoid, is synthesized and degraded by the enzymes, NAPE-PLD and FAAH respectively. It binds to cannabinoid and vanilloid receptors (CB1, CB2 and TRPV1). Anandamide promotes sperm release from bovine oviductal epithelial cells and sperm capacitation, by activation of CB1 and TRPV1. Also, an increase of nitric oxide (NO) levels is involved in these pathways.We first studied the expression of Fn in the bovine oviduct during the estrous cycle. We observed that bovine oviductal epithelial cells differentially express all mRNA isoforms of Fn (sFn and cFn). Fibronectin was localized in the apical region of the epithelial cells and sFn levels fluctuated during the estrous cycle in the oviductal fluid. Using in vitro co-cultures of oviductal epithelial cells with spermatozoa, we found that spermatozoa are attached to the oviductal epithelium through alpha 5 beta 1 sperm integrins. The incubation of co-cultures with sFn induced sperm release from the oviductal cells. In addition sFn induced sperm capacitation evaluated by CTC assay, LPC-induced acrosome reaction and induction of PKA substrates and p-Tyr phosphorylation. Then, we investigated the endocannabinoid system activation during capacitation by Fn in bovine spermatozoa.We have found that sFn induces sperm capacitation through CB1 and TRPV1 receptors. Immunocytochemistry assays revealed a co-localization of TRPV1 and alpha 5 beta 1 integrin in bovine sperm. We also observed changes in TRPV1 localization in spermatozoa during capacitation induced by sFn. Furthermore, FAAH activity was modulated in spermatozoa capacitated in vitro with sFn. We also measured the NO levels in spermatozoa incubated with sFn and capsazepin (a TRPV1 antagonist). Soluble Fn increased sperm NO levels during capacitation; the pre-incubation with capsazepin reversed the glycoprotein effect. Finally, sFn up-regulated the phosphorylation of the PKA substrates through TRPV1 activation.In conclusion, our results indicated that Fn regulates sperm-oviduct interaction and induces events associated to sperm capacitation through the activation of endocannabinoid system in bovines.