Apical membranes prepared by peeling from whole porcine oviducts interact with homologous sperm

Abstract

In mammals, interaction between sperm and oviductal epithelial cells provides the formation of a sperm reservoir and sperm selection at the isthmus of the oviduct. Several in vitro methods are used to study this interaction. Apical plasma membranes (APM) have been prepared by peeling from culture and differentiated kidney cells. In this work, we modify this method, using it for the preparation of APM directly from the whole oviduct, proving purity of the apical plasma membranes obtained by western blot for proteins of known specific locations. The obtained APM correspond only to the most differentiated cells, exposed at the lumen of the organ. Also, the prepared APM are shown by biotinylation to interact with sperm. The binding is at the head of sperm and induces on them prolonged motility and tyrosine phosphorylation of proteins of masses 92, 97, 210 and 220 kDa. The tyrosine phosphorylation of p97 has been previously described as an effect of the apical membrane exposed sperm binding glycoprotein (SBG), which is shown to be present in the preparations described here. Upon treatment with APM, the tyrosine phosphorylation pattern of sperm changes from heads to tail. Thus, we describe an easy method for APM preparation directly from organs that allows the study of oviductal proteins in their context and permits sperm-oviduct interaction studies. This method renders APM specifically from the cells located at the lumen of the oviduct.

Characterizations of annexin A1-interacting proteins in apical membrane and cytosolic compartments of renal tubular epithelial cells

“…Moreover, as the peeling method is free of any chemicals during the isolation processes, the physico-chemical properties of apical membrane proteins should be better preserved as compared with the precipitation-based methods, in which chemicals may introduce modifications to the proteins and interfere with PPI [41] , [42] . In addition, the peeling method has been proven to be very effective for many other studies to isolate and investigate the apical membrane proteins in several various aspects [34] , [37] , [43] , [44] , [45] , [46] , [47] .…”

Review: The epic journey of sperm through the female reproductive tract

“…The reservoir also releases a finite number of stored sperm, acting as a buffer for sperm number to prevent polyspermy but still provide an appropriate number of fertile sperm to the upper oviduct (Hunter and Leglise, 1971). The isthmic epithelium binds and retains preferentially sperm that have intact acrosomes and normal morphology (Teijeiro et al, 2011;Teijeiro and Marini, 2012). All together, the isthmus functions to increase the probability that a suitable number of fertile sperm are present at the site of fertilization.…”