Apical membranes prepared by peeling from whole porcine oviducts interact with homologous sperm
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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.
KeywordsBoar sperm Peeling Oviduct Apical membrane Biotinylation
We thank Dr. Laura Trumper for providing antibodies anti-Na+/K+-ATPase α1 and Dr. Volker Gerke for providing anti-annexin A2 antibodies. We also thank Frigorífico Paladini SA for the oviducts. This work was supported in part by the ANPCyT-BID program PICT 01–15092 of Argentina. Patricia E. Marini is a member of the research career CIC-UNR.
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(AVI 27909 kb)
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