Extracellular ATP and its hydrolysis product adenosine modulate various reproductive functions such as those taking place
in oviducts, including contraction, beating of cilia, and maintenance of fluid composition that, in turn, influences sperm
capacitation and hyperactivation, as well as oocyte and embryo nourishing. Ecto-nucleotidases are the enzymes that regulate
extracellular ATP and adenosine levels, thus playing a role in reproduction. We have optimized a convenient method for
characterizing ecto-nucleotidases that simultaneously localizes the protein and its associated enzyme activity in the same
tissue slice and characterizes ecto-nucleotidases in human oviducts. The technique combines immunofluorescence and in situ
histochemistry, allowing precise identification of ecto-nucleotidases at a subcellular level. In oviducts, remarkably, ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) and NTPDase3, with the ability to hydrolyze ATP to AMP, are
expressed in ciliated epithelial cells but with different subcellular localization. Ecto-5′nucleotidase/CD73 is also expressed
apically in ciliated cells. CD73, together with alkaline phosphatase, also expressed apically in oviductal epithelium, complete
the hydrolysis sequence by dephosphorylating AMP to adenosine. The concerted action of these enzymes would contribute
to the local increase of adenosine concentration necessary for sperm capacitation. The use of this method would be an asset
for testing new potential therapeutic drugs with inhibitory potential, which is of great interest presently in the field of oncology and in other clinical disciplines.

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• Characterization of ecto-nucleotidases in human oviducts with an improved approach simultaneously identifying protein expression and in situ enzyme activity