Abstract
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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Abbreviations
- ADA:
-
Adenosine deaminase
- AP:
-
Alkaline phosphatase
- α,β-meADP:
-
Alpha, beta-methylene adenosine 5′-diphosphate
- E-NTPDase:
-
Ectonucleoside triphosphate diphosphohydrolase
- PLAP:
-
Placental alkaline phosphatase
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Acknowledgements
This work was supported by a grant from the Instituto de Salud Carlos III (FIS PI15/00036), co-funded by FEDER funds/European Regional Development Fund (ERDF) “a Way to Build Europe”-// FONDOS FEDER “una manera de hacer Europa”, and a grant from the Fundación Merck Salud (Ayuda Merck de Investigación 2016-Fertilidad). ARM was awarded a fellowship from the Asociación Española Contra el Cáncer (AECC). JS received support from the Canadian Institutes of Health Research (CIHR) and was the recipient of a Chercheur National Scholarship from the Fonds de Recherche du Québec–Santé (FRQS). We are grateful for the technical support of Serveis Científics i Tecnològics, Campus Bellvitge, Universitat de Barcelona. The authors thank Tom Yohannan for language editing.
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418_2017_1627_MOESM1_ESM.tif
Supplementary material 1. Supplementary Fig. 1 A Secondary antibody controls for immunofluorescence (a) and immunohistochemistry (b) experiments. In these experiments primary antibody was omitted to determine the background level due to nonspecific secondary antibody binding. Nuclei were labeled with DAPI (a) or hematoxylin (b). Insets show the results of the experiments that included primary antibodies (anti-NTPDase2 in a and anti-CD73 in b) Note the clean background obtained with both techniques. Scale bars 25 µm (a) and 100 µm (b). B Histochemistry controls in consecutive slides incubated with the same buffers and washing steps but omitting the nucleotide to visualize the background of the technique when samples included PBS (a) on not (b) in the initial steps. Note the clean background obtained in both cases. Scale bars 100 µm (TIF 5565 KB)
418_2017_1627_MOESM2_ESM.tif
Supplementary material 2. Supplementary Fig. 2 Confocal fluorescence images of the mucosa of human oviducts with antibodies against CD26 (a) and CD73 (b). a CD26 receptor expression was detected in the secretory, non-ciliated, epithelial cells. b CD73 was abundantly immunodetected on the apical side of ciliated epithelial cells. Nuclei were labeled with To-Pro-3 (c). Merge image shows that CD26 staining and CD73 staining were mutually exclusive (d). Scale bar 40 µm (TIF 5766 KB)
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Villamonte, M.L., Torrejón-Escribano, B., Rodríguez-Martínez, A. et al. Characterization of ecto-nucleotidases in human oviducts with an improved approach simultaneously identifying protein expression and in situ enzyme activity. Histochem Cell Biol 149, 269–276 (2018). https://doi.org/10.1007/s00418-017-1627-8
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DOI: https://doi.org/10.1007/s00418-017-1627-8