Abstract
Extracellular ATP and its hydrolysis product adenosine modulate various reproductive functions such as those requiring contraction, steroidogenesis, and maintenance of fluid composition. Interestingly, adenosine might act as a key capacitative effector for mammalian spermatozoa to acquire the capacity for fertilisation. Extracellular nucleotide levels are affected by cell surface ectonucleotidases, amongst which the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family regroups the most abundant and effective enzymes to hydrolyse ATP and ADP to AMP in physiological conditions. In the male reproductive tract three members of this family have been indentified: NTPDase1, NTPDase2 and NTPDase3 (Martín-Satué et al. in Histochem Cell Biol 131:615–628, 2009). The purpose of the present study was to characterize in the male reproductive tract the expression profile of the main enzyme responsible for the generation of adenosine from AMP, namely the ecto-5′-nucleotidase (CD73). The enzyme was identified by immunological techniques and by in situ enzymatic assays, including inhibition experiments with α,β-methylene-ADP, a specific CD73 inhibitor. High levels of ecto-5′-nucleotidase were detected in testes in association with both germinal and somatic cells, in smooth muscle cells throughout the tract, in secretory epithelia from exocrine glands, and remarkably, in principal cells of epididymis, where co-localization with NTPDase3 was found. The relevance of this co-expression on nucleotide hydrolysis in these cells directly involved in the control of sperm fluid composition was addressed biochemically. This study suggests close regulation of extracellular nucleoside and nucleotide levels in the genital tract by ecto-5′-nucleotidase that, in concurrence with NTPDases, may impact male fertility.
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Acknowledgements
This work was supported by grants to J. Sévigny from the Canadian Institutes of Health Research (CIHR) and to M. Martín-Satué from the University of Barcelona (ACESB09). M. Martín-Satué was recipient of a fellowship from the Spanish Ministry of Education and Science (MEC-Programa José Castillejo), E.G. Lavoie of a scholarship from the Fonds de Recherche en Santé du Québec (FRSQ) and J. Sévigny of a New Investigator award from the CIHR and of a Junior 2 scholarship from the FRSQ. Authors thank Benjamín Torrejón-Escribano from the Microscopy Unit of Serveis Cientificotècnics of the University of Barcelona (Bellvitge Campus) for his technical assistance. MF was a recipient of a scholarship from the government of Gabon.
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M. Martín-Satué and E. G. Lavoie contributed equally to the work.
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Supplementary Figure 1. Immunofluorescence (A) and western blot (B) of ecto-5’-nucleotidase in mouse sperm. A) Ecto-5’-nucleotidase was not detected in sperm smears (a). DAPI was used to label the nuclei (b). Scale bar 20 μm. B) Ecto-5’-nucleotidase was detected in total sperm homogenate and in the soluble fraction but not in spermatozoa alone. Five μg of particulate membrane protein fraction of epididymis were used as control for the presence of the protein (JPEG 64 kb)
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Supplementary Figure 2. Immunolocalization of ecto-5’-nucleotidase in mouse epididymis. Ecto-5’-nucleotidase was detected by immunofluorescence at the luminal surface of principal cells from epithelium in caput (A), corpus (B) and cauda (C) epididymis. Scale bar 75 μm. (JPEG 110 kb)
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Martín-Satué, M., Lavoie, E.G., Fausther, M. et al. High expression and activity of ecto-5′-nucleotidase/CD73 in the male murine reproductive tract. Histochem Cell Biol 133, 659–668 (2010). https://doi.org/10.1007/s00418-010-0704-z
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DOI: https://doi.org/10.1007/s00418-010-0704-z