Localization of plasma membrane bound NTPDases in the murine reproductive tract

Abstract

Extracellular nucleotides might influence aspects of the biology of reproduction in that ATP affects smooth muscle contraction, participates in steroidogenesis and spermatogenesis, and also regulates transepithelial transport, as in oviducts. Activation of cellular nucleotide purinergic receptors is influenced by four plasma membrane-bound members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family, namely NTPDase1, NTPDase2, NTPDase3, and NTPDase8 that differ in their ecto-enzymatic properties. The purpose of this study was to characterize the expression profile of the membrane-bound NTPDases in the murine female and male reproductive tracts by immunological techniques (immunolabelling, Western blotting) and by enzymatic assays, in situ and on tissue homogenates. Other than the expected expression on vascular endothelial and smooth muscle cells, NTPDase1 was also detected in Sertoli cells and interstitial macrophages in testes, in ovarian granulosa cells, and in apical cells from epididymal epithelium. NTPDase2 was largely expressed by cells in the connective tissue; NTPDase3 in secretory epithelia, and finally, NTPDase8 was not detected in any of the tissues studied here. In addition, NTPDase6 was putatively detected in Golgi-phase acrosome vesicles of round spermatids. This descriptive study suggests close regulation of extracellular nucleotide levels in the genital tract by NTPDases that may impact specific biological functions.

Ecto-nucleotidases distribution in human cyclic and postmenopausic endometrium

“…Maximal score is found in secretory endometria NTPDase3 is expressed in luminal and glandular epithelia. NTPDase3 was already identified in other secretory epithelial cells from mouse reproductive organs such as epididymis, prostate and oviducts [27,43]. We report here for the first time the expression of NTPDase3 in relation to blood vessels.…”

“…A few studies have been conducted in mice; NTPDase1 and 2 were identified in myometrium [27], and ecto-5′-nucleotidase in endometrium, where the expression and activity fluctuate along the estrous cycle and with pregnancy, pointing to hormonal regulation of extracellular adenosine levels in this organ [28,29]. However, to our knowledge, there are no available data concerning the expression of ecto-nucleotidases in human endometrium and their possible changes along the cycle.…”

Purinergic signalling in the reproductive system in health and disease

“…There are earlier reviews that include discussion of the involvement of nucleotides and nucleosides in the complex innervation of the male reproductive system ( [14,157]; see also [66]). There are also studies of P2X receptor immunoreactivity in male reproductive organs [227] and of the localisation of plasma membrane-bound NTPDases [260]. A high expression and activity of ecto-5′ nucleotidase (CD73) in the male murine reproductive tract has also been reported that may impact male fertility [261].…”

Identification of the ectonucleotidases expressed in mouse, rat, and human Langerhans islets: potential role of NTPDase3 in insulin secretion

“…All primary antibodies used in this study have previously been validated: rabbit C9F (14,21) and guinea pig mN1-2 C (40) to mouse NTPDase1, rabbit mN2-36 L to mouse NTPDase2 (3), rabbit BZ3-4F to rat NTPDase2 (21,53), guinea pig mN3-3C to mouse NTPDase3 (40), rabbit rN3-1L to rat NTPDase3 (57), mouse monoclonal hN3-H10S to human NTPDase3 (42), guinea pig rN8-8C to rat NTPDase8 (15), rabbit rNU-9L (30) and guinea pig rNU-4C to rat ecto-5=-nucleotidase/CD73 (16), a commercially available mouse monoclonal antibody to human ecto-5=-nucleotidase (clone 4G4; HyCult Biotechnology, Uden, The Netherlands) and polyclonal antibodies specific for pancreatic hormones, both guinea pig anti-insulin and anti-pancreatic polypeptide (Millipore, St. Charles, MO), rabbit anti-glucagon (BioGenex, San Ramon, CA), and goat anti-somatostatin (Santa Cruz Biotechnology, Santa Cruz, CA). Secondary antibodies used were biotinylated goat anti-guinea pig and goat anti-rabbit (Jackson ImmunoResearch Laboratories, West Grove, PA) as well as goat anti-mouse (Vector Laboratories, Burlington, ON, Canada); Alexa fluor 594 donkey anti-rabbit, Alexa fluor 594 goat anti-guinea pig, Alexa fluor 633 goat anti-mouse, Alexa fluor 488 goat anti-rabbit, and Alexa fluor 488 rabbit anti-goat (Invitrogen); FITC-conjugated goat anti-guinea pig (Jackson ImmunoResearch Laboratories) and goat anti-rabbit (Santa Cruz Biotechnology); and finally, an unconjugated goat antiguinea pig (Vector Laboratories).…”

“…Ectonucleotidase activities in pancreas sections were visualized using the Wachstein/Meisel lead phosphate precipitation method, performed as described (40). Briefly, fixed tissue sections were preincubated for 30 min at 25°C in Tris-maleate buffer (2 mM CaCl 2, 250 mM sucrose, 50 mM Tris-maleate, pH 7.4) supplemented with 2.5 mM levamisole as an inhibitor of alkaline phosphatases.…”