Strong inhibition of neutrophil-sperm interaction in cattle by selective phosphatidylinositol 3-kinase inhibitors

The vast majority of sperm are lost from the female reproductive tract in hours following natural mating or artificial insemination in mammals. Multiple complex processes including uterine contractions, mucus barriers, and phagocytosis of sperm by neutrophils have been reported to be involved in the sperm loss, although the contribution of each process is uncertain. If phagocytosis by neutrophils has a significant role in sperm loss, inhibition of neutrophil response to sperm could potentially reduce the dose of sperm required for artificial insemination. Through the development of a quantitative in vitro assay, we have screened 74 candidate compounds for their ability to inhibit the neutrophil-sperm interaction in cattle. Nine inhibitors (GSK2126458, wortmannin, ZSTK474, PIK294, CAL-101, GSK 1059615, GDC-0941, PIK 90 and PI103) active against phosphatidylinositol 3-kinase (PI3-kinase) were most potent, and strongly reduced neutrophil-sperm interaction with an IC50 of 10 nM or less. These inhibitors did not significantly modify sperm motility, and five of the inhibitors did not affect in vitro fertilization. Examination of neutrophil-sperm interaction by time-lapse video microscopy and cell tracking analysis revealed that GSK2126458 may prevent sperm phagocytosis through inhibition of neutrophil movement and/or attachment. Twenty-four other compounds exhibited weaker inhibition (IC50 < 115 mu M), and the rest did not inhibit the neutrophil-sperm interaction. Strong PI3-kinase inhibitors identified in this study may be useful to determine the contribution of neutrophil phagocytosis in the clearance of sperm from the female reproductive tract. Summary Sentence Neutrophil-sperm interaction is strongly inhibited by selective PI3-kinase inhibitors at nanomolar concentrations.