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 µM), 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.

Gain-of-function phenotypes of many CLAVATA3/ESR genes, including four new family members, correlate with tandem variations in the conserved CLAVATA3/ESR domain.

Secreted peptide ligands are known to play key roles in the regulation of plant growth, development, and environmental responses. However, phenotypes for surprisingly few such genes have been identified via loss-of-function mutant screens. To begin to understand the processes regulated by the CLAVATA3 (CLV3)/ESR (CLE) ligand gene family, we took a systems approach to gene identification and gain-of-function phenotype screens in transgenic plants. We identified four new CLE family members in the Arabidopsis (Arabidopsis thaliana) genome sequence and determined their relative transcript levels in various organs. Overexpression of CLV3 and the 17 CLE genes we tested resulted in premature mortality and/or developmental timing delays in transgenic Arabidopsis plants. Overexpression of 10 CLE genes and the CLV3 positive control resulted in arrest of growth from the shoot apical meristem (SAM). Overexpression of nearly all the CLE genes and CLV3 resulted in either inhibition or stimulation of root growth. CLE4 expression reversed the SAM proliferation phenotype of a clv3 mutant to one of SAM arrest. Dwarf plants resulted from overexpression of five CLE genes. Overexpression of new family members CLE42 and CLE44 resulted in distinctive shrub-like dwarf plants lacking apical dominance. Our results indicate the capacity for functional redundancy of many of the CLE ligands. Additionally, overexpression phenotypes of various CLE family members suggest roles in organ size regulation, apical dominance, and root growth. Similarities among overexpression phenotypes of many CLE genes correlate with similarities in their CLE domain sequences, suggesting that the CLE domain is responsible for interaction with cognate receptors.