Disrupted intercellular bridges and spermatogenesis in fatty acyl-CoA reductase 1 knockout mice: A new model of ether lipid deficiency.

Peroxisomal fatty acyl-CoA reductase 1 (FAR1) is a rate-limiting enzyme for ether lipid (EL) synthesis. Gene mutations in FAR1 cause a rare human disease. Furthermore, altered EL homeostasis has also been associated with various prevalent human diseases. Despite their importance in human health, the exact cellular functions of FAR1 and EL are not well-understood. Here, we report the generation and initial characterization of the first Far1 knockout (KO) mouse model. Far1 KO mice were subviable and displayed growth retardation. The adult KO male mice had smaller testes and were infertile. H&E and immunofluorescent staining showed fewer germ cells in seminiferous tubules. Round spermatids were present but no elongated spermatids or spermatozoa were observed, suggesting a spermatogenesis arrest at this stage. Large multi-nucleated giant cells (MGC) were found lining the lumen of seminiferous tubules with many of them undergoing apoptosis. The immunofluorescent signal of TEX14, an essential component of intercellular bridges (ICB) between developing germ cells, was greatly reduced and mislocalized in KO testis, suggesting the disrupted ICBs as an underlying cause of MGC formation. Integrative analysis of our total testis RNA-sequencing results and published single-cell RNA-sequencing data unveiled cell type-specific molecular alterations underlying the spermatogenesis arrest. Many genes essential for late germ cell development showed dramatic downregulation, whereas genes essential for extracellular matrix dynamics and cell-cell interactions were among the most upregulated genes. Together, this work identified the cell type-specific requirement of ELs in spermatogenesis and suggested a critical role of Far1/ELs in the formation/maintenance of ICB during meiosis.

Acidosis and proteolysis in the tumor microenvironment.

The glycolytic phenotype of the Warburg effect is associated with acidification of the tumor microenvironment. In this review, we describe how acidification of the tumor microenvironment may increase the invasive and degradative phenotype of cancer cells. As a template of an extracellular acidic microenvironment that is linked to proteolysis, we use the resorptive pit formed between osteoclasts and bone. We describe similar changes that have been observed in cancer cells in response to an acidic microenvironment and that are associated with proteolysis and invasive and metastatic phenotypes. This includes consideration of changes observed in the intracellular trafficking of vesicles, i.e., lysosomes and exosomes, and in specialized regions of the membrane, i.e., invadopodia and caveolae. Cancer-associated cells are known to affect what is generally referred to as tumor proteolysis but little direct evidence for this being regulated by acidosis; we describe potential links that should be verified.

Trophoblast adhesion of the peri-implantation mouse blastocyst is regulated by integrin signaling that targets phospholipase C.

Integrin signaling modulates trophoblast adhesion to extracellular matrices during blastocyst implantation. Fibronectin (FN)-binding activity on the apical surface of trophoblast cells is strengthened after elevation of intracellular Ca(2+) downstream of integrin ligation by FN. We report here that phosphoinositide-specific phospholipase C (PLC) mediates Ca(2+) signaling in response to FN. Pharmacological agents used to antagonize PLC (U73122) or the inositol phosphate receptor (Xestospongin C) inhibited FN-induced elevation of intracellular Ca(2+) and prevented the upregulation of FN-binding activity. In contrast, inhibitors of Ca(2+) influx through either voltage-gated or non-voltage-gated Ca(2+) channels were without effect. Inhibition of protein tyrosine kinase activity by genistein, but not G-protein inhibition by suramin, blocked FN-induced intracellular Ca(2+) signaling and upregulation of adhesion, consistent with involvement of PLC-gamma. Confocal immunofluorescence imaging of peri-implantation blastocysts demonstrated that PLC-gamma2, but not PLC-gamma1 nor PLC-beta1, accumulated near the outer surface of the embryo. Phosphotyrosine site-directed antibodies revealed phosphorylation of PLC-gamma2, but not PLC-gamma1, upon integrin ligation by FN. These data suggest that integrin-mediated activation of PLC-gamma to initiate phosphoinositide signaling and intracellular Ca(2+) mobilization is required for blastocyst adhesion to FN. Signaling cascades regulating PLC-gamma could, therefore, control a critical feature of trophoblast differentiation during peri-implantation development.

Integrin signaling regulates blastocyst adhesion to fibronectin at implantation: intracellular calcium transients and vesicle trafficking in primary trophoblast cells.

Accumulating evidence indicates that the endometrial extracellular matrix (ECM) modulates trophoblast adhesion during mouse blastocyst implantation. In previous studies of adhesion-competent mouse blastocysts, we have demonstrated that integrin-mediated fibronectin (FN)-binding activity on the apical surface of trophoblast cells is initially low, but becomes strengthened after embryos are exposed to FN. In the present study, we have examined whether the ligand-induced upregulation of trophoblast adhesion to FN is mediated by integrin signaling. The strengthening of adhesion to FN required integrin ligation, which rapidly elevated cytoplasmic-free Ca(2+). Chelation of intracellular Ca(2+) using BAPTA-AM, or inhibition of the Ca(2+)-dependent proteins, protein kinase C or calmodulin, significantly attenuated the effect of FN on binding activity. Furthermore, direct elevation of cytoplasmic Ca(2+) levels with ionomycin upregulated FN-binding activity, demonstrating that Ca(2+) signaling is required and sufficient for strong adhesion to FN. Ca(2+) signaling may induce protein trafficking, a known requirement for ligand-induced upregulation of FN-binding activity. Indeed, intracellular vesicles accumulated in adhesion-competent blastocysts, but were absent after exposure to either FN or ionomycin. These findings suggest that, during implantation, contact between peri-implantation blastocysts and FN elevates intracellular Ca(2+), which strengthens trophoblast adhesion to ECM through protein redistribution.