Dithiothreitol prevents the spontaneous release of cortical granules in in vitro aged mouse oocytes by protecting regulatory proteins of cortical granules exocytosis and thickening the cortical actin cytoskeleton.

In assisted fertility protocols, in vitro culture conditions mimic physiological conditions to preserve gametes in the best conditions. After collection, oocytes are maintained in a culture medium inside the incubator until in vitro fertilization (IVF) is performed. This time outside natural and physiological conditions exposes oocytes to an oxidative stress that renders in vitro aging. It has been described that in vitro aging produces a spontaneous cortical granule (CG) release decreasing the fertilization rate of oocytes. Nevertheless, this undesirable phenomenon has not been investigated, let alone prevented. In this work, we characterized the spontaneous CG secretion in in vitro aged oocytes. Using immunofluorescence indirect, quantification, and functional assays, we showed that the expression of regulatory proteins of CG exocytosis was affected. Our results demonstrated that in vitro oocyte aging by 4 and 8 h altered the expression and localization of alpha-SNAP and reduced the expression of NSF and Complexin. These alterations were prevented by supplementing culture medium with dithiothreitol (DTT), which in addition to having a protective effect on those proteins, also had an unexpected effect on the actin cytoskeleton. Indeed, DTT addition thickened the cortical layer of fibrillar actin. Both DTT effects, together, prevented the spontaneous secretion of CG and recovered the IVF rate in in vitro aged oocytes. We propose the use of DTT in culture media to avoid the spontaneous CG secretion and to improve the success rate of IVF protocols in in vitro aged oocytes.

Dehydroleucodine and xanthatin, two natural anti-inflammatory lactones, inhibit mast cell degranulation by affecting the actin cytoskeleton.

Actin remodeling is a critical regulator of mast cell secretion. In previous work, we have shown that dehydroleucodine and xanthatin, two natural α,ß-unsaturated lactones, exhibit anti-inflammatory and mast cell stabilizing properties. Based on this background, this study aimed to determine whether the mast cell stabilizing action of these lactones is associated with changes in the actin cytoskeleton. Rat peritoneal mast cells were preincubated in the presence of dehydroleucodine or xanthatin before incubation with compound 48/80. Comparative studies with sodium cromoglycate and latrunculin B were also made. After treatments, different assays were performed on mast cell samples: ß-hexosaminidase release, cell viability studies, quantification of mast cells and their state of degranulation by light microscopy, transmission electron microscopy, and actin staining for microscopy observation. Results showed that dehydroleucodine and xanthatin inhibited mast cell degranulation, evidenced by the inhibition of ß-hexosaminidase release and decreased degranulated mast cell percentage. At the same time, both lactones altered the F-actin cytoskeleton in mast cells resulting, similarly to Latrunculin B, in a higher concentration of nuclear F-actin when activated by compound 48/80. For the first time, this study describes the biological properties of dehydroleucodine and xanthatin concerning to the rearrangement of actin filaments during stimulated exocytosis in mast cells. These data have important implications for developing new anti-inflammatory and mast cell stabilizing drugs and for designing new small molecules that may interact with the actin cytoskeleton.

The light chain of tetanus toxin bound to arginine-rich cell-penetrating peptide inhibits cortical reaction in mouse oocytes.

Introduction: Cortical reaction is a secretory process that occurs after a spermatozoon fuses with the oocyte, avoiding the fusion of additional sperm. During this exocytic event, the cortical granule membrane fuses with the oocyte plasma membrane. We have identified several molecular components involved in this process and confirmed that SNARE proteins regulate membrane fusion during cortical reaction in mouse oocytes. In those studies, we microinjected different nonpermeable reagents to demonstrate the participation of a specific protein in the cortical reaction. However, the microinjection technique has several limitations. In this work, we aimed to assess the potential of cell-penetrating peptides (CPP) as biotechnological tools for delivering molecules into oocytes, and to evaluate the functionality of the permeable tetanus toxin (bound to CPP sequence) during cortical reaction. Methods: Arginine-rich cell-penetrating peptides have demonstrated the optimal internalization of small molecules in mammalian cells. Two arginine-rich CPP were used in the present study. One, labeled with 5-carboxyfluorescein, to characterize the factors that can modulate its internalization, and the other, the permeable light chain of tetanus toxin, that cleaves the SNAREs VAMP1 and VAMP3 expressed in mouse oocytes. Results: Results showed that fluorescent CPP was internalized into the oocyte cytoplasm and that internalization was dependent on the concentration, time, temperature, and maturation stage of the oocyte. Using our functional assay to study cortical reaction, the light chain of tetanus toxin bound to arginine-rich cell-penetrating peptide inhibited cortical granules exocytosis. Discussion: Results obtained from the use of permeable peptides demonstrate that this CPP is a promising biotechnological tool to study functional macromolecules in mouse oocytes.

Live imaging of cortical granule exocytosis reveals that in vitro matured mouse oocytes are not fully competent to secrete their content.

Oocyte in vitro maturation does not entirely support all the nuclear and cytoplasmic changes that occur physiologically, and it is poorly understood whether in vitro maturation affects the competence of cortical granules to secrete their content during cortical reaction. Here, we characterize cortical granule exocytosis (CGE) in live mouse oocytes activated by strontium chloride using the fluorescent lectin FITC-LCA. We compared the kinetic of CGE between ovulated (in vivo matured, IVO) and in vitro matured (IVM) mouse oocytes. Results show that: (1) IVM oocytes have a severely reduced response to strontium chloride; (2) the low response was confirmed by quantification of remnant cortical granules in permeabilized cells and by a novel method to quantify the exudate in non-permeabilized cells; (3) the kinetic of CGE in IVO oocytes was rapid and synchronous; (4) the kinetic of CGE in IVM oocytes was delayed and asynchronous; (5) cortical granules in IVM oocytes show an irregular limit in regards to the cortical granule free domain. We propose the analysis of CGE in live oocytes as a biological test to evaluate the competence of IVM mouse oocytes.This article has an associated First Person interview with the first author of the paper.