Effects of Porcine Immature Oocyte Vitrification on Actin Microfilament Distribution and Chromatin Integrity During Early Embryo Development in vitro

Vitrification is mainly used to cryopreserve female gametes. This technique allows maintaining cell viability, functionality, and developmental potential at low temperatures into liquid nitrogen at −196°C. For this, the addition of cryoprotectant agents, which are substances that provide cell protection during cooling and warming, is required. However, they have been reported to be toxic, reducing oocyte viability, maturation, fertilization, and embryo development, possibly by altering cell cytoskeleton structure and chromatin. Previous studies have evaluated the effects of vitrification in the germinal vesicle, metaphase II oocytes, zygotes, and blastocysts, but the knowledge of its impact on their further embryo development is limited. Other studies have evaluated the role of actin microfilaments and chromatin, based on the fertilization and embryo development rates obtained, but not the direct evaluation of these structures in embryos produced from vitrified immature oocytes. Therefore, this study was designed to evaluate how the vitrification of porcine immature oocytes affects early embryo development by the evaluation of actin microfilament distribution and chromatin integrity. Results demonstrate that the damage generated by the vitrification of immature oocytes affects viability, maturation, and the distribution of actin microfilaments and chromatin integrity, observed in early embryos. Therefore, it is suggested that vitrification could affect oocyte repair mechanisms in those structures, being one of the mechanisms that explain the low embryo development rates after vitrification.

F-actin microfilament polymerized by jasplakinolide affects the expression of aquaporin-4 in astrocytic swelling after oxygen-glucose deprivation and reoxygenation

This study was to determine whether Jasplakinolide (JSK), an actin microfilament polymerization, reduced the astrocytic swelling induced by oxygen-glucose deprivation and reoxygenation (OGD/Reox) and whether this effect could be mediated through the modulation of aquaporin-4 (AQP4) expression. Astrocytes were obtained from rat spinal cord. Cells were subjected to reoxygenation after 5 h OGD. JSK with different concentrations (20, 50, 100, 200 and 400 nM) was added to astrocytes for 12 h before and during OGD/Reox. Then the cell volume and AQP4 expression were analyzed. The results showed that astrocytic swelling occurred with cell volume increasing after OGD/Reox and the severity was significantly attenuated by JSK. A strong increase of AQP4 protein expression was observed after OGD/Reox, reaching a peak at 1.5 h into reoxygenation. The increase in levels of AQP4 protein was significantly reduced by treatment with JSK. In addition, confocal microscopy analysis showed that the F-actin microfilament polymerization by JSK accounted for AQP4 downregulation in the plasma membrane. This study demonstrated that JSK could attenuate astrocytes swelling caused by OGD/Reox, an ischemia and reperfusion-like injury, and part of its protective effects might be related to the reorganization the F-actin microfilament and inhibiting the abundance expression of AQP4 protein.

Matrix metalloproteinases: an emerging role in regulation of actin microfilament system

Matrix metalloproteinases (MMPs) are implicated in many physiological and pathological processes, including contraction, migration, differentiation, and proliferation. These processes all involve cell phenotype changes, known to be accompanied by reorganization of actin cytoskeleton. Growing evidence indicates a correlation between MMP activity and the dynamics of actin system, suggesting their mutual regulation. Here, data on the influence of MMPs on the actin microfilament system, on the one hand, and the dependence of MMP expression and activation on the organization of actin structures, on the other hand, are reviewed. The different mechanisms of putative actin-MMP regulation are discussed.

Involvement of Actin Microfilament in Regulation of Pacemaking Activity Increased by Hypotonic Stress in Cultured ICCs of Murine Intestine

Distension is a regular mechanical stimulus in gastrointestinal (GI) tract. This study was designed to investigate the effect of hypotonic stress on pacemaking activity and determine whether actin microfilament is involved in its mechanism in cultured murine intestinal interstitial cells of Cajal (ICCs) by using whole-cell patch-clamp and calcium imaging techniques. Hypotonic stress induced sustained inward holding current from the baseline to –650±110 pA and significantly decreased amplitudes of pacemaker current. Hypotonic stress increased the intensity of basal fluorescence ratio (F/F0) from baseline to 1.09±0.03 and significantly increased Ca2+ oscillation amplitude. Cytochalasin-B (20 μM), a disruptor of actin microfilaments, significantly suppressed the amplitudes of pacemaker currents and calcium oscillations, respectively. Cytochalasin-B also blocked hypotonic stress-induced sustained inward holding current and hypotonic stress-induced increase of calcium oscillations. Phalloidin (20 μM), a stabilizer of actin microfilaments, significantly enhanced the amplitudes of pacemaker currents and calcium oscillations, respectively. Despite the presence of phalloidin, hypotonic stress was still able to induce an inward holding current and increased the basal fluorescence intensity. These results suggest that hypotonic stress induces sustained inward holding current via actin microfilaments and the process is mediated by alteration of intracellular basal calcium concentration and calcium oscillation in cultured intestinal ICCs.