Reversible Disassembly of the Actin Cytoskeleton Improves the Survival Rate and Developmental Competence of Cryopreserved Mouse Oocytes

Effective cryopreservation of oocytes is critically needed in many areas of human reproductive medicine and basic science, such as stem cell research. Currently, oocyte cryopreservation has a low success rate. The goal of this study was to understand the mechanisms associated with oocyte cryopreservation through biophysical means using a mouse model. Specifically, we experimentally investigated the biomechanical properties of the ooplasm prior and after cryopreservation as well as the consequences of reversible dismantling of the F-actin network in mouse oocytes prior to freezing. The study was complemented with the evaluation of post-thaw developmental competence of oocytes after in vitro fertilization. Our results show that the freezing-thawing process markedly alters the physiological viscoelastic properties of the actin cytoskeleton. The reversible depolymerization of the F-actin network prior to freezing preserves normal ooplasm viscoelastic properties, results in high post-thaw survival and significantly improves developmental competence. These findings provide new information on the biophysical characteristics of mammalian oocytes, identify a pathophysiological mechanism underlying cryodamage and suggest a novel cryopreservation method

Microbubbles reveal chiral fluid flows in bacterial swarms

Flagellated bacteria can swim within a thin film of fluid that coats a solid surface, such as agar; this is a means for colony expansion known as swarming. We found that micrometer-sized bubbles make excellent tracers for the motion of this fluid. The microbubbles form explosively when small aliquots of an aqueous suspension of droplets of a water-insoluble surfactant (Span 83) are placed on the agar ahead of a swarm, as the water is absorbed by the agar and the droplets are exposed to air. Using these bubbles, we discovered an extensive stream (or river) of swarm fluid flowing clockwise along the leading edge of an Escherichia coli swarm, at speeds of order 10 μm/s, about three times faster than the swarm expansion. The flow is generated by the action of counterclockwise rotating flagella of cells stuck to the substratum, which drives fluid clockwise around isolated cells (when viewed from above), counterclockwise between cells in dilute arrays, and clockwise in front of cells at the swarm edge. The river provides an avenue for long-range communication in the swarming colony, ideally suited for secretory vesicles that diffuse poorly. These findings broaden our understanding of swarming dynamics and have implications for the engineering of bacterial-driven microfluidic devices

Oocyte survival after cryopreservation.

<p>(A) The percentage of mouse oocytes that survived slow-cooling with (left) and without (right) LATA pretreatment. LATA pretreatment increased the cryosurvival rate by 26.2% (<i>p</i><0.05). (B) The percentage of blastocysts developed from 2-cell embryos with (left) and without (right) LATA pretreatment. LATA pretreatment increased developmental competence by 81% (<i>p</i><0.05). Error bars represent the SEM.</p

The oocyte viscoelastic parameters.

<p>Viscoelastic parameters before freezing without (Fresh, n  =  29 cells) or with LATA treatment (Fresh-LATA, n  =  28 cells) and after freezing without (Thawed, n  =  16 cells) or with (Thawed LATA, n  =  19 cells) LATA pretreatment. (A) Relaxation time. (B) Friction coefficient <i>µ</i>₁. (C) Elastic coefficient <i>k</i>. (D) Friction coefficient <i>µ</i>. All viscoelastic parameters show significant statistical difference (see <i>p</i> values in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002787#pone-0002787-t001" target="_blank">Table 1</a>) between the Fresh and Thawed groups and most show statistical difference between Fresh and Fresh LATA groups. There is no significant statistical difference between the Fresh and the Thawed LATA groups. Bars represent geometric mean±geometric SE (panel A) and mean±SEM (panels B, C and D).</p

The cytoplasmic trajectory of a magnetic bead.

<p>A.Typical bead trajectory under a 2-second constant force pulse (filled circles: bead position; gray line: magnetic pulse; “0” on the time axis represents the moment when the force pulse was applied). B. Best fit to the trajectory during creep (<i>F</i>≠0). Filled circles: experimental data points; black line: best fit with the linear viscoelastic model; gray line: best fit with the power law. Inset: bead trajectory on a log-log scale. The numbers along both axes are powers of e, the base of natural logarithm.</p

The viscoelastic model for the ooplasmic environment.

<p>The ooplasmic environment is modeled as a Voight body (<i>k</i>,<i>µ</i>) in series with a dashpot (<i>µ</i>₁). The ideal trajectory of the bead under constant force (creep, <i>F</i>≠0) and during relaxation (<i>F</i> = 0). <i>F</i> = magnetic force; <i>k</i> = elastic coefficient; <i>µ</i> and <i>µ</i>₁ = friction coefficients.</p

A magnetic bead inside of a mouse oocyte.

<p>A photomicrograph of a CD1 mouse oocyte having been injected with a 5 µm superparamagnetic bead, and subsequently embedded in fibrin gel, is shown.</p