Effect of cryoprotectant concentration on bovine oocyte permeability and comparison of two membrane permeability modelling approaches

Generalitat de Catalunya 2019 FI_B2 00055The plasma membrane permeability to water and cryoprotectant (CPA) significantly impacts vitrification efficiency of bovine oocytes. Our study was designed to determine the concentration-dependent permeability characteristics for immature (GV) and mature (MII) bovine oocytes in the presence of ethylene glycol (EG) and dimethyl sulphoxide (MeSO), and to compare two different modeling approaches: the two parameter (2P) model and a nondilute transport model. Membrane permeability parameters were determined by consecutively exposing oocytes to increasing concentrations of MeSO or EG. Higher water permeability was observed for MII oocytes than GV oocytes in the presence of both MeSO and EG, and in all cases the water permeability was observed to decrease as CPA concentration increased. At high CPA concentrations, the CPA permeability was similar for MeSO and EG, for both MII and GV oocytes, but at low concentrations the EG permeability of GV oocytes was substantially higher. Predictions of cell volume changes during CPA addition and removal indicate that accounting for the concentration dependence of permeability only has a modest effect, but there were substantial differences between the 2P model and the nondilute model during CPA removal, which may have implications for design of improved methods for bovine oocyte vitrification

Micro-computed tomography (μ-CT) as a potential tool to assess the effect of dynamic coating routes on the formation of biomimetic apatite layers on 3D-plotted biodegradable polymeric scaffolds

This work studies the influence of dynamic biomimetic coating procedures on the growth of bonelike apatite layers at the surface of starch/polycaprolactone (SPCL) scaffolds produced by a 3D-plotting technology. These systems are newly proposed for bone Tissue Engineering applications. After generating stable apatite layers through a sodium silicate-based biomimetic methodology the scaffolds were immersed in Simulated Body Fluid solutions (SBF) under static, agitation and circulating flow perfusion conditions, for different time periods. Besides the typical characterization techniques, Micro-Computed Tomography analysis (μ-CT) was used to assess scaffold porosity and as a new tool for mapping apatite content. 2D histomorphometric analysis was performed and 3D virtual models were created using specific softwares for CT reconstruction. By the proposed biomimetic routes apatite layers were produced covering the interior of the scaffolds, without compromising their overall morphology and interconnectivity. Dynamic conditions allowed for the production of thicker apatite layers as consequence of higher mineralizing rates, when comparing with static conditions. μ-CT analysis clearly demonstrated that flow perfusion was the most effective condition in order to obtain well-defined apatite layers in the inner parts of the scaffolds. Together with SEM, this technique was a useful complementary tool for assessing the apatite content in a non-destructive way

Zebrafish Ciliopathy Screen Plus Human Mutational Analysis Identifies C21orf59 and CCDC65 Defects as Causing Primary Ciliary Dyskinesia

Primary ciliary dyskinesia (PCD) is caused when defects of motile cilia lead to chronic airway infections, male infertility, and situs abnormalities. Multiple causative PCD mutations account for only 65% of cases, suggesting that many genes essential for cilia function remain to be discovered. By using zebrafish morpholino knockdown of PCD candidate genes as an in vivo screening platform, we identified c21orf59, ccdc65, and c15orf26 as critical for cilia motility. c21orf59 and c15orf26 knockdown in zebrafish and planaria blocked outer dynein arm assembly, and ccdc65 knockdown altered cilia beat pattern. Biochemical analysis in Chlamydomonas revealed that the C21orf59 ortholog FBB18 is a flagellar matrix protein that accumulates specifically when cilia motility is impaired. The Chlamydomonas ida6 mutant identifies CCDC65/FAP250 as an essential component of the nexin-dynein regulatory complex. Analysis of 295 individuals with PCD identified recessive truncating mutations of C21orf59 in four families and CCDC65 in two families. Similar to findings in zebrafish and planaria, mutations in C21orf59 caused loss of both outer and inner dynein arm components. Our results characterize two genes associated with PCD-causing mutations and elucidate two distinct mechanisms critical for motile cilia function: dynein arm assembly for C21orf59 and assembly of the nexin-dynein regulatory complex for CCDC65

An Outer Arm Dynein Conformational Switch Is Required for Metachronal Synchrony of Motile Cilia in Planaria

Here we use the motile ventral cilia of the planarian S. mediterranea to examine the role of outer arm dynein in the generation and maintenance of metachronal synchrony. We demonstrate that a single dynein light chain plays a mechanosensory role necessary to entrain and maintain the metachronal synchrony of motile cilia