Studies on cryopreservation of zebrafish (Danio Rerio) oocytes using controlled slow cooling

A thesis submitted to the University of Bedfordshire in partial fulfillment of the requirements for the degree of Doctor of PhilosophyCryopreservation of fish germ cells has important applications in aquaculture, conservation of endangered species and human genomic studies. Although investigations on cryopreservation of fish sperm and embryos have been carried out extensively, cryopreservation of fish oocytes has not been studied systematically. The objective of the present study was to develop successful cryopreservation protocol for zebrafish oocytes at temperature of liquid nitrogen (-196°C), or if unachieved, to investigate the limiting factors associated with fish oocytes cryopreservation. In this study, the effects of cryoprotectants exposure and enzymatic treatments on oocytes survival were studied, and new viability tests for zebrafish oocytes were developed. The effects of controlled slow cooling with different cryoprotective agents, in different freezing media and at different cooling rates on cryosurvival of zebrafish (D. rerio) oocytes were investigated. Cryomicroscopic observations on zebrafish oocytes were also carried out. Three reliable vital tests -trypan blue (TB) staining, ATP assay, and in vitro maturation followed by germinal vesicle breakdown observation (GVBD) were found suitable for assessment of oocytes viability. Vitellogenesis (stage III) was found to be the optimal developmental stage for cryopreservation. Methanol was found to be the best CPA for zebrafish oocytes. Combination of 4M methanol and 0.2M glucose in potassium chloride (KCI) buffer was found to be the optimal cryoprotective solution. Controlled slow cooling at 0.3°C/min rate, combined with seeding at -12.5°C and plunge to liquid nitrogen (LN) at-40°C were found to be the optimal conditions for cryopreservation of stage III oocytes. However, even with the optimal protocol, TB-assessed viability, Le. the ratio of oocytes with intact plasma membrane after cooling to -196°C was 19.6±8%. Furthermore, GVBD experiments showed that none of the cryopreserved oocytes can be matured in vitro, and their ATP levels were decreased dramatically, indicating that successful cryopreservation of fish oocytes at liquid nitrogen temperature still remains elusive. Cryomicroscopic observations demonstrated, that the damages of oocytes are associated with intracellular ice formation (lIF). IIF occurred simultaneously with extracellular ice formation (ElF) in nearly 100% of the cases, and formation of lethal hexagonal type of ice was observed. This study was the first systematic attempt to cryopreserve fish oocytes at liquid nitrogen temperature. The results provided will undoubtedly assist successful protocol design for cryopreservation of fish oocytes in the future

Cryo-EM, X-ray diffraction, and atomistic simulations reveal determinants for the formation of a supramolecular myelin-like proteolipid lattice

Myelin protein P2 is a peripheral membrane protein of the fatty acid?binding protein family that functions in the formation and maintenance of the peripheral nerve myelin sheath. Several P2 gene mutations cause human Charcot-Marie-Tooth neuropathy, but the mature myelin sheath assembly mechanism is unclear. Here, cryo-EM of myelin-like proteolipid multilayers revealed an ordered three-dimensional (3D) lattice of P2 molecules between stacked lipid bilayers, visualizing supramolecular assembly at the myelin major dense line. The data disclosed that a single P2 layer is inserted between two bilayers in a tight intermembrane space of ?3 nm, implying direct interactions between P2 and two membrane surfaces. X-ray diffraction from P2-stacked bicelle multilayers revealed lateral protein organization, and surface mutagenesis of P2 coupled with structure-function experiments revealed a role for both the portal region of P2 and its opposite face in membrane interactions. Atomistic molecular dynamics simulations of P2 on model membrane surfaces suggested that Arg-88 is critical for P2-membrane interactions, in addition to the helical lid domain. Negatively charged lipid headgroups stably anchored P2 on the myelin-like bilayer surface. Membrane binding may be accompanied by opening of the P2 ?-barrel structure and ligand exchange with the apposing bilayer. Our results provide an unprecedented view into an ordered, multilayered biomolecular membrane system induced by the presence of a peripheral membrane protein from human myelin. This is an important step toward deciphering the 3D assembly of a mature myelin sheath at the molecular level.Peer reviewe

Intracellular ice formation in adult stem cells in the presence of polyvinyl pyrrolidone

The main objective of this work was to assess the effect of 10% (w/v) polyvinylpyrrolidone (PVP) on the pattern of intracellular ice formation (IIF) in human adipose tissue derived adult stem cells (ASCs) in the absence of serum and other cryoprotective agents (CPAs). Passage 1 (P1) ASCs were cultured, washed and suspended in either 1x PBS (Phosphate Buffered Saline) or 10% w/v solution of PVP in 1x PBS. The freezing experiments were carried out using a fluorescence microscope equipped with a Linkam™ cooling stage using two different temperature/time cooling protocols. Both the cooling protocols had a common cooling ramp: cells were cooled from 20 ˚C to –8 ˚C at 20 ˚C/min and then further cooled to –13 ˚C at 1 ˚C/min (during which the extra-cellular medium froze very rapidly and was accompanied by the formation of intracellular ice in ~96% of the cells, as noted by visible flashing/darkening ). At this point we employed either, cooling protocol 1: the cells were cooled from –13 ˚C to – 40 ˚C at a pre-determined cooling rate of 1, 5, 10, 20 or 40 ˚C/min and then thawed back to 20 ˚C at 20 ˚C/min; or cooling protocol 2: the cells were re-warmed from –13 ˚C to –5 ˚C at 20 ˚C/min and then re-cooled at a pre-determined rate of 1, 5, 10, 20 or 40 ˚C/min to –40 ˚C. Almost all (\u3e95%) of the ASCs frozen in 1x PBS and protocol 1 exhibited IIF whereas almost none (\u3c5%) of the ASCs frozen in 1x PBS and protocol 2 exhibited IIF. The lack of IIF in cells cooled in 1x PBS and protocol 2 was due to the initial loss of cell viability (confirmed through an additional membrane dye exclusion study) that was associated with the formation of IIF in the common cooling ramp, described earlier. Similarly, almost all (\u3e95%) of the ASCs frozen in 10% PVP in PBS and protocol 1 exhibited IIF where as ~0, ~40, ~47, ~67 and ~100% of the ASCs frozen in 10% PVP in PBS and protocol 2 exhibited IIF at a cooling rate of 1, 5, 10, 20 or 40 ˚C/min, respectively. The observed increase in the % of ASCs exhibiting IIF when frozen in 10% PVP and protocol 2, is presumably due to PVP mitigating the damaging effects of IIF during the common cooling ramp

Structural and functional investigation of the essential secondary active transporter LicB from the human pathogen S. pneumoniae

Streptococcus pneumoniae is a human pathogen that can cause mild symptoms but also exhibits the potential to cause severe infections in the lungs or the brain. As a Gram-positive bacteria it has teichoic acids attached to its surface, where there are two types which are either embedded in the membrane or bound to the peptidoglycan. The synthesis of those teichoic acids is unique for S. pneumoniae, as it shares the same biosynthetic pathway for both types. Another unique feature is their modification with phosphorylcholine. The attachment of phosphorylcholine happens at the inner leaflet during the synthesis of the nascent teichoic acid chain and only those modified teichoic acids are exported to the surface. Choline is the substrate for the phosphorylcholine moieties and is essential for the survival of S. pneumoniae. The substrate cannot be synthesized by the bacteria and can only be imported by a secondary active transporter denoted as LicB. This transporter belongs to the drug/metabolite transporter superfamily, which is comprised of transporters with ten transmembrane helices of two inverted repeats arising from internal gene duplication. The structure and function of LicB has not been described before and known structures of other members of the drug/metabolite transporter superfamily are sparse and mostly describe only a small part of the families. This study describes the functional and structural features of the pneumococcal choline importer LicB. LicB exhibits promiscuous transport behavior, where it shows to transport not only choline but also arsenocholine and acetylcholine. The half maximal effective concentrations have been determined by solid supported membrane electrophysiology to 47 ± 15 μM for choline, 170 ± 9 μM for arsenocholine and 740 ± 84 μM for acetylcholine. Radiolabeled acetylcholine was supplemented in choline reduced media to grow S. pneumoniae cells and to subsequently extract the teichoic acids. The extracted teichoic acids exhibited a signal which provides evidence of the import and catabolization of acetylcholine as an alternative choline source. Proton-coupling as the driving force for the alternating access mechanism during the transport cycle has been confirmed with a fluorescence-based assay. Additionally, a library of synthetic nanobodies, selected against LicB, was characterized based on their inhibitory potential resulting in the determination of several unique inhibitors. In this case solid supported membrane electrophysiology has proven to be a robust and fast technique to screen inhibitory nanobodies and presents an application for other potential drug targets. The structure of the transporter LicB was solved in the substrate bound occluded state at a resolution of 3.8 Å via x-ray crystallography and in the outward facing state, reconstituted into nanodiscs and bound to a synthetic nanobody at a resolution of 3.75 Å via cryo-EM. The transporter plays a crucial role for the survival of the human pathogen and the knowledge about the structure, the function and the identification of inhibitory synthetic nanobodies can help to provide a platform for antimicrobial drug development and for novel alternatives to combat pathogens

Three-dimensional reconstruction of Heterocapsa circularisquama RNA virus by cryo-electron microscopy

Heterocapsa circularisquama RNA virus is a non-enveloped icosahedral ssRNA virus infectious to the harmful bloom-forming dinoflagellate, H. circularisquama, and which is assumed to be the major natural agent controlling the host population. The viral capsid is constructed from a single gene product. Electron cryo-microscopy revealed that the virus has a diameter of 34 nm and T53 symmetry. The 180 quasi-equivalent monomers have an unusual arrangement in that each monomer contributes to a ‘bump’ on the surface of the protein. Though the capsid protein probably has the classic ‘jelly roll’ b-sandwich fold, this is a new packing arrangement and is distantly related to the other positive-sense ssRNA virus capsid proteins. The handedness of the structure has been determined by a novel method involving high resolution scanning electron microscopy of the negatively stained viruses and secondary electron detection

Mechanisms of cell damage and recovery in cryopreserved freshwater protists

SIGLEAvailable from British Library Document Supply Centre-DSC:DXN015370 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Cryoelectron Tomography of Bacteria and their Macromolecular Machines

Cryoelectron tomography (CET) fills a glaring gap in the imaging capabilities of biology by reconstructing cells to medium resolution. The technique was applied in three areas to understand biology’s macromolecular machines: (1) the quaternary structure of the octahedrally-cored E. coli pyruvate dehydrogenase (PDHC) and 2-oxoglutarate dehydrogenase (OGDHC) complexes in vitro; (2) the ultrastructure of the spirochete Treponema primitia; and (3) the structure of the in situ flagellar motors from T. primitia, Hylemonella gracilis, Caulobacter crescentus, and Vibrio cholerae. Whereas the complexes PDHC and OGDHC were thought to have their subunit proteins E1 and E3 bound directly to the octahedral E2 core—the so-called face/edge model—it was discovered that the subunits are flexibly tethered 11 nm from the corners of the core. Several novel structures were discovered in the spirochete T. primitia. Spirochetes are spiral-shaped cells that propel themselves with periplasmic, not external, flagella. Bowl-shaped structures dot its surface and hook-like appendages that form arcades stripe the length of the cell. Fibrils extend from its cell tips that might help attach the cells to surfaces. Inside the periplasm, porous, cone-shaped structures reside at each cell tip and a second periplasmic layer undergirds its outer membrane, which might prevent the periplasmic flagella from rupturing the cell. Previous imaging of the flagellar motor produced either high-resolution reconstructions of the purified basal body removed from its context or low-resolution images of the in situ motor. Our in situ 3-D reconstructions described for the first time the structure of the stators, the membrane embedded component that spins the rotor. Novel shapes were discovered that indicate there are various attachments and versions of the flagellar motor that were never expected

DEVELOPMENT OF A MICROFLUIDICS INTEGRATED MICROVASCULARISED HUMAN SKIN-ON-A-CHIP TISSUE MODEL

Tissue engineered skin constructs have been under development since the 1980s as a replacement for cadaverous human skin and animal models. These have evolved from simple confluent single cell-type arrangements to models with integrated dermal equivalents and often multiple cell types. Concomitantly, formation of stable self-assembled nanofibrous peptide amphiphile (PA) membranes upon contact with hyaluronic acid (HA) in aqueous solution, with comparable ultrastructure to the apical skin basement membrane (BM) have been reported. With the rise of microfluidic cell culture, scientists have scaled down these technologies; however no group to our knowledge has yet published a full thickness microfluidic skin equivalent with a physiologically mimetic tubular microvasculature or investigated integration of a skin model PA-based BM equivalent. This project aimed to integrate these features into a full thickness human skin-on-a-chip model, with a focus on microvasculature. We report the formation of interfacial self-assembled nanofibrous membranes between fibrin and collagen hydrogels and various PAs including C16V3A3K3. We exploit this in the stabilisation of the dermoepidermal interface in a macroscale fibrin-based skin equivalent model. We then design a novel skin-on-a-chip model integrating human-derived cells in a full-thickness arrangement. The model features a stratified epidermal equivalent featuring correct spatial localisation of keratinocyte basal and differentiation markers, dermoepidermal interfacial deposition of laminin 332 and a dermal compartment populated with human fibroblasts expressive of vimentin. The extent of expression of basal and corneal keratinocyte markers, and the sizes of dermally localised vimentin positive bodies, are comparable between our model and ex vivo human skin. The vascularised model incorporates either HUVECs alone or alongside primary pericytes and displays formation of tubular microvessels positive for CD31 surrounded by a laminin positive basement membrane. Microvessel diameters are comparable to those reported in literature for ex vivo human skin vasculature. The skin-equivalent is also tested in 3D extrusion bioprinted devices. We believe that this is a novel development in the field of microfluidic skin models, with a complex microvascular component displaying anastomosis and lumen formation unlike other vascularised skin-chip models. This model has potential utility in the fields of preclinical drugs testing and disease modellin

Wheat pollen viability and physiological, biochemical and biophysical factors impacting pollen storability

Pollen has high potential to preserve and exchange nuclear genes of plant genetic resources. To facilitate breeding programs, short- and more importantly long-term pollen preservation protocols have been established for many species. Long-term pollen preservation, particularly in wheat breeding programs, and especially with spatially and temporally isolated parents, would be of great interest to expand genetic diversity and to facilitate hybrid seed production. Wheat sheds tricellular pollen at maturity which loses the ability to germinate under ambient conditions within one hour. So far, neither short- nor long-term storage protocols for wheat pollen have been established, and physiological, ultrastructural and biochemical processes in pollen after shedding are hardly understood. To gain a comprehensive overview of processes contributing to fast viability loss, in this thesis, three consecutive studies on the viability and storability of wheat pollen were conducted. First, a comparison of different viability tests and their reliability for use in wheat pollen was made (Chapter 2, Manuscript 1). Second, two different environmental factors (temperature and relative humidity, RH) were investigated for their influence on wheat pollen longevity, physiological properties, pollen ultrastructure and metabolism (Chapter 3, Manuscript 2). Finally, experiments were conducted to investigate the feasibility of cryopreservation for wheat pollen (Chapter 4, Manuscript 3). In the first study a consistent semi-solid in vitro pollen germination medium containing raffinose, boric acid, calcium chloride and gelrite was formulated and compared against existing media (Jian et al. 2014; Jayaprakash et al. 2015; Cheng and McComb 1992) for wheat pollen. The medium formulation resulted in consistent germination percentages for fresh pollen of > 87%. The germination was correlated with pollen viability assessed by impedance flow cytometry (IFC viability, r = 0.67, P < 0.001) and fluorescein diacetate (FDA, r = 0.54, P < 0.05) staining (Chapter 2, Manuscript 1, Figure 5). However, when the medium was used with other Poaceae pollen species, germination was low and assumed to require further adaptation. Although, FDA and IFC viability can be easily applied both, FCR and IFC, seem to overestimate pollen viability (Chapter 2, Manuscript 1, Figure 5). Therefore, two viability tests, in vitro germination and IFC viability, were applied in consecutive studies. The second work revealed that low temperature (~ 4 °C) and high RH (> 90%) could keep pollen viable with a maximum of > 70% pollen germination after 60 minutes storage. The metabolic changes were most pronounced in unfavourable conditions (low RH and room temperature) were pollen lost most of its viability (pollen germination reached only 10%) after short storage of only 20 to 30 minutes. Under these conditions, wheat pollen showed extensive and deleterious changes in the ultra-structure (intine and cytoplasmic organization), fluctuations in primary metabolite concentration, and changes in water content (WC) (Chapter 3, Manuscript 2, Figures 1, 2, 6, 7). Overall, metabolic status, ultrastructural and WC changes lead to irreversible damages and viability loss suggesting that wheat pollen is not equipped with sufficient protection mechanisms to survive longer periods. Additionally, in these two studies we found differences in germination percentage (Manuscript 1, Figure 6; Manuscript 2, Supplemental Figure S4) and metabolite concentrations of specific compounds (Manuscript 2, Figure 4) between different genotypes tested. Thus, it is suggested that the genotype may have an important influence on pollen survival. Further research with a wide range of genotypic implications could reveal marker genes that might influence wheat pollen viability under different conditions. In the third study, it was tested if wheat pollen may be able to survive cryopreservation. Therefore, wheat pollen had been dried and cooled under different regimes. Rapidly dried wheat pollen to WC above the unfrozen water content (0.91 ± 0.11 mg H2O mg-1 DW) for 5 min retained IFC viability of around 6.1 ± 8.8% after fast cooling and warming but were not able to germinate. Nevertheless, damages induced by dehydration and cryo-injury during ultra-low temperature exposure seemed to occur to a lesser extent in the rapidly dried pollen compared to fresh pollen and pollen dried at ambient conditions for both, slow and rapid cooling/warming. Thus, within a very small window of a specific pollen WC and further adjustment pollen may survive cryopreservation storage. Future research and amendment of fast-drying and an optimisation of the cooling/warming rate will reveal whether the survival rate of pollen can be increased after exposure to cryo-storage. The use of cryoprotection may have favourable effects on the survival. Further suggestions for possible improvements of cryopreservation will be discussed in one of the sections of discussion