Identification and Development of Enzymes Associated With the Sperm Inner Acrosomal Membrane and Their Function during Fertilization

In order for mammalian fertilization to transpire, spermatozoa must transit through the female reproductive tract and penetrate the outer investments of the oocyte: the cumulus oophorus and the zona pellucida. In order to penetrate the oocyte, spermatozoa must undergo the acrosome reaction. The acrosome reaction results in the exposure of the inner acrosomal membrane (IAM) and proteins that coat it to the extracellular environment. After the acrosome reaction, the IAM becomes the leading edge of spermatozoa undergoing progressive movement. Thus the enzymes which effect lysis of the oocyte investments ought to be located on the IAM. An objective of this study was to identify and characterize enzymatic activity detected on the IAM and provide evidence that they play a role in fertilization. This study also describes procedures for fractionating spermatozoa and isolating the IAM and proteins on its intra- and extra-vesicular surfaces, and describes their development during male gametogenesis. Since the IAM is exposed to the extracellular environment and oviductal milieu after the acrosome reaction, this study also sought to characterize interactions and relationships between factors in the oviductal environment and the enzymes identified on the IAM. The data presented provide evidence that MMP2 and acrosin are co-localized on the IAM, originate from the Golgi apparatus in gametogenesis, and suggest they cooperate in their function. Their localization and results of in vitro fertilization suggests they have a function in zona pellucida penetration. The data also provide evidence that plasminogen, originating from the oviductal epithelium and/or cumulus-oocyte complex, is present in the immediate environment of sperm-egg initial contact and penetration. Additionally, plasminogen interacts with MMP2 and enhances its enzymatic action on the IAM. The data also provide evidence that MMP2 has an important function in penetration of the cumulus oophorus. Holistically, this thesis provides evidence that enzymes on the IAM, originating from the Golgi apparatus in development, have an important function in penetration of the outer investments of the oocyte, and are aided in penetration by plasminogen in the female reproductive tract.

BLOCK COPOLYMERS CONTAINING A LOW-SURFACE-ENERGY BLOCK AND THEIR APPLICATIONS

This thesis reports the synthesis and/or applications of three types of block copolymers that each bear a low-surface-energy block. First, poly(dimethylsiloxane)-block-poly(2-cinnamoyloxyethyl acrylate) (PDMS-b-PCEA) was synthesized and characterized. Cotton coating using a micellar solution of this block copolymer yielded superhydrophobic cotton fabrics. X-ray photoelectron spectroscopy (XPS) and surface property analyses indicated that the PDMS block topped the polymer coating. Photocuring the cotton swatches crosslinked the underlying PCEA layer and yielded permanent coatings. More interestingly, hydrophilically patterned superhydrophobic cotton fabrics were produced using photolithography that allowed the crosslinking of the coating around irradiated fibers but the removal, by solvent extraction, of the coating on fibers that were not irradiated. Since water-based ink only permeated the uncoated regions, such patterned fabric was further used to print ink patterns onto substrates such as fabrics, cardboard, paper, wood, and aluminum foil. Then, another PDMS-based diblock copolymer poly(dimethylsiloxane)-block-poly(glycidyl methacrylate) (PDMS-b-PGMA) was prepared. Different from PCEA that photocrosslinked around cotton fibers, PGMA reacted with hydroxyl groups on cotton fiber surfaces to get covalently attached. Further, different PGMA chains crosslinked with each other. PDMS-b-PGMA-coated cotton fabrics have been used for oil-water separations. In addition, polymeric nanoparticles were grafted onto cotton fiber surface before PDMS-b-PGMA was used to cover the surfaces of the grafted spheres and the residual surfaces of the cotton fibers. These two types of fabrics, coated by the block copolymer alone or by the polymer nanospheres and then the copolymer, were characterized by scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FT-IR), and water repellency analyses. A comprehensive comparative study was made of their performances in oil-water separation. Finally, a fluorinated ABC triblock copolymer poly(acrylic acid)-block-poly(2-cinnamoyloxyethyl methacrylate)-block-poly(2-perfluorooctylethyl methacrylate) (PAA-b-PCEMA-b-PFOEMA) was used to iii encapsulate air nanobubbles. The produced air nanobubbles were thermodynamically stable in water and were some 100 times more stable than commercially available perfluorocarbon-filled microbubbles under ultrasound. These nanobubbles, due to their small sizes and thus ability to permeate the capillary networks of organs and to reach tumors, may expand the applications of microbubbles in diagnostic ultrasonography and find new applications in ultrasound-regulated drug delivery.

Ice-binding proteins adsorb to their ligand via anchored clathrate waters

The main success of my thesis has been to establish the mechanism by which antifreeze proteins (AFPs) bind irreversibly to ice crystals, and hence prevent their growth. AFPs organize ice-like water on their ice-binding site, which then merges and freezes with the quasi-liquid layer of ice. This was revealed from studying the exceptionally large (ca. 1.5-MDa) Ca 2+-dependent AFP from the Antarctic bacterium Marinomonas primoryensis (MpAFP). The 34-kDa antifreeze- active region of MpAFP was predicted to fold as a novel Ca 2+-binding β-helix. Site-directed mutagenesis confirmed the model and demonstrated that its ice-binding site (IBS) consisted of solvent-exposed Thr and Asx parallel arrays on the Ca 2+-binding turns. The X-ray crystal structure of the antifreeze region was solved to a resolution of 1.7 Å. Two of the four molecules within the unit cell of the crystal had portions of their IBSs freely exposed to solvent. Identical clathrate-like cages of water molecules were present on each IBS. These waters were organized by the hydrophobic effect and anchored to the protein via hydrogen bonds. They matched the spacing of water molecules in an ice lattice, demonstrating that anchored clathrate waters bind AFPs to ice. This mechanism was extended to other AFPs including the globular type III AFP from fishes. Site-directed mutagenesis and a modified ice-etching technique demonstrated this protein uses a compound ice-binding site, comprised of two flat and relatively hydrophobic surfaces, to bind at least two planes of ice. Reinvestigation of several crystal structures of type III AFP identified anchored clathrate waters on the solvent-exposed portion of its compound IBS that matched the spacing of waters on the primary prism plane of ice. Ice nucleation proteins (INPs), which can raise the temperature at which ice forms in solution to just slightly below 0oC, have the opposite effect to AFPs. A novel dimeric β-helical model was proposed for the INP produced by the bacterium Pseudomonas borealis. Molecular dynamics simulations showed that INPs are also capable of ordering water molecules into an ice- like lattice. However, their multimerization brings together sufficient ordered waters to form an ice nucleus and initiate freezing.