The overall goal of the proposed research is to develop a novel cryopreservation technique for zebrafish oocytes. The zebrafish is a valuable model system for human diseases and other research areas such as embryogenesis, organ development, and aging. Successful cryopreservation of zebrafish oocytes or embryos is urgently needed to maintain exponentially growing numbers of mutant and transgenic lines. Successful banking of zebrafish gametes or embryos would not only significantly reduce costs, but also avoid risks of infection and genetic drift associated with continuous breeding. Furthermore, development of a successful cryopreservation technique for zebrafish oocytes would be a major stepping-stone towards conservation of other fish species, many of which are rapidly declining. Cryopreservation of fish sperm is currently practiced albeit with limited success, whereas fish embryos remain non-freezable due to several major obstacles such as their large size and multi-compartmental structure, extremely low membrane permeability to water and cryoprotective chemicals, and susceptibility to chilling injury and intracellular ice formation. To circumvent these obstacles, this project proposes to cryopreserve unfertilized zebrafish oocytes using a multidisciplinary approach. This proposal predicts that zebrafish oocytes will be more amenable to cryopreservation than embryos, based on their smaller size, much simpler single-cell structure, and higher permeability and tolerance to cryoprotectants. Intellectual merit: This project is significant and innovative because it proposes to tackle a fundamental scientific problem (i.e., cryopreservation of fish oocytes) by combining engineering (e.g., modeling membrane permeability and intracellular ice formation, mathematical and experimental optimization of CPA loading/removal and cooling profiles) and cell/molecular biology approaches (e.g., molecular manipulation of the membrane transport). The proposed mechanistic approach is novel and inspired by adaptation schemes observed in nature (e.g., survival strategies by organisms such as frogs, tardigrades, brine shrimp, bacteria, and yeast, which are able to adapt to extreme conditions including freezing and desiccation); a key adaptation is the accumulation of intra- and extracellular sugars. Recent studies have demonstrated beneficial effect of sugars during cryopreservation and desiccation of mammalian cells, whereas the membrane permeability barrier to sugars must be overcome for this strategy to be effective. Preliminary studies have demonstrated that the combination of sugars with low concentrations of conventional penetrating cryoprotectants can compensate for the low permeation of the former wile reducing the cytotoxicity of the latter. Thus, the central hypothesis of this project is that combination of intra- and extracellular sugars with small amounts of a penetrating cryoprotectant will protect zebrafish oocytes against freezing-associated stresses. Using engineering models of mass transport and non-equilibrium phase transformation, in combination with molecular biology techniques, this proposal will test the central hypothesis by pursuing three specific aims: (1) test the working hypothesis that osmotic limitations of zebrafish oocytes can be overcome by a multi-disciplinary strategy including biological, physico-chemical, and engineering approaches; (2) test working hypothesis that an optimized combination of intra- and extracellular sugars with a penetrating CPA permits successful cryopreservation of zebrafish oocytes; (3) test working hypothesis that to maximize viability, zebrafish oocytes should be cooled as rapidly as possible without causing formation of deleterious intracellular ice crystals. The interdisciplinary approach proposed here is expected to overcome the obstacles associated with cryopreservation of zebrafish germplasm, and lead to significant advances towards successful cryopreservation of zebrafish oocytes. The proposed work is also expected to lead to advances in the state-of-the-art of bioheat/mass-transfer modeling of cryobiological phenomena, and in computer-aided optimization of biothermal process design. Broader impacts of the proposed research will be attained by: (1) educational outreach activities in local high schools using zebrafish as a teaching tool to awaken curiosity/interest among the students in science and technology; (2) developing teaching materials for K-12 as well as undergraduate bioengineering students; (3) creating a summer research program for high school students; (4) training graduate students and postdoctoral fellows. The proposed outreach activities are expected to broaden and strengthen the ongoing partnership of the Medical College of Georgia with local high school and communities. The proposed development of educational materials and course modules will enhance the bioengineering curriculum at Villanova University and beyond.
