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Title: Cell membrane permeability coefficients of murine and human oocytes : fluxes of water and cryoprotectants during cryopreservation procedures
Author: Hunter, Jocelyn Elena
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 1992
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The aim of this thesis was to determine the cell membrane permeability characteristics of human pre-ovulatory oocytes and to apply this information to the development of a successful cryopreservation protocol. Currently, although there is no reliable method for the cryopreservation of human oocytes, there is an urgent need for such a technique in clinical I.V.F. programmes. The permeability of cells to both water and cryoprotectants is important in determining whether a cell will survive the cryopreservation procedure, and these basic parameters are unknown for human oocytes. A microscope diffusion chamber was developed to allow direct measurement of cell membrane permeability under controlled environmental conditions of temperature and solute exposure. Volumetric responses of individual oocytes were recorded using video microscopy, and cell volume data processed using a computer algorithm based on the Kedem- Katchalsky membrane transport equations. From these values the membrane water permeability ( Lp ) , the temperature-dependent Arrhenius activation energy ( Ea ) of the Lp, and the solute permeability ( w ) were calculated. The values of Lp and Ea for fresh pre-ovulatory human oocytes were compared to those calculated for mouse oocytes for which values have previously been determined and failed-to-fertilise human oocyte ( Ff ). Values for Lp and Ea in human oocytes were of the same range as those determined for the mouse, but human pre-ovulatory oocytes were inherently more diverse than mouse oocytes. The most reliable value for Ea of the human oocytes was obtained by studying individual fresh oocytes at several temperatures, and this yielded a mean value for Ea similar to other mammalian cells. At low temperatures, both mouse and human oocytes, responded to hypertonic perfusion with an altered morphology and non-spherical shrinkage. Although this response was morphologically reversible, it led to a reduction in the subsequent fertilisation and embryonic development. However, exposure of oocytes to cryoprotectants before cold shrinkage provided some protection to the oocytes as judged by subsequent fertilisation and reduced the damage to the developmental potential. Taking the values for Lp and Ea into account, protocols were chosen to investigate cryopreservation of human oocytes using glycerol or dimethyl sulphoxide as cryoprotectants. Fertilisation was achieved using both systems following cryopreservation, but further embryonic development was inhibited.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available