Two major areas are addressed in this proposal. The first concerns the development of better methods for the culture of mammalian preimplantation embryos, particularly methods which support normal differentiation of the blastocyst in vitro. The inability to successfully culture blastocysts is one of the remaining gaps in our methodology. This methodology is needed to experimentally analyze the early stages of organogenesis in mammals. The main technique to be used will be simplex optimization to find the optimum concentrations of the components of culture solution.
The specific aims are: (1) To test whether optimum concentrations of components in a medium for the culture of preimplantation embryos depend on the response parameter, (2) To devise methods for the optimization of media with large numbers of components, such as 20 amino acids, (3) To obtain evidence that preimplantation embryos contain small organic molecules that act as osmolytes, and (4) To examine whether the response to growth factors is dependent on the background provided by other components of the medium. The second area concerns the construction of efficient ways of producing non-human mature oocytes in vitro. Studies of preimplantation development in primates are limited by the small number of embryos that can be produced by conventional means. In biotechnology the production of transgenic primates will be severely hampered by this problem. An alternative approach is to tap the large store of oocytes in the ovary by culture methods to produce large numbers of mature oocytes for fertilization in vitro.
The specific aims are: (1) To assemble a database on relevant properties of viable rhesus and tamarin follicles and oocytes that can be recovered from animals of different ages, (2) To construct culture conditions which support the development of granulosa cell enclosed oocytes obtained from rhesus and tamarin antral follicles to the fertilizable mataphase II state, and (3) To construct culture conditions which support the development of oocytes in preantral follicles obtained from rhesus and tamarin ovaries to the fertilizable metaphase II state.
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