Oocyte quality is an essential factor for successful fertilization, preimplantation development and production of viable offspring. Oocyte quality is determined by the environment of oocyte development and genetic programs intrinsic to the oocyte. This proposal focuses on the construction of serum-free culture systems for oocytes that promote successful embryogenesis. Oocyte culture systems are extremely valuable as experimental tools for studying the mechanisms regulating oogenesis and subsequent embryogenesis. In addition, oocyte culture systems could be used to expand populations of valuable experimental animals, or important agricultural animals. The objectives are to construct systems for complete oogenesis in vitro and to resolve the effects of the developmental stage of the oocytes at the time of isolation, the culture environment, and the genotype of the oocyte on the quality of the embryo produced.
Specific Aim 1 is to construct serum-free culture systems for complete oocyte development in vitro. The critical importance of granulosa cells in oocyte development is well documented. Therefore, the hypothesis that conditions that promote the development and function of granulosa cells will be beneficial for oocyte development in vitro will be tested. The emphasis of experiments will be to determine whether various growth factors or hormones known to promote granulosa cell development and function also encourage oocyte development in vitro. Endpoints of various experiments will include (a) percentage of primordial oocytes initiating growth, (b) oocyte size, (c) percentage that cleave to the 2-cell stage after maturation and fertilization in vitro, (d) percentage of ova that complete the 2-cell stage to blastocyst transition, and (e) birth of live young. After maturation and insemination in vitro, oocytes isolated from follicles at a specific stage of development cleave at high frequency but fail to develop significantly beyond the 2-cells stage.
Specific Aim 2 is to test the hypothesis that this restriction in competence for embryogenesis is a manifestation of failure to appropriately initiate embryonic transcription at the 1-2-cell stage. The synthesis of heat shock protein 70 will be used as one of the markers of an activated embryonic genome.
Specific Aim 3 is to determine whether the restriction in development of embryos that occurs when fully developed oocytes mature in suboptimal media also reflects a failure to appropriately activate the embryonic genome at the 1-2-cell stage.
Specific Aim 4 is to assess the influence of the genotype of the oocyte on oocyte development in vitro. By assessing the development of oocytes from various genotypes in vitro, the general applicability of the oocyte culture systems will be tested and mechanisms of atypical oocyte development will be assessed.
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