A nonhuman primate model is desirable for the study of reproductive and developmental processes because of the similarities to man, the need for improved disease models for medical research and the desire to preserve endangered nonhuman primate species. Toward this end, the PI has established an in vitro fertilization-embryo transfer (IVF-ET) laboratory at the Oregon Regional Primate Research Center (ORPRC) which routinely conducts IVF-ET using rhesus macaque gametes and embryos. The ORPRC maintains a large colony of rhesus monkeys and has the resources, experience and expertise required to support an organized study of the culture conditions required for successful in vitro reproductive processes. Improvements in our ability to mature ovarian oocytes in vitro and to culture IVF-produced embryos through preimplantation development are critical to further application of this nonhuman primate animal resource.
The specific aims of this proposal are: (1) To analyze the effect of different culture media on the in vitro maturation of rhesus monkey oocytes.
This aim will evaluate the maturational efficiency of media formulations using the attainment of metaphase II and the oocyte's ability to fertilize as measurable endpoints. Since zona hardening may occur in vitro and limit sperm access to the oocyte, strategies are included that assay for hardening and that violate the integrity of the zona pellucida so that the capacity to fertilize and the developmental potential of the in vitro matured oocyte can be directly measured. (2) To formulate improved culture media for the preimplantation development of rhesus monkey embryos. The ability of different culture conditions to support development will be analyzed with emphasis on co-culture approaches and the addition of defined growth factors. The developmental potential of in vitro produced embryos will be evaluated by timed embryo transfers to synchronized recipients in natural menstrual cycles. Specific endpoints, implantation and development to live term birth will be compared with those obtained from similar transfers involving in vivo-produced blastocysts recovered nonsurgically by uterine lavage. (3) To characterize rhesus monkey preimplantation development in vitro. Dramatic changes in biochemical activity accompany preimplantation development and activation of the embryonic genome. In order to identify potential biochemical markers of embryo quality and to define the initiation, during development, of zygotic gene activity, an investigation of cytoplasmic and nuclear changes that occur during development is proposed.
