The overall objective is to improve a chemically-defined culture medium (HECM) to support normal development in vitro of oocytes and of in vitro fertilized embryos, from hamsters and rhesus monkeys. A strong underlying premise is that improved ability to sustain preimplantation development in vitro will reveal new information about the epigenetic regulation of embryogenesis. These two goals are interdependent. A key strategy is the use of protein-free culture media, avoiding unknown factors associated with most commercial protein (serum albumin) preparations. This allows examination of effects of particular regulators (hormones, energy substrates, etc.) on embryo development without extraneous factors compromising responses. AS well as increasing the proportions of developing embryos, reduction of experimental variance is a goal, in order to make studies more efficient and cost-effective, including smaller numbers of animals required. The experimental strategy is rigorous and systematic, with each experiment building on preceding ones to form a firm conceptual framework. A variety of technical approaches will be tested on oocytes and embryos, including: measurement of intracellular pH [pHi], respirometry, fatty acid uptake, and the novel """"""""simplex optimization"""""""" technology using computer-designed media formulations. Developmental endpoints include embryo transfers to validate culture medium improvements. Discrete developmental events will be recorded by time-lapse video and image-processing technology, providing a baseline to reveal temporal changes in embryo development responses, and to correlate morphological events with changes in [pHi]. Knowledge of the normal [pHi] of the preimplantation embryo is important for enhancing development in vitro; at present, it is now known if conventional culture media maintain normal [pHi]. A model for amino acid buffering of [pHi] will be tested that could provide insights into the mode of [pHi] control in vivo, as well as leading to further improvements in culture medium technology, including the possibility of dispensing with the need for gaseous CO2 for embryo culture. A major experiment will examine different energy substrates for embryos, in light of growing evidence that carbohydrate substrates are inadequate. Respirometry applied to embryos will be more efficient than embryo culture in determining effectiveness of different substrates. Other potentially important factors to be examined include vitamins, trace metals and hormones. The outcome of this research will be increased knowledge of the regulation of primate, including human, oocyte maturation and preimplantation embryo development, that could be applied towards the treatment of infertility as well as fertility regulation; and an improved, chemically-defined culture medium suitable for human and other primate oocytes and embryos. This medium will (i) permit considerable numbers of germinal vesicle stage oocytes harvested for human in vitro fertilization to be fully utilized in this infertility treatment; (ii) provide a means to recruit large numbers of non-human primate oocytes for production of preimplantation embryos by IVF, thus breaking down the principal barrier to progress in research on primate early development. This advance will have significant indirect benefits to human reproductive health problems.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project--Cooperative Agreements (U01)
Project #
5U01HD022023-09
Application #
3552562
Study Section
Special Emphasis Panel (SRC (12))
Project Start
1991-09-01
Project End
1996-08-31
Budget Start
1993-09-01
Budget End
1994-08-31
Support Year
9
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Type
Schools of Earth Sciences/Natur
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Squirrell, J M; Schramm, R D; Paprocki, A M et al. (2003) Imaging mitochondrial organization in living primate oocytes and embryos using multiphoton microscopy. Microsc Microanal 9:190-201
Zheng, Ping; Si, Wei; Bavister, Barry D et al. (2003) 17Beta-estradiol and progesterone improve in-vitro cytoplasmic maturation of oocytes from unstimulated prepubertal and adult rhesus monkeys. Hum Reprod 18:2137-44
Bavister, B D; Kinsey, D L; Lane, M et al. (2003) Recombinant human albumin supports hamster in-vitro fertilization. Hum Reprod 18:113-6
Zheng, P; Wang, H; Bavister, B D et al. (2001) Maturation of rhesus monkey oocytes in chemically defined culture media and their functional assessment by IVF and embryo development. Hum Reprod 16:300-5
Squirrell, J M; Lane, M; Bavister, B D (2001) Altering intracellular pH disrupts development and cellular organization in preimplantation hamster embryos. Biol Reprod 64:1845-54
Ludwig, T E; Lane, M; Bavister, B D (2001) Differential effect of hexoses on hamster embryo development in culture. Biol Reprod 64:1366-74
Zheng, P; Si, W; Wang, H et al. (2001) Effect of age and breeding season on the developmental capacity of oocytes from unstimulated and follicle-stimulating hormone-stimulated rhesus monkeys. Biol Reprod 64:1417-21
Steeves, C L; Lane, M; Bavister, B D et al. (2001) Differences in intracellular pH regulation by Na(+)/H(+) antiporter among two-cell mouse embryos derived from females of different strains. Biol Reprod 65:14-22
Ludwig, T E; Squirrell, J M; Palmenberg, A C et al. (2001) Relationship between development, metabolism, and mitochondrial organization in 2-cell hamster embryos in the presence of low levels of phosphate. Biol Reprod 65:1648-54
Zheng, P; Bavister, B D; Ji, W (2001) Energy substrate requirement for in vitro maturation of oocytes from unstimulated adult rhesus monkeys. Mol Reprod Dev 58:348-55

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