56 SOMATIC CELL NUCLEAR TRANSFER IN NON-HUMAN PRIMATES: THE POSSIBILITY OF USING OOCYTES MATURED IN VITRO FOR UP TO 3 DAYS AS HOST OOPLASTS

Abstract

Production of cloned nonhuman primate embryos has been reported using mature oocytes obtained from donors treated in vivo with a high dose of recombinant human FSH (r-hFSH, 35 IU per day for 10 days). The disadvantages of this approach are the high cost of hormones and the need to use the oocytes shortly after collection. Our study aimed to investigate the possibility of using initial in vivo treatment with a reduced FSH dose followed by in vitro culture for long periods of up to 3 days to produce mature monkey oocytes as host ooplasts for somatic cell nuclear transfer (SCNT). Adult female long-tailed Macaque (Macaca fascicularis) monkeys were treated with r-hFSH (Serono, Aubonne, Switzerland, 35 IU per day, i.m.) either for 10 days with an injection of hCG (1000 IU, i/m) 34 h before oocyte collection (G.I) or with only r-hFSH for 7 days (G.II). Cumulus oocyte complexes (COCs) were collected by follicular aspiration and then cultured in TCM-199 medium (GIBCO) supplemented with estradiol-17β, FSH, LH, and 10% FCS at 39°C in an incubator with 5% CO₂ in air. The maturation rate based on the level of cumulus expansion and the presence of the first polar body was recorded at the moment of collection and during 24 h, 48 h, and 72 h of in vitro maturation (IVM). For SCNT, the mature Metaphase II oocytes were separated from cumulus cells and selected for enucleation in the presence of cytochalasin B (Sigma, St. Louis, MO, USA). Skin fibroblasts obtained from adult monkeys were cultured in DMEM+ 10% FCS and induced to quiescence in DMEM 0% FCS 2 days before use. A single cell was transferred under the zona of each enucleated oocyte. Couplets were fused with two direct current (DC) pulses of 220 V/mm for 25 μs in Zimmerman medium. Fused oocytes were cultured in medium containing cyclohexamide for 6 h before placing them into monkey culture medium (Cook, Brisbane, Australia). The average number of oocytes collected per animal were 21.2 (n = 18) and 18.6 (n = 12) for the G.I and G.II treatments, respectively. For G.I, the rate of COCs with fully expanded cumulus was 42% at collection and was maximal (80%) at Day 1 of IVM. For G.II, fully expanded cumulus was not observed at the time of collection and during the first 2 days of IVM, but 75% of COCs had full cumulus expansion by Day 3 of IVM. The rates of intact and fused oocytes were 50.3% for G.I and 55.4% for G.II. From the fused oocytes, 67.8% and 64.4% developed to the 4- to 8-cell stages at Days 2–3 after nuclear transfer for G.I and G.II, respectively. From these data, it can be concluded that this approach can be applied to optimize production of mature oocytes for non-human primate SCNT and ART (assisted reproductive technologies) programs.

This work was supported by AIRE-Development.