60 INFLUENCE OF HOECHST STAINING FOR NUCLEAR TRANSFER ON PARTHENOGENETIC EMBRYOS IN CYNOMOLGUS MONKEYS (MACACA FASCICULARIS)

Abstract

Nonhuman primates are valuable animal models for the study of human diseases, and somatic cell nuclear transfer (SCNT) is an important method for establishing tailor-made embryonic stem (ES) cells and transgenic animals in these model species. However, there have been few reports on SCNT in nonhuman primates. Moreover, the development of cloned blastocysts could be influenced by any chemical reagents and manipulations used in this technique. In this study we compared blastocyst developmental rates with and without Hoechst staining. Metaphase II (MII) oocytes were collected from hormone-treated adult female cynomolgus monkeys (Macaca fascicularis) under laparoscopic observation (Torii et al. 2000 Primates 41, 39–47). A pseudo-SCNT procedure, which consisted of cytochalasin B treatment, cytoplasm removal, and dissection of the oocyte membrane, was performed on MII oocytes either in the presence of (Experiment 1; Ex1) or in the absence of Hoechst 33342 (Experiment 2; Ex2). Hoffman modulation contrast microscopy was used in Ex1 and Nomarski differential interference contrast (DIC) was used in Ex2. In Ex1, cumulus-free MII oocytes were treated with Hoechst 33342 (5 mg mL^–1; Sigma Chemical Co., St. Louis, MO, USA) for 5 min and the following pseudo-SCNT procedure was carried out: cytochalasin B (CB, 5 µg mL^–1; Sigma) for 20 min, removal of a small amount of cytoplasm (pseudo-EN), and then dissection of the oocyte cytoplasmic membrane (pseudo-IN) under Hoffman modulation contrast microscopy. In Ex2, CB treatment, pseudo-EN, and pseudo-IN were performed under Nomarski DIC microscopy. After treatment, these oocytes were activated by parthenogenetic stimulation. Parthenogenesis was induced by 5-m ionomycin (Sigma) for 2 min and 2 mm 6-dimethylaminopurine (Sigma) for 4 h. As a control, cumulus-free MII oocytes were activated by only parthenogenetic stimulation, without the above manipulations. These activated oocytes were cultured in CMRL-1066 medium containing 20% calf serum at 38°C in 5% CO₂, 5% O₂, and 90% N₂ for 7–8 days. The rates of development to blastocyst stage were 14% (1/7) in Ex1, 30% (3/10) in Ex2, and 29% (2/7) in the control. The developmental rate of parthenotes to the blastocyst stage in Ex2 was greater than that in Ex1 and similar to the control. These results suggest that treatment of cynomolgus monkey oocytes with Hoechst staining possibily decreases development to the blastocyst stage. Therefore, enucleation under Nomarski DIC will be a good alternative to Hoechst staining and could improve the potential development of nonhuman primate SCNT embryos.