Inducing and Harvesting Xenopus Oocytes

Xenopus laevis oocytes were collected using the in vitro fertilization technique. Ovulation of the female frog was previously induced by subcutaneous injection of the hormone Human Chorionic Gonadotropin (hCG). 48 h before fertilization (pre-prime), the female was injected with 50 units of hCG. 24 h later the female was injected again with 250–300 units of hCG (prime). Embryos were maintained in Marc’s modified Ringer’s solution (10% MMR, pH 7.4), containing 1 M NaCl, 20 mM KCl, 10 mM MgSO₄, 20 mM CaCl₂, and 50 mM HEPES. The male gonads were obtained through abdominal dissection. The testes were stored in 1× MMR medium supplemented with 20% FBS. 12 h after the second injection of hCG, the oocytes were collected in 1× MMR solution, later they were fertilized with X. laevis testis extract and incubated for 1 h at room temperature in 10% MMR solution. Afterward, the medium was replaced by a 2% cysteine solution in 10% MMR, pH 7.8–7.9 for 5 min, to dissolve the gelatinous layer “degelatinization” [22 (link)]. Subsequently, the embryos were washed and incubated in 10% MMR at room temperature and finally harvested at different neurulation stages: 12.5 (early neurulation), 14 (middle neurulation), and stage 20 (late neurulation); and stage 40–45 (tadpole) [2 (link),105 ]. We observed the expression of Cxs in stages 12.5–20 and the analysis of the closure of the neural tube and morphological effects in stages 40–45. Stage 10 (gastrulation) was used as a negative control for Cxs expression and the adult brain as a positive control to validate Cxs expression in molecular biology assays.