A whole-embryo culture system proposed to study mouse sexual determination

Standard procedures to culture rodent embryos (2-4 somite explanted embryos and a 48-hr culture period), do not allow assessment of genital crest differentiation. Cultures of older embryos have to be used for this objective. The proposed method uses glass apparatus derived from those initially decribed by New (1967) and Cockroft (1973). Each apparatus allows the culture of six embryos in 80ml of a permanently gassed (95% O(2)) and circulating culture medium [Waymouth's medium-Hanks' balanced salt solution-rat serum (40:40:20, by vol.)]. In this system, 24 embryos (four groups of six) can be cultured under the same experimental conditions. In the mouse, the genital crest begins to develop on gestation day (GD) 9 and differentiation can be observed between GD12 and GD13 [GD0 = middle of the mating period (09.00-11.00 hr)]. GD12 mouse embryos were cultured for 30 hr. An in vitro /itin vivo comparison of survival rate, development and morphology was performed. Serial sections of cultured embryos were taken for microscopic examination. Survival rate proved to be 82% using this method. No delay in general development was observed. Histological examination demonstrated that gonadal determination in cultured embryos also paralleled differentiation in vivo. The results clearly demonstrate that a 30-hr culture period of GD12 mouse embryos enables the study of the murine sexual determination.