A WHOLE-EMBRYO CULTURE SYSTEM PROPOSED TO STUDY MOUSE SEXUAL DETERMINATION

Standard procedures to culture rodent embryos (2-4 somite explanted em bryos and a 48-hr culture period), do not allow assessment of genital crest differentiation. Cultures of older embryos have to be used for t his objective. The proposed method uses glass apparatus derived from t hose initially decribed by New (1967) and Cockroft (1973). Each appara tus allows the culture of six embryos in 80 ml of a permanently gassed (95% O-2) and circulating culture medium [Waymouth's medium-Hanks' ba lanced salt solution-rat serum (40:40:20, by vol.)]. In this system, 2 4 embryos (four groups of six) can be cultured under the same experime ntal 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 mo use embryos were cultured for 30 hr. An in vitro/in vivo comparison of survival rate, development and morphology was performed. Serial secti ons of cultured embryos were taken for microscopic examination. Surviv al rate proved to be 82% using this method. No delay in general develo pment 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 G D12 mouse embryos enables the study of the murine sexual determination .