Extracts of rat amniotic fluid contain a potent inducer of intestinal crypt formation

The effect of acid, basic, and organic extracts from Long Evans rat amniotic fluid (RAF) and from Swiss ICR mouse amniotic fluid (MAF) was studied on 15-day fetal mouse duodenal mucosa in organ culture. Amniotic fluids (20 ml) were lyophilized and extracted 1) with CHCl3:MeOH and the organic phase was evaporated; then 2), the residue was acidified with a solution of 0.1 N HCl in 10% acetic acid and the liquid phase was lyophilized; finally 3), 0.01 M NH4OH was added to the residue and the liquid phase was lyophilized. The product of each extraction was added to 20 ml of Trowell T8 medium. Acid and basic extracts of RAF and MAF have no effect on the formation of duodenal villi and crypts after 48 hours of culture. With the organic extract of MAF, small villi are present after 48 hours of culture and absorptive cells are poorly differentiated. With the organic extract of RAF, well-developed villi have differentiated after 48 hours of culture; moreover, crypts are present at the same stage and Paneth cells are identified within these crypts. During the 8-10 hour period, the explants cultured with the Trowell T8 medium supplemented with RAF or MAF organic extracts show a 35% increase in 3H-thymidine incorporation over the controls cultured with Trowell T8 medium alone. These results indicate that organic extracts from MAF and RAF are able to promote villus formation in undifferentiated explants from 15-day fetal mouse duodenum in organ culture. Furthermore, RAF organic extract contains a factor that can induce the formation of duodenal crypts and the differentiation of Paneth cells in culture at least 2 days before their normal appearance.