The sexual cycle of Toxoplasma gondii is limited to the feline intestine where millions of oocysts are formed and subsequently excreted in their feces. The goal of our previous R21 was to recapitulate the T. gondii sexual cycle in tissue culture. We achieved this goal by generating cat intestinal organoids in collaboration with Jason Spence at the University of Michigan and supplementing the T. gondii infected organoids with saturated fatty acids, leading us to hypothesize that saturated fatty acids are used as a signaling molecules and not for nutrition. Polyunsaturated fatty acids are often processed to signaling molecules by oxygenation of their double bonds through the action of lipoxygenases. The T. gondii genome encodes four lipoxygenases, one of which, annotation TGME49_315970 called TgLOX1, is 600-fold upregulated in cat intestine compared to asexual stages. We hypothesize that TgLOX1 oxygenates linoleic acid to signal sexual development. One of the goals of this R01 is to test this hypothesis. Another goal of this R01 is to test the hypothesis that 3D culture and low oxygen conditions will maximize oocyst production. While we can generate sporulated oocysts that are infectious to mice, the overall yield is low because most of the oocysts in the culture do not sporulate. Currently, we plate our feline intestinal cells on snapwell inserts so that the apical side of the intestine is easy to infect, but the cells are in 2D, which may affect T. gondii development. To model 3D intestines, we will test two methods in this R01, 1) microinjection of the whole intestinal organoid, 2) a simple biomedical engineering device that creates intestinal tubes. These new 3D models as well as lowering the oxygen concentration will more closely model the cat small intestine and likely produce more mature oocysts that sporulate with greater efficiency. The final goal for this R01 is to determine the species specificity for T. gondii sexual development. The sexual cycle of T. gondii is restricted to the cat, but it is unclear why. Overall, creation of cell culture sexual development conditions will allow classical genetic crosses to become a routine technique for the T. gondii field. It will also allow for a molecular analysis of the complete life cycle of a protozoan parasite, as T. gondii asexual development can already be performed in tissue culture.