The Lycat homologue in zebrafish maps to the deletion interval of the cloche mutant in which hematopoietic and endothelial cell lineages are affected. However, its definitive relationship to cloche is inconclusive, partly due to inadequate expression data of Lycat from any organisms. We precisely examined the temporal and spatial expression patterns of Lycat in mouse using RNA in situ hybridization, immunostaining, and BAC transgenesis. Lycat is initially expressed in developing heart, lung, and somites, and later becomes progressively restricted to all vascular smooth muscle cells. In adult ovaries, Lycat turns on in oocytes during the transition from primary to secondary follicles. Expression of the Lycat/reporter transgene in the extraembryonic mesoderm, cardiogenic mesoderm, and primitive streak, but not extraembryonic endoderm at E7.5, suggests its potential roles in regulating cardiac, smooth muscle, hematopoietic and endothelial lineages. Promoter mapping assay by transient transgenesis identifies a novel cardiac-specific regulatory region in the Lycat locus.

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A 2-step culture system was designed and tested for the <i>in vitro</i> maturation efficiency of oocytes from pre-puberty preantral follicles of FVB/N inbred mice. The following modifications were made: 1) The concentration of ITS was reduced by half in the basal MIF medium to minimize uncoordinated growth between oocyte and GC cells; 2) Heterogeneous preantral follicles were cultured in groups of 3 - 5 follicles in hanging drops of medium with reduced concentration of ITS for six days to induction follicular aggregation. This hanging drop method mimics a 3-D IVM culture system at the early stage of cultivation in which the sphere structure of each follicle is well maintained. It also enables follicles in each aggregate to communicate with each other, synchronize their growth, and thus prevent immature follicular rupture. 3) Medium was further supplemented with retinoic acid to enhance developmental capacity of meiotically arrested oocytes. After a 14-day culture in vitro, ~37% of the collected inbred preantral follicles completed nuclear maturation. Approximately 94% of the mature oocytes tested were able to be fertilized; and 77% of them developed into healthy embryos. These results demonstrate that our IVM system is reliable to produce a satisfactory number of high quality oocytes. In addition, multiple cytoplasmic parameters, including gene expression of key regulators, chromosome/spindle organization, mitochondrial proliferation and distribution, and total ATP content were explored to characterize the supportive and limiting components of our IVM system so that the culture system can be further optimized