AbstractSponges contain an astounding diversity of lipids that serve in several biological functions, including yolk formation in their oocytes and embryos. The study of lipid metabolism during reproduction can provide information on food-web dynamics and energetic needs of the populations in their habitats, however, there are no studies focusing on the lipid metabolism of sponges during their seasonal reproduction. In this study, we used histology, lipidome profiling (UHPLC-MS), and transcriptomic analysis (RNA-seq) on the deep-sea sponge Phakellia ventilabrum (Demospongiae, Bubarida), a key species of North-Atlantic sponge grounds, with the goal to (i) assess the reproductive strategy and seasonality of this species, (ii) examine the relative changes in the lipidome signal and the gene expression patterns of the enzymes participating in lipid metabolism during oogenesis. Phakellia ventilabrum is an oviparous and most certainly gonochoristic species, reproducing in May and September in the different studied areas. Half of the specimens were reproducing, generating two to five oocytes per mm2. Oocytes accumulated lipid droplets and as oogenesis progressed, the signal of most of the unsaturated and monounsaturated triacylglycerides increased, as well as of a few other phospholipids. In parallel, we detected upregulation of genes in female tissues related to triacylglyceride biosynthesis and others related to fatty acid beta-oxidation. Triacylglycerides are likely the main type of lipid forming the yolk in P. ventilabrum since this lipid category has the most marked changes. In parallel, other lipid categories were engaged in fatty acid beta-oxidation to cover the energy requirements of female individuals during oogenesis. In this study, the reproductive activity of the sponge P. ventilabrum was studied for the first time uncovering their seasonality and revealing 759 lipids, including 155 triacylglycerides. Our study has ecological and evolutionary implications providing essential information for understanding the molecular basis of reproduction and the origins and formation of lipid yolk in early-branching metazoans.