Conservation and contrast in cell states of echinoderm ovaries

In contrast to women, echinoderms have the amazing ability to keep producing functional gametes throughout their lifespan, in some cases exceeding 200 years. The histology and ultrastructure of echinoderm ovaries has been described but how these ovaries function and maintain the production of high-quality gametes is still a mystery. Here, we present the first single cell RNA sequencing (scRNAseq) datasets of two sea urchin species (Strongylocentrotus purpuratus and Lytechinus variegatus) and one sea star species (Patiria miniata). We find 14 cell states in the Sp ovary, 16 cell states in the Lv ovary and 13 cell states in the ovary of the sea star. This resource is essential to understand the structure and functional biology of the ovary in echinoderms, and better informs decisions in the utilization of in situ RNA hybridization probes selective for various cell types. We link key genes with cell clusters of the feature plots in validation of this approach. This resource also aids in the identification of the stem cells for prolonged and continuous gamete production, is a foundation for testing changes in the annual reproductive cycle, and is essential for understanding the evolution of reproduction of this important phylum. Highly selective gene expression revealed by this dataset also divulges gene targets of highest priority for interrogating gene activities by Cas9-targeted gene knock-out and knock-in approaches and in dissociated ovarian cell cultures to test the function of each cell type identified. Adult Strongylocentrotus purpuratus were obtained from Pete Halmay of Pt. Loma Marine Invertebrate Lab (Lakeside, CA, e-mail: [e-mail]) and housed in aquaria with artificial seawater (ASW) at 16°C (Coral Life Scientific Grade Marine Salt; Carson, CA). Adult Lytechinus variegatus were obtained from the Pelagic Corporation ([e-mail]). They were housed in aquaria with ASW at room temperature (Coral Life Scientific Grade Marine Salt; Carson, CA). Adults Patiria miniata were collected by Josh Ross (South Coast Bio-Marine) off the Californian coast housed in aquaria with artificial seawater (ASW) at 16°C. Ovaries were collected from adult echinoderms and washed twice with calcium-free seawater (For 1 L: NaCl 26.5 gm; KCl 0.7 gm; MgSO4-7H2O 11.9 gm; NaHCO3 0.5 gm; pH to 8.0, salinity should be 34 ppt; McClay, 2004), and then suspended in hyalin-extraction media (HEM, for 1 L: NaCl 18.5 gm; KCl 0.7 gm; MgSO4-7H2O 11.9 gm; glycine 22.5 gm; Tris base 1.21 gm; EGTA 0.76 gm; pH to 8.0; 56) for 15 min. When cells were beginning to dissociate, the ovaries were collected and washed in 0.5M NaCl, gently sheared with a pipette, run through a 40 micron Nitex mesh, counted on a hemocytometer, and diluted to reach the appropriate concentration for the scRNA-seq protocol.