Cryopreservation of Hydractinia symbiolongicarpus Sperm to Support Community-Based Repository Development for Preservation of Genetic Resources.

Hydractinia symbiolongicarpus is an emerging model organism in which cutting-edge genomic tools and resources are being developed for use in a growing number of research fields. One limitation of this model system is the lack of long-term storage for genetic resources. The goal of this study was to establish a generalizable cryopreservation approach for Hydractinia that would support future repository development for other cnidarian species. Specific objectives were to: (1) characterize basic parameters related to sperm quality; (2) develop a generalizable approach for sperm collection; (3) assess the feasibility of in vitro fertilization (IVF) with sperm after refrigerated storage; (4) assess the feasibility of IVF with sperm cryopreserved with various sperm concentrations; (5) evaluate feasibility of cryopreservation with various freezing conditions, and (6) explore the feasibility of cryopreservation by use of a 3-D printed open-hardware (CryoKit) device. Animal husbandry and sperm collection were facilitated by use of 3-D printed open hardware. Hydractinia sperm at a concentration of 2 × 107 cells/mL stored at 4 °C for 6 d were able to achieve 50% fertilization rate. It appeared that relatively higher sperm concentration (>5 × 107 cells/mL) for cryopreservation could promote fertilization. A fertilization rate of 41−69% was observed using sperm equilibrated with 5, 10, or 15% (v/v) cryoprotectant (dimethyl sulfoxide or methanol) for 20 min, cooled at a rate of 5, 10, or 20 °C/min from 4 °C to −80 °C, at a cell concentration of 108/mL, in 0.25 mL French straws. Samples cryopreserved with the CryoKit produced a fertilization rate of 72−82%. Establishing repository capabilities for the Hydractinia research community will be essential for future development, maintenance, protection, and distribution of genetic resources. More broadly, these generalizable approaches can be used as a model to develop germplasm repositories for other cnidarian species.

A family of unusual immunoglobulin superfamily genes in an invertebrate histocompatibility complex.

Most colonial marine invertebrates are capable of allorecognition, the ability to distinguish between themselves and conspecifics. One long-standing question is whether invertebrate allorecognition genes are homologous to vertebrate histocompatibility genes. In the cnidarian Hydractinia symbiolongicarpus, allorecognition is controlled by at least two genes, Allorecognition 1 (Alr1) and Allorecognition 2 (Alr2), which encode highly polymorphic cell-surface proteins that serve as markers of self. Here, we show that Alr1 and Alr2 are part of a family of 41 Alr genes, all of which reside in a single genomic interval called the Allorecognition Complex (ARC). Using sensitive homology searches and highly accurate structural predictions, we demonstrate that the Alr proteins are members of the immunoglobulin superfamily (IgSF) with V-set and I-set Ig domains unlike any previously identified in animals. Specifically, their primary amino acid sequences lack many of the motifs considered diagnostic for V-set and I-set domains, yet they adopt secondary and tertiary structures nearly identical to canonical Ig domains. Thus, the V-set domain, which played a central role in the evolution of vertebrate adaptive immunity, was present in the last common ancestor of cnidarians and bilaterians. Unexpectedly, several Alr proteins also have immunoreceptor tyrosine-based activation motifs and immunoreceptor tyrosine-based inhibitory motifs in their cytoplasmic tails, suggesting they could participate in pathways homologous to those that regulate immunity in humans and flies. This work expands our definition of the IgSF with the addition of a family of unusual members, several of which play a role in invertebrate histocompatibility.

Cell Aggregation Assays for Homophilic Interactions Between Cell Surface Proteins.

Many proteins expressed on the cellular surface provide signaling and cell adhesion properties required for vital cellular functions. These binding interactions can occur between different but complementary proteins such as a ligand and receptor, or between the same protein acting as both ligand and receptor. The cell aggregation assay is a straightforward technique to identify homophilic interactions from such proteins. Here we describe the procedure for testing proteins via cell aggregation assays in HEK293T cells.

New binding specificities evolve via point mutation in an invertebrate allorecognition gene.

Many organisms use genetic self-recognition systems to distinguish themselves from conspecifics. In the cnidarian, Hydractinia symbiolongicarpus, self-recognition is partially controlled by allorecognition 2 (Alr2). Alr2 encodes a highly polymorphic transmembrane protein that discriminates self from nonself by binding in trans to other Alr2 proteins with identical or similar sequences. Here, we focused on the N-terminal domain of Alr2, which can determine its binding specificity. We pair ancestral sequence reconstruction and experimental assays to show that amino acid substitutions can create sequences with novel binding specificities either directly (via one mutation) or via sequential mutations and intermediates with relaxed specificities. We also show that one side of the domain has experienced positive selection and likely forms the binding interface. Our results provide direct evidence that point mutations can generate Alr2 proteins with novel binding specificities. This provides a plausible mechanism for the generation and maintenance of functional variation in nature.