Cold storage and cryopreservation methods for spermatozoa of the sea urchins Lytechinus pictus and Strongylocentrotus purpuratus

• Published in: Developmental dynamics Vol. 253; no. 8; pp. 781 - 790
• Main Authors: Vacquier, Victor D., Hamdoun, Amro
• Format: Journal Article
• Language: English
• Published: Hoboken, USA John Wiley & Sons, Inc 01.08.2024; Wiley Subscription Services, Inc
• Subjects: Animals; Brittleness; Cell culture; Cold storage; Cryopreservation; Cryopreservation - methods; echinoderm; Echinoidea; Embryogenesis; Fertilization; Gene manipulation; genetic models; Genetic modification; Larvae; Lytechinus - physiology; Lytechinus pictus; Male; Metamorphosis; sea urchin; Sea urchins; Semen Preservation - methods; Sperm; sperm cryopreservation; Spermatozoa; Spermatozoa - cytology; Spermatozoa - physiology; Strongylocentrotus purpuratus; Strongylocentrotus purpuratus - embryology; Strongylocentrotus purpuratus - physiology; sea urchin; spermatozoa; genetic models; echinoderm; sperm cryopreservation
• ISSN: 1058-8388; 1097-0177; 1097-0177
• DOI: 10.1002/dvdy.691

Background Sea urchins have contributed greatly to knowledge of fertilization, embryogenesis, and cell biology. However, until now, they have not been genetic model organisms because of their long generation times and lack of tools for husbandry and gene manipulation. We recently established the sea urchin Lytechinus pictus, as a multigenerational model Echinoderm, because of its relatively short generation time of 4–6 months and ease of laboratory culture. To take full advantage of this new multigenerational species, methods are needed to biobank and share genetically modified L. pictus sperm. Results Here, we describe a method, based on sperm ion physiology that maintains L. pictus and Strongylocentrotus purpuratus sperm fertilizable for at least 5–10 weeks when stored at 0°C. We also describe a new method to cryopreserve sperm of both species. Sperm of both species can be frozen and thawed at least twice and still give rise to larvae that undergo metamorphosis. Conclusions The simple methods we describe work well for both species, achieving >90% embryo development and producing larvae that undergo metamorphosis to juvenile adults. We hope that these methods will be useful to others working on marine invertebrate sperm. Key Findings Sperm can maintain fertilizing capacity ex vivo for 5‐10 weeks when stored at 0oC. When freezing in liquid nitrogen no stepwise addition of cryoprotectant, or stepwise drop in temperature are required. A standard fertilization assay is presented to score cleavage stage sea urchin embryos produced by cryopreserved sperm. Sperm frozen and thawed more than once can produce larvae.