Cryoprotection and cryodamage in sea urchin eggs
- Autores: Sara Campos Rosende
- Directores de la Tesis: Estefanía Paredes Rosendo (dir. tes.), Jesús Souza Troncoso (dir. tes.)
- Lectura: En la Universidade de Vigo ( España ) en 2025
- Idioma: inglés
- Tribunal Calificador de la Tesis: Miguel Angel Silvestre Camps (presid.), María Aranguren Gassis (secret.), Tiantian Zhang (voc.)
- Programa de doctorado: Programa de Doctorado en Ciencias Marinas, Tecnología y Gestión por la Universidad de A Coruña; la Universidad de Santiago de Compostela; la Universidad de Vigo; Universidade de Aveiro(Portugal); Universidade de Trás-os-Montes e Alto Douro(Portugal) y Universidade do Minho (Portugal)
- Menciones:
- Internacional
- Calificación: Sobresaliente Cum Laude
- Materias:
- Enlaces
- Tesis en acceso abierto en: Investigo
- Resumen
This thesis covers the study of cryopreservation in eggs of the sea urchin Paracentrotus lividus, focusing on determining the factors that, so far, have not allowed it to be successfully carried out. It also defines the damage caused by cryopreservation and its complexity, highlighting the need for more advanced and personalized approaches to overcome current limitations and laying the foundations for future research in cryobiology of marine organisms.
Chapter I comprehensively addresses the characteristics, ecological importance, methods of study and conservation of echinoderms, with an emphasis on sea urchins and the species Paracentrotus lividus. In addition, cryopreservation techniques are presented as an essential tool for the conservation and sustainable management of these species, ensuring their availability for research and human consumption without compromising natural populations.
Echinoderms are a phylum of invertebrates recognized for their diversity, ecological importance and scientific value. These organisms are part of the deuterostome group, which also includes the chordates, and within the phylum are included stars, urchins, cucumbers and sea lilies, classified into five main classes: Crinoidea, Asteroidea, Ophiuroidea, Echinoidea and Holothuroidea. Despite their wide diversity, echinoderms share several common characteristics. Adults show a body organization based on pentaradial symmetry, in contrast to the bilateral symmetry exhibited by their larvae. They also have an aquifer vascular system that uses water for locomotion functions (through tubular feet connected to this system), feeding, breathing and sensory perception. Its internal skeleton is composed of calcareous plates or ossicles that provide structural support and protection. They have a decentralized nervous system, without a central brain, but with a nerve ring and radial nerves. The digestive system is composed of the mouth, stomach, intestine and anus.
Within marine ecosystems, echinoderms are key players in the regulation of benthic communities. For example, sea urchins play an essential role as herbivores that control the growth of algae, contributing to nutrient recycling and ecosystem balance. They are also the object of study in evolutionary and developmental biology due to their relevance in the understanding of embryonic processes.
Sea urchins are a dioecious species, with separate sexes, whose reproductive system includes five gonads that release gametes into the water through gonopores (spawn). Embryonic development is highly studied due to its scientific importance and is highly influenced by environmental factors such as temperature and food availability. It includes the following stages: 1. Fertilization and cell segmentation. Developmental patterns are established in these first cell divisions.
2. Blastula. Early stage characterized by a focal structure.
3. Gastrula. At this stage the germinal layers (ectoderm, mesoderm and endoderm) are formed.
4. Larva pluteus. Pelagic form with arms that will transform through metamorphosis into an adult urchin, marking a transition to the juvenile, benthic state.
The sea urchin Paracentrotus lividus, known as the purple urchin, is a species of ecological, economic and scientific interest that inhabits coastal waters of the north-east Atlantic and the Mediterranean, preferring rocky bottoms and areas with an abundance of seaweed on which it feeds. It is a massive spawner with external fertilization, its larvae are pelagic and go through a metamorphosis before settling on the seabed as juveniles. The gonads of this species are in high demand in Europe and Japan, reaching prices of up to 150€/kg. However, overexploitation has drastically reduced their natural populations, leading to the implementation of measures such as catch quotas, seasonal restrictions and minimum size limits. Aquaculture is presented as a sustainable alternative to meet demand and reduce pressure on wild populations.
Cryopreservation is an essential technique for the preservation of cells, tissues and gametes. It allows long-term storage at extremely low temperatures, stopping all biological activity. There are two methods of cryopreservation, slow cooling and vitrification. In the former the temperature is gradually reduced, minimizing the formation of ice crystals, while vitrification rapidly cools the material to a glassy state preventing the formation of ice crystals but requiring high concentrations of cryoprotective agents (CPAs). These CPAs are components of various classes that play a role in protecting cells during storage at low temperatures. They act by inhibiting the formation of intracellular ice thus mitigating possible damage caused by dehydration. They can be classified according to their specific site of action as permeable (if they are able to penetrate cells) or non-permeable (when they act outside the cell stabilizing the membranes and reducing toxicity). The main risks of cryopreservation are osmotic and mechanical damage caused by cell dehydration and ice crystal formation, respectively.
The cryopreservation of marine organisms has progressed mainly in sperm and larvae of those species that have the greatest economic importance for aquaculture. Eggs have specific characteristics (large volumes, low surface/volume ratio, low permeability to CPAs, high sensitivity to cold and CPA toxicity) that prevent successful cryopreservation so far.
Chapter II details the methodology and procedures used for gamete collection, toxicity testing, cryopreservation, incubation and statistical analyzes in the study of Paracentrotus lividus.
The gametes (eggs and spermatozoa) were obtained by transverse dissection, taking as quality criteria the spherical shape and homogeneous brown color in the eggs and mobility under the microscope in the spermatozoa. In addition, only eggs from females with a fertilization rate greater than 90% were included.
Toxicity tests allowed to evaluate the toxicity of cryoprotective agents and to determine the optimal concentrations that do not cause irreversible damage. Two methods were used for this, one-step exposure where direct mixing of cells and CPA is performed and allowed to incubate for 15 minutes, and 15-step exposure using stepwise addition of CPA in equimolar steps every minute. After exposure, the samples were washed with filtered sea water (FSW) and incubated for 48h at 18°C. Evaluation was performed by measuring normal larval development (4 arms) under the microscope, and the results obtained were used to calculate No Observed Effect Concentrations (NOEC) and Lowest Observed Effect Concentrations (LOEC).
In terms of cryopreservation, five methods have been studied, each with specific characteristics. The first of these was slow cooling using a controlled freezer (Cryologic LLD) with an initial cooling to 4°C followed by a decrease in temperature of -1°C/minute until reaching -35°C and seeding at 12°C. The samples were finally immersed in liquid nitrogen and thawed in a water bath at 20°C. Another method used was contact vitrification where the samples were placed in a Fibreplug and vitrified by contact with a cooled block immersed in liquid nitrogen. Thawing was performed in the same way as in the previous method. On the other hand, a drop vitrification was performed, where a mixture of CPA and cells was deposited in small drops (3-5 mm) directly in liquid nitrogen, and a vitrification in straws of 0.25 µl immersed directly in LN2. The last method used was vitrification with laser thawing by depositing the samples in a Cryotop and placing it directly in LN2 for subsequent thawing with a high-speed laser.
Measurement of toxicity and success of cryopreservation was performed by observing formaldehyde-fixed samples to calculate the percentage of normal larvae (typical 4-armed pluteus larval form) and larval size in a subgroup (35 cells per replicate). The most observed abnormalities were retraction or malformation of arms, delayed growth or lack of fertilization.
Chapter III covers the main challenges present in the cryopreservation of sea urchin eggs, particularly of Paracentrotus lividus. It also addresses fundamental aspects of the current state of research and the reasons behind the technical and biological obstacles that prevent success in this area.
The cryopreservation of gametes, embryos and larvae of marine invertebrates has been studied for decades, with important advances in the preservation of spermatozoa, but with great challenges in the case of eggs. This procedure has great potential in species conservation, aquaculture and genetic selection programs. However, until now, no effective protocols have been developed for the cryopreservation of sea urchin eggs, especially in P. lividus, which is considered a model species due to its ecological and commercial relevance. The main obstacles that prevent the cryopreservation of these cells are defined by their specific characteristics: 1. Cell size and structure. Eggs are large cells with a low surface/volume ratio that hinders the efficient transport of water and CPAs across the membrane. The low permeability of the cell membrane increases the risk of intracellular ice formation during cryopreservation, which can be lethal to the cell.
2. Composition of the membrane. P. lividus eggs have a unique lipid composition, which includes phospholipids such as phosphatidylethanolamine and phosphatidylinositol, in addition to an unusual cholesterol-phospholipid balance (1:1). This composition affects their behavior towards CPAs. On the other hand, eggs have the presence of microvilli which, although important for fertilization, do not have a clear function in cryopreservation, but could influence permeability.
3. Water and lipid content. The high water and lipid content in eggs hinders the cellular dehydration necessary to prevent ice formation. Lipids also interact with CPAs and can increase their toxicity.
4. Sensitivity to cryoprotectants. CPAs can be toxic to cells due to their ability to interact with cell membranes and other internal structures. This toxicity may be CPA-specific or non-specific (related to its chemical nature). The combination of permeable and non-permeable CPAs has shown potential to reduce toxicity in P. lividus eggs but requires further investigation.
Attempts have been made to cryopreserve eggs from starfish, oysters and mussels with a certain percentage of success, but the results have not been ideal. For example, in starfish a combination of Me2SO and trehalose resulted in a post-thaw survival of 34% but only a small percentage continued embryo development. Post-thaw fertilizations were achieved in oysters and mussels but with reduced developmental capacity compared to controls. As for P. lividus, the only viable stage so far for cryopreservation has been the embryo in the blastula state (8 hours post-fertilization), which has been shown to be able to fully develop after the process.
Studies on membrane permeability in P. lividus eggs are ongoing, and preliminary research indicates that low permeability to water and CPAs is a critical challenge that needs to be addressed. In addition, the effects of osmolarity and salinity must be considered since, as osmoconformers, they adjust their osmotic balance according to that of the external environment, which can affect the interaction with CPAs. The toxicity they present affects differently according to the stage of development, with unfertilized eggs being the most sensitive.
Future research strategies and areas should include studies of the cell and its environment by determining permeability parameters for water and CPAs, as well as evaluating the role of microvilli and other structural components in membrane permeability. Likewise, establish a control of the toxicity of CPAs by investigating combinations of permeable and non-permeable CPAs to minimize toxic effects. And finally, design optimal protocols with optimized cooling and thawing rates, exploring vitrification as an alternative to avoid slow freeze damage.
Chapter IV studies the structural damage caused by different concentrations of CPAs and cryopreservation techniques using Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM), providing valuable information on the mechanisms of damage, laying the foundations for future research in cryobiology of marine organisms. It highlights the need for more advanced and personalized approaches to preserve highly sensitive cells like P. lividus eggs.
In this study, different CPAs (dimethyl sulfoxide (Me2SO), ethylene Glycol (EG) and propylene glycol (PG)) were tested at different concentrations (0.5 to 3M) and addition methods (in a single step or in 15 equimolar steps of 1 minute each). The cryopreservation methods used were the following: 1. Slow controlled cooling (1 °C/min to -35 °C) followed by immersion in liquid nitrogen. In this case Me2SO was used in concentrations from 0.5 to 3M with and without non-permeable CPAs (trehalose, PVP, sucrose, BSA).
2. Vitrification. Three types were used: by contact where the samples touch a surface in contact with LN2, by dripping by depositing drops directly in LN2, or on straws immersed in LN2. In all cases, Me2SO and EG were used in a concentration of 0.5 to 3M.
Unfertilized control eggs had a spherical shape, intact and organized microvilli, as well as clearly organized cortical granules under the plasma membrane. On the other hand, in the fertilized eggs these granules disappeared, due to the formation of the clearly visible fertilization membrane. The effects of CPAs varied depending on the concentration used. Thus, low concentrations (0.5M) caused minimal damage, the cells kept the microvilli intact and the internal organization similar to the controls. In contrast, high concentrations (3M) caused severe and irreversible damage, with membrane ruptures, intracellular disorganization and loss of microvilli. This damage occurred regardless of the CPA or addition method used, although stepwise addition caused less structural damage than single-step addition, minimizing the osmotic impact. In slow cooling all treatments caused severe damage, with membrane rupture, extrusion of cell material and non-specific formation of fertilization membranes. Furthermore, the addition of non-permeable CPAs did not improve the results. Likewise, all vitrification methods resulted in severe damage, although 1.5M EG partially preserved the cytoplasm according to TEM images, but the cell surface showed obvious damage (loss of microvilli and membrane rupture) in SEM images.
The study identified several critical factors in cryopreservation of P. lividus eggs. The impact of CPAs showed that although low concentrations caused minimal damage, these are not sufficient to protect eggs during cryopreservation. High concentrations required for vitrification resulted in irreversible damage, highlighting the challenge of balancing cryopreservation protection and toxicity. Regarding the effects of cryopreservation methods, slow cooling caused massive deterioration due to the formation of intracellular ice crystals and osmotic shock. Vitrification, although more promising, requires high concentrations of CPAs that the eggs are not able to tolerate.
Future research should focus on optimizing ultra-rapid cooling and thawing protocols, the addition and removal of CPAs that minimize osmotic damage, and the combination of CPAs that offer protection while causing the least possible or no toxicity.
Chapter V explores the challenges and possibilities of cryopreserving P. lividus eggs, recognized for their high sensitivity to cryoprotective agents and their vulnerability to damage from low temperatures (cold injury). He also investigates the potential of advanced techniques such as vitrification to preserve cell viability and support marine conservation, genetic resource management and aquaculture. Thus, the research aims to design protocols that allow its preservation without compromising viability.
In order to address these limitations, the study focuses on exploring the combination of different CPAs, osmotic adjustments, and advanced techniques such as vitrification and rapid laser thawing. CPAs tested included dimethyl sulfoxide (Me2SO), ethylene glycol (EG); propylene glycol (PG), methanol (METH), N,N-dimethylformamide (DMF) and sugars such as sucrose and fructose. The tests were carried out both stepwise and single-step to evaluate the impact of the methodology on toxicity and cell viability. In addition, it was investigated how eggs respond to variations in salinity and osmolarity, considering that these conditions are crucial to minimize damage due to osmotic stress during cryopreservation.
The results showed that the eggs tolerate brief exposures (2 5 minutes) to reduce salinities between 45% and 60%, allowing rates of embryonic development above 50%. However, prolonged exposure to these conditions, over 48h, eliminated cell viability. Regarding the toxicity of CPAs, it was observed that low concentrations of Me2SO (up to 1.4M) were tolerable, but higher concentrations drastically reduced embryonic development. The combination of Me2SO with DMF or methanol showed better results in terms of toxicity reduction, while combinations with sugars did not provide significant benefits. Gradual addition of CPAs was more effective than one-step addition, improving both viability and larval development. With respect to cold injury, eggs briefly exposed to 4°C maintained 75% viability after 5 minutes. However, prolonged exposures of more than one hour resulted in a complete loss of viability. This result reinforces the importance of limiting cooling time to avoid irreversible damage.
Vitrification, considered a promising technique, was evaluated along with rapid laser thawing. For this, solutions with gold particles and India Ink as energy absorbing media were tested. Although these particles did not show adverse effects in short exposures, their effectiveness in cryopreserving eggs was limited due to aggregation problems in salt water. None of the solutions tested reached the consistent vitrified state, and thawed eggs showed severe damage that prevented any further embryonic development.
Overall, the study highlights that while current cryopreservation and vitrification techniques have shown promising advances, they still have significant limitations. The toxicity of CPAs, osmotic stress and chilling damage remain major obstacles. Adjustments in CPA concentrations, stepwise addition and combination of permeable and non-permeable CPAs offer some improvement but are not sufficient to ensure viability. Vitrification, although a potential alternative, requires further improvements in the solutions used and the cooling and thawing rates to prevent ice crystal formation and preserve cell integrity.
The work concludes that it is essential to continue research on the interactions between toxicity, osmolarity and vitrification to advance the cryopreservation of sea urchin eggs. In addition, it suggests that the implementation of new technologies, such as rapid thawing by laser, and the adaptation of specific protocols for marine organisms, will be essential to overcome the current challenges.
Chapter VI brings together the findings of this doctoral thesis, where not only the complexities inherent in the preservation of sea urchin eggs are highlighted, but also provides a solid basis for future research in marine cryobiology and its application in conservation and aquaculture. Finally, the main overall conclusions and possible future lines of research to be pursued to advance towards successful cryopreservation of sea urchin eggs are summarized.
