Dry storage of mammalian spermatozoa and cells: state-of-the-art and possible future directions.

This review provides a snapshot of the current state-of-the-art of drying cells and spermatozoa. The major successes and pitfalls of the most relevant literature are described separately for spermatozoa and cells. Overall, the data published so far indicate that we are closer to success in spermatozoa, whereas the situation is far more complex with cells. Critical for success is the presence of xeroprotectants inside the spermatozoa and, even more so, inside cells to protect subcellular compartments, primarily DNA. We highlight workable strategies to endow gametes and cells with the right combination of xeroprotectants, mostly sugars, and late embryogenesis abundant (LEA) or similar 'intrinsically disordered' proteins to help them withstand reversible desiccation. We focus on the biological aspects of water stress, and in particular cellular and DNA damage, but also touch on other still unexplored issues, such as the choice of both dehydration and rehydration methods or approaches, because, in our view, they play a primary role in reducing desiccation damage. We conclude by highlighting the need to exhaustively explore desiccation strategies other than lyophilisation, such as air drying, spin drying or spray drying, ideally with new prototypes, other than the food and pharmaceutical drying strategies currently used, tailored for the unique needs of cells and spermatozoa.

Late Embryogenesis Abundant (LEA) proteins confer water stress tolerance to mammalian somatic cells.

Late Embryogenesis Abundant (LEA) proteins are commonly found in plants and other organisms capable of undergoing severe and reversible dehydration, a phenomenon termed "anhydrobiosis". Here, we have produced a tagged version for three different LEA proteins: pTag-RAB17-GFP-N, Zea mays dehydrin-1dhn, expressed in the nucleo-cytoplasm; pTag-WCOR410-RFP, Tricum aestivum cold acclimation protein WCOR410, binds to cellular membranes, and pTag-LEA-BFP, Artemia franciscana LEA protein group 3 that targets the mitochondria. Sheep fibroblasts transfected with single or all three LEA proteins were subjected to air drying under controlled conditions. After rehydration, cell viability and functionality of the membrane/mitochondria were assessed. After 4 h of air drying, cells from the un-transfected control group were almost completely nonviable (1% cell alive), while cells expressing LEA proteins showed high viability (more than 30%), with the highest viability (58%) observed in fibroblasts expressing all three LEA proteins. Growth rate was markedly compromised in control cells, while LEA-expressing cells proliferated at a rate comparable to non-air-dried cells. Plasmalemma, cytoskeleton and mitochondria appeared unaffected in LEA-expressing cells, confirming the protection conferred by LEA proteins on these organelles during dehydration stress. This is likely to be an effective strategy when aiming to confer desiccation tolerance to mammalian cells.

Towards storage of cells and gametes in dry form.

We review published data on cell/gamete lyophilization. Most studies have utilized the same established protocols for cryopreservation (storage in liquid nitrogen) as for cell lyophilization (dehydration of frozen samples by water sublimation). Surveying natural lyoprotectants, we suggest trehalose and late embryogenesis abundant (LEA) proteins as ideal candidates for the reversible desiccation of mammalian cells/gametes. We find that despite the numerous water subtraction techniques, scientists have relied almost exclusively on lyophilization. There is thus room for improvement in both medium formulation and water subtraction strategies for dry cell/gamete storage. We believe the development of dry processing protocols for use in biobanks of cells/gametes, at reduced cost and with minimal carbon footprint, is within our grasp.

High levels of anandamide, an endogenous cannabinoid, block the growth of sheep preimplantation embryos by inducing apoptosis and reversible arrest of cell proliferation.

BACKGROUND: The process of implantation is mediated by various molecules, one of which is anandamide (AEA), a lipid signalling ligand belonging to the family of endocannabinoids. AEA exerts its effects on implantation by binding to the Type 1 Cannabinoid Receptor (CB1-R), expressed in both blastocysts and uterus. We wanted to know whether the endocannabinoid signalling system was present also in the sheep reproductive tract and which kind of effect(s) AEA had on the development of sheep blastocysts in vitro. METHODS: We analysed the expression and activity of the endocannabinoid system in sheep reproductive tracts and blastocysts. Hatched sheep blastocysts were then exposed to AEA and its effect(s) were determined by TUNEL assay and by measuring the rate of necrosis and 5-bromo-deoxyuridine incorporation. RESULTS: We show that the AEA signalling system is present in sheep and that high concentrations of AEA induce apoptosis and inhibit cell proliferation via a CB1-R-dependent mechanism. Indeed, AEA effects were blocked when sheep blastocysts were cultured in the presence of the CB1-R antagonist SR161417A. Moreover, AEA inhibition of cell proliferation was reversible, as arrested embryos resumed a normal growth rate upon AEA removal from the medium. CONCLUSIONS: Our results suggest that disturbed regulation of AEA signalling via CB1-R may be associated with pregnancy failure. AEA could lower the quality of blastocysts by inducing apoptosis and inhibiting cell proliferation, thus making them incompetent for implantation.