41 Antifreeze proteins as cryoprotectants in reproductive biology: a systematic review

Antifreeze proteins (AFP) are synthesised by diverse non-mammalian species, allowing them to survive at harshly cold environments. The use of diverse types of AFP for low-temperature preservation of germ cells and tissues have been explored once AFP protect live organisms from freezing. AFP protective mechanisms include creating a thermal hysteresis gap and preventing ice crystal formation. The aim of this systematic review was to compile results from the use of AFP as cryoprotectants for reproductive specimens, taking into account the species (tissue/cell source), cryopreservation method, AFP type, and concentration. Internet databases were consulted using the terms “antifreeze protein” OR “AFP” OR “antifreeze glycoprotein” OR “AFGP” OR “ice-binding protein” OR “IBP” OR “thermal hysteresis protein” AND “cryopreservation”. A total of 56 scientific articles, containing 87 experiments testing AFP in media for low-temperature preservation of gametes (34 experiments of sperm and 18 of oocytes), embryos (24), or somatic reproductive tissues or cells (11) were fully evaluated. Among the 66 experiments conducted in mammals, 77.3% resulted in positive, and 28.8% in negative outcomes upon the use of AFP. In fishes, positive and negative outcomes were observed in, respectively, 71.4% and 33.3% of 21 experiments. Most positive outcomes included improved post-warming cell survival. The AFP concentrations used in mammalian semen ranged from 0.001 µg mL⁻¹ to 500 µg mL⁻¹ and often resulted in reduction of loss of motility and kinetic parameters and improvement in fertility. Vitrification of mammalian oocytes was carried out with AFP at concentrations varying from 0.1 µg mL⁻¹ to 50 mg mL⁻¹, and this was also the most common method studied for preservation of mammalian embryos, within the same concentration range as for oocytes. The use of AFP for oocyte vitrification frequently enhanced the protection of oolemma structure, antioxidant defences, and/or subsequently embryo development while the use of AFP for embryo vitrification increased their survivability/viability after cryopreservation. For mammalian ovaries vitrification, AFP ranged from 0.1 mg mL⁻¹ to 20 mg mL⁻¹ and it often led to reduction of apoptotic follicles and maintenance of survivability after transplantation. In fishes, AFP was used at concentrations of 10 mg mL⁻¹ for blastomeres, 10 mg mL⁻¹ to 20 mg mL⁻¹ for primordial germ cells and gonadal ridges, and 0.1 µg mL⁻¹ to 10 µg mL⁻¹ for testes. Concentration of AFP in fish semen ranged from 0.1 µg mL⁻¹ to 10 mg mL⁻¹ and in fish embryos, from 40 µg mL⁻¹ to 10 mg mL⁻¹. The number of investigations using AFP as cryoprotectant for reproductive specimens has increased over the past decade. Discrepancy in the outcomes varied clearly due to AFP concentrations but were also associated with species. Overall, the beneficial effects of AFP support their use in cryobiological approaches. Moreover, a combination of different AFP types, or of AFP with antioxidants, or even the use of AFP-like synthetic molecules, represent some promising approaches to be further explored in cryopreservation.

This research was supported by CAPES, CNPq, and FAPERJ.