Novel Apoplastic Antifreeze Proteins of Deschampsia antarctica as Enhancer of Common Cell Freezing Media for Cryobanking of Genetic Resources, a Preliminary Study

Abstract

Antifreeze proteins (AFPs) are natural biomolecules found in cold-adapted organisms that lower the freezing point of water, allowing survival in icy conditions. These proteins have the potential to improve cryopreservation techniques by enhancing the quality of genetic material postthaw. Deschampsia antarctica, a freezing-tolerant plant, possesses AFPs and is a promising candidate for cryopreservation applications. In this study, we investigated the cryoprotective properties of AFPs from D. antarctica extracts on Atlantic salmon spermatozoa. Apoplastic extracts were used to determine ice recrystallization inhibition (IRI), thermal hysteresis (TH) activities and ice crystal morphology. Spermatozoa were cryopreserved using a standard cryoprotectant medium (C+) and three alternative media supplemented with apoplastic extracts. Flow cytometry was employed to measure plasma membrane integrity (PMI) and mitochondrial membrane potential (MMP) postthaw. Results showed that a low concentration of AFPs (0.05 mg/mL) provided significant IRI activity. Apoplastic extracts from D. antarctica demonstrated a cryoprotective effect on salmon spermatozoa, with PMI comparable to the standard medium. Moreover, samples treated with apoplastic extracts exhibited a higher percentage of cells with high MMP. These findings represent the first and preliminary report that suggests that AFPs derived from apoplastic extracts of D. antarctica have the potential to serve as cryoprotectants and could allow the development of novel freezing media.

Keywords: antifreeze proteins; apoplastic extract; cryoprotectant; ice recrystallization inhibition; thermal hysteresis.

Figure 1

Ice recrystallization inhibition (IRI) activity of controls and apoplastic extracts of D. antarctica. C−: Negative control (30% Sucrose); C+: Positive control (type I AFP); AE: Apoplastic extracts. The extracts were serially diluted with the protein concentration shown above each panel. The scale bar represents 25 μm.

Figure 2

Effect of apoplastic protein concentration on the ice crystal morphology and thermal hysteresis (TH) activity. C−: Negative control (30% Sucrose); C+: Positive control (type I AFP); AE: Apoplastic extracts at two different concentrations.

Figure 3

Effect of freezing medium on the antifreeze activity of apoplastic extracts of D. antarctica. (A) Ice recrystallization inhibition (IRI) activity. (B) Thermal hysteresis (TH) activity. (C) Ice crystal morphology in presence of apoplastic proteins. C− (FM): Freezing medium with Me₂SO, glucose and BSA in Cortland^® diluent; AE: apoplast extract obtained from plants cold acclimated for two weeks; AE + FM: apoplast extract obtained from plants cold acclimated for two weeks supplemented with freezing medium. One-way ANOVA and Tukey’s post hoc tests were applied for multiple comparisons. Different letters indicate significant differences, p < 0.05, n = 3.

Figure 4

Effect of the cryopreservation on fish sperm quality. Postthawing plasma membrane integrity (PMI) and mitochondrial membrane potential (MMP) analysis. (A) Percentage of spermatozoa that maintained PMI postthawing. (B) Percentage of spermatozoa with PMI that maintained high MMP postthawing. C−: semen without cryoprotectant; C+: standard freezing medium; T1: 1.3 M Me₂SO, 0.3 M Glucose, 2% (w/v) BSA and 0.05 mg/mL apoplastic extract; T2: 1.3 M Me₂SO, 0.3 M Glucose and 0.05 mg/mL apoplastic extract; T3: 0.05 mg/mL apoplastic extract. One-way ANOVA and Tukey post hoc tests were applied for multiple comparisons. Significance levels are marked with a different letter, where a, b and c are significantly different, p < 0.05, n = 3.