The beneficial effects of antifreeze proteins in the vitrification of immature mouse oocytes

Abstract

Antifreeze proteins (AFPs) are a class of polypeptides that permit organismal survival in sub-freezing environments. The purpose of this study was to investigate the effect of AFP supplementation on immature mouse oocyte vitrification. Germinal vesicle-stage oocytes were vitrified using a two-step exposure to equilibrium and vitrification solution in the presence or absence of 500 ng/mL of AFP III. After warming, oocyte survival, in vitro maturation, fertilization, and embryonic development up to the blastocyst stage were assessed. Spindle and chromosome morphology, membrane integrity, and the expression levels of several genes were assessed in in vitro matured oocytes. The rate of blastocyst formation was significantly higher and the number of caspase-positive blastomeres was significantly lower in the AFP-treated group compared with the untreated group. The proportion of oocytes with intact spindles/chromosomes and stable membranes was also significantly higher in the AFP group. The AFP group showed increased Mad2, Hook-1, Zar1, Zp1, and Bcl2 expression and lower Eg5, Zp2, Caspase6, and Rbm3 expression compared with the untreated group. Supplementation of the vitrification medium with AFP has a protective effect on immature mouse oocytes, promoting their resistance to chilling injury. AFPs may preserve spindle forming ability and membrane integrity at GV stage. The fertilization and subsequent developmental competence of oocytes may be associated with the modulation of Zar1, Zp1/Zp2, Bcl2, Caspase6, and Rbm3.

Figure 1. Microphotographs showing fluorescent caspase staining and differential staining of blastocysts derived from vitrified-warmed immature mouse oocytes.

(I) Apoptotic blastomeres appear green; (II) red-pink indicates trophectoderm and blue indicates inner cell mass (400×). (I) a: DAPI, b: FITC, C: merged; (II) a: Hoechst 33342, b: propidium iodide, c: merged.

Figure 2. Representative microphotographs showing morphology of meiotic spindle organization and chromosome alignment in vitrified-warmed oocytes.

AFP  =  Antifreeze protein. (x400).

Figure 3. Vitrified-warmed oocytes stained with 1 µg/ml fluorescein diacetate (FDA, green) and 50 µg/ml propidium iodide (PI, red).

Upper panel; live and intact oocytes showing positive FDA and negative PI. Under panel; live but damaged oocytes showing positive FDA and PI.

Figure 4. The expression of genes associated with meiotic or mitotic checkpoints (Mad2, Eg5), microtubule microstructure (Hook1), maternal effects (Mater, Hsf1, and Zar1), oxidative stress (Sod1), zona pellucida (Zp1, Zp2, and Zp3), apoptosis (Bcl2, Bax, Caspase3, and Caspase6) and cold-stress (cold-inducible RNA-binding protein [CIRP] and cold-shock protein RNA-binding motif protein-3 [Rbm3]) in vitrified-warmed, in vitro matured mouse oocytes (fresh, non-treated, and AFP-treated).