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.
Highlights
Oocyte cryopreservation is an important strategy of fertility preservation
Enhancing vitrification protocol at germinal vesicle (GV) stage might be a potential method from the perspective of preserving spindle forming ability
We assumed that properly organized GV oocyte vitrification protocol helps to preserve normal spindle forming ability after warming
Summary
Oocyte cryopreservation is an important strategy of fertility preservation. The improvement of oocyte vitrification protocol has significantly increased oocyte survival and contributed to increased pregnancy rates [1,2,3,4,5]. Vitrification at the germinal vesicle (GV) stage is an alternative strategy that can prevent spindle depolymerization These immature oocytes are arrested in the diplotene stage of prophase I. The use of immature GV-stage oocytes reduces the risk of polyploidy and aneuploidy because the chromosomes are diffuse and surrounded by a nuclear membrane [8,9]. This method could enhance the GV survival rate, the maturation, fertilization, and subsequent embryonic development up to the blastocyst stage are impaired; this is considered the main problem associated with GV oocyte vitrification [10,11,12]. More progress of oocyte developmental stage is needed to enhance the developmental potential of vitrified GV oocytes to achieve good clinical results
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