卵母细胞冷冻保存尚未被认为是一种可靠的技术,因为它可以降低几种物种中卵母细胞的质量和存活率。本研究确定了不同浓度的抗冻蛋白I(AFPI)对未成熟猫卵母细胞玻璃化溶液的影响。为此,将卵母细胞随机分为三组,并以0μg/mL(G0,0μM);0.5μg/mL(G0.5,0.15μM)玻璃化,或1μg/mL(G1,0.3μM)的AFPI.解冻后,评估卵母细胞的形态质量,并与关于肌动蛋白完整性的新鲜组(FG)进行比较,线粒体活性和质量,活性氧(ROS)和谷胱甘肽(GSH)水平,核成熟,GDF9,BMP15,ZAR-1,PRDX1,SIRT1和SIRT3基因的表达(通过ACTB和YWHAZ基因标准化),和超微结构。G0.5和G1的COC分级为I的比例较高,而G0的质量明显较低。G1在COCs中完整肌动蛋白的百分比高于G0和G0.5(P<0.05)。FG和G1之间的线粒体活性没有差异(P>0.05),它们均高于G0和G0.5(P<0.05)。G1的线粒体质量明显低于FG和G0(P<0.05),FG之间没有差异。G0和G0.5。G1组ROS高于各组(P<0.05),玻璃化组之间的GSH水平无差异(P>0.05)。为了核成熟,G1和G0.5之间无差异(P>0.05),但两者均高于G0(P<0.05),低于FG(P<0.05)。关于基因表达,在G0和G0.5中,大多数基因与FG相比下调,G0中的SIRT1和SIRT3以及G0.5中的SIRT3除外。此外,G1保持与FG更相似的表达。不管浓度如何,在未成熟猫卵母细胞的玻璃化溶液中补充AFPI提高了成熟率,形态质量,和肌动蛋白完整性,不影响GSH水平。在测试的最高浓度(1μg/mL)中,AFP维持线粒体活性,线粒体质量减少,ROS水平增加,基因表达与FG更相似。总之,这些数据表明,玻璃化期间的AFP补充似乎减轻了冷冻保存改善猫卵母细胞的完整性和冷冻存活的一些负面影响。
Oocyte cryopreservation is not yet considered a reliable technique since it can reduce the quality and survival of oocytes in several species. This study determined the effect of different concentrations of antifreeze protein I (AFP I) on the vitrification solution of immature cat oocytes. For this, oocytes were randomly distributed in three groups and vitrified with 0 μg/mL (G0, 0 μM); 0.5 μg/mL (G0.5, 0.15 μM), or 1 μg/mL (G1, 0.3 μM) of AFP I. After thawing, oocytes were evaluated for morphological quality, and compared to a fresh group (FG) regarding actin integrity, mitochondrial activity and mass, reactive oxygen species (ROS) and glutathione (GSH) levels, nuclear maturation, expression of GDF9, BMP15, ZAR-1, PRDX1, SIRT1, and SIRT3 genes (normalized by ACTB and YWHAZ genes), and ultrastructure. G0.5 and G1 presented a higher proportion of COCs graded as I and while G0 had a significantly lower quality. G1 had a higher percentage of intact actin in COCs than G0 and G0.5 (P < 0.05). There was no difference (P > 0.05) in the mitochondrial activity between FG and G1 and they were both higher (P < 0.05) than G0 and G0.5. G1 had a significantly lower (P < 0.05) mitochondrial mass than FG and G0, and there was no difference among FG, G0, and G0.5. G1 had higher ROS than all groups (P < 0.05), and there was no difference in GSH levels among the vitrified groups (P > 0.05). For nuclear maturation, there was no difference between G1 and G0.5 (P > 0.05), but these were both higher (P < 0.05) than G0 and lower (P < 0.05) compared to FG. Regarding gene expression, in G0 and G0.5, most genes were downregulated compared to FG, except for SIRT1 and SIRT3 in G0 and SIRT3 in G0.5. In addition, G1 kept the expression more similar to FG. Regardless of concentration, AFP I supplementation in vitrification solution of immature cat oocytes improved maturation rates, morphological quality, and actin integrity and did not impact GSH levels. In the highest concentration tested (1 μg/mL), AFP maintained the mitochondrial activity, reduced mitochondrial mass, increased ROS levels, and had the gene expression more similar to FG. Altogether these data show that AFP supplementation during vitrification seems to mitigate some of the negative impact of cryopreservation improving the integrity and cryosurvival of cat oocytes.