Faculdade de Veterinária, Universidade Federal Fluminense, Av. Vital Brazil Filho, 64, CEP 24230-340, Niterói, RJ, Brazil.
Faculdade de Veterinária, Universidade Federal Fluminense, Av. Vital Brazil Filho, 64, CEP 24230-340, Niterói, RJ, Brazil.
Theriogenology. 2024 Nov;229:108-117. doi: 10.1016/j.theriogenology.2024.08.002. Epub 2024 Aug 3.
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.
卵母细胞冷冻保存尚未被认为是一种可靠的技术，因为它会降低几种物种中卵母细胞的质量和存活率。本研究旨在确定不同浓度的抗冻蛋白 I（AFP I）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，并分别用 0μg/mL（G0，0μM）、0.5μg/mL（G0.5，0.15μM）或 1μg/mL（G1，0.3μM）的 AFP I 进行玻璃化处理。解冻后，评估卵母细胞的形态质量，并与新鲜组（FG）的肌动蛋白完整性、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 基因归一化）以及超微结构进行比较。G0.5 和 G1 表现出更高比例的 COCs 被评为 I 级，而 G0 的质量明显较低。G1 的 COC 中完整的肌动蛋白比例高于 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），且均高于 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、G0 和 G0.5 之间没有差异。G1 的 ROS 水平高于所有组（P<0.05），且玻璃化组之间的 GSH 水平没有差异（P>0.05）。对于核成熟，G1 和 G0.5 之间没有差异（P>0.05），但均高于 G0，且低于 FG（P<0.05）。关于基因表达，在 G0 和 G0.5 中，与 FG 相比，大多数基因下调，除了 G0 中的 SIRT1 和 SIRT3 以及 G0.5 中的 SIRT3。此外，G1 保持与 FG 更相似的表达。无论浓度如何，在未成熟的猫卵母细胞玻璃化溶液中添加 AFP I 均能提高成熟率、形态质量和肌动蛋白完整性，且不影响 GSH 水平。在测试的最高浓度（1μg/mL）下，AFP 保持了线粒体活性，减少了线粒体质量，增加了 ROS 水平，并且基因表达与 FG 更相似。综上所述，玻璃化过程中 AFP 的补充似乎减轻了冷冻保存的一些负面影响，提高了猫卵母细胞的完整性和冷冻保存能力。
