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竹琳, 林子媛, 刘燕燕, 等. 母体年龄相关的卵母细胞老化对生育力的影响机制:斑马鱼模型的转录组学测序分析[J]. koko体育app 学报(医学版), 2024, 55(3): 588-595. DOI:
引用本文: 竹琳, 林子媛, 刘燕燕, 等. 母体年龄相关的卵母细胞老化对生育力的影响机制:斑马鱼模型的转录组学测序分析[J]. koko体育app 学报(医学版), 2024, 55(3): 588-595. DOI:
ZHU Lin, LIN Ziyuan, LIU Yanyan, et al. Mechanisms of the Effect of Maternal Age-Related Oocyte Aging on Fertility: Transcriptomic Sequencing Analysis of a Zebrafish Model[J]. Journal of Sichuan University (Medical Sciences), 2024, 55(3): 588-595. DOI:
Citation: ZHU Lin, LIN Ziyuan, LIU Yanyan, et al. Mechanisms of the Effect of Maternal Age-Related Oocyte Aging on Fertility: Transcriptomic Sequenꦆcing Analysis of a Zebrafish Model[J]. Journal of Sichuan University (Medical Sciences), 2024, 55(3): 588-595. DOI:

母体年龄相关的卵母细胞老化对生育力的影响机制:斑马鱼模型的转录组学测序分析

Mechanisms of the Effect of Maternal Age-Related Oocyte Aging on Fertility: Transcriptomic Sequencing Analysis of a Zebrafish Model

  • 摘要:
    目的 以斑马鱼为模型,研究母体年龄相关的卵母细胞老化对生育力的影响,寻找随年龄增长的生育力下降的潜在分子机制。
    方法 随机选取6月龄、12月龄、18月龄雌性斑马鱼各8条,均与6月龄雄性斑马鱼交配产卵。对各月龄组的结局指标进行比较。主要结局指标为胚胎受精率,次要结局指标为雌性斑马鱼单次产卵数量、胚胎死亡率和胚胎畸形率。收集不同月龄雌性斑马鱼的卵母细胞以及后代囊胚期胚胎进行转录组学测序分析。
    结果 18月龄组受精率(86.8±5.5)%低于6月龄组(94.9±3.6)%及12月龄组(92.3±4.2)%,差异有统计学意义(P均<0.05)。与6月龄组相比,12月龄与18月龄组雌性斑马鱼胚胎死亡率均升高(P均<0.05)。各组单次产卵数量和胚胎畸形率之间的差异无统计学意义。母体年龄相关的囊胚期胚胎差异性表达基因主要富集于MAPK信号通路以及脂肪酸降解等通路途径。母体年龄相关的卵母细胞差异性表达基因主要富集于细胞黏附分子,蛋白质消化吸收等通路途径。
    结论 母体年龄可能是卵母细胞受精能力降低及早期胚胎死亡率升高的影响因素,母体年龄相关的卵母细胞老化影响生育力及后代胚胎的发育。
     
    Abstract:
    Objective Female fertility gradually decreases with the increase in women’s age. The underlying reasons include the decline in the quantity and quality of oocytes. Oocyte aging is an important manifestation of the decline in oocyte quality, including in vivo oocyte aging before ovulation and in vitro oocyte aging after ovulation. Currently, few studies have been done to examine oocyte aging, and the relevant molecular mechanisms are not fully understood. Therefore, we used zebrafish as a model to investigate oocyte aging. Three different age ranges of female zebrafish were selected to mate with male zebrafish of the best breeding age. In this way, we studied the effects of maternal age-related oocyte aging on fertility and investigated the potential molecular mechanisms behind maternal age-related fertility decline.
    Methods Eight female zebrafish aged between 158 and 195 d were randomly selected for the 6-month age group (180±12) d, 8 female zebrafish aged between 330 and 395 d were randomly selected for the 12-month age group (360±22) d, and 8 female zebrafish aged between 502 and 583 d were randomly selected for the 18-month age group (540±26) d. Male zebrafish of (180±29) d were randomly selected from zebrafish aged between 158 and 195 d and mated with female zebrafish in each group. Each mating experiment included 1 female zebrafish and 1 male zebrafish. Zebrafish embryos produced by the mating experiments were collected and counted. The embryos at 4 hours post-fertilization were observed under the microscope, the total number of embryos and the number of unfertilized embryos were counted, and the fertilization rate was calculated accordingly. The numbers of malformed embryos and dead embryos were counted 24 hours after fertilization, and the rates of embryo malformation and mortality were calculated accordingly. The primary outcome measure was the embryo fertilization rate, and the secondary outcome measures were the number of embryos per spawn (the total number of embryos laid within 1.5 hours after the beginning of mating and reproduction of the zebrafish), embryo mortality, and embryo malformation rate. The outcome measures of each group were compared. The blastocyst embryos of female zebrafish from each group born after mating with male zebrafish in their best breeding period were collected for transcriptomics analysis. Fresh oocytes of female zebrafish in each group were collected for transcriptomics analysis to explore the potential molecular mechanisms of maternal age-related fertility decline.
    Results Compared with that of the 6-month group (94.9%±3.6%), the embryo fertilization rate of the 12-month group (92.3%±4.2%) showed no significant difference, but that of the 18-month group (86.8%±5.5%) decreased significantly (P<0.01). In addition, the fertilization rate in the 18-month group was significantly lower than that in the 12-month group (P<0.05). Compared with that of the 6-month group, the embryo mortality of the female zebrafish in the 12-month group and that in the 18-month group were significantly higher than that in the 6-month group (P<0.000 1, P<0.001). There was no significant difference in the number of embryos per spawn or in the embryo malformation rate among the three groups. The results of the transcriptomics analysis of blastocyst embryos showed that some genes, including dusp5, bdnf, ppip5k2, dgkg, aldh3a2a, acsl1a, hal, mao, etc, were differentially expressed in the 12-month group or the 18-month group compared with their expression levels in the 6-month group. According to the KEGG enrichment analysis, these differentially expressed genes (DEGs) were significantly enriched in the MAPK signaling pathway, the phosphatidylinositol signaling system, and the fatty acid degradation and histidine metabolism pathway (P<0.05). The analysis of the expression trends of the genes expressed differentially among the three groups (the 6-month group, the 12-month group, and the 18-month group in turn) showed that the gene expression trends of fancc, fancg, fancb, and telo2, which were involved in Fanconi anemia pathway, were statistically significant (P<0.05). In the results of oocyte transcriptomics analysis, the genes that were differentially expressed in the 12-month group or the 18-month group compared with the 6-month group were mainly enriched in cell adhesion molecules and the protein digestion and absorption pathway (P<0.05). The results of the trends of gene expression in the zebrafish oocytes of the three groups (the 6-month group, the 12-month group, and the 18-month group in turn) showed that three kinds of gene expression trends of declining fertility with growing maternal age had significant differences (P<0.05). Further analysis of the three significantly differential expression trends showed 51 DEGs related to mitochondria and 5 DEGs related to telomere maintenance and DNA repair, including tomm40, mpc2, nbn, tti1, etc.
    Conclusion With the increase in the maternal age of the zebrafish, the embryo fertilization rate decreased significantly and the embryo mortality increased significantly. In addition, with the increase in the maternal age of the zebrafish, the expression of mitochondria and telomere-related genes, such as tomm40, mpc2, nbn, and tti1, in female zebrafish oocytes decreased gradually. Maternal age may be a factor contributing to the decrease in oocyte fertilization ability and the increase in early embryo mortality. Maternal age-related oocyte aging affects the fertility and embryo development of the offspring.
     

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