■女性生育率随着女性年龄的增加而逐渐下降。其根本原因包括卵母细胞数量和质量的下降。卵母细胞老化是卵母细胞质量下降的重要表现,包括排卵前体内卵母细胞老化和排卵后体外卵母细胞老化。目前,很少有研究来检查卵母细胞的衰老,和相关的分子机制尚未完全了解。因此,我们使用斑马鱼作为研究卵母细胞衰老的模型。选择三种不同年龄范围的雌性斑马鱼与最佳繁殖年龄的雄性斑马鱼交配。这样,我们研究了母亲年龄相关的卵母细胞老化对生育能力的影响,并探讨了母亲年龄相关的生育能力下降的潜在分子机制.
■随机选择年龄在158至195d之间的8条雌性斑马鱼作为6月龄(180±12)d组,随机选取年龄在330~395d的雌性斑马鱼8只作为12月龄组(360±22)d,随机选择年龄在502至583d之间的8只雌性斑马鱼作为18月龄组(540±26)d。从年龄在158至195d之间的斑马鱼中随机选择(180±29)d的雄性斑马鱼,并与雌性斑马鱼交配。每个交配实验包括1只雌性斑马鱼和1只雄性斑马鱼。收集并计数通过交配实验产生的斑马鱼胚胎。在显微镜下观察受精后4小时的胚胎,计算胚胎总数和未受精胚胎的数量,并据此计算了受精率。受精后24小时计数畸形胚胎和死亡胚胎的数量,并据此计算胚胎畸形率和死亡率。主要结局指标是胚胎受精率,次要结果指标是每个产卵的胚胎数(斑马鱼开始交配和繁殖后1.5小时内产下的胚胎总数),胚胎死亡率,和胚胎畸形率。比较各组的结局指标。收集各组雌性斑马鱼在最佳繁殖期与雄性斑马鱼交配后出生的囊胚进行转录组学分析。收集各组雌性斑马鱼的新鲜卵母细胞进行转录组学分析,以探讨母亲年龄相关生育力下降的潜在分子机制。
■与6个月组(94.9%±3.6%)相比,12个月组胚胎受精率(92.3%±4.2%)差异无统计学意义,但18个月组(86.8%±5.5%)显着降低(P<0.01)。此外,18个月组受精率明显低于12个月组(P<0.05)。与6个月组相比,12个月组和18个月组雌性斑马鱼的胚胎死亡率明显高于6个月组(P<0.0001,P<0.001)。三组之间每个菌种的胚胎数量或胚胎畸形率没有显着差异。囊胚胚胎的转录组学分析结果表明,包括dusp5,bdnf,ppip5k2,dgkg,aldh3a2a,acsl1a,哈尔,毛,etc,与6个月组的表达水平相比,在12个月组或18个月组中差异表达。根据KEGG富集分析,这些差异表达基因(DEGs)在MAPK信号通路中显著富集,磷脂酰肌醇信号系统,脂肪酸降解和组氨酸代谢途径(P<0.05)。三组间差异表达基因的表达趋势分析(6个月组,12个月组,反过来,18个月组)显示,fancc的基因表达趋势,Fancg,Fancb,和telo2,涉及范可尼贫血途径,有统计学意义(P<0.05)。在卵母细胞转录组学分析的结果中,与6个月组相比,12个月组或18个月组差异表达的基因主要富集在细胞粘附分子和蛋白质消化吸收途径(P<0.05)。三组斑马鱼卵母细胞基因表达变化趋势的结果(6个月组,12个月组,18个月组)显示,随着母亲年龄的增长,生育力下降的三种基因表达趋势具有显着差异(P<0.05)。进一步分析三种显著差异表达趋势,结果显示51个DEGs与线粒体相关,5个DEGs与端粒维持和DNA修复相关,包括tomm40,mpc2,nbn,tti1等.
■随着斑马鱼母亲年龄的增加,胚胎受精率显著下降,胚胎死亡率显著上升。此外,随着斑马鱼母亲年龄的增加,线粒体和端粒相关基因的表达,如tomm40,mpc2,nbn,和tti1,在雌性斑马鱼卵母细胞中逐渐减少。母亲年龄可能是导致卵母细胞受精能力下降和早期胚胎死亡率增加的因素。母亲年龄相关的卵母细胞老化影响后代的生育能力和胚胎发育。
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.