METHODS: Placental BDNF knockout mice were created using CRISPR/CAS9. Homozygous knockout (cKO(HO)) mice didn\'t survive, while heterozygous knockout (cKO(HE)) mice did. BDNF reduction in cKO(HE) mice was confirmed via immunohistochemistry and Western blots. Ovaries were collected from cKO(HE) mice at various ages, analyzing ovarian metrics, FSH expression, and litter sizes. In one-month-old mice, oocyte numbers were assessed using super-ovulation, and oocyte gene expression was analyzed with smart RNAseq. Ovaries of P7 mice were studied with SEM, and gene expression was confirmed with RT-qPCR. Alkaline phosphatase staining at E11.5 and immunofluorescence for cyclinD1 assessed germ cell number and cell proliferation.
RESULTS: cKO(HE) mice had decreased ovarian function and litter size in adulthood. They were insensitive to ovulation induction drugs manifested by lower oocyte release after superovulation in one-month-old cKO(HE) mice. The transcriptome and SEM results indicate that mitochondria-mediated cell death or aging might occur in cKO(HE) ovaries. Decreased placental BDNF led to diminished primordial germ cell proliferation at E11.5 and ovarian reserve which may underlie POI in adulthood.
CONCLUSIONS: The current results showed decreased placental BDNF diminished primordial germ cell proliferation in female fetuses during pregnancy and POI in adulthood. Our findings can provide insights into understanding the underlying mechanisms of POI.
方法:使用CRISPR/CAS9创建胎盘BDNF敲除小鼠。纯合敲除(cKO(HO))小鼠没有存活,而杂合子敲除(cKO(HE))小鼠。通过免疫组织化学和Western印迹证实cKO(HE)小鼠中的BDNF减少。从不同年龄的cKO(HE)小鼠收集卵巢,分析卵巢指标,FSH表达,和垃圾大小。一个月大的老鼠,使用超排卵评估卵母细胞数量,用smartRNAseq分析卵母细胞基因表达。用SEM研究了P7小鼠的卵巢,用RT-qPCR确认基因表达。E11.5的碱性磷酸酶染色和cyclinD1的免疫荧光评估了生殖细胞数量和细胞增殖。
结果:cKO(HE)小鼠在成年期卵巢功能和产仔数减少。它们对排卵诱导药物不敏感,表现为一个月大的cKO(HE)小鼠超排卵后卵母细胞释放减少。转录组和SEM结果表明,线粒体介导的细胞死亡或衰老可能发生在cKO(HE)卵巢中。胎盘BDNF减少导致E11.5的原始生殖细胞增殖和卵巢储备减少,这可能是成年期POI的基础。
结论:目前的结果显示,胎盘BDNF减少了女性胎儿在怀孕期间的原始生殖细胞增殖和成年后的POI。我们的发现可以为理解POI的潜在机制提供见解。