Panobinostat (PB), a histone deacetylase (HDAC) inhibitor drug, is clinically used in the treatment of cancers. We investigated the effects of PB on murine ovarian functions in embryos and adult animals. C57BL/6J mice were treated with 5 mg/kg PB on alternate days from embryonic day (E) 6.5 to E15.5. We analysed the effects of PB on the ovaries by using immunofluorescence, gene expression analysis and DNA methylation analysis techniques. At E15.5, we observed increases in histone H3K9Ac, H4Ac and H3K4me3 marks, while the level of the silencing H3K9me3 mark decreased. Synaptonemal complex examination at E15.5, E17.5 and E18.5 showed a delay in meiotic progression characterized by the absence of synaptonemal complexes at E15.5 and the persistence of double-strand breaks (DSBs) at E17.5 and E18.5 in PB-exposed oocytes. We found that exposure to PB led to changes in the expression of 1169 transcripts at E15.5. Genes regulated by the male-specific factors SRY-Box Transcription Factor 9 (SOX9) and Doublesex and Mab-3-related Transcription factor 1 (DMRT1) were among the most upregulated genes in the ovaries of PB-exposed mice. In contrast, PB treatment led to decreases in the expression of genes regulated by the WNT4 pathway. Notably, we observed 119 deregulated genes encoding Zn-finger proteins. The observed alterations in epigenetic marks and gene expression correlated with decreases in the numbers of germ cells at E15.5. After birth, PB-exposed ovaries showed increased proliferation of primary and secondary follicles. We also observed decreases in the numbers of primordial, primary and secondary follicles in adult ovaries from mice that were exposed to PB in utero. Finally, epigenetic alterations such as decreased H3K4me3 and increased H4 acetylation levels were also detected in somatic cells surrounding fully grown oocytes. Our data suggest that inhibition of histone deacetylase by PB during a critical developmental window affects reprogramming and germ cell specification via alteration of epigenetic marks.

中文翻译：

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组蛋白去乙酰化酶抑制导致调节组蛋白标记改变并损害卵母细胞的减数分裂

Panobinostat (PB) 是一种组蛋白去乙酰化酶 (HDAC) 抑制剂药物，临床上用于治疗癌症。我们研究了 PB 对胚胎和成年动物小鼠卵巢功能的影响。C57BL/6J 小鼠在胚胎日 (E) 6.5 至 E15.5 的隔天接受 5 mg/kg PB 治疗。我们通过使用免疫荧光、基因表达分析和 DNA 甲基化分析技术分析了 PB 对卵巢的影响。在 E15.5，我们观察到组蛋白 H3K9Ac、H4Ac 和 H3K4me3 标记的增加，而沉默 H3K9me3 标记的水平降低。在 E15.5、E17.5 和 E18.5 的联会复合体检查显示减数分裂进程延迟，其特征是在 E15.5 没有联会复合体，在 E17.5 和 E18 持续存在双链断裂 (DSB)。 5 在暴露于 PB 的卵母细胞中。我们发现接触 PB 会导致 E15.5 的 1169 个转录本的表达发生变化。受雄性特异性因子 SRY-Box 转录因子 9 (SOX9) 和 Doublesex 和 Mab-3 相关转录因子 1 (DMRT1) 调节的基因是 PB 暴露小鼠卵巢中上调最多的基因之一。相反，PB 处理导致受 WNT4 途径调节的基因表达降低。值得注意的是，我们观察到 119 个失调的编码锌指蛋白的基因。观察到的表观遗传标记和基因表达的变化与 E15.5 时生殖细胞数量的减少相关。出生后，暴露于 PB 的卵巢显示初级和次级卵泡增殖增加。我们还观察到原始数量的减少，子宫内暴露于 PB 的小鼠成年卵巢中的初级和次级卵泡。最后，在完全生长的卵母细胞周围的体细胞中也检测到表观遗传改变，例如 H3K4me3 降低和 H4 乙酰化水平升高。我们的数据表明，PB 在关键发育窗口期间对组蛋白脱乙酰酶的抑制会通过改变表观遗传标记来影响重编程和生殖细胞规格。