Department of Biomedicine, Aarhus University, Department of Biomedicine, Aarhus University, Wilhelm Meyers Allé 4, DK-8000 Aarhus C, Denmark.
Fertility Clinic, Herlev Hospital, Herlev Ringvej 75, DK-2730 Herlev, Denmark.
Hum Reprod. 2017 Aug 1;32(8):1684-1700. doi: 10.1093/humrep/dex238.
Do specific transcriptome dynamics in human oocytes from primordial and primary follicles identify novel pathways in oocyte activation?
The transcriptomic profiles in oocytes from primordial and primary follicles, respectively, revealed several new canonical pathways as putative mediators of oocyte dormancy and activation.
Cellular signaling pathways including PI3K/AKT and AKT/mTOR as well as TGF-β and IGF signaling are known to regulate the primordial-to-primary transition in mammalian follicle development.
STUDY DESIGN, SIZE, DURATION: We performed a class comparison study on human oocytes from primordial (n = 436) and primary (n = 182) follicles donated by three women having ovarian tissue cryopreserved before chemotherapy.
PARTICIPANTS/MATERIALS, SETTING, METHODS: RNA was extracted from oocytes from primordial and primary follicles isolated by Laser Capture Microdissection, and submitted to the HiSeq Illumina platform. Data mapping, quality control, filtering and expression analysis were performed using Tophat (2.0.4), Cufflinks (2.0.2), BWA (0.6.2) and software R. Modeling of complex biological systems was performed using the IPA® software. Finally, qPCR and immunohistochemistry were employed to explore expression and localization of selected genes and products in human ovarian tissue.
We found 223 and 268 genes down-regulated and up-regulated, respectively, in the oocytes during the human primordial-to-primary follicle transition (P < 0.05 and/or FPKM fold-change >2). IPA® enrichment analysis revealed known pathways ('mTOR Signaling', 'PI3K/AKT Signaling' and 'PTEN Signaling') as well as enriched canonical pathways not previously associated with human ovarian follicle development such as 'ErB Signaling' and 'NGF Signaling' in the down-regulated category and 'Regulation of eIF4 and P70S6K Signaling' and 'HER-2 Signaling in Breast Cancer' in the up-regulated group. Additionally, immunohistochemistry on human ovarian tissue explored the intraovarian localization of VASA, FOXO1 and eIF4E.
http://users-birc.au.dk/biopv/published_data/ernst_2017/.
LIMITATIONS, REASONS FOR CAUTION: This is a descriptive analysis and no functional studies were performed. The study was based on a limited number of patients and the experimental design could not take into account the natural biological variance in human samples. Therefore, qPCR was used to confirm selected genes alongside immunohistochemical stainings.
This study shows, for the first time, a detailed molecular description of global gene transcription activities in oocytes from primordial and primary follicles, respectively. Knowing the global transcription profiles of human oocyte dormancy and activation are important in developing new clinical applications.
STUDY FUNDING/COMPETING INTEREST(S): E.H.E. was supported by Health Faculty, Aarhus University and Kong Christian Den Tiendes Fond. K.H. and S.F. were supported by an MRC (UK) project grant MR/M012638/1. K.L.H. was supported by grants from Fonden til Lægevidenskabens Fremme, Kong Christian Den Tiendes Fond. K.L.H. and L.S. were supported by the IDEAS grant from Aarhus University Research Foundation (AUFF). There are no conflicts of interest.
原始卵泡和初级卵泡中的人类卵母细胞的特定转录组动态是否能确定卵母细胞激活的新途径？
分别来自原始卵泡和初级卵泡的卵母细胞的转录组谱揭示了几个新的经典途径，可能作为卵母细胞休眠和激活的介体。
包括 PI3K/AKT 和 AKT/mTOR 以及 TGF-β 和 IGF 信号在内的细胞信号通路，已知可调节哺乳动物卵泡发育中的原始卵泡向初级卵泡的转变。
研究设计、大小、持续时间：我们对三位接受化疗前卵巢组织冷冻保存的女性捐献的原始卵泡（n = 436）和初级卵泡（n = 182）中的卵母细胞进行了类别比较研究。
参与者/材料、设置、方法：使用激光捕获显微切割从原始卵泡和初级卵泡中提取卵母细胞的 RNA，并提交给 HiSeq Illumina 平台。使用 Tophat（2.0.4）、Cufflinks（2.0.2）、BWA（0.6.2）和软件 R 进行数据映射、质量控制、过滤和表达分析。使用 IPA®软件对复杂生物系统进行建模。最后，进行 qPCR 和免疫组织化学染色以探索所选基因和产物在人类卵巢组织中的表达和定位。
我们发现人类原始卵泡向初级卵泡过渡期间分别有 223 和 268 个基因下调和上调（P < 0.05 和/或 FPKM 倍数变化 >2）。IPA®富集分析显示了已知的途径（“mTOR 信号”、“PI3K/AKT 信号”和“PTEN 信号”）以及与人类卵巢卵泡发育无关的丰富经典途径，例如下调组中的“ErB 信号”和“NGF 信号”以及上调组中的“eIF4 和 P70S6K 信号的调节”和“HER-2 信号在乳腺癌中”。此外，对人类卵巢组织进行免疫组织化学染色研究了 VASA、FOXO1 和 eIF4E 的卵巢内定位。
http://users-birc.au.dk/biopv/published_data/ernst_2017/。
局限性、谨慎的原因：这是一项描述性分析，没有进行功能研究。该研究基于有限数量的患者，实验设计无法考虑人类样本中的自然生物学差异。因此，使用 qPCR 来确认选定的基因，同时进行免疫组织化学染色。
这项研究首次详细描述了原始卵泡和初级卵泡中卵母细胞的全局基因转录活性。了解人类卵母细胞休眠和激活的全局转录谱对于开发新的临床应用非常重要。
研究资金/利益冲突：E.H.E. 得到了奥胡斯大学健康学院和 Kong Christian Den Tiendes 基金会的支持。K.H. 和 S.F. 得到了英国医学研究理事会（UK）项目资助 MR/M012638/1 的支持。K.L.H. 得到了 Fonden til Lægevidenskabens Fremme、Kong Christian Den Tiendes 基金会的资助。K.L.H. 和 L.S. 得到了奥胡斯大学研究基金会（AUFF）IDEAS 赠款的支持。没有利益冲突。
