E H Ernst1, M L Grøndahl2, S Grund3, K Hardy4, A Heuck1, L Sunde1,5, S Franks4, C Y Andersen6, P Villesen3,7, K Lykke-Hartmann1,5,7,8. 1. Department of Biomedicine, Aarhus University, Department of Biomedicine, Aarhus University, Wilhelm Meyers Allé 4, DK-8000 Aarhus C, Denmark. 2. Fertility Clinic, Herlev Hospital, Herlev Ringvej 75, DK-2730 Herlev, Denmark. 3. Bioinformatics Research Centre (BiRC), Aarhus University, C.F.Møllers Allé 8, DK-8000 Aarhus C, Denmark. 4. Institute of Reproductive and Developmental Biology, Imperial College London, South Kensington Campus, London SW7 2AZ, UK. 5. Department of Clinical Genetics, Aarhus University Hospital, Brendstrupgårdsvej 21, DK-8200 Aarhus N, Denmark. 6. Laboratory of Reproductive Biology, Copenhagen University Hospital, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen N, Denmark. 7. Department of Clinical Medicine, Aarhus University, Wilhelm Meyers Allé 4, DK-8000 Aarhus C, Denmark. 8. Aarhus Institute of Advanced Studies, Aarhus University, Høegh-Guldbergs Gade 6B, DK-8000 Aarhus C, Denmark.
Abstract
STUDY QUESTION: Do specific transcriptome dynamics in human oocytes from primordial and primary follicles identify novel pathways in oocyte activation? SUMMARY ANSWER: The transcriptomic profiles in oocytes from primordial and primary follicles, respectively, revealed several new canonical pathways as putative mediators of oocyte dormancy and activation. WHAT IS KNOWN ALREADY: 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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. LARGE SCALE DATA: 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. WIDER IMPLICATIONS OF THE FINDINGS: 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.
STUDY QUESTION: Do specific transcriptome dynamics in human oocytes from primordial and primary follicles identify novel pathways in oocyte activation? SUMMARY ANSWER: The transcriptomic profiles in oocytes from primordial and primary follicles, respectively, revealed several new canonical pathways as putative mediators of oocyte dormancy and activation. WHAT IS KNOWN ALREADY: 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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. LARGE SCALE DATA: 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. WIDER IMPLICATIONS OF THE FINDINGS: 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.
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