1 Reconstitution of the oocyte transcriptional network with transcription factors 2 3 Nobuhiko Hamazaki*1, Hirohisa Kyogoku2, Hiromitsu Araki3, Fumihito Miura3, Chisako 4 Horikawa1, Norio Hamada1,4, So Shimamoto1, Orie Hikabe1, Kinichi Nakashima1, 5 Tomoya Kitajima2, Takashi Ito3, Harry G Leitch5,6, Katsuhiko Hayashi*1 6 7 1 Department of Developmental Stem Cell Biology, Graduate School of Medical 8 Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan. 9 2 Laboratory for Chromosome Segregation, RIKEN Center for Biosystems Dynamics 10 Research, Kobe, Japan. 11 3 Department of Biochemistry, Graduate School of Medical Sciences, Kyushu University, 12 Fukuoka, Japan 13 4 Department of Obstetrics and Gynecology, Graduate School of Medical Sciences, 14 Kyushu University, Fukuoka, Japan 15 5 MRC London Institute of Medical Sciences (LMS), London, UK 16 6 Institute of Clinical Sciences (ICS), Faculty of Medicine, Imperial College London, 17 London, UK 18 19 Abstract 20 During female germline development oocytes become a highly specialized cell type, and 21 form a maternal cytoplasmic store of crucial factors during oocyte growth. Oocyte 22 growth is triggered at the primordial-primary follicle transition accompanied with dynamic 23 changes in gene expression1, yet the gene regulatory network underpinning oocyte 24 growth remains elusive. Here we identified a set of transcription factors sufficient to 25 trigger oocyte growth. By dissection of the change in gene expression and functional 26 screening using an in vitro oocyte development system, we identified 8 transcription 27 factors, each of which was essential for the primordial-primary follicle transition. 28 Surprisingly, enforced expression of these transcription factors swiftly converted 29 pluripotent stem cells to oocyte-like cells that were competent for fertilization and 30 subsequent cleavage. These transcription factor-induced oocyte-like cells were formed 31 without PGC specification, epigenetic reprogramming or meiosis, establishing that 32 oocyte growth and lineage-specific de novo DNA methylation is separable from the 33 preceding epigenetic reprogramming in PGCs. This study identifies a core set of 34 transcription factors for orchestrating oocyte growth, and also provides an alternative 35 source of ooplasm, which is a unique material for reproductive biology and medicine. 36 37 Main 38 In mouse germline, primordial germ cells (PGCs) go through a sequential 39 differentiation process beginning with PGC specification, followed by migration to the 40 gonad, epigenetic reprogramming (including genome-wide DNA demethylation)2, fate 41 determination3 and sex determination4. Following sex determination, female PGCs enter 42 meiosis thereby becoming oocytes. Oocytes are arrested in the diplotene stage of 43 meiotic prophase I and most are maintained in primordial follicles. Cytoplasmic 44 expansion is triggered upon activation of primordial follicles. Once oocyte growth is 45 triggered, maternal RNAs and proteins are stored in the cytoplasm5,6. Meiosis resumes 46 in full-grown oocytes, establishing metaphase II (MII) oocytes, and is not completed until 47 after fertilization. 48 Oocyte differentiation therefore entails two key processes: oocyte growth and 49 meiosis. Although concurrent, these two features are separable, as evidenced by a 50 seminal study in which Stra8-knockout mice were shown to develop oocyte-like cells 51 that do not enter meiosis. A number of genes essential for early oocyte growth have 52 been identified, including Figla7, Sohlh18, Sohlh29, Lhx810, Nobox11, Taf4b12,13, Yy114 53 and Tbpl215. Transcriptome analysis using oocytes lacking these genes8,10,15-19 and 54 identification of direct target sequences/genes7,8,20-22 has revealed downstream gene 55 cascades involved in early oocyte growth. In addition, a previous microarray analysis 56 uncovered highly dynamic gene expression changes between postnatal day 2 (P2) 57 oocytes in primordial follicles and P6 oocytes in the primary follicles (Primordial to 58 primary follicle transition: PPT)1. Genes enriched at PPT were involved in protein 59 synthesis and transcription1, suggesting a role in oocyte growth. 60 However, there has been limited further advance towards a comprehensive 61 description and functional dissection of the gene regulatory network specifically 62 orchestrating oocyte growth. Here, we identify a set of transcription factors that 63 comprise the underlying gene regulatory network and validate these findings with 64 functional screening. Furthermore, we successfully reconstitute the network in 65 pluripotent stem cells, thereby generating oocyte-like cells that are competent for 66 fertilization and subsequent cleavage divisions. 67 68 Characterization of PPT in oogenesis 69 To identify the gene regulatory network, we utilized our recently established culture 70 system that recapitulated female germline differentiation using embryonic stem cells 71 (ESCs)23. In this culture system, ESCs are differentiated into PGC-like cells (PGCLCs) 72 and then undergo oogenesis to give rise to MII oocytes in the presence of supporting 73 gonadal somatic cells. Using female germline cells in this culture system and their in 74 vivo counterparts, we mapped the trajectory of the female germline cycle by RNA-seq 75 analysis (Fig. 1a and Extended Data Fig.1a). Principal component analysis (PCA) 76 revealed that a highly dynamic gene expression change occurred in oocytes between in 77 vitro differentiation culture day 11 (IVD.D11) and day 13 (IVD.D13), which corresponded 78 to the period between postnatal day 1 (P1) and P8 in vivo. As previous microarray 79 analysis identified PPT between P2 and P6 oocytes1, we considered the transition 80 between IVD11 and IVD13 to represent PPT in vitro. Oocytes also exhibited a rapid 81 increase in their cytoplasmic and nuclear volumes between IVD11 and IVD13, as 82 observed in cytoplasmic expansion at PPT in vivo24, along with a decrease in their 83 nucleus/cytoplasmic (N/C) ratios (Fig. 1b and c). Notably, the magnitude of gene 84 expression changes in PPT was as large as the transition between MII oocytes and 85 early stage preimplantation embryos, a period known as oocyte-to-embryo transition 86 (OET), during which zygotic genome activation (ZGA) occurs25 (Fig. 1a). 87 During PPT, the upregulated genes included those encoding oocyte-specific 88 structural proteins as previously reported1. However, we also identified genes involved 89 in preimplantation development and DNA methylation of the maternal genome (Fig.1d). 90 In addition, upregulation of a subset of long terminal repeat (LTR) retrotransposon 91 families23 and the decline of the ratio of mean X chromosome transcripts in relation to 92 mean transcripts from the autosomes (X:A ratio)1 were observed at PPT (Extended 93 Data Fig.1b and c). As previous studies have described that growing oocytes use TATA 94 promoters, while ZGA is governed by a reciprocal switch from TATA promoter usage 95 back to GC promoter usage26,27, we hypothesised that the gene expression changes 96 observed during PPT might also be coupled with a change in promoter usage. We found 97 that GC nucleotides were enriched in the promoter region of genes highly transcribed 98 until IVD.D11, whereas AT nucleotides were enriched in that of genes highly transcribed 99 from IVD.D13 (Extended Data Fig. 1d). The AT nucleotides were specifically enriched at 100 20-30 bp upstream from the transcriptional start sites, and included TATA-box motifs 101 (Extended Data Fig. 1e-g). These results demonstrate that promoter usage was shifted 102 from GC promoter to TATA promoter usage upon PPT. 103 104 Functional screening of PPT genes 105 To identify key transctiption factors responsible for PPT, we applied weighted gene 106 co-expression network analysis (WGCNA) to our RNA-seq data and identified 34 107 modules (Extended Data Fig. 2a). Among these modules, we focused on five modules 108 (MEcyan, MEdarkred, MEmagenta, MEwhite and MEpurple) whose genes were 109 specifically expressed around PPT. From these modules, we extracted 27 transcription- 110 related genes that fulfilled the GO term “regulation of transcription, DNA-templated 111 (GO:0006355)” (Fig. 1e), and then investigated the functional requirement of these 112 genes for PPT by loss-of-function analysis using ESCs harboring the Blimp1-mVenus 113 (BV) and Stella-ECFP (SC) reporter genes23 (Extended Data Fig. 2b and c). Of the total 114 26 gene-knockout (KO) ESC lines, 11 differentiated normally beyond IVD13, 8 were 115 arrested between IVD11 and IVD13, and 7 were arrested at an earlier stage than 116 IVD.D3 (Extended Data Fig. 2c-e). Transcriptome analysis of these KO oocytes, except 117 for those arrested before IVD3, revealed that the 8 KO-oocytes (Figla, Sohlh1, Lhx8, 118 Nobox, Stat3, Tbpl2, Dynll1 or Sub1-KO oocytes) were arrested before or around PPT 119 (Fig. 1f). These 8 KO-oocytes did not show the representative features of PPT, such as 120 a decrease in the X/A ratio, propensity for TATA promoter usage and upregulation of 121 specific retrotransposons, with the single exception that Dynll1-KO oocytes showed a 122 decreased X/A ratio (Extended Data Fig. 3a-c). Analysis of reciprocal gene expression 123 in each line of KO-oocytes revealed that the 8 genes had a mutual effect on the 124 expression, and an imputed transcriptional network illustrated that Lhx8, Sohlh1, Nobox, 125 and Tbpl2 formed a core network to which Stat3, Dynll1, Sub1 and Figla were tightly 126 connected (Extended Data Fig. 3d and e). 127 128 PPT induction by transcription factors 129 Next we tested whether these 8 genes (hereinafter PPT8) are sufficient to drive 130 PPT and induce competence for oocyte growth. We overexpressed PPT8 in 131 BVSC“NCh”-ESCs, which consisted of BVSC ESCs with the mCherry gene inserted into 132 the Npm2 locus (Extended Data Fig. 4a). Consistent with evidence that Npm2 is 133 specifically expressed at later oogenesis stages in vivo28 and from IVD.D13 in vitro (Fig. 134 1d), Npm2-mCherry expression became visible in the nucleus of BVSCNCh-ESC- 135 derived oocytes from 15 days of IVD culture (Extended Data Fig.