Transcriptome Landscape of Human Folliculogenesis Reveals Oocyte and Granulosa Cell Interactions.

The dynamic transcriptional regulation and interactions of human germlines and surrounding somatic cells during folliculogenesis remain unknown. Using RNA sequencing (RNA-seq) analysis of human oocytes and corresponding granulosa cells (GCs) spanning five follicular stages, we revealed unique features in transcriptional machinery, transcription factor networks, and reciprocal interactions in human oocytes and GCs that displayed developmental-stage-specific expression patterns. Notably, we identified specific gene signatures of two cell types in particular developmental stage that may reflect developmental competency and ovarian reserve. Additionally, we uncovered key pathways that may concert germline-somatic interactions and drive the transition of primordial-to-primary follicle, which represents follicle activation. Thus, our work provides key insights into the crucial features of the transcriptional regulation in the stepwise folliculogenesis and offers important clues for improving follicle recruitment in vivo and restoring fully competent oocytes in vitro.

Differentially expressed circular RNAs in maternal and neonatal umbilical cord plasma from SGA compared with AGA.

Small for gestational age (SGA) has a high risk of mortality and morbidity and is common in obstetrics. To date, no effective prediction and treatment tools are available. Acting as microRNA (miRNA) sponges and disease biomarkers are clear functions of circular RNAs (circRNAs). However, it is still unknown what role circRNAs act in SGA. To explore the role of circRNAs in SGA, circRNA expression patterns of the umbilical cord and maternal plasma in SGA was assessed. We first evaluated circRNAs in umbilical cord blood of the SGA and appropriate for gestational age (AGA) groups by microarray sequencing. In total, 170 340 circRNAs were sequenced, and 144 circRNAs were significantly upregulated while 977 were markedly downregulated. Has_circRNA15994-13, has_circ_0001359, and has_circ_0001360 were abundant and differentially expressed between the SGA and AGA groups, and confirmed in the umbilical cord and maternal blood specimens by reverse transcription polymerase chain reaction. By combining miRNA microarray data of the SGA placenta tissue in NCBI, it was found that two miRNAs were both hsa_circRNA15994-13 targets and differentially expressed, including hsa-miR-3619-5p and hsa-miR-4741. Further KEEG analysis revealed that the most significant pathway enriched by hsa-miR-3619-5p was Wnt signaling that is closely related to SGA; meanwhile, previous reports demonstrated that hsa-miR-3619-5p directly binds to ß-catenin to accommodate the Wnt/ß-catenin pathway, whereby the suggestive hsa_circRNA15994-13 → hsa-miR-3619-5p → ß-catenin signaling pathway may play an important part in SGA.

A MAPK cascade couples maternal mRNA translation and degradation to meiotic cell cycle progression in mouse oocytes.

Mammalian oocyte maturation depends on the translational activation of stored maternal mRNAs upon meiotic resumption. Cytoplasmic polyadenylation element binding protein 1 (CPEB1) is a key oocyte factor that regulates maternal mRNA translation. However, the signal that triggers CPEB1 activation at the onset of mammalian oocyte maturation is not known. We provide evidence that a mitogen-activated protein kinase (MAPK) cascade couples maternal mRNA translation to meiotic cell cycle progression in mouse oocytes by triggering CPEB1 phosphorylation and degradation. Mutations of the phosphorylation sites or ubiquitin E3 ligase binding sites in CPEB1 have a dominant-negative effect in oocytes, and mimic the phenotype of ERK1/2 knockout, by impairing spindle assembly and mRNA translation. Overexpression of the CPEB1 downstream translation activator DAZL in ERK1/2-deficient oocytes partially rescued the meiotic defects, indicating that ERK1/2 is essential for spindle assembly, metaphase II arrest and maternal-zygotic transition (MZT) primarily by triggering the translation of key maternal mRNAs. Taken together, ERK1/2-mediated CPEB1 phosphorylation/degradation is a major mechanism of maternal mRNA translational activation, and is crucial for mouse oocyte maturation and MZT.

Value of transferring embryos derived from monopronucleated (1PN) zygotes at the time of fertilization assessment.

This paper is a retrospective analysis of the sole transfer of monopronucleated zygotes (1PN) embryos both in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) to determine the value of transferring embryos formed from 1PN. In fresh cycles, 1PN cleavage-stage embryos (1PN cleavage fresh) were transferred. In frozen-thawed cycles, 1PN blastocyst-stage embryos (1PN blast frozen) were transferred. We used comparison groups: for fresh cycles, 2PN cleavage-stage embryos (2PN cleavage fresh) were transferred; and for frozen-thawed cycles, 2PN blastocyst-stage embryos (2PN blast frozen) were transferred. Comparison groups were matched for cycle and patient characteristics to the 1PN group. Finally, for fresh cycles, live birth rates (LBR) in the 1PN cleavage group were significantly lower than those in 2PN cleavage group, both for IVF [LBR = 7.64% vs. pregnancy rate (PR) = 22.12%, P = 0.003, respectively] and ICSI (LBR = 0% vs. LBR = 20.00%, P < 0.001, respectively). For frozen-thawed IVF cycles, the PR in the 1PN blastocyst group were comparable with those of the 2PN blastocyst group (1PN: LBR = 33.14% vs. 2PN: LBR = 37.24%, P = 0.289, respectively), while in ICSI, the PR in the 1PN blastocyst group were lower than those in the 2PN blastocyst group (LBR = 15.25% vs. LBR = 40.68%, P = 0.002, respectively). So, for IVF, blastocyst culture was capable of selecting normal 1PN embryos for transfer and achieves satisfying outcomes. However, for ICSI, blastocyst culture was not effective enough to eliminate abnormal embryos and 1PN embryo transfer needed to be treated with caution.

Promoting reprogramming by FGF2 reveals that the extracellular matrix is a barrier for reprogramming fibroblasts to pluripotency.

Leukemia inhibitory factor and bone morphogenetic protein signaling pathways play important roles in maintaining the self-renewal of mouse embryonic stem cells (ESCs). In contrast, the supplementation of fibroblast growth factor 2 (FGF2) in culture promotes mouse ESC differentiation. It has been proposed that factors that are adverse for maintaining the self-renewal of ESCs might play detrimental roles in the transcription factor-mediated reprogramming of somatic cells to pluripotency. However, recent evidence has revealed that reprogramming efficiency could be improved by FGF2, while the underlying molecular mechanism remains unknown. In this study, we dissected the roles of FGF2 in promoting mouse fibroblast reprogramming and disclosed the molecular mechanism behind this process. We used both primary induction and secondary inducible reprogramming systems and demonstrated that supplementation with FGF2 in the early phase of induced pluripotent stem cell induction could significantly increase reprogramming efficiency. Moreover, we discovered that many extracellular matrix candidate genes were significantly downregulated in fibroblasts treated with FGF2, and in particular, the synthesis of collagen could be greatly reduced by FGF2 treatment. Subsequently, we demonstrated that collagen is a barrier for reprogramming fibroblast cells to pluripotency, and the decreasing of collagen either by collagenase treatment or downregulation of collagen gene expression could significantly improve the reprogramming efficiency. Our results reveal a novel role of the extracellular matrix in mediating fibroblasts reprogramming.

Obstetric and neonatal outcomes after the transfer of vitrified-warmed blastocysts developing from nonpronuclear and monopronuclear zygotes: a retrospective cohort study.

OBJECTIVE: To evaluate the obstetric and neonatal outcomes after the transfer of vitrified-warmed single blastocysts developing from nonpronuclear (0PN) and monopronuclear (1PN) zygotes. DESIGN: Cohort study. SETTING: Affiliated hospital. PATIENT(S): This study was a retrospective analysis of 435 0PN and 281 1PN vitrified-warmed single blastocyst transfers, and 151 0PN and 75 1PN singletons, compared with 13,167 two-pronuclear (2PN) vitrified-warmed single blastocyst transfers and 4,559 2PN singletons, respectively. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Pregnancy rate (PR), abortion rate (AR), live birth rate (LBR), and singleton birthweight were the primary outcome measures. RESULT(S): PR, AR, and LBR were similar when compared between the 0PN and 2PN groups after vitrified-warmed blastocyst transfer. However, the 0PN group had a higher birthweights, higher z scores, and a greater proportion of very large for gestational age newborns. When comparing the 1PN and 2PN groups, we found that the PR was similar whereas the AR was higher and the LBR was lower. No differences were detected in the other neonatal outcomes. CONCLUSION(S): The results of the present study show that the transfer of 2PN blastocysts should be prioritized because of a higher AR and a lower LBR after 1PN blastocyst transfers and a higher birthweight after 0PN blastocyst transfers when compared with 2PN blastocyst transfers. Our data indicate the need for concern about the safety of 1PN and 0PN embryo transfers.

Maintenance of Nucleolar Homeostasis by CBX4 Alleviates Senescence and Osteoarthritis.

CBX4, a component of polycomb repressive complex 1 (PRC1), plays important roles in the maintenance of cell identity and organ development through gene silencing. However, whether CBX4 regulates human stem cell homeostasis remains unclear. Here, we demonstrate that CBX4 counteracts human mesenchymal stem cell (hMSC) aging via the maintenance of nucleolar homeostasis. CBX4 protein is downregulated in aged hMSCs, whereas CBX4 knockout in hMSCs results in destabilized nucleolar heterochromatin, enhanced ribosome biogenesis, increased protein translation, and accelerated cellular senescence. CBX4 maintains nucleolar homeostasis by recruiting nucleolar protein fibrillarin (FBL) and heterochromatin protein KRAB-associated protein 1 (KAP1) at nucleolar rDNA, limiting the excessive expression of rRNAs. Overexpression of CBX4 alleviates physiological hMSC aging and attenuates the development of osteoarthritis in mice. Altogether, our findings reveal a critical role of CBX4 in counteracting cellular senescence by maintaining nucleolar homeostasis, providing a potential therapeutic target for aging-associated disorders.

ATF6 safeguards organelle homeostasis and cellular aging in human mesenchymal stem cells.

Loss of organelle homeostasis is a hallmark of aging. However, it remains elusive how this occurs at gene expression level. Here, we report that human mesenchymal stem cell (hMSC) aging is associated with dysfunction of double-membrane organelles and downregulation of transcription factor ATF6. CRISPR/Cas9-mediated inactivation of ATF6 in hMSCs, not in human embryonic stem cells and human adipocytes, results in premature cellular aging, characteristic of loss of endomembrane homeostasis. Transcriptomic analyses uncover cell type-specific constitutive and stress-induced ATF6-regulated genes implicated in various layers of organelles' homeostasis regulation. FOS was characterized as a constitutive ATF6 responsive gene, downregulation of which contributes to hMSC aging. Our study unravels the first ATF6-regulated gene expression network related to homeostatic regulation of membrane organelles, and provides novel mechanistic insights into aging-associated attrition of human stem cells.

Low Cell-Matrix Adhesion Reveals Two Subtypes of Human Pluripotent Stem Cells.

We show that a human pluripotent stem cell (hPSC) population cultured on a low-adhesion substrate developed two hPSC subtypes with different colony morphologies: flat and domed. Notably, the dome-like cells showed higher active proliferation capacity and increased several pluripotent genes' expression compared with the flat monolayer cells. We further demonstrated that cell-matrix adhesion mediates the interaction between cell morphology and expression of KLF4 and KLF5 through a serum response factor (SRF)-based regulatory double loop. Our results provide a mechanistic view on the coupling among adhesion, stem cell morphology, and pluripotency, shedding light on the critical role of cell-matrix adhesion in the induction and maintenance of hPSC.

Oocyte-expressed yes-associated protein is a key activator of the early zygotic genome in mouse.

In early mammalian embryos, the genome is transcriptionally quiescent until the zygotic genome activation (ZGA) which occurs 2-3 days after fertilization. Despite a long-standing effort, maternal transcription factors regulating this crucial developmental event remain largely elusive. Here, using maternal and paternal mouse models of Yap1 deletion, we show that maternally accumulated yes-associated protein (YAP) in oocyte is essential for ZGA. Maternal Yap1-knockout embryos exhibit a prolonged two-cell stage and develop into the four-cell stage at a much slower pace than the wild-type controls. Transcriptome analyses identify YAP target genes in early blastomeres; two of which, Rpl13 and Rrm2, are required to mediate maternal YAP's effect in conferring developmental competence on preimplantation embryos. Furthermore, the physiological YAP activator, lysophosphatidic acid, can substantially improve early development of wild-type, but not maternal Yap1-knockout embryos in both oviduct and culture. These observations provide insights into the mechanisms of ZGA, and suggest potentials of YAP activators in improving the developmental competence of cultured embryos in assisted human reproduction and animal biotechnology.

BTG4 is a meiotic cell cycle-coupled maternal-zygotic-transition licensing factor in oocytes.

The mRNAs stored in oocytes undergo general decay during the maternal-zygotic transition (MZT), and their stability is tightly interconnected with meiotic cell-cycle progression. However, the factors that trigger decay of maternal mRNA and couple this event to oocyte meiotic maturation remain elusive. Here, we identified B-cell translocation gene-4 (BTG4) as an MZT licensing factor in mice. BTG4 bridged CNOT7, a catalytic subunit of the CCR4-NOT deadenylase, to eIF4E, a key translation initiation factor, and facilitated decay of maternal mRNA. Btg4-null females produced morphologically normal oocytes but were infertile, owing to early developmental arrest. The intrinsic MAP kinase cascade in oocytes triggered translation of Btg4 mRNA stored in fully grown oocytes by targeting the 3' untranslated region, thereby coupling CCR4-NOT deadenylase-mediated decay of maternal mRNA with oocyte maturation and fertilization. This is a key step in oocyte cytoplasmic maturation that determines the developmental potential of mammalian embryos.

Tracing the expression of circular RNAs in human pre-implantation embryos.

BACKGROUND: PolyA- RNAs have not been widely analyzed in human pre-implantation embryos due to the scarcity of materials. In particular, circular RNA (circRNA), a novel type of polyA- RNA, has not been characterized during human pre-implantation development. RESULTS: We systematically analyze polyA+ messenger RNAs (mRNAs) and polyA- RNAs in individual human oocytes and pre-implantation embryos using SUPeR-seq. We de novo identify 10,032 circRNAs from 2974 hosting genes. Most of these circRNAs are developmentally stage-specific and dynamically regulated. Many of them are maternally expressed, implying their potentially important regulatory functions in oogenesis and the formation of totipotent zygotes. Comparison between human and mouse embryos reveals both high conservation and clear distinction between these two species. Human pre-implantation embryos generate more types of circRNA compared with mouse embryos and this is associated with a striking increase of the length of the circRNA flanking introns in humans. We also perform RNA de novo assembly and identify novel transcript units, many of which are potentially novel long non-coding RNAs. CONCLUSIONS: This study reports the first analysis of the whole transcriptome comprising both polyA+ mRNAs and polyA- RNAs including circRNAs during human pre-implantation development. It provides an invaluable resource for analyzing the unique function and complex regulatory mechanisms of circRNAs during this process.