Ten measures of experimental electron-density-map quality are examined and the skewness of electron density is found to be the best indicator of actual map quality. A Bayesian approach to estimating map quality is developed and used in the PHENIX AutoSol wizard to make decisions during automated structure solution.

Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX autosol wizard

MTOR (mechanistic target of rapamycin) is a widely recognized integrator of signals and pathways key for cellular metabolism, proliferation, and differentiation. Here we show that conditional knockout (cKO) of Mtor in either primordial or growing oocytes caused infertility but differentially affected oocyte quality, granulosa cell fate, and follicular development. cKO of Mtor in nongrowing primordial oocytes caused defective follicular development leading to progressive degeneration of oocytes and loss of granulosa cell identity coincident with the acquisition of immature Sertoli cell-like characteristics. Although Mtor was deleted at the primordial oocyte stage, DNA damage accumulated in oocytes during their later growth, and there was a marked alteration of the transcriptome in the few oocytes that achieved the fully grown stage. Although oocyte quality and fertility were also compromised when Mtor was deleted after oocytes had begun to grow, these occurred without overtly affecting folliculogenesis or the oocyte transcriptome. Nevertheless, there was a significant change in a cohort of proteins in mature oocytes. In particular, down-regulation of PRC1 (protein regulator of cytokinesis 1) impaired completion of the first meiotic division. Therefore, MTOR-dependent pathways in primordial or growing oocytes differentially affected downstream processes including follicular development, sex-specific identity of early granulosa cells, maintenance of oocyte genome integrity, oocyte gene expression, meiosis, and preimplantation developmental competence.

/pdf/oocyte-stage-specific-effects-of-mtor-determine-granulosa-3xdwa64q3w.pdf

Oocyte stage-specific effects of MTOR determine granulosa cell fate and oocyte quality in mice

Prior to ovulation, the mammalian oocyte undergoes a process of differentiation within the ovarian follicle that confers on it the ability to give rise to an embryo. Differentiation comprises two phases-growth, during which the oocyte increases more than 100-fold in volume as it accumulates macromolecules and organelles that will sustain early embryogenesis; and meiotic maturation, during which the oocyte executes the first meiotic division and prepares for the second division. Entry of an oocyte into the growth phase appears to be triggered when the adjacent granulosa cells produce specific growth factors. As the oocyte grows, it elaborates a thick extracellular coat termed the zona pellucida. Nonetheless, cytoplasmic extensions of the adjacent granulosa cells, termed transzonal projections (TZPs), enable them to maintain contact-dependent communication with the oocyte. Through gap junctions located where the TZP tips meet the oocyte membrane, they provide the oocyte with products that sustain its metabolic activity and signals that regulate its differentiation. Conversely, the oocyte secretes diffusible growth factors that regulate proliferation and differentiation of the granulosa cells. Gap junction-permeable products of the granulosa cells prevent precocious initiation of meiotic maturation, and the gap junctions also enable oocyte maturation to begin in response to hormonal signals received by the granulosa cells. Development of the oocyte or the somatic compartment may also be regulated by extracellular vesicles newly identified in follicular fluid and at TZP tips, which could mediate intercellular transfer of macromolecules. Oocyte differentiation thus depends on continuous signaling interactions with the somatic cells of the follicle. WIREs Dev Biol 2018, 7:e294. doi: 10.1002/wdev.294 This article is categorized under: Gene Expression and Transcriptional Hierarchies > Cellular Differentiation Signaling Pathways > Cell Fate Signaling Early Embryonic Development > Gametogenesis.

/pdf/regulation-of-germ-cell-development-by-intercellular-5cxs55tgr4.pdf

Regulation of germ cell development by intercellular signaling in the mammalian ovarian follicle.

CpG dinucleotides are suppressed in most vertebrate RNA viruses, including HIV-1, and introducing CpGs into RNA virus genomes inhibits their replication. The zinc finger antiviral protein (ZAP) binds regions of viral RNA containing CpGs and targets them for degradation. ZAP does not have enzymatic activity and recruits other cellular proteins to inhibit viral replication. We found that KHNYN, a protein with no previously known function, interacts with ZAP. KHNYN overexpression selectively inhibits HIV-1 containing clustered CpG dinucleotides and this requires ZAP and its cofactor TRIM25. KHNYN requires both its KH-like domain and NYN endonuclease domain for antiviral activity. Crucially, depletion of KHNYN eliminated the deleterious effect of CpG dinucleotides on HIV-1 RNA abundance and infectious virus production and also enhanced the production of murine leukemia virus. Overall, we have identified KHNYN as a novel cofactor for ZAP to target CpG-containing retroviral RNA for degradation.

KHNYN is essential for the zinc finger antiviral protein (ZAP) to restrict HIV-1 containing clustered CpG dinucleotides

Maternal mRNA clearance is an essential process that occurs during maternal-to-zygotic transition (MZT). However, the dynamics, functional importance, and pathological relevance of maternal mRNA decay in human preimplantation embryos have not yet been analyzed. Here we report the zygotic genome activation (ZGA)-dependent and -independent maternal mRNA clearance processes during human MZT and demonstrate that subgroups of human maternal transcripts are sequentially removed by maternal (M)- and zygotic (Z)-decay pathways before and after ZGA. Key factors regulating M-decay and Z-decay pathways in mouse have similar expression pattern during human MZT, suggesting that YAP1-TEAD4 transcription activators, TUT4/7-mediated mRNA 3'-oligouridylation, and BTG4/CCR4-NOT-induced mRNA deadenylation may also be involved in the regulation of human maternal mRNA stability. Decreased expression of these factors and abnormal accumulation of maternal transcripts are observed in the development-arrested embryos of patients who seek assisted reproduction. Defects of M-decay and Z-decay are detected with high incidence in embryos that are arrested at the zygote and 8-cell stages, respectively. In addition, M-decay is not found to be affected by maternal TUBB8 mutations, although these mutations cause meiotic cell division defects and zygotic arrest, which indicates that mRNA decay is regulated independent of meiotic spindle assembly. Considering the correlations between maternal mRNA decay defects and early developmental arrest of in vitro fertilized human embryos, M-decay and Z-decay pathway activities may contribute to the developmental potential of human preimplantation embryos.

/pdf/dynamics-and-clinical-relevance-of-maternal-mrna-clearance-8vf2qg25y1.pdf

Dynamics and clinical relevance of maternal mRNA clearance during the oocyte-to-embryo transition in humans.

Communication between oocytes and their companion somatic cells promotes the healthy development of ovarian follicles, which is crucial for producing oocytes that can be fertilized and are competent to support embryogenesis. However, how oocyte-derived signaling regulates these essential processes remains largely undefined. Here, we demonstrate that oocyte-derived paracrine factors, particularly GDF9 and GDF9-BMP15 heterodimer, promote the development and survival of cumulus-cell-oocyte complexes (COCs), partly by suppressing the expression of Ddit4l, a negative regulator of MTOR, and enabling the activation of MTOR signaling in cumulus cells. Cumulus cells expressed less Ddit4l mRNA and protein than mural granulosa cells, which is in striking contrast to the expression of phosphorylated RPS6 (a major downstream effector of MTOR). Knockdown of Ddit4l activated MTOR signaling in cumulus cells, whereas inhibition of MTOR in COCs compromised oocyte developmental competence and cumulus cell survival, with the latter likely to be attributable to specific changes in a subset of transcripts in the transcriptome of COCs. Therefore, oocyte suppression of Ddit4l expression allows for MTOR activation in cumulus cells, and this oocyte-dependent activation of MTOR signaling in cumulus cells controls the development and survival of COCs.

Oocyte-dependent activation of MTOR in cumulus cells controls the development and survival of cumulus-oocyte complexes.

Producing normal eggs for fertilization and species propagation requires completion of meiosis and protection of the genome from the ravages of retrotransposons. Mutation of Marf1 (meiosis regulator and mRNA stability factor 1) results in defects in both these key processes in mouse oocytes and thus in infertility. MARF1 was predicted to have ribonuclease activity, but the structural basis for the function of MARF1 and the contribution of its putative ribonuclease domain to the mutant oocyte phenotype was unknown. Therefore, we resolved the crystal structures of key domains of MARF1 and demonstrated by biochemical and mutagenic analyses that the ribonuclease activity of MARF1 controls oocyte meiotic progression and retrotransposon surveillance. The N-terminal NYN domain of MARF1 resembles the nuclease domains of Vpa0982, T4 RNase H, and MCPIP1 and contains four conserved aspartate residues, D178, D215, D246, and D272. The C-terminal LOTUS domain of MARF1 adopts a winged helix-turn-helix fold and binds ssRNA and dsRNA. Purified MARF1 cleaved ssRNAs in vitro, but this cleavage activity was abolished by mutations of conserved aspartates in its NYN domain and truncation of the LOTUS domain. Furthermore, a point mutation in the D272 residue in vivo caused a female-only infertile phenotype in mice, with failure of meiotic resumption and elevation of Line1 and Iap retrotransposon transcripts and DNA double-strand breaks in oocytes. Therefore, the ribonuclease activity of MARF1 controls oocyte meiosis and genome integrity. This activity depends upon conserved aspartic residues in the catalytic NYN domain and the RNA-binding activity of the LOTUS domain.

https://www.pnas.org/content/pnas/115/44/11250.full.pdf

Xiaoyun Zhang

Papers

Oocyte stage-specific effects of MTOR determine granulosa cell fate and oocyte quality in mice

Oocyte-dependent activation of MTOR in cumulus cells controls the development and survival of cumulus-oocyte complexes.

Ribonuclease activity of MARF1 controls oocyte RNA homeostasis and genome integrity in mice.

Oocyte-dependent activation of MTOR in cumulus cells controls the development and survival of cumulus–oocyte complexes