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Abstract Aneuploidy seriously compromises female fertility and increases incidence of birth defects. Rates of aneuploidy in human eggs from even young women are significantly higher than those in other mammals. However, intrinsic genetic factors underlying this high incidence of aneuploidy in human eggs remain largely unknown. Here, we found that ectopic expression of human TUBB8 in mouse oocytes increases rates of aneuploidy by causing kinetochore–microtubule (K–MT) attachment defects. Stretched bivalents in mouse oocytes expressing TUBB8 are under less tension, resulting in continuous phosphorylation status of HEC1 by AURKB/C at late metaphase I that impairs the established correct K–MT attachments. This reduced tension in stretched bivalents likely correlates with decreased recruitment of KIF11 on meiotic spindles. We also found that ectopic expression of TUBB8 without its C-terminal tail decreases aneuploidy rates by reducing erroneous K–MT attachments. Importantly, variants in the C-terminal tail of TUBB8 were identified in patients with recurrent miscarriages. Ectopic expression of an identified TUBB8 variant in mouse oocytes also compromises K–MT attachments and increases aneuploidy rates. In conclusion, our study provides novel understanding for physiological mechanisms of aneuploidy in human eggs as well as for pathophysiological mechanisms involved in recurrent miscarriages.

Abstract Oocyte maturation arrest is one of the important causes of female infertility, but the genetic factors remain largely unknown. PABPC1L, a predominant poly(A)‐binding protein in Xenopus, mouse, and human oocytes and early embryos prior to zygotic genome activation, plays a key role in translational activation of maternal mRNAs. Here, we identified compound heterozygous and homozygous variants in PABPC1L that are responsible for female infertility mainly characterized by oocyte maturation arrest in five individuals. In vitro studies demonstrated that these variants resulted in truncated proteins, reduced protein abundance, altered cytoplasmic localization, and reduced mRNA translational activation by affecting the binding of PABPC1L to mRNA. In vivo, three strains of Pabpc1l knock‐in (KI) female mice were infertile. RNA‐sequencing analysis showed abnormal activation of the Mos‐MAPK pathway in the zygotes of KI mice. Finally, we activated this pathway in mouse zygotes by injecting human MOS mRNA, and this mimicked the phenotype of KI mice. Our findings reveal the important roles of PABPC1L in human oocyte maturation and add a genetic potential candidate gene to be screened for causes of infertility.

Abstract Background Oocyte maturation arrest and early embryonic arrest are important reproductive phenotypes resulting in female infertility and cause the recurrent failure of assisted reproductive technology (ART). However, the genetic etiologies of these female infertility-related phenotypes are poorly understood. Previous studies have mainly focused on inherited mutations based on large pedigrees or consanguineous patients. However, the role of de novo mutations (DNMs) in these phenotypes remains to be elucidated. Results To decipher the role of DNMs in ART failure and female infertility with oocyte and embryo defects, we explore the landscape of DNMs in 473 infertile parent–child trios and identify a set of 481 confident DNMs distributed in 474 genes. Gene ontology analysis reveals that the identified genes with DNMs are enriched in signaling pathways associated with female reproductive processes such as meiosis, embryonic development, and reproductive structure development. We perform functional assays on the effects of DNMs in a representative gene Tubulin Alpha 4a (TUBA4A), which shows the most significant enrichment of DNMs in the infertile parent–child trios. DNMs in TUBA4A disrupt the normal assembly of the microtubule network in HeLa cells, and microinjection of DNM TUBA4A cRNAs causes abnormalities in mouse oocyte maturation or embryo development, suggesting the pathogenic role of these DNMs in TUBA4A. Conclusions Our findings suggest novel genetic insights that DNMs contribute to female infertility with oocyte and embryo defects. This study also provides potential genetic markers and facilitates the genetic diagnosis of recurrent ART failure and female infertility.

Preimplantation embryo arrest (PREMBA) is a common cause of female infertility and recurrent failure of assisted reproductive technology. However, the genetic basis of PREMBA is largely unrevealed. Here, using whole-exome sequencing data from 606 women experiencing PREMBA compared with 2,813 controls, we performed a population and gene–based burden test and identified a candidate gene, karyopherin subunit α7 (KPNA7). In vitro studies showed that identified sequence variants reduced KPNA7 protein levels, impaired KPNA7 capacity for binding to its substrate ribosomal L1 domain-containing protein 1 (RSL1D1), and affected KPNA7 nuclear transport activity. Comparison between humans and mice suggested that mouse KPNA2, rather than mouse KPNA7, acts as an essential karyopherin in embryonic development. Kpna2–/– female mice showed embryo arrest due to zygotic genome activation defects, recapitulating the phenotype of human PREMBA. In addition, female mice with an oocyte-specific knockout of Rsl1d1 recapitulated the phenotype of Kpna2–/– mice, demonstrating the vital role of substrate RSL1D1. Finally, complementary RNA (cRNA) microinjection of human KPNA7, but not mouse Kpna7, was able to rescue the embryo arrest phenotype in Kpna2–/– mice, suggesting mouse KPNA2 might be a homologue of human KPNA7. Our findings uncovered a mechanistic understanding for the pathogenesis of PREMBA, which acts by impairing nuclear protein transport, and provide a diagnostic marker for PREMBA patients.

Oocyte maturation and fertilization are fundamental processes for successful human reproduction, and abnormalities in these processes will cause infertility. Recently, we identified biallelic mutations in CDC20 that are responsible for human oocyte maturation arrest, fertilization failure, and early embryonic development arrest. In this study, we screened for further CDC20 mutations in a new cohort of patients with abnormalities in oocyte maturation, fertilization, and early embryonic development. Through whole-exome sequencing, we identified the four novel mutations c.887G > A (p. Arg296Gln), c.964C > T (p.Arg322∗), c.1155G > C (p.Trp385Cys), and c.330 + 1G > A (p. Glu111Ilefs∗36) and one previously reported mutation c.965G > A (p.Arg322Gln) in CDC20 in four infertile individuals from three independent families. The patients had different phenotypes of oocyte maturation arrest and fertilization failure resulting from the different mutations. This study confirms our previous research and expands the spectrum of known mutations in CDC20, providing new evidence supporting the function of CDC20 in the genetic etiology of female infertility characterized by oocyte maturation arrest and fertilization failure.

BACKGROUND: Klebsiella pneumoniae is a clinically significant species of bacterium which causes a variety of diseases. Clinical treatment of this bacterial infection is greatly hindered by the emergence of multidrug-resistant strains. The resistance is largely due to the acquisition of plasmids carrying drug-resistant as well as pathogenic genes, and its conjugal transfer facilitates the spread of resistant phenotypes. METHODOLOGY/PRINCIPAL FINDINGS: The 70,057 bp plasmid pKF3-70, commonly found in Klebsiella pneumoniae, is composed of five main functional modules, including regions involved in replication, partition, conjugation, transfer leading, and variable regions. This plasmid is more similar to several Escherichia coli plasmids than any previously reported K. pneumoniae plasmids and pKF3-70 like plasmids share a common and conserved backbone sequence. The replication system of the pKF3-70 is 100% identical to that of RepFII plasmid R100 from E. coli. A beta-lactamase gene ctx-m-14 with its surrounding insertion elements (ISEcp1, truncated IS903 and a 20 bp inverted repeat sequence) may compose an active transposon which is directly bordered by two putative target repeats "ATTAC." CONCLUSIONS/SIGNIFICANCE: The K. pneumoniae plasmid pKF3-70 carries an extended-spectrum beta-lactamase gene, ctx-m-14. The conjugative characteristic makes it a widespread plasmid among genetically relevant genera which poses significant threat to public health.

Mitochondria play diverse roles in mammalian physiology. The architecture, activity, and physiological functions of mitochondria in oocytes are largely different from those in somatic cells, but the mitochondrial proteins related to oocyte quality and reproductive longevity remain largely unknown. Here, using whole-exome sequencing data from 1,024 women (characterized by oocyte maturation arrest and degenerated or morphologically abnormal oocytes) and 2,868 healthy controls, we performed a population and gene-based burden test for mitochondrial genes and identified a candidate gene, cytochrome c oxidase assembly protein 15 (COX15). We report that biallelic COX15 pathogenic variants cause human oocyte ferroptosis and female infertility in a recessive inheritance pattern. COX15 variants impaired mitochondrial respiration in Saccharomyces cerevisiae and led to reduced protein levels in HeLa cells. Oocyte-specific deletion of Cox15 led to impaired Fe2+ and reactive oxygen species homeostasis that caused mitochondrial dysfunction and ultimately sensitized oocytes to ferroptosis. In addition, ferrostatin-1 (an inhibitor of ferroptosis) could rescue the oocyte ferroptosis phenotype in vitro and ex vivo. Our findings not only provide a genetic diagnostic marker for oocyte development defects but also expand the spectrum of mitochondrial disorders to female infertility and contribute to unique insights into the role of ferroptosis in human oocyte defects. [ABSTRACT FROM AUTHOR]

Sperm with normal morphology and motility are essential for successful fertilization, and the strong attachment of the sperm head-tail coupling apparatus to the nuclear envelope during spermatogenesis is required to ensure the integrity of sperm for capacitation and fertilization. Here, we report that Arrdc5 is associated with spermatogenesis. The Arrdc5 knockout mouse model showed male infertility characterized by a high bent-head rate and reduced motility in sperm, which led to capacitation defects and subsequent fertilization failure. Through mass spectrometry, we found that ARRDC5 affects spermatogenesis by affecting NDC1 and SUN5. We further found that ARRDC5 might affect the vesicle-trafficking protein SEC22A-mediated transport and localization of NDC1, SUN5 and other head-tail coupling apparatus-related proteins that are responsible for initiating the attachment of the sperm head and tail. We finally performed intracytoplasmic sperm injection as a way to explore therapeutic strategies. Our findings demonstrate the essential role and the underlying molecular mechanism of ARRDC5 in anchoring the sperm head to the tail during spermatogenesis. [ABSTRACT FROM AUTHOR]

The accumulation and storage of maternal mRNA is crucial for oocyte maturation and embryonic development. PATL2 is an oocyte-specific RNA-binding protein, and previous studies have confirmed that PATL2 mutation in humans and knockout mice cause oocyte maturation arrest or embryonic development arrest, respectively. However, the physiological function of PATL2 in the process of oocyte maturation and embryonic development is largely unknown. Here, we report that PATL2 is highly expressed in growing oocytes and couples with EIF4E and CPEB1 to regulate maternal mRNA expression in immature oocytes. The germinal vesicle oocytes from Patl2-/- mice exhibit decreasing maternal mRNA expression and reduced levels of protein synthesis. We further confirmed that PATL2 phosphorylation occurs in the oocyte maturation process and identified the S279 phosphorylation site using phosphoproteomics. We found that the S279D mutation decreased the protein level of PATL2 and led to subfertility in Palt2S279D knock-in mice. Our work reveals the previously unrecognized role of PATL2 in regulating the maternal transcriptome and shows that phosphorylation of PATL2 leads to the regulation of PATL2 protein levels via ubiquitin-mediated proteasomal degradation in oocytes. [ABSTRACT FROM AUTHOR]

Background Abnormal pronuclear formation during fertilisation and subsequent early embryonic arrest results in female infertility. In recent years, with the prevalence of assisted reproductive technology, a few genes have been identified that are involved in female infertility caused by abnormalities in oocyte development, fertilisation and embryonic development. However, the genetic factors responsible for multiple pronuclei formation during fertilisation and early embryonic arrest remain largely unknown. Objective We aim to identify genetic factors responsible for multiple pronuclei formation during fertilisation or early embryonic arrest. Methods Whole-exome sequencing was performed in a cohort of 580 patients with abnormal fertilisation and early embryonic arrest. Effects of mutations were investigated in HEK293T cells by western blotting and immunoprecipitation, as well as minigene assay. Results We identified a novel homozygous missense mutation (c.397T>G, p.C133G) and a novel homozygous donor splice-site mutation (c.546+5G>A) in the meiotic gene REC114. REC114 is involved in the formation of double strand breaks (DSBs), which initiate homologous chromosome recombination. We demonstrated that the splice-site mutation affected the normal alternative splicing of REC114, while the missense mutation reduced the protein level of REC114 in vitro and resulted in the loss of its function to protect its partner protein MEI4 from degradation. Conclusions Our study has identified mutations in REC114 responsible for human multiple pronuclei formation and early embryonic arrest, and these findings expand our knowledge of genetic factors that are responsible for normal human female meiosis and fertility. [ABSTRACT FROM AUTHOR]

Background Successful human reproduction requires normal spermatogenesis, oogenesis, fertilisation and early embryonic development, and abnormalities in any of these processes will result in infertility. Early embryonic arrest is commonly observed in infertile patients with recurrent failure of assisted reproductive technology (ART). However, the genetic basis for early embryonic arrest is largely unknown. Objective We aim to identify genetic causes of infertile patients characterised by early embryonic arrest. Methods We pursued exome sequencing in a proband with embryonic arrest from the consanguineous family. We further screened candidate genes in a cohort of 496 individuals diagnosed with early embryonic arrest by Sanger sequencing. Effects of mutations were investigated in HeLa cells, oocytes and embryos. Results We identified five independent individuals carrying biallelic mutations in NLRP2. We also found three individuals from two families carrying biallelic mutations in NLRP5. These mutations in NLRP2 and NLRP5 caused decreased protein expression in vitro and in oocytes and embryos. Conclusions NLRP2 and NLRP5 are novel mutant genes responsible for human early embryonic arrest. This finding provides additional potential diagnostic markers for patients with recurrent failure of ART and helps us to better understand the genetic basis of female infertility characterised by early embryonic arrest. [ABSTRACT FROM AUTHOR]

Study Question: Are there any new type of mutations and novel phenotypes in patients with arrest in oocyte maturation, fertilization or early embryonic development having tubulin beta eight class VIII (TUBB8) mutations?Summary Answer: We identified new types of mutations in TUBB8 associated with maturation, fertilization and developmental arrest.What Is Known Already: We previously found heterozygous mutations and a homozygous frameshift/internal seven amino acid deletion in TUBB8 that are responsible for oocyte maturation arrest.Study Design, Size, Duration: We recruited 10 new primary infertility patients from 9 families from December 2015 to May 2016, most of which exhibited failures in oocyte maturation.Participants/materials, Setting, Methods: Ten primary infertility patients were recruited from the reproduction centers in local hospitals. Genomic DNA samples from the affected individuals, their family members and healthy controls were extracted from peripheral blood. TUBB8 in the DNA samples were sequenced by Sanger sequencing. TUBB8 sequence was then aligned by CodonCode software to identify rare variants. ExAC database was used to search frequency of corresponding mutations. In silico analysis of mutations was used by Polyphen and PROVEAN. Phenotypes of oocytes and embryos were evaluated by light microscopy, polarization microscopy or immunolabeling.Main Results and the Role Of Chance: Besides several novel heterozygous missense mutations, we also identified other new types of genetic variants, including homozygous mutations and a de novo compound heterozygous mutation. We also found a patient with a homozygous deletion of the whole TUBB8 gene, which is the first reported case of a large structural variation in this gene. In addition, we found different mutations in TUBB8 that could result in variability in oocyte/embryo phenotypes, including oocyte maturation arrest, first polar body (PB1) oocytes that cannot be fertilized, and PB1 oocytes that can be fertilized but arrest at an early embryonic stage.Limitations, Reasons For Caution: The exact molecular mechanism has not been analyzed and should be further investigated in the future. In addition, immunostaining of more oocytes with mutations and checking spindle status of oocytes with mutations non-invasively by polarization microscopy needs to be done in order to determine exact stage of PB1 oocytes and the functional differences of these mutations.Wider Implications Of the Findings: The results not only emphasize the important role of TUBB8 in oocyte maturation, fertilization and early embryonic development but they also provide a basis for determining the genetic variations in TUBB8 as a potential additional criterion for evaluating the quality of patients' functional PB1 oocytes.Study Funding/competing Interests: National Key R&D Program of China (2016YFC1000600); Basic Research Program of China (2015CB943300); National Natural Science Foundation of China (81270747 and 81571501). No competing interests declared. [ABSTRACT FROM AUTHOR]

Background TUBB8 is a primate-specific β-tubulin isotype whose expression is confined to oocytes and the early embryo. We previously found that mutations in TUBB8 caused oocyte maturation arrest. The objective was to describe newly discovered mutations in TUBB8 and to characterise the accompanying spectrum of phenotypes and modes of inheritance. Methods and results Patients with oocyte maturation arrest were sequenced with respect to TUBB8. We investigated the effects of identified mutations in vitro, in cultured cells and in mouse oocytes. Seven heterozygous missense and two homozygous mutations were identified. These mutations cause a range of folding defects in vitro, different degrees of microtubule disruption upon expression in cultured cells and interfere to varying extents in the proper assembly of the meiotic spindle in mouse oocytes. Several of the newly discovered TUBB8 mutations result in phenotypic variability. For example, oocytes harbouring any of three missense mutations (I210V, T238M and N348S) could extrude the first polar body. Moreover, they could be fertilised, although the ensuing embryos became developmentally arrested. Surprisingly, oocytes from patients harbouring homozygous TUBB8 mutations that in either case preclude the expression of a functional TUBB8 polypeptide nonetheless contained identifiable spindles. Conclusions Our data substantially expand the range of dysfunctional oocyte phenotypes incurred by mutation in TUBB8, underscore the independent nature of human oocyte meiosis and differentiation, extend the class of genetic diseases known as the tubulinopathies and provide new criteria for the qualitative evaluation of meiosis II (MII) oocytes for in vitro fertilization (IVF). [ABSTRACT FROM AUTHOR]

Background: Antigenic variation in Plasmodium falciparum involves switching among multicopy var gene family and is responsible for immune evasion and the maintenance of chronic infections. Current understanding of var gene expression and switching patterns comes from experiments conducted on long laboratory-adapted strains, with little known about their wild counterparts. Methods: Genome sequencing was used to obtain 50 var genes from a parasite isolated from the China-Myanmar border. Four clones with different dominant var genes were cultured in vitro in replicates for 50 generations. Transcription of the individual var gene was detected by real-time PCR and then the switching process was analysed. Results: The expression of multicopy var genes is mutually exclusive in clones of a wild P. falciparum isolate. The activation of distinct primary dominant var genes leads to different and favoured switching patterns in the four clones. The on/off rates of individual var genes are variable and the choice of subsequent dominant var genes are random, which results in the different switching patterns among replicates of each clonal wild P. falciparum isolate with near identical initial transcription profiles. Conclusions: This study suggests that the switching patterns of var genes are abundant, which consist of both conserved and random parts. [ABSTRACT FROM AUTHOR]

Arthrospira platensis is an ideal organism for a proteome study under conditions of environmental stress. Herein, we report the differential expression of proteins in A. platensis strain YZ responding to 48 h of continuous dark or light conditions by two-dimensional gel electrophoresis and peptide mass fingerprinting techniques. There were 102 proteins retrieved with significant differential expression between light and dark incubation, among which 96 protein spots were found with high homology to Arthrospira genus (67 proteins in A. platensis strain Paraca and 29 in A. maxima strain CS-328). The other six proteins belonged to other microorganisms. Using a clusters of orthologous groups database, we classified the 102 differentially expressed positive proteins into 19 types based on their function and localized them in their respective KEGG metabolic pathways. The proteins were involved in 47 metabolic pathways, such as photosynthesis, glycolysis/gluconeogenesis pathway, oxidative phosphorylation, and amino acid and fatty acid synthesis. These results implied that the proteins performed predictable roles in rendering A. platensis strain YZ resistant against dark stress. Additionally, we determined the transcriptional level of 26 genes in response to dark stress in vivo by quantitative reverse-transcription polymerase chain reaction, and we found that the expression of six differential proteins, representing 23.08% of the total target genes, was consistent between transcriptional and translational levels. The remaining 20 genes showed inconsistent protein expression compared with the transcriptional level, which accounted for 76.92% of the total target genes. [ABSTRACT FROM AUTHOR]

Background: The piggyBac transposon system provides a powerful forward genetics tool to study gene function in Plasmodium parasites via random insertion mutagenesis and phenotypic screening. The identification of genotype of piggyBac mutants in the Plasmodium genome is thus an indispensable step in forward genetic analysis. Several PCR-based approaches have been used to identify the piggyBac insertion sites in Plasmodium falciparum and Plasmodium berghei, but all are tedious and inefficient. Next generation sequencing can produce large amounts of sequence data and is particularly suitable for genome-wide association studies. In this study, the Next generation sequencing technology was employed to efficiently identify piggyBac insertion sites in the genome of P. berghei. Methods: Plasmodium berghei parasites were co-transfected with piggyBac donor and helper plasmids. Initially, the classical inverse PCR method was used to identify the existence of piggyBac insertions in the P. berghei genome. The whole genome of post-transfection parasites was subsequently sequenced with a PCR-free paired-end module using the Illumina HiSeq sequencing system. The two distinct methods ('BLAST method' and 'SOAP method') were employed to identify piggyBac insertion sites in the P. berghei genome with Illumina sequencing data. All the identified piggyBac insertions were further tested by half-nested PCR. Results: The inverse PCR method resulted in a very low yield of ten individual insertions identified. Conversely, 47 piggyBac insertions were identified from about 1 Gb of Illumina sequencing data via the two distinct analysis methods. The majority of identified piggyBac insertions were confirmed by half-nested PCR. In addition, 1,850 single nucleotide polymorphisms were identified through alignment of the Illumina sequencing data of the P. berghei ANKA strain used in this study with the reference genome sequences. Conclusion: This study demonstrates that a high-throughput genome sequencing approach is an efficient tool for the identification of piggyBac-mediated insertions in Plasmodium parasites. [ABSTRACT FROM AUTHOR]

Fertilization is a fundamental process of development and is a prerequisite for successful human reproduction. In mice, although several receptor proteins have been shown to play important roles in the process of fertilization, only three genes have been shown to cause fertilization failure and infertility when deleted in vivo. In clinical practice, some infertility case subjects suffer from recurrent failure of in vitro fertilization and intracytoplasmic sperm injection attempts due to fertilization failure, but the genetic basis of fertilization failure in humans remains largely unknown. Wee2 is a key oocyte-specific kinase involved in the control of meiotic arrest in mice, but WEE2 has not been associated with any diseases in humans. In this study, we identified homozygous mutations in WEE2 that are responsible for fertilization failure in humans. All four independent affected individuals had homozygous loss-of-function missense mutations or homozygous frameshift protein-truncating mutations, and the phenotype of fertilization failure was shown to follow a Mendelian recessive inheritance pattern. All four mutations significantly decreased the amount of WEE2 protein in vitro and in affected individuals' oocytes in vivo, and they all led to abnormal serine phosphorylation of WEE2 and reduced tyrosine 15 phosphorylation of Cdc2 in vitro. In addition, injection of WEE2 cRNA into affected individuals' oocytes rescued the fertilization failure phenotype and led to the formation of blastocysts in vitro. This work presents a novel gene responsible for human fertilization failure and has implications for future therapeutic treatments for infertility cases. [ABSTRACT FROM AUTHOR]