Live birth derived from a markedly large polar body oocyte: a rare case report.

Oocytes with excessively large first polar bodies (PB1) often occur in assisted reproductive procedures. Many times these oocytes are discarded without insemination and, as a result, the application of this portion of oocytes has scarcely been reported to date. Few studies have examined large PB1 oocytes in infertile women and have virtually entirely studied genetic variations for large PB1 oocyte abnormalities. Here, we describe an unusual case of a live birth from a remarkably large PB1 oocyte in a frozen embryo transfer (FET) cycle. This is the first instance of a successful live birth resulting from a PB1 oocyte with an extremely large polar body measuring 80 µM × 40 µM in size. The large PB1 oocyte was performed by an early rescue intracytoplasmic sperm injection (r-ICSI) and was formed into a blastocyst on day 5. Following FET, a healthy boy baby weighing 3100 g was finally delivered by caesarean section at 37 weeks and 5 days after conception. Additionally, there were no complications throughout the antenatal period or the perinatal phase of this following full-term delivery. In this study, it is revealed for the first time that a huge PB1 oocyte can be fertilized, resulting in the growth of a blastocyst, a subsequent pregnancy, and a live birth. This new information prompts us to reconsider the use of large PB1 oocytes. More insightful talks should be given attention to prevent the waste of embryos because not all oocytes with aberrant morphology are unavailable.

Aneuploidy detection in pooled polar bodies using rapid nanopore sequencing.

PURPOSE: Various screening techniques have been developed for preimplantation genetic testing for aneuploidy (PGT-A) to reduce implantation failure and miscarriages in women undergoing in vitro fertilisation (IVF) treatment. Among these methods, the Oxford nanopore technology (ONT) has already been tested in several tissues. However, no studies have applied ONT to polar bodies, a cellular material that is less restrictively regulated for PGT-A in some countries. METHODS: We performed rapid short nanopore sequencing on pooled first and second polar bodies of 102 oocytes from women undergoing IVF treatment to screen for aneuploidy. An automated analysis pipeline was developed with the expectation of three chromatids per chromosome. The results were compared to those obtained by array-based comparative genomic hybridisation (aCGH). RESULTS: ONT and aCGH were consistent for 96% (98/102) of sample ploidy classification. Of those samples, 36 were classified as euploid, while 62 were classified as aneuploid. The four discordant samples were assessed as euploid using aCGH but classified as aneuploid using ONT. The concordance of the ploidy classification (euploid, gain, or loss) per chromosome was 92.5% (2169 of 2346 of analysed chromosomes) using aCGH and ONT and increased to 97.7% (2113/2162) without the eight samples assessed as highly complex aneuploid using ONT. CONCLUSION: The automated detection of the ploidy classification per chromosome and shorter duplications or deletions depending on the sequencing depth demonstrates an advantage of the ONT method over standard, commercial aCGH methods, which do not consider the presence of three chromatids in pooled polar bodies.

Evaluation of Nanopore Sequencing on Polar Bodies for Routine Pre-Implantation Genetic Testing for Aneuploidy.

BACKGROUND: Preimplantation genetic testing for aneuploidy (PGT-A) using polar body (PB) biopsy offers a clinical benefit by reducing the number of embryo transfers and miscarriage rates but is currently not cost-efficient. Nanopore sequencing technology opens possibilities by providing cost-efficient and fast sequencing results with uncomplicated sample preparation work flows. METHODS: In this comparative experimental study, 102 pooled PB samples (99 passing QC) from 20 patients were analyzed for aneuploidy using nanopore sequencing technology and compared with array comparative genomic hybridization (aCGH) results generated as part of the clinical routine. Samples were sequenced on a Nanopore MinION machine. Whole-chromosome copy-numbers were called by custom bioinformatic analysis software. Automatically called results were compared to aCGH results. RESULTS: Overall, 96/99 samples were consistently detected as euploid or aneuploid in both methods (concordance = 97.0%, sensitivity = 0.957, specificity = 1.0, positive predictive value = 1.0, negative predictive value = 0.906). On the chromosomal level, concordance reached 98.7%. Chromosomal aneuploidies analyzed in this trial covered all 23 chromosomes with 98 trisomies, and 97 monosomies in 70 aCGH samples.The whole nanopore work flow is feasible in under 5 h (for one sample) with a maximum time of 16 h (for 12 samples), enabling fresh PB-euploid embryo transfer. A material cost of US$ 165 (EUR 150)/sample possibly enables cost-efficient aneuploidy screening. CONCLUSIONS: This is the first study systematically comparing nanopore sequencing with standard methods for the detection of PB aneuploidy. High concordance rates confirmed the feasibility of nanopore technology for this application. Additionally, the fast and cost-efficient work flow reveals the clinical utility of this technology, making it clinically attractive for PB PGT-A.

Impact of oxygen tension during in vitro maturation: a sibling-oocyte prospective double-blinded study.

OBJECTIVE: To determine whether oxygen (O2) tension (20% vs. 5%) has an impact on oocyte maturation rates and morphology during in vitro maturation (IVM). DESIGN: A prospective, observational, monocentric, sibling-oocyte study. SETTING: University Hospital. PATIENTS: A total of 143 patients who underwent IVM for fertility preservation purposes from November 2016 to April 2021 were analyzed. Patients were included when ≥2 cumulus-oocyte complexes (COCs) were retrieved. The cohort of COCs obtained for each patient was randomly split into two groups: group 20% O2 and group 5% O2. INTERVENTION: Cumulus-oocyte complexes were incubated for 48 hours either under 5% O2 or 20% O2. After 24 and 48 hours of culture, every oocyte was assessed for maturity and morphology, to estimate oocyte quality. Morphology was evaluated considering six parameters (shape, size, ooplasm, perivitelline space, zona pellucida, and polar body characteristics), giving a total oocyte score ranging from -6 to +6. MAIN OUTCOME MEASURES: Maturation rates and total oocyte scores were compared using paired-sample analysis between group 20% O2 and group 5% O2. RESULTS: Patient median age was 31.4 [28.1-35.2] years-old. The mean serum antimüllerian hormone levels and antral follicle count were 3.2 ± 2.3 ng/mL and 27.2 ± 16.0 follicles, respectively. A mean of 10.7 COCs per cycle were retrieved, leading to 6.1 ± 2.4 metaphase II oocytes vitrified (total maturation rate = 57.3%; 991 metaphase II oocytes/1,728 COCs). A total of 864 COCs were included in each group. Oocyte maturation rates were not different between the two groups (group 20% O2: 56.82% vs. group 5% O2: 57.87%, respectively). Regarding oocyte morphology, the mean total oocyte score was significantly higher in group 5% O2 compared with group 20% O2 (3.44 ± 1.26 vs. 3.16 ± 1.32, P=.014). CONCLUSION: As culture under low O2 tension (5% O2) improves oocyte morphology IVM, our results suggest that culture under hypoxia should be standardized. Additional studies are warranted to assess the impact of O2 tension on oocyte maturation and the benefit of IVM under low O2 tension for embryo culture after utilization of frozen material.

An oocyte meiotic midbody cap is required for developmental competence in mice.

Embryo development depends upon maternally derived materials. Mammalian oocytes undergo extreme asymmetric cytokinesis events, producing one large egg and two small polar bodies. During cytokinesis in somatic cells, the midbody and subsequent assembly of the midbody remnant, a signaling organelle containing RNAs, transcription factors and translation machinery, is thought to influence cellular function or fate. The role of the midbody and midbody remnant in gametes, in particular, oocytes, remains unclear. Here, we examined the formation and function of meiotic midbodies (mMB) and mMB remnants using mouse oocytes and demonstrate that mMBs have a specialized cap structure that is orientated toward polar bodies. We show that that mMBs are translationally active, and that mMB caps are required to retain nascent proteins in eggs. We propose that this specialized mMB cap maintains genetic factors in eggs allowing for full developmental competency.

Microtubules oppose cortical actomyosin-driven membrane ingression during C. elegans meiosis I polar body extrusion.

During C. elegans oocyte meiosis I cytokinesis and polar body extrusion, cortical actomyosin is locally remodeled to assemble a contractile ring that forms within and remains part of a much larger and actively contractile cortical actomyosin network. This network both mediates contractile ring dynamics and generates shallow ingressions throughout the oocyte cortex during polar body extrusion. Based on our analysis of requirements for CLS-2, a member of the CLASP family of proteins that stabilize microtubules, we recently proposed that a balance of actomyosin-mediated tension and microtubule-mediated stiffness limits membrane ingression throughout the oocyte during meiosis I polar body extrusion. Here, using live cell imaging and fluorescent protein fusions, we show that CLS-2 is part of a group of kinetochore proteins, including the scaffold KNL-1 and the kinase BUB-1, that also co-localize during meiosis I to structures called linear elements, which are present within the assembling oocyte spindle and also are distributed throughout the oocyte in proximity to, but appearing to underlie, the actomyosin cortex. We further show that KNL-1 and BUB-1, like CLS-2, promote the proper organization of sub-cortical microtubules and also limit membrane ingression throughout the oocyte. Moreover, nocodazole or taxol treatment to destabilize or stabilize oocyte microtubules leads to, respectively, excess or decreased membrane ingression throughout the oocyte. Furthermore, taxol treatment, and genetic backgrounds that elevate the levels of cortically associated microtubules, both suppress excess membrane ingression in cls-2 mutant oocytes. We propose that linear elements influence the organization of sub-cortical microtubules to generate a stiffness that limits cortical actomyosin-driven membrane ingression throughout the oocyte during meiosis I polar body extrusion. We discuss the possibility that this regulation of sub-cortical microtubule dynamics facilitates actomyosin contractile ring dynamics during C. elegans oocyte meiosis I cell division.

Focusing on the accumulation of chromatin/chromosomes around nucleoli and optimizing the timing of ICSI to facilitate the rescue in vitro maturation of denuded GV stage oocytes.

PURPOSE: This study aims to achieve the methodological improvement of rescue IVM by predicting germinal vesicle breakdown (GVBD) and optimizing the timing of ICSI. METHODS: Time lapse analysis was performed retrospectively to evaluated the relationship between the presence of AC around the nucleoli and GVBD. To find the optimal timing of ICSI, the time from the initiation of the first polar body extrusion to ICSI were measured, and the rates of fertilization at each point were calculated. RESULTS: The GVBD rate of GV stage oocytes with AC around the nucleoli was significantly higher than that of GV stage oocytes without AC. The GV stage oocytes required more time for nuclear maturation after polar body extrusion than MI oocytes, with GV stage oocytes taking 400-600 min from polar body extrusion to the optimal timing of ICSI, while the MI stage oocytes took 200-400 min. The GV stage oocytes resulted in the birth of healthy babies with the appropriate timing of ICSI. CONCLUSION: It was found that GV stage oocytes with AC around nucleoli can initiate GVBD and reach the MII stage with a high rate, and that GV stage oocytes required more time than MI stage oocytes to reach the optimal timing of ICSI. Considering these factors, ART laboratories may employ immature GV stage oocytes in routine ART procedures rather than discarding them.

Cell fragmentation in mouse preimplantation embryos induced by ectopic activation of the polar body extrusion pathway.

Cell fragmentation is commonly observed in human preimplantation embryos and is associated with poor prognosis during assisted reproductive technology (ART) procedures. However, the mechanisms leading to cell fragmentation remain largely unknown. Here, light sheet microscopy imaging of mouse embryos reveals that inefficient chromosome separation due to spindle defects, caused by dysfunctional molecular motors Myo1c or dynein, leads to fragmentation during mitosis. Extended exposure of the cell cortex to chromosomes locally triggers actomyosin contractility and pinches off cell fragments. This process is reminiscent of meiosis, during which small GTPase-mediated signals from chromosomes coordinate polar body extrusion (PBE) by actomyosin contraction. By interfering with the signals driving PBE, we find that this meiotic signaling pathway remains active during cleavage stages and is both required and sufficient to trigger fragmentation. Together, we find that fragmentation happens in mitosis after ectopic activation of actomyosin contractility by signals emanating from DNA, similar to those observed during meiosis. Our study uncovers the mechanisms underlying fragmentation in preimplantation embryos and, more generally, offers insight into the regulation of mitosis during the maternal-zygotic transition.

Genome transfer technique for bovine embryo production using the metaphase plate and polar body.

Despite many studies in humans and mice using genome transfer (GT), there are few reports using this technique in oocytes of wild or domestic animals. Therefore, we aimed to establish a GT technique in bovine oocytes using the metaphase plate (MP) and polar body (PB) as the sources of genetic material. In the first experiment, GT was established using MP (GT-MP), and a sperm concentration of 1 × 106 or 0.5 × 106 spermatozoa/ml gave similar fertilization rates. The cleavage rate (50%) and blastocyst rate (13.6%) in the GT-MP group was lower than that of the in vitro production control group (80.2% and 32.6%, respectively). The second experiment evaluated the same parameters using PB instead of MP; the GT-PB group had lower fertilization (82.3% vs. 96.2%) and blastocyst (7.7% vs. 36.8%) rates than the control group. No differences in the amount of mitochondrial DNA (mtDNA) were observed between groups. Finally, GT-MP was performed using vitrified oocytes (GT-MPV) as a source of genetic material. The cleavage rate of the GT-MPV group (68.4%) was similar to that of the vitrified oocytes (VIT) control group (70.0%) and to that of the control IVP group (81.25%, P < 0.05). The blastocyst rate of GT-MPV (15.7) did not differ neither from the VIT control group (5.0%) nor from the IVP control group (35.7%). The results suggested that the structures reconstructed by the GT-MPV and GT-PB technique develop in embryos even if vitrified oocytes are used.

A novel approach toward the generation of oocytes by direct diploid cell haploidization.

Oocyte-mediated somatic cell haploidization is a process in which a diploid cell halves its chromosomal content by segregating its homologue within the ooplasm. Replacing the donor oocyte nucleus with a patient's female diploid somatic nucleus can generate patient-genotyped oocytes. Insemination of these resulting constructs enables their activation and induces a reductive meiotic division, haploidizing the diploid female donor cell that can subsequently support syngamy with the male genome and create a zygote. So far, experimental data for this method have been limited and have not consistently proven the generation of chromosomally normal embryos. Overall, we achieved reconstruction of murine oocytes with a micromanipulation survival rate of 56.5%, and a correct haploidization and fertilization rate of 31.2%, resulting in a 12.7% blastocyst rate. Time-lapse analysis revealed that reconstructed embryos underwent a timely polar body extrusion and pronuclear appearance followed by a satisfactory embryonic cleavage, comparable with the control. Whole genome sequencing of the analyzed embryos indicated that 27.3% (6/22) were properly diploid. Our findings suggest that diploid cell haploidization may be a feasible technique for creating functional gametes in mammals.

MOS mutation causes female infertility with large polar body oocytes.

The Moloney sarcoma oncogene (MOS) encodes a protein serine/threonine kinase and MOS is expressed at high levels in oocytes undergoing meiotic maturation. The MOS/MAPK pathway is normally required for the maintenance of microtubules and chromatin in a metaphasic state during the meiotic divisions. To determine the pathogenic genes in a female infertile patient due to large polar body oocytes, whole-exome sequencing was performed on the patient and available family members. We identified a novel homozygous missense mutation c.591T > G in MOS. Bioinformatics analysis showed that the mutation is harmful. These findings suggest that MOS mutation results in oocytes with a large polar body and poor embryonic development in patients. The MOS variant may regulate oocyte asymmetric division by MAPK/WAVE2/Arp2/3/actin signaling pathway. This will help to understand the comprehensive role of MOS in early human reproductive process and provide genetic markers for future genetic counseling for more individualized treatments.

Cell Repolarization: A Bifurcation Study of Spatio-Temporal Perturbations of Polar Cells.

The intrinsic polarity of migrating cells is regulated by spatial distributions of protein activity. Those proteins (Rho-family GTPases, such as Rac and Rho) redistribute in response to stimuli, determining the cell front and back. Reaction-diffusion equations with mass conservation and positive feedback have been used to explain initial polarization of a cell. However, the sensitivity of a polar cell to a reversal stimulus has not yet been fully understood. We carry out a PDE bifurcation analysis of two polarity models to investigate routes to repolarization: (1) a single-GTPase ("wave-pinning") model and (2) a mutually antagonistic Rac-Rho model. We find distinct routes to reversal in (1) vs. (2). We show numerical simulations of full PDE solutions for the RD equations, demonstrating agreement with predictions of the bifurcation results. Finally, we show that simulations of the polarity models in deforming 1D model cells are consistent with biological experiments.

A localized calcium transient and polar body abscission.

Polar body emission is a special form of cytokinesis in oocyte meiosis that ensures the correct number of chromosomes in reproduction-competent eggs. The molecular mechanism of the last step, polar body abscission, is poorly understood. While it has been proposed that Ca2+ signaling plays important roles in embryonic cytokinesis, to date transient increases in intracellular free Ca2+ have been difficult to document in oocyte meiosis except for the global Ca2+ wave induced by sperm at fertilization. Here, we find that microinjection of the calcium chelator dibromo-BAPTA inhibits polar body abscission in Xenopus laevis oocytes. Using a novel, microtubule-targeted ratio-metric calcium sensor, we detected a calcium transient that is focused at the contractile ring-associated plasma membrane and which occurred after anaphase and constriction of the contractile ring but prior to abscission. This calcium transient was confirmed by mobile calcium probes. Further, the Ca2+-sensitive protein kinase Cß C2 domain transiently translocated to the contractile ring-associated membrane simultaneously with the calcium transient. Collectively, these results demonstrate that a calcium transient, apparently originating at the contractile ring-associated plasma membrane, promotes polar body abscission.

Biallelic variants in MOS cause large polar body in oocyte and human female infertility.

STUDY QUESTION: What is the genetic basis of female infertility involving abnormal oocyte morphology with the production of a large first polar body (PB1)? SUMMARY ANSWER: The homozygous missense variant (c.791C>G) and compound missense variants (c.596A>T and c.875C>T) in MOS proto-oncogene, serine/threonine kinase (MOS) (Online Mendelian Inheritance in Man (OMIM) reference: 190060; NM_005372.1) are responsible for abnormal oocyte morphology with the production of a large PB1 to cause infertility in women. WHAT IS KNOWN ALREADY: MOS, an oocyte-specific gene, encodes a serine/threonine-protein kinase that directly phosphorylates mitogen-activated protein kinase (MAPK) kinase (MEK) to activate MAPK (also called extracellular-signal-regulated kinase (ERK)) signal cascade in the oocyte. Female mice lacking Mos remained viable, but infertile because of oocyte symmetric division, spontaneous parthenogenetic activation and early embryonic arrest. Recently, two independent studies demonstrated that female infertility with early embryonic arrest and fragmentation can be caused by biallelic mutations in MOS. However, so far, MOS variants have not been associated with the phenotype of large PB1 extrusion in human oocytes to contribute to female infertility. STUDY DESIGN, SIZE, DURATION: Two independent infertile families characterized by the presence of large PB1 in oocytes were recruited between December 2020 and February 2022. PARTICIPANTS/MATERIALS, SETTING, METHODS: Genomic DNA was extracted from the peripheral blood samples of the subjects for whole-exome sequencing. Pedigree analysis was validated by Sanger sequencing. Then, the pathogenic effects of the MOS variants on MOS protein properties and ERK1/2 activation were determined in HEK293 cells and mouse oocytes. MAIN RESULTS AND THE ROLE OF CHANCE: We identified three rare missense variants in MOS, including a homozygous missense variant (c.791C>G) from Patient 1 in Family 1 and two compound missense variants (c.596A>T and c.875C>T) from twin sisters in Family 2. The MOS variants followed a recessive inheritance pattern in infertile patients. All three patients displayed a high percentage of large PB1 extrusion in the oocytes. The three MOS variants could not activate MEK1/2 and ERK1/2 in oocytes and HEK293 cells. In addition, when compared with wild-type MOS, the MOS variants decreased the MOS protein level and attenuated the binding capacity with MEK1. Microinjection of wild-type human MOS complementary RNAs (cRNAs) reversed the symmetric division of oocytes after siMos treatment. In contrast, the three MOS variants demonstrated no rescuing ability. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Owing to the scarcity of human oocyte samples and the associated ethical restrictions, we could not perform the rescue attempt for the study patients. WIDER IMPLICATIONS OF THE FINDINGS: Our findings expand the genetic and phenotypic spectrum of MOS variants in causing female infertility. Our study findings facilitate the early genetic diagnosis of abnormal oocyte morphology characterized as large PB1 that eventually causes infertility in women. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Natural Science Foundation of China (82071640 and 82001633), Natural Science Foundation of Zhejiang Province (LD22C060001), the Key Projects Jointly Constructed by the Ministry and the Province of Zhejiang Medical and Health Science and Technology Project (WKJ-ZJ-2005), China Postdoctoral Science Foundation (2020M682575 and 2021T140198), the Changsha Municipal Natural Science Foundation (kq2007022) and Hunan Provincial Grant for Innovative Province Construction (2019SK4012). None of the authors declare any competing interests. TRIAL REGISTRATION NUMBER: N/A.

Mettl3 downregulation in germinal vesicle oocytes inhibits mRNA decay and the first polar body extrusion during maturation†.

In oocytes, mRNA decay is essential for maturation and subsequent events, such as maternal-zygotic transition, zygotic genomic activation, and embryo development. Reversible N6-methyladenosine RNA methylation directly regulates transcription, pre-mRNA splicing, mRNA export, mRNA stability, and translation. Here, we identified that downregulation of N6-methyladenosine modification by microinjecting a methyltransferase-like 3 (Mettl3)-specific small interfering RNA into mouse germinal vesicle oocytes led to defects in meiotic spindles and the first polar body extrusion during maturation in vitro. By further quantitative real-time polymerase chain reaction and Poly(A)-tail assay analysis, we found that N6-methyladenosine methylation mainly acts by reducing deadenylation of mRNAs mediated by the carbon catabolite repression 4-negative on TATA less system, thereby causing mRNA accumulation in oocytes. Meanwhile, transcriptome analysis of germinal vesicle oocytes revealed the downregulation of transcripts of several genes encoding ribosomal subunits proteins in the Mettl3 small interfering RNA-treated group, suggesting that N6-methyladenosine modification might affect translation. Together, our results indicate that RNA methylation accelerates mRNA decay, confirming the critical role of RNA clearance in oocyte maturation.

Topoisomerase II dysfunction causes metaphase I arrest by activating Aurora B, SAC and MPF and prevents PB1 abscission in mouse oocytes†.

Oocyte aneuploidy is caused mainly by chromosome nondisjunction and/or unbalanced sister chromatid pre-division. Although studies in somatic cells have shown that topoisomerase II (TOP2) plays important roles in chromosome condensation and timely separation of centromeres, little is known about its role during oocyte meiosis. Furthermore, because VP-16, which is a TOP2 inhibitor and induces DNA double strand breaks, is often used for ovarian cancer chemotherapy, its effects on oocytes must be studied for ovarian cancer patients to recover ovarian function following chemotherapy. This study showed that inhibiting TOP2 with either ICRF-193 or VP-16 during meiosis I impaired chromatin condensation, chromosome alignment, TOP2α localization, and caused metaphase I (MI) arrest and first polar body (PB1) abscission failure. Inhibiting or neutralizing either spindle assembly checkpoint (SAC), Aurora B or maturation-promoting factor (MPF) significantly abolished the effect of ICRF-193 or VP-16 on MI arrest. Treatment with ICRF-193 or VP-16 significantly activated MPF and SAC but the effect disappeared when Aurora B was inhibited. Most of the oocytes matured in the presence of ICRF-193 or VP-16 were arrested at MI, and only 11-27% showed PB1 protrusion. Furthermore, most of the PB1 protrusions formed in the presence of ICRF-193 or VP-16 were retracted after further culture for 7 h. In conclusion, TOP2 dysfunction causes MI arrest by activating Aurora B, SAC, and MPF, and it prevents PB1 abscission by promoting chromatin bridges.

Deletion of Orc4 during oogenesis severely reduces polar body extrusion and blocks zygotic DNA replication†.

Origin recognition complex subunit 4 (ORC4) is a DNA-binding protein required for DNA replication. During oocyte maturation, after the last oocyte DNA replication step and before zygotic DNA replication, the oocyte undergoes two meiotic cell divisions in which half the DNA is ejected in much smaller polar bodies. We previously demonstrated that ORC4 forms a cytoplasmic cage around the DNA that is ejected in both polar body extrusion (PBE) events. Here, we used ZP3 activated Cre to delete exon 7 of Orc4 during oogenesis to test how it affected both predicted functions of ORC4: its recently discovered role in PBE and its well-known role in DNA synthesis. Orc4 deletion severely reduced PBE. Almost half of Orc4-depleted germinal vesicle (GV) oocytes cultured in vitro were arrested before anaphase I (48%), and only 25% produced normal first polar bodies. This supports the role of ORC4 in PBE and suggests that transcription of the full-length Orc4 during oogenesis is required for efficient PBE. Orc4 deletion also abolished zygotic DNA synthesis. Fewer Orc4-depleted oocytes developed to the metaphase II (MII) stage, and after activation these oocytes were arrested at the two-cell stage without undergoing DNA synthesis. This confirms that transcription of full-length Orc4 after the primary follicle stage is required for zygotic DNA replication. The data also suggest that MII oocytes do not have a replication licensing checkpoint as cytokinesis progressed without DNA synthesis. Together, the data confirm that oocyte ORC4 is important for both PBE and zygotic DNA synthesis.

Developmental trajectory of monopronucleated zygotes after in vitro fertilization when they include both male and female genomes.

OBJECTIVE: To examine the cause of monopronucleated zygote (1PN) formation that includes both maternal and paternal genomes. DESIGN: Retrospective cohort study. SETTING: Private fertility clinic. PATIENT(S): A total of 44 1PN and 726 2-pronuclear zygotes from 702 patients were observed using 2 different time-lapse observation systems. INTERVENTION(S): Previously recorded time lapse data were reviewed to examine the mechanism of 1PN formation. MAIN OUTCOME MEASURE(S): The distance between the position of the second polar body extrusion and the fertilization cone or epicenter/starting position of the cytoplasmic wave was measured, and the consequent data were analyzed. Cytoplasmic waves were confirmed using vector analysis software. RESULT(S): The cut-off value for the difference in the distance between the position of the second polar body extrusion and the fertilization cone or the epicenter/starting position of the cytoplasmic wave was 17 µm (AUC: 0.987, 95% CI: 0.976-0.999) for the Embryo Scope and 18 µm (AUC: 0.972, 95% CI: 0.955-0.988) for the iBIS time-lapse observation systems. CONCLUSION(S): In this study, it was found with a high degree of accuracy that a monopronucleus is formed when the fusion of the sperm takes place within 18 µm from the point of the second polar body extrusion. The theoretical chance of 1PN occurrence after in vitro fertilization is 2.7% when the sperm is considered to be fused anywhere in the plasma membrane of an oocyte.

The Lon protease temporally restricts polar cell differentiation events during the Caulobacter cell cycle.

The highly conserved protease Lon has important regulatory and protein quality control functions in cells from the three domains of life. Despite many years of research on Lon, only a few specific protein substrates are known in most organisms. Here, we used a quantitative proteomics approach to identify novel substrates of Lon in the dimorphic bacterium Caulobacter crescentus. We focused our study on proteins involved in polar cell differentiation and investigated the developmental regulator StaR and the flagella hook length regulator FliK as specific Lon substrates in detail. We show that Lon recognizes these proteins at their C-termini, and that Lon-dependent degradation ensures their temporally restricted accumulation in the cell cycle phase when their function is needed. Disruption of this precise temporal regulation of StaR and FliK levels in a Δlon mutant contributes to defects in stalk biogenesis and motility, respectively, revealing a critical role of Lon in coordinating developmental processes with cell cycle progression. Our work underscores the importance of Lon in the regulation of complex temporally controlled processes by adjusting the concentrations of critical regulatory proteins. Furthermore, this study includes the first characterization of FliK in C. crescentus and uncovers a dual role of the C-terminal amino acids of FliK in protein function and degradation.

Two mutations in TUBB8 cause developmental arrest in human oocytes and early embryos.

RESEARCH QUESTION: How can the effect of genetic mutations that may cause primary female infertility be evaluated? DESIGN: Patients and their family members underwent whole-exome sequencing and Sanger sequencing to detect the infertility-causing gene and inheritance pattern. To study the function of mutant proteins in vitro, vectors containing wild-type or mutant TUBB8 cDNA were constructed for transient expression in HeLa cells, and in-vitro transcribed mRNA were used for microinjection in germinal vesicle-stage mouse oocytes. Immunofluorescence staining was used to observe the microtubule structure in HeLa cells or meiotic spindle in mouse oocytes. RESULTS: A maternally inherited TUBB8 (Tubulin beta 8 class VIII) mutation (NM_177987.2: c. 959G>A: p. R320H) and a previously reported (NM_177987.2: c. 161C>T: p. A54V) recessive mutation from two infertile female patients were identified. The oocytes from the patient carrying p.A54V mutation failed fertilization, whereas oocytes with p.R320H mutation could be fertilized but showed heavy fragmentation during early development. In vitro, functional assays showed that p. A54V mutant disrupted the microtubule structure in HeLa cells (49.3% of transfected cells) and caused large polar body extrusion in mouse oocytes (27.5%), whereas the p.R320H mutant caused a higher abnormal rate (69.7%) in cultured cells and arrested mouse oocytes at meiosis I (38.7%). CONCLUSION: Two TUBB8 mutations (p.A54V and p.R320H) were identified and their pathogeny was confirmed by in-vitro functional assays.