Mad2 is dispensable for accurate chromosome segregation but becomes essential when oocytes are subjected to environmental stress.

Accurate chromosome segregation, monitored by the spindle assembly checkpoint (SAC), is crucial for the production of euploid cells. Previous in vitro studies by us and others showed that Mad2, a core member of the SAC, performs a checkpoint function in oocyte meiosis. Here, through an oocyte-specific knockout approach in mouse, we reconfirmed that Mad2-deficient oocytes exhibit an accelerated metaphase-to-anaphase transition caused by premature degradation of securin and cyclin B1 and subsequent activation of separase in meiosis I. However, it was surprising that the knockout mice were completely fertile and the resulting oocytes were euploid. In the absence of Mad2, other SAC proteins, including BubR1, Bub3 and Mad1, were normally recruited to the kinetochores, which likely explains the balanced chromosome separation. Further studies showed that the chromosome separation in Mad2-null oocytes was particularly sensitive to environmental changes and, when matured in vitro, showed chromosome misalignment, lagging chromosomes, and aneuploidy with premature separation of sister chromatids, which was exacerbated at a lower temperature. We reveal for the first time that Mad2 is dispensable for proper chromosome segregation but acts to mitigate environmental stress in meiotic oocytes.

MEIKIN expression and its C-terminal phosphorylation by PLK1 is closely related the metaphase-anaphase transition by affecting cyclin B1 and Securin stabilization in meiotic oocyte.

Oocyte meiotic maturation failure and chromosome abnormality is one of the main causes of infertility, abortion, and diseases. The mono-orientation of sister chromatids during the first meiosis is important for ensuring accurate chromosome segregation in oocytes. MEIKIN is a germ cell-specific protein that can regulate the mono-orientation of sister chromatids and the protection of the centromeric cohesin complex during meiosis I. Here we found that MEIKIN is a maternal protein that was highly expressed in mouse oocytes before the metaphase I (MI) stage, but became degraded by the MII stage and dramatically reduced after fertilization. Strikingly, MEIKIN underwent phosphorylation modification after germinal vesicle breakdown (GVBD), indicating its possible function in subsequent cellular event regulation. We further showed that MEIKIN phosphorylation was mediated by PLK1 at its carboxyl terminal region and its C-terminus was its key functional domain. To clarify the biological significance of meikin degradation during later stages of oocyte maturation, exogenous expression of MEIKIN was employed, which showed that suppression of MEIKIN degradation resulted in chromosome misalignment, cyclin B1 and Securin degradation failure, and MI arrest through a spindle assembly checkpoint (SAC)-independent mechanism. Exogenous expression of MEIKIN also inhibited metaphase II (MII) exit and early embryo development. These results indicate that proper MEIKIN expression level and its C-terminal phosphorylation by PLK1 are critical for regulating the metaphase-anaphase transition in meiotic oocyte. The findings of this study are important for understanding the regulation of chromosome segregation and the prevention meiotic abnormality.

SRSF2 is required for mRNA splicing during spermatogenesis.

BACKGROUND: RNA splicing plays significant roles in fundamental biological activities. However, our knowledge about the roles of alternative splicing and underlying mechanisms during spermatogenesis is limited. RESULTS: Here, we report that Serine/arginine-rich splicing factor 2 (SRSF2), also known as SC35, plays critical roles in alternative splicing and male reproduction. Male germ cell-specific deletion of Srsf2 by Stra8-Cre caused complete infertility and defective spermatogenesis. Further analyses revealed that deletion of Srsf2 disrupted differentiation and meiosis initiation of spermatogonia. Mechanistically, by combining RNA-seq data with LACE-seq data, we showed that SRSF2 regulatory networks play critical roles in several major events including reproductive development, spermatogenesis, meiotic cell cycle, synapse organization, DNA recombination, chromosome segregation, and male sex differentiation. Furthermore, SRSF2 affected expression and alternative splicing of Stra8, Stag3 and Atr encoding critical factors for spermatogenesis in a direct manner. CONCLUSIONS: Taken together, our results demonstrate that SRSF2 has important functions in spermatogenesis and male fertility by regulating alternative splicing.

Maternal SMC2 is essential for embryonic development via participating chromosome condensation in mice.

During the oocyte growth, maturation and zygote development, chromatin structure keeps changing to regulate different nuclear activities. Here, we reported the role of SMC2, a core component of condensin complex, in oocyte and embryo development. Oocyte-specific conditional knockout of SMC2 caused female infertility. In the absence of SMC2, oocyte meiotic maturation and ovulation occurred normally, but chromosome condensation showed defects and DNA damages were accumulated in oocytes. The pronuclei were abnormally organized and micronuclei were frequently observed in fertilized eggs, their activity was impaired, and embryo development was arrested at the one-cell stage, suggesting that maternal SMC2 is essential for embryonic development.

Mitochondrial E3 ubiquitin ligase MARCH5 is required for mouse oocyte meiotic maturation†.

As the most abundant organelles in oocytes, mitochondria play an important role in maintaining oocyte quality. Here, we report that March5, encoding a mitochondrial ubiquitin ligase that promotes mitochondrial elongation, plays a critical role in mouse oocyte meiotic maturation via regulating mitochondrial function. The subcellular localization of MARCH5 was similar to the mitochondrial distribution during mouse oocyte meiotic progression. Knockdown of March5 caused decreased ratios of the first polar body extrusion. March5-siRNA injection resulted in oocyte mitochondrial dysfunctions, manifested by increased reactive oxygen species, decreased ATP content as well as decreased mitochondrial membrane potential, leading to reduced ability of spindle formation and an increased ratio of kinetochore-microtubule detachment. Further study showed that the continuous activation of the spindle assembly checkpoint and the failure of Cyclin B1 degradation caused MI arrest and first polar body (PB1) extrusion failure in March5 knockdown oocytes. Taken together, our results demonstrated that March5 plays an essential role in mouse oocyte meiotic maturation, possibly via regulation of mitochondrial function and/or ubiquitination of microtubule dynamics- or cell cycle-regulating proteins.

MAPRE2 regulates the first meiotic progression in mouse oocytes.

Microtubule plus-end tracking proteins (+TIPs) associate with growing microtubule plus ends and control microtubule dynamics and interactions with different cellular structures during cell division, cell migration and morphogenesis. Microtubule-associated RP/EB family member 2 (MAPRE2/EB2) is a highly conserved core component of +TIPs networks, but whether this molecule is required for mammalian meiotic progression is unknown. In this study, we investigated the expression and function of MAPRE2 during oocyte maturation. Our results showed that MAPRE2 was consistently expressed from germinal vesicle (GV) to metaphase II (MII) stages and that MAPRE2 was distributed in the cytoplasm of oocytes at GV stage and along the spindle at metaphase I (MI) and MII stages. Small interfering RNA-mediated knockdown of Mapre2 severely impaired microtubule stability, kinetochore-microtubule attachment, and chromosome alignment and subsequently caused spindle assembly checkpoint (SAC) activation and cyclin B1 nondegradation, leading to failure of chromosome segregation and first polar body extrusion. This study demonstrates for the first time that MAPRE2 plays an important role during mouse oocyte meiosis.

Effects of titanium dioxide microparticles and nanoparticles on cytoskeletal organization, cell adhesion, migration, and proliferation in human gingival fibroblasts in the presence of lipopolysaccharide.

BACKGROUND AND OBJECTIVE: Titanium wear particles may participate in the etiology of peri-implantitis. However, the influence of titanium wear particles on biological behavior of human gingival fibroblasts (HGFs) in the presence of LPS is still not clear. The present study demonstrated the effects of titanium dioxide micro- and nanoparticles (TiO2  MPs and NPs) on HGF cell viability, cytoskeletal organization, adhesion, migration, and proliferation in vitro, and LPS was used to mimic the in vivo condition. METHODS: Primary HGFs were treated with TiO2 MPs (primary particle size <5 µm, 0.1 mg/ml) and NPs (primary particle size <100 nm, 0.1 mg/ml) with or without 1 µg/ml LPS. The effects of TiO2 MPs and NPs on HGFs cell viability was measured by CCK-8 assay. The proliferation of HGF was detected by Ki67 nuclear staining. The confocal laser scanning microscope (CLSM) was used to detect the internalization of TiO2 MPs and NPs in HGFs as well as the arrangement of F-actin, vinculin, and vimentin organization. Wound healing assay and transwell assay were performed to measure the migration of HGFs induced by TiO2 MPs and NPs. Cell adhesion was measured using fibronectin-coated plates. The relative mRNA and protein expression of adhesion relative protein such as focal adhesion kinase (FAK), fibronectin (FN), and type I collagen (COL1) were measured using quantitative RT-PCR and western blot analysis. One-way analysis of variance (ANOVA) and Student's t-test were used to analyze the statistical significance, and p < .05 was considered statistically significant. RESULTS: TiO2 NPs significantly inhibited HGF cell viability, proliferation, and migration compared with TiO2 MPs group and control group. Compared with control group (2.64 ± 0.09), the mean absorbance of the cells in 1 mg/ml TiO2 MPs group and 0.25 mg/ml TiO2 NPs group were significantly decreased to 1.93 ± 0.33 (p < .05) and 2.22 ± 0.18 (p < .01), respectively. The cytoskeleton disruption was found in TiO2 NPs group. The mRNA and protein expression were significantly downregulated by TiO2 NPs. Furthermore, both TiO2 NPs and MPs induced more adverse effects on HGFs in the presence of LPS. CONCLUSION: Our results indicate that TiO2 NPs but not TiO2 MPs significantly disrupt the cytoskeletal organization and inhibited cell adhesion, migration, and proliferation of HGFs. However, in the presence of LPS, TiO2 MPs, and TiO2 NPs enhance these negative effects in HGFs. Titanium wear particles are probably involved in the initiation and progression of peri-implant diseases.

PTHrP promotes development of mouse preimplantation embryos through the AKT/cyclin D1 pathway and nuclear translocation of HDAC4.

Parathyroid hormone-related protein (PTHrP), the main cause of humoral hypercalcemia in malignancies, promotes cell proliferation and delays terminal cell maturation during embryonic development. Our previous study reported that PTHrP plays important roles in blastocyst formation, pluripotency gene expression, and histone acetylation during mouse preimplantation embryonic development. In this study, we further investigated the mechanism of preimplantation embryonic development regulated by PTHrP. Our results showed that Pthrp depletion decreased both the developmental rate of embryos at the cleavage stage and the cell number of morula-stage embryos. Pthrp-depleted embryos had significantly decreased levels of cyclin D1, phospho (p)-AKT (Thr308) and E2F1. However, Pthrp depletion did not cause significant changes in CDK4, ß-catenin or RUNX2 expression. In addition, our results indicated that Pthrp depletion promoted HDAC4 translocation from the cytoplasm to the nucleus in cleavage-stage embryos by stimulating the activity of protein phosphatase 2A (PP2A), which resulted in dephosphorylation of HDAC4. Taken together, these results suggest that PTHrP regulates cleavage division progression and blastocyst formation through the AKT/cyclin D1 pathway and that PTHrP modulates histone acetylation patterns through nuclear translocation of HDAC4 via PP2A-dependent HDAC4 dephosphorylation during preimplantation embryonic development in mice.

Nuclear and cytoplasmic quality of oocytes derived from serum-free culture of secondary follicles in vitro.

In vitro culture of follicles is a promising technology to generate large quantities of mature oocytes and it could offer a novel option of assisted reproductive technologies. Here we described a 2-dimensional follicular serum-free culture system with 3-dimensional effect that can make secondary follicles develop into antral follicles (78.52%), generating developmentally mature oocytes in vitro (66.45%). The oocytes in this serum-free system completed the first meiosis; spindle assembly and chromosome congression in most oocytes matured from follicular culture were normal. However, these oocytes showed significantly lower activation and embryonic development rates, and their ability to produce Ca2+ oscillations was also lower in response to parthenogenetic activation, after which a 2-cell embryonic developmental block occurred. Oocytes matured from follicular culture displayed increased abnormal mitochondrial distribution and increased reactive oxygen species levels when compared to in vivo matured oocytes. These data are important for understanding the reasons for reduced developmental potential of oocytes matured from follicular culture, and for further improving the cultivation system.

Cell division cycle 23 is required for mouse oocyte meiotic maturation.

Precise regulation of chromosome segregation during oocyte meiosis is of vital importance to mammalian reproduction. Anaphase promoting complex/cyclosome (APC/C) is reported to play an important role in metaphase-to-anaphase transition. Here we report that cell division cycle 23 (Cdc23, also known as APC8) plays a critical role in regulating the oocyte chromosome separation. Cdc23 localized on the meiotic spindle, and microinjection of Cdc23 siRNA caused decreased ratios of metaphase-to-anaphase transition. Loss of Cdc23 resulted in abnormal spindles, misaligned chromosomes, errors of homologous chromosome segregation, and production of aneuploid oocytes. Further study showed that inactivation of spindle assembly checkpoint and degradation of Cyclin B1 and securin were disturbed after Cdc23 knockdown. Furthermore, we found that inhibiting spindle assembly checkpoint protein Msp1 partly rescued the decreased polar body extrusion and reduced the accumulation of securin in Cdc23 knockdown oocytes. Taken together, our data demonstrate that Cdc23 is required for the chromosome segregation through regulating the spindle assembly checkpoint activity, and cyclin B1 and securin degradation in meiotic mouse oocytes.

Degradation of Ccnb3 is essential for maintenance of MII arrest in oocyte.

Before fertilization, ovulated mammalian oocytes are arrested at the metaphase of second meiosis (MII), which is maintained by the so-called cytostatic factor (CSF). It is well known that the continuous synthesis and accumulation of cyclin B is critical for maintaining the CSF-mediated MII arrest. Recent studies by us and others have shown that Ccnb3 is required for the metaphase-to-anaphase transition during the first meiosis of mouse oocytes, but whether Ccnb3 plays a role in MII arrest and exit remains unknown. Here, we showed that the protein level of Ccnb3 gradually decreased during oocyte meiotic maturation, and exogenous expression of Ccnb3 led to release of MII arrest, degradation of securin, separation of sister chromatids, extrusion of the second polar body (PB2), and finally entry into interphase. These phenotypes could be rescued by inhibition of Wee1B or CDK2. Our results indicate that Ccnb3 plays a critical regulatory role in MII arrest and exit in mouse oocytes.

Deletion of BAF250a affects oocyte epigenetic modifications and embryonic development.

BRG1-associated factor 250a (BAF250a) is a component of the SWI/SNF adenosine triphosphate-dependent chromatin remodeling complex, which has been shown to control chromatin structure and transcription. BAF250a was reported to be a key component of the gene regulatory machinery in embryonic stem cells controlling self-renewal, differentiation, and cell lineage decisions. Here we constructed Baf250aF/F ;Gdf9-cre (Baf250aCKO ) mice to specifically delete BAF250a in oocytes to investigate the role of maternal BAF250a in female germ cells and embryo development. Our results showed that BAF250a deletion did not affect folliculogenesis, ovulation, and fertilization, but it caused late embryonic death. RNA sequencing analysis showed that the expression of genes involved in cell proliferation and differentiation, tissue morphogenesis, histone modification, and nucleosome remodeling were perturbed in Baf250aCKO MII oocytes. We showed that covalent histone modifications such as H3K27me3 and H3K27ac were also significantly affected in oocytes, which may reduce oocyte quality and lead to birth defects. In addition, the DNA methylation level of Igf2r, Snrpn, and Peg3 differentially methylated regions was decreased in Baf250aCKO oocytes. Quantitative real-time polymerase chain reaction analysis showed that the relative messenger RNA (mRNA) expression levels of Igf2r and Snrpn were significantly increased. The mRNA expression level of Dnmt1, Dnmt3a, Dnmt3l, and Uhrf1 was decreased, and the protein expression in these genes was also reduced, which might be the cause for impaired imprinting establishment. In conclusion, our results demonstrate that BAF250a plays an important role in oocyte transcription regulation, epigenetic modifications, and embryo development.

Dimethyl Sulfoxide Wet-bonding Technique May Improve the Quality of Dentin Bonding.

PURPOSE: To investigate the effect of the dimethyl-sulfoxide wet-bonding technique on composite-dentin bonds and to explore its potential mechanism. MATERIALS AND METHODS: Thirty human third molars were segmented, ground, etched, and randomly divided into three groups according to the following pretreatments: 1. water; 2. ethanol; 3. 50% (v/v) dimethyl sulfoxide (DMSO). Then, Single Bond 2 was applied and composite buildups were constructed. After 24 h of water storage or 10,000 cycles of thermocycling, the microtensile bond strength (MTBS) and nanoleakage were measured. Contact angle measurement, Masson's trichrome staining, and in situ zymography were used to explore the possible action mechanism of DMSO on adhesive-dentin interfaces. RESULTS: DMSO pretreatment prevented the decline of thermocycled MTBS (p < 0.05) without affecting the immediate MTBS (p > 0.05) compared to the water wet-bonded group. Nanoleakage expression in the thermocycled DMSO wet-bonded group was also less than that in the thermocycled water-wet group (p < 0.05). Moreover, DMSO decreased the contact angle of the dentin surfaces (p < 0.05), reduced the amount of collagen exposure (p < 0.05), and decreased the collagenolytic activity in the hybrid layer (p < 0.05). CONCLUSION: The 50% DMSO pretreatment was effective in increasing the wettability of the etched dentin surface, promoting the penetration of the adhesive monomer, and enhancing the stability of the dentin collagen at the adhesive- dentin interface. All these changes may lead to higher quality dentin bonds, suggesting that DMSO wet bonding is a viable alternative to improve the durability of dentin bonding.

Comparative Evaluation of Mechanical Properties and Color Stability of Dental Resin Composites for Chairside Provisional Restorations.

Resin composites have become the preferred choice for chairside provisional dental restorations. However, these materials may undergo discoloration, changes in surface roughness, and mechanical properties with aging in the oral cavity, compromising the aesthetics, functionality, and success of dental restorations. To investigate the color and mechanical stability of chairside provisional composite resins, this study evaluated the optical, surface, and mechanical properties of four temporary restoration resin materials before and after aging, stimulated by thermal cycling in double-distilled water. Measurements, including CIE LAB color analysis, three-point bending test, nanoindentation, scanning electron microscopy (SEM), and atomic force microscopy (AFM), were conducted (n = 15). Results showed significant differences among the materials in terms of optical, surface, and mechanical properties. Revotek LC (urethane dimethacrylate) demonstrated excellent color stability (ΔE00 = 0.53-Black/0.32-White), while Artificial Teeth Resin (polymethyl methacrylate) exhibited increased mechanical strength with aging (p < 0.05, FS = 68.40 MPa-non aging/87.21 MPa-aging). Structur 2 SC (Bis-acrylic) and Luxatemp automix plus (methyl methacrylate bis-acrylate) demonstrated moderate stability in optical and mechanical properties (Structur 2 SC: ΔE00 = 1.97-Black/1.38-White FS = 63.20 MPa-non aging/50.07 MPa-aging) (Luxatemp automix plus: ΔE00 = 2.49-Black/1.77-White FS = 87.72 MPa-non aging/83.93 MPa-aging). These results provide important practical guidance for clinical practitioners, as well as significant theoretical and experimental bases for the selection of restorative composite resins.

A telomere-to-telomere reference genome of ficus (Ficus hispida) provides new insights into sex determination.

A high-quality reference genome is indispensable for resolving biologically essential traits. Ficus hispida is a dioecious plant. A complete Ficus reference genome will be crucial for understanding their sex evolution and important biological characteristics, such as aerial roots, mutualistic symbiosis with ficus-wasps, and fruiting from old stems. Here, we generated a telomere-to-telomere (T2T) genome for F. hispida using PacBio HiFi and Oxford Nanopore Ultra-long sequencing technologies. The genome contiguity and completeness has shown improvement compared with the previously released genome, with the annotation of six centromeres and 28 telomeres. We have refined our previously reported 2-Mb male-specific region into a 7.2-Mb genomic region containing 51 newly predicted genes and candidate sex-determination genes AG2 and AG3. Many of these genes showed extremely low expression, likely attributed to hypermethylation in the gene body and promoter regions. Gene regulatory networks (GRNs) revealed that AG2 and AG3 are related to the regulation of stamen development in male flowers, while the AG1 gene is responsible for regulating female flowers' defense responses and secondary metabolite processes. Comparative analysis of GRNs showed that the NAC, WRKY, and MYB transcription factor families dominate the female GRN, whereas the MADS and MYB transcription factor families are prevalent in the male GRN.

The transgenerational effects of maternal low-protein diet during lactation on offspring.

Environmental factors such as diet and lifestyle can influence the health of both mothers and offspring. However, its transgenerational transmission and underlying mechanisms remain largely unknown. Here, using a maternal lactation-period low-protein diet (LPD) mouse model, we show that maternal LPD during lactation causes decreased survival and stunted growth, significantly reduces ovulation and litter size, and alters the gut microbiome in the female LPD-F1 offspring. The transcriptome of LPD-F1 metaphase II (MII) oocytes shows that differentially expressed genes are enriched in female pregnancy and multiple metabolic processes. Moreover, maternal LPD causes early stunted growth and impairs metabolic health, which is transmitted over two generations. The methylome alteration of LPD-F1 oocytes can be partly transmitted to the F2 oocytes. Together, our results reveal that LPD during lactation transgenerationally affects offspring health, probably via oocyte epigenetic changes.

Sequencing and Assembly of Polyploid Genomes.

Polyploidy has been observed throughout major eukaryotic clades and has played a vital role in the evolution of angiosperms. Recent polyploidizations often result in highly complex genome structures, posing challenges to genome assembly and phasing. Recent advances in sequencing technologies and genome assembly algorithms have enabled high-quality, near-complete chromosome-level assemblies of polyploid genomes. Advances in novel sequencing technologies include highly accurate single-molecule sequencing with HiFi reads, chromosome conformation capture with Hi-C technique, and linked reads sequencing. Additionally, new computational approaches have also significantly improved the precision and reliability of polyploid genome assembly and phasing, such as HiCanu, hifiasm, ALLHiC, and PolyGembler. Herein, we review recently published polyploid genomes and compare the various sequencing, assembly, and phasing approaches that are utilized in these genome studies. Finally, we anticipate that accurate and telomere-to-telomere chromosome-level assembly of polyploid genomes could ultimately become a routine procedure in the near future.

5mC DNA methylation modification-mediated regulation in tissue functional differentiation and important flavor substance synthesis of tea plant (Camellia sinensis L.).

In plants, 5mC DNA methylation is an important and conserved epistatic mark involving genomic stability, gene transcriptional regulation, developmental regulation, abiotic stress response, metabolite synthesis, etc. However, the roles of 5mC DNA methylation modification (5mC methylation) in tea plant growth and development (in pre-harvest processing) and flavor substance synthesis in pre- and post-harvest processing are unknown. We therefore conducted a comprehensive methylation analysis of four key pre-harvest tissues (root, leaf, flower, and fruit) and two processed leaves during oolong tea post-harvest processing. We found that differential 5mC methylation among four key tissues is closely related to tissue functional differentiation and that genes expressed tissue-specifically, responsible for tissue-specific functions, maintain relatively low 5mC methylation levels relative to non-tissue-specifically expressed genes. Importantly, hypomethylation modifications of CsAlaDC and TS/GS genes in roots provided the molecular basis for the dominant synthesis of theanine in roots. In addition, integration of 5mC DNA methylationomics, metabolomics, and transcriptomics of post-harvest leaves revealed that content changes in flavor metabolites during oolong tea processing were closely associated with transcription level changes in corresponding metabolite synthesis genes, and changes in transcript levels of these important synthesis genes were strictly regulated by 5mC methylation. We further report that some key genes during processing are regulated by 5mC methylation, which can effectively explain the content changes of important aroma metabolites, including α-farnesene, nerolidol, lipids, and taste substances such as catechins. Our results not only highlight the key roles of 5mC methylation in important flavor substance synthesis in pre- and post-harvest processing, but also provide epimutation-related gene targets for future improvement of tea quality or breeding of whole-tissue high-theanine varieties.

SRSF1-mediated alternative splicing is required for spermatogenesis.

Alternative splicing (AS) plays significant roles in a multitude of fundamental biological activities. AS is prevalent in the testis, but the regulations of AS in spermatogenesis is only little explored. Here, we report that Serine/arginine-rich splicing factor 1 (SRSF1) plays critical roles in alternative splicing and male reproduction. Male germ cell-specific deletion of Srsf1 led to complete infertility by affecting spermatogenesis. Mechanistically, by combining RNA-seq data with LACE-seq data, we showed that SRSF1 affected the AS of Stra8 in a direct manner and Dazl, Dmc1, Mre11a, Syce2 and Rif1 in an indirect manner. Our findings demonstrate that SRSF1 has crucial functions in spermatogenesis and male fertility by regulating alternative splicing.

The highly improved genome of Ixodes scapularis with X and Y pseudochromosomes.

Ixodes scapularis, the black-legged tick, is the principal vector of the Lyme disease spirochete, Borrelia burgdorferi, and is responsible for most of the ∼470,000 estimated Lyme disease cases annually in the USA. Ixodes scapularis can transmit six additional pathogens of human health significance. Because of its medical importance, I. scapularis was the first tick genome to be sequenced and annotated. However, the first assembly, I. scapularis Wikel (IscaW), was highly fragmented because of the technical challenges posed by the long, repetitive genome sequences characteristic of arthropod genomes and the lack of long-read sequencing techniques. Although I. scapularis has emerged as a model for tick research because of the availability of new tools such as embryo injection and CRISPR-Cas9-mediated gene editing yet the lack of chromosome-scale scaffolds has slowed progress in tick biology and the development of tools for their control. Here we combine diverse technologies to produce the I. scapularis Gulia-Nuss (IscGN) genome assembly and gene set. We used DNA from eggs and male and female adult ticks and took advantage of Hi-C, PacBio HiFi sequencing, and Illumina short-read sequencing technologies to produce a chromosome-level assembly. In this work, we present the predicted pseudochromosomes consisting of 13 autosomes and the sex pseudochromosomes: X and Y, and a markedly improved genome annotation compared with the existing assemblies and annotations.