Interspecies Chimerism with Mammalian Pluripotent Stem Cells.

Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.

A Surveillance Mechanism Ensures Repair of DNA Lesions during Zygotic Reprogramming.

Sexual reproduction culminates in a totipotent zygote with the potential to produce a whole organism. Sperm chromatin reorganization and epigenetic reprogramming that alter DNA and histone modifications generate a totipotent embryo. Active DNA demethylation of the paternal genome has been proposed to involve base excision and DNA repair-based mechanisms. The nature and consequence of DNA lesions generated during reprogramming are not known. Using mouse genetics and chemical biology, we discovered that Tet3-dependent zygotic reprogramming generates paternal DNA lesions that are monitored by a surveillance mechanism. In vivo structure-function rescue assays revealed that cohesin-dependent repair of paternal DNA lesions prevents activation of a Chk1-dependent checkpoint that delays mitotic entry. Culturing conditions affect checkpoint stringency, which has implications for human in vitro fertilization. We propose the zygotic checkpoint senses DNA lesions generated during paternal DNA demethylation and ensures reprogrammed loci are repaired before mitosis to prevent chromosome fragmentation, embryo loss, and infertility.

Optimized PAR-2 RING dimerization mediates cooperative and selective membrane binding for robust cell polarity.

Cell polarity networks are defined by quantitative features of their constituent feedback circuits, which must be tuned to enable robust and stable polarization, while also ensuring that networks remain responsive to dynamically changing cellular states and/or spatial cues during development. Using the PAR polarity network as a model, we demonstrate that these features are enabled by the dimerization of the polarity protein PAR-2 via its N-terminal RING domain. Combining theory and experiment, we show that dimer affinity is optimized to achieve dynamic, selective, and cooperative binding of PAR-2 to the plasma membrane during polarization. Reducing dimerization compromises positive feedback and robustness of polarization. Conversely, enhanced dimerization renders the network less responsive due to kinetic trapping of PAR-2 on internal membranes and reduced sensitivity of PAR-2 to the anterior polarity kinase, aPKC/PKC-3. Thus, our data reveal a key role for a dynamically oligomeric RING domain in optimizing interaction affinities to support a robust and responsive cell polarity network, and highlight how optimization of oligomerization kinetics can serve as a strategy for dynamic and cooperative intracellular targeting.

Polarity establishment in the plant zygote at a glance.

The complex structures of multicellular organisms originate from a unicellular zygote. In most angiosperms, including Arabidopsis thaliana, the zygote is distinctly polar and divides asymmetrically to produce an apical cell, which generates the aboveground part of the plant body, and a basal cell, which generates the root tip and extraembryonic suspensor. Thus, zygote polarity is pivotal for establishing the apical-basal axis running from the shoot apex to the root tip of the plant body. The molecular mechanisms and spatiotemporal dynamics behind zygote polarization remain elusive. However, advances in live-cell imaging of plant zygotes have recently made significant insights possible. In this Cell Science at a Glance article and the accompanying poster, we summarize our understanding of the early steps in apical-basal axis formation in Arabidopsis, with a focus on de novo transcriptional activation after fertilization and the intracellular dynamics leading to the first asymmetric division of the zygote.

Dynamic metabolism during early mammalian embryogenesis.

Dynamic metabolism is exhibited by early mammalian embryos to support changing cell fates during development. It is widely acknowledged that metabolic pathways not only satisfy cellular energetic demands, but also play pivotal roles in the process of cell signalling, gene regulation, cell proliferation and differentiation. Recently, various new technological advances have been made in metabolomics and computational analysis, deepening our understanding of the crucial role of dynamic metabolism during early mammalian embryogenesis. In this Review, we summarize recent studies on oocyte and embryo metabolism and its regulation, with a particular focus on its association with key developmental events such as fertilization, zygote genome activation and cell fate determination. In addition, we discuss the mechanisms of certain metabolites that, in addition to serving as energy sources, contribute to epigenetic modifications.

CHK1 controls zygote pronuclear envelope breakdown by regulating F-actin through interacting with MICAL3.

CHK1 mutations could cause human zygote arrest at the pronuclei stage, a phenomenon that is not well understood at the molecular level. In this study, we conducted experiments where pre-pronuclei from zygotes with CHK1 mutation were transferred into the cytoplasm of normal enucleated fertilized eggs. This approach rescued the zygote arrest caused by the mutation, resulting in the production of a high-quality blastocyst. This suggests that CHK1 dysfunction primarily disrupts crucial biological processes occurring in the cytoplasm. Further investigation reveals that CHK1 mutants have an impact on the F-actin meshwork, leading to disturbances in pronuclear envelope breakdown. Through co-immunoprecipitation and mass spectrometry analysis of around 6000 mouse zygotes, we identified an interaction between CHK1 and MICAL3, a key regulator of F-actin disassembly. The gain-of-function mutants of CHK1 enhance their interaction with MICAL3 and increase MICAL3 enzymatic activity, resulting in excessive depolymerization of F-actin. These findings shed light on the regulatory mechanism behind pronuclear envelope breakdown during the transition from meiosis to the first mitosis in mammals.

Donor template delivery by recombinant adeno-associated virus for the production of knock-in mice.

BACKGROUND: The ability of recombinant adeno-associated virus to transduce preimplantation mouse embryos has led to the use of this delivery method for the production of genetically altered knock-in mice via CRISPR-Cas9. The potential exists for this method to simplify the production and extend the types of alleles that can be generated directly in the zygote, obviating the need for manipulations of the mouse genome via the embryonic stem cell route. RESULTS: We present the production data from a total of 13 genetically altered knock-in mouse models generated using CRISPR-Cas9 electroporation of zygotes and delivery of donor repair templates via transduction with recombinant adeno-associated virus. We explore the efficiency of gene targeting at a total of 12 independent genetic loci and explore the effects of allele complexity and introduce strategies for efficient identification of founder animals. In addition, we investigate the reliability of germline transmission of the engineered allele from founder mice generated using this methodology. By comparing our production data against genetically altered knock-in mice generated via gene targeting in embryonic stem cells and their microinjection into blastocysts, we assess the animal cost of the two methods. CONCLUSIONS: Our results confirm that recombinant adeno-associated virus transduction of zygotes provides a robust and effective delivery route for donor templates for the production of knock-in mice, across a range of insertion sizes (0.9-4.7 kb). We find that the animal cost of this method is considerably less than generating knock-in models via embryonic stem cells and thus constitutes a considerable 3Rs reduction.

CRISPR-mediated megabase-scale transgene de-duplication to generate a functional single-copy full-length humanized DMD mouse model.

BACKGROUND: The development of sequence-specific precision treatments like CRISPR gene editing therapies for Duchenne muscular dystrophy (DMD) requires sequence humanized animal models to enable the direct clinical translation of tested strategies. The current available integrated transgenic mouse model containing the full-length human DMD gene, Tg(DMD)72Thoen/J (hDMDTg), has been found to have two copies of the transgene per locus in a tail-to-tail orientation, which does not accurately simulate the true (single) copy number of the DMD gene. This duplication also complicates analysis when testing CRISPR therapy editing outcomes, as large genetic alterations and rearrangements can occur between the cut sites on the two transgenes. RESULTS: To address this, we performed long read nanopore sequencing on hDMDTg mice to better understand the structure of the duplicated transgenes. Following that, we performed a megabase-scale deletion of one of the transgenes by CRISPR zygotic microinjection to generate a single-copy, full-length, humanized DMD transgenic mouse model (hDMDTgSc). Functional, molecular, and histological characterisation shows that the single remaining human transgene retains its function and rescues the dystrophic phenotype caused by endogenous murine Dmd knockout. CONCLUSIONS: Our unique hDMDTgSc mouse model simulates the true copy number of the DMD gene, and can potentially be used for the further generation of DMD disease models that would be better suited for the pre-clinical assessment and development of sequence specific CRISPR therapies.

Transcriptional integration of paternal and maternal factors in the Arabidopsis zygote.

In many plants, the asymmetric division of the zygote sets up the apical-basal axis of the embryo. Unlike animals, plant zygotes are transcriptionally active, implying that plants have evolved specific mechanisms to control transcriptional activation of patterning genes in the zygote. In Arabidopsis, two pathways have been found to regulate zygote asymmetry: YODA (YDA) mitogen-activated protein kinase (MAPK) signaling, which is potentiated by sperm-delivered mRNA of the SHORT SUSPENSOR (SSP) membrane protein, and up-regulation of the patterning gene WOX8 by the WRKY2 transcription factor. How SSP/YDA signaling is transduced into the nucleus and how these pathways are integrated have remained elusive. Here we show that paternal SSP/YDA signaling directly phosphorylates WRKY2, which in turn leads to the up-regulation of WOX8 transcription in the zygote. We further discovered the transcription factors HOMEODOMAIN GLABROUS11/12 (HDG11/12) as maternal regulators of zygote asymmetry that also directly regulate WOX8 transcription. Our results reveal a framework of how maternal and paternal factors are integrated in the zygote to regulate embryo patterning.

DNA- and Selectable-Marker-Free Genome-Editing System Using Zygotes from Recalcitrant Maize Inbred B73.

Genome-editing tools such as the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system have become essential tools for increasing the efficiency and accuracy of plant breeding. Using such genome-editing tools on maize, one of the most important cereal crops of the world, will greatly benefit the agriculture and the mankind. Conventional genome-editing methods typically used for maize involve insertion of a Cas9-guide RNA expression cassette and a selectable marker in the genome DNA; however, using such methods, it is essential to eliminate the inserted DNA cassettes to avoid legislative concerns on gene-modified organisms. Another major hurdle for establishing an efficient and broadly applicable DNA-free genome-editing system for maize is presented by recalcitrant genotypes/cultivars, since cell/tissue culture and its subsequent regeneration into plantlets are crucial for producing transgenic and/or genome-edited maize. In this study, to establish a DNA-free genome-editing system for recalcitrant maize genotypes/cultivars, Cas9-gRNA ribonucleoproteins were directly delivered into zygotes isolated from the pollinated flowers of the maize-B73 cultivar. The zygotes successfully developed and were regenerated into genome-edited plantlets by co-culture with phytosulfokine, a peptide phytohormone. The method developed herein made it possible to obtain DNA- and selectable-marker-free genome-edited recalcitrant maize genotypes/cultivars with high efficiency. This method can advance the molecular breeding of maize and other important cereals, regardless of their recalcitrant characteristics.

Maternal Smc3 protects the integrity of the zygotic genome through DNA replication and mitosis.

Aneuploidy is frequently observed in oocytes and early embryos, begging the question of how genome integrity is monitored and preserved during this crucial period. SMC3 is a subunit of the cohesin complex that supports genome integrity, but its role in maintaining the genome during this window of mammalian development is unknown. We discovered that, although depletion of Smc3 following meiotic S phase in mouse oocytes allowed accurate meiotic chromosome segregation, adult females were infertile. We provide evidence that DNA lesions accumulated following S phase in SMC3-deficient zygotes, followed by mitosis with lagging chromosomes, elongated spindles, micronuclei, and arrest at the two-cell stage. Remarkably, although centromeric cohesion was defective, the dosage of SMC3 was sufficient to enable embryogenesis in juvenile mutant females. Our findings suggest that, despite previous reports of aneuploidy in early embryos, chromosome missegregation in zygotes halts embryogenesis at the two-cell stage. Smc3 is a maternal gene with essential functions in the repair of spontaneous damage associated with DNA replication and subsequent chromosome segregation in zygotes, making cohesin a key protector of the zygotic genome.

Electroporation-mediated genome editing in vitrified/warmed porcine zygotes obtained in vitro.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) system is the most efficient and widely used technology for genome editing in all sorts of organisms, including livestock animals. Here, we examined the feasibility of CRISPR/Cas9-derived genome editing (GE) in vitrified porcine zygotes, where the flexible planning of experiments in time and space is expected. OCT4 and CD46 genes were targeted, and the Cas9/sgRNA ribonucleoprotein complexes (RNP) were electroporated into zygotes at 2 h after warming. Vitrification or GE alone did not significantly reduce the developmental rates to the blastocyst stage. However, vitrification followed by GE significantly reduced blastocyst development. Sequencing analysis of the resultant blastocysts revealed efficient GE for both OCT4 (nonvitrified: 91.0%, vitrified: 95.1%) and CD46 (nonvitrified: 94.5%, vitrified: 93.2%), with no significant difference among them. Immunocytochemical analysis showed that GE-blastocysts lacked detectable proteins. They were smaller in size, and the cell numbers were significantly reduced compared with the control (p < 0.01). Finally, we demonstrated that double GE efficiently occurs (100%) when the OCT4-RNP and CD46-RNP are simultaneously introduced into zygotes after vitrification/warming. This is the first demonstration that vitrified porcine zygotes can be used in GE as efficiently as nonvitrified ones.

Effect of ejaculatory abstinence period on fertilization and clinical outcomes in ICSI cycles: a retrospective analysis.

RESEARCH QUESTION: Does ejaculatory abstinence impact fertilization outcomes in intracytoplasmic sperm injection (ICSI) cycles in infertile couples? DESIGN: This single-centre retrospective observational study included 6919 ICSI cycles from 2013 to 2022. The primary outcome was the assessment of oocyte fertilization, measured in terms of the rate of formation of two-pronuclear (2PN), 3PN and 1PN zygotes. Secondary outcomes were blastulation, cumulative positive ß-human chorionic gonadotrophin test and clinical pregnancy rates. Relationships between ejaculatory abstinence and fertilization outcomes, and ejaculatory abstinence and clinical outcomes were evaluated with multivariable analysis, including possible confounders. RESULTS: A positive association was observed between ejaculatory abstinence and semen sample volume (P < 0.001), sperm concentration (P < 0.001) and total motile sperm count (P < 0.001). No association was found between the 1PN zygote rate and ejaculatory abstinence (P = 0.97). Conversely, for each additional day of ejaculatory abstinence, the likelihood of obtaining 2PN zygotes from all inseminated oocytes decreased by 3% [adjusted odds ratio (aOR) 0.97, 95% CI 0.94-0.99], whilst the likelihood of obtaining 3PN zygotes from all inseminated oocytes increased significantly by 14% (aOR 1.14, 95% CI 1.07-1.22). No significant associations were found between ejaculatory abstinence and blastulation, cumulative pregnancy or miscarriage rates. CONCLUSIONS: A longer ejaculatory abstinence period significantly decreases the rate of 2PN zygotes, and increases the rate of 3PN zygotes without directly affect blastulation and pregnancy rates.

Chromatin structure in totipotent mouse early preimplantation embryos.

Totipotency refers to the ability of a single cell to give rise to all the different cell types in the body. Terminally differentiated germ cells (sperm and oocytes) undergo reprogramming, which results in the acquisition of totipotency in zygotes. Since the 1990s, numerous studies have focused on the mechanisms of totipotency. With the emergence of the concept of epigenetic reprogramming, which is important for the undifferentiated and differentiated states of cells, the epigenomes of germ cells and fertilized oocytes have been thoroughly analyzed. However, in early immunostaining studies, detailed epigenomic information was difficult to obtain. In recent years, the explosive development of next-generation sequencing has made it possible to acquire genome-wide information and the rise of genome editing has facilitated the analysis of knockout mice, which was previously difficult. In addition, live imaging can effectively analyze zygotes and 2-cell embryos, for which the number of samples is limited, and provides biological insights that cannot be obtained by other methods. In this review, the progress of our research using these advanced techniques is traced back from the present to its earliest years.

Genome editing of porcine zygotes via lipofection of two guide RNAs using a CRISPR/Cas9 system.

CRISPR/Cas9-based multiplex genome editing via electroporation is relatively efficient; however, lipofection is versatile because of its ease of use and low cost. Here, we aimed to determine the efficiency of lipofection in CRISPR/Cas9-based multiplex genome editing using growth hormone receptor (GHR) and glycoprotein alpha-galactosyltransferase 1 (GGTA1)-targeting guide RNAs (gRNAs) in pig zygotes. Zona pellucida-free zygotes were collected 10 h after in vitro fertilization and incubated with Cas9, gRNAs, and Lipofectamine 2000 (LP2000) for 5 h. In Experiment 1, we evaluated the mutation efficiency of gRNAs targeting either GHR or GGTA1 in zygotes transfected using LP2000 and cultured in 4-well plates. In Experiment 2, we examined the effects of the culture method on the development, mutation rate, and mutation efficiency of zygotes with simultaneouslydouble-edited GHR and GGTA1, cultured using 4-well (group culture) and 25-well plates (individual culture). In Experiment 3, we assessed the effect of additional GHR-targeted lipofection before and after simultaneous double gRNA-targeted lipofection on the mutation efficiency of edited embryos cultured in 25-well plates. No significant differences in mutation rates were observed between the zygotes edited with either gRNA. Moreover, the formation rate of blastocysts derived from GHR and GGTA1 double-edited zygotes was significantly increased in the 25-well plate culture compared to that in the 4-well plate culture. However, mutations were only observed in GGTA1 when zygotes were transfected with both gRNAs, irrespective of the culture method used. GHR mutations were detected only in blastocysts derived from zygotes subjected to GHR-targeted lipofection before simultaneous double gRNA-targeted lipofection. Overall, our results suggest that additional lipofection before simultaneous double gRNA-targeted lipofection induces additional mutations in the zygotes.

Coordinate Normalization of Live-Cell Imaging Data Reveals Growth Dynamics of the Arabidopsis Zygote.

Polarization of the zygote defines the body axis during plant development. In Arabidopsis (Arabidopsis thaliana), the zygote becomes polarized and elongates in the longitudinal direction, ultimately forming the apical-basal axis of the mature plant. Despite its importance, the mechanism for this elongation remains poorly understood. Based on live-cell imaging of the zygote, we developed new image analysis methods, referred to as coordinate normalization, that appropriately fix and align positions in an image, preventing fluctuation across a temporal sequence of images. Using these methods, we discovered that the zygote elongates only at its apical tip region, similar to tip-growing cells such as pollen tubes and root hairs. We also investigated the spatiotemporal dynamics of the apical tip contour of the zygote and observed that the zygote tip retains its isotropic, hemispherical apical shape during cell elongation. By looking at the elliptical fitting of the contour over time, we further discovered that the apical cell tip becomes thinner at first and then thickens, with a transient increase in growth speed that is followed by the first cell division. We performed the same series of analyses using root hairs and established that both the hemispherical tip shape and the changes in growth rate associated with changes in tip size are specific to the zygote. In summary, the Arabidopsis zygote undergoes directional elongation as a tip-growing cell, but its tip retains an unusual isotropic shape, and the manner of growth changes with the developmental stage.

Somatic embryo initiation by rice BABY BOOM1 involves activation of zygote-expressed auxin biosynthesis genes.

Plant embryogenesis results from the fusion of male and female gametes but can also be induced in somatic cells. The molecular pathways for embryo initiation are poorly understood, especially in monocots. In rice, the male gamete expressed BABY BOOM1 (OsBBM1) transcription factor functions as an embryogenic trigger in the zygote and can also promote somatic embryogenesis when ectopically expressed in somatic tissues. We used gene editing, transcriptome profiling, and chromatin immunoprecipitation to determine the molecular players involved in embryo initiation downstream of OsBBM1. We identify OsYUCCA (OsYUC) auxin biosynthesis genes as direct targets of OsBBM1. Unexpectedly, these OsYUC targets in zygotes are expressed only from the maternal genome, whereas the paternal genome exclusively provides functional OsBBM1 to initiate embryogenesis. Induction of somatic embryogenesis by exogenous auxin requires OsBBM genes and downstream OsYUC targets. Ectopic OsBBM1 initiates somatic embryogenesis without exogenous auxins but requires functional OsYUC genes. Thus, an OsBBM-OsYUC module is a key player for both somatic and zygotic embryogenesis in rice. Zygotic embryo initiation involves a partnership of male and female genomes, through which paternal OsBBM1 activates maternal OsYUC genes. In somatic embryogenesis, exogenous auxin triggers OsBBM1 expression, which then activates endogenous auxin biosynthesis OsYUC genes.

The destinies of human embryos reaching blastocyst stage between Day 4 and Day 7 diverge as early as fertilization.

STUDY QUESTION: What clinical and laboratory differences emerge from parallel direct comparison of embryos reaching the blastocyst stage between Days 4, 5, 6, and 7 (Days 4-7)? SUMMARY ANSWER: Increasing times to blastocyst formation are associated with a worse clinical outcome and perturbations in developmental patterns appear as early as the fertilization stage. WHAT IS KNOWN ALREADY: Previous evidence indicates that later times to blastocyst development are associated with a worse clinical outcome. However, the vast majority of these data concern Day 5 and Day 6 blastocysts, while Day 4 and Day 7 blastocysts remain less thoroughly investigated. In addition, studies comparing in parallel the developmental patterns and trajectories of Day 4-7 blastocysts are lacking. This leaves unanswered the question of when and how differences among such embryos emerge. Acquisition of such knowledge would significantly contribute to understanding the relative impact of intrinsic and extrinsic causes of embryo developmental kinetics and competence. STUDY DESIGN, SIZE, DURATION: This retrospective study involved time-lapse technology (TLT) monitoring of Day 4 (N = 70), Day 5 (N = 6147), Day 6 (N = 3243), and Day 7 (N = 149) blastocysts generated in 9450 ICSI cycles. Oocyte retrievals were carried out after clomiphene citrate-based minimal ovarian stimulation, between January 2020 and April 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: Couples included in the study presented with different diagnoses, mainly male factor and unexplained infertility. Cases involving cryopreserved gametes or surgically retrieved sperm were excluded. Microinjected oocytes were assessed by a combined TLT-culture system. Day 4-7 blastocyst groups were compared in terms of morphokinetics (pronuclear dynamics, cleavage patterns and timings, and embryo quality) and clinical outcome. Clinically usable blastocysts were cryopreserved and transferred in single vitrified-warmed blastocyst transfers (SVBT). MAIN RESULTS AND THE ROLE OF CHANCE: From 19 846 microinjected oocytes, 17 144 zygotes (86.4%) were obtained. Overall, the blastocyst development rate was 56.0%. Rates of blastocysts formation on Days 4, 5, 6, and 7 were 0.7%, 64.0%, 33.8%, and 1.6%, respectively. The average expanded blastocyst development times were 98.4 ± 0.4, 112.4 ± 0.1, 131.6 ± 0.1, and 151.2 ± 0.5 h in the Day 4-7 groups, respectively. Female age was positively associated with longer times to blastocyst development. Rates of both inner cell mass (ICM) and trophectoderm (TE) morphological grade A blastocysts were negatively associated with the day of blastocyst development (P < 0.0001). The differences in development times and intervals increased progressively until blastocyst expansion (P < 0.0001 for all development times). Strikingly, such differences were already markedly evident as early as the time of pronuclear fading (tPNf) (20.6 ± 0.3, 22.5 ± 0.0, 24.0 ± 0.0, 25.5 ± 0.3; Days 4-7, respectively; P < 0.0001). Rates of cleavage anomalies (tri-/multi-chotomous mitosis or rapid cleavage) occurring at the first or second/third division cycles were also positively associated with longer times to blastocyst development. Implantation, ongoing pregnancy, and live birth rates were progressively reduced with increasing blastocyst development times (P < 0.0001), even after stratification for maternal age. When controlled for female age, male age, number of previous embryo transfer cycles, morphological grade of the ICM and TE, and progesterone supplementation, the probabilities of implantation, clinical, and ongoing pregnancy and live birth were significantly decreased in Day 6 blastocysts in comparison to Day 5 blastocysts. Follow-up data on birth length, weight, and malformations were comparable among the four blastocyst groups. LIMITATIONS, REASONS FOR CAUTION: The study is limited by its retrospective design. Having been obtained from a single centre, the data require independent validation. WIDER IMPLICATIONS OF THE FINDINGS: This study extends previous data on the relation between time of blastocyst formation and clinical outcome. It also indicates that differences in developmental times and patterns of Day 4-7 blastocysts occur as early as the fertilization stage, possibly dictated by intrinsic gamete-derived factors. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the participating institutions. The authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Revisiting ZAR proteins: the understudied regulator of female fertility and beyond.

Putative RNA-binding proteins (RBPs), zygote arrested-1 (ZAR1), and ZAR2 (also known as ZAR1L), have been identified as maternal factors that mainly function in oogenesis and embryogenesis. Despite divergence in their spatio-temporal expression among species, the CxxC structure of the C-terminus of ZAR proteins is highly conserved and is reported to be the functional domain for the activity of the RBPs of ZAR proteins. In oocytes from Xenopus laevis and zebrafish, ZAR proteins have been reported to bind to maternal transcripts and inhibit translation in immature growing oocytes, whereas in fully grown mouse oocytes, they promote the translation during meiotic maturation. Thus, ZAR1 and ZAR2 may be required for the maternal-to-zygotic transition by stabilizing the maternal transcriptome in oocytes with partial functional redundancy. In addition, recent studies have suggested non-ovarian expression and function of ZAR proteins, particularly their involvement in tumorigenesis. ZAR proteins are potentially associated with tumor suppressors and can serve as epigenetically inactivated cancer biomarkers. In this review, studies on Zar1/2 are systematically summarized, and some issues that require discussion and further investigation are introduced as perspectives.

Successful production of offspring derived from mouse zygotes vitrified with carboxylated ε-poly-L-lysine and polyvinyl alcohol without serum.

The vitrification of zygotes is important for their use as donors for generating genome-edited mice. We previously reported the successful vitrification of mouse zygotes using carboxylated ε-poly-L-lysine (COOH-PLL). However, this vitrification solution contains fetal calf serum (FCS), which contains unknown factors and presents risks of pathogenic viral and microbial contamination. In this study, we examined whether polyvinyl alcohol (PVA) can be used as an alternative to FCS in vitrification solutions for mouse zygotes. When COOH-PLL was added to the vitrification solutions, zygotes vitrified with solutions containing 0.01% PVA (PV0.01) and those vitrified in a control solution containing FCS (75.6%) developed into blastocysts (78.4%). In addition, there were no significant differences in the ability to develop to term between the control solution (46.6%) and PV0.01 (44.1%) groups. In conclusion, we clearly demonstrated that PVA can replace FCS in our vitrification solution supplemented with COOH-PLL for mouse zygotes.