Could the presence and proportion of three/multiple pronuclei (3PN/MPN) zygotes indicate the cytoplasmic maturation state of oocyte cohort in conventional IVF patients?

Although abnormally fertilized zygotes with three or multiple pronuclei (3 PN/MPN) are commonly believed to be associated with improper maturation of the oocyte cytoplasm in conventional IVF cycles, no studies investigated the association between the proportion of MPN zygotes and the maturation state of the oocyte cohort. We compared the cytoplasmic maturity of oocytes from conventional IVF cycles with different proportions of 3 PN/MPN zygotes. A total of 1428 conventional IVF patients with ≥6 oocytes retrieved and fresh embryos transferred were divided into 4 groups according to the proportions of 3 PN/MPN zygotes. The pregnancy outcomes and the proportion of nuclear immature oocytes were analyzed to suggest the cytoplasmic maturation state of the oocyte cohort. Our results showed that the group with a low proportion of 3 PN/MPN zygotes had a higher clinical pregnancy rate (CPR) than those without 3 PN/MPN zygotes (P < 0.05). However, the live birth rate (LBR) was not significantly different between the two groups. The implantation rate (IR), CPR, and LBR did not differ between the low-proportion and high-proportion 3 PN/MPN groups. The proportion of nuclear immature oocytes on day 1 was highest in the group without 3 PN/MPN zygotes (23.8 %) and gradually decreased with an increased proportion of 3 PN/MPN zygotes (P < 0.001). Therefore, the presence of 3 PN/MPN zygotes after conventional IVF may indicate a more mature cytoplasmic state of the oocyte cohort, and the increased proportion of 3 PN/MPN zygotes is associated with an increased maturation state of the whole oocyte cohort. The occurrence and proportion of 3 PN/MPN zygotes may serve as an indicator for the cytoplasmic maturity of the oocyte cohort and help clinicians evaluate the efficiency of ovarian stimulation and optimize the stimulation protocols in subsequent cycles.

Dynamic methylation pattern of H19DMR and KvDMR1 in bovine oocytes and preimplantation embryos.

PURPOSE: This study aimed to evaluate the epigenetic reprogramming of ICR1 (KvDMR1) and ICR2 (H19DMR) and expression of genes controlled by them as well as those involved in methylation, demethylation, and pluripotency. METHODS: We collected germinal vesicle (GV) and metaphase II (MII) oocytes, and preimplantation embryos at five stages [zygote, 4-8 cells, 8-16 cells, morula, and expanded blastocysts (ExB)]. DNA methylation was assessed by BiSeq, and the gene expression was evaluated using qPCR. RESULTS: H19DMR showed an increased DNA methylation from GV to MII oocytes (68.04% and 98.05%, respectively), decreasing in zygotes (85.83%) until morula (61.65%), and ExB (63.63%). H19 and IGF2 showed increased expression in zygotes, which decreased in further stages. KvDMR1 was hypermethylated in both GV (71.82%) and MII (69.43%) and in zygotes (73.70%) up to morula (77.84%), with a loss of methylation at the ExB (36.64%). The zygote had higher expression of most genes, except for CDKN1C and PHLDA2, which were highly expressed in MII and GV oocytes, respectively. DNMTs showed increased expression in oocytes, followed by a reduction in the earliest stages of embryo development. TET1 was downregulated until 4-8-cell and upregulated in 8-16-cell embryos. TET2 and TET3 showed higher expression in oocytes, and a downregulation in MII oocytes and 4-8-cell embryo. CONCLUSION: We highlighted the heterogeneity in the DNA methylation of H19DMR and KvDMR1 and a dynamic expression pattern of genes controlled by them. The expression of DNMTs and TETs genes was also dynamic owing to epigenetic reprogramming.

An FGF timer for zygotic genome activation.

Zygotic genome activation has been extensively studied in a variety of systems including flies, frogs, and mammals. However, there is comparatively little known about the precise timing of gene induction during the earliest phases of embryogenesis. Here we used high-resolution in situ detection methods, along with genetic and experimental manipulations, to study the timing of zygotic activation in the simple model chordate Ciona with minute-scale temporal precision. We found that two Prdm1 homologs in Ciona are the earliest genes that respond to FGF signaling. We present evidence for a FGF timing mechanism that is driven by ERK-mediated derepression of the ERF repressor. Depletion of ERF results in ectopic activation of FGF target genes throughout the embryo. A highlight of this timer is the sharp transition in FGF responsiveness between the eight- and 16-cell stages of development. We propose that this timer is an innovation of chordates that is also used by vertebrates.

Regulation of the Actin Cytoskeleton-Linked Ca2+ Signaling by Intracellular pH in Fertilized Eggs of Sea Urchin.

In sea urchin, the immediate contact of the acrosome-reacted sperm with the egg surface triggers a series of structural and ionic changes in the egg cortex. Within one minute after sperm fuses with the egg plasma membrane, the cell membrane potential changes with the concurrent increases in intracellular Ca2+ levels. The consequent exocytosis of the cortical granules induces separation of the vitelline layer from the egg plasma membrane. While these cortical changes are presumed to prevent the fusion of additional sperm, the subsequent late phase (between 1 and 4 min after fertilization) is characterized by reorganization of the egg cortex and microvilli (elongation) and by the metabolic shift to activate de novo protein and DNA syntheses. The latter biosynthetic events are crucial for embryonic development. Previous studies suggested that the early phase of fertilization was not a prerequisite for these changes in the second phase since the increase in the intracellular pH induced by the exposure of unfertilized sea urchin eggs to ammonia seawater could start metabolic egg activation in the absence of the cortical granule exocytosis. In the present study, we have demonstrated that the incubation of unfertilized eggs in ammonia seawater induced considerable elongations of microvilli (containing actin filaments) as a consequence of the intracellular pH increase, which increased the egg's receptivity to sperm and made the eggs polyspermic at fertilization despite the elevation of the fertilization envelope (FE). These eggs also displayed compromised Ca2+ signals at fertilization, as the amplitude of the cortical flash was significantly reduced and the elevated intracellular Ca2+ level declined much faster. These results have also highlighted the importance of the increased internal pH in regulating Ca2+ signaling and the microvillar actin cytoskeleton during the late phase of the fertilization process.

Cell orientation characteristics of the natural combs of honey bee colonies.

The cell orientation characteristics of the natural combs of honey bees have received much research attention. Although natural combs have been shown to be composed of cells with three orientations-vertical, intermediate (oblique), and horizontal-the proportion of comb cells in these three orientations varies. Knowledge of the comb-building preferences of honey bees is essential for the installation of wax comb foundations, and clarification of the cell orientation characteristics of natural honey bee combs is important for beekeeping. The purpose of this study was to determine the cell orientation characteristics of natural combs of Eastern honey bees (Apis cerana cerana) and Western honey bees (Apis mellifera ligustica). Newly built combs were used to measure the orientation of hexagonal cells and calculate the proportion of cells in different orientations relative to the total number of cells. The number of eggs laid by queens in the cells of different orientations was also determined. The orientation of cells in the natural combs of Eastern and Western honey bees was determined based on the value of the minimum included angle between the pair of parallel cell walls and a vertical line connecting the top and bottom bars of the movable frame in the geometric plane of the comb: 0°≤θ≤10°, 10°<θ≤20°, and 20°<θ≤30° for vertical, intermediate, and horizontal orientations, respectively. Natural combs were composed of cells with at least one orientation (vertical or horizontal), two orientations (vertical + intermediate (oblique) or vertical + horizontal), or three orientations (vertical + intermediate + horizontal), and the proportions of combs with the three aforementioned configurations differed. Both Eastern honey bees and Western honey bees preferred building combs with cells in a vertical orientation. Queens showed no clear preference for laying eggs in cells of specific orientations. The results of this study provide new insight that could aid the production and cutting of wax comb foundations of Eastern and Western honey bees. Our study highlights the importance of installing wax comb foundations compatible with the comb-building preferences of bees.

Maternal age and maternal environment affect egg composition, yolk testosterone, offspring growth and behaviour in laying hens.

Maternal effects have been reported to alter offspring phenotype in laying hens. In this study, we investigated the effects of maternal environment and maternal age on egg traits and offspring development and behaviour. For this, we ran two experiments. First (E1), commercial hybrid hens were reared either in aviary or barren brooding cages, then housed in aviary, conventional cages or furnished (enriched) cages, thus forming different maternal housing treatments. Hens from each treatment were inseminated at three ages, and measures of egg composition, yolk testosterone concentration and offspring's development, anxiety and fearfulness were assessed. In experiment 2 (E2), maternal age effects on offspring's growth and behaviour were further investigated using fertile eggs from commercial breeder flocks at three different ages. Results from E1 showed that Old hens laid heavier eggs with less yolk testosterone and produced offspring with fewer indicators of anxiety and fearfulness. Maternal rearing and housing affected egg traits, offspring weight and behaviour, but not in a consistent way. Effects of maternal age were not replicated in E2, possibly due to differences in management or higher tolerance to maternal effects in commercial breeders. Overall, our research confirms that maternal age and maternal environment affects egg composition, with maternal age specifically affecting yolk testosterone concentration, which may mediate physical and behavioural effects in offspring.

The embryonic transcriptome of Parhyale hawaiensis reveals different dynamics of microRNAs and mRNAs during the maternal-zygotic transition.

Parhyale hawaiensis has emerged as the crustacean model of choice due to its tractability, ease of imaging, sequenced genome, and development of CRISPR/Cas9 genome editing tools. However, transcriptomic datasets spanning embryonic development are lacking, and there is almost no annotation of non-protein-coding RNAs, including microRNAs. We have sequenced microRNAs, together with mRNAs and long non-coding RNAs, in Parhyale using paired size-selected RNA-seq libraries at seven time-points covering important transitions in embryonic development. Focussing on microRNAs, we annotate 175 loci in Parhyale, 88 of which have no known homologs. We use these data to annotate the microRNAome of 37 crustacean genomes, and suggest a core crustacean microRNA set of around 61 sequence families. We examine the dynamic expression of microRNAs and mRNAs during the maternal-zygotic transition. Our data suggest that zygotic genome activation occurs in two waves in Parhyale with microRNAs transcribed almost exclusively in the second wave. Contrary to findings in other arthropods, we do not predict a general role for microRNAs in clearing maternal transcripts. These data significantly expand the available transcriptomics resources for Parhyale, and facilitate its use as a model organism for the study of small RNAs in processes ranging from embryonic development to regeneration.

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.

Epigenetic Reprogramming in Early Animal Development.

Dramatic nuclear reorganization occurs during early development to convert terminally differentiated gametes to a totipotent zygote, which then gives rise to an embryo. Aberrant epigenome resetting severely impairs embryo development and even leads to lethality. How the epigenomes are inherited, reprogrammed, and reestablished in this critical developmental period has gradually been unveiled through the rapid development of technologies including ultrasensitive chromatin analysis methods. In this review, we summarize the latest findings on epigenetic reprogramming in gametogenesis and embryogenesis, and how it contributes to gamete maturation and parental-to-zygotic transition. Finally, we highlight the key questions that remain to be answered to fully understand chromatin regulation and nuclear reprogramming in early development.

Ovicidal, larvicidal and pupicidal efficacy of silver nanoparticles synthesized by Bacillus marisflavi against the chosen mosquito species.

Microbial synthesis of silver nanoparticles is more advantageous and is eco-friendly to combat the various vectors that cause diseases in humans. Hence, in the present study a Bacillus strain is isolated from marine habitat and is evaluated for its ability to synthesize silver nanoparticles (AgNPs) and its efficacy evaluated against the immature stages of selected mosquito species. The effective candidate was confirmed to be Bacillus marisflavi after 16S rRNA sequencing. The synthesis of AgNPs was confirmed by UV-Vis spectrophotometer. Atomic Force Microscopic (AFM) analysis showed spherical nanoparticles. Size analysis using Scanning Electron Microscope (SEM) showed particles of nano size averaging 78.77 nm. The diameter of the particles analyzed by Dynamic Light Scattering (DLS) showed 101.6 nm with a poly-dispersive index of 0.3. Finally the elemental nature of the nanoparticles was identified by Fourier-transform infrared spectroscopy (FTIR). LC50 and LC90 values for the ovicidal, larvicidal and pupicidal efficacy of the AgNPs against the egg, larvae and pupae of Aedes aegypti, Culex quinquefasciatus and Anopheles stephensi respectively were evaluated. The present study revealed that the nanoparticles have an excellent toxic effect against the disease transmitting vector mosquitoes. Hence, the rapid synthesis of AgNPs would be an appropriate eco-friendly tool for biocontrol of vector mosquitoes.

Dietary Micronutrients from Zygote to Senility: Updated Review of Minerals' Role and Orchestration in Human Nutrition throughout Life Cycle with Sex Differences.

Micronutrients such as selenium, fluoride, zinc, iron, and manganese are minerals that are crucial for many body homeostatic processes supplied at low levels. The importance of these micronutrients starts early in the human life cycle and continues across its different stages. Several studies have emphasized the critical role of a well-balanced micronutrient intake. However, the majority of studies looked into or examined such issues in relation to a specific element or life stage, with the majority merely reporting the effect of either excess or deficiency. Herein, in this review, we will look in depth at the orchestration of the main element requirements across the human life cycle beginning from fertility and pregnancy, passing through infancy, childhood, adolescence, and reaching adulthood and senility, with insight on the interactions among them and underlying action mechanisms. Emphasis is given towards approaches to the role of the different minerals in the life cycle, associated symptoms for under- or overdoses, and typical management for each element, with future perspectives. The effect of sex is also discussed for each micronutrient for each life stage as literature suffice to highlight the different daily requirements and or effects.

Inhibition of LINE-1 retrotransposition represses telomere reprogramming during mouse 2-cell embryo development.

PURPOSE: To investigate whether inhibition of LINE-1 affects telomere reprogramming during 2-cell embryo development. METHODS: Mouse zygotes were cultured with or without 1 µM azidothymidine (AZT) for up to 15 h (early 2-cell, G1/S) or 24 h (late 2-cell, S/G2). Gene expression and DNA copy number were determined by RT-qPCR and qPCR respectively. Immunostaining and telomeric PNA-FISH were performed for co-localization between telomeres and ZSCAN4 or LINE-1-Orf1p. RESULTS: LINE-1 copy number was remarkably reduced in later 2-cell embryos by exposure to 1 µM AZT, and telomere lengths in late 2-cell embryos with AZT were significantly shorter compared to control embryos (P = 0.0002). Additionally, in the absence of LINE-1 inhibition, Dux, Zscan4, and LINE-1 were highly transcribed in early 2-cell embryos, as compared to late 2-cell embryos (P < 0.0001), suggesting that these 2-cell genes are activated at the early 2-cell stage. However, in early 2-cell embryos with AZT treatment, mRNA levels of Dux, Zscan4, and LINE-1 were significantly decreased. Furthermore, both Zscan4 and LINE-1 encoded proteins localized to telomere regions in 2-cell embryos, but this co-localization was dramatically reduced after AZT treatment (P < 0.001). CONCLUSIONS: Upon inhibition of LINE-1 retrotransposition in mouse 2-cell embryos, Dux, Zscan4, and LINE-1 were significantly downregulated, and telomere elongation was blocked. ZSCAN4 foci and their co-localization with telomeres were also significantly decreased, indicating that ZSCAN4 is an essential component of the telomere reprogramming that occurs in mice at the 2-cell stage. Our findings also suggest that LINE-1 may directly contribute to telomere reprogramming in addition to regulating gene expression.

Knockdown of Dnmt1 and Dnmt3a gene expression disrupts preimplantation embryo development through global DNA methylation.

PURPOSE: DNA methylation is one of the epigenetic mechanisms that plays critical roles in preimplantation embryo development executed by DNA methyltransferase (Dnmt) enzymes. Dnmt1, responsible for the maintenance of methylation, and Dnmt3a, for de novo methylation, are gradually erased from the zygote in succeeding stages and then reestablished in the blastocyst. This study was designed to address the vital role of Dnmt1 and Dnmt3a enzymes by silencing their gene expressions in embryonic development in mice. METHODS: Groups were (i) control, (ii) Dnmt1-siRNA, (iii) Dnmt3a-siRNA, and (iv) non-targeted (NT) siRNA. Knockdown of Dnmt genes using siRNAs was confirmed by measuring the targeted proteins using Western blot and immunofluorescence. Following knockdown of Dnmt1 and Dnmt3a in zygotes, the developmental competence and global DNA methylation levels were analyzed after 96 h in embryo cultures. RESULTS: A significant number of embryos arrested at the 2-cell stage or had undergone degeneration in the Dnmt1 and Dnmt3a knocked-down groups. By 3D observations in super-resolution microscopy, we noted that Dnmt1 was exclusively found in juxtanuclear cytoplasm, while the Dnmt3a signal was preferentially localized in the nucleus, both in trophoblasts (TBs) and embryoblasts (EBs). Interestingly, the global DNA methylation level decreased in the Dnmt1 knockdown group, while it increased in the Dnmt3a knockdown group. CONCLUSION: Precisely aligned expression of Dnmt genes is highly essential for the fate of an embryo in the early developmental period. Our data indicates that further analysis is mandatory to designate the specific targets of these methylation/demethylation processes in mouse and human preimplantation embryos.

Parthenogenesis and the Evolution of Anisogamy.

Recently, it was pointed out that classic models for the evolution of anisogamy do not take into account the possibility of parthenogenetic reproduction, even though sex is facultative in many relevant taxa (e.g., algae) that harbour both anisogamous and isogamous species. Here, we complement this recent analysis with an approach where we assume that the relationship between progeny size and its survival may differ between parthenogenetically and sexually produced progeny, favouring either the former or the latter. We show that previous findings that parthenogenesis can stabilise isogamy relative to the obligate sex case, extend to our scenarios. We additionally investigate two different ways for one mating type to take over the entire population. First, parthenogenesis can lead to biased sex ratios that are sufficiently extreme that one type can displace the other, leading to de facto asexuality for the remaining type that now lacks partners to fuse with. This process involves positive feedback: microgametes, being numerous, lack opportunities for syngamy, and should they proliferate parthenogenetically, the next generation makes this asexual route even more prominent for microgametes. Second, we consider mutations to strict asexuality in producers of micro- or macrogametes, and show that the prospects of asexual invasion depend strongly on the mating type in which the mutation arises. Perhaps most interestingly, we also find scenarios in which parthenogens have an intrinsic survival advantage yet facultatively sexual isogamous populations are robust to the invasion of asexuals, despite us assuming no genetic benefits of recombination. Here, equal contribution from both mating types to zygotes that are sufficiently well provisioned can outweigh the additional costs associated with syngamy.

Direct evidence of sex and a hypothesis about meiosis in Symbiodiniaceae.

Dinoflagellates in the family Symbiodiniaceae are obligate endosymbionts of diverse marine invertebrates, including corals, and impact the capacity of their hosts to respond to climate change-driven ocean warming. Understanding the conditions under which increased genetic variation in Symbiodiniaceae arises via sexual recombination can support efforts to evolve thermal tolerance in these symbionts and ultimately mitigate coral bleaching, the breakdown of the coral-Symbiodiniaceae partnership under stress. However, direct observations of meiosis in Symbiodiniaceae have not been reported, despite various lines of indirect evidence that it occurs. We present the first cytological evidence of sex in Symbiodiniaceae based on nuclear DNA content and morphology using Image Flow Cytometry, Cell Sorting and Confocal Microscopy. We show the Symbiodiniaceae species, Cladocopium latusorum, undergoes gamete conjugation, zygote formation, and meiosis within a dominant reef-building coral in situ. On average, sex was detected in 1.5% of the cells analyzed (N = 10,000-40,000 cells observed per sample in a total of 20 samples obtained from 3 Pocillopora colonies). We hypothesize that meiosis follows a two-step process described in other dinoflagellates, in which diploid zygotes form dyads during meiosis I, and triads and tetrads as final products of meiosis II. This study sets the stage for investigating environmental triggers of Symbiodiniaceae sexuality and can accelerate the assisted evolution of a key coral symbiont in order to combat reef degradation.

Dual spindles assemble in bovine zygotes despite the presence of paternal centrosomes.

The first mitosis of the mammalian embryo must partition the parental genomes contained in two pronuclei. In rodent zygotes, sperm centrosomes are degraded, and instead, acentriolar microtubule organizing centers and microtubule self-organization guide the assembly of two separate spindles around the genomes. In nonrodent mammals, including human or bovine, centrosomes are inherited from the sperm and have been widely assumed to be active. Whether nonrodent zygotes assemble a single centrosomal spindle around both genomes or follow the dual spindle self-assembly pathway is unclear. To address this, we investigated spindle assembly in bovine zygotes by systematic immunofluorescence and real-time light-sheet microscopy. We show that two independent spindles form despite the presence of centrosomes, which had little effect on spindle structure and were only loosely connected to the two spindles. We conclude that the dual spindle assembly pathway is conserved in nonrodent mammals. This could explain whole parental genome loss frequently observed in blastomeres of human IVF embryos.

Dual role of Fam208a during zygotic cleavage and early embryonic development.

Maintenance of genome stability is essential for every living cell as genetic information is repeatedly challenged during DNA replication in each cell division event. Errors, defects, delays, and mistakes that arise during mitosis or meiosis lead to an activation of DNA repair processes and in case of their failure, programmed cell death, i.e. apoptosis, could be initiated. Fam208a is a protein whose importance in heterochromatin maintenance has been described recently. In this work, we describe the crucial role of Fam208a in sustaining genome stability during cellular division. The targeted depletion of Fam208a in mice using CRISPR/Cas9 led to embryonic lethality before E12.5. We also used the siRNA approach to downregulate Fam208a in zygotes to avoid the influence of maternal RNA in the early stages of development. This early downregulation increased arresting of the embryonal development at the two-cell stage and the occurrence of multipolar spindles formation. To investigate this further, we used the yeast two-hybrid (Y2H) system and identified new putative interaction partners Gpsm2, Svil, and Itgb3bp. Their co-expression with Fam208a was assessed by RT-qPCR profiling and in situ hybridization [1] in multiple murine tissues. Based on these results we proposed that Fam208a functions within the HUSH complex by interaction with Mphosph8 as these proteins are not only able to physically interact but also co-localise. We are bringing new evidence that Fam208a is a multi-interacting protein affecting genome stability on the cell division level at the earliest stages of development and by interaction with methylation complex in adult tissues. In addition to its epigenetic functions, Fam208a appears to have an important role in the zygotic division, possibly via interaction with newly identified putative partners Gpsm2, Svil, and Itgb3bp.