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Naive pluripotency exists in epiblast cells of mouse pre-implantation embryos. However, whether the naive pluripotency is transient or nonexistent in primate embryos remains unclear. Using RNA-seq in single blastomeres from 16-cell embryos through to hatched blastocysts of rhesus monkey, we constructed the lineage segregation roadmap in which the specification of trophectoderm, epiblast, and primitive endoderm is initiated simultaneously at the early blastocyst stage. Importantly, we uncovered the existence of distinct pluripotent states in monkey pre-implantation embryos. At the early- and middle-blastocyst stages, the epiblast cells have the transcriptome features of naive pluripotency, whereas they display a continuum of primed pluripotency characteristics at the late and hatched blastocyst stages. Moreover, we identified potential regulators that might play roles in the transition from naive to primed pluripotency. Thus, our study suggests the transient existence of naive pluripotency in primates and proposes an ideal time window for derivation of primate embryonic stem cells with naive pluripotency., (© 2018 Liu et al.; Published by Cold Spring Harbor Laboratory Press.)

Intracellular Ca 2+ signaling regulates cellular activities during embryogenesis and in adult organisms. We generated stable Tg[βactin2:GCaMP6s] stl351 and Tg[ubi:GCaMP6s] stl352 transgenic lines that combine the ubiquitously-expressed Ca 2+ indicator GCaMP6s with the transparent characteristics of zebrafish embryos to achieve superior in vivo Ca 2+ imaging. Using the Tg[βactin2:GCaMP6s] stl351 line featuring strong GCaMP6s expression from cleavage through gastrula stages, we detected higher frequency of Ca 2+ transients in the superficial blastomeres during the blastula stages preceding the midblastula transition. Additionally, GCaMP6s also revealed that dorsal-biased Ca 2+ signaling that follows the midblastula transition persisted longer during gastrulation, compared with earlier studies. We observed that dorsal-biased Ca 2+ signaling is diminished in ventralized ichabod/β-catenin2 mutant embryos and ectopically induced in embryos dorsalized by excess β-catenin. During gastrulation, we directly visualized Ca 2+ signaling in the dorsal forerunner cells, which form in a Nodal signaling dependent manner and later give rise to the laterality organ. We found that excess Nodal increases the number and the duration of Ca 2+ transients specifically in the dorsal forerunner cells. The GCaMP6s transgenic lines described here enable unprecedented visualization of dynamic Ca 2+ events from embryogenesis through adulthood, augmenting the zebrafish toolbox., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: To investigate the blastocoelic fluid (BF) for the presence of DNA that could be amplified and analyzed; the extent to which its chromosomal status corresponds to that found in trophectoderm (TE) cells, polar bodies (PBs), or blastomeres; and the identification of segmental abnormalities., Design: Longitudinal cohort study., Setting: In vitro fertilization unit., Patient(s): Fifty-one couples undergoing preimplantation genetic screening or preimplantation genetic diagnosis for translocations by array-comparative genomic hybridization on PBs (n = 21) or blastomeres (n = 30)., Intervention(s): BFs and TE cells were retrieved from 116 blastocysts, whose chromosome status had already been established by PB or blastomere assessment. Separate chromosome analysis was performed in 70 BFs., Main Outcome Measure(s): Presence of DNA in BFs, evaluation of the chromosome condition, and comparison with the diagnosis made in TE cells and at earlier stage biopsies., Result(s): DNA detection was 82%, with a net improvement after refinement of the procedure. In 97.1% of BFs, the ploidy condition corresponded to that found in TE cells, with one false positive and one false negative. The rate of concordance per single chromosome was 98.4%. Ploidy and chromosome concordance with PBs were 94% and 97.9%, respectively; with blastomeres, the concordances were 95% and 97.7%, respectively. Segmental abnormalities, which were detected in PBs or blastomeres of 16 blastocysts, were also identified in the corresponding BFs., Conclusion(s): BF represents to a good extent the blastocyst ploidy condition and chromosome status when compared with TE cells. If the proportion of clinically useful BFs is improved, blastocentesis could become the preferred source of DNA for chromosomal testing., (Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Study Question: Could selected pluripotency-enhancing small molecules (SMs) lead to efficient derivation of human embryonic stem cells (hESCs) from cleavage embryos-derived single blastomeres (SBs)?, Summary Answer: Inhibition of glycogen synthase kinase β (GSK3β) and Rho-associated kinase (ROCK) signaling can enhance the derivation of hESCs from cleavage embryo-derived SBs., What Is Known Already: Parameters involved in sustaining the pluripotency of biopsied blastomeres for generating hESCs without causing injury to a viable embryo have remained obscure. This research seeks to improve the culture conditions for increasing the efficiency of deriving hESCs from SBs from cleavage-stage embryos by using SMs., Study Design, Size, Duration: In order to identify SMs which may enhance hESC generation from SBs, 11 pluripotency-enhancing SMs were screened and CHIR99021 (CH), a GSK3β inhibitor, was selected. To optimize culture condition in hESC generation from SMs, we used ROCK inhibitor Y27632 (Y) and basic fibroblast growth factor in combination with CH or its alternative, Kenpaullone, in different time courses over 12 days. We also assessed a critical time point for CH + Y treatment of cleavage embryos from 4- to 8-cell embryo. In total, 224 embryos and 1607 SBs were used in the study., Participants/materials, Setting, Methods: Blastomeres of fair and poor-quality from 6- to 8-cell stage human embryos were mechanically dispersed and individually seeded into a 96-well plate that was precoated with mitotically inactivated feeder cells. Derivation of hESC line from each SB was carried out in hESC defined medium supplemented with SMs. Randomly selected hESC lines were evaluated by immunostaining for pluripotency markers, karyotype analysis and differentiation potential into the three embryonic germ layer derivatives., Main Results and the Role of Chance: We found that 3 μM CH was the only SM that was capable of directing SBs from fair and poor-quality 6-8-cell embryos into hESC lines. The application of hESC-conditioned medium had no additive effect on hESC establishment from SBs. Also, we indicated that CH combined with Y improved hESC generation efficiency by up to 31%. By using of Kenpaullone as an alternative to CH, we confirmed the involvement of GSK3 inhibition in hESC derivation from SBs. Interestingly, by treatment of 4-cell embryos, these SMs could enhance the derivation efficiency of SB-derived hESC lines up to 73% and the maximum number of hESC lines from SBs of one embryo was achieved in this state., Limitations, Reasons for Caution: The low quality of the embryos used in this study most likely had an effect on hESC generation. Furthermore, although we attempted to minimize any differences in inter-embryo quality, we cannot exclude the possibility that small differences in starting quality between embryos may have contributed to the differences observed, other than the addition of SMs., Wider Implications of the Findings: This approach would allow the establishment of autogeneic or allogeneic matched cells from embryos fertilized in vitro without destroying them., Study Funding/competing Interest(s): This study was financially supported by the National Elite Foundation and the Royan Institute for Stem Cell Biology and Technology. The authors have no conflict of interest to declare.

The nature and pace of genome mutation is largely unknown. Because standard methods sequence DNA from populations of cells, the genetic composition of individual cells is lost, de novo mutations in cells are concealed within the bulk signal and per cell cycle mutation rates and mechanisms remain elusive. Although single-cell genome analyses could resolve these problems, such analyses are error-prone because of whole-genome amplification (WGA) artefacts and are limited in the types of DNA mutation that can be discerned. We developed methods for paired-end sequence analysis of single-cell WGA products that enable (i) detecting multiple classes of DNA mutation, (ii) distinguishing DNA copy number changes from allelic WGA-amplification artefacts by the discovery of matching aberrantly mapping read pairs among the surfeit of paired-end WGA and mapping artefacts and (iii) delineating the break points and architecture of structural variants. By applying the methods, we capture DNA copy number changes acquired over one cell cycle in breast cancer cells and in blastomeres derived from a human zygote after in vitro fertilization. Furthermore, we were able to discover and fine-map a heritable inter-chromosomal rearrangement t(1;16)(p36;p12) by sequencing a single blastomere. The methods will expedite applications in basic genome research and provide a stepping stone to novel approaches for clinical genetic diagnosis.

Objective: To evaluate the impact of multinucleation of a sibling blastomere of day 2 embryos on the rate of aneuploidy detected by day 3 preimplantation genetic screening (PGS) biopsy and the effect on subsequent implantation and pregnancy rates., Design: Retrospective cohort study., Setting: University-based IVF center., Patient(s): A total of 141 couples undergoing their first IVF-PGS cycle for idiopathic recurrent pregnancy loss (RPL) or multiple failed IVF implantations., Intervention(s): Biopsy of single-nucleated blastomeres for PGS analysis of chromosomes X, Y, 13, 15, 16, 17, 18, 21, and 22 by fluorescence in situ hybridization., Main Outcome Measure(s): Aneuploidy, implantation, and pregnancy rates., Result(s): PGS revealed an increased incidence of aneuploidy when comparing multinucleated day 2 embryos with single-nucleated embryos (85% vs. 78%; relative risk 0.92 (95% confidence interval 0.84-1.00). Transfer of single-nucleated euploid embryos resulted in clinical pregnancy and implantation rates of 28% and 24%. Transfer of multinucleated euploid embryos resulted in no implantations., Conclusion(s): The presence of multinucleated blastomeres on day 2 of embryo development, 1 day before biopsy, predicts an increase of aneuploidy and poor pregnancy outcomes in IVF-PGS cycles., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

In invertebrates and amphibians, informational macromolecules in egg cytoplasm are organized to provide direction to the formation of embryonic lineages, but it is unclear whether vestiges of such prepatterning exist in mammals. Here we examined whether twin blastomeres from 2-cell stage mouse embryos differ in mRNA content. mRNA from 26 blastomeres derived from 13 embryos approximately mid-way through their second cell cycle was subjected to amplification. Twenty amplified samples were hybridized to arrays. Of those samples that hybridized successfully, 12 samples in six pairs were used in the final analysis. Probes displaying normalized values >0.25 (n = 4573) were examined for consistent bias in expression within blastomere pairs. Although transcript content varied between both individual embryos and twin blastomeres, no consistent asymmetries were observed for the majority of genes, with only 178 genes displaying a >1.4-fold difference in expression across all six pairs. Although class discovery clustering showed that blastomere pairs separated into two distinct groups in terms of their differentially expressed genes, when the data were tested for significance of asymmetrical expression, only 39 genes with >1.4-fold change ratios in six of six blastomere pairs passed the two-sample t-test (P < 0.05). Transcripts encoding proteins implicated in RNA processing and cytoskeletal organization were among the most abundant, differentially distributed mRNA, suggesting that a stochastically based lack of synchrony in cell cycle progression between the two cells might explain at least some and possibly all of the asymmetries in transcript composition.

Mutations in mitochondrial DNA (mtDNA) are a common cause of genetic disease. Pathogenic mutations in mtDNA are detected in approximately 1 in 250 live births and at least 1 in 10,000 adults in the UK are affected by mtDNA disease. Treatment options for patients with mtDNA disease are extremely limited and are predominantly supportive in nature. Mitochondrial DNA is transmitted maternally and it has been proposed that nuclear transfer techniques may be an approach for the prevention of transmission of human mtDNA disease. Here we show that transfer of pronuclei between abnormally fertilized human zygotes results in minimal carry-over of donor zygote mtDNA and is compatible with onward development to the blastocyst stage in vitro. By optimizing the procedure we found the average level of carry-over after transfer of two pronuclei is less than 2.0%, with many of the embryos containing no detectable donor mtDNA. We believe that pronuclear transfer between zygotes, as well as the recently described metaphase II spindle transfer, has the potential to prevent the transmission of mtDNA disease in humans.

The present study was conducted to evaluate the effect of accessory sperm cells that adhered to the zona pellucida or blastomeres on the accuracy of genetic diagnosis of preimplantation embryos. The properties of sperm cells as a template for DNA amplification were examined using bovine sperm cells frozen-thawed or incubated in PBS after thawing for 7 days at 39 C. Sexing by loop-mediated isothermal amplification (LAMP) and claudin-16 genotyping by polymerase chain reaction (PCR) were performed using 10, 50 and 100 sperm cells. When sexing based on LAMP was performed, no amplified DNA was detected in 10 sperm-derived samples, whereas male-specific (10-60%) and gender-natural DNA (30-100%) sequences were detected in 50 and 100 sperm-derived samples. The detection rates for gender-natural DNA sequences were higher in incubated sperm samples than in sperm samples immediately after freeze-thawing. The detection rates for claudin-16 were low (7-13%) regardless of the concentration of sperm cells and the period of incubation after thawing. The present results showed that male-specific DNA, gender-natural DNA and claudin-16 sequences were not usually amplified from a small number of sperm cells (< or =10 cells). However, when a large number of sperm cells (> or =50 cells) were present, male-specific and gender-natural DNA sequences were amplified at a high rate, and claudin-16 DNA sequences were also occasionally detected. These results raise the possibility that accessory sperm cells may reduce the accuracy of the genetic diagnosis of bovine embryos. Therefore, steps to prevent the contamination of sperm cells, such as removal of the zona pellucida and washing of sample blastomeres, are necessary to obtain an accurate result.

Genomic imbalances are a major cause of constitutional and acquired disorders. Therefore, aneuploidy screening has become the cornerstone of preimplantation, prenatal and postnatal genetic diagnosis, as well as a routine aspect of the diagnostic workup of many acquired disorders. Recently, array comparative genomic hybridization (array CGH) has been introduced as a rapid and high-resolution method for the detection of both benign and disease-causing genomic copy-number variations. Until now, array CGH has been performed using a significant quantity of DNA derived from a pool of cells. Here, we present an array CGH method that accurately detects chromosomal imbalances from a single lymphoblast, fibroblast and blastomere within a single day. Trisomy 13, 18, 21 and monosomy X, as well as normal ploidy levels of all other chromosomes, were accurately determined from single fibroblasts. Moreover, we showed that a segmental deletion as small as 34 Mb could be detected. Finally, we demonstrated the possibility to detect aneuploidies in single blastomeres derived from preimplantation embryos. This technique offers new possibilities for genetic analysis of single cells in general and opens the route towards aneuploidy screening and detection of unbalanced translocations in preimplantation embryos in particular.

This study assessed the interactive effects of carbohydrate type (fibre vs starch) and fatty acid (FA) supplementation (0% vs 6% calcium soaps of palm oil FA) on the post-fertilisation development of oocytes recovered from low and moderate body condition score (BCS) heifers. A secondary objective was to compare the FA composition of plasma to that of granulosa cells (GCs) and cumulus-oocyte complexes (COCs) from these animals, and to relate these findings to the developmental potential of oocytes. Plasma, GCs and COCs were recovered from 32 heifers on day 5 of a synchronised oestrous cycle for FA analyses. Oocytes were also recovered on days 10 and 15 of the same cycle after short-term ovarian stimulation (FSH + GnRH), and matured, fertilised and cultured to the blastocyst stage in vitro. High levels of dietary starch increased (P < 0.01) plasma insulin but, together with dietary FA, reduced (P < 0.05) blastocyst yields in low, but not in moderate, BCS heifers. Diet-induced alterations to the FA content of plasma were less apparent in GCs and COCs. In summary, although dietary lipids increased the FA content of COCs, the selective uptake of saturated FAs at the expense of mainly polyunsaturated FAs within the follicular compartment ensured that the FA composition of COCs was largely unaffected by diet. However, the concentration of saturated FAs within COCs was inherently high, and so further increases in FA content may have impaired post-fertilisation development. The data establish a robust nutritional framework for more detailed studies into the mechanistic effects of dietary composition on the post-fertilisation developmental potential of oocytes.

Purpose: We evaluated methods of preparation of DNA from single cells for amplification and preimplantation genetic diagnosis (PGD), including our "spanning protocol.", Methods: Dystrophin gene exons 45 and 51 were amplified by nested polymerase chain reaction (PCR) from a single lymphocyte or blastomere. Amplification efficiencies were compared between DNA extraction by (A) lysis in distilled water with freeze-thawing and boiling; (B) two-step lysis involving potassium hydroxide and dithiothreitol; and (C) the spanning protocol, using N-lauroylsarcosine., Results: With method A, amplification efficiency was 66/120 (55%) and false-positive such as amplification failure or allele drop out was 42/120 (35%); with B, 96/120 (80%) and 21/120 (17.5%); and with C, 111/120 (92%) and 5/120 (4.2%), using single blastomeres and unaffected lymphocytes from male. Occurrence of false-negative such as contamination of another DNA with method A was 4/120 (3.3%); with B, 10/120 (8.3%); and with C, 2/120 (1.7%) from using single lymphocytes from affected males., Conclusion: The spanning protocol was most efficient for extracting DNA from a single cell and should be particularly useful for preimplantation genetic diagnosis.

The establishment of body axes and specification of early embryonic cells depend on maternally supplied transcripts and/or proteins, several of which are localized at specific regions of fertilized eggs and early embryos. The ascidian is known to exhibit a mosaic mode of development, and this mode is largely dependent on localized maternal factors. Using blastomere isolation, microarray and whole-mount in situ hybridization, the present study of Ciona intestinalis demonstrates that maternal transcripts of a total of 17 genes are localized at the posterior-most region of fertilized eggs and early embryos. Ten of them are newly identified in the present study, while the remaining seven genes have already been characterized in previous studies. In addition, maternal transcripts of two genes, in addition to 14 genes encoded by the mitochondrial genome, showed a mitochondria-like distribution. Despite the present comprehensive approach, we could not identify maternal transcripts that are clearly localized to the animal-pole side, the vegetal-pole side, the anterior-side or other specific regions of the early embryo. Therefore, we concluded that the posterior-most localization and mitochondria-like distribution appear to be major specialized patterns of maternal transcripts in early Ciona embryos.

Background: Specification of primordial germ cells in mice depends on instructive signalling events, which act first to confer germ cell competence on epiblast cells, and second, to impose a germ cell fate upon competent precursors. fragilis, an interferon-inducible gene coding for a transmembrane protein, is the first gene to be implicated in the acquisition of germ cell competence., Results: Here, we describe four additional fragilis-related genes, fragilis2-5, which are clustered within a 68 kb region in the vicinity of the fragilis locus on Chr 7. These genes exist in a number of mammalian species, which in the human are also clustered on the syntenic region on Chr 11. In the mouse, fragilis2 and fragilis3, which are proximate to fragilis, exhibit expression that overlaps with the latter in the region of specification of primordial germ cells. Using single cell analysis, we confirm that all these three fragilis-related genes are predominant in nascent primordial germ cells, as well as in gonadal germ cells., Conclusion: The Fragilis family of interferon-inducible genes is tightly associated with germ cell specification in mice. Furthermore, its evolutionary conservation suggests that it probably plays a critical role in all mammals. Detailed analysis of these genes may also elucidate the role of interferons as signalling molecules during development.

The abnormally high number of centrosomes found in many human tumor cells can lead directly to aneuploidy and genomic instability through the formation of multipolar mitotic spindles. To facilitate investigation of the mechanisms that control centrosome reproduction, a frog egg extract arrested in S phase of the cell cycle that supported repeated assembly of daughter centrosomes was developed. Multiple rounds of centrosome reproduction were blocked by selective inactivation of cyclin-dependent kinase 2-cyclin E (Cdk2-E) and were restored by addition of purified Cdk2-E. Confocal immunomicroscopy revealed that cyclin E was localized at the centrosome. These results demonstrate that Cdk2-E activity is required for centrosome duplication during S phase and suggest a mechanism that could coordinate centrosome reproduction with cycles of DNA synthesis and mitosis.

Tobacco smoking is deleterious to reproduction. Benzo[a]pyrene (B[a]P) is a potent carcinogen in cigarette smoke. Its reactive metabolite induces DNA-adducts, which can cause mutations. We investigated whether B[a]P diol epoxide (BPDE) DNA adducts are detectable in preimplantation embryos in relation to parental smoking. A total of 17 couples were classified by their smoking habits: (i) both partners smoke; (ii) wife non-smoker, husband smokes; and (iii) both partners were non-smokers. Their 27 embryos were exposed to an anti-BPDE monoclonal antibody that recognizes BPDE-DNA adducts. Immunostaining was assessed in each embryo and an intensity score was calculated for embryos in each smoking group. The proportion of blastomeres which stained was higher for embryos of smokers than for non-smokers (0.723 versus 0.310). The mean intensity score was also higher for embryos of smokers (1.40+/-0.28) than for non-smokers (0.38+/-0.14; P = 0.015), but was similar for both types of smoking couples. The mean intensity score was positively correlated with the number of cigarettes smoked by fathers (P = 0.02). Increased mean immunostaining in embryos from smokers, relative to non-smokers, indicates a relationship with parental smoking. The similar levels of immunostaining in embryos from both types of smoking couples suggest that transmission of modified DNA is mainly through spermatozoa. We confirmed paternal transmission of modified DNA by detection of DNA adducts in spermatozoa of a smoker father and his embryo.

Polarisation of cells during mouse preimplantation development first occurs within blastomeres at the eight-cell stage, as part of a process called compaction. Cell-cell contact mediated by the cell adhesion molecule uvomorulin (E-cadherin) and the activity of the microfilament cytoskeleton are important in the development of compaction, which is crucial for establishment of trophoblast and pluriblast (inner cell mass) lineages and for subsequent development. Members of the Rho family of p21 GTPases have been shown to regulate the organisation of the actin cytoskeleton and adhesion in other cell types. The potential role of these proteins in compaction was investigated. Inhibition of Rho with Clostridium botulinum C3-transferase disturbed intercellular flattening at compaction and prevented cytocortical microfilament polarisation of eight-cell blastomeres, in contrast to cytochalasin D which inhibited only adhesion. Microinjection of a constitutively activated recombinant Rho protein into four-cell blastomeres induced cortical microfilament disruption and apical displacement of nuclei associated with polarised clustering of microtubules. Interblastomere adhesion was reduced and E-cadherin was aberrently clustered at remaining cell-cell contacts. Similarly, activated Cdc42 protein induced nuclear displacement with additional cytoplasmic actin bundle formation between nucleus and cell-cell contacts. The effects produced by both of the activated GTPase proteins are indicative of prematurely induced but aberrently organised polarity. These results suggest that Rho family GTPases are involved in the polarisation of early mouse blastomeres., (Copyright 1999 Academic Press.)

During the first few cleavages of the zebrafish embryo, daughter blastomeres are loosely associated immediately after furrow ingression, but then gradually cohere. Cohesion appears to be cadherin-dependent, as cadherin and beta-catenin are found at the membrane between cohering blastomeres, and blastomeres fail to cohere in calcium-free medium. Cadherin and beta-catenin are not initially found on the blastomere surface, but are deposited specifically at the furrow surface. An array of parallel microtubules is present on either side of the furrow tip during ingression, as seen by confocal microscopy of alpha-tubulin labelled embryos. Transient incubation of embryos in 1 microg/ml nocodazole at the start of furrowing, which causes a loss of the furrow array, inhibits the localization of beta-catenin to the furrow surface but does not prevent furrow ingression. During ingression, intracellular membranes are transported to the furrow, as shown by labelling with DiD or DiOC6(3). Concentration of these membranes near the furrow surface is microtubule-dependent. These findings suggest that microtubules are required for cohesion of blastomeres because they mediate trafficking of intracellular membranes to the furrow surface, where they are exocytosed and allow cohesion via cadherins.

Purpose: The mechanisms by which the sex ratio might be altered at fertilization were reviewed, following a case of preimplantation gender analysis revealing a significantly skewed proportion of male-to-female embryos., Methods: The case of a known carrier of X-linked hydrocephalus with a history of three affected male pregnancies is presented. Her husband's family history consisted of a strong increase in the number of males relative to females. She had four cycles of stimulated in vitro fertilization, with sex chromosome analysis using fluorescent in situ hybridization (FISH) on suitable cleavage-stage embryos. The difference in the sex ratio of normal male-to-female embryos was compared using a significance probabilities test for sex ratio. The sex ratio of sperm from a semen sample from the male partner was determined by FISH., Results: Fifty embryos were suitable for analysis. A significantly higher number of normal male (n = 20) than normal female (n = 8) embryos was obtained (P < 0.05). The FISH assessment of the husband's semen analysis revealed no alteration in the normal X:Y ratio., Conclusions: As the sperm analysis revealed a normal X:Y ratio, an alteration in the embryo sex ratio might be explained by the preferential binding of Y-bearing sperm to the oocyte, an oocyte-related "discouragement" of binding of X-bearing sperm, or a postfertilization event.

Purpose: Our purpose was to evaluate the utility of spectral imaging for multicolor, multichromosome enumeration in human interphase cell nuclei., Methods: Chromosome-specific probes labeled with different fluorochromes or nonfluorescent haptens were obtained commercially or prepared in-house. Metaphase spreads, interphase lymphocytes, or blastomeres cells were hybridized with either 7 or 11 distinctly different probes. Following 46 hr of hybridization, slides were washed and detected using either a filter-based quantitative image processing system (QUIPS) developed in-house or a commercial spectral imaging system., Results: The filter-based fluorescence microscope system is preferred for simultaneous detection of up to seven chromosome targets because of its high sensitivity and speed. However, this approach may not be applicable to interphase cells when 11 or more targets need to be discriminated. Interferometer-based spectral imaging with a spectral resolution of approximately 10 nm allows labeling of chromosome-specific DNA probes with fluorochromes having greatly overlapping emission spectra. This leads to increases in the number of fluorochromes or fluorochrome combinations available to score unambiguously chromosomes in interphase nuclei., Conclusions: Spectral imaging provides a significant improvement over conventional filter-based microscope systems for enumeration of multiple chromosomes in interphase nuclei, although further technical development is necessary in its application to embryonic blastomeres. When applied to preconception/preimplantation genetic diagnosis, presently available probes for spectral imaging are expected to detect abnormalities responsible for 70-80% of spontaneous abortions caused by chromosomal trisomies.

Purpose: Our purpose was to achieve preimplantation genetic diagnosis (PGD) of the dominant cancer predisposition syndrome, familial adenomatous polyposis coli (FAPC), as an alternative to prenatal diagnosis., Methods: The affected patient was superovulated and oocytes were retrieved and fertilized by intracytoplasmic sperm injection (ICSI). Two cells were biopsied from each embryo and the whole genome was amplified by primer extension preamplification (PEP). Nested PCR was then used to amplify two APC fragments: one including the APC mutation site and the other an informative intragenic polymorphism. Both were detected by simultaneous single-strand conformation polymorphism and heteroduplex analysis., Results: Four normally fertilized embryos were biopsied on day 3 post ICSI, and two cells were successfully removed from each embryo. Following PEP the APC mutation was successfully amplified in 7 of 8 cells, and the polymorphism in 6 of 8 cells. The APC mutation was detected in three embryos. This result was confirmed by identification of the mutation associated polymorphism in two cases. A single embryo was diagnosed as homozygous normal for the mutation and the polymorphism in both cells sampled. This unaffected embryo was transferred to the mother, but no pregnancy resulted., Conclusions: We report here the first diagnosis of a cancer predisposition syndrome in human preimplantation embryos. Our results indicate that difficulties associated with single-cell PCR, allele-specific amplification failure in particular, need not prevent preimplantation diagnosis of diseases with a dominant mode of inheritance, provided appropriate strategies are applied.

Duplications or deletions are present in a high percentage of the gametes produced by individuals carrying balanced translocations. Preimplantation genetic diagnosis was used to examine chromosome balance in embryos from a patient having a reciprocal translocation within the short arms of chromosomes 5 and 8 (46,XX,t(5;8)(p13;p23)). This woman has two sisters with the translocation unbalanced, resulting in a partial trisomy for chromosome 5 and partial monosomy for chromosome 8 (46,XX,-8, +der(8)t(5;8)(p13;p23)) with associated mental retardation and physical abnormalities. The patient and her husband desired to have children without the abnormal chromosome balance and wished to reduce the likelihood of spontaneous abortion or need for therapeutic abortion. Fluorescence in-situ hybridization (FISH) probes for the alpha-satellite region of chromosome 8 and for a region on the short arm of chromosome 5 (5p15.2) were tested initially on lymphocytes from the patient and her sisters. The hybridization signal for chromosome 5 was detected in the expected two copies for the patient and three copies for the sisters in 87% of the cells. Two hybridization signals for chromosome 8 were detected in 96% of the cells from all individuals. Additional probe testing was done using blastomeres from polyspermic embryos. The couple then proceeded with a stimulated in-vitro fertilization (IVF) cycle and biopsies were done on 13 embryos at the 7-10-cell stage using a method of zona drilling and fluid displacement. Diagnosis was possible on at least one blastomere for nine embryos. Three embryos had nuclei with three hybridization signals for chromosome 5, three had fewer than two signals for one or both chromosomes, one was mosaic, and two had two signals for each chromosome. The latter were transferred to the patient, but pregnancy was not achieved. The results demonstrate that preimplantation genetic diagnosis for patients with reciprocal translocations can be used to identify embryos having normal chromosome balance. The potential advantages and limitations of this approach are discussed.

Blastomeres in C. elegans embryos execute lineage programs wherein the fate of a cell is correlated reproducibly with the division sequence by which that cell is born. We provide evidence that the pop-1 gene functions to link anterior-posterior cell divisions with cell fate decisions. Each anterior cell resulting from an anterior-posterior division appears to have a higher level of nuclear POP-1 protein than does its posterior sister. Genes in the C. elegans Wnt pathway are required for this inequality in POP-1 levels. We show that loss of pop-1(+) activity leads to several types of anterior cells adopting the fates of their posterior sisters. These results suggest a mechanism for the invariance of blastomere lineages.

Carriers of chromosomal inversions or other balanced rearrangements represent a significant fraction of patients in in-vitro fertilization (IVF) programmes due to recurrent reproductive problems. In most cases, chromosomal imbalance in fertilized oocytes is incompatible with embryo survival leading to increased rates of spontaneous abortions. Assuming that a fraction of the germ cells is karyotypically normal, these patients would greatly benefit from efficient procedures for generation and use of breakpoint-specific DNA hybridization probes in preconception and preimplantation genetic diagnosis (PGD). We describe the generation of such patient-specific probes to discriminate between normal and aberrant chromosomes in interphase cells. First, a large insert DNA library was screened for probes that bind adjacent to the chromosomal breakpoints or span them. Then, probe and hybridization parameters were optimized using white blood cells from the carrier to increase in hybridization signal intensity and contrast. Finally, the probes were tested on target cells (typically polar bodies or blastomeres) and a decision about the colour labelling scheme was made, before the probes can be used for preconception or preimplantation genetic analysis. Thus, it was demonstrated that cells with known structural abnormalities could be detected, based on hybridization of breakpoint spanning yeast artificial chromosome (YAC) DNA probes in interphase cells.

The E proteins of mammals, and the related Daughterless (DA) protein of Drosophila, are ubiquitously expressed helix-loop-helix (HLH) transcription factors that play a role in many developmental processes. We report here the characterization of a related C. elegans protein, CeE/DA, which has a dynamic and restricted distribution during development. CeE/DA is present embryonically in neuronal precursors, some of which are marked by promoter activity of a newly described Achaete-scute-like gene hlh-3. In contrast, we have been unable to detect CeE/DA in CeMyoD-positive striated muscle cells. In vitro gel mobility shift analysis detects dimerization of CeE/DA with HLH-3 while efficient interaction of CeE/DA with CeMyoD is not seen. These studies suggest multiple roles for CeE/DA in C. elegans development and provide evidence that both common and alternative strategies have evolved for the use of related HLH proteins in controlling cell fates in different species.

Objective: To describe an investigation of the feasibility of carrying out polymerase chain reaction (PCR) and fluorescent in situ hybridization on the same single human cell for sex determination., Design: To develop protocol for a clinical diagnostic test in preclinical trials., Setting: Infertile human volunteers in a clinical environment., Patients: Polyploid embryos were obtained from patients treated by IVF at the Advanced Institute of Fertility., Interventions: Seventeen blastomeres biopsied from human polyploid embryos were analyzed for sex determination by simultaneous application of PCR and fluorescent in situ hybridization., Main Outcome Measure: Presence of an X- or X and Y-chromosome band after agarose gel electrophoresis of PCR products and presence of an X- or X and Y-chromosome fluorescent probe signal after fluorescent in situ hybridization following PCR analysis., Results: By PCR, all 17 blastomeres were amplified and, by fluorescent in situ hybridization, 12 (70.6%) blastomeres produced signals that were consistent with PCR results. Two blastomeres showed only X signals, although by PCR they had both X and Y-chromosome bands., Conclusions: The sequential use of PCR and fluorescent in situ hybridization on the same blastomere can be applied to improve the accuracy of sex determination before fresh ET.

Objective: To determine aneuploidy for chromosome 16 by recycling nuclei of cells already analyzed for chromosomes X, Y, 18, 13, and 21 using multiple fluorescence in situ hybridization in preimplantation human embryos in a time frame compatible with clinical IVF and to asssess the incidence of chromosome 16 aneuploidy in embryos related to maternal age., Design: Prospective experimental study., Setting: In vitro fertilization program in a tertiary center., Patients: One hundred four consenting patients undergoing IVF., Main Outcome Measures: Chromosome 16 ploidy was analyzed in a total of 195 embryos. In 89 embryos, a standard multiple-probe fluorescence in situ hybridization was used for chromosomes X, Y, 18 and 16 (series 1). The remaining 106 embryos (series 2) were reanalyzed with a new procedure for chromosome 16, which involves rehybridization with a digoxigenin-labeled alpha satellite probe after the standard analysis for chromosomes X, Y, 18, 13, and 21 was completed. The embryos were assigned to one of three groups according to the women's age; group 1: /= 40 years (n = 23)., Results: Successful analysis, including biopsy, fixation, and fluorescence in situ hybridization was achieved in 86% of the blastomeres within approximately 10 hours. A significant relationship was found between the rate of aneuploidy for chromosome 16 and increasing maternal age: group 1: 0%, group 2: 6.3%, and group 3: 11.7%. Monosomy for chromosome 16 was found in 72.7% of the 11 embryos carrying chromosome 16 anomalies, with the remaining three embryos having two trisomies and one tetrasomy. This new protocol was applied clinically to five patients undergoing preimplantation aneuploidy assessment. Aneuploidy for chromosome 16 was found in five embryos from three of those patients., Conclusions: This study demonstrates that preimplantation genetic diagnosis of the major human aneuploidies is achievable within a time frame compatible with IVF. In addition, this study confirms, for embryos, the existing data from spontaneous abortions suggesting that chromosome 16 aneuploidy increases with maternal age. The high prevalence of embryonic monosomy, which is rarely found in spontaneous abortions, suggests that monosomy 16 could be a factor associated with failure of implantation, as well as pointing to a different mechanism involved in the generation of chromosome 16 aneuploidy.

An understanding of the relationship between nuclear morphology and DNA function is important in cytology and preimplantation diagnosis. In this study, direct polymerase chain reaction (PCR) amplification was used to diagnose the common delta F508 mutation of cystic fibrosis in 62 biopsied human embryo cells. The nuclei were photographed and classified into three categories depending on their microscopic appearance; these were further correlated with the results of PCR amplification. The normal nucleus group (42 embryo cells, with clear and regular nuclear membrane, transparent nucleoplasm and prominent nucleoli) showed 100% PCR amplification, with normal amplification results, i.e. bright DNA bands. These were considered to be the living cells. Only half of the cells (10 embryo cells) which contained abnormal nuclei (with abnormal nuclear membranes or nucleoplasm) showed PCR amplification, often with abnormal amplification results, i.e. weak DNA bands. These cells were considered to be either degenerate or to be undergoing degeneration. The anuclear cells (10 embryo cells) were composed of living (metaphase) and degenerated cells and showed about 30% PCR amplification. These results demonstrated that one of the important signs of early visible cell degeneration is the partial or total degeneration of the nucleus. Abnormal morphological changes of the nuclear membrane and nucleoplasm are usually accompanied with functional and structural DNA alteration. It is suggested that base degradation occurs earlier than the breakage of base-sugar bonds and phosphodlester bonds during the course of DNA degradation. The selection of optimal cells with a normal nucleus for single cell embryo biopsy is important for the precision and safety of preimplantation diagnosis.

The par-3 gene is required for establishing polarity in early C. elegans embryos. Embryos from par-3 homozygous mothers show defects in segregation of cytoplasmic determinants and in positioning of the early cleavage spindles. We report here that the PAR-3 protein is asymmetrically distributed at the periphery of the zygote and asymmetrically dividing blastomeres of the germline lineage. The PAR-3 distribution is roughly the reciprocal of PAR-1, another protein required for establishing embryonic polarity in C. elegans. Analysis of the distribution of PAR-3 and PAR-1 in other par mutants reveals that par-2 activity is required for proper localization of PAR-3 and that PAR-3 is required for proper localization of PAR-1. In addition, the distribution of the PAR-3 protein correlates with differences in cleavage spindle orientation and suggests a mechanism by which PAR-3 contributes to control of cleavage pattern.

Two injection methods were examined for making chimeras between Chinese pigs (Meishan) and European pigs (Landrace or Landrace x Large White). Furthermore, mitochondrial DNA (mtDNA) polymorphism was detected as a cell marker for the analysis of chimerism. In the first experiment, blastomeres were transplanted into embryos at the 4-16-cell stage. Of 41 transplanted embryos transferred into 3 females, 12 were single-colored, but no overt chimeras were obtained. Judging from coat color and mtDNA in white blood cells, 2 piglets in 2 litters were derived from injected blastomeres, and 10 piglets in 3 litters were derived from recipient blastomeres. In the second experiment, inner cell mass cells of Day 6 Landrace embryos were injected into blastocoels of Day 6 Meishan embryos. Of 35 injected embryos transferred into 3 females, 2 overt chimeras of each sex were obtained in a single litter. In the overt male chimera, mtDNA clearly showed chimerism in spleen, pancreas, brain, kidney, lung, liver, heart, testis, and small intestine. The overt female chimera showed chimerism not only in blood but also in germ line according to a progeny test. No chimerism was detected in any of the 21 single-colored piglets in the second experiment.

The luminal environment of the estrogen (E2)-dominated mammalian oviduct generates and sustains the environment in which the first embryonic cleavages take place. The objective of this study was to determine, by use of an antiserum against an E2-dependent sheep oviduct secretory glycoprotein (M(r) 90,000-92,000), whether the E2-dominated and pregnant oviduct of the Djungarian hamster (Phodopus sungorus) releases an antigenically related protein. If the protein was present, a secondary objective was to define its fate and association with filamentous-actin (f-actin) and chromatin patterns in early cleavage-stage embryos. Oviduct flushings containing embryos (1-cell fertilized, 2-, 4-, and 8-cells), and uterine flushings (> 16 cell embryos) were obtained from pregnant hamsters. Embryos were removed from flushings, and oviduct secretions were analyzed by Western blotting. The zona pellucida was removed with acid Tyrode's solution from approximately half of the 2-, 4-, and 8-cell embryos. Zona-intact and zona-free embryos were then fixed and subjected to triple immunofluorescence staining with an antiserum to the sheep oviduct protein, rhodamine phalloidin, and Hoechst 33258. An antigenically related protein M(r) 200,000) was detected in oviduct secretions of E2-treated, ovariectomized, and pregnant hamsters, and not in secretions from ovariectomized controls. In the zona pellucida of 1- and 2-cell embryos, the oviduct protein displayed an intertwining, reticular organization that was replaced by a diffuse and more intense accumulation in 4-, 8-, and > 16-cell embryos. In 2-cell embryos, punctate foci of the oviduct protein were distributed unevenly over the apical blastomere plasma membrane, forming patches in regions of f-actin exclusion, which were absent at later development stages. At the 4- and 8-cell stage of development, as blastomeres lost their spherical form by minimizing intercellular spaces, the oviduct protein took on a polarized arrangement and was intensely concentrated on membrane areas involved in cell-cell contact that were also the focus of f-actin. These data show that in early cleavage-stage hamster embryos, the intensity and pattern of staining for an E2-dependent oviduct protein (M(r) 200,000) that is released into the oviduct lumen during embryo transport can be distinguished on the basis of membrane f-actin display and blastomere number and shape. These events may mediate cellular processes related to blastomere cleavage, shaping, and/or adhesion that occur in the oviduct.

The isolation of the cortex of the sea urchin blastomere by detergent lysis was explored with the aim of analyzing components important in the structure and function of the cortical cytoskeleton, and their relationship to such phenomena as contraction. Buffered EGTA medium supplemented with isotonic glycerol and with magnesium, at a level close to the reported internal cellular concentration, yields stable cytoskeletal cortices that retain their spherical shape. Cortices prepared this way contain actin, myosin, fascin and spectrin, components normally associated with the cortical cytoskeleton in a similar distribution to that in intact zygotes. They retain the organized cortical filamentous structure, including the actin-fascin bundles that form cores of microvilli. ATP and NaCl caused changes in cortical shape, described as either contraction or expansion, respectively. Spectrin, but not myosin, was partially extracted by NaCl, resulting in expansion of the cortex that suggests a role for spectrin in maintenance of cortical structure. ATP (but not ADP nor ATP gamma S), which caused the partial removal of myosin and spectrin, led to the contraction of the cortex, consistent with a role for myosin in cortical tension. In cortices isolated from dividing eggs, the zygotes retained their cleavage furrows and ATP induced continuation of furrow progression. This preparation appears to be a useful in vitro model for cytokinesis.

Two homeobox-containing genes that belong to different homeodomain classes have been isolated from a sea urchin genomic library. One, PlHbox11, is the sea urchin homologue of the human and mouse Hox B3 gene, the other, PlHbox12, shows about 55% identity with paired class genes. Expression profile analysis of the two sea urchin Hbox genes suggests that they play different roles during embryogenesis. In fact, PlHbox11 transcripts are rare and are detected only in the pluteus larva and in the Aristotle's lantern and intestine of the adult. The PlHbox12 gene is, on the contrary, transiently expressed in the very early embryo already at the four cell stage; it accumulates at the 64 cell stage and disappears at later stages of development. In situ hybridization experiments to 16 and 32 cell stage whole mount embryos showed localization of the PlHbox12 mRNA to part of the mesomere-macromere region of the early cleavage embryo. These observations suggest a possible role of this gene in early events of cell specification.

The V-region represents one of three alternatively spliced segments in Xenopus fibronectin. Here, we identify this V-region as binding epitope of the monoclonal antibody (MAb 6D9) that we generated against Xenopus plasma fibronectin. By the use of this antibody we obtained new results that change the present view of the fibronectin expression pattern before gastrulation: (1) the V(+)-fibronectin is the major isoform expressed during early development since only a single fibronectin band is found in Western blots up to tadpole stages. (2) In contrast to previously published data we demonstrate that fibronectin expression is induced by progesterone during oocyte maturation. (3) During cleavage stages the protein is stored in the cytoplasm where it is predominantly associated with plasma membranes. Immunoelectronmicroscopy reveals that V(+)-fibronectin is present at the surface of animal pole blastomeres and secreted into intercellular spaces. This extracellular localization of fibronectin is predominantly observed in the marginal zone, surrounding single cells of the outer cell layer baso-laterally. In the vegetal hemisphere V(+)-fibronectin is restricted to the cytoplasm and accumulated at plasma membranes. With the onset of gastrulation the intracellular and membrane associated fibronectin disappears and fibronectin becomes detectable at the blastocoel roof. Since reaggregation of dissociated blastula cells was not blocked by addition of GRGDS peptide or antibodies against fibronectin, we assume that the early expression and secretion of fibronectin serves as store to allow a rapid matrix assembly with onset of mesodermal cell migration.

The objectives of the present study were to develop an antibody probe to the porcine estrogen-dependent oviductal glycoproteins and to determine, by use of immunogold electron microscopy, whether these glycoproteins become associated with oviductal and uterine oocytes and early embryos. Polyclonal antibody, prepared using the M(r) 75,000-85,000 glycoprotein, separated from other proteins by two-dimensional SDS-PAGE, specifically recognized all three estrogen-dependent glycoproteins (acidic 75,000-85,000 M(r); acidic 100,000 M(r); basic 100,000 M(r)). In ampullary tissue collected from ovariectomized and estrogen-treated gilts and from gilts at Day 1 of estrus, gold particles were clustered over putative secretory granules restricted to the apical region of secretory epithelial cells. While follicular oocytes did not react with immunoreactive colloidal gold, oviductal and uterine unfertilized oocytes were found to be densely and uniformly labeled by colloidal gold throughout the zona pellucida, associated with flocculent material in the perivitelline space, and associated with microvilli and vitelline membrane. Similarly, in oviductal (1-4-cell) and unhatched uterine (4-cell/blastocyst) embryos, colloidal gold particles were distributed throughout the zona pellucida, heavily associated with flocculent material in the perivitelline space, and associated with the plasma membrane of the blastomeres. Immunoreactive colloidal gold remained detectable within Day 7 hatched uterine embryos, but not with embryos from later days. These results further support the proposal that porcine estrogen-dependent oviductal glycoproteins are released into the oviductal lumen, become associated with oviductal and uterine oocytes and early embryos, and are retained by oocytes and early embryos in the uterus.

Detection of genes known to be present on the mammalian Y chromosome was adapted for sexing mouse early embryos using the polymerase chain reaction (PCR) method. Sry and Zfy genes located in the sex-determining region of the Y chromosome were chosen for Y-specific target sequences, and DXNds3 sequence on the X chromosome was chosen for control. The two-step PCR method using two pairs of primers for each of the target sequences was employed for detecting the sequences. When DNAs of male and female mice were amplified with these primers, male-specific fragments were detected even in DNAs that were equivalent in amount to two cells. Mouse embryos at the two-cell stage were separated into two individual blastomeres, and one blastomere was karyotyped at the second cleavage. The remaining blastomere was subjected to PCR amplification immediately or after having been cultured for 48 h up to the morula stage. The Sry and Zfy sequences were detected in about half the embryos; detection of the Sry and Zfy sequences corresponded exactly to the presence of the Y chromosome, except in one sample of male morula in which embryos may have been lost before the PCR amplification. It is concluded that the sex of mouse preimplantation embryos can be accurately determined through detection of the Y-specific sequences using the two-step PCR method, even with the single blastomeres separated at the two-cell stage.

A gastrula cDNA library was screened using a cDNA probe encoding the cytoplasmic domain of uvomorulin, a mouse Ca(2+)-dependent cell adhesion molecule. A Xenopus cDNA clone was isolated, which shares an amino acid sequence identity with uvomorulin of 91% in the transmembrane and 89% in the cytoplasmic domain. A restriction fragment of 397 bp representing the lowest degree of identity to all other known cadherin sequences was used to study the expression pattern of this Xenopus cadherin gene on RNA and protein level. The 397 bp restriction fragment was expressed bacterially as fusion protein, against which polyclonal antibodies were raised. An mRNA of 3.9 kb and a corresponding 125 kDa glycoprotein could be identified. Both molecules are present throughout oogenesis and early embryogenesis. When cleavage starts, the protein becomes integrated into the newly formed membranes. This polypeptide is found at cell membranes of all blastomeres except those at the outer surface of the embryo. Immunoblots and immunohistological analyses of adult organs reveal that this protein is expressed in pituitary gland, lung and kidney. It could not be detected in liver, heart and skeletal muscle. Since this cadherin differs in its tissue distribution from that of U-cadherin and in sequence alignments from ep-cadherin, it was termed XBcad for Xenopus blastomere cadherin.