Four-cell Stage Research Articles

Expression patterns and biological function of BCL2L10 during mouse preimplantation development

BCL2-like 10 (BCL2L10) is abundantly expressed in mammalian oocytes and plays a crucial role in the completion of oocyte meiosis. However, the expression patterns of BCL2L10 and its biological functions during preimplantation development have not been well characterized. Here, we investigated the spatiotemporal expressions of Bcl2l10 during mouse preimplantation development using RT-qPCR and immunofluorescence and its biological function using siRNA and morpholino injection into pronuclear embryos. Results from RT-qPCR showed that Bcl2l10 was highly expressed in the metaphase Ⅱ-stage oocytes and pronuclear-stage embryos, but expression markedly decreased from the two-cell stage onwards and was no longer detected at the four-cell stage and beyond. Immunofluorescence staining showed that BCL2L10 was detectable throughout preimplantation development and localized in the cytoplasm and nuclei. Knocking down Bcl2l10 resulted in a reduced blastocyst formation rate (P < 0.01) and decreased expression of OCT4, NANOG, and SOX17 (P < 0.05). We concluded that the role of BCL2L10 is strongly associated with developmental competence of preimplantation mouse embryos.

Nucleation status of Day 2 pre-implantation embryos, acquired by time-lapse imaging during IVF, is associated with live birth.

The primary purpose of this time-lapse data analysis was to identify the association between the nucleation status of a Day 2 preimplantation embryo and live births following in vitro fertilization (IVF). The retrospective data analysis was based on 2769 transferred embryos from 1966 treatment cycles and utilised only Known Implantation Data (KID) for live births. Nucleation errors (NE) such as micronucleation, binucleation, multinucleation and minor error groups, were annotated in the time-lapse images which were taken every 15 minutes for a minimum of 44 hours post insemination. Further, factors that may impact NE and the relationship of early morphological attributes and morphokinetic variables with NE occurrence were explored. The frequency of NE among the transferred embryos was 23.8%. The reversibility of NE evidenced by their presence at the two-cell stage, but absence at the four-cell stage was 89.6%. Embryos exhibiting nucleation errors at the two-cell stage had significantly lower live birth rates compared to embryos with no nucleation errors, constituting a significant predictor. A Generalized Additive Mixed Model was used to control for confounders and for controlling clustering effects from dual embryo transfers. Increased incidences of NE were observed with increasing age, with delayed occurrence of cell divisions and in oocytes inseminated with surgically retrieved spermatozoa. NE assessment and their impact on live birth provides valuable markers for early preimplantation embryo selection. In addition, the high incidence of reversibility of NE and their possible impact on live birth suggest that incorporating two-cell nuclear status annotations in embryo selection, alongside morphology and morphokinetics, is of value.

Dynamic and aberrant patterns of H3K4me3, H3K9me3, and H3K27me3 during early zygotic genome activation in cloned mouse embryos.

Somatic cell nuclear transfer (NT) is associated with aberrant changes in epigenetic reprogramming that impede the development of embryos, particularly during zygotic genome activation. Here, we characterized epigenetic patterns of H3K4me3, H3K9me3, and H3K27me3 in mouse NT embryos up to the second cell cycle (i.e. four-celled stage) during zygotic genome activation. In vivo fertilized and parthenogenetically activated (PA) embryos served as controls. In fertilized embryos, maternal and paternal pronuclei exhibited asymmetric H3K4me3, H3K9me3, and H3K27me3 modifications, with the paternal pronucleus showing delayed epigenetic modifications. Higher levels of H3K4me3 and H3K9me3 were observed in NT and PA embryos than in fertilized embryos. However, NT embryos exhibited a lower level of H3K27me3 than PA and fertilized embryos from pronuclear stage 3 to the four-celled stage. Our finding that NT embryos exhibited aberrant H3K4me3, H3K9me3, and H3K27me3 modifications in comparison with fertilized embryos during early zygotic genome activation help to unravel the epigenetic mechanisms of methylation changes in early NT reprogramming and provide an insight into the role of histone H3 in the regulation of cell plasticity during natural reproduction and somatic cell NT.

High oocyte immaturity rates affect embryo morphokinetics: lessons of time-lapse imaging system

Research questionDoes oocyte immaturity rate affect morphokinetic events in a time-lapse imaging (TLI) system? DesignHistorical cohort study carried out in a private university-affiliated IVF centre. Injected oocytes (n = 3368) cultured in a TLI incubator, from intracytoplasmic sperm injection (ICSI) cycles (n = 474) carried out between March 2019 and December 2020, were analysed. The effects of immature oocyte rates (the number of germinal-vesicle and metaphase I oocytes by the number of retrieved oocytes in each cycle, on morphokinetic events) were investigated considering clustering of data using mixed models. Evaluated kinetic markers were pronuclei appearance (tPNa), timing to pronuclei fading (tPNf), timing to two (t2), three (t3), four (t4), five (t5), six (t6), seven (t7), and eight cells (t8), timing to morulae (tM) and timing to start of blastulation (tSB) and to blastulation (tB). Durations of the second (t3-t2) and third (t5-t3) cell cycles (cc2 and cc3, respectively) and timing to complete synchronous divisions s1 (t2-tPNf), s2 (t4-t3) and s3 (t8-t5) were also evaluated. ResultsPositive relationships were observed between oocyte immaturity rates and slower tPNa, tPNf, t2, t3, t4, t5, t6, t7, t8, tSB, tB and cc3. Multinucleation at two- and four-cell stages were positively correlated with oocyte immaturity rate. The KIDScore ranking was negatively correlated with oocyte immaturity rate. No associations were found between oocyte immaturity rate and clinical outcomes. ConclusionsIncreasing oocyte immaturity rate correlates with delayed cell cleavage and blastulation. These findings highlight the importance of TLI for the identification and de-selection of slow-growing embryos for transfer, in cycles with high oocyte immaturity rate.

Morphological and morphokinetic associations with aneuploidy: a systematic review and meta-analysis.

A time lapse system (TLS) is utilized in some fertility clinics with the aim of predicting embryo viability and chance of live birth during IVF. It has been hypothesized that aneuploid embryos display altered morphokinetics as a consequence of their abnormal chromosome complement. Since aneuploidy is one of the fundamental reasons for IVF failure and miscarriage, attention has focused on utilizing morphokinetics to develop models to non-invasively risk stratify embryos for ploidy status. This could avoid or reduce the costs associated with pre-implantation genetic testing for aneuploidy (PGT-A). Furthermore, TLS have provided an understanding of the true prevalence of other dysmorphisms. Hypothetically, the incorporation of morphological features into a model could act synergistically, improving a model's discriminative ability to predict ploidy status. The aim of this systematic review and meta-analysis was to investigate associations between ploidy status and morphokinetic or morphological features commonly denoted on a TLS. This will determine the feasibility of a prediction model for euploidy and summarize the most useful prognostic markers to be included in model development. Five separate searches were conducted in Medline, Embase, PubMed and Cinahl from inception to 1 July 2021. Search terms and word variants included, among others, PGT-A, ploidy, morphokinetics and time lapse, and the latter were successively substituted for the following morphological parameters: fragmentation, multinucleation, abnormal cleavage and contraction. Studies were limited to human studies. Overall, 58 studies were included incorporating over 40000 embryos. All except one study had a moderate risk of bias in at least one domain when assessed by the quality in prognostic studies tool. Ten morphokinetic variables were significantly delayed in aneuploid embryos. When excluding studies using less reliable genetic technologies, the most notable variables were: time to eight cells (t8, 1.13 h, 95% CI: 0.21-2.05; three studies; n = 742; I2 = 0%), t9 (2.27 h, 95% CI: 0.5-4.03; two studies; n = 671; I2 = 33%), time to formation of a full blastocyst (tB, 1.99 h, 95% CI 0.15-3.81; four studies; n = 1640; I2 = 76%) and time to expanded blastocyst (tEB, 2.35 h, 95% CI: 0.06-4.63; four studies; n = 1640; I2 = 83%). There is potentially some prognostic potential in the degree of fragmentation, multinucleation persisting to the four-cell stage and frequency of embryo contractions. Reverse cleavage was associated with euploidy in this meta-analysis; however, this article argues that these are likely spurious results requiring further investigation. There was no association with direct unequal cleavage in an embryo that progressed to a blastocyst, or with multinucleation assessed on Day 2 or at the two-cell stage. However, owing to heterogeneous results and poor-quality evidence, associations between these morphological components needs to be investigated further before conclusions can be reliably drawn. This first systematic review and meta-analysis of morphological and morphokinetic associations with ploidy status demonstrates the most useful morphokinetic variables, namely t8, t9 and tEB to be included in future model development. There is considerable variability within aneuploid and euploid embryos making definitively classifying them impossible; however, it is feasible that embryos could be prioritized for biopsy. Furthermore, these results support the mechanism by which algorithms for live birth may have predictive ability, suggesting aneuploidy causes delayed cytokinesis. We highlight significant heterogeneity in our results secondary to local conditions and diverse patient populations, therefore calling for future models to be robustly developed and tested in-house. If successful, such a model would constitute a meaningful breakthrough when accessing PGT-A is unsuitable for couples.

Evaluation of porcine urine-derived cells as nuclei donor for somatic cell nuclear transfer.

BackgroundSomatic cell nuclear transfer (SCNT) is used widely in cloning, stem cell research, and regenerative medicine. The type of donor cells is a key factor affecting the SCNT efficiency.ObjectivesThis study examined whether urine-derived somatic cells could be used as donors for SCNT in pigs.MethodsThe viability of cells isolated from urine was assessed using trypan blue and propidium iodide staining. The H3K9me3/H3K27me3 level of the cells was analyzed by immunofluorescence. The in vitro developmental ability of SCNT embryos was evaluated by the blastocyst rate and the expression levels of the core pluripotency factor. Blastocyst cell apoptosis was examined using a terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. The in vivo developmental ability of SCNT embryos was evaluated after embryo transfer.ResultsMost sow urine-derived cells were viable and could be cultured and propagated easily. On the other hand, most of the somatic cells isolated from the boar urine exhibited poor cellular activity. The in vitro development efficiency between the embryos produced by SCNT using porcine embryonic fibroblasts (PEFs) and urine-derived cells were similar. Moreover, The H3K9me3 in SCNT embryos produced from sow urine-derived cells and PEFs at the four-cell stage showed similar intensity. The levels of Oct4, Nanog, and Sox2 expression in blastocysts were similar in the two groups. Furthermore, there is a similar apoptotic level of cloned embryos produced by the two types of cells. Finally, the full-term development ability of the cloned embryos was evaluated, and the cloned fetuses from the urine-derived cells showed absorption.ConclusionsSow urine-derived cells could be used to produce SCNT embryos.

Differences in Morphokinetic Parameters and Incidence of Multinucleations in Human Embryos of Genetically Normal, Abnormal and Euploid Embryos Leading to Clinical Pregnancy.

The selection of the best embryo for embryo transfer (ET) is one of the most important steps in IVF (in vitro fertilisation) treatment. Preimplantation genetic testing (PGT) is an invasive method that can greatly facilitate the decision about the best embryo. An alternative way to select the embryo with the greatest implantation potential is by cultivation in a time-lapse system, which can offer several predictive factors. Non-invasive time-lapse monitoring can be used to select quality embryos with high implantation potential under stable culture conditions. The embryo for ET can then be selected based on the determined morphokinetic parameters and morphological features, which according to our results predict a higher implantation potential. This study included a total of 1027 morphologically high-quality embryos (552 normal and 475 abnormal PGT-tested embryos) from 296 patients (01/2016–06/2021). All embryos were cultivated in a time-lapse incubator and PGT biopsy of trophectoderm cells on D5 or D6 was performed. Significant differences were found in the morphological parameters cc2, t5 and tSB and the occurrence of multinucleations in the stage of two-cell and four-cell embryos between the group of genetically normal embryos and abnormal embryos. At the same time, significant differences in the morphological parameters cc2, t5 and tSB and the occurrence of multinucleations in the two-cell and four-cell embryo stage were found between the group of genetically normal embryos that led to clinical pregnancy after ET and the group of abnormal embryos. From the morphokinetic data found in the PGT-A group of normal embryos leading to clinical pregnancy, time intervals were determined based on statistical analysis, which should predict embryos with high implantation potential. Out of a total of 218 euploid embryos, which were transferred into the uterus after thawing (single frozen embryo transfer), clinical pregnancy was confirmed in 119 embryos (54.6%). Our results show that according to the morphokinetic parameters (cc2, t5, tSB) and the occurrence of multinucleations during the first two cell divisions, the best euploid embryo for ET can be selected with high probability.

O-223 Fatty acid supplementation into warming solutions improve the developmental competence of mouse, bovine, and human oocytes and embryos after vitrification

Abstract Study question Does fatty acid (FA) supplementation into vitrification and warming solutions influence the developmental competence of oocyte and embryo after vitrification and warming? Summary answer FA supplementation during the warming process improves the developmental competence of vitrified-warmed mouse oocytes and embryonic-morphologies after vitrification at the cleavage-stage in bovines and humans. What is known already Vitrified metaphase II stage oocytes exhibit a diminished ability to develop into blastocysts and live births. Previous studies have shown reduction in intracellular lipid content as one of the factors associated with reduced developmental competence of oocytes after vitrification as the intracellular lipid content of oocytes is affected by vitrification. FAs derived from break down of lipids are primarily transferred to the mitochondria, where it plays a crucial role in cellular metabolism. However, the effects of FA supplementation in warming solutions on the cytoplasmic lipid content and subsequent embryo development are unknown. Study design, size, duration A chemically defined FA mixture was added to the vitrification and/or warming solutions. Oocytes collected from C57BL6/N (n = 80) were randomly divided into three groups (fresh, n = 634; non-FA (control), n = 961; FA, n = 1,686), and were vitrified-warmed with/without FA. Lipid composition, developmental competence, and gene expression levels were compared among the groups. Bovine embryos (fresh, n = 420; control, n = 524; FA, n = 492) and discarded human day-2 embryos (control, n = 87; FA, n = 92) were used to examine the developmental competence of embryos. Participants/materials, setting, methods Lipids in the ooplasm were stained with Nile red and the fluorescence intensity was analysed. The developmental competence of mouse oocytes was examined by performing intracytoplasmic sperm injection. Expressions of FA metabolism-related genes were measured. The bovine embryos were vitrified at the four-cell stage and cultured to the blastocyst stage after warming. Cryopreserved discarded human embryos were warmed and cultured. The obtained blastocysts were then placed on fibronectin-coated dishes to examine the outgrowth formation. Main results and the role of chance Lipid content of mouse oocytes was significantly lower in the control group compared to that in the fresh group (P &amp;lt; 0.05). On the contrary, lipid contents of FA and fresh groups were comparable (P = 0.24). Blastocyst formation rate was significantly higher in the FA group than that in the control group (55.7% and 44.8%, respectively; P &amp;lt; 0.05). To examine the optimal timing for FA supplementation, FA was added to the vitrification solution (FAvit), warming solution (FAthaw), and/or both solutions (FAvit-thaw). Blastocyst formation rate was significantly higher in the FAthaw group than that in the control group (59.8% and 50.0%, respectively; P &amp;lt; 0.05). The mRNA expressions of Acaa2 and Hadha in mouse embryos were significantly higher in the FAthaw group compared to that in the control group (P &amp;lt; 0.05). Moreover, FA supplemented warming solutions significantly improved the blastocyst formation rate in bovines (control, 53.5%; FAthaw, 64.5%; P &amp;lt; 0.05). Developmental rate to the expanded blastocyst stage was slightly improved in human embryos (control, 53.7%; FAthaw, 63%; P = 0.38) and the proportion of Grade A in inner cell mass and trophectoderm was significantly higher in the FAthaw group than that in the control group (P &amp;lt; 0.05). There were no differences in the outgrowth abilities between the control and FAthaw groups. Limitations, reasons for caution Since the experiments of the current study on human embryos were performed in vitro using discarded embryos, in vivo developmental ability was not evaluated. Therefore, to validate the application of our findings in human assisted reproductive technologies, further clinical trials (ART) are warranted. Wider implications of the findings FA supplementation into the warming solutions improved the developmental competence of vitrified–warmed oocytes and cleaved embryos by activating the β-oxidation pathway. These results indicate that FA supplementation into warming solutions is a potential strategy to improve clinical outcomes in human ART. Trial registration number not applicable

Clathrin-mediated endocytosis is essential for the selective degradation of maternal membrane proteins and preimplantation development.

Fertilization triggers significant cellular remodeling through the oocyte-to-embryo transition. In this transition, the ubiquitin-proteasome system and autophagy are essential for the degradation of maternal components; however, the significance of degradation of cell surface components remains unknown. In this study, we show that multiple maternal plasma membrane proteins, such as the glycine transporter GlyT1a, are selectively internalized from the plasma membrane to endosomes in mouse embryos by the late two-cell stage and then transported to lysosomes for degradation at the later stages. During this process, large amounts of ubiquitylated proteins accumulated on endosomes. Furthermore, the degradation of GlyT1a with mutations in potential ubiquitylation sites was delayed, suggesting that ubiquitylation may be involved in GlyT1a degradation. The clathrin inhibitor blocked GlyT1a internalization. Strikingly, the protein kinase C (PKC) activator triggered the heterochronic internalization of GlyT1a; the PKC inhibitor markedly blocked GlyT1a endocytosis. Lastly, clathrin inhibition completely blocked embryogenesis at the two-cell stage and inhibited cell division after the four-cell stage. These findings demonstrate that PKC-dependent clathrin-mediated endocytosis is essential for the selective degradation of maternal membrane proteins during oocyte-to-embryo transition and early embryogenesis.

Regulation of the mammalian maternal-to-embryonic transition by eukaryotic translation initiation factor 4E.

ABSTRACTEukaryotic translation initiation factor 4E (eIF4E) mediates cap-dependent translation. Genetic and inhibitor studies show that eIF4E expression is required for the successful transition from maternal to embryonic control of mouse embryo development. eIF4E was present in the oocyte and in the cytoplasm soon after fertilization and during each stage of early development. Functional knockout (Eif4e−/−) by PiggyBac [Act-RFP] transposition resulted in peri-implantation embryonic lethality because of the failure of normal epiblast formation. Maternal stores of eIF4E supported development up to the two- to four-cell stage, after which new expression occurred from both maternal and paternal inherited alleles. Inhibition of the maternally acquired stores of eIF4E (using the inhibitor 4EGI-1) resulted in a block at the two-cell stage. eIF4E activity was required for new protein synthesis in the two-cell embryo and Eif4e−/− embryos had lower translational activity compared with wild-type embryos. eIF4E-binding protein 1 (4E-BP1) is a hypophosphorylation-dependent negative regulator of eIF4E. mTOR activity was required for 4E-BP1 phosphorylation and inhibiting mTOR retarded embryo development. Thus, this study shows that eIF4E activity is regulated at key embryonic transitions in the mammalian embryo and is essential for the successful transition from maternal to embryonic control of development.

Totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage

In multicellular organisms, oocytes and sperm undergo fusion during fertilization and the resulting zygote gives rise to a new individual. The ability of zygotes to produce a fully formed individual from a single cell when placed in a supportive environment is known as totipotency. Given that totipotent cells are the source of all multicellular organisms, a better understanding of totipotency may have a wide-ranging impact on biology. The precise delineation of totipotent cells in mammals has remained elusive, however, although zygotes and single blastomeres of embryos at the two-cell stage have been thought to be the only totipotent cells in mice. We now show that a single blastomere of two- or four-cell mouse embryos can give rise to a fertile adult when placed in a uterus, even though blastomere isolation disturbs the transcriptome of derived embryos. Single blastomeres isolated from embryos at the eight-cell or morula stages and cultured in vitro manifested pronounced defects in the formation of epiblast and primitive endoderm by the inner cell mass and in the development of blastocysts, respectively. Our results thus indicate that totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage.

Distinct dynamics and functions of H2AK119ub1 and H3K27me3 in mouse preimplantation embryos.

Polycomb repressive complexes 1 and 2 (PRC1/2) maintain transcriptional silencing of developmental genes largely by catalyzing the formation of mono-ubiquitinated histone H2A at lysine 119 (H2AK119ub1) and trimethylated histone H3 at lysine 27 (H3K27me3), respectively. How Polycomb domains are reprogrammed during mammalian preimplantation development remains largely unclear. Here we show that, although H2AK119ub1 and H3K27me3 are highly colocalized in gametes, they undergo differential reprogramming dynamics following fertilization. H3K27me3 maintains thousands of maternally biased domains until the blastocyst stage, whereas maternally biased H2AK119ub1 distribution in zygotes is largely equalized at the two-cell stage. Notably, while maternal PRC2 depletion has a limited effect on global H2AK119ub1 in early embryos, it disrupts allelic H2AK119ub1 at H3K27me3 imprinting loci including Xist. By contrast, acute H2AK119ub1 depletion in zygotes does not affect H3K27me3 imprinting maintenance, at least by the four-cell stage. Importantly, loss of H2AK119ub1, but not H3K27me3, causes premature activation of developmental genes during zygotic genome activation (ZGA) and subsequent embryonic arrest. Thus, our study reveals distinct dynamics and functions of H3K27me3 and H2AK119ub1 in mouse preimplantation embryos.

SIN3A Regulates Porcine Early Embryonic Development by Modulating CCNB1 Expression.

SIN3A is the central scaffold protein of the SIN3/histone deacetylase (HDAC) transcriptional repressor complex. SIN3A participates in the mouse preimplantation development by fine-tuning HDAC1 expression. However, it remains unresolved if this functional significance of SIN3A was conserved in other mammals. Herein, RNA-seq results show a large amount of SIN3A mRNA is present in oocytes and early embryos prior to embryonic genome activation and a low amount thereafter, suggesting a maternal origin of SIN3A in pigs, cattle, mice, and humans. Interestingly, immunofluorescence data show that SIN3A protein level peaks at four-cell stage in pigs compared with morula stage in cattle. SIN3A depletion in early embryos causes a developmental arrest at two-cell stage in pigs but does not affect bovine early embryonic development. In contrast with mouse data, SIN3A depletion results in only a slight decrease and even no difference in HDAC1 expression in porcine and bovine early embryos, respectively. In addition, HDAC1 knockdown does not cause two-cell block but leads to a reduced blastocyst rate. By using unbiased RNA-seq approach, we found that Cyclin B1 (CCNB1) transcript level is dramatically reduced. Moreover, CCNB1 knockdown results in a similar phenotype as SIN3A depletion. Injection of exogenous CCNB1 mRNA into SIN3A-depleted embryos could partly rescue embryonic development to pass two-cell stage. In conclusion, our results indicate SIN3A plays an essential role in porcine early embryonic development, which probably involves the regulation of CCNB1 expression.

Morphokinetics of embryos after IMSI versus ICSI in couples with sub-optimal sperm quality: A time-lapse study.

An investigation was conducted to determine the influence of two sperm selection modalities, IMSI and ICSI, on the morphokinetics, dynamic development and ploidy status of embryos derived from males with sub-optimal sperm profiles during IVF program. A total of 209 PGTA-tested top-quality blastocysts (IMSI=129, ICSI=80) from 84 couples (IMSI=51, ICSI=33) were assessed retrospectively. This study found that both IMSI and ICSI yielded comparable embryo morphokinetics, except for the T7, TEB and CC3 parameters (p<0.05). A significant lower incidence of multinucleation was observed in the IMSI group when compared to the ICSI group (48.8% vs. 71.3%, p=0.002), while other parameters of embryo development such as direct cleavage, distorted cytoplasmic movement, reverse cleavage and vacuole(s) appearance did not differ (p>0.05). No differences were noticed in the proportion of generating chromosomally euploid embryos (44.2% vs. 51.3%, p=0.394, respectively, for IMSI and ICSI). The implementation of IMSI or ICSI in couples with sub-optimal sperm profiles resulted in embryos with comparatively similar morphokinetics. Furthermore, the incidence of multinucleation at the two- to four-cell stage was lower following the practice of IMSI, although the method did not improve the proportion of gaining euploid embryos.

Evolutionary modification of gastrulation in Parvulastra exigua, an asterinid seastar with holobenthic lecithotrophic development.

Gastrulation is a fundamental morphogenetic process in development. In echinoderms with ancestral-type development through feeding larvae, gastrulation involves radially symmetrical invagination of cells around the blastopore. Gastrulation in the seastar Parvulastra exigua, a species with non-feeding larvae deviates from this pattern. Microinjection of cells with fluorescent lineage tracer dye revealed that early blastomeres contribute unequally to ectoderm and endoderm. In embryos injected at the two-cell stage, asymmetry was evident in the fluorescence at the top of the archenteron and animal pole ectoderm. Archenteron elongation is driven by asymmetrical involution of cells with more cells crossing the blastopore on one side. Lineages of cells injected at the four-cell stage also differed in allocation to endoderm and ectoderm. In embryos injected at the eight-cell stage ectodermal and endodermal fates were evident reflecting the animal and vegetal fates determined by third cleavage as typical of echinoderms. Modification of gastrulation associated with evolution of development in P. exigua shows that this foundational morphogenetic process can be altered despite its importance for subsequent development. However, observations of slight asymmetry in the lineage fates of blastomeres in asterinids with planktotrophic development indicates that gastrulation by asymmetrical involution in P. exigua may be a hypertrophic elaboration of a pre-existing state in ancestral-type development. As for echinoids with lecithotrophic development, involution as a mechanism to contribute to archenteron elongation may be associated with the impact of extensive maternal nutritive reserves on the mechanics of cell movement and a novel innovation to facilitate early development of the adult rudiment.

Time-lapse imaging derived morphokinetic variables reveal association with implantation and live birth following in vitro fertilization: A retrospective study using data from transferred human embryos.

The purpose of this retrospective time-lapse data analysis from transferred preimplantation human embryos was to identify early morphokinetic cleavage variables that are related to implantation and live birth following in vitro fertilization (IVF). All embryos were monitored from fertilization check until embryo transfer for a minimum of 44 hours. The study was designed to assess the association between day 2 embryo morphokinetic variables with implantation and live birth based on Known Implantation Data (KID). The kinetic variables were subjected to quartile-based analysis. The predictive ability for implantation and live birth was studied using receiver operator characteristic (ROC) curves. Three morphokinetic variables, time to 2-cells (t2), duration of second cell cycle (cc2) below one threshold and cc2 above another threshold had the highest predictive value with regards to implantation and live birth following IVF treatment. The predictive pre-transfer information has little divergence between fetal heartbeat and live birth data and therefore, at least for early morphokinetic variables up to the four-cell stage (t4), conclusions and models based on fetal heartbeat data can be expected to be valid for live birth datasets as well. The three above mentioned variables (t2, cc2 below one threshold and cc2 above another threshold) may supplement morphological evaluation in embryo selection and thereby improve the outcome of in vitro fertilization treatments.

Timing of cleavage divisions determined with time-lapse imaging is linked to blastocyst formation rates and quality of in vitro-produced ovine embryos

Time-lapse (TL) imaging provides a practical and safe tool to constantly monitor the development of in vitro-derived embryos. TL may help develop novel methods of predicting the timing of embryo cleavage that will lead to optimizing blastocyst cryopreservation or transfer. The primary objective of the present study was to employ TL imaging to examine associations among the division kinetics of ovine embryos, their quality and rates of development to the blastocyst stage. Oocytes were collected by ovary scarification from 78 Longwool ewes slaughtered in the breeding season (November–March). Cumulus oocyte complexes (COCs) were matured for 24 h in TCM 199 media containing 0.1 IU/mL LH/FSH and 10% FBS. In-vitro fertilization was carried out by co-incubation of semen and COCs for 19 h. Presumptive zygotes were placed in microwells, in droplets of Cult medium (Gynemed, Lensahn, Germany). Digital images of developing embryos were captured every 10 min by Primo Vision TL system (EVO+; Vitrolife, Göteburg, Sweden). The following time intervals were recorded: from IVF to the attainment of two-cell (t2), three-cells (t3) or four-cell (t4) stage, to morula detection (tM), blastulation (tSB) and blastocyst formation (tB). Lastly, the duration of the second cell cycle (cc2; t3-t2) and complete synchronous cell division (s2; t4-t3) were calculated, and the incidence of developmental anomalies noted. Out of 147 embryos selected for TL observations, 55 (37.4%) developed to the blastocyst stage (normally developing embryos, NE) and 92 (62.6%) failed to reach the blastocyst stage (arrested embryos, AE; P < 0.05). Mean t2, tM, s2 and cc2 were all less (P ≤ 0.02) in NE compared with AE. Approximately 61.9% of embryos exhibited developmental anomalies (35.5% in the NE group and 78.2% in the AE group; P < 0.05) and AE exceeded (P < 0.05) NE in the proportion of FRG (blastomeric fragmentation), IRR (blastomeres of irregular size after cleavage), DC (direct cleavage) and MA (multi-morphological aberrations). Of all NE, 63.6% were classified as good quality and 36.4% as poor quality blastocysts (P < 0.05). Good quality ovine blastocysts attained t2, t3, t4, tSB and tB stages earlier (P ≤ 0.03) than poor quality blastocysts and none of the poor quality blastocysts was seen to hatch. To recapitulate, the present results indicate that the kinetics of early ovine embryo development are significant predictors of their potential to develop to the blastocyst stage and the markers of blastocyst quality. Time-lapse imaging may serve as a useful technique for predicting the outcome and enhancing efficacy of in vitro embryo production in sheep.

Caffeine improves the developmental competence of parthenogenetic embryos derived from aging porcine oocytes

Oocytes are committed to deterioration in quality as they aged due to a long duration manipulation which leads to the reduced success rate of somatic cell nuclear transfer (SCNT). Caffeine with an effect to maintain the maturation-promoting factor (MPF) from the metaphase of oocytes is expected to enhance the quality of the aging oocytes. To investigate the timely treatment of caffeine to rescue aging oocytes, caffeine was supplemented after in vitro maturation (IVM) or during metaphase I – metaphase II (MI – MII) transition. First, the effect of caffeine after IVM of oocytes was examined. After IVM for 42 h, oocytes were left for aging within 6 or 8 hours in supplement with various concentrations of caffeine (0, 5 and 10 mM), and then, examined the quality of embryo from aged oocyte through parthenogenesis activation. We found that 5 mM caffeine for the first 6 hours of aging process was suggested to improve the early development of parthenogenetic diploid embryos. However, the cytoplasmic homogeneity is significantly reduced in aging oocyte compared to fresh oocyte and it could not be improved by caffeine treatment. Next, the effect of caffeine during MI – MII transition of oocyte was examined. Caffeine was supplemented during MI – MII transition (27 – 42 h) of IVM. Then mature oocytes were left for aging within 6 h to examine on aging porcine oocyte quality via parthenogenesis embryos. The results indicated that 5 mM caffeine during MI-MII transition could efficiently rescue aged oocytes and improve the development of embryos derived from aging oocytes to four-cell, eight-cell and blastocyst stage as compared to fresh oocytes. Especially, these aged oocytes treated by caffeine could improve the cytoplasmic homogeneity in embryos and the quality of blastocysts by increasing cell number similar to fresh oocytes.

Nuclear control of early embryonic development in domestic pigs

In mammals, growing oocytes have characteristically high levels of RNA synthesis. After the initiation of meiosis, that is germinal vesicle breakdown, this RNA synthesis ceases. Although there is limited evidence for RNA synthesis by the zygote, significant amounts of RNA synthesis do not occur until a species-specific cell stage. In pigs, significant amounts of mRNA synthesis cannot be detected before the four-cell stage. There appear to be three qualitatively different periods of transcription during the four-cell stage. The first occurs during a short (< 2 h) G1 phase. The second, occurs after completion of DNA synthesis (S phase) about 16 h after cleavage to the four-cell stage, and the third occurs about 24 h after cleavage to the four-cell stage. Correlated with these changes in RNA synthesis are changes in nucleolar morphology, amino acid transport characteristics, protein production, mitochondrial morphology, and metabolism of the embryo. The mechanisms that regulate initiation of RNA synthesis in early mammalian embryos appear to repress transcription. A state of transcription permissiveness then follows that sets into motion the differentiation programme.

In vitro fertilization and embryo development in pigs

Considerable progress has been made in the in vitro production of pig embryos using improved methods for in vitro maturation (IVM) and fertilization (IVF). Despite the progress, polyspermic penetration remains a problem for in vitro-matured oocytes. Variation among boars, ejaculates and IVF protocols used in different laboratories appears to influence the incidence of polyspermy. Recent studies indicate that oviduct cells and their secretions play a role in reducing polyspermy. Very early attempts to culture in vivo-derived pig embryos met with little success and most were arrested at the four-cell stage. At present, many culture media are available that can overcome the four-cell block and support development to the blastocyst stage. In contrast, blastocyst development of in vitro-produced (IVP) embryos in these culture media varies significantly. Significant differences in morphology and numbers of cells have been observed in in vitro-produced blastocysts compared with in vivo-derived blastocysts. Surgical transfer of in vitro-produced embryos to recipient animals has resulted in acceptable pregnancy rates with moderate litter sizes. Although several systems are available for the generation of in vitro-produced embryos, the problems of polyspermy and poor embryo survival prevent large-scale production of embryos. Further research should be directed to improve oocyte and embryo quality, and to develop methods to minimize polyspermy through development of better IVM, IVF and embryo culture techniques.