Studies using animal models demonstrated the importance of autocrine/paracrine factors secreted by preimplantation embryos and reproductive tracts for embryonic development and implantation. Although in vitro fertilization-embryo transfer (IVF-ET) is an established procedure, there is no evidence that present culture conditions are optimal for human early embryonic development. In this study, key polypeptide ligands known to be important for early embryonic development in animal models were tested for their ability to improve human early embryo development and blastocyst outgrowth in vitro. We confirmed the expression of key ligand/receptor pairs in cleavage embryos derived from discarded human tri-pronuclear zygotes and in human endometrium. Combined treatment with key embryonic growth factors (brain-derived neurotrophic factor, colony-stimulating factor, epidermal growth factor, granulocyte macrophage colony-stimulating factor, insulin-like growth factor-1, glial cell-line derived neurotrophic factor, and artemin) in serum-free media promoted >2.5-fold the development of tri-pronuclear zygotes to blastocysts. For normally fertilized embryos, day 3 surplus embryos cultured individually with the key growth factors showed >3-fold increases in the development of 6-8 cell stage embryos to blastocysts and >7-fold increase in the proportion of high quality blastocysts based on Gardner’s criteria. Growth factor treatment also led to a 2-fold promotion of blastocyst outgrowth in vitro when day 7 surplus hatching blastocysts were used. When failed-to-be-fertilized oocytes were used to perform somatic cell nuclear transfer (SCNT) using fibroblasts as donor karyoplasts, inclusion of growth factors increased the progression of reconstructed SCNT embryos to >4-cell stage embryos. Growth factor supplementation of serum-free cultures could promote optimal early embryonic development and implantation in IVF-ET and SCNT procedures. This approach is valuable for infertility treatment and future derivation of patient-specific embryonic stem cells.
Studies on new arthropod models such as the beetle Tribolium castaneum are shifting our knowledge of embryonic patterning and morphogenesis beyond the Drosophila paradigm. In contrast to Drosophila, Tribolium embryos exhibit the short-germ type of development and become enveloped by extensive extra-embryonic membranes, the amnion and serosa. The genetic basis of these processes has been the focus of active research. Here, we complement genetic approaches with live fluorescence imaging of Tribolium embryos to make the link between gene function and morphogenetic cell behaviors during blastoderm formation and differentiation, germband condensation and elongation, and extra-embryonic development. We first show that transient labeling methods result in strong, homogeneous and persistent expression of fluorescent markers in Tribolium embryos, labeling the chromatin, membrane, cytoskeleton or combinations thereof. We then use co-injection of fluorescent markers with dsRNA for live imaging of embryos with disrupted caudal gene function caused by RNA interference. Using these approaches, we describe and compare cell and tissue dynamics in Tribolium embryos with wild-type and altered fate maps. We find that Tribolium germband condensation is effected by cell contraction and intercalation, with the latter being dependent on the anterior-posterior patterning system. We propose that germband condensation drives initiation of amnion folding, whereas expansion of the amniotic fold and closure of the amniotic cavity are likely driven by contraction of an actomyosin cable at the boundary between the amnion and serosa. Our methodology provides a comprehensive framework for testing quantitative models of patterning, growth and morphogenetic mechanisms in Tribolium and other arthropod species.
Upon activation, mammalian eggs release billions of zinc ions in an exocytotic event termed the “zinc spark.” The zinc spark is dependent on and occurs coordinately with intracellular calcium transients, which are tightly associated with embryonic development. Thus, we hypothesized that the zinc spark represents an early extracellular physicochemical marker of the developmental potential of the zygote. To test this hypothesis, we monitored zinc exocytosis in individual mouse eggs following parthenogenetic activation or in vitro fertilization (IVF) and tracked their development. Retrospective analysis of zinc spark profiles revealed that parthenotes and zygotes that developed into blastocysts released more zinc than those that failed to develop. Prospective selection of embryos based on their zinc spark profile significantly improved developmental outcomes and more than doubled the percentage of embryos that reached the blastocyst stage. Moreover, the zinc spark profile was also associated with embryo quality as the total cell number in the resulting morulae and blastocysts positively correlated with the zinc spark amplitude (R = 0.9209). Zinc sparks can thus serve as an early biomarker of zygote quality in mouse model.
Islet transplantation is an established therapy for diabetes. We have previously shown that rat pancreata can be created from rat pluripotent stem cells (PSCs) in mice through interspecies blastocyst complementation. Although they were functional and composed of rat-derived cells, the resulting pancreata were of mouse size, rendering them insufficient for isolating the numbers of islets required to treat diabetes in a rat model. Here, by performing the reverse experiment, injecting mouse PSCs into Pdx-1-deficient rat blastocysts, we generated rat-sized pancreata composed of mouse-PSC-derived cells. Islets subsequently prepared from these mouse-rat chimaeric pancreata were transplanted into mice with streptozotocin-induced diabetes. The transplanted islets successfully normalized and maintained host blood glucose levels for over 370 days in the absence of immunosuppression (excluding the first 5 days after transplant). These data provide proof-of-principle evidence for the therapeutic potential of PSC-derived islets generated by blastocyst complementation in a xenogeneic host.
Tight control over the segregation of endoderm, mesoderm, and ectoderm is essential for normal embryonic development of all species, yet how neighboring embryonic blastomeres can contribute to different germ layers has never been fully explained. We postulated that microRNAs, which fine-tune many biological processes, might modulate the response of embryonic blastomeres to growth factors and other signals that govern germ layer fate. A systematic screen of a whole-genome microRNA library revealed that the let-7 and miR-18 families increase mesoderm at the expense of endoderm in mouse embryonic stem cells. Both families are expressed in ectoderm and mesoderm, but not endoderm, as these tissues become distinct during mouse and frog embryogenesis. Blocking let-7 function in vivo dramatically affected cell fate, diverting presumptive mesoderm and ectoderm into endoderm. siRNA knockdown of computationally predicted targets followed by mutational analyses revealed that let-7 and miR-18 down-regulate Acvr1b and Smad2, respectively, to attenuate Nodal responsiveness and bias blastomeres to ectoderm and mesoderm fates. These findings suggest a crucial role for the let-7 and miR-18 families in germ layer specification and reveal a remarkable conservation of function from amphibians to mammals.
Increasing evidence supports an association between exposure to endocrine disruptors, such as the xenoestrogen bisphenol A (BPA), a commonly used plasticiser, and the developmental programming of offspring health. To date however animal studies to investigate a direct causal have mainly focussed on supra-environmental BPA concentrations, without investigating the effect on the early embryo. In this study we investigated the effect of acute BPA exposure (days 3.5 to 7.5 post-fertilisation) at environmentally relevant concentrations (1 and 10 ng/mL) on in vitro bovine embryo development, quality and metabolism. We then examined whether culturing embryos in the presence of the oestrogen receptor inhibitor fulvestrant could negate effects of BPA and 17β-oestradiol (E2). Exposure to BPA or E2 (10 ng/mL) decreased blastocyst rate and the percentage of transferrable quality embryos, without affecting cell number, lineage allocation or metabolic gene expression compared to untreated embryos. Notably, blastocysts exposed to BPA and E2 (10 ng/mL) displayed an increase in glucose consumption. The presence of fulvestrant however negated the adverse developmental and metabolic effects, suggesting BPA elicits its effects via oestrogen-mediated pathways. This study demonstrates that even acute exposure to an environmentally relevant BPA concentration can affect early embryo development and metabolism. These may have long-term health consequences on an individual.
The epiblast (EPI) and the primitive endoderm (PE), which constitute foundations for the future embryo body and yolk sac, build respectively deep and surface layers of the inner cell mass (ICM) of the blastocyst. Before reaching their target localization within the ICM, the PE and EPI precursor cells, which display distinct lineage-specific markers, are intermingled randomly. Since the ICM cells are produced in two successive rounds of asymmetric divisions at the 8→16 (primary inner cells) and 16→32 cell stage (secondary inner cells) it has been suggested that the fate of inner cells (decision to become EPI or PE) may depend on the time of their origin. Our method of dual labeling of embryos allowed us to distinguish between primary and secondary inner cells contributing ultimately to ICM. Our results show that the presence of two generations of inner cells in the 32-cell stage embryo is the source of heterogeneity within the ICM. We found some bias concerning the level of Fgf4 and Fgfr2 expression between primary and secondary inner cells, resulting from the distinct number of cells expressing these genes. Analysis of experimental aggregates constructed using different ratios of inner cells surrounded by outer cells revealed that the fate of cells does not depend exclusively on the timing of their generation, but also on the number of cells generated in each wave of asymmetric division. Taking together, the observed regulatory mechanism adjusting the proportion of outer to inner cells within the embryo may be mediated by FGF signaling.
In vitro culture conditions and certain events during the earliest stages of development are linked to embryonic survival, possibly in a sex-related manner. In vitro-produced (IVP) porcine embryos in vitro cultured with glucose (IVC-Glu) or pyruvate-lactate (IVC-PL) were tested for any relationship between the timing of the first embryonic cleavage and development and sex ratio. The embryos were assigned to IVC-Glu or IVC-PL groups and classified depending on the timing of their first cleavage: 24, 26, 30, and 48 hours post-insemination (hpi). They were cultured separately and evaluated for cleavage rate and pattern, blastocyst rate and stage, cell number, apoptosis, and sex ratio. Regardless of energy source, the percentage of 2-cell stage and fragmented embryos at the time of their first cleavage was respectively higher and lower in early-cleaving embryos. Those embryos cleaved by 24 hpi developed to blastocysts at a higher rate (IVC-Glu: 37.90 ± 3.06%; IVC-PL: 38.73 ± 4.08%) than those cleaved between 30-48 hpi (IVC-Glu: 5.87 ± 3.02%; IVC-PL: 8.41 ± 3.50%). Furthermore, a shift toward males was seen among embryos first cleaved before 30 hpi, versus towards females among those cleaved later. The early-cleaving embryos, only from the IVC-PL group, had a higher proportion of expanded blastocysts (81.05 ± 6.54% vs. 13.33 ± 13.33%) with higher cell numbers than their late-cleaving counterparts. Moreover, a shift toward males only appeared at the blastocyst stage in IVC-PL embryos. These findings confirm that the timing of the first cleavage influences development of IVP porcine embryos in a sex-related manner, and it depends on the main energy source of the in vitro culture medium. Mol. Reprod. Dev. © 2013 Wiley Periodicals, Inc.
What is the impact on clinical pregnancy rates when vitrified cleavage stage Day 3 embryos, warmed and cultured overnight to Day 4, are transferred on the 3rd or 5th day of progesterone administration in an artificial cycle?
Stereotypic cleavage patterns play a crucial role in cell fate determination by precisely positioning early embryonic blastomeres. Although misplaced cell divisions can alter blastomere fates and cause embryonic defects, cleavage patterns have been modified several times during animal evolution. However, it remains unclear how evolutionary changes in cleavage impact the specification of blastomere fates. Here, we analyze the transition from spiral cleavage - a stereotypic pattern remarkably conserved in many protostomes - to a biradial cleavage pattern, which occurred during the evolution of bryozoans.