Embryonic Discs Research Articles

Human peri-gastruloids: a significant advancement in embryology research.

The peri-gastruloids comprise both embryonic (epiblast) and extraembryonic (hypoblast) tissues, faithfully mirroring crucial developmental events spanning from the immediate post-implantation phase to early organogenesis, encompassing the emergence of amniotic and yolk sac cavities, as well as the progression from bilaminar to trilaminar embryonic discs.

Modeling post-implantation stages of human development into early organogenesis with stem-cell-derived peri-gastruloids

Invitro stem cell models that replicate human gastrulation have been generated, but they lack the essential extraembryonic cells needed for embryonic development, morphogenesis, and patterning. Here, we describe a robust and efficient method that prompts human extended pluripotent stem cells to self-organize into embryo-like structures, termed peri-gastruloids, which encompass both embryonic (epiblast) and extraembryonic (hypoblast) tissues. Although peri-gastruloids are not viable due to the exclusion of trophoblasts, they recapitulate critical stages of human peri-gastrulation development, such as forming amniotic and yolk sac cavities, developing bilaminar and trilaminar embryonic discs, specifying primordial germ cells, initiating gastrulation, and undergoing early neurulation and organogenesis. Single-cell RNA-sequencing unveiled transcriptomic similarities between advanced human peri-gastruloids and primary peri-gastrulation cell types found in humans and non-human primates. This peri-gastruloid platform allows for further exploration beyond gastrulation and may potentially aid in the development of human fetal tissues for use in regenerative medicine.

Culture of pig embryos

Pig embryos can be cultured using a number of different strategies including complex approaches like culture in vivo in a surrogate oviduct (rabbit, sheep, mouse), culture in mouse oviducts in organ culture, and co-culture of embryos with cells in addition to simple approaches like culture in defined media or salt solutions. Addition of serum to medium has been of particular importance where blastocyst development and hatching are required. Pig conceptuses (day 10-15), embryonic discs or cell lines derived from conceptuses can be cultured in complex media like Eagle's minimal essential medium or Dulbecco's modified Eagle's medium with serum, although embryonic discs can be cultured in the absence of serum. In contrast, early stage pig embryos (one-cell to blastocyst) are best cultured in simpler media such as those used for mouse embryos. The media that have been used are all relatively similar in composition. They contain salts and one or more energy sources such as glucose, lactate, or pyruvate with BSA as a macromolecular component. Early attempts to culture pig embryos were not very successful, but some embryos did develop to the blastocyst stage. More recent reports indicate a much higher rate of development with relatively little or no change in media composition. Some workers have reported improved development in medium lacking glucose, which is consistent with findings with laboratory animals such as hamsters. Glutamine can serve as the sole exogenous energy source in medium lacking glucose, lactate and pyruvate. Addition of taurine and hypotaurine to culture medium enhances development of pig embryos in vitro. We suggest, where possible, adoption of a standard medium that could be used by different laboratories and, perhaps, with different species. Use of one medium for different species would simplify experimental protocols, enhance studies of comparative embryonic physiology and metabolism, and expedite transfer of information obtained in different species.

Effects of Erythrosine on Neural Tube Development in Early Chicken Embryos

Erythrosine (E127), a synthetic food dye containing iodine and sodium, has often been used inside packaged foods and beverages in Turkey and many other countries. We evaluated the effects of erythrosine on neural tube development in early-stage chicken embryos. The study included 4 groups, with a total of 80 embryos: a control group, a normal saline group, a half-dose group, and a high-dose group. After 30 hours of incubation, saline and erythrosine solution was injected under the embryonic discs. At the end of 72 hours, the embryos were excised and evaluated macroscopically and histopathologically. Neural tube defects were detected in the erythrosine-administered groups with statistically significant differences. In contrast, the embryos in the control and saline groups displayed normal development. Erythrosine increased the risk of neural tube defects in early-stage chicken embryos, even at half of the approved dose.

Intragenic sequences in the trophectoderm harbour the greatest proportion of methylation errors in day 17 bovine conceptuses generated using assisted reproductive technologies

BackgroundAssisted reproductive technologies (ART) are widely used to treat fertility issues in humans and for the production of embryos in mammalian livestock. The use of these techniques, however, is not without consequence as they are often associated with inauspicious pre- and postnatal outcomes including premature birth, intrauterine growth restriction and increased incidence of epigenetic disorders in human and large offspring syndrome in cattle. Here, global DNA methylation profiles in the trophectoderm and embryonic discs of in vitro produced (IVP), superovulation-derived (SOV) and unstimulated, synchronised control day 17 bovine conceptuses (herein referred to as AI) were interrogated using the EmbryoGENE DNA Methylation Array (EDMA). Pyrosequencing was used to validate four loci identified as differentially methylated on the array and to assess the differentially methylated regions (DMRs) of six imprinted genes in these conceptuses. The impact of embryo-production induced DNA methylation aberrations was determined using Ingenuity Pathway Analysis, shedding light on the potential functional consequences of these differences.ResultsOf the total number of differentially methylated loci identified (3140) 77.3 and 22.7% were attributable to SOV and IVP, respectively. Differential methylation was most prominent at intragenic sequences within the trophectoderm of IVP and SOV-derived conceptuses, almost a third (30.8%) of the differentially methylated loci mapped to intragenic regions. Very few differentially methylated loci were detected in embryonic discs (ED); 0.16 and 4.9% of the differentially methylated loci were located in the ED of SOV-derived and IVP conceptuses, respectively. The overall effects of SOV and IVP on the direction of methylation changes were associated with increased methylation; 70.6% of the differentially methylated loci in SOV-derived conceptuses and 57.9% of the loci in IVP-derived conceptuses were more methylated compared to AI-conceptuses. Ontology analysis of probes associated with intragenic sequences suggests enrichment for terms associated with cancer, cell morphology and growth.ConclusionBy examining (1) the effects of superovulation and (2) the effects of an in vitro system (oocyte maturation, fertilisation and embryo culture) we have identified that the assisted reproduction process of superovulation alone has the largest impact on the DNA methylome of subsequent embryos.

The Effects of Lacosamide in the Early Stages of Neural Tube Development in Chick Embryos

Antiepileptic drugs (AED) are teratogens that confer a risk of various congenital malformations including neural tube defects. Lacosamide (LCM) is a novel third-generation AED and its effects on neural tube (NT) development remain unclear. Thus, the aim of the present study was to investigate the effects of LCM in the early stages of NT development in the chicken embryo. Three different doses of LCM were applied under the embryonic disks of chicken embryos after they were incubated for 30 hours. Incubation was continued for an additional 80 hours and then all embryos were obtained for routine histology. In the LCM-treated groups, there was occlusion in the middle and ventral levels of the cavity. In the group treated with 1.60 mg of LCM, the NT cavity was closed, the notochord exhibited deterioration, and cellular association appeared abnormal. In the groups treated with 0.12 and 0.5 mg of LCM, the ectoderm layer surrounding the embryo was hypertrophic and the number of pyknotic cells increased in a dose-dependent manner. Thus, different doses of LCM had different effects. The results of this study demonstrated that LCM causes histopathological alterations during the neurulation stage that might ultimately result in the development of congenital defects and/or malformations.

Principles of early human development and germ cell program from conserved model systems.

Human primordial germ cells (hPGCs), the precursors of sperm and eggs, originate during week 2-3 of early postimplantation development1. Using in vitro models of hPGC induction2–4, recent studies suggest striking mechanistic differences in specification of human and mouse PGCs5. This may partly be due to the divergence in their pluripotency networks, and early postimplantation development6–8. Since early human embryos are inaccessible for direct studies, we considered alternatives, including porcine embryos that, as in humans, develop as bilaminar embryonic discs. Here we show that porcine PGCs (pPGCs) originate from the posterior pre-primitive streak competent epiblast by sequential upregulation of SOX17 and BLIMP1 in response to WNT and BMP signalling. Together with human and monkey in vitro models simulating peri-gastrulation development, we show conserved principles for epiblast development for competency for PGC fate, followed by initiation of the epigenetic programme9–11, regulated by a balanced SOX17–BLIMP1 gene dosage. Our combinatorial approach using human, porcine and monkey in vivo and in vitro models, provides synthetic insights on early human development.

Anatomical basis of femoral component sizing of total knee arthroplasty

Conjoined twins are a rare congenital anomaly of twinning which borders on monstrosity. Of the various types of conjoined twins, thoraco-omphalopagus is a commonly occurring type. A recent theory postulates fusion of two embryonic discs as more of a cause for conjoined twinning than fission of a single embryonic plate. In this paper, the findings of a pair thoraco-omphalopagus twins are outlined with a review of the embryological theories that surround their formation.

Effects of lacosamide "a novel antiepileptic drug" in the early stages of chicken embryo development.

Antiepileptic drugs (AEDs) are teratogens and confer a risk of congenital malformation. The estimated prevalence of major congenital malformations such as cardiac defects, facial clefts, hypospadias, and neural tube defects in epileptic women is 4-10%, which represents a two- to fourfold increase in pregnant women compared to the general population. However, there are no clear data for newer drugs. Lacosamide (LCM), a novel AED, is the first of the third-generation AEDs to be approved as adjunctive therapy for the treatment of partial-onset seizures. There are no data on the pharmacokinetics of LCM during pregnancy, and only some published data have reported on its effects during pregnancy. In this study, three different doses of LCM (0.12, 0.5, and 1.60mg in 0.18mL) were applied under the embryonic disks of specific pathogen-free Leghorn chicken embryos after a 30-h incubation. Incubation was continued for 80h, at which time all embryos were evaluated macroscopically and microscopically. There was growth retardation in all of the LCM-treated groups. Major malformations increased in a dose-dependent manner and were mostly observed in the supratherapeutic group. Based on our data, LCM may cause growth retardation or major congenital malformations. Nevertheless, more extensive investigations of its reliability are needed.

Conjoined twins – Thoraco-omphalopagus: A case report

Conjoined twins are a rare congenital anomaly of twinning which borders on monstrosity. Of the various types of conjoined twins, thoraco-omphalopagus is a commonly occurring type. A recent theory postulates fusion of two embryonic discs as more of a cause for conjoined twinning than fission of a single embryonic plate. In this paper, the findings of a pair thoraco-omphalopagus twins are outlined with a review of the embryological theories that surround their formation.

Monocephalus thoracopagus conjoined twins in a goat

Monocephalus thoracopagus is a kind of attached symmetrical twin in which two nearly complete individuals joined front-to-front in the thoracic region with a single head, fused chests, one or two hearts and two lower bodies. A died female kid goat with one head, four ears, four paired limbs and two more or less complete body was referred to the Veterinary Faculty of the Shahid Bahonar University of Kerman. Detailed dissection was done on it and the progress of the duplication process of the internal organs was studied precisely. In the presented case, the way of the articulation of the ribs to the sterna and the progress of the duplication process in the alimentary canal can support the Spencer's spherical theory, saying that “a group of conjoined twins resulting from the union of two embryonic discs (or incomplete fission in one disc) over a common yolk sac”.

Pluripotency Network in Porcine Embryos and Derived Cell Lines

Huge amounts of work have been dedicated to the establishment of embryonic stem cell lines from farm animal species since the successful isolation of embryonic stem cells from the mouse and from the human. However, no conclusive results have been obtained so far, and validated lines have yet to be established in domestic animals. Many limiting factors have been suggested and need to be studied further to isolate truly pluripotent cell lines from livestock. In this review, we will discuss the difficulties in deriving and maintaining embryonic stem cell lines from farm animal embryos and how can this lack of success be explained. We will summarize results obtained in our laboratory regarding derivation of pluripotent cells in the pigs. Problems related to the identification of standard methods for derivation, maintenance and characterization of cell lines will also be examined. We will focus our attention on the need for appropriate stemness-related marker molecules that can be used to reliably investigate pluripotency in domestic species. Finally, we will review data presently available on functional key pluripotency-maintaining pathways in farm animals.

Study of the first blastomeres in Coho salmon (Oncorhynchus kisutch)

Summary There is a lack of information on the morphology of the first blastomeres that could be used as a diagnostic tool for the first stages of embryonic development for Coho salmon. The purpose of this investigation, therefore, was to characterize morphometrically the first blastomeres of Coho salmon (Oncorhynchus kisutch). In total, 660 embryonic discs from a pool of eggs that had been fertilized and incubated at 5°C and after 19 h of incubation were extracted and photographed. Of these, 20 microphotographs of blastodiscs of normal appearance were analyzed morphologically (control blastodiscs: CB) and 100 random microphotographs from the whole group were classified as either symmetrical or asymmetrical according to their morphology and then compared with the CB. The length and width of each blastomere and the proportions of length and width were measured to determine symmetry in the embryos at the 4-cell stage. Seven categories were created to characterize the blastomeres: 38% normal (G1); 26% unequal (G2); 10% 'pie-shaped' (G3); 10% amorphous (G4); 8% with three equal and one unequal blastomere (G5); 6% 'clover-shaped' blastomeres (G7), and 3% with inclusions. The mean of the proportions of lengths and widths of the groups of blastomeres that were measured was 0.87 ± 0.08 and 0.85 ± 0.07, respectively. The morphometric results that were obtained in this investigation are compared with the results observed by other authors for teleostei and are discussed.

Monochorionic diamniotic conjoined twins

Monochorionic diamniotic (MCDA) conjoined twins are extremely rare. In monochorionic monoamniotic (MCMA) conjoined twins, the embryonic disc failed to undergo complete separation at 15-17th day of gestation. Conversely, MCDA conjoined...

Morphometric characterization of the first blastomeres of rainbow trout (Oncorhynchus mykiss)

In the following investigation the morphometric characteristics of the first two blastomeres of rainbow trout (Oncorhynchus mykiss) were determined. Embryos were incubated at 9°C and then fixed in a Stockard solution every 30 min starting from 8.5 to 12.5 h of incubation post fertilization. Embryonic discs were extracted and microphotographs were taken with Q Capture Pro 5.0 software using a stereomicroscope Olympus SZX7. The average size of the blastodiscs was 941.22 ± 160.42 μm. The first cleavage finished after approximately 12 h of incubation. The first two blastomeres were regularly symmetrical in their morphology. Blastomere 1 had an average length (L) of 942.68 ± 105.56 μm and width (W) of 467.34 ± 64.33 μm. Blastomere 2 had an average length of 887.60 ± 101.65 and width of 454.49 ± 47.25 μm (n = 91). Significant differences were found between the length and width of blastomeres 1 and 2. The proportion between the length of blastomeres 1 and 2 was 0.94 ± 0.07 (n = 91); between the width of blastomeres 1 and 2 it was 0.88 ± 0.11 (n = 91); and the width/length ratio was 0.51 ± 0.09 (n = 182). It was concluded that rainbow trout blastomeres tend to be asymmetrical in length with a higher dispersion of widths.

Transforming growth factor β controls CCN3 expression in nucleus pulposus cells of the intervertebral disc

To investigate transforming growth factor β (TGFβ) regulation of CCN3 expression in cells of the nucleus pulposus. Real-time reverse transcription-polymerase chain reaction and Western blot analyses were used to measure CCN3 expression in the nucleus pulposus. Transfections were used to measure the effect of Smad3, MAPKs, and activator protein 1 (AP-1) on TGFβ-mediated CCN3 promoter activity. Lentiviral knockdown of Smad3 was performed to assess the role of Smad3 in CCN3 expression. CCN3 was expressed in embryonic and adult intervertebral discs. TGFβ decreased the expression of CCN3 and suppressed its promoter activity in nucleus pulposus cells. DN-Smad3, Smad3 small interfering RNA, or DN-AP-1 had little effect on TGFβ suppression of CCN3 promoter activity. However, p38 and ERK inhibitors blocked suppression of CCN3 by TGFβ, suggesting involvement of these signaling pathways in the regulation of CCN3. Interestingly, overexpression of Smad3 in the absence of TGFβ increased CCN3 promoter activity. We validated the role of Smad3 in controlling CCN3 expression in Smad3-null mice and in nucleus pulposus cells transduced with lentiviral short hairpin Smad3. In terms of function, treatment with recombinant CCN3 showed a dose-dependent decrease in the proliferation of nucleus pulposus cells. Moreover, CCN3-treated cells showed a decrease in aggrecan, versican, CCN2, and type I collagen expression. The opposing effect of TGFβ on CCN2 and CCN3 expression and the suppression of CCN2 by CCN3 in nucleus pulposus cells further the paradigm that these CCN proteins form an interacting triad, which is possibly important in maintaining extracellular matrix homeostasis and cell numbers.

Fine Structures of Embryonic Discs of In Vivo Post‐hatching Porcine Blastocysts at the Pre‐Primitive Streak Stage

To date, reports about the ultrastructure of porcine embryonic discs have not shown details of the primitive streak. The main objective of this study was to examine the ultrastructure of interior and exterior embryonic discs in porcine in vivo blastocysts with diameters of 1, 3 and 9 mm using scanning electron microscopy and transmission electron microscopy. For the first time, we revealed the ultrastructure of the unusual group of cells in the pre-primitive streak area of embryonic discs. The cells were 1-2 μm in diameter, had high electron density and contained abundant, free ribosomes and endoplasmic reticulum. These primitive streak cells could represent original embryonic stem cells or represent a stem cell niche. The results also showed three types of cells on the exterior surface of the embryonic discs. Moreover, our results provided morphological evidence of condensed nuclei in the smooth cells on the surface of the embryonic disc.

Establishment of goat embryonic stem cells from in vivo produced blastocyst‐stage embryos

Embryonic stem (ES) cells with the capacity for germ line transmission have only been verified in mouse and rat. Methods for derivation, propagation, and differentiation of ES cells from domestic animals have not been fully established. Here, we describe derivation of ES cells from goat embryos. In vivo-derived embryos were cultured on goat fetal fibroblast feeders. Embryos either attached to the feeder layer or remained floating and expanded in culture. Embryos that attached showed a prominent inner cell mass (ICM) and those that remained floating formed structures resembling ICM disks surrounded by trophectodermal cells. ICM cells and embryonic disks were isolated mechanically, cultured on feeder cells in the presence of hLIF, and outgrown into ES-like colonies. Two cell lines were cultured for 25 passages and stained positive for alkaline phosphatase, POU5F1, NANOG, SOX2, SSEA-1, and SSEA-4. Embryoid bodies formed in suspension culture without hLIF. One cell line was cultured for 2 years (over 120 passages). This cell line differentiated in vitro into epithelia and neuronal cells, and could be stably transfected and selected for expression of a fluorescent marker. When cells were injected into SCID mice, teratomas were identified 5-6 weeks after transplantation. Expression of known ES cell markers, maintenance in vitro for 2 years in an undifferentiated state, differentiation in vitro, and formation of teratomas in immunodeficient mice provide evidence that the established cell line represents goat ES cells. This also is the first report of teratoma formation from large animal ES cells.

The effects of diesel exhaust particles on neural tube development in the early stage chicken embryo

Particulate matter is an important air-pollutant and its toxicity has been reported. Diesel exhaust particles (DEP) constitute a large portion of particulate matter. Therefore, we established our study to investigate the effects of DEP on neural tissue in early stage chicken embryos. MATERIAL and Four study groups and one control group, each of which included 24 objects were designed. Eggs were incubated for 30 hours. Solutions of DEP containing 10, 50, 100, and 200 μg/0.1 ml were prepared with serum saline. At the end of thirty hours diesel exhaust particle solutions were administered under the embryonic discs. After 72nd hour of the incubation, embryos were excised and evaluated macroscopically and histopathologically. The difference between the embryos that were defined as poorly and well developed, was found statistically significant (p < 0.05). Neural tube defects were detected in 16 of 104 embryos. Statistically significant association between the administration of DEP and development of neural tube defect was identified (p=0.037). Thus, the direct neurotoxic effects of DEP, which the whole population encounters inevitably, have been shown in the early stages of embryonic development. Further studies are needed to identify the effects of these particles in the later stages of embryonic development.

145 EFFECT OF TRANSFER OF CATTLE ELONGATING EMBRYO TO A REPEAT BREEDER COW ON PREGNANCY RATE AND INCIDENCE OF A RETURN TO ESTRUS

In cattle, a low pregnancy rate directly affects calf and milk production and brings about economic loss to livestock producers. The number of repeat breeder cows in herds decreases the overall pregnancy rate. Therefore, many studies have focused on improving the cows’ reproductive performance. On the other hand, it has been demonstrated that the major embryonic loss occurs in the first or second week of pregnancy, after which mother cows recognize the presence of their embryos via a signal protein secreted by the embryos, interferon-τ. Therefore, to bypass the period with a high incidence of pregnancy loss in cattle, we used elongating embryos for embryo transfer (ET) to repeat breeder cows. Elongating embryos on Day 14 postoestrus were used for the present study because embryos of this stage have the ability to produce interferon-τ and embryos of later stages are easily damaged when they are collected from the uterus by flushing. In this study, Japanese Black cows were superovulated and inseminated when they showed standing oestrus. Elongating embryos were collected by nonsurgical uterine flushing. Collected embryos that had evident embryonic discs and intact trophectoderms were used for ET. These embryos were transferred, using plastic catheters or ET guns to ipsilateral uterine horns, to the corpus luteum of synchronized crossbred (Japanese Black × Holstein-Friesian) recipient cows. Recipient cows were divided into 2 groups according to the previous reproductive history of each cow: reproductively normal cows that were pregnant with fewer than 3 times of AI or regular ET (using Day 7 embryos) after every parturition (n = 33) and repeat breeder (RB) cows (n = 42). Elongating embryos were transfer on Day 14 after oestrus, and all cows were monitored for signs of standing oestrus twice a day until Day 80. Pregnancy was diagnosed with ultrasonography every 10 days from Day 30 to 80 postoestrus. The data were analysed by Fisher’s exact test. The pregnancy rate of reproductively normal cows was 54.5%. However, in repeat breeder cows, the pregnancy rate was significantly decreased (2.4%; P &lt; 0.01). When the repeat breeder cows did not become pregnant, transfer of the elongating embryos was repeated another 5 times continuously and the incidence of a return of oestrus was monitored daily. At the first ET period, the average interestrus interval in nonpregnant repeat breeder cows was 50 ± 7.2 days. As the ET period proceeded, the interestrus intervals were gradually decreased, and at a sixth ET period, it approximated the normal oestrus cycle length (22.8 ± 0.8). These results indicated that although transfer of elongating embryos could bypass the critical period of high incidence of embryo loss, it did not improve the pregnancy rate of repeat breeder cows. Moreover, it suggests that the uterine environment of repeat breeder cows was gradually deteriorating in a time-dependent manner.