Transcript profiling for early stages during embryo development in Scots pine

Background: Characterization of the expression and function of genes regulating embryo development in conifers is interesting from an evolutionary point of view. However, our knowledge about the regulation of embryo development in conifers is limited. During early embryo development in Pinus species the proembyo goes through a cleavage process, named cleavage polyembryony, giving rise to four embryos. One of these embryos develops to a dominant embryo, which will develop further into a mature, cotyledonary embryo, while the other embryos, the subordinate embryos, are degraded. The main goal of this study has been to identify processes that might be important for regulating the cleavage process and for the development of a dominant embryo. Results: RNA samples from embryos and megagametophytes at four early developmental stages during seed development in Pinus sylvestris were subjected to high-throughput sequencing. A total of 6.6 million raw reads was generated, resulting in 121,938 transcripts, out of which 36.106 contained ORFs. 18,638 transcripts were differentially expressed (DETs) in embryos and megagametophytes. GO enrichment analysis of transcripts up-regulated in embryos showed enrichment for different cellular processes, while those up-regulated in megagametophytes were enriched for accumulation of storage material and responses to stress. The highest number of DETs was detected during the initiation of the cleavage process. Transcripts related to embryogenic competence, cell wall modifications, cell division pattern, axis specification and response to hormones and stress were highly abundant and differentially expressed during early embryo development. The abundance of representative DETs was confirmed by qRT-PCR analyses. Conclusion: Based on the processes identified in the GO enrichment analyses and the expression of the selected transcripts we suggest that (i) processes related to embryogenic competence and cell wall loosening are involved in activating the cleavage process; (ii) apical-basal polarization is strictly regulated in dominant embryos but not in the subordinate embryos; (iii) the transition from the morphogenic phase to the maturation phase is not completed in subordinate embryos. This is the first genome-wide transcript expression profiling of the earliest stages during embryo development in a Pinus species. Our results can serve as a framework for future studies to reveal the functions of identified genes

Development and germination of Sandersonia aurantiaca (Hook.) seeds : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Plant Biology and Biotechnology at Massey University

Sandersonia aurantiaca (Hook.) has recently become an important horticultural crop through its economic value for export of its cut flowers and tubers. Little information however is available on seed structure, morphology, development and propagation. The main objectives of this study were to investigate the pattern of seed development, to find satisfactory methods of improving the seed germination and to assess possible mechanisms of seed dormancy of Sandersonia aurantiaca (Hook.). Seed development was investigated by fixing plant material in FAA solution, embedding in paraffin, and staining with safranin-fast green. A series of sections were examined and photographed under a microscope. Both embryo and endosperm development in Sandersonia show close similarity to development in Allium fistulosum (Alliaceae). Embryo development passes through early globular, late globular, elongated spheroidal and linear embryo development stages. Endosperm development conforms to the Nuclear type. Freely-growing walls between the endosperm nuclei may be associated with the embryo sac wall as projections. The structure of the mature seeds is very similar to that of Iris (Iridaceae) seeds. The small, linear embryo is embedded in the endosperm which constitutes most of the seed volume. Such small, linear embryos may be one reason for embryo dormancy in Sandersonia seed. A special structure (a conical or cylindrical protuberance) is observed in the inner part of the seed coat, which may combine with a lignified layer (and perhaps including the endosperm) to contribute to the coat-imposed domancy in this species. Eighty five treatments were firstly used to improve the germination percentage of Sandersonia seed. Only the treatment in which seeds scarified firstly with sandpaper for 1 min and then nicked near the radicle end showed increased germination from 0 to 10.6% by 30 days, at 20°C. Based on this result, 31 new treatment methods were designed in germination experiment 2. Water uptake patterns, allelopathic effect on lettuce seeds and embryo rescue of Sandersonia seed were also studied for assessing the possible mechanisms of dormancy. The findings of the present study suggest that the Sandersonia seeds have double dormancy. The dormancy mechanism is located in both the seed coat and the embryo and it consists of at least two steps that must be activated in sequence before germination can occur. The first step can be activated prematurely by scarifying and nicking the seeds, thus allowing the seed coat to become permeable to water, oxygen or to reduced mechanical restriction. The second step can be activated directly GA3 which stimulates embryo growth. This germination-promoting technique has great potential for Sandersonia for improvement of the germination percentage of seeds from 0 to about 70%, but development on a commercial scale needs further studies

The chicken embryo and its micro environment during egg storage and early incubation

When egg storage periods are prolonged (>7 days), hatchability and chick quality declines. The reason for this decline has been investigated, but is still not completely understood. At oviposition the developmental stage of the chicken embryo varies and so do the total number of viable cells. During storage, changes can occur in the embryo. Embryo viability at the end of storage seems to be dependent on the number of viable cells and the developmental stage of the embryo at oviposition. When the hypoblast is completely formed (during the quiescent developmental stage), the embryo seems to be more able to endure prolonged storage periods than embryos that are less or more advanced. During storage, changes also occur in egg characteristics such as albumen viscosity, albumen pH and yolk pH. There appears to be an interaction between albumen pH and embryo viability during early incubation and perhaps also during storage. An albumen pH of 8.2 seems to be optimal for embryo development. Albumen pH may influence embryo viability, but embryo viability may in turn, affect albumen pH. It has been hypothesised that an embryo in which the hypoblast is completely formed is better able to provide an effective barrier between the internal embryo and the exterior (yolk and albumen) and/or is better able to produce sufficient amount of carbon dioxide, which will reduce the pH level in the micro environment of the embryo to the optimal pH of 8.2. It appears that, to maintain hatchability and chick quality after prolonged storage periods, embryonic development should be advanced to the stage in which the hypoblast is completely formed or the atmosphere during storage and early incubation should be altered in such a way that albumen pH is maintained at the optimal level of 8.2

Role of the Mitochondrial Genome During Early Development in Mice

The role of the mitochondrial genome in early development and differentiation was studied in mouse embryos cultured in vitro from the two to four cell stage to the blastocyst (about 100 cells). During this period the mitochondria undergo morphological differentiation: progressive enlargement followed by an increase in matrix density, in number of cristae, and in number of mitochondrial ribosomes. Mitochondrial ribosomal and transfer RNA synthesis occurs from the 8 to 16 cell stage on and contributes to the establishment of a mitochondrial protein-synthesizing system. Inhibition of mitochondrial RNA- and protein-synthesis by 0.1 µg/ml of ethidium bromide or 31.2 µg/ml of chloramphenicol permits essentially normal embryo development and cellular differentiation. Mitochondrial morphogenesis is also nearly normal except for the appearance of dilated and vesicular cristae in blastocyst mitochondria. Such blastocysts are capable of normal postimplantation development when transplanted into the uteri of foster mothers. Higher concentrations of these inhibitors have general toxic effects and arrest embryo development. It is concluded that mitochondrial differentiation in the early mouse embryo occurs through the progressive transformation of the preexisting mitochondria and is largely controlled by the nucleocytoplasmic system. Mitochondrial protein synthesis is required for the normal structural organization of the cristae in blastocyst mitochondria. Embryo development and cellular differentiation up to the blastocyst stage are not dependent on mitochondrial genetic activity

A novel method for transmission electron microscopy study of cytoplasmic fragments from preimplantation human embryos

Transmission electron microscopy (TEM) is the main tool for exploring the intracellular damage and organelle distribution. The cause of producing embryo cytoplsamic fragmentation is not completely understood. Since the fragments have detrimental effects on embryo development, the ultrastructural analysis of fragments may play an important role in fragmentation etiology and in embryo development as well. There are no studies regarding the ultrastructure of fragments in transferable embryos, because the preparation for TEM is not vital and embryos are discarded inevitably. This study aims to introduce a new method for ultrastructural evaluation of fragments without damaging the human cleaving embryos

The Plant Ontology facilitates comparisons of plant development stages across species

The Plant Ontology (PO) is a community resource consisting of standardized terms, definitions, and logical relations describing plant structures and development stages, augmented by a large database of annotations from genomic and phenomic studies. This paper describes the structure of the ontology and the design principles we used in constructing PO terms for plant development stages. It also provides details of the methodology and rationale behind our revision and expansion of the PO to cover development stages for all plants, particularly the land plants (bryophytes through angiosperms). As a case study to illustrate the general approach, we examine variation in gene expression across embryo development stages in Arabidopsis and maize, demonstrating how the PO can be used to compare patterns of expression across stages and in developmentally different species. Although many genes appear to be active throughout embryo development, we identified a small set of uniquely expressed genes for each stage of embryo development and also between the two species. Evaluating the different sets of genes expressed during embryo development in Arabidopsis or maize may inform future studies of the divergent developmental pathways observed in monocotyledonous versus dicotyledonous species. The PO and its annotation databasemake plant data for any species more discoverable and accessible through common formats, thus providing support for applications in plant pathology, image analysis, and comparative development and evolution

In vitro production of bovine embryos derived from individual donors in the Corral® dish

Background: Since the identity of the embryo is of outmost importance during commercial in vitro embryo production, bovine oocytes and embryos have to be cultured strictly per donor. Due to the rather low yield of oocytes collected after ovum pick-up (OPU) per individual cow, oocyte maturation and embryo culture take place in small groups, which is often associated with inferior embryo development. The objective of this study was to improve embryonic development in small donor groups by using the Corral (R) dish. This commercial dish is designed for human embryo production. It contains two central wells that are divided into quadrants by a semi-permeable wall. In human embryo culture, one embryo is placed per quadrant, allowing individual follow-up while embryos are exposed to a common medium. In our study, small groups of oocytes and subsequently embryos of different bovine donors were placed in the Corral (R) dish, each donor group in a separate quadrant. Results: In two experiments, the Corral (R) dish was evaluated during in vitro maturation (IVM) and/or in vitro culture (IVC) by grouping oocytes and embryos of individual bovine donors per quadrant. At day 7, a significantly higher blastocyst rate was noted in the Corral (R) dish used during IVM and IVC than when only used during IVM (12.9% +/- 2.10 versus 22.8% +/- 2.67) (P < 0.05). However, no significant differences in blastocyst yield were observed anymore between treatment groups at day 8 post insemination. Conclusions: In the present study, the Corral (R) dish was used for in vitro embryo production (IVP) in cattle; allowing to allocate oocytes and/or embryos per donor. As fresh embryo transfers on day 7 have higher pregnancy outcomes, the Corral (R) dish offers an added value for commercial OPU/IVP, since a higher blastocyst development at day 7 is obtained when the Corral (R) dish is used during IVM and IVC

Embryo donation families : a follow-up in middle childhood

Couples who conceive using donated embryos rear a child to whom they are genetically unrelated. It has been suggested that this may have negative consequences for parenting and child development. Findings are presented of the 2nd phase of an exploratory study of families with a child conceived through embryo donation. Seventeen embryo donation families with a 5- to 9-year-old child were compared with 24 adoptive families and 28 in vitro fertilization families. The quality of the mother's parenting and the child's social and emotional development were assessed using standardized interviews and questionnaires administered to mothers and teachers. Embryo donation children were not at increased risk of psychological problems during middle childhood, and the families were generally functioning well. However, higher emotional overinvolvement of embryo donation parents was found, along with more reluctance to disclose the method of family creation. These preliminary findings are discussed in terms of implications regarding the importance of genetic and gestational relationships between parents and children

Embryo Development of Tree Frog Polypedates Leucomystax at Campus of State University of Malang

Tree frogs live in natural places which are unpolluted. Regarding their role as an ecological indicator, the decrease of frogs population in a particular habitat indicates the danger of environment quality decrease. Moreover, this condition can harm the frogs themselves. All kinds of frogs breed in aqueous environment such as ponds, marshes, and farming fields. One of the tree frogs, Polypedates leucomystax, which belongs to Familia Rachophoridae, is widely spread in Indonesia. This frog has yellowish brown skin with black spots or six lines extending from head to the posterior tip of body. A breeding couple of the frog produces foam nests on the water or plants around water body, where they will nest their fertilized eggs. This species produces over a hundred embryos in one spawning season. These embryos require appropriate conditions to develop normally in the nature. Frog embryo development may becomes a reference to understand how the frog population survives. This study focused on P. leucomystax with regards to its decrease in number due to the drying up of the environment and a lot lost of trees in Campus of State University of Malang. The development of P. leucomystax embryos in the reproduction foam was observed until it reached a tadpole stage. The result showed that the embryos developed in the foam until they hatched then they move out of the foam into the water under which they would continue their development. Considering that water body is a critical requirement for the development of P. leucomystax embryos, it is our responsibility to make any efforts to conserve not only the trees but also any type of water bodies including ponds, marshes, and farming fields as well

Embryonic POU5F1 is Required for Expanded Bovine Blastocyst Formation.

POU5F1 is a transcription factor and master regulator of cell pluripotency with indispensable roles in early embryo development and cell lineage specification. The role of embryonic POU5F1 in blastocyst formation and cell lineage specification differs between mammalian species but remains completely unknown in cattle. The CRISPR/Cas9 system was utilized for targeted disruption of the POU5F1 gene by direct injection into zygotes. Disruption of the bovine POU5F1 locus prevented blastocyst formation and was associated with embryonic arrest at the morula stage. POU5F1 knockout morulas developed at a similar rate as control embryos and presented a similar number of blastomeres by day 5 of development. Initiation of SOX2 expression by day 5 of development was not affected by lack of POU5F1. On the other hand, CDX2 expression was aberrant in embryos lacking POU5F1. Notably, the phenotype observed in bovine POU5F1 knockout embryos reveals conserved functions associated with loss of human embryonic POU5F1 that differ from Pou5f1- null mice. The similarity observed in transcriptional regulation of early embryo development between cattle and humans combined with highly efficient gene editing techniques make the bovine a valuable model for human embryo biology with expanded applications in agriculture and assisted reproductive technologies