Embryos Of Different Stages Research Articles

Occurrence and the state of a tudor repeat protein in the development of Xenopus laevis

The Xenopus tudor repeat (Xtr) protein of the unfertilized and fertilized eggs at different intervals, and embryos of different stages was compared by immunoprecipitating with anti-Xtr monoclonal antibody, followed by Western blotting using anti-Xtr antibody. Besides the Xtr of 270 kDa, another band of Xtr below 150 kDa, was detected reproducibly after fertilization of the eggs. This result suggested that a part of Xtr was degraded after fertilization. The screening profile of the existence of Xtr and the distribution of degradation from unfertilized eggs to the nerula stage embryos showed that in the unfertilized eggs there was no occurrence of degradation. But it was visualized just after 20-minutes of fertilization and lasted up to the gastrula stage. Interestingly, the degradation could not be detected beyond this stage of development. Probably, the Xtr protein was degraded following the activation of the eggs because previous investigations suggested that Xtr acted after fertilization or activation of the eggs.Jahangirnagar University J. Biol. Sci. 3(2): 1-8, 2014 (December)

Effect of recombinant-LH and hCG in the absence of FSH on in vitro maturation (IVM) fertilization and early embryonic development of mouse germinal vesicle (GV)-stage oocytes

During in vitro maturation (IVM), intrinsic and extrinsic factors must co-operate properly in order to ensure cytoplasmic and nuclear maturation. We examined the possible effect of LH/hCG in the process of oocyte maturation in mice with the addition of recombinant LH (r-LH) and hCG in our IVM cultures of mouse germinal vesicle (GV)-stage oocytes. Moreover, the effects of these hormones on fertilization, early embryonic development and the expression of LH/hCG receptor were examined. Nuclear maturation of GV-stage oocytes was evaluated after culture in the presence of r-LH or hCG. Fertilization rates and embryonic development were assessed after 24h. Total RNA was isolated from oocytes of different stages of maturation and from zygotes and embryos of different stages of development in order to examine the expression of LH/hCG receptor, using RT-PCR. The in vitro nuclear maturation rate of GV-stage oocytes that received hCG was significantly higher compared to the control group. Early embryonic development was increased in the hCG and LH cultures of GV oocytes when LH was further added. The LH/hCG receptor was expressed in all stages of in vitro matured mouse oocytes and in every stage of early embryonic development. Addition of hCG in IVM cultures of mouse GV oocytes increased maturation rates significantly. LH, however, was more beneficial to early embryonic development than hCG. This suggests a promising new technique in basic science research or in clinical reproductive medicine.

200 STAGE-SPECIFIC EXPRESSION PATTERNS OF THE INSULIN-LIKE GROWTH FACTOR 1 RECEPTOR DURING BOVINE PRE-IMPLANTATION EMBRYONIC DEVELOPMENT

Insulin-like growth factor 1 (IGF1) is a key regulator in early embryonic development, influencing physiological processes and stimulating growth and development (Fowden et al. 2003). Supplementing IGF1 during in vitro culture of bovine embryos improved cleavage and developmental rates while it reduced apoptosis (Byrne et al. 2002). The signal transduction of IGF1 is performed by its binding to the insulin-like growth factor 1 receptor (IGF1R). At the mRNA level, IGF1R is expressed throughout pre-implantation embryonic development and was identified as a potential marker of good quality embryos (Yaseen et al. 2001). However, information on protein level is rare. Therefore, protein expression of the IGF1R during early embryonic development in vitro was analysed in the present study by immunofluorescence staining. Furthermore, the mRNA expression of the IGF1R was investigated by RT-qPCR. In vitro derived embryos of different stages (2-cell, 4-cell, 8-cell, 16-cell stage, morula, blastocyst, and expanded blastocyst) were either directly subjected to immunofluorescence staining or frozen at –80°C for use in RT-qPCR. Staining was performed with a peptide antibody against two peptide sequences of the bovine IGF1R α unit, which was specifically produced. Pixel intensity of immunofluorescence was measured and a mean grey value was calculated using the cellsens® software (Olympus, Hamburg, Germany). Data were analysed by one-way ANOVA followed by a Tukey's test using SigmaStat 3.5 Software (Systat Software GmbH, Erkrath, Germany). The detection of the IGF1R mRNA and protein was possible in all stages of embryonic development beginning at the 2-cell stage up to the expanded blastocyst. The maximal mRNA expression could be observed in 2- and 4-cell embryos. It significantly decreased to the 8-cell stage, followed by an increase up to the expanded blastocyst. The IGF1R protein was mainly localised in the plasma membrane of single blastomeres and also weakly in the cytoplasm. Mean grey values are highest in the 2-cell stage, showing a significant decline up to the 16-cell stage and an increase again until the expanded blastocyst. The mRNA and protein expression showed similar patterns during early embryonic development. IGF1R expression started to increase at the 8-cell stage (mRNA) and 16-cell stage (protein) indicating a link to the maternal-embryonic transition. For the first time, these results show that in bovine embryos, the IGF1R expression is related to the activation of the embryonic genome. We gratefully acknowledge the financial support of the H. Wilhelm Schaumann Foundation (Hamburg, Germany).

High efficiency cyclic production of secondary somatic embryos and ISSR based assessment of genetic fidelity among the emblings in Calliandra tweedii (Benth.)

A high frequency secondary somatic embryogenesis and plant regeneration have been achieved in Calliandra tweedii (Benth.) through internodal segments excised from 5-year-old shrub on Murashige and Skoog (MS) medium enriched with different concentrations of 2-isopentenyl adenine (2iP). Somatic embryos were differentiated directly from cut ends, epidermal crevices as well as indirectly through callus. Such primary embryos when subcultured on MS medium augmented with 2-isopentenyl adenine (2iP), N6 benzyladenine (BA), 2,4-dichlorophenoxy acetic acid (2,4-D), α-naphthaleneacetic acid (NAA) in different concentrations (0.1, 0.5, 1, 1.5, 5, 10, and 20μM) individually, redifferentiated numerous secondary somatic embryos of different stages, i.e., globular, heart-shaped, torpedo and cotyledonary types within 6 weeks of culture. The somatic embryos originated either from radicular or plumular parts or from all over the surfaces. Of the aforesaid growth regulators, 1μM 2iP proved to be the best for recurrent production of an average of 19.44±0.52 cotyledonary somatic embryos per culture within 6 weeks. The embryogenic clumps (100mg per culture tube) comprising the pre cotyledonary stages were transferred to different concentrations (1, 2.5, 5, 7, 10, 20, and 50μM) of abscisic acid (ABA) for their maturation and preventing precocious germination. ABA at 5μM proved optimum for developing an average of 15.87±0.31 normal cotyledonary embryos per embryogenic clumps within 4 weeks. These bipolar mature embryos after transfer to half-strength MS medium germinated into plantlets. The genetic uniformity of the emblings was established employing 14 inter simple sequence repeat (ISSR) marker where 96.1% of the bands generated were monomorphic and similar with those of stock plant with only 2% variation among the plantlets.

Generation of parthenogenetic goat blastocysts: effects of different activation methods and culture media

The present study was carried out to investigate the effects of different activation methods and culture media on the in vitro development of parthenogenetic goat blastocysts. Calcium (Ca2+) ionophore, ethanol or a combination of the two, used as activating reagents, and embryo development medium (EDM), modified Charles Rosenkrans (mCR2a) medium and research vitro cleave (RVCL) medium were used to evaluate the developmental competence of goat blastocysts. Quantitative expression of apoptosis, stress and developmental competence-related genes were analysed in different stages of embryos. In RVCL medium, the cleavage rate of Ca2+ ionophore-treated oocytes (79.61 ± 0.86) was significantly (P < 0.05) higher than in ethanol (74.90 ± 1.51) or in the combination of both Ca2+ ionophore and ethanol. In mCR2a or EDM, hatched blastocyst production rate of Ca2+ ionophore-treated oocytes (8.33 ± 1.44) was significantly higher than in ethanol (6.46 ± 0.11) or in the combined treatment (6.70 ± 0.24). In ethanol, the cleavage, blastocyst and hatched blastocyst production rates in RVCL medium (74.90 ± 1.51, 18.30 ± 1.52 and 8.24 ± 0.15, respectively) were significantly higher than in EDM (67.81 ± 3.21, 14.59 ± 0.27 and 5.59 ± 0.42) or mCR2a medium (65.09 ± 1.57, 15.36 ± 0.52 and 6.46 ± 0.11). The expression of BAX, Oct-4 and GlUT1 transcripts increased gradually from 2-cell stage to blastocyst-stage embryos, whereas the transcript levels of Bcl-2 and MnSOD were significantly lower in blastocysts. In addition, different activation methods and culture media had little effect on the pattern of variation and relative abundance of the above genes in different stages of parthenogenetic activated goat embryos. In conclusion, Ca2+ ionophore as the activating agent, and RVCL as the culture medium are better than other tested options for development of parthenogenetic activated goat blastocysts.

Localization of parathyroid hormone-related protein in the preimplantation mouse embryo is associated with events of blastocyst hatching

To determine the pattern of expression of parathyroid hormone-related protein (PTHrP) and its receptor, parathyroid hormone receptor 1 (PTHR1), in mouse embryos in different stages of preimplantation development. Embryos were cultured from the pronuclear zygote stage and harvested as 2-cell, 4-cell and 8-cell embryos, morulae and blastocysts. RT-PCR was carried out on mRNAs of these and of trophoblast outgrowths for detection of PTHrP and PTHR1. Whole mounted embryos intact or stripped of zonae pellucidae were immunofluorescently stained for PTHrP and PTH receptor and observed with confocal microscopy. PTHrP mRNA was present in the pronuclear zygote, not present in 2-cell, 4-cell and uncompacted 8-cell embryos, present in the 8-cell compacting embryo, and not detected in 16-cell morulae or blastocysts. The mRNA was present in trophoblasts growing on fibronectin beds. mRNA for PTHR1 was detected in the pronuclear zygote, then undetected until the compacted 8-cell stage and thereafter. PTH receptor protein was observed in 2-cell embryos, morulae and in the inner cell mass and trophectoderm of blastocysts. PTHrP was observed dispersed in the cytoplasm of 2-cell, 4-cell and uncompacted 8-cell embryos, and in distinct foci near the nuclei of morulae. In blastocysts, PTHrP appeared on the apical surface of only trophoblast cells which had extruded from the zona pellucida. Fully hatched blastocysts expressed the protein on the apical side of all trophoblasts. When morulae were prematurely stripped of their zonae, PTHrP was observed on the embryos' outer surface. PTHrP protein is expressed throughout early embryo development, and its receptor PTHR1 is expressed from the morula stage. Embryo hatching is associated with translocation of PTHrP to the apical plasma membrane of trophoblasts. PTHrP may thus have autocrine effects on the developing blastocyst.

Effects of copper exposure on the hatching status and antioxidant defense at different developmental stages of embryos and larvae of goldfish Carassius auratus

This study aims to assess the effects of copper exposure on hatching status and antioxidant defense at different stages of embryos and larvae of goldfish Carassius auratus. In this study, day-old embryos were randomly grouped after fertilization and then exposed to copper concentrations of 0, 0.1, 0.4, 0.7, and 1.0mgL−1. Copper-exposed fish embryos were sampled every 24h to determine superoxide dismutase (SOD), and catalase (CAT) activities, as well as malondialdehyde (MDA) content. In addition, cumulative mortality and larval deformity were also investigated. The findings showed that cumulative mortality and larval deformity rate increased gradually with copper concentration increase. SOD and CAT activities were inhibited at higher copper concentrations. At a lower concentration (0.1mgL−1), SOD activity increased in larvae, whereas CAT activity showed no significant change (p>0.05). MDA, as the lipid peroxidation product, gradually accumulated in embryos and larvae with increasing copper concentration and the extension of exposure time. At 0.4mgL−1 and more, copper toxicity was shown in embryos and larvae. In conclusion, copper-exposed effects on hatching status and antioxidant defense in C. auratus embryos and larvae showed concentration- and time-dependent patterns. The biochemical parameters in this study can be used as effective indicators for evaluating the responses of copper-exposed fish embryos. In addition, this study demonstrates that 0.4mgL−1 copper (corresponding to 1mgL−1 copper sulfate), used to kill parasites in aquaculture, is not safe concentration, because it can result in toxicity to larvae. Therefore, the copper concentration to kill pathogen should be less than 0.4mgL−1 for C. auratus.

Estudios preliminares sobre congelación de embriones de yamú (Brycon amazonicus) en diferentes estadios de desarrollo

Reproductive seasonality in fish has limited its commercial farming production due to the restriction in the fingerling availability. The cryopreservation is an alternative to solve this problem since facilitates the fish reproduction throughout the year. Therefore, this study evaluated the yamu embryos (Brycon amazonicus) viability conserved at -14 °C as a possible strategy to improve its production. For this purpose, sexually mature broodstocks were induced to reproduction with carp pituitary extract [females 0.25 (0 h), 0.5 (24 h) and 5 (36 h) mg/kg and males (4 mg/kg)]. The embryos in different stages of embryonic development such as blastulation (2 hours post-fertilization, hpf), gastrulation (6 hpf) and segmentation (8 hpf) were kept at 6 °C during 10 min and then storaged in falcon tubes at -14 °C and subjected (1:1) in a solution of 10 and 15% of dimethyl sulfoxide (DMSO) or methanol (MET). After one hour of storage, the embryos were immersed in a water bath at 27 °C for 10 min and then transferred again to the incubators to evaluate viability and hatching. The viability and hatching depended to the embryonic development stage as well as the storage solution type. The embryos of the control group showed an average viability and hatching of 96.2±0.1 and 96.3±0.7%, respectively. No hatching was observed in the treatments with 2 hpf embryos; in contrast, 8-hpf embryos subjected to MET 15% showed the best hatching results (54.1±4.1%). In conclusion, yamu embryos in later stages of development are susceptible to preserved at -14 °C at least for 1 hour.

Identification of Stable Reference Genes for Gene Expression Studies Using Quantitative Real Time PCR in Buffalo Oocytes and Embryos

The present study was aimed to validate expression stability of 6 housekeeping genes (viz. YWHAZ, SDHA, GAPDH, RPS15, RPS18 and RN18S1) in the oocytes and embryos of different stages in buffalo. A modified Trizol protocol was optimized for RNA isolation from as few as five oocytes. The expression level of selected genes was studied by an optimized real time PCR using DCT method and their stability of expression was evaluated by Microsoft Excel based visual application, geNORM. The analysis revealed that the RPS15 and GAPDH were the most stable genes across different samples. Also, the geometric mean of three genes (i.e. RPS15,RPS18 and GAPDH) were found suitable for normalization of real time PCR data from buffalo oocytes⁄embryos. The information would help in more accurate interpretation of gene expression data from oocytes⁄embryos towards understanding the molecular events in these cells during development.

Small-conductance calcium-activated K+ channels 3 (SK3) regulate blastocyst hatching by control of intracellular calcium concentration

The present study was designed to investigate the expression of small-conductance calcium-activated K(+) channels 3 (SK3) in preimplantation embryos and to explore their role in the underlying mechanism of blastocyst hatching. Human preimplantation embryos were donated by patients who achieved successful pregnancy with in vitro fertilization. Mouse preimplantation embryos in different stages were collected and cultured with or without siRNA cell injection. The expression of SK3 was examined by RT-PCR, quantitative real-time PCR, western blot and immunofluorescence. Functional expression of SK3 was investigated using the patch-clamp technique. [Ca(2+)]i was measured by fluorescent imaging. Embryos were cultured in vitro to investigate the effect of SK3 knockdown or apamin, an SK3 inhibitor, on blastocyst hatching and F-actin formation. In human blastocysts, the level of SK3 expression was significantly lower in blastocysts that failed to hatch than in blastocysts that hatched successfully. In mouse embryos, SK3 mRNA and protein were not found in zygotes, but were detected from the 2-cell stage onward, with the highest levels observed in blastocysts. SK3 was predominately located in the trophectoderm cell membrane of expanded blastocysts. SK3 knockdown in trophectoderm cells not only suppressed the SK3 current, but also reduced [Ca(2+)]i elevation and membrane potential hyperpolarization induced by thapsigargin. Although the formation of expanded blastocysts was not affected, blastocyst hatching and F-actin formation were significantly inhibited after SK3 knockdown in trophectoderm cells. SK3-mediated [Ca(2+)]i elevation and membrane potential hyperpolarization in trophectoderm cells are important for blastocyst hatching, and defects in SK3 expression may contribute to infertility.

VARIATION IN OFFSPRING SIZE WITH BIRTH ORDER IN PLACENTAL FISH: A ROLE FOR ASYMMETRIC SIBLING COMPETITION?

Asymmetric sibling competition arises when siblings with different competitive abilities share a limited resource. Such competition occurs in species with postnatal parental care and may also occur when mothers provision embryos between fertilization and birth (matrotrophy). We hypothesized that the combination of matrotrophy and the simultaneous provisioning of embryos in different stages of development (superfetation) leads to asymmetric competition between sibling embryos. Moreover, we expect the intensity of this competition to increase with the level of superfetation as high levels of superfetation result in greater temporal overlap between broods. This hypothesis predicts that offspring from early broods, which predominantly compete with less-developed siblings, will be larger at birth than offspring from later broods, which experience competition from more and less-developed siblings. Data on offspring size at birth from two populations of the highly matrotrophic fish, Heterandria formosa, and similar studies of poeciliid fish spanning a range of life histories are consistent with our hypothesis. Together these results suggest that sibling competition is a direct consequence of the evolution of matrotrophy and superfetation in poeciliid fish.

The first spontaneous spawning of European hakeMerluccius merlucciusL.: characteristics of eggs and early larval stages

For the first time, a spontaneous spawning of hake was recorded in Spain in April 2009. The spawn was obtained from broodstock kept in captivity for two years at the facilities of the Spanish Institute of Oceanography in Vigo (NW Spain). Eggs were transparent, spherical and had an average diameter of 1.067 ± 0.024 mm; yolk occupied the majority of egg volume. The oil droplet had a diameter of 0.27 ± 0.03 mm. The incubation period of the eggs lasted for 4 days at 14°C and the duration from hatching to the total absorption of the yolk sac was between 5–7 days after hatching, at the same temperature. Newly hatched larvae had an average total length of 3.20 ± 0.13 mm and began feeding 6 days after hatching; a daily growth rate of 0.158 mm day-1 was observed from hatching to yolk sac consumption. This paper describes the daily evolution of biometric and morphological characteristics of the different stages of embryos and larvae of European hake up to the age of 19 days.

Propagation and Conservation of Native Forest Genetic Resources of Medicinal Use by Means of In Vitro and ex vitro Techniques

In Argentina, there are numerous native species which are an important source of natural products and which are traditionally used in medicinal applications. Some of these species are going through an intense extraction process in their natural habitat which may affect their genetic diversity. The aim of this study was to establish vegetative propagation systems for three native forestal species of medicinal interest. This will allow the rapid obtainment of plants to preserve the germplasm. This study included the following species which are widely used in folk medicine and its applications: Erythrina crista-galli or "seibo" (astringent, used for its cicatrizant properties and for bronchiolitic problems); Acacia caven or "espinillo" (antirheumatic, digestive, diuretic and with cicatrizant properties) and Salix humboldtiana or "sauce criollo" (antipyretic, sedative, antispasmodic, astringent). The methodology included the micropropagation of seibo, macro and micropropagation of Salix humboldtiana and the somatic embryogenesis of Acacia caven. The protocol for seibo regeneration was adjusted from nodal sections of seedlings which were obtained from seeds germinated in vitro. The macropropagation through rooted cuttings of "sauce criollo" was achieved and complete plants of this same species were obtained through both direct and indirect organogenesis using in vitro cultures. The somatic embryogenesis for Acacia caven was optimized and this led to obtain a high percentage of embryos in different stages of development. We are able to support the conservation of native forest resources of medicinal use by means of vegetative propagation techniques.

Characterization of the Zona Pellucida in Canine Embryos Using Schiff Periodic Acid (PAS).

The zona pellucida (ZP) is the extracellular matrix that surrounds the oocyte. The biosynthesis of ZP requires an integrate participation of the oocyte and the granulosa cells, being that canine ovaries have oocytes that synthesize ZP proteins in response to the expression of specific genes responsible for the folliculogenesis. A structural model of the zona pellucida, based on the biochemical and ultrastructural analysis of the mouse ZP, involves long filaments of a repeating ZP2 and ZP3 heterodimer. ZP1 provides structural integrity for the zona pellucida by cross-linking with disulphide bonds the ZP2/ZP3 filaments, however, it is not known if the model proposed for the mouse can be applied for others mammals, since differences between species were often observed and few specific studies was to carried with canine embryos. The objective in this study was to identify and verify the composition of the zona pellucida in canine embryos in different stages of development using Schiff periodic acid (PAS). The embryos collection was performed by flushing the oviducts and uterine horns after ovariohysterectomy using PBS with heparin and polyvinyl alcohol. All embryos were fixed in 2.5% glutaraldehyde and included in historesin. Histological sections with 3 μm were stained with hematoxilin-eosin (HE) and Schiff periodic acid (PAS). Light microscopy observation showed that canine ZP has two defined acellular stratus. The inner part was PAS positive indicating that this stratus was formed by glycoproteins and, the outer stratus was PAS negative and barely stained with HE indicating neutral proteins. It is known that many sugars are associated with ZP which can be added during its formation in the ovarian follicle or during the oocyte transport through the oviduct. In rabbits, a mucoprotein cover is added to the oocyte by the oviduct cells, which have an important role in the relationship between ZP and the endometrium during implantation. The bilaminar structure observed in this experiment of the canine ZP can be related to the addition of components during oviductal transport, since this feature was not observed on oocytes that were harvested from sliced ovaries in previous studies performed at the same laboratory. Financial support: FAPESP. (poster)

The Effect of Endometriosis on the DNA Integrity of Different Stages of Embryos' Development and the Role of L- Carnitine in Preventing Damage to Early Stages

The Effect of Endometriosis on the DNA Integrity of Different Stages of Embryos' Development and the Role of L- Carnitine in Preventing Damage to Early Stages

Temporal expression pattern of insulin-like growth factors (IGF-1 and IGF-2) ligands and their receptors (IGF-1R and IGF-2R) in buffalo ( Bubalus bubalis) embryos produced in vitro

The objective of the study was to assess the temporal expression of genes for IGF-1, IGF-2 and their receptors in different pre-implantation stage buffalo embryos (from oocytes to blastocyst) and to evaluate the effect of IGF-1 on culture media during in vitro buffalo embryo development. Buffalo oocytes were retrieved from slaughterhouse derived ovaries. In vitro matured oocytes were fertilized with frozen buffalo bull semen. Presumptive zygotes were cultured in modified synthetic oviductal fluid (mSOF supplemented with β-mercaptoethanol (100 μM) and without (control) or with IGF-1 (100 ng/ml) till blastocyst stage. For each treatment five replicates were taken. Supplementation of IGF-1 to embryo culture media resulted in significantly higher blastocyst formation rate (29.7% versus 25.6%; p < 0.05) than the control group. In vitro produced embryos of different stages were stored in small amount of PBS at − 70 °C till used for RNA isolation. Total RNA isolated from oocytes and pre-implantation embryos was reverse transcribed to cDNA in total volume of 25 μl reaction mixture using Moloney-Murine Leukemia Virus Reverse Transcriptase (MMLV-RT). Polymerase chain reaction (PCR) was performed using cDNA from different pools of oocytes and embryos. RT-PCR revealed that IGF-1 mRNA was neither expressed in oocytes (immature, matured and matured vitrified oocytes) nor at any pre-implantation stage embryos while mRNA for IGF-1R, IGF-2 and IGF-2R were detected in all oocytes as well as in all pre-implantation stage embryos. It can be concluded that supplementation of IGF-1 is useful for in vitro oocyte maturation and embryo development in buffalo.

Elevated glucose induces congenital heart defects by altering the expression of tbx5, tbx20, and has2 in developing zebrafish embryos

Maternal diabetes increases the risk of congenital heart defects in infants, and hyperglycemia acts as a major teratogen. Multiple steps of cardiac development, including endocardial cushion morphogenesis and development of neural crest cells, are challenged under elevated glucose conditions. However, the direct effect of hyperglycemia on embryo heart organogenesis remains to be investigated. Zebrafish embryos in different stages were exposed to D-glucose for 12 or 24 hr to determine the sensitive window during early heart development. In the subsequent study, 6 hr post-fertilization embryos were treated with either 25 mmol/liter D-glucose or L-glucose for 24 hr. The expression of genes was analyzed by whole-mount in situ hybridization. The highest incidence of cardiac malformations was found during 6-30 hpf exposure periods. After 24 hr exposure, D-glucose-treated embryos exhibited significant developmental delay and diverse cardiac malformations, but embryos exposed to L-glucose showed no apparent phenotype. Further investigation of the origin of heart defects showed that cardiac looping was affected earliest, while the specification of cardiac progenitors and heart tube assembly were complete. Moreover, the expression patterns of tbx5, tbx20, and has2 were altered in the defective hearts. Our data demonstrate that elevated glucose alone induces cardiac defects in zebrafish embryos by altering the expression pattern of tbx5, tbx20, and has2 in the heart. We also show the first evidence that cardiac looping is affected earliest during heart organogenesis. These research results are important for devising preventive and therapeutic strategies aimed at reducing the occurrence of congenital heart defects in diabetic pregnancy.

Effect of Insulin on Development of ICR Mouse Embryos in Vitro

Objective To study the effects and the appropriate concentration of insulin on the development of mouse embryos in vitro, and the effects of insulin on the development of different stages of embryos. Methods Mouse embryos were cultured in vitro in the mKSOM media supplemented with insulin at different concentrations and with insulin during different stages of embryos. The blastocyst rates and the cell numbers were counted. Results Additions of insulin significantly increased the rates of blastocyst and the total cell numbers. The concentrations of 0.005 µg/ml and 0.05 µg/ml insulin caused a significant increase in the total cell numbers compared with the control and experimental groups. Addition of insulin from 2-cell to 4-cell stage or from the 4-cell to morula stage, significantly increased the blastocyst rates compared with control and experimental groups. Conclusion Insulin can promote the development of mouse embryos in vitro. The appropriate concentration of insulin added in mKSOM was 0.005 µg/ml and 0.05 µg/ml. Exposure of embryos to insulin, beginning at the 2-cell and extending to the 4-cell stage or beginning at the 4-cell and extending to the morula stage, is important for the development of ICR mouse embryos in vitro.

Opened brood chamber with embryos of different stages from the demosponge Amphimedon queenslandica.

AbstractOpened brood chamber with embryos of different stages from the demosponge Amphimedon queenslandica. This poriferan species has its genome sequenced, assembled, and annotated and serves as a model organism for sponge developmental biology. Image supplied by Bernie Degnan (University of Queensland).

Somatic embryogenesis and two embryo specific proteins (38 and 33 kD) in Catharanthus roseus

In the present study, the regeneration pathway, especially the different events of somatic embryogenesis (SE) have been studied morphologically and biochemically in Catharanthus roseus. Firstly, the calluses were induced from different explant sources (hypocotyl, epicotyl and root) by using various auxins. Embryogenic and non-embryogenic calluses were identified based on their morphology, colour and dry weight. Embryogenic callus was later cultivated on MS added with 0.45 μM 2,4-D, 6.62 μM BAP and 1.44 μM GA3 for obtaining various developmental stages of embryos. Different stages of embryos have been assayed for the establishment of marker based embryogenesis, particularly on embryo specific proteins whose presence or absence will ensure a rapid and efficient production of embryos that has a special application to clonal biotechnology. Two embryo specific proteins (38 and 33 kD) have been identified for the first time in C. roseus during torpedo stage of embryogenesis. Besides, multiple shoot formation from in vitro raised emblings was also attempted to examine the role of BAP and kinetin for shoot proliferation. The shoots were rooted with 5.37 μM NAA and 5.71 μM IAA before transplantation.