Early Stages Of Embryo Development Research Articles

Short communication: Effects of serum obtained from dairy cows with low or high body condition score on in vitro embryo development

The objective of the study was to determine whether the serum obtained from animals differing in body condition score (BCS) affects in vitro embryo development. After in vitro fertilization, serum obtained from dairy cows of either low (L-BCS; 2.1±0.14 on a scale of 1 to 5) or high BCS (H-BCS; 4.0±0.0), or commercially available bovine serum (control) was added at 5% concentration to the in vitro culture medium. Use of serum obtained from H-BCS cows increased the cleavage rates compared with control serum at both 24 and 48h after in vitro fertilization (78.3 vs. 71.9% and 79.9 vs. 75.1%, respectively), whereas use of serum obtained from L-BCS cows increased the blastocyst rate compared with control serum at 7d (23.8 vs. 19.1%), but this difference was not evident at 8 or 9d after in vitro fertilization. As nonesterified fatty acid concentrations were highest in control serum, followed by serum from L-BCS and H-BCS cows (621, 559, and 272μEq/L, respectively), a high concentration of nonesterified fatty acids might adversely affect the very early stages of embryo development, and its negative effects might be greater immediately after fertilization compared with developmental stages after morula formation. Our findings also indicate that factors promoting early stage embryo development do not necessarily promote blastocyst development. Serum obtained from animals under different physiological conditions may be used for in vitro embryo culture to study the effects of nutritional management of dairy cattle on embryo development.

Identification and characterization ofPlAlix, theAlix homologue from theMediterranean sea urchinParacentrotus lividus

The sea urchin provides a relatively simple and tractable system for analyzing the early stages of embryo development. Here, we use the sea urchin species, Paracentrotus lividus, to investigate the role of Alix in key stages of embryogenesis, namely the egg fertilization and the first cleavage division. Alix is a multifunctional protein involved in different cellular processes including endocytic membrane trafficking, filamentous (F)-actin remodeling, and cytokinesis. Alix homologues have been identified in different metazoans; in these organisms, Alix is involved in oogenesis and in determination/differentiation events during embryo development. Herein, we describe the identification of the sea urchin homologue of Alix, PlAlix. The deduced amino acid sequence shows that Alix is highly conserved in sea urchins. Accordingly, we detect the PlAlix protein cross-reacting with monoclonal Alix antibodies in extracts from P. lividus, at different developmental stages. Focusing on the role of PlAlix during early embryogenesis we found that PlAlix is a maternal protein that is expressed at increasingly higher levels from fertilization to the 2-cell stage embryo. In sea urchin eggs, PlAlix localizes throughout the cytoplasm with a punctuated pattern and, soon after fertilization, accumulates in larger puncta in the cytosol, and in microvilli-like protrusions. Together our data show that PlAlix is structurally conserved from sea urchin to mammals and may open new lines of inquiry into the role of Alix during the early stages of embryo development.

Granulocyte macrophage colony stimulating factor supplementation in culture media for subfertile women undergoing assisted reproduction technologies: a systematic review.

Granulocyte macrophage colony stimulating factor (GM-CSF) is a cytokine/growth factor produced by epithelial cells that exerts embryotrophic effects during the early stages of embryo development. We performed a systematic review, and six studies that were performed in humans undergoing assisted reproduction technologies (ART) were located. We wanted to evaluate if embryo culture media supplementation with GM-CSF could improve success rates. As the type of studies and the outcome parameters investigated were heterogeneous, we decided not to perform a meta-analysis. Most of them had a trend favoring the supplementation with GM-CSF, when outcomes were measured in terms of increased percentage of good-quality embryos reaching the blastocyst stage, improved hatching initiation and number of cells in the blastocyst, and reduction of cell death. However, no statistically significant differences were found in implantation and pregnancy rates in all apart from one large multicenter trial, which reported favorable outcomes, in terms of implantation and live birth rates. We propose properly conducted and adequately powered randomized controlled trials (RCTs) to further validate and extrapolate the current findings with the live birth rate to be the primary outcome measure.

Cloning, characterization and expression analysis of a cold shock domain family member YB-1 in turbot Scophthalmus maximus

The Y-box proteins are a family of highly conserved nucleic acid binding proteins. In this report we have identified a new member, YB-1 from turbot (Scophthalmus maximus) spleen cDNA library. The full-length cDNA sequence of turbot YB-1 was obtained and then the expression at transcriptional level was researched by qRT-PCR. In normal organs, the expression of YB-1 was higher in liver, brain, gill and heart, respectively. YB-1 had the highest expression level at gastrula stage during the early stages of embryo development. In the liver, kidney and spleen, the turbot YB-1 expression level was the highest at 72 h after challenge with lymphocystis disease virus (LCDV) and the highest at 12 h after challenge with Vibrio anguillarum (V. anguillarum). Furthermore, the expression of turbot YB-1 also distinctly increased in turbot kidney cells (TK) at 24 h after challenge with V. anguillarum and LCDV. These results indicated that the turbot YB-1 protein may play a significant role in the immune response of turbot.

Effect of LIF on human embryos

Effect of LIF on human embryos

Development of a procedure for production of haploid plants through microspore culture of roselle (Hibiscus sabdariffa L.)

Attempts to obtain new variants of roselle through conventional breeding were unsuccessful for 358 trials of (UKMR-3×UKMR-2) and 63 trials of (UKMR-3×accession 3). Roselle is an autotetraploid plant (2n=4x=72). Therefore the objectives of this study were to obtain new variants in roselle through the establishment of a plant regeneration system via microspore culture followed by detection of the ploidy level of regenerants based on their distinctive PCR patterns. The effects of different media and hormonal combinations were evaluated on microspore culture of roselle. This method was effective for callus induction, early-stage embryo development and subsequently efficient plant regeneration. Pretreatment of microspores at 4°C and 35°C for 3 days in the dark and without pretreatment, showed significant results on percentage of callus induction. Thirty regenerants were evaluated for their ploidy level using flow cytometry combined with propidium iodide and only 1 was detected as haploid, 4 mixploids and 25 diploids. Polymerase chain reaction (PCR) analysis using M13 universal primer (5′-TTATGAAACGACGGCCAGT-3′) showed the regenerated haploid plant having four unique bands at loci M13-01, M13-03, M13-04, and M13-06, which were absent in the microspore donor plant UKMR-1. To our knowledge, this is the first report of the development of haploid plants in roselle via microspore culture.

Oviductal, endometrial and embryonic gene expression patterns as molecular clues for pregnancy establishment

In higher animals, the beginning of new life and transfer of genetic material to the next generation occurs in the oviduct when two distinct gametes cells unite resulting in the formation of a zygote. The zygote then undergoes serial developmental processes in the oviduct and enters into the uterus where it faces challenges and scrutiny from the endometrial ecosystem. Thus, embryos that are able to establish an appropriate embryo–maternal dialogue are capable of developing to term whereas the incompetent ones can perish any time during the gestation period. Although several lines of evidences indicated that pregnancy loss is a multi-factorial phenomenon, the biochemical composition of the embryo and maternal environment are the main players to determine pregnancy outcome. Indeed, expression patterns of the genes are the driving forces that induce biochemical composition changes in embryo, oviduct and uterine environment. Thus, examining the molecular signals that are associated with oviductal or endometrial receptivity and embryo implantation is essential for establishing strategies to improve pregnancy success. Therefore, this review focuses on the contribution of oviduct and its transcriptome profile on early stage embryo development and the impact of endometrium and its transcriptome changes on peri and post embryo implantation. In addition, this paper integrates established facts about hormonal and molecular signatures associated with endometrial receptivity. Finally, the blastocyst and pre-conception endometrial gene expression profiles have been discussed in relation to the pregnancy outcome to highlight the potentials of blastocyst and pre-transfer endometrial transcriptome profile approach for selecting appropriate recipient and developmentally competent embryo.

The Physiology of Bicarbonate Transporters in Mammalian Reproduction1

HCO(3)(-) plays critically important roles during virtually the entire process of reproduction in mammals, including spermatogenesis, sperm capacitation, fertilization, and development of early stage embryos. Therefore, the acid-base balance in the male and female reproductive tracts must be finely modulated. The fluid milieu in the epididymis is acidic, containing very low concentration of HCO(3)(-). In this acidic low HCO(3)(-) environment, mature sperm are rendered quiescent in the epididymis. In contrast, the luminal fluid in the female uterus and oviduct is alkaline, with very high concentration of HCO(3)(-) that is essential for sperm to fulfill fertilization. HCO(3)(-) transporter of solute carrier 4 (SLC4) and SLC26 families represent the major carriers for HCO(3)(-) transport across the plasma membrane. These transporters play critical roles in intracellular pH regulation and transepithelial HCO(3)(-) transport. The physiological roles of these transporters in mammalian reproduction are of fundamental interest to investigators. Here we review recent progress in understanding the expression of HCO(3)(-) transporters in reproductive tract tissues as well as the physiological roles of these transporters in mammalian reproduction.

Is caprine arthritis encephalitis virus (CAEV) transmitted vertically to early embryo development stages (morulae or blastocyst) via in vitro infected frozen semen?

The aim of this study was to determine, in vivo, whether in vitro infected cryopreserved caprine sperm is capable of transmitting caprine arthritis-encephalitis virus (CAEV) vertically to early embryo development stages via artificial insemination with in vitro infected semen. Sperm was collected from CAEV-free bucks by electroejaculation. Half of each ejaculate was inoculated with CAEV-pBSCA at a viral concentration of 104 TCID50/mL. The second half of each ejaculate was used as a negative control. The semen was then frozen. On Day 13 of superovulation treatment, 14 CAEV-free does were inseminated directly into the uterus under endoscopic control with thawed infected semen. Six CAEV-free does, used as a negative control, were inseminated intrauterine with thawed CAEV-free sperm, and eight CAEV-free does were mated with naturally infected bucks. Polymerase chain reaction (PCR) was used to detect CAEV proviral-DNA in the embryos at the D7 stage, in the embryo washing media, and in the uterine secretions of recipient does. At Day 7, all the harvested embryos were PCR-negative for CAEV proviral-DNA; however, CAEV proviral-DNA was detected in 8/14 uterine smears, and 9/14 flushing media taken from does inseminated with infected sperm, and in 1/8 uterine swabs taken from the does mated with infected bucks. The results of this study confirm that (i) artificial insemination with infected semen or mating with infected bucks may result in the transmission of CAEV to the does genital tack seven days after insemination, and (ii) irrespective of the medical status of the semen or the recipient doe, it is possible to obtain CAEV-free early embryos usable for embryo transfer.

Differentially expressed genes in white egg 2 mutant of silkworm, Bombyx mori, at early embryo development stages

White egg 2 is one of white egg mutants in silkworm, whose molecular mechanism remains unknown so far. In order to obtain an overall view on gene expression profiles at early embryo development stages, the white egg 2 near-isogenic line was constructed and the whole-genome of silkworm microarray system containing 21375 predicted genes from the silkworm whole genome sequence was employed to investigate gene expression profiles at 0, 24 and 48 h post oviposition between white egg 2 mutant and normal black egg strain. At 24 h post oviposition, 49 genes exhibited at least 2.0 fold differences at expression level, including 24 up-regulated genes and 25 down-regulated genes while at 48 h post oviposition, 52 genes, including 23 up-regulated genes and 29 down-regulated genes were expressed differentially over 2.0 change fold. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that nine differentially expression genes were involved in nine significant (p<0.05) pathways at 24 h post oviposition and 24 significant pathways at 48 h post oviposition, respectively. These pathways were related to amino acid metabolism, sugar metabolism, and series of major physiological metabolism. Our results hopefully shed light on the further study of molecular mechanism of white egg 2 mutant. Key words : Bombyx mori , white egg 2 mutant, microarray, embryo, differentially expressed gene.

Are there symplastic connections between the endosperm and embryo in some angiosperms?—a lesson from the Crassulaceae family

It is believed that there is symplastic isolation between the embryo (new sporophyte) and the endosperm (maternal-parental origin tissue, which nourishes the embryo) in angiosperms. However, in embryological literature there are rare examples in which plasmodesmata between the embryo suspensor and endosperm cells have been recorded (three species from Fabaceae). This study was undertaken in order to test the hypothesis that plasmodesmata between the embryo suspensor and the endosperm are not so rare but also occur in other angiosperm families; in order to check this, we used the Crassulaceae family because embryogenesis in Crassulaceae has been studied extensively at an ultrastructure level recently and also we tread members of this family as model for suspensor physiology and function studies. These plasmodesmata even occurred between the basal cell of the two-celled proembryo and endosperm cells. The plasmodesmata were simple at this stage of development. During the development of the embryo proper and the suspensor, the structure of plasmodesmata changes. They were branched and connected with electron-dense material. Our results suggest that in Crassulaceae with plasmodesmata between the endosperm and suspensor, symplastic connectivity at this cell–cell boundary is still reduced or blocked at a very early stage of embryo development (before the globular stage). The occurrence of plasmodesmata between the embryo suspensor and endosperm cells suggests possible symplastic transport between these different organs, at least at a very early stage of embryo development. However, whether this transport actually occurs needs to be proven experimentally. A broader analysis of plants from various families would show whether the occurrence of plasmodesmata between the embryo suspensor and the endosperm are typical embryological characteristics and if this is useful in discussions about angiosperm systematic and evolution.

Formation of Embryoid Bodies with Controlled Sizes and Maintained Pluripotency in Three‐Dimensional Inverse Opal Scaffolds

AbstractEmbryoid bodies (EBs) are aggregates of cells derived from embryonic stem (ES) cells, which can serve as a good model system to investigate molecular and cellular interactions in the earliest stages of embryo development. Current methods for producing EBs mainly rely on the use of hanging drops or suspensions in non‐tissue culture treated plates, microwells, and spinner flasks. The capability of these methods is limited in terms of size uniformity and distribution as well as scalability. Here, a new platform based on three‐dimensional alginate inverse opal scaffolds with uniform pores is presented, where uniform EBs with controllable sizes could be produced in the pores and then recovered after disintegration of the scaffolds. The size of the EBs could be readily controlled by varying the culture time and/or by using scaffolds with different pore sizes. The EBs maintained their viability and undifferentiated state, and they were able to differentiate into specific lineages upon stimulation.

Herpes simplex virus infection of human spermatozoa correlates with decreased frequency of blastocyst formation and frequency of embryo implantation during in vitro fertilization

We have conducted a comparative analysis of developing human embryos in the course of in vitro fertilization (IVF) as a method of sterility treatment of two groups of patients: herpes simplex virus (HSV) was detected in the fraction of motile sperm of male partners in group I (n = 28) and no HSV was found in sperm in group II (n = 103). W assessed number of fertilized ova, embryos during cleavage, and blastocysts as well as such parameters as frequency of implantation and frequency of pregnancy in IVF cycles. It was established that the presence of HSV in spermatozoa did not affect the efficiency of fertilization or cleavage of zygotes. At the same time, in cases of virus-infected male gametes, the frequency ofblastocyst formation was two times less (p = 0.015), and the frequency of embryo implantation and pregnancy was, on average, five times lower (p = 0.01 andp = 0.016, respectively). Based on the obtained results, a conclusion was made about the negative influence of HSV-virus in male gametes at the early stages of embryo development and the frequency of achievement of pregnancy in respect to the methods of assisted reproductive technologies (ART).

Toxicity of Chlorine-Produced Oxidants to Arabian Killifish Embryos for Acute Developmental Stages Exposures

Background: Fish embryos are excellent models for studies aimed at the understanding of toxic mechanisms and the indication of possible acute and chronic effects. Therefore, we present the use of Arabian killifish (Aphanius dispar) embryos, an indigenous species to the Arabian Gulf, to study the effect of chlorine-produced oxidants to marine organisms in the Qatari coastal area. Objective: The objective of this study is to develop chlorine toxicity data for the marine Fish Embryo Toxicity (mFET) test. The test is designed as a means to replace or refine the use of marine juvenile and/or adult fish in standard approaches evaluating toxicity of chemicals and effluents. Methods: Embryos were collected from a breeding stock of sexually mature Arabian killifish. Testing was initiated as soon as possible after fertilization of the eggs with exposure to aqueous concentrations of calcium hypochlorite (0.10 – 12.3 mg/l) for up to 240 hours. The investigated endpoints included; coagulated eggs, somite development, heartbeat, tail detachment, hatchability and post hatch mortality. Results: The results showed a developmental stage-dependent response to chlorine. During earlier developmental stages, chlorine had reduced effects on the embryos and the survival rate and hatchability were high, even at relatively high concentrations. In later developmental stages (pre to post hatch, eleutheroembryo), the embryos were significantly more sensitive to chlorine than in the early stages of development. The chorion, (membrane surrounding the egg), is believed to provide a barrier against chlorine in early stages of embryo development. Conclusions: Taking into account the findings presented here, Killifish embryos exhibit the ability to be an indicator organism for environmental risk assessments of the Qatari coastal area. Benefits include, animal alternative, ease of fish breeding, clarity of the embryos, reduced sample size, reduced waste generation and shorter study duration.

Expression analysis of regulatory microRNAs in bovine cumulus oocyte complex and preimplantation embryos

MicroRNAs (miRNAs) are small endogenous molecules that are involved in a diverse of cellular process. However, little is known about their abundance in bovine oocytes and their surrounding cumulus cells during oocyte development. To elucidate this situation, we investigated the relative expression pattern of sets of miRNAs between bovine oocyte and the surrounding cumulus cells during in vitro maturation using miRNA polymerase chain reaction (PCR) array. Results revealed that a total of 47 and 51 miRNAs were highly abundant in immature and matured oocytes, respectively, compared with their surrounding cumulus cells. Furthermore, expression analysis of six miRNAs enriched in oocyte miR-205, miR-150, miR-122, miR-96, miR-146a and miR-146b-5p at different maturation times showed a dramatic decrease in abundance from 0 h to 22 h of maturation. The expression of the same miRNAs in preimplantation stage embryos was found to be highly abundant in early stages of embryo development and decreased after the 8-cell stage to the blastocyst stage following a typical maternal transcript profile. Similar results were obtained by localization of miR-205 in preimplantation stage embryos, in which signals were higher up to the 4-cell stage and reduced thereafter. miR-205 and miR-210 were localized in situ in ovarian follicles and revealed a spatio-temporal expression during follicular development. Interestingly, the presence or absence of oocytes or cumulus cells during maturation was found to affect the expression of miRNAs in each of the two cell types. Hence, our results showed the presence of distinct sets of miRNAs in oocytes or cumulus cells and the presence of their dynamic degradation during bovine oocyte maturation.

Important processes during differentiation and early development of somatic embryos of Norway spruce as revealed by changes in global gene expression

The aim of this study has been to identify important processes that regulate early stages of embryo development in conifers. Somatic embryogenesis in Picea abies has become a model system for studying embryology in conifers, providing a well-characterized sequence of developmental stages, resembling zygotic embryogeny, which can be synchronized by specific treatments, making it possible to collect a large number of somatic embryos at specific developmental stages. We have used this model to analyze global changes in gene expression during early stages of embryo development by generating an expression profile of 12,536 complementary DNA clones. This has allowed us to identify molecular events regulating putative processes associated with pattern formation during the earliest stages of embryogenesis which have not been identified on the molecular level in conifers before. We recognize notable changes in the expression of genes involved in regulating auxin biosynthesis and auxin response, gibberellin-mediated signaling, signaling between the embryo and the female gametophyte, tissue specification including the formation of boundary regions, and the switch from embryonic to vegetative development. In addition, our results confirm the involvement of previously described processes, including stress, differentiation of a protoderm, and programmed cell death.

Involvement of ribosomal protein RPL27a in meristem activity and organ development

The plant shoot apical meristem is established early during embryogenesis and subsequently gives rise to a shoot through reiterative generation of lateral organs and axillary meristems. In our recent manuscript we reported identification and characterization of a semi-dominant mutation in ribosomal protein RPL27a, which disrupts plant growth and shoot development.1 rpl27ac-1d effects on the shoot are evident from an early stage of embryo development. During embryogenesis rpl27-1d mutants are slow growing and are defective in apical patterning with a delay in establishment of the shoot meristem and outgrowth of cotyledons. Concomitant with this disturbed patterning, the shoot meristem genes SHOOT MERISTEMLESS (STM) and CUP-SHAPED COTYLEDON2 (CUC2) are misexpressed in outer cell layers of the rpl27ac-1d embryo and there is a delay in expression of the organ-patterning gene FILAMENTOUS FLOWER (FIL). Genetic interactions between rpl27ac-1d and other ribosomal protein mutants indicates rpl27ac-1d has reduced ribosome function. Our results highlight a role for ribosomal proteins in growth and development and we propose that the ribosome regulates specific patterning events during development.

Isolated microspore culture of chickpea (Cicer arietinum L.): induction of androgenesis and cytological analysis of early haploid divisions

Routine production of haploid plants has not been reported for any legume, despite the major role these species play in sustainable farming systems and human nutrition. It is within this context that we report a protocol for the induction of haploid development in chickpea (Cicer arietinum L.) using isolated microspore culture. The cultivars “Rupali”, “Narayen”, and “Kimberley Large” were identified as responsive to isolated microspore culture. Flower bud length and microspore developmental stage were correlated for these cultivars. Depending on the cultivar, buds 2.85–3.5 mm in length contained uninucleate microspores. Microspores from donor plants grown in winter and spring were more responsive than those grown in summer. A cold treatment (4°C) of between 24 and 48 h enhanced microspore response in winter- and spring-grown material but was not effective in summer-grown material. A medium developed by the authors was effective for microspore induction and early-stage embryo development. The addition of hormones to this medium was promotive of microspore induction in winter- and spring-grown material, but not in summer-grown material. The initial haploid division predominantly occurred via symmetrical division of the vegetative nucleus. Further research is under way to convert pro-embryos into plants.

Maternal Intake of Astaxanthin Improved Hatchability of Fertilized Eggs Stored at High Temperature

This work aimed at examining the interactive effect of maternal intake of astaxanthin (AX) and storage conditions (temperature and period) on the hatchability of fertilized eggs. Single comb White Leghorn laying hens were allocated to 4 groups with cocks. Experimental diets with 4 levels of AX were prepared by supplementing AX to a basal diet. AX levels were 0, 5, 10 and 20ppm. Eggs from each treatment group were stored for 0, 4, 7, 14 and 21 days at either 10 or 21°C of storage temperature with 70% of relative humidity. The number of hatched chicks was counted and un-hatched eggs were used to check the age of embryo death. When fertilized eggs were stored at 10°C, the hatchability was kept at high level regardless of storage period. At 10°C of storage temperature, maternal intake of AX had no effect on the hatchability of fertilized eggs. On the other hand, the hatchability of eggs stored at 21°C was significantly lower than that at 10°C, and lowered hatchability was restored by maternal intake of AX. Elevated levels of dietary AX from 5 to 20ppm gradually restrained the decrease in hatchability at 21°C. On the mortality of embryos from 0 to 7 days of incubation, the interaction between dietary AX levels and storage temperature was significant. However, after 7 days of incubation, there was no significant interaction between dietary AX levels and storage temperature, and the effect of AX was not significant. These results suggest that the lowered hatchability of eggs stored at high temperature was restored by maternal feeding of dietary AX, and the beneficial effect of AX may be effective at early stage of embryo development.

A Novel Transcript of Oil Palm (Elaeis guineensis Jacq.), Eg707, is Specifically Upregulated in Tissues Related to Totipotency

In this study, we report the molecular characterization of clone Eg707 isolated from cell suspension culture of the oil palm. The deduced polypeptide of clone Eg707 is highly similar to an unknown protein from Arabidopsis thaliana. The presence of an Ald-Xan-dh-C2 superfamily domain in the deduced protein sequence suggested that Eg707 protein might be involved in abscisic acid biosynthesis. Eg707 might be present as a single copy gene in the oil palm genome. This gene is highly expressed in tissue cultured materials compared to vegetative and reproductive tissues, suggesting a role of this gene during oil palm somatic embryogenesis or at the early stages of embryo development. Expression analysis of Eg707 by RNA in situ hybridization showed that Eg707 transcripts were present throughout somatic embryo development starting from proembryo formation at the embryogenic callus stages till the maturing embryo stages. Since proembryo formation within the embryogenic callus is one of the first key factors in oil palm somatic embryo development, it is suggested that Eg707 could be used as a reliable molecular marker for detecting early stage of oil palm somatic embryogenesis.