Early Stages Of Embryo Development Research Articles

Infantile spasms in a mosaic monocentric and duplicated SMC 15 patient

ObjectiveTo report detail of a patient with infantile spasms whose cytogenetic analysis revealed mosaic monocentric and duplicated supernumerary marker chromosome (SMC) 15. Subject and methodsThe subject for this case was a 13-month-old girl with infantile spasms and delayed developmental milestones. Chromosomal analysis with G-band showed the presence of SMC in mosaic. Further investigations using in situ hybridization, methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), microsatellite marker, and single nucleotide polymorphism (SNP) array analysis were performed. ResultsHer karyotype was noted as mosaic 47,XX,+mar[26]/46,XX[4], ish der(15)(D15Z1+, SNRPN++, PML−) de novo. MS-MLPA analysis showed that the Prader–Willi syndrome/Angelman syndrome critical region is highly methylated, and microsatellite marker analysis proved that the 15q11.2 region of the patient comprises three kinds of alleles: one paternal and two maternal. SNP array analysis suggested an asymmetric structure of SMC(15) composed of 15q11–q13 recombination at breakpoint (BP) 4:BP5. ConclusionsThis is the first report of SMC(15) with monocentric and duplicated proximal 15q. The clinical presentations are quite similar to those of isodicentric chromosome 15 syndrome. The results of microsatellite and SNP array analysis suggest two possibilities regarding the timing of the mosaic SMC(15) formation. One possibility is that it occurred during maternal meiosis, and the other possibility is formation during a very early stage of embryo development that was initially trisomic of chromosome 15.

Factors associated with congenital anomalies in Addis Ababa and the Amhara Region, Ethiopia: a case-control study

BackgroundThe early stage of embryo development is extremely vulnerable to various teratogenic factors, leading to congenital anomalies. In Ethiopia, a significant number of babies are born with congenital anomalies, but the risk factors for the anomalies have never been studied. Understanding the specific risk factors for congenital anomalies is very essential to provide health education that aims at creating awareness and establishing preventive strategic plan/s. The main objective of this study was to assess the risk factors associated with congenital anomalies in Addis Ababa and the Amhara Region, Ethiopia.MethodsA case-control study was conducted from January 1- June 30, 2015. The participants were recruited at the purposively selected hospitals in Addis Ababa and the Amhara Region. A total of 207 cases and 207 controls were included in the study. Cases were neonates, infants, and children 0-11 months of age with external and internal major congenital anomalies diagnosed by pediatricians. Controls were neonates, infants, and children 0-11 months of age without external and internal anomalies. Data on sociodemographic characteristics, exposure to risk factors, and reproductive history were collected by face to face interviews with children’s mothers/caregivers using a structured questionnaire. Binary logistic regression was employed to explore risk factors associated with the occurrence of the problems.ResultsAbout 87.4% of the children were below 6 months, and 12.6% were between 6 and 11 months. The majority (59.9%) of the children were male, with the M: F sex ratio of 1.49. The mean age of the mothers was 26 years (16-45 years). Unidentified medication use during early pregnancy (AOR = 4.595; 95% CI: 1.868-11.301, P-value = 0.001), maternal alcohol drinking (AOR = 2.394; 95% CI: 1.212-4.726, P-value = 0.012), and exposure to chemicals (AOR = 9.964; 95% CI = 1.238-80.193, P-value = 0.031) were significantly associated with the occurrence of congenital anomalies. Iron folate use (AOR = 0.051; 95% CI: 0.010-0.260, P-value = < 0.001) before and during early pregnancy had a protective effect on congenital anomaly.ConclusionUnidentified medication use, alcohol drinking during early pregnancy, and exposure to chemicals had a significant association with the occurrence of congenital anomalies, whereas iron folate use before and during early pregnancy had a protective effect from congenital anomalies.

Laminarin improves developmental competence of porcine early stage embryos by inhibiting oxidative stress

Laminarin (LMA), a β-glucan mixture with good biocompatibility, improves the growth performance and immune response when used as food additives and nutraceuticals. The aim of the present research was to explore the effects of LMA on porcine early stage embryo development, as well as the underlying mechanisms. The results showed that the developmental competence of porcine early stage embryos was dramatically improved after LMA supplementation during the in vitro culture period. The presence of 20 μg/mL LMA during the in vitro culture period significantly improved cleavage rate, blastocyst formation rates, hatching rate, and total cell number in the blastocyst compared to that in the control group. Notably, LMA attenuated the intracellular reactive oxygen species generation induced by H2O2. Furthermore, LMA not only increased intracellular glutathione levels, but also ameliorated mitochondrial membrane potential. In addition, the expression of a zygotic genome activation related gene (YAP1), pluripotency-related genes (OCT4, NANOG, and SOX2), and hatching-related genes (COX2, GATA4, and ITGA5) were up-regulated following LMA supplementation during porcine early stage embryo development. These results demonstrate that LMA has beneficial effects on the development of porcine early stage embryos via regulation of oxidative stress. This evidence provides a novel method for embryo development improvement associated with exposure to LMA.

Evaluation of the early developmental neural toxicity of F-53B, as compared to PFOS, with an in vitro mouse stem cell differentiation model

F-53B, as an alternative to the persistent organic pollutant perfluorooctane sulfonate (PFOS), is amply used in the electric plating industry. F-53B and PFOS have similar physicochemical, biochemical and physiological properties, due to the similarity in their chemical structure. Thus, they may also possess similar toxicities. Although epidemiological studies and in vivo assays have shown that prenatal exposure to PFOS may impair the development of the nervous system, toxicity data for F-53B are still scarce. In this study, we employed an embryonic stem cell (ESC) in vitro differentiation system, to detect the potential developmental neural toxicity of F-53B and PFOS, at human exposure relevant doses. We demonstrated that during early mouse ESC (mESC) neural differentiation, F-53B and PFOS disrupted the expression of neural marker genes and affected the morphology of the differentiated cells. However, the very same treatments did not cause any cytotoxic effects. In conclusion, our ESC in vitro differentiation system was able to prove for the first time that F-53B and PFOS at human exposure relevant concentrations, could alter the expression of differentiation biomarkers, indicating a potential developmental neural toxicity. Based on our findings, it is reasonable to deduce that excessive exposure to F-53B and PFOS may cause severe dysfunctions during early stages of embryo development.

A Lexicon of DNA Modifications: Their Roles in Embryo Development and the Germline.

5-methylcytosine (5mC) on CpG dinucleotides has been viewed as the major epigenetic modification in eukaryotes for a long time. Apart from 5mC, additional DNA modifications have been discovered in eukaryotic genomes. Many of these modifications are thought to be solely associated with DNA damage. However, growing evidence indicates that some base modifications, namely 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), 5-carboxylcytosine (5caC), and N6-methadenine (6mA), may be of biological relevance, particularly during early stages of embryo development. Although abundance of these DNA modifications in eukaryotic genomes can be low, there are suggestions that they cooperate with other epigenetic markers to affect DNA-protein interactions, gene expression, defense of genome stability and epigenetic inheritance. Little is still known about their distribution in different tissues and their functions during key stages of the animal lifecycle. This review discusses current knowledge and future perspectives of these novel DNA modifications in the mammalian genome with a focus on their dynamic distribution during early embryonic development and their potential function in epigenetic inheritance through the germ line.

L-Carnitine affects the early stage embryo development

l-Carnitine affects the early stage embryo development

Blighted ovum in subfertile patients undergoing assisted reproductive technology

To explore the incidence and risk factors of blighted ovum in subfertile patients undergoing assisted reproductive technology (ART). This retrospective analysis was conducted among 2378 patients who were pregnant following embryo transfer at our center from January, 2012 to December, 2015, including cases of early pregnancy losses and simultaneous live births. The cases with early pregnancy losses were divided into embryonic pregnancy and blighted ovum groups based on the presence or absence of an embryonic pole before dilation and curettage. The clinical data of the 3 groups were analyzed for comparisons of the maternal age, paternal age, BMI, AFC, basal FSH, bFSH/bLH, duration of infertility, Gn dosage, Gn days, serum estradiol on the day of HCG administration, endometrium thickness, number of oocyte retrieved, proportion of high-quality embryos transferred, serum β-HCG value on the 10th to 14th days of embryo transfer, infertility type and miscarriage times. The incidences of blighted ovum were compared between cases with different cycles, embryo stages, infertile factors and methods of fertilization. Maternal age and paternal age, BMI, duration of infertility, infertility type and miscarriage times differed significantly between cases with blighted ovum and those with live births. Serum β-HCG level was the lowest in blighted ovum group followed by embryonic pregnancy group and then by live birth group. Blastocyst transfer was associated with a significantly higher incidence of blighted ovum as compared with cleavage embryo transfer (11.6% vs 5.6%, P=0.000). No significant difference was found in the other parameters among the 3 groups (P>0.05). Adjusted logistic regression analysis showed that maternal age, β-HCG level and blastocyst transfer were risk factors of blighted ovum. Advanced maternal age, low β-HCG level and blastocyst transfer may increase the risk of blighted ovum possibly in association with gene imprinting errors during the early stage of embryo development.

Peptone and tomato extract induced early stage of embryo development of Dendrobium phalaenopsis Orchid

Germination and growth of orchid seeds can be accelerated by the addition of organic supplement and plant extract in culture medium. The objective of this study was to determine the effect of peptone and tomato extract on early stage of embryo development of Dendrobium phalaenopsis orchids. Orchid seeds were sown on NP and VW medium with addition of 10% of CW (NPCW and VWCW). Five weeks after seed germination, about 58.03% seed germination was observed on VWCW medium, and only 37.45% seed germination on NPCW. Tomato extract and peptone were added in VWCW, resulting VWCWTP medium. After 4-8 weeks on VWCWTP, 94.42% seeds was germinated into plantlet, but only 67.30% germinated seeds on VWCW. To get optimal growth and development of D. phalaenopsis orchids embryos in the in vitro condition, supplement of 100 ml.L-1 coconut water, 100 mg.L-1 tomato extract and 2 mg.L-1 peptone into VW basic medium is required.

Temporal Petal Closure Benefits Reproductive Development of Magnolia denudata (Magnoliaceae) in Early Spring.

The Magnoliaceae shows strong phylogenetic niche conservatism, in which temporal petal closure has been extensively reported. However, it is yet elusive whether temporal petal closure is an idle floral character inherited from their ancestors or an adaptive trait to their habitats. Here, we monitored the process of temporal floral closure and re-opening in a thermogenic plant, Magnolia denudata (Magnoliaceae). Furthermore, we artificially interrupted temporal petal closure and investigated its effects on development of female and male gametophytes. Intriguingly, we found considerable anatomical changes in the anthers shortly after temporal closure of petals: disintegration of tapeta, crack of anther walls, and release of matured pollens. In comparison with normal flowers, artificially interrupted flowers (no petal closure) showed delayed anther development and slower pollen germination on stigmas, while little difference in embryo morphology was observed during the early stage of embryo development. Moreover, seed set and quality were significantly decreased when petal closure was prevented. In addition, we found pollination accelerated floral closure in M. denudata. Taken together, temporal floral closure benefits reproduction of M. denudata in early spring by promoting anther development and pollen function, which suggests that it is an adaptive floral trait to its specific habitat.

The Arabidopsis homolog of Scc4/MAU2 is essential for embryogenesis

Factors regulating dynamics of chromatin structure have direct impact on expression of genetic information. Cohesin is a multi-subunit protein complex crucial for pairing sister chromatids during cell division, DNA repair and regulation of gene transcription and silencing. In non-plant species cohesin is loaded on chromatin by the Scc2/Scc4 (NIBPL/MAU2) complex. Here we identify AtSCC4, the Arabidopsis homolog of Scc4, and show that it forms a functional complex with AtSCC2, the homolog of Scc2. We demonstrate that AtSCC2 and AtSCC4 act in the same pathway and that both proteins are indispensable for cell fate determination during early stages of embryo development. Mutant embryos lacking either of these proteins develop only up to the globular stage, and show suspensor over-proliferation phenotype preceded by ectopic auxin maxima distribution. We further establish a new assay to reveal the AtSCC4-dependent dynamics of cohesin loading on chromatin in vivo . Our findings define Scc2/Scc4 complex as an evolutionary conserved machinery controlling cohesin loading and chromatin structure maintenance and provide new insight into plant-specific role of this complex in controlling cell fate during embryogenesis.

The Arabidopsis homolog of Scc4/MAU2 is essential for embryogenesis.

Factors regulating dynamics of chromatin structure have direct impact on expression of genetic information. Cohesin is a multi-subunit protein complex that is crucial for pairing sister chromatids during cell division, DNA repair and regulation of gene transcription and silencing. In non-plant species, cohesin is loaded on chromatin by the Scc2-Scc4 complex (also known as the NIBPL-MAU2 complex). Here, we identify the Arabidopsis homolog of Scc4, which we denote Arabidopsis thaliana (At)SCC4, and show that it forms a functional complex with AtSCC2, the homolog of Scc2. We demonstrate that AtSCC2 and AtSCC4 act in the same pathway, and that both proteins are indispensable for cell fate determination during early stages of embryo development. Mutant embryos lacking either of these proteins develop only up to the globular stage, and show the suspensor overproliferation phenotype preceded by ectopic auxin maxima distribution. We further establish a new assay to reveal the AtSCC4-dependent dynamics of cohesin loading on chromatin in vivo Our findings define the Scc2-Scc4 complex as an evolutionary conserved machinery controlling cohesin loading and chromatin structure maintenance, and provide new insight into the plant-specific role of this complex in controlling cell fate during embryogenesis.

DYRK2 displays muscle fiber type specific function during zebrafish early somitogenesis.

Dual specificity tyrosine-phosphorylation regulated kinase 2 (DYRK2) is a serine/threonine kinase. In zebrafish, DYRK2 is expressed in the lateral somites and adaxial cells at the early stage of embryo development. However, its role in early myogenesis had not been elucidated yet. Here, we report that DYRK2 mRNA and MyoD mRNA were colocalized in the muscle progenitor cells in somites, including both the posterior compartment of the lateral somites and adaxial cells. Knockdown of DYRK2 reduced the levels of MyoD transcripts in the muscle progenitor cells in somites. In contrast, overexpression of DYRK2 increased the levels of MyoD transcripts in the muscle progenitor cells in somites. The effects of knockdown and overexpression of DYRK2 on the expression of MyoD in the posterior compartment of the lateral somites were much greater than in the adaxial cells. Further studies indicated that forced expression of DYRK2 increased the levels of fast-twitch skeletal myosin RNA. Moreover, knockdown or forced expression of DYRK2 affected the levels of fast-twitch skeletal myosin protein. Together, these data indicate that DYRK2 is expressed in the developing muscle progenitor cells in somites and that it positively regulates fast-twitch muscle differentiation, at least at the early stages.

88 EFFECT OF BICARBONATE/CO2 LEVEL DURING EMBRYO CULTURE ON EQUINE BLASTOCYST RATE AFTER INTRACYTOPLASMIC SPERM INJECTION

Equine embryos appear to require a high glucose concentration for development to the blastocyst stage. The complete cell-culture medium, DMEM/F-12 (DM), which contains 17 mM glucose, has been widely used for equine embryo culture; however, in other species, high glucose during the early stages of embryo development is detrimental. To avoid this, we initiated a 2-step system using a low-glucose human embryo culture medium (Global) from Days 0 to 5 [Day 0 = day of intracytoplasmic sperm injection (ICSI)], with glucose (20 mM) added to the medium in the second step (Days 5 to 10; Choi et al. 2015 Reproduction 150, 31–41). We noted a high pregnancy loss rate (20%) in our clinical ICSI program (Hinrichs et al. 2014 J. Equine Vet. Sci. 34, 176), which used this 2-step Global system. Limited data are available on pregnancy with DM-produced embryos, but in one study, the loss rate was 1/13 (7.7%; Choi et al. 2011 Reproduction 142, 529–538). It is possible that use of DM in the second step of culture would better support normal blastocyst development than does Global with added glucose. However, DM is typically used at 5% CO2, and Global at 6% CO2, so use of both media would necessitate 2 sets of incubators. In the present study, we explored the use of DM in the second step of a two-step equine embryo culture system, under different CO2 environments. Oocytes were collected from research mares via follicle aspiration and were held overnight before being matured in vitro for 30 h. All media included 10% fetal bovine serum. On Days 0 to 5 after ICSI, all embryos were cultured in Global under 6% CO2 in mixed gas (5% O2 and remainder N2) at 38.2°C. In Experiment 1, on Day 5, embryos were transferred to DM prepared according to our standard method, with 14.3 mM NaHCO3 and 5 mM NaOH, and were cultured in mixed gas at either 5% CO2 or 6% CO2. Five replicates were performed. In Experiment 2, DM was prepared by our standard method, or with 24.2 mM bicarbonate and no NaOH. When pH was measured using a pH meter after media were equilibrated overnight, this higher bicarbonate provided the same pH at 6% CO2 (pH 7.3), as was achieved with the standard DM preparation at 5% CO2. Six replicates were performed. In both experiments, blastocyst development was assessed on Days 7 to 10, and blastocyst rates were compared between treatments by Fisher’s exact test. In Experiment 1, blastocyst rates were 43%, 13/30 and 27%, 8/30 for the standard DM preparation in 5% and 6% CO2, respectively (P &gt; 0.05). In Experiment 2, the blastocyst rates were 34%, 14/44 for the standard DM preparation at 5% CO2 and 43%, 19/44 for the high-bicarbonate DM at 6% CO2 (P &gt; 0.05). We conclude that a 2-step Global-DM system can support equine blastocyst production under a consistent CO2 environment (6%) if DM bicarbonate levels are adjusted to balance the increased CO2. This work was supported by the Clinical Equine ICSI Program, Texas A&amp;M University, and by the Link Equine Research Endowment Fund, Texas A&amp;M University.

Matrix-assisted laser desorption/ionization imaging mass spectrometry for the spatial location of feline oviductal proteins.

With the purpose of identifying factors involved in early stages of embryo development in the domestic cat, matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) was used for the first time to describe the spatial localization of proteins in the oviducts of queens. Oviducts were obtained from two 2 and 4years old cross-bred queens, divided into three segments, snap-frozen in liquid nitrogen and then stored at -80°C until use. Next, they were sectioned in a cryostat, fixed on ITO (indium tin oxide) conductive glass slides for MALDI-IMS and serial sections were collected on microscope slides for histology. As confirmed by histology, MALDI-IMS was able to show contrasting protein distributions in the oviductal infundibulum, ampulla and isthmus. Mass spectra were characterized by abundant ions of m/z 1,259, 4,939, 4,960 and 10,626, which have been tentatively attributed to keratin, thymosin β10, thymosin β4 and S100, respectively. Keratin and thymosins are involved in the biological response to tissue damage. S100 proteins are calcium-modulated proteins implicated in a variety of cellular activities, including cell differentiation and regulation of cell motility. These results suggest that protein composition differs between segments of the cat oviduct, which corresponds to morphological changes within these sections. Further functional studies could elucidate the effects of these proteins on feline reproductive physiology.

L-Carnitine affects preimplantation embryo development toward infertility in mice.

l-Carnitine (l-Cn), despite the beneficial role as energy-generating substance delivering long-chain fatty acids to the β-oxidation pathway in mitochondria, has been accused to cause an endometriosis-like state to BALB/c mice manifested by increased inflammatory cytokines in serum and peritoneal fluid, accumulation of immune cells in the peritoneal cavity and uterine walls and most importantly, correlating to infertility. Exploring this type of infertility, the effect of l-Cn on preimplantation embryo development, ovarian integrity and systemic maternal immunity was studied. Using nonlinear microscopy analysis, which was shown to be a powerful tool for determining embryo quality by quantitatively estimating the lipid body (LB) content of the cells, it was shown that in vitro and in vivo administration of l-Cn significantly decreased LB mean area in zygotes. Daily intraperitoneal administration of 2.5mg l-Cn for 3, 4 and 7days to mice significantly decreased the percent of normal zygotes. However, only the 7-day treatment persisted by affecting 2- and 8-cell stage embryos, while almost abolishing blastocyst development. Such effects were accompanied by abnormal ovarian histology, showing increased numbers of corpora luteus and elevated progesterone concentration in the serum. In addition, it was shown that the 7-day l-Cn treatment pushed maternal systemic immunity toward inflammation and immunosuppression by increasing CD11b-, CD25- and CD11bGr1-positive cells in spleen, which opposed the necessity for immunostimulation at these early stages of pregnancy. In conclusion, the results presented here demonstrated that elevated doses of l-Cn affect early stages of embryo development, leading to infertility.

Target genes of Dpp/BMP signaling pathway revealed by transcriptome profiling in the early D.melanogaster embryo

In the early Drosophila melanogaster embryo, the gene regulatory network controlled by Dpp signaling is involved in the subdivision of dorsal ectoderm into the presumptive dorsal epidermis and amnioserosa. In this work, we aimed to identify new Dpp downstream targets involved in dorsal ectoderm patterning. We used oligonucleotide D. melanogaster microarrays to identify the set of genes that are differential expressed between wild type embryos and embryos that overexpress Dpp (nos-Gal4>UAS-dpp) during early stages of embryo development. By using this approach, we identified 358 genes whose relative abundance significantly increased in response to Dpp overexpression. Among them, we found the entire set of known Dpp target genes that function in dorsal ectoderm patterning (zen, doc, hnt, pnr, ush, tup, and others) in addition to several up-regulated genes of unknown functions. Spatial expression pattern of up-regulated genes in response to Dpp overexpression as well as their opposing transcriptional responses to Dpp loss- and gain-of-function indicated that they are new candidate target genes of Dpp signaling pathway. We further analyse one of the candidate genes, CG13653, which is expressed at the dorsal-most cells of the embryo during a restricted period of time. CG13653 orthologs were not detected in basal lineages of Dipterans, which unlike D. melanogaster develop two extra-embryonic membranes, amnion and serosa. We characterized the enhancer region of CG13653 and revealed that CG13653 is directly regulated by Dpp signaling pathway.

49 - Summary of 262 Samples from 46 Patients Received for Preimplantation Genetic Screening by Array CGH: Hints at Early Stage Embryo Development

49 - Summary of 262 Samples from 46 Patients Received for Preimplantation Genetic Screening by Array CGH: Hints at Early Stage Embryo Development

Dormancy induction by summer temperatures and/or desiccation in imbibed seeds of trumpet daffodils Narcissus alcaracensis and N.longispathus (Amaryllidaceae).

We analysed the effects of summer temperatures (28/14°C) and/or desiccation (from 48% to 8% humidity) on imbibed Narcissus alcaracensis and N.longispathus seeds with an elongating embryo. In the N.alcaracensis seeds that overcame dormancy (embryo elongation=27.14%), exposure to high temperatures induced secondary dormancy and reduced subsequent embryo growth. A further 3-month cold stratification (5°C) was required to break secondary dormancy. Desiccation in early embryo growth stages (elongation=11.42%) also reduced germination. Desiccation in the seeds in a more advanced growth stage (i.e. embryo elongation=27.14%) induced secondary dormancy, which the further 3-month cold stratification did not overcome. When desiccation was preceded by high temperatures, seeds better overcame secondary dormancy (i.e. longer embryo elongation and seed germination). Treatments did not affect seed viability. In the N.longispathus seeds that overcame dormancy (embryo elongation=59.21%), exposure to high temperatures induced secondary dormancy and they needed a further 1-month stratification at 15/4°C+2months at 5°C to reactivate the germination process. When embryo elongation was 42.10%, seed desiccation totally impeded subsequent germination. When embryo elongation reached 59.21%, desiccation induced secondary dormancy, which was not overcome by the above-described stratification treatment. When desiccation was preceded by high temperatures, seeds better overcame dormancy. Stress treatments killed 5-10% of seeds. This study suggests that the seeds of species with complex morphophysiological dormancy (MPD) levels are sensitive to desiccation in early embryo development stages, as opposed to the seeds of species with deep simple epicotyl MPD, which better tolerate water stress.

Female gametophyte and embryo development in Helleborus bocconei Ten. (Ranunculaceae).

In this study, we investigated cytohistochemistry, cycle progression, and relative DNA content of the female gametophyte cells of Helleborus bocconei Ten. before and after fertilization process. The early stages of embryo development were also investigated. H. bocconei possesses a monosporic seven-celled/eight-nucleate Polygonum type female gametophyte, characterized by a morpho-functional polarity. The cells of the embryo sac showed abundant reserves of polysaccharides, strongly increasing in the egg cell just before fertilization. With different timing in DNA replication during cell cycle progression, synergids, egg cells, and polar nuclei showed a haploid DNA content at the end of their differentiation, while antipodes underwent three DNA endoreduplication cycles. Programmed cell death symptoms were detectable in synergid and antipodal cells. After double fertilization, the central cell quickly underwent many mitotic cycles forming the endosperm, which exhibited a progressive increase in protein bodies and starch grains. Close to the developing embryo, the endosperm differentiated a well-defined region rich in a fibrillar carbohydrate matrix. The zygote, that does not start immediately to divide after double fertilization, developed in to an embryo that reached the heart stage at fruit maturation time. A weakly differentiated embryo at this time indicates a morpho-physiological dormancy of seeds, as a survival strategy imposed by the life cycle of this plant with seed dispersal in spring and their germination in the following winter.

First Morphological and Molecular Evidence of the Negative Impact of Diatom-Derived Hydroxyacids on the Sea Urchin Paracentrotus lividus.

Oxylipins (including polyunsaturated aldehydes [PUAs], hydoxyacids, and epoxyalcohols) are the end-products of a lipoxygenase/hydroperoxide lyase metabolic pathway in diatoms. To date, very little information is available on oxylipins other than PUAs, even though they represent the most common oxylipins produced by diatoms. Here, we report, for the first time, on the effects of 2 hydroxyacids, 5- and 15-HEPE, which have never been tested before, using the sea urchin Paracentrotus lividus as a model organism. We show that HEPEs do induce developmental malformations but at concentrations higher when compared with PUAs. Interestingly, HEPEs also induced a marked developmental delay in sea urchin embryos, which has not hitherto been reported for PUAs. Recovery experiments revealed that embryos do not recover following treatment with HEPEs. Finally, we report the expression levels of 35 genes (involved in stress, development, differentiation, skeletogenesis, and detoxification processes) to identify the molecular targets affected by HEPEs. We show that the 2 HEPEs have very few common molecular targets, specifically affecting different classes of genes and at different times of development. In particular, 15-HEPE switched on fewer genes than 5-HEPE, upregulating mainly stress-related genes at a later pluteus stage of development. 5-HEPE was stronger than 15-HEPE, targeting 24 genes, mainly at the earliest stages of embryo development (at the blastula and swimming blastula stages). These findings highlight the differences between HEPEs and PUAs and also have important ecological implications because many diatom species do not produce PUAs, but rather these other chemicals are derived from the oxidation of fatty acids.