Embryo Tissues Research Articles

Influence of fixation method and duration of archiving on immunohistochemical staining intensity in embryonic and fetal tissues

IntroductionIn this study we detail the effect of different fixation agents and the duration of storage has on the immunohistochemical staining positivity of samples of archival embryonic and fetal tissues. Materials and methodsThe samples were stained by indirect two-step immunohistochemistry (IHC) method for Ki-67, cyclin A and β-actin. ResultsIrrespective of the length of tissue archiving, tissue fixation with 10% neutral buffered formalin had better IHC intensity results in all cases when compared to methacarn-fixed tissues. In the case of β-actin, this difference was statistically significant, while differences in Ki-67 and cyclin A were not. The second aspect studied was which effect tissue block archiving duration has on the IHC reactivity. We demonstrated a statistically significant decrease in IHC positivity for all studied antigens between the samples that were archived for 10–19 or 20–45 years, regardless the fixative solution. ConclusionTo the best of our knowledge, the influence that the duration of tissue block archiving has on IHC positivity in human embryo and fetal tissue material has not yet been studied. Although the causes of the IHC positivity decline in archived tissue blocks are not well understood, a possible decrease in IHC over time should be considered, particularly in retrospective studies.

SUN-208 Rare Case of 47XXY/46XX Mosaic Klinefelter Syndrome

Introduction: Klinefelter syndrome (KS) is the most common X chromosome abnormality and genetic cause of male infertility. Nondisjunction of paired X chromosomes during meiosis or gametogenesis gives rise to the karyotype 47XXY seen in 80 % of cases. The most common form of mosaicism results from post-fertilization nondisjunction of the XXY zygote in two cell lines 47XXY/46XY in 15-20 % of KS. The 47XXY/46XX mosaic form is very rare with only 20-25 cases described in the literature to date. Phenotype varies with distribution of the two cell lineages in developing embryo tissues, including KS male, true hermaphroditism, ovarian hypoplasia and normal female. Only 10 cases have been reported of 47XXY/46XX mosaic KS male phenotype . Our case is the 11th. Case: A 39 year old male presented with fatigue, weight gain, poor concentration and loss of libido. He reported no secondary sexual development during puberty, including hair, muscle mass, penis and testicles, and minimal erections. On exam, both testes were less than 2 cm with a normal penis. He had abdominal obesity, reduced muscle mass, and gynecomastia, as well as male pattern frontal baldness. Total testosterone (T) was 125 ng/dl (250-1100), Free T was 16 ng/dl (46-224), albumin was 3.9, FSH was 25 mIU/ml, and LH was 15.9 mIU/ml. Chromosome analysis: 25/30 (83%) cells 47XXY and 5/30 (17 %) cells 46XX. FISH study on 100 peripheral blood cells showed 87 % 47XXY and 14 % 46XX. The patient did not desire fertility and started testosterone replacement. Conclusion: Our case demonstrated the common features of 47XXY/46XX mosaic KS, including small testes, gynecomastia, hypergondotropic hypogonadism, and normal IQ. Interestingly, our patient had male pattern baldness, which was also common in his family. The average age of diagnosis for cases reported to date was 12-62 years, similar to ours, suggesting a delay in diagnosis, as can occur in many KS cases. Distribution of cell lines can vary between different tissues, but the presence of a Y chromosome in the gonad determines the phenotype. Mediastinal germ cell tumor, testicular teratoma, and radioulnar synostosis have been noted in 3 cases but not in this case. Mosaic cases tend to be more androgenic, and are more likely to have spermatogenesis than non-mosaic KS. This along with the failure to detect the 46XX cell line in routine blood cytogenetic tests (as opposed to gonadal tissue) is the reason for delayed detection in many cases. KS mosaicism should be considered in all cases of male infertility.

The Effects of Electromagnetic Fields on Mitochondria: An Ultra‐structural and Biochemical Study

Smart phones use non‐ionizing low frequency electromagnetic waves (RFW) for communication. Excessive use of smart phones may cause adverse health effects. We studied the effects of these RFW on the developing chick embryo. 40 ‘Cobb’ (Gallus gallus domesticus) breed zero‐day fertilized chicken eggs were randomly divided into control and exposed groups. A popular mobile phone and service provider was selected with 1800 MHz frequency, power of 0.47 W/kg and SAR 1.10 w/KG. The mobile phone was placed inside the 30‐egg incubator in call receiving mode in the exposed group, while in the control group, mobile phone was not used. Mobile phone was called by another mobile from outside for 50 minutes daily starting from day one of incubation. The embryos were sacrificed at day 10 (total exposure time 500 minutes) and day 15 (total exposure time 750 minutes), liver and heart tissues were dissected for electron microscopy, heat shock protein 70 (Hsp70) and its mRNA. Results revealed increase in the number of mitochondria in the hepatocytes at day 10 in the exposed group. At day 15, the mitochondria were rounded, elongated, or dumbbell shaped, and cristae were disorganization with areas of degeneration. The mitochondria were found surrounded by fragments of rough endoplasmic reticulum and free ribosomes and an increase number of lipid filled vacuoles were observed in the cytoplasm of the hepatocytes. In the heart muscle, mitochondria became elongated with areas of degeneration of the cristae, however, rough endoplasmic reticulum or free ribosomes were not seen surrounding the mitochondria. Geoffry N et al. reported that mitochondria are the source of free radicals produced in response to electromagnetic wave exposure in human sperms. It was further reported that potential causative mechanism of electron leakage from the mitochondrial electron transport chain caused oxidative DNA damage. Hsin‐Chen LEE reported that oxidative stress induced by H2O2 leads to increase number of mitochondria and mtDNA in human lung. Gorczynska and Wegrzynowicz reported that mitochondria was the most sensitive organelle to cell stress leading to its swelling, source of free radicals, which was due to electron leakage from the mitochondrial electron transport chain. Increase levels of mRNA and Hsp70 were observed in the exposed groups at both day 10 and 15. This increase was 14% at day 10 and became 39% at day 15 demonstrating cellular mechanism in attempting to express housekeeping genes and Hsp70 for survival. Hsp70 is a multifaceted chaperon protein that is conserved across species and is involved in many cellular metabolic processes. We conclude that RFW exposure caused early damage to mitochondria in liver and heart tissues of chick embryo and resulted in reactive oxygen species (ROS) production which caused marked increase in mRNA and Hsp70.Support or Funding InformationInternal medical college grantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization of Cks2 localization and interaction with Cdk1 in mitotic cells of Xenopus laevis embryos

Although mitotic regulation has been extensively studied, several of the mechanisms behind this process have yet to be fully understood. Our group is particularly interested in mechanisms regulating the timing of mitotic exit and how such mechanisms coordinate with other mitotic cues such as spindle orientation. Recent work in our lab has shown that spindle movements in epithelial cells of the developing frog embryo correlate with mitotic exit and perturbations in normal spindle movements lengthen metaphase. Further, these changes in mitotic progression are accompanied by increases in Cdk1 Tyr15 phosphorylation, specifically at cell junctions. We propose that the link between spindle movement and mitotic progression is an important part of the mechanisms influencing spindle orientation in the frog epithelium. Our current work examines the role of Cks2 in the aforementioned mechanism. Cks2 serves as a targeting subunit for the mitotic kinase Cdk1. Specifically, work in yeast suggests Cks2 may direct Cdk1 toward the anaphase‐promoting complex (APC) and interact with both complexes, facilitating the phosphorylation of Cdk1 and activation of the APC and thus help control mitotic exit. In this study, we characterize Cks2's mitotic function in Xenopus laevis. Our aim is to characterize Cks2 distribution throughout mitosis and determine whether perturbing Cks2 function alters mitotic progression in the gastrula‐stage epithelium. We hypothesize that if Cks2 influences Cdk1 function in mitosis, then the two proteins will exhibit similar localization patterns in mitotic cells. We visualized endogenous Cks2 and also expressed GFP‐tagged Cks2 in fixed epithelial tissues of Xenopus laevis embryos through confocal microscopy. Our early data show that Cks2 localization overlaps with that of a phosphorylated Cdk1(pY15) at cell junctions and overexpression of GFP‐tagged Cks2 results in increased levels of Cdk1‐pY15. In addition, the assay expressing a dominant negative version of Cks2 suggests that depletion of endogenous Cks2 activity may contribute to decreased Cdk1‐pY15 at cell junctions and increased cell area. Therefore, these data suggest a link between Cks2 and Cdk1 in mitotic regulation and are a first step toward a greater characterization of Cks2's role in regulating mitotic progression in vertebrate organisms.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tuftelin Is Required for NGF-Induced Differentiation of PC12 Cells.

Nerve growth factor (NGF) promotes pleiotropic gene transcription-dependent biological effects, in neuronal and non-neuronal cells, including survival, proliferation, differentiation, neuroprotection, pain, and angiogenesis. It is hypothesized that during odontogenesis, NGF may be implicated in morphogenetic and mineralization events by affecting proliferation and/or differentiation of dental cells. Tuftelin belongs to the enamel associated teeth proteins and is thought to play a role in enamel mineralization. We previously reported that tuftelin transcript and protein, which are ubiquitously expressed in various tissues of embryos, adults, and tumors, were significantly upregulated during NGF-induced PC12 differentiation. To further confirm the involvement of tuftelin in the differentiation process, we established a tuftelin-knockdown neuronal PC12 cell model, using a non-cytotoxic siRNA directed towards sequences at the 3' UTR of the tuftelin gene. Using real-time PCR, we quantified tuftelin mRNA expression and found that tuftelin siRNA, but not scrambled siRNA or transfection reagents, efficiently depleted about 60% of NGF-induced tuftelin mRNA transcripts. The effect of tuftelin siRNA was quantified up to 6days of NGF-induced differentiation. Using immunofluorescence and western blot analyses, we also found a direct correlation between reduction of 60-80% in tuftelin protein expression and inhibition of about 50-70% in NGF-induced differentiation of the cells, as was detected after 3-6days of treatment. These results demonstrate an important role for tuftelin in NGF-induced differentiation of PC12 cells. Tuftelin could be a useful target for drug development in disease where neurotrophin therapy is required.

Vascular leakage caused by loss of Akt1 is associated with impaired mural cell coverage.

Angiogenesis plays a critical role in embryo development, tissue repair, tumor growth and wound healing. In the present study, we investigated the role of the serine/threonine kinase Akt in angiogenesis. Silencing of Akt1 in human umbilical vein endothelial cells significantly inhibited vascular endothelial growth factor (VEGF)‐induced capillary‐like tube formation. Mice lacking Akt1 exhibited impaired retinal angiogenesis with delayed endothelial cell (EC) proliferation. In addition, VEGF‐induced corneal angiogenesis and tumor development were significantly inhibited in mice lacking Akt1. Loss of Akt1 resulted in reduced angiogenic sprouting, as well as the proliferation of ECs and mural cells. Addition of culture supernatant of vascular smooth muscle cells (VSMCs) in which Akt1 was silenced suppressed tube formation, the stability of preformed tubes and the proliferation of ECs. In addition, attachment of VSMCs to ECs was significantly reduced in cells in which Akt1 was silenced. Mural cell coverage of retinal vasculature was reduced in mice lacking Akt1. Finally, mice lacking Akt1 showed severe retinal hemorrhage compared to the wild‐type. These results suggest that the regulation of EC function and mural cell coverage by Akt1 is important for blood vessel maturation during angiogenesis.

Similarities and differences in tissue distribution of DLK1 and DLK2 during E16.5 mouse embryogenesis.

DLK1 and DLK2 are transmembrane proteins belonging to the EGF-like repeat-containing family that function as non-canonical NOTCH inhibitory ligands. DLK1 is usually downregulated after embryo development and its distribution in some adult and embryonic tissues has been described. However, the expression and role of DLK2 in embryo and adult tissues remains unclear. To better understand the relevance of both proteins during embryo development, we analyzed the expression pattern of DLK1 and DLK2 in 16.5-day-old mouse embryos (E16.5) and evaluated the possible relationship between these two proteins in embryo tissues and cell types. We found that DLK1 and DLK2 proteins exhibited a broad distribution pattern, which was detected in developing mouse organs from each of the three germ layers: ectoderm (brain, salivary glands), mesoderm (skeletal muscle, vertebral column, kidney, cartilage), and endoderm (thymus, lung, pancreas, intestine, liver). The expression pattern of DLK1 and DLK2 indicates that both proteins could play a synergistic role during cell differentiation. This study provides additional information for understanding temporal and site-specific effects of DLK1 and DLK2 during embryo morphogenesis and cell differentiation.

Lanthanides compete with calcium for binding to cadherins and inhibit cadherin-mediated cell adhesion.

Lanthanides are rare-earth metals with a broad range of applications in biological research and medicine. In addition to their unique magnetic and spectroscopic properties, lanthanides are also effective mimics of calcium and can stimulate or inhibit the function of calcium-binding proteins. Cadherins are a large family of calcium-binding proteins that facilitate cell adhesion and play key roles in embryo development, tissue homeostasis and tumour metastasis. However, whether lanthanides can bind cadherins and functionally replace calcium binding has not been comprehensively explored. In this study, we investigated the effect of lanthanide binding on cadherin structure and function using terbium, which is a commonly used lanthanide for protein spectroscopy and a proposed anti-cancer agent. We demonstrate that terbium can compete with calcium for binding to calcium-binding sites in cadherins. Terbium binding to cadherins abolished their cell adhesive activity and rendered cadherins sensitive to proteolysis by trypsin. Molecular dynamics simulations indicate that replacement of calcium by terbium results in structural rearrangements and increases the flexibility of the cadherin ectodomain. These changes in structure and dynamics are likely to underlie the inability of lanthanide-bound cadherins to support cell adhesion. Taken together, our findings further knowledge on lanthanide interactions with calcium-binding proteins and provide new insight into the influence of metal chemistry on cadherin structure, dynamics and function.

Abstract P4-16-02: Fertility preservation before neoadjuvant or adjuvant chemotherapy for breast cancer: Final results of PRESAGE trial

Abstract Background: Breast cancer is the most frequent form of cancer in young women. For these patients, breast cancer is generally more aggressive and chemotherapy is more often needed. Chemotherapy is commonly associated with amenorrhea and a decrease of ovarian reserve depending on the patient's age, agents and dose. Embryo, oocyte and ovarian tissue cryopreservation are the three options to preserve fertility. Embryo and oocyte cryopreservation require controlled ovarian stimulation (COS). The use of COS is associated with an increase of estradiol levels. It led to develop protocols using Tamoxifen or Letrozole combined with FSH to protect patients of the potential deleterious effects of the COS. PRESAGE is the first French prospective multicenter feasibility study about fertility preservation by COS combined with Tamoxifen and oocyte +/- embryo cryopreservation before neoadjuvant (NAC) or adjuvant (AC) chemotherapy for breast cancer. Material and method: Prospective multicenter study for patients of less than 40 years, with a breast cancer, for whom a treatment of NAC or AC is indicated and who wish to preserve their fertility. The main objective was to evaluate the feasibility of a COS associating Tamoxifen with FSH followed by an oocyte+/- embryo cryopreservation. The secondary objectives were to evaluate the average deadline prior to the beginning of the chemotherapy and the impact of the type of COS (depending on the phase of the menstrual cycle, conventional-start or random-start COS protocol) on the number and the quality of oocytes harvested. Statistical analysis was performed using SAS statistical software version 9.4 (SAS Institute, Cary, NC). Results: 101 patients were included between February 2014 and July 2017 and 97 patients were eligible for statistical analysis. Mean age of the patients was 31,5 +/- 4 years, the half of them was nulliparous (53/97) and 23,7 % (23/97) were single. They presented mainly SBR II or III (91/96, 94,8 %) lesions, ER + (66/96, 68,7 %). 38 patients benefited from a NAC and 59 of an AC. We have found a significant shorter care (time between the first oncologist's consultation and the beginning of the chemotherapy) according to the type of chemotherapy: 29,7 +/- 15,6 days in NAC group vs 45,2 +/- 21,5 days in AC group (p=0,003) with the same duration of ovarian stimulation in the two groups (10,5 +/-2 days). The success rate of the COS procedure was 90,7 % (88/97) with no significant difference between the groups according to the type of COS (p = 0.06) or the type of chemotherapy (AC vs. NAC p= 0,3). In the 88 patients who had oocyte retrieval, the number of oocytes harvested per patient was 12,8 +/- 7,8 , the number of oocytes preserved was 9,7 +/- 6,1 and an IVF was performed in 12,5% of patients (11/88) with 5,1 +/- 3,1 embryos obtained. We have found no impact of the type of chemotherapy or the type of COS on the number of oocytes or embryos preserved. Conclusion: with a high success rate (90,7%), our study suggests that COS with Tamoxifen and FSH is feasible before adjuvant or neoadjuvant chemotherapy in breast cancer patients. We also show that COS procedure before neoadjuvant chemotherapy can be realized without increasing the time before introducing chemotherapy. Citation Format: Bordes V, Palpacuer C, FRICK C, Leperlier F, Dezellus A, De Blay P, Delay F, Sauterey B, Augereau P, Duros S, Lefeuvre-Plesse C, Lavau S, Durand LM, Mouret MA, Gremeau AS, Campone M, Mirallie S. Fertility preservation before neoadjuvant or adjuvant chemotherapy for breast cancer: Final results of PRESAGE trial [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-16-02.

Concentrations of perfluoroalkyl substances (PFASs) in human embryonic and fetal organs from first, second, and third trimester pregnancies

BackgroundThe persistent environmental contaminants perfluoroalkyl substances (PFASs) have gained attention due to their potential adverse health effects, in particular following early life exposure. Information on human fetal exposure to PFASs is currently limited to one report on first trimester samples. There is no data available on PFAS concentrations in fetal organs throughout all three trimesters of pregnancy. MethodsWe measured the concentrations of perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoroundecanoic acid (PFUnA), and perfluorohexane sulfonic acid (PFHxS) in human embryos and fetuses with corresponding placentas and maternal serum samples derived from elective pregnancy terminations and cases of intrauterine fetal death. A total of 78 embryos and fetuses aged 7–42 gestational weeks were included and a total of 225 fetal organs covering liver, lung, heart, central nervous system (CNS), and adipose tissue were analyzed, together with 71 placentas and 63 maternal serum samples. PFAS concentrations were assayed by liquid chromatography/triple quadrupole mass spectrometry. ResultsAll evaluated PFASs were detected and quantified in maternal sera, placentas and embryos/fetuses. In maternal serum samples, PFOS was detected in highest concentrations, followed by PFOA > PFNA > PFDA = PFUnA = PFHxS. Similarly, PFOS was detected in highest concentrations in embryo/fetal tissues, followed by PFOA > PFNA = PFDA = PFUnA. PFHxS was detected in very few fetuses. In general, PFAS concentrations in embryo/fetal tissue (ng/g) were lower than maternal serum (ng/ml) but similar to placenta concentrations. The total PFAS burden (i.e. the sum of all PFASs) was highest in lung tissue in first trimester samples and in liver in second and third trimester samples. The burden was lowest in CNS samples irrespective of fetal age. The placenta:maternal serum ratios of PFOS, PFOA and PFNA increased across gestation suggesting bioaccumulation in the placenta. Further, we observed that the ratios were higher in pregnancies with male fetuses compared to female fetuses. ConclusionsHuman fetuses were intrinsically exposed to a mixture of PFASs throughout gestation. The compounds were detected in all analyzed tissues, suggesting that PFASs reach and may affect many types of organs. Collectively, our results demonstrate that PFASs pass the placenta and deposit to embryo and fetal tissues, calling for risk assessment of gestational exposures.

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection.

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major pathogens in BAI. Current experimental BAI mammalian animal models such as mouse models are costly and time-consuming, and therefore not suitable for high throughput analysis. Thus, novel animal models as complementary systems for investigating BAI in vivo are desired. In the present study, we aimed to develop a zebrafish embryo model for in vivo visualization and intravital analysis of bacterial infection in the presence of biomaterials based on fluorescence microscopy. In addition, the provoked macrophage response was studied. To this end, we used fluorescent protein-expressing S. aureus and transgenic zebrafish embryos expressing fluorescent proteins in their macrophages and developed a procedure to inject bacteria alone or together with microspheres into the muscle tissue of embryos. To monitor bacterial infection progression in live embryos over time, we devised a simple but reliable method of microscopic scoring of fluorescent bacteria. The results from microscopic scoring showed that all embryos with more than 20 colony-forming units (CFU) of bacteria yielded a positive fluorescent signal of bacteria. To study the potential effects of biomaterials on infection, we determined the CFU numbers of S. aureus with and without 10 µm polystyrene microspheres (PS10) as model biomaterials in the embryos. Moreover, we used the ObjectJ project file "Zebrafish-Immunotest" operating in ImageJ to quantify the fluorescence intensity of S. aureus infection with and without PS10 over time. Results from both methods showed higher numbers of S. aureus in infected embryos with microspheres than in embryos without microspheres, indicating an increased infection susceptibility in the presence of the biomaterial. Thus, the present study shows the potential of the zebrafish embryo model to study BAI with the methods developed here.

Tissue-specific differences in metabolites and transcripts contribute to the heterogeneity of ricinoleic acid accumulation in Ricinus communis L. (castor) seeds.

Castor (Ricinus communis L.) seeds are valued for their production of oils which can comprise up to 90% hydroxy-fatty acids (ricinoleic acid). Castor oil contains mono-, di- and tri- ricinoleic acid containing triacylglycerols (TAGs). Although the enzymatic synthesis of ricinoleic acid is well described, the differential compartmentalization of these TAG molecular species has remained undefined. To examine the distribution of hydroxy fatty acid accumulation within the endosperm and embryo tissues of castor seeds. Matrix assisted laser desorption/ionization mass spectrometry imaging was used to map the distribution of triacylglycerols in tissue sections of castor seeds. In addition, the endosperm and embryo (cotyledons and embryonic axis) tissues were dissected and extracted for quantitative lipidomics analysis and Illumina-based RNA deep sequencing. This study revealed an unexpected heterogeneous tissue distribution of mono-, di- and tri- hydroxy-triacylglycerols in the embryo and endosperm tissues of castor seeds. Pathway analysis based on transcript abundance suggested that distinct embryo- and endosperm-specific mechanisms may exist for the shuttling of ricinoleic acid away from phosphatidylcholine (PC) and into hydroxy TAG production. The embryo-biased mechanism appears to favor removal of ricinoleic acid from PC through phophatidylcholine: diacylglycerol acyltransferase while the endosperm pathway appears to remove ricinoleic acid from the PC pool by preferences of phospholipase A (PLA2α) and/or phosphatidylcholine: diacylglycerol cholinephosphotransferase. Collectively, a combination of lipidomics and transcriptomics analyses revealed previously undefined spatial aspects of hydroxy fatty acid metabolism in castor seeds. These studies underscore a need for tissue-specific studies as a means to better understand the regulation of triacylglycerol accumulation in oilseeds.

Oocyte embryo and ovarian tissue freezing in endometriosis: Food for thought

Fertility preservation by oocyte, embryo, or ovarian tissue in females is an established technique now. In addition to oncology patients, the indications have been extended to other benign diseases such as endometriosis which carry a significant risk of decrease in ovarian reserve due to disease or therapy instituted. Fertility preservation is not without its pros and cons. All young women need to be counseled regarding the decrease in ovarian reserve with endometriosis, age and/or surgery. Fertility preservation options need to be discussed whenever immediate childbearing is not possible or desired.

Structural and functional damage to the hippocampal neurovascular unit in diabetes-related depression

Previous studies have shown that models of depression exhibit structural and functional changes to the neurovascular unit. Thus, we hypothesized that diabetes-related depression might be associated with damage to the hippocampal neurovascular unit. To test this hypothesis, neurons, astrocytes and endothelial cells were isolated from the brain tissues of rat embryos and newborn rats. Hippocampal neurovascular unit co-cultures were produced using the Transwell chamber co-culture system. A model of diabetes-related depression was generated by adding 150 mM glucose and 200 µM corticosterone to the culture system and compared with the neuron + astrocyte and astrocyte + endothelial cell co-culture systems. Western blot assay was used to measure levels of structural proteins in the hippocampal neurovascular unit co-culture system. Levels of basic fibroblast growth factor, angiogenic factor 1, glial cell line-derived neurotrophic factor, transforming growth factor β1, leukemia inhibitory factor and 5-hydroxytryptamine in the hippocampal neurovascular unit co-culture system were measured by enzyme-linked immunosorbent assay. Flow cytometry and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling staining was used to assess neuronal apoptosis in the hippocampal neurovascular unit. The neurovascular unit triple cell co-culture system had better barrier function and higher levels of structural and secretory proteins than the double cell co-culture systems. In comparison, in the model of diabetes-related depression, the neurovascular unit was damaged with decreased barrier function, poor structural integrity and impaired secretory function. Moreover, neuronal apoptosis was markedly increased, and 5-hydroxytryptamine levels were reduced. These results suggest that diabetes-related depression is associated with structural and functional damage to the neurovascular unit. Our findings provide a foundation for further studies on the pathogenesis of diabetes-related depression.

Potential Technological Use of Reserves of <i>Jatropha curcas</i> and <i>J. macrocarpa</i> Griseb. Seeds

J. curcas and J. macrocarpa are useful for restoring degraded areas and their seeds contain oils for biodiesel production. The aim of the work was to determine the reserve substances in the endosperm and the embryo of J. curcas and J. macrocarpa which is important in understanding the germination process, the establishment of these species and its industrial employment. Seeds were imbibed in distilled water for 24 h, to facilitate removal of seed coat with the aim to separate the embryo and nutritive tissues. In both species, the endosperm contained aleurone grains consisting of a crystalloid and globoid, lipids of red color and the starch was not observed. Four major fatty acids were determined in J. curcas seed: oleic, palmitic, stearic, palmitoleic and oleic fatty acid represents about 70% oil content. Oleic acid was the most abundant in J. macrocarpa seeds, while, there was not palmitoleic acid. Seed with predominantly unsaturated fatty acids is ideal for biodiesel industry. The means of the sugar content were: 14.3 μg/mg in endosperm and 104.76 μg/mg in embryo of J. curcas and 6.48 μg/mg in endosperm and 59.20 μg/mg in embryo of J. macrocarpa. The means of the protein content were: 4.2 μg/mg in endosperm and 45.02 μg/mg in embryo of J. curcas and 3.26 μg/mg in endosperm and 31.08 μg/mg in embryo of J. macrocarpa. Sugar and protein contents of Jatropha seeds were significantly higher in embryo in both species (p 0.1), which suggests early mobilization towards the embryo during imbibition period.

The novel model: Experimental optical coherence tomography-guided anterior segment imaging chick embryo model.

Purpose:The aim of this study was to present an experimental optical coherence tomography (OCT)–guided anterior segment (AS) imaging chick embryo model. Through this model, we aimed to reveal similarities and differences between human cornea, AS tissues, and chick embryo tissues by quantitative image analysis.Methods:Ex vivo, the chick embryos’ globes were determined by detailed AS camera of spectral-domain (SD)-OCT in 10 fertilized specific pathogen-free eggs on the 20th day. Quantitative image analysis of anterior chamber tissues was performed with SD-OCT in detail. After imaging, cross sections of the chick embryo globes containing cornea with anterior chamber were histologically examined and compared with human tissues. The similarities of our model with data in the human cornea and AS studies in the literature were compared.Results:SD-OCT imaging was able to successfully delineate the AS tissues of chick embryos such as the cornea, iris, lens, pupil, conjunctiva, ciliary body, anterior chamber, and lens. Quantitative semi-automated measurements showed the following: mean central corneal thickness: 213.4 ± 7.05 μm (197–223 μm), mean anterior chamber depth: 878.9 ± 41.74 (804–919 μm), mean anterior chamber area: 2.43 ± 0.16 mm2 (2.17–2.73 mm2), mean corneoscleral junction (limbal) thickness: 322.8 ± 20.05 μm (289–360 μm), and mean iris thickness: 230.4 ± 13.27 μm (203–245 μm). In addition, detailed histological comparisons of the AS tissues with human tissues were evaluated to be very similar.Conclusion:In conclusion, this chick embryo model mimics human tissues and it can be considered as a platform for the study of teratogen-induced malformations and AS dysgenesis during gestation of AS tissues. In addition, this study demonstrates the feasibility of SD-OCT in the quantitative assessment of AS structures in chick embryo model.

High-Spatial-Resolution Multi-Omics Atlas Sequencing of Mouse Embryos via Deterministic Barcoding in Tissue

Spatial gene expression heterogeneity plays an essential role in a range of biological, physiological and pathological processes but it remains a scientific challenge to conduct high-spatial-resolution, genome-wide, unbiased biomolecular profiling over a large tissue area. Herein, we present a fundamentally new approach – microfluidic Deterministic Barcoding in Tissue for spatial omics sequencing (DBiT-seq). Parallel microfluidic channels (10μm, 25μm, or 50μm in width) are used to deliver molecular barcodes to the surface of a formaldehyde fixed tissue slide in a spatially confined manner. Crossflow of two sets of barcodes A1-A50 and B1-B50 followed by ligation in situ yields a 2D mosaic of tissue pixels, each containing a unique combination of full barcode AiBj (i=1-50, j=1-50). It permits simultaneous barcoding of mRNAs, proteins, or even other omics on a fixed tissue slide, enabling the construction of a high-spatial-resolution multi-omics atlas by NGS sequencing. Applying it to mouse embryo tissues revealed all major tissue types in early organogenesis, distinguished brain microvascular networks, discovered new developmental patterning in forebrain, and demonstrated the ability to detect a single-cell-layer of melanocytes lining an optical vesicle and asymmetric expression of Rorb and Aldh1a1 within it, presumably associated with the onset of retina and lens, respectively. Automated feature identification using spatial differential expression further identified dozens of developmental features. DBiT-seq is a highly versatile technology that may become a universal method for spatial barcoding and sequencing of a range of molecular information at a high resolution and the genome scale. It can be readily adopted by biologists with no experience in microfluidics or advanced imaging, and could be quickly disseminated for broader impacts in a variety of fields including developmental biology, cancer biology, neuroscience, and clinical pathology.

Using ATAC-seq and RNA-seq to increase resolution in GRN connectivity.

Echinoderms have some of the most complete reconstructed developmental gene regulatory networks (GRN) of any embryo, accounting for the formation of most embryo tissues and organs. Yet, many nodes (genes and regulators) and their regulatory interactions are still to be uncovered. Traditionally, knockdown/knockout experiments are performed to determine regulator-gene interactions, which are individually validated by cis-regulatory analysis. Differential RNA-seq, combined with perturbation analysis, allows for genome-wide reconstruction of a GRN around given regulators; however, this level of resolution cannot determine direct interactions. ChiP-chip or ChIP-seq is better equipped for determining, genome-wide, whether binding of a given transcription factor (TF) to cis-regulatory elements occurs. Antibodies for the TFs of interest must be available, and if not, this presents a limiting factor. ATAC-seq identifies regions of open chromatin, that are typically trimethylated at H3K4, H3K36 and H3K79 (Kouzarides, 2007), for a given time point, condition, or tissue. This technology combined with RNA-seq and perturbation analysis provides high resolution of the possible functional interactions occurring during development. Additionally, ATAC-seq is less expensive than ChIP-seq, requires less starting material, and provides a global view of regulatory regions. This chapter provides detailed steps to identify potential regulatory relationships between the nodes of a GRN, given a well assembled genome, annotated with gene models, and ATAC-seq data combined with RNA-seq and knockdown experiments.

Cytotoxic and Antiproliferative Activity of Polyisoprenoids in Seventeen Mangroves Species Against WiDr Colon Cancer Cells

Background:Secondary metabolites from the group of isoprenoid compounds are widely distributed in mangrove plants. Polyisoprenoids (dolichol and polyprenol) are known to have benefits as anticancer agents. The present study was conducted to determine the cytotoxic potential of polyisoprenoids in leaves from seventeen selected mangrove species against colon cancer (WiDr) cells.Methods:Cytotoxic activity was evaluated by MTT assay in vitro using WiDr human colon cancer cells and 3T3 fibroblasts from Swiss albino mouse embryo tissue as controls. Mechanisms of action were approached by assessing apoptosis and the cell cycle using flow cytometry and fluorescence microscopy with annexin V-FITC, as well as expression of Bcl-2 and cyclin D1 by immunocytochemistry.Results:Polyisoprenoids from N. fruticans leaves demonstrated the highest anticancer activity, with an IC50 of 180.2 µg/mL, as compared to 397.7 µg/mL against 3T3 normal cells. Significant decrease in the expression of Bcl-2 and cyclin D1 was also noted, facilitating apoptosis and arrest of the cell cycle in the G0-G1 phase in WiDr cells. The present study showed for the first time that polyisoprenoids from N. fruticans exhibit concrete anticancer activity in vitro, decreasing cell proliferation and inducing apoptosis in colon cancer cells.Conclusions:Polyisoprenoids isolated from N. fruticans leaves may have promise as a source of anticancer agents.

Uterine RGS2 expression is regulated by exogenous estrogen and progesterone in ovariectomized mice, and downregulation of RGS2 expression in artificial decidualized ESCs inhibits trophoblast spreading in vitro.

Embryo implantation is a complicated event that relies on two critical factors: the competent blastocyst and the receptive uterus. Successful implantation results from tight coordination of these two factors. The maternal hormone environment of the uterus and molecular cross-talk between the embryo and uterine tissue play pivotal roles in implantation. Here we showed that regulator of G-protein signaling 2 (RGS2), a member of ubiquitous family of proteins that regulate G-protein activation, plays an important role in embryo implantation by interfering in the cross-talk between the embryo and uterine tissue. RGS2 expression increased during the implantation process, and was higher in the implant site than at the nonimplantation site. Meanwhile, ovariectomized (OVX) mice exhibited higher expression of RGS2 in the uterus. Exogenous 17β-estradiol and progesterone in OVX mice downregulated the expression of RGS2. Treatment with exogenous 17β-estradiol alone caused uterine RGS2 messenger RNA levels of OVX mice to return to those of normal female mice; when these mice were treated with progesterone or 17β-estradiol plus progesterone, RGS2 levels rose. Downregulation of Rgs2 by small interfering RNA in an in vitro coculture system of decidualized endometrial stromal cells and blastocysts inhibited blastocyst outgrowth by restricting trophoblast spreading, suggesting a mechanism by which RGS2 regulates embryo implantation.