Expression In Day Research Articles

Effect of Preimplantation Bisphenol-A Exposure on Uterine Receptivity in Mice.

Uterine receptivity is a transient state in which the uterus can accept an embryo to implant into the uterine wall. It is controlled by the ovarian hormones progesterone (P4) and estrogen (E2). P4 and E2 exert their uterine effects mainly through their respective receptors, PR and ER. Bisphenol-A (BPA) is an endocrine disruptor that has been widely used in polycarbonate plastics and epoxy resins. It has been demonstrated that BPA has adverse effects on uterine development and can alter uterine expression of PR and ER. In addition, it has been reported that BPA treatment at ~400 mg/kg/day in early pregnancy is detrimental to embryo implantation. However, it is unknown whether the adverse effect is on the embryo or the uterus or both during embryo implantation. To investigate the potential effect of BPA on uterine receptivity, wild type C57B6/129 mixed background young adult females (4-6 each group) were mated with untreated wild type stud males. The timed pregnant mice were treated with 0, 40, 100, and 400 mg/kg/day BPA (dissolved in sesame oil) daily via subcutaneously injection from embryonic day 0.5 to 3.5. Implantation sites were visualized with Evans blue dye injection at day 4.5 (mating night as day 0, implantation normally initiates ~ day 4.0 in mice). No implantation sites were detected in both100 and 400 mg/kg/day BPA treated groups. No embryos were flushed from these uteri either, indicating that BPA at these high doses are toxic to the early stage embryos. Reduced number of implantation sites were detected in the 40 mg/kg/day BPA treated group. However, the appearance of the blue bands (implantation sites) was not as sharp as those seen in the uteri of the control group. Immunohistochemistry of PR indicated that there was no decidual zone at the implantation sites in the 40 mg/kg/day BPA treated group, and PR was highly expressed in the luminal epithelium, suggesting that implantation was abnormal in this group. PR expression disappeared from the luminal epithelium but was strong in the primary decidal zone in the implantation sites from the control group. PR expression in day 4.5 uteri from both100 and 400 mg/kg/day BPA treated groups was also dysregulated. To further define the effect of BPA on uterine receptivity at these high doses, we will transfer embryos from untreated females into BPA-treated pseudo-pregnant uterus. Our results demonstrate that high doses of BPA are detrimental to early stage embryos and their effect on uterine receptivity awaits further clarification. (Supported by startup funding from The University of Georgia and funding from the Interdisciplinary Toxicology Program at The University of Georgia). (poster)

Silkworm Coatomers and Their Role in Tube Expansion of Posterior Silkgland

BackgroundCoat protein complex I (COPI) vesicles, coated by seven coatomer subunits, are mainly responsible for Golgi-to-ER transport. Silkworm posterior silkgland (PSG), a highly differentiated secretory tissue, secretes fibroin for silk production, but many physiological processes in the PSG cells await further investigation.Methodology/Principal FindingsHere, to investigate the role of silkworm COPI, we cloned six silkworm COPI subunits (α,β,β′, δ, ε, and ζ-COP), determined their peak expression in day 2 in fifth-instar PSG, and visualized the localization of COPI, as a coat complex, with cis-Golgi. By dsRNA injection into silkworm larvae, we suppressed the expression of α-, β′- and γ-COP, and demonstrated that COPI subunits were required for PSG tube expansion. Knockdown of α-COP disrupted the integrity of Golgi apparatus and led to a narrower glandular lumen of the PSG, suggesting that silkworm COPI is essential for PSG tube expansion.Conclusions/SignificanceThe initial characterization reveals the essential roles of silkworm COPI in PSG. Although silkworm COPI resembles the previously characterized coatomers in other organisms, some surprising findings require further investigation. Therefore, our results suggest the silkworm as a model for studying intracellular transport, and would facilitate the establishment of silkworm PSG as an efficient bioreactor.

Integrin expression and function in the response of primary culture hepatic stellate cells to connective tissue growth factor (CCN2)

Production of connective tissue growth factor (CCN2, also known as CTGF) is a hallmark of hepatic fibrosis. This study examined early primary cultures of hepatic stellate cells (HSC) for (i) CCN2 regulation of its cognate receptor integrin subunits; and (ii) interactions between CCN2 and integrin α5β1, heparan sulphate proteoglycans (HSPG) or fibronectin (FN) in supporting cell adhesion. HSC were isolated from healthy male Balb/c mice. mRNA levels of CCN2 or α5, β1, αv or β3 integrin subunits were measured in days 1–7 primary culture HSC, and day 3 or day 7 cells treated with recombinant CCN2 or CCN2 small interfering RNA. Interactions between CCN2 and integrin α5β1, HSPG or FN were investigated using an in vitro cell adhesion assay. Co-incident with autonomous activation over the first 7 days, primary culture HSC increasingly expressed mRNA for CCN2 or integrin subunits. Addition of exogenous CCN2 or knockdown of endogenous CCN2 differentially regulated integrin gene expression in day 3 versus day 7 cells. Either full length CCN2 (‘CCN21–4’) or residues 247–349 containing module 4 alone (‘CCN24’) supported day 3 cell adhesion in an integrin α5β1- and HSPG-dependent fashion. Adhesion of day 3 cells to FN was promoted in an integrin α5β1-dependent manner by CCN21–4 or CCN24, whereas FN promoted HSPG-dependent HSC adhesion to CCN21–4 or CCN24. These findings suggest CCN2 regulates integrin expression in primary culture HSC and supports HSC adhesion via its binding of cell surface integrin α5β1, a novel CCN2 receptor in primary culture HSC which interacts co-operatively with HSPG or FN.

ESTIMATION COMBINING ABILITY AND HETEROSIS FOR GRAIN YIELD AND ITS RELATED TRAITS IN RICE UNDER WATER STRESS CONDITION

Combining ability analysis was carried out in rice through A 8 x 8 diallel set analysis excluding reciprocals involving 8 diverse parents for grain yield and some yield related traits at the Experimental Farm of Rice Research and Training Center, Sakha, Kafr El-Sheikh, Egypt during 2007 and 2008 summer seasons. The ratio of variances due to general and specific combining abilities (o2 GCA / o2 SCA) was more than unity, indicating the preponderance of additive genes in controlling for 100-grain weight (g). On the contrary, predominance of non-additive gene action chiefly controlled the expression of days to 50% heading, plant height, panicle length, number of panicles/plant, sterility (%) and grain yield/plant. Among the parents, IET 1444, Wab 450 and Balado were found to be significantly superior general combiners for grain yield/plant. The genotypes IET 1444 and Wab 878 were good general combiners for plant height, panicle length and sterility (%). Moreover, Balado, Wab 450 and Wab 878 were the best general combiners for 100-grain weight. On the other hand, Sakha 102, Balado and Gaori rice varieties were good combiners for earliness. In addition, a total of 14 crosses exhibited positive and highly significant SCA for grain yield /plant. The promising combinations for grain yield and most of the studied traits were Sakha 102 X Sakha 104, Wab 450 X IET 1444, Wab 450 X GZ 1368 and IET 1444 X Wab 878. It is observed that majority of the crosses with high SCA for grain yield were resulted from low / low or high / high or high / low combining parents. Highly significant negative estimates of SCA for number of days to 50% heading (earliness) were recorded for Wab 450 X Wab 878, Sakha 102 X Sakha 104 and Wab 450 X GZ 1368 cross combinations. Moreover, 16 cross combinations were found to be the best specific combinations for plant height. The cross combinations, Sakha 102 X IET 1444, Sakha 104 X IET 1444, Sakha 104 X Wab 878 and Wab 450 X GZ 1368 were the best specific combinations for panicle length and number of panicles /plant. Ten cross combinations exhibited highly significant and negative SCA effects for sterility %, while, nineteen crosses were the best cross combinations for 100-grain weight. The results also revealed that, a greater magnitude of heterosis when it measured as a deviation from mid-parent and better parent was observed in Wab 450 X Wab 878, Wab 878 X Gaori, Sakha 104 X Wab 878, IET 1444 X Wab 878, Wab 450 X Gaori and Sakha 102 X GZ 1368 rice crosses for grain yield /plant. Grain yield was positively and strongly correlated with each of number of panicles/plant (0.563), 100 grain weight (0.659) and panicle length (0.330). However, a highly significant and positive estimate of phenotypic correlation coefficient (0.574) was recorded between plant height and panicle length. On the other hand, significant negative estimates of phenotypic correlation coefficients were recorded between number of panicles/plant and each of panicle length (-0.445) and 100-grain weight (-0.295), and between the last ones and panicle length (-0.312).

Effect of estrus induction on prostaglandin content and prostaglandin synthesis enzyme expression in the uterus of early pregnant pigs

Prostaglandins (PGs) play a pivotal role in maternal recognition of pregnancy and implantation in pigs. In the present study, PGE2, PGF2α, and PGFM (PGF2α metabolite) content, as well as PGE2 synthase (mPGES-1) and PGF2α synthase (PGFS) expression was investigated in early pregnant gilts with natural (n=21) and PMSG/hCG-stimulated (n=19) estrus. Endometrial tissue samples, uterine luminal flushings (ULFs), and blood serum were collected on days 10–11, 12, and 15 after insemination. Additionally, day 15 conceptuses were collected for mPGES-1 and PGFS protein expression. Effect of estrus induction was observed on day 15 of pregnancy, when the content of PGE2 in the uterine lumen was fourfold lower in gonadotropin-stimulated gilts in comparison to controls (P<0.001). Decreased PGE2 content in ULFs of gonadotropin-treated pigs was preceded by lower endometrial mPGES-1 gene expression in hormonally-stimulated animals in comparison to control gilts (P<0.01). On the other hand, estrus induction with PMSG/hCG resulted in higher PGE2 accumulation in the endometrial tissue on day 15 of pregnancy (P<0.01). Furthermore, PGF2α content in the endometrium and PGFM levels in blood serum were lower in gonadotropin-treated gilts, especially on day 12 after insemination when compared to control gilts (P<0.01). Finally, PGFS expression in day 15 conceptuses was decreased in animals with hormonally-induced estrus. We conclude that PMSG/hCG stimulation of prepubertal gilts to induce estrus results in changes of PG production and secretion during early pregnancy, which, in turn, may affect conceptus development, implantation, and the course of pregnancy.

152 POST-HATCHING EMBRYO DEVELOPMENT IN VITRO UNTIL DAY 15 IN AGAR GEL DILUTED IN PBS OR IN MilliQ WATER

Post-hatching (PH) in vitro embryo culture is a procedure that allows the establishment of more accurate tools for evaluating embryo developmental potential. An important step in the PH system is the tunnels preparation, since they will hold the developing embryo in advanced stages during culture. However, if the diluent used to dissolve agarose for tunnels preparation can influence the diffusion of nutrients from culture medium to the gel compromising embryo development, is not known. The aim of this study was to compare developmental kinetics and gene expression of Day 15 embryos cultured in the PHD system using PBS and MilliQ water to dilute the agar gel used for tunnels construction. Bovine oocytes obtained from slaughterhouse ovaries were matured, fertilized, and cultured in vitro for 8 days in SOFaaci with 5% fetal bovine serum (FBS) at 39°C in 5% CO2 in air. On Day 9, PHD (Brandão et al. 2004 Biol. Reprod. 71, 2048-2055) medium was added in culture droplets and on Day 11 embryos were measured. Only morphologically normal with ≥0.5 mm of diameter Day 11 blastocysts were placed individually in the tunnels. The tunnels were produced using agar gel 2.4% diluted either in PBS or MilliQ water, supplemented with 10% FBS. Morphology and length of embryos were evaluated on Day 11, Day 12.5, Day 14, and Day 15. Elongated embryos in Day 15 were used for extraction of total RNA using Trizol Reagent to verify gene expression by qPCR. A total of 4 pools containing 3 Day 15 embryos in each pool were used to evaluate the expression of genes involved in glucose metabolism [glucose-6-phosphate dehydrogenase (G6PDH), solute carrier family 2 member 1 (SLC2A1), solute carrier family 2 member 3 (SLC2A3), phosphoglycerate kinase 1 (PGK1)], placenta development [placenta-specific 8 (PLAC8), keratin proteins 8 (KRT8)], heat stress [heat shock transcription factors 1(HSF1)], and maternal recognition of pregnancy [interferon tau (IFNT)]. Embryo growth and gene expression data were examined by the t-test for parametric or Mann-Whitney for non-parametric (P &lt; 0.05). From a total of 2,933 oocytes used, 1,216 (41%) reached the blastocyst stage on Day 8 and of those 72% hatched on Day 9 (n = 873). On Day 11, 39.5% (345/873) of the hatched blastocysts had diameter ≥0.5 mm and 286 were loaded into the MilliQ water (154) and PBS tunnels (132). No difference was observed in the proportion of elongated embryos between PBS (54%, n = 71) and MilliQ water treatment (42%, n = 65) by chi-square analysis. Final size at Day 15 as well as increase in length of the embryos between Days 11 and 15 were similar in PBS (2.37 ± 0.13 mm; 1.68 ± 0.13 mm) and MilliQ water tunnels (3.03 ± 0.23 mm; 2.33 ± 0.22 mm). Regarding gene expression analysis, only SLC2A1 had a tendency (P = 0.08) to be higher expressed in embryos cultured in PBS tunnels. The results suggested that water can be use to construct agar gel tunnels for the PHD system without interfering in embryo developmental kinetics until Day 15 of culture. Embrapa, CNPq, CAPES.

Capecitabine, Oxaliplatin and Radiotherapy: A Phase IB Neoadjuvant Study for Esophageal Cancer with Gene Expression Analysis

Purpose: To determine the maximal tolerated dose of capecitabine with oxaliplatin + radiotherapy in a phase I study of localized esophageal cancer. Patients and Methods: Oxaliplatin (85 mg/m2) administered on days 1, 15, and 29. Capecitabine administered twice daily 5 days weekly; dose levels (DL) were 1, 1000; 2, 1250; and 3, 1500 mg/m2 with 50.4 Gy radiation. Results: Dose-limiting toxicity was reached at DL 3. Carboxylesterase expression in day 2 tumor specimens and induction correlated with response (p ≤ 0.05). Conclusion: The maximal tolerated dose was 85 mg/m2 of oxaliplatin, 1,250 mg/m2/day of capecitabine, and 50.4 Gy of radiation.

Expression and Hormonal Regulation of IL-11Rα in Canine Uterus During Early Pregnancy

Embryo implantation is critical for the successful establishment of pregnancy. Interleukin-11 (IL-11) is essential for adequate decidualization in the mouse and human via binding to the specific IL-11 receptor alpha (IL-11Ralpha). But the expression and regulation of IL-11 and IL-11Ralpha in the canine endometrium remain unknown. The aim of this study was to investigate the differential expression of IL-11Ralpha in canine uterus during early pregnancy and its regulation under different conditions by in situ hybridization. Interleukin-11Ralpha mRNA was mainly localized in glandular epithelium in canine uterus. There was a low level of IL-11Ralpha expression in the glandular epithelium on days 6, 12 and 17 of pregnancy. On day 20 of pregnancy when embryo implanted, IL-11Ralpha mRNA was highly expressed in the glandular epithelium surrounding the embryo, but not in the luminal epithelium and stroma. On day 23 of pregnancy, the expression of IL-11Ralpha mRNA maintained a constant level compared with the expression of day 20 and increased on day 28 of pregnancy. During the oestrous cycle, a high level of IL-11Ralpha mRNA expression was seen in the oestrous uterus. Progesterone slightly induced the expression of IL-11Ralpha mRNA in the ovariectomized canine uterus. These results suggest that IL-11Ralpha expression is closely related to canine implantation and up-regulated by progesterone.

Analysis of the expression of putatively imprinted genes in bovine peri-implantation embryos

The application of assisted reproductive technologies (ART) has been shown to induce changes in the methylation of the embryonic genome, leading to aberrant gene expression, including that of imprinted genes. Aberrant methylation and gene expression has been linked to the large offspring syndrome (LOS) in bovine embryos resulting in increased embryonic morbidity and mortality. In the bovine, limited numbers of imprinted genes have been studied and studies have primarily been restricted to pre-implantation stages. This study reports original data on the expression pattern of 8 putatively imprinted genes ( Ata3, Dlk1, Gnas, Grb10, Magel2, Mest- 1, Ndn and Sgce) in bovine peri-implantation embryos. Two embryonic developmental stages were examined, Day 14 and Day 21. The gene expression pattern of single embryos was recorded for in vivo, in vitro produced (IVP) and parthenogenetic embryos. The IVP embryos allow us to estimate the effect of in vitro procedures and the analysis of parthenogenetic embryos provides provisional information on maternal genomic imprinting. Among the 8 genes investigated, only Mest- 1 showed differential expression in Day 21 parthenogenetic embryos compared to in vivo and IVP counterparts, indicating maternal imprinting of this gene. In addition, our expression analysis of single embryos revealed a more heterogeneous gene expression in IVP than in in vivo developed embryos, adding further to the hypothesis of transcriptional dysregulation induced by in vitro procedures, either by in vitro maturation, fertilization or culture. In conclusion, effects of genomic imprinting and of in vitro procedures for embryo production may influence the success of bovine embryo implantation.

MicroRNA Regulation of Cell Lineages in Mouse and Human Embryonic Stem Cells

Cell fate decisions of pluripotent embryonic stem (ES) cells are dictated by activation and repression of lineage-specific genes. Numerous signaling and transcriptional networks progressively narrow and specify the potential of ES cells. Whether specific microRNAs help refine and limit gene expression and, thereby, could be used to manipulate ES cell differentiation has largely been unexplored. Here, we show that two serum response factor (SRF)-dependent muscle-specific microRNAs, miR-1 and miR-133, promote mesoderm formation from ES cells but have opposing functions during further differentiation into cardiac muscle progenitors. Furthermore, miR-1 and miR-133 were potent repressors of nonmuscle gene expression and cell fate during mouse and human ES cell differentiation. miR-1's effects were in part mediated by translational repression of the Notch ligand Delta-like 1 (Dll-1). Our findings indicate that muscle-specific miRNAs reinforce the silencing of nonmuscle genes during cell lineage commitment and suggest that miRNAs may have general utility in regulating cell-fate decisions from pluripotent ES cells.

Bcl2 enhances induced hematopoietic differentiation of murine embryonic stem cells

Bcl2 is a potent antiapoptotic gene that can increase resistance of adult bone marrow hematopoietic progenitor cells to lethal irradiation, and thereby preserve their ability to differentiate. However, the effect of Bcl2 on murine embryonic stem (ES) cells induced to undergo hematopoietic differentiation in the absence of a toxic stress is not known. To test this, murine CCE-ES cells that can be induced to undergo hematopoietic differentiation in a two-step process that results in upregulation of Bcl2 were used. Upregulation of Bcl2 precedes formation of hematopoietic embryoid bodies (EB) and their further differentiation into hematopoietic colony-forming units, when plated as single cells in methylcellulose. ES cells stably expressing a Bcl2 siRNA plasmid to "knock-down" endogenous expression or cells expressing wild-type (WT) Bcl2 or phosphomimetic Bcl2 mutants were examined. ES cells expressing the Bcl2 siRNA or those expressing a dominant-negative, nonphosphorylatable Bcl2 display a strikingly reduced capacity to form hematopoietic EBs and colony-forming units compared to cells expressing WT or phosphomimetic Bcl2 that demonstrate an increased capacity. Bcl2's effect on induced-hematopoietic differentiation of ES cells does not result from either decreased apoptosis or a reduced number of cells. Rather, Bcl2-enhances hematopoietic differentiation of ES cells by upregulating p27, which results in retardation of the cell cycle at G1/G 0. Thus siRNA silencing of p27 reverts Bcl2's enhancement phenotype in a manner similar to that of Bcl2 "silencing" or expression of a nonphosphorylable Bcl2. In addition to Bcl2's well-described antiapoptotic and cell-cycle retardant effect on somatic cells, Bcl2 may also function to enhance induced hematopoietic cell differentiation of murine ES cells. These findings may have potential relevance for expanding hematopoietic stem/progenitor cell numbers from an ES cell source for stem cell transplantation applications.

Prevention of ochratoxin A‐induced neural tube defects by folic acid in the genetic polydactyly/arhinencephaly mouse,Pdn/Pdn

The gene responsible for the polydactyly/arhinencephaly (Pdn/Pdn) mouse, which exhibits polysyndactyly and arhinencephaly and has a 13.2% risk of neural tube defects (NTD), has been identified as Gli3. Ochratoxin A (OTA) is a teratogen causing NTD in mice. When Pdn/Pdn embryos were exposed to 2 mg/kg of OTA on day 7.5, the incidence of NTD in Pdn/Pdn fetuses increased to 51.6%. Pre-treatment with folinic acid (FA), metabolically the most active form of folic acid, before OTA-treatment decreased the incidence of NTD to 20.8%. We investigated the effect of OTA and FA on gene expression in day 9 embryos using whole-mount in situ hybridization and real-time PCR. Over-expression of Fgf8 was observed at the anterior neural ridge (ANR) in the non-treated Pdn/Pdn. Over-expression at the ANR expanded in the OTA-treated Pdn/Pdn, and it was ameliorated by pretreatment with FA. Emx2 signal was observed in the dorsal forebrain in the non-treated +/+, but disappeared in the OTA-treated +/+, and was recovered by FA. The Emx2 signal was pale and the expression amount was depressed in the non-treated and OTA-treated Pdn/Pdn embryos. It was suggested that down-regulation of Gli3 induced the over-expression of Fgf8 at the ANR, that OTA treatment accelerated the over-expression, and that pretreatment with FA ameliorated the OTA-induced over-expression of Fgf8 in the Pdn/Pdn. It was also suggested that down-regulation of Gli3 induced the down-regulation of Emx2 in the Pdn/Pdn. It was further speculated that the over-expression of Fgf8 at the ANR and down-regulation of Emx2 in the dorsal forebrain may contribute to NTD induction.

Maternal low protein diet restricted to the preimplantation period induces a gender‐specific change on hepatic gene expression in rat fetuses

It has been shown previously that maternal low protein diet (LPD) throughout rat gestation altered hepatic gene expression and enzyme activities in offspring. Here, we investigate the effect of maternal LPD (9% casein vs. 18% control) exclusively during the preimplantation period (switched diet group) or provided throughout gestation on hepatic gene expression in day 20 fetuses. Using quantitative competitive PCR, we found that switched diet induced a two-fold increase (P = 0.008) in hepatic gene expression of phosphoenolpyruvate carboxykinase (PEPCK, a rate limiting enzyme for gluconeogenesis) in male fetuses and a 17% increase (P = 0.005) in 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1, acts primarily as a reductase to produce active glucocorticoid) in female liver compared with control fetuses. Maternal LPD administered throughout gestation increased 11beta-HSD1 expression in male fetal liver by 27% (P = 0.042) compared with controls. However, maternal LPD fed for either period did not affect fetal hepatic insulin receptor (IR), glucocorticoid receptor (GR), glycogen synthase (GS) nor placental glucose transporter 1 (Glut1) and 3 (Glut3) transcript levels. The alteration in fetal hepatic gene expression could not be attributed specifically to known regulators including insulin or glucose concentrations in fetal blood nor alteration in cAMP in fetal liver, although a combination of these regulatory factors may be responsible. Fetal hepatic glycogen level was unaffected by maternal diet. The present findings show that the long term potential of the preimplantation embryo is sensitive to maternal LPD such that basal levels of hepatic gene expression in day 20 fetuses are altered in a gender-specific manner.

Imprinted gene expression in the rat embryo–fetal axis is altered in response to periconceptional maternal low protein diet

In our previous study, we have shown that maternal low protein diet (LPD, 9% casein vs 18% casein control) fed exclusively during the rat preimplantation period (0-4.25 day postcoitum) induced low birth weight, altered postnatal growth and hypertension in a gender-specific manner. In this study, we investigated the effect of maternal LPD restricted only to the preimplantation period (switched diet) or provided throughout gestation on fetal growth and imprinted gene expression in blastocyst and fetal stages of development. Male, but not female, blastocysts collected from LPD dams displayed a significant reduction (30%) in H19 mRNA level. A significant reduction in H19 (9.4%) and Igf2 (10.9%) mRNA was also observed in male, but not in female, fetal liver at day 20 postcoitum in response to maternal LPD restricted to the preimplantation period. No effect on the blastocyst expression of Igf2R was observed in relation to maternal diet. The reduction in H19 mRNA expression did not correlate with an observed alteration in DNA methylation at the H19 differentially methylated region in fetal liver. In contrast, maternal LPD throughout 20 days of gestation did not affect male or female H19 and Igf2 imprinted gene expression in fetal liver. Neither LPD nor switched diet treatments affected H19 and Igf2 imprinted gene expression in day 20 placenta. Our findings demonstrate that one contributor to the alteration in postnatal growth induced by periconceptional maternal LPD may derive from a gender-specific programming of imprinted gene expression originating within the preimplantation embryo itself.

Effect of culture conditions on the expression and function of Bsep, Mrp2, and Mdr1a/b in sandwich-cultured rat hepatocytes

Rat hepatocytes cultured in a sandwich configuration form functional canalicular networks. The influence of extracellular matrix configuration, medium composition, and confluency on the expression and function of Bsep, Mrp2, and Mdr1a/b in sandwich-cultured (SC) rat hepatocytes was examined. Primary rat hepatocytes were: (1) maintained in various extracellular matrix sandwich configurations, (2) cultured in Dulbecco's modified Eagle's medium (DMEM), Modified Chee's medium (MCM) or Williams’ E medium (WME), and/or (3) plated at decreasing cell density. Bsep, Mrp2, and Mrdr1a/b expression in day 4 SC rat hepatocytes was assessed by Western blot; function was measured by accumulation of taurocholate, 5(and 6)-carboxy-2′,7′-dichlorofluorescein, and rhodamine 123, respectively, in canalicular networks. In general, the extracellular matrix conditions examined resulted in similar protein expression and function. Function of Bsep, Mrp2, and Mdr1a/b was higher in SC rat hepatocytes maintained in DMEM or WME. Mrp2 and Mdr1a/b expression, representative of total cellular content, did not always correlate directly with function, which should be reflective of canalicular membrane expression. Mrp2 expression decreased significantly as cell density decreased in SC hepatocytes. Low plating density in Biocoat™ plates resulted in poor canalicular network formation and reduced function of Mrp2 and Mdr1a/b. Expression and/or function of Mrp2 and Mdr1a/b in rat hepatocytes cultured in a sandwich configuration may be influenced by plating density and media type.

Gene Expression in Leukemic Blasts Persisting at Day 8 of Induction Therapy in Childhood ALL Displays a Common Shift towards Terminally Differentiated B Cells and a Cytoreduction-Associated Impairment of the Translational Machinery.

In childhood acute lymphoblastic leukemia (ALL), persistence of leukemic blasts during therapy is of crucial prognostic significance. To approach the mechanisms of therapy resistance, we addressed genome-wide gene expression in blasts persisting after one week of induction therapy (day 8 blasts) and their molecular signatures as compared with blast cells at initial diagnosis (day 0 blasts). In order to approach this issue experimentally, a procedure has been established including flow sorting of leukemic blasts by their leukemia-associated immunophenotype and preparation of cRNA, starting from a small number of cells. Blast cells from 12 patients with precursor B-cell ALL were investigated using Affymetrix HG U133A microarrays, and genes commonly up- or down-regulated in blast cells under therapy were identified in matched pairs of day 8 and day 0 samples. In spite of the heterogeneous clinical features of the patients (mean rate of cytoreduction after 7 days of initial therapy = 82%, range between 33% and 99%), we were able to determine a set of 310 genes whose expression was commonly changed between day 8 and day 0 with an estimated false discovery rate of 0.05. The identified set of genes indicated inhibited cell cycling, reduced metabolism, and expression changes of multiple factors related to B-cell differentiation. These changes collectively suggested that gene expression in day 8 blasts is shifted towards resting mature B cells. To test this hypothesis, we isolated normal B cells from peripheral blood samples of leukemic patients and compared their gene expression to that of leukemic blasts using Principal Component Analysis (PCA). PCA revealed that day 8 samples are positioned between day 0 samples and normal B-cell samples, and statistical significance of this observation could be established using the Jonckheere-Terpstra test. Changes of B-cell differentiation markers on protein level supported this finding. In addition, we analyzed all genes with regard to the correlation of their expression changes with the rates of cytoreduction in peripheral blood. We observed differential impairment of the key components of the translational machinery including ribosome, eukaryotic 43S preinitiation complex and eukaryotic 48S initiation complex. Overall, expression levels of these factors decreased in therapy-sensitive patients but did not change in therapy-resistant patients. Taken together, investigation of leukemia cells persisting during therapy identifies common and individual expression changes which may potentially affect sensitivity towards anti-leukemic agents and offers new insights into the mechanisms of therapy resistance in ALL.

68 GENE EXPRESSION COMPARISONS BETWEEN BOVINE IN VIVO AND CLONED EMBRYOS PRODUCED BY THREE DIFFERENT METHODS

Developmental pathways in the mammalian embryo are profoundly influenced by the epigenetic interaction of the environment and the genome. Loss of epigenetic control has been implicated in aberrant gene expression and altered imprinting patterns with consequence to the physiology and viability of the conceptus. Bovine somatic cell nuclear transfer (SCNT) is contingent on in vitro culture, and both SCNT and culture conditions are known to induce changes in embryonic gene expression patterns. Using these experimental models, this study compared gene expression of Day 7 cloned blastocysts created from three different SCNT protocols using the same cell line, with Day 7 in vivo blastocysts to elucidate mechanisms responsible for variations in phenotypic outcomes. SCNT methods included: (1) traditional SCNT by subzonal injection (SI); (2) handmade cloning (HMC); and (3) modified serial nuclear transfer (SNT), developed within the group. Four imprinted genes (Grb10, Ndn, Nnat, and Ube3a), four chromatin remodeling genes (Cbx1, Cbx3, Smarca4, and Smarcb1) and two genes implicated in polycystic liver disease (Prkcsh and Sec63) were analyzed in single blastocysts from each treatment (n = 5). All blastocysts expressed Actin, Oct-4 and Ifn-tau. All genes were sequence verified. Several genes were expressed ubiquitously across all groups, including Ndn, Ube3a, Cbx1, Cbx3, and Smarcb1. Interestingly, Grb10 was not expressed in two HMCs and one SNT blastocyst. Nnat was weakly expressed in one in vivo blastocyst and in the majority of cloned blastocysts in all groups. Prkcsh and Sec63 were expressed in all but one HMC blastocyst. While gene expression patterns were mostly maintained following SCNT, the imprinted genes Nnat and Grb10 showed instances of differential or abnormal expression in SCNT embryos. The chromatin remodeling genes were maintained in all SCNT treatments. Prkcsh and Sec63 were both absent in one HMC blastocyst, with implications for liver dysfunction, a condition previously reported in abnormal cloned offspring. The variable mRNA expression following SCNT provides an insight into genetic and environmental factors controlling implantation, placentation, organ formation, and fetal growth.

Gene Expression Analysis of Leukemic Blasts Persisting at Day 8 of Induction Therapy in Childhood ALL.

In childhood acute lymphoblastic leukemia (ALL), early response to therapy is of crucial prognostic significance. In the frontline ALL-BFM (Berlin-Frankfurt-Münster) trial, treatment stratification is based on blast count estimation in peripheral blood at day 8 of induction prephase with prednisone and one dose of intrathecal methotrexate at day 1. To approach yet unknown mechanisms of therapy resistance and to characterize cells persisting under therapy on molecular level, we investigated gene expression profiles of leukemic blasts at day 8 of therapy (“day 8” cells) and their changes as compared with blast cells at initial diagnosis (“day 0” cells). To this end, an experimental procedure has been established including flow sorting of leukemic cells by their leukemia-associated immunophenotype and preparation of cRNA, starting from a small number of cells and using an additional amplification step. Experiments have shown that flow sorting procedure does not affect RNA quality, and this experimental approach facilitates investigation of patient samples with blast cell counts as low as 50 blast cells/μl. Blast cells from ten patients with B-cell precursor ALL were investigated using Affymetrix HG U133A microarrays, and gene expression data were processed by normalization procedure on probe level by variance stabilization. Genes commonly up- or downregulated in blast cells under therapy were identified in matched pairs of day 8 and day 0 samples using Significance Analysis of Microarrays (SAM) and a filtering criterion of at least two-fold mean change. By this procedure a group of 84 genes with a false discovery rate of less than 10 % was identified. In this group, 24 genes, reportedly involved at different levels of the cell cycle regulation, including cell cycle progression (CDC2, cyclin B2), DNA replication (e.g. thymidylate synthetase TYMS, ribonucleotide reductase RRM2, proteins MCM 4 and 6) and execution of mitosis (e.g. cell cycle checkpoint kinases CHEK1 and BUB1B, kinesin-like proteins 1 and 7, and MAD2L1), were found to be downregulated in the day 8 cells. In contrast, genes (n=7) encoding for proteins involved in survival signaling (e.g. IL-4/IL-13 common receptor chain, membrane-spanning 4-domains MS4A1 and CD20) showed increased expression in day 8 blasts. Taken together, the described experimental approach enabled gene expression analysis of ALL cells persisting under therapy and pointed to increased survival signaling in day 8 blasts and their preferential positioning in the G1 cell cycle phase.

Specification of vertebral identity is coupled to Notch signalling and the segmentation clock.

To further analyse requirements for Notch signalling in patterning the paraxial mesoderm, we generated transgenic mice that express in the paraxial mesoderm a dominant-negative version of Delta1. Transgenic mice with reduced Notch activity in the presomitic mesoderm as indicated by loss of Hes5 expression were viable and displayed defects in somites and vertebrae consistent with known roles of Notch signalling in somite compartmentalisation. In addition, these mice showed with variable expressivity and penetrance alterations of vertebral identities resembling homeotic transformations, and subtle changes of Hox gene expression in day 12.5 embryos. Mice that carried only one functional copy of the endogenous Delta1 gene also showed changes of vertebral identities in the lower cervical region, suggesting a previously unnoticed haploinsufficiency for Delta1. Likewise, in mice carrying a null allele of the oscillating Lfng gene, or in transgenic mice expressing Lfng constitutively in the presomitic mesoderm, vertebral identities were changed and numbers of segments in the cervical and thoracic regions were reduced, suggesting anterior shifts of axial identity. Together, these results provide genetic evidence that precisely regulated levels of Notch activity as well as cyclic Lfng activity are critical for positional specification of the anteroposterior body axis in the paraxial mesoderm.

Placental vascular endothelial growth factor receptor system mRNA expression in pigs selected for placental efficiency.

Selecting Yorkshire breeding stock for increased placental efficiency (PE; piglet weight divided by its placental weight) results in larger litters (i.e. approximately 3 more piglets litter-1) and reduced placental sizes. Placental vessel density increases progressively after day 50 of gestation in the pig, and is positively correlated with PE and placental expression of vascular endothelial growth factor (VEGF), a potent angiogenic and permeability factor. To elicit its vascular effects, VEGF must bind to its receptors (R), VEGF-R1 and VEGF-R2. The objective of this study was to compare placental and endometrial blood vessel density and placental VEGF, VEGF-R1 and VEGF-R2 mRNA expression in day 70 and 90 conceptuses from Yorkshire females selected for high PE, low PE, and from unselected controls. A greater (P < 0.05) PE was observed for conceptuses in the high PE selection group when compared to the low PE selection group. Placental blood vessel density increased (P < 0.05) from day 70 to 90 (1.8 +/- 0.1 versus 2.8 +/- 0.2) in association with increases (P < 0.05) in placental VEGF mRNA expression. No selection group differences were observed in expression of VEGF, VEGF-R1, or VEGF-R2 on day 70. By day 90, however, placentae of conceptuses from the high PE group expressed greater (P < 0.05) amounts of VEGF and VEGF-R1 mRNA than the unselected controls and the low PE group. These data demonstrate that increased placental expression of the VEGF receptor system is associated with increased placental vascular density observed with the advancement of gestation in the pig. Although placental blood vessel density was not increased in the high PE selection group, elevated levels of the VEGF receptor system suggest an effect on increasing placental and endometrial blood vessel permeability and/or proximity in the high PE group.