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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.  相似文献   

3.
Male factor infertility has been associated with abnormal DNA methylation at imprinted genes. Little information is available on the status of imprinting in the sperm of men with azoospermia, including the association between aberrant imprinting and obstructive azoospermia (OA) or non-OA (NOA). Analysis of DNA methylation at imprinted genes in the sperm of men undergoing vasectomy reversal would aid determination of whether aberrant imprinting is associated with obstruction. Testicular sperm was retrieved from testicular biopsies obtained from men with azoospermia (N=18), including OA (N=10), NOA (N=5), and unknown pathology (N=3), and from men undergoing vasectomy reversal (N=17). Sperm was also obtained from proven fertile men (N=9). DNA methylation was investigated at multiple CpG sites within the differentially methylated regions (DMRs) of three imprinted genes, H19, IG-GTL2 and MEST, using bisulphite sequencing. Unique clones representative of single cells were analyzed. We found a significant decrease in DNA methylation at the H19 DMR in testicular sperm of azoospermic men compared with proven fertile men. The decrease was also significant between OA and proven fertile men, and between men undergoing vasectomy reversal and proven fertile men, suggesting that aberrant DNA methylation may be associated with obstruction. Changes in DNA methylation at IG-GTL2 and MEST DMRs among groups were not significant. Our data suggest that imprinting abnormalities may be associated with obstruction and may occur in response to changes in testicular environment and not only spermatogenesis failure, as previously reported. Methylation at the H19 DMR was particularly prone to modification in testicular sperm.  相似文献   

4.
Endocrine-disrupting chemicals (EDCs), among which methoxychlor (MXC), have been reported to affect the male reproductive system. This study evaluates the possible deleterious effects of MXC on imprinted genes. After administration of the chemical in adult male mice or in pregnant mice we analyzed by pyrosequencing possible methylation defects in two paternally imprinted (H19 and Meg3 (Gtl2)) and three maternally imprinted (Mest (Peg1), Snrpn, and Peg3) genes in the sperm and in the tail, liver, and skeletal muscle DNAs of the adult male mice and of the male offspring. MXC treatment of adult mice decreased the percentages of methylated CpGs of Meg3 and increased those of Mest, Snrpn, and Peg3 in the sperm DNA. MXC treatment of pregnant mice decreased the mean sperm concentrations by 30% and altered the methylation pattern of all the imprinted genes tested in the F1 offspring. In the latter case, MXC effects were transgenerational but disappeared gradually from F1 to F3. MXC did not affect imprinting in the somatic cells, suggesting that it exerts its damaging effects via the process of reprogramming that is unique to gamete development. A systematic analysis at the CpG level showed a heterogeneity in the CpG sensitivity to MXC. This observation suggests that not only DNA methylation but also other epigenetic modifications can explain the transgenerational effects of MXC. The reported effects of EDCs on human male spermatogenesis might be mediated by complex imprinting alterations analogous to those described in this study.  相似文献   

5.
The preimplantation embryos of a range of mammals can be susceptible to disruptions in genomic imprinting mechanisms, resulting in loss of imprinting. Such disruptions can have developmental consequences involving foetal and placental growth such as Beckwith-Wiedemann syndrome in humans and large offspring syndrome in sheep. Our objective was to investigate the dynamics of establishing monoallelic expression of individual sheep imprinted genes post-fertilisation. Semi-quantitative RT-PCR was used to amplify cDNA from the sheep blastocyst, day 21 foetus and day 21 chorioallantois, to compare expression levels between biparental and parthenogenetic embryos in order to indicate allelic expression status. In common with other mammals, IGF2, PEG1 and PEG3 were paternally expressed in the day 21 conceptus, while H19, IGF2R, GRB10 and p57KIP were maternally expressed. Interestingly, GNAS was maternally expressed in the foetus, but paternally expressed in the chorioallantois at day 21. Overall, the imprinting of ovine GRB10 and IGF2R was comparable with mouse but not with human. Contrary to the trophoblast-restricted maternal expression in both mouse and human, SASH2 (sheep homologue of Mash2/HASH2) was expressed in the ovine foetus and was biallelically expressed in the chorioallantois. Differential methylation of the H19 CTCF III upstream region and IGF2R DMR2 in the chorioallantois revealed predominantly paternal and maternal methylation respectively, indicating conservation of these imprinting regulatory regions. In blastocysts, IGF2R, GRB10 and SASH2 were expressed biallelically, while the other genes were not detected. Thus, for the majority of ovine imprinted genes examined, monoallelic expression does not occur until after the blastocyst stage.  相似文献   

6.
Scope : DNA methylation patterns are tissue specific and may influence tissue‐specific gene regulation. Human studies investigating DNA methylation in relation to environmental factors primarily use blood‐derived DNA as a surrogate for DNA from target tissues. It is therefore important to know if DNA methylation changes in blood in response to environmental changes reflect those in target tissues. Folate intake can influence DNA methylation, via altered methyl donor supply. Previously, manipulations of maternal folate intake during pregnancy altered the patterns of DNA methylation in offspring but, to our knowledge, the consequences for maternal DNA methylation are unknown. Given the increased requirement for folate during pregnancy, mothers may be susceptible to aberrant DNA methylation due to folate depletion. Methods and results : Female mice were fed folate‐adequate (2 mg folic acid/kg diet) or folate‐deplete (0.4 mg folic acid/kg diet) diets prior to mating and during pregnancy and lactation. Following weaning, dams were killed and DNA methylation was assessed by pyrosequencing® in blood, liver, and kidney at the Esr1, Igf2 differentially methylated region (DMR)1, Igf2 DMR2, Slc39a4CGI1, and Slc39a4CGI2 loci. We observed tissue‐specific differences in methylation at all loci. Folate depletion reduced Igf2 DMR1 and Slc39a4CGI1 methylation across all tissues and altered Igf2 DMR2 methylation in a tissue‐specific manner (p<0.05). Conclusion : Blood‐derived DNA methylation measurements may not always reflect methylation within other tissues. Further measurements of blood‐derived and tissue‐specific methylation patterns are warranted to understand the complexity of tissue‐specific responses to altered nutritional exposure.  相似文献   

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Scope : Epidemiological evidence supports the developmental origins of health and disease hypothesis that developmental under/over‐nutrition increases adulthood disease risk. Epigenetic markings are one potential mechanism mediating these effects. Altered folate supply may influence methyl group availability for DNA methylation. We reported low folate supply in utero was associated with reduced global DNA methylation in the murine small intestine of adult offspring. We hypothesised that aberrant methylation would be observed during early development. Methods and results : Female C57BL/6J mice were fed diets containing 2 mg folic acid/kg or 0.4 mg folic acid/kg 4 wk before mating and during pregnancy. At 17.5 day gestation, gene methylation in fetal gut was analysed by Pyrosequencing®. Low folate reduced overall methylation of Slc394a by 3.4% (p=0.038) but did not affect Esr1 or Igf2 differentially methylated region (DMR) 1. There were sex‐specific differences in Slc394a and Esr1 methylation (2.4% higher in females (p=0.002); 4% higher in males (p=0.0014), respectively). Conclusion : This is the first study reporting causal effects of maternal folate depletion on gene‐specific methylation in fetal gut. These observations support reports that altered methyl donor intake during development affects DNA methylation in the offspring. The consequences of epigenetic changes for health throughout the life course remain to be investigated.  相似文献   

9.
The aim of this study was to determine the effects of maternal undernutrition, applied during physiologically relevant stages of development of the reproductive system, on reproductive development in male sheep fetuses. Groups of ewes (n = 11-19) were fed rations providing either 100% (high; H) or 50% (low; L) of metabolizable energy requirements for live weight maintenance during selected 'windows', bounded by days 0, 30, 50, 65 and 110 after mating. Ewes of control groups (HH (Expts 1 and 2) and HHH (Expt 3)) were fed the H ration from mating until they were killed at day 50 (Expt 1), day 65 (Expt 2) or day 110 (Expt 3) of gestation, whereas ewes of other groups were fed the L ration for the periods days 0-30 of gestation (LH and LHH), days 31-50 or days 31-65 of gestation (HL and HLH), days 65-110 of gestation (HHL), or day 0 to day 50, day 65 or day 110 of gestation (LL and LLL) when the animals were killed. At day 50 of gestation, there was no effect of nutritional treatment on mean fetal mass or fetal testicular mass, but there was increased expression of mRNA for steroidogenic acute regulatory protein (StAR) in the testes of LL animals (P < 0.05) compared with HH controls. Compared with HH animals, the mean plasma testosterone concentrations of LL fetuses tended to be higher, but this result did not reach significance. At day 65 of gestation there were no significant differences between treatments in mean fetal masses, testicular masses, mean plasma testosterone concentrations or StAR mRNA content. At day 110 of gestation, fetal masses in the LLL group were lower (P < 0.01) than those of control fetuses, although no differences in testicular size or fetal plasma testosterone concentrations were recorded. It is concluded that the effects of undernutrition on reproductive development of male sheep fetuses are dependent on the timing of the period of undernutrition.  相似文献   

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This study investigated the effects of maternal body condition on fetal growth. Fetal and placental parameters from Dorset ewes of body condition score 2.0 (lean, n = 5), 3.5 (moderate, n = 7) and 5.0 (fat, n = 4) at mating were studied on day 65 of gestation. The fetal weight and fetal weight:crown-rump length ratio were greater in fat ewes than in ewes of moderate condition. The raised total and mean placentome weight in fat ewes compared with ewes of moderate condition may have contributed to their increased fetal growth. However, the fetal crown-rump length was not affected. With in situ hybridization, insulin-like growth factor II (IGF-II) mRNA and insulin-like growth factor binding protein 2 (IGFBP-2), -3 and -6 were all detected in the placentome capsule; IGF-II mRNA was also found in the mesoderm of the fetal villi and IGFBP-3 and IGFBP-6 were present in the caruncular stroma of the maternal villi. Ewes of moderate condition, which had the smallest placentae, had the greatest placental expression of IGF-II, IGFBP-2 and IGFBP-3. In the intercotyledonary endometrium, IGFBP-3, IGFBP-5 and uterine milk protein (UTMP) mRNA were all expressed in the glandular epithelium. IGFBP-3 and IGFBP-5 absorbance values were lowest in the lean ewes, whereas UTMP values were highest. Maternal insulin concentrations were greater in fat ewes, whereas plasma glucose and IGF-I concentrations in the fetal compartment were lowest in fat ewes. Therefore, in obese ewes, fetal and placental growth is increased in mid-gestation in association with higher maternal insulin concentrations and lower expression of IGFBPs in the maternal placentomes. Placental and fetal development in lean ewes may be promoted by reduced IGFBP expression in the placentomes and enhanced UTMP production by the endometrial glands. The ewes of moderate condition had the smallest fetuses and placentae coupled with the highest placental expression of IGF-II and IGFBPs.  相似文献   

12.
This study aimed to determine whether reduced fetal ovary folliculogenesis in ewes undernourished during early/midpregnancy is associated with altered ovarian cell proliferation and/or the expression of apoptosis-regulating genes. Groups of ewes (n = 11-19) were fed either 100% (high; H) or 50% (low; L) of metabolisable energy requirements for live-weight maintenance during selected windows of gestation. All animals were killed at days 50, 65 or 110 of gestation. Between mating and slaughter, control animals were fed the H ration, while animals of other subgroups were fed the L ration from (a) mating to slaughter at 50, 65 or 110 days; (b) 0 to 30 days; (c) 31 to 50 or 65 days; or (d), in the day 110 slaughter group only, from 66 to 110 days. Bouin's-fixed fetal ovaries were examined for (a) Ki67 immunoexpression (proliferation) and (b) Bax and Mcl-1 (apoptosis-regulating genes) expression by in situ hybridisation (day 110) and immunohistochemistry (days 50, 65 and 110). At day 50, maternal nutrition had no effect on Ki67, predominant in germ cells, or Bax and Mcl-1, predominant in the oocytes. Restricted maternal food intake from 0 to 30 days significantly reduced staining for Ki67 in germ cells at day 65 (P < 0.05) but increased staining in granulosa cells at day 110 (P < 0.05). In animals fed the L ration for 110 days, primordial follicle Bax and Mcl-1 were significantly increased (Bax: P < 0.01; Mcl-1: P < 0.05). Granulosa cell Bax was also increased (P < 0.05). When the L ration was fed from 66 to 110 days, granulosa cell Bax (P < 0.05) and primordial follicle Mcl-1 (P < 0.01) were also significantly increased. In the fetal ovarian vasculature, animals underfed for 0-110 days had significantly elevated perivascular Mcl-1 (P < 0.001) and endothelial Bax expression (P < 0.05). Moreover, at day 110, endothelial Mcl-1 was increased by underfeeding from 0 to 30 days (P < 0.05). These data indicate that maternal undernutrition alters proliferation and the expression of apoptosis-regulating genes in the developing fetal ovary. The precise mechanism depends on the window of maternal food restriction.  相似文献   

13.
《Journal of dairy science》2023,106(1):769-782
Aberrant epigenetic nuclear reprogramming, especially imprinting pattern disorders, is one of the major causes of failure of clone development from somatic cell nuclear transfer (SCNT). Previous studies showed that ZFP57 is a key protein required for imprint maintenance after fertilization. In this study, we found that imprinting control regions in several imprinted genes were significantly hypomethylated in cloned embryos compared with in vitro fertilization embryos, indicating a loss of imprinted gene methylation. The ZFP57 expression was capable of maintaining the correct degree of methylation at several imprinting control regions and correcting abnormal hypomethylation. Moreover, we successfully obtained bovine fetal fibroblasts overexpressing ZFP57, which were used as donors for SCNT. Our results demonstrated that overexpression of ZFP57 increased total and trophectoderm cell numbers and the ratio of inner cell mass to total cells, reduced the apoptosis rate and significantly enhanced the development of SCNT blastocysts in vitro, ultimately achieving a degree of methylation similar to that in in vitro fertilization embryos. We concluded that overexpression of ZFP57 in donor cells provided an effective method for enhancing nuclear reprogramming and developmental potential in SCNT embryos. The ZFP57 protein played a key role in maintaining the methylation of imprinted genes during early embryonic development, which may be effective for enhanced SCNT in cattle.  相似文献   

14.
The aims of the present study were: (i) to evaluate verotoxin-producing Escherichia coli (VTEC) prevalence in pork cutting meat; (ii) to determine the effects of cutting process on pork meat contamination by VTEC; (iii) to characterise the VTEC strains isolated from pork and pork cutting plants (virulence genes and serotype); and (iv) to compare the strains isolated the same day in the same cutting plant in order to identify the routes of contamination inside the cutting plant. Pork carcasses from three French cutting plants were sampled before carcass cutting (carcass samples), after carcasses were divided into big portions (untrimmed cuts) and after preparation of primal cuts (rindless boneless cuts), and different environmental sites in each cutting plant were sampled at three different times in the work day. Potable water was also collected. PCR detection of stx genes was performed on a total of 2042 samples. In addition, a second PCR specific for E. coli O157:H7 detection was carried out on the stx-positive samples. VTEC strains were recovered from positive samples by colony hybridisation or immunoconcentration, then serotyped, genetically characterised (eae, ehx, stx1, stx2, stx2e, uidA and genes which are associated with virulence) and pulsotyped. No E. coli O157:H7 was detected. Meat contamination decreased from carcass (12%) and primary cuts (19%) to secondary cuts (5%), whereas environmental contamination increased after 2 h of activity (from 3% before the commencement of the work day to 25% and 20%, 2 and 6 h after commencement of cutting). No VTEC isolates harboured eae, ehx and uidA genes. VTEC contamination routes were not clearly identified.  相似文献   

15.
The recent birth of a camel-llama hybrid, after numerous failed attempts, has prompted an investigation into the glycosylation of apposing fetal and maternal tissues of pregnant camels and alpacas. This study was undertaken to determine whether interspecies differences in glycans are factors that may account in part for the difficulty in producing a viable hybrid. Specimens of camel placentae from day 60 to day 375 of gestation and alpaca placentae from day 22 to term (approximately 345 days) were fixed and embedded in resin, and sections were stained with a panel of 19 biotinylated lectins and an avidin--peroxidase revealing system. Several qualitative interspecies differences in tissue glycosylation were found, mainly in the trophoblast, and especially with respect to bi/tri-antennary bisected N-glycan, fucosylated structures, beta-galactosyl residues and sialyl termini. In the maternal uterine epithelium, differences were found mainly in bi/tri-antennary bisected complex N-glycan and beta-galactosyl residues, indicating that there is more conservation of glycosylation in maternal tissues compared with trophoblast. There were also many quantitative differences in the distribution of glycans. It is possible that a failure to effect the normal glycan--glycan complementation that occurs at the cell surface between maternal and fetal tissues during the implantation processes of apposition and adhesion may account in part for the difficulty in establishing a viable pregnancy between these two species.  相似文献   

16.
The survival of Staphylococcus aureus (St. aureus), Bacillus cereus, Yersinia enterocolitica, Eschericia coli O157:H7, Shigella flexneri (Sh. flexneri) and Salmonella enteritidis (Sa. enteritidis) in urfa cheese (a traditional white‐brined Turkish cheese) which was stored in brine concentrations varying from 12.5 to 17.5% (wt/v) was tested. Two sets of cheeses were made, namely scalded and unscalded cheeses (scalding was done by heating at 95 °C for 3 min). The variations in the counts of pathogenic colonies were monitored throughout a 90‐day storage period at <10 °C. Results indicated that scalding caused statistically significant reductions in the colony counts of Y. enterocolitica, E. coli O157:H7, Sh. flexneri and Sa. enteritidis during the early periods of storage. In contrast, St. aureus and B. cereus were not generally affected by scalding and brine concentrations, although B. cereus in 17.5% (wt/v) brine was affected. In the unscalded cheeses, 12.5 and 15.0% (wt/v) brine concentrations seemed to be insufficient to eradicate the pathogenic organisms examined.  相似文献   

17.
Enucleated oocytes matured in vitro, from which chromosomes were removed by treatment with ionomycin and demecolcine, were used as recipient oocytes for nuclear transfer of fibroblast cells from a mature male rabbit. The enucleated oocytes with donor nuclei were electrically activated 2 h after fusion. The potential of nuclear-transferred oocytes matured in vitro and ovulated oocytes to develop into blastocysts was high (33-55%), except for oocytes cultured for 8.0 (19%) and 8.5 h (25%) in vitro. After transfer of nuclear-transferred oocytes to recipients, ten of 62 (16%) and one of eight (13%) recipients that received in vitro-matured and ovulated oocytes, respectively, had 19 (1%) and one (0.6%) implantation sites at the time of laparotomy on days 8-17 after transfer. Four fetuses, including two with beating hearts, were obtained on day 15 of gestation after transfer of nuclear-transferred oocytes matured in vitro. The reason for the low efficiency of fetus production was not clear. One possibility is chromosomal abnormalities of nuclear-transferred oocytes, as most (21 of 22) of the oocytes had chromosomes dispersed along the spindle fibre at the first cell cycle. This is the first report of successful production of fetuses after nuclear transfer of rabbit somatic cells.  相似文献   

18.
In the placenta, cortisol is inactivated by NADP(+)- and NAD(+)-dependent isoforms of 11beta-hydroxysteroid dehydrogenase (11betaHSD). Decreased placental 11betaHSD activities have been implicated in intrauterine growth restriction (IUGR) and fetal programming of adult diseases. The objective of this study was to investigate whether placental 11betaHSD activities and fetal plasma cortisol:cortisone ratios could be affected by nutritional restriction of ewes (70% maintenance diet) throughout gestation, for specific stages of gestation, or prior to mating. Chronic nutritional restriction from day 26 of gestation onwards decreased NAD(+)-dependent 11betaHSD activities by 52 +/- 4% and 45 +/- 6% on days 90 and 135 of gestation respectively. Although the decreases in enzyme activities were associated with fetal IUGR, the cortisol:cortisone ratio in fetal plasma was unaffected by chronic nutritional restriction throughout pregnancy. Nutritional restriction confined to early (days 26-45), mid- (days 46-90) and late gestation (days 91-135), or the 30 days prior to mating, had no significant effect on NAD(+)-dependent, placental 11betaHSD activities, nor was there evidence of IUGR. However, nutritional restriction at each stage of pregnancy and prior to mating was associated with significant decreases in the fetal plasma cortisol:cortisone ratio (3.2 +/- 0.7 in control fetuses; 1.0 to 1.6 in fetuses carried by nutritionally restricted ewes). We conclude that nutritional restriction of pregnant ewes for more than 45 consecutive days can significantly decrease NAD(+)-dependent placental 11betaHSD activities in association with IUGR. While the cortisol:cortisone ratio in fetal plasma is sensitive to relatively acute restriction of nutrient intake, even prior to mating, this ratio does not reflect direct ex vivo measurements of placental 11betaHSD activities.  相似文献   

19.
The rate of 2-chlorobiphenyl dechlorination by palladized iron (Fe/ Pd) decreased with increasing pH until pH > 12.5. Iron corrosion potential (Ec) and current (jc), obtained from polarization curves of a rotating disk electrode of iron, followed the Tafel equation at pH < or = 5.5 and pH > or = 9.5. The pH dependence of the dechlorination rate constant (k1) suggests four pH regimes. In the low pH regime (3-5.5), /Ec/ and je decreased with increasing pH and k1 was linearly correlated to /Ec/ and jc0.5. The correlation between k1 and jc0.5 indicates direct involvement of active hydrogen species (on the Pd surface) in PCB dechlorination. In the mid pH regime (5.5-9.5), no significant effect of pH was evident on the values of k1, je, and Ec, a combined result of limiting anodic oxidation of iron to an intermediate product (iron hydroxide) and a proton-independent overall reaction. Both /Ec/ and jc increased significantly as pH increased from 9.5 to 14. A cleartrough of the k1 values in solutions of pH between 12 and 13 and the mismatch between the kinetic and corrosion data suggest two pH regimes (9.5-12.5 and 12.5-14) of different corrosion mechanisms.  相似文献   

20.
轻烧氧化镁及其前驱体的制备   总被引:2,自引:0,他引:2  
以水氯镁石(MgCl2·6H2O)为镁源,Na2CO3为沉淀剂,利用沉淀法得到碳酸镁水合物,然后通过低温煅烧得到高纯氧化镁。研究了沉淀过程中反应温度和初始反应溶液的pH值对轻烧氧化镁及其前驱体的组成和晶型的影响,结果表明在室温~323 K时,得到了棒状的三水碳酸镁;在333 K~363 K之间,得到了碱式碳酸镁。333 K温度下,pH值在9.5~12.5范围内,得到形状不同的碱式碳酸镁颗粒。在1 073 K下煅烧上述前驱体得到高纯度(99%以上)的MgO,为后续高纯镁砂的制备提供了优质原料,同时为盐湖镁资源制备高纯镁砂工艺的进一步深入研究奠定了基础。  相似文献   

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