016.Role of cited genes in placental morphogenesis: studies in null mutant mice
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Cited1 and Cited2 interact with CBP and p300. CBP/p300 bind numerous proteins and evidence exists, for Cited2 at least, that Cited binding prevents the binding of other proteins to CBP/p300. Since CBP/p300 interact with many proteins, can acetylate protein and DNA, and act as a ubiquitin ligase, it is likely that Cited1 and Cited2 function at a number of sites during development. We have generated mice that carry a null mutant allele for each of these genes. Analysis of null mutant embryos demonstrates that both Cited1 and Cited2 are required for normal embryonic development and survival. Although both Cited1 and Cited2 are expressed in the developing embryo and placenta, it appears that abnormal placental development and function is the cause of embryonic death. The defect that develops in the placentas of Cited1 null mutants is not apparent until late in gestation (16.5dpc). Cited1 null mutants are smaller than controls at birth and die during the early postnatal period. The placentas of these mutants are disorganised, with spongiotrophoblasts projecting in to the labyrinthine layer. In addition, resin casts of the maternal blood spaces within these placentas revealed extremely enlarged blood sinuses. We are searching for factors that could result in the increased size of the maternal blood sinuses. Cited2 null placentas and embryos are significantly smaller than controls; mutants die 3/4 the way through gestation (15.5dpc). The null mutant placentas have proportionally fewer spongiotrophoblasts, trophoblast giant cells and invasive trophoblasts. In addition, resin casts of fetal vasculature of the placenta reveal that the capillary network is underdeveloped. Through the isolation of trophoblast stem (TS) cells we are exploring the possibility that TS cell proliferation and/or differentiation is impaired due to a lack of Cited2. We suspect that the development of the phenotype may relate to the Hypoxia Inducible Factor-1a (HIF1a) transcription factor as Cited2 expression is induced by HIF1 and it acts to negatively regulate its activity.Keywords:
Null allele
Trophoblast
Chorioallantoic membrane
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The mature and immature Panax quinquefolium seeds were used as test materials,and the zygotic embryo were dissected out aseptically and placed on solid MS medium supplemented with different concentrations of 2,4-D in the light and dark culture conditions,effect of 2,4-D on the somatic embryogenesis of american ginseng were studied.The results showed that somatic embryogenesis could occur on the mature embryos at different concentrations of 2,4-D and the optimal concentration were 0.5 mg/L,and illumination could stimulate the process;The immature embryo could form somatic embryo.The conclusion were that 2,4-D stimulated somatic embryogenesis on the mature embryos,the responses between mature and immature embryo were apparently different;The developmental control mechanism in zygotic embryo of Panax quinquefolium was from that in Panax ginseng.
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The present study analyzed variation of protein components and contents in Antheraea pernyi embryos of different developmental stages by SDS-PAGE and 2D-PAGE,so as to accumulate basic information on sequential expression of embryonic development genes in A.pernyi at the protein level.SDS-PAGE results showed that 32 major protein bands were isolated during the embryo development,including high abundance proteins with molecular weight of around 70,60 and 30 kD respectively.As the embryo development advanced,contents of these three proteins declined,while those of 40 and 15 kD proteins increased gradually.Proteins of 70 and 60 kD almost disappeared in newly-hatched larvae(324 h).Further analysis to 2D-PAGE images of embryonic proteins from different developmental stages showed that the protein spots increased gradually with embryo development.At 12 h of embryo development,there were 370 protein spots being detected.And at 300 h of embryo development,the number of protein spots reached 422.The matching rates of protein spots at 132 h and 276 h of embryo development with protein spots at 36 h of early embryo development was 52.4% and 28.4% respectively.More-over,2D-PAGE image of total proteins in newly-hatched larvae varied greatly with those at the embryonic stage.These results suggest that during the early stage of embryo development,i.e.from the embryo formation stage to appendage appearing stage(12～60 h),number of protein species were relatively low and did not change obviously;and after the trachea formation stage(228～276 h),protein species changed greatly,with increased number of acidic proteins and low matching rate of protein spots with previous stages.
Antheraea pernyi
Spots
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The ability of the baby to receive nutrients and oxygen in utero depends on the healthy development of the placenta. For maternal blood to adequately perfuse the placenta, it dramatically alters the arteries in the uterus that supply it with nutrient-rich blood right from the start of pregnancy. Placental cells (trophoblasts) invade both into the tissue of the uterus and into the maternal blood vessels nearest to the site of implantation (the spiral arteries (SAs)) and transform these allowing a relatively high and steady flow of nutrient-rich blood to perfuse the placenta. Trophoblasts also form plugs that occlude SAs, preventing maternal blood flow to the placenta until the late first trimester, at which point these plugs dislodge or disintegrate. Here we present an agent-based model of trophoblast migration within plugged SAs to tease apart the impact of chemical signals and mechanical factors on trophoblast behaviour. The model supports our previous in vitro hypothesis that plugging of the maternal arteries in early pregnancy can act to promote trophoblast invasion by providing a ‘low flow’ environment and extends our understanding by suggesting ‘weak spots’ in plug structure can lead to plug degeneration, allowing increased blood flow through the materno-fetal circulation.
Trophoblast
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The effects of calcium and ionophore A23187 on in vitro development of early embryo in rice(Oryza sativa L.)were studied.The main results were as follows:(1) the influence of Ca2+ on the in vitro development of 3 to 5dayold embryos was related to the concentration of Ca2+ and the age of embryos.Without Ca2+ or with the high concentration of Ca2+(10-1 mol/L),the growth of 3dayold embryos was inhibited completely and the development of 4 to 5dayold embryos was affected in certain degree.With 10-3 mol/L Ca2+,3 to 5dayold embryos grew rapidly and the highest frequency of embryogenesis appeared.In the same concentration of Ca2+,the older the embryos were,the higher the frequency of embryogenesis and the total induction appeared.(2) A23187 influenced in vitro embryo development and morphosis.With increasing A23187 concentration,the in vitro development of the embryos in rice was inhibited more violently.The younger embryos were,the more obvious inhibition.
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The morphological changes of embryo of Hyriopsis cumingii in its nurturing pouch of outer gill were examined.It was found that,when the water temperature ranged from 20 ℃ to 23 ℃,the embryonic development from oosperm to mature glochidia took 11 days.After the oosperm sucked the water,the membrance swelt up and its size varied little,while the embryo bulged distinctly.The embryonic development of Hyriopsis cumingii might be divided into four stages: oosperm,cleavage,gastrulae and glochidia.The length of embryo increased 82.8% during the whole embryonic development.The growth rate of late embryo was higher than that of early embryo.The highest growth rate was observed in gastrulae stage and glochidia stage.There were relations between different stages of the embryonic development and the character of the outer gill of the female.
Cleavage (geology)
Pouch
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An association study was performed in rabbits between early embryo survival and development, and the nonconservative SNP 12944C>G located in exon 11 and the triallellic microsatellite [(GT)(15)T(G)(5), (GT)(14)T(G)(5), and (GT)(11)T(G)(7))] located in the promoter region of the oviductal glycoprotein 1 (OVGP1) gene. We analyzed an F(2) cross of 2 lines of rabbits divergently selected for uterine capacity. A total of 172 and 159 females were slaughtered at 48 and 72 h of gestation, respectively, to determine whether OVGP1 influences ovulation rate, fertilization rate, early embryo survival, and embryonic stage of development. The results of the SNP indicated that all genotypes showed similar early embryo survival and a similar embryonic stage of development at 48 h of gestation. However, at 72 h of gestation, the GG genotype showed greater early embryo survival than the CC genotype (0.56 embryos) and their embryos presented less embryonic development. Analysis of the microsatellite was performed to ascertain the presence or absence of the allele (GT)(14)T(G)(5). At both stages of gestation, the (GT)(14)T(G)(5)/(GT)(14)T(G)(5) genotype showed greater early embryo survival (0.94 and 1.54 embryos at 48 and 72 h of gestation, respectively) and less embryonic development than the homozygous genotypes without the allele (GT)(14)T(G)(5).
SNP
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Objective: The embryonic stem cell(ES) culture system(feeder layer+RH-CM medium) was used in this experiment to coculture mouse early stage embryos in order to study whether this system could ensure the development of the embryos and increase the rate of the embryo development.Methods: Five hundred and twenty-one embryos were divided into 10 groups according to the different feeder layers(mouse embryonic fibroblast,MEF;SIM mouse embryo-derived thioquanine and ouabain(resistant) cells,STO; oviduct epithelial cell,OEC) and conditioned medium.The effect of different culture conditions on the rate of embryo development in vitro was compared.Results: The ES cell culture system was better than the conventional culture system for the embryo development(P0.05).When embryos were cultured in ES culture system,STO feeder achieved a(higher) rate of embryo development than MEF,while both STO and MEF were significantly better than OEC for the embryo development(P0.05).Conclusions: The results suggest that this system can apparently increases the development rate of the mouse early stage embryos.Using ES cell culture system to coculture early stage embryos,STO is a better feeder than MEF for the embryo development.
Oviduct
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Ruminants have a semi-invasive placenta, which possess highly vascularized placentomes formed by maternal endometrial caruncles and fetal placental cotyledons and required for fetal development to term. The synepitheliochorial placenta of cattle contains at least two trophoblast cell populations, including uninucleate (UNC) and binucleate (BNC) cells that are most abundant in the cotyledonary chorion of the placentomes. The interplacentomal placenta is more epitheliochorial in nature with the chorion developing specialized areolae over the openings of uterine glands. Of note, the cell types in the placenta and cellular and molecular mechanisms governing trophoblast differentiation and function are little understood in ruminants. To fill this knowledge gap, the cotyledonary and intercotyledonary areas of the mature day 195 bovine placenta were analyzed by single nuclei analysis. Single-nuclei RNA-seq analysis found substantial differences in cell type composition and transcriptional profiles between the two distinct regions of the placenta. Based on clustering and cell marker gene expression, five different trophoblast cell types were identified in the chorion, including proliferating and differentiating UNC and two different types of BNC in the cotyledon. Cell trajectory analyses provided a framework for understanding the differentiation of trophoblast UNC into BNC. The upstream transcription factor binding analysis of differentially expressed genes identified a candidate set of regulator factors and genes regulating trophoblast differentiation. This foundational information is useful to discover essential biological pathways underpinning the development and function of the bovine placenta.
Trophoblast
Cell type
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Objective. To assess the influence of different concentrations of recombinant human leukemia inhibitory factor (LIF) on mouse embryo development.
Methods. All 2-4 cell embryos obtained from ICR mice were cultured in human tubal fluid media containing different concentrations of LIE. Mouse embryos were then divided into 6 groups according to the amount of LIF added: 1) 2500 IU/ml LIF; 2) 2000 IU/ml LIF; 3) 1500 IU/ml LIF; 4) 1000 IU/ml LIF; 5) 500 IU/ml LIF; 6) HTF. The embryonic development at different stages in each group was then recorded. Results the percentage of early embryo stage development in all groups was similar. However, higher formation rates of pre-implantation embryos were found in groups 1,2,3,4 and 5.
Conclusions. LIF has positive effects on pre-implantation embryo development and produces no significant influence upon early embryo development. Therefore, the concentration of 500 IU/ml LIF could provide for optimal embryogenesis.
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