Xenopus RCOR2 (REST corepressor 2) interacts with ZMYND8, which is involved in neural differentiation

Regulation of neuronal gene expression is critical to nervous system development. REST (RE1-silencing transcription factor) regulates neuronal gene expression through interacting with a group of corepressor proteins including REST corepressors (RCOR). Here we show that Xenopus RCOR2 is predominantly expressed in the developing nervous system. Through a yeast two-hybrid screen, we isolated Xenopus ZMYND8 (Zinc finger and MYND domain containing 8) as an XRCOR2 interacting factor. XRCOR2 and XZMYND8 bind each other in co-immunoprecipitation assays and both of them can function as transcriptional repressors. XZMYND8 is co-expressed with XRCOR2 in the nervous system and overexpression of XZMYND8 inhibits neural differentiation in Xenopus embryos. These data reveal a RCOR2/ZMYND8 complex which might be involved in the regulation of neural differentiation. (C) 2010 Elsevier Inc. All rights reserved.

A new observation on large scale disintegration of eggs of Indian major carps during development

While carp themselves may destroy carp eggs by sucking from outside the happa, destruction in the absence of carp also occurs. During observations of the effect of electrical field on developing carp eggs it was seen that the egg membrane ruptured prematurely under certain potential differences. In natural waters and mud potential differences occur in the presence of high concentrations of certain salts compared with the internal concentration. Temperature and pH fluctuations following strong sun or heavy rain accelerates the diffusion of ions along side water through the egg membrane. The egg cells expand rapidly and the embryos were rapidly dropped.

The giant panda (Ailurapoda melanoleuca) somatic nucleus can dedifferentiate in rabbit ooplasm and support early development of the reconstructed egg

The giant panda skeletal muscle cells, uterus epithelial cells and mammary gland cells from an adult individual were cultured and used as nucleus donor for the construction of interspecies embryos by transferring them into enucleated rabbit eggs. All the three kinds of somatic cells were able to reprogram in rabbit ooplasm and support early embryo development, of which mammary gland cells were proven to be the Lest, followed by uterus epithelial cells and skeletal muscle cells. The experiments showed that direct injection of mammary gland cell into enucleated rabbit ooplasm, combined with in vivo development in ligated rabbit oviduct, achieved higher blastocyst development than in vitro culture after the somatic cell was injected into the perivitelline space and fused with the enucleated egg by electrical stimulation. The chromosome analysis demonstrated that the genetic materials in reconstructed blastocyst cells were the same as that in panda somatic cells. In addition, giant panda mitochondrial DNA (mtDNA) was shown to exist in the interspecies reconstructed blastocyst. The data suggest that (i) the ability of ooplasm to dedifferentiate somatic cells is not species-specific; (ii) there is compatibility between interspecies somatic nucleus and ooplasm during early development of the reconstructed egg.

Embryonic and larval development of Mystus gulio (Ham.)

Mystus gulio eggs are strongly adhesive and contain relatively small yolk (0.75-1.0 mm). The egg envelop is thick and transparent. First cleavage (two cells), four cells, eight cells, sixteen cells and multi cells stages were found 20, 25, 35-40, 60 and 70 minutes after fertilization, respectively. The morula stage was visualized within 1.5 h after fertilization. The heart beat visible and the circulatory system commenced after 16 h of fertilization. Embryos hatched 18-20h after activation of egg. The newly hatched larva measured 2.82±0.03 mm in length and 0.32±0.06 mg in weight. The yolk sac was fully absorbed by the third day though larvae commenced exogenous feeding even before completion of yolk absorption. A 5-day old post larva began wandering in search of food. Ten-day old post larvae endowed with eight branched rays in dorsal fin and seven in caudal fin. Fifteen-day old post larvae had the pectm:al spine become stout though the embryonic fin folds had to be disappeared. The length of fingerlings ranged from 25-30 mm after 30 days, and their external features were just like those of an adult except that they were not sexually matured.

吗啡对鸡胚胎发育的影响

目的:研究吗啡对胎动、心率、孵化率、孵化时间、雏鸡体重等的影响.方法:以气室给药的方式给鸡胚注射吗啡,记录胎动、心率、孵化率、孵化时间、雏鸡体重.结果:吗啡可以缩短雏鸡的孵化时间,降低雏鸡的孵化率,并导致雏鸡出现运动障碍;20 mg/kg吗啡剂量和12-16胚龄的给药时间,鸡胚孵化率最高,残疾率最低;吗啡导致胚胎心率加快,胎动减少(P＜0.05).结论:吗啡对胚胎发育有损伤作用,损伤程度与吗啡剂量和给药时间有关.

Cloning and expression of zebra fish ferroportin and investigating its influence on anemia

Iron is required for many microbes and pathogens for their survival and proliferation including Leishmania which cause leishmaniasis. Leishmaniasis is an increasingly serious infectious disease with a wide spectrum of clinical manifestations. These range from localized cutaneous leishmaniasis (CL) lesions to a lethal visceral form. Certain strains such as BALB/c mice fail to control L. major infection and develop progressive lesions and systemic disease. These mice are thought to be a model of non-healing forms of the human disease such as kala-azar or diffuse cutaneous leishmaniasis. Progression of disease in BALB/c mice has been associated with the anemia, in last days of their survival, the progressive anemia is considered to be one of the reasons of their death. Ferroportin (Fpn), a key regulator of iron homeostasis is a conserved membrane protein that exports iron across the duodenal enterocytes as well as macrophages and hepatocytes into the blood circulation. Fpn has also critical influence on survival and proliferation of many microorganisms whose growth is dependent upon iron, thus preparation of Fpn is needed to study the role of iron in immune responses and pathogenesis of micoorganisms. To prepare and characterize a recombinant ferroportin, total RNA was extracted from Indian zebrafish duodenum, and used to synthesize cDNA by RT-PCR. PCR product was first cloned in Topo TA vector and then subcloned into the GFP expression vector pEGFP–N1. The final resulted plasmid (pEGFP-ZFpn) was used for expression of FPN-EGFP protein in Hek 293T cells. The expression was confirmed by fluorescence microscopy and flow cytometery. Recombinant Fpn was further characterized by submission of its predicted amino acid sequences to the TMHMM V2.0 prediction server (hidden Markov model), NetOGlyc 3.1 server and NetNGlyc 3.1 server. Data emphasised that obtained Fpn from indian zebrafish contained eight transmembrane domains with N- and C-termini inside the cytoplasm and harboured 78 mucin-type glycosylated amino acid. The results indicate that the prepared and characterized recombinant Fpn protein has no membrane topology difference compared to other Fpn described by other researcher. Our next aim was to deliver recombinant plasmid (pEGFP-ZFpn) to entrocyte cells. However, naked therapeutic genes are rapidly degraded by nucleases, showing poor cellular uptake, nonspecificity to the target cells, and low transfection efficiency. The development of safe and efficient gene carriers is one of the prerequisites for the success of gene therapy. Chitosan and alginate 139 polymers were used for oral gene carrier because of their biodegradability, biocompatibility and their mucoadhesive and permeability-enhancing properties in the gut. Nanoparticles comprising Alginate/Chitosan polymers were prepared by pregel preparation method. The resulting nanoparticles had a loading efficiency of 95% and average size of 188 nm as confirmed by PCS method and SEM images had showed spherical particles. BALB/c mice were divided to three groups. The first and second group were fed with chitosan/alginate nanoparticles containing the pEGFP-ZFpn and pEGFP plasmid, respectively (30 μgr/mice) and the third group (control) didn’t get any nanoparticles. The result showed BALB/c mice infected by L.major, resulted in higher hematocryte and iron level in pEGFP-ZFpn fed mice than that in other groups. Consentration of cytokines determined by ELISA showed lower levels of IL-4 and IL-10 and higher levels of IFN-γ/IL-4 and IFN-γ/IL-10 ratios in pEGFP-ZFpn fed mice than that in other groups. Morover more limited increase of footpad thickness and significant reduction of viable parasites in lymph node was seen in pEGFP-ZFpn fed mice. The results showed the first group exhibited a highr hematocryte and iron compared to the other groups. These data strongly suggests the in vivo administration of chitosan/alginate nanoparticles containing pEGFP-ZFpn suppress Th2 response and may be used to control the leishmaniasis .

全放牧婆罗门牛和BMY牛胚胎回收适期的探讨

选用婆罗门牛和BMY牛供体超数排卵,以第1次人工授精(AI)当日为0 d,分别在AI第6～6.5天、7～7.5天和8～8.5天回收胚胎,统计≤16细胞胚、桑椹胚、囊胚和孵化胚的数量,计算各发育阶段胚胎所占的百分率,分析婆罗门牛和BMY牛的胚胎回收适期.结果表明:婆罗门牛AI第6～6.5天的桑椹胚率比BMY牛高43.54个百分点(P＜0.01),AI第8～8.5天的孵化胚率比BMY牛高35.94个百分点(P＜0.05),胚胎发育速度比BMY牛较快.罗门牛和BMY牛在AI第6～6.5天回收胚胎的囊胚率低,而第8～8.5天的孵化胚率高,胚胎回收以AI第7～7.5天为宜.

哺乳动物胚胎冷冻技术的研究进展

胚胎冷冻保存已广泛用于胚胎移植、动物克隆以及动物资源保护。此技术的应用需保证胚胎在冷冻—解冻后具有较高的成活率。自从1972年小鼠胚胎冷冻保存获得成功以来,许多学者在简化冷冻程序、缩短冷冻时间等方面进行了深入的研究。文章就胚胎冷冻的原理、保护剂、冷冻方法以及解冻方法等方面进行了综述。

小鼠四倍体半克隆胚胎发育研究

半克隆U-9/G;7Q":9V2胚胎是通过注射体细胞核到未去核的卵母细胞中产生的# 在半克隆胚胎中!体细胞被用来作为精子的替代物# 然而!由于异常的染色体分离!构建的半克隆胚胎在激活后形成了非整倍体而导致胚胎发育受到严重影响!不能发育到期# 本研究通过抑制小 鼠半克隆胚胎在激活过程中染色体数目减半!避免非整倍体胚胎形成!研究四倍体半克隆$W9XKMY#"GV -9/G;Z#":9V!W-7%胚胎的发育和体细 胞核的掺入对胚胎发育的影响& 结果显示!W-7 胚胎的体外发育率显著高于二倍体半克隆胚胎!与正常受精卵及孤雌激活对照无显著性差异! 但W-7 胚胎的细胞数在桑椹胚和囊胚期比正常二倍体受精胚胎和孤雌激活胚胎少& 通过AZX;> 染色发现!W-7 胚胎囊胚期内细胞团$5::9K 79## [M\\]57[%细胞很少或者没有& 移植1& 个四倍体半克隆胚胎到& 只假孕母鼠体内!得到%* 个胎盘!但没有得到胎儿& 组蛋白乙酰化和 4’@ 甲基化检测显示!部分W-7 胚胎在囊胚期没有形成正常受精胚胎在57[ 和滋养外胚层$WK"Y89ZX"V9K/!WE%之间的差异分布& W-7 胚 胎的基因表达不依赖于细胞分裂次数而依赖于发育时间& 虽然W-7 胚胎避免了二倍体半克隆胚胎形成非整倍体现象!但由于W-7 胚胎没有 57[ 细胞或57[ 细胞很少!所以只能形成胎盘而不能形成胎儿& 本实验第一次较为全面地研究了W-7 胚胎的发育!同时也为研究体细胞核再 程序化’基因打靶技术提供了一种新的途径#

Development of zona-free hamster ova to blastocysts in vitro

The zona pellucida (ZP) enclosing the mammalian ovum is important for its protection and for initial stages of fertilization, but the role of the ZP during embryo development is less clear. This study was designed to investigate if the hamster ZP is needed for embryo development from 1-cell to blastocyst in vitro, and to compare methods for removing the ZP. A total of 395 hamster pronucleate ova were collected 10 h post activation from superovulated, mated female hamsters. The ZP was removed from some ova using either 0.05% pronase, 0.05% trypsin or acid Tyrode's solution. To prevent ZP-free ova from sticking together, they were cultured singly in 30-50 muL drops of HECM-6 culture medium together with ZP-intact ova as controls. There was no significant difference among treatment groups in embryo development to blastocyst: 36/87 (42%) in the ZP intact group; 35/75 (47%) in the pronase-treated ZP-free group; 37/74 (50%) in the trypsin-treated ZP-free group; and 37/71 (52%) in the acid-treated ZP-free group. These results indicate that 1) the ZP is unnecessary for hamster embryo development in vitro from the pronucleate ovum stage to blastocyst; 2) none of the three ZP-removal methods was detrimental to embryo development; 3) embryos do not need to be cultured in groups during in vitro development from 1-cell to blastocyst. (C) 2000 by Elsevier Science Inc.

Effect of antibiotics on development in vitro of hamster pronucleate ova

Antibiotics are commonly added to embryo culture media, but effects on embryo development have not been examined thoroughly. Hamster ova were used to investigate whether penicillin, streptomycin or gentamicin affect embryo development in vitro. Ova were collected 10 h post activation by spermatozoa in vivo and cultured in five treatments: 1) Control: chemically-defined medium HECM-9 with no antibiotics; 2) HECM-9 with 100 IU/mL, penicillin; 3) HECM-9 with 50 mug/mL streptomycin; 4) HECM-9 with 10 mug/mL,gentamicin and 5) HECM-9 with both 100 IU/mL penicillin and 50 mug/mL streptomycin. Individually, penicillin, streptomycin and gentamicin did not affect embryo development to the 8-cell stage at 58 h post oocyte activation, or morula/blastocyst stages, or blastocysts alone at 82 h post activation. However, when penicillin and streptomycin were both present in the culture medium the percentages of 8-cell embryos at 58 h and blastocysts at 82 h were significantly lower than the control. No antibiotic treatment improved hamster embryo development in vitro. We caution against the use of penicillin and streptomycin together for hamster embryo culture, and show that it is not necessary to include any antibiotics in embryo culture media for up to 72 h if proper sterile technique is used with an oil overlay. (C) 2000 by Elsevier Science Inc.

The natural life history of milkfish

Following brief descriptions of the various phases in the natural life history of the milkfish (Chanos chanos), namely adults, eggs and embryos, larvae, fry and metamorphosis, juveniles and sub-adults, a summary is provided of the life history, providing also a schematic diagram.

Murine MyaK, a member of a family of yeast YAK1-related genes, is highly expressed in hormonally modulated epithelia in the reproductive system and in the embryonic central nervous system

We have cloned a mouse homologue (designated Myak) of the yeast protein kinase YAK1. The 1210 aa open reading frame contains a putative protein kinase domain, nuclear localization sequences and PEST sequences. Myak appears to be a member of a growing family of YAK1-related genes that include Drosophila and human Minibrain as well as a recently identified rat gene ANPK that encode a steroid hormone receptor interacting protein. RNA blot analysis revealed that Myak is expressed at low levels ubiquitously but at high levels in reproductive tissues, including testis, epididymis, ovary, uterus, and mammary gland, as well as in brain and kidney. In situ hybridization analysis on selected tissues revealed that Myak is particularly abundant in the hormonally modulated epithelia of the epididymis, mammary gland, and uterus, in round spermatids in the testis, and in the corpora lutea in the ovary, Myak is also highly expressed in the aqueduct of the adult brain and in the brain and spinal cord of day 12.5 embryos, Mol. Reprod. Dev. 55:372-378, 2000. (C) 2000 Wiley-Liss, Inc.

Regulation of zygotic gene activation in the preimplantation mouse embryo: Global activation and repression of gene expression

Superimposed on the activation of the embryonic genome in the preimplantation mouse embryo is the formation of a transcriptionally repressive state during the two-cell stage. This repression appears mediated at the level of chromatin structure, because it is reversed by inducing histone hyperacetylation or inhibiting the second round of DNA replication. We report that of more than 200 amplicons analyzed by mRNA differential display, about 45% of them are repressed between the two-cell and four-cell stages. This repression is scored as either a decrease in amplicon expression that occurs between the two-cell and four-cell stages or on the ability of either trichostatin A tan inhibitor of histone deacetylases) or aphidicolin tan inhibitor of replicative DNA polymerases) to increase the level of amplicon expression. Results of this study also indicate that about 16% of the amplicons analyzed likely are novel genes whose sequence doesn't correspond to sequences in the current databases, whereas about 20% of the sequences expressed during this transition likely are repetitive sequences. Lastly, inducing histone hyperacetylation in the two-cell embryos inhibits cleavage to the four-cell stage. These results suggest that genome activation is global and relatively promiscuous and that a function of the transcriptionally repressive state is to dictate the appropriate profile of gene expression that is compatible with further development.

Using drift nets to capture early life stages and monitor spawning of the Yangtze River Chinese sturgeon (Acipenser sinensis)

P>A sampling system for capturing sturgeon eggs using a D-shaped bottom anchored drift net was used to capture early life stages (ELS) of Chinese sturgeon, Acipenser sinensis, and monitor annual spawning success at Yichang on the Yangtze River, 1996-2004, before and just after the Three Gorges Dam began operation. Captured were 96 875 ELS (early life stages: eggs, yolk-sac larvae = eleuthero embryos, and larvae); most were eggs and only 2477 were yolk-sac larvae. Most ELS were captured in the main river channel and inside the bend at the Yichang spawning reach. Yolk-sac larvae were captured for a maximum of 3 days after hatching began, indicating quick dispersal downstream. The back-calculated day of egg fertilization over the eight years indicated a maximum spawning window of 23 days (20 October-10 November). Spawning in all years was restricted temporally, occurred mostly at night and during one or two spawning periods, each lasting several days. The brief temporal spawning window may reduce egg predation by opportunistic predators by flooding the river bottom with millions of eggs. During 1996-2002, the percentage of fertilized eggs in an annual 20-egg sample was between 63.5 to 94.1%; however, in 2003 the percentage fertilized was only 23.8%. This sudden decline may be related to the altered environmental conditions at Yichang caused by operation of the Three Gorges Dam. Further studies are needed to monitor spawning and changes in egg fertilization in this threatened population.