Advances in cell lineage reprogramming

As a milestone breakthrough of stem cell and regenerative medicine in recent years,somatic cell reprogramming has opened up new applications of regenerative medicine by breaking through the ethical shackles of embryonic stem cells.However,induced pluripotent stem(iPS) cells are prepared with a complicated protocol that results in a low reprogramming rate.To obtain differentiated target cells,iPS cells and embryonic stem cells still need to be induced using step-by-step procedures.The safety of induced target cells from iPS cells is currently a further concerning matter.More broadly conceived is lineage reprogramming that has been investigated since 1987.Adult stem cell plasticity,which triggered interest in stem cell research at the end of the last century,can also be included in the scope of lineage reprogramming.With the promotion of iPS cell research,lineage reprogramming is now considered as one of the most promising fields in regenerative medicine,will hopefully lead to customized,personalized therapeutic options for patients in the future.     

The fertilization—induced Ca^2＋ oscillation in mouse oocytes is cytoplasmic maturation dependent

Mature eggs (at metaphase II stage) produce a series of Ca^2 oscillation at fertilization.To define whether the fertilization-induced Ca^2 oscillation is restrict to the metaphase II eggs and cell cycle dependent,mouse oocytes at prophase I (arrested at germinal vesicle stage),metaphase I,metaphase II,as well as the pronuclear embryos at interphase of the first mitotic division derived from fertilization of parthenogenetic activation were inseminated after removal of zona pellucida,The results show that the fertilization-induced Ca^2 oscillation is not specific to metaphase II eggs.This is supported by the fact that immature oocytes generated the Ca^2 oscillations at fertilization regardless of their nuclear progression from prophase I to metaphase I (in vitro matured) stage.More interestingly,it was first found that pronuclear embryos at interphase derived from parthenogenetic activation showed Ca^2 oscillations in response to fertilization while the zygotes at interphase did not after reinsemination or intracytoplasmic injection of sperm extracts which induce Ca^2 oscillations in MII eggs.This suggests that the ability of oocytes to generate Ca^2 oscillation in response to sperm penetration is not regulated in a cell cycle dependent manner but dependent on the cytoplasmic maturation.     

Generation of cloned calves from different types of somatic cells

Remarkable progress has been made in animal cloning research since the first mammal was success-fully cloned[1], and the technique of SCNT is now widely used in biological studies. In theory, successful development of live offspring from SCNT embryos demonstrates that a fully differentiated somatic cell can be reprogrammed and restore its totipotency; in practice, animal cloning can be applied for duplication of elite animals, production of transgenic animals, rescue of endangered species …     

ES cells derived from cloned embryos in monkey--a jump toward human therapeutic cloning

Therapeutic cloning refers to the derivation of embryonic stem cells （ntESC） from embryos derived from somatic cell nuclear transfer （SCNT） also known as cloning. Cloning involves transplanting a differentiated cell into an oocyte that has had its nucleus （DNA） removed. The reconstructed oocyte can be activated to divide and develop into an embryo. The process that allows this to happen is termed nuclear reprogramming, and is defined as the mechanism through which a differentiated cell de-differentiates or returns to a totipotent state （capable of giving rise to any cell type, including extra-embryonic） and directs embryonic development [1]. Cells from blastocyst stage cloned embryos can be used to generate ntESC lines. Such cell lines can differentiate into any adult cell type, and have tremendous potential for patient-specific disease therapy [2].     

Insights into epigenetic patterns in mammalian early embryos

Mammalian fertilization begins with the fusion of two specialized gametes,followed by major epigenetic remodeling leading to the formation of a totipotent embryo.During the development of the pre-implantation embryo,precise reprogramming progress is a prerequisite for avoiding developmental defects or embryonic lethality,but the underlying molecular mechanisms remain elusive.For the past few years,unprecedented breakthroughs have been made in mapping the regulatory network of dynamic epigenomes during mammalian early embryo development,taking advantage of multiple advances and innovations in low-input genome-wide chromatin analysis technologies.The aim of this review is to highlight the most recent progress in understanding the mechanisms of epigenetic remodeling during early embryogenesis in mammals,including DNA methylation,histone modifications,chromatin accessibility and 3D chromatin organization.     

Oil Body Biogenesis during Brassica napus Embryogenesis

Although the oil body is known to be an important membrane enclosed compartment for oil storage in seeds, we have little understanding about its biogenesis during embryogenesis. In the present study we investigated the oil body emergence and variations in Brassica napus cv. Topas. The results demonstrate that the oil bodies could be detected already at the heart stage, at the same time as the embryos began to turn green, and the starch grains accumulated in the chloroplast stroma. In comparison, we have studied the development of oil bodies between Arabidopsis thaliana wild type （Col） and the low-seed-oil mutant wrinkled1-3. We observed that the oil body development in the embryos of Col is similar to that of B. napus cv. Topas, and that the size of the oil bodies was obviously smaller in the embryos of wrinkled1-3. Our results suggest that the oil body biogenesis might be coupled with the embryo chloroplast.     

Establishment of customized mouse stem cell lines by sequential nuclear transfer

Therapeutic cloning, whereby embryonic stem cells （ESCs） are derived from nuclear transfer （NT） embryos, may play a major role in the new era of regenerative medicine. In this study we established forty nuclear transfer-ESC （NTESC） lines that were derived from NT embryos of different donor cell types or passages. We found that NT-ESCs were capable of forming embryoid bodies. In addition, NT-ESCs expressed pluripotency stem cell markers in vitro and could differentiate into embryonic tissues in vivo. NT embryos from early passage RI donor cells were able to form full term developed pups, whereas those from late passage RI ES donor cells lost the potential for reprogramming that is essential for live birth. We subsequently established sequential NT-RI-ESC lines that were developed from NT blastocyst of late passage R 1 ESC donors. However, these NT-R I-ESC lines, when used as nuclear transfer donors at their early passages, failed to result in live pups. This indicates that the therapeutic cloning process using sequential NT-ESCs may not rescue the developmental deficiencies that resided in previous donor generations.     

蟋蟀精子表面LCA及ConA结合糖复合物的分布变化

LCA and ConA-binding glycoconjugates on cricket (Teleogryllus emma) sperm surface were detected with fluorescence microscope after FITC labelling for better understanding of the distribution of glycoconjugates during spermatogenesis and spermiogenesis. FITC-LCA and FITC-ConA were bound on the spermatocytes, and their distribution changes in the process of spermiogenesis were observed .In the testis sperm, FITC-LCA and FITC-ConA were mainly bound on the head and neck region. That is different from the mark pattern of spermatophore sperm, in which the nucleus, neck region and front of the tail showed obvious fluorescence mark, especially the acrosome complex and neck region exhibited stronger mark. The mark patterns of FITC-LCA and FITC-ConA were similar,though the former was distinctly clearer than the latter. But a little difference still exists in both of them. For example in the ninth stage of spermatid, FITC-LCA mark is located on the spermatid head and neck region, and FITC-ConA mark on the spermatid head, neck and front of the tail region. When fixed germ cells were treated with PBS instead of lectin solution, or fixed cells were incubated with lectin solution, which have been treated with 0.1 mol/L specific sugar inhibitor, i.e.α-D-mannose for FITC-LCA and FITC-ConA, and α-D-glucose for FITC-ConA, no mark was observed on the cells. Those results indicate that FITC-LCA conjugated glycoconjugates has the α-D- mannose residue, and FITC-ConA conjugated glycoconjugates has the α-D-mannose and α-D-glucose residue. The investigations show that the changes in glycoconjugates distribution of cricket sperm is similar to those of other insects and mammals. The evidence exhibit that a common rule of the glycoconjugates distribution on the sperm surface is followed by most of animal sperm which may relate to the function of sperm physiology.     

猪卵巢体外保存温度对卵母细胞染色质构型和成熟能力的影响

Most porcine oocytes used in studies on embryo biotechnology and the in vitro production of embryos are currently obtained from the ovaries of slaughtered gilts. The duration and temperature during ovary transportation and handling might, therefore, affect the recovery of culturable COCs, chromatin configuration and developmental competence of oocytes. The effects of ovary storage temperature on chromatin configuration and in vitro maturation of porcine oocytes were examined in this study. Ovaries collected from a slaughterhouse were stored in vitro for 8 h under different temperatures. The results showed that more culturable COCs were isolated from the ovares stored at 39℃ than that from ovaries stored at 31℃ or 20℃ and before storage. Thirty-one centidegree was the best storage temperature in terms of cumulus expansion, nuclear maturation and morphology of the first polar body after in vitro maturation culture. The ability of cumulus expansion was completely lost in COCs derived from ovaries stored at 39℃ for 8 hours. Ovary storage （at both 31℃ and at 20℃ ） increased the proportion of oocytes with the GVc configuration in which chromatin condensed into a single big clump at the nucleolus and the functional significance of this configuration needs further investigations [ Acta Zoologica Sinica 51 （5）： 919 923, 2005].     

Effect of antikeratin microinjection on the embryonic development of Xenopus laevis

Anti-keratin monoclonal antibody AF5 was introduced into fertilized eggs of Xenopus laevis.,and its effects on embryonic development were studied.Survival rate of the antikeratin-injected embryos was much lower(only 35.67% at gastrula)than that of the control(74.85% at gastrula),in which embryos were injected with mouse IgG.Most of survivors in the experimental series showed aberrant external appearance.On the other hand,in cleavage stage,ie 2-7h after fertilization,immunohistochemical staining of embryos showed that the expermental embryos were mostly keratin negative,while embryos of the control ones were keratin positive.When introducing this antikeratin into one cell of a 2-cell embryo,only the uninjected half of the embryo continued its development while the other half could not develop at all.These results suggested that intact keratin cytoskeleton in early embryos is indispensable to the embryonic development of Xenopus laevis.     

Crossability Barriers in the Interspecific Hybridization between Oryza sativa and O. meyeriana

Oryza meyeriana Baill （GG genome） is a precious germplasm in the tertiary gene pool of cultivated rice （AA genome）, and possesses important traits such as resistance and tolerance to biotic and abiotic stress. However, interspeciflc crossability barrier, a critical bottleneck restricting genes transfer from O. meyeriana to cultivars has led to no hybrids through conventional reproduction. Therefore, the reasons underlying incrossability were investigated in the present report. The results showed that： （i） at 3-7 d after pollination （DAP）, many hybrid embryos degenerated at the earlier globular-shaped stage, and could not develop into the later pear-shaped stage. Meanwhile, free endosperm nuclei started to degenerate at 1 DAP, and cellular endosperm could not form at 3 DAP, leading to nutrition starvation for young embryo development; （ii） at 11-13 DAP, almost all hybrid ovaries aborted. Even though 72.22% of hybrid young embryos were produced in the interspecific hybridization between O. sativa and O. meyeriana, young embryos were not able to further develop into hybrid plantlets via culturing in vitro. The main reason for the incrossability was hybrid embryo inviability, presenting as embryo development stagnation and degeneration since 3 DAP. Some possible approaches to overcome the crossability barriers in the interspecific hybridization between O. sativa and O. meyeriana are discussed.     

Delay of ZGA initiation occurred in 2-cell blocked mouse embryos

One-cell mouse embryos from KM strain and B6C3F1 strain were cultured in M16 medium, in which2-cell block generally occurs. Embryos of KM strain exhibited 2-cell block, whereas B6C3F1 embryos,which are regarded as a nonblocking strain, proceeded to the 4-cell stage in our culture condition. It is oftenassumed that the block of early development is due to the failure of zygotic gene activation (ZGA) in culturedembryos. In this study we examined protein synthesis patterns by two-dimensional gel electrophoresis of[35^S] methionine radiolabeled 2-cell embryos. Embryos from the blocking strain and the nonblocking strainwere compared in their development both in vitro and in vivo. The detection of TRC expression, a markerof ZGA, at 42 h post hCG in KM embryos developed in vitro suggested that ZGA was also initiated even inthe 2-cell arrested embryos. Nevertheless, a significant delay of ZGA was observed in KM strain as comparedwith normally developed B6C3F1 embryos. At the very beginning of major ZGA as early as 36 h post hCG,TRC has already been expressed in B6C3F1 embryos developed in vitro and KM embryos developed in vivo.But for 2-cell blocked KM embryos, TRC was still not detectable even at 38 h post hCG. These evidences suggest that 2-cell-blocked embryos do initiate ZGA, and that 2-cell block phenomenon is due not to the disability in initiating ZGA, but to a delay of ZGA.     

Embryonic Growth and Yolk Depletion during Incubation in the Chinese Skink,Plestiodon chinensis

We collected 24 gravid female Chinese skinks(Plestiodon chinensis) to study embryonic growth and yolk depletion during incubation. Females laid eggs between late May and mid-June. Eggs were incubated at 24(± 0.3) ℃. One egg from each clutch was dissected at 5-d intervals starting at laying. Embryonic stages at laying varied from Dufaure and Hubert's(1961) Stage 30-35, with a mean stage of 32.6. Incubation lengths at 24 ℃ varied from 35.1 to 48.3 d, with a mean of 41.5 d. Based on the derived functions describing instantaneous changes in embryo dry mass and yolk dry mass, we identified three phases of embryonic growth or yolk depletion in P. chinensis. Phase 1, from Day 0(at laying) to Day 15(～36% of the way through incubation), was one of minimal transfer of material from yolk to embryo. Phase 2, from Day 15 to Day 32-33(～77%-80% of the way through incubation), was characterized by increasingly rapid embryonic growth or yolk depletion. Phase 3, from Day 32-33 to hatching, was characterized by reduced embryonic growth or yolk depletion. The length of the last embryonic stage(Stage 40 = completely differentiated embryos) accounted for about 28% of incubation length, and the dry mass of the smallest embryos of Stage 40 accounted for only ～48% of the hatchling dry mass. Our study adds evidence to the idea that oviposition is not timed to coincide with the onset of rapid embryonic growth in oviparous reptiles, and is first to demonstrate that ～50% embryonic growth occurs in the last quarter of incubation in P. chinensis.     

Genome wide abnormal DNA methylome of human blastocyst in assisted reproductive technology

Proper reprogramming of parental DNA methylomes is essential for mammalian embryonic development.However,it is unknown whether abnormal methylome reprogramming occurs and is associated with the failure of embryonic development.Here we analyzed the DNA methylomes of 57 blastocysts and 29 trophectoderm samples with different morphological grades during assisted reproductive technology(ART) practices.Our data reveal that the global methylation levels of high-quality blastocysts are similar(0.30 ± 0.02,mean ± SD).while the methylation levels of low-quality blastocysts are divergent and away from those of high-quality blastocysts.The proportion of blastocysts with a methylation level falling within the range of 0.30 ± 0.02 in different grades correlates with the live birth rate for that grade.Moreover,abnormal methylated regions are associated with the failure of embryonic development.Furthermore,we can use the methylation data of cells biopsied from trophectoderm to predict the blastocyst methylation level as well as to detect the aneuploidy of the blastocysts.Our data indicate that global abnormal methylome reprogramming often occurs in human embryos,and suggest that DNA methylome is a potential biomarker in blastocyst selection in ART.     

Assessment of Genetic Stability Among In Vitro Plants of Arachis retusa Using RAPD and AFLP Markers for Germplasm Preservation

Arachis retusa Krapov. et W. C. Gregory et Valls is endemic in the West-central region of Brazil, occurring In areas endangered by human actions. The establishment of in vitro preservation methods for wild species of Arachis is an alternative to seed banks for germplasm storage, multiplication and distribution. The risk of genetic changes Induced by tissue culture and the monitoring of the genetic stability of the biological material before, during and after storage must be considered In the context of conservation. Random amplified polymorphlc ONA （RAPO） and amplified fragment length polymorphism （AFLP） fingerprinting were used to evaluate the genetic stability of in vitro plants originated from cotyledons and embryo axes of A. retusa. Cotyledons originated shoots through direct organogenesls and embryo axes displayed muItishoot formation Induced by 110 mmol/L and 8.8 mmol/L BAP, respectively. Ninety genomlc regions （loci） generated from RAPO and 372 from AFLP analyses were evaluated. All amplified fragments detected by both techniques in plants derived from the two explant types were monomorphic. The results Indicate that the recovered shoots are genetically stable at the assessed genomic regions.     

Effects of uranium poisoning on cultured preimplantation embryos

The toxic effect of uranium in cultured preimplantation embryos of the mouse is presented. Embryos were obtained from hybrid females CBA×C57 BL following induction of superovulation and were incubated in M16 cultured medium. Two different experiments were performed. In one, embryos in a one-cell stage were placed in culture media with final concentrations of uranyl nitrate of 104 and 208 μg/mL during 120 h in the same dish. In the other experiment, embryos in a one-cell stage were placed in culture medium with uranyl nitrate with final U concentrations of 26, 52, 104, and 208 μg/mL. At 24 h, those embryos which had reached the two-cell stage were transferred to another culture dish to which fresh solutions with uranyl nitrate were added. The percentage of embryos in two-cell stage, morula, early blastocyst, expanded blastocyst, and hatched blastocyst were recorded at 24, 72, 96 and 120 h of culture. The results obtained showed that concentrations as from 26 μg U/mL induced the delay of embryo development and the impairment of blastomere proliferation. The toxic effect of uranium increased in those experiments in which the embryos were transferred to a new medium. This embryo-culture system appears to be appropriate to evaluate the toxic effect of uranium on embryos removed from maternal influences and represents a suitable test system for environmental pollutants.