Developmental competence and mRNA expression of preimplantation in vitro-produced embryos from prepubertal and postpubertal cattle and their relationship with apoptosis after intraovarian administration of IGF-1

Recombinant human Insulin-like growth factor-I (hIGF-1) was administered to one ovary of prepubertal and postpubertal cattle to determine its effects on (1) oocyte developmental competence, (2) the expression pattern of six developmentally important genes (GLUT3, GLUT8, AKT1, BCL-XL, BAD, and BAX), and (3) its relationship with apoptosis (female Holstein-Friesian). Oocytes were retrieved from 7- to 10-mo-old prepubertal dairy calves (preP), 11- to 18-mo-old postpubertal heifers (postP), and cows via ultrasound-guided follicular aspiration. Immature oocytes were matured in vitro then fertilized and cultured up to the blastocyst stage. Apoptosis was determined by terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) in 8-d blastocysts. Similar low blastocyst yields were observed in the IGF-1-treated preP group (11.2 ± 2.4%), the control preP group (10.4 ± 3.0%), and in the IGF-1 postP group (10.9 ± 2.3%). These were lower (P ≤ 0.01) compared with the control postP group (21.2 ± 3.8%) and with cows (23 ± 3.7%). The expression profile of the six genes was partly affected by age and IGF-1 treatment. Apoptosis was correlated with the age of the oocyte donors and was increased in blastocysts derived from prepubertal heifers. Results show that apoptosis is a critical feature of the acquisition of developmental competence of oocytes from prepubertal cattle and that IGF-1 did not beneficially affect oocyte developmental competence.     

Distribution of GABA-like immunoreactivity during post-metamorphic development and regeneration of the central nervous system in the ascidian Ciona intestinalis

During regeneration of the neural ganglion in Ciona intestinalis, the pattern of reappearance of some peptidergic cells is similar to the ontogenetic patterns exhibited by these cell types during normal post-metamorphic development. Using a specific antiserum to gamma-aminobutyric acid (GABA), we describe here the appearance of GABA-ergic cells in Ciona during both post-metamorphic development and regeneration of the neural ganglion following total ablation. Post-metamorphic animals were divided into the categories: 1, 3–5, 6–10, 11–15 and 23–27 mm in body length. Regeneration was monitored at 12, 15, 18, 21, 28 and 56 days post ablation. The first appearance of GABA-like immunoreactive cells during normal development were at the 3 to 5-mm stage where they were seen as discrete cells, without processes, evenly distributed in the cortical region throughout the ganglion. Fibres were first seen at the 6 to 10-mm stage. As development proceeded, GABA-like immunoreactive cells became more concentrated near the nerve root exits and along the dorsal rind of the ganglion. In regenerating ganglia, GABA was first detected at 18–21 days post ablation, in cells that lacked any obvious processes and that were distributed in all regions of the ganglion. At 28 days post ablation, processes could be detected in the neuropile, and after 56 days GABA cells were found predominantly in the same regions as in the normally developing adult ganglion. Although the overall pattern reflects that in a normal adult, a few differences were detectable. For example, rather more GABAergic cells were concentrated ventrally in the ganglion close to the neural gland.     

Somatic embryogenesis in Simarouba glauca

Frequency of somatic embryogenesis from callus cultures derived from immature cotyledon explants of Simarouba glauca Linn. was highest on solid MS medium supplemented with 11.1 M benzyladenine and 13.42 M -naphthaleneacetic acid. On transfer of the somatic embryos into maturation medium containing half-strength MS medium supplemented with 1.89 M abscisic acid (ABA) and 2% (w/v)sucrose, 20–25 % of embryos germinated within 20 days of culture with distinct cotyledon, hypocotyl and radicle.     

Plant regeneration and somaclonal variation from cultured immature embryos of sister lines of rye and triticale differing in their content of heterochromatin

Summary Plants were regenerated from cultured immature embryos of two pairs of sister lines of triticale (X Triticosecale) cvs Rosner and Drira and five sister lines of rye (Secale cereale). The triticale lines differ in heterochromatic content of a particular rye chromosome (6R or 7R), while the rye lines differ in only one heterochromatic band. Variation in morphogenetic response was present between the triticale cultivars and between the rye lines. One of the rye lines (7RL+ +) showed a distinctive superior response in terms of somatic embryogenesis. These findings are discussed in relation to factors affecting morphogenetic response and genetic stability in culture.     

Precise recapitulation of methylation change in early cloned embryos

Change of DNA methylation during preimplantation development is very dynamic, which brings this term to the most attractive experimental target for measuring the capability of cloned embryo to reprogram its somatic genome. However, one weak point is that the preimplantation stage carries little information on genomic sequences showing a site-specific re-methylation after global demethylation; these sequences, if any, may serve as an advanced subject to test how exactly the reprogramming/programming process is recapitulated in early cloned embryos. Here, we report a unique DNA methylation change occurring at bovine neuropeptide galanin gene sequence. The galanin gene sequence in early bovine embryos derived by in vitro fertilization (IVF) maintained a undermethylated status till the morula stage. By the blastocyst, certain CpG sites became methylated specifically, which may be an epigenetic sign for the galanin gene to start a differentiation programme. The same sequence was moderately methylated in somatic donor cell and, after transplanted into an enucleated oocyte by nuclear transfer (NT), came rapidly demethylated to a completion, and then, at the blastocyst stage, re-methylated at exactly the same CpG sites, as observed so in normal blastocysts. The precise recapitulation of normal methylation reprogramming and programming at the galanin gene sequence in bovine cloned embryos gives a cue for the potential of cloned embryo to superintend the epigenetic states of foreign genome, even after global demethylation.     

The role of gap junction membrane channels in development

In most developmental systems, gap junction-mediated cell-cell communication (GJC) can be detected from very early stages of embryogenesis. This usually results in the entire embryo becoming linked as a syncytium. However, as development progresses, GJC becomes restricted at discrete boundaries, leading to the subdivision of the embryo into communication compartment domains. Analysis of gap junction gene expression suggests that this functional subdivision of GJC may be mediated by the differential expression of the connexin gene family. The temporal-spatial pattern of connexin gene expression during mouse embryogenesis is highly suggestive of a role for gap junctions in inductive interactions, being regionally restricted in distinct developmentally significant domains. Using reverse genetic approaches to manipulate connexin gene function, direct evidence has been obtained for the connexin 43 (Cx43) gap junction gene playing a role in mammalian development. The challenges in the future are the identification of the target cell populations and the cell signaling processes in which Cx43-mediated cell-cell interactions are critically required in mammalian development. Our preliminary observations suggest that neural crest cells may be one such cell population.     

Further characterization of the cathepsin L-associated protein and its gene in two species of the brine shrimp, Artemia

The major cysteine protease in embryos and larvae of the brine shrimp Artemia franciscana is a heterodimer composed of a cathepsin L-like polypeptide of 28.5 kDa and a 31.5 kDa polypeptide called the cathepsin L-associated protein or CLAP. In a previous study, CLAP was shown to be a cell adhesion protein containing two Fas I domains and two GTP/ATP binding sites known as Walker A and B motifs. Here, we have characterized CLAP and its genes to better understand the role of this protein in Artemia development. The polymerase chain reaction was used to investigate the structure of the CLAP gene in two species of Artemia, the New World bisexual diploid A. franciscana and the Old World parthenogenetic tetraploid Artemia parthenogenetica. The protein coding region of the CLAP gene from each species was 99.5% identical for a protein of 332 amino acids, while the 3' non-coding region, representing nearly 45% of the gene, was only 86% identical between the two related species. However, while the CLAP gene is intronless in A. franciscana, in A. parthenogenetica the gene contained a mini-intron of 30 base pairs in the 3' non-coding region. The sequences representing the CLAP gene in A. franciscana and A. parthenogenetica have been entered into the NCBI database as AY757920 and DQ100385, respectively. Northern blot analysis showed that while the cathepsin L gene is expressed constitutively in Artemia franciscana embryos and young larvae, the CLAP gene is not expressed in late embryos and young larvae. In contrast, Western blots indicated that CLAP is present in developing embryos and young larvae, at least to the first larval molt, supporting results obtained previously showing CLAP's resistance to degradation by its dimeric partner, cathepsin L. At the protein level we showed that the GTP/ATP binding sites in CLAP are functional with rate constants of 0.024 and 0.022 for GTP and ATP hydrolase activity, respectively. GTP but not ATP also had a slight stimulatory effect on cathepsin L activity of the heterodimeric protease containing CLAP. Our results support the hypothesis that CLAP plays an important role in targeting and expression regulation of cathepsin L activity during early development of Artemia.     

裸燕麦胚性愈伤组织培养及悬浮系的建立

裸燕麦成熟胚在IN_６培养基上诱导培养,初始愈伤组织为白色、瘤状、外软内硬、易分化植株的非松脆类型。当在IM_１～IM₄培养基上进行循环式调控培养,经7～8个月,初始愈伤组织转变为浅黄色、小颗粒状、生活力强的松脆型胚性愈伤组织。将胚性愈伤组织置于液体培养基中悬浮培养,得到分散性好、生长快的悬浮细胞系。悬浮系经分化培养获得了再生植株,移栽成活率达95%以上。     

Communicating junctions and calmodulin: Inhibition of electrical uncoupling inXenopus embryo by calmidazolium

Summary This paper reports the inhibitory effects of calmidazolium (CDZ), a calmodulin inhibitor, on electrical uncoupling by CO₂. Membrane potential and coupling ratio (V ₂/V₁) are measured in two neighboring cells ofXenopus embryos (16 to 64 cell stage) for periods as long as 5.5 hr. Upon exposure to 100% CO₂, control cells consistently uncouple even if the CO₂ treatments are repeated every 15 min for 2.5 hr. CDZ (5×10^–8–1×10^–7 m) strongly inhibits uncoupling. The inhibition starts after 30, 50 and 60 min of treatment with 1×10^–7, 7×10^–8 and 5×10^–8 m CDZ, respectively, is concentration-dependent and partially reversible. In the absence of CO₂, CDZ also improves electrical coupling. CDZ has no significant effect on membrane potential and nonjunctional membrane resistance. These data suggest that calmodulin or a calmodulin-like protein participates in the uncoupling mechanism.     

Nuclear structure in early mouse embryos: A comparative ultrastructural and immunocytochemical study with special emphasis on the "2-cell block in vitro"

The embryos from many outbred and inbred strains of mice are arrested at the late 2-cell stage when cultured in vitro in simple culture media. This phenomenon is referred to as the "2-cell block in vitro". The ultrastructural morphology of the nuclei of the blocked embryos is not yet well described. In the present paper we documented the results of a comparative study on the nuclei of mouse embryos, both normally developing and arrested at the 2-cell stage. The blocked embryos maintain the morphological integrity of their nuclei. Main nuclear domains (nucleolus precursor bodies, interchromatin granule clusters, perichromatin granules, and perichromatin fibrils), typical for the control embryos, are observed in the blocked ones. A number and morphological characteristics of these nuclear substructures are not changed significantly in the blocked embryos. At the same time, RNA polymerase II and pre-mRNA splicing factors are redistributed in the nucleus of the blocked embryos. Although something goes to show that nuclear organization of the blocked embryos differ from that of the control, we could not reveal in the blocked embryos distinct signs of degeneration which might characterize aged or dying cells. Our data confirm a peculiar functional state of the 2-cell blocked embryos.