The genetic characterisation of novel multi-addition doubled haploid lines derived from triticale x wheat hybrids

Two novel 46-chromosome doubled haploid lines, W66 and M17, derived from separate hexaploid triticale x bread wheat crosses, were characterised using cytological and biochemical markers. Both lines were shown to be relatively stable cytologically, over 11 and 8 generations of selfing, respectively. By examining mitotic and meiotic chromosomes, the stabilities of the two lines were shown to be similar with frequencies of 2n=46 in 74.2–85.5% of cells. However, over selfed generations, the rye chromosomes were shown to have lost some of their heterochromatin, which made it difficult to establish their continued presence using cytological techniques, such as C-banding alone. Cytological evidence from pairing studies, C-banding, and fluorescence in-situ hybridization, showed that both M17 and W66 are wheat/rye multi-addition lines with rye chromosome constitutions of 1R+6R, and 1R+4R, respectively. These conclusions were confirmed by isozyme and storage-protein analysis.     

Effect of the cumulus on in vitro fertilization of bovine matured oocytes

The effect of the cumulus on in vitro fertilization in bovines was examined. Follicular oocytes were cultured in medium 199 plus OCS and extra granulosa cells. Frozen-thawed bovine spermatozoa was separated by the swim-up technique, suspended in Talp medium and capacitated with heparin. Fresh sheep and goat semen was incubated for 4 h at room temperature, washed and spermatozoa were then suspended in Talp medium and capacitated by incubation at 38.5 °C and 5% CO₂ in air and heparin.

In experiment 1, cumulus-enclosed oocytes, denuded oocytes and denuded oocytes plus additional cumulus cells were incubated with a reduced concentration of bovine spermatozoa for 8 or 18 h. In Experiment 2, cumulus enclosed and denuded oocytes were incubated with bovine spermatozoa for 4, 6, 8 and 18 h using a sperm concentration adjusted to secure high fertilization rates. In Experiment 3, cumulus-enclosed and denuded bovine oocytes were incubated with either sheep or goat spermatozoa for 18 h. Fertilization rates were then calculated and compared statistically. The results showed that 1) the cumulus improved the fertilization rate only when cumulus cells were associated with the oocytes 2) the timing of sperm penetration was not modified by the cumulus and started at 4 h after sperm incubation and 3) the presence of the cumulus improved the heterologous fertilization rate only when sheep spermatozoa were used. The results suggest that the cumulus improves fertilization rate by providing a capacitation-inducing mechanism and by facilitating the interaction between capacitated spermatozoa and the zona pellucida surface.     

大鼠前列腺上皮细胞角蛋白8mRNA表达调节的定位研究

本文应用反义RNA探针原位杂交法,研究雄激素对大鼠腹侧前列腺(VP)上皮细胞角蛋白(CK)8 mRNA表达的影响。发现1.在任何VP组织切片中,CK 8探针专一、大量定位于VP腺上皮细胞中,CK 8 mRNA是前列腺上皮细胞特异而灵敏的标志。2.去睾大鼠VP CK 8 mRNA染色增强,提示CK 8mRNA有过度表达,注射雄激素又可抑制其过度表达。3.与已知受雄激素抑制性基因不同,即使大鼠VP完全萎缩之后达2个月之久,其存留腺上皮细胞CK 8 mRNA表达仍持续增高。4.前列腺发育早期,迅速增殖的幼稚腺上皮细胞高度表达CK 8 mRNA,以后随着体内雄激素水平升高,VP上皮CK 8 mRNA表达下降,分布转移。以上结果进一步支持前列腺CK 8基因是新的一类受雄激素抑制性基因的推测,同时表明前列腺CK 8基因的表达与前列腺干细胞的增殖分化有密切联系,CK 8 mRNA高度表达是前列腺干细胞一个重要特征。     

Development of IVM-IVF produced 8-cell bovine embryos in simple,serum-free media after conditioning or Co-culture with buffalo rat liver cells

Development of 8-cell bovine embryos derived from in vitro matured/in vitro fertilized (IVM/IVF) oocytes was evaluated in two simple, serum-free media (CZB and SOM) with buffalo rat liver cells co-culture (BRLC) or after conditioning compared to a commonly used, serum-supplemented complex medium TCM-199. In a 3 x 4 factorial design, 578 eight-cell embryos were randomly assigned to 12 treatment groups. The factors were: first, type of culture medium (M199/FBS, CZBg and SOM), and second, the use of BRLC (as co-culture or to condition media for 24 hr and 48 hr) and unconditioned media. Development to morula was not affected by the type of medium, but co-culture and 48 hr conditioning within media type resulted in better development when compared to the 24-hr conditioned or unconditioned groups. Blastocyst development in SOM (38.9%) was different (P < 0.05) than in CZBg (46.6%) and M199/FBS (48.7%) and was lowest in the unconditioned group (27.8%) followed by 24 hr conditioned (33.3%), 48 hr (56.3%), and co-culture (59.6%). No blastocyst expansion was observed with unconditioned media and 24 hr conditioned SOM. Significant differences (P < 0.05) were found among all treatment groups except the co-culture and 48-hr conditioned groups. Hatching occurred only with co-culture and 48-hr conditioned groups of M199/FBS and CZBg media. These data show that CZB with glucose conditioned by BRLC monolayers for 48 hr can support the development of IVM/IVF produced bovine embryos to blastocyst compared to culture in TCM-199 with serum. © 1994 Wiley-Liss, Inc.     

Amplified somatic embryogenesis from male flowers of triploid banana and plantain cultivars (Musa spp.)

Summary Somatic embryogenesis and plant regeneration of banana and plantain cultivars (Musa spp.) were obtained by culturing young male flowers. Multiplication and maintenance of embryogenic cultures were achieved by culturing somatic embryos in a temporary immersion system (SIT). A multiplication rate of 40 allowed us to obtain more than 6000 somatic embryos after 6 mo. of subculture. Plant recovery frequencies were 60 to 70%. This method was expanded to different banana and plantain genomic groups.     

Altered free calcium transients in pig kidney cells (LLC-PK1) cultured with penicillin/streptomycin

Summary The effect of a conventional antibiotic (penicillin/streptomycin) mixture on the widely used kidney epithelial cell line, LLC-PK₁, was investigated by measuring growth and intracellular free calcium. Free calcium concentration was the same in cells cultured for 3 to 7 wk with (“plus”) and without (“minus”) antibiotics both at rest and when challenged with high (14 mM) external calcium. When exposed to vasopressin, minus cells exhibited significantly smaller calcium transients than plus cells. A similar difference existed for transients elicited by a calcium ionophore, 4-br-A23187. After longer periods of culture (>20 wk), minus cells grew slower than plus cells but on reaching confluence (minus cells took 1 day longer) the morphologies and viabilities were indistinguishable. The finding that culture with penicillin/streptomycin reversibly modified some properties of LLC-PK₁ cells, at least partly through altered calcium homeostasis, is of importance for workers using this cell model to study drug effects and raises the general possibility of similar effects on other cultured cells.     

Immature mouse uterine tissue in organ culture: Estrogen-induced growth,morphology and biochemical parameters

Summary Although estrogens have been shown to stimulate a variety of morphologic and biochemical changes in the uterus in vivo, no clear consistent demonstration of similar responses in vitro have been made; thus, a defined organ culture system using the immature mouse uterus was established to study the possibility of demonstrating estrogenic responses in vitro. Uterine tissue from immature outbred mice (17 to 24 days of age) were cut crosswise in 1-mm³ coins and cultured in a defined medium in the absence of serum, phenol red, or growth factor supplements. Diethylstilbestrol (DES), a synthetic estrogen, was added to the media at doses ranging from 1 to 100 ng/ml. The effect of DES on uterine cell proliferation was assessed by morphologic changes in uterine epithelial and stromal cells, increase in number of epithelial cells per unit basement membrane, increase in height of luminal epithelial cells, and [³H]thymidine incorporation. Functional changes were determined by measuring the amounts of the estrogen-inducible uterine protein, lactoferrin, that was localized in the epithelial cells and secreted into the media, and the localization of the estrogen receptor in the cultured tissues. Results indicate that under the described conditions of culture, estrogens like DES can induce morphologic and biochemical responses in the uterus that are similar to those seen in vivo. This organ culture system will aid in the investigation of various mechanisms involved in the hormonal regulation of growth and differentiation of estrogen target tissues.     

A Novel Method for Generating Nested Deletions Using the in Vitro Bacteriophage T3 DNA Packaging System

To sequence a DNA segment inserted into a cosmid vector underthe directed sequencing strategy, we established a simple andrapid method for generating nested deletions which uses thein vitro packaging system of bacteriophage T3 DNA. The principleis based on the previous finding that this system can translocateany linear double-stranded DNA up to 40 kb into the phage capsidin a time-dependent manner and the encapsulated DNA becomesDNase-resistant. For this purpose, we constructed a cosmid vectorthat carries two different antibiotic selection markers at bothsides of the multiple cloning site, and after insertion of aDNA segment, the clone was linearized by -terminase at the cossite. After the packaging reaction in vitro followed by DNasetreatment, the encapsulated DNA was introduced into Escherichiacoli cells to give clones with unidirectional deletions by differentialantibiotic selection. Restriction and sequence analyses of deletionclones demonstrated that an ordered set of clones with nesteddeletions, ranging from less than 1 kb to 25 kb, was createdfrom either the end of the DNA segment. Thus, nested deletionclones that cover the entire region of a 40-kb cosmid insertcan be obtained by a single packaging reaction, and its restrictionmap can be simultaneously obtained.     

Cellular Localization of Transforming Growth Factor-α mRNA in Rat Forebrain

Abstract: The cellular localization of transforming growth factor-α (TGFa) mRNA in juvenile and adult rat forebrain was examined using in situ hybridization with a ³⁵S-labeled cRNA probe. TGFα cRNA-labeled neuronal perikarya were distributed across many forebrain regions including the olfactory bulb, caudate-putamen, nucleus accumbens, olfactory tubercle, ventral pallidum, amygdala, hippocam-pal stratum granulosum and CA3 stratum pyramidale, and piriform, entorhinal, and retrosplenial cortices. TGFα cRNA-hybridizing cells were also localized to several thalamic nuclei and to the suprachiasmatic, dorsomedial, and ventromedial nuclei of the hypothalamus. In addition, labeled cells were present in regions of white matter including the corpus callosum, anterior commissure, internal and external capsules, optic tract, and lateral olfactory tract. Thus, both neurons and glia appear to synthesize TGFα in normal brain. Hybridization densities were greater in neuronal fields at 2 weeks of age compared with the adult, suggesting a role for TGFα in the development of several forebrain systems. Our results demonstrating the prominent and widespread expression of TGFα mRNA in forebrain, combined with the extremely low abundance of epidermal growth factor mRNA in brain, support the argument that TGFα is the principal endogenous ligand for the epidermal growth factor receptor in normal brain.     

Clearance of Exogenous Dopamine in Rat Dorsal Striatum and Nucleus Accumbens: Role of Metabolism and Effects of Locally Applied Uptake Inhibitors

In vivo electrochemistry was used to investigate the mechanisms contributing to the clearance of locally applied dopamine in the dorsal striatum and nucleus accumbens of urethane-anesthetized rats. Chronoamperometric recordings were continuously made at 5 Hz using Nafion-coated carbon fiber electrodes. When a finite amount of dopamine was pressure-ejected at 5-min intervals from a micropipette adjacent to the electrode, transient and reproducible dopamine signals were detected. Substitution of L-a-methyldopamine, a substrate for the dopamine transporter but not for monoamine oxidase, for dopamine in the micropipette did not substantially alter the time course of the resulting signals. This indicates that metabolism of locally applied dopamine to 3,4-dihydroxyphenylacetic acid is not responsible for the decline in the dopamine signal. Similarly, changing the applied oxidation potential from ±0.45 to ±0.80 V, which allows for detection of 3-methoxytyramine formed from dopamine via catechol-O-methyltransferase, had little effect on signal amplitude or time course. In contrast, lesioning the dopamine terminals with 6-hydroxydopamine, or locally applying the dopamine uptake inhibitors cocaine or nomifensine before pressure ejection of dopamine, significantly increased the amplitude and time course of the dopamine signals in both regions. The effects of cocaine and nomifensine were greater in the nucleus accumbens than in the dorsal striatum. Local application of lidocaine and procaine had no effect on the dopamine signals. Initial attempts at modeling resulted in curves that were in qualitative agreement with our experimental findings. Taken together, these data indicate that (1) uptake of dopamine by the neuronal dopamine transporter, rather than metabolism or diffusion, is the major mechanism for clearing locally applied dopamine from the extracellular milieu of the dorsal striatum and nucleus accumbens, and (2) the nucleus accumbens is more sensitive to the effects of inhibitors of dopamine uptake than is the dorsal striatum.