Co-expression of the proto-oncogene fos (c-fos) and an embryonic interferon (ovine trophoblastin) by sheep conceptuses during implantation.

Expression of the c-fos proto-oncogene by ovine conceptuses was analyzed by Northern and slot blots and indirect immunohistofluorescence in relation to the expression of the embryonic interferon-alpha (oTP) during implantation. c-fos was expressed initially in the trophoblast, and then in the allantois, when this tissue began to develop (day 17). In the embryonic tissues, the c-fos proto-oncogene was weakly expressed up to day 22 and increased thereafter. In the trophoblast, the expression of c-fos proto-oncogene was transient, occurring when the oTP gene was transcribed at a maximal level at the beginning of implantation (days 14-15), and decreased thereafter, following the pattern of oTP gene expression. This decline is due essentially to the arrest of c-fos and oTP gene expression by the trophoblastic cells which established cellular contacts with the uterine epithelium during the implantation process.     

Embryonic development times of entomostracan zooplankton from Lake le Roux (Orange River,South Africa), and their possible relationships to seasonal succession

Embryonic development times of the principal entomostracan zooplankton of Lake le Roux, Orange River, South Africa, are described. Durations decreased monotonically with temperature over the thermal tolerance range and are described using Blehrádek's equation. The present data are compared with other observations on rate-temperature responses of warm-water and temperate zooplankton. Small, but ecologically significant, differences are discerned. The possible significance of species-specific rate-temperature responses to the phenomenon of seasonal succession is explored.     

Mobility of the conceptus and uterine contractions in the mare

Location of the embryonic vesicle within the uterus of mares was recorded every. five minutes for two consecutive hours (25 location determinations per trial) in three experiments. In Experiment 1 (n=7), the number of location changes among nine uterine segments (three body segments and three segments for each horn) was greater (P<0.05) on Day 13 than on Day 10. The vesicle was located in the body more frequently (P<0.05) and tended (P<0.1) to move to a more caudal position more frequently on Day 10 than on Day 13. Fixation occurred on Day 15 in four of seven mares and on Day 16 in the remaining three mares. The number of location changes was not significantly different between two days prior to fixation and one day prior to fixation. In Experiment 2, the effect of clenbuterol, a B₂ sympathomimetic blocker of uterine contractions, was studied on Days 12 or 13 of pregnancy. Location changes occurred less frequently (P<0.05) in treated mares (n=9) than in controls (n=10), indicating involvement of uterine contractions in the mobility of the embryonic vesicle. In Experiment 3, when the initial direction of location changes was caudal within a horn and cranial within the uterine body, the vesicle was more likely (P<0.05) to continue moving in the same direction than in the opposite direction. However, when the direction within a horn was cranial, the next location change was as likely to be in the opposite direction as in the same direction (not significantly different from equality). When the direction within the uterine body was caudal, the next location change was more likely (P<0.05) to be in the opposite direction.     

Ecdysteroid levels during adult reproductive and embryonic developmental stages of Sarcophaga bullata (Sarcophagidae: Diptera)

Ecdysteroid levels were determined during the period of the adult reproductive cycle in the ovovivparous fleshfly Sarcophaga bullata. Low levels were found in males during the 10 days following eclosion while the entire adult female showed a significant peak of ecdysteroid activity at 190 h post eclosion (i.e. during embryogenesis). When the female reproductive tract was analyzed it was observed that the ovaries became vitellogenic a short time after a protein meal was offered at 96 h post eclosion: at 140 h, ecdysteroid activity was recorded in the developing oöcytes. The major peak of ecdysteroids during the reproductive cycle was found in developing embryos at 190 h. The significance of these releases of ecdysteroids is discussed in relation to major embryonic developmental events.     

Effects of non-24-hour days on reproductive efficacy and embryonic development in mice

ICR female mice were exposed to either 22 (L11, D11) or 26 hour day (L13, D13) light/dark cycles for at least 2 weeks before mating and/or during pregnancy. The mating rates of these animals decreased considerably. When pregnant females were examined at gestation days 12.0 or 17.5, resorption rates were increased, the embryos weighed less, and development was retarded in the experimental groups with preconceptional exposure to non-24-hour days. We speculate that in mice maternal and paternal pre- and periconceptional environment of daily light/dark cycles is important for normal reproductive efficacy and normal embryonic development during pregnancy.     

In vitro differentiation of rabbit blastocyst cells

Summary Six- and seven-day post-coitus (p.c.) rabbit embryos have been cultured in an attempt to establish a trophectodermal cell line. Results indicate that cells with epithelial characteristics (i.e. positive staining for cytokeratin) will survive in culture until Passage 3. At that time a fibroblastlike cell becomes predominant. In addition, we have found that the presence of the inner cell mass is required for embryo explants often results in the development of cells that spontaneously contract. These cells stain positively for myosin, which indicates that they may be precardiac cells. Maximum diastolic potential was −59±1.2 mV and the threshold potential was −53±2.3 mV. Spontaneously contracting cells did not respond to atropine, acetylcholine, epinephrine, isoproterenol, or propranolol. Action potential seems to be a result of an inward calcium current, because the beating rate is decreased in a dose-related manner with the calcium channel blocker verapamil, whereas the voltage-sensitive sodium channel blocker tetrodotoxin was without effect. This work was supported by grants HD21302, HD07069, DK31091, and HL37320 from the National Institutes of Health, Bethesda, MD, with additional support from a University of Alabama at Birmingham Cardviovascular Research and Training Center Award.     

Two classes of continuous cell lines established from Syrian hamster 9 day gestation embryos: Preneoplastic cells and progenitor cells

Summary Primary cultures of 9-d-gestation Syrian hamster embryo (E9) cells are distinct from primary cultures of later gestational age in terms of their growth and differentiation. First, primary E9 cell cultures express multiple mesenchymal differentiation lineages (e.g., adipocyte, myoblast) only rarely seen in cultures of 13-d-gestation fetal (F13) cells. Second, although most primary E9 cultures have a limited in vitro proliferative life span and exhibit cellular senescence similar to primary cultures of F13 cells, E9 cultures seem to have higher frequency of escape from senescence and conversion to continuous cell lines compared to F13 cells. Moreover, this frequency can be further increased 4- to 5-fold by continuous exposure of the E9 cells to tumor promoters or epidermal growth factor. Eleven continuous cell lines have been isolated from unreated, promoter-treated, or epidermal growth factor-treated primary E9 cultures. Seven of these are neoplastic or preneoplastic. However, the remaining four do not show any evidence of being in neoplastic progression and three of these continue to express the same differentiated phenotype observed in ther parental primary cell cultures. These studies were supported in part by grants from the National Institutes of Health (AG 01998), Bethesda, MD, and the U.S. Department of Energy (DE-A-C02-76-EVO-3280), Washington, DC.     

Approaching the multifaceted nature of energy metabolism: inactivation of the cytosolic creatine kinases via homologous recombination in mouse embryonic stem cells

To study the physiological role of the creatine kinase/phosphocreatine (CK/PCr) system in cells and tissues with a high and fluctuating energy demand we have concentrated on the site-directed inactivation of the B- and M-CK genes encoding the cytosolic CK protein subunits. In our approach we used homologous recombination in mouse embryonic stem (ES) cells from strain 129/Sv. Using targeting constructs based on strain 129/Sv isogenic DNA we managed to ablate the essential exons of the B-CK and M-CK genes at reasonably high frequencies. ES clones with fully disrupted B-CK and two types of M-CK gene mutations, a null (M-CK^–) and leaky (M-CK¹) mutation, were used to generate chimaeric mutant mice via injection in strain C57BL/6 derived blastocysts. Chimaeras with the B-CK null mutation have no overt abnormalities but failed to transmit the mutation to their offspring. For the M-CK^– and M-CK¹ mutations successful transmission was achieved and heterozygous and homozygous mutant mice were bred. Animals deficient in MM-CK are phenotypically normal but lack muscular burst activity. Fluxes through the CK reaction in skeletal muscle are highly impaired and fast fibres show adaptation in cellular architecture and storage of glycogen. Mice homozygous for the leaky M-CK allele, which have 3-fold reduced MM-CK activity, show normal fast fibres but CK fluxes and burst activity are still not restored to wildtype levels.     

A channel that allows inwardly directed fluxes of anions in protoplasts derived from wheat roots

An anion channel that only allows outward current flow (anion influx) has been identified in protoplasts derived from wheat (Triticum aestivum L., Triticum turgidum L.) roots. The anion outward rectifier (anion OR) measured by patch-clamp of whole cells activated very quickly, usually reaching a steady-state level in less than 100 ms and was easily distinguished from the cation outward rectifier (cation OR) which activated more slowly during membrane depolarisation. The anion OR is permeable to NO ₃ ^– and Cl^–, moderately permeable to I^–, and relatively impermeable to H₂PO₄/^– and ClO₄/^–. An anomalous mole-fraction effect between ClO_4/ ^– and Cl^– was observed on the outward current, indicating that the channel is a multi-ion pore. The anion OR is gated by both voltage and external anion concentration such that it activates near to the equilibrium potential for the permeant anion. It activated at more negative membrane potentials when NO ₃ ^– was substituted for Cl^– in the external medium, indicating that the channel may function to allow NO ₃ ^– influx under luxuriant external NO ₃ ^– concentrations. For most experiments, K⁺ and Cl^– were the main cation and anion in solution, and under these conditions it appeared likely that the anion OR functioned in membrane-potential regulation by facilitating a Cl^– influx at membrane potentials more positive than the chloride reversal potential (E_Cl). If E_Cl was more negative than the K⁺ reversal potential (E_K) then the anion OR dominated but both the anion and cation ORs occurred together when the membrane potential difference (V_m) was positive of both E_Cl and E_K. The cation OR was inhibited by increasing external Cl^– concentrations, but the anion OR was not affected by external K⁺ or Na⁺ concentration. The anion-transport inhibitors, zinc and phenylglyoxal were ineffective in blocking the anion OR. 4,4-Di-isothiocyanostilbene-2, 2-disulfonic acid (DIDS) irreversibly blocked about 34% of the current when applied extracellularly at a concentration of 25 M, and about 69% at a concentration of 200 M. However, DIDS (200 M) also occasionally acted as an irreversible blocker of the cation OR. Perchlorate blocked irreversibly 75% of the current at an external concentration of 10 mM and did not block the cation OR. Whole-cell currents also indicated that the anion OR was insensitive to external pH (pH=5–7) and calcium concentration ([Ca²⁺]=0.1–10 mM). Increasing intracellular calcium concentration significantly increased the occurrence of the fast outward current in whole cells (P < 0.005, X² test). With approximately 10 nM calcium inside the cell the anion outward current was observed in 64% (n = 45) of cells and with 50 nM calcium inside the cell the anion current was observed in 88% (n = 69) of cells. Single-anion OR channels observed in outside-out patches had a conductance in 300 mM KCl (external) of about 4 pS. When voltage pulses were applied to outside-out patches the average currents were similar to those observed in whole cells. The significance of the anion OR as a likely route for Cl uptake in high salinities is discussed.Abbreviations Bath solution bathing the extracellular face of the membrane - DIDS (4,4-diisothiocyanostilbene-2,2-disulfonic acid) - E_x reversal potential for ion x - OR outward rectifier - Pip solution inside the pipette - TEACl (tetraethyl-ammonium chloride) - V_m membrane potential difference We thank the Australian Research Council for financial support, G.P. Findlay and A. Garrill for helpful discussions, and K. Morris and D. Mackenzie for expert technical assistance. M.S. was supported by an Australian Postgraduate Research Award.     

玉米胚性愈伤组织的长期继代及其染色体分析

对５种基因型幼胚诱导的愈伤组织继代培养表明,玉米胚性愈伤组织的长期继代受基因型,培养基成分,激素,培养条件的影响。适时继代,逐代筛选对胚性保持起重要作用。适当降低培养温度（１２±２℃）有利于愈伤组织的保存和胚性保持,可以减少愈伤组织长期继代所需的物质和工作量。长期继代培养的胚性愈伤组织,胚状体发生能力和植株再生率无显著变化,但正常苗的再生频率显著下降。观察愈伤组织细胞染色体发现：（１）基因型对不同倍性细胞的比例有明显影响。（２）随着继代时间的延长,二倍体细胞下降,四倍体和非二倍体细胞增多。（３）愈伤组织中出现多种染色体结构变异,这些结构变异有可能导致非整倍体细胞的形成。