Development of mouse embryos in media containing lectins

Lectins known to stimulate mitosis in cultured cells were evaluated for effects on development of mouse embryos in vitro. Two-cell mouse embryos were cultured in one of the following treatments: Whitten's medium as the control medium; Whitten's medium with 1, 10 or 100 mug/ml concanavalin A; Whitten's medium with 1, 10 or 100 mug/ml leucoagglutinin; Whitten's medium with 1, 10 or 100 mug/ml phytohemagglutinin; Whitten's medium with 1, 10 or 100 mug/ml pokeweed-mitogen; and Whitten's medium with 1, 10 or 100 mug/ml wheat germ agglutinin. Development to the morula stage was blocked in media with 100 mug/ml concanavalin A and 10 and 100 mug/ml wheat germ agglutinin, whereas blastocyst formation was blocked in all pokeweed-mitogen supplemented media. Embryos incubated in 10 and 100 mug/ml wheat germ agglutinin underwent premature cavitation or vacuolation at 24 to 48 h of culture. More embryos formed blastocysts in media with 1 and 100 mug/ml phytohemagglutinin and 10 mug/ml leucoagglutinin than in Whitten's medium (P<0.05). The percentage of embryos hatching was greatest in 1 mug/ml phytohemagglutinin (P<0.05), but it was the same in Whitten's medium, 1 mug/ml concanavalin A and 1 mug/ml leucoagglutinin (P>0.05). Cell division was not stimulated by the lectins; however, it was significantly suppressed in media with 10 and 100 mug/ml concanavalin A, 100 mug/ml phytohemagglutinin, 1, 10 and 100 mug/ml pokeweed-mitogen, and 10 and 100 mug/ml wheat germ agglutinin. Solubility of the zona pellucida in sodium isothicyanate (NaSCN) was reduced in 100 mug/ml phytohemagglutinin, 100 mug/ml leucoagglutinin and 1 mug/ml wheat germ agglutinin media (P<0.05) when compared to Whitten's medium and may have accounted for the reduced hatching observed in these treatments. Development of isolated blastomeres into blastocysts was reduced in media with 1 mug/ml wheat germ agglutinin, 1 mug/ml concanavalin A, and 10 and 100 mug/ml leucoagglutinin (P<0.05) but was similar in media with 1 mug/ml leucoagglutinin and 1, 10 and 100 mug/ml phytohemagglutinin when compared to Whitten's medium (P>0.05). The extent of embryo development in media with lectins depended upon the degree of cytotoxicity and potential biochemical modifications induced in the zona pellucida. Greatest embryo development took place in medium with 1 mug/ml phytohemagglutinin; however, the mechanism was not that of stimulation of cell division or a change in zona pellucida solubility.     

The effects of mannose on rat embryos grown in vitro

Rat embryos have been cultured in vitro from 9.5 days of gestation for different times in serum containing mannose, and the embryos have been observed by scanning electron microscopy. Embryos cultured in 3 mg/ml (1.7 X 10(-2) M) or 6 mg/ml (3.3 X 10(-2) M) mannose for 48 h showed inhibition of the expansion of the yolk sac and were smaller than the control embryos. Mannose-treated embryos also showed delayed development according to morphological criteria, and a range of abnormalities including abnormalities of the neural tube. Embryos cultured in 6 mg/ml mannose for 24 h also showed significant inhibition of yolk-sac expansion and were smaller and less advanced than the control embryos. Abnormalities were seen, including a delay in the closure of the neural folds. Abnormalities were also observed in embryos cultured in mannose for 10 h; embryos at the neural groove stage showed irregularities in the neural groove. Mannose did not inhibit the re-elevation of neural folds which had been caused to collapse by exposure to medium containing low calcium. These results are compatible with the idea that mannose retards development and thereby perturbs the morphogenesis of the neural tube.     

Differential inhibition of trophoblast outgrowth and inner cell mass growth by actinomycin D in cultured mouse embryos.

Treatment of preimplantation mouse embryos in vitro with 10(-3) to 10(-1) mug actinomycin D/ml for 2 hr showed that (i) postimplantation development in vitro was inhibited most when embryos were treated at the morula stage and (ii) after the morula stage actinomycin D inhibited trophoblast outgrowth less than inner cell mass development.     

Inactivation of bovine herpesvirus-1 and bovine viral diarrhea virus in association with preimplantation bovine embryos using photosensitive agents

Hematoporphyrin (HP), hematoporphyrin derivative (HPD), and thiopyronine (TP) are photosensitive agents (PSA) that have a germicidal effect when they are activated by light: helium neon laser (He Ne ) light (HP, HPD), white light (HP, HPD), and yellow-green light (TP). Experiments were conducted with appropriate controls to determine the effect of photosensitive agents a) for inactivating bovine herpesvirus-1 (BHV-1; titre 10(6) TCID(50) /ml) and bovine viral diarrhea virus (BVDV; titre 10(6) TCID(50) /ml); b) for disinfecting Day-7, zona pellucida-intact (ZP-I) bovine embryos that had been exposed to BHV-1 (titre 10(6) TCID(50) /ml) or BVDV (titre 10(6) TCID(50) /ml); and c) on the in vitro development of embryos. Exposure to HP, HPD and TP followed by light irradiation inactivated BHV-1 and BVDV. Embryos exposed to BHV-I were disinfected by HP or HPD (5 mug/ml) in combination with He Ne light, or by HP or HPD (10 mug/ml) in combination with white light. Embryos exposed to BVDV were disinfected by HPD (5 and 10 mug/ml) followed by He Ne or white light irradiation. Exposure of embryos to light alone or to light and HP or HPD had no detrimental effect on their in vitro development; however, exposure of embryos to TP (5 mug/ml) followed by irradiation caused embryonic degeneration. Exposure of embryos to 5 mug of HPD followed by He Ne light, or 10 mug/ml of HP or HPD, followed by white light, is simple methods of disinfecting them of BHV-I and BVDV.     

Effects of culture duration and time of gonadotropin addition on in vitro maturation and fertilization of red deer (Cervus elaphus) oocytes

Immature red deer (Cervus elaphus ) oocytes (n = 1208) were collected from 1 to 4 - mm diameter follicles on ovaries and then cultured for 16, 20, 24 or 28 h (Groups I to IV) in TCM 199 supplemented with 10% FCS, 1 x 10(6) granulosa cells/ml and 1 mug/ml estradiol at 39 degrees C under 5% CO(2) in air. Gonadotropins (10 mug/ml, FSH and LH) were added to the culture medium at the start of culture (0 h) or after 6 h. Approximately one-third of the oocytes were examined for maturation, and the remainder were fertilized in vitro with frozen-thawed semen collected from a stag by electroejaculation. In vitro fertilized oocytes (n = 309) from four of the maturation treatment (Groups II and III in both gonadotropin treatments) were cultured for 7 d and examined for cleavage. Oocytes cultured for 16 h (Group I) had lower (P < 0.001) maturation rates (4.7%) than those in the longer culture durations (Groups II to IV: 68.9%). Culture for 20 (Group II) and 24 h (Group III) resulted in higher (P <0.001) fertilization rates than culture for 16 (Group I) and 28 h (Group IV) (18.3, 20.5, 7.1, 7.8%, respectively). The time of gonadotropin addition did not affect maturation or fertilization rates, but its addition at 6 h increased (P < 0.05) the percentage of oocytes cleaving (5.7 vs 12.5%). Oocytes cultured for 20 h (Group II) and with the delayed addition of gonadotropins cleaved most readily (18.2%). No embryos developed beyond eight-cell stage.     

Effect of gossypol on bovine embryo development during the preimplantation period

The purpose of this study was to evaluate the effect of varying doses of gossypol acetic acid on early bovine embryo development in vitro. One hundred and forty-eight excellent and good quality bovine morulae were randomly cultured in 0, 1.0, 5.0, 10.0, 30.0 mug gossypol acetic acid (GAA) in normal steer serum and Ham's F-10 media. Bovine embryo development was assessed at 12-h intervals for 96 h. Sixty-seven percent of embryos developed in 0 mug GAA to the hatched blastocyst stage, while 43, 19, 4 and 0% had comparable development in 1.0, 5.0, 10.0 and 30.0 mug GAA, respectively. Embryos in 5.0 mug GAA had a delayed development to the blastocyst stage compared to embryos in 1.0 mug GAA. Development time to expanded blastocyst stage was longer for 10.0 mug GAA embryos than 0, and 1.0 GAA-treated embryos. No embryo cultured in 30.0 mug GAA advanced past the morula stage. Final developmental scores were highest for embryos in 0 mug GAA (4.06) and lowest for embryos cultured in 10.0 and 30.0 mug GAA (0.44 and -0.02, respectively). Embryos cultured in higher doses of GAA degenerated sooner than embryos cultured in 0 mug GAA. These data show a dose-dependent detrimental action of GAA on early bovine embryo development and suggest a direct action on the embryo itself.     

Effect of Selected Herbicides on Bacterial Growth Rates

Specific growth rate constants were used to evaluate the effects of selected herbicides on Erwinia carotovora, Pseudomonas fluorescens, and Bacillus sp. Comparison of growth rate constants permitted the identification of either stimulatory or inhibitory effects of these substances. E. carotovora was inhibited by 6,7-dihydrodipyrido(1,2-a:2'-c)pyrazinediium (diquat) and 4-hydroxy-3,5-diiodobenzonitrile (ioxynil) at 25 mug/ml; 1,1'-dimethyl-4,4'-bipyridinium (paraquat) at 50 mug/ml; and pentachlorophenol (PCP) at 10 mug/ml. P. fluorescens was inhibited by paraquat and PCP at 25 mug/ml and by 4-amino-3,5,6-trichloropicolinic acid (picloram) at 50 mug/ml. Stimulation of P. fluorescens was observed with 4-(methylsulfonyl)-2,6-dinitro-N,N-dipropylaniline (nitralin) at 25 mug/ml. The Bacillus species was inhibited by diquat (25 mug/ml), ioxynil (10 mug/ml), and paraquat and PCP (5 mug/ml). No significant effect of 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (atrazine), 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron), alpha,alpha,alpha-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine (trifluralin), or 1,1-dimethyl-3-(alpha,alpha,alpha-trifluoro-m-tolyl)urea (fluometuron) on growth rates of the bacteria was observed at 25 and 50 mug/ml.     

Mechanism of Action of the Fungicide Thiabendazole, 2-(4′-Thiazolyl) Benzimidazole

Thiabendazole, 2-(4'-thiazolyl) benzimidazole (TBZ) inhibited the growth of Penicillium atrovenetum at 8 to 10 mug/ml. Oxygen consumption with exogenous glucose was inhibited at 20 mug/ml, but endogenous respiration required more than 100 mug/ml. TBZ inhibited completely the following systems of isolated heart or fungus mitochondria: reduced nicotinamide adenine dinucleotide oxidase, succinic oxidase, reduced nicotinamide adenine dinucleotide-cytochrome c reductase, and succinic-cytochrome c reductase at concentrations of 10, 167, 10, and 0.5 mug/ml, respectively. Cytochrome c oxidase was not inhibited. Antimycin A and sodium azide caused the usual inhibition patterns for both fungus and heart terminal electron transport systems. In the presence of antimycin, the fungicide inhibited completely succinate-dichloro-phenolindophenol reductase and succinate-2, 2-di-p-nitrophenyl-(3, 3-dimethoxy-4, 4-biphenylene-5, 5-diphenylditetrazolium)-reductase at 2 and 4 mug of TBZ per ml, respectively. Coenzyme Q reductase required 15 mug/ml. TBZ reduced the uptake by P. atrovenetum of glucose and amino acids and decreased the synthesis of various cell components. At 120 mug/ml, the incorporation of labeled carbon from amino acids-U-(14)C was decreased: lipid, 73%; nucleic acids, 80%; protein, 80%; and a residual fraction, 89%. TBZ did not inhibit peptide synthesis in a cell-free protein-synthesizing system from Rhizoctonia solani. Probably the primary site of inhibition is the terminal electron transport system and other effects are secondary.     

Stage-specific response of the mesenchyme to excess vitamin A in developing rat facial processes

The effects of excess retinol (vitamin A alcohol) on facial process formation were examined in cultured rat embryos. The embryos were explanted at day 11 of gestation (plug day = 0) and cultured for 72 hr in rat serum containing an additional 1 or 10 micrograms/ml retinol. The reduction of outgrowth in the facial processes was observed in 1 microgram/ml retinol-treated embryos, and this type of malformation was found to be more severe in 10 micrograms/ml retinol-treated embryos. Histological findings of 10 micrograms/ml retinol-treated embryos at the 50-somite stage showed that the nasal epithelium was developed but folded. In the mesenchyme, there were necrotic cells. Thymidine incorporation by mesenchymal cells in the facial processes was also determined. At the 50-somite stage, the uptake was decreased to 66.4% of control value at 1 microgram/ml retinol, whereas the addition of the same dose of retinol did not cause the inhibition at the 36-, 40-, and 42-somite stages. The uptake at the 50-somite stage was decreased to 23.0% as a result of the 10 micrograms/ml retinol treatment. These results show that the response of the facial mesenchyme to excess retinol is dependent on the development stage and the critical stage of the facial mesenchyme for excess retinol in cultured rat embryos is the 42-somite stage.     

A serum-free, cell-free culture system for development of bovine one-cell embryos up to blastocyst stage with improved viability

With the aim of developing a serum-free, cell-free culture system for embryo development, in vitro-matured (IVM) and -fertilized (IVF) bovine oocytes were cultured in TCM 199 with the following supplements: 1) BSA alone (10 mg/ml); 2) BSA with ITS (5 mug/ml insulin, 5 mug/ml transferrin and 5 ng/ml selenium; BSAITS medium); 3) estrous cow serum alone (ECS; 10%); or 4) ECS with BOEC (bovine oviduct epithelial cells) (Experiment 1). In Experiment 2, embryos were cultured in BSAITS medium with or without feeding with fresh medium on Day 4 (day of insemination = Day 0). Embryos were evaluated on Day 2 for first cleavage, on Day 7 for morulae and blastocysts, and on Day 8 for blastocysts. Blastocysts from Experiment 1 were frozen in 10% glycerol in PBS, thawed and further cultured in ECS medium with BOEC for 48 h, and evaluated for formation of a distinct blastocoel, or expansion and hatching of blastocysts. In vivo-developed, Grade-1 and Grade-2, 7-d-old embryos served as control for the freezing, thawing and subsequent culture procedures. The percentage of first cleavage did not differ between the treatments (74 to 79% in Experiment 1 and 80 to 83% in Experiment 2). The percentage of blastocysts developed in BSAITS medium did not differ from that in ECS medium whether BOEC were present or not. However, medium with BSA alone had fewer blastocysts than any other culture system (P<0.05). Feeding embryos with fresh BSAITS medium on Day 4 did not lead to any further increase in the proportion of blastocysts. The culture systems had a significant effect on the post-thaw viability of blastocysts developed in them (P<0.001). Blastocysts developed in BSAITS medium had better (P<0.05) viability (14/38) than those from medium with ECS alone (1/27) or with ECS and BOEC (3/37). The post-thaw survival of control embryos was 80% (n=30). One of the three transfers of BSAITS-treated, frozen-thawed blastocysts resulted in a pregnancy. The results indicate that a serum-free, cell-free culture system can support the development of IVM-IVF bovine oocytes up to the blastocyst stage with better viability than a complex co-culture system.     

Influences of epidermal growth factor and insulin-like growth factor-I on bovine blastocyst development in vitro

Experiments were carried out to investigate putative beneficial effects of adding epidermal growth factor (EGF) or insulin-like growth factor-I (IGF-I) for bovine embryo culture in chemically defined media. Presumptive zygotes (18 h post-insemination) were randomly assigned to culture treatments. In experiment 1, treatments involved additions of recombinant human EGF to provide concentrations of 0 ng (control), 1, 5, and 25 ng/ml. No differences were seen in numbers of 4-cell stage embryos between groups. A concentration of 5 ng/ml EGF but not 1 or 25 ng/ml during embryo culture improved percentages of 4-cell stage embryos reaching blastocysts compared to the control (P<0.05). Numbers of inner cell mass (ICM) cells and trophoblast cells of day 8 blastocysts were similar for the control and 5 ng/ml EGF-treated groups. In experiment 2, culture with recombinant human IGF-I in concentrations of 0 ng (control), 2, 10, and 50 ng/ml resulted in no differences in numbers of 4-cell stage embryos between groups. When compared to controls, IGF-I treatments at 10 and 50 ng/ml improved proportions of 4-cell stage embryos that reached blastocysts (P<0.05). In experiment 3, numbers of ICM cells of day 8 blastocysts were significantly higher after being cultured with 50 ng/ml of IGF-I compared to those of the controls (P<0.05). No additive effect of combining EGF (5 ng/ml) and IGF-I (50 ng/ml) was seen when results were compared to those following supplementation of the media with either EGF or IGF-I alone. In conclusion, both EGF and IGF-I could independently enhance bovine preimplantational development in chemically defined media and IGF-I but not EGF may play a mitogenic role during early bovine development.     

Phase shifting the circadian rhythm of neuronal activity in the isolated Aplysia eye with puromycin and cycloheximide. Electrophysiological and biochemical studies

The effects of pulse application of puromycin (PURO) or cycloheximide (CHX) were tested on the circadian rhythm (CR) of spontaneous compound action potential (CAP) activity in the isolated Aplysia eye. CAP activity was recorded from the optic nerve in constant darkness at 15degreesC. PURO pulses (6, 12 h; 12--134 mug/ml) and CHX pulses (12 h, 500--2,000 mug/ml) caused dose-dependent phase delays in the CR when administered during projected night. PURO pulses (6 h, 125 mug/ml) caused phase advances when given during projected day and caused phase delays when given during projected night. In biochemical experiments PURO (12 h, 20 mug/ml) and CHX (12 h, 500 mug/ml) inhibited leucine incorporation into the eye by about 50%. PURO (12 h; 50, 125 mug/ml) also changed the molecular weight distribution of proteins synthesized by the eye during the pulse. The effect of PURO (12 h, 125 mug/ml) on the level of incorporation was almost completely reversible within the next 12 h but the phase-shifted eye showed an latered spectrum of proteins for up to 28 h after the pulse. In electrophysiological experiments spontaneous CAP activity and responses to light were measured before, during, and after drug treatments. In all, eight parameters in three periods were analyzed quantitatively. Of these 24 indices, only 3 showed significant changes. PURO increased spontaneous CAP frequency by 67% 0-7 h after the drug pulse and increased the CAP amplitude of the tonic light response by 23% greater than 7 h after the pulse. CHX increased the intraburst spontaneous CAP frequency by 33% during the pulse and CAP frequency of the tonic light response by 32% 0- 7 h after the pulse. The above data indicate that phase-shifting doses of PURO and CHX inhibit protein synthesis in the eye without causing adverse electrophysiological effects, and suggest that protein synthesis is involved in the production of the CR of the isolated Aplysia eye.     

Platelet-activating factor alters the dynamics of prostaglandin and protein synthesis by endometrial explants from pregnant and cyclic cows at day 17 following estrus

Factors produced by bovine conceptuses alter prostaglandin (PG) and protein secretion by endometrial explants from cyclic cows and induce an intracellular inhibitor of PG synthesis. Endometrial explants from cyclic (n = 4) and pregnant (n = 3) cows at Day 17 following estrus were incubated for 24 h with 0, 0.1, 0.5, 1 and 5 mug platelet-activating factor (PAF)/ml. Cotyledonary microsomes from parturient cows were utilized to determine levels of an intracellular/cytosolic inhibitor of PG synthesis. Endometrial explants from additional cyclic cows (n = 4) were incubated for 24 h with 0 or 5 mug PAF/ml with and without 50 muCi [(3)H]leucine. Endometrial explants (cyclic cows, n = 3) were also incubated for 12 h with each of the following treatments: 1) Control; 2) PAF (1 mug/ml); 3) lyso-PAF (2 to 10 mug/ml); 4) PAF-receptor antagonist (2 to 10 mug/ml); 5) PAF (1 mug/ml) + antagonist (2 to 10 mug/ml); 6) bovine conceptus secretory proteins (bCSP; 25 mug/ml); and 7) bCSP (25 mug/ml) + antagonist (5 mug/ml). Platelet-activating factor had distinct negative and positive dose effects on PGF and PGE-2 secretion, respectively, by explants from cyclic cows, whereas PG secretion was not altered by PAF in the endometrium of pregnant cows. Platelet-activating factor did not alter the level of an intracellular inhibitor of prostaglandin synthesis, whereas, bCSP increased the level of this inhibitor. Platelet-activating factor decreased the incorporation of [(3)H]leucine into tissue and secreted proteins for explants from cyclic cows. Lyso-PAF did not alter endometrial prostaglandin secretion. The effects of PAF but not of bCSP were blocked by the PAF-receptor antagonist. Platelet-activating factor altered PG and protein secretion by the endometrium from cyclic cows, and it may be a potential regulatory factor during early pregnancy if secreted by the bovine conceptus.     

Mutagenesis in cultured human diploid cells. III. Induction of 8-azaguanine-resistant mutations by furylfuramide.

Trans-2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (furylfuramide: FF or AF2) was tested for ability to induce 8-azaguanine (8AG) resistant mutations in cultured human diploid cells. FF had a relatively severe cytotoxic effect on the cells. From the concentration-survival curve, the D0 value for 2-h treatment with FF was estimated to be 11 mug/ml. When cells were treated with FF at various concentrations for 2 h, cultured in normal medium for 48 h, and then selected in medium containing 8AG at 30 mug/ml, the induced mutation frequency increased gradually with increase in concentration of FF. When cells were treated with FF at 10 mug/ml for 2 h, cultured in normal medium for various periods of mutation expression time, and selected with 8AG at 30 mug/ml, the highest induced mutation frequency was obtained with 48 h of mutation expression time. Microscopic examination of the numbers of cells in colonies indicated that the total number of cells increased by half during this mutation expression time of 48 h.     

Term development of caprine embryos derived from immature oocytes in vitro

Ovaries were surgically removed from female goats (Toggenburg, Nubian and Saanen breeds). Oocytes were collected by follicular aspiration or after ovaries were minced, then matured in mTCM-199 with 100 mug LH + 0.5 mug FSH + 1.0 mug estradiol 17-beta/ml for 27 h prior to in vitro fertilization (17). Although more oocytes were made available by mincing than by aspiration, higher proportions of aspirated oocytes were fertilized and developed to morulae. Proportions that fertilized and reached morulae were 82 102 (80.4%) and 50 102 (49.0%) versus 77 126 (61.1%) and 27 126 (21.4%) for oocytes obtained by aspiration and after ovarian mincing, respectively (P<0.05). Proportions of inseminated ova undergoing cleavage and continuing development to the morula stage differed significantly (P<0.05) among 5 co-culture treatment groups, with higher proportions of cleavage (23 27 , 85.2%) and morulae (14 27 , 51.9%) obtained by co-culture on caprine cumulus cells (cCC). Some oocytes reached the blastocyst stage (4 54 , 7.4%) following oocyte collection by aspiration and culture on caprine oviduct epithelial cells (cOEC). After 4- and 8-cell stage embryos obtained by aspiration and culture on cCC were transferred pregnancy resulted. Twin male kids (developed from different embryos) were born on August 6, 1993, and have developed into normal bucks. Conditions reported here provided an adequate environment for support of oocyte maturation, fertilization and early embryonic development in vitro (IVMFC) with normal development after embryo transfer.     

Increased reactivity of cultured chicken blastodermal cells to anti-stage-specific embryonic antigen-1 antibody after exposure to bone morphogenetic proteins

We evaluated whether bone morphogenetic proteins (BMPs) increased the reactivity of chicken stage X blastodermal cells to the germ cell marker, anti-stage-specific embryonic antigen (SSEA)-1 antibody. In Experiment 1, blastodermal cells cultured on a feeder layer of SIM mouse embryo-derived thioguanine and ouabain resistant (STO) cells were treated with different doses of BMP-2 and/or BMP-4, and the anti-SSEA-1 antibody reactivity of cultured cells was examined 48 h later. A significant (P < 0.05) increase in the number of anti-SSEA-1 antibody-positive cells was detected after the addition of 75 or 100 ng/ml BMP-2. Neither 0-20 ng/ml BMP-4 nor the combined addition of 75 ng/ml BMP-2 with either 10 or 15 ng/ml BMP-4 increased reactivity more than that induced by 75 ng/ml BMP-2 alone. Results of the qualification and quantification of BMP receptor kinase (BRK)-1, BRK-2, and BRK-3 using RT-PCR and real-time PCR showed that all three receptors were detected in blastodermal cells treated with BMPs, intact stage X embryos and 5.5-day-old embryonic gonads, but no expression was detected in STO feeder cells. In Experiment 2, the treatment of stage X embryos with different doses of BMP-2 (0.15-3 ng/embryo) or BMP-4 (0.02-0.4 ng/embryo) did not affect the reactivity of 5.5-day-old embryonic gonadal cells to the anti-SSEA-1 antibody. BRK-1 expression was selectively increased in stage X embryos after the infusion of 3ng BMP-2 than after no infusion, but no changes in other BRKs' expression were detected. In conclusion, the addition of BMP-2 to culture medium in the presence of STO feeder cells promoted the reactivity of blastodermal cells to anti-SSEA-1 antibody, which might contribute to the generation of chicken primordial germ cell precursor or germ cell-like cells. The relationship between BMP action and BRK expression was further discussed.     

Influence of glucagon, 6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate and triamcinolone on the arginine synthetase system in perinatal rat liver

1. The administration of triamcinolone (19-190mug/animal) to postnatal rats increased the arginine synthetase system activity 1.2-2.5-fold above control values 24h after exposure to the hormone. Cortisol (hydrocortisone), however, increased the arginine synthetase system activity only when larger (190mug/animal) or repeated daily doses were given. Glucagon (100mug/animal) stimulated arginine synthetase system activity only after the second postnatal day. None of these agents increased the activity in 19.5-21.5-day foetuses after intrauterine administration. 2. The viability of foetal rat liver explants maintained in organ culture for up to 54h was validated both by ultramicroscopic examination and by incorporation of radioactive leucine and orotic acid. 3. In organ cultures of foetal rat liver explants (18.5 days to term), triamcinolone (20mug/ml of medium) evoked a 2.8-4.3-fold increase after 24h of incubation. This increase was completely inhibited by actinomycin D (25mug/ml) or cycloheximide (10mug/ml). Cortisol (5-50mug/ml) or glucagon (0.067-67mug/ml) also increased the arginine synthetase system activity above the respective control values, but there was no increase in activity with insulin (0.05-0.25i.u./ml). 4. Maximum concentrations of glucagon (67mug/ml), dibutyryl cyclic AMP (6-N,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate) (0.1mm) and triamcinolone (20mug/ml) incubated for 24h with foetal rat liver explants each produced between a two-and three-fold increase in the activity of the arginine synthetase system. Combinations of maximum amounts of glucagon and the cyclic nucleotide did not produce a greater effect than either agent alone. However, the combination of dibutyryl cyclic AMP with triamcinolone appeared to produce somewhat less than additive effects. 5. The effects of the cyclic nucleotide and triamcinolone were evident after 12h of incubation and increased steadily throughout the 24h of observation. This time-course of increased enzyme activity is very much slower than that reported for the induction of other enzymes in explant cultures of foetal rat liver.     

Mutagenesis in cultured human diploid cells. IV. Induction of 8-azaguanine resistant mutations by phloxine, a mutagenic red dye.

Disodium 9-(3',4',5',6'-tetrachloro-o-carboxyphenyl)-6-hydroxy-2,4,5,7-tetrabromo-3-isoxanthone (phloxine), a red dye used as a food additive, was tested for its activity to induce 8-azaguanine (8AG) resistant mutations in cultured human embryonic cells. Phloxine had a severe cytotoxic effect on the cells at concentrations of 1 to 10 mug/ml. At concentrations of more than 30 mug/ml of phloxine no further decrease in cell survival was found. This cytotoxic effect of phloxine was not dependent on the duration of treatment. After treatment with phloxine for 2 h division of cells in normal medium was inhibited for 120 h. When cells were treated with phloxine at various concentrations for 2 h, cultured in normal medium for 48 h, and then selected with 30 mug/ml of 8AG, an increase in the induced mutation frequency was found. This increase in mutation frequency was dependent on the concentration of phloxine used as a mutagen and treatment with 100 mug/ml of phloxine increased the frequency to six times that in untreated cultures.     

Effect of prostaglandins on cell function and multiplication of parainfluenza 3 viruses in WISH cells.

Cytotoxic effect of prostaglandins E2 and F2alpha on cells grown in vitro and the influence of these compounds on multiplication of myxovirus parainfluenza 3 were investigated. The prostaglandins were added to culture medium (0-01-10 mug/ml) 24 hr before virus infection, or for 2 and 48 hr after inoculation with viruses. WISH cells and monkey kidney cell cultures were used. No direct cytotoxic effect of prostaglandins at concentrations 0-01-1 mug/ml was found (viability, supravital staining, phase-contrast system, Nitro-BT reduction and succinic dehydrogenase tests), whereas the concentration of 10 mug/ml within 48 hr led to reduction and succinic dehydrogenase tests), whereas the concentration of 10 mug/ml within 48 hr led to partial injury of the cell population with symptoms of damage to mitochondria. Prostaglandins E2 and F2alpha inhibited multiplication of parainfluenza 3 virus at concentrations 0-1-10 mug/ml. The inhibitory effect was most pronounced if prostaglandins were added to medium for the whole period of virus multiplication i.e. for 48 hr but little or no effect was found if they were added prior to inoculation or for 2 hr after it. Inhibitory effect of prostaglandins on replication phase of viruses is suggested.     

Effect of Inoculum Size on In Vitro Susceptibility Testing with Lincomycin

There is disagreement in the literature as to whether lincomycin is primarily a bacteriostatic or a bactericidal agent against gram-positive cocci and also regarding the levels of activity of this agent against susceptible microorganisms. These questions were examined in a study of the effect of inoculum size on the results of tube dilution susceptibility determinations with lincomycin against 49 clinical isolates of Staphylococcus aureus and 25 strains of streptococci and pneumococci. Lincomycin was both highly active and bactericidal when tested against 40 strains of S. aureus with inocula containing a maximum of 10(4) cells per ml [median minimal inhibitory concentration (MIC), 0.78 mug/ml; median minimal bactericidal concentration (MBC), 1.56 mug/ml]. With inocula of 10(5) cells per ml, lincomycin was primarily bacteriostatic (median MIC, 1.56 mug/ml; median MBC, 12.5 mug/ml). There were further decreases in inhibitory levels and significant losses of bactericidal activity when inocula containing more than 10(7) cells were tested (median MIC, 3.13 mug/ml; median MBC > 100 mug/ml). Similar measurements with streptococci and pneumococci revealed a lesser effect of inoculum size. The mean MBC value for alpha-hemolytic streptococci increased from 0.40 to 1.05 mug/ml with an increase in inocula from 10(4) to 10(6) cells per ml, but without a marked increase in MIC values. Similar results were obtained for beta-hemolytic streptococci and pneumococci.