Cinematographic Observation of “Post-Fertilization waves” (PFW) on the zygote ofXenopus laevis

Summary In fertilizedXenopus eggs, shortly after egg rotation but well before the occurrence of the cleavage-associated surface contraction waves, two circular dark zones originate consecutively from the pigment spot marking the site of sperm entrance. They expand and travel centrifugally over the egg surface.     

Single cell analysis of G1 check points-the relationship between the restriction point and phosphorylation of pRb

Several recent reports suggest that there is far more plasticity that previously believed in the developmental potential of bone-marrow-derived cells (BMCs) that can be induced by extracellular developmental signals of other lineages whose nature is still largely unknown. In this study, we demonstrate that bone-marrow-derived mesenchymal stem cells (MSCs) co-cultured with mouse proliferating or fixed (by paraformaldehyde or methanol) neural stem cells (NSCs) generate neural stem cell-like cells with a higher expression of Sox-2 and nestin when grown in NS-A medium supplemented with N2, NSC conditioned medium (NSCcm) and bFGF. These neurally induced MSCs eventually differentiate into beta-III-tubulin and GFAP expressing cells with neuronal and glial morphology when grown an additional week in Neurobasal/B27 without bFGF. We conclude that juxtacrine interaction between NSCs and MSCs combined with soluble factors released from NSCs are important for generation of neural-like cells from bone-marrow-derived adherent MSCs.     

Critical role of Erythrocyte Binding-Like protein of the rodent malaria parasite Plasmodium yoelii to establish an irreversible connection with the erythrocyte during invasion

Plasmodium malaria parasites multiply within erythrocytes and possess a repertoire of proteins whose function is to recognize and invade these vertebrate host cells. One such protein involved in erythrocyte invasion is the micronemal protein, Erythrocyte Binding-Like (EBL), which has been studied as a potential target of vaccine development in Plasmodium vivax (PvDBP) and Plasmodium falciparum (EBA-175). In the rodent malaria parasite model Plasmodium yoelii, specific substitutions in the EBL regions responsible for intracellular trafficking (17XL parasite line) or receptor recognition (17X1.1pp. parasite line), paradoxically increase invasion ability and virulence rather than abolish EBL function. Attempts to disrupt the ebl gene locus in the 17XL and 17XNL lines were unsuccessful, suggesting EBL essentiality. To understand the mechanisms behind these potentially conflicting outcomes, we generated 17XL-based transfectants in which ebl expression is suppressed with anhydrotetracycline (ATc) and investigated merozoite behavior during erythrocyte invasion. In the absence of ATc, EBL was secreted to the merozoite surface, whereas following ATc administration parasitemia was negligible in vivo. Merozoites lacking EBL were unable to invade erythrocytes in vitro, indicating that EBL has a critical role for erythrocyte invasion. Quantitative time-lapse imaging revealed that with ATc administration a significant number of merozoites were detached from the erythrocyte after the erythrocyte deformation event and no echinocytosis was observed, indicating that EBL is required for merozoites to establish an irreversible connection with erythrocytes during invasion.     

Evaluation of a short-incubation-time small-volume radiocarbon-uptake algal toxicity test

A short term small volume¹⁴C-assimilation algal toxicity test usingSelenastrum capricomutum CCAP 278/4 as test organism was used to determine the potential toxicity of a few selected compounds. Generally the sensitivity of the method increased with increasing exposure time to the substances tested. It was found that the method complied with the prerequisites of an acute test, because in most instances toxicity could be detected within 0.5 h exposure. Copper, cadmium, mercury and atrazine were the most toxic of the compounds tested.     

The Super-Spinner: A low cost animal cell culture bioreactor for the CO2 incubator

The production of small quantities of monoclonal antibodies and recombinant proteins was carried out using a new low cost production system, the Super Spinner. Into a 1 1 standard Duran^® flask a membrane stirrer equipped with a polypropylene hollow fiber membrane was installed to improve the oxygen supply by bubble-free aeration. The aeration was facilitated by using the CO₂ conditioned incubator gas, which was pumped through the membrane stirrer via a small membrane pump. The maximal oxygen transfer rate (OTR_max) of the Super Spinner was detected. For this purpose one spinner flask was equipped with an oxygen electrode. The OTR_max was measured by the dynamic method. The ratio of membrane length to culture volume was adapted corresponding to the oxygen uptake rate of the cells according to the desired cell density. A balanced nutrient supply resulted in an optimal formation and yield of products.     

The direction of cleavage waves and the regional variation in the duration of cleavage cycles on the dorsal side of the Xenopus laevis blastula

Summary The animal and the dorsal side of five embryos of Xenopus laevis were studied in detail from the 7th to the 13th cleavage by means of time-lapse cinematography. At each cleavage the regionally ordered sequence of blastomere divisions is visible in the films as a cleavage wave, propagating about three times slower in the dorsal than in the animal view. In the dorsal view the waves run in an animal-vegetal direction, initially with a left-to-right deviation and in later cleavages converging on the region of the future blastopore. The lengthening of cleavage cycles begins at cycle 8 on the dorsal side, just above the future blastopore. From cycle 9 to 11 nearly equal lengthening occurs in each cycle at all animal-vegetal levels. In general, cycles lengthen a little more in median than in lateral sectors and a little more in right than in left sectors. Cycle 12 is longest in the sector above the future blastopore and shortest in the animal region. The results show that the initial pattern of a regionally ordered sequence of cleavage cycles of equal duration changes into a pattern of cycles of different durations as a result of gradual cycle lengthening, starting in the region just above the future blastopore and spreading in animal direction. The results are compared with data on the cleavage cycles of isolated blastomeres, and the possible relation with the induction of the mesoendoderm occurring during the stages studied is discussed.     

A comparison of primary production measurements using two laboratory systems with differences in light quality

Primary production measurements were carried out simultaneously, using two laboratory systems with different light conditions: (1) a'classical' incubator and (2) a Laboratory Scale Enclosure. The model used for calculating primary production (STEELE, 1965) does not correct for spectral changes caused by high phytoplankton biomass. In the incubator, light of almost all wavelengths decreased more or less according to the attenuation of total PhAR in water. In the LSE, high absorption was found of the blue light and some of the red light, which was due to the high sestonic concentration. The Steele function provided a good fit for both sets of data. The depth integrated gross production values derived from the simultaneous measurements were not significantly different.     

Relationship between pregnancy,embryo development,and sperm deoxyribonucleic acid fragmentation dynamics

The way the dynamics of DNA fragmentation affects the growth of embryos in real time, and effectiveness of infertility treatment using the ICSI procedure were determined in 148 couples treated with the ICSI technique. The percentage of sperm with fragmented DNA (known as the DNA fragmentation index [DFI]) in semen samples was determined at 3, 6 and 12 h. Embryo culture was assessed continuously during 12 h of observation monitoring.Statistically significant difference was found in DFI at 12 h and outcome of treatment. For the remaining time intervals, no statistically significant differences were noted. An analysis of relationship between the DFI dynamics over time at individual measurements and achievement of pregnancy, confirmed a statistically significant relationship between the rate measured at 6–12 h of observations of DFI changes (DFI 12 h%/h), and achieving pregnancy. Correlation was observed between DFI (during 0, 3, 6 and 12 h), the growth rate in DFI, and time of embryo development. A statistically significant relationship was found between the rate from the start to the end of observations of the DFI, and outcome of treatment.Intensity level regarding fragmentation of sperm DNA and its growth rate affected the time of embryo development in the ICSI procedure. The most significant prognostic factor for achieving pregnancy was intensification of sperm DNA fragmentation after 12 h.     

Investigation of morphological change of Aulacoseira baicalensis using a small desktop incubator controlling light and temperature

A desktop incubator with temperature control over the range 1–20°C (±0.5°C) was designed to hold two microtitre plates. Illumination of individual wells in the plate was by a matrix of 96 light-emitting diodes, whose intensity, period and pulsation could be controlled individually in each of 12 rows of eight chambers. The incubator was used to test how the length of light period affected cell length of the planktonic diatom Aulacoseira baicalensis, which only grows below 4°C. Short cells (mean length 26 µm) were formed under a 8-h : 16-h light–dark cycle, intermediate cells (mean length 35 µm) under a 4-h : 20-h light–dark cycle and longer cells (mean 38 µm) under a 2-h : 22-h light–dark cycle. These were similar to changes in mean cell length from 28 to 49 µm found in Lake Baikal during a decrease in light period caused by increased convective mixing from 15 m under-ice to 94 m after ice break-up. The laboratory experiments confirmed that decreasing light period was the environmental cue that initiated the production of long cells that then developed into resting stages.     

Time-lapse videography of human oocytes following intracytoplasmic sperm injection: Events up to the first cleavage division

A total of 341 fertilized and 37 unfertilized oocytes from 63 intracytoplasmic sperm injection (ICSI) treatment cycles were included for retrospective assessment using the Embryoscope™ time-lapse video system. The second polar body (pb2) extrusion occurred at 2.9 ± 0.1 h (range 0.70–10.15 h) relative to sperm injection. All oocytes reduced in size following sperm injection (p < 0.05) with shrinkage ceasing after 2 h in the unfertilized and at pb2 extrusion in the fertilized oocytes. Pb2 extrusion was significantly delayed for women aged >38 years compared to those <35 years (3.4 ± 0.2 vs. 2.8 ± 0.1, p < 0.01) or 35–38 years (3.4 ± 0.2 vs. 2.8 ± 0.1, p < 0.01), but timing was not related to the Day 3 morphological grades (1–4) of subsequent embryos (2.9 ± 0.1, 2.9 ± 0.1, 2.8 ± 0.2 and 3.0 ± 0.1; p > 0.05 respectively). A shorter time of first cleavage division relative to either sperm injection or pb2 extrusion is associated with both top grade (AUC = 0.596 or 0.601, p = 0.006 or 0.004) and usable embryos (AUC = 0.638 or 0.632, p = 0.000 respectively) on Day 3. In summary, (i) pb2 of human oocytes extrudes at various times following sperm injection, (ii) the timing of pb2 extrusion is significantly delayed when female age >38 years, but not related to subsequent embryo development, (iii) all human oocytes reduce in size following sperm injection, (iv) completion of pb2 extrusion in the fertilized oocytes is a pivotal event in terminating shrinkage of the vitellus, and (v) time to first cleavage division either from sperm injection or pb2 extrusion is a significant predictive marker for embryo quality on Day 3.