ALBERT

Notes: Ten embryonic stem (ES) cell lines from mink blastocysts were isolated and characterized. All the lines had a normal diploid karyotype; of the ten lines studied, five had the XX and five had the XY constitution. Testing of the pluripotency of the ES-like cells demonstrated that (1) among four lines of genotype XX, an X was late-replicating in three; both Xs were active in about one-third of cells of line MES8, and analysis of glucose-6-phosphate dehydrogenase revealed no dosage compensation for the X-linked gene; (2) when cultured in suspension, the majority of lines were capable of forming “simple” embryoid bodies (EB), and two only showed the capacity for forming “cystic” multilayer EBs. However, formation of ectoderm or foci of yolk sac hematopoiesis, a feature of mouse ES ceils, was not observed in the “cystic” EB; (3) when cultured as a monolayer without feeder, the ES cells differentiated into either vimentin-positive fibroblast-like cells or cytokeratin-positive epithelial-like cells (less frequently); neural cells appeared in two lines; (4) when injected into athymic mice, only one of the four tested lines gave rise to tumors. These were fibrosarcomas composed of fibrobalst-like cells, with an admixture of smooth muscular elements and stray islets of epithelial tissue; (5) when the ES cells of line MES1 were injected into 102 blastocyst cavities and subsequently transplanted into foster mathers, we obtained 30 offspring. Analysis of the biochemical markers and coat color did not demonstrate the presence of chimaeras among offspring. Thus the cell lines derived from mink blastocysts are true ES cells. However, their pluripotential capacities are restricted. © 1992 Wiley-Liss, Inc.

Notes: We have used two monoclonal antibodies to demonstrate the presence and localization of actin in interphase and mitotic vegetative cells of the green alga Chlamydomonas reinhardtii. Commercially available monoclonal antibodies raised against smooth muscle actin (Lessard: Cell Motil. Cytoskeleton 10:349-362, 1988; Lin: Proc. Natl. Acad. Sci. USA 78:2335-2339, 1981) identify Chlamydomonasactin as a ∼43,000-Mr protein by Western immunoblot procedures. In an earlier study, Detmers and coworkers (Cell Motil. 5:415-430, 1985) first identified Chlamydomonas actin using NBD-phallacidin and an antibody raised against Dictyostelium actin; they demonstrated that F-actin is localized in the fertilization tubule of mating gametes. Here, we show by immunofluorescence that vegetative Chlamydomonas cells have an array of actin that surrounds the nucleus in interphase cells and undergoes dramatic reorganization during mitosis and cytokinesis. This includes the following: reorganization of actin to the ante- rior of the cell during preprophase; the formation of a cruciate actin band in prophase; reorganization to a single anterior actin band in metaphase; rearrange- ment forming a focus of actin anterior to the metaphase plate; reextension of the actin band in anaphase; presence of actin in the forming cleavage furrow during telophase and cytokinesis; and finally reestablishment of the interphase actin array. The studies presented here do not allow us to discriminate between G and F-actin. None the less, our observations, demonstrating dynamic reorganization of actin during the cell cycle, suggest a role for actin that may include the movement of basal bodies toward the spindle poles in mitosis and the formation of the cleavage furrow during cytokinesis. © 1992 Wiley-Liss, Inc.

Notes: Our results show that an insulin-like growth factor binding protein, IGFBP-3, purified from rat serum, is an inhibitor of chick embryo fibroblast (CEF) growth. It abolished DNA synthesis in CEF stimulated by IGF-l as well as by human serum. Rat IGFBP-3 and IDF45 (an inhibitory diffusible factor secreted by mouse cells) had the same activities, confirming that they have an intrinsic capacity to inhibit serum stimulation and may be considered as growth inhibitors. Our data show that inhibition by IGFBP-3 of serum stimulation was not simply the result of its inhibition of IGF present in the serum: 1) While anti-I lgG was able to completely inhibit stimulation induced by added IGF-I, it did not decrease stimulation induced by 1% human serum. Anti-IGF-II IgG inhibited the stimulation induced by added IGF-II, but only 25% decreased the stimulation induced by 0.7% serum. The percent inhibition was not significantly increased when the concentration of serum was decreased to 0.2%, which induced 140% stimulation of DNA synthesis; 2) stimulation by 0.2% serum was much more inhibited by IGFBP-3 than by IgG anti IGF-II; 3) after separation of IGF-I and IGF-II from serum by chromatography of acidified serum proteins on BioGel P150, the remaining serum proteins (with a molecular mass greater than 45 kDa) which were depleted in IGF-I and -II (verified by RIA determination) still stimulated DNA synthesis, and this stimulation was 80% inhibited by IGFBP-3. © 1992 Wiley-Liss, Inc.

Notes: A method previously used in this laboratory for entrapment of tumor cells in alginate beads has been extended to provide a slow release delivery system for growth factors with known in vivo angiogenic activity. Protein growth factors were entrapped in alginate beads in amounts sufficient to cause incorporation of 3H-thymidine by COMMA-D cells in vitro, and in vivo neovascularization when injected subcutaneously into Balb/c mice. Entrapment of 125I-labelled growth factors showed that the amount of molecule entrapped in alginate beads may vary with the charge of the molecule. In vitro cell proliferation studies showed that entrapment in alginate beads may provide a slow-release system or a stabilizing environment for the protein. In some cases biological activity of the growth factor in solution was increased by the presence of control alginate beads. When alginate-entrapped growth factors were injected into Balb/c mice, induction of new blood vessels could be monitored qualitatively by macroscopic photography and assessed quantitatively by measuring the pooling of radiolabelled red blood cells at the experimental site. Subcutaneous injection of purified angiogenic factors not entrapped in alginate beads did not cause neovascularization. Diffusion of 125I-labelled growth factors from alginate beads in the animal showed that release in vivo may depend on the charge of the protein molecule. These results indicate that injection of purified molecules entrapped in alginate beads provides an effective localized and slow-release delivery of biologically active molecules. This delivery system may extend the time of effectiveness of biologically active molecules in vivo compared to direct injection without alginate entrapment. The method of entrapment and injection has potential for identifying active factors in tumor-induced angiogenesis and testing new compounds as modulators of neovascularization. © 1992 Wiley-Liss, Inc.

Notes: Endothelial cells (EC) are very responsive to the proinflammatory cytokine inter-leukin-1 (IL-1). EC are induced by IL-1 to secrete chemotactic factors and to increase expression of cell surface adhesion molecules leading to increased leukocyte adhesion. Activated EC turther contribute to the inflammatory response by secreting additional cytokines. IL-1 interacts with EC through high-affinity cell-surface receptors. However, the low number of receptors present on EC has made characterization difficult. Further, recent evidence has suggested diversity in the responses of EC from different regions of the vascular system. Interested in the effect of IL-1 on early atherosclerotic lesion formation, we have characterized the IL-1 receptors on human aortic endothelial cells (HAEC). Using a direct binding assay, we found that HAEC have 1,000-3,000 IL-1 receptors per cell and bind IL-1α with a Kd of 3.5 × 10-10 M. We found that a monoclonal antibody specific for the type I receptor completely blocks IL-1α binding. The blocking antibody also completely inhibits the IL-1 induced increase in intracellular adhesion molecule 1 (ICAM-1) expression by HAEC. Using solution hybridization and ribonuclease protection with an antisense probe, a sensitive method for detection of low abundance mRNA species we found that HAEC as well as human umbilical vein EC (HUVEC) have significant levels of mRNA for the type I IL-1 receptor. To test whether HAEC might also contain transcripts for the type II IL-1 receptor, we compared levels of mRNAs by polymerase chain reaction (PCR) amplification of cDNAs reverse-transcribed from total RNA. We found only transcripts for the type I receptor and not the type II receptor in HAEC. Based on this data, we conclude that aortic endothelial cells respond to IL-1 through the type I receptor. © 1992 Wiley-Liss, Inc.

Notes: Our goal was to investigate effects of long-term exposure to pulsed microwave radiation. The major emphasis was to expose a large sample of experimental animals throughout their lifetimes and to monitor them for effects on general health and longevity.An exposure facility was developed that enabled 200 rats to be maintained under specific-pathogen-free (SPF) conditions while housed individually in circularly-polarized waveguides. The exposure facility consisted of two rooms, each containing 50 active waveguides and 50 waveguides for sham (control) exposures. The experimental rats were exposed to 2,450-MHz pulsed microwaves at 800 pps with a 10-μs pulse width. The pulsed microwaves were square-wave modulated at 8-Hz. Whole body calorimetry, thermographic analysis, and power-meter analysis indicated that microwaves delivered at 0.144 W to each exposure waveguide resulted in an average specific absorption rate (SAR) that ranged from 0.4 W/kg for a 200-g rat to 0.15 W/kg for an 800-g rat.Two hundred male, Sprague-Dawley rats were assigned in equal numbers to radiation-exposure and sham-exposure conditions. Exposure began at 8 weeks of age and continued daily, 21.5 h/day, for 25 months. Animals were bled at regular intervals and blood samples were analyzed for serum chemistries, hematological values, protein electrophoretic patterns, thyroxine, and plasma corticosterone levels. In addition to daily measures of body mass, food and water consumption by all animals, O2 consumption and CO2 production were periodically measured in a sub-sample (N=18) of each group. Activity was assessed in an open-field apparatus at regular intervals throughout the study. After 13 months, 10 rats from each group were euthanatized to test for immunological competence and to permit whole-body analysis, as well as gross and histopathological examinations. At the end of 25 months, the survivors (11 sham-exposed and 12 radiation-exposed rats) were euthanatized for similar analyses. The other 157 animals were examined histopathologically when they died spontaneously or were terminated in extremis.

Notes: Epon labeled with bromide was used to embed ejaculated and freeze-thawed spermatozoa, with the hypothesis that it replaces most of cell water. Image analysis of relative contrasts between sperm nuclei and the surrounding medium revealed that when used in low concentrations, bromide is mostly absorbed to the nuclear structures. For higher concentrations, the chromatin is saturated, and the increase in contrast can be used to calculate relative differences in the hydration of nuclei. Boar sperm nuclei are more hydrated after freeze-thawing than before. © 1992 Wiley-Liss, Inc.

Notes: This study was designed to determine what effect electropulse parameters would have on rate of fusion, lysis, and embryo viability when embryos were subjected to electrofusion treatment in nonelectrolyte or electrolyte pulse media. Previous experiments have shown electrolyte medium (i.e., phosphate-buffered saline; PBS) to have a positive effect on electric pulse-induced murine oocyte activation. In addition, these results also indicated that pulse media containing 0.9 mM Ca2+ induced a dramatic increase in the rate of murine oocyte activation compared with oocytes pulsed in media containing 0.0 or 0.05 mM Ca2+. Pronuclear or two-cell-stage embryos were obtained from superovulated prepubertal randomly bred Swiss (albino) female mice. Embryos were randomly assigned to three nonelectrolyte and three electrolyte treatment media. Nonelectrolyte media consisted of 0.3 M mannitol (T1), 0.3 M mannitol + 0.05 mM CaCl2 (T2), and 0.3 M mannitol + 0.9 mM CaCl2 (T3). Electrolyte media consisted of Ca2+ -free PBS (T4), PBS containing 0.05 mM CaCl2 (T5), and PBS containing 0.9 mM CaCl2 (T6). Three experiments were carried out; the objective of the first was to determine the rate of fusion and rate of lysis in murine two-cell embryos placed in the two types of (0.3 M mannitol, T1-T3; and PBS, T4-T6) fusion media and subjected to a fusion procedure (3 V, 5 sec AC alignment pulse, followed by a 1.56 kV · cm-1, 99 μsec DC fusion pulse). Control two-cell embryos were placed in T1 for 2 min and did not receive a fusion pulse. The objective of experiment 2 was to evaluate the development of pronuclear stage embryos to the blastocyst stage in vitro after receiving the fusion pulse in T1, T3, and T6. Control embryos were not subjected to fusion treatment. In experiment 3, T3 and T6 were used to test the rate of fusion and rate of development for pronuclear-stage karyoplasts fused to enucleated pronuclear-stage cytoplasts. Micromanipulations were carried out, and all pronuclear embryos were placed into culture and the number developing to the four-cell stage and subsequently to the blastocyst stage was assessed. Two-cell-stage embryos pulsed in T5 and T6 exhibited significantly higher rates of fusion (96.9 and 92.9%, respectively) compared with T1 (83.7%), T2 (84.7%), or T3 (77.6%) ( P 〉 0.05). There was no significant difference (P 〉 0.05) in rate of lysis between any of the treatment groups. Pronuclear-stage embryos placed in T1, T3, and T6 and subjected to the electrofusion procedure resulted in 55.5%, 52.3%, 50.0%, and 54.2% of T1, T3, T6, and control embryos developing to the blastocyst stage, respectively. There was no difference (P 〉 0.05) between treatment groups in development to the blastocyst stage after 98 hr in culture. Finally, nuclear transplant results indicated no difference in the rate of pronuclear karyoplast-cytoplast fusion between T3 (79.7%) and electrolyte T6 (85.5%) media (P 〉 0.05). There was also no difference in the rate of development to the four-cell stage (78% vs. 72.9%) or blastocyst stage (59.3% vs. 54.2%, T3 and T6, respectively) (P 〉 0.05). However, a difference was observed in rate of development to the four-cell stage and blastocyst stage between nonpulsed control pronuclear stage embryos and T6-treated karyoplast-cytoplast constructs (86.3% vs. 72.9% and 68.5% vs. 54.2%, respectively) (P 〈 0.05). In addition, there was no difference in the rate of lysis observed between T3, T6, and control embryos. These data indicate that the application of a 3 V, 5 sec AC alignment pulse prior to a single 1.56 kV · cm-1 DC fusion pulse to electrolyte PBS results in successful fusion of murine two-cell blastomeres at a rate equal to that of nonelectrolyte 0.3 M mannitol. In vitro development of pronuclear-stage or fused pronuclear transferred karyoplast-cytoplasts after an AC alignment pulse followed by a DC fusion pulse in either 0.3 M mannitol containing 0.9 mM Ca2+ or PB1 does not adversely effect early murine embryonic development in vitro.

Notes: We have previously reported that a heterodimeric protein secreted by rat Sertoli cells is antigenically related to a protein associated with outer dense fibers of the sperm tail. Therefore, we have explored the possibility that Sertoli and spermatogenic cells express a similar gene encoding a homologous protein. A Sertoli cell heterodimeric protein cDNA probe recognizes specific mRNA in pachytene and round spermatids fractionated by centrifugal elutriation; however, this specific mRNA was less prominent than in cultured Sertoli cells. In agreement with these observations, in situ hybridization experiments show that Sertoli cells are predominantly engaged in active heterodimeric protein mRNA synthesis, while meiotic prophase spermatocytes and spermatids also show significant but less abundant specific mRNA. Immunoblotting experiments demonstrate that, while Sertoli cells synthesize a heterodimeric protein consisting of two disulfidelinked components with molecular masses of 45 and 35 kD, both primary spermatocytes and round spermatids synthesize single 30 kD monomers not associated by disulfide linkage but recognized by antisera to Sertoli cell heterodimeric protein Immunoblotting and immunogold electron microscopic studies show that antisera to Sertoli cell heterodimeric protein recognize a protein associated with outer dense fibers. This immunoreactivity was abolished by a 5-min pronase treatment, without affecting the integrity of outer dense fibers. Results of this study and previous studies demonstrate that both Sertoli and spermatogenic cells express a similar gene and that an antigenically related product encoded by this gene becomes associated with outer dense fibers during their assembly at spermiogenesis. © 1992 Wiley-Liss, Inc.

Notes: These experiments were designed to monitor influx of extracellular Ca2+ into the murine ooplasm following a 1.56 kV · cm-1 direct current (DC) electrofusion pulse and subsequently to determine its effect on rate of activation. Pulse media consisted of nonelectrolyte (0.3 M mannitol) and electrolyte (phosphatebuffered saline; PBS) media each containing 0.0, 0.05, or 0.9 mM Ca2+ (groups T1-T3 and T4-T6, respectively). Cumulus-free oocytes were incubated in 100 μl drops of PBS containing 2 μM of the calcium indicator fluo-3/AM for 60 min at 37°C. Fluo-3/AM-loaded oocytes were equilibrated for 7 min in assigned treatment media (T1-T6) prior to application of DC pulse. Change in fluorescent intensity was monitored for 6.5 min after DC pulse by photon counting spectrofluorometry. Fluorometric measurements demonstrate a dramatic rise in intracellular free Ca2+ (Ca2+i) following DC pulse is associated with Ca2+ ion concentration in the pulse medium. Significantly (P 〈 0.01) higher Ca2+i levels were observed when 0.9 mM Ca2+ was added to the pulse medium (T3 and T6) compared with pulse medium containing lower Ca2+ ion concentrations (T1, T2, T4, and T5; P 〉 0.05). Differences (P 〈 0.01) were observed in peak Ca2+i levels 18 sec after pulse with mean percent change in fluorescence of 5.1%a, 33.9%b, 112.7%c, 1.2%a, 9.3%a, and 99.9%c for T1-T6, respectively (values with different superscripts are significantly different at P 〈 0.01). Increased oocyte membrane permeability to Ca2+ ion after DC pulse was observed for a minimum of 5 min after delivery of the 1.56kV · cm-1 pulse. Nonloaded oocytes receiving the same pulse treatments were cultured in 100 μl drops of Whitten's medium for 8 h prior to evaluation of oocyte activation. Activation was defined as the formation of one pronucleus and one polar body, two or more pronuclei, or two cells each containing a nuclear structure. Activation rates of 28.2%b, 31.1%b, 70.4%d, 35.6%b, 57.3%c, 71.8%d 8.7%a (P 〈 0.01) were observed for T1-T6 and control nonpulsed oocytes, respectively. These data demonstrate that supplementation of 0.9 mM of Ca2+ ion to pulse medium results in a dramatic rise in Ca2+i after DC pulse. This influx of Ca2+ ion has a positive effect on oocyte activation rate.