B6CBF1 Mice Research Articles

Nitric oxide modulates mitochondrial activity and apoptosis through protein S-nitrosylation for preimplantation embryo development

Previous studies reported that patients with endometriosis had excess nitric oxide (NO) in the reproductive tract and poor embryo development in IVF cycles. This study aims to elucidate the effects of NO on early embryo development. Zygotes from superovulated B6CBF1 mice were cultured to blastocysts in a variety of media. Sodium nitroprusside (SNP) and N(G)-nitro-L-arginine (LNA) were added to the culture medium as a NO donor and a NO synthase inhibitor, respectively. The localization and fluorescence intensity of S-nitrosylated (SNO) proteins within 2-cell stage embryos were analyzed with confocal microscopy. Apoptosis and ATP production in the blastocysts were measured. Subsequent to NO exposure, the SNO proteins mainly colocalized with the mitochondria and endoplasmic reticulum and the intensity of SNO proteins increased. The addition of a quanylate cyclase inhibitor and a cyclic GMP mimic agent induced nonsignificant changes in SNO proteins, whereas addition of a superoxide scavenger or a reduced form of glutathione rescued the embryos from the effects of NO. However, superoxide scavenger supplementation resulted in decreased blastocyst ATP production. Elevated NO exerts deleterious effects on embryo development, possibly through protein S-nitrosylation in the mitochondria and endoplasmic reticulum. Including glutathione as a component in the culture medium might counteract this effect.

Cyclic QDE peptide increases fertilization rates and provides healthy pups in mouse

To evaluate the effects of cyclic QDE peptide (cQDE), derived from sperm fertilin beta (ADAM2), in mouse in vitro fertilization (IVF) and its harmlessness on pups after embryo transfer. Prospective in vivo and in vitro study in mice. Murine model in an academic research environment in France. Normal B6CBF1 mice. Indirect immunofluorescence was used to evaluate cQDE binding to oolemma. The IVF assays were run in presence of the species-specific tripeptide (cQDE at 100 microM), and embryos were transferred in pseudopregnant mice. Controls were obtained by natural mating and IVF in regular medium followed by embryo transfer. Evaluation of fertilization, litter size, pup fertility and health. Biotinylated cQDE peptide binds to oocyte plasma membrane and increases gamete fusion in cumulus-intact IVF assay. Litter size as well as pup development after embryo transfer showed no statistically significant differences compared with controls. Pups born from embryos that were incubated with the peptide were as healthy and normally fertile as control mice over at least three generations. Cyclic QDE is harmless and improves mouse IVF pregnancy rates. A randomized prospective clinical trial to evaluate the beneficial effect of cyclic FEE on human IVF is required before its clinical use.

Culture of Zygotes Increases p53 Expression in B6 Mouse Embryos, which Reduces Embryo Viability1

The expression of TRP53 in blastocysts that had been cultured from the zygote stage in vitro for 90 h was compared with that in blastocysts collected from the uterus in C57BL6 (B6) and in F1 hybrid (B6CBF1) strain mice. In both strains, there was little TRP53 detected in blastocysts collected from the uterus. There was some increased expression in cultured embryos from B6CBF1 mice and marked increased expression in cultured B6 blastocysts. In cultured B6 embryos, there was obvious accumulation of TRP53 within the nuclear region of embryonic cells. Cultured B6 zygotes had significantly poorer rates of blastocyst formation and of capacity to undergo implantation or form viable fetuses than cultured zygotes from B6CBF1 mice or B6 blastocysts collected from the uterus. Trp53-/- zygotes (B6 background) were significantly more likely to form blastocysts than sibling wild-type embryos, with Trp53+/- embryos having an intermediate level of viability (P<0.01). On transfer of blastocysts to recipient females, Trp53-/- blastocysts were more likely to form viable fetuses than wild-type or heterozygous sibling blastocysts when the embryos resulted from culture of zygotes (P<0.001). This shift in viability did not occur when embryos were only subjected to 24 h of culture from the compacted embryo stage. Culture in vitro in the B6 strain caused a marked increase in the expression and nuclear accumulation of TRP53. This expression was a significant cause of the loss of viability that occurs on culture of zygotes from this strain in vitro.

Follicle-Stimulating Hormone Affects Metaphase I Chromosome Alignment and Increases Aneuploidy in Mouse Oocytes Matured in Vitro1

Follicle-Stimulating Hormone (FSH) at a wide range of doses is routinely added to culture media during in vitro maturation (IVM) of oocytes, but the effects on oocyte health are unclear. The suggestion that superovulation may cause aneuploidy and fetal abnormalities prompted us to study the potential role of FSH in the genesis of chromosomal abnormalities during meiosis I. Mouse cumulus-oocyte complexes (COCs) isolated from the antral follicles of unprimed, sexually immature B6CBF1 mice were cultured in increasing concentrations of FSH. Following culture, matured oocytes were isolated, spread, stained with DAPI, and the numbers of chromosomes counted. Significantly increased aneuploidy, arising during the first meiotic division, was observed in metaphase II oocytes matured in higher concentrations of FSH (> or =20 ng/ml). The effect of FSH on spindle morphology and chromosome alignment during metaphase I was then explored using immunocytochemistry and three-dimensional reconstruction of confocal sections. High FSH had no effect on gross spindle morphology but did alter chromosome congression during prometaphase and metaphase, with the spread of chromosomes across the spindle at this time being significantly greater in oocytes cultured in 2000 ng/ml compared with 2 ng/ml FSH. Analysis of three-dimensional reconstructions of spindles in oocytes matured in 2000 ng/ml FSH shows that chromosomes are more scattered and farther apart than they are following maturation in 2 ng/ml FSH. These results demonstrate that exposure to high levels of FSH during IVM can accelerate nuclear maturation and induce chromosomal abnormalities and highlights the importance of the judicious use of FSH during IVM.

Anxiety-related behaviour in C57BL/6↔BALB/c chimeric mice

In a previous study on anxiety-related behaviours of the genetically and behaviourally distant inbred mouse strains C57BL/6 and BALB/c using the Elevated plus-maze (EPM) and Open-field (OF) apparatuses, we identified a number of variables, the factorial scores of which were grouped by principal component analysis (PCA) into factors specifically describing each inbred strain [4]. We have now studied the effect of C57BL/6 and BALB/c haploid sets of genes on this behaviour by comparing EPM and OF variables of C57BL/6 and BALB/c versus C57BL/6×BALB/c F1 hybrids (B6CBF1) and chimeric C57BL/6×BALB/c (CHIM) mice. CHIM mice were made by embryo aggregation and the chimerism degree of their brain was inferred from coat black/white distribution. Discriminant analysis of EPM and OF factorial scores of C57BL/6, BALB/c and CHIM mice showed that CHIM mice with an exceeding (≥80%) C57BL/6 or BALB/c coat component had behaviours similar to those of the predominant strain, whereas CHIM mice with intermediate chimerism differed from both inbred strains. Additional MANOVA analysis showed that the anxiety behaviour of CHIM mice with intermediate chimerism was similar to that of B6CBF1 mice as for factors not describing the inbred strains, including a motor activity mostly limited to protected areas, with attempts to approach the anxiogenic areas while processing/storing the external information. We conclude that the balanced presence of both C57BL/6 and BALB/c genetic backgrounds, either when carried by the same cell or by different cells, gives rise to a novel stress coping strategy described by factors different from those of the inbred strains.

Effects of varying gonadotrophin dose and timing on antrum formation and ovulation efficiency of mouse follicles in vitro.

This study tested factors affecting mouse follicle growth in vitro, to determine end-points marking follicle function in vitro. Pre-antral follicles (mean 137 microm) from B6CBF1 mice were cultured in a substrate-adherent system for < or = 14 days. FSH (0-1000 mIU/ml) day of HCG (1.5 IU/ml days 9-14) protein supplement [fetal calf serum (FCS) (x2) mouse serum (x2) hypogonadal (hpg) mouse serum or human serum albumin (HSA)] were varied. Follicle survival timing of antrum formation incidence of ovulation within 16,24,40,48 h of HCG oocyte growth were assessed. FSH (100 mIU/ml) produced the best antral development (P < 0.001 versus 10 and 1000 mIU/ml). Antra were observed from day 5. Transient antra formed occasionally in the absence of FSH. By 14 days significant senescence had occurred (P < 0.001) but the proportion of follicles ovulating within 16 h of HCG declined from day 9 onwards indicating this to be a more sensitive marker of follicle responsiveness. Optimal growth occurred in 5% FCS (x2) or hpg mouse serum although fewer follicles ovulated in hpg serum (P < 0.05). No normal growth occurred in normal mouse serum (x2) or HSA. Oocytes grew to full size within 9 days with 100 mIU/ml FSH FCS. These data provide sensitive end-points for assessing follicle growth in vitro.

Parthenogenetic Activation of Mouse Oocytes by Strontium.

The purposes of this study were to determine the optimal conditions for inducing parthenogenetic activation by strontium and to evaluate the developmental ability of the activated oocytes in mice. MII oocytes collected from B6CBF1 and CD-1 mice were cultured for 2-60 min in medium containing 1.7 mM strontium. The proportion of oocytes activated increased in a time-dependent manner, with an apparent difference between the B6CBF1 and CD-1 strains in sensitivity to strontium. In oocytes derived from B6CBF1 mice more than 90% of oocytes were activated by the treatment with strontium for 30 min, whereas more than 80% of the oocytes from CD-1 mice were activated by the treatment for 5 min. The majority of oocytes formed the second polar body and a single pronucleus in both cases. Of the parthenogenetically activated 1-cell embryos with the haploid genome, around 39% developed to the blastocyst stage in the B6CBF1, and in contrast the developmental ability was low (11%) in the CD-1 strain. The ability to develop to blastocysts was significantly improved in diploid parthenogenetic embryos of both the strains; 93% and 58% were developed to the blastocyst stage, respectively. After transfer of the diploid parthenogenetic embryos to recipient mice, 11% of the embryos developed to day 10 of gestation. The record of intracytoplasmic Ca2+ concentrations showed that the exposure of oocytes to medium containing 1.7 mM strontium induced repetitive intracellular Ca2+ transients. These data clearly showed that strontium is a potent stimulus for inducing parthenogenetic activation and supporting in vitro and in vivo development in mouse oocytes.

Radioprotection by Polyethylene Glycol-Protein Complexes in Mice

Polyethylene glycol of about 5000 D was activated with cyanuric chloride, and the activated compound was complexed to each of three proteins. Polyethylene glycol-superoxide dismutase and polyethylene glycol-catalase were each radioprotectants when administered prophylactically to female B6CBF1 mice before irradiation. The dose reduction factor for these mice was 1.2 when 5000 units of polyethylene glycol-catalase was administered before 60Co irradiation. Female B6CBF1 mice administered prophylactic intravenous injections of catalase, polyethylene glycol-albumin, or heat-denatured polyethylene glycol-catalase had survival rates similar to phosphate-buffered saline-injected control mice following 60Co irradiation. Polyethylene glycol-superoxide dismutase and polyethylene glycol-catalase have radioprotective activity in B6CBF1 mice, which appears to depend in part on enzymatic activities of the complex. However, no radioprotective effect was observed in male C57BL/6 mice injected with each polyethylene glycol-protein complex at either 3 or 24 hr before irradiation. The mechanism for radioprotection by these complexes may depend in part on other factors.

Reduced toxicity of irradiated endotoxin in mice compromised by irradiation, tumors, or infections.

Endotoxin (LPS) exposed to 100–200 kGy of γ radiation no longer induced rabbit platelet aggregation in vitro and it had a reduced ability to cause mitogenesis of B-lymphocytes from C57BL/6 mice. When tested in B6CBF1 mice exposed to 10 Gy 60Co radiation 3 or 7 days previously, 300 μg of unirradiated LPS, which caused no mortality in unirradiated mice, killed 60 and 44% of the mice when given on days 3 or 7 post-radiation, respectively. Similarly, 60% of C57BL/6 mice engrafted with 5 × 106 Lewis lung carcinoma cells 3 days before challenge with 300 μg of normal LPS died, but no animals given irradiated LPS died. One hundred micrograms of either LPS preparation caused 80 to 100% mortality of NMRI mice when given simultaneously with 105 live K. pneumoniae. No deaths were obtained if 50 μg of irradiated LPS were administered with 105 bacteria, but 50 μg of unirradiated LPS caused 100% mortality. Similar mortalities were obtained when 100 μg of either toxin preparation were given 24 hr postinfection, but these were lower than those obtained with the simultaneous challenge. Endotoxin given 24 hr before 105 organisms was less toxic than that given at the other times, and irradiated LPS caused fewer deaths than did unirradiated LPS. Similar results were seen when 106 cells were used to challenge mice. Treatment 24 hr before challenge caused 70% mortality with unirradiated LPS compared to 30% mortality with irradiated LPS. All mice challenged with 107 bacteria 24 hr after treatment with saline or unirradiated LPS died by 72 hr postinfection, but only 50% mortality was obtained in mice given irradiated LPS. Furthermore, none of these mice died until after 72 hr. Our data indicate that under some circumstances irradiated LPS can be more safely administered to animals with compromised resistance than can unirrudiated LPS.

Plasma attenuation of endotoxin toxicity in irradiated and tumor-bearing mice

We tested the hypothesis that citrated plasma from normal or irradiated mice and from normal rabbits can protect mice against endotoxin-induced lethality. In contrast to saline-endotoxin mixtures, challenge i.p. with 0.3 mg of Salmonella typhi endotoxin mixed with 1 ml of plasma from either normal or irradiated donors was not lethal for B6CBF1 mice irradiated 7 days previously with 1000 rads [ 60Co]. In other experiments unirradiated mice were simultaneously inoculated i.p. with 0.8 mg of endotoxin and 1 ml of saline or normal rabbit plasma. This plasma also protected recipient animals challenged with endotoxin. Furthermore, rabbit plasma protected C57BL/6 mice whose sensitivity to 0.3 mg of endotoxin was enhanced due to implantation of the animals with a 3LL carcinoma 3 days previously. The evidence thus obtained confirms the hypothesis that plasma interferes with the lethal effects of the endotoxin.

Association of leukopenia and intestinal permeability with radiation-induced sensitivity to endotoxin

Sensitivity to the lethal effects of endotoxin is increased after exposure to ionizing radiation in the hematopoietic death range. We hypothesized that leukopenia and altered intestinal permeability may be causative factors for increased sensitivity to endotoxin after irradiation. The presence of leukocytes and platelets was correlated with sensitivity of male B6CBF1 mice to 0.25 mg of Salmonella typhosa endotoxin administered intraperitoneally. This study was conducted over the 10-day period following irradiation (1000 rads Co-60) and the beginning of deaths due to radiation alone. These data were then associated with the status of tight junction barriers (zonula occludens) between epithelial cells of the ileum. A biphasic pattern of sensitivity to endotoxin was observed in irradiated mice. Mice were resistant to endotoxin through day 2 after radiation, but sensitivity increased greatly (80% mortality) by day 3. Resistance increased by day 6 (6.7% mortality) and then dropped again by days 9 and 10 (100% mortality). Leukocyte numbers decreased 91% by day 2 (from 10,880 to 1,020 per mm 3) and further by day 3 (300 per mm 3). Leukopenia persisted through the duration of the experiment. Platelets began to decrease in number by day 5, and levels continued to drop until day 9. Disruption of some intestinal tight junctions was observed on days 1–5 after radiation. Junctional repair was evident by day 5. Repair was noted as extensive junctional elements on ablumenal membrane fracture faces. A combination of leukopenia and leakage of endotoxin from the intestine may account for increased sensitivity to endotoxin seen at day 3. Repair of the intestinal permeability barrier on day 5 coincided with reduced sensitivity to endotoxin. Later (7–10 days) bacteremia was present in host tissues, and mortality due to challenge with endotoxin was again increased. Disruption of some tight junctional barriers was seen again on days 9 and 10 after radiation.

Biologic properties of bacterial lipopolysaccharides treated with chromium chloride

Addition of small amounts of chromium chloride to a saline suspension of Salmonella typhosa lipopolysaccharide (LPD; Difco) caused a marked reduction in several of the biologic activities of this substance including toxicity, B-cell mitogenicity, plasma colony-stimulating activity (CSA), radioprotective effect, and induction of the dermal Shwartzman reaction. Nevertheless, LPS treated with chromium chloride was found to be at least as effective as untreated LPS in enhancing resistance of B6CBF1 mice to the lethal effects of Klebsiella pneumoniae infection.

Mitigation of graft-versus-host disease in lethally irradiated mice grafted with spleen cells adherent to glass beads.

Murine spleen cells were separated on the basis of adherence to glass beads into distinct subpopulations that differ in their ability to produce acute graft-versus-host disease (GVHD). Nonadherent CBA spleen cells produce acute GVHD in 6-10 days in lethally irradiated (C57BL/6 X CBA)F1 mice as do unfractionated spleen cells. Spleen cells which are adherent to glass beads, however, enable 71% of the mice to survive without symptomatology of acute GVHD. The low proliferative response of these cells to phytohemagglutinin (PHA) correlated with the mitigated GVHD seen in animals grafted with this fraction. Proliferative cells as determined by the spleen colony assay and the in vitro agar colony-forming assay are present in this fraction as are cells responsive to mitogenic stimulation with lipopolysaccharide (LPS). B6CBF1 mice grafted with CBA adherent cells exhibit a gradual return over a period of 5 months to normal PHA and LPS stimulation levels as shown by splenic cell responses of these mice to mitogens. Surviving mice grafted with adherent cells were chimeric as determined by electrophoretic hemoglobin pattern analysis and serial bone marrow transplantation.

Aseptic Endotoxemia in Radiation Injury and Graft-vs-Host Disease

to clear endotoxin from the circulation and detoxify it in the liver of immunosuppressed animals. To determine if intestinal endotoxin may contribute to pathogenesis in immunosuppressed individuals, B6CBF1 mice were exposed to 850 rads X-rays, and transplanted with allogeneic CBA spleen cells. Aseptic endotoxemia, detected with the limulus lysate assay, was found at 24 and 72 hr postirradiation in livers of mice irradiated only. Contrary to this, endotoxin was not found in mice on days 5, 7, and 8 after X-irradiation. Mice died between 11 and 13 days after radiation exposure, at which time bacteria and endotoxin were detected in the liver. In contrast, endotoxin was demonstrable on days 1 and 5 in mice undergoing GVHD. Mice receiving allogeneic grafts after 850 rads survived only 7 days, while gram-negative organisms were detected frequently in liver fragments from 24 hr to day 7. By day 4, hepatosplenic localization of iv-injected [6ECr]endotoxin was reduced 50% in these animals and 2- to 3-fold increases in endotoxin levels were found in lung, kidney, heart, and brain. Hepatosplenic endotoxin concentration was not altered dramatically in mice receiving radiation alone. Endotoxin levels in other organs were not altered. Thus, endotoxin is present in detectable amounts in the liver of immunosuppressed mice.