Morphological appearance of the cryopreserved mouse blastocyst as a tool to identify the type of cryoinjury

BACKGROUND: If it were possible to deduce the mechanism of injury in cryopreserved embryos by their appearance, it would help to optimize cryopreservation protocols. METHODS: Mouse blastocysts were treated so that they were damaged by the six types of cryoinjuries listed below, and their appearance was observed at recovery in sucrose solution and a modified phosphate-buffered saline (PB1), and after culture for 1 and 24 h. RESULTS: (i) Intracellular ice: the embryos shrank normally in sucrose solution, but swelled in PB1 and collapsed after culture. (ii) Chemical toxicity of the cryoprotectant: the embryos looked normal in sucrose solution and PB1. After 1 h of culture, however, the blastomeres showed decompaction and degenerated thereafter. If the toxicity was extremely high, embryos looked nearly normal in PB1, but the surface of the cytoplasm was wrinkled as if they were "fixed". (iii) Osmotic swelling: the embryos looked normal in PB1, but after culture they shrank. (iv) Osmotic shrinkage: the embryos swelled in PB1, and then collapsed. (v) Fracture damage: the zona pellucida of the embryos was dissected. (vi) Extracellular ice: the zona of the embryos was elongated. CONCLUSIONS: It was often possible to deduce the type of injury that had occurred in cryopreserved embryos from their appearance at recovery and during subsequent culture. This may help to improve cryopreservation protocols for embryos of many species, including man.     

The plane of first cleavage is not related to the distribution of sperm components in the mouse

BACKGROUND: Marking experiments using phytohaemagglutinin (PHA) and Concanavalin A (ConA) have suggested that the first cleavage plane is related to the point of sperm entry. Because of concerns about the specificity of lectin binding, the distribution of sperm components has been investigated directly. METHODS AND RESULTS: The sperm tail could be identified in cleaving zygotes and early 2-cell stages following their permeabilization and exposure to Oregon Green Paclitaxel. At neither stage did the anterior end of the tail, which lies initially at the site of sperm entry, bear a consistent relationship to the first cleavage plane, even when it had clearly retained its original location. Moreover, using artificial insemination with MitoTracker-labelled sperm, the midpiece was found to remain associated with anterior end of the tail through to the 2-cell stage. Lectins showed no discernible binding to the fertilization cone of mechanically denuded zygotes and very strong binding to the zona pellucida. Moreover, after general labelling of zygotes with either ConA or PHA, persisting surface lectin tended to be concentrated towards the cleavage plane. CONCLUSION: The present findings challenge the claim that the sperm specifies the plane of first cleavage, and also question the methodology on which it was based.     

Prophylaxis and treatment of influenza encephalitis in an experimental mouse model

A mouse model study using mouse brain-adapted influenza A virus was performed to establish the prophylaxis and treatment of influenza encephalitis and encephalopathy. All mice died after intranasal inoculation of the brain-adapted influenza A virus (H7N3), and the pathological findings indicated the presence of significant encephalitis. Viral antigen was also detected in the brain, both pathologically and virologically. By contrast, infected mice immunized with inactivated vaccine of the same strain did not lose weight, which is an indicator of the overall condition of the mice, and all of them survived. Similarly, antiserum treatment in the early period (0-1 day post-infection) resulted in 100% survival, and no pathological findings were observed in the brain. However, mice treated with antiserum 3 days post-infection showed encephalitis with viral antigens in both glial cells and neurocytes. Although amantadine treatment for 4 days delayed weight loss, it did not prevent death from encephalitis. These results show vaccination and early antiserum treatment to be highly effective, whereas 4-day treatment of amantadine was not very effective in treating or preventing influenza encephalitis. The life-prolonging effect of amantadine, however, suggests that use of amantadine together with other treatments may inhibit the progression of encephalitis.     

Pulmonary pathologies in pallid mice result from nonhematopoietic defects

Several single gene pigment mutants of inbred C57BL/6J mice display a triad of subcellular granule-associated defects: oculocutaneous pigment dilution, prolonged bleeding due to defects in platelet dense granules, and abnormal lysosomes. These features also characterize Hermansky-Pudlak Syndrome (HPS), making these mice relevant animal models for HPS. Mice of one mutant strain, pallid, in addition to the hallmark triad of signs, also exhibit age-dependent lung pathology. Respiratory system mechanics showed that the age-dependent histopathology of pallid mice was accompanied by a decrease in lung reactance. Furthermore, the possibility that pallid pulmonary pathology may result from persistent inflammation due to microhemorrhage owing to the platelet defect was examined. Hematopoietic reconstitution of pallid mice with marrow from normal C57BL/6J donors did not prevent the development of the pulmonary histopathology or respiratory system mechanics characteristic of the pallid genotype. Similarly, wild-type mice 12 months after engraftment with pallid marrow did not develop pallid-like pulmonary histopathology or respiratory system mechanics. Thus, pallid-associated pulmonary functional and structural pathologies are not linked to the marrow (bleeding) genotype, but instead are the result of an age-dependent process resulting from a defect(s) in one or more nonhematopoietic cell types.     

Changes in the expression of claudins and transepithelial electrical resistance of mouse Sertoli cells by Leydig cell coculture

In the testis, tight junctions (TJs) between adjacent Sertoli cells are important for the formation of blood-testis barrier (BTB). To verify the role of paracrine interactions between the Sertoli and Leydig cells in the structure and function of BTB in testis, the expression of claudin-1 and -11, and transepithelial electrical resistance (TER) of the mouse Sertoli cells were examined under the Leydig cell coculture. TER of Sertoli cell monolayer was significantly larger under the Leydig cell coculture in comparison with the control culture. Meanwhile, the expression of claudin-1 slightly decreased and claudin-11 significantly increased in the Sertoli cells in the Leydig cell coculture compared with control. Testosterone significantly increased claudin-11 expression in cultured Sertoli cells. Taken together, it suggested that Leydig cell coculture changed the structure and functions of inter-Sertoli TJs in vitro. Interactions between Leydig and Sertoli cells might be involved in the development of functional blood testis barrier in mouse testis.     

Transabdominal testicular descent is disrupted in mice with deletion of insulinlike factor 3 receptor

Background: Several factors are implicated in transabdominal testicular descent, including insulinlike factor 3 (INSL3) hormone and Müllerian-inhibiting substance (MIS). A transgene insertional mutation found on chromosome 5 in the mouse, known as crsp, causes deletion of a transmembrane G protein-coupled receptor gene, Great, which is highly expressed in the gubernaculum. The authors describe here a detailed analysis of the testicular descent and gubernacular development in crsp mice to determine the role of the Great gene in this process.Methods: Homozygous (crsp/crsp) mutant and wild-type heterozygous (crsp/+) mice were examined at birth (D 0) and at days 10 (D 10) and 30 (D 30) postnatally. Serial sagittal or coronal sections were analyzed for position of the gonads and cremaster sac development.Results: Transabdominal testicular descent was absent at D 0 in crsp/crsp homozygous mice with no swelling reaction in the gubernacula. By D 10 the cremaster sac was significantly thinner (P < .05) and contained less collagen in the mutants than in the wild-type controls. On D 30 the cremaster sacs of mutant males were similar in thickness to those in females.Conclusions: Disruption of the Great gene causes failure of the transabdominal phase of descent, identical to that seen in the Insl3-deficient mutants, consistent with the recent data suggesting that Great gene encodes the Insl3 transmembrane receptor. No differences between D 30 mutant males and females were found in the gubernacula, suggesting that Insl3/Great signaling regulates gubernacular development.     

Intracellular survival of Staphylococcus aureus within cultured enterocytes

BACKGROUND: Candida albicans is a polymorphic fungus that frequently causes systemic infection in postsurgical and trauma patients. Others have reported that Escherichia coli lipopolysaccharide (LPS) acts as a copathogen to enhance the virulence of parenteral C. albicans. Experiments were designed to clarify the effect of parenteral LPS on systemic candidiasis initiated via the oral route. MATERIALS AND METHODS: Antibiotic-treated mice were orally inoculated with C. albicans CAF2 (wild-type) or mutant HLC54 (defective in filament formation), and were given 100 microg parenteral LPS 16 h before sacrifice. Separate groups of mice were additionally exposed to intermittent hypoxia prior to LPS. At sacrifice, cecal flora and microbial translocation to the mesenteric lymph nodes were quantified. C. albicans adherence to cultured HT-29 and Caco-2 enterocytes (pretreated with LPS, or calcium-free medium to expose the enterocyte lateral surface, or both) was quantified by enzyme-linked immunoabsorbent assay. RESULTS: All mice had high numbers of cecal C. albicans, and LPS was associated with an additional increase in cecal concentrations of HLC54 but not CAF2. Translocation of HLC54, but not CAF2, appeared facilitated by hypoxia, but LPS did not facilitate translocation in any treatment group. Exposure of the lateral surface of cultured enterocytes had no effect on C. albicans adherence, although LPS consistently decreased adherence of both C. albicans strains. CONCLUSIONS: In contrast to experiments where systemic candidiasis was initiated by the parenteral route, parenteral LPS did not act as a copathogen in mice with systemic candidiasis initiated by the oral route, and these results might be related to LPS-induced alterations in C. albicans adherence to host enterocytes.     

The freshwater cyanobacterium Lyngbya aerugineo-coerulea produces compounds toxic to mice and to mammalian and fish cells

Despite a growing awareness of the presence of cyanobacterial toxins, knowledge about the ability of specific species to produce toxic compounds is still rather limited. It was the overall goal of the current work to investigate if probes derived from the freshwater species Lyngbya aerugineo-coerulea (Kutz.) Gomont, a cyanobacterium frequently found in southern Europe and not previously investigated for the presence of bioactive compounds, were capable of eliciting in vivo and in vitro toxicity. The cyanobacterial extract revealed signs of neuro- as well as hepatotoxicity in mice, although these signs could not be explained by the well-known respective cyanobacterial neuro- and hepatotoxins saxitoxin and microcystin. Cytotoxicity was elicited by the cyanobacterial extract in all mammalian cell lines tested. As well, the rainbow trout liver cell line, RTL-W1, was found to be susceptible to the cytotoxic effects of the extract, although the cytotoxicity was dependent on temperature. In contrast, the cyanobacterial growth medium elicited cytotoxicity independent of temperature, leading to morphological changes indicative of alterations to the cytoskeleton. Overall, the results suggest that Lyngbya aerugineo-coerulea is an important cyanobacterium to be considered for its potential to cause health risks on environmental exposure of it to mammals and fish. Applying a combination of mammalian and piscine cell line bioassays is a unique approach that, combined with chemical analysis, could be used in the future to identify the structure and cellular mechanisms of the as-yet-unknown toxic Lyngbya aerugineo-coerulea metabolites in particular and to screen cyanobacterial extracts for their toxicity in general.