Partial deletion of chromosome 8 ß-defensin cluster confers sperm dysfunction and infertility in male mice.

ß-defensin peptides are a family of antimicrobial peptides present at mucosal surfaces, with the main site of expression under normal conditions in the male reproductive tract. Although they kill microbes in vitro and interact with immune cells, the precise role of these genes in vivo remains uncertain. We show here that homozygous deletion of a cluster of nine ß-defensin genes (DefbΔ9) in the mouse results in male sterility. The sperm derived from the mutants have reduced motility and increased fragility. Epididymal sperm isolated from the cauda should require capacitation to induce the acrosome reaction but sperm from the mutants demonstrate precocious capacitation and increased spontaneous acrosome reaction compared to wild-types but have reduced ability to bind the zona pellucida of oocytes. Ultrastructural examination reveals a defect in microtubule structure of the axoneme with increased disintegration in mutant derived sperm present in the epididymis cauda region, but not in caput region or testes. Consistent with premature acrosome reaction, sperm from mutant animals have significantly increased intracellular calcium content. Thus we demonstrate in vivo that ß-defensins are essential for successful sperm maturation, and their disruption leads to alteration in intracellular calcium, inappropriate spontaneous acrosome reaction and profound male infertility.

Explanation of the Colour Change in Alexandrites.

Alexandrites are remarkable and rare gemstones. They display an extraordinary colour change according to the ambient lighting, from emerald green in daylight to ruby red in incandescent light from tungsten lamps or candles. While this colour change has been correctly attributed to chromium impurities and their absorption band in the yellow region of the visible light spectrum, no adequate explanation of the mechanism has been given. Here, the alexandrite effect is fully explained by considering the von Kries model of the human colour constancy mechanism. This implies that our colour constancy mechanism is real (objective) and primarily attuned to correct for the colour temperature of black-body illuminants.

Theoretical investigation into the dissolved oxygen levels in follicular fluid of the developing human follicle using mathematical modelling.

Oxygen levels in the follicle are likely to be critical to follicle development. However, a quantitative description of oxygen levels in the follicle is lacking. Mathematical modelling was used to predict the dissolved oxygen levels in the follicular fluid of the developing human follicle. The model predictions showed that follicular fluid dissolved oxygen levels are highly variable among follicles, due to the unique geometry of individual follicles. More generally, predictions showed that oxygen levels in follicular fluid increase rapidly during the initial early antral stages of follicle growth before peaking in the later early antral phase. Follicular fluid dissolved oxygen levels then decline through to the beginning of the pre-ovulatory phase, from which they increase through to ovulation. Based on the best available parameter estimates, the model predictions suggest that the mean dissolved oxygen levels in human follicular fluid during the late antral and pre-ovulatory phases range between 11 and 51 mmHg (approximately 1.5-6.7 vol%). These predictions suggest that the human ovarian follicle is a low-oxygen environment that is often challenged by hypoxia, and are in agreement with only some published data on follicular fluid oxygen levels. Predictions are discussed in relation to follicle health and oocyte culture.

Evaluation of different strategies for the development of amperometric biosensors for L-lactate.

Two strategies were investigated for the development of lactate biosensors based on sol-gel matrixes and polysulfone composite films, both containing L-lactate dehydrogenase (LDH). Firstly, reagentless disposable screen-printed electrodes (SPE's) with Meldola's Blue (MB) and the cofactor NAD(+) inside a sol-gel matrix were prepared. These showed relatively low sensitivities (260 microA/M). Secondly, mediator-modified-polysulfone-graphite composite films deposited over both cylindrical epoxy-graphite and SPE's. These electrodes showed enhanced performance characteristics: improved sensitivity (80 mA/M), detection limit (0.87 microM) and reproducibility (2%). Reagentless electrodes, incorporating NAD(+) in the polysulfone film, had a decreased sensitivity, although better than that achieved by the sol-gel electrodes. While sol-gel electrodes showed a linear range between 1.25 x 10(-4) and 2.48 x 10(-3)M, the epoxy-graphite composite electrodes based on polysulfone composite films allowed the detection of lactate at a linear range of lower concentrations from 1 x 10(-6) to 1.2 x 10(-5)M. Finally, the performance of the LDH-MB-polysulfone-composite film-based SPE's in a flow system was studied. Short response times were obtained (t<30s). Furthermore, repeatability and reproducibility values were notably improved, especially when working with electrodes covered with a polyamide layer prepared with N-(2-aminoethyl)-piperazine.

Presence of endocrine and exocrine markers in EGFP-positive cells from the developing pancreas of a nestin/EGFP mouse.

In order to purify and characterize nestin-positive cells in the developing pancreas a transgenic mouse was generated, in which the enhanced green fluorescent protein (EGFP) was driven by the nestin second intronic enhancer and upstream promoter. In keeping with previous studies on the distribution of nestin, EGFP was expressed in the developing embryo in neurones in the brain, eye, spinal cord, tail bud and glial cells in the small intestine. In the pancreas there was no detectable EGFP at embryonic day 11.5 (E11.5). EGFP expression appeared at E12.5 and increased in intensity through E14.5, E18.5 and post-natal day 1. Flow cytometry was used to quantify and purify the EGFP positive population in the E15.5 pancreas. The purified (96%) EGFP-expressing cells, which represent 20% of the total cell population, were shown by RT/PCR to express exocrine cell markers (amylase and P48) and endocrine cell markers (insulin 1, insulin 2, and Ngn3). They also expressed, at a lower level, PDX-1, Isl-1, and the islet hormones pancreatic polypeptide, glucagon and somatostatin as well as GLUT2, the stem cell marker ABCG2 and PECAM, a marker of endothelial cells. It was further shown by immunocytochemistry of the E15.5 pancreas that EGFP colocalised in separate subpopulations of cells that expressed nestin, insulin and amylase. These results support the conclusion that nestin expressing cells can give rise to both endocrine and exocrine cells. The ability to purify these putative progenitor cells may provide further insights into their properties and function.

Genotype-phenotype correlation of mouse pde6b mutations.

PURPOSE: To identify the underlying molecular defects causing retinal degeneration in seven N-ethyl-N-nitrosourea (ENU) induced mutant alleles of the Pde6b gene and to analyze the timescale of retinal degeneration in these new models of retinitis pigmentosa. METHODS: Conformation sensitive capillary electrophoresis and DNA sequencing were used to identify the mutations in the Pde6b gene. Visual acuity testing was performed with a visual-tracking drum at ages ranging from postnatal day 25 to week 10. Retinal examination was performed with an indirect ophthalmoscope. Animals were killed and eyes were prepared for histologic analysis. RESULTS: Point mutations in the seven new alleles of Pde6b were identified: Three generated premature stop codons, two were missense mutations, and two were splice mutations. The three stop codon mutants and one of the splice mutants had phenotypes indistinguishable from the Pde6b(rd1) mouse in rapidity of onset of retinal degeneration, suggesting that they are null alleles. However, the remaining alleles showed slower onset of retinal degeneration, as determined by visual acuity testing, fundus examination, and histology, indicating that they are hypomorphic alleles. CONCLUSIONS: These data demonstrate a correlation between genotype and phenotype. Four of the mutants with severe genetic lesions have rapid onset of retinal degeneration, as determined by fundus examination. These mice were indistinguishable from Pde6b(rd1) mice, which are effectively blind by 3 weeks of age. In contrast, the milder genetic lesions show a slower progression of the disease and provide the community with models that more closely mimic human retinitis pigmentosa.

The developmental regulator Pax6 is essential for maintenance of islet cell function in the adult mouse pancreas.

The transcription factor Pax6 is a developmental regulator with a crucial role in development of the eye, brain, and olfactory system. Pax6 is also required for correct development of the endocrine pancreas and specification of hormone producing endocrine cell types. Glucagon-producing cells are almost completely lost in Pax6-null embryos, and insulin-expressing beta and somatostatin-expressing delta cells are reduced. While the developmental role of Pax6 is well-established, investigation of a further role for Pax6 in the maintenance of adult pancreatic function is normally precluded due to neonatal lethality of Pax6-null mice. Here a tamoxifen-inducible ubiquitous Cre transgene was used to inactivate Pax6 at 6 months of age in a conditional mouse model to assess the effect of losing Pax6 function in adulthood. The effect on glucose homeostasis and the expression of key islet cell markers was measured. Homozygous Pax6 deletion mice, but not controls, presented with all the symptoms of classical diabetes leading to severe weight loss requiring termination of the experiment five weeks after first tamoxifen administration. Immunohistochemical analysis of the pancreata revealed almost complete loss of Pax6 and much reduced expression of insulin, glucagon, and somatostatin. Several other markers of islet cell function were also affected. Notably, strong upregulation in the number of ghrelin-expressing endocrine cells was observed. These findings demonstrate that Pax6 is essential for adult maintenance of glucose homeostasis and function of the endocrine pancreas.

A CNS-specific hypomorphic Pdgfr-beta mutant model of diabetic retinopathy.

PURPOSE: A mouse mutant identified during a recessive N-ethyl-N-nitrosourea (ENU) mutagenesis screen exhibited ocular hemorrhaging resulting in a blood-filled orbit, and hence was named "redeye." We aimed to identify the causal mutation in redeye, and evaluate it as a model for diabetic retinopathy (DR). METHODS: The causative gene mutation in redeye was identified by haplotype mapping followed by exome sequencing. Glucose tolerance tests, detailed histologic and immunofluorescence analyses, and vascular permeability assays were performed to determine the affect of redeye on glucose metabolism, pericyte recruitment, and the development of the retinal vasculature and blood-retinal barrier (BRB). RESULTS: A mutation was identified in the Pdgfrb gene at position +2 of intron 6. We show that this change causes partial loss of normal splicing resulting in a frameshift and premature termination, and, therefore, a substantial reduction in normal Pdgfrb transcript. The animals exhibit defective pericyte recruitment restricted to the central nervous system (CNS) causing basement membrane and vascular patterning defects, impaired vascular permeability, and aberrant BRB development, resulting in vascular leakage and retinal ganglion cell apoptosis. Despite exhibiting classic features of diabetic retinopathy, redeye glucose tolerance is normal. CONCLUSIONS: The Pdgfrb(redeye/redeye) mice exhibit all of the features of nonproliferative DR, including retinal neurodegeneration. In addition, the perinatal onset of the CNS-specific vascular phenotype negates the need to age animals or manage diabetic complications in other organs. Therefore, they are a more useful model for diseases involving pericyte deficiencies, such as DR, than those currently being used.

Functional characterization of the human RPGR proximal promoter.

PURPOSE: Mutations in the retinitis pigmentosa (RP) GTPase regulator (RPGR) gene account for more than 70% of X-linked RP cases. This study aims to characterize the proximal promoter region of the human RPGR gene. METHODS: The 5'-flanking region (5 kb) of human RPGR was cloned and sequenced. A potential transcription start site and transcription factor binding motifs were identified by bioinformatic analysis. Constructs containing the putative human RPGR promoter region upstream of a luciferase reporter gene were generated and analyzed by transient transfection and luciferase assays. Transgenic mouse lines carrying a 3-kb human RPGR promoter sequence fused to lacZ were generated and RPGR proximal promoter activity was analyzed by X-gal staining. RESULTS: Bioinformatic analyses of the human RPGR 5'-flanking region uncovered potential transcription factor binding sites and a CpG island. Transient transfection assays with RPGR promoter/luciferase reporter constructs revealed a 980-bp fragment (-952 to +28) that produced higher levels of luciferase activity. Mutagenesis identified a putative Sp1 binding site that was critical for regulating transcriptional activity. We generated transgenic mice in which a lacZ reporter gene was controlled by the 3-kb upstream region of RPGR. ß-galactosidase expression was predominantly found in mouse retina, brain, and kidney. In the retina, the photoreceptor cell layer showed the strongest ß-galactosidase staining. CONCLUSIONS: Our study defined the human RPGR proximal promoter region in which a 3-kb fragment contained sufficient regulatory elements to control RPGR expression in mouse retina and other tissues. Characterization of the RPGR promoter will facilitate understanding of the functional role of RPGR in the retina and gene therapy of X-linked RP.

The occurrence and nature of electrochemical activity in milk from a herd of dairy cows.

Amperometric sensors may be used in milk analysis but electrochemical interference from compounds other than the analyte is an on-going problem. A survey was made of the level of electrochemical activity (potential interference) in milk from a herd of dairy cows grazing on summer pasture. It was a ubiquitous feature of the aqueous phase of whey and de-proteinized milk over about 3 months. The nature of the interference was studied by differential pulse voltammetry and responses to ascorbic acid oxidase and uricase. The principal source of interference appeared to be uric acid.

The effects of IVF aspiration on the temperature, dissolved oxygen levels, and pH of follicular fluid.

PURPOSE: To investigate the effects of IVF aspiration on the temperature, pH, and dissolved oxygen of bovine follicular fluid. METHODS: The temperature was monitored at various positions in an aspiration kit. Dissolved oxygen and pH were measured before and after aspiration. RESULTS: The temperature of follicular fluid dropped by 7.7 +/- 1.3 degrees C upon aspiration. Dissolved oxygen levels rose by 5 +/- 2 vol.%. The pH increased by 0.04 +/- 0.01. CONCLUSIONS: The temperature change was attributed mainly to evaporation of fluid in the collection tube. Changes in dissolved oxygen levels and pH were due to contact with air. Standard vacuum-based aspiration may induce changes in follicular fluid, which could be detrimental to oocyte health and affect attempts to correlate chemical characteristics with oocyte quality.

Cardiac malformations and midline skeletal defects in mice lacking filamin A.

The X-linked gene filamin A (Flna) encodes a widely expressed actin-binding protein that crosslinks actin into orthogonal networks and interacts with a variety of other proteins including membrane proteins, integrins, transmembrane receptor complexes and second messengers, thus forming an important intracellular signalling scaffold. Heterozygous loss of function of human FLNA causes periventricular nodular heterotopia in females and is generally lethal (cause unknown) in hemizygous males. Missense FLNA mutations underlie a spectrum of disorders affecting both sexes that feature skeletal dysplasia accompanied by a variety of other abnormalities. Dilp2 is an X-linked male-lethal mouse mutation that was induced by N-ethyl-N-nitrosourea. We report here that Dilp2 is caused by a T-to-A transversion that converts a tyrosine codon to a stop codon in the Flna gene (Y2388X), leading to absence of the Flna protein and male lethality because of incomplete septation of the outflow tract of the heart, which produces common arterial trunk. A proportion of both male and female mutant mice have other cardiac defects including ventricular septal defect. In addition, mutant males have midline fusion defects manifesting as sternum and palate abnormalities. Carrier females exhibit milder sternum and palate defects and misshapen pupils. These results define crucial roles for Flna in development, demonstrate that X-linked male lethal mutations can be recovered from ENU mutagenesis screens and suggest possible explanations for lethality of human males hemizygous for null alleles of FLNA.

Fgf10 maintains notch activation, stimulates proliferation, and blocks differentiation of pancreatic epithelial cells.

The pancreas is an endodermally derived organ that initially appears as a dorsal and ventral protrusion of the primitive gut epithelium. The pancreatic progenitor cells present in these early pancreatic anlagen proliferate and eventually give rise to all pancreatic cell types. The fibroblast growth factor receptor (FGFR) 2b high-affinity ligand FGF10 has been linked to pancreatic epithelial cell proliferation, and we have shown previously that Notch signalling controls pancreatic cell differentiation by means of lateral inhibition. In the developing pancreas, activated intracellular Notch appears to be required for maintaining cells in the progenitor state, in part by blocking the expression of the pro-endocrine gene neurogenin 3 (ngn3), and hence endocrine cell differentiation. Here, we show that persistent expression of Fgf10 in the embryonic pancreas of transgenic mice also inhibits pancreatic cell differentiation, while stimulating pancreatic epithelial cell proliferation. We provide evidence that one of the effects of the persistent expression of Fgf10 in the developing pancreas is maintained Notch activation, which results in impaired expression of ngn3 within the pancreatic epithelium. Together, our data suggest a role for FGF10/FGFR2b signalling in regulation of pancreatic cell proliferation and differentiation and that FGF10/FGFR2b signalling affects the Notch-mediated lateral inhibition pathway.

Haploinsufficiency for Phox2b in mice causes dilated pupils and atrophy of the ciliary ganglion: mechanistic insights into human congenital central hypoventilation syndrome.

Dilp1 is a semi-dominant mouse mutation that causes dilated pupils when heterozygous and is lethal when homozygous. We report here that it is caused by a point mutation that introduces a stop codon close to the start of the coding sequence of the paired-like homeobox transcription factor Phox2b. Mice carrying a targeted allele of Phox2b also have dilated pupils and the two alleles do not complement. Phox2b is necessary for the development of the autonomic nervous system and when absent one of the consequences is that all parasympathetic ganglia fail to form. Constriction of the pupil is a parasympathetic response mediated by the ciliary ganglion and we find that in Phox2b heterozygous mutants it is highly atrophic. The development of other parasympathetic and sympathetic ganglia appears to be largely unaffected indicating that the ciliary ganglion is exquisitely sensitive to a reduction in dose of this transcription factor. PHOX2B has been implicated in human disease. Mutations, principally leading to polyalanine expansions within the protein, have been found in patients with congenital central hypoventilation syndrome (CCHS), the cardinal feature of which is an inability to breathe unassisted when asleep. Additionally, some CCHS patients have ocular abnormalities, including pupillary defects, although they principally have constricted rather than dilated pupils. The apparent phenotypic differences observed between mice carrying a loss-of-function mutation of Phox2b and CCHS patients indicate that PHOX2B mutations found in CCHS patients, all of which can produce proteins with intact DNA-binding domains, are gain-of-function mutations that alter rather than abolish protein function.

Milk L-lactate concentration is increased during mastitis.

A study was undertaken in cattle to evaluate changes in milk L-lactate in relation to mastitis. A healthy, rear quarter of the udder of each of ten cows in mid-lactation was infused with 1000 colony-forming units (cfu) of Streptococcus uberis following an afternoon milking. Foremilk samples were taken at each milking from control and treated quarters and antibiotic treatment was applied following the onset of clinical mastitis or after 72 h. One cow did not become infected. Six quarters showed clinical symptoms of mastitis within 24-40 h and this was associated with a more than 30-fold increase in milk L-lactate (to 3.3 mM) and an increase in somatic cell count (SCC) from 4.5 x 10(3) to 1 x 10(7) cells/ml. Three cows were subclinical, with cell counts ranging from 1.5 x 10(6) to 1 x 10(7) cells/ml. In these animals, milk lactate ranged from 0.7 to 1.5 mM in the infected quarters up to 40 h post-infection, compared with less than 0.1 mM in control quarters. Milk was examined from 137 cows in mid-lactation which were known to have mastitis. Foremilk samples were taken aseptically from control and infected quarters of cows on commercial farms. Mean milk L-lactate concentrations and SCC were 0.14 +/- 0.02 mM and 1.85 +/- 0.3 x 10(5) cells/ml, respectively, in control (bacteriologically negative) samples. However, L-lactate concentrations exceeded 2.5 mM in the presence of some types of infection, the level of the lactate response being closely related to the impact of the infection on SCC. L-Lactate concentrations were relatively elevated in milk samples taken post partum, declining from 0.8 to 0.14 mM oyer the first few days of lactation. In conclusion, milk L-lactate has potential as an indicator of clinical and subclinical mastitis in dairy cows.