p70 lupus autoantigen binds the enhancer of the T-cell receptor beta-chain gene.

The p70 (Ku) autoantigen has been described as a nonhistone nuclear protein recognized by antibodies from lupus patients. In our studies on the regulation of T-cell receptor (TCR) beta-chain gene expression we have identified the p70 lupus autoantigen as a DNA-binding protein that binds the enhancer of the TCR beta-chain gene. This enhancer is essential for expression of the TCR beta gene. The core TCR beta enhancer contains the E3 motif, which we show here is essential for enhancer activity. The protection of the E3 motif in T cells and the marked reduction in enhancer activity when the E3 motif is mutated underline its physiological importance in regulating beta enhancer activity. The p70 lupus autoantigen gene was identified by screening T-cell lambda gt11 libraries with an E3 probe. The gene encodes a protein which binds the E3 motif in a sequence-specific manner. The identification of a 70-kDa protein as a major E3-binding protein by UV crosslinking is consistent with the conclusion that the p70 lupus autoantigen binds the beta enhancer. Finally, we have shown that T-cell nuclear proteins which bind the E3 motif bear p70 (Ku) lupus autoantigenic determinants. Together these data suggest that the p70 autoantigen binds a critical motif in the beta enhancer and probably regulates TCR beta gene expression.     

Relative carriage rates of nuclear dehydrogenating clostridia in two populations of different colorectal cancer risk

Carriage of nuclear dehydrogenating clostridia has been associated with colon cancer and implicated in its aetiology. This study has compared the carriage of these organisms in a British population at high risk for the development of colon cancer with a low risk Nigerian population. Clostridia were found in all of the stools from both populations. Nuclear dehydrogenating clostridia were only found in the stools of the British subjects (32%). These results support the suggestion that the carriage rate of nuclear dehydrogenating clostridia in a population is related to the risk of colon cancer.     

Development of hatching blastocysts from immature human oocytes following in-vitro maturation and fertilization using a co-culture system

Recently, in-vitro maturation (IVM) of immature human oocytes recovered from non-stimulated follicles has been applied in the treatment of infertility. However, in previous reports, very few embryos cultured in conventional medium have reached the expanded blastocyst stage following in-vitro maturation and fertilization (IVM/IVF). The objective of this study was to investigate whether the developmental competence of human embryos following IVM/IVF could be enhanced by the use of a human ampullary cell co-culture system. Immature human oocytes were aspirated from small follicles at Caesarean section and then cultured in medium containing human menopausal gonadotrophin for 36 to 48 h, followed by insemination. Zygotes were randomly cultured either in conventional culture medium alone or in the co-culture system. Of 48 embryos cultured in conventional medium alone, all arrested at the 2-16- cell stage on day 3 after insemination. Of 46 embryos cultured in the co-culture system, 26 embryos (56.5%) arrested at the 2-16-cell stage. Six embryos (13%) developed to the morula stage. Fourteen embryos (30.4%) developed to expanded blastocysts and two blastocysts were hatching on day 7 after insemination. We conclude that co-culture significantly enhances the development of blastocysts in embryos resulting from IVM/IVF.      

Detection of chromosomes and estimation of aneuploidy in human spermatozoa using fluorescence in-situ hybridization

The development and application of fluorescence in-situ hybridization (FISH) has opened the way for comprehensive studies on numerical chromosome abnormalities in human spermatozoa. FISH can be rapidly applied to large numbers of spermatozoa and thus overcomes the major limitation of karyotyping spermatozoa after penetration of zona-free hamster oocytes. The simultaneous hybridization of two or more chromosome-specific probes to spermatozoa and subsequent detection of the bound probes using different fluorescent detection systems enables two or more chromosomes to be localized simultaneously in the same spermatozoon and provides a technique for undertaking reasonable estimates of aneuploidy. The most commonly used probes are those which bind to the centromeric region of specific chromosomes. Most studies to date have concentrated on estimating aneuploidy in spermatozoa from normospermic men, although reports are beginning to appear on aneuploidy in spermatozoa from subfertile and infertile men. Multi- probe FISH studies have generally reported disomy (hyperhaploidy) estimates of 0.05-0.2% per chromosome. There is preliminary evidence that some chromosomes such as X, Y and 21 are predisposed towards higher rates of non-disjunction during spermatogenesis. There are also suggestions of inter-donor variability in aneuploidy frequencies for specific chromosomes, although this requires confirmation in larger studies. While FISH is clearly a powerful technique that has many applications in reproductive medicine, it must also be realized that it does have limitations and the technology itself is still evolving and has yet to be fully validated on spermatozoa.      

Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography

In this study, we have used time-lapse video cinematography to study fertilization in 50 human oocytes that had undergone intracytoplasmic sperm injection (ICSI). Time-lapse recording commenced shortly after ICSI and proceeded for 17-20 h. Oocytes were cultured in an environmental chamber which was maintained under standard culture conditions. Overall, 38 oocytes (76%) were fertilized normally, and the fertilization rate and embryo quality were not significantly different from 487 sibling oocytes cultured in a conventional incubator. Normal fertilization followed a defined course of events, although the timing of these events varied markedly between oocytes. In 35 of the 38 fertilized oocytes (92%), there were circular waves of granulation within the ooplasm which had a periodicity of 20-53 min. The sperm head decondensed during this granulation phase. The second polar body was then extruded, and this was followed by the central formation of the male pronucleus. The female pronucleus formed in the cytoplasm adjacent to the second polar body at the same time as, or slightly after, the male pronucleus, and was subsequently drawn towards the male pronucleus until the two abutted. Both pronuclei then increased in size, the nucleoli moved around within the pronuclei and some nucleoli coalesced. During pronuclear growth, the organelles contracted from the cortex towards the centre of the oocyte, leaving a clear cortical zone. The oocyte decreased in diameter from 112 to 106 microm (P < 0.0001) during the course of the observation period. The female pronucleus was significantly smaller in diameter than the male pronucleus (24.1 and 22.4 microm respectively, P = 0.008) and contained fewer nucleoli (4.2 and 7.0 respectively, P < 0.0001). After time-lapse recording, oocytes were cultured for 48 h prior to embryo transfer or cryopreservation. Embryo quality was related to fertilization events and periodicity of the cytoplasmic wave, and it was found that good quality embryos arose from oocytes that had more uniform timing from injection to pronuclear abuttal and tended to have a longer cytoplasmic wave. In conclusion, we have shown that time-lapse video cinematography is an excellent tool for studying fertilization and early embryo development, and have demonstrated that human fertilization comprises numerous complex dynamic events.      

Contribution of cholinergic and gabaergic functions to memory processes in BALB/cANnCrlBR mice

Several lines of evidence indicate that glucose influences on memory depend on interactions between glucose, glucoregulation and hippocampal cholinergic function. We previously demonstrated that glucose and scopolamine differentially affected memory consolidation for an operant bar pressing task in two closely-related BALB/c mouse strains. Whereas glucose normally improves memory in several animal strains, memory consolidation was not effected by systemic glucose injections in BALB/cANnCrlBR mice. Moreover, these mice were relatively insensitive to the normally observed amnestic effects of scopolamine. We therefore sought to determine whether cholinergic mechanisms in the dorsal hippocampus were involved in such atypical drug effects on memory processing in that strain of mice. In Experiment 1, we examined whether post-training oxotremorine would also atypically influence memory consolidation for an appetitively reinforced operant bar pressing task following microinjection in the dorsal hippocampus. In Experiment 2, we examined the effects of intrahippocampal GABAA drugs on memory consolidation. The non-selective muscarinic agonist, oxotremorine, dose-dependently impaired memory and the GABAA antagonist, bicuculline, improved retention in BALB/cANnCrlBR mice. It was concluded that GABA-mediated influences on hippocampal pyramidal output in BALB/cANnCrlBR mice and other strains are similar; but the amnestic effects of oxotremorine from the dorsal hippocampus were opposite to facilitating effects normally observed in other animal strains. Results are discussed relative to possible altered septo-hippocampal cholinergic neurotransmission in BALB/cANnCrlBR mice.