Development of a well-defined medium for the in vitro maturation of immature bovine cumulus-oocyte complexes.

PURPOSE: Our purpose was to develop a well-defined medium for the in vitro maturation (IVM) of immature bovine cumulus-oocyte complexes (COC). METHODS: The COC were cultured in the presence of three protein supplementations: fetal bovine serum (FBS), bovine serum albumin, and Synthetic Serum Substitute. The embryos obtained after in vitro fertilization of IVM oocytes were cocultured with Vero cells and their development to the morula and blastocyst stages was studied. RESULTS: When FBS was absent from the IVM medium, a significantly lower fertilization rate was observed, followed by a decrease in the percentage of embryos reaching the blastocyst stage. When FBS was replaced by a defined protein supplementation, the best results were obtained with Synthetic Serum Substitute. CONCLUSIONS: Adequate protein supplementation of the IVM medium optimizes the fertilization rate and the development of bovine IVM oocytes. The implication of these results in the human field is discussed.

Ly49D-mediated ITAM signaling in immature thymocytes impairs development by bypassing the pre-TCR checkpoint.

Activating and inhibitory NK receptors regulate the development and effector functions of NK cells via their ITAM and ITIM motifs, which recruit protein tyrosine kinases and phosphatases, respectively. In the T cell lineage, inhibitory Ly49 receptors are expressed by a subset of activated T cells and by CD1d-restricted NKT cells, but virtually no expression of activating Ly49 receptors is observed. Using mice transgenic for the activating receptor Ly49D and its associated ITAM signaling DAP12 chain, we show in this article that Ly49D-mediated ITAM signaling in immature thymocytes impairs development due to a block in maturation from the double negative (DN) to double positive (DP) stages. A large proportion of Ly49D/DAP12 transgenic thymocytes were able to bypass the pre-TCR checkpoint at the DN3 stage, leading to the appearance of unusual populations of DN4 and DP cells that lacked expression of intracellular (ic) TCRβ-chain. High levels of CD5 were expressed on ic TCRβ(-) DN and DP thymocytes from Ly49D/DAP12 transgenic mice, further suggesting that Ly49D-mediated ITAM signaling mimics physiological ITAM signaling via the pre-TCR. We also observed unusual ic TCRβ(-) single positive thymocytes with an immature CD24(high) phenotype that were not found in the periphery. Importantly, thymocyte development was completely rescued by expression of an Ly49A transgene in Ly49D/DAP12 transgenic mice, indicating that Ly49A-mediated ITIM signaling can fully counteract ITAM signaling via Ly49D/DAP12. Collectively, our data indicate that inappropriate ITAM signaling by activating NK receptors on immature thymocytes can subvert T cell development by bypassing the pre-TCR checkpoint.

Expression of SCG10 and stathmin proteins in the rat olfactory system during development and axonal regeneration.

The membrane-associated protein SCG10 is expressed specifically by neuronal cells. Recent experiments have suggested that it promotes neurite outgrowth by increasing microtubule dynamics in growth cones. SCG10 is related to the ubiquitous but neuron-enriched cytosolic protein stathmin. To better understand the role played by SCG10 and stathmin in vivo, we have analyzed the expression and localization of these proteins in both the olfactory epithelium and the olfactory bulb in developing and adult rats, as well as in adult bulbectomized rats. The olfactory epithelium is exceptional in that olfactory receptor neurons constantly regenerate and reinnervate the olfactory bulb throughout animal life-span. SCG10 and stathmin expression in the olfactory receptor neurons was found to be regulated during embryonic and postnatal development and to correlate with neuronal maturation. Whereas SCG10 expression was restricted to immature olfactory receptor neurons (GAP-43-positive, olfactory marker protein-negative), stathmin was also expressed by the basal cells. In the olfactory bulb of postnatal and adult rats, a moderate to strong SCG10 immunoreactivity was present in the olfactory nerve layer, whereas no labeling was detected in the glomerular layer. Olfactory glomeruli also showed no apparent immunoreactivity for several cytoskeletal proteins such as tubulin and microtubule-associated proteins. In unilaterally bulbectomized rats, SCG10 and stathmin were seen to be up-regulated in the regenerating olfactory epithelium at postsurgery stages corresponding to olfactory axon regeneration. Our data strongly suggest that, in vivo, both SCG10 and stathmin may play a role in axonal outgrowth during ontogenesis as well as during axonal regeneration.

Aggregating Brain Cell Cultures as a Model to Study Ischaemia-induced Neurodegeneration.

Rotation-mediated aggregating brain cell cultures at two different maturational stages (DIV 11 and DIV 20) were subjected for 1 or 2 hours to ischaemic conditions by transient immobilization (arrest of media circulation). During recovery, cell damage was evaluated by measuring changes in cell type-specific enzyme activities and total protein content. It was found that in immature cultures (DIV 11), immobilization for 1 or 2 hours did not affect the parameters measured. By contrast, at DIV 20, ischaemic conditions for 1 hour caused a pronounced decrease in the activities of glutamic acid decarboxylase and choline acetyltransferase. A significant decrease in these neuron-specific enzyme activities was found at post-ischaemic days 1-14, indicating immediate and irreversible neuronal damage. The activity of the astrocyte-specific enzyme, glutamine synthetase, was significantly increased at 4 days post-treatment; equal to control values at 6 days; and significantly decreased at 14 days after the ischaemic insult. Immobilization of DIV 20 cultures for 2 hours caused a drastic reduction in all the parameters measured at post-ischaemic day 6. Generally, the ischaemic conditions appeared to be more detrimental to neurons than to astrocytes, and GABAergic neurons were more affected than cholinergic neurons.

Visual binding abilities in the initial and advanced stages of schizophrenia

OBJECTIVE: The study tests the hypothesis that intramodal visual binding is disturbed in schizophrenia and should be detectable in all illness stages as a stable trait marker. METHOD: Three groups of patients (rehospitalized chronic schizophrenic, first admitted schizophrenic and schizotypal patients believed to be suffering from a pre-schizophrenic prodrome) and a group of normal control subjects were tested on three tasks targeting visual 'binding' abilities (Muller-Lyer's illusion and two figure detection tasks) in addition to control parameters such as reaction time, visual selective attention, Raven's test and two conventional cortical tasks of spatial working memory (SWM) and a global local test. RESULTS: Chronic patients had a decreased performance on the binding tests. Unexpectedly, the prodromal group exhibited an enhanced Gestalt extraction on these tests compared both to schizophrenic patients and to healthy subjects. Furthermore, chronic schizophrenia was associated with a poor performance on cortical tests of SWM, global local and on Raven. This association appears to be mediated by or linked to the chronicity of the illness. CONCLUSION: The study confirms a variety of neurocognitive deficits in schizophrenia which, however, in this sample seem to be linked to chronicity of illness. However, certain aspects of visual processing concerned with Gestalt extraction deserve attention as potential vulnerability- or prodrome- indicators. The initial hypothesis of the study is rejected.

Unexpectedly late expression of intracellular CD3epsilon and TCR gammadelta proteins during adult thymus development.

During adult thymus development immature CD4(-)CD8(-) [double-negative (DN)] precursor cells pass through four phenotypically distinct stages defined by expression of CD44 and CD25: CD44(hi)CD25(-) (DN1), CD44(hi)CD25(+) (DN2), CD44(lo)CD25(+) (DN3) and CD44(lo)CD25(-) (DN4). Although it is well established that the TCR beta, gamma and delta genes are rearranged and expressed in association with the CD3 components in DN thymocytes, the precise timing of expression of the TCR and CD3 proteins has not been determined. In this report we have utilized a sensitive intracellular (ic) staining technique to analyze the expression of ic CD3epsilon, TCR beta and TCR gammadelta proteins in immature DN subsets. As expected from previous studies of TCR beta rearrangement and mRNA expression, icTCR beta(+) cells were first detected in the DN3 subset and their proportion increased thereafter. Surprisingly, however, both icCD3epsilon(+) and icTCR gammadelta(+) cells were detected at later stages of development than was predicted by molecular studies. In particular icCD3epsilon protein expression coincided with the transition from the DN2 to DN3 stage of development, whereas icTCR gammadelta protein expression was only detected in a minor subset of DN4 cells. The implications of these findings for alphabeta lineage divergence will be discussed.

Variation in the intensity of inbreeding depression among successive life-cycle stages and generations in gynodioecious Silene vulgaris (Caryophyllaceae).

Inbreeding depression is one of the hypotheses explaining the maintenance of females within gynodioecious plant populations. However, the measurement of fitness components in selfed and outcrossed progeny depends on life-cycle stage and the history of inbreeding. Comparative data indicate that strong inbreeding depression is more likely to occur at later life-cycle stages. We used hermaphrodite individuals of Silene vulgaris originating from three populations located in different valleys in the Swiss Alps to investigate the effect of two generations of self- and cross-fertilization on fitness components among successive stages of the life cycle in a glasshouse experiment. We detected significant inbreeding depression for most life-cycle stages including: the number of viable and aborted seeds per fruit, probability of germination, above ground biomass, probability of flowering, number of flowers per plant, flower size and pollen viability. Overall, the intensity of inbreeding depression increased among successive stages of the life cycle and cumulative inbreeding depression was significantly stronger in the first generation (delta approximately 0.5) compared with the second generation (delta approximately 0.35). We found no evidence for synergistic epistasis in our experiment. Our finding of more intense inbreeding depression during later stages of the life cycle may help to explain the maintenance of females in gynodioecious populations of S. vulgaris because purging of genetic load is less likely to occur.

A homing procedure for studying spatial memory in immature and adult rodents

In this procedure, subjects learn the spatial position of one hole out of many, that allows them to escape from a large open-field into their home cage. The arena is circular and can be rotated between trials so that no proximal landmark is permanently associated with the target hole. This task is thus similar to the Morris water maze procedure, since subjects must remember the position of the escape hole relative to extra-arena cues only. In addition it allows studying the importance of olfactory cues such as scent marks in or around a hole. Since the motivation is to reach home and the motor requirement is low, this task provides a useful alternative to the Morris place navigation task for studying spatial orientation in weanling or senescent rats. Examples are given showing that various behavioural parameters provide a good estimation as how subjects learn this task.

Biased V beta usage in immature thymocytes is independent of DJ beta proximity and pT alpha pairing.

During thymus development, the TCR beta locus rearranges before the TCR alpha locus. Pairing of productively rearranged TCR beta-chains with an invariant pT alpha chain leads to the formation of a pre-TCR and subsequent expansion of immature pre-T cells. Essentially nothing is known about the TCR V beta repertoire in pre-T cells before or after the expression of a pre-TCR. Using intracellular staining, we show here that the TCR V beta repertoire is significantly biased at the earliest developmental stage in which VDJ beta rearrangement has occurred. Moreover (and in contrast to the V(H) repertoire in immature B cells), V beta repertoire biases in immature T cells do not reflect proximity of V beta gene segments to the DJ beta cluster, nor do they depend upon preferential V beta pairing with the pT alpha chain. We conclude that V gene repertoires in developing T and B cells are controlled by partially distinct mechanisms.

Experimental peripheral arterial disease: new insights into muscle glucose uptake, macrophage, and T-cell polarization during early and late stages.

Peripheral arterial disease (PAD) is a common disease with increasing prevalence, presenting with impaired walking ability affecting patient's quality of life. PAD epidemiology is known, however, mechanisms underlying functional muscle impairment remain unclear. Using a mouse PAD model, aim of this study was to assess muscle adaptive responses during early (1 week) and late (5 weeks) disease stages. Unilateral hindlimb ischemia was induced in ApoE(-/-) mice by iliac artery ligation. Ischemic limb perfusion and oxygenation (Laser Doppler imaging, transcutaneous oxygen pressure assessments) significantly decreased during early and late stage compared to pre-ischemia, however, values were significantly higher during late versus early phase. Number of arterioles and arteriogenesis-linked gene expression increased at later stage. Walking ability, evaluated by forced and voluntary walking tests, remained significantly decreased both at early and late phase without any significant improvement. Muscle glucose uptake ([18F]fluorodeoxyglucose positron emission tomography) significantly increased during early ischemia decreasing at later stage. Gene expression analysis showed significant shift in muscle M1/M2 macrophages and Th1/Th2 T cells balance toward pro-inflammatory phenotype during early ischemia; later, inflammatory state returned to neutrality. Muscular M1/M2 shift inhibition by a statin prevented impaired walking ability in early ischemia. High-energy phosphate metabolism remained unchanged (31-Phosphorus magnetic resonance spectroscopy). Results show that rapid transient muscular inflammation contributes to impaired walking capacity while increased glucose uptake may be a compensatory mechanisms preserving immediate limb viability during early ischemia in a mouse PAD model. With time, increased ischemic limb perfusion and oxygenation assure muscle viability although not sufficiently to improve walking impairment. Subsequent decreased muscle glucose uptake may partly contribute to chronic walking impairment. Early inflammation inhibition and/or late muscle glucose impairment prevention are promising strategies for PAD management.

The naturally occurring food mycotoxin fumonisin B1 impairs myelin formation in aggregating brain cell culture.

The effects of subchronical applications of the mycotoxin Fumonisin B1 (FB1) were analyzed in vitro, using aggregating cell cultures of fetal rat telencephalon as a model. As cells in the aggregates developed from an immature state to a highly differentiated state, with synapse and compact myelin formation, it was possible to study the effects of FB1 at different developmental stages. The results showed that FB1 did not cause cell loss and it had no effects on neurons. However it decreased strongly the total content of myelin basic protein, the main constituent of the myelin sheath, during the myelination period (DIV 18-28). The loss of myelin was not accompanied by a loss of oligodendrocytes, the myelinating cells. However FB1 had effects on the maturation of oligodendrocytes, as revealed by a decrease in the expression of galactocerebroside, and on the compaction of myelin, as shown by a reduction of the expression of the mnyelin/oligodendrocyte glycoprotein MOG. The content of the cytoskeletal component glial fibrillary acidic protein (GFAP) was decreased in differentiated astrocytes, exclusively, while neurons were not affected by 40 microM of FB1 applied continuously for 10 days. In summary, FB1 selectively affected glial cells. In particular, FB1 delayed oligodendrocyte development and impaired myelin formation and deposition.

Reduction in the expression of glucose transporter protein GLUT 2 in preneoplastic and neoplastic hepatic lesions and reexpression of GLUT 1 in late stages of hepatocarcinogenesis.

Expression of two important glucose transporter proteins, GLUT 2 (which is the typical glucose transporter in hepatocytes of adult liver) and the erythroid/brain type glucose transporter GLUT 1 (representing the typical glucose transporter in fetal liver parenchyma), was studied immunocytochemically during hepatocarcinogenesis in rats at different time points between 7 and 65 wk after cessation of 7-wk administration of 12 mg/kg of body weight of N-nitrosomorpholine p.o. (stop model). Foci of altered hepatocytes excessively storing glycogen (GSF) and mixed cell foci (MCF) composed of both glycogenotic and glycogen-poor cells were present at all time points studied. Seven wk after withdrawal of the carcinogen, GSF were the predominant type of focus of altered hepatocytes. Morphometrical evaluation of the focal lesions revealed that the number and volume fraction of GSF increased steadily until Wk 65. MCF were rare at 7 wk, increased slightly in number and size until Wk 37, but showed a pronounced elevation in their number and volume fraction from Wk 37 to Wk 65. In both GSF and MCF, GLUT 2 was generally decreased or partially absent at all time points. Consequently, foci of decreased GLUT 2 expression showed a steady increase in number and volume fraction from Wk 7 to Wk 65. GLUT 1 was lacking in GSF but occurred in some MCF from Wk 50 onward. The liver type glucose transporter GLUT 2 was decreased in all adenomas and hepatocellular carcinomas (HCC). In three of seven adenomas and 10 of 12 carcinomas, expression of GLUT 1 was increased compared with normal liver parenchyma. In two cases of adenoid HCC, cells of ductular formations coexpressed GLUT 2 and GLUT 1. In contrast, normal bile ducts, bile duct proliferations, and cystic cholangiomas expressed only GLUT 1. Seven of 12 HCC contained many microvessels intensely stained for GLUT 1, a phenomenon never observed in normal liver. Whenever adenoid tumor formations occurred, GLUT 1-positive microvessels were located in the immediate vicinity of these formations. Only in one HCC were such microvessels found in the absence of adenoid formations. Our studies indicate that a reduction of GLUT 2 expression occurs already in early preneoplastic hepatic foci and is maintained throughout hepatocarcinogenesis, including benign and malignant neoplasms. Reexpression of GLUT 1, however, appears in a few MCF and in the majority of adenomas and carcinomas.

Expression and developmental regulation of Ehk-1, a neuronal Elk-like receptor tyrosine kinase in brain.

Protein tyrosine kinases are pivotal in central nervous tissue development and maintenance. Here we focus on the expression of Ehk-1, a novel Elk-related receptor tyrosine kinase. Ehk-1 gene expression is observed in the developing and adult central nervous system and is highly regulated throughout development at both the messenger RNA and protein levels. Three messenger RNA transcripts of 8.5, 5.9 and 5.1 kb are detectable in the rat brain and a variety of splice possibilities have been identified. However, a major protein species of around M(r) 120,000 predominates throughout development. Ehk-1 messenger RNA and protein levels are highest in the first postnatal week. By in situ messenger RNA hybridization the gene is expressed by all neurons of the adult brain, but mostly in the hippocampus, cerebral cortex and large neurons of the deep cerebellar nuclei, as well as the Purkinje and granular cells of the cerebellum. At earlier stages of development, transcripts are most prominent in the periventricular germinal layers of the brain. Immunohistochemistry reveals a pronounced membrane associated protein expression in immature neurons. In the adult animal, peak reactivity was found in the neuropil with sparing of most perikarya. The spatial and temporal pattern of ehk-1 gene expression suggests a role in both the development and maintenance of differentiated neurons of the central nervous system.

Cycles de vie d'une femme et tabac [The health consequences of smoking for women at different stages of their life].

Women who smoke underestimate the risks of smoking on their health, especially the impact of the anti-estrogenic and toxic effects of tobacco at the different stages of their life. The risk of female infertility related to tobacco is now well-proven, as is the risk of arterial and venous thrombotic event when tobacco is associated with oral contraception. Many foetal and maternal pathologies are linked to maternal smoking. Regarding post-menopausal women, smoking is associated with an increased osteoporotic and cardio-vascular risk. Doctors are at the first line to advise women and propose them help and assistance in their quit smoking process in a way adapted to their situation.

Survival-independent function of NF-kappaB/Rel during late stages of thymocyte differentiation.

Transcription factors of the NF-kappaB/Rel family are important mediators of extracellular signals. Their implication in positive selection of thymocytes is suggested by a defective thymic development in transgenic mice that over-express IkappaB in thymocytes. These mice exhibit an accumulation of an unusually prominent population of TCRhigh/CD4/CD8 double positive cells in the thymus and a dramatic reduction of CD4+ and CD8+ cells in the periphery. The present study addresses the role of NF-kappaB in survival and differentiation processes of maturing thymocytes using IkappaB/bcl-2 and IkappaB/HY double-transgenic mice. Neither the introduction of the anti-apoptosis gene bcl-2 nor the positively selecting background in female HY transgenic mice resulted in a rescue of the maturational defects observed in the thymus of IkappaB transgenic mice. Thus, rather than promoting survival the main role of NF-kappaB/Rel proteins during positive selection of thymocytes appears to be the mediation of differentiation signals.