In vivo and in vitro development of alpha-MSH and ACTH in the embryonic and postnatal rat brain.

The appearance of immunoreactive alpha-melanotropin (alpha-MSH) and adrenocorticotropin (ACTH) during development was studied in 3 areas of the rat brain--cerebral hemispheres, midbrain and hindbrain--from embryonic day (ED) 13-14 until day 21 postnatally. The alpha-MSH content in vivo was always highest in the midbrain; a peak content at birth was followed by a transient decline and a later, higher plateau from postnatal day 7 onwards. The alpha-MSH content in the cerebral hemispheres rose progressively after birth reaching a peak at day 21. Values in the hindbrain rose at day 3 and changed relatively sue taken at ED 15-16 showed a gradual increase in alpha-MSH content over the 20 days. The alpha-MSH content of hindbrain cultures remained at constant low levels, while no alpha-MSH was detectable in cerebral hemisphere cultures. ACTH appeared in vivo earlier than alpha-MSH and was detectable in embryonic brains at ED 13-14. A transient rise was seen at ED 17-18 and major peaks at birth, day 2 and day 3, in the midbrain, hemispheres and hindbrain, respectively. In vitro, the ACTH content increased in all brain regions during the first 5 days in culture and showed no further change thereafter. Comparisons of the in vivo and in vitro development of alpha-MSH and ACTH demonstrate that (i) these two peptide systems are independent in respect to their localization and time of appearance; (ii) they undergo maturation both in vivo and in vitro; (iii) epigenetic factors, such as interactions with other neurotransmitter systems may modulate the developmental pattern of these two peptides.

Regulation of sodium transport in the cortical collecting duct : physiological role of GILZ in vitro and in vivo : characterisation of Na+ transport in the mCCDcl1 cell line

SUMMARY Regulation of sodium excretion by the kidney is a key mechanism in the long term regulation of blood pressure, and when altered it constitutes a risk factor for the appearance of arterial hypertension. Aldosterone, which secretion depends upon salt intake in the diet, is a steroid hormone that regulates sodium reabsorption in the distal part of the nephron (functional unit of the kidney) by modulating gene transcription. It has been shown that it can act synergistically with the peptidic hormone insulin through the interaction of their signalisation pathways. Our work consisted of two distinct parts: 1) the in vitro and in vivo characterisation of Glucocorticoid-Induced Leucine Zipper (GILZ) (an aldosterone-induced gene) mechanism of action; 2) the in vitro characterisation of insulin mechanism of action and its interaction with aldosterone. GILZ mRNA, coded by the TSC22D3 gene, is strongly induced by aldosterone in the cell line of principal cells of the cortical collecting duct (CCD) mpkCCDc14, suggesting that GILZ is a mediator of aldosterone response. Co-expression of GILZ and the amiloride-sensitive epithelial sodium channel ENaC in vitro in the Xenopus oocyte expression system showed that GILZ has no direct effect on the ENaC-mediated Na+ current in basal conditions. To define the role of GILZ in the kidney and in other organs (colon, heart, skin, etc.), a conditional knock-out mouse is being produced and will allow the in vivo study of its role. Previous data showed that insulin induced a transepithelial sodium transport at supraphysiological concentrations. Insulin and the insulin-like growth factor 1 (IGF-1) are able to bind to each other receptor with an affinity 50 to 100 times lower than to their cognate receptor. Our starting hypothesis was that the insulin effect observed at these supraphysiological concentrations is actually mediated by the IGF receptor type 1 (IGF-1R). In a new cell line that presents all the characteristics of the principal cells of the CCD (mCCDc11) we have shown that both insulin and IGF-1 induce a physiologically significant increase of Na+ transport through the activation of IGF-1R. Aldosterone and insulin/IGF-1 have an additive effect on Na+ transport, through the activation of the PI3-kinase (PI3-K) pathway and the phosphorylation of the serum- and glucocorticoid-induced kinase 1 (Sgk1) by the IGF-1R, and the induction of Sgk1 expression by aldosterone. Thus, Sgk1 integrates IGF-1/insulin and aldosterone effects. We suggest that IGF-1 is physiologically relevant in the modulation of sodium balance, while insulin can only regulate Na+ transport at supraphysiological conditions. Both hormones would bind to the IGF-1R and induce Na+ transport by activating the PI3-K PDK1/2 - Sgk1 pathway. We have shown for the first time that Sgk1 is expressed and phosphorylated in principal cells of the CCD in basal conditions, although the mechanism that maintains Sgk1 phosphorylation is not known. This new role for IGF-1 suggests that it could be a salt susceptibility gene. In effect, IGF-1 stimulates Na+ and water transport in the kidney in vivo. Moreover, 35 % of the acromegalic patients (overproduction of growth hormone and IGF-1) are hypertensives (higher proportion than in normal population), and genetic analysis suggest a link between the IGF-1 gene locus and blood pressure. RÉSUMÉ La régulation de l'excrétion rénale de sodium (Na+) joue un rôle principal dans le contrôle à long terme de la pression sanguine, et ses altérations constituent un facteur de risque de l'apparition d'une hypertension artérielle. L'aldosterone, dont la sécrétion dépend de l'apport en sel dans la diète, est une hormone stéroïdienne qui régule la réabsorption de Na+ dans la partie distale du nephron (unité fonctionnelle du rein) en contrôlant la transcription de gènes. Elle peut agir de façon synergistique avec l'hormone peptidique insuline, probablement via l'interaction de leurs voies de signalisation cellulaire. Le but de notre travail comportait deux volets: 1) caractériser in vitro et in vivo le mécanisme d'action du Glucocorticoid Induced Leucine Zipper (GILZ) (un gène induit par l'aldosterone); 2) caractériser in vitro le mécanisme d'action de l'insuline et son interaction avec l'aldosterone. L'ARNm de GILZ, codé par le gène TSC22D3, est induit par l'aldosterone dans la lignée cellulaire de cellules principales du tubule collecteur cortical (CCD) mpkCCDc14, suggérant que GILZ est un médiateur potentiel de la réponse à l'aldosterone. La co-expression in vitro de GILZ et du canal à Na+ sensible à l'amiloride ENaC dans le système d'expression de l'oocyte de Xénope a montré que GILZ n'a pas d'effet sur les courants sodiques véhiculées par ENaC en conditions basales. Une souris knock-out conditionnelle de GILZ est en train d'être produite et permettra l'étude in vivo de son rôle dans le rein et d'autres organes. Des expériences préliminaires ont montré que l'insuline induit un transport transépithelial de Na+ à des concentrations supraphysiologiques. L'insuline et l'insulin-like growth factor 1 (IGF-1) peuvent se lier à leurs récepteurs réciproques avec une affinité 50 à 100 fois moindre qu'à leur propre récepteur. Nous avons donc proposé que l'effet de l'insuline soit médié par le récepteur à l'IGF type 1 (IGF-1R). Dans une nouvelle lignée cellulaire qui présente toutes les caractéristiques des cellules principales du CCD (mCCDc11) nous avons montré que les deux hormones induisent une augmentation physiologiquement significative du transport du Na+ par l'activation des IGF-1 R. Aldosterone et insuline/IGF-1 ont un effet additif sur le transport de Na+, via l'activation de la voie de la PI3-kinase et la phosphorylation de la serum- and glucocorticoid-induced kinase 1 (Sgk1) par l'IGF-1R, dont l'expression est induite par l'aldosterone. Sgk1 intègre les effets de l'insuline et l'aldosterone. Nous proposons que l'IGF-1 joue un rôle dans la modulation physiologique de la balance sodique, tandis que l'insuline régule le transport de Na+ à des concentrations supraphysiologiques. Les deux hormones agissent en se liant à l'IGF-1R et induisent le transport de Na+ en activant la cascade de signalisation PI3-K - PDK1/2 - Sgk1. Nous avons montré pour la première fois que Sgk1 est exprimée et phosphorylée dans des conditions basales dans les cellules principales du CCD, mais le mécanisme qui maintient sa phosphorylation n'est pas connu. Ce nouveau rôle pour l'IGF-1 suggère qu'il pourrait être un gène impliqué de susceptibilité au sel. Aussi, l'IGF-1 stimule le transport rénal de Na+ in vivo. De plus, 35 % des patients atteints d'acromégalie (surproduction d'hormone de croissance et d'IGF-1) sont hypertensifs (prévalence plus élevée que la population normale), et des analyses génétiques suggèrent un lien entre le locus du gène de l'IGF-1 et la pression sanguine. RÉSUMÉ GRAND PUBLIC Nos ancêtres se sont génétiquement adaptés pendant des centaines de millénaires à un environnement pauvre en sel (chlorure de sodium) dans la savane équatoriale, où ils consommaient moins de 0,1 gramme de sel par jour. On a commencé à ajouter du sel aux aliments avec l'apparition de l'agriculture (il y a 5000 à 10000 années), et aujourd'hui une diète omnivore, qui inclut des plats préparés, contient plusieurs fois la quantité de sodium nécessaire pour notre fonction physiologique normale (environ 10 grammes par jour). Le corps garde sa concentration constante dans le sang en s'adaptant à une consommation très variable de sel. Pour ceci, il module son excrétion soit directement, soit en sécrétant des hormones régulatrices. Le rein joue un rôle principal dans cette régulation puisque l'excrétion urinaire de sel change selon la diète et peut aller d'une quantité dérisoire à plus de 36 grammes par jour. L'attention qu'on prête au sel est liée à sa relation avec l'hypertension essentielle. Ainsi, le contrôle rénal de l'excrétion de sodium et d'eau est le principal mécanisme dans la régulation de la pression sanguine, et une ingestion excessive de sel pourrait être l'un des facteurs-clé déclenchant l'apparition d'un phénotype hypertensif. L'hormone aldosterone diminue l'excrétion de sodium par le rein en modulant l'expression de gènes qui pourraient être impliqués dans la sensibilité au sel. Dans une lignée cellulaire de rein l'expression du gène TSC22D3, qui se traduit en la protéine Glucocorticoid Induced Leucine Zipper (GILZ), est fortement induite par l'aldosterone. Ceci suggère que GILZ est un médiateur potentiel de l'effet de l'aldosterone, et pourrait être impliqué dans la sensibilité au sel. Pour analyser la fonction de GILZ dans le rein plusieurs approches ont été utilisées. Par exemple, une souris dans laquelle GILZ est spécifiquement inactivé dans le rein est en train d'être produite et permettra l'étude du rôle de GILZ dans l'organisme. De plus, on a montré que GILZ, en conditions basales, n'a pas d'effet direct sur la protéine transportant le sodium à travers la membrane des cellules, le canal sodique épithélial ENaC. On a aussi essayé de trouver des protéines qui interagissent directement avec GILZ utilisant une technique appelée du « double-hybride dans la levure », mais aucun candidat n'a émergé. Des études ont montré que, à de hautes concentrations, l'insuline peut aussi diminuer l'excrétion de sodium. A ces concentrations, elle peut activer son récepteur spécifique, mais aussi le récepteur d'une autre hormone, l'Insulin-Like Growth Factor 1 (IGF-1). En plus, l'infusion d'IGF-1 augmente la rétention rénale de sodium et d'eau, et des mutations du gène codant pour l'IGF-1 sont liées aux différents niveaux de pression sanguine. On a utilisé une nouvelle lignée cellulaire de rein développée dans notre laboratoire, appelée mCCDc11, pour analyser l'importance relative des deux hormones dans l'induction du transport de sodium. On a montré que les deux hormones induisent une augmentation significative du transport de sodium par l'activation de récepteurs à l'IGF-1 et non du récepteur à l'insuline. On a montré qu'à l'intérieur de la cellule leur activation induit une augmentation du transport sodique par le biais du canal ENaC en modifiant la quantité de phosphates fixés sur la protéine Serumand Glucocorticoid-induced Kinase 1 (Sgk1). On a finalement montré que l'IGF-1 et l'aldosterone ont un effet additif sur le transport de sodium en agissant toutes les deux sur Sgk1, qui intègre leurs effets dans le contrôle du transport de sodium dans le rein.

Selective inhibition of JNK with a peptide inhibitor attenuates pain hypersensitivity and tumor growth in a mouse skin cancer pain model.

Cancer pain significantly affects the quality of cancer patients, and current treatments for this pain are limited. C-Jun N-terminal kinase (JNK) has been implicated in tumor growth and neuropathic pain sensitization. We investigated the role of JNK in cancer pain and tumor growth in a skin cancer pain model. Injection of luciferase-transfected B16-Fluc melanoma cells into a hindpaw of mouse induced robust tumor growth, as indicated by increase in paw volume and fluorescence intensity. Pain hypersensitivity in this model developed rapidly (<5 days) and reached a peak in 2 weeks, and was characterized by mechanical allodynia and heat hyperalgesia. Tumor growth was associated with JNK activation in tumor mass, dorsal root ganglion (DRG), and spinal cord and a peripheral neuropathy, such as loss of nerve fibers in the hindpaw skin and induction of ATF-3 expression in DRG neurons. Repeated systemic injections of D-JNKI-1 (6 mg/kg, i.p.), a selective and cell-permeable peptide inhibitor of JNK, produced an accumulative inhibition of mechanical allodynia and heat hyperalgesia. A bolus spinal injection of D-JNKI-1 also inhibited mechanical allodynia. Further, JNK inhibition suppressed tumor growth in vivo and melanoma cell proliferation in vitro. In contrast, repeated injections of morphine (5 mg/kg), a commonly used analgesic for terminal cancer, produced analgesic tolerance after 1 day and did not inhibit tumor growth. Our data reveal a marked peripheral neuropathy in this skin cancer model and important roles of the JNK pathway in cancer pain development and tumor growth. JNK inhibitors such as D-JNKI-1 may be used to treat cancer pain.

In vitro organogenesis from internodal segments of adult sweet orange plants

The objective of this work was to optimize in vitro plant regeneration via organogenesis from tissues of adult 'Hamlin', 'Pêra', and 'Valência' sweet orange plants. Explants were grown in EME culture medium with different concentrations of 6-benzylaminopurine (BAP) and naphthaleneacetic acid (NAA), at 27ºC in the absence of light for 50 days, followed by a 16-hour photoperiod for 20 days. Regeneration was assessed 50 and 70 days after in vitro culture. Organogenesis in cultivars Hamlin and Valência was promoted by EME supplemented with BAP, while NAA showed no apparent effect.

Differential gene expression in the pathogenic dermatophyte Arthroderma benhamiae in vitro versus during infection.

Although dermatophytes are the most common agents of superficial mycoses in humans and animals, the molecular basis of the pathogenicity of these fungi is largely unknown. In vitro digestion of keratin by dermatophytes is associated with the secretion of multiple proteases, which are assumed to be responsible for their particular specialization to colonize and degrade keratinized host structures during infection. To investigate the role of individual secreted proteases in dermatophytosis, a guinea pig infection model was established for the zoophilic dermatophyte Arthroderma benhamiae, which causes highly inflammatory cutaneous infections in humans and rodents. By use of a cDNA microarray covering approximately 20-25 % of the A. benhamiae genome and containing sequences of at least 23 protease genes, we revealed a distinct in vivo protease gene expression profile in the fungal cells, which was surprisingly different from the pattern elicited during in vitro growth on keratin. Instead of the major in vitro -expressed proteases, others were activated specifically during infection. These enzymes are therefore suggested to fulfil important functions that are not exclusively associated with the degradation of keratin. Most notably, the gene encoding the serine protease subtilisin 6, which is a known major allergen in the related dermatophyte Trichophyton rubrum and putatively linked to host inflammation, was found to be the most strongly upregulated gene during infection. In addition, our approach identified other candidate pathogenicity-related factors in A. benhamiae, such as genes encoding key enzymes of the glyoxylate cycle and an opsin-related protein. Our work provides what we believe to be the first broad-scale gene expression profile in human pathogenic dermatophytes during infection, and points to putative virulence-associated mechanisms that make these micro-organisms the most successful aetiological agents of superficial mycoses.

Short-term storage in vitro and large-scale propagation of grapevine genotypes

The objective of this work was to evaluate the large-scale propagation of grapevine genotypes after short-term storage in vitro. Microshoots from ten grapevine genotypes were used. The following storage temperatures were evaluated: 10, 20, and 25°C. After short-term storage, the shoots were propagated in up to five successive subcultures, to assess the large-scale propagation of the germplasm maintained under conditions of minimal growth. The propagated shoots were rooted in different concentrations of indolbutiric acid (IBA) and acclimatized in greenhouse. The best temperature for short-term storage in vitro and survival of the genotypes was 20°C. In the propagation phase, the highest number of shoots per explant was found in the subcultures 4 and 5, with averages of 4.9 and 4.8 shoots per explant, respectively. In the rooting phase, the best results for number of roots were obtained using a culture medium supplemented with 0.4 µmol L-1 of IBA, with an average of three roots per shoot. During the acclimation phase, a survival rate higher than 95% was achieved after 30 days in the greenhouse. Grapevine genotypes maintained for six months in vitro, at 20ºC, can be micropropagated in large scale.

Nonactivated versus thrombin-activated platelets on wound healing and fibroblast-to-myofibroblast differentiation in vivo and in vitro.

Background: Platelet preparations for tissue healing are usually preactivated before application to deliver concentrated growth factors. In this study, the authors investigated the differences between nonactivated and thrombin-activated platelets in wound healing.Methods: The healing effects (i.e., wound closure, myofibroblast formation, and angiogenesis) of nonactivated and thrombin-activated platelets were compared in experimental wounds in diabetic (db/db) animals. In vitro, fibroblast phenotype and function were tested in response to platelets and activated platelets. No treatment served as a negative control.Results: Wounds treated with platelets reached 90 percent closure after 15 days, faster than activated platelets (26 days), and with higher levels of myofibroblasts and angiogenesis. In vitro, platelets enhanced cell migration and induced twofold higher myofibroblast differentiation and contraction compared with activated platelets.Conclusions: Platelets stimulate wound healing more efficiently compared with activated platelets by enhancing fibroblast differentiation and contractile function. Similar levels of growth factors may induce different biological effects when delivered "on demand" rather than in an initial bolus. (Plast. Reconstr. Surg. 129: 46e, 2012.)

Effect of RasGAP N2 fragment-derived peptide on tumor growth in mice.

Peptides that interfere with the natural resistance of cancer cells to genotoxin-induced apoptosis may improve the efficacy of anticancer regimens. We have previously reported that a cell-permeable RasGAP-derived peptide (TAT-RasGAP(317-326)) specifically sensitizes tumor cells to genotoxin-induced apoptosis in vitro. Here, we examined the in vivo stability of a protease-resistant D-form of the peptide, RI.TAT-RasGAP(317-326), and its effect on tumor growth in nude mice bearing subcutaneous human colon cancer HCT116 xenograft tumors. After intraperitoneal injection, RI.TAT-RasGAP(317-326) persisted in the blood of nude mice for more than 1 hour and was detectable in various tissues and subcutaneous tumors. Tumor-bearing mice treated daily for 7 days with RI.TAT-RasGAP(317-326) (1.65 mg/kg body weight) and cisplatin (0.5 mg/kg body weight) or doxorubicin (0.25 mg/kg body weight) displayed reduced tumor growth compared with those treated with either genotoxin alone (n = 5-7 mice per group; P = .004 and P = .005, respectively; repeated measures analysis of variance [ANOVA, two-sided]). This ability of the RI.TAT-RasGAP(317-326) peptide to enhance the tumor growth inhibitory effect of cisplatin was still observed at peptide doses that were at least 150-fold lower than the dose lethal to 50% of mice. These findings provide the proof of principle that RI.TAT-RasGAP(317-326) may be useful for improving the efficacy of chemotherapy in patients.

In vitro selection of bacteria with potential for use as probiotics in marine shrimp culture

The objective of this work was to isolate strains of lactic acid bacteria with probiotic potential from the digestive tract of marine shrimp (Litopenaeus vannamei), and to carry out in vitro selection based on multiple characters. The ideotype (ideal proposed strain) was defined by the highest averages for the traits maximum growth velocity, final count of viable cells, and inhibition halo against nine freshwater and marine pathogens, and by the lowest averages for the traits duplication time and resistance of strains to NaCl (1.5 and 3%), pH (6, 8, and 9), and biliary salts (5%). Mahalanobis distance (D²) was estimated among the evaluated strains, and the best ones were those with the shortest distances to the ideotype. Ten bacterial strains were isolated and biochemically identified as Lactobacillus plantarum (3), L. brevis (3), Weissella confusa (2), Lactococcus lactis (1), and L. delbrueckii (1). Lactobacillus plantarum strains showed a wide spectrum of action and the largest inhibition halos against pathogens, both Gram-positive and negative, high growth rate, and tolerance to all evaluated parameters. In relation to ideotype, L. plantarum showed the lowest Mahalanobis (D²) distance, followed by the strains of W. confusa, L. brevis, L. lactis, and L. delbrueckii. Among the analyzed bacterial strains, those of Lactobacillus plantarum have the greatest potential for use as a probiotic for marine shrimp.

Hormonal protocols for in vitro production of Zebu and taurine embryos

The objective of this work was to evaluate the effects of hormonal synchronization protocols, associated or not with follicular development stimulation, on the recovery of oocytes and on in vitro production of Bos indicus and B. taurus embryos, in different seasons. Ultrasound-guided follicular aspirations (n=237) were performed without pre-treatment (G1, control group) and after follicular wave synchronization (G2), or after follicular wave synchronization and follicle growth induction (G3). Bos indicus produced more oocytes and embryos than B. taurus (18.7±0.9 vs. 11.9±0.6 oocytes and 4.8±0.3 vs. 2.1±0.2 embryos). On average, oocyte and embryo yields were higher in G3 than in G2, and both were greater than in G1, which lead to a higher conversion of oocytes to embryos in these treatments. The hot or the cold season did not affect the B. indicus outcomes, whereas, in B. taurus, both oocyte recovery and embryo production were higher in the cold season. Follicular wave synchronization improves ovum pick-up and in vitro production of embryos in both cattle subspecies evaluated.

Identification of in vitro HSC fate regulators by differential lipid raft clustering.

Most hematopoietic stem cells (HSC) in the bone marrow reside in a quiescent state and occasionally enter the cell cycle upon cytokine-induced activation. Although the mechanisms regulating HSC quiescence and activation remain poorly defined, recent studies have revealed a role of lipid raft clustering (LRC) in HSC activation. Here, we tested the hypothesis that changes in lipid raft distribution could serve as an indicator of the quiescent and activated state of HSCs in response to putative niche signals. A semi-automated image analysis tool was developed to map the presence or absence of lipid raft clusters in live HSCs cultured for just one hour in serum-free medium supplemented with stem cell factor (SCF). By screening the ability of 19 protein candidates to alter lipid raft dynamics, we identified six factors that induced either a marked decrease (Wnt5a, Wnt3a and Osteopontin) or increase (IL3, IL6 and VEGF) in LRC. Cell cycle kinetics of single HSCs exposed to these factors revealed a correlation of LRC dynamics and proliferation kinetics: factors that decreased LRC slowed down cell cycle kinetics, while factors that increased LRC led to faster and more synchronous cycling. The possibility of identifying, by LRC analysis at very early time points, whether a stem cell is activated and possibly committed upon exposure to a signaling cue of interest could open up new avenues for large-scale screening efforts.

Dopamine affects parvalbumin expression during cortical development in vitro.

This study was undertaken to determine how dopamine influences cortical development. It focused on morphogenesis of GABAergic neurons that contained the calcium-binding protein parvalbumin (PV). Organotypic slices of frontoparietal cortex were taken from neonatal rats, cultured with or without dopamine, harvested daily (4-30 d), and immunostained for parvalbumin. Expression of parvalbumin occurred in the same regional and laminar sequence as in vivo. Expression in cingulate and entorhinal preceded that in lateral frontoparietal cortices. Laminar expression progressed from layer V to VI and finally II-IV. Somal labeling preceded fiber labeling by 2 d. Dopamine accelerated PV expression. In treated slices, a dense band of PV-immunoreactive neurons appeared in layer V at 7 d in vitro (DIV), and in all layers of frontoparietal cortex at 14 DIV, whereas in control slices such labeling did not appear until 14 and 21 DIV, respectively. The laminar distribution and dendritic branching of PV-immunoreactive neurons were quantified. More labeled neurons were in the superficial layers, and their dendritic arborizations were significantly increased by dopamine. Treatment with a D1 receptor agonist had little effect, whereas a D2 agonist mimicked dopamine's effects. Likewise, the D2 but not the D1 antagonist blocked dopamine-induced changes, indicating that they were mediated primarily by D2 receptors. Parvalbumin expression was accelerated by dopaminergic reinnervation of cortical slices that were cocultured with mesencephalic slices. Coapplication of the glutamate NMDA receptor antagonist MK801 or AP5 blocked dopamine-induced increases in dendritic branching, suggesting that changes were mediated partly by interaction with glutamate to alter cortical excitability.

Imaging techniques applied for detection of secretion, transgene delivery and G proteincoupled receptors in reproductive and endocrine cells in vivo and in vitro

Post-testicular sperm maturation occurs in the epididymis. The ion concentration and proteins secreted into the epididymal lumen, together with testicular factors, are believed to be responsible for the maturation of spermatozoa. Disruption of the maturation of spermatozoa in the epididymis provides a promising strategy for generating a male contraceptive. However, little is known about the proteins involved. For drug development, it is also essential to have tools to study the function of these proteins in vitro. One approach for screening novel targets is to study the secretory products of the epididymis or the G protein-coupled receptors (GPCRs) that are involved in the maturation process of the spermatozoa. The modified Ca2+ imaging technique to monitor release from PC12 pheochromocytoma cells can also be applied to monitor secretory products involved in the maturational processes of spermatozoa. PC12 pheochromocytoma cells were chosen for evaluation of this technique as they release catecholamines from their cell body, thus behaving like endocrine secretory cells. The results of the study demonstrate that depolarisation of nerve growth factor -differentiated PC12 cells releases factors which activate nearby randomly distributed HEL erythroleukemia cells. Thus, during the release process, the ligands reach concentrations high enough to activate receptors even in cells some distance from the release site. This suggests that communication between randomly dispersed cells is possible even if the actual quantities of transmitter released are extremely small. The development of a novel method to analyse GPCR-dependent Ca2+ signalling in living slices of mouse caput epididymis is an additional tool for screening for drug targets. By this technique it was possible to analyse functional GPCRs in the epithelial cells of the ductus epididymis. The results revealed that, both P2X- and P2Y-type purinergic receptors are responsible for the rapid and transient Ca2+ signal detected in the epithelial cells of caput epididymides. Immunohistochemical and reverse transcriptase-polymerase chain reaction (RTPCR) analyses showed the expression of at least P2X1, P2X2, P2X4 and P2X7, and P2Y1 and P2Y2 receptors in the epididymis. Searching for epididymis-specific promoters for transgene delivery into the epididymis is of key importance for the development of specific models for drug development. We used EGFP as the reporter gene to identify proper promoters to deliver transgenes into the epithelial cells of the mouse epididymis in vivo. Our results revealed that the 5.0 kb murine Glutathione peroxidase 5 (GPX5) promoter can be used to target transgene expression into the epididymis while the 3.8 kb Cysteine-rich secretory protein-1 (CRISP-1) promoter can be used to target transgene expression into the testis. Although the visualisation of EGFP in living cells in culture usually poses few problems, the detection of EGFP in tissue sections can be more difficult because soluble EGFP molecules can be lost if the cell membrane is damaged by freezing, sectioning, or permeabilisation. Furthermore, the fluorescence of EGFP is dependent on its conformation. Therefore, fixation protocols that immobilise EGFP may also destroy its usefulness as a fluorescent reporter. We therefore developed a novel tissue preparation and preservation techniques for EGFP. In addition, fluorescence spectrophotometry with epididymal epithelial cells in suspension revealed the expression of functional purinergic, adrenergic, cholinergic and bradykinin receptors in these cell lines (mE-Cap27 and mE-Cap28). In conclusion, we developed new tools for studying the role of the epididymis in sperm maturation. We developed a new technique to analyse GPCR dependent Ca2+ signalling in living slices of mouse caput epididymis. In addition, we improved the method of detecting reporter gene expression. Furthermore, we characterised two epididymis-specific gene promoters, analysed the expression of GPCRs in epididymal epithelial cells and developed a novel technique for measurement of secretion from cells.

Cytokine-induced sleep: Neurons respond to TNF with production of chemokines and increased expression of Homer1a in vitro.

Interactions of neurons with microglia may play a dominant role in sleep regulation. TNF may exert its somnogeneic effects by promoting attraction of microglia and their processes to the vicinity of dendrites and synapses. We found TNF to stimulate neurons (i) to produce CCL2, CCL7 and CXCL10, chemokines acting on mononuclear phagocytes and (ii) to stimulate the expression of the macrophage colony stimulating factor (M-CSF/Csf1), which leads to elongation of microglia processes. TNF may also act on neurons by affecting the expression of genes essential in sleep-wake behavior. The neuronal expression of Homer1a mRNA, increases during spontaneous and enforced periods of wakefulness. Mice with a deletion of Homer1a show a reduced wakefulness with increased non-rapid eye movement (NREM) sleep during the dark period. Recently the TNF-dependent increase of NREM sleep in the dark period of mice with CD40-induced immune activation was found to be associated with decreased expression of Homer1a. In the present study we investigated the effects of TNF and IL-1β on gene expression in cultures of the neuronal cell line HT22 and cortical neurons. TNF slightly increased the expression of Homer1a and IL-1β profoundly enhanced the expression of Early growth response 2 (Egr2). The data presented here indicate that the decreased expression of Homer1a, which was found in the dark period of mice with CD40-induced increase of NREM sleep is not due to inhibitory effects of TNF and IL-1β on the expression of Homer1a in neurons.