First Hours Of Culture Research Articles

Cluster-Assembled Zirconia Substrates Accelerate the Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells

Due to their high mechanical strength and good biocompatibility, nanostructured zirconia surfaces (ns-ZrOx) are widely used for bio-applications. Through supersonic cluster beam deposition, we produced ZrOx films with controllable roughness at the nanoscale, mimicking the morphological and topographical properties of the extracellular matrix. We show that a 20 nm ns-ZrOx surface accelerates the osteogenic differentiation of human bone marrow-derived MSCs (bMSCs) by increasing the deposition of calcium in the extracellular matrix and upregulating some osteogenic differentiation markers. bMSCs seeded on 20 nm ns-ZrOx show randomly oriented actin fibers, changes in nuclear morphology, and a reduction in mitochondrial transmembrane potential when compared to the cells cultured on flat zirconia (flat-ZrO2) substrates and glass coverslips used as controls. Additionally, an increase in ROS, known to promote osteogenesis, was detected after 24 h of culture on 20 nm ns-ZrOx. All the modifications induced by the ns-ZrOx surface are rescued after the first hours of culture. We propose that ns-ZrOx-induced cytoskeletal remodeling transmits signals generated by the extracellular environment to the nucleus, with the consequent modulation of the expression of genes controlling cell fate.

Low-intensity continuous ultrasound to inhibit cancer cell migration.

In recent years, it has been verified that collective cell migration is a fundamental step in tumor spreading and metastatic processes. In this paper, we demonstrate for the first time how low-intensity ultrasound produces long-term inhibition of collective migration of epithelial cancer cells in wound healing processes. In particular, we show how pancreatic tumor cells, PANC-1, grown as monolayers in vitro respond to these waves at frequencies close to 1MHz and low intensities (<100mWcm-2) for 48-72h of culture after some minutes of a single ultrasound irradiation. This new strategy opens a new line of action to block the spread of malignant cells in cancer processes. Despite relevant spatial variations of the acoustic pressure amplitude induced in the assay, the cells behave as a whole, showing a collective dynamic response to acoustic performance. Experiments carried out with samples without previous starving showed remarkable effects of the LICUs from the first hours of culture, more prominent than those with experiments with monolayers subjected to fasting prior to the experiments. This new strategy to control cell migration demonstrating the effectiveness of LICUS on not starved cells opens a new line of action to study effects of in vivo ultrasonic actuation on tumor tissues with malignant cells. This is a proof-of-concept study to demonstrate the physical effects of ultrasound stimulation on tumor cell migration. An in-depth biological study of the effects of ultrasounds and underlying biological mechanisms is on-going but out of the scope of this article.

The Role of MAPK3/1 and AKT in the Acquisition of High Meiotic and Developmental Competence of Porcine Oocytes Cultured In Vitro in FLI Medium.

The developmental potential of porcine oocytes cultured in vitro was remarkably enhanced in a medium containing FGF2, LIF and IGF1 (FLI) when compared to a medium supplemented with gonadotropins and EGF (control). We analyzed the molecular background of the enhanced oocyte quality by comparing the time course of MAPK3/1 and AKT activation, and the expression of genes controlled by these kinases in cumulus-oocyte complexes (COCs) cultured in FLI and the control medium. The pattern of MAPK3/1 activation in COCs was very similar in both media, except for a robust increase in MAPK3/1 phosphorylation during the first hour of culture in the FLI medium. The COCs cultured in the FLI medium exhibited significantly higher activity of AKT than in the control medium from the beginning up to 16 h of culture; afterwards a deregulation of AKT activity occurred in the FLI medium, which was not observed in the control medium. The expression of cumulus cell genes controlled by both kinases was also modulated in the FLI medium, and in particular the genes related to cumulus-expansion, signaling, apoptosis, antioxidants, cell-to-cell communication, proliferation, and translation were significantly overexpressed. Collectively, these data indicate that both MAPK3/1 and AKT are implicated in the enhanced quality of oocytes cultured in FLI medium.

Antibacterial effect of hyaluronan/chitosan nanofilm in the initial adhesion of Pseudomonas aeruginosa wild type, and IV pili and LPS mutant strains

Materials coated with nanofilms obtained by polyelectrolytes assembled layer-by-layer are promising as antibacterial surfaces. Nanofilms of hyaluronan/chitosan (HA/CHI) have satisfactory antibacterial effects against human pathogenic bacteria, such as Escherichia coli and Staphylococcus aureus, but are not efficient for Pseudomonas aeruginosa. To better understand the interaction between P. aeruginosa and HA/CHI nanofilms, this work evaluates the role of type IV pili (T4P) and lipopolysaccharide (LPS) structures in the initial adhesion of this opportunistic pathogen on a bioinert silica substrate and a 20 nm-thick HA/CHI nanofilm using two genetically modified strains: ΔpilA and LPS–. ΔpilA cannot twitch and LPS– lacks the O-antigen structures of LPS molecules. Our results indicate that each strain presented a different adhesion on both surfaces according to their particular features. For the silica substrate, the PA14 wild-type strain exhibited motility because formed interconnected rings, as a result of cell motility; however, in the case of ΔpilA strain, nonconfluent aggregates were generated by the lack of twitching motility in cells. For the LPS– strain, bacteria completely covered the silica, demonstrating a significantly higher rate of adhesion and growth when compared to the other strains. The HA/CHI nanofilm produced membrane damage and lysis on all the used strains, confirming its antibacterial effect during the first hours of culture. However, the lack of LPS seemed to protect the bacteria partially from the HA/CHI nanofilm, probably due to their autoaggregative phenotype, preventing the exposure of part of the cells to the nanofilm. Moreover, in the case of PA14 wild-type, cells were able to adhere on top of the lysed bacteria, using them as a new surface. This behavior may explain why this antibacterial material has not been so efficient against P. aeruginosa for longer culture times.

Membrane Elastic Properties During Neural Precursor Cell Differentiation.

Neural precursor cells differentiate into several cell types that display distinct functions. However, little is known about how cell surface mechanics vary during the differentiation process. Here, by precisely measuring membrane tension and bending modulus, we map their variations and correlate them with changes in neural precursor cell morphology along their distinct differentiation fates. Both cells maintained in culture as neural precursors as well as those plated in neurobasal medium reveal a decrease in membrane tension over the first hours of culture followed by stabilization, with no change in bending modulus. During astrocyte differentiation, membrane tension initially decreases and then increases after 72 h, accompanied by consolidation of glial fibrillary acidic protein expression and striking actin reorganization, while bending modulus increases following observed alterations. For oligodendrocytes, the changes in membrane tension are less abrupt over the first hours, but their values subsequently decrease, correlating with a shift from oligodendrocyte marker O4 to myelin basic protein expressions and a remarkable actin reorganization, while bending modulus remains constant. Oligodendrocytes at later differentiation stages show membrane vesicles with similar membrane tension but higher bending modulus as compared to the cell surface. Altogether, our results display an entire spectrum of how membrane elastic properties are varying, thus contributing to a better understanding of neural differentiation from a mechanobiological perspective.

Partial inhibition of nitric oxide synthase activity stimulates the nuclear maturation progression of bovine cumulus-oocyte complex in vitro in the presence of hemisections of the follicular walls.

This study aimed to assess the effects of the inhibition of nitric oxide synthase (NOS) on events that modulate bovine in vitro oocyte maturation. Cumulus-oocyte complexes (COCs) were cultured with hemisections (HSs) of the follicular walls in a maturation medium supplemented with different concentrations (0.1-10.0 mM) of Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME). Controls consisted of COCs cultured in the presence (+HSs) or absence of HSs (-HSs) with no additional l-NAME supplementation. The following parameters were assessed: oocyte nuclear maturation stage; cumulus cell (CC) membrane integrity; nitrate/nitrite, progesterone, and estradiol concentrations in the culture medium at 22 h of cultivation; and the concentrations of cGMP and cAMP in COCs during the first hour of maturation. The addition of 1.0 mM l-NAME increased the percentage of oocytes that reached metaphase II (MII) and the percentage of intact CCs (P < 0.05). All l-NAME concentrations reduced the nitrate/nitrite concentrations (P < 0.05), but none affected steroid concentrations compared with control +HSs (P > 0.05). The addition of 1.0 mM l-NAME reduced cGMP concentrations at 3 h and increased cAMP concentrations in the first hour of culture (P < 0.05). Our findings suggest that the NOS/NO/cGMP pathway participates in meiosis progression (MI to MII) of the bovine oocytes matured in vitro in the presence of hemisections of the follicular walls. Lastly, the mechanisms that lead to the progression of meiosis after NOS inhibition do not involve changes in steroid production.

Hormonal and gene dynamics in de novo shoot meristem formation during adventitious caulogenesis in cotyledons of Pinus pinea

Key messageSeveral members of WOX and KNOX gene families and several plant growth regulators, basically cytokinins and auxins, play a key role during adventitious caulogenesis in the conifer Pinus pinea.Similar to Arabidopsis thaliana, Pinus pinea shoot organogenesis is a multistep process. However, there are key differences between both species, which may alter the underlying physiological and genetic programs. It is unknown if the genic expression models during angiosperm development may be applicable to conifers. In this work, an analysis of the endogenous content of different plant growth regulators and the expression of genes putatively involved in adventitious caulogenesis in P. pinea cotyledons was conducted. A multivariate analysis of both datasets was also realized through partial least squares regression and principal component analysis to obtain an integral vision of the mechanisms involved in caulogenesis in P. pinea. Analyses show that cotyledons cultured in the presence of benzyladenine during long times (2–6 days) cluster separately from the rest of the samples, suggesting that the benzyladenine increase observed during the first hours of culture is sufficient to trigger the caulogenic response through the activation of specific developmental programs. In particular, the most relevant factors involved in this process are the cytokinins trans-zeatin, dihydrozeatin, trans-zeatin riboside and isopentenyl adenosine; the auxin indoleacetic acid; and the genes PpWUS, PpWOX5, PpKN2, PpKN3 and PipiRR1. WUS is functional in pines and has an important role in caulogenesis. Interestingly, WOX5 also seems to participate in the process, although its specific role has not been determined.

Cystathionine Promotes the Proliferation of Human Astrocytoma U373 Cells.

In certain cancers, accumulation of cystathionine has been observed. The present study investigated the effect of cystathionine on astrocytoma (U373) cell proliferation, the activity of γ-cystathionase (CTH) and changes in thiols levels. The methods used in the study included cytotoxicity assay, crystal violet staining method, CTH activity assay and reverse phase-high performance liquid chromatography (RP-HPLC). The addition of cystathionine to the culture medium resulted in an increase of cystathionine level in U373 cells after 24 h of culture. Reduction of intracellular cystathionine level after 48 and 72 h of culture was associated with increased L-cysteine and L-cystine levels and stimulation of cell proliferation. Interestingly, a decrease in intracellular L-cysteine and L-cystine levels during the first hours of culture was observed. Elevated levels of cystathionine resulted in increased U373 cell proliferation by increasing the L-cysteine levels and GSH/GSSG ratio (especially after 72 h of the culture), but not with a simultaneous increase in the levels of total glutathione.

Nanotextured titanium surfaces stimulate spreading, migration, and growth of rat mast cells.

Titanium is a biomaterial widely used in dental and orthopedic implants. Since tissue-implant interactions occur at the nanoscale level, nanotextured titanium surfaces may affect cellular activity and modulate the tissue response that occurs at the tissue-implant interface. Therefore, the characterization of diverse cell types in response to titanium surfaces with nanotopography is important for the rational design of implants. Mast cells are multifunctional cells of the immune system that release a range of chemical mediators involved in the inflammatory response that occurs at the tissue-implant interface. Therefore, the aim of this study was to investigate the effects of the nanotopography of titanium surfaces on the physiology of mast cells. The results show that the nanotopography of titanium surfaces promoted the spreading of mast cells, which was accompanied by the reorganization of the cytoskeleton. Also, the nanotopography of titanium surfaces enhanced cell migration and cell growth, but did not alter the number of adherent cells in first hours of culture or affect focal adhesions and mediator release. Thus, the results show that nanotopography of titanium surfaces can affect mast cell physiology, and represents an improved strategy for the rational production of surfaces that stimulate tissue integration with the titanium implants. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2150-2161, 2017.

Peripheral mineralization of a 3D biodegradable tubular construct as a way to enhance guidance stabilization in spinal cord injury regeneration

Spinal cord injuries (SCI) present a major challenge to therapeutic development due to its complexity. Combinatorial approaches using biodegradable polymers that can simultaneously provide a tissue scaffold, a cell vehicle, and a reservoir for sustained drug delivery have shown very promising results. In our previous studies we have developed a novel hybrid system consisting of starch/poly-e-caprolactone (SPCL) semi-rigid tubular porous structure, based on a rapid prototyping technology, filled by a gellan gum hydrogel concentric core for the regeneration within spinal-cord injury sites. In the present work we intend to promote enhanced osteointegration on these systems by pre-mineralizing specifically the external surfaces of the SPCL tubular structures, though a biomimetic strategy, using a sodium silicate gel as nucleating agent. The idea is to create two different cell environments to promote axonal regeneration in the interior of the constructs while inducing osteogenic activity on its external surface. By using a Teflon cylinder to isolate the interior of the scaffold, it was possible to observe the formation of a bone-like poorly crystalline carbonated apatite layer continuously formed only in the external side of the tubular structure. This biomimetic layer was able to support the adhesion of Bone Marrow Mesenchymal Stem Cells, which have gone under cytoskeleton reorganization in the first hours of culture when compared to cells cultured on uncoated scaffolds. This strategy can be a useful route for locally stimulate bone tissue regeneration and facilitating early bone ingrowth.

Activation of oxidative burst induces antioxidant response and indole alkaloid production in Uncaria Tomentosa root cultures

Uncaria tomentosa (cat's claw), an indigenous plant from the Amazon rainforest, is the source of monoterpenoid oxindole alkaloids (MOA) with immunomodulatory and antitumor activities. In cell cultures, production of these alkaloids has been stimulated by hydrodynamic stress via oxidative burst, while in root cultures, the role of reactive oxygen species in alkaloid production has not been fully examined. Roots were grown in 200 mL airlift bioreactors using a 4% (w/v) inoculum, reaching a total biomass of 21.1±0.8g dry wt L-1 after 65 days. Within the first hour of culture, a H2O2 peak (0.34µmol g-1 dry wt) was observed, indicating that oxidative burst occurred. In order to register the root redox state, activity of antioxidant enzymes was monitored. SOD and POD showed activity peaks of 340±5 U mg prot-1 and 546±8µM min-1 mg prot-1 5 days after inoculation, while GR and GPx activities remain constant. The oxidative burst and enzyme response were associated with 14 and 6-fold increase of MOA and 3α-dihydrocadambine (DHC) at day five (1.4±0.1 and 6.2±0.7mg L-1), also preceded by a two-fold increase of strictosidine synthase and strictosidine glucosidase activities. Enhancement of both activities showed correlation with mRNA transcript level. MOA and DHC production was 12 and 14-fold higher than those found in roots grown in Erlenmeyer flasks.

Quantitative study of lipase secretion, extracellular lipolysis, and lipid storage in the yeast Yarrowia lipolytica grown in the presence of olive oil: analogies with lipolysis in humans

Lipase secretion, extracellular lipolysis, and fatty acid uptake were quantified in the yeast Yarrowia lipolytica grown in the presence of olive oil and/or glucose. Specific lipase assays, Western blot analysis, and ELISA indicated that most of the lipase activity measured in Y. lipolytica cultures resulted from the YLLIP2 lipase. Lipase production was triggered by olive oil and, during the first hours of culture, most of the lipase activity and YLLIP2 immunodetection remained associated with the yeast cells. YLLIP2 was then released in the culture medium before it was totally degraded by proteases. Olive oil triglycerides were largely degraded when the lipase was still attached to the cell wall. The fate of lipolysis products in the culture medium and inside the yeast cell, as well as lipid storage, was investigated simultaneously by quantitative TLC-FID and GC analysis. The intracellular levels of free fatty acids (FFA) and triglycerides increased transiently and were dependent on the carbon sources. A maximum fat storage of 37.8% w/w of yeast dry mass was observed with olive oil alone. A transient accumulation of saturated FFA was observed whereas intracellular triglycerides became enriched in unsaturated fatty acids. So far, yeasts have been mainly used for studying the intracellular synthesis, storage, and mobilization of neutral lipids. The present study shows that yeasts are also interesting models for studying extracellular lipolysis and fat uptake by the cell. The quantitative data obtained here allow for the first time to establish interesting analogies with gastrointestinal and vascular lipolysis in humans.

Effects of colchicine on anther and microspore culture of bread wheat (Triticum aestivum L.)

The aim of this work was to study the effects of colchicine application on chromosome doubling and androgenic response in anther and microspore culture of different bread wheat genotypes. Colchicine was applied during a mannitol stress pretreatment or during the first 48 h of culture at concentrations of 0, 150 and 300 mg l−1. When colchicine was applied during stress pretreatment, the percentage of doubling depended on genotype and concentration. A significant increase in doubling was observed with 300 mg l−1 in the low androgenic responding cv. Caramba. Colchicine incorporation during the first hours of culture improved percentage of doubling in all genotypes, in both anther and microspore culture. Application of 300 mg l−1 colchicine improved the percentage of doubling in the two low responding genotypes, to 118% of control in DH24033, and 75% in Caramba in microspore and anther culture, respectively. Concerning the androgenic response, the effect of colchicine on embryo formation and percentage of green plants depended on the genotype and on the culture method. In cv. Pavon, a 2- and a 3-fold increase in percentage of embryogenesis and green plants, respectively, were obtained with 300 mg l−1 colchicine in microspore culture. However, no significant differences in these two variables were observed in anther culture. The number of green doubled haploid (DH) plants reflects the index of success of the procedure. Regardless of the culture method, when colchicine was incorporated during the first hours of culture, the number of green DH plants increased significantly in three of four genotypes. These results confirm the usefulness of colchicine application during the first hours of culture in wheat breeding programs.

Accumulation of deoxynivalenol and its 15-acetylated form is significantly modulated by oxidative stress in liquid cultures of Fusarium graminearum

Liquid cultures of Fusarium graminearum were supplemented with H2O2 or other oxidative compounds. The accumulation kinetics of the resulting trichothecenes were monitored. At non-lethal concentrations, the H2O2 treatments modulated toxin accumulation, dependent on the method of supplementation. When H2O2 was added at the same time as the inoculation, higher levels of toxins accumulated 30 days later. Conversely, adding H2O2 2 or 7 days after inoculation had little effect. When H2O2 was added daily over the course of the culture, the accumulation of trichothecenes was rapidly and strongly enhanced. The fungus may adapt to oxidative stress when the first exposure to H2O2 occurs at the beginning of the culture course. The highest toxin levels were measured when the H2O2 was added daily. The importance of the first hours of culture was confirmed: pre-treating conidia with H2O2 does not affect their germination kinetics but leads to a reduction in the yield of trichothecenes 40 days later. The H2O2 regulation of this trichothecene accumulation may be specific, as paraquat, another pro-oxidant compound, inhibits their production. Since H2O2 is a major component of the oxidative burst occurring in pathogen/host interactions, these data support the theory that trichothecenes may act as virulence factors.

Leptospira interrogans activation of peripheral blood monocyte glycolipoprotein demonstrated in whole blood by the release of IL-6

Glycolipoprotein (GLP) from pathogenic serovars of Leptospira has been implicated in the pathogenesis of leptospirosis by its presence in tissues of experimental animals with leptospirosis, the inhibition of the Na,K-ATPase pump activity, and induced production of cytokines. The aims of the present study were to investigate the induction of IL-6 by GLP in peripheral blood mononuclear cells (PBMC) and to demonstrate monocyte stimulation at the cellular level in whole blood from healthy volunteers. PBMC were stimulated with increasing concentrations (5 to 2500 ng/ml) of GLP extracted from the pathogenic L. interrogans serovar Copenhageni, lipopolysaccharide (positive control) or medium (negative control), and supernatants were collected after 6, 20/24, and 48 h, and kept at -80 degrees C until use. Whole blood was diluted 1:1 in RPMI medium and cultivated for 6 h, with medium, GLP and lipopolysaccharide as described above. Monensin was added after the first hour of culture. Supernatant cytokine levels from PBMC were measured by ELISA and intracellular IL-6 was detected in monocytes in whole blood cultures by flow-cytometry. Monocytes were identified in whole blood on the basis of forward versus side scatter parameters and positive reactions with CD45 and CD14 antibodies. GLP ( > or = 50 ng/ml)-induced IL-6 levels in supernatants were detected after 6-h incubation, reaching a peak after 20/24 h. The percentage of monocytes staining for IL-6 increased with increasing GLP concentration. Thus, our findings show a GLP-induced cellular activation by demonstrating the ability of GLP to induce IL-6 and the occurrence of monocyte activation in whole blood at the cellular level.

LESS NO PRODUCTION AND BETTER MOTION PARAMETER IN HUMAN SPERM BY SWIM-UP PROCESSING

Fifteen semen specimens were obtained from men for semen analysis; each was divided into two aliquots for prepararation. The motile sperm recovery rate, percentage motility, and motion parameters were measured for each semen specimen (n = 15) before and after preparation with the use of the two methods, and cultured with different time courses (1 hr, 3 hr, and 6 hr). Nitric oxide (NO) was measured using the chemiluminscence method after centrifugation. Recovery rate of motile cell was significantly higher in direct swim-up method (69.5 +/- 42.4% versus 49.3 +/- 29.3%, p < 0.05). In motility, direct swim-up method in the different time courses was significantly better than IxaPrep method. (1 hr: 91.1 +/- 5.2% vs 65.6 +/- 16.4%, 3 hr: 87.2 +/- 7.9% vs 65.2 +/- 16.5%, 6 hr: 86.1 +/- 7.5% vs 60.8 +/- 17.6% and prewash: 61.6 +/- 16.2%, p < 0.05). In VAP and VSL, the sperm prepared by the above two methods all improved compared to pre-wash sperm (p < 0.05), but there was no statistical significance between the two methods. NO production in the direct swim-up group was significantly lower than IxaPrep group in the first hour of culture (0.09 +/- 0.09 uM vs 0.15 +/- 0.09 uM, p < 0.05). NO production increased as the culture time increased in swim-up group, but conversed in IxaPrep group. The lower level of NO produced in the swim-up group may suggest that better sperm quality achieved is due to the decreased NO production.

Basis of coffee seed sensitivity to liquid nitrogen exposure: oxidative stress or imbibitional damage?

Two hypotheses, namely the occurrence of post‐thaw oxidative stress or imbibitional damage, were tested to explain the high sensitivity of coffee seeds to liquid nitrogen (LN) exposure. Oxidative stress was studied by measuring primary and secondary products of lipid peroxidation in seeds during the desiccation and rehydration periods. The 4‐hydroxynonenal (4‐HNE) content of seeds remained constant throughout the desiccation step. No significant difference was observed between desiccated seeds and seeds desiccated and exposed to LN for the evolution of their 4‐HNE and hydroperoxide contents during rehydration. In both cases, an increase in 4‐HNE and hydroperoxide contents of seeds was observed during the first hours of culture under germination conditions, followed by a progressive decrease down to values comparable to those observed in desiccated seeds. The hydroperoxide composition of frozen seeds was not significantly different from that of control seeds. The (S)/(R) enantiomeric ratios of 9‐ and 13‐hydroxy‐octadecadienoic acid extracted from rehydrating seeds were chiral, suggesting that they originated from lipoxygenase activity. These results suggest that the high sensitivity of coffee seeds to LN exposure is not directly associated with the occurrence of an oxidative stress during post‐thaw rehydration. The effect on seed viability of different rehydration procedures previously identified to reduce membrane imbibitional injury was studied after desiccation and LN exposure. Desiccation tolerance increased with, by increasing order, seed osmoconditioning, pre‐heating and pre‐humidifying prior to their culture under germination conditions. Among the four combinations of pre‐humidification durations (24 or 48 h) and temperatures (25 or 37°C) tested, pre‐humidification for 24 h at 37°C gave the highest level of desiccation tolerance. This rehydration procedure also dramatically increased seed viability after LN exposure. Seed desiccation sensitivity modelling in combination with the calculation of the decrease in seed water activity during cooling facilitated the explanation of the beneficial effect of controlled rehydration after desiccation and LN exposure. These results support the hypothesis that imbibitional membrane damage is involved in the sensitivity of coffee seeds to LN exposure.

Functional role of hepatocyte growth factor receptor during sperm maturation.

Mammalian spermatozoa acquire motility and fertilizing capacity during their transit through the epididymis. Hepatocyte growth factor (HGF) is a pleiotropic cytokine with potent motogenic capacities that has been identified in different organs, including the mammalian male genital tract. In mice, HGF is present in the testis and, in large amounts, in the distal part of the epididymis. In prepuberal rats, we have demonstrated that HGF is synthesized by the peritubular myoid cells and in men, HGF is present in significant quantities in seminal plasma. It has been suggested that in mice, HGF has a role in initiating sperm motility, whereas in men, no significant correlations between HGF concentration and sperm motility have been found. In the present paper we report that in rats, HGF receptor, c-met, is expressed in testicular and epididymal spermatozoa. Through immunocytochemistry, we have found that c-met is exclusively localized on the head in testicular sperm. A different localization of c-met has been found in sperm isolated from caput and cauda epididymidis. Cells isolated from epididymal caput show a c-met localization exclusively restricted to the head in most cells. In a minority of caput epididymis spermatozoa the receptor is localized both in the cell head and along the flagellum. Spermatozoa isolated from the epididymal cauda were quite homogeneous, showing the receptor localized along the entire cell surface. We also report that HGF is synthesized and secreted by the rat epididymis as indicated by the scatter effect of epididymal cell homogenate and culture medium on MDCK cells. To clarify whether HGF is involved in the acquisition of sperm motility in the epididymis, its maintenance, or both, spermatozoa isolated from caput epididymidis have been cultured in medium alone or supplemented with HGF. The results obtained indicated that HGF has a positive effect on the maintenance of sperm motility which, in the absence of HGF, significantly decreases during the first hour of culture, whereas it is maintained for at least 3 hours when HGF is present in the culture medium. We also report that HGF does not influence spermatozoa viability as indicated by the cytometrical analysis of propidium iodide-labeled sperm; an equal number of dead cells appeared in control and in HGF-treated preparations. In conclusion, our data strongly support the hypothesis that HGF positively influences sperm motility maintenance during sperm transit through the epididymis, indicating that c-met receptor and its ligand, HGF, have a role in male fertility.

Immunocytochemical location of endogenous cytokinins in buds of kiwifruit (Actinidia deliciosa) during the first hours of in vitro culture.

Post-embedding immunocytochemical techniques using peroxidase-antiperoxidase as markers for light microscopy or immunoglobulin G-gold for electron microscopy respectively were used for the localization of cytokinins [9-beta-D-ribofuranosil-N6-(delta2-isopentenil) adenina ([9R]iP), 9-beta-D-ribofuranosyl-zeatin ([9R]Z) and 9-beta-D-ribofuranosyl-dihydrozeatin ([9R](diH)Z)] in kiwifruit (Actinidia deliciosa) meristematic cells of the second nodal segment. Immunolocation at the cellular level was carried out in cells from explants grown during 16 and 72 h in liquid medium. Subcellular immunolocalization was performed in cells from explants grown for 35 d on agar solidified-medium and for 30 min, 4 and 16 h in liquid medium with cellulose plugs as explant support. Taken as a whole, the results obtained for Actinidia deliciosa show that the studied cytokinins change their location during the culture period, although they can always be found to a greater or lesser extent in the nucleus and the cytoplasm. For instance, [9R]Z appears in the cytoplasm and in the nucleus during the first hours of culture and later is the only one that appears located mainly in nucleus. On the other hand, [9R](diH)Z changes from being predominantly located in the nucleus to practically appearing only in the cytoplasm at the end of the culture period. [9R]iP is principally found up to 4 h of culture in the cytoplasm, and at 16 h is evenly distributed in all the subcellular compartments except in the chloroplast. The existence of a large amount of cytokinins in the nucleus during the first hours of culture compared with the immunolabelling density at 35 d is probably due to the activation of cell cycle mechanisms leading to organogenic development at the beginning of culture.

HA and double-layer HA-P2O5/CaO glass coatings: influence of chemical composition on human bone marrow cells osteoblastic behavior.

Human osteoblastic bone marrow derived cells were cultured for 28 days onto the surface of a glass reinforced hydroxyapatite (HA) composite and a commercial type HA plasma sprayed coatings, both in the "as-received" condition and after an immersion treatment with culture medium during 21 days. Cell proliferation and differentiation were analyzed as a function of the chemical composition of the coatings and the immersion treatment. Cell attachment, growth and differentiation of osteoblastic bone marrow cells seeded onto "as-received" plasma sprayed coatings were strongly affected by the time-dependent variation of the surface structure occurring during the first hours of culture. Initial interactions leading to higher amounts of adsorbed protein and zeta potential shifts towards negative charges appeared to result in surface structures with better biological performance. Cultures grown onto the pretreated coatings showed higher rate of cell proliferation and increased functional activity, as compared to those grown onto the corresponding "as-received" materials. However, the cell behavior was similar in the glass composite and HA coatings. The results showed that the glass composites present better characteristics for bone cell growth and function than HA. In addition, this work also provide evidence that the biological performance of the glass composites can be modulated and improved by manipulations in the chemical composition, namely in the content of glass added to HA.