Percoll Gradient Centrifugation Research Articles

Physiologically distinct subpopulations formed in Escherichia coli cultures in response to heat shock

Bacteria can form heterogeneous populations containing phenotypic variants of genetically identical cells. The heterogeneity of populations can be considered a bet-hedging strategy allowing adaptation to unknown environmental changes – at least some individual subpopulations or cells might be able to withstand future adverse conditions. Using Percoll gradient centrifugation, we demonstrated that in an Escherichia coli culture exposed to heat shock at 50 °C, two physiologically distinct subpopulations were formed. A high-density subpopulation (HD50) demonstrated continued growth immediately after its transfer to LB medium, whereas the growth of a low-density subpopulation (LD50) was considerably postponed. The LD50 subpopulation contained mainly viable but non-culturable bacteria and exhibited higher tolerance to sublethal concentrations of antibiotics or H2O2 than HD50 cells. The levels of aggregated proteins and main molecular chaperones were comparable in both subpopulations; however, a decreased number of ribosomes and a significant increase in protein oxidation were observed in the LD50 subpopulation as compared with the HD50 subpopulation. Interestingly, under anaerobic heat stress, the formation of the HD50 subpopulation was decreased and culturability of the LD50 subpopulation was significantly increased. In both subpopulations the level of protein aggregates formed under anaerobic and aerobic heat stress was comparable. We concluded that the formation of protein aggregates was independent of oxidative damage induced by heat stress, and that oxidative stress and not protein aggregation limited growth and caused loss of LD50 culturability. Our results indicate that heat stress induces the formation of distinct subpopulations differing in their ability to grow under standard and stress conditions.

Molecular mechanisms of tributyltin-induced alterations in cholesterol homeostasis and steroidogenesis in hamster testis: In vivo and in vitro studies.

Tributyltins (TBT) are ubiquitous and persistent environmental contaminants that disturb normal endocrine function including gonadal function in humans and marine organisms. TBT was administered through oral route to male Syrian hamsters at daily doses of 50, 100, and 150 ppm/kg for 65 days. Changes in testis morphology, immunohistochemistry of iNOS, 3β-HSD, and 17β-HSD, cholesterol transport receptor, nuclear receptors, and transcription factors were analyzed. TBT treatment affected each of these parameters to significant levels in a dose-dependent manner compared to vehicle treated control. Real-time PCR and protein analyses revealed that expression levels of ApoE and LDL-R mRNA were up-regulated in the testis of TBT-treated animals while the expression levels of SR-B1, LXR, PPARs α, β, and γ, SCAP, SREBP 1 and 2, 3β-HSD, 17β-HSD, CYP17A1, and P450SCC were down-regulated. Leydig cells were isolated and separated adopting percoll gradient centrifugation under aseptic condition. The viability of Leydig cell was affected by TBT treatment in a dose- and time-dependent manner. Further, the mechanism of action of TBT was ascertained by siRNA transfection of ApoE, which was upregulated, and SREBP, which was down-regulated. These observations led us to infer that exposure to TBT hinders intracellular cholesterol transport resulting in abnormal sex steroid biosynthesis and alteration of steroidogenic enzyme activities. Finally, we could recognize ApoE and SREBP as the major factors regulating genes that control cholesterol biosynthesis and steroidogenesis that ultimately inhibit the synthesis of testosterone. Therefore, ApoE is one of the important molecular targets that can be intercepted in context of male infertility/male contraception.

192 Culture Conditions Supporting Long-Term Expansion of Bovine Spermatogonial Stem Cells Isolated from Adult and Immature Testes

Spermatogonial stem cells (SSC) self-renew and differentiate into spermatocytes to produce haploid sperm. Because SSC are a small population of adult stem cells in the testis, numerous studies have been reported to derive cell lines from cultured SSC. It has been reported that neonatal and adult mouse SSC can be cultured in vitro over the long term. Male germline stem (GS) cells, embryonic stem (ES)-like cells, and multipotent male germline stem (MGS) cells were derivated from mouse SSC. However, in domestic species including cattle, information about in vitro culture of SSC is mainly available in the neonatal and immature animal. To our knowledge, there are no reports about long-term culture of SSC isolated from adult bovine testis. In this report, we established culture conditions to maintain SSC isolated from adult and immature testes. The SSC were isolated by 3-step enzymatic digestion and enriched by Percoll gradient centrifugation. For adult testicular cell suspensions, SSC were further enriched by differential plating on precoated gelatin dish. After Percoll gradient centrifugation, we found differential expression of SSC markers (GFRα-1 and UCHL-1) in the isolated cells from immature and adult testis. The RT-PCR results also confirmed the expression of differentiated spermatogonia markers (SYCP3 and STRA-8) in adult testicular cell suspensions. It suggests that isolated testicular germ cell population from adult testis are more heterogeneous than those of immature testis. The SSC isolated from adult testes were cultured in low-serum media containing 6-bromoindirubin-3′-oxime (BIO), an inhibitor of glycogen synthase kinase-3α (GSK3), and subsequently the cultures were maintained in the medium containing glial cell line-derived neurotropic factor (GDNF). The cell lines have characteristics resembling mouse GS cell lines as confirmed by their grape-like shape morphology, the expression of SSC markers (UCHL-1, DBA, and GFRa-1), and pluripotent stem cell markers (POU5F1, SOX2, KLF4). The SSC from immature testes were proliferated for more than 3 months in serum-free culture conditions in the presence of GDNF and bovine leukemia inhibitory factor (LIF). The cell lines had ES-like cell morphology, expressed pluripotent stem cell markers and SSC-specific markers. They differentiated in vitro into 3 germ layers confirmed by the expression of ectoderm (NESTIN), mesoderm (BMP4), and endoderm (GATA-6) markers by RT-PCR and neuron like-cells confirmed by the expression of glial fibrillary acidic protein (GFAP) by immunofluorescence analysis. In conclusion, these findings indicate an efficient method to enrich SSC without cell sorting method and different long-term culture systems subsequently established to maintain SSC from adult and immature testes. Furthermore, our data would be useful for further studies that aim to preserve endangered species and improve livestock production through genome editing technology.

Concentration of Plasmodium falciparum gametocytes in whole blood samples by magnetic cell sorting enhances parasite infection rates in mosquito feeding assays

BackgroundMosquito-feeding assays are important tools to guide the development and support the evaluation of transmission-blocking interventions. These functional bioassays measure the sporogonic development of gametocytes in blood-fed mosquitoes. Measuring the infectivity of low gametocyte densities has become increasingly important in malaria elimination scenarios. This will pose challenges to the sensitivity and throughput of existing mosquito-feeding assay protocols. Here, different gametocyte concentration methods of blood samples were explored to optimize conditions for detection of positive mosquito infections.MethodsMature gametocytes of Plasmodium falciparum were diluted into whole blood samples of malaria-naïve volunteers. Standard centrifugation, Percoll gradient, magnetic cell sorting (MACS) enrichment were compared using starting blood volumes larger than the control (direct) feed.ResultsMACS gametocyte enrichment resulted in the highest infection intensity with statistically significant increases in mean oocyst density in 2 of 3 experiments (p = 0.0003; p ≤ 0.0001; p = 0.2348). The Percoll gradient and standard centrifugation procedures resulted in variable infectivity. A significant increase in the proportion of infected mosquitoes and oocyst density was found when larger volumes of gametocyte-infected blood were used with the MACS procedure.ConclusionsThe current study demonstrates that concentration methods of P. falciparum gametocyte-infected whole blood samples can enhance transmission in mosquito-feeding assays. Gametocyte purification by MACS was the most efficient method, allowing the assessment of gametocyte infectivity in low-density gametocyte infections, as can be expected in natural or experimental conditions.

Two hemocyte sub-populations of kuruma shrimp Marsupenaeus japonicus

Hemocytes in the circulating hemolymph play important roles for immune responses in shrimp. Previous studies on immune responses by hemocytes in penaeid shrimp were based on gene expression analyses of the entire population of hemocytes and thus may have missed different immune responses of different hemocyte sub-populations. In this study, we separated hemocytes into two sub-populations by Percoll gradient centrifugation, morphological characteristics of each population were then analyzed by May-Giemsa staining, flow cytometry, and FACSCalibur. Results showed hemocytes were divided into an upper layer basophilic, and lower layer of eosinophilic hemocytes. Basophilic hemocytes were larger in size compared to eosinophilic hemocytes, which were more granulated than the basophilic hemocytes. Transcriptome analysis was then conducted through RNA-seq analysis by Miseq, which revealed 16 differentially-expressed transcripts between the two sub-populations. In the upper-layer, the highly expressed transcripts that were homologous to immune-related genes that suggest hemocytes from this layer may play as the regulator of immune system and control the action of other cells to eliminate pathogen. On the other hand, transcripts that were highly expressed in the lower-layer were homologous to the antimicrobial peptide (AMP) crustin, which supports that hemocytes on this layer have granules as crustins are normally secreted from hemocyte granules. The high expression of crustin in the lower-layer also provides insight on the mechanism of the anti-microbial function, where hemocytes produce and store AMPs in its granules. These differentially expressed genes are potential hemocyte molecular markers, and among them we identified one of the highly expressed genes in the hemocytes from the upper-layer (c11736_g1) to be a promising candidate molecular marker predicted to be a surface molecule, which is a common characteristic for molecular markers.

Cation (Ca2+ and Mn2+) Partitioning Assays with Intact Arabidopsis Chloroplasts.

Determination of the relative distribution of Ca2+ and Mn2+ is an important tool for analyzing mutants showing altered levels of calcium and/or manganese transporters in the chloroplast envelope or thylakoid membrane. The method described in this protocol allows quantitative analyses of the relative distribution of calcium and manganese ions between chloroplast stroma and thylakoids using the isotopes [45Ca] and [54Mn] as radioactive tracers. To avoid contaminations with non chloroplastidic membrane systems, the method is designed for isolating pure and intact chloroplasts of Arabidopsis thaliana. Intact chloroplasts are isolated via Percoll gradient centrifugation. Chloroplasts are then allowed to take up [45Ca] or [54Mn] during a light incubation step. After incubation, chloroplasts are either kept intact or osmotically/mechanically treated to release thylakoids. The amount of incorporated [45Ca] or [54Mn] can be determined by liquid scintillation counting and the relative distribution calculated.

Isolation and Structural Studies of Mitochondria from Pea Roots.

For structural and respiratory studies, isolation of intact and active mitochondria is essential. Here, we describe an isolation method which gave good yield and intact mitochondria from 2-week-old pea (Pisum sativum) roots grown hydroponically under standard growth conditions. We used Percoll gradient centrifugation for this isolation procedure. The yield of purified mitochondria was 50μg/g FW. Isolated mitochondria maintained their structure which was observed by using MitoTracker green in confocal microscope and scanning electron microscopy(SEM). Intact mitochondria are clearly visible in SCM images. Taken together this isolation method can be used for physiological and microscopic studies on mitochondria.

Differential protein abundance in promastigotes of nitric oxide-sensitive and resistant Leishmania chagasi strains.

Leishmania chagasi is the causative agent of zoonotic visceral leishmaniasis in Brazil. Domestic and stray dogs are the main reservoirs. The life cycle of the parasite involves two stages. Promastigotes are extracellular and develop within the sand fly gut. Amastigotes survive inside the harsh environment of the phagolysosome of mammalian host phagocytes, which display the nitric oxide defense mechanism. Surprisingly, we were able to isolate promastigotes that are also resistant to NO. This finding may be explained by the preadaptative hypothesis. An insight into the proteome of NO-sensitive and resistant promastigotes is presented herein. Total protein extracts were prepared from promastigote cultures of an NO-sensitive and a resistant strain at early-logarithmic, mid-logarithmic and stationary phase. A population enriched in metacyclic promastigotes was also isolated by Percoll gradient centrifugation. In vitro infectivity of both strains was compared. Differential protein abundance was analyzed by 2DE-MALDI-TOF/TOF. The most striking results were tested at the mRNA level by qRT-PCR. Three biological replicates were performed in all cases. NO-resistant L. chagasi promastigotes are more infective than NO-sensitive ones. Among the differentially abundant spots, 40 proteins could be successfully identified in the sensitive strain and 38 in resistant promastigotes. The increase of G6PD and the decrease of ARG and GPX transcripts and proteins contribute to NO resistance in L. chagasi promastigotes. These proteins may be studied as potential drug targets and/or vaccine candidates in the future.

Analysis and Purification of Mouse Intestinal Dendritic Cell and Macrophage Subsets by Flow Cytometry.

The unit presents a method for analysis of intestinal dendritic cell (DC) and macrophage subsets by flow cytometry in the single cell suspension prepared from the mouse small and large intestine (Basic Protocol). describes a strategy to enrich the hematopoietic cell fraction in the sample by Percoll gradient centrifugation, and describes preparation of single cell suspensions from specific tissue layers of the small intestine, such as the epithelium, villi mucosa, submucosa, and muscularis externa. Finally, Support Protocol explains how to purify specific intestinal DC and macrophage subsets by flow-cytometry-based cell sorting. © 2016 by John Wiley & Sons, Inc.

Effects of T-2 toxin on the regulation of steroidogenesis in mouse Leydig cells.

T-2 toxin is one of the mycotoxins, a group of type A trichothecenes produced by several fungal genera including Fusarium species, which may lead to the decrease of testosterone secretion in primary Leydig cells derived from mouse testis. The previous study demonstrated T-2 toxin decrease the testosterone biosynthesis in the primary Leydig cells derived from the mouse testis directly. In this study, we further examined the direct biological effects of T-2 toxin on the process of steroidogenesis, primarily in Leydig cells of mice. Leydig cells of mature mouse were purified by Percoll gradient centrifugation and the cell purity was determined by 3β-hydroxysteroid dehydrogenase (3β-HSD) staining. To examine the decrease in T-2 toxin-induced testosterone secretion, we measured the transcription level of three key steroidogenic enzymes including 3β-HSD-1, cytochrome P450 side-chain cleavage (P450scc) enzyme, and steroidogenic acute regulatory (StAR) protein in T-2 toxin/human chorionic gonadotropin (hCG) co-treated cells. Our previous study showed that T-2 toxin (10(-7), 10(-8), and 10(-9) M) significantly suppressed hCG (10 ng/ml)-induced testosterone secretion. The studies demonstrated that the suppressive effect is correlated with a decrease in the level of transcription of 3β-HSD-1, P450scc, and StAR (p < 0.05).

37 1,25(OH)2D3 suppresses oxidative stress – Induced microparticle release by placental trophoblasts

Introduction Emerging evidence showed that microparticles (MPs) derived from placental trophoblasts (TC) contribute to maternal vascular dysfunction and promote endothelial inflammatory response in preeclampsia (PE). Vitamin D insufficiency has been linked to placental dysfunction in pregnancy disorders such as PE and IUGR. Objective To determine: (1) if TC from PE placentas release more MPs than TC from normal placentas; (2) if vitamin D could suppress oxidative stress-induced MP release by placental TC; and (3) potential mechanism of vitamin D protective effects on placental TCs. Methods TC were isolated from normal and PE placentas by trypsin digestion and purified with Percoll gradient centrifugation. Freshly isolated TC (5×06cells/well) were seeded into 6 well plates and cultured with DMEM containing 5% FBS and antibiotics for 72h. For the oxidative stress experiment, cobalt chloride (CoCl 2 , a hypoxic mimicking agent) was used to induce TC oxidative stress and 1,25(OH)2D3 was used as bioactive vitamin D. MPs were isolated from culture supernatants by a two-step centrifugation procedure (420g for 20min and then 20,000g for 60min) and quantified by annexin V positive staining measured by flow cytometry with TruCount beads to determine absolute counts of MP for each sample. Data are expressed as annexin V positive MP/μg cell protein/well. Total cellular protein and MP protein were also collected and protein expression for caveolin-1, eNOS, pro-caspase-3, cleaved caspase-3, and ROCK1 were determined by Western blot. Data were analyzed by paired or unpaired t-test or ANOVA with Newman-Keuls test as a post hoc test. A p level Results TC isolated from PE placentas ( n =6) released significantly more MP than cells isolated from normal placentas ( n =6), 295.20±47.67 vs. 117.20±18.02/μg protein/well, p 2 compared to control TC, p 2 was significantly reduced in TC treated with 1,25(OH)2D3+CoCl 2 , p 2 . This phenomenon could be reversed when cells were treated with 1,25(OH)2D3. Moreover, reduced pro-caspase-3 and ROCK1 expression in TC induced by CoCl 2 could also be attenuated in cells treated with1,25(OH)2D3. Conclusions MP release was significantly increased in TC from PE placenta. 1,25(OH)2D3 suppresses oxidative stress-induced MP release by placental TC. Caveolin-1 is an essential structural component of caveolae and eNOS is associated with plasma membrane caveolae. Therefore, reduced caveolin-1 and increased eNOS expression together with reduced MP release in TC treated with 1,25(OH)2D3 demonstrate that vitamin D could protect placental TC from oxidative stress-induced injury during pregnancy. Since caspase-3 cleavage and ROCK1 activation are key steps of MP formation and release, suppression of caspase-3 cleavage and ROCK1 activation by vitamin D could be a plausible mechanism of vitamin D inhibition of increased MP release induced by oxidative stress.

Dehydroepiandrosterone inhibits cell proliferation and improves viability by regulating S phase and mitochondrial permeability in primary rat Leydig cells.

Dehydroepiandrosterone (DHEA) is widely used as a nutritional supplement and exhibits putative anti-aging properties. However, the molecular basis of the actions of DHEA, particularly on the biological characteristics of target cells, remain unclear. The aim of the current study was to investigate the effects of DHEA on cell viability, cell proliferation, cell cycle and mitochondrial function in primary rat Leydig cells. Adult Leydig cells were purified by Percoll gradient centrifugation, and cell proliferation was detected using a Click-iT® EdU Assay kit and cell cycle assessment performed using flow cytometry. Mitochondrial membrane potential was detected using JC-1 staining assay. The results of the current study demonstrate that DHEA decreased cell proliferation in a dose-dependent manner, whereas it improved cell viability in a time-dependent and dose-dependent manner. Flow cytometry analysis demonstrated that DHEA treatment increased the S phase cell population and decreased the G2/M cell population. Cyclin A and CDK2 mRNA levels were decreased in primary rat Leydig cells following DHEA treatment. DHEA treatment decreased the transmembrane electrical gradient in primary Leydig cells, whereas treatment significantly increased succinate dehydrogenase activity. These results indicated that DHEA inhibits primary rat Leydig cell proliferation by decreasing cyclin mRNA level, whereas it improves cells viability by modulating the permeability of the mitochondrial membrane and succinate dehydrogenase activity. These findings may demonstrate an important molecular mechanism by which DHEA activity is mediated.

IL-1β enhances inflammatory TH2 differentiation

IL-1β enhances inflammatory TH2 differentiation

Isolation of functionally active and highly purified neuronal mitochondria from human cortex

BackgroundFunctional and structural properties of mitochondria are highly tissue and cell dependent, but isolation of highly purified human neuronal mitochondria is not currently available. New methodWe developed and validated a procedure to isolate purified neuronal mitochondria from brain tissue. The method combines Percoll gradient centrifugation to obtain synaptosomal fraction with nitrogen cavitation mediated synaptosome disruption and extraction of mitochondria using anti mitochondrial outer membrane protein antibodies conjugated to magnetic beads. The final products of isolation are non-synaptosomal mitochondria, which are a mixture of mitochondria isolated from different brain cells (i.e. neurons, astrocytes, oligodendrocytes, microglia) and synaptic mitochondria, which are of neuronal origin. This method is well suited for preparing functional mitochondria from human cortex tissue that is surgically extracted. ResultsThe procedure produces mitochondria with minimal cytoplasmic contaminations that are functionally active based on measurements of mitochondrial respiration as well as mitochondrial protein import. The procedure requires approximately four hours for the isolation of human neuronal mitochondria and can also be used to isolate mitochondria from mouse/rat/monkey brains. Comparison with existing methods and conclusionsThis method will allow researchers to study highly enriched neuronal mitochondria without the confounding effect of cellular and organelle contaminants.

Immortalization of porcine placental trophoblast cells through reconstitution of telomerase activity

Placental trophoblast cells (PTCs) play a critical role in histotrophic nutrient absorption, gaseous exchange, endocrine activities, and barrier function between the maternal and fetal systems. Establishment of immortalized porcine PTCs will help us to investigate the potential effects of different viruses on porcine trophoblast. In the present study, primary porcine PTCs were isolated from healthy gilts at Day 30 to Day 50 of gestation through collagenase digestion, percoll gradient centrifugation, and anti-CD9 immunomagnetic negative selection. To provide stable and long lifespan cells, primary PTCs were transfected with human telomerase reverse transcriptase (hTERT) gene. One porcine placental trophoblast cell line, named as hTERT-PTCs, was chosen for characterization. Human telomerase reverse transcriptase-PTCs achieved an extended replicative lifespan without exhibiting any neoplastic transformation signs in vivo or in vitro. The morphologic and key physiological characteristics of the immortalized PTCs were similar to primary PTCs. The immortalized PTCs retained original cell polarity and normal karyotype, expressed trophoblast-specific marker cytokeratin 7 and E-cadherin but did not express vimentin and major histocompatibility complex class I antigens as well as primary PTCs. Human telomerase reverse transcriptase-PTCs secreted low levels of chorionic gonadotrophin β-subunit and placental lactogen that were coincident with primary PTCs. Taken together, our results demonstrated that the porcine PTCs could be immortalized through reconstitution of telomerase activity. The immortalized PTCs maintained its original characteristics and can be used as a model cells line to study the pathologic changes of porcine placental trophoblast in viruses infectious diseases.

Efficient pig ICSI using Percoll-selected spermatozoa; evidence for the essential role of phospholipase C-ζ in ICSI success.

In pigs, the damaged sperm membrane leads to leakage of phospholipase C-ζ (PLCζ), which has been identified as a sperm factor, and a reduction of oocyte-activating ability. In this study, we investigated whether sperm selected by Percoll gradient centrifugation (Percoll) have sufficient PLCζ, and whether the efficiency of fertilization and blastocyst formation after intracytoplasmic sperm injection (ICSI) using Percoll-selected sperm can be improved. Percoll-selected sperm (Percoll group) or sperm without Percoll selection (Control group) were used. A proportion of the oocytes injected with control sperm were subjected to electrical stimulation at 1 h after ICSI (Cont + ES group). It was found that the Percoll group showed a large amount of PLCζ in comparison with the Control group. Furthermore, application of Percoll-selected sperm for ICSI increased the efficiency of fertilization and embryo development. Thus, these results indicate the Percoll-selected sperm have sufficient PLCζ and high oocyte-activating ability after ICSI in pigs.

151 COMPARATIVE ANALYSIS OF FRESH AND CRYOPRESERVED BOAR SPERMATOZOA USING RNA SEQUENCING

Fertility of cryopreserved spermatozoa is significantly reduced compared with that of their fresh counterparts, which is certainly due to the inflicted sublethal damage to spermatozoa that is observed at various molecular and cellular levels. The identification and characterisation of this damage will help us better understand sperm cryobiology and therefore develop suitable media and procedures to improve sperm cryopreservation and fertility outcomes, especially in swine. Here, we present our preliminary assessment of RNA pools of fresh and frozen‐thawed spermatozoa using RNA-sequencing technology. Semen ejaculates of 8 fertile boars were harvested and divided into 2 fractions for each ejaculate. Fraction 1 was freshly extended in commercial diluent (FD) and fraction 2 was frozen in 5-mL plastic straws (FT). Both specimens were shipped to our laboratory for analyses. The samples were purified through Percoll gradient centrifugation and resulting motile spermatozoa were washed in cold PBS. Pelleted spermatozoa were used for total RNA extraction, followed by an in-column DNase digestion. Purity and integrity of RNA samples were checked and rRNA depleted. After random priming, 40 million short cDNA reads were produced using Illumina RNA-Seq technology (Illumina Inc., San Diego, CA, USA). All reads were aligned to the pig reference genome and the produced genome-scale transcription maps consisted of both the transcript structure and the expression level of each gene mapped. Analysis of FD sperm RNA revealed a total of 18 357 sequence tags that were successfully mapped to all pig chromosomes and the mitochondrial genome. Frozen‐thawed spermatozoa showed only 16 864 sequence tags. In both FD and FT samples, chromosomes 1, 2, 6, 7, and 13 contained, in total, the highest density of mapped transcripts (&gt;42%). Chromosome Y and mitochondrial RNAs had the lowest sequence tags mapped (&lt;0.08%). A comparative analysis of FD and FT datasets revealed a net decrease in the total number of sequence tags (1493) with each chromosome being affected, except mitochondria. Chromosomes of FT samples showed a strong (&gt;10%; 17, 7, 4, Y, and X) to moderate (10 to 5%) or weak (≤5%) reduction in RNA numbers. Structural annotation revealed a diverse population of sperm transcripts comprising both coding (mRNA) and noncoding (rRNA, snRNA, and mtRNA) RNAs. In both FD and FT samples, noncoding RNAs were among the most abundant sequence tags. Approximately 12 355 of sequence tags in FD v. 10 948 in FT spermatozoa were annotated with ENSEMBL and the selected genes are under investigation for comparative analyses using RT-PCR. In conclusion, mature boar spermatozoa contain a large pool of coding and non-coding RNAs that can be affected by the freezing-thawing procedure. Inflicted damage affects RNAs of all chromosomes with a great effect being seen on chromosome X. Generated datasets have the potential to lead to further study of the cryo-damage associated with reduced fertility of cryopreserved spermatozoa. Study was supported by USDA-ARS Biophotonics initiative grant # 58-6402-3-0120 and MAFES-SRI grants.

Myometrial contractility influences oxytocin receptor (OXTR) expression in term trophoblast cells obtained from the maternal surface of the human placenta.

BackgroundOxytocin (OXT) acts through its specific receptor (OXTR) and increased density of OXTR and/or augmented sensitivity to OXT were postulated as prerequisites of normal onset of labor. Expression of OXTR in the placental term trophoblast cells has not yet been analyzed in the context of contractile activity of the uterus. Here we examine comparatively OXT contents in the placental tissue adjacent to the uterine wall and expressions of OXTR in this tissue and corresponding isolated placental trophoblast cells.MethodsTwenty eight placentae after normal labors at term (group I, N = 14) and after cesarean sections performed without uterine contractile activity (group II, N = 14) have been collected. Tissue excised from the maternal surface of examined placenta was used for OXT concentration measurement, cytotrophoblast cell cultures preparation and immunohistochemistry of OXTR. Concentration of OXT was estimated in the tissue homogenates by an enzyme immunoassay with colorimetric detection. Cytotrophoblast cells were isolated using Kliman’s method based on trypsin, DNase, and a 5–70 % Percoll gradient centrifugation. The cultures were incubated for 5 days in normoxia. Both placental specimens and terminated cytotrophoblast cultures were fixed and embedded in paraffin before being immunostained for OXTR. Using light microscopy with computed morphometry for quantitative analysis, OXTR expressions were estimated in calibrated areas of the paraffin sections.ResultsThere were not significant differences between the groups in respect to the mean OXT concentration. However, in both groups the median value of OXT concentration was significantly (p < 0.05) higher in the tissue obtained from the peripheral regions of the maternal surface of the placenta, compared to the samples from the central region of this surface. In placental tissue the mean expression of OXTR in group I was significantly (p < 0.05) increased by approximately 3.2-fold and 3.45-fold (the samples collected from central and peripheral regions, respectively) compared to the values obtained in group II.In the isolated primary trophoblast cultures the differences were even more evident (p < 0.02) and the mean change in OXTR expression in group I comprised approximately 6.9-fold increase and 6.5-fold increase (the samples collected from central and peripheral regions, respectively) compared to the values obtained in group II.ConclusionsUpregulation of OXTR within placental trophoblast cells localized close or adherent to uterine wall may play a crucial role in labor with efficient contractile activity (vaginal delivery). Further studies may disclose if this local OXT/OXTR signaling is utilized in the third stage of labor to elicit placental detachment or contribute in a more versatile way throughout the labor period.

Yellow head virus infection in black tiger shrimp reveals specific interaction with granule-containing hemocytes and crustinPm1 as a responsive protein

Yellow head virus (YHV) causes acute infections and mass mortality in black tiger shrimp culture. Our study aims to investigate molecular interaction between YHV and circulating hemocytes of Penaeus monodon at early infection. Total shrimp hemocytes were isolated by Percoll gradient centrifugation and identified by flow cytometric analysis. At least three types of hemocyte cells were identified as hyaline, semi-granular, and granular hemocytes. Experimental infection of YHV in shrimp culture demonstrated drastic changes in total and each hemocyte cell counts. Immunohistochemistry analysis demonstrated interaction and replication of YHV mainly with the granule-containing hemocytes and little to none in hyaline cell. These granule-containing hemocytes are proposed to be YHV targets providing the first line of defense to viral infection. Protein expression profiling of granule-containing hemocytes revealed several immune-responsive proteins including antimicrobial protein crustins (crustinPm1 and crustinPm4), alpha-2-macroglobulin, and kazal-type serine proteinase inhibitor. During an early phase of YHV infection at 6 hpi crustinPm1 illustrated a significant increase of mRNA and protein expression level in plasma. The results suggest that an antimicrobial crustinPm1 may participate in shrimp defense mechanism against YHV, especially on the granule-containing hemocytes.

NrCAM modulates sonic hedgehog signalling by controlling smoothened translocation in the cilium

Objective: Cerebellar development involves a spurt of proliferation in external granule layer (EGL) in response to shh, causing granule neuron precursors (GNPs) to proliferate. These cells subsequently differentiate into granule neurons in the inner granule layer (IGL). F3, a CNTN family molecule, can interact with NrCAM to switch GNPs from proliferation to differentiation. We aim to identify the role of NrCAM in the sonic hedgehog response in GNPs. Methods: GNPs were extracted from wildtype and NrCAM mutant P5 cerebella using Percoll gradient centrifugation. Proliferation response to shh was measured using EdU in presence/absence of F3-Fc. GNPs treated with shh/SAG were stained with antibodies against Arl13b and smo to look for differences in cilia size and smo occupancy after different treatment times. Results: NrCAM-/- and wildtype GNPs both proliferated equally in response to shh. F3 was found to block the proliferation response in wildtype but not in NrCAM-/- GNPs. F3 also failed to affect proliferation in SmoA1 GNPs with a constitutively active smo suggesting that the F3-NrCAM mediated block lay upstream of Smo. NrCAM was detected in wildtype cilia and Smo localization was affected in NrCAM-/- GNPs. No differences in cilia length were observed. Conclusion: Our results suggest that NrCAM affects shh-mediated proliferation by controlling smo movement into the cilium.