Discontinuous Gradient Centrifugation Research Articles

Deciphering the influence of mare oviductal organoid secretions on equine spermatozoa

The oviductal milieu assists fertilization by modulating sperm survival, supporting capacitation, and facilitating transport of early embryos. In many mammals, the direct relationship between the spermatozoa and the oviductal epithelial cells prolong sperm survival by inducing quiescence and suppressing metabolic activity. However, the detailed mechanisms remain unknown. In this study we sought to develop an equine organoid model to examine sperm-oviduct interactions. We aimed to determine whether oviductal secretions alone can induce quiescence, or alternatively, drive capacitation in preparation for fertilization. This was done by exposing spermatozoa to oviductal organoid-conditioned media and assessing sperm motility, viability, mitochondrial function, and capacitation. Oviductal organoids were cultured from single cells derived from the enzymatic digestion of freshly collected mare (n=3) infundibulum. Mature organoids (>21 days) with ciliated and secretory cells were cultured for 24 h with either fresh oviductal epithelial (OE) culture medium (containing Advanced DMEM-F12, Glutamax, HEPES, penicillin-streptomycin and WNT3A-RSPO3-NOGGIN) or Biggers-Whitten-Whittingham medium (BWW). Thereafter, culture media were collected and centrifuged (conditioned medium). Spermatozoa were collected from a stallion of known fertility, isolated using discontinuous gradient centrifugation and diluted to 20 × 106 cells/ml. Spermatozoa were then co-incubated with conditioned and non-conditioned (control) OE, and conditioned and non-conditioned BWW medium. Total (TM) and progressive (PM) motility were assessed at 1h and 24h; viability (via Live-Deadfluorescent stain/flow cytometry), mitochondrial membrane potential (MMP; via JC-1 probe/flow cytometry) and capacitation (via anti-tyrosine phosphorylation immunocytochemistry) were assessed at 24 h. Spermatozoa incubated with conditioned OE media showed significantly reduced motility at 24h (TM: conditioned vs control; 17.1±2.6% vs 43.2±5.5%; p=.025. and PM: conditioned vs control; 3.7±0.4% vs 27.1±2.8%; p=.008). Similar results were seen for BWW. Spermatozoa viability was not significantly affected (conditioned vs control; OE: 58.2±5.8% vs 84.6±5.9%; p>.05) nor was MMP (conditioned vs control; OE: 63.8±6.1% vs 66.9±7.5% of live cells with high MMP; p>.05). For BWW, similar results were recorded for viability (conditioned vs control; 84.5±6.2% vs 75.6±0.05%; p>.05), and MMP (conditioned vs control; 31.3±10.2% vs 56.2±13.5% of live cells with high MMP; p>.05). Observationally, MMP was suppressed in organoid-conditioned BWW exposed spermatozoa in 2 of the 3 replicates (from 41.3% to 6.3%, and from 89.1% to 42% cells with high MMP, concurrent with higher viability in exposed samples). Tyrosine phosphorylation, indicative of capacitation, showed no difference between organoid-conditioned medium exposed spermatozoa and controls. In summary, spermatozoa exposed to oviductal organoid-conditioned medium respond with suppressed motility but remain viable. These findings are consistent with the hypothesis that oviductal epithelial secretions can induce quiescence in the spermatozoa reservoir awaiting arrival of the oocyte.

Synchronization isolation method for multiple types of cells from mouse liver

Objective: To explore a simple and feasible method for the isolation and purification of hepatocytes, hepatic stellate cells (HSC), and lymphocytes from mice. Methods: The cell suspension was obtained from male C57bl/6 mice by hepatic perfusion through the portal vein digestion method and then isolated and purified by discontinuous Percoll gradient centrifugation. Trypan blue exclusion was used to determine cell viability. Glycogen staining, cytokeratin 18, and transmission electron microscopy were used to identify hepatic cells. Immunofluorescence was used to detect α-smooth muscle actin combined with desmin in HSCs. Flow cytometry was used to analyze lymphocyte subsets in the liver. Results: After isolation and purification, about 2.7×10(7) hepatocytes, 5.7×10(5) HSCS, and 4.6×106 hepatic mononuclear cells were obtained from the liver of mice with a body weight of about 22g. The cell survival rate in each group was > 95%. Hepatocytes were apparent in glycogen deposited purple-red granules and cytokeratin 18. Electron microscopy showed that there were abundant organelles in hepatocytes and tight junctions between cells. HSC had expressed α-smooth muscle actin and desmin. Flow cytometry showed hepatic mononuclear cells, including lymphocyte subsets such as CD4, CD8, NKs, and NKTs. Conclusion: The hepatic perfusion through the portal vein digestion method can isolate multiple primary cells from the liver of mice at once and has the features of simplicity and efficiency.

Cryoprotective effect of sericin on re-freezing of bull spermatozoa and subsequent embryonic development after in vitro fertilization

Sericin, a protein produced by silk worm, has been used to improve quality of frozen-thawed spermatozoa in several species. However, the cryoprotective effect of sericin on re-freezing of spermatozoa has not been reported. This study was conducted to investigate the putative cryoprotective effect of sericin on re-freezing of bull spermatozoa followed by an assessment of in vitro fertilizing capacity of sericin-supplemented re-frozen spermatozoa. In experiment 1, the effect of 0.5% sericin supplemented in semen extender on re-freezing of spermatozoa was examined. Highly motile frozen-thawed spermatozoa were prepared with Percoll discontinuous gradient centrifugation prior to re-freezing. Refrozen-thawed spermatozoa were examined for motility, viability, membrane integrity, and acrosome integrity. In experiment 2, the effect of sericinsupplemented re-frozen spermatozoa on fertilizing capacity and subsequent embryonic development were determined using in vitro embryo production approach. Bovine oocytes were fertilized with three groups spermatozoa, re-frozen/control, re-frozen/sericin, and once frozen. The results showed that the total motility of re-frozen thawed spermatozoa in the sericin supplemented group was higher (53.9 % vs. 41.5%, p<0.05) than that of the control group whereas viability, membrane integrity, and acrosome integrity were unaffected. The proportion of cleaved oocytes in re-frozen/sericin-supplemented group was comparable to those in the re-frozen/control and once frozen groups. Additionally, the percentages of blastocyst development from cleaved oocytes were similar among the three groups. In conclusion, the results of this study demonstrated that pretreatment of frozen-thawed spermatozoa with 0.5% sericin improved motility after re-frozen thawed without affecting the in vitro fertilizing capacity and subsequent embryonic development.

Isolation and Measurement of Respiration and Structural Studies of Purified Mitochondria from Heterotrophic Plant Tissues.

Mitochondria are the power houses of eukaryotic cells. These organelles contain various oxidoreductase complexes. Electron transfer from different reducing equivalents channeled via these complexes drives proton translocation across the inner mitochondrial membrane, leading to ATP generation. Plant mitochondria contain alternative NAD(P)H dehydrogenases, alternative oxidase, and uncoupling protein, and TCA cycle enzymes are located in their matrix. Apart from ATP production, mitochondria are also involved in synthesis of vitamins and cofactors and participate in fatty acid, nucleotide, photorespiratory, and antioxidant metabolism. Recent emerging evidence suggests that mitochondria play a role in redox signaling and generation of reactive oxygen and nitrogen species. For mitochondrial studies, it is essential to isolate physiologically active mitochondria with good structural integrity. In this article, we explain a detailed procedure for isolation of mitochondria from various heterotrophic tissues, such as germinating chickpea seeds, potato tubers, and cauliflower florets. This procedure requires discontinuous Percoll gradient centrifugation and can give a good yield of mitochondria, in the range of 4 to 8 mg per 50 g tissue with active respiratory capacity. After MitoTracker staining, isolated mitochondria can be visualized by using a confocal microscope. The structure of mitochondria can be monitored by scanning electron microscopy. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Isolation of mitochondria from germinating chickpea seeds, potato tubers, and cauliflower florets Basic Protocol 2: Quantification of mitochondrial protein concentration by Bradford assay Basic Protocol 3: Quantification of mitochondrial respiration using single-channel free-radical analyzer Basic Protocol 4: Staining of mitochondria and confocal imaging Basic Protocol 5: Visualization of isolated mitochondria under scanning electron microscope.

Influence of maggot excretions/secretions on the anti-Pseudomonas aeruginosa effect of neutrophils in patients with diabetic foot ulcer

Objective: To investigate the effects of medical maggot excretions/secretions (ES) on neutrophils phagocytosis and bactericidal effect in patients with diabetic foot ulcer (DFU). Methods: The experimental research method was used. Thirty DFU patients (16 males and 14 females, aged (64±7) years)who were admitted to the Diabetes Foot Center, the Department of Endocrinology of Air Force Hospital of Eastern Theater Command from June to December 2020 and met the inclusion criteria were recruited. Discontinuous percoll gradient centrifugation method was used to separate the neutrophils. Cells from each patient were enrolled into normal saline group and maggot ES group (30 wells in each group), respectively; sterile normal saline and ES with a final mass concentration of 357 μg/mL (the same as below) were added, respectively. After 1 and 2 hour(s) of culture, the phagocytosis rate and phagocytic index of cells were observed and counted under Wright's staining. Ten patients were selected, then the cells of each patient were enrolled into Pseudomonas aeruginosa+neutrophils group and Pseudomonas aeruginosa+neutrophils+maggot ES group (10 wells in each group) and were treated corresponding, respectively. Pseudomonas aeruginosa alone group and Pseudomonas aeruginosa+maggot ES group (10 wells in each group) were set up respectively; Pseudomonas aeruginosa+RPMI 1640 culture medium+sterile normal saline and Pseudomonas aeruginosa+RPMI 1640 culture medium+maggot ES were added, respectively. After 2 hours of culture, the number of viable bacteria colony was counted by plate colony number method. Six, six, and three patients were selected respectively, and the cells of each patient were respectively enrolled into maggot ES group and normal saline group (6, 6, and 3 wells in each group, respectively) and treated accordingly. After 6 hours of culture, real-time fluorescent quantitative reverse transcription polymerase chain reaction was used to detect the mRNA expressions of interleukin 1β (IL-1β), IL-6, and lysozyme in cells, the content of IL-1β and IL-6 in cell culture supernatant were determined by enzyme-linked immunosorbent assay, and the positive cells expressing lysozyme were observed with immunofluorescence method. Data were statistically analyzed with one-way analysis of variance, paired sample t test, least significant difference test, and Wilcoxon rank sum test. Results: After 1 hour of culture, the phagocytosis rate and phagocytic index of cells in maggot ES group (53.5% (49.7%, 58.0%) and 3.18 (2.96, 3.32)) were similar to 52.0% (47.5%, 55.2%) and 3.15 (2.96, 3.25) of normal saline group (Z=-1.701, -1.092, P>0.05). After 2 hours of culture, the phagocytosis rate and phagocytic index of cells in maggot ES group (70.0% (66.7%, 72.0%) and 4.47 (4.22, 4.96)) were significantly higher than 58.0% (55.0%, 60.0%) and 4.11 (3.52, 4.24) in normal saline group (Z=-4.786, -4.279, P<0.01). After 2 hours of culture, the number of viable bacteria colony in Pseudomonas aeruginosa+neutrophils group was significantly lower than that in Pseudomonas aeruginosa alone group (P<0.01), and the number of viable bacteria colony in Pseudomonas aeruginosa+neutrophils+maggot ES group was significantly lower than that in Pseudomonas aeruginosa+maggot ES group and Pseudomonas aeruginosa+neutrophils group (P<0.01). After 6 hours of culture, the mRNA expressions of IL-1β, IL-6, and lysozyme of cells in maggot ES group were significantly higher those in normal saline group (t=-3.279, -4.273, -4.763, P<0.05 or P<0.01); the concent of IL-1β and IL-6 in cell culture supernatant of maggot ES group were significantly higher than those of normal saline group (t=-9.526, -6.447, P<0.01); there were significantly more positive cells expressing lysozyme in maggot ES group than in normal saline group. Conclusions: Maggot ES can enhance the phagocytosis and bactericidal effect of neutrophils on Pseudomonas aeruginosa by promoting the production of neutrophils immune defense related cytokines and lysozyme in DFU patients.

Isolation, in vitro study, and stem cell markers for type A spermatogonia in a Characiformes species.

The objective of this study was to establish a protocol for the characterization, isolation, and culture of type A spermatogonia using specific molecular markers for these cells in fish. To this end, adult Prochilodus lineatus testes were collected and digested enzymatically and the resulting testicular suspension was separated using a discontinuous Percoll gradient, followed by differential plating. The cell cultures obtained were monitored for 15 days and analyzed using the immunofluorescence method with anti-Vasa, anti-GFRα1, and anti-OCT4 antibodies. Spermatogonial enrichment was also performed using flow cytometry. Although discontinuous Percoll gradient centrifugation followed by differential plating enabled the removal of differentiated germ cells and somatic cells, enriching the pool of type A spermatogonia, the enrichment of type A spermatogonia through flow cytometry of samples without Percoll proved to be more efficient. Prominent cell agglomerates that were characterized according to different stem cell markers as type A spermatogonia were observed during the 15 days of the cell culture. The use of immunoperoxidase and western blot analysis methods confirmed the specificity of the markers for type A spermatogonia of P. lineatus. When combined with specific cell culture conditions, the positive characterization of these molecular markers clarified certain aspects of spermatogonial regulation, such as survival and proliferation. Finally, understanding the regulation of the in vitro germ cell maintenance process may contribute to the enhancement of in vivo and in vitro reproduction techniques of endangered or aquaculture fish species.

Cushioned centrifugation during sperm selection increases the fertilization and cleavage rates of cattle embryos produced in vitro

This study was conducted to evaluate the effect of utilization of an iodixanol-based solution as a cushioning method during the sperm selection utilizing discontinuous Percoll gradient centrifugation in in vitro production (IVP) of cattle embryos. In Experiment I, all aliquots of thawed semen were subjected to sperm selection using the same discontinuous Percoll® gradients, except for the following four conditions: presence of cushioning solution (Cushion Fluid, Minitube) during the first centrifugation process (C1), presence of cushioning solution during the second centrifugation process (C2), inclusion of cushioning solution in both centrifugation steps (C1-2), and no addiction of cushioning solution (C; control group). Recovery rates, sperm kinetics, and reactive oxygen species (ROS) production were evaluated. In Experiment II, sperm cells were processed using sperm selection conditions C and C1, and fertilization rates and embryonic development kinetics were compared between experimental groups. With use of condition C1, there was improvement in fertilization and cleavage rates when compared to use of condition C (56.4% compared with 45.5% and 80.0% compared 64.7%, respectively). In conclusion, results indicate the use of a cushioning solution during sperm selection positively affects the developmental potential of embryos.

Isolation and High Throughput Flow Cytometric Apoptosis Assay of Human Neutrophils to Enable Compound Library Screening.

The study of human neutrophils in vitro is challenging due to their short half-life and propensity for activation. However, with careful handling and manipulation in the laboratory, they can be a powerful tool to investigate immune responses in health and disease. Here we describe a method for the isolation of human neutrophils from peripheral blood samples, followed by a high-throughput screen to assess the efficacy of a library of compounds in inducing neutrophil apoptosis, which may have therapeutic potential in neutrophil-driven diseases. This protocol is based on previously-published neutrophil isolation methods utilizing Dextran sedimentation of red blood cells followed by the separation of granulocytes with plasma/Percoll discontinuous gradient centrifugation. Yields of ~1 x 106 neutrophils per millilitre of blood, and purities of > 95% neutrophils are typical. Neutrophils are treated with a library of kinase inhibitors, followed by flow cytometry to assess the rate of neutrophil apoptosis. This protocol allows for the high-throughput screening of primary human immune cells to identify compounds with a potential to modify neutrophil function, and could be modified to assess other phenotypes if required.

An Efficient Method of Mitochondrial DNA Isolation from Vigna radiata for Genomic Studies.

Isolation of mitochondrial DNA from root tissues of mung bean (Vigna radiata) is quite tedious, complex, and often results in low yield. Hence here we show a simple, rapid, and improved protocol for isolation of mitochondrial DNA from root tissues of hydroponically grown mung bean plants. This method involves purification of mitochondria and subsequent isolation of DNA from obtained purified mitochondria. For this purpose, mitochondria were isolated using a discontinuous Percoll gradient centrifugation followed by RNase I treatment to the isolated DNA to remove any traces of RNA contamination. The mitochondrial DNA was isolated from mitochondrial samples by commonly used CTAB method. The specificity of isolated mitochondrial DNA was confirmed using mtDNA-specific genes (NAD1 and COX3). β-Actin primer was used to check the nuclear DNA contamination. PCR amplification was observed in mtDNA specific genes NAD1 and COX3 except nuclear encoded β-actin gene suggesting that mitochondrial DNA was not contaminated by nuclear DNA.

Cryptosporidium Oocyst Purification Using Discontinuous Gradient Centrifugation.

Many laboratory studies in cryptosporidial research require a source of purified oocysts. Sources can include experimentally infected laboratory animals or from samples collected from naturally infected animals and from clinical cases of human cryptosporidiosis. Purification of oocysts can be accomplished with readily available laboratory equipment including tabletop centrifuges and microcentrifuges. Following purification, oocysts can be stored in antibiotic-supplemented buffers or in 2.5% aqueous potassium dichromate for over 6months. Ultimately, oocyst viability and infectivity decline to less than 10% after 1year, so if isolates are expected to be maintained, serial passage in a suitable host at ≤6-month intervals is recommended. Oocysts purified as described in this chapter are suitable for animal infection studies, cell culture studies, and a wide range of molecular biological studies, environmental studies, drug testing, and disinfection studies.

The effects of sevoflurane and propofol on human sperm motility and capacitation in vitro

Objective To investigate the effects of sevoflurane and propofol on human sperm motility and capacitation at the clinic concentration in vitro. Methods Sperm samples were obtained from normal adults and prepared with discontinuous percoll gradient centrifugation technique, adjusting the sperm density at (30-50)×106/ml. The sperm samples were randomly divided into control group (C group), sevoflurane group (S group) and propofol group (P group), according to the concentration of sevoflurane, S group was divided into low concentration group (S1 group, 1.8% sevoflurane) and high concentration group (S2 group, 2.2% sevoflurane), according to the concentration of propofol, P group was divided into low concentration group (P1 group, 1.8 μg/ml) and high concentration group (P2 group, 2.4 μg/ml), all the samples were incubated for 5 h in an airtight glass container filled with air at 37 ℃. Then human sperm motility waft examined in vitro at 37 ℃ and analyzed by the computer-assisted sperm analysis (CASA), including sperm motility (a+ b)%, curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP) and amplitude of lateral head displacement (ALH). The capacitation effect was assessed by using the chlortetracycline (CTC) staining, (B+ AR)% indicates the proportion of sperm capacitation has occurred. Results Compared with control group, after incubation of the sperm samples with 2% and 4% sevoflurane, 4 μg/ml and 8 μg/ml propofol for 5 h, the motility of sperm (a+ b)% and VCL, VSL, VAP, ALH has decreased, and the impact on sperm motility in a dose-dependent manner ; both sevoflurane and propofol at clinical concentrations could inhibit sperm capacitation in a dose-dependent manner(P=0.017); at the same clinical effect concentrations, inhibitory effects of sevoflurane on sperm motility was stronger than propofol (P=0.026); about sperm capacitation, at the same clinical effect concentrations, inhibitory effects of sevoflurane on sperm capacitation was significantly stronger than the propofol (P=0.011). Conclusion Sevoflurane and propofol at clinically relevant concentrations have significant inhibitory effects on human sperm motility and capacitation in a dose-dependent manner. Sevoflurane has stronger inhibitory effect than propofol. Key words: Sevoflurane; Propofol; Sperm motility; Sperm capacitation

SLDP: a novel protein related to caleosin is associated with the endosymbiotic Symbiodinium lipid droplets from Euphyllia glabrescens.

Intracellular lipid droplets (LDs) have been proposed to play a key role in the mutualistic endosymbiosis between reef-building corals and the dinoflagellate endosymbiont Symbiodinium spp. This study investigates and identifies LD proteins in Symbiodinium from Euphyllia glabrescens. Discontinuous Percoll gradient centrifugation was used to separate Symbiodinium cells from E. glabrescens tentacles. Furthermore, staining with a fluorescent probe, Nile red, indicated that lipids accumulated in that freshly isolated Symbiodinium cells and lipid analyses further showed polyunsaturated fatty acids (PUFA) was abundant. The stable LDs were purified from endosymbiotic Symbiodinium cells. The structural integrity of the Symbiodinium LDs was maintained via electronegative repulsion and steric hindrance possibly provided by their surface proteins. Protein extracts from the purified LDs revealed a major protein band with a molecular weight of 20kDa, which was termed Symbiodinium lipid droplet protein (SLDP). Interestingly, immunological cross-recognition analysis revealed that SLDP was detected strongly by the anti-sesame and anti-cycad caleosin antibodies. It was suggested that the stable Symbiodinium LDs were sheltered by this unique structural protein and was suggested that SLDP might be homologous to caleosin to a certain extent.

Isolation and cultivation of metabolically competent alveolar epithelial cells from A/J mice

The A/J mouse strain is used in lung cancer studies. To enable mechanistic investigations the isolation and cultivation of alveolar epithelial cells (AECs) is desirable. Based on four different protocols dispase digestion of lung tissue was best and yielded 9.3±1.5×106 AECs. Of these 61±13% and 43±5% were positive for AP and NBT staining, respectively. Purification by discontinuous Percoll gradient centrifugation did not change this ratio; however, reduced the total cell yield to 4.4±1.1×106 AECs. Flow cytometry of lectin bound AECs determined 91±7% and 87±5% as positive for Helix pomatia and Maclura pomifera to evidence type II pneumocytes. On day 3 in culture the ethoxyresorufin-O-demethylase activity was 251±80pmol/4h×1.5×106 and the production of androstenedione proceed at 243.5±344.4pmol/24h×1.5×106 AECs.However, 6-α, 6-β and 16-β-hydroxytestosterone were produced about 20-fold less as compared to androstenedione and the production of metabolites depended on the culture media supplemented with 2% mouse serum or 10% FCS. Finally, by RT-PCR expression of CYP genes was confirmed in lung tissue and AECs; a link between testosterone metabolism and CYP2A12, 3A16 and 2B9/10 expression was established. Taken collectively, AECs can be successfully isolated and cultured for six days while retaining metabolic competence.

First Birth after Sperm Selection through Discontinuous Gradient Centrifugation and Artificial Insemination from a Chromosomal Translocation Carrier

Introduction. Balanced chromosomal carriers, though usually healthy, are confronted with recurrent spontaneous abortions and malformations in the offspring. Those are related to the transmission of an abnormal, chromosomally unbalanced genotype. We evidenced that the proportion of unbalanced spermatozoa can be significantly decreased through a sperm preparation process called discontinuous gradient centrifugation (DGC). We therefore started offering intrauterine inseminations with this procedure to couples with a male translocation carriers. Case Presentation. We report the case of a 37-year-old man carrying a t(3;10)(q25;p13) reciprocal translocation. He and his partner had had trouble conceiving for ten years and had four spontaneous abortions. DGC in this patient decreased the proportion of unbalanced spermatozoa from 63.6% to 52.3%. They were therefore offered intrauterine insemination with DGC, which eventually led to the birth of a healthy female child carrying the paternal translocation. Conclusion. We showed that translocation carriers could be offered intrauterine inseminations with DGC. Before this, the only two options were natural conception with prenatal diagnosis and termination of chromosomally unbalanced fetuses or preimplantation genetic diagnosis, which is a much heavier and costly procedure. We are currently offering this option through a multicentric program in France, and this is the first birth originating from it.

Exposure to cocaine regulates inhibitory synaptic transmission from the ventral tegmental area to the nucleus accumbens

Synaptic projections from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) make up the backbone of the brain reward pathway, a neural circuit that mediates behavioural responses elicited by natural rewards as well as by cocaine and other drugs of abuse. In addition to the well-known modulatory dopaminergic projection, the VTA also provides fast excitatory and inhibitory synaptic input to the NAc, directly regulating NAc medium spiny neurons (MSNs). However, the cellular nature of VTA-to-NAc fast synaptic transmission and its roles in drug-induced adaptations are not well understood. Using viral-mediated in vivo expression of channelrhodopsin 2, the present study dissected fast excitatory and inhibitory synaptic transmission from the VTA to NAc MSNs in rats. Our results suggest that, following repeated exposure to cocaine (15 mg kg(-1) day(-1) × 5 days, i.p., 1 or 21 day withdrawal), a presynaptic enhancement of excitatory transmission and suppression of inhibitory transmission occurred at different withdrawal time points at VTA-to-NAc core synapses. In contrast, no postsynaptic alterations were detected at either type of synapse. These results suggest that changes in VTA-to-NAc fast excitatory and inhibitory synaptic transmissions may contribute to cocaine-induced alteration of the brain reward circuitry.

Teratozoospermic sperm with highly fragmented DNA subjected to Discontinuous Gradient Centrifugation+Annexin V-MACS have similar fertilization and pregnancy rates than non-apoptotic controls

To evaluate fertilization (FR) and pregnancy (PR) rates in patients with sperm showing high levels of DNA fragmentation and treated with ICSI after Annexin V-Magnetic Activated Cell Sorting (AV-MACS) sperm selection.

Failure in beta-amyloid degradation in the endosomal/lysosomal pathway results in intracellular beta-amyloid accumulation within lipid rafts

Endothelin-converting enzymes (ECE)-1 and -2 are members of the M13 family of zinc-metalloproteases that plays a major role in Aβ catabolism. We have recently established that ECE-1 and ECE-2 are found in Aβ-generating compartments and degrade the peptide at intracellular sites of production. Based on the localization of different ECE-1 isoforms to the plasma membrane, recycling endosomes, and late endosomes, ECE-1 activity influences extracellular Aβ levels (mainly by degrading the peptide prior to secretion) as well as intracellular levels. ECE-2, however, is restricted to the endosomal/lysosomal pathway in neurons and regulates a pool of Aβ produced within the vesicles of this pathway, including autophagosomes. Treatment of neuronal cells with ECE inhibitors produces increases in both extracellular and intracellular Aβ. Levels of intracellular Aβ, however, stabilize over time after ECE inhibition suggesting that additional proteases contribute to intracellular Aβ removal SH-SY5Y cells stably transfected with wild type APP were used as a neuronal model. Inhibition of ECE and lysosomal enzymes was produced with specific protease inhibitors. Autophagic flux was inhibited with lysosomotropic agents. Levels of Aβ were measured by ELISA and immunofluorescence. Detergent resistant membranes were isolated by discontinuous gradient centrifugation after incubation in cold 1% Triton X-100. Western blot analysis was used to confirm the presence of specific lipid raft markers in the obtained fractions. Inhibition of ECEs with phosphoramidon produced intracellular accumulation of Aβ in the lumen of endosomal/lysosomal vesicles. Simultaneous inhibition of ECEs and cysteine protease activity withE64 doubled intracellular Aβ accumulation compared to ECE inhibition alone, without further altering secreted levels. With the combined treatment, there was also a selective accumulation of Aβ in CTFs-enriched lipid rafts. Interestingly,E64 alone did not have any impact in either extracellular or intracellular Aβ concentration in these cells. Our findings demonstrate that failure in Aβ degradation in the endosomal/lysosomal pathway can result in an accumulation of intracellular Aβ in lipid rafts. As intracellular acidic compartments and lipid rafts have been proposed as potential sites for Aβ aggregation, our cell model may prove helpful in understanding the initial of steps of the disease.

Estradiol potentiates 8-OH-DPAT-induced sumoylation of 5-HT1A receptor: Characterization and subcellular distribution of sumoylated 5-HT1A receptors

Sumoylation is a recently described post-translational modification and only a few sumoylated neurotransmitter receptors are known. Through the present studies, we discovered that serotonin1A receptors (5-HT1A-Rs) can be sumoylated by SUMO1 (small-ubiquitin-related modifier 1) protein. The SUMO1-5-HT1A-R is ∼55kDa, is located in the membrane fraction, but not the cytosol, and is distributed in all of the brain regions expressing 5-HT1A-Rs examined. Acute stimulation of 5-HT1A-Rs significantly increased SUMO1-5-HT1A-R in rat hypothalamus. Pre-treatment with estradiol for 2 days, which causes a partial desensitization of 5-HT1A-R signaling, potentiated agonist-induced increases in SUMO1-5-HT1A-Rs in the hypothalamus of ovariectomized rats. Using discontinuous gradient centrifugation followed by digitonin treatment, we found that the majority of SUMO1-5-HT1A-Rs is co-localized with endoplasmic-reticulum and trans-Golgi-network markers. Although a small proportion of SUMO1-5-HT1A-Rs are located in the detergent resistant microdomain (DRM) that contain active G-protein coupled receptors, their distribution was different from that of the Gαz protein that couples to the receptors. These data suggest that the SUMO1-5-HT1A-Rs are an inactive form of 5-HT1A-Rs, a finding further supported by results showing minimal 5-HT1A-R agonist binding to SUMO1-5-HT1A-Rs. Furthermore, SUMO1-5-HT1A-Rs in the DRM were increased by treatment with a 5-HT1A-R agonist, 8-OH-DPAT ((+)8-hydroxy-2-dipropylaminotetralin). Together, these data suggest that sumoylation of 5-HT1A-Rs may be related to 5-HT1A-R trafficking and internalization, which may contribute to 5-HT1A-R desensitization. Since 5-HT1A-Rs play an important role in mood regulation, the present results significantly impact on the understanding of the pathogenesis of affective disorders and development of better therapeutic approaches for these diseases.

Discontinuous gradient centrifugation (DGC) decreases the proportion of chromosomally unbalanced spermatozoa in chromosomal rearrangement carriers

Can the proportion of unbalanced spermatozoa in chromosomal rearrangement carriers be decreased through the use of discontinuous gradient centrifugation (DGC)? DGC significantly decreases the proportion of genetically unbalanced spermatozoa in chromosomal rearrangement carriers. Chromosomal rearrangement carriers present with a certain proportion of unbalanced gametes, which can lead to miscarriages or malformations in the offspring. There is presently no known way to select the balanced spermatozoa and use them for IVF. The proportion of unbalanced spermatozoa after DGC was compared with that before DGC in 21 patients with a chromosomal rearrangement. At least 500 spermatozoa were analysed per observation. Twenty-one male patients with a chromosomal rearrangement were included in this prospective study. They initially consulted for infertility, recurrent miscarriages or a history of abnormal pregnancy. The samples were split into two, with one part undergoing DGC and the other being immediately fixed. Fluorescence in situ hybridization was performed to establish the chromosome segregation pattern of each spermatozoon. DGC significantly decreased the proportion of unbalanced spermatozoa in all but 1 of the 21 chromosomal rearrangement carriers (P < 0.05). Although DGC reduces the proportion of unbalanced spermatozoa in ejaculates from patients with chromosome rearrangements this elimination is only partial and some abnormal spermatozoa remain. Means to exclude these spermatozoa to ensure that only balanced ones are used in IVF remain to be discovered. The motility and morphology of the sperm before and after DGC were not measured. Used in IVF or intrauterine insemination, DGC could decrease the chance that a man carrying a chromosomal rearrangement will father an abnormal fetus.

Differential expression of protamine 1 and 2 genes in mature spermatozoa of normal and motility impaired semen producing crossbred Frieswal (HF × Sahiwal) bulls

Mature spermatozoa contain thousands of mRNA transcripts. These untranslated mRNA may perhaps serve as a “footprint” of spermatogenesis since many of them might directly or indirectly be involved in fertilization, early embryo cleavage, poor semen quality and fertility. In this study, we tried to isolate high-quality RNA from mature spermatozoa and to monitor the expression profile of protamine 1 (PRM1) and protamine 2 (PRM2) gene in ejaculated spermatozoa of normal (good, % initial progressive motility: 57.61±1.41, n=9) and motility impaired (poor, % initial progressive motility: 18.45±1.61, n=8) crossbred Frieswal (HF×Sahiwal) bulls semen using real time quantitative PCR. Semen samples were subjected to discontinuous (45:90) Percoll gradient centrifugation, specifically to eliminate damaged spermatozoa and contaminating somatic cells. Total RNA was extracted from sperm pellets and cDNA was synthesized. Furthermore, the absence of contamination of germ cells, epithelial cells and leucocytes in all the RNA extractions was tested by RT-PCR targeting specific molecular markers like KIT, CDH1 and CD4, respectively. The presence of transcripts like PRM1, PRM2, DAZL, and PPIA were demonstrated in ejaculated spermatozoa using appropriate PCR primers without RNA amplification. Expression of PRM1 and PRM2 genes were evaluated by real time quantitative PCR using TaqMan chemistry, where PPIA was used as internal control. The cDNA synthesized from normal buffalo testicular tissue was served as positive control. The good quality semen producing group showed significantly higher level of PRM1 mRNAs expression as compared to the poor quality semen producers (P<0.05) indicating putative role of the gene and semen quality parameters especially initial progressive motility. However, PRM2 transcript levels were not significantly different between the groups (P>0.05).