Paternal cocaine taking elicits epigenetic remodeling and memory deficits in male progeny.

This corrects the article DOI: 10.1038/mp.2017.8.

Paternal cocaine taking elicits epigenetic remodeling and memory deficits in male progeny.

Paternal environmental perturbations including exposure to drugs of abuse can produce profound effects on the physiology and behavior of offspring via epigenetic modifications. Here we show that adult drug-naive male offspring of cocaine-exposed sires have memory formation deficits and associated reductions in NMDA receptor-mediated hippocampal synaptic plasticity. Reduced levels of the endogenous NMDA receptor co-agonist d-serine were accompanied by increased expression of the d-serine degrading enzyme d-amino acid oxidase (Dao1) in the hippocampus of cocaine-sired male progeny. Increased Dao1 transcription was associated with enrichment of permissive epigenetic marks on histone proteins in the hippocampus of male cocaine-sired progeny, some of which were enhanced near the Dao1 locus. Finally, hippocampal administration of d-serine reversed both the memory formation and synaptic plasticity deficits. Collectively, these results demonstrate that paternal cocaine exposure produces epigenetic remodeling in the hippocampus leading to NMDA receptor-dependent memory formation and synaptic plasticity impairments only in male progeny, which has significant implications for the male descendants of chronic cocaine users.

Monitoring the Foetal Phase of Gestation in the Queen With a 12.5-MHz Ultrasound Probe and Prediction of the Parturition by Combining the Measurements of Head and Abdominal Diameters.

Ten gestations in six domestic shorthair cats (Europeans) were monitored daily during the foetal phase of gestation, from the 28th day after the first mating until parturition, using ultrasound with a 12.5-MHz probe. The development of the various organs over this period was recorded. The diameters of the head (HD) and abdomen (AD) were measured. Skeletal calcification visible on ultrasound occurred in a defined order between the 34th and 40th day of gestation. During the last 30 days of gestation, there was a significant correlation between HD and days before parturition (DBP) (r(2)  = 0.99) and between AD and DBP (r(2)  = 0.98). The following equations were obtained: DBP = -2.10*HD (mm) + 50.74; DBP = -1.01*AD (mm) + 42.19. The confidence intervals were stable over the last 30 days of gestation. For the HD, the confidence interval was ±1 day in 53% of cases and ±2 days in 85% of cases. For the AD, the confidence interval was ±1 day in 45% of cases and ±2 days in 77% of cases. A table obtained by combining the HD and AD measurements made it possible to estimate the date of parturition within 2 days with a reliability of over 85%.

Canine-chilled sperm: study of a semen extender made with low-density lipoproteins from hen egg yolk supplemented with glutamine.

The objective of this study was to evaluate the effects of a combination of 6% low-density lipoproteins (LDL) and 20 mm glutamine in comparison with other extenders used for the refrigeration of canine semen: Tris egg yolk (EY) 20% and 6% LDL. The percentages of mobile spermatozoa after 4 days storage in a domestic refrigerator at +4 °C were 53.1%, 44.2% and 52.2% for the 6% LDL + 20 mm glutamine, 20% EY and 6% LDL extenders respectively for 100% of the dogs. After 7 days of storage, these percentages fell to 37.8%, 26.4% and 33.6% in the same extenders for 50% of the dogs. In vitro fertility tests were performed with all of the extenders following the mobility results. These tests were conducted on the day of sampling (D0), and 48 and 96 h after sampling. The results of the hypo-osmotic swelling test were 82.6%, 81.2% and 85.7% on D0, 75.2%, 74.1% and 78.5% on D2, and 70.8%, 71% and 76.1% on D4 for the 6% LDL + 20 mm glutamine, 20% EY and 6% LDL extenders, respectively. For the FITC/pisum sativum agglutinin (PSA) test, the results were 81.5%, 70.2% and 84.8% on D0, 78.9%, 62.3% and 84.2% on D2, and 72.7%, 59.6% and 73.7% on D4 for the 6% LDL + 20 mm glutamine, 20% EY and 6% LDL extenders, respectively. The acridine orange test was positive; in nearly 100% of cases, none of the spermatozoa had been denatured on D0, D2 and D4. The 6% LDL + 20 mm glutamine and the 6% LDL extenders are capable of preserving spermatozoa that have been stored in a domestic refrigerator at +4°C for at least 4 days. This means that the spermatozoa retain good cytoplasmic membrane integrity, had not capacitated and contained intact DNA in comparison with spermatozoa preserved in the egg yolk extender. The duration of storage is a very important consideration when faced with the problem of sending semen over ever-greater distances.

Effect of semen dilution to low-sperm number per dose on motility and functionality of cryopreserved bovine spermatozoa using low-density lipoproteins (LDL) extender: comparison to Triladyl and Bioxcell.

Artificial insemination with doses containing low-sperm numbers has been utilized to optimize the use of elite bulls. Hen egg yolk is widely used as a cryoprotective agent in semen freezing extender protecting the spermatozoa. Its action is due to the presence of low-density lipoproteins (LDL) in the hen egg yolk. The objectives of the present study were to evaluate the effects of the semen dilution to low-sperm number/dose on sperm motility and integrity of sperm plasma membrane in the cryopreservation process, using two commercial extenders (Triladyl, Bioxcell and LDL extender prepared in our laboratory, 97% purity. Fifteen ejaculates were collected from five fertile crossbred bulls (Bos taurusxBos indicus). After collection, sperm motility was examined by Computer-Assisted Semen Analysis (Hamilton Thorne), morphological sperm characteristics were evaluated by differential interference microscopy and the integrity of plasma membranes was determined using the hypo-osmotic swelling test. The semen was subsequently divided into three aliquots and diluted with the three extenders into 120 x 10(6), 60 x 10(6) and 20 x 10(6)sperm/mL, corresponding to 30 x 10(6), 15 x 10(6) and 5 x10(6) sperm/dose, respectively. This study revealed that LDL extender was more effective in preservation of motility and integrity of the plasma membrane of spermatozoa than Bioxcell and Triladyl (p<0.05), but no significant difference was observed between Triladyland Bioxcell. Therefore we can conclude that LDL extender could be used instead of Triladyl or Bioxcellat low semen concentration per dose for elite bulls, it also could be envisaged for the industry of sex-stored semen.

Effect of glutamine on post-thaw motility of bull spermatozoa after association with LDL (low density lipoproteins) extender: preliminary results.

Glutamine has been used in the composition of semen extenders in several species, but never in the bull. The aim of our study is to demonstrate the cryoprotective role of glutamine for freezing bovine semen and to determine concentration of the latter to improve the motility and trajectory characteristics of spermatozoa. Three experiments were undertaken with 21 ejaculates from three different bulls. In the first experiment, glutamine was added to 40, 80, and 120 mM of basic medium (BM) which consisted of Tris+glycerol 6.4% (v/v). In the second experiment glutamine was added to the 8% low density lipoprotein (LDL) diluent at 40, 80, and 120 mM. In the third experiment, the best concentration of glutamine was determined; this was then added to the LDL extender at 10, 20, 30, and 40 mM. The semen was diluted then frozen in the different media. Motility parameters were assessed using an image analyser following thawing. Experiment 1 demonstrated that glutamine had a cryoprotective effect; at 40 mM it gave superior motility parameters to those obtained with the basic medium (p<0.05). Experiment 2 demonstrated that the combination of LDL-glutamine 40 mM and 80 mM did not improve motility and even deteriorated it in comparison with the glutamine-free LDL extender. Experiment 3 demonstrated that the addition of 10mM of glutamine to the LDL medium lead to a significant improvement (p<0.05) in the motility of bull spermatozoa and could be used to improve bovine semen extenders.

Impaired object recognition following prolonged withdrawal from extended-access cocaine self-administration.

Cocaine addicts have a number of cognitive deficits that persist following prolonged abstinence. These include impairments in executive functions dependent on the prefrontal cortex, as well as deficits on learning and memory tasks sensitive to hippocampal function. Recent preclinical studies using non-human animals have demonstrated that cocaine treatment can produce persistent deficits in executive functions, but there is relatively little evidence that treatment with cocaine produces persistent deficits in performance on hippocampal-dependent tasks. We recently demonstrated that extended (but not limited) access to self-administered cocaine is especially effective in producing persistent deficits on a test of cognitive vigilance, and therefore, we used this procedure to examine the effects of limited or extended access to cocaine self-administration on recognition memory performance, which is sensitive to hippocampal function. We found that extended access to cocaine produced deficits in recognition memory in rats that persisted for at least 2 weeks after the cessation of drug use. We conclude that the deficits in learning and memory observed in cocaine addicts may be at least in part due to repeated drug use, rather than just due to a pre-existing condition, and that in studying the neural basis of such deficits procedures involving extended access to self-administered cocaine may be especially useful.

Use of glutamine and low density lipoproteins isolated from egg yolk to improve buck semen freezing.

To improve the results obtained with a reference cryopreservation extender (control extender: Triladyl + 20% (v/v) egg yolk + 6.4% (v/v) glycerol) for freezing caprine semen, glutamine was added to 18 split ejaculates at concentrations of 0, 20, 40, 80 and 120 mM (experiment 1). In experiment 2, glutamine was added to 18 split ejaculates at concentrations of 20, 25, 30, 35 and 40 mM. In the third experiment, the egg yolk was replaced with the low-density lipoprotein (LDL) fraction of egg yolk. The quality of frozen then thawed spermatozoa in each extender was compared using computer-assisted semen analysis. In experiment 1, glutamine at concentrations of 20 mm and 40 mm significantly improved sperm motility compared with the control extender. However, at 120 mM, a significant decrease in motility and velocity was observed. In experiment 2, motility, curvilinear velocity and amplitude of lateral head displacement (ALH) were improved in glutamine at 25 mM compared with the control. In experiment 3, 8% LDL and 25 mM glutamine significantly improved sperm motility, straight line velocity and ALH. In the fourth experiment, the quality of the previously defined freezing extender (Triladyl + 8% (v/v) LDL + 25 mM glutamine + 6.4% (v/v) glycerol) was tested by comparing acrosome, tail membrane, plasma membrane and DNA integrity in 18 split ejaculates of frozen then thawed spermatozoa with spermatozoa that had been frozen then thawed in the control extender, and with spermatozoa from fresh, unfrozen sperm. The percentage of spermatozoa with intact acrosomes and tail membranes was significantly higher with the newly defined extender than that observed with the control extender. There was no significant difference in the percentage of spermatozoa with intact DNA between the frozen and fresh semen.

A self-inducible heterologous protein expression system in Escherichia coli.

Escherichia coli is an important experimental, medical and industrial cell factory for recombinant protein production. The inducible lac promoter is one of the most commonly used promoters for heterologous protein expression in E. coli. Isopropyl-ß-D-thiogalactoside (IPTG) is currently the most efficient molecular inducer for regulating this promoter's transcriptional activity. However, limitations have been observed in large-scale and microplate production, including toxicity, cost and culture monitoring. Here, we report the novel SILEX (Self-InducibLe Expression) system, which is a convenient, cost-effective alternative that does not require cell density monitoring or IPTG induction. We demonstrate the broad utility of the presented self-inducible method for a panel of diverse proteins produced in large amounts. The SILEX system is compatible with all classical culture media and growth temperatures and allows protein expression modulation. Importantly, the SILEX system is proven to be efficient for protein expression screening on a microplate scale.

Sequestration of bovine seminal plasma proteins by different assemblies of phosphatidylcholine: A new technical approach.

Binder of SPerm (BSP) proteins, the main proteins from bovine seminal plasma, are known to partially intercalate into the outer leaflet of the spermatozoa membrane and bind to choline-containing lipids being present therein. This insertion generates a negative effect on semen quality after cryopreservation by inducing an early-stage capacitation of spermatozoa. The assumption of surface properties exhibited by BSP proteins was checked by tensiometry measurements: BSP proteins are highly surface active. This suggests that BSP proteins can reach the interface covered by phospholipids not only by interactions between one and each other but also due to their own surface activity. The insertion of BSP proteins into the lipid domains outer leaflet of spermatozoa was reproduced on a biomimetic system such as Langmuir monolayers. The insertion of BSP proteins can be performed in the compressible fluid domains which contain choline-bearing lipids. Monolayer films were used as well to study the complexation of BSP proteins by two phospholipid assemblies: low density lipoprotein (LDLs) from egg yolk or liposomes produced from egg phospholipids. Irrespective of the phospholipid structure (lipoprotein or liposome), BSP was hindered to alter the structure of the membrane. Only the overall ratio BSP proteins:phosphatidylcholine was important. The difference between the two sequestering agents lies on their surface properties: LDL have a strong tendency to merge with the outer layer whereas liposomes mainly remain in the bulk on the same time scale.

Cloning and expression of a queen pheromone-binding protein in the honeybee: an olfactory-specific, developmentally regulated protein.

Odorant-binding proteins (OBPs) are small abundant extracellular proteins thought to participate in perireceptor events of odor-pheromone detection by carrying, deactivating, and/or selecting odor stimuli. The honeybee queen pheromone is known to play a crucial role in colony organization, in addition to drone sex attraction. We identified, for the first time in a social insect, a binding protein called antennal-specific protein 1 (ASP1), which binds at least one of the major queen pheromone components. ASP1 was characterized by cDNA cloning, expression in Pichia pastoris, and pheromone binding. In situ hybridization showed that it is specifically expressed in the auxiliary cell layer of the antennal olfactory sensilla. The ASP1 sequence revealed it as a divergent member of the insect OBP family. The recombinant protein presented the exact characteristics of the native protein, as shown by mass spectrometry, and N-terminal sequencing and exclusion-diffusion chromatography showed that recombinant ASP1 is dimeric. ASP1 interacts with queen pheromone major components, opposite to another putative honeybee OBP, called ASP2. ASP1 biosynthetic accumulation, followed by nondenaturing electrophoresis during development, starts at day 1 before emergence, in concomitance with the functional maturation of olfactory neurons. The isobar ASP1b isoform appears simultaneously to ASP1a in workers, but only at approximately 2 weeks after emergence in drones. Comparison of in vivo and heterologous expressions suggests that the difference between ASP1 isoforms might be because of dimerization, which might play a physiological role in relation with mate attraction.

Impact of esterification on the folding and the susceptibility to peptic proteolysis of beta-lactoglobulin.

beta-Lactoglobulin was esterified and the differences between unmodified and ethylated beta-lactoglobulin were studied by microcalorimetry, circular dichroism and limited proteolysis. Microcalorimetric studies and circular dichroic spectra in aromatic regions revealed changes of esterified beta-lactoglobulin tertiary structure compared with native beta-lactoglobulin conformation in aqueous media. These changes are characteristic of molten globule state. While beta-lactoglobulin is resistant to peptic hydrolysis in aqueous and physiological conditions, a study of peptic action on esterified (ethylated, approximately 40% of the carboxyl groups substituted) beta-lactoglobulin in aqueous conditions showed that it is hydrolysed rapidly by this enzyme. The main part of the obtained peptic peptides has been purified and identified. Their analysis shows that 22 new sites of pepsin cleavage are induced by esterification of beta-lactoglobulin. Fourteen cleavage sites are pepsin specific and their unveiling is due to imposed tertiary structure changes. Eight of the observed new cleavage targets are entirely atypical containing either one or two distal dicarboxylic acid moieties. Apparently, the ethylation of beta- and/or gamma-carboxylates removing charges and grafting hydrophobic ethyl groups adapts substituted dicarboxylic amino-acid side chains for the recognition by pepsin.

Non-touch fittings in hospitals: a procedure to eradicate Pseudomonas aeruginosa contamination.

Non-touch taps, now common in hospitals, can easily be contaminated with Pseudomonas aeruginosa. We report our experience with 87 non-touch taps in a newly built wing of our teaching hospital contaminated with P. aeruginosa from the central pipe water system. Serotyping and genotyping of strains revealed genetic diversity of isolates, but also showed that major clones were able to persist for long periods of time in non-touch taps despite chlorination. It is notoriously difficult to decontaminate such taps with biocides and disinfectants. We describe an easy and economical procedure for the eradication of P. aeruginosa contamination from non-touch taps that does not require their removal.

Temporal evolution of hepatic anatomy during gestation and growth in the sheep.

In mammals, the liver undergoes a series of spectacular anatomical changes during development, particularly in domestic ruminants. In all domestic mammals, the liver retracts cranially until it reaches its definitive diaphragmatic position; however, in the sheep, it also withdraws from the entire left side of the diaphragm and seems to rotate through 180°. An anatomical study reveals that the hepatic conformation evolves very little during this topographical change. The latter occurs in two phases: an initial phase of marked regression of the left lobe, which starts from the beginning of the foetal period (44th day of gestation), followed by marked regression of the entire liver, which starts between the 90th and 117th days and ends between the 2nd and 3rd month of life. The path of hepatic regression is dictated by the particular layout of the liver's attachments in the sheep. The left triangular ligament, which holds the L lobe to the left in other species, is almost completely absent in the sheep, whilst the right lobe is fixed to the top of the diaphragm. As the liver regresses, the right lobe therefore draws the left lobe with it to the right-hand side. A statistical study shows constant regression of the hepatic surface area during the topographical evolution of the liver, with a particularly marked and sudden reduction between the end of the 4th month and the middle of the 5th month of gestation. It also shows that the regression of the left lobe is consistently greater than that of the right lobe and that the topographical regression of the liver cannot be predicted by measuring the weight of the liver, which behaves independently to the surface area of the liver.

Chemosensory signalling pathways involved in sensing of amino acids by the ghrelin cell.

Taste receptors on enteroendocrine cells sense nutrients and transmit signals that control gut hormone release. This study aimed to investigate the amino acid (AA) sensing mechanisms of the ghrelin cell in a gastric ghrelinoma cell line, tissue segments and mice. Peptone and specific classes of amino acids stimulate ghrelin secretion in the ghrelinoma cell line. Sensing of L-Phe occurs via the CaSR, monosodium glutamate via the TAS1R1-TAS1R3 while L-Ala and peptone act via 2 different amino acid taste receptors: CaSR &TAS1R1-TAS1R3 and CaSR &GPRC6A, respectively. The stimulatory effect of peptone on ghrelin release was mimicked ex vivo in gastric but not in jejunal tissue segments, where peptone inhibited ghrelin release. The latter effect could not be blocked by receptor antagonists for CCK, GLP-1 or somatostatin. In vivo, plasma ghrelin levels were reduced both upon intragastric (peptone or L-Phe) or intravenous (L-Phe) administration, indicating that AA- sensing is not polarized and is due to inhibition of ghrelin release from the stomach or duodenum respectively. In conclusion, functional AA taste receptors regulate AA-induced ghrelin release in vitro. The effects differ between stomach and jejunum but these local nutrient sensing mechanisms are overruled in vivo by indirect mechanisms inhibiting ghrelin release.

Effect of pea and bovine trypsin inhibitors on wild-type and modified trypsins.

In order to modify the catalytic properties of trypsin, lysine-188 (S1) of the substrate binding pocket was substituted by an aromatic amino acid residue (Phe, Tyr, Trp) or by a histidyl residue. Two other mutants were obtained by displacement or elimination of the negative charge of aspartic acid-189 (K188D/D189K and G187W/K188F/D189Y, respectively). The high affinity inhibitors, like PSTI II and BPTI, behaved as specific substrates of the trypsin and its mutants. Their inhibiting effect toward modified trypsins was studied. The bovine inhibitor had a higher affinity for all tested enzymes than pea inhibitor. The inhibition constants differed according to the mutations on the protease.

Impact of the lysine-188 and aspartic acid-189 inversion on activity of trypsin.

The impact of the charge rearrangement on the specificity of trypsin was tested by an inversion of sequence K188D/D189K maintaining the integrity of the charges of the substrate binding pocket when switching their polarity. In native trypsin, aspartate 189 situated at the bottom of the primary substrate binding pocket interacts with arginine and lysine side chains of the substrate. The kinetic parameters of the wild-type trypsin and K188D/D189K mutant were determined with synthetic tetrapeptide substrates. Compared with trypsin, the mutant K188D/D189K exhibits a 1.5- to 6-fold increase in the Km for the substrates containing arginine and lysine, respectively. This mutant shows a approximately 30-fold decrease of its k(cat) and its second-order rate constant k(cat)/Km decreases approximately 40- and 150-fold for substrates containing arginine and lysine, respectively. Hence, trypsin K188D/D189K displays a large increase in preference for arginine over lysine.

Odorant and pheromone binding by aphrodisin, a hamster aphrodisiac protein.

Aphrodisin is a soluble glycoprotein of hamster vaginal discharges, which stimulates male copulatory behavior. Natural aphrodisin was purified and its post-translational modifications characterized by MALDI-MS peptide mapping. To evaluate its ability to bind small volatile ligands, the aphrodisiac protein was expressed in the yeast Pichia pastoris as two major isoforms differing in their glycosylation degree, but close in conformation to the natural protein. Dimeric recombinant aphrodisins were equally able to efficiently bind odors (2-isobutyl-3-methoxypyrazine and methyl thiobutyrate) and a pheromone (dimethyl disulfide), suggesting that they could act as pheromone carriers instead of, or in addition to, direct vomeronasal neuron receptor activators.

Organization of lipids in the artificial outer membrane of bull spermatozoa reconstructed at the air-water interface.

Cryopreservation is widely used to preserve the quality of bull spermatozoa over time. Sequestration of seminal plasma proteins by low density lipoproteins and formation of a protective film around the spermatozoa membrane by low density lipoproteins were the main mechanisms proposed. However, the organization of lipids in the outer leaflet of the spermatozoa membrane has been never considered as a possible parameter. This study evaluated whether a change in the organization of the outer leaflet of the spermotozoa membrane could occur during cooling down. The organization of the main components of the spermatozoa membrane's outer layer at the liquid-gas interface was analysed. Cryopreservative media (at 8° and 34°C) were used to study the miscibility of the spermatozoa membrane lipids using epifluorescence imaging and by tensiometry on Langmuir films. The results show that the four lipids: sphingomyelin, cholesterol, 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PC) and plasmalogen 1-(1Z-octadecenyl)-2-docosahexaenoyl-sn-glycero-3-phosphocholine (P-PC) were not fully miscible and their organization was controlled by temperature. Cholesterol and sphingomyelin form condensed domains surrounded by a mixture of PC and P-PC at 34°C while these condensed domains are surrounded by separated domains of pure PC and pure P-PC at 8°C. The organization of the outer membrane lipids, in particular the separation of PC and P-PC lipids during cooling down, must be considered to fully understand preservation of membrane integrity during cryopreservation.

Running-induced anxiety is dependent on increases in hippocampal neurogenesis.

Exercise, specifically voluntary wheel running, is a potent stimulator of hippocampal neurogenesis in adult mice. In addition, exercise induces behavioral changes in numerous measures of anxiety in rodents. However, the physiological underpinnings of these changes are poorly understood. To investigate the role of neurogenesis in exercise-mediated anxiety, we examined the cellular and behavioral effects of voluntary wheel running in mice with a reduction in hippocampal neurogenesis, achieved through conditional deletion of ataxia telangiectasia-mutated and rad-3-related protein (ATR), a cell cycle checkpoint kinase necessary for normal levels of neurogenesis. Following hippocampal microinjection of an adeno-associated virus expressing Cre recombinase to delete ATR, mice were exposed to 4 weeks of voluntary wheel running and subsequently evaluated for anxiety-like behavior. Wheel running resulted in increased cell proliferation and neurogenesis, as measured by bromodeoxyuridine and doublecortin, respectively. Wheel running also resulted in heightened anxiety in the novelty-induced hypophagia, open field and light-dark box tests. However, both the neurogenic and anxiogenic effects of wheel running were attenuated following hippocampal ATR deletion, suggesting that increased neurogenesis is an important mediator of exercise-induced anxiety.