Identification of sperm subpopulations in canine ejaculates: effects of cold storage and egg yolk concentration

The aim of this study was to evaluate the effects of cold storage and egg yolk concentration on the distribution of spermatozoa within the different subpopulations. Twenty ejaculates from 4 dogs were collected, diluted in either TRIS buffer containing 20% (TEY20) or 10% centrifuged egg yolk (TEY10) and cooled following a conventional protocol. The kinematic parameters of individual spermatozoa were evaluated in fresh ejaculates and after 24 and 72 h of preservation at 5°C. A multivariate clustering procedure separated 54,261 motile spermatozoa into four subpopulations: Subpopulation 1 consisting of poorly active and non-progressive spermatozoa (19.80%), Subpopulation 2 consisting of slow and low-linear spermatozoa (25.21%), Subpopulation 3 consisting of high speed and progressive spermatozoa (23.88%), and Subpopulation 4 consisting of highly active but non-progressive spermatozoa (31.11%). Although, cold storage had a significant (P<0.05) effect on both the frequency distribution of spermatozoa within subpopulations and the motion characteristics of each subpopulation, the sperm subpopulation structure was perfectly maintained after cold storage. Subpopulations 1 and 2 significantly (P<0.001) decreased during cold storage (Subpopulation 1: 26.6, 16.9 and 18.4%; and Subpopulation 2: 33.6, 21.3 and 24.0%, respectively, for fresh, 24 and 72 h post-cooled), whereas Subpopulations 3 and 4 significantly (P<0.05) increased (Subpopulation 3: 16.7, 27.6 and 24.3%, and Subpopulation 4: 23.1, 34.1 and 33.4%, respectively, for fresh, 24 and 72 h post-cooled). Regarding the relative percentage of spermatozoa within each extender, Subpopulation 3 was more frequently observed in TEY20 after both 24 and 72 h of cold storage. Significant correlations (P<0.05) were found between the proportions of spermatozoa assigned to Subpopulation 3 in the fresh ejaculates and those in stored samples after 24 h (r=0.48498). In conclusion, cold storage significantly modified both the specific parameters and the distribution of spermatozoa within subpopulations. These changes did not affect the general motile sperm structure present in dog, which is conserved during cold storage. The analysis of the changes observed in structures of subpopulations also suggests that the TEY20 provide more effective preservation of dog semen during cold storage.     

The disulfiram metabolites S-methyl-N,N-diethyldithiocarbamoyl sulfoxide and S-methyl-N,N-diethylthiocarbamoyl sulfone irreversibly inactivate betaine aldehyde dehydrogenase from Pseudomonas aeruginosa, both in vitro and in situ, and arrest bacterial growth

Betaine aldehyde dehydrogenase from the human opportunistic pathogen Pseudomonas aeruginosa (PaBADH) catalyzes the irreversible, NAD(P)⁺-dependent oxidation of betaine aldehyde, producing glycine betaine, an osmoprotectant. PaBADH participates in the catabolism of choline and likely in the defense against the osmotic and oxidative stresses to which the bacterium is exposed when infecting human tissues. Given that choline or choline precursors are abundant in infected tissues, PaBADH is a potential drug target because its inhibition will lead to the build up of the toxic betaine aldehyde inside bacterial cells. We tested the thiol reagents, disulfiram (DSF) and five DSF metabolites—diethyldithiocarbamic acid (DDC), S-methyl-N,N-diethyldithiocarbamoyl sulfoxide (MeDDTC-SO) and sulfone (MeDDTC-SO₂), and S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO) and sulfone (MeDTC-SO₂)—as inhibitors of PaBADH and P. aeruginosa growth. As in vitro PaBADH inhibitors, their order of potency was: MeDDTC-SO₂ > DSF > MeDTC-SO₂ > MeDDTC-SO > MeDTC-SO. DDC did not inactivate the enzyme. PaBADH inactivation by DSF metabolites (i) was not affected by NAD(P)⁺, (ii) could not be reverted by dithiothreitol, and (iii) did not affect the quaternary structure of the enzyme. Of the DSF metabolites tested, MeDTC-SO₂ and MeDDTC-SO produced significant in situ PaBADH inactivation and arrest of P. aeruginosa growth in choline containing media, in which the expression of PaBADH is induced. They had no effect in media lacking choline, indicating that PaBADH is their main intracellular target, and that arrest of growth is due to accumulation of betaine aldehyde. The in vitro and in situ kinetics of enzyme inactivation by these two compounds were very similar, indicating no restriction on their uptake by the cells. MeDDTC-SO₂ and DSF have no inhibitory effects in situ, probably because their high reactivity towards intracellular nonessential thiols causes their depletion. Our results support that PaBADH is a promising target to treat P. aeruginosa infections, and that some DSF metabolites might be of help in this aim.     

Development and characterization of a mouse floxed <Emphasis Type="Italic">Bmp2</Emphasis> osteoblast cell line that retains osteoblast genotype and phenotype

Bone morphogenetic protein 2 (Bmp2) is essential for osteoblast differentiation and osteogenesis. Generation of floxed Bmp2 osteoblast cell lines is a valuable tool for studying the effects of Bmp2 on osteoblast differentiation and its signaling pathways during skeletal metabolism. Due to relatively limited sources of primary osteoblasts, we have developed cell lines that serve as good surrogate models for the study of osteoblast cell differentiation and bone mineralization. In this study, we established and characterized immortalized mouse floxed Bmp2 osteoblast cell lines. Primary mouse floxed Bmp2 osteoblasts were transfected with pSV3-neo and clonally selected. These transfected cells were verified by PCR and immunohistochemistry. To determine the genotype and phenotype of the immortalized cells, cell morphology, proliferation, differentiation and mineralization were analyzed. Also, expression of osteoblast-related gene markers including Runx2, Osx, ATF4, Dlx3, bone sialoprotein, dentin matrix protein 1, osteonectin, osteocalcin and osteopontin were examined by quantitative RT-PCR and immunohistochemistry. These results showed that immortalized floxed Bmp2 osteoblasts had a higher proliferation rate but preserved their genotypic and phenotypic characteristics similar to the primary cells. Thus, we, for the first time, describe the development of immortalized mouse floxed Bmp2 osteoblast cell lines and present a useful model to study osteoblast biology mediated by BMP2 and its downstream signaling transduction pathways.     

Biomarkers of Alzheimer′s Disease in the Cerebrospinal Fluid of Spanish Patients With Mild Cognitive Impairment

The study of biomarkers in the cerebrospinal fluid (CSF) of patients with mild cognitive impairment (MCI) is a technique used with increasing frequency in the early diagnosis of Alzheimers disease (AD). Our objectiv was to gain an own experience while evaluating the reliability, sensitivity, and reproducibility of this technique in Spanish patients. Thirty-seven patients with MCI and twenty-four control subjects were studied by means of AD biomarker analysis in CSF. xMAP Luminex and INNO-BIA Alzbio3 reagents of Innogenetics were used. The study variables assessed were levels of Aβ_1–42, T-tau and P-tau_181p proteins as well as the ratios of T-tau/Aβ_1–42 and P-tau_181p/Aβ_1–42. Samples from nineteen patients were examined twice. Intra-class correlation coefficients for the three biomarkers used showed values higher than 0.95. We observed significant differences between the control group and the MCI groups. In the 6 months following lumbar puncture (LP), eleven (29%) patients with MCI developed AD. These patients showed significant lower levels in Aβ_1–42 protein (276.35 ± 78 vs. 367.13 ± 123.49, P < 0.03) and higher ratios (T-tau/Aβ_1–42 [0.38 ± 0.2 vs. 0.22 ± 0.14, P < 0.01] and P-tau_181p/Aβ_1–42 [0.27 ± 0.13 vs. 0.16 ± 0.1, P < 0.008]) to those in the same group who remained stable. We obtained similar results to those in the most recent reliable literature with our ROC curves, especially with our P-tau_181p values and T-tau/Aβ_1–42 ratio in order to differentiate between control and AD groups. Our experience showed that the analysis of CSF-AD biomarkers in patients with MCI is reliable, sensitive and reproducible. In our knowledge, this is the first experience in Spanish patients.     

Chilling tolerance in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> induced by seed priming with putrescine

Chilling stress is one of primary constraints to tobacco production in many parts of the world. The present study was conducted to induce chilling tolerance in tobacco by seed priming with putrescine (Put) in relation to physiological changes, using seeds from two tobacco varieties, MSk326 (chilling sensitive variety) and Honghuadajinyuan (HHDJY, chilling tolerant variety). Seed germination, seedling antioxidant enzyme activities and malondialdehyde (MDA) concentration, as well as polyamine concentration were determined under low temperature. During chilling stress at 11°C, seed priming with 0.01 mM Put for 48 h (Put0.01mM48 h) and seed priming with 0.1 mM Put for 48 h (Put0.1mM48 h) significantly increased germination percentage, germination index, seedling length and dry weight of both varieties compared to the controls without Put treatment. When seedlings of 4-leaf stage suffered a short chilling stress (5°C), Put 0.1 mM 48 h improved the activities of antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX), increased endogenous Put, Spd and Spm concentration and decreased the MDA concentration. The results showed that Put priming treatments were available to enhance the chilling tolerance of tobacco seedlings. The optimal treatment of Put was Put0.1 mM48 h.     

Male barn swallows use different signalling rules to produce ornamental tail feathers

The evolution of secondary sexual characters is the subject of controversial debate between those defending their role as ‘viability indicators’ and those arguing that ornaments are purely ‘attractive traits’ selected by females. Recent theoretical studies suggest that these hypotheses are not mutually exclusive, as both viability and attractiveness can contribute to improve the reproductive success of progeny and could thus simultaneously underlie female choices. If that is the case, strategies of cheaper advertisement, allowing the expression of larger ornaments for the same cost, could proliferate even in species in which honest signalling of viability prevails. Under this scenario, different males could invest a different amount of resources per ornament unit of expression, thus using different signalling rules. We studied the relationship between tail feather length (a trait that is the subject of a female mate preference) and feather mass (a measure of investment in feather production) in a barn swallow Hirundo rustica population. Different males used different and consistent signalling rules when developing ornamental feathers. That is, to produce a feather of a given length, each male used a constant amount of resources across different years, but this amount varied between males. Although the amount of material invested in feathers (feather mass) is a condition-dependent trait, the organization of this material in ornamental feathers (i.e. the signalling rules) was not. Neither survival nor risk of feather breakage was related to the signalling rules. Thus, these results suggest that both ‘viability’ and ‘runaway’ mechanisms are independent determinants of the evolution of ornamental sexual feathers in the barn swallow. A preference for long tails will ensure that females either obtain a sire with high viability, or one transferring the capability to produce longer and more attractive tails at a lower cost of production to its offspring.     

Role of vesicle pools in action potential pattern-dependent dopamine overflow in rat striatum in vivo

Action potential (AP) patterns and dopamine (DA) release are known to correlate with rewarding behaviors, but how codes of AP bursts translate into DA release in vivo remains elusive. Here, a given AP pattern was defined by four codes, termed total AP number, frequency, number of AP bursts, and interburst time [N, f, b, i].. The 'burst effect' was calculated by the ratio (γ) of DA overflow by multiple bursts to that of a single burst when total AP number was fixed. By stimulating the medial forebrain bundle using AP codes at either physiological (20 Hz) or supraphysiological (80 Hz) frequencies, we found that DA was released from two kinetically distinct vesicle pools, the fast-releasable pool (FRP) and prolonged-releasable pool (PRP), in striatal dopaminergic terminals in vivo. We examined the effects of vesicle pools on AP-pattern dependent DA overflow and found, with given 'burst codes' [b=8, i=0.5 s], a large total AP number [N = 768, f = 80 Hz] produced a facilitating burst-effect (γ[b8/b1] = 126 ± 3%), while a small total AP number [N=96, 80 Hz] triggered a depressing-burst-effect (γ[b8/b1] = 29 ± 4%). Furthermore, we found that the PRP (but not the FRP) predominantly contributed to the facilitating-burst-effect and the FRP played an important role in the depressing-burst effect. Thus, our results suggest that striatal DA release captures pre-synaptic AP pattern information through different releasable pools.     

Biphasic effects of copper on neurotransmission in rat hippocampal neurons

The importance of copper in the CNS is well documented, but the mechanisms related to its brain functions are poorly understood. Copper is released at the synaptic cleft, where it may modulate neurotransmission. To understand the functional impact of copper on the neuronal network, we have analyzed the synaptic activity of primary rat hippocampal neurons by using different approaches including whole cell patch clamp, recording of calcium transients, immunofluorescence and western blot. Here, we show that copper produces biphasic changes in neurotransmission. When copper is acutely applied to the plate it blocks neurotransmission. Interestingly, when it is applied for 3 h to hippocampal neurons it mainly increases the frequency and amplitude of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)ergic currents (control: 0.21 ± 0.05 Hz/22.9 ± 1.3 pA; copper: 0.68 ± 0.16 Hz/30.5 ± 2.5 pA), intracellular calcium transients (control: 0.05 ± 0.013 Hz; copper: 0.11 ± 0.02 Hz) and evoked AMPA currents (control: EC50 8.3 ± 0.5?μM; copper: EC50 2.9 ± 0.2 μM). Moreover, our results suggest that copper increases GluA1 subunit levels of the AMPA receptor through the anchorage of AMPA receptors to the plasma membrane as a result of PSD-95 accumulation. We also found that copper-treated neurons displayed an undistinguishable neurotransmission to control neurons after 24 h of treatment, indicating that changes in neurotransmission induced by copper at 3 h of incubation are homeostatically regulated after long-term exposure to the metal. Together, our data reveal an unexpected biphasic effect of copper on neurotransmission, which may be relevant to understand the effects of this ion in brain diseases that display copper dyshomeostasis such as that observed in Alzheimer's disease (AD).