The Impact of Bacteriocenoses on Sperm Vitality, Immunological and Oxidative Characteristics of Ram Ejaculates: Does the Breed Play a Role?

Bacterial contamination of semen is an often overlooked, yet important, factor contributing to decreased sperm vitality. Understanding the impact of bacterial presence on sperm structural integrity and functional activity may assist the development of effective strategies to prevent, or manage, bacteriospermia in the breeding practice. The aim of this study was to describe the bacterial profiles of ram semen (n = 35), and we also focused on the associations between bacteriospermia, sperm structure, and function, as well as oxidative and inflammatory characteristics of semen. For a better insight, the samples were divided into three groups, according to the breeds used in the study: native Wallachian (NW), improved Wallachian (IW), and Slovak dairy (SD) breeds. The results showed a significantly lower motility and membrane integrity in the NW group in comparison to the IW and SD groups, which was accompanied by a significantly higher concentration of leukocytes, increased reactive oxygen species (ROS) generation, and subsequent oxidative insults to the sperm lipids and proteins. Accordingly, the NW group presented with the highest bacterial load, in which Staphylococcus and Escherichia were the predominant representatives. The Pearson correlation analysis uncovered positive relationships amongst the bacterial load and leukocytospermia (r = 0.613), the extent of lipid peroxidation (r = 0.598), protein oxidation (r = 0.514), and DNA fragmentation (r = 0.638). Furthermore, positive correlations were found between the bacterial load and pro-inflammatory molecules, such as the C-reactive protein (r = 0.592), interleukin 1 (r = 0.709), and interleukin 6 (r = 0.474), indicating a possible involvement of the immune response in the process of bacteriospermia. Overall, our data indicate that ram semen quality may be equally affected by the bacterial load and diversity. Furthermore, we can assume that the presence of bacteria in ejaculates triggers inflammatory processes, causes ROS overproduction, and, thereby, contributes to alterations in the sperm structure, while at the same time compromising the fertilization ability of male gametes.

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Codon harmonization reduces amino acid misincorporation in bacterially expressed P. falciparum proteins and improves their immunogenicity

AMB Express ◽

10.1186/s13568-019-0890-6 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Neeraja Punde ◽

Jennifer Kooken ◽

Dagmar Leary ◽

Patricia M. Legler ◽

Evelina Angov

Keyword(s):

Protein Structure ◽

Amino Acid ◽

Codon Usage ◽

Dna Sequences ◽

Structural Integrity ◽

Host Cells ◽

Loss Of Function ◽

Species Specific ◽

And Function ◽

The Impact

Abstract Codon usage frequency influences protein structure and function. The frequency with which codons are used potentially impacts primary, secondary and tertiary protein structure. Poor expression, loss of function, insolubility, or truncation can result from species-specific differences in codon usage. “Codon harmonization” more closely aligns native codon usage frequencies with those of the expression host particularly within putative inter-domain segments where slower rates of translation may play a role in protein folding. Heterologous expression of Plasmodium falciparum genes in Escherichia coli has been a challenge due to their AT-rich codon bias and the highly repetitive DNA sequences. Here, codon harmonization was applied to the malarial antigen, CelTOS (Cell-traversal protein for ookinetes and sporozoites). CelTOS is a highly conserved P. falciparum protein involved in cellular traversal through mosquito and vertebrate host cells. It reversibly refolds after thermal denaturation making it a desirable malarial vaccine candidate. Protein expressed in E. coli from a codon harmonized sequence of P. falciparum CelTOS (CH-PfCelTOS) was compared with protein expressed from the native codon sequence (N-PfCelTOS) to assess the impact of codon usage on protein expression levels, solubility, yield, stability, structural integrity, recognition with CelTOS-specific mAbs and immunogenicity in mice. While the translated proteins were expected to be identical, the translated products produced from the codon-harmonized sequence differed in helical content and showed a smaller distribution of polypeptides in mass spectra indicating lower heterogeneity of the codon harmonized version and fewer amino acid misincorporations. Substitutions of hydrophobic-to-hydrophobic amino acid were observed more commonly than any other. CH-PfCelTOS induced significantly higher antibody levels compared with N-PfCelTOS; however, no significant differences in either IFN-γ or IL-4 cellular responses were detected between the two antigens.

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Abstract 126: Peroxisomal Proliferator Activated Receptor Alpha Overexpression in Hypertrophied Cardiomyocytes Restores Mitochondrial Integrity and Function

Circulation Research ◽

10.1161/res.121.suppl_1.126 ◽

2017 ◽

Vol 121 (suppl_1) ◽

Author(s):

Sagartirtha Sarkar ◽

Santanu Rana

Keyword(s):

Energy Demand ◽

Cardiac Tissue ◽

Structural Integrity ◽

Heart Function ◽

Ros Generation ◽

Peroxisome Proliferator ◽

Peroxisome Proliferator Activated Receptor ◽

Atp Production ◽

Receptor Alpha ◽

And Function

Cardiac tissue engineering is an interdisciplinary field that engineers modulation of viable molecular milieu to restore, maintain or improve heart function. Myocardial workload (energy demand) and energy substrate availability (supply) are in continual flux to maintain specialized cellular processes, yet the heart has a limited capacity for substrate storage and utilization during pathophysiological conditions. Damage to heart muscle, acute or chronic, leads to dysregulation of cardiac metabolic processes associated with gradual but progressive decline in mitochondrial respiratory pathways resulting in diminished ATP production. The Peroxisome Proliferator Activated Receptor Alpha ( PPARα ) is known to regulate fatty acid to glucose metabolic balance as well as mitochondrial structural integrity. In this study, a non-canonical pathway of PPARα was analyzed by cardiomyocyte targeted PPARα overexpression during cardiac hypertrophy that showed significant downregulation in p53 acetylation as well as GSK3β activation levels. Targeted PPARα overexpression during hypertrophy resulted in restoration of mitochondrial structure and function along with significantly improved mitochondrial ROS generation and membrane potential. This is the first report of myocyte targeted PPARα overexpression in hypertrophied myocardium that results in an engineered heart with significantly improved function with increased muscle mitochondrial endurance and reduced mitochondrial apoptotic load, thus conferring a greater resistance to pathological stimuli within cardiac microenvironment.

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Biomolecular Perturbations in In-Cell Dynamic Nuclear Polarization Experiments

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.743829 ◽

2021 ◽

Vol 8 ◽

Author(s):

Sarah A. Overall ◽

Alexander B. Barnes

Keyword(s):

Structural Integrity ◽

Membrane Integrity ◽

Embryonic Cell ◽

Nmr Studies ◽

Intact Cells ◽

Biomolecular Structure ◽

Glass Forming ◽

The Rich ◽

And Function ◽

Cryogenic Conditions

In-cell DNP is a growing application of NMR to the study of biomolecular structure and function within intact cells. An important unresolved question for in-cell DNP spectroscopy is the integrity of cellular samples under the cryogenic conditions of DNP. Despite the rich literature around cryopreservation of cells in the fields of stem cell/embryonic cell therapeutics, cell line preservation and in cryo-EM applications, the effect of cryopreservation procedures on DNP parameters is unclear. In this report we investigate cell survival and apoptosis in the presence of cryopreserving agents and DNP radicals. We also assess the effects of these reagents on cellular enhancements. We show that the DNP radical AMUPol has no effect on membrane permeability and does not induce apoptosis. Furthermore, the standard aqueous glass forming reagent, comprised of 60/30/10 d8-glycerol/D2O/H2O (DNP juice), rapidly dehydrates cells and induces apoptosis prior to freezing, reducing structural integrity of the sample prior to DNP analysis. Preservation with d6-DMSO at 10% v/v provided similar DNP enhancements per √unit time compared to glycerol preservation with superior maintenance of cell size and membrane integrity prior to freezing. DMSO preservation also greatly enhanced post-thaw survival of cells slow-frozen at 1°C/min. We therefore demonstrate that in-cell DNP-NMR studies should be done with d6-DMSO as cryoprotectant and raise important considerations for the progression of in-cell DNP-NMR towards the goal of high quality structural studies.

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111 ESTIMATION OF SPERM QUALITY IN FRESH AND FROZEN - THAWED SEMEN FROM ASTURIANA DE LOS VALLES BULLS

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab111 ◽

2006 ◽

Vol 18 (2) ◽

pp. 163

Author(s):

C. Tamargo ◽

M. Carbajo ◽

C. Diez ◽

D. Martin ◽

C. O. Hidalgo

Keyword(s):

Sperm Motility ◽

Semen Quality ◽

Sperm Quality ◽

Membrane Integrity ◽

Daily Routine ◽

Morphological Abnormalities ◽

Hypoosmotic Swelling Test ◽

Motion Characteristics ◽

The Impact ◽

Casa System

Artificial insemination and semen cryopreservation have significantly improved the breeding potential of male animals. However, current freezing techniques commonly result in reduced semen quality (Januskauskas et al. 1999 Theriogenology 52, 641-658), and surviving cells are affected post-thaw either structurally or functionally (Nagy et al. 2004 Anim. Reprod. Sci. 80, 225-235). In this work we analyze the impact of cryopreservation on Asturiana de los Valles bull sperm. Ejaculates (n = 373) from seven adult bulls were weekly collected by means of artificial vagina. Immediately after collection, routine parameters including volume (V), mass motility (MM), and concentration (C) of sperm cells were evaluated. Then the semen was extended with a commercial extender, loaded into 0.25-mL plastic straws at a concentration of 23 � 106 per straw, frozen and stored for further analysis. Four straws per ejaculate were thawed, pooled and analyzed for motion characteristics by means of a CASA system (Sperm Class Analyzer, SCA 2002� Microptic S. L., Barcelona, Spain) added to an optical phase-contrast microscope with heatable (37�C) stage. Immediately after thawing, we analyzed the % of motile spermatozoa (MS) and the % of progressively motile spermatozoa (PMS); then samples were incubated for 3 h at 37�C and MS and PMS were measured again (MS3 and PMS3, respectively). Functional integrity of the plasmallema was evaluated by the hypoosmotic swelling test (HOST) together with the % of typical tail coiling/swelling (percentage of HOST-positive spermatozoa, HOST-PS). The % of viable spermatozoa (VS) [membrane integrity was evaluated by fluorescence microscopy with a dual staining system (propidium iodide (PI) and 6-carboxyfluorescein diacetate (CFDA)]. Sperm showing partial or complete red fluorescence (PI staining) were considered nonviable, whereas sperm showing complete green fluorescence were considered viable. Altered acrosomes (AA) and morphological abnormalities were also determined. The % of morphological abnormalities was classified according to their location in head (HA), midpiece (MA), and tail (TA). Proximal and distal cytoplasmic droplets were counted as separate abnormalities (CD). Data were analyzed by the MEANS procedure of SAS (SAS Institute, Inc., Cary, NC, USA). A significant (P < 0.05) decrease in the sperm motility was observed after freezing/thawing (MS: 80.20 � 0.75 vs. 47.36 � 1.04, and PMS: 68.73 � 0.73 vs. 42.14 � 0.96 for fresh and frozen-thawed semen, respectively). Also, the frozen-thawed sperm showed increased % of HA, MA, AA, HOST-PS, and VS (P < 0.05). These morphological abnormalities could contribute to decreasing sperm motility. The new computer and video technologies provide useful information about sperm quality and can be used in the daily routine of processing semen. This work was performed in collaboration with ASEAVA.

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Effect of inbreeding depression on bull sperm quality and field fertility

Reproduction Fertility and Development ◽

10.1071/rd15324 ◽

2017 ◽

Vol 29 (4) ◽

pp. 712 ◽

Cited By ~ 6

Author(s):

Jesús Dorado ◽

Rosa Morales Cid ◽

Antonio Molina ◽

Manuel Hidalgo ◽

Julia Ariza ◽

...

Keyword(s):

Inbreeding Depression ◽

Sperm Morphology ◽

Semen Quality ◽

Sperm Quality ◽

Membrane Integrity ◽

Calving Interval ◽

Bull Semen ◽

Head Displacement ◽

Highly Active ◽

And Function

The present study investigated the effect of inbreeding depression on sperm quality using automated and objective methods and subsequent effects on beef bull field fertility. Individual inbreeding coefficient (F) values and field fertility data were determined using a dataset of AI bulls belonging to the Spanish Retinta Breeders Association (Asociación Nacional de Criadores de Ganado Vacuno Selecto de Raza Retinta (ANCRE)). Animals were clustered in two groups according to the F values as follows: (1) a high inbreeding group (HI; F ≥ 13.5%, mean 16.3); and (2) a non-inbreeding group (NI; F = 0%). In total, 17 different assessments were performed in both experimental groups, including evaluation of sperm morphology, acrosomal and DNA status, sperm plasma membrane integrity and function (hypo-osmotic swelling test), 10 kinetic parameters and the structure of sperm subpopulations. Sperm morphology, acrosomal and DNA status and osmotic tolerance were similar in both groups. Three velocity parameters (curvilinear velocity, straight line velocity and average path velocity) and the amplitude of lateral head displacement were higher in HI (P < 0.05). Cluster analysis of kinematic parameters revealed three different sperm subpopulations (sP1, sP2 and sP3), with the proportion of the sP1 population (highly active but non-progressive spermatozoa) being significantly (P < 0.05) higher in the HI group. Field fertility was assessed using two calving record datasets. In a smaller database including only bulls evaluated in the present study, there was a significant increase in the calving interval of cows sired with HI bulls. Conversely, in an extended genetic analysis of the ANCRE database, inbreeding only explained a small part of the variation in calving interval, and the results of regression analysis were not significant among bulls. The findings of the present study suggest that high inbreeding levels have a moderate effect on bull semen quality, with an increased percentage of highly active but non-progressive spermatozoa, but only when F values reached a certain threshold. This motility pattern could explain, in part, the higher calving interval produced by inbred bulls under field conditions.

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Addition of Reduced Glutathione (GSH) to Freezing Medium Reduces Intracellular ROS Levels in Donkey Sperm

Veterinary Sciences ◽

10.3390/vetsci8120302 ◽

2021 ◽

Vol 8 (12) ◽

pp. 302

Author(s):

Iván Yánez-Ortiz ◽

Jaime Catalán ◽

Ariadna Delgado-Bermúdez ◽

Augusto Carluccio ◽

Jordi Miró ◽

...

Keyword(s):

Reduced Glutathione ◽

Membrane Integrity ◽

Redox Balance ◽

Low Fertility ◽

Ros Generation ◽

Enzymatic Antioxidants ◽

Freezing Medium ◽

Exogenous Addition ◽

Sperm Plasma Membrane ◽

The Impact

In donkeys, the use of frozen-thawed sperm for artificial insemination (AI) leads to low fertility rates. Furthermore, donkey sperm produce a large amount of reactive oxygen species (ROS), and post-AI inflammation induces the formation of neutrophil extracellular traps (NETosis), which further generates many more ROS. These high ROS levels may induce lipid peroxidation in the sperm plasma membrane, thus affecting its integrity. Enzymatic and non-enzymatic antioxidants, mainly found in the seminal plasma (SP), are responsible for maintaining the redox balance. However, this fluid is removed prior to cryopreservation, thereby exposing sperm cells to further oxidative stress. The exogenous addition of antioxidants to the freezing medium can reduce the detrimental effects caused by ROS generation. Therefore, the aim of this study was to evaluate how the addition of different reduced glutathione (GSH) concentrations (control, 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM) to fresh sperm affect their cryotolerance. Total and progressive motility, kinematic parameters and motile sperm subpopulations were significantly (p < 0.05) different from the control in treatments containing 8 mM and 10 mM GSH, but not at lower concentrations. Plasma and acrosome membrane integrity, mitochondrial membrane potential (MMP) and intracellular superoxide levels (O2−) were not affected (p > 0.05) by any GSH concentration. Interestingly, however, the addition of 8 mM or 10 mM GSH reduced (p < 0.05) the percentages of viable sperm with high overall ROS levels compared to the control. In conclusion, frozen-thawed donkey sperm are able to tolerate high GSH concentrations, which differs from what has been observed in other species. This antioxidant capacity suggests that ROS could be important during post-AI and that the impact of using exogenous antioxidants like GSH to improve the sperm resilience to freeze-thawing is limited in this species.

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Mutational and structural study of RipA, a key enzyme inMycobacterium tuberculosiscell division: evidence for theL-to-Dinversion of configuration of the catalytic cysteine

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004714013674 ◽

2014 ◽

Vol 70 (9) ◽

pp. 2295-2300 ◽

Cited By ~ 12

Author(s):

Flavia Squeglia ◽

Alessia Ruggiero ◽

Maria Romano ◽

Luigi Vitagliano ◽

Rita Berisio

Keyword(s):

Structural Integrity ◽

Cysteine Proteases ◽

Enzyme Inactivation ◽

Side Chain ◽

Catalytic Cysteine ◽

Key Enzyme ◽

And Function ◽

The Impact ◽

Daughter Cell Separation

RipA is a key cysteine protease ofMycobacterium tuberculosisas it is responsible for bacterial daughter-cell separation. Although it is an important target for antimicrobial development, its mechanism of action and its interaction pattern with its substrate are hitherto unknown. By combining crystallographic and mutational studies with functional assays and molecular modelling, it is shown that the catalytic activity of the enzyme relies on a Cys–His–Glu triad and the impact of the mutation of each residue of the triad on the structure and function of RipA is analysed. Unexpectedly, the crystallographic analyses reveal that mutation of the glutamic acid to alanine results in inversion of the configuration of the catalytic cysteine. The consequent burial of the catalytic cysteine side chain explains the enzyme inactivation upon mutation. These data point to a novel role of the acidic residue often present in the triad of cysteine proteases as a supervisor of cysteine configuration through preservation of the local structural integrity.

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Effect of crocin and naringenin supplementation in cryopreservation medium on post-thawed rooster sperm quality and expression of apoptosis associated genes

10.1101/846758 ◽

2019 ◽

Cited By ~ 1

Author(s):

Mahdieh Mehdipour ◽

Hossein Daghigh Kia ◽

Abouzar Najafi

Keyword(s):

Lipid Peroxidation ◽

Mrna Expression ◽

Caspase 3 ◽

Semen Quality ◽

Sperm Quality ◽

Membrane Integrity ◽

Control Group ◽

Mitochondrial Activity ◽

Ros Generation ◽

Hatching Rate

AbstractThe aim of our research was to examine the effects of crocin (0.5 (C0.5), 1 (C1) and 1.5 (C1.5) mM) and naringenin (50 (N50), 100 (N100) and 150 (N150) µM) in cryopreservation extender for freezing rooster semen. Sperm motility, viability, abnormalities, membrane integrity, mitochondrial activity, apoptosis status, lipid peroxidation (LP), GPX, SOD, TAC, the mRNA expression of pro-apoptotic (CASPASE 3) and anti-apoptotic (Bcl-2) genes, fertility and hatchability rate were investigated following freeze-thawing. C1 and N100 resulted in the higher (P < 0.05) total motility and progressive motility in comparison to the control group. C1 and N100 improved viability, membrane integrity and reduced lipid peroxidation. We found much higher values for mitochondria activity with C1 and N100 respect to the control group. The C1 and N100 showed lower percentages of early apoptosis when compared with control group. Also, C1 and N100 had higher TAC when compared with control group. The mRNA expression of BCL-2 in the C1 and N100 group were significantly higher than that of other treatments. The expression of CASPASES 3 was significantly reduced in C1 and N100 group (P < 0.05) when compared to control group. Significantly higher percentage of fertility and hatching rate were observed in C1 and N100 compared to the control group. In conclusion, crocin at 1 mM and naringenin at 100 µM seem to improve the post-thawing rooster semen quality, fertility and could protect the sperm against excessive ROS generation by reducing the pro-apoptotic (CASPASE 3) and increasing anti-apoptotic (Bcl-2) genes.

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Neutrophils contribute to severity of tuberculosis pathology and recovery from lung damage pre- and post-treatment

Clinical Infectious Diseases ◽

10.1093/cid/ciab729 ◽

2021 ◽

Author(s):

Caleb Nwongbouwoh Muefong ◽

Olumuyiwa Owolabi ◽

Simon Donkor ◽

Salome Charalambous ◽

Abhishek Bakuli ◽

...

Keyword(s):

Flow Cytometry ◽

Bacterial Load ◽

Load Flow ◽

Lung Damage ◽

Ros Generation ◽

Lung Pathology ◽

X Rays ◽

Whole Cell Lysate ◽

Specific Antigens ◽

And Function

Abstract Background Despite microbiological cure, about 50% of TB patients have poor lung recovery. Neutrophils are associated with lung pathology, however, CD16|CD62L-defined subsets have not been studied in TB. Using flow cytometry, we monitored frequencies, phenotype and function of neutrophils following stimulation with Mtb whole cell lysate (WCL) and ESAT-6/CFP-10 fusion protein (EC)) in relation to lung pathology. Methods Fresh blood from 42 adult, HIV-negative TB patients were analysed pre- and post-therapy, with disease severity determined using Chest X-Rays and bacterial load. Flow cytometry was used to monitor frequencies, phenotype and function (generation of ROS, together with CD11b, TNF and IL10 expression) of neutrophils following 2-hour stimulation with Mtb-specific antigens. Results We show that total neutrophils decrease by treatment completion compared to baseline (p=0.0059); however, CD16 brCD62L br (segmented) neutrophils increase (p=0.0031) and CD16 dimCD62L br (banded) neutrophils decrease (p=0.038). Moreover, banded neutrophils are lower in patients with severe lung damage at baseline (p=0.035). Furthermore, we demonstrate that following WCL stimulation, ROS from segmented neutrophils is higher in patients with low Mtb loads even after adjusting for sex (p=0.038) while IL-10-expressing CD16 dimCD62L lo are higher in patients with mild damage (p=0.0397) at baseline. Patients showing good recovery from lung damage possess higher baseline granulocyte frequencies following WCL (p=0.042) and EC stimulation (p=0.011). Conclusion Hence, our results suggest that high ROS generation, low levels of banded neutrophils and high levels of IL10-expressing CD16 dimCD62L lo neutrophils are associated with reduced lung pathology at TB diagnosis. Hence, neutrophils are potential early indicators of TB severity and promising targets for TB host-directed therapy.

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Microtubule dynamics in cytoskeleton, neurodegenerative and psychiatric disease

STEMedicine ◽

10.37175/stemedicine.v2i6.81 ◽

2021 ◽

Vol 2 (6) ◽

pp. e81

Author(s):

Simone Mortal

Keyword(s):

Neurodegenerative Diseases ◽

Structural Integrity ◽

Neuronal Cell ◽

Psychiatric Disease ◽

Psychiatric Illnesses ◽

Associated Proteins ◽

Dynamics And Function ◽

And Function ◽

The Impact ◽

Β Tubulin

Microtubules (MTs) are fundamental polymers composed by α and β tubulin, they provide integrity to neuronal cell and are necessaries in intracellular trafficking and organization. The extension and retraction of MTs occur with the addition or removal of α and β tubulin subunits and the binding with microtubule associated proteins (MAPs) that selectively target specific tubulin regions, manipulating the MT dynamics and function. Altered MT homeostasis can compromise the function of MTs in the structural integrity and axonal transport inside the neuron. Here I review the evidence of MT anomalies in several neurodegenerative diseases, including Alzheimer’s disease, Parkinson disease, amyotrophic lateral sclerosis and traumatic brain injury and psychiatric disorders, such as depression, schizophrenia, and bipolar disorder. The focus of this review is to point out which can be the impact of MT issues in the major neurodegenerative diseases and discuss which MT abnormalities can lead to psychiatric illnesses.

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