Nuclear Chromatin Decondensation Research Articles

Protective Effect of Aloe vera (L.) on Diabetes-Induced Oxidative Stress Linked Spermiological Co-Morbidity in Human and Rat: An In-Vitro Analysis.

Diabetes linked reproductive complications are rising problems nowadays. The study focused on the protective efficacy of Aloe vera (L.) on sperm cell damage in an oxidative stress milieu encumbered by a chronic diabetes in human and streptozotocin treated Wistar rat (Rattus norvegicus). Spermatozoa from rat's epididymal washing, and human semen after 3-4days of abstinence of mating or masturbation were collected from control and diabetes groups. Spermatozoa of human and rat were incubated for 1 or 2h at 370C in an in-vitro medium separately and considered as normo-glycemic control and diabetes sub-groups. Dose of 1 or, 2 or, 4mg/ml of Aloe vera (L.) hydro-ethanolic (40:60) extract (AVHE) was given to diabetes samples, considered as sub-sub-group for assessing its protective effect on spermiological and oxidative stress parameters. The motility, viability, plasma membrane integrity, nuclear chromatin decondensation for DNA fragmentation, acrosome cap status, and antioxidative status of human and rat spermatozoa were decreased whereas spermatozoal apoptosis was elevated significantly (p < 0.05), noted by TUNEL assay in diabetes samples compared to the duration-matched control group. Exposure of AVHE to diabetes samples resulted significant rectification (p < 0.05) in the said parameters than the unexposed diabetes group. In control group, AVHE exposure has significant protective effect from spermiological deterioration compared to unexposed control group. Identification of major phytomolecules in AVHE was done by LC-MS study. Diabetes-induced oxidative stress-mediated spermatozoal injuries can be protected by AVHE extract, raise the possibility for potentiating sperm of human for increasing the success rate of in-vitro fertilization-blastocyst implantation.

In Vitro Study on Spermicidal Action of Hydro-methanol Extract of Tinospora cordifolia (Willd.) Stem in Rat and Human Sperm: a Comparative Analysis.

Human fertility regulation is a major way to control overpopulation. In this perspective, this study emphasized the in vitro effect of hydro-methanol extract of Tinospora cordifolia (TCHME) stem for spermicidal and reproductive hypo-functions using human and rat samples. Control, 0.5-, 1-, and 2-mg TCHME-charged groups were considered to assess the relevant parameters. Levels of spermiological parameters like sperm motility, viability, the integrity of plasma and acrosomal membrane, and nuclear chromatin decondensation were significantly reduced (p < 0.05) in the dose- and duration-dependent TCHME-charged groups compared to the control. The inhibitory concentration 50 (IC50) of TCHME on motile human and rat sperms were 0.8 and 0.4 mg/ml, respectively. Testicular androgenic key enzymes and antioxidant enzymes (human sperm pellet, testes, and epididymis of rat)' activities were significantly diminished (p < 0.05), while antioxidant enzymes' activities were significantly elevated (p < 0.05) in renal and insignificantly (p > 0.05) elevated in hepatic tissues of rat in TCHME-charged groups compared to the control. Significant elevation (p < 0.05) of thiobarbituric acid reactive substances (TBARS)' level in human sperm pellet, testes, and epididymis of rats and significant diminution (p < 0.05) in TBARS levels of liver and kidney were observed in TCHME-charged groups. It focused that TCHME is more potent for stress imposition on reproductive tissues and sperm compared to the other tested tissues. Non-significant alterations (p > 0.05) in glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) activities in the said organs of rat indicated its non-toxic effect. It highlighted that TCHME possesses spermicidal and reproductive tissue-specific effects which strengthen the possibilities of male contraceptive development from it.

Formulation and evaluation of solid lipid nanoparticles loaded with papaya seed chloroform extract for long-term antifertility effects on the male rat, Bandicota bengalensis

The study is the first to formulate and investigate potential of papaya seed chloroform extract based solid lipid nanoparticles (PSCEN) as antifertility agents on male Bandicota bengalensis. The prepared nanoparticles were spherical of size 300–600 nm. The release kinetics showed a controlled release of the drug with major release over 48 h. To assess the antifertility effects of PSCEN, adult male rats were fed a diet containing two different concentrations of PSCEN (5% and 10%) for 15 days under bi-choice conditions. The mean total active ingredient ingestion of the rats in the two treated groups ranged from 2.13–3.31 and 3.92–5.87 g/100g body weight, respectively. No adverse effects of treatment on body weight were observed. Also, no mortality of rats was observed. The treatment had a significant effect on the weight of the testis and the epididymis, but not on the other organs. Sperm motility (%), sperm viability (%), sperm count (millions/ml), sperm mitochondrial activity (%), sperm nuclear chromatin de-condensation (%) and sperm hypo-osmotic swelling (%) were significantly decreased, and sperm abnormality (%) significantly increased compared to the vehicle control group. The reproductive success rates of male rats treated with 5% and 10% PSCEN and mated with untreated female rats were 20.00–66.67% and 16.67%, respectively, while in untreated female rats mated with male rats of vehicle control group, reproductive success rate was 33.33 to 80%. The study found a maximal antifertility effect of the 10% PSCEN containing bait, which was irreversible up to 105 days after stopping treatment, suggesting long-term efficacy.

RISUG® offers early contraception: An experience during Phase III clinical trials

Objectives: An early contraceptive efficacy with reasonable assurance of reversibility has been a challenge in male contraception. With nearly four decades of research in reversible inhibition of sperm under guidance (RISUG®) as an intravasal male contraceptive, including pre-clinical trials in rats, rabbits, langur monkeys, and three phases of clinical trials, the present study aims to evaluate the additional parameters of a center of Phase III clinical trials. Material and Methods: Subjects were recruited following ICMR guidelines of inclusion and exclusion criteria. Samples were analyzed for sperm functional tests, namely, hypo-osmotic swelling, acrosomal intactness, nuclear chromatin decondensation, and sperm mitochondrial activity index. Furthermore, seminal biochemistry and serum hormones such as follicle-stimulating hormone, luteinizing hormone, testosterone, cortisol, and prolactin were assessed along with levels of anti-sperm antibodies and prostate-specific antigen (PSA). Results: The present study, on human subjects, emphasizes the efficacy of RISUG® with early onset of contraception and indication of a greater possibility of reversal. A significant decrease in all sperm functional parameters was observed following RISUG® injection along with increased sperm abnormalities. Semen biochemistry revealed no marked alterations in the concentration of fructose and acid phosphatase, while significantly decreased levels of glycerophosphorylcholine and neutral α-glucosidase were observed. No significant changes in the circulatory levels of hormones and the levels of PSA were observed. In addition, the development of anti-sperm antibodies, an adverse effect of other vas occlusive methods, was not indicated after RISUG® administration, implying the potential of reversibility in humans as observed earlier in different animal models. Conclusion: RISUG® presenting deleterious effects on spermatozoa and marked alterations in epididymal markers provides early contraception with a greater possibility of reversal. Although the progress of RISUG® toward development as an ideal male contraceptive is slow, the study implies a strong future possibility.

Mechanical stretch facilitates tenomodulin expression to induce tenocyte migration via MAPK signaling pathway

Tenomodulin (Tnmd) is a type II transmembrane glycoprotein that regulates tendon development and maturation. Our previous study indicated that mechanical stretch could induce Tnmd expression to promote tenocyte migration, associated with reinforcement of fibrous actin (F-actin) stress fibers and chromatin decondensation. However, the detailed molecular mechanisms of this processes are far from clear. Activation of mitogen-activated protein kinase (MAPK) signaling occurs in response to various extracellular stimuli and controls a large number of fundamental cellular processes. The present study we investigated the influence of MAPK signaling on mechanical stretch-induced Tnmd expression and its action way. Expression and activities of extracellular signal-related kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinases (JNK) and p38 MAPK (p38) were determined by Western blot. Cell migration was detected by Transwell assay. Immunofluorescence staining was used to detect F-actin stress fibers. Nuclear chromatin decondensation was detected by in situ DNaseI sensitivity assay. It was found that mechanical stretch promoted Tnmd expression by activating ERK1/2, JNK and p38 signaling. The inhibition of the ERK1/2, JNK or p38 repressed mechanical stretch-promoted tenocyte migration and mechanical stretch-induced reinforcement of F-actin stress fibers. However, only ERK1/2 and p38 inhibitor could repress mechanical stretch-induced chromatin decondensation, and the JNK inhibitor had no significant effect. Moreover, latrunculin (Lat A), the most widely used reagent to depolymerize actin filaments, could inhibit the stretch-induced chromatin decondensation. Taken together, our findings elucidated a molecular pathway by which a mechanical signal is transduced via activation of MAPK signaling to influence reinforcement of F-actin stress fibers and chromatin decondensation, which could further lead Tnmd expression to promote tenocyte migration.

Deleterious impact of short duration UV-A exposure on the human sperm cell –An in vitro study

BackgroundEnvironmental stressors like radiation are a cause for male infertility. The quantum of UV-A in solar ultraviolet (UV) radiation surpasses the amount of UVB by 10 to 100 times, penetrates deeper than UV-B into tissue and mediates possible molecular damage, which warrants evaluation of the impact of UV-A on human spermatozoa. AimTo determine the differences in effects and responses of UV-A exposure on sperm cells from fertile volunteers and infertile men. Materials and MethodsThe study subjects were divided into Group I (Control) and Group II (Infertile). The impact of UV-A on sperm viability, nuclear integrity, chromatin maturity and DNA fragmentation (pre-and post-exposure) was evaluated using standard techniques. Antioxidant enzyme activity assays viz. Lipid Peroxidation (LPO), Superoxide Dismutase (SOD) and Catalase were also analysed. Statistical significance was considered at p < 0.05. ResultsThis study revealed significant decline in%viability in Group II after UV-A exposure when compared with pre-exposed Group I. The percent sperm with disrupted nuclear toroidal assembly significantly increased in infertile group after exposure, leaving the DNA vulnerable to damage. A significant elevation was observed in the percent immature forms correlated with the significant reduction in% halo formation (impaired nuclear chromatin decondensation) in both post-treated groups as compared to pre-treated groups, suggesting that disruption of sperm toroid triggers DNA damage. UV-A exposure resulted in increased LPO with decrease in antioxidant enzymes in these groups. The percent decline in viability, chromatin decondensation (halo) and antioxidant enzymes were higher in Group II than in Group I. In addition, the percent increase in disrupted toroids and immature chromatin in Group II suggested that the deleterious impact of UV-A on spermatozoa from the infertile group is significantly greater than that of fertile individuals. ConclusionThis study thus revealed that short term UV-A exposure adversely impacts spermatozoa of infertile males to a greater extent than that of the fertile group. Thus, specific awareness and precautions are imperative for occupationally exposed individuals.

In vitro spermicidal effect of Thevetia Peruviana leaves on human spermatozoa.

An in vitro spermicidal effect of aqua-methanolic (2:3) extract of Thevetiaperuviana leaves on human spermatozoa was evaluated in a dose-dependent manner (20, 40, 80 and 160mg/ml) at a 1:1 ratio. Sperm motility, viability, hypo-osmotic swelling (HOS) and acrosomal status and function tests were performed immediately (20s), and after 5 and 10min of exposure of the spermatozoa to the extract of Thevetiaperuviana leaves at different dose concentrations. Nuclear chromatin decondensation (NCD) test, DNA fragmentation test and sperm revival test were also evaluated. The sperm motility was affected immediately at a dose of 20mg/ml and reduced gradually at doses of 40 and 80mg/ml of Thevetiaperuviana extract. Complete immobilisation of spermatozoa was observed at 160mg /ml dose of this extract treatment within 5min. 50% immobilisation of spermatozoa (EC50) was observed at 28mg/ml dose of Thevetiaperuviana extract within 20s. The sperm viability decreased significantly at a higher concentration of extract, and all spermatozoa were found to be non-viable after 10min when treated with 160mg/ml dose of Thevetiaperuviana extract. HOS and NCD of spermatozoa also reduced gradually at a higher concentration of extract administration. The percentage of DNA damage in spermatozoa was four times greater than in the control group. The findings indicate that the hydro-methanolic extract of Thevetiaperuviana leaves possesses appreciably potent spermicidal activity through an in vitro model, which may explore an effective vaginal contraceptive.

Application of the FISH Technique to Visualize Sex Chromosomes in Domestic Cat Spermatozoa.

Simple SummaryThe study was conducted with the aim of visualizing sex chromosomes in domestic cat sperm. Our research was carried out using fluorescence in situ hybridization (FISH) technique and the microscopic analysis showed the presence of X and Y chromosomes in domestic cat spermatozoa. The procedure used for sperm decondensation and FISH is adequate to visualize chromosomes in domestic cat sperm.Fluorescence in situ hybridization is a molecular cytogenetics technique that enables the visualization of chromosomes in cells via fluorescently labeled molecular probes specific to selected chromosomes. Despite difficulties in carrying out the FISH technique on sperm, related to the need for proper nuclear chromatin decondensation, this technique has already been used to visualize chromosomes in human, mouse, cattle, swine, horse, and dog spermatozoa. Until now, FISH has not been performed on domestic cat sperm; therefore, the aim of this study was to visualize sex chromosomes in domestic cat sperm. The results showed the presence of X and Y chromosomes in feline spermatozoa. The procedure used for sperm decondensation and fluorescence in situ hybridization was adequate to visualize chromosomes in domestic cat spermatozoa and, in the future, it may be used to determine the degree of chromosomal abnormalities in these gametes.

Recent progress in the mechanistic understanding of NET formation in neutrophils.

Neutrophils are the most abundant circulating white blood cells and one of the major cell types of the innate immune system. Neutrophil extracellular traps (NETs) are a result of the extracellular release of nuclear chromatin from the ruptured nuclear envelope and plasma membrane. The externalized chromatin is an ancient defense weapon for animals to entrap and kill microorganisms in the extracellular milieu, thus protecting animals ranging from lower invertebrates to higher vertebrates. Although the externalized chromatin has the advantage of acting as anti-infective to protect against infections, extracellular chromatin might be problematic in higher vertebrate animals as they have an adaptive immune system that can trigger further immune or autoimmune responses. NETs and their associated nuclear and/or cytoplasmic components may induce sterile inflammation, immune, and autoimmune responses, leading to various human diseases. Though important in human pathophysiology, the cellular and molecular mechanisms of NET formation (also called NETosis) are not well understood. Given that nuclear chromatin forms the backbone of NETs, the nucleus is the root of the nuclear DNA extracellular traps. Thus, nuclear chromatin decondensation, along with the rupture of nuclear envelope and plasma membrane, is required for nuclear chromatin extracellular release and NET formation. So far, most of the literature focuses on certain signaling pathways, which are involved in NET formation but without explanation of cellular events and morphological changes described above. Here, we have summarized emerging evidence and discuss new mechanistic understanding, with our perspectives, in NET formation in neutrophils.

Citrullination Licenses Calpain to Decondense Nuclei in Neutrophil Extracellular Trap Formation.

Neutrophils respond to various stimuli by decondensing and releasing nuclear chromatin characterized by citrullinated histones as neutrophil extracellular traps (NETs). This achieves pathogen immobilization or initiation of thrombosis, yet the molecular mechanisms of NET formation remain elusive. Peptidyl arginine deiminase-4 (PAD4) achieves protein citrullination and has been intricately linked to NET formation. Here we show that citrullination represents a major regulator of proteolysis in the course of NET formation. Elevated cytosolic calcium levels trigger both peptidylarginine deiminase-4 (PAD4) and calpain activity in neutrophils resulting in nuclear decondensation typical of NETs. Interestingly, PAD4 relies on proteolysis by calpain to achieve efficient nuclear lamina breakdown and chromatin decondensation. Pharmacological or genetic inhibition of PAD4 and calpain strongly inhibit chromatin decondensation of human and murine neutrophils in response to calcium ionophores as well as the proteolysis of nuclear proteins like lamin B1 and high mobility group box protein 1 (HMGB1). Taken together, the concerted action of PAD4 and calpain induces nuclear decondensation in the course of calcium-mediated NET formation.

Inhibition of RNA synthesis during Scriptaid exposure enhances gene reprogramming in SCNT embryos.

Insufficient epigenetic reprogramming is incompatible with normal development of embryos produced by somatic cell nuclear transfer (SCNT), but treatment with histone deacetylases inhibitors (HDACi) enhances development of SCNT embryos. However, the mechanisms underpinning HDACi benefits in SCNT embryos remain largely uncharacterized. We hypothesized that, in addition to enhancing reprogramming, HDACi treatment may promote expression of genes not required for early development of SCNT embryos. To test this hypothesis, RNA synthesis was inhibited by treating bovine SCNT embryos with 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside (DBR), which were concomitantly treated or not with Scriptaid (Scrip; an HDACi). Development to the blastocyst stage was significantly increased by treatment with Scrip alone (26.6%) or associated with DRB (28.6%) compared to Control (17.9%). The total number of nuclei was significantly improved only in embryos that were treated with both Scrip + DRB. Nuclear decondensation after SCNT was significantly increased by DRB treatment either alone or associated with Scrip. The relative mRNA expression, evaluated during the embryo genome activation (EGA) transition, revealed that some KDMs (KDM1A, KDM3A, KDM4C and KDM6A) and DNMT1 where prematurely expressed in Scrip-treated embryos. However, treatment with Scrip + DRB inhibited early mRNA expression of those genes, as well as several other KDMs (KDM4A, KDM4B, KDM5A, KDM5B, KDM5C and KDM7A) compared to embryos treated with Scrip alone. These findings revealed that HDACi improved development in SCNT embryos compared to Control, but altered the expression of genes involved in epigenetic regulation and did not improve embryo quality. Inhibition of RNA synthesis during HDACi treatment enhanced nuclear chromatin decondensation, modulated gene expression and improved SCNT embryo quality.

Padi4 Regulates NET Formation and Inflammatory Cell Death Downstream of Mlkl

Neutrophil extracellular trap (NET) formation can generate short-term functional anucleate cytoplasts and can trigger loss of cell viability. We examined the role of non-apoptotic cell death signaling in NET formation by studying necroptotic human and mouse neutrophils. Necroptotic cell death signaling was activated by caspase-8 inhibition and pharmacological targeting of inhibitor of apoptosis proteins (IAPs). The specificity of cell death was confirmed using neutrophils from mice deficient in receptor-interacting protein kinase-3 (Ripk3), Ripk1 kinase activity (Ripk1D138N/D138N), caspase-8, or the plasma membrane-disrupting effector protein mixed lineage kinase domain-like (Mlkl). NETs were investigated with a combination of imaging and quantitative flow cytometry, immunogold electron microscopy, immunofluorescence microscopy, and ex vivo microbicidal assays to demonstrate functionality of NETs.In response to necroptotic stimuli, Mlkl translocates to the plasma membrane in neutrophils, and is required for downstream NADPH oxidase-independent reactive oxygen species production, loss of cytoplasmic granules, breakdown of the nuclear membrane, chromatin decondensation, histone hypercitrullination, and extrusion of bacteriostatic NETs. Neutrophils expressing a kinase-dead form of Ripk1 (Ripk1D138N/D138N) were unable to generate Ripk3/Mlkl-dependent NETs, or to undergo necroptosis or caspase-8-dependent apoptosis. NET formation that is triggered by necroptotic stimuli is dependent on TNF production, can be differentially modulated by the actions of G-CSF and IFNγ, and occurs concomitantly with the production, processing and release of IL-1α/β. Human necroptotic neutrophils also release NETs that kill S.aureus. Necroptotic NETs contain components of canonical NETs including dsDNA, hypercitrullinated histones, and neutrophil elastase, but also non-canonical components including Mlkl and membranes.Peptidylarginine deiminase 4 (Padi4) is required for extrusion of necroptotic NETs but not for chromatin decondensation. Padi4-deficient neutrophils are hypersensitive to necroptotic stimuli despite normal levels of phosphorylated Mlkl, indicating that Padi4 acts downstream of Mlkl activation. The failure of Padi4-deficient neutrophils to generate necroptotic NETs in the presence of membrane-associated Mlkl, suggests that the removal of Mlkl membrane-disrupting complexes by NETs can facilitate membrane repair and control the kinetics of neutrophil necroptosis. This work defines a distinct cell death signaling network downstream of Mlkl that promotes Padi4-dependent necroptotic NET release. DisclosuresNo relevant conflicts of interest to declare.

A transient pool of nuclear F-actin at mitotic exit controls chromatin organization.

Re-establishment of nuclear structure and chromatin organization after cell division is integral for genome regulation or development and is frequently altered during cancer progression. The mechanisms underlying chromatin expansion in daughter cells remain largely unclear. Here, we describe the transient formation of nuclear actin filaments (F-actin) during mitotic exit. These nuclear F-actin structures assemble in daughter cell nuclei and undergo dynamic reorganization to promote nuclear protrusions and volume expansion throughout early G1 of the cell cycle. Specific inhibition of this nuclear F-actin assembly impaired nuclear expansion and chromatin decondensation after mitosis and during early mouse embryonic development. Biochemical screening for mitotic nuclear F-actin interactors identified the actin-disassembling factor cofilin-1. Optogenetic regulation of cofilin-1 revealed its critical role for controlling timing, turnover and dynamics of F-actin assembly inside daughter cell nuclei. Our findings identify a cell-cycle-specific and spatiotemporally controlled form of nuclear F-actin that reorganizes the mammalian nucleus after mitosis.

RESOLVING THE ENIGMA OF EFFECT OF MOBILE PHONE USAGE ON SPERMATOGENESIS IN HUMANS IN SOUTH INDIAN POPULATION

Objective: This study was aimed at to evaluate the possible risk of radiofrequency and electromagnetic waves of mobile phones on spermatogenic impairment and functional capacity of the spermatozoa along with oxidative stress, DNA damages, and hormone profile among mobile phone users.Methods: Mobile phone users were classified into three groups are 1-5, 6-10, and above 10 hrs/day, respectively, based on the exposure to electromagnetic radiation. Blood and semen samples are collected with informed consent letter. The semen samples used to carry out to the physical examination such as volume, liquefaction time, color, odor, pH, and viscosity, and functional status of the spermatozoa was carried out such as nuclear chromatin decondensation test, hypo-osmotic swelling test, and acrosomal intactness test. Seminal plasma was used for to evaluate the oxidative stress markers superoxide dismutase (SOD) activity, reactive oxygen species (ROS) levels, and total antioxidant capacity (TAC). Blood serum was used to estimate the level of follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone. DNA collected from blood used for DNA ladder assay.Results: In the present investigation, both physical and microscopic examinations were negatively correlated with mobile phone usage. No variation exists in functional status of spermatozoa. Oxidative stress markers such as the presence of ROS, enzymatic scavengers such as SOD and TAC showed no statistical variations between control group and mobile phone users and even no variations in hormone profile such as testosterone, FSH, and LH of users of mobile phone compared to normal reference values. Conclusion: In conclusion, though the literature has suggested that mobile phone use alters semen parameters, functional status of spermatozoa, increased oxidative stress, with subsequent sperm DNA damage in humans. The present study deviates from previous study stating nil impact of mobile phones on spermatogenetic impairment in humans.Keywords: Association of mobile phone usage, Male infertility.

Effect of ultrasmall gold nanoparticles on the murine native sperm chromatin

The effects of ultrasmall (2-3 nm) gold nanoparticles on native epididymal sperm chromatin of CBAxC57BL/6 hybrid mice and 129/IMG mice with a mutation in the DNA-polymerase iota gene were studied. It is shown that for both mouse strains after sperm incubation in a solution containing Au nanoparticles, at 23, 37 and 60 degrees C for 30 min followed by 1 hour treatment in dithiothreitol solution, a decrease in the number of nuclei with fully decondensed chromatin was observed compared with the control. Though, the manifestation of this effect in the population of 129/IMG mice mature sperm, was weaker. Also we have demonstrated that sperm of both strains that were incubated in a sol of Au nanoparticles at 60 degrees C behave differently under the action of dithiothreitol. A considerable part (-80%) of sperm of CBAxC57BL/6 hybrid mice treated with Au nanoparticles showed high resistance to the action of dithiothreitol, whereas in the case of 129/IMG mice only -30% did, and a partial or complete chromatin decondensation takes place in the remaining sperm. In general, using the method of nuclear chromatin decondensation in vitro for the native sperm, the patterns that we have identified in earlier studies on previously demembranized sperm are confirmed.

Relationship between sperm aneuploidy, sperm DNA integrity, chromatin packaging, traditional semen parameters, and recurrent pregnancy loss

To study the possible relationship between sperm aneuploidy, sperm DNA integrity, chromatin packaging, traditional semen parameters, and recurrent pregnancy loss (RPL). Descriptive study. University-affiliated tertiary teaching. A total of 22 couples with history of RPL and 20 fertile men. Semen samples from case and control men were examined for differences in semen parameters, DNA fragmentation, chromatin condensation, and sperm aneuploidy. Sperm DNA and chromatin integrity and sperm aneuploidy. Sperm progressive motility (30.2% vs. 51.5%) was significantly lower and abnormal morphology (74.8% vs. 54.2%) was significantly higher in the RPL group versus the control group, respectively. The percentage of fragmented DNA was significantly increased in the RPL group (17.1% vs. 10.2%) as well as the rate of spermatozoa with nuclear chromatin decondensation (23.6% vs. 11.8%). There was a significantly higher sperm aneuploidy rate among the RPL group as well. The increase in abnormal sperm parameters, sperm DNA fragmentation, nuclear chromatin decondensation, and sperm aneuploidy suggest possible causes of unexplained RPL.

Antiapoptotic efficacy of seed of Eugenia jambolana on testicular germ cell in experimental diabetic rat: a genomic study.

This study was designed to focus the genetic regulation of diabetes-induced testicular hypofunction and its amelioration by ethyl acetate fraction of seed of Eugenia jambolana. In this regard, we have assessed relevant biosensors such as biochemical, spermiological, histological and gene expression of antioxidant enzymes, germ cell apoptosis and androgenic key enzymes along with in situ end labelling and DNA fragmentation study. After 60 days administration of said fraction, significant recovery in the glycated haemoglobin, serum testosterone, sperm viability, hypo-osmotic swelling and nuclear chromatin decondensation were noted in fraction-treated diabetic group in comparison with diabetic control. Besides this, a significant recovery in the expression of Bax, Bcl-2, caspase-9, caspase-3, catalase, peroxidase, ∆(5) , 3β-hydroxy steroid dehydrogenase and 17β-hydroxy steroid dehydrogenase genes was noted towards the control in ethyl acetate fraction-treated group. Testicular histology focused a significant recovery in the number of different generation of germ cells at stage VII of spermatogenesis in fraction-treated group. In situ end labelling and DNA fragmentation study of testicular tissues also showed a significant recovery in fraction-treated group towards the control. These findings indicate that the ethyl acetate fraction showed outstanding antiapoptotic activity by neutralising oxidative stress as well as by the improvement in glycaemic sensors.

Extraction of histone H1 and decondensation of nuclear chromatin with various Mg-dependent organization levels under treatment with polyglutamic acid and distamycin.

Chromatin in rat liver nuclei under conditions of low ionic strength (20-25mM) and [Mg2+] from 2 to 5mM has a condensed structure (100-200nm globules) and gives the same CD signal (320-340nm) at interaction with the antibiotic distamycin A (DM). Reducing [Mg2+] to 1mM leads to chromatin decondensation to 30nm structures and increases the CD signal. Poly-L-glutamic acid (PG) at weight ratio PG/DNA= 6 and in the presence of 5mM Mg2+ extracts only about 1/8 of nuclear histone H1, preserving a condensed chromatin structure. Removal of about 1/4 of H1 at 3mM Mg2+ leads to chromatin decondensation to 30nm fibrils. Extraction of about half of histone H1 at [Mg2+] ≤ 2mM results in chromatin refolding to nucleosome fibrils. PG-decondensation leads to a significant increase in the CD signal. The main H1 extraction occurs in 1-2min, but at all Mg2+ concentrations the more slowly PG extracted fraction is found comprising 5-7% of nuclear H1. About 25% of leaving nuclear H1 can be extracted by PG in the presence of saturating DM concentration (molar DM/DNA= 0.1). H1 release depends significantly on the PG concentration. However, even at high weight ratio PG/DNA= 30 and DM/DNA= 0.1, about 5-10% of histone H1 remained in the nuclei. Decondensation of chromatin in the nucleus is not always proportional to the yield of extracted histone H1 and is weakened in the presence of positively charged DM or high concentrations of PG. Our results show that the interaction of DM with chromatin depends primarily on chromatin packaging, while PG extraction depends on [Mg2+] supporting this packaging.

Investigation of the association between the outcomes of sperm chromatin condensation and decondensation tests, and assisted reproduction techniques.

The main purpose of this prospective study is to examine possible influences of abnormalities of sperm nuclear condensation and chromatin decondensation with sodium dodecyl sulphate (SDS)-EDTA on outcomes of intrauterine insemination (IUI) or intracytoplasmic sperm injection (ICSI) cycles. Semen samples from 122 IUI and 236 ICSI cycles were evaluated. Before semen preparation for IUI or ICSI, basic semen analysis was performed and a small portion from each sample was spared for fixation. The condensation of sperm nuclear chromatin was evaluated with acidic aniline blue, followed by sperm chromatin decondensation by SDS-EDTA and evaluation under light microscope. Ongoing pregnancy rate was 24% and 26.2% in the IUI and ICSI groups respectively. The chromatin condensation rate was significantly higher in the ongoing pregnancy-positive group compared to the negative group, both in IUI (P=0.042) and ICSI groups (P=0.027), and it was positively correlated with ongoing pregnancy rate in both IUI and ICSI groups (P=0.015, r=0.214 and P=0.014, r=0.312 respectively). Chromatin decondensation rates were not significantly different in neither of the groups. These results indicate that IUI and ICSI outcome is influenced by the rate of spermatozoa with abnormal chromatin condensation. Sperm chromatin condensation with aniline blue is useful for selecting assisted reproduction techniques (ART) patients.

NADPH Oxidase Promotes Neutrophil Extracellular Trap Formation in Pulmonary Aspergillosis

NADPH oxidase is a crucial enzyme in antimicrobial host defense and in regulating inflammation. Chronic granulomatous disease (CGD) is an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates. Aspergillus species are ubiquitous, filamentous fungi, which can cause invasive aspergillosis, a major cause of morbidity and mortality in CGD, reflecting the critical role for NADPH oxidase in antifungal host defense. Activation of NADPH oxidase in neutrophils can be coupled to the release of proteins and chromatin that comingle in neutrophil extracellular traps (NETs), which can augment extracellular antimicrobial host defense. NETosis can be driven by NADPH oxidase-dependent and -independent pathways. We therefore undertook an analysis of whether NADPH oxidase was required for NETosis in Aspergillus fumigatus pneumonia. Oropharyngeal instillation of live Aspergillus hyphae induced neutrophilic pneumonitis in both wild-type and NADPH oxidase-deficient (p47(phox-/-)) mice which had resolved in wild-type mice by day 5 but progressed in p47(phox-/-) mice. NETs, identified by immunostaining, were observed in lungs of wild-type mice but were absent in p47(phox-/-) mice. Using bona fide NETs and nuclear chromatin decondensation as an early NETosis marker, we found that NETosis required a functional NADPH oxidase in vivo and ex vivo. In addition, NADPH oxidase increased the proportion of apoptotic neutrophils. Together, our results show that NADPH oxidase is required for pulmonary clearance of Aspergillus hyphae and generation of NETs in vivo. We speculate that dual modulation of NETosis and apoptosis by NADPH oxidase enhances antifungal host defense and promotes resolution of inflammation upon infection clearance.