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1.
Int J Biol Macromol ; 116: 633-647, 2018 Sep.
Article in English | MEDLINE | ID: mdl-29723624

ABSTRACT

Motility of spermatozoa is a crucial factor for determining semen quality. Here we report motility inhibitory factor (MIF-II) from goat epididymal plasma, revealing its structure, function, localization and motility inhibitory pathway. Structural characterization with MALDI revealed novelty of this protein while circular dichroism data confirmed its alpha helical nature. Higher dilutions of MIF-II antibody increased cauda sperm motility and induced immature/immotile caput sperm motility as tested microscopically. Higher number of sperm cells and lower dilutions of antibody induced agglutination in cauda sperm showing surface localization. Indirect immuno-fluorescence showed MIF-II localization throughout the caput sperm surface which relocated more towards acrosomal region with maturation. ELISA assay revealed gradual increase and decrease in concentration of MIF-II in epididymal plasma and plasma membrane respectively from caput to cauda. Signaling cascade that leads to sperm motility inhibition elevates nitric oxide levels through cAMP dependent pathway. MIF-II treatment doesn't alter sperm surface morphology. Expression pattern of MIF-II during epididymal maturation goes hand-in-hand with gaining motility potential as well as dormancy of spermatozoa before ejaculation. Both MIF-II and its antibody inhibit fertilization in-vitro thus expected to open new gateway for future male infertility and contraceptive development research.


Subject(s)
Proteins , Second Messenger Systems/physiology , Sperm Motility/physiology , Spermatozoa , Animals , Cyclic AMP/metabolism , Epididymis/chemistry , Epididymis/metabolism , Humans , Male , Proteins/chemistry , Proteins/metabolism , Rabbits , Rats , Spermatozoa/chemistry , Spermatozoa/metabolism
2.
Cell Biochem Biophys ; 70(2): 1177-83, 2014 Nov.
Article in English | MEDLINE | ID: mdl-24880438

ABSTRACT

Sperm motility can be maintained in vitro by incubation in a defined medium under specific conditions. In most studies, the exact role of various constituents of epididymal fluid, including calcium, has remained obscure. Most of the culture media have included millimolar concentrations of calcium, but previous reports have indicated that millimolar calcium inhibits sperm motility. In this present study, we sought the optimum concentration of extracellular calcium required for optimum sperm motility. This study showed that extracellular calcium has a concentration-dependent biphasic role in motility regulation. It promoted motility and velocity at lower (10 µM) concentration whereas notably inhibited it at higher concentrations. When external membrane-bound calcium was removed by ethylene glycol tetraacetic acid, motility decreased considerably. To confirm the motility-inhibiting role of calcium above 10 µM, a sperm motility-stimulating protein (MSP) recently reported from our laboratory was used which at 0.9 µM induces motility in 60-70 % cells. Calcium at 10 µM had no appreciable effect on the motility-promoting activity of the MSP but depressed the activity above 10 µM. Thus, our present results emphasize the biphasic role of extracellular calcium and the importance of its optimum concentration in different buffers and media used for sperm motility initiation.


Subject(s)
Calcium/pharmacology , Sperm Motility/drug effects , Animals , Calcium/metabolism , Dose-Response Relationship, Drug , Egtazic Acid/pharmacology , Extracellular Space/drug effects , Extracellular Space/metabolism , Goats , Male , Spermatozoa/cytology , Spermatozoa/drug effects
3.
Nanomedicine (Lond) ; 9(3): 441-56, 2014 Mar.
Article in English | MEDLINE | ID: mdl-23742256

ABSTRACT

AIM: Hepatocellular carcinoma (HCC) has no successful pharmacotherapeutic remedy. The aim of this study was to ascertain whether ginkgolide B (GB)-loaded polymeric nanocapsules can prevent diethylnitrosamine (DEN)-induced HCC in rats. MATERIALS & METHODS: GB was fabricated in two types of nanocapsules of which one was polyethylene glycol coated (N1GB) and the other was uncoated (N2GB). These nanocapsules were orally gavaged during DEN-induced HCC development in rats. RESULTS: Nanocapsulation of GB enabled aqueous suspension and slow time-dependent release of the compound. Anticarcinogenic potential of N2GB was reflected by its ability in the management of DEN-induced reactive oxygen species generation, mitochondrial dysfunction, p53, NF-κB, inducible nitric oxide synthase, COX-2 and VEGF expressions, and induction of apoptosis in cancer cells in the rat liver. CONCLUSION: Positive zeta-potential on N2GB surface might have offered higher hepatic accumulation of GB, especially at the electron-dense organelle mitochondria. Mitochondria protection against DEN-induced oxidative damage ensured HCC prevention.


Subject(s)
Anticarcinogenic Agents/therapeutic use , Carcinoma, Hepatocellular/chemically induced , Carcinoma, Hepatocellular/prevention & control , Ginkgolides/therapeutic use , Lactones/therapeutic use , Liver Neoplasms/chemically induced , Liver Neoplasms/prevention & control , Mitochondria/drug effects , Animals , Anticarcinogenic Agents/administration & dosage , Carcinoma, Hepatocellular/pathology , Diethylnitrosamine , Ginkgolides/administration & dosage , Lactones/administration & dosage , Liver/drug effects , Liver/pathology , Liver Neoplasms/pathology , Male , Mitochondria/pathology , Nanocapsules/chemistry , Polymers/chemistry , Rats
4.
Toxicon ; 74: 138-50, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23981271

ABSTRACT

BF-CT1, a 13 kDa protein isolated from Bungarus fasciatus snake venom through CM cellulose ion exchange chromatography at 0.02 M NaCl salt gradient showed cytotoxicity in in vitro and in vivo experimental models. In in vivo Ehrlich ascites carcinoma (EAC) induced BALB/c mice model, BF-CT1 treatment reduced EAC cell count significantly through apoptotic cell death pathway as evidenced by FACS analysis, increased caspase 3, 9 activity and altered pro, antiapoptotic protein expression. BF-CT1 treatment caused cell shrinkage, chromatin condensation and induced apoptosis through increased caspase 3, caspase 9 activity, PARP cleavage and down regulation of heat shock proteins in U937 leukemic cell line. Cytosolic cytochrome C production was increased after BF-CT1 treatment upon U937 cell line. BF-CT1 treated U937 cell showed cell cycle arrest at sub G1 phase through cyclin D and CDK down regulation with up regulation of p15 and p16. It also down regulated PI3K/AKT pathway and MAPkinase pathway and promoted apoptosis and regulated cell proliferation in U937 cells. BF-CT1 prevented angiogenesis in in vitro U937 cell line through decreased VEGF and TGF-ß1 production.


Subject(s)
Apoptosis/drug effects , Bungarus , Cell Cycle Checkpoints/drug effects , Reptilian Proteins/pharmacology , Signal Transduction/drug effects , Snake Venoms/pharmacology , Angiogenesis Inducing Agents/pharmacology , Animals , Annexin A5/pharmacology , Caspase 3/metabolism , Caspase 8/metabolism , Caspase 9/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin D/genetics , Cyclin D/metabolism , Cyclin-Dependent Kinases/genetics , Cyclin-Dependent Kinases/metabolism , Disease Models, Animal , Down-Regulation , Humans , Lethal Dose 50 , Male , Mice , Mice, Inbred BALB C , Molecular Weight , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Reactive Oxygen Species/metabolism , Reptilian Proteins/isolation & purification , Snake Venoms/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Transforming Growth Factor beta1/metabolism , U937 Cells , Up-Regulation , Vascular Endothelial Growth Factor A/metabolism
5.
Exp Parasitol ; 135(1): 126-33, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23830987

ABSTRACT

Leishmaniasis threatens more than 350 million people worldwide specially in tropical and subtropical region. Antileishmanial drugs that are currently available have various limitations. The search of new drugs from natural products (plants, animals) possessing antileishmanial activity is ventured throughout the world. The present study deals with the antileishmanial activity of Bungarus caeruleus snake venom (BCV) on in vitro promastigotes and amastigotes of Leishmania donovani parasite and leishmania infected BALB/c mice. The effect of BCV on peritoneal macrophage, release of cytokines from the activated macrophages, production of nitric oxide, reactive oxygen species and cytokines were studied in vivo and in vitro. IC50 value of BCV on L. donovani promastigote was 14.5 µg/ml and intracellular amastigote was 11.2 µg/ml. It activated peritoneal macrophages, significantly increased cytokines and interleukin production. BCV (20 µg/kg and 40 µg/kg body weight of mice) decreased parasite count by 54.9% and 74.2% in spleen and 41.4% and 60.4% in liver of infected BALB/c mice. BCV treatment significantly increased production of TNF-α, IFN-γ, ROS, NO in infected mice. Histological studies showed decreased granuloma formation in treated liver as compared with control. Liver and spleen structure was partially restored due to BCV treatment in infected mice. The present study revealed that BCV possessed antileishmanial activity against L. donovani parasite in vivo and in vitro and this activity was partly mediated through immunomodulatory activity involving macrophages.


Subject(s)
Antiprotozoal Agents/pharmacology , Bungarotoxins/pharmacology , Leishmania donovani/drug effects , Leishmaniasis, Visceral/drug therapy , Animals , Antimony Sodium Gluconate/pharmacology , Antimony Sodium Gluconate/therapeutic use , Antiprotozoal Agents/therapeutic use , Bungarotoxins/therapeutic use , Bungarus , Cricetinae , Dose-Response Relationship, Drug , Female , Inhibitory Concentration 50 , Interferon-gamma/metabolism , Leishmaniasis, Visceral/immunology , Liver/parasitology , Liver/pathology , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/immunology , Macrophages, Peritoneal/parasitology , Male , Mesocricetus , Mice , Mice, Inbred BALB C , Nitric Oxide/metabolism , Phagocytosis/drug effects , Reactive Oxygen Species/metabolism , Spleen/parasitology , Spleen/pathology , Tumor Necrosis Factor-alpha/metabolism
6.
Fertil Steril ; 100(1): 269-79.e1-5, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23548940

ABSTRACT

OBJECTIVE: To identify and characterize a novel sperm motility-stimulating protein (MSP) from caprine serum. DESIGN: Prospective experimental study. SETTING: Research laboratory. ANIMAL(S): Rabbits and male and female BALB/c mice. INTERVENTION(S): Protein purification by conventional methods and functional and immunological characterization. MAIN OUTCOME MEASURE(S): MSP was purified, purity was checked, and molecular weight was determined. Surface localization, tissue distribution, and IVF for contraceptive efficacy were studied. RESULT(S): MSP is a heat-stable 66-kDa monomeric novel protein. At 0.9 µM, it showed much higher forward motility and longer motility maintenance than other known activators. Studies with the motility analyzers CASA and SPERMA showed an increase in horizontal and vertical velocities, respectively. MSP action was cyclic AMP independent. Its occurrence was higher in testis, although blood was the richest source. MSP was localized throughout the cell surface of spermatozoa. Its antibody caused significant inhibition of sperm motility and inhibited fertilization up to 100% at 1:25 dilution, which showed its contraceptive efficacy. CONCLUSION(S): MSP has high efficacy to stimulate sperm forward motility and thus may be used in biomedical application in infertility clinics, animal breeding centers, poultries, and animal conservation centers.


Subject(s)
Blood Proteins/isolation & purification , Blood Proteins/physiology , Sperm Motility/physiology , Animals , Female , Goats/blood , Male , Mice , Mice, Inbred BALB C , Prospective Studies , Rabbits
7.
Free Radic Biol Med ; 51(10): 1893-902, 2011 Nov 15.
Article in English | MEDLINE | ID: mdl-21914470

ABSTRACT

Chronic arsenic exposure causes oxidative stress and mitochondrial dysfunction in the liver and brain. The ideal treatment would be to chelate arsenic and prevent oxidative stress. meso-2,3-Dimercaptosuccinic acid (DMSA) is used to chelate arsenic but its hydrophilicity makes it membrane-impermeative. Conversely, quercetin (QC) is a good antioxidant with limited clinical application because of its hydrophobic nature and limited bioavailability, and it is not possible to solubilize these two compounds in a single nontoxic solvent. Nanocapsules have emerged as a potent drug delivery system and make it feasible to incorporate both hydrophilic and lipophilic compounds. Nanoencapsulated formulations with QC and DMSA either alone or coencapsulated in polylactide-co-glycolide [N(QC+DMSA)] were synthesized to explore their therapeutic application in a rat model of chronic arsenic toxicity. These treatments were compared to administration of quercetin or DMSA alone using conventional delivery methods. Both nanoencapsulated quercetin and nanoencapsulated DMSA were more effective at decreasing oxidative injury in liver or brain compared to conventional delivery methods, but coencapsulation of quercetin and DMSA into nanoparticles had a marked synergistic effect, decreasing liver and brain arsenic levels from 9.5 and 4.8µg/g to 2.2 and 1.5µg/g, respectively. Likewise, administration of coencapsulated quercetin and DMSA virtually normalized changes in mitochondrial function, formation of reactive oxygen species, and liver injury. We conclude that coencapsulation of quercetin and DMSA may provide a more effective therapeutic strategy in the management of arsenic toxicity and also presents a novel way of combining hydrophilic and hydrophobic drugs into a single delivery system.


Subject(s)
Arsenic Poisoning/drug therapy , Arsenic/metabolism , Brain/drug effects , Liver/drug effects , Nanoparticles/therapeutic use , Animals , Antioxidants/administration & dosage , Arsenic Poisoning/pathology , Arsenic Poisoning/physiopathology , Brain/metabolism , Brain/pathology , Chelating Agents/administration & dosage , Disease Models, Animal , Drug Compounding/methods , Drug Delivery Systems , Drug Synergism , Female , Humans , Hydrophobic and Hydrophilic Interactions , Lactic Acid/chemistry , Liver/metabolism , Liver/pathology , Nanoparticles/chemistry , Oxidative Stress/drug effects , Polyglycolic Acid/chemistry , Polylactic Acid-Polyglycolic Acid Copolymer , Quercetin/administration & dosage , Rats , Rats, Wistar , Succimer/administration & dosage
8.
PLoS One ; 5(8): e12039, 2010 Aug 10.
Article in English | MEDLINE | ID: mdl-20706623

ABSTRACT

Several studies have been reported on the occurrence of sperm motility inhibiting factors in the male reproductive fluids of different mammalian species, but these proteins have not been adequately purified and characterized. A novel sperm motility inhibiting factor (MIF-II) has been purified from caprine epididymal plasma (EP) by Hydroxylapatite gel adsorption chromatography, DEAE-Cellulose ion-exchange chromatography and chromatofocusing. The MIF-II has been purified to apparent homogeneity and the molecular weight estimated by Sephacryl S-300 gel filtration is 160 kDa. MIF-II is a dimeric protein, made up of two subunits each having a molecular mass of 80 kDa as shown by SDS-PAGE. The isoelectric point of MIF-II is 5.1 as determined by chromatofocusing and isoelectric focusing. It is a heat labile protein and maximal active at the pH 6.9 to 7.5. The sperm motility inhibiting protein factor at 2 microg/ml (12.5 nM) level showed maximal motility-inhibiting activity. The observation that the epididymal plasma factor lowered the intracellular cAMP level of spermatozoa in a concentration-dependent manner suggests that it may block the motility of caprine cauda spermatozoa by interfering the cAMP dependent motility function. The results revealed that the purified protein factor has the potential of sperm motility inhibition and may serve as a vaginal contraceptive. The antibody raised against the MIF-II has the potential for enhancement of forward motility of cauda-spermatozoa. This antibody may thus be useful for solving some of the problems of male infertility due to low sperm motility.


Subject(s)
Chemical Fractionation/methods , Epididymis/chemistry , Goats , Sperm Motility/drug effects , Animals , Cyclic AMP/metabolism , Humans , Intracellular Space/drug effects , Intracellular Space/metabolism , Male , Semen Analysis , Spectrophotometry , Spermatozoa/cytology , Spermatozoa/drug effects
9.
J Cell Physiol ; 222(1): 254-63, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19795390

ABSTRACT

Numerous reports have appeared on the occurrence of undefined protein factors in male reproductive fluids that promote motility of mature sperm and initiate forward motility in the immature (immotile) caput-epididymal sperm. This study reports for the first time purification to apparent homogeneity of a motility initiating protein (MIP) from epididymal plasma and its characterization using the caprine sperm model. It is a 125 kDa (approximately) dimeric protein made up of two subunits: 70 and 54 kDa. MIP is an acidic protein with an isoelectric point of 4.75. The motility protein at 30 microg/ml (240 nM) level showed nearly maximal motility-promoting activity. MIP is heat stable and it is maximally active at pH 8. It is a glycoprotein that binds with high affinity to concanavalin A and it contains mannose, galactose, and N-acetyl glucosamine approximately in the ratios of 6:1:6. It is sensitive to the actions of alpha-mannosidase and beta-N-acetylglucoseaminidase thereby demonstrating that the sugar side chain of the glycoprotein is essential for its biological activity. Epididymal plasma is its richest source. It is also capable of enhancing forward motility of mature cauda-sperm. Its antibody markedly inhibits sperm motility. MIP antibody is highly immunospecific and it recognizes both the subunits. MIP causes significant increase of the intrasperm level of cyclic AMP. MIP: the physiological motility-activating protein has potential for use as a contraceptive vaccine and for solving some of the problems of human infertility and animal breeding.


Subject(s)
Epididymis/metabolism , Goats/metabolism , Molecular Motor Proteins/isolation & purification , Molecular Motor Proteins/metabolism , Semen/metabolism , Animals , Antibodies/immunology , Antibodies/pharmacology , Antibody Specificity/drug effects , Blotting, Western , Chromatography, Affinity , Chromatography, Gas , Chromatography, High Pressure Liquid , Cyclic AMP/metabolism , Electrophoresis, Polyacrylamide Gel , Epididymis/drug effects , Glycoproteins/isolation & purification , Glycoproteins/metabolism , Glycoside Hydrolases/pharmacology , Hydrogen-Ion Concentration/drug effects , Isoelectric Focusing , Male , Organ Specificity/drug effects , Reference Standards , Sperm Motility/drug effects
10.
Exp Cell Res ; 312(12): 2299-308, 2006 Jul 15.
Article in English | MEDLINE | ID: mdl-16631739

ABSTRACT

Testicular immotile sperm undergo maturation during epididymal transit when these cells pass through caput, corpus, and cauda-epididymal regions. Maturing goat spermatozoa specifically at the distal corpus epididymal stage show head-to-head autoagglutination when incubated in vitro in a modified Ringer's solution. Here, we show the biochemical mechanism of autoagglutination event and its functional significance. A lectin-like molecule located on sperm surface specifically interacts with its receptor of the neighboring homologous cells to cause autoagglutination. Lectin is a Ca++-dependent galactose-specific protein. Failure of the pre- and post-distal corpus sperm to show autoagglutination is due to lack of lectin-like molecule and its receptors, respectively. Maturing sperm at distal corpus stage acquire lectin-like molecule followed by sharp disappearance of its receptor, and this event is synchronously associated with the initiation of sperm forward motility that is essential for fertilization in vivo. Lectin and its receptor isolated from sperm plasma membrane showed high efficacy for blocking autoagglutination phenomenon. The data are consistent with the view that synchronous modulation of homologous cell surface lectin and their receptors constitutes a novel mechanism for cellular regulation by generating waves of signals by manipulating lectin-sugar-dependent "self-talk" and cell-cell "cross-talk".


Subject(s)
Cell Communication/physiology , Lectins/metabolism , Membrane Glycoproteins/metabolism , Receptors, Cell Surface/metabolism , Sperm Agglutination/physiology , Animals , Asialoglycoproteins/metabolism , Asialoglycoproteins/pharmacology , Binding, Competitive , Cations, Divalent/pharmacology , Cell Aggregation/drug effects , Cell Aggregation/physiology , Cell Communication/drug effects , Cell Membrane/immunology , Cell Membrane/metabolism , Disaccharides/pharmacology , Erythrocytes/cytology , Erythrocytes/immunology , Fetuins , Galactose/pharmacology , Goats , Hemagglutination Tests , Lectins/pharmacology , Male , Membrane Glycoproteins/immunology , Membrane Glycoproteins/pharmacology , Monosaccharides/pharmacology , Protein Binding , Rabbits , Sperm Agglutination/drug effects , Sperm Maturation/drug effects , Sperm Maturation/physiology , Spermatozoa/cytology , Spermatozoa/drug effects , Spermatozoa/physiology , alpha-Fetoproteins/metabolism , alpha-Fetoproteins/pharmacology
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