Lead and calcium crosstalk tempted acrosome damage and hyperpolarization of spermatozoa: signaling and ultra-structural evidences.

BACKGROUND: Exposure of humans and animals to heavy metals is increasing day-by-day; thus, lead even today remains of significant public health concern. According to CDC, blood lead reference value (BLRV) ranges from 3.5 µg/dl to 5 µg/dl in adults. Recently, almost 2.6% decline in male fertility per year has been reported but the cause is not well established. Lead (Pb2+) affects the size of testis, semen quality, and secretory functions of prostate. But the molecular mechanism(s) of lead toxicity in sperm cells is not clear. Thus, present study was undertaken to evaluate the adverse effects of lead acetate at environmentally relevant exposure levels (0.5, 5, 10 and 20 ppm) on functional and molecular dynamics of spermatozoa of bucks following in vitro exposure for 15 min and 3 h. RESULTS: Lead significantly decreased motility, viable count, and motion kinematic patterns of spermatozoa like curvilinear velocity, straight-line velocity, average path velocity, beat cross frequency and maximum amplitude of head lateral displacement even at 5 ppm concentration. Pb2+ modulated intracellular cAMP and Ca2+ levels in sperm cells through L-type calcium channels and induced spontaneous or premature acrosome reaction (AR) by increasing tyrosine phosphorylation of sperm proteins and downregulated mitochondrial transmembrane potential. Lead significantly increased DNA damage and apoptosis as well. Electron microscopy studies revealed Pb2+ -induced deleterious effects on plasma membrane of head and acrosome including collapsed cristae in mitochondria. CONCLUSIONS: Pb2+ not only mimics Ca2+ but also affects cellular targets involved in generation of cAMP, mitochondrial transmembrane potential, and ionic exchange. Lead seems to interact with Ca2+ channels because of charge similarity and probably enters the sperm cell through these channels and results in hyperpolarization. Our findings also indicate lead-induced TP and intracellular Ca2+ release in spermatozoa which in turn may be responsible for premature acrosome exocytosis which is essential feature of capacitation for fertilization. Thus, lead seems to reduce the fertilizing capacity of spermatozoa even at 0.5 ppm concentrations.

Calcium signaling cascades differentially regulate PGF2α-induced myometrial contractions in water buffaloes (Bubalus bubalis).

This study unravels the differential involvement of calcium signaling pathway(s) in PGF2α-induced contractions in myometrium of nonpregnant (NP) and pregnant buffaloes. Compared to the myometrium of pregnant animals, myometrium of NP buffaloes was more sensitive to PGF2α as manifested by changes in mean integral tension (MIT) and tonicity. In the presence of nifedipine, myometrial contraction to PGF2α was significantly attenuated in both NP and pregnant uteri; however, mibefradil and NNC 55-0396 produced inhibitory effects only in uterus of pregnant animals, thus suggesting the role of extracellular Ca2+ influx through nifedipine-sensitive L-type Ca2+-channels both in NP and pregnant, but T-type Ca2+ channels seem to play a role only during pregnancy. Entry of extracellular Ca2+ is triggered by enhanced functional involvement of Pyr3-sensitive TRPC3 channels and Rho-kinase pathways as evidenced by a significant rightward shift of the concentration-response curve of PGF2α in the presence of Pyr3 and Y-27632 in NP myometrium. But significant down-expressions of TRPC3 and Rho-A proteins during pregnancy apparently facilitate uterine quiescence. In the presence of Ca2+-free solution and cyclopiazonic acid (SERCA blocker), feeble contraction to PGF2α was observed in both NP and pregnant myometrium which suggests minor role of intracellular source of Ca2+ in mediating PGF2α-induced contractions in these tissues.

Collapsed mitochondrial cristae in goat spermatozoa due to mercury result in lethality and compromised motility along with altered kinematic patterns.

Earlier we have reported mercury-induced alterations in functional dynamics of buck spermatozoa through free radicals-mediated oxidative stress and spontaneous acrosome reaction. Based on our earlier findings, we aimed to investigate the effect of mercury exposure on motility, kinematic patterns, DNA damage, apoptosis and ultra-structural alterations in goat spermatozoa following in vitro exposure to different concentrations (0.031-1.25 µg/ml) of mercuric chloride for 15 min and 3 h. Following exposure of sperm cells to 0.031 µg/ml of mercuric chloride for 3 h, livability and motility of sperms was significantly reduced along with altered kinematic patterns, significant increase in per cent necrotic sperm cells and number of cells showing DNA damage; and this effect was dose- and time-dependent. Contrary to up-regulation of Bax gene after 3 h in control group, there was significant increase in expression of Bcl-2 in mercury-treated groups. Transmission electron microscopy studies revealed rifts and nicks in plasma and acrosomal membrane, mitochondrial sheath, and collapsed mitochondria with loss of helical organization of mitochondria in the middle piece of spermatozoa. Our findings evidently suggest that mercury induces necrosis instead of apoptosis and targets the membrane, acrosome, mid piece of sperms; and the damage to mitochondria seems to be responsible for alterations in functional and kinematic attributes of spermatozoa.

Chitosan and gelatin biopolymer supplemented with mesenchymal stem cells (Velgraft®) enhanced wound healing in goats (Capra hircus): Involvement of VEGF, TGF and CD31.

AIM: Cell-based therapy has emerged as promising strategy for chronic and impaired wounds treatment. Current research is focused on developing biomaterial systems that act as a niche for mesenchymal stem cells (MSCs) to promote wound healing through paracrine molecular cascading. This study was aimed to evaluate the wound healing potential of Velgraft, a ready-to-use biodegradable artificial skin substitute, on excision wound in goats. MATERIALS AND METHODS: Twelve male goats were randomized divided in to three groups of four animals each. After infliction of surgical wound, Velgraft and Soframycin were applied on wounds of the animals of Groups II and III while Group I (sham operated) served as control. Wound diameters were measured at pre-defined time-points for determination of progressive wound healing up to 28 days. Skin sections were stained using Hematoxylin and eosin (H&E) for examining the histoarchitectural changes, Masson trichome staining for ascertaining collagen synthesis and immunohistochemistry for expression of CD31, VEGF and TGF-ß1 proteins to determine post-treatment angiogenesis in the inflicted wounds. RESULTS: Velgraft application appreciably enhanced wound closure by day 21 which was confirmed through restoration of the normal skin architecture as evident based on histopathological examination and characterized by complete regeneration of epidermal layers, collagen fibers, blood capillaries and hair follicular formation. Stimulation of angiogenesis markers was also observed at different time-points post-Velgraft application; which is suggestive of the improved angiogenesis and vasculogenesis. CONCLUSION: Velgraft facilitates wound healing by augmenting early wound closure, enhancing collagen synthesis and deposition, trichosis development and promoting revascularization and epidermal layers restoration.

In vitro and in vivo effects of flubendiamide and copper on cyto-genotoxicity, oxidative stress and spleen histology of rats and its modulation by resveratrol, catechin, curcumin and α-tocopherol.

BACKGROUND: Living organisms are frequently exposed to more than one xenobiotic at a time either by ingestion of contaminated food/fodder or due to house-hold practices, occupational hazards or through environment. These xenobiotics interact individually or in combination with biological systems and act as carcinogen or produce other toxic effects including reproductive and degenerative diseases. Present study was aimed to investigate the cyto-genotoxic effects of flubendiamide and copper and ameliorative potential of certain natural phyotconstituent antioxidants. METHOD: In vitro cytogenotoxic effects were evaluated by employing battery of assays including Propidium iodide staining, Tunel assay, Micronuclei, DNA fragmentation and Comet assay on isolated splenocytes and their prevention by resveratrol (5 and 10 µM), catechin (10 and 20 µM), curcumin (5 and 10 µM) and α-tocopherol (5, 10 and 20 µM). In vivo study was also undertaken daily oral administration of flubendiamide (200 mg/kg) or copper (33 mg/kg) and both these in combination, and also all these concurrently with of α-tocopherol to Wistar rats for 90 days. RESULTS: Flubendiamide and copper produced concentration-dependent cytotoxic effects on splenocytes and at median lethal concentrations, flubendiamide (40 µM) and copper (40 µM) respectively produced 71 and 81% nonviable cells, higher number of Tunel+ve apoptotic cells, 7.86 and 9.16% micronucleus and 22.90 and 29.59 comets/100 cells and DNA fragmentation. In vivo study revealed significant (P < 0.05) increase in level of lipid peroxidation (LPO) and decrease in glutathione peroxidase (GPx), glutathione-S-transferase (GST) and superoxide dismutase (SOD) activities in groups exposed to flubendiamide or copper alone or both these in combination. Histopathological examination of rat spleens revealed depletion of lymphoid tissue, separation of splenocytes and rarification in splenic parenchyma of xenobiotic(s) treated groups. CONCLUSION: Flubendiamide and copper induce oxidative stress and produce cytogenotoxic effects along with histoarchitectural changes in spleen. All four tested natural antioxidants (resveratrol, catechin, curcumin and α-tocopherol) reduced flubendiamide and copper-induced cytotoxic effects in rat splenocytes. Rat splenocytes are very sensitive to flubendiamide and copper-induced cytogenotoxicity, therefore, these can be effectively employed for screening of compounds for their cytogenotoxic potential. α-tocopherol was effective in restoring alterations in oxidative stress biomarkers and preventing histoarchitectural lesions in spleen.

Myometrial Calcium and Potassium Channels Play a Pivotal Role in Chromium-Induced Relaxation in Rat Uterus: an In Vitro Study.

Hexavalent chromium, a well-known environmental toxicant, adversely affects female reproduction and results in abnormal implantation, fetal resorption, and reduction in litter size. Uterine myogenic activity is under control of number of receptors and ion channels, and it regulates fetal-implantation and feto-maternal communication. Despite several known adverse effects of chromium on female reproduction, direct action of chromium on myometrial activity is yet to be understood. In the present study, the effect of in vitro exposure of hexavalent chromium (Cr-VI) on the myogenic activity of isolated myometrial strips of rats was evaluated after mounting the tissue in thermostatically (37 ± 0.5 °C) controlled organ bath under a resting tension of 1 g. Chromium produced concentration-dependent (0.1 nM-0.1 mM) inhibitory effect on myometrial activity. Following pre-treatment of the myometrial strips with glibenclamide (a KATP channel blocker) and 4-aminopyridine (a Kv channel blocker), the concentration-response curve (CRC) of chromium was significantly (P < 0.05) shifted towards right with decrease in the maximum relaxant effect. Contractile effects of CaCl2 and BAY K-8644 (a selective opener of L-type Ca2+ channel) were significantly (P < 0.05) attenuated in the presence of chromium. Chromium-induced myometrial relaxation was also significantly (P < 0.05) reduced in the presence of ICI 118,551 (a selective ß2-antagonist) and SR 59230A (a selective ß3-antagonist). These findings evidently suggest that chromium produced relaxant effect on rat myometrium by interfering with Ca2+ entry through voltage-dependent Ca2+ channels, and by interacting with beta-adrenoceptors (ß2 and ß3) and potassium channels (especially KATP and Kv channels). Graphical Abstract Proposed signaling pathway(s) of chromium (VI)-induced myometrial relaxations in rats. KATP: ATP-sensitive K+ channel; KV: voltage-dependent K+ channel; VDCC: voltage-dependent Ca2+ channel; [Ca2+]i: intracellular calcium concentration, stimulatory mechanism, inhibitory mechanism.

Mercury-Induced Inhibition of Tyrosine Phosphorylation of Sperm Proteins and Altered Functional Dynamics of Buck Spermatozoa: an In Vitro Study.

Present study was undertaken on buck spermatozoa to investigate the effect of mercuric chloride on functional dynamics of buck spermatozoa. Four different concentrations (0.031, 0.125, 0.25 and 1.25 µg/mL) of mercuric chloride, which were 1/40th, 1/10th, 1/5th and equivalent to the LC50 value of HgCl2, were selected for studying their effect following in vitro exposure for 15 min and 3 h. Exposure of spermatozoa to 0.031 µg/mL mercuric chloride for 3 h resulted in significant (p < 0.05) decrease in sperm motility, sperm having intact membrane, intact acrosome and high mitochondrial trans-membrane potential. However, following exposure to higher concentrations (0.25, 1.25 µg/mL), similar results were observed even after 15 min of exposure. HgCl2 significantly (p < 0.05) increased the levels of malondialdehyde and reactive oxygen species and significantly (p < 0.05) decreased total antioxidant capacity and superoxide dismutase activity in spermatozoa within 15 min of exposure. Mercuric chloride-treated spermatozoa did not show capacitation, rather exhibited spontaneous acrosome reaction along with significant increase in intracellular Ca2+ and cAMP levels. Immuno-blotting of semen samples of control and 0.031 µg/mL mercury-treated groups showed low intensity bands of p55, p70, p80, p105 and p190 kDa tyrosine phosphorylation proteins while higher concentration-treated groups showed no such bands. Our findings evidently suggest that mercuric chloride even at 0.031 µg/mL adversely affected sperm functions, inhibited tyrosine phosphorylation proteins and capacitation due to oxidative stress. Spontaneous acrosome reaction (AR) in mercury-treated spermatozoa may possibly be due to increase in intracellular Ca2+ and cAMP levels, and capacitation failure may be due to inhibition of tyrosine phosphorylation of proteins.

Functional and molecular characterization of endothelium-dependent and endothelium-independent relaxant pathways in uterine artery of non-pregnant buffaloes.

Present study was undertaken to unravel the endothelium-dependent and endothelium-independent relaxant pathways in uterine artery of non-pregnant buffaloes. Isometric tension of arterial rings was recorded using data acquisition system based polyphysiograph. Acetylcholine (ACh) produced endothelium-dependent vasorelaxation by releasing nitric oxide (NO), and inhibition of nitric oxide synthase (NOS) by L-NAME (300 µM) significantly (P < 0.05) reduced the NO release and thereby the vasorelaxant effect of ACh. However, L-NMMA, another NOS inhibitor, and PTIO, a NO scavenger, did not have any additional inhibitory effect on NO and ACh-induced vasorelaxation. Cyclooxygenase (COX) inhibitor (indomethacin) alone did not have any inhibitory action on vasorelaxant response to ACh; however, simultaneous inhibition of COX and NOS enzymes significantly (P < 0.05) attenuated the relaxant response indicating the concurrent release of these two mediators in regulating ACh-induced relaxation. Besides NOS and COX-derived metabolites (EDRF), small (SKCa) and intermediate (IKCa) conductance K+ channels being the members of EDHF play predominant role in mediating ACh-induced vasorelaxation. Using different molecular tools, existence of eNOS, COX-1, and,IKCa in the endothelium, BKCa in vascular smooth muscle, and SKCa in both endothelium and vascular smooth muscle was demonstrated in buffalo uterine artery. Gene sequencing of COX-1 and SKCa genes in uterine artery of buffaloes showed more than 97% structural similarity with ovine (Ovis aries), caprine (Capra hircus), and Indian cow (Bos indicus). Endothelium-independent nitrovasodilator, sodium nitroprusside (SNP), produced vasorelaxation which was sensitive to blockade by soluble guanylate cyclase (sGC) inhibitor (ODQ), thus suggesting the important role of cGMP/PKG pathways in uterine vasorelaxation in buffaloes. Taken together, it is concluded that both endothelium-dependent (EDHF and EDRF) and endothelium-independent (sGC-cGMP) relaxant pathways are present in uterine arteries of non-pregnant buffaloes, and they differently contribute to vasorelaxation during non-pregnant state.

Oleic Acid Prevents Isoprenaline-Induced Cardiac Injury: Effects on Cellular Oxidative Stress, Inflammation and Histopathological Alterations.

The present study was designed to assess the cardio-protective role of oleic acid in myocardial injury (MI) induced by intra-peritoneal injection of isoprenaline (ISO) in rats for 2 consecutive days. Oleic acid (OA) was administered orally (@ 5 mg/kg b.wt and 10 mg/kg b.wt) for 21 days before inducing MI. Pre-exposure to OA at higher dose significantly improved the HW/BW ratio, myocardial infarct size, lipid profiles (total cholesterol, HDL-C) and cardiac injury biomarkers (LDH, CK-MB, cardiac troponin-I, MMP-9), thus suggesting its cardio-protective role. The ameliorative potential of the higher dose of OA was further substantiated by its ability to reduce the cardiac oxidative stress as evidenced by significant decrease in lipid peroxidation coupled with increase in superoxide dismutase activity and reduced glutathione level. Significant decrease in heart rate as well as increase in RR and QT intervals in oleic acid pre-exposed rats were also observed. OA pre-treatment also reduced the histopathological alterations seen in myocardial injury group rats. The mRNA expression of cardiac UCP-2 gene, a regulator of reactive oxygen species (ROS) generation, was significantly increased in oleic acid pre-exposure group compared to the ISO-induced myocardial injury group. Thus increase in expression of UCP-2 gene in cardiac tissue seems to be one of the protective measures against myocardial injury. Based on the above findings, it may be inferred that oleic acid possesses promising cardio-protective potential against myocardial injury due to its anti-oxidative property and ability to modulate cardiac metabolic processes.

Functional insights into voltage gated proton channel (Hv1) in bull spermatozoa.

Acid extrusion and intracellular alkalisation are the key events during sperm capacitation and these are mediated through proton gated channels (Hv1). Role of Hv1 in regulating sperm motility, capacitation and acrosome reaction has been documented in human spermatozoa; but no such data is available in bull spermatozoa; therefore, the present study was undertaken in Hariana bull spermatozoa. Sixty four ejaculates were collected from four Hariana bulls to investigate the functional involvement of Hv1 in regulation of sperm motility, capacitation and acrosome reaction in bull spermatozoa. Immunoblotting revealed the presence of a single band of 31.8 kDa corresponding to Hv1 in Hariana bull spermatozoa and immunoflourescence confirmed the positive immune-reactivity at principal piece of spermatozoa for Hv1. Functional study was carried out using 200 µM 2-Guanidinobenzimidazole (2-Guanidinobenzimidazole,selective Hv1 blocker) and 1 mM zinc chloride (potent Hv1 blocker), and 0.3 µM Anandamide (AEA), an activator of Hv1. Blocking of Hv1 resulted in significant (P < 0.05) reduction in progressive sperm motility (PSM). Activation of Hv1 with AEA also resulted in significant (P < 0.05) reduction in PSM till 1 h and thereafter, the PSM was restored and reduction was almost similar to that in control. However, during activation and inactivation of Hv1, per cent spermatozoa showing hyperactive motility were found to be markedly increased (20-30%) compared to 10-20% in control. 2-Guanidinobenzimidazole, zinc and AEA treated spermatozoa revealed significant (P < 0.05) increase in B-pattern of spermatozoa indicating induction of capacitation. Downstream signalling of Hv1 activation or inactivation seemed to be mediated through cAMP and PKA pathways. Catsper channels were found to be intimately associated with Hv1 function in regulating sperm motility. Blocking as well as activation of Hv1 resulted in significant (P < 0.05) reduction in sperm livability, spermatozoa having intact membrane, intact acrosome, and high mitochondrial transmembrane potential (MTP). Our findings evidently suggest that Hv1 channels are present in bull spermatozoa and these regulate sperm functions like hypermotility, capacitation and acrosome reaction through complex interplay between different pathways involving cAMP, PKC, and Catsper. Further studies are required to find out the possible relationship between Hv1 channels and other channels in regulating spermatozoa functions.

Molecular and functional insights into Transient Receptor Potential Vanilloid 1 (TRPV1) in bull spermatozoa.

In view of the limited information available on functional significance of TRPV1 in regulating sperm functions, present study was undertaken on bull spermatozoa. Sixty four ejaculates were collected from four Hariana bulls and were used for molecular and functional characterisation of TRPV1. Immunoblotting using TRPV1 specific antibody revealed the presence of a single band of 104 kDa corresponding to TRPV1 in Hariana bull spermatozoa. Indirect immuno fluorescence revealed positive immune-reactivity to TRPV1 at acrosomal, pre-acrosomal, post acrosomal and flagellar regions of spermatozoa. Based on the results of pilot study dose-response analysis, doses of anandamide (AEA; 0.3 µM) and capsazepine (Cp; 10 µM) were selected for further studies. Three groups of semen samples (control 100 µL diluted semen having 1 × 106 spermatozoa; anandamide (3 µL AEA+97 µL of diluted semen containing 1 × 106 spermatozoa and Cp (1 µL Cp+99 µL of diluted semen containing 1 × 106 spermatozoa) were used to study the functional involvement of TRPV1 in bull spermatozoa. Blocking of TRPV1 with Cp resulted in significant (P < 0.05) reduction in progressive sperm motility (PSM) as compared to control. With activation of TRPV1 using AEA also, PSM was significantly (P < 0.05) decreased till 1h and thereafter the PSM was sustained to the level as observed in control. However, both during blocking and activation of TRPV1, per cent spermatozoa showing hyperactive motility were significantly (P < 0.05) increased (20-30%) compared to the control. Treatment with both Cp and AEA revealed significant (P < 0.05) increase in B-pattern of spermatozoa in chlortetracycline hydrochloride (CTC) staining indicating induction of capacitation. Inhibition of soluble adenyl cyclase (sAC) with 99 nM KH7and protein kinase A (PKA) with 3 µM P9115 significantly (P < 0.05) decreased PSM both in the presence of Cp and AEA. Blocking as well as activation of TRPV1 showed significant (P < 0.05) reduction in sperm livability, intact membrane, intact acrosome, high mitochondrial transmembrane potential; hence indicating the involvement of TRPV1 in regulation of sperm functions in bulls. From the study-it was concluded that TRPV1 channels are found in bull spermatozoa and mediate number of sperm functions like motility, hypermotility, capacitation and acrosome reaction. Further studies are required to find out the possible relationship between TRPV1 channels and other channels in regulating spermatozoa function and possible mechanisms associated with TRPV1 activation as well as its role in sperm function regulation.

Endocannabinoid-mediated modulation of Gq protein-coupled receptor mediates vascular hyporeactivity to nor-adrenaline during polymicrobial sepsis.

BACKGROUND: Endocannabinoids level are reported to increase in sepsis, however, the role of vascular cannabinoid receptor-1 (CB1R) in sepsis-induced vascular hyporeactivity is yet to be unravelled. METHODS: Polymicrobial sepsis was induced by caecal ligation and puncture in mice. Isometric tension in isolated aortic rings during early (6 h) and late (20 h) phases of sepsis was recorded and expression of mRNA of monoacylglycerol lipase (MAGL) and cannabinoid receptor-1 (CB1R) was investigated. RESULTS: Sepsis significantly (p < 0.001) reduced the mean survival time in mice along with increase in bacterial load in blood and peritoneal lavage. Compared to Sham-operated (SO) mice, vascular reactivity to nor-adrenaline (NA) was significantly (p < 0.05) attenuated in both early and late phases of sepsis. NA-induced vasoconstriction was significantly (p < 0.05) potentiated by inhibition of diacylglycerol lipase (DAGL) and attenuated by inhibition of MAGL in SO mice. Pre-incubation with KT 109, a DAGL inhibitor, significantly (p < 0.05) improved the vascular hypo-reactivity to NA during both the phases of sepsis. mRNA expression of MAGL in aorta was significantly (p < 0.05) attenuated during both the phases of sepsis. But in the presence of AM 251, specific antagonist of CB1R, vascular reactivity to NA was significantly (p < 0.05) restored along with significant (p < 0.05) increase in mRNA expression of CB1R in aortic rings from both early and late phases of septic mice. CONCLUSION: 2-AG regulates vascular response to NA and increased aortic expression of CB1R is responsible for vascular hyporeactivity to NA in sepsis, and in vitro inhibition of this receptor by AM 251 restored the vascular reactivity.

Molecular characterization of voltage-gated potassium channel (Kv) and its importance in functional dynamics in bull spermatozoa.

Present study was undertaken to characterize the voltage gated potassium channel (Kv 1.1) in bull spermatozoa using sixty four ejaculates collected from four Hariana bulls. Functional characterization was undertaken using a selective blocker of Kv channel, 4-Aminopyridine (4-AP) while molecular presence of Kv on bull spermatozoa by immunoblotting and indirect immunofluorescence. Three sets of 100 µL diluted sperm samples namely-negative control (100 µL of sperm dilution medium (SDM) containing 10 × 106 cells), vehicle control (99 µL of SDM containing 10 × 106 cells, and DMSO- 1  µL) and 4-AP treatment group (99 µL of SDM containing 10 × 106 cells, and drug 1 µL 4-AP) were used in the study. Immunoblotting identified a single band of 56 kDa corresponding to Kv1.1 in Hariana bull spermatozoa. Immunolocalization showed the positive immunoreactivity at head, middle piece and principal piece of the spermatozoa for Kv 1.1. Blocking of Kv using 4-AP resulted in significant (p < 0.05) reduction in sperm progressive motility, per cent capacitated spermatozoa (B-pattern) and acrosome reacted (AR-pattern) spermatozoa, while significant (P < 0.05) increase in per cent swollen spermatozoa. Blocking of Kv channels resulted in significantly (P < 0.05) increased percentage of spermatozoa with lower mitochondrial transmembrane potential. Computer assisted semen analysis (CASA) of motion and kinematic parameters in 4-AP treated spermatozoa indicated reduction in sperm motion parameters like LIN, STR, VSL and VAP and higher ALH, VCL, and BCF indicating hyperactivity of spermatozoa. Based on our findings, it may be concluded that voltage-gated potassium channel (Kv) are present on bull spermatozoa and these are associated with functional dynamics of spermatozoa. However, based on our limited study, it is not possible to deduce that how these channels are associated with induction of hyperactivity. Therefore, further studies are warranted to unravel the mechanistic signaling pathways involved in Kv-mediated alterations in functional dynamics of spermatozoa.

Modulation of voltage-gated sodium channels induces capacitation in bull spermatozoa through phosphorylation of tyrosine containing proteins.

In our previous study, we have reported the molecular presence of Nav 1.8 in bull spermatozoa and its potential involvement in regulation of sperm functions. With the selective blocking of Nav 1.8 using A-803467, alterations in sperm functions were observed, therefore, we envisaged of investigating the involvement of Nav in regulating sperm function and the mechanism(s) involved in it using veratridine, a selective opener of Nav channels. Forty ejaculates were collected from four Hariana bulls and semen samples were pooled in view of the non-significant variations between the different ejaculates. Treatment of sperm cells with veratridine (6, 8, and 10 µM) resulted in concentration- and time-dependent increase in forward progressive sperm motility and it persisted up to 6 h. However, hyperactive motility was induced by veratridine at higher concentrations (8 and 10 µM) and after 2 h of incubation, which was confirmed by subjective assessment followed by chlortetracycline staining showing the increased B-pattern spermatozoa, and thereby suggesting the involvement of Nav in regulation of capacitation in spermatozoa. To substantiate the functional study observations especially veratridine-induced capacitation, immunoblotting and indirect immune fluorescence assays were performed for detection of the tyrosine-phosphorylated proteins. The immune blot study revealed the presence of five tyrosine phosphorylated proteins, namely-p17, p30, p54, p90 and p100. The p17 protein showed the highest band intensity compared to other protein bands indicating its potential involvement in the process of capacitation. Immunolocalization study revealed positive immunoreactivity for tyrosine phosphorylated proteins in the middle piece, post acrosomal region (high fluorescence) and tail of the spermatozoa (low fluorescence). From the results of present study, it is evident that activation of NaV by veratridine, especially at higher concentrations, induced capacitation which is evidently mediated through phosphorylation of the tyrosine containing proteins localized in the post acrosomal regions, middle piece and tail of the spermatozoa. However, further studies will help in unraveling the involvement of Nav and other ion channels regulating different physiological functions of sperm.

ATP-sensitive and maxi potassium channels regulate BRL 37344-induced tocolysis in buffaloes-an in vitro study.

Cellular coupling of beta3-adrenoceptors (ß3-ADR) to potassium channels in myometrium is largely unknown. In vitro study was undertaken to unravel the presence of ß3-adrenergic receptors (ADR) and the role of K+-channels in mediating ß3-ADR-induced relaxation in isolated myometrial strips from cyclic non-pregnant water buffaloes. Isometric tension was recorded in isolated myometrial strips using data acquisition system based physiograph. Compared to SR 59230A, BRL 37344 was found to be more potent in inducing ß3-dependent myometrial relaxation which was significantly (p < 0.05) inhibited in the presence of ß3 antagonist, SAR 150640. The immunoreactive protein to ß3-ADR was also detected in membrane fraction of myometrial protein. Further, incubation with BRL 37344 (10 µM) significantly (p < 0.05) increased c-AMP accumulation (37.58 ± 9.52 pmol/mg protein; n = 4) in the myometrial strips compared to basal c-AMP level (16.85 ± 3.87 pmol/mg protein; n = 4). The concentration response curves (CRC) of BRL 37344 were significantly (p < 0.05) shifted towards right in the presence of KATP channels specific blocker, glibenclamide (10 µM) and maxi K+-channels (BKCa) specific blocker, iberiotoxin (100 nM), with decrease in both efficacy and potency as compared to control. However, 4-aminopyridine (4-AP), a specific blocker of the voltage gated K+-channels (Kv), failed to alter the CRC of BRL 37344. Existence of immunoreactive protein to Kir6.1, α-subunit of BKCa and Kv1.1 channels were also detected in the membrane fraction of myometrial protein. Based on the above findings, it can be concluded that BRL 37344 is a potent stimulator of ß3-adrenoceptors in buffalo myometrium and besides mediating their effect through rise in c-AMP, they are coupled to KATP and BKCa channels in inducing tocolytic effects.

The functional and molecular studies on involvement of hydrogen sulphide in myometrial activity of non-pregnant buffaloes (Bubalus bubalis).

BACKGROUND: Hydrogen sulphide (H2S), a member of the gasotransmitters family, is known to play patho-physiological role in different body systems including during pregnancy. But its involvement in myometrial spontaneity and associated signalling pathways in uterus in non-pregnant animals is yet to be studied. Present study describes the effect of L-cysteine, an endogenous H2S donor, on isolated myometrial strips of non-pregnant buffaloes and the underlying signaling mechanism(s). RESULTS: L-cysteine (10 nM-30 mM) produced concentration-dependent contractile effect on buffalo myometrium which was extracellular Ca2+ and L-type calcium channels-dependent. Significant rightward shift of dose-response curve of L-cysteine was observed with significant decrease in maxima in the presence of amino-oxyacetic acid (AOAA; 100 µM) and d, l-propargylglycine (PAG; 100 µM), the specific blockers of cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE), respectively. Existence of CBS enzyme of 63 kDa and CSE of 45 kDa molecular weights was confirmed by western blot using specific antibodies and also by immunohistochemistry. CONCLUSIONS: Endogenous H2S along with its biosynthetic enzymes (CBS and CSE) is evidently present in uteri of non-pregnant buffaloes and it regulates spontaneity in uteri of non-pregnant buffaloes and this effect is dependent on extracellular Ca2+ influx through nifedipine-sensitive L-type calcium channels. Thus H2S-signalling pathway may be a potential target to alter the uterine activities in physiology and patho-physiolgical states.

WITHDRAWN: Differential involvement of L- and T-type Ca2+ channels, store-operated calcium channel (TRPC) and Rho-kinase signaling pathway(s) in PGF2α-induced contractions in myometrium of non-pregnant and pregnant buffaloes (Bubalus bubalis).

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Extra and intracellular calcium signaling pathway(s) differentially regulate histamine-induced myometrial contractions during early and mid-pregnancy stages in buffaloes (Bubalus bubalis).

This study examines the differential role of calcium signaling pathway(s) in histamine-induced uterotonic action during early and mid-pregnancy stages in buffaloes. Compared to mid pregnancy, tonic contraction, amplitude and mean-integral tension were significantly increased by histamine to produce myometrial contraction during early pregnancy with small effects on phasic contraction and frequency. Although uterotonic action of histamine during both stages of pregnancy is sensitive to nifedipine (a L-type Ca2+ channels blocker) and NNC55-0396 (T-type Ca2+ channels blocker), the role of extracellular calcium seems to be more significant during mid-pregnancy as in this stage histamine produced only 9.38±0.96% contraction in Ca2+ free-RLS compared to 21.60±1.45% in uteri of early pregnancy stage. Intracellular calcium plays major role in histamine-induced myometrial contraction during early pregnancy as compared to mid pregnancy, as in the presence of cyclopiazonic acid (CPA) Ca2+-free RLS, histamine produced significantly higher contraction in myometrial strips of early-pregancy in comparison to mid-pregnancy (10.59±1.58% and 3.13±0.46%, respectively). In the presence of U-73122, the DRC of histamine was significantly shifted towards right with decrease in maximal effect (Emax) only in early pregnancy suggesting the predominant role of phospholipase-C (PL-C) in this stage of pregnancy.

Functional involvement of protein kinase C, Rho-kinase and TRPC3 decreases while PLC increases with advancement of pregnancy in mediating oxytocin-induced myometrial contractions in water buffaloes (Bubalus bubalis).

Present study unravels the involvement of different calcium signaling pathways in oxytocin-induced contractions in myometrium of non-pregnant and pregnant buffaloes during early and mid-pregnancy stages. Uteri of pregnant animals were more sensitive than of non-pregnant buffaloes. Phasic contractions and frequency of contraction significantly increased with advancement of pregnancy, while tonic contractions non-significantly and amplitude significantly decreased from six months pregnancy onward. Oxytocin produced concentration-dependent-contraction on isolated myometrial strips of pregnant and non-pregnant buffaloes and the dose response curves (DRCs) of oxytocin were significantly (P < 0.05) shifted to right in the presence of nifedipine (1 µM), in Ca2+-free Ringer Locke solution (RLS), ruthenium red (30 µM), ruthenium red + nifedipine, cyclopiazonic acid (CPA; Ca2+ free RLS as well as RLS), CPA (10 µM)+nifedipine, U-73122 (1 µM) + nifedipine and SKF96365 (25 µM) on uteri of non-pregnant and pregnant (early and mid) animals. The DRCs were also significantly shifted towards right in the presence of Y-27632 (10 µM), GF109203X (5 µM) and Pyr3 (10 µM) on uteri of non-pregnant and early pregnancy stage buffaloes while only in the presence of U-73122 (1 µM) on uteri of mid-pregnancy stage buffaloes. Our finding suggest that and L-type Ca2+ channels, IP3-RyR-gated, and store-operated calcium channels including transient receptor potential channel (TRPC) pathways play significant role in mediating oxytocin-induced contractions in myometrium of pregnant and non-pregnant buffaloes. SERCA plays major role only during early-pregnancy while functional role of protein kinase C (PKC), Rho-kinase and TRPC3 pathways decreased and role of G-protein coupled receptor-phospholipase C (GPCR-PLC) pathway increased with advancement of pregnancy.

Functional and molecular characterization of voltage gated sodium channel Nav 1.8 in bull spermatozoa.

The aim of our study was to characterize the voltage gated sodium channel Nav 1.8 in bull spermatozoa. Forty ejaculates were collected from four Hariana bulls and semen samples were pooled in view of the nonsignificant variations between different ejaculates. Functional characterization was undertaken using A-803467, a selective blocker of Nav1.8, and veratridine as an opener of the voltage gated sodium channels while molecular characterization was done using western blotting and indirect immunofluorescence assays. In vitro capacitation was induced using heparin, and to study the functional involvement of Nav 1.8 in regulation of capacitation induced hyper sperm motility, A-803467 was used. Selective blocking of NaV 1.8 by A-803467 at 6 and 8 µM concentration significantly (P < 0.05) decreased the forward progressive sperm motility in a time-dependent manner, while, blocking at higher concentrations (10 and 15 µM) resulted in fast forward motility in spermatozoa after 2 h of incubation and it was observed up to 3 h. Treatment of sperm cells with veratridine (6, 8, 10, 15, 20 µM) resulted in concentration- and time-dependent increase in forward progressive sperm motility and it persisted up to 4 h. However, hyperactive motility was induced by veratridine at higher concentrations (10 and 15 µM) after 2 h of incubation. In vitro capacitated spermatozoa treated with A-803467 revealed significant (P < 0.05) reduction in forward progressive motility after 2 h of incubation. Both A-803467 and veratridine altered the percentage of spermatozoa showing high mitochondrial transmembrane potential in concentration- and time-dependent manner. High concentrations (10 and 15 µM) of A-803467 and veratridine resulted in bent neck condition in spermatozoa along with significant (P < 0.05) reduction in membrane integrity (HOST negative). Immunoblot revealed the presence of a single protein band of 260 kDa molecular weight along with positive immunoreactivity (IR) in head, neck, middle piece and tail of the spermatozoa. Strongest IR was observed in the neck and middle piece whereas weak IR was observed in tail and acrosomal region of the spermatozoa. Results of our present study evidently revealed the presence of voltage gated sodium channel Nav1.8 in bull spermatozoa and its functional involvement in regulation of spermatozoa dynamics in terms of motility, membrane integrity, acrosome integrity, capacitation and mitochondrial transmembrane potential. Further studies are warranted to unravel their mechanistic pathways and/or their interaction with other ion channels in regulating sperm dynamics.