Melatonin regulates nitric oxide synthase expression in ischemic brain injury

Nitric oxide (NO) is produced by three NO synthases (NOS), iNOS, eNOS, and nNOS. Production of NO by iNOS plays key roles in neurodegeneration, while eNOS is a protective enzyme. This study investigated the neuroprotective effect of melatonin and the levels of NOS isoforms induced by melatonin in ischemic brain injury. Adult male rats were treated with melatonin (5 mg/kg) or vehicle prior to middle cerebral artery occlusion (MCAO). Brain samples were collected at 24 hr after the onset of occlusion. Results confirmed that melatonin significantly reduces infarct area. Western blot analysis was used to evaluate the expression levels of iNOS, eNOS, and nNOS. The level of iNOS and nNOS increased in vehicle-treated animals, while melatonin prevented injury-induced increase of iNOS. In contrast to iNOS levels, eNOS levels decreased in vehicle-treated animals, while melatonin prevented the injury-induced decrease of eNOS. This study provides further evidence that melatonin exerts neuroprotective effects, and the regulation of NOS isoforms by melatonin may contribute to the neuroprotective effects.     

Involvement of neuronal nitric oxide synthase (nNOS) in the regulation of migrating motor complex (MMC) in sheep

The objectives of this study were to evaluate the role of nitric oxide (NO) synthase isoforms (nNOS, eNOS, and iNOS) in the regulation of the migrating motor complex (MMC) in sheep using electromyography and their expression in the gastrointestinal (GI) tract by Western blot (WB) and immunohistochemistry. Intravenous administration of L-NAME or the nNOS inhibitor 7-nitroindazole (7-NI) decreased the MMC interval. Myoelectric activity of intestinal phase II was increased, whereas antral activity was reduced. These effects were blocked by L-arginine. Inhibitors of either iNOS (aminoguanidine and S-methylisothiourea) or eNOS (L-NIO) were ineffective. The NO donor sodium nitroprusside decreased GI myoelectric activity, inhibited the MMC pattern, and prevented the effects induced by L-NAME and 7-NI in the intestine. Intracerebroventricular administration of these agents did not modify GI motility. In the rumen, abomasal antrum, duodenum, and jejunum, WB showed three bands at about 155, 145, and 135kDa corresponding to nNOS, and a 140-kDa band (eNOS); however iNOS was not detected. Positive nNOS immunostaining was observed in neurons of the myenteric and submucous plexus of all GI tissues, while eNOS was found in the endothelial cells, ruminal and intestinal epithelium, as well as in some enteric neurons and in endocrine-like cells of the duodenal Brunner's glands. In contrast, only weak iNOS immunoreactivity was found in ruminal epithelium. Taken together, our results suggest that NO, synthesized at a peripheral level by nNOS, is tonically inhibiting the MMC pattern and intestinal motility in sheep.     

Pseudorabies virus induces a rapid up-regulation of nitric oxide synthases in the nervous system of swine

Nitric oxide (NO) is a free radical gas with important roles in the host's immune response against viral infections. In this study, we examined the kinetics and distribution of nitric oxide synthase (NOS) expression during the early steps of infection of the porcine nervous system by the alphaherpesvirus pseudorabies virus (PRV). To this end, we examined changes in the expression of the three major NOS isoforms, neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS), by immunohistochemistry in the trigeminal ganglia and brain of pigs inoculated intranasally with a virulent PRV strain. The results obtained show that infection of the porcine nervous system by PRV induced a rapid and progressive increment in NOS expression that coincided in timing, location, and magnitude with those of virus propagation in the nervous tissue. A major finding of this study was that PRV caused not only nNOS and iNOS induction in a variety of cell types, but also eNOS up-regulation in endothelial cells and neurons; therefore, all possible sources of NO are activated and probably contribute to the overproduction of NO during infection with the neurotropic alphaherpesvirus PRV in its natural host.     

Purification and localization of nitric oxide synthases from hybrid tilapia (Nile tilapia x Mozambique tilapia)

The aims of this study were to purify and localize the nitric oxide synthases (NOSs) from hybrid tilapia (Nile tilapia Oreochromis niloticus x Mozambique tilapia O. mossambicus). The purification procedures involved affinity chromatography with a 2', 5'-ADP-agarose 4B column and ion exchange with a diethylaminoethanol Bio-Gel A column. The results from gel filtration assays showed that the molecular weights of neuronal NOS (nNOS) and inducible NOS (iNOS) were 178 and 120 kDa, respectively. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis results showed that there were three bands with molecular weights of 89, 47, and 29 kDa from the purified nNOS. However, only one band, with a molecular weight of 120 kDa, appeared on the gel from the purified iNOS. Hybrid tilapia nNOS was a dimer structure, while iNOS appeared to be a monomer structure. Moreover, our results revealed that the activities of nNOS and iNOS were significantly higher after the addition of Ca+2 or Mg+2 ions individually. However, when L-arginine and NADPH were present, the addition of 1 mM of either ion did not further increase the activity. The chemical L-N(G)-methyl-L-arginine could inhibit the activities of the purified NOSs with or without L-arginine. Western blot analyses showed only an 89-kDa immunoreactive band from the extracts of cerebrum; however, we did not find the specific bands in other tissues, such as gill, intestine, liver, spleen, and anterior kidney. We found another 120-kDa immunoreactive protein band with the rabbit antirat iNOS serum against iNOS from the extracts of anterior kidney and spleen. The results of immunohistochemistry with the rabbit antihuman nNOS serum indicated that the nNOS existed in the cerebellum, olfactory bulb, diencephalons, and nerve cell bodies and neuronal fibers of the spinal cord. Interestingly, only macrophages from anterior kidney and spleen showed positive reactions with the rabbit antirat iNOS serum. In the same way, the endothelial NOS (eNOS) located in the heart and epithelial cells of the blood vessels reacted positively with the rabbit antibovine eNOS serum.     

Increased expression of both constitutive and inducible forms of nitric oxide synthase in the delayed phase of acute experimental testicular torsion

To elucidate the roles of both constitutive endothelial nitric oxide synthase (eNOS) and neuronal NOS (nNOS), and inducible NOS (iNOS) in acute experimental testicular torsion, the expression of iNOS and constitutive eNOS and nNOS were studied in the rat testis with ischemia/reperfusion (I/R) injury. Western blot analysis showed that all three isoforms of NOS increased significantly at 24-48 hr after I/R and declined slightly thereafter. After I/R, immunoreactivity for both iNOS and nNOS was detected, mainly in the interstitial space around damaged tubules, while germ cells in the damaged tubules were immunostained intensely for eNOS. We postulate that increased expression of the three NOS isoforms in the testis after I/R, which might generate nitric oxide, affects delayed germ cell death following I/R via paracrine or autocrine fashion.     

Endometrial nitric oxide production and nitric oxide synthases in the equine endometrium: Relationship with microvascular density during the estrous cycle

Nitric oxide (NO) plays an important role in angiogenesis and in the regulation of the blood flow. This study was carried out to investigate (i) the effects of endogenous estrogens and progestins and exogenous progesterone (P₄) (5 ng/ml or 1 μg/ml) or estradiol 17β (E₂β) (50 pg/ml or 1 μg/ml) on in vitro endometrial NO synthesis; (ii) the presence of different isoforms of NO synthase; (iii) and their relationship to microvascular density in the equine endometrium during the estrous cycle. NOS expression was also evaluated in the myometrium. Expression of endothelial and inducible forms of NOS in the uterus was assessed by Western blot and immunocytochemistry. Vascular density in endometrial tissue was determined on histologic sections. In the luteal phase, compared to the follicular phase, endometrial NO production increased without exogenous hormones and with exogenous E₂β (1 μg/ml). Although immunocytochemistry revealed iNOS and eNOS expression in the endometrium, no positive signal for iNOS was detected by Western blot. Endothelial NOS was observed in endometrial glands, endothelial cells, fibroblasts, blood and lymphatic vessels. Endometrial eNOS expression was the highest in the follicular and mid-luteal phases while it was found to be the lowest in the early luteal phase. In the follicular phase, hyperplasia of endometrial tissue with respect to myometrium was detected. No difference in vascular density was present between phases. All together, NO may play some roles in both proliferative and secretory phases of endometrial development in the mare.     

Expression Patterns of Endothelial and Inducible Nitric Oxide Isoforms in the Porcine Umbilical Cord

Stable fetal–placental blood pressure and flow are extremely important in fetal growth and development. Uncontrolled and long-standing increased or decreased vascular blood pressure in the umbilical cord (UC) affects hyperaemia or ischaemia and consequently causes fetal death. Nitric oxide (NO) is one of the most active factors controlling blood flow through relaxation of the vascular smooth muscle. In this study, we investigated endothelial (eNOS) and inducible (iNOS) nitric oxide synthase expression and NADPH-diaphorase activity (NADPH-d) in the porcine UC at various stages of pregnancy. The UCs were collected from pigs on days 40, 60, 75 and 90 of pregnancy and postpartum. Western blot analysis as well as immunohistchemical staining revealed protein presence for eNOS and iNOS in the UC of the pig. The eNOS expression was maintained at a significantly higher level in all analysed days of pregnancy when compared with postnatal stage. Additionally, a significant protein increase for eNOS was observed in a periplacental part of UC on day 90. There were no obvious differences in iNOS protein level in UC samples derived from different stages of pregnancy. NADPH-diaphorase histochemical activity was correlated with NOS immunoreactivity during all analysed days of pregnancy. These results suggest that NOS isoforms are responsible for regulation of blood circulation in UC and immune responses.     

Localization of eNOS in the olfactory epithelium of the rat

Nitric oxide (NO) is a free radical and produced from L-arginine by nitric oxide synthase (NOS). Since NO is recently suggested to be involved in olfactory perception, the expression of eNOS, an isoform of NOS, was examined in the rat olfactory epithelium. The activity of NADPH-diaphorase was also examined as a marker of NOS. In the dorsomedial region of the nasal cavity, intensely positive reactions for NADPH-diaphorase were observed in the entire cytoplasm of sensory cells (olfactory cells). By immunohistochemistry, intensely positive reactions for eNOS were also found in the dorsomedial region of the nasal cavity. These reactions were observed on the free border of the olfactory epithelium. By immunoelectron microscopy, positive reactions for eNOS were found in the cilia of olfactory cells. In addition, in situ hybridization analysis of the olfactory epithelium revealed the expression of eNOS mRNA in the olfactory cells. These results indicate the presence of eNOS in the olfactory cells of the rat, and differential expression of eNOS in the olfactory epithelium depending on the regions of the nasal cavity. In addition, NO produced by eNOS may be involved in olfactory perception in the cilia of olfactory cells.     

Expression of nitric oxide synthase isoforms in the testes of pigs

This study examined the expression of three isoforms of nitric oxide synthase (NOS) in the testes of pigs. Immunohistochemical studies demonstrated the presence of nNOS, eNOS and iNOS in interstitial cells, primary spermatocytes and spermatids. Positive immunoreactions for eNOS and iNOS were detected in peritubular myoid cells. Some vascular endothelial cells were positive for nNOS and eNOS. The expression of nitrotyrosine was detected in interstitial cells. In addition, the histochemical study revealed that all the interstitial cells were stained positively for NADPH-diaphorase, although some spermatids and vascular endothelial cells displayed moderate enzymatic activity. These findings suggest that three isoforms of NOS are expressed in the testis of pig and that they play important roles in the biology of interstitial cells that produce testosterone, as well as in spermatogenesis in the seminiferous tubules.     

Presence of nitric oxide synthase immunoreactivity and mRNA in buffalo (Bubalus bubalis) oocytes and embryos

This study examined the presence of immunoreactivity and mRNA for different nitric oxide synthase (NOS) isoforms in immature and in vitro matured oocytes and in embryos at two‐, four‐ and eight‐cell, and morula and blastocyst stages in buffalo. Oocytes obtained from slaughterhouse buffalo ovaries were subjected to in vitro maturation in TCM‐199 + 10% FBS + 5 μg/ml pFSH + 1 μg/ml estradiol‐17β + 0.81 mm sodium pyruvate + 10% buffalo follicular fluid + 50 μg/ml gentamycin sulphate for 24 h in a CO₂ incubator (5% CO₂ in air) at 38.5°C. Following in vitro fertilization carried out by incubating them with 2–4 million spermatozoa/ml for 18 h, the presumed zygotes were cultured in mCR2aa medium containing 0.6% BSA and 10% FBS for up to 8 days post insemination. Immunofluorescence staining of NOS using antibodies that cross‐reacted either with all the NOS isoforms i.e., universal (uNOS) or specifically with inducible (iNOS) or endothelial (eNOS) isoforms revealed that NOS was present in oocytes and embryos at all the stages examined. Examination of the semi‐quantitative expression of NOS genes by RT‐PCR revealed that the iNOS, eNOS and nNOS mRNA was present in the immature and mature oocytes and in all the embryonic stages examined. In conclusion, it was demonstrated in the present study that immunoreactivity and mRNA for different NOS isoforms was present in buffalo oocytes and pre‐implantation stage embryos.     

一氧化氮与卵母细胞发育

一氧化氮(NO)是一种在生物体内具有多种生物学效应的气体自由基。论文根据国内外研究资料,介绍了NO的发现、生物学特性、作用途径,着重阐述了NO在卵巢组织中的分布以及NO与卵母细胞的发生、成熟、排卵和卵泡细胞凋亡与卵泡闭锁的关系。     

Expression and localization of NO synthase isoenzymes (iNOS and eNOS) in development of the rabbit placenta

Nitric oxide synthase (NOS) is a key regulator of angiogenesis and embryogenesis in the mammalian reproductive process. Here, we attempted to clarify the expression and localization of inducible and endothelial NOS (iNOS and eNOS) in the developing rabbit placenta. Real-time RT-PCR analysis indicated that iNOS mRNA was significantly upregulated till the complete development of the placenta (d18), and then significantly decreased at the end of fetal growth stage (d28) during successful pregnancy. The eNOS mRNA was also enhanced in the pregnant uteri and gradually decreased near the term of pregnancy. Western blot analysis also showed elevation of the iNOS and eNOS protein levels during the course of successful pregnancy till the functional maturation of the placenta (d18). Immunohistochemical study revealed distinct localizations of iNOS along the radial arteries and eNOS at the spiral arteries and arterial sinuses in the developing placenta. This may reflect that iNOS and eNOS participate in pregnancy success through placentation-specific vascular formation and by supporting adequate blood circulation in the rabbit placenta.     

Expression of constitutive endothelial, neuronal and inducible nitric oxide synthase in the testis and epididymis of horse

The expression of three isoforms of nitric oxide synthase (NOS) were examined in the testis and epididymis of a thoroughbred horse. Immunohistochemical studies demonstrated the presence of eNOS immunostaining in some germ cells in the seminiferous tubules and in vascular endothelial cells in the interstitial tissues. Interstitial cells, most likely Leydig cells, were also intensely immunopositive for eNOS. The pattern of immunostaining for nNOS was similar to that for eNOS in the testis. Weak expression of iNOS was detected in the seminiferous tubules of the testis, but intense expression was found in interstitial cells. Inducible NOS was also strongly detected in stereocilia, sperm, epithelium and connective tissue of the epididymis of normal horses. These findings suggest that three isoforms of NOS are expressed in the testis and epididymis of horse and that they play important roles in the biology of interstitial cells that produce testosterone, as well as in spermatogenesis in the seminiferous tubules.     

Direct measurements of nitric oxide release in relation to expression of endothelial nitric oxide synthase in isolated porcine mitral valves

The aim of this study was to measure the direct release of nitric oxide (NO) from the porcine mitral valve using a NO microelectrode. Furthermore, the expression and localization of endothelial nitric oxide synthase (eNOS) in the mitral valve was studied using immunohistochemistry, Western blotting and RT-PCR. Results show that bradykinin increases NO release from mitral valves (DeltaBradykinin: 33.71 +/- 10.41 nm NO, P < 0.001, n = 10), whereas N-nitro-l-arginine methyl esther (l-NAME) decreases NO release when compared with basal level (Deltal-NAME: 82.69 +/- 15.66 nm NO, P < 0.005, n = 4). Both protein and mRNA expression of eNOS in mitral valves and in isolated valvular endothelial cells suggest that the NO release is mainly associated with the mitral valve endothelium. It is concluded that direct NO release from porcine mitral valves coincides with eNOS expression. This study documents useful techniques for investigations into the role of local NO release in mitral valve diseases.     

Detection and localization of regucalcin in spermatozoa of water buffalo (Bubalus bubalis): A calcium‐regulating multifunctional protein

Regucalcin (RGN) is a calcium‐regulating, anti‐apoptotic, antioxidative and antiproliferative multifunctional protein predominantly seen in liver and kidney. All these functions are very crucial during spermatogenesis and sperm maturation process until fertilization of the ovum. Although many studies have reported the wide distribution of regucalcin in the male reproductive tract of the rat, human and bovine, its presence in spermatozoa is yet to be demonstrated wherein calcium has a pivotal role in the transport, capacitation, acrosomal reaction and further fusion with ova. Here, we detected the expression of regucalcin mRNA and protein in buffalo spermatozoa using real‐time PCR and Western blot, respectively. The study detected two new regucalcin isoforms of 44 kDa and 48 kDa size along with the reported 34‐kDa, 28‐kDa and 24‐kDa isoforms, wherein the 34‐kDa isoform was found to be membrane associated in spermatozoa. Further, immunocytochemistry study localized the regucalcin protein in the acrosomal region of the caudal and ejaculated buffalo spermatozoa while it was detected in both cytoplasm and acrosomal region of testicular spermatozoa. This discovery of RGN in spermatozoa and localization in the acrosomal region will help to focus researchers to see its role in calcium‐related functions like capacitation, acrosomal reaction and membrane fusion. Overall, regucalcin may be a new fertility marker in buffalo and can be utilized for infertility treatments.     

Localization of CD9 Molecule on Bull Spermatozoa: Its Involvement in the Sperm–Egg Interaction

Tetraspanin CD9 is one of the egg membrane proteins known to be essential in fertilization process. The presence and localization of CD9 molecule in spermatozoa and its possible function in reproduction are still unclear. In our study, we describe the localization of CD9 on bull spermatozoa. In the immunofluorescence assay, the positive signal has been observed in the high proportion of sperm cells as a fine grains either on the apical part or through the entire anterior region of sperm head. CD9 recognized by monoclonal antibody IVA‐50 was detected on freshly ejaculated (83.4 ± 3.7%) and frozen‐thawed (84.3 ± 2.3%) sperm. The same reaction pattern was observed on sperm capacitated for 1 h, 2 h, 3 h and 4 h (83.6 ± 2.0%; 84.0 ± 1.5%; 85.7 ± 0.8%; 77.5 ± 10.8%). The presence of CD9 exclusively on plasma membrane of the bovine sperm has been detected by Western blot analysis of the protein fractions after the discontinuous sucrose gradient fractionation of the bull sperm. Moreover, probable role of the sperm CD9 molecule in fertilization process of cattle has been suggested as sperm treatment with anti‐CD9 antibody significantly reduced (by 25%, p ≤ 0.001) the number of fertilized oocytes compared to control group in fertilization assay in vitro.     

Failure to detect bovine immunodeficiency virus contamination of stud bull spermatozoa, blood leukocytes, or semen leukocytes in samples supplied by artificial insemination centers

OBJECTIVE: To determine whether bovine immunodeficiency virus (BIV) infection could be detected in spermatozoa, blood leukocytes, or semen leukocytes from stud bulls in artificial insemination centers. ANIMALS: 30 bulls at 3 artificial insemination centers. PROCEDURE: Polymerase chain reaction testing that used 3 sets of primer pairs targeting pol and env regions of the BIV proviral genome was performed on DNA extracted from semen leukocytes, spermatozoa, and blood leukocytes from each bull. Southern blot analysis was performed to increase sensitivity of detection. Western blot analysis of plasma samples was used to detect antibodies against BIV. RESULTS: BIV provirus was not detected in DNA samples obtained from semen leukocytes, spermatozoa, or blood leukocytes, and antibodies against BIV were not detected. CONCLUSIONS AND CLINICAL RELEVANCE: Contrary to our report of high point prevalence of BIV contamination of semen from a single artificial insemination center, bulls of the study reported here did not appear to be infected. Maximum risk of BIV infection in similar bulls was estimated at 10% with a confidence level of 95%.