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.     

Nitric oxide synthase expression in foetal placentas of cows with retained fetal membranes

The objectives of this study were to investigate relationship of retained fetal membranes (RFM) to expression of NOS and NOS mRNA and to analyze pathohistological changes and the distribution of nitric oxide synthase (NOS) in foetal placentas of cows with RFM. Twenty cows were assigned to two groups, a control group (no retained fetal membranes, NRFM, n = 10) and a diseased group (RFM, n = 10). The endpoint method was used to detect the nitric oxide (NO) content and nitric oxide synthase (NOS) activity in foetal placental tissue fluid and the fluorescent quantitation PCR was used to measure the expression of NOS mRNA. Immunohistochemistry and hematoxylin-eosin staining were used to observe pathohistological changes. Tissue from RFM cows showed fibronecrosis of the chorionic villi, and a decreased number of trophoblastic cells. The majority of trophoblastic cells displayed vacuolar degeneration. Interstitium vessels were distended and congested. Expression of induced nitric oxide synthase (iNOS) protein and iNOS mRNA was significantly higher (P < 0.05) in the cytoplasm of placental villus trophoblastic cells in the RFM group. But expression of endothelial nitric oxide synthase (eNOS) protein and eNOS mRNA was significantly lower (P<0.05) in the RFM group. The NO content and NOS activity of cows with RFM were significantly higher (P < 0.05). A high expression of iNOS protein and iNOS mRNA in the cow foetal placenta could produce high content of NO, which might inhibit uterine contraction. So over expression of iNOS protein and iNOS mRNA might be an important agent of retained fetal membranes in cows, and it may be a potential diagnosis biomarker.     

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.     

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.     

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.     

一氧化氮合酶在猪早期胚胎发育中表达的初步研究

本研究运用分子生物学手段探究了一氧化氮(NO)在猪早期胚胎发育过程中的表达情况及其相关的规律。应用RT-PCR方法将猪各发育阶段的早期胚胎的内皮型一氧化氮合酶(eNOS)和诱导型一氧化氮合酶(iNOS）进行检测,然后对产物进行半定量分析。结果表明,iNOS在猪早期胚胎发育的整个过程中都有表达,其相对表达量随着胚胎发育进程呈现上升的趋势,在桑葚/囊胚阶段达到最高;eNOS仅仅是在2-细胞期和4-细胞期有表达,其相对表达量在2～4-细胞期间的差异不明显;在猪2～4-细胞胚胎发育过程中,iNOS的相对表达量高于eNOS的相对表达量。结果表明,在猪早期胚胎发育中NO的产量主要由iNOS调节。     

Expression of the Vascular Endothelial Growth Factor (VEGF‐A) and its Receptors in the Umbilical Cord in the Course of Pregnancy in the Pig

The umbilical cord (UC) and the placenta are important organs through which respiratory gases, nutrients, wastes and biologically active substances are exchanged between the maternal and the foetal system. A rapid placental vascularization observed in the second half of pig pregnancy is positively correlated with the mRNA expression of the vascular endothelial growth factor (VEGF). Based on these findings, we hypothesized that VEGF may have a stimulatory effect in the dynamically growing UC. To further understand the role of the VEGF–VEGFR system during UC development, mRNA and protein expression as well as the cellular localization of VEGF‐A, VEGFR‐1 and VEGFR‐2 in UC were examined on days 40, 60, 75 and 90 of pregnancy and after physiological delivery in the pig (day 114 of pregnancy). Real Time RT‐PCR analysis showed an increase in the mRNA levels of VEGF120 and VEGF164 from day 90 of pregnancy. VEGFR‐1 mRNA expression was significantly increased on day 75 of pregnancy. No significant changes in VEGFR‐2 mRNA expression were detected. In turn, western blot analysis revealed an increase in VEGF‐A protein expression on day 40, compared to the later days of pregnancy. A rapid increase in the VEGFR‐1 protein level was noted on day 75 and 90 of gestation. No significant changes in VEGFR‐2 protein expression were detected on any of the analysed days of pregnancy. Immunohistochemical staining enabled detection of VEGF–VEGFR system, in endothelial and tunica media cells of the umbilical vessels and in allantoic duct and amniotic epithelium on all analysed days of pregnancy. Positive reactions for VEGF‐A and VEGFR‐1, but not VEGFR‐2, were also observed in myofibroblasts. In conclusion, this data shows that members of the VEGF–VEGFR system are temporally and spatially well localized for playing key roles during umbilical cord formation and its intensive growth observed after day 75 of pregnancy.     

Immunohistochemical localization of endothelial and inducible nitric oxide synthase within neurons of cattle with rabies

The expression of constitutive endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) in the brains of cattle with natural rabies was studied. Increased expression of eNOS was detected in neurons of the brain stem and Purkinje cells of cerebellum. By contrast, iNOS was diffusely localized in the cytoplasm of affected neurons, and some inflammatory cells were positive. eNOS and rabies antigen were co-localized in inclusion bodies (Negri bodies) in neurons. The specific localization of eNOS, but not iNOS, in the Negri bodies suggests that eNOS is involved in the formation of rabies virus inclusion bodies.     

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 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.     

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.     

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.     

Expression of NOS and VEGF in feline mammary tumours and their correlation with angiogenesis

In order to define the role of nitric oxide (NO) in feline mammary tumours, the expression of endothelial or inducible nitric oxide synthase (e/iNOS) and vascular endothelial growth factor (VEGF), and their relationship with angiogenesis, was investigated in 23 feline mammary tumours (two hyperplastic, 19 adenocarcinoma, one osteosarcoma and one squamous cell carcinoma) by immunohistochemistry. Tumour angiogenesis was assessed by CD31 immunostaining and was expressed as microvessel density (MVD). In general, iNOS immunoreactivity was localised in tumour cells and occasionally in stromal myofibroblasts, whereas eNOS and VEGF were localised in the cytoplasm of tumour epithelial cells and endothelium. In malignancy, expression of iNOS increased from well- to less-differentiated phenotypes (Grades 1-3) and was significantly higher in G3 and G2 when compared with G1 cases. However, increasing eNOS expression was limited only in hyperplastic lesions and showed no significant changes among the grades. In addition, expression of iNOS was positively correlated with VEGF and MVD in feline mammary tumours and both measures were significantly greater in less differentiated phenotypes (P<0.05). In conclusion, the expression of NOS isoforms in feline mammary tumours depended on tumour grade, and the positive correlations between iNOS and angiogenic markers suggests that iNOS synthesised by tumour cells promotes tumour growth. Thus, iNOS can be used as an important immunohistochemical marker to determine the degree of malignancy and prognosis of feline mammary carcinoma.     

Nitric oxide stimulates progesterone and prostaglandin E2 secretion as well as angiogenic activity in the equine corpus luteum

Cytokines and nitric oxide (NO) are potential mediators of luteal development and maintenance, angiogenesis, and blood flow. The aim of this study was to evaluate (i) the localization and protein expression of endothelial and inducible nitric oxide synthases (eNOS and iNOS) in equine corpora lutea (CL) throughout the luteal phase and (ii) the effect of a nitric oxide donor (spermine NONOate, NONOate) on the production of progesterone (P4) and prostaglandin (PG) E(2) and factor(s) that stimulate endothelial cell proliferation using equine luteal explants. Luteal tissue was classified as corpora hemorrhagica (CH; n = 5), midluteal phase CL (mid-CL; n = 5) or late luteal phase CL (late CL; n = 5). Both eNOS and iNOS were localized in large luteal cells and endothelial cells throughout the luteal phase. The expression of eNOS was the lowest in mid-CL (P < 0.05) and the highest in late CL (P < 0.05). However, no change was found for iNOS expression. Luteal explants were cultured with no hormone added or with NONOate (10(-5) M), tumor necrosis factor-α (TNFα; 10 ng/mL; positive control), or equine LH (100 ng/mL; positive control). Conditioned media by luteal tissues were assayed for P4 and PGE(2) and for their ability to stimulate proliferation of bovine aortic endothelial cells (BAEC). All treatments stimulated release of P4 in CH, but not in mid-CL. TNFα and NONOate treatments also increased PGE(2) levels and BAEC proliferation in CH (P < 0.05). However, in mid-CL, no changes were observed, regardless of the treatments used. These data suggest that NO and TNFα stimulate equine CH secretory functions and the production of angiogenic factor(s). Furthermore, in mares, NO may play a role in CL growth during early luteal development, when vascular development is more intense.     

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。     

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.     

Immunohistochemical Expression of Nitric Oxide Synthase Enzymes (iNOS,eNOS, nNOS) in the Estrual and Luteal Phases of the Sexual Cycle in the Cow Oviduct

This study was aimed to determine staining intensity, cellular localization and distribution of the nitric oxide synthase (NOS) enzymes during the sexual cycle in the cow oviduct. Oviduct samples belonging to 20 cows, 10 of which were in the estrual phase and 10 in the luteal phase of the sexual cycle, were examined by an immunohistochemical procedure to determine the presence of the NOS enzymes. In the epithelial cells of the isthmus, endothelial NOS (eNOS) expression showed a strong positive reaction during the estrual phase and a weak positive reaction during the luteal phase in the endothelium and smooth muscle of the blood vessels found in the serosa and lamina propria. eNOS expression was not observed in the epithelium of either the ampulla or the fimbria in the two particular phases of the sexual cycle. The eNOS reactions observed in the blood vessel wall in these regions were stronger during the estrual phase. eNOS activity was not observed in the tunica muscularis in any of the regions of the oviduct. During the estrual phase, it was observed that inducible NOS expression showed a stronger positive reaction in the epithelium and muscle layer of the isthmus and ampulla and in the epithelium of the fimbria, compared to the luteal phase. Neuronal NOS immunoreactivity was observed in the epithelial cells of all oviduct regions and in the muscle layer of the isthmus and ampulla and did not display any significant difference between the estrual and luteal phases.     

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.