Influence of Nitric Oxide Synthase Inhibitors on Embryo Destruction and Morphological Features in Pre- and Postimplantation Mouse Embryos

Nitric oxide is an important intraovarian regulatory factor. The periimplantation period is a critical phase in mouse development. Although it was shown that nitric oxide plays an essential role during gestation, its role in the preimplantation period is not yet fully clear. We studied the involvement of nitric oxide in developmental competence (embryonic defects and morphology of pre- and postimplantation embryos) using nitric oxide synthase inhibitors, which suppress all forms of nitric oxide synthase, and female mice, to which the inhibitors had been administered before their mating with intact males. The level of mortality of pre- and postimplantation embryos in females mated to intact males increased soon after the administration of inhibitors. Studies of the morphology of embryos have shown that there was a delay in embryogenesis at the stages of cleavage and gastrulation. The results obtained suggest that nitric oxide is a potent regulator of embryonic differentiation, specifically in pre- and postimplantation mouse embryos.     

Participation of the mouse implanting trophoblast in nitric oxide production during pregnancy

While considerable progress has been made in elucidating nitric oxide (NO) regulatory mechanisms in the later stages of gestation, much less is known about its synthesis and role during embryo implantation. Thus, to evaluate the participation of the trophoblast in the production of NO during this phase, this study focused on NADPH-diaphorase activity and the distribution of NO synthase isoforms (NOS) using immunohistochemistry in pre- and postimplantation mouse embryos in situ and in vitro, as well as on NO production itself, measured as total nitrite, in trophoblast culture supernatants (Griess reaction). No NADPH-diaphorase activity was found in preimplanting embryos except after culturing for at least 48 h, when a few trophoblastic giant cells were positive. Conversely, postimplantation trophoblast cells either lodged into the implantation chamber (in situ) or after culturing (in vitro) showed intense NADPH-diaphorase activity. Also in the postimplantation trophoblast, the endothelial and inducible NOS (eNOS and iNOS) isoforms were immunodetected, under both in situ and in vitro conditions, although in different patterns. Extracts of ectoplacental cone also revealed bands of 135 and 130 kDa on SDS-PAGE that reacted with anti-eNOS and anti-iNOS, respectively, on Western blot. Analysis of the culture supernatant demonstrated that the nitrite concentration was 1) proportional to the number of cultured trophoblast cells, 2) almost completely abolished in the presence of N(omega)-nitro-L-arginine methyl ester, and 3) increased 2-fold in cultures stimulated with gamma-interferon. These results strongly suggest the production of NO from constitutive and inducible isoforms of NOS by the implanting mouse trophoblast. They also emphasize the possibility of the participation of these cells in vasodilatation and angiogenesis, and in cytotoxic mechanisms involved in the intense phagocytosis of injured maternal cells, which occur during the implantation process.     

Temporal/spatial expression and efflux activity of ABC transporter, P-glycoprotein/Abcb1 isoforms and Bcrp/Abcg2 during early murine development

ABC transporters pump out from cells a large number of endo- and xenobiotics including signal molecules and toxins; they are molecular markers of stem/progenitor cells as well. Here, we present the study of temporal/spatial patterns of Abcb1 isoforms and Abcg2 transporter expression and efflux activity in pre- and early postimplantation murine embryos. We found in 2-cell embryos abcb1a, abcb1b and abcg2 mRNAs which were believed to be maternally inherited. The expression of abcb1b and abcg2 genes was found in blastocysts and in 7 days postcoitum (dpc) embryos, while in 9dpc embryos beside of abcb1b/abcg2, the abcb1a gene was expressed. The abcb2 mRNA was detectable neither in pre- nor in postimplantation embryos. Moreover, we analysed temporal/spatial patterns of rhodamine 123/Hoechst 33342 efflux, which mirrors the ABC transporter phenotype, from individual cells of pre- and postimplantation murine embryos. The blastomeres of 2-, 4- and 8-cell embryos had efflux-inactive phenotype. Single, efflux-active cells emerged first in the morulae and their number increased in blastocyst inner cell mass. In 6 and 7 dpc embryos, all embryonic cells hold the efflux-active phenotype. Proximal embryonic endoderm of 6-8 dpc embryos contained two sub-domains: one consisted of efflux-active cells and another one of efflux-inactive cells reflecting polarity of an embryo. Between 7 and 8 dpc, at the onset of organogenesis, the vehement surge of efflux-inactive embryonic cells occurred, and their number increased in 9 dpc embryos, which consequently contained few efflux-active cells.     

Brugia malayi: localization of nitric oxide synthase in a lymphatic filariid

Nitric oxide synthase converts L-arginine to citrulline and nitric oxide, a gaseous signaling molecule critical to multiple physiological responses. Nitric oxide synthase was detected by Western blot analysis of Brugia malayi extracts using an antibody raised against a peptide from murine brain nitric oxide synthase. Using NADPH diaphorase staining and immunohistochemistry, nitric oxide synthase was localized in the parasitic nematode B. malayi. As in Ascaris suum, nitric oxide synthase was detected in the body wall muscles of adult B. malayi. This localization pattern is in agreement with the role of nitric oxide in the control of muscle tone in other invertebrates and in vertebrates. A novel finding was the localization of nitric oxide synthase in the oocytes, in developing embryos, and in spermatozoa. B. malayi nitric oxide synthase may play a role in developmental signaling, as has been suggested for Drosophila and Ilyanassa, a marine mud snail.     

Role of different isoforms of nitric oxide synthase in development of tumor mutants in Drosophila melanogaster

We studied the role of nitric oxide synthase during tumor growth in oncovirus-induced tumor mutants of Drosophila melanogaster. The lines with different capacity for malignancy differed reliably in the level of enzymatic activity. It was shown using specific inhibitors of neuronal and inducible isoforms that the neuronal isoform was not involved in tumor formation, while the inducible one appears to play an important role in tumor growth inhibition. This isoform was identified with the help of immunoblotting and monoclonal antibodies against inducible nitric oxide synthase.     

Nitric oxide affects preimplantation embryonic development in a rotating wall vessel bioreactor simulating microgravity

Microgravity was simulated with a rotating wall vessel bioreactor (RWVB) in order to study its effect on pre-implantation embryonic development in mice. Three experimental groups were used: stationary control, rotational control and clinostat rotation. Three experiments were performed as follows. The first experiment showed that compared with the other two (control) groups, embryonic development was significantly retarded after 72 h in the clinostat rotation group. The second experiment showed that more nitric oxide (NO) was produced in the culture medium in the clinostat rotation group after 72 h (P<0.05), and the nitric oxide synthase (NOS) activity in this group was significantly higher than in the controls (P<0.01). In the third experiment, we studied apoptosis in the pre-implantation mouse embryos after 72 h in culture and found that Annexin-V staining was negative in the normal (stationary and rotational control) embryos, but the developmentally retarded (clinostat rotation) embryos showed a strong green fluorescence. These results indicate that microgravity induced developmental retardation and cell apoptosis in the mouse embryos. We presume that these effects are related to the higher concentration of NO in the embryos under microgravity, which have cause cytotoxic consequences.     

Inhibition of oxidative metabolism by nitric oxide restricts EMCV replication selectively in pancreatic beta-cells

Environmental factors, such as viral infection, are proposed to play a role in the initiation of autoimmune diabetes. In response to encephalomyocarditis virus (EMCV) infection, resident islet macrophages release the pro-inflammatory cytokine IL-1β, to levels that are sufficient to stimulate inducible nitric oxide synthase (iNOS) expression and production of micromolar levels of the free radical nitric oxide in neighboring β-cells. We have recently shown that nitric oxide inhibits EMCV replication and EMCV-mediated β-cell lysis and that this protection is associated with an inhibition of mitochondrial oxidative metabolism. Here we show that the protective actions of nitric oxide against EMCV infection are selective for β-cells and associated with the metabolic coupling of glycolysis and mitochondrial oxidation that is necessary for insulin secretion. Inhibitors of mitochondrial respiration attenuate EMCV replication in β-cells, and this inhibition is associated with a decrease in ATP levels. In mouse embryonic fibroblasts (MEFs), inhibition of mitochondrial metabolism does not modify EMCV replication or decrease ATP levels. Like most cell types, MEFs have the capacity to uncouple the glycolytic utilization of glucose from mitochondrial respiration, allowing for the maintenance of ATP levels under conditions of impaired mitochondrial respiration. It is only when MEFs are forced to use mitochondrial oxidative metabolism for ATP generation that mitochondrial inhibitors attenuate viral replication. In a β-cell selective manner, these findings indicate that nitric oxide targets the same metabolic pathways necessary for glucose stimulated insulin secretion for protection from viral lysis.     

Role of Different Isoforms of Nitric Oxide Synthase in Development of Tumor Mutants in Drosophila melanogaster

We studied the role of nitric oxide synthase during tumor growth in oncovirus-induced tumor mutants of Drosophila melanogaster. The lines with different capacity for malignancy differed reliably in the level of enzymatic activity. It was shown using specific inhibitors of neuronal and inducible isoforms that the neuronal isoform was not involved in tumor formation, while the inducible one appears to play an important role in tumor growth inhibition. This isoform was identified with the help of immunoblotting and monoclonal antibodies against inducible nitric oxide synthase.     

Paracrine action of FGF4 during periimplantation development maintains trophectoderm and primitive endoderm

FGF4, a member of the fibroblast growth factor (FGF) family, is absolutely required for periimplantation mouse development, although its precise role at this stage remains unknown. The nature of the defect leading to postimplantation lethality of embryos lacking zygotic FGF4 is unclear and little is known about downstream targets of FGF4-initiated signaling within the various cellular compartments of the blastocyst. Here we report that postimplantation lethality of Fgf4(-/-) embryos is unlikely to reflect strictly mitogenic requirements for FGF4. Rather, our results suggest that FGF4 is required to maintain trophectoderm and primitive endoderm identity at embryonic day 4.5. This result is consistent with the reported in vitro activity of FGF4 in maintaining trophoblast stem cells and with the requirement for receptor tyrosine kinase signaling in primitive endoderm formation. Thus, postimplantation lethality of Fgf4(-/-) embryos likely results from the failure of proper differentiation and function of extraembryonic cell types.     

Postimplantation development of tetraploid mouse embryos produced by electrofusion

Despite the fact that a variety of experimental techniques have been devised over the years to induce tetraploid mammalian embryonic development, success rates to date have been limited. Apart from the early study by Snow, who obtained development to term of a limited number of cytochalasin B-induced tetraploid mouse embryos, no other researchers have achieved development of tetraploid embryos beyond the early postimplantation period. We now report advanced postimplantation development of tetraploid mouse embryos following electrofusion of blastomeres at the 2-cell stage, and subsequent transfer of these 1-cell 'fused' embryos to appropriate recipients. Cytogenetic analysis of the extraembryonic membranes of all of the postimplantation embryos encountered in the present study has provided an unequivocal means of confirming their tetraploid chromosome constitution. A preliminary morphological and histological analysis of the tetraploid embryos obtained by this technique has revealed that characteristic craniofacial abnormalities particularly involving the forebrain and eyes were consistently observed, and these features were often associated with abnormalities of the vertebral axis and heart. The most advanced viable embryo in this series was recovered on the 15th day of gestation, and its morphological features suggest that it was developmentally equivalent to a normal embryo of about 13.5-14 days p.c.     

Astrocytic production of nerve growth factor in motor neuron apoptosis: implications for amyotrophic lateral sclerosis

Reactive astrocytes frequently surround degenerating motor neurons in patients and transgenic animal models of amyotrophic lateral sclerosis (ALS). We report here that reactive astrocytes in the ventral spinal cord of transgenic ALS-mutant G93A superoxide dismutase (SOD) mice expressed nerve growth factor (NGF) in regions where degenerating motor neurons expressed p75 neurotrophin receptor (p75(NTR)) and were immunoreactive for nitrotyrosine. Cultured spinal cord astrocytes incubated with lipopolysaccharide (LPS) or peroxynitrite became reactive and accumulated NGF in the culture medium. Reactive astrocytes caused apoptosis of embryonic rat motor neurons plated on the top of the monolayer. Such motor neuron apoptosis could be prevented when either NGF or p75(NTR) was inhibited with blocking antibodies. In addition, nitric oxide synthase inhibitors were also protective. Exogenous NGF stimulated motor neuron apoptosis only in the presence of a low steady state concentration of nitric oxide. NGF induced apoptosis in motor neurons from p75(NTR +/+) mouse embryos but had no effect in p75(NTR -/-) knockout embryos. Culture media from reactive astrocytes as well as spinal cord lysates from symptomatic G93A SOD mice-stimulated motor neuron apoptosis, but only when incubated with exogenous nitric oxide. This effect was prevented by either NGF or p75(NTR) blocking-antibodies suggesting that it might be mediated by NGF and/or its precursor forms. Our findings show that NGF secreted by reactive astrocytes induce the death of p75-expressing motor neurons by a mechanism involving nitric oxide and peroxynitrite formation. Thus, reactive astrocytes might contribute to the progressive motor neuron degeneration characterizing ALS.     

Cardiac malformations are associated with altered expression of vascular endothelial growth factor and endothelial nitric oxide synthase genes in embryos of diabetic mice

The aim of this study was to investigate the role of nitric oxide (NO), and the expression of endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) genes in developing hearts at embryonic day 13.5 of embryos from diabetic mice. The protein and mRNA expression levels of eNOS and VEGF were significantly altered in the developing hearts of embryos from diabetic mice. The NO level was significantly decreased, whereas the VEGF concentration was significantly increased in the developing hearts of the embryos from diabetic mice. In vitro study showed a significant reduction in eNOS expression and cell proliferation in cardiac myoblast cells exposed to high glucose concentrations. Further, high glucose induced apoptosis in myoblast cells. Ultrastructural changes characteristics of apoptosis, including cell blebbing, aggregation of ribosomes and vacuoles in the cytoplasm were also evident in myoblast cells exposed to high glucose. It is suggested that hyperglycemia alters the expression of eNOS and VEGF genes that are involved in the regulation of cell growth and vasculogenesis, thereby contributing to the cardiac malformations seen in embryos from diabetic mice.     

Nitric oxide synthase in filariae: demonstration of nitric oxide production by embryos in Brugia malayi and Acanthocheilonema viteae

The radical gas nitric oxide (NO) is synthesized by nitric oxide synthase (NOS) from l-arginine and molecular oxygen. Nitric oxide is an important signaling molecule in invertebrate and vertebrate systems. Previously we have shown that NOS is localized to more tissues in Brugia malayi than has been reported in Ascaris suum. In this paper, we analyze the distribution of NOS in Acanthocheilonema viteae, a filarial nematode that differs from B. malayi in that A. viteae females release microfilariae without a sheath. A. viteae is also one of a few filarial parasites without the Wolbachia intracellular endosymbiont. By use of a specific antibody, NOS was demonstrated in extracts of A. viteae and Dirofilaria immitis. The localization pattern of NOS in A. viteae was similar to that seen in B. malayi, with the enzyme localized to the body wall muscles of both sexes, developing spermatozoa, intrauterine sperm, and early embryos. By use of DAF-2, a fluorescent indicator specific for nitric oxide, the embryos of B. malayi and A. viteae were demonstrated to produce NO ex utero. The near identical staining patterns seen in A. viteae and B. malayi argue that NO is not produced by Wolbachia, nor is it produced by the nematodes in response to the infection. Localization of NOS to the sperm of filarial nematodes suggests a role for NO during fertilization as has been described for sea urchin and ascidian fertilization. Demonstration of the activity of embryonic NOS supports our earlier hypothesis that NO is a signaling molecule during embryogenesis in filarial nematodes.     

一氧化氮合酶与脓毒症的研究进展

脓毒症在外科临床工作中较常见,治疗相当困难;本文主要概述了一氧化氮合酶的基因定位、结构特点以及一氧化氮合酶与脓毒症的关系,进一步说明由一氧化氮合酶介导的一氧化氮生成与脓毒症关系密切,而选择性一氧化氮合酶抑制剂在脓毒症各阶段恰当的应用可能是有效治疗脓毒症、降低病死率的一个重要途径,也将成为今后研究的热点。     

一氧化氮合酶与脓毒症的研究进展

脓毒症在外科临床工作中较常见,治疗相当困难;本文主要概述了一氧化氮合酶的基因定位、结构特点以及一氧化氮合酶与脓毒症的关系,进一步说明由一氧化氮合酶介导的一氧化氮生成与脓毒症关系密切,而选择性一氧化氮合酶抑制剂在脓毒症各阶段恰当的应用可能是有效治疗脓毒症、降低病死率的一个重要途径,也将成为今后研究的热点。     

NADPH-diaphorase-positive nerve cells in heterotopic spinal cord transplants

The method of ectopic transplantation of embryonic CNS rudiments makes it possible to study the mechanisms underlying adaptation of the transplanted embryonic rudiments. The production of nitric oxide by cells is considered as one of such mechanisms. NADPH-diaphorase is an index of the presence of nitric oxide synthase in cells. It was shown that the nerve cells of rat embryonic spinal cord transplants preserved their capacity to express NADPH-diaphorase after transplantation in the sciatic nerve of an adult animal for six months. The dynamics of NADPH-diaphorase-positive neurons of rat embryonic spinal cord developing after transplantation and in situ were studied. In spinal cord neck region, small bipolar NADPH-diaphorase-positive neurons were visualized on day 17 of prenatal development. After transplantation of the embryonic (day 15) spinal cord in the nerve, NADPH-diaphorase-positive neurons were formed later than in situ: within seven days. The results of histochemical studies carried out within six months after the operation suggest a protective role of NADPH-diaphorase in the neurons of allotransplants developing under the conditions of altered microenvironment and insufficient innervation and also suggest that nitric oxide can cause the death of neurons in long surviving transplants.     

Endothelial nitric oxide synthase modulates gastric ulcer healing in rats

Nitric oxide has been shown to be beneficial for gastric ulcer healing. We determined the relative effects of endothelial and inducible nitric oxide synthases on gastric ulcer healing in rats. Ulcers were induced by serosal application of acetic acid. Ulcer severity, angiogenesis, and nitric oxide synthase expression were assessed 3-10 days later. The effects of inhibitors of nitric oxide synthase were also examined. Inducible nitric oxide synthase mRNA was only detected in ulcerated tissue (maximal at day 3), whereas the endothelial isoform mRNA was detected in normal tissue and increased during ulcer healing. Inducible nitric oxide synthase was expressed in inflammatory cells in the ulcer bed, whereas endothelial nitric oxide synthase was found in the vascular endothelium and in some mucosal cells in both normal and ulcerated tissues. Angiogenesis changed in parallel with endothelial nitric oxide synthase expression. N(6)-(iminoethyl)-L-lysine did not affect angiogenesis or ulcer healing, while N(G)-nitro-L-arginine methyl ester significantly reduced both. In conclusion, endothelial nitric oxide synthase, but not the inducible isoform, plays a significant role in gastric ulcer healing.     

The modulation of neuroinflammation by inducible nitric oxide synthase

The accumulation and propagation of misfolded proteins in the brain is a pathological hallmark shared by many neurodegenerative diseases, such as the depositions of β-amyloid and hyperphosphorylated tau proteins in Alzheimer''s disease. Initial evidence shows the role of nitric oxide synthases in the development of neurodegenerative diseases. A recent, in an exciting paper (Bourgognon et al. in Proc Natl Acad Sci USA 118, 1–11, 2021. 10.1073/pnas.2009579118) it was shown that the inducible nitric oxide synthase plays an important role in promoting oxidative and nitrergic stress leading to neuroinflammation and consequently neuronal function impairments and decline in synaptic strength in mouse prion disease. In this context, we reviewed the possible mechanisms of nitric oxide synthase in the generation of neurodegenerative diseases.