scholarly journals Localization of nitric oxide synthase activity in unfertilized oocytes and fertilized embryos during preimplantation development in mice

Reproduction ◽  
2001 ◽  
pp. 957-963 ◽  
Author(s):  
A Nishikimi ◽  
T Matsukawa ◽  
K Hoshino ◽  
S Ikeda ◽  
Y Kira ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Preimplantation Development ◽  
Cell Stage ◽  
Nos Inhibitors ◽  
Development In Vitro ◽  
Oxide Synthase

Changes in the activities of nitric oxide synthase (NOS) during embryonic development, and the distribution of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) isoforms were examined in unfertilized mouse oocytes at the second meiotic metaphase (MII) stage and in fertilized mouse embryos during preimplantation development. In addition, the effects of NOS inhibitors on mouse preimplantation development in vitro were investigated. The activities of NOS in MII oocytes and fertilized embryos during the preimplantation period were determined by NADPH-diaphorase staining. Although NOS activity was detected in unfertilized MII oocytes, the intensity of staining was much weaker than that of fertilized embryos at the one-cell stage. There was a decrease in NOS activity in embryos from the four-cell to the eight-cell stage; however, NOS activity increased again in embryos at the morula stage, particularly in the inner cell population. In the expanded blastocysts, staining was confined to the inner cell mass. Immuno-cytochemical staining showed that eNOS and iNOS were expressed in the cytoplasm of oocytes and embryos during the preimplantation period, and eNOS was also distributed in the nuclei of the embryos. When one-cell embryos were treated with 1 mmol N(omega)-nitro-L-arginine methyl ester (L-NAME) l(-1), their development in vitro was arrested at the two-cell stage. This inhibition of development was overcome by the addition of 1 mmol L-arginine l(-1) to the medium. These observations indicate that nitric oxide plays an important role as a diffusible regulator of cell proliferation and differentiation, especially at the developmental transition from the two-cell to the four-cell stage during preimplantation development of mice.

scholarly journals Development of Preimplantation Mouse Embryos in vivo and in vitro

1982 ◽  
Vol 35 (2) ◽  
pp. 187 ◽  
Author(s):  
GM Harlow ◽  
P Quinn
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Cell Stage ◽  
Preimplantation Mouse ◽  
Development In Vitro

The culture conditions for the development in vitro of (C57BL/6 X CBA) F2 hybrid two-cell embryos to the blastocyst stage have been optimized. Commercially available pre-sterile disposable plastic culture dishes supported more reliable development than re-usable washed glass tubes. The presence of an oil layer reduced the variability in development. An average of 85 % of blastocysts developed from hybrid two-cell embryos cultured in drops of Whitten's medium under oil in plastic culture dishes in an atmosphere of 5% O2 : 5% CO2 : 90% N2 ? The time taken for the total cell number to double in embryos developing in vivo was 10 h, and in cultured embryos 17 h. Embryos cultured in vitro from the two-cell stage to blastocyst stage were retarded by 18-24 h in comparison with those remaining in vivo. Day-4 blastocysts in vivo contained 25-70 cells (mean 50) with 7-28 (mean 16) of these in the inner cell mass. Cultured blastocysts contained 19-73 cells (mean 44) with 8-34 (mean 19) of these in the inner cell mass. In the uterine environment, inner-cell-mass blastomeres divided at a faster rate than trophectoderm blastomeres and it is suggested that a long cell cycle is associated with terminal differentiation. Although cultured blastocysts and inner cell masses contained the same number of cells as blastocysts and inner cell masses in vivo, the rate of cell division in cultured inner cell masses was markedly reduced.

Development in vitro of mouse embryos from the two-cell egg stage to the early somite stage

Development ◽  
1974 ◽  
Vol 31 (1) ◽  
pp. 235-245
Author(s):  
Yu-Chih Hsu ◽  
John Baskar ◽  
Leroy C. Stevens ◽  
John E. Rash
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Calf Serum ◽  
Defined Medium ◽  
Chemically Defined Medium ◽  
Cell Stage ◽  
Cord Serum ◽  
Somite Stage ◽  
Development In Vitro

About 1–3% of mouse blastocysts, which had initially been cultured from the two-cell stage in chemically defined medium or about 3–5% of blastocysts which were explanted from the uterus, developed to the early somite stage when cultured in vitro on collagen. Two-cell eggs were initially cultivated in chemically defined medium to the blastocyst stage. Blastocysts were then transferred to Eagle's minimal essential medium (MEM) plus 10% heat-inactivated calf serum. Two barriers to further development were overcome. First, the formation of endoderm and ectoderm from the inner cell mass immediately after attachment to collagen. Second, formation of the embryo proper from the embryonic region. Both barriers were overcome by using heat-inactivated human cord serum after the blastocysts hatched from the zona pellucida and attached to collagen. After attachment, embryos were cultured in MEM plus 20% heat-inactivated human cord serum which was changed daily until early somite stages. Apparently normal healthy development in vitro occurred, as judged by light and electron microscopic examination.

Erhöhte NF-κB- und iNOS-Expression in Keratozyten von Keratokonuspatienten – Hinweise auf eine entzündliche Komponente?

2019 ◽  
Author(s):  
Tanja Stachon ◽  
Lorenz Latta ◽  
Krasimir Kolev ◽  
Berthold Seitz ◽  
Achim Langenbucher ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Nuclear Factor Kappa B ◽  
No Synthase ◽  
Inos Expression ◽  
Nuclear Factor ◽  
Nos Inhibitors ◽  
Inos Inhibitor ◽  
Oxide Synthase

Zusammenfassung Hintergrund In den letzten Jahren mehren sich Hinweise auf eine entzündliche Komponente beim Keratokonus (KC). Ein Schlüsselgen bei entzündlichen Prozessen ist der Nuclear Factor Kappa B (NF-κB). NF-κB ist ein Transkriptionsfaktor, der unter anderem das Enzym Nitric Oxide Synthase (NOS), das mit dem konkurrierenden Enzym Arginase (Arg) bei entzündlichen Prozessen involviert ist, aktiviert. Ziel dieser Studie war es, die Isotypen von NOS und Arginase zu analysieren, die Expression NF-κB, NOS und Arginase sowie den regulativen Mechanismus von NOS und Arginase in Keratozyten von Keratokonuspatienten mithilfe des Inhibitors 1400W in vitro zu untersuchen. Methoden Primäre humane Keratozyten wurden durch enzymatische Behandlung mit Kollagenase A aus humanen Korneoskleralscheiben (n = 8) und von Explantaten von geplanten perforierenden Keratoplastiken (KC-Patienten) isoliert (n = 8) und in DMEM/F12-Kulturmedium, versetzt mit 5% fetalem Kälberserum, kultiviert. Die Expression von NF-κB, NOS und Arginase wurden mit quantitativer PCR (qPCR) und Westernblot-Analyse (WB) untersucht. Nitrit- und Ureakonzentrationen im Zellkulturüberstand wurden nach Zugabe des NOS-Inhibitors 1400W (0 – 40 µM) analysiert. Ergebnisse In den Keratozyten wurden ausschließlich die Isotypen iNOS (induzierbare NO-Synthase) und Arg-II nachgewiesen. Die mRNA-Expression von NF-κB und iNOS waren in KC-Keratozyten höher als in normalen Zellen (p = 0,0135 und p = 0,0001), während in der Arg-II-Expression keine Unterschiede messbar waren. Im WB war bei NF-κB eine höhere Bandenintensität messbar (p = 0,0012), bei iNOS konnten keine Unterschiede in der Bandenintensität nachgewiesen werden. Im Überstand der KC-Keratozyten wurden geringere Konzentrationen von Nitrit und Urea nach Zugabe des Inhibitors 1400W gemessen (p = ≤ 0,014), nicht jedoch bei normalen Zellen (p ≥ 0,178). Schlussfolgerung Aufgrund der erhöhten Expression von NF-κB und iNOS muss von einer inflammatorischen Komponente beim Keratokonus ausgegangen werden. Die unterschiedliche Regulation der KC-Keratozyten durch den iNOS-Inhibitor 1400W legt eine veränderte metabolische Aktivität nahe, die durch entzündliche Prozesse hervorgerufen werden kann.

scholarly journals Totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marino Maemura ◽  
Hiroaki Taketsuru ◽  
Yuki Nakajima ◽  
Ruiqi Shao ◽  
Ayaka Kakihara ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Mouse Embryos ◽  
Primitive Endoderm ◽  
Cell Stage ◽  
Supportive Environment ◽  
Mouse Zygotes ◽  
Single Blastomeres ◽  
Multicellular Organisms

AbstractIn multicellular organisms, oocytes and sperm undergo fusion during fertilization and the resulting zygote gives rise to a new individual. The ability of zygotes to produce a fully formed individual from a single cell when placed in a supportive environment is known as totipotency. Given that totipotent cells are the source of all multicellular organisms, a better understanding of totipotency may have a wide-ranging impact on biology. The precise delineation of totipotent cells in mammals has remained elusive, however, although zygotes and single blastomeres of embryos at the two-cell stage have been thought to be the only totipotent cells in mice. We now show that a single blastomere of two- or four-cell mouse embryos can give rise to a fertile adult when placed in a uterus, even though blastomere isolation disturbs the transcriptome of derived embryos. Single blastomeres isolated from embryos at the eight-cell or morula stages and cultured in vitro manifested pronounced defects in the formation of epiblast and primitive endoderm by the inner cell mass and in the development of blastocysts, respectively. Our results thus indicate that totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage.

Nitric oxide synthase inhibitors 7- and 6-nitroindazole relax smooth muscle in vitro

1994 ◽  
Vol 256 (1) ◽  
pp. R5-R6 ◽  
Author(s):  
Andrew D. Medhurst ◽  
Carol Greenlees ◽  
Andrew A. Parsons ◽  
Susan J. Smith
Keyword(s):  
Nitric Oxide ◽  
Smooth Muscle ◽  
Nitric Oxide Synthase ◽  
Nitric Oxide Synthase Inhibitors ◽  
Oxide Synthase

Abstract 085: CHIP: E3 Ubiquitin Ligase Mediates Proteasomal Degradation of Neuronal Nitric Oxide Synthase in the Paraventricular Nucleus of Rats with Heart Failure

Hypertension ◽  
2017 ◽  
Vol 70 (suppl_1) ◽  
Author(s):  
Neeru M Sharma ◽  
Kenichi Katsurada ◽  
Xuefei Liu ◽  
Kaushik P Patel
Keyword(s):  
Nitric Oxide ◽  
Heart Failure ◽  
Nitric Oxide Synthase ◽  
Paraventricular Nucleus ◽  
Ubiquitin Ligase ◽  
Protein A ◽  
Neuronal Nitric Oxide Synthase ◽  
Proteasomal Degradation ◽  
Oxide Synthase

The exaggerated sympathetic drive is a characteristic of heart failure (HF) due to reduced neuronal nitric oxide synthase (nNOS) within the paraventricular nucleus (PVN). Previously we have shown that there were increased accumulation of nNOS-ubiquitin (nNOS-Ub) conjugates in the PVN of rats with HF (1.0±0.05 Sham vs. 1.29±0.06 HF) due to the increased levels of PIN (a protein inhibitor of nNOS, known to dissociate nNOS dimers into monomers) (0.76±0.10 Sham vs. 1.12±0.09 HF) and decreased levels of tetrahydrobiopterin (BH4): a cofactor required for stabilization of nNOS dimers (0.62±0.02 Sham vs. 0.44±0.03 HF). We also showed that there is blunted nitric oxide-mediated inhibition of sympathetic tone via the PVN in HF. Here we examined whether CHIP(C-terminus of Hsp70 -interacting protein), a chaperone-dependent E3 ubiquitin-protein isopeptide ligase known to ubiquitylate Hsp90-chaperoned proteins could act as an ubiquitin ligase for nNOS in the PVN. Immunofluorescence studies revealed colocalization of nNOS and CHIP in the PVN indicating their possible interaction. CHIP expression was increased by 50% in the PVN of rats with HF(0.96±0.08 Sham vs.1.44±0.10* HF). It is shown that Hsp90 protects nNOS from ubiquitination while Hsp70 promotes the ubiquitination and degradation. We observed significant upregulation of Hsp70 (0.49±0.03 Sham vs. 0.65±0.02* HF) with a trend toward the decrease in Hsp90 expression (0.90±0.07 Sham vs. 0.71±0.06 HF). The opposing effects of the two chaperones could account for the increased CHIP-mediated ubiquitination and degradation of dysfunctional nNOS monomers in the PVN of rats with HF. Furthermore, neuronal NG108-15 cell line transfected with the pCMV3-CHIP-GFP spark (CHIP overexpression plasmid) showed approximately 74% increase in CHIP with concomitant 49% decrease in nNOS expression. In vitro ubiquitination assay in NG108 cells transfected with pCMV-(HA-Ub) 8 and pCMV3-CHIP-GFP spark plasmid reveal increased HA-Ub-nNOS conjugates (1.13 ± 0.09 Scramble vs. 1.65 ± 0.12* CHIP plasmid). Taken together, our results identify CHIP as an E3 ligase for ubiquitination of dysfunctional nNOS and CHIP expression is augmented during HF leading to increased proteasomal degradation of nNOS in the PVN.

A radiometric analysis of nitric oxide synthase activity in Hymenolepis diminuta

Parasitology ◽  
2000 ◽  
Vol 120 (1) ◽  
pp. 91-95 ◽  
Author(s):  
N. B. TERENINA ◽  
M. V. ONUFRIEV ◽  
N. V. GULYAEVA ◽  
A. M. LINDHOLM ◽  
M. K. S. GUSTAFSSON
Keyword(s):  
Nitric Oxide ◽  
Free Radical ◽  
Nitric Oxide Synthase ◽  
Nadph Diaphorase ◽  
Hymenolepis Diminuta ◽  
Complete Reduction ◽  
Radiometric Analysis ◽  
Nos Inhibitors ◽  
Oxide Synthase ◽  
Nitric Oxide Synthase Activity

The free radical nitric oxide (NO) is a neuronal messenger which is synthesized from L-arginine and O2 by nitric oxide synthase (NOS). In the synthesis NO and L-citrulline are produced in a stoichiometric 1[ratio ]1 relation. The activity of NOS was analysed in homogenates of the rat tapeworm Hymenolepis diminuta by measuring the formation of L-[3H]citrulline after incubation with L-[3H]arginine. The nature of NOS in H. diminuta was determined by studying the effect of 3 types of NOS inhibitors: (1) L-NAME, (2) EGTA, (3) 7-nitro-indazole. All inhibitors caused a significant but not complete reduction in the formation of L-[3H]citrulline. The results are discussed against the background of nerve cells and fibres positive for NADPH-diaphorase staining in H. diminuta.

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