Partial inhibition of nitric oxide synthase activity stimulates the nuclear maturation progression of bovine cumulus–oocyte complex in vitro in the presence of hemisections of the follicular walls

Zygote ◽  
2020 ◽  
Vol 28 (5) ◽  
pp. 388-396
Author(s):  
Diego Fernando Dubeibe ◽  
Maria Clara Caldas-Bussiere ◽  
Valter Luiz Maciel ◽  
Wlaisa Sampaio ◽  
Paulo B.D. Gonçalves ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Cumulus Cell ◽  
Membrane Integrity ◽  
Maturation Stage ◽  
Nuclear Maturation ◽  
Cumulus Oocyte Complex ◽  
Maturation Medium ◽  
Oxide Synthase

SummaryThis study aimed to assess the effects of the inhibition of nitric oxide synthase (NOS) on events that modulate bovine in vitro oocyte maturation. Cumulus–oocyte complexes (COCs) were cultured with hemisections (HSs) of the follicular walls in a maturation medium supplemented with different concentrations (0.1–10.0 mM) of Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME). Controls consisted of COCs cultured in the presence (+HSs) or absence of HSs (–HSs) with no additional l-NAME supplementation. The following parameters were assessed: oocyte nuclear maturation stage; cumulus cell (CC) membrane integrity; nitrate/nitrite, progesterone, and estradiol concentrations in the culture medium at 22 h of cultivation; and the concentrations of cGMP and cAMP in COCs during the first hour of maturation. The addition of 1.0 mM l-NAME increased the percentage of oocytes that reached metaphase II (MII) and the percentage of intact CCs (P < 0.05). All l-NAME concentrations reduced the nitrate/nitrite concentrations (P < 0.05), but none affected steroid concentrations compared with control +HSs (P > 0.05). The addition of 1.0 mM l-NAME reduced cGMP concentrations at 3 h and increased cAMP concentrations in the first hour of culture (P < 0.05). Our findings suggest that the NOS/NO/cGMP pathway participates in meiosis progression (MI to MII) of the bovine oocytes matured in vitro in the presence of hemisections of the follicular walls. Lastly, the mechanisms that lead to the progression of meiosis after NOS inhibition do not involve changes in steroid production.

scholarly journals Effects of inhibiting nitric oxide synthase on cumulus expansion and nuclear maturation of sheep oocytes

2011 ◽  
Vol 56 (No. 6) ◽  
pp. 284-291 ◽  
Author(s):  
Heidari Amale M ◽  
Zare Shahne A ◽  
A. Abavisani ◽  
S. Nasrollahi
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
No Donor ◽  
Cumulus Expansion ◽  
Maturation Medium ◽  
Nos Inhibitor ◽  
Oxide Synthase

Nitric oxide (NO) is a biological signaling molecule that plays a crucial role in oocyte maturation of mammalians. It is generated by the nitric oxide synthase (NOS) enzyme from l-arginine. Although the effect of NO has been shown in oocyte maturation of some species, there is no report about its effect on the in vitro maturation of sheep oocyte. So, this study aimed to investigate the importance of NO/NOS system in the in vitro maturation of ovine oocytes. Different concentrations of L-NAME (a NOS inhibitor) (0.1, 1 and 10mM) were added to maturation medium to evaluate the effect of inhibiting NOS on cumulus expansion and meiotic resumption of sheep oocytes. After 26 h culture, low and medium concentrations of L-NAME (0.1 and 1mM) had no significant effect on cumulus expansion, however, its higher concentration (10mM) decreased percentage of oocytes with total cumulus expansion as compared to control (P &lt; 0.05). The extrusion of the first polar body was also suppressed in a dose-dependent manner, so that the addition of 10mM L-NAME to maturation medium significantly stopped oocytes in GV stage (P &lt; 0.05). Moreover, to confirm the results and to evaluate if this effect is reversible, 0.1mM sodium nitroprusside (SNP, a NO donor) was added only to the maturation medium which had the highest concentration of L-NAME (10mM). The concomitant addition of NOS inhibitor with NO donor reversed the inhibitory effect of L-NAME on cumulus expansion and meiotic maturation. These results indicated that NO/NOS system is involved in the maturation of sheep oocytes.

283 ROLE OF iNOS/NO/cGMP PATHWAY ON IN VITRO MATURATION OF BOVINE COCs CO-CULTURED WITH HEMI-SECTIONS OF FOLLICULAR WALL

2015 ◽  
Vol 27 (1) ◽  
pp. 230
Author(s):  
N. F. Torres ◽  
M. C. C. Bussiere ◽  
K. S. Nogueira ◽  
D. F. Dubeibe ◽  
C. L. M. Souza
Keyword(s):  
Nitric Oxide ◽  
Plasma Membrane ◽  
In Vitro Maturation ◽  
Membrane Integrity ◽  
Cumulus Cells ◽  
Plasma Membrane Integrity ◽  
Nuclear Maturation ◽  
Maturation Medium ◽  
Cgmp Pathway

Nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) works by stimulating the activity of the enzyme soluble guanylate cyclase (sGC) to synthesise cGMP, which has action in metabolism of PDE3A. Thus, NO controls the concentration of cAMP in the oocyte. The intraoocyte concentration of these cyclic nucleotides is directly linked to the control of maturation in rodents. The follicular wall hemi-sections (HS) in maturation medium partially inhibit nuclear maturation of oocytes in culture, which allows us to study the mechanism of meiosis resumption in bovines. The aim of this study is to evaluate the effect of iNOS and sGC inhibition in the nuclear maturation. Twenty cumulus-oocytes complexes (COC)/treatment were cultured with 8 HS of follicular wall at 38.5°C and 5% CO2 in 200 µL of maturation medium (TCM 199/BSA) supplemented with different concentrations of aminoguanidine (AG), iNOS inhibitor (1,10, 50, 100, and 150 mM; n = 840), and the sGC inhibitor, 1H-[1,2,4] oxadiazole-[4,3-a] quinoxalin-1-one (ODQ; 10–3, 10–4, and 10–5 nM; n = 600). The COC groups cultured in the presence (control – ve) or absence of HS (control + ve) were used as controls. The stage of nuclear maturation of oocytes was assessed by staining with 2% acetic orcein and plasma membrane integrity of cumulus cells assessed with propidium iodite (PI) and hoechst (H33342). Statistical analyses of the 6 replicates were performed by ANOVA followed by Tukey test (P < 0.05) using SAEG software (Fundação Arthur Bernardes-UFV-Viçosa, Brazil). The integrity of cumulus cells from the group of oocytes cultured without HS (control + ve; 85.9 ± 2.3%) differed from control – ve (71.2 ± 3.7%) and other treatments, 1, 10, 50, 100, and 150 mM AG, (57.8 ± 12.1; 66.3 ± 4.2; 58.2 ± 4.6; 55.3 ± 4.3; 48.3 ± 3.3, respectively; P < 0.05). The same occurred when ODQ was used, the control + ve showed the highest cellular integrity (81.1 ± 1.6), differing from the control – ve (68.1 ± 1.8) and treatment with 10–5, 10–4, and 10–3 mM ODQ (72.0 ± 2.2; 64.6 ± 4.6; 49.6 ± 6.8, respectively; P < 0.05). The presence of HS (control – ve) decreased the percentage of oocytes that reached the metaphase II (MII) in both experiments (AG and ODQ; 41.0 ± 4.0; 39.1 ± 1.7, respectively) compared to control + ve (78.5 ± 3.9; 71.9 ± 16.6, respectively; P < 0.05). The addition of 100 and 150 mM AG inhibited the resumption of meiosis and progression to MII compared with other concentrations of AG and the controls + ve and – ve. The addition of ODQ stimulated resumption of meiosis, but at the concentration 10–3 nM there was a decrease in the number of COC that reached MII (21.8 ± 3.4) compared to control + ve (71.9 ± 16.6) and – ve (39.1 ± 1.7) and the other treatments (10–4 and 10–5 nM; 33.0 ± 1.8; 35.7 ± 2.5, respectively; P < 0,05). Using the model of in vitro maturation in which partial inhibition of meiosis resumption occurs, the results of this experiment show that (1) the iNOS/NO/cGMP pathway modulates plasma membrane integrity of cumulus cells and (2) that the activity of iNOS/NO pathway is important for the maintenance of the COC at the stage of germinal vesicle (GV) and progression of meiosis to MII.The authors acknowledge FAPERJ E-26/103.080/2011 for financial support.

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.

Sign in / Sign up

Export Citation Format

Share Document