Nitric oxide (NO) accounts for the biologic activity of endothelium-derived relaxing factor (EDRF), discovered by Furchgott and Zawadzki (1980). NO is synthesized in endothelial cells from L-arginine by nitric oxide synthase (NOS). EDRF is important in regulation of vasomotor tone and blood flow by inhibiting smooth muscle contraction and platelet aggregation. Janssens et al. (1992) showed that the cDNA encoded a calcium-regulated, constitutively expressed endothelial NOS, capable of producing EDRF in blood vessels. Nitric oxide synthases have been assigned to 2 classes: a constitutively expressed, calcium-regulated class identified in brain, neutrophils, and endothelial cells, and a calcium-independent class identified in endotoxin- or cytokine-induced macrophages and endothelial cells.
NCBI Summary:
Nitric oxide is a reactive free radical which acts as a biologic mediator in several processes, including neurotransmission and antimicrobial and antitumoral activities. Nitric oxide is synthesized from L-arginine by nitric oxide synthases. Variations in this gene are associated with susceptibility to coronary spasm. Alternative splicing and the use of alternative promoters results in multiple transcript variants. [provided by RefSeq, Oct 2016]
General function
Comment
Nitric oxide (NO) acts as a neuronal messenger in both the central and peripheral nervous systems and has been implicated in reproductive physiology and behavior (Drazen, et al., 1999).
Cellular localization
Cytoplasmic
Comment
Ovarian function
Initiation of primordial follicle growth, Ovulation, Luteolysis, Oogenesis
Comment
The endothelial nitric oxide synthase/cyclic guanosine monophosphate/protein kinase G pathway activates primordial follicles. Zhao P et al. (2020) In mammals, the well-organized activation of quiescent primordial follicles is pivotal for female reproductive reserve. In the present study, we examined the mechanisms underlying primordial follicle activation in mice. We found that endothelial nitric oxide synthase (eNOS) and its downstream effectors, cyclic guanosine monophosphate (cGMP) and cGMP-dependent protein kinase G (PKG), were expressed in pre-granulosa cells and promoted primordial follicle activation, oocyte growth and granulosa cell proliferation in neonatal ovaries. Mammalian target of rapamycin (mTOR) colocalized with PKG in pre-granulosa cells and was essential for eNOS/cGMP/PKG pathway-induced primordial follicle activation. The eNOS/cGMP/PKG pathway was found to stabilize mTOR protein. The mRNA levels of F-box and WD repeat domain containing 7 (FBXW7), an E3 ubiquitin ligase, correlated negatively with mTOR protein levels in neonatal ovaries. FBXW7 bound to and destabilized mTOR protein in pre-granulosa cells in a ubiquitin/proteasome-dependent manner. However, agonists of the eNOS/cGMP/PKG pathway reduced FBXW7 mRNA levels. FBXW7 overexpression suppressed primordial follicle activation and prevented the eNOS/cGMP/PKG pathway from activating primordial follicles and stabilizing mTOR protein. These findings demonstrate that the eNOS/cGMP/PKG pathway activates primordial follicles by suppressing FBXW7-induced ubiquitination of mTOR in mice.//////////////////
Jablonka-Shariff et al. (1999) concluded that Ovarian nitric oxide synthesis is required for maximal ovulation, and a lack of nitric oxide during the periovulatory period results in severe defects in oocyte maturation.
Dunnam et al. (1999) concluded that chronic NO inhibition produces constant estrus due to increased estradiol production and that NO acts to inhibit estradiol and androstenedione production.
Friden BE, et al 2000 reported evidence for nitric oxide acting as a luteolytic factor in the
human corpus luteum.
Immunoblotting analyses revealed that endothelial MOS (eNOS)
is the most abundant isoform in human CL with highest values during the late
luteal phase. Immunoreactive eNOS was localized predominantely in the theca
lutein layer, being particularly abundant in endothelial cells, but with
positive staining also in some steroidogenic cells. Immunoreactive inducible
NOS (iNOS) was also detected, but to lesser degree, and did not display
apparent phase-specific changes. The effect of NO on CL steroid synthesis was
examined using human chorionic gonadotrophin (HCG)-stimulated dispersed CL
cells cultured in vitro, Progesterone production was significantly decreased
by the MO donor spermine NONOate (10(-5) mol/l) in cells of the
late, but not mid-, luteal phase.
Expression regulated by
LH
Comment
Nitric oxide synthase activity Is critical for the preovulatory epidermal growth factor-like cascade induced by luteinizing Hormone in bovine granulosa cells. Zamberlam G 2014 et al.
In rabbits and rodents, nitric oxide (NO) is generally considered to be critical for ovulation. In monovulatory species, however, the importance of NO has not been determined, nor is it clear where in the preovulatory cascade NO may act. The objectives of the present study were (1) to determine if nitric oxide synthase (NOS) enzymes are regulated by luteinizing hormone (LH) and (2) to determine if and where endogenous NO is critical for expression of genes essential for the ovulatory cascade in bovine granulosa cells in serum-free culture. Time- and dose-response experiments demonstrated that LH had a significant stimulatory effect on endothelial NOS (NOS3) mRNA abundance but in a prostaglandin-dependent manner. NO production was stimulated by LH before a detectable increase in NOS3 mRNA levels was observed. Pretreatment of cells with the NOS inhibitor, L-NAME, blocked the effect of LH on the epidermal growth factor (EGF)-like ligands epiregulin and amphiregulin, as well as prostaglandin-endoperoxide synthase-2 (PTGS2) mRNA abundance and protein levels. Similarly, EGF treatment increased mRNA encoding epiregulin, amphiregulin and the early response gene EGR1, and this was inhibited by pretreatment with L-NAME. Interestingly, pretreatment with L-NAME had no effect on either ERK1/2 or AKT activation. Taken together, these results suggest that endogenous NOS activity is critical for LH-induced ovulatory cascade in granulosa cells of a monotocous species and acts downstream of EGF receptor activation but upstream of the EGF-like ligands.
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The involvement of nitric oxide in the ovulatory process in the rat. Shukovski L et al. Nitric Oxide (NO) is now recognized as a mediator of several biological functions. In the present study we examined the effects of NO synthase (NOS) inhibitors on the ovulatory process in vivo, and whether this effect can be reversed by a NO generator. Immature eCG-hCG treated rats were injected intraperitonealy (ip) or unilaterally into the periovarian sac (intrabursal injection; ib) with inhibitors of the inducible form of NOS. Aminoguanidine (AG) suppressed ovulation in a dose-dependent manner, reaching a 54% inhibition at a dose of 20 mg/kg when injected ip (p < 0.001 vs. saline control). Likewise, local ib administration inhibited ovulation from the treated ovary; thus a dose of 2 mg/kg resulted in 48% inhibition, as compared to the contralateral ovary (p < 0.01). Similar results were obtained whether AG was administered 2 h prior to the stimulation of ovulation by hCG or deferred up to 4 h afterwards. An additional NOS inhibitor, NG-methyl-L-arginine (L-NMA) suppressed ovulation, albeit to a lower extent. Intrabursal administration of L-NMA (0.1 and 1 mg/kg) resulted in 34% and 32% inhibition, respectively (p < 0.05 vs. the saline treated control). The same doses of NG-methyl-D-arginine (D-NMA) did not inhibit ovulation significantly compared to the saline treated control. When sodium nitroprusside (0.5 mg/kg), a NO generator, was injected concomitantly with AG, it completely reversed its inhibitory action on ovulation. Thus, we have demonstrated the ability of NOS inhibitors to suppress hCG-induced ovulation in the rat in vivo. The specificity of this effect is confirmed by the ability of a NO generator to reverse the inhibitory action of AG. In conclusion, the ovarian NO/NOS system seems to be necessary for follicle rupture during ovulation.
Ovarian localization
Oocyte, Theca, Luteal cells
Comment
Cardiac Nitric Oxide Synthases and Na+/K+-ATPase in the Rat Model of Polycystic Ovary Syndrome Induced by Dihydrotestosterone. Tepavčević S et al. (2015) Nitric oxide synthases (NOSs) and Na(+)/K(+)-ATPase are enzymes essential for regular functioning of the heart. Since both enzymes are under insulin and androgen regulation and since insulin action and androgen level were disturbed in polycystic ovary syndrome (PCOS), we hypothesized that cardiac nitric oxide (NO) production and sodium/potassium transport would be deteriorated in PCOS. To test our hypothesis we introduced animal model of PCOS based on dihydrotestosterone (DHT) treatment of female Wistar rats and analyzed protein expression, phosphorylation or subcellular localization of endothelial NOS (eNOS), inducible NOS (iNOS) and alpha subunits of Na(+)/K(+)-ATPase in the heart. Obtained results indicate that DHT treatment significantly decreased cardiac eNOS protein level and activating phosphorylation at serine 1 177, while inhibitory phosphorylation at threonine 495 was increased. In contrast to expression of eNOS, iNOS protein level in the heart of DHT-treated rats was significantly elevated. Furthermore, cardiac protein level of alpha 1 subunit of the ATPase, as well as its plasma membrane content, were decreased in rats with PCOS. In line with this, alpha 2 subunit protein level in fraction of plasma membranes was also significantly below control level. In conclusion, DHT treatment impaired effectiveness of NOSs and Na(+)/K(+)-ATPase in the female rat heart. Regarding the importance of NO production and sodium/potassium transport in the cardiac contraction and blood flow regulation, it implicates strong consequences of PCOS for heart functioning.//////////////////
In control ovaries, eNOS was detected in the theca cell layer, ovarian stroma, and on the surface of oocytes. During follicular development, eNOS staining was still expressed in the theca cell layer and was also present in mural granulosa cells. After ovulation, homogenous eNOS staining was observed within cells of the corpus luteum (CL) (Jablonka-Shariff, et al., 1997).
Strong positive staining for eNOS was observed in the cytoplasm of ovarian and ovulated oocytes (Jablonka-Shariff, et al., 1999).
Hattori MA et al 2000 reported that the endothelial type of NO
synthase (eNOS) (130 kDa) was detected in the porcine oocyte, but not in the
proliferated cumulus cells, by immunoblotting.
Follicle stages
Primordial, Primary, Secondary, Antral, Preovulatory, Corpus luteum
Comment
Expression of nitric oxide synthase-3 in porcine oocytes obtained at different follicular development Takesue K, et al .
The present study was designed to determine the localization of nitric oxide synthase-3 (NOS-3) in porcine follicles during follicular development. A 130-kDa NOS-3 protein was found with greater frequency much in the oocytes than in the cumulus cells, as revealed by Western blotting analysis. The content of NOS-3 in the oocyte was higher in large follicles (> 7-mm diameter) than in small follicles (< 2-mm). The data by Western blotting showed the same pattern as the observations obtained from the immunohistochemical studies, in which the periphery of the oocyte stained strong positive. The inner surface cell layer of granulosa cells and cumulus cells were positive staining, especially in large antral follicles. In the primordial follicles, NOS-3 was restricted to the cytoplasm of oocytes, and no stained product was observed in the nucleus of oocytes or granulosa cells. A significant synthesis of NO by oocytes was observed in the presence of ionomycin, but not in the absence of ionomycin, indicating that oocyte NOS-3 functions in response to transient elevations in the intracellular calcium level. We concluded that NOS-3 is expressed in the oocyte from the primordial follicular stage to antral follicular stage, and that it is functional at least in the antral follicles.
Expression of endothelial nitric oxide synthase in the ovine ovary throughout the estrous cycle. Grazul-Bilska AT et al. This study was conducted to evaluate the expression of endothelial nitric oxide synthase (eNOS) in ovarian follicles and corpora lutea (CL) throughout the estrous cycle in sheep. Three experiments were conducted to (1) immunolocalize eNOS protein, (2) determine expression of mRNA for eNOS and its receptor guanylate cyclase 1 soluble beta3 (GUCY1B3), and (3) co-localize eNOS and vascular endothelial growth factor (VEGF) proteins in the follicles and/or CL throughout the estrous cycle. In experiment 1, ovaries were collected from ewes treated with FSH, to induce follicular growth or atresia. In experiment 2, ovaries were collected from ewes treated with FSH and hCG to induce follicular growth and ovulation. In experiment 3, ovaries were collected from superovulated ewes to generate multiple CL on days 2, 4, 10, and 15 of the estrous cycle. In experiments 1 and 2, the expression of eNOS protein was detected in the blood vessels of the theca externa and interna of healthy ovarian follicles. However, in early and advanced atretic follicles, eNOS protein expression was absent or reduced. During the immediate postovulatory period, eNOS protein expression was detected in thecal-derived cells that appeared to be invading the granulosa layer. Expression of eNOS mRNA tended to increase in granulosa cells at 12 and 24 h, and in theca cells 48 h after hCG injection. In experiment 3, eNOS protein was located in the blood vessels of the CL during the estrous cycle. Dual localization of eNOS and VEGF proteins in the CL demonstrated that both were found in the blood vessels.
Phenotypes
Mutations
3 mutations
Species: mouse
Mutation name: None
type: null mutation fertility: fertile Comment:Huang, et al. (1995) disrupted the gene encoding eNOS in mice by homologou recombination. Homozygous mutant mice were found to be viable, fertile and indistinguishible from wildtype and heterozygous littermates in apperance or routine behaviour. Endothelium-derived relaxing factor activity, as assayed by acetylcholine-induced relaxation, is absent, and the eNOS mutant mice are hypertensive. Thus eNOS mediates basal vasodilation. Responses to NOS blockade in the mutant mice suggest that non-endothelial isoforms of NOS may be involved in maintaining blood pressure.
Species: mouse
Mutation name: None
type: null mutation fertility: subfertile Comment:Drazen et al. (1999) reported that pharmacological inhibition of nitric oxide synthase (NOS) with the nonspecific NOS inhibitor, L-N-G-nitro-Arg-methyl ester (L-NAME), induced deficits in both the number of ovarian rupture sites and the number of oocytes recovered in the oviducts of mice. Female neuronal NOS knockout (nNOS-/-) mice have normal numbers of rupture sites, but reduced numbers of oocytes recovered following systemic injections of gonadotropins, suggesting that NO produced by nNOS accounts, in part, for deficits in ovulatory efficiency observed after L-NAME administration. Additionally, endothelial NOS knockout (eNOS-/-) mice have reduced numbers of ovulated oocytes after superovulation.
Drazen et al. (1999) reported that estrous cycle length and variability were consistently reduced in eNOS-/- females. The number of rupture sites was normal in eNOS-/- mice under natural conditions and after administration of exogenous GnRH. After exogenous gonadotropin administration, eNOS-/-females displayed a significant reduction in the number of ovarian rupture sites. Female eNOS-/- mice also produced fewer pups/litter compared to WT mice.
These data suggest that NO from endothelial sources might play a role in mediating rodent ovulation and may be involved in regulation of the timing of the estrous cycle.
Species: mouse
Mutation name: None
type: null mutation fertility: subfertile Comment: Disruption of the Endothelial Nitric Oxide Synthase gene affects ovulation, fertilization and early embryo survival in a knockout mouse model. Pallares P et al. Two consecutive experiments determined whether disruption of the endothelial NOS (NOS3) gene affects ovulation, fertilization, implantation and embryo development. In the first trial, Nos3-knockout mice (groups Nos3-/-) and wild-type mice (groups Nos3+/+) showed significant differences in mean number of corpora lutea (9.7 +/- 1.2 in Nos3-/- vs 14.2 +/- 1.2 in Nos3+/+; P < 0.01), rate of anovulation (48.3 +/- 7.3 % in Nos3-/- vs 29.7 +/- 6.3 in Nos3+/+; P < 0.05), total mean number of recovered oocytes/zygotes (4.0 +/- 1.1 in Nos3-/- vs 10.4 +/- 1.6 in Nos3+/+; P < 0.01) and non-fertilization rates (50.7 in Nos3-/- vs 3.3% in Nos3+/+; P < 0.001). In the second trial, implantation and early pregnancy losses in Nos3-knockout and wild-type dams were detected by real-time ultrasound imaging. The number of embryos reaching implantation was higher in Nos3+/+ than in Nos3-/- mice (7.5 +/- 0.4 vs 4.0 +/- 0.4; P < 0.005); thereafter, embryo losses were detected between Days 8.5 and 13.5, in 62.5% of the Nos3-knockout dams and, at Days 10.5 and 11.5, in 16.7% of the control females (P < 0.005). Thus, NO and NOS3 deficiencies affect reproductive and developmental features in the Nos3-knockout mouse model.