Nitric Oxide Synthase Mutant Mice Research Articles

Sertoli cell numbers and spermatogenic efficiency are increased in inducible nitric oxide synthase mutant mice

Nitric oxide (NO) is produced via oxidation of l-arginine by nitric oxide synthases (NOSs), and is known as inducible (iNOS), neuronal, endothelial or testis-specific. Suggesting important functions for NOS in the normal rat and mouse testis, iNOS is reported to be constitutively expressed in Leydig cells (LC), Sertoli cells (SC) and germ cells. In our study, we sought to provide further insights into the roles of iNOS in the adult mouse testis using iNOS(-/-) mice. Perfusion-fixed testes from wild type (WT) and iNOS(-/-) mice were used for histological and stereological evaluations. Some of the mice had been injected with (3) H-thymidine to label proliferating cells and to determine the duration of spermatogenesis that was unaffected in iNOS(-/-) mice. Both LC nuclear volume and individual cell size were significantly decreased in iNOS(-/-) mice, but the total number of LC per testis was increased (p < 0.05) by approximately 16%. The number of SC per testis was strikingly increased (approximately twofold) in iNOS(-/-) mice, and testis weight and DSP per gram of testis (spermatogenic efficiency) were similarly increased. The anogenital distance was also significantly increased in iNOS(-/-) mice, and this key endpoint suggests that the augmentation observed for the SC number may be related to increased foetal T-exposure during the masculinization programming window. Compared with WT testes, the numbers of spermatocytes and spermatids and SC per tubule cross sections were significantly increased in iNOS(-/-) mice. Except for stages V-VI and VII-VIII, iNOS(-/-) mice exhibited approximately 3.5-fold fewer apoptotic germ cells than in WT mice. Taken together, our results provide new evidence that iNOS plays an important role in numerical and functional regulation of key somatic cells in the testis, which in turn impacts on germ cells and their survival and thus on daily sperm production.

NMDA-evoked release of striatal neurotransmitters in vivo is differentially affected by low and high doses in neuronal nitric oxide synthase mutant mice

NMDA-evoked release of striatal neurotransmitters in vivo is differentially affected by low and high doses in neuronal nitric oxide synthase mutant mice

Regulation of nerve growth factor release by nitric oxide through cyclic GMP pathway in cortical glial cells.

In the present study, we found that S-nitroso-N-acetyl-DL-penicillamine, a spontaneous nitric oxide (NO) generator, dose-dependently inhibited basal nerve growth factor (NGF) release from mixed glial cells. To elucidate the function of endogenous NO in the regulation of NGF release, the mixed glial cells were stimulated with lipopolysaccharide (LPS) or LPS plus interferon-gamma (IFNgamma). The results showed that LPS alone induced NGF release and moderate NO production. However, costimulation with LPS plus IFNgamma greatly enhanced NO production but significantly suppressed LPS-induced NGF release. When N(G)-monomethyl-L-arginine, an NOS inhibitor, was added to the culture, the suppression of NGF release by IFNgamma was significantly reduced. Quantitative reverse transcription-polymerase chain reaction demonstrated S-nitroso-N-acetyl-DL-penicillamine was also able to inhibit the LPS-induced NGF mRNA expression. To understand the different contributions of astroglia and microglia to this phenomenon, both cell types were purified. We found purified astroglia produced high amounts of NGF but low amounts of NO. However, purified microglia produced a large amount of NO but very low amounts of NGF after stimulation with LPS or LPS plus IFNgamma. Our data also indicated the second messenger cyclic GMP, but not cyclic AMP, was able to inhibit basal NGF release. In vivo experiments confirmed that NGF protein level was significantly enhanced in rats treated with L-N(omega)-nitro-arginine methyl ester and in endothelial NO synthase mutant mice. Taken together, we conclude NO derived mainly from microglia down-regulates NGF release from astroglia at the transcriptional level by stimulating cyclic GMP pathway.

Brain distribution of nitric oxide synthase in neuronal or endothelial nitric oxide synthase mutant mice using [3H]L-NG-nitro-arginine autoradiography.

The regional distribution of nitric oxide synthase in the central nervous system was assessed by quantitative autoradiography using [3H]L-NG-nitro-arginine binding in wild-type mice (SV-129 and C57black/6) and in mice lacking expression of the neuronal (type 1) and endothelial (type 3) nitric oxide synthase gene. The distribution of nitric oxide synthase binding sites in wild-type mice was similar to that described for rat brain by nicotinamide adenine dinucleotide phosphate-diaphorase staining and immunohistochemistry, and as determined by quantitative autoradiography. In the wild-type mice, the densest labelling was observed in the granular layer of the olfactory bulb, tenia tecta, rhinal fissure, amygdaloid complex and molecular layer of cerebellum. The islands of Calleja, the hippocampal CA1 and CA3 subfields, dentate gyrus, cortical layers I-II, the superficial gray layer of superior colliculus and the granule layer of cerebellum displayed intermediate binding. Cortical layers III-VI, the striatum and the thalamus were only weakly labelled. Binding was saturable and of high affinity, and was displaced by 7-nitroindazole (100 microM), a potent and selective inhibitor of type 1 nitric oxide synthase, and by unlabelled L-NG-nitro-arginine (10 microM). The density of [3H]L-NG-nitro-arginine binding was dramatically reduced in all brain regions in type 1 mutant mice, whereas there were no detectable binding differences between wild-type and type 3 nitric oxide synthase mutant mice. Hence, type 1 nitric oxide synthase is the major source of [3H]L-NG-nitro-arginine binding in the mouse brain. [3H]L-NG-Nitro-arginine autoradiography may be a useful tool to quantify nitric oxide synthase in different brain areas after pharmacological or physiological manipulations.

L-NNA-sensitive regional cerebral blood flow augmentation during hypercapnia in type III NOS mutant mice.

The effect of NG-nitro-L-arginine (L-NNA) on regional cerebral blood flow (rCBF) response to hypercapnia (5% CO2 inhalation) was studied in urethan-anesthetized wild-type (SV-129) and type III nitric oxide (NO) synthase (NOS)-deficient mice, using laser-Doppler flowmetry and the closed cranial window technique. Resting rCBF during normocapnia decreased by approximately 25% after L-NNA superfusion in wild-type mice only (n = 18), suggesting a role for type III NOS in baseline blood flow. Hypercapnia augmented rCBF approximately 50% in both wild-type and type III NOS mutant mice. L-NNA superfusion (1 mM) inhibited this increase by approximately 60% in both strains. Hence, synthesis of NO by the constitutively expressed type I NOS contributes to blood flow augmentation during hypercapnia.

Genetic analysis of nitric oxide synthase isoforms: targeted mutation in mice.

Since the discovery that nitric oxide is an endogenous vasodilator responsible for endothelium-derived relaxing factor activity, nitric oxide has been found in many different cell types and implicated in many diverse biological processes. Because pharmacological blockade does not distinguish between the three major isoforms of nitric oxide synthase, the tissue and enzyme source of nitric oxide is unclear in many situations. Targeted disruption of the genes for the various isoforms of nitric oxide synthase offers a useful genetic approach to study the roles of each isoform and to examine the effects of their deletion on physiological processes in intact animals. Here we review the phenotypes of the various nitric oxide synthase mutant mice and examine what they reveal about the complexities of the nitric oxide signaling system and about molecular and physiological compensations brought into play in the absence of individual isoforms.

L-NA-sensitive rCBF augmentation during vibrissal stimulation in type III nitric oxide synthase mutant mice.

Regional cerebral blood flow (rCBF) was studied in type III nitric oxide (NO) synthase (endothelial, eNOS) mutant and wild type mice during mechanical whisker stimulation before and after nitro-L-arginine (L-NA) superfusion using the closed cranial window technique. rCBF increased equally in cortical barrel fields in both strains during stimulation, as measured by laser Doppler-flowmetry, and was inhibited by L-NA superfusion (1 mM) in both groups. Hence, coupling of blood flow and metabolism appears neuronal NOS-(nNOS) but not eNOS-dependent in cortical barrel fields of the mouse.