In eNOS knockout mice skeletal muscle arteriolar dilation to acetylcholine is mediated by EDHF

The mechanisms that account for acetylcholine (ACh)-induced responses of skeletal muscle arterioles of mice lacking endothelial nitric oxide (NO) synthase (eNOS-KO) were investigated. Isolated, cannulated, and pressurized arterioles of gracilis muscle from male eNOS-KO (74.1 +/- 2.3 microm) and wild-type (WT, 87.2 +/- 2.1 microm) mice developed spontaneous tone accounting for 63 and 61% of their passive diameter (116.8 +/- 3.4 vs. 143.2 +/- 2.8 microm, respectively) and dilated dose-dependently to ACh (10(-9)-10(-7) M). These dilations were significantly smaller in vessels of eNOS-KO compared with WT mice (29.2 +/- 2.0 microm vs. 46.3 +/- 2.1 microm, at maximum concentration) but responses to the NO donor, sodium nitrite (NaNO(2), 10(-6)-3 x 10(-5) M), were comparable in the vessels of the two strains. N(G)-nitro-L-arginine (L-NNA, 10(-4) M), an inhibitor of eNOS, inhibited ACh-induced dilations by 60-90% in arterioles of WT mice but did not affect responses in those of eNOS-KO mice. In arterioles of eNOS-KO mice, dilations to ACh were not affected by indomethacin but were essentially abolished by inhibitors of cytochrome P-450, clotrimazole (CTZ, 2 x 10(-6) M) or miconazole (MCZ, 2 x 10(-6) M), as well as by either high K(+) (40 mM) or iberiotoxin [10(-7) M, a blocker of Ca(2+)-dependent K(+) channels (K(Ca) channels)]. On the other hand, in WT arterioles CTZ or MCZ inhibited ACh-induced dilations only by approximately 10% and only in the presence of L-NNA. These results indicate that in arterioles of eNOS-KO mice, endothelium-derived hyperpolarizing factor (EDHF), synthesized via cytochrome P-450, accounts entirely for the mediation of ACh-induced dilation via an increase in K(Ca)-channel activity. In contrast, in arterioles of WT mice, endothelium-derived NO predominantly mediates ACh-induced dilation in which participation of EDHF becomes apparent only after inhibition of NO synthesis.     

Impairment of endothelial nitric oxide synthase causes abnormal fat and glycogen deposition in liver

Nitric oxide (NO) affects fatty acid synthesis and biogenesis of fatty acid consuming mitochondria. However, whether NO generated by the endothelial NO synthase isoform (eNOS) has significant impact on the synthesis and deposition of fat in liver remained unclear. We analyzed the quantity and distribution of mitochondria and fat in liver of wild-type (WT) mice and mice lacking eNOS (eNOS-KO). The livers of eNOS-KO mice contained tenfold more fat close (zone 1) and twenty fold more distal (zone 3) to the artery. The fat was deposited as droplets co-localized with mitochondria. Additionally, the livers of eNOS-KO mice contained 1.5-fold more homogenously distributed glycogen. No difference in the quantity of mitochondria was found between liver homogenates of eNOS-KO mice and WT animals. Mitochondria from liver homogenates of eNOS-KO mice exhibited a higher ratio of citrate synthase (CS) and NADH-cytochrome c oxidoreductase (KI+III) activity. We conclude that lack of eNOS-derived NO stimulates citrate- and lipid synthesis in liver thus contributing to the development of overweight. In support of this view, more visceral fat and 70% higher body weight was determined in one year old eNOS-KO mice in comparison to WT animals.     

Effects of nitric oxide synthase inhibitors on porcine oocyte meiotic maturation

As an important biological messenger, nitric oxide (NO) exhibits a wide range of effects during physiological and pathophysiological processes, including mammalian oocyte meiotic maturation. The present study investigated whether NO derived from two nitric oxide synthase (NOS) isoforms, inducible NOS (iNOS) or endothelial NOS (eNOS), is involved in the meiotic maturation of porcine oocytes. Meanwhile, the cumulus cells' function in meiotic maturation and their interaction with oocyte development and degeneration were also investigated using cumulus-enclosed oocytes (CEOs) and denuded oocytes (DOs). Different inhibitors for NOS were supplemented to the medium. Cumulus expansion, cumulus cell DNA fragmentation and oocyte meiotic resumption were evaluated 48 h after incubation. Aminoguanidine (AG), a selective inhibitor for iNOS, suppressed cumulus expansion and inhibited CEOs to resume meiosis (p < 0.05), but did not inhibit cumulus cell DNA fragmentation. Both Nomega-nitro-L-arginine (L-NNA) and Nomega-nitro-L-arginine methyl ester (L-NAME), inhibitors for both iNOS and eNOS, delayed cumulus expansion, inhibited cumulus cell DNA fragmentation and inhibited CEOs to resume meiosis. Such effects were not seen in DOs. These results indicate that iNOS-derived NO is necessary for cumulus expansion and meiotic maturation by mediating the function of the surrounding cumulus cells, and eNOS-derived NO is also involved in porcine meiotic maturation.     

Activation of the cGMP signaling pathway is essential in delaying oocyte aging in diabetes mellitus

Uncontrolled diabetes mellitus (DM) adversely affects oocyte maturation and embryo development via mechanisms that are yet unclear. Nonetheless, DM may cause uncoupling of nitric oxide synthases (NOSs) with reduction in the bioavailability of nitric oxide (NO), which is critical to maintain oocyte viability and prevent aging. The current study investigates the role of NO-mediated signaling related to oocyte aging in diabetic and nondiabetic mice. Age-related alterations in the oocytes, including ooplasmic microtubule dynamics (OMD), cortical granule (CG) status, and zona pellucida (ZP) hardening as well as the integrity of the spindle/chromatin were studied using confocal microscopy. Oocytes obtained from diabetic mice exhibited accelerated aging compared to that from nondiabetic mice. Moreover, oocytes from diabetic animals were exquisitely sensitive to NOS and guanylate cyclase (GC) inhibitors (L-NAME, ODQ), which induced aging and relatively resistant to its delay by the cGMP derivative (8-Br-cGMP). Oocytes from nondiabetic control mice displayed similar sensitivity to L-NAME in older oocytes, although to a significantly lower extent than that of DM (P < 0.04-0.0001). Despite the differences in response between DM and nonDM mice, the activation of cGMP pathway is essential to maintain the integrity of oocytes and delay oocyte aging. These findings not only indicate the role of NO signaling in the prevention of oocyte aging but also suggest enhanced aging and NO insufficiency in oocytes from diabetic mice. A comprehensive model incorporating our current findings with NOS, GC, and G kinase cycles is presented.     

Effect of inhibition of synthesis of inducible nitric oxide synthase-derived nitric oxide by aminoguanidine on the in vitro maturation of oocyte-cumulus complexes of cattle

The aim of the present study was to investigate the effects of inhibition of the enzyme inducible nitric oxide synthase (iNOS) by aminoguanidine (AG) on the in vitro maturation of oocyte-cumulus cell complex(es) (COC) of cattle. COC were cultured with different concentrations of AG (0, 1, 10, and 100mM) for 24h. In Experiment 1, the extent of cumulus complex expansion, nuclear maturation status and plasma membrane integrity of oocytes and cumulus cells from each treatment were assessed. Nitrate/nitrite (NO(3)(-)/NO(2)(-)) concentrations were determined in culture medium by the Griess method. Addition of different concentrations of AG to maturation medium promoted a dose-response inhibitory effect on cumulus expansion (P<0.05). Addition of 1 and 10mM AG to IVM medium did not affect plasma membrane integrity of oocytes or nuclear maturation rates (P>0.05), but it did reduce plasma membrane integrity in cumulus cells. One hundred millimolar inhibited pre-metaphase I (pre-MI) to metaphase II (MII) transition, promoted plasma membrane damage in oocytes (P<0.05), and increased NO(3)(-)/NO(2)(-) concentration when compared to controls (P<0.05). To evaluate if this effect was reversible, 10(-5)M sodium nitroprusside (SNP, NO donor) was added, only in the treatment with 100mM AG that inhibited the nuclear maturation. However, association of 10(-5)M SNP to 100mM AG did not reverse the effects of AG, but increased NO(3)(-)/NO(2)(-)concentration (P<0.05). In Experiment 2, the effect of different AG concentrations on cytoplasmic maturation in vitro was assessed based on cortical granule migration, and embryonic development. There was a dose effect on cortical granule migration rate, in which 1mM AG (83.9+/-6.2%) did not differ from control oocytes (83.6+/-8.2%; P>0.05), but 10mM partially inhibited migration (3.8+/-6.4%) and 100mM totally inhibited migration (P<0.05). SNP (10(-5)M) did not revert this inhibitory effect on cortical granules migration in oocytes treated with 100mM AG. Only those concentrations that did not inhibit IVM were used to assess cleavage and blastocyst development. Addition of 10mM AG to IVM medium reduced (73.0+/-8.1%, 15.0+/-8.9%; P<0.05) cleavage and blastocyst development, respectively when compared with controls (89.1+/-3.4%, 37.6+/-7.3%, respectively), but did not differ, (P>0.05), from the group treated with 1mM AG (80.9+/-8.4%, 41.5+/-10.5%, respectively). The results from the present study demonstrate that NO derived from iNOS affects the in vitro maturation of bovine COC, modulating the viability of cumulus cells and of oocyte, the progression of meiosis after GVBD, the migration of cortical granules, and cleavage and blastocyst development.     

Requirement of nitric oxide for murine oocyte maturation, embryo development, and trophoblast outgrowth in vitro

We investigated the extent to which NO participates in the developmental competence (oocyte maturation, fertilization and embryo development to blastocyst) using an in vitro culture system adding sodium nitroprusside (SNP), NO donor, and NOS inhibitor (N-omega-nitro-L-arginine methyl ester, L-NAME). We also assessed the effects of NO/NOS system on blastocyst implantation using an in vitro trophoblast outgrowth assay. The treatment of low concentrations of SNP (10(-7) M) significantly stimulated meiotic maturation to metaphase II stages in cumulus enclosed oocytes. In contrast, 10(-3) and 10(-5) M L-NAME demonstrated a significant suppression in resumption of meiosis. This inhibition was reversed by the addition of SNP. No development beyond the four-cell stage was observed by the addition of high concentration of SNP (10(-3) M). Inhibition of embryo development, especially the conversion of morulae to blastocysts, was also observed in the treatment of lower doses of SNP (10(-5) and 10(-7) M). Similarly, inhibition of NO by NOS inhibitor resulted in the dose-dependent inhibition of embryo development and hatching rates, but the concomitant addition of SNP with L-NAME reversed the inhibitory effect by each SNP or L-NAME treatment. Furthermore, low concentration of SNP (10(-7) M) but not high concentration of SNP (10(-3) M) significantly stimulated trophoblast outgrowth, whereas the addition of L-NAME suppressed the spreading of blastocysts in a dose-dependent manner. These results suggest that NO may have crucial roles in oocyte maturation and embryogenesis including the process of implantation. The observed differences in required amount of NO and the sensitivity to cytotoxicity of NO in each developmental stage embryos may also suggest that NO/NOS system is tightly regulated in developmental stage specific manner.     

Endothelial and inducible nitric oxide synthases in oocytes of cattle

Nitric oxide (NO) is a chemical messenger generated by the activity of the nitric oxide synthases (NOS). The NOS/NO system appears to be involved in oocyte maturation, but there are few studies on gene expression and protein activity in oocytes of cattle. The present study aimed to investigate gene expression and protein activity of NOS in immature and in vitro matured oocytes of cattle. The influence of pre-maturation culture with butyrolactone I in NOS gene expression was also assessed. The following experiments were performed: (1) detection of the endothelial (eNOS) and inducible (iNOS) isoforms in the ovary by immunohistochemistry; (2) detection of eNOS and iNOS in the oocytes before and after in vitro maturation (IVM) by immunofluorescence; (3) eNOS and iNOS mRNA and protein in immature and in vitro matured oocytes, with or without pre-maturation, by real time PCR and Western blotting, respectively; and (4) NOS activity in immature and in vitro matured oocytes by NADPH-diaphorase. eNOS and iNOS were detected in oocytes within all follicle categories (primary, secondary and tertiary), and other compartments of the ovary and in the cytoplasm of immature and in vitro matured oocytes. Amount of mRNA for both isoforms decreased after IVM, but was maintained after pre-maturation culture. The NOS protein was detected in immature (pre-mature or not) and was still detected in similar amount after pre-maturation and maturation for both isoforms. NOS activity was detected only in part of the immature oocytes. In conclusion, isoforms of NOS (eNOS and iNOS) are present in oocytes of cattle from early folliculogenesis up to maturation; in vitro maturation influences amount of mRNA and NOS activity.     

Chromosomal analysis of mammalian oocytes matured in vitro with various culture systems

The objective of this study was to evaluate oocyte maturation in vitro. Ten virgin CD-1 mice were used with 3 replications for in vitro with 4 different culture media. Media were minimal essential medium (MEM) with Earl's salt, Waymouth MB 752/1 (MB 752/1), BGjb medium (BGjb), and tissue culture medium-199 (TCM-199). The oocyte chromosomes were C-banded to enable an objective analysis of the chromosome abnormality and number. There was a percentage of blockage at metaphase I (M I), in matured oocytes in all culture media. Metaphase II (M II) was reached by 70.9 to 87.3% of oocytes in 4 different culture media. The frequencies of hyperploid M II oocytes were 0.0, 1.1, 2.8 and 2.6% for TCM-199, MEM, MB 752/1 and BGjb, respectively. A small proportion of oocytes was also found to be polyploid in 4 different culture media. There was an occurrence of premature centromere separation among oocytes. It was concluded that the chromosomes of the oocytes matured in vitro were not all in the normal condition (being at M II). The media used in this study for oocyte maturation caused maturation delay (being blocked at M I), premature centromere separation, polyploidy, and aneuploidy (such as, hyperploid, hypoploid).     

Effect of time during transport of excised mare ovaries on oocyte recovery rate and quality after in vitro maturation

In the mare only a limited number of oocytes can be successfully collected in vivo, so that when large numbers of oocytes are needed for experimentation, ovaries harvested from slaughtered mares must be used. The resulting temperature changes and time intervals mandated by handling and transport of ovaries from the slaughterhouse to the laboratory adversely affect the rate of oocyte recovery and their quality after IVF and maturation. We chose to study the effect of temperature and time in transit of excised ovaries by evaluating rate of oocyte recovery, nuclear maturation stage reached before, and cleavage rate reached after IVF, following short (1.5 to 4 h) and long (6 to 8 h) storage. Temperatures in the storage container decreased from 37-C to 32 degrees and 27.5 degrees C during the short and long interval, respectively. The cumulus-oocytes complexes (COCs) were classified as having a compact cumulus, completely or partially surrounding the oocyte (compact); those having only a corona radiata surrounding the oocyte (corona); those having a completely or partially expanded cumulus, showing a cellular or sparsely cellular, gelatinous cloud around the oocyte (expanded); and those that were completely denuded of both cumulus and corona cells (denuded). All COCs, except the denuded ones, which were discarded, were matured in vitro for 30 h at 38.5 degrees C in 5% CO2. The recovery rate of oocytes was significantly higher after long vs short storage (48 vs 35%; P < 0.01), but the distribution of the collected COCs into the 4 classes was not affected by the storage time. After in vitro maturation nuclear maturity was not affected by the storage time, but oocytes with intact cytoplasmic membranes were more frequently found after short than after long storage (54 vs 34%; P = 0.07), and fully matured oocytes were more often seen with intact membrane (P < 0.01). Moreover, oocytes with intact membranes in metaphase II (MII) were associated with short storage intervals and the corona COC class, while damaged membranes and incomplete maturation were associated with the long storage and the compact COC class.     

EDHF mediates flow-induced dilation in skeletal muscle arterioles of female eNOS-KO mice

Vasodilation to increases in flow was studied in isolated gracilis muscle arterioles of female endothelial nitric oxide synthase (eNOS)-knockout (KO) and female wild-type (WT) mice. Dilation to flow (0-10 microl/min) was similar in the two groups, yet calculated wall shear stress was significantly greater in arterioles of eNOS-KO than in arterioles of WT mice. Indomethacin, which inhibited flow-induced dilation in vessels of WT mice by approximately 40%, did not affect the responses of eNOS-KO mice, whereas miconazole and 6-(2-proparglyoxyphenyl)hexanoic acid (PPOH) abolished the responses. Basal release of epoxyeicosatrienonic acids from arterioles was inhibited by PPOH. Iberiotoxin eliminated flow-induced dilation in arterioles of eNOS-KO mice but had no effect on arterioles of WT mice. In WT mice, neither N(omega)-nitro-L-arginine methyl ester nor miconazole alone affected flow-induced dilation. Combination of both inhibitors inhibited the responses by approximately 50%. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) alone inhibited flow-induced dilation by approximately 49%. ODQ + indomethacin eliminated the responses. Thus, in arterioles of female WT mice, nitric oxide and prostaglandins mediate flow-induced dilation. When eNOS is inhibited, endothelium-derived hyperpolarizing factor substitutes for nitric oxide. In female eNOS-KO mice, metabolites of cytochrome P-450, via activation of large-conductance Ca2+-activated K+ channels of smooth muscle, mediate entirely the arteriolar dilation to flow.     

Capacity of adult and prepubertal mouse oocytes to undergo embryo development in the presence of cysteamine

The present study was carried out to study de novo glutathione (GSH) synthesis and to evaluate the effect of stimulating GSH synthesis during in vitro maturation (IVM) of adult and prepubertal mouse oocytes on the embryo developmental rate. Adult (8 weeks old) and prepubertal mice (24-26 days old) were primed with 5 IU of PMSG and oocytes were retrieved from the ovary 48 hr later for IVM. After IVM (18 hr) Cumulus oocyte complexes (COC) were in vitro fertilized (IVF) and in vitro culture (IVC) in order to observe embryo development. The IVM medium was supplemented with: 0, 25, 50, 100, or 200 microM of cysteamine. To study the novo GSH synthesis, 5 mM BSO was added during IVM of adult or prepubertal oocyte. Developmental rates up to blastocyst were recorded for each group. Experiments also included a group of ovulated oocytes (in vivo matured) after priming with PMSG and HCG. After IVM of adult mice oocytes, an improvement was observed on embryo development in all the supplemented groups when compared with the untreated group (P < 0.05). No differences were observed in blastocyst rate among IVM oocytes with cysteamine and ovulated oocytes. Prepubertal IVM mouse oocytes had a lower cleavage rate compared with ovulated oocytes (P < 0.05). Cysteamine failed to improve prepubertal oocytes developmental rates (P > 0,05). 2-cell embryos, coming from IVM prepubertal oocytes and ovulated oocytes had the same preimplantation developmental rate up to the blastocyst stage. In prepubertal, and adult oocytes an inhibition of embryo development was observed when buthionine sulfoximide (BSO), a specific inhibitor of the gamma-glutamylcysteine synthetase, was added during oocyte maturation (P < 0.01). In conclusion, an improvement in mouse embryo development was observed when cysteamine was added to the IVM medium of adult mice oocytes. In prepubertal oocytes cysteamine addition during oocyte maturation failed to improve embryo developmental rates. The presence of BSO lowered or completely blocked blastocyst development. This proves that, de novo GSH synthesis during oocyte maturation of adult and prepubertal oocytes undoubtedly plays an important role in embryo development. The improvement on oocyte competence observed in adult mice oocytes is probably related to intracellular GSH synthesis stimulated by cysteamine. Nevertheless the reason why cysteamine failed to improve prepubertal oocytes competence remains as an open question.     

Distinct subtypes of zona pellucida morphology reflect canine oocyte viability and cumulus-oocyte complex quality

The aim of this study was to analyze surface morphology of the zona pellucida (ZP) and assess its relationship with oocyte viability, cumulus-oocyte complex (COC) quality, and oocyte donor age in dogs. Canine ovaries were sliced to release COCs for use in three experiments. In Experiment 1, oocytes from high-quality (grade I) COCs were viewed with scanning electron microscopy to visualize the zona surface. Four zonae, classified as types I, II, III, and IV, were detectable on high-quality oocytes. Most (95.5%) dog donors had oocytes with two or three ZP types. The ZP type I had a smooth compact surface with few pores. The ZP type II was less compact with many distinct circular or elliptical pores. The ZP type III had a rough surface with folds and many irregular shaped pores and hollows. The ZP type IV also had a rough surface with folds, but in addition, stringy filaments obscured the pores and hollows. The frequency of ZP type I in the oocyte population was low (2.7%), whereas ZP types II, III, and IV each occurred in approximately one-third of the oocyte population. In Experiment 2, oocytes from high-quality COCs were stained with propidium iodide (PI) before scanning electron microscopy to investigate the relationship of oocyte viability with ZP morphology. In Experiment 3, oocytes were collected from low-quality (grade 2) and high-quality (grade 1) COCs to investigate the role of COC quality on zona structure. Zonae types I and II were characteristic of PI-positive (dead) oocytes and oocytes from low-quality COCs, whereas ZP types III and IV were prevalent on PI-negative (living) oocytes and oocytes from high-quality COCs. We concluded that the heterogeneous ZP surface underwent structural rearrangements related to oocyte viability and COC quality. This warrants further investigation into ZP structure and may be useful for canine-assisted reproduction.     

Development of porcine embryos reconstituted with somatic cells and enucleated metaphase I and II oocytes matured in a protein-free medium

Background

Many cloned animals have been created by transfer of differentiated cells at G0/G1 or M phase of the cell cycle into enucleated M II oocytes having high maturation/meiosis/mitosis-promoting factor activity. Because maturation/meiosis/mitosis-promoting factor activity during oocyte maturation is maximal at both M I and M II, M I oocytes may reprogram differentiated cell nuclei as well. The present study was conducted to examine the developmental ability in vitro of porcine embryos reconstructed by transferring somatic cells (ear fibroblasts) into enucleated M I or M II oocytes.

Results

Analysis of the cell cycle stages revealed that 91.2 ± 0.2% of confluent cells were at the G0/G1 phase and 54.1 ± 4.4% of nocodazole-treated cells were at the G2/M phase, respectively. At 6 h after activation, nuclear swelling was observed in 50.0-88.9% and 34.4-39.5% of embryos reconstituted with confluent cells and nocodazole-treated cells regardless of the recipient oocytes, respectively. The incidence of both a swollen nucleus and polar body was low (6.3-10.5%) for all nocodazole-treated donor cell regardless of the recipient oocyte. When embryos reconstituted with confluent cells and M I oocytes were cultured, 2 (1.5%) blastocysts were obtained and this was significantly (P < 0.05) lower than that (7.6%) of embryos produced by transferring confluent cells into M II oocytes. No reconstructed embryos developed to the blastocyst stage when nocodazole-treated cells were used as donors.

Conclusions

Porcine M I oocytes have a potential to develop into blastocysts after nuclear transfer of somatic cells.     

Renal cortical and medullary blood flow responses to L-NAME and ANG II in wild-type, nNOS null mutant, and eNOS null mutant mice

Experiments in wild-type (WT; C57BL/6J) mice, endothelial nitric oxide synthase null mutant [eNOS(-/-)] mice, and neuronal NOS null mutant [nNOS(-/-)] mice were performed to determine which NOS isoform regulates renal cortical and medullary blood flow under basal conditions and during the infusion of ANG II. Inhibition of NOS with N(omega)-nitro-l-arginine methyl ester (l-NAME; 50 mg/kg iv) in Inactin-anesthetized WT and nNOS(-/-) mice increased arterial blood pressure by 28-31 mmHg and significantly decreased blood flow in the renal cortex (18-24%) and the renal medulla (13-18%). In contrast, blood pressure and renal cortical and medullary blood flow were unaltered after l-NAME administration to eNOS(-/-) mice, indicating that NO derived from eNOS regulates baseline vascular resistance in mice. In subsequent experiments, intravenous ANG II (20 ng x kg(-1) x min(-1)) significantly decreased renal cortical blood flow (by 15-25%) in WT, eNOS(-/-), nNOS(-/-), and WT mice treated with l-NAME. The infusion of ANG II, however, led to a significant increase in medullary blood flow (12-15%) in WT and eNOS(-/-) mice. The increase in medullary blood flow following ANG II infusion was not observed in nNOS(-/-) mice, in WT or eNOS(-/-) mice pretreated with l-NAME, or in WT mice administered the nNOS inhibitor 5-(1-imino-3-butenyl)-l-ornithine (1 mg x kg(-1) x h(-1)). These data demonstrate that NO from eNOS regulates baseline blood flow in the mouse renal cortex and medulla, while NO produced by nNOS mediates an increase in medullary blood flow in response to ANG II.     

Nitric Oxide Synthase (NOS) Inhibition during Porcine In Vitro Maturation Modifies Oocyte Protein S-Nitrosylation and In Vitro Fertilization

Nitric oxide (NO) is a molecule involved in many reproductive processes. Its importance during oocyte in vitro maturation (IVM) has been demonstrated in various species although sometimes with contradictory results. The objective of this study was to determine the effect of NO during IVM of cumulus oocyte complexes and its subsequent impact on gamete interaction in porcine species. For this purpose, IVM media were supplemented with three NOS inhibitors: NG-nitro-L-arginine methyl ester (L-NAME), NG-monomethyl-L-arginine (L-NMMA) and aminoguanidine (AG). A NO donor, S-nitrosoglutathione (GSNO), was also used. The effects on the cumulus cell expansion, meiotic resumption, zona pellucida digestion time (ZPdt) and, finally, on in vitro fertilization (IVF) parameters were evaluated. The oocyte S-nitrosoproteins were also studied by in situ nitrosylation. The results showed that after 42 h of IVM, AG, L-NAME and L-NMMA had an inhibitory effect on cumulus cell expansion. Meiotic resumption was suppressed only when AG was added, with 78.7% of the oocytes arrested at the germinal vesicle state (P<0.05). Supplementation of the IVM medium with NOS inhibitors or NO donor did not enhance the efficiency of IVF, but revealed the importance of NO in maturation and subsequent fertilization. Furthermore, protein S-nitrosylation is reported for the first time as a pathway through which NO exerts its effect on porcine IVM; therefore, it would be important to determine which proteins are nitrosylated in the oocyte and their functions, in order to throw light on the mechanism of action of NO in oocyte maturation and subsequent fertilization.     

Endothelial nitric oxide synthase is predominantly involved in angiotensin II modulation of renal vascular resistance and norepinephrine release

Nitric oxide (NO) is mainly generated by endothelial NO synthase (eNOS) or neuronal NOS (nNOS). Recent studies indicate that angiotensin II generates NO release, which modulates renal vascular resistance and sympathetic neurotransmission. Experiments in wild-type [eNOS(+/+) and nNOS(+/+)], eNOS-deficient [eNOS(-/-)], and nNOS-deficient [nNOS(-/-)] mice were performed to determine which NOS isoform is involved. Isolated mice kidneys were perfused with Krebs-Henseleit solution. Endogenous norepinephrine release was measured by HPLC. Angiotensin II dose dependently increased renal vascular resistance in all mice species. EC(50) and maximal pressor responses to angiotensin II were greater in eNOS(-/-) than in nNOS(-/-) and smaller in wild-type mice. The nonselective NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 0.3 mM) enhanced angiotensin II-induced pressor responses in nNOS(-/-) and wild-type mice but not in eNOS(-/-) mice. In nNOS(+/+) mice, 7-nitroindazole monosodium salt (7-NINA; 0.3 mM), a selective nNOS inhibitor, enhanced angiotensin II-induced pressor responses slightly. Angiotensin II-enhanced renal nerve stimulation induced norepinephrine release in all species. L-NAME (0.3 mM) reduced angiotensin II-mediated facilitation of norepinephrine release in nNOS(-/-) and wild-type mice but not in eNOS(-/-) mice. 7-NINA failed to modulate norepinephrine release in nNOS(+/+) mice. (4-Chlorophrnylthio)guanosine-3', 5'-cyclic monophosphate (0.1 nM) increased norepinephrine release. mRNA expression of eNOS, nNOS, and inducible NOS did not differ between mice strains. In conclusion, angiotensin II-mediated effects on renal vascular resistance and sympathetic neurotransmission are modulated by NO in mice. These effects are mediated by eNOS and nNOS, but NO derived from eNOS dominates. Only NO derived from eNOS seems to modulate angiotensin II-mediated renal norepinephrine release.     

The mycotoxin beauvericin induces oocyte mitochondrial dysfunction and affects embryo development in the juvenile sheep

Beauvericin (BEA) is a mycotoxin produced by Beauveria bassiana and Fusarium species recently reported as toxic on porcine oocyte maturation and embryo development. The aim of this study was to assess, in the juvenile sheep, whether its effects are due to alterations of oocyte and/or embryo bioenergetic/oxidative status. Cumulus‐oocyte‐complexes (COCs) were exposed to BEA during in vitro maturation (IVM), evaluated for cumulus cell (CC) apoptosis, oocyte maturation and bioenergetic/oxidative status or subjected to in vitro fertilization (IVF) and embryo culture (IVEC). Oocyte nuclear maturation and embryo development were assessed after Hoechst staining and CC apoptosis was analysed by terminal deoxynucleotidyl transferase‐mediated dUTP nick‐End labeling assay and chromatin morphology after Hoechst staining by epifluorescence microscopy. Oocyte and blastocyst bioenergetic/oxidative status were assessed by confocal microscopy after mitochondria and reactive oxygen species labelling with specific probes. BEA showed various toxic effects, that is, short‐term effects on somatic and germinal compartment of the COC (CCs and the oocyte) and long‐term carry‐over effects on developing embryos. In detail, at 5 µM, it significantly reduced oocyte maturation and immature oocytes showed increased late‐stage (Type C) CC apoptosis and DNA fragmentation while matured oocytes showed unaffected CC viability but abnormal mitochondrial distribution patterns. At lower tested concentrations (3–0.5 µM), BEA did not affect oocyte maturation, but matured oocytes showed reduced mitochondrial activity. At low concentrations, BEA impaired embryo developmental capacity and blastocyst quality after IVF and IVEC. In conclusion, in the juvenile sheep, COC exposure to BEA induces CC apoptosis and oocyte mitochondrial dysfunction with negative impact on embryo development.     

Survival of oocytes recovered from vitrified sheep ovarian tissues

The objective of this work was to develop an effective vitrification technique for cryopreserving oocytes in sheep ovarian tissues. Ovaries were surgically recovered from 15 pubertal ewes and the ovarian cortex was cut into sections. Ovarian tissues were placed in equilibration medium consisting of 4% (v/v) ethylene glycol (EG) and 20% (v/v) FBS in TCM-199 on ice for 30 min and transferred to vitrification solution (35% EG, 5% polyvinylpyrrolidone, 0.4M trehalose and 20% FBS in TCM-199) for 5 min. Ovarian tissues were vitrified by dropping the tissue on the surface of a steel cube cooled by liquid nitrogen. Cumulus-enclosed oocyte complexes (COC) were also collected and vitrified following the procedure used for ovarian tissues. After 2-3 weeks of storage in liquid nitrogen, ovarian tissues and COC were thawed at 37 degrees C in 0.3M trehalose and COC in ovarian tissues were mechanically and enzymatically isolated. Vitrified COC and freshly collected COC were washed twice in maturation medium (TCM-199 supplemented with 0.255 mM pyruvate and 10% heat-treated estrus cow serum) and cultured in 50 microl drops of maturation medium under paraffin oil for 23-25h at 39 degrees C in a humidified atmosphere of 5% CO(2) in air. After culture, cumulus cells were removed by hyaluronidase treatment and vortexing and oocytes were fixed and stained. No significant differences were observed between vitrified oocytes, oocytes recovered from vitrified ovarian tissues and non-vitrified control oocytes in the percentage of oocytes with acceptable staining per total number of oocytes fixed or with visible chromatin per total number of oocytes with acceptable staining. However, fewer (P<0.05) oocytes obtained from vitrified ovarian tissues (70%) reached metaphase II compared to vitrified oocytes (88%) and non-vitrified control oocytes (90%). In contrast, when oocytes with at least 3-5 layers of cumulus cells were considered from each of the three groups, no differences (P>0.05) were observed due to treatment in the percentages of oocytes developing to metaphase II. These results demonstrate that sheep oocytes can be successfully cryopreserved by vitrification of ovarian tissues and exhibit in vitro maturation rates similar to that of vitrified and non-vitrified oocytes.     

Nitric oxide extends the oocyte temporal window for optimal fertilization

Deteriorating oocyte quality is a critical hurdle in the management of infertility, especially one associated with advancing age. In this study, we explore the role of nitric oxide (NO) on the sustenance of oocyte quality postovulation. Sibling oocytes from superovulated mice were subjected to intracytoplasmic sperm injection (ICSI) with cauda-epididymal spermatozoa following exposure to either the NO donor, S-nitroso-N-acetylpenicillamine (SNAP, 0.23 microM/min), an NO synthase (NOS) inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME, 1 mM), or an inhibitor of soluble guanylyl cyclase (sGC), 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ, 100 microM); while their sibling oocytes were subjected to ICSI either before (young) or after culture for the corresponding period of time (old). Outcomes of normal fertilization, cleavage, and development to the morula and blastocyst stages were compared. Embryos from each subgroup were also subjected to TUNEL assay for apoptosis. A significant deterioration in the ability of the oocytes to undergo normal fertilization and development to morula and blastocyst stages occurred among oocytes aged in culture medium compared to their sibling cohorts subjected to ICSI immediately after ovulation (P<0.05). This deterioration was prevented in oocytes exposed to SNAP. In contrast, exposure to L-NAME or ODQ resulted in a significant compromise in fertilization and development to the morula and blastocyst stages (P<0.05). Finally, apoptosis was noted in embryos derived from aged oocytes and those exposed to L-NAME or ODQ, but not in embryos derived from young oocytes or oocytes exposed to SNAP. Thus, NO is essential for sustenance of oocyte quality postovulation.