The authors have declared that no competing interests exist.
Conceived and designed the experiments: JR-A CM. Performed the experiments: JR-A APS CM. Analyzed the data: JR-A APS FAG-V CM. Contributed reagents/materials/analysis tools: JR-A FAG-V PC CM. Wrote the paper: JR-A FAG-V PC CM.
Nitric oxide (NO) is a molecule involved in many reproductive processes. Its importance during oocyte
One of the problems that affect in vitro fertilization (IVF) in mammals is polyspermy
A key role in regulating oocyte maturation has been demonstrated for nitric oxide (NO)
In contrast to many other molecules whose signaling mechanisms and biological effects have been studied for many years, the NO-signaling processes have only recently begun to be studied. Despite its molecular simplicity, NO acts as a biological signal in a number of ways
Nitric oxide plays a dual role in reproduction, depending on its concentration. At low concentrations it stimulates or enhances early reproductive events, but both an excess and a lack of NO have negative consequences
The literature contains several studies on the effect of NO on oocyte maturation. However, in porcine species, such studies are very limited and do not take into account the repercussions on IVF parameters. IVM in pig is a long process, during which free radicals are generated
Furthermore, NO has the potential to induce protein S-nitrosylation
This study was developed following institutional approval from the Bioethics Committee of the University of Murcia, and was performed in accordance with the Animal Welfare regulations of that institution.
Unless otherwise stated, chemicals and reagents were purchased from Sigma-Aldrich Química S.A. (Madrid, Spain). Equine chorionic gonadotropin (eCG; Foligon) was supplied by Intervet International BV (Boxmeer, Holland), human chorionic gonadotropin (hCG; VeterinCorion) by Divasa Farmavic (Barcelona, Spain) and Percoll by GE Healthcare (Uppsala, Sweden). Texas Red-2-sulphonamidoethyl methanethiosulphonate (MTSEA-Texas Red) was obtained from Toronto Research Chemicals (North York, Ontario, Canada) and the prolonged antifade mounting medium (SlowFadeAntifade Kit) by Invitrogen (Paisley, United Kingdom). NG-nitro-L-arginine methyl ester (L-NAME; 483125) and NG-monomethyl-L-arginine (L-NMMA; 475886) were purchased from Calbiocherm (distributed by Merck Chemicals, Beeston, Notthingan, UK)
The medium used for the IVM of pig oocytes was NCSU-37
The basic medium used for pig IVF was TALP medium
Ovaries from Landrace x Large White gilts were collected at a local slaughterhouse (El Pozo Alimentación S.A., Alhama de Murcia, Murcia, Spain) and transported to the laboratory in saline solution containing 100 µg/ml kanamycin sulfate at 38.5°C, washed once in 0.04% cetrimide solution and twice in saline within 30 min of slaughter. Cumulus-oocyte complexes (COCs) were collected from antral follicles (3–6 mm diameter), washed twice with Dulbecco's PBS (DPBS) supplemented with 1 mg/ml PVA and 0.005 mg/ml red phenol, and twice more in maturation medium previously equilibrated for a minimum of 3 h at 38.5°C under 5% CO2 in air. Only COCs with complete and dense cumulus oophorus were used for the experiments. Groups of 50 COCs were cultured in 500 µl maturation medium for 22 h at 38.5°C under 5% CO2 in air. After culture, oocytes were washed twice in fresh maturation medium without dibutyryl cAMP, eCG, and hCG and cultured for an additional period of 20–22 h. The media were supplemented with NOS inhibitors, NO donor or not supplemented, as described below in experimental design.
After removing the CCs, the in vitro matured porcine oocytes with different treatments were washed quickly in DPBS and transferred into drops of 50 µl of 0.5% (w/v) pronase solution in DPBS. ZPs were continuously observed for dissolution under an inverted microscope equipped with a warm plate at 38.5°C (
In vitro matured porcine oocytes, after removing the CCs, were washed and transferred into drops of 0.5% (w/v) pronase solution in DPBS. ZPs were continuously observed for dissolution under an inverted microscope at 38.5°C. Representative pictures of porcine oocytes before (A) and after (B) pronase digestion.
COCs cultured for a total of 44 h in maturation medium from each treatment were stripped of CCs by pipetting and washed three times with TALP medium; groups of 50 oocytes were transferred into each well of a 4-well multidish containing 250 µl IVF medium previously equilibrated at 38.5°C under 5% CO2. The sperm-rich fraction of semen from mature, fertility-tested boars was collected by the gloved hand method, immediately transported to the laboratory and diluted at 1∶8 in Beltsville thawing solution
Oocytes after IVM and putative zygotes were fixed for 15 min (0.5% glutaraldehyde in DPBS), stained for 30 min (1% Hoechst 33342 in DPBS), washed in DPBS containing 1 mg/ml polyvinylpyrrolidone and mounted on glass slides. Oocytes were examined under an epifluorescence microscope at 200X and 400X magnifications.
To visualize the S-nitrosylation of oocyte proteins a method adapted from Lefièvre et al.
All images were taken using the AxioVision imaging system (Rel. 4.8) with an AxioCamHRc camera (Carl Zeiss, Göttingen, Germany) attached to a Leica DMR fluorescence microscope (Leica Microsystems, Wetzlar, Germany) equipped with fluorescent optics (N2.1 filter; excitation BP 515–560 nm). Fluorescence was measured in each oocyte using the Leica QWin image analysis software (Leica Microsystems, Barcelona, Spain) in a blind analysis.
The data are presented as the mean ± standard error of the mean (SEM) and were tested for normality using the Kolmogorov-Smirnov test, and the homogeneity of variance was determined using the Levene test. ANOVA was used for the statistical analysis of COC diameter, ZPdt and IVF parameters and the means were separated using the Tukey test at P<0.05. To assess protein S-nitrosylation, the intensity of fluorescence in each oocyte was measured and transformed into grey values. Since the data did not satisfy the Kolmogorov-Smirnov and Levene tests, the Kruskal-Wallis test was applied, and treatment average ranks were separated using the stepwise step-down multiple comparisons method
To evaluate the effect of the NO synthesis inhibition on porcine oocyte IVM and the subsequent impact on gamete interaction, five experimental groups were used: 1) CONTROL: oocytes matured without treatment; 2) GSNO: oocytes matured with 100 µM GSNO; 3) L-NAME: oocytes matured with 10 mM L-NAME; 4) L-NMMA: oocytes matured with 10mM L-NMMA and 5) AG: oocytes matured with 10 mM AG. These concentrations were chosen based on a literature review. Morphologic criteria were followed to determine whether the given NOS inhibitors or NO donor concentration caused oocyte degeneration. Oocytes with a cytoplasm of irregular appearance and dark areas were classified as degenerated
The three NOS inhibitors and the NO donor were used to assess the effect of NO on COC maturation. To this end, CC expansion, oocyte nuclear maturation and ZP digestion were evaluated.
To evaluate the expansion of CCs, images of COC groups were takenusing a Nikon SMZ-10A stereomicroscope at 10X magnification and COC diameter was measured in each oocyte using the ImageJ software (
To assess nuclear maturation, oocytes from the different experimental groups were denuded, fixed and stained. When the nucleus was in germinal vesicle or metaphase I, the oocyte was considered as immature, while it was considered as mature when the metaphasic plate and polar body were present. This experiment was repeated 5 times with a total of 709 oocytes.
The dissolution time of the ZP of each oocyte was registered as the time between the placement of the oocytes in the pronase solution and the time when the ZP was no longer visible at 200X magnification. This time was referred to as ZPdt. This experiment was repeated 5 times with a total number of 605 oocytes.
The IVF was used as a tool to determine the role of NO during oocyte IVM. For this purpose, 20–25 denuded oocytes for each experimental group were used in each trial. Maturation (%), PEN (percentage of mature oocytes with decondensed sperm heads or male pronuclei in the oolemma), SPZ-ZP, SPZ-OO and MON were assessed in each oocyte. This experiment was repeated 5 times to evaluate a total of 609 oocytes.
To elucidate whether the effect of NO on IVM was through the S-nitrosylated protein pathway, the
Oocytes processed without MMTS (all thiol groups available) were taken as positive control, while, for negative controls oocytes processed without ascorbate (absence of thiol groups) or without MTSEA-Texas Red (no fluorescent labeling) were used.
The addition of the NO donor during IVM did not affect cumulus expansion since COC diameters in the GSNO-treated oocytes did not differ significantly from those of the control group (299.22±7.39 µm in GSNO group vs. 293.60±12.87 µm in control group,
(A) Immature (left) and mature (right) porcine COCs. Images were taken with a Nikon SMZ-10A stereomicroscope (Magnification10X). Scale bar = 250 µm. (B) The average diameter (mean ± SEM in µm, n of COCs) of immature porcine COCs (167.72±2.63, n = 150) was compared to COCs after 44 h in maturation medium under different experimental conditions: CONTROL (293.60±12.87, n = 141), GSNO (299.22±7.39, n = 147), AG (192.17±9.25, n = 149), L-NAME (157.66±3.79, n = 140) and L-NMMA (230.43±6.03, n = 144). The letters a,b,c in different bars denote significant differences (p<0.05).
In contrast, the addition of any of the inhibitors resulted in a significantly lower degree of CCs expansion compared with the control group (192.17±9.25 µm, 157.66±3.79 µm and 230.43±6.03 µm, for AG,L-NAME and L-NMMA inhibitors, respectively P<0.05,
Meiotic resumption was not affected by the treatment with GSNO or the inhibitors L-NAME and L-NMMA (
(A) Histogram showing maturation percentage (dots) and ZPdt (lines) in porcine oocytes after IVM under different experimental conditions. Letters a,b and α,β,γ in different bars denote significant differences (p<0.05). (B) Porcine oocytes were fixed, Hoechst 33342-stained, mounted on glass slides and examined under an epifluorescence microscope at 200X and 400X magnifications. Representative pictures of porcine immature (left) and matured (right) oocytes.
When ZP solubility was assessed a significant increase in ZPdt was observed for all the experimental groups (277.6±9.5 s, 371.8±11.3 s, 304.3±8.1 sand 295.6±10.1 sfor GSNO, L-NAME, AG and L-NMMA, respectively) compared to the control (235.5±8.8 s) (
The only NOS inhibitor significantly affecting the percentage of oocytes penetrated after IVF was L-NAME (
Group | N | Maturation (%) | Penetration (%) | Sperm/OO (n) | Sperm/ZP (n) | Monospermy (%) |
CONTROL | 119 | 96.64±1.66a | 73.04±4.16a | 3.11±0.27 | 25.02±1.83a | 27.38±4.90 |
GSNO | 106 | 94.34±2.26a | 63.00±4.85a,b | 2.67±0.27 | 14.36±1.07b | 36.51±6.11 |
AG | 132 | 18.94±3.42b | 52.00±10.20a,b | 3.31±0.80 | 35.28±5.72c | 46.15±14.39 |
L-NAME | 123 | 91.06±2.58a | 45.54±4.73b | 2.14±0.19 | 12.76±1.45b | 33.33±6.67 |
L-NMMA | 129 | 89.92±2.66a | 61.21±4.54a,b | 2.72±0.32 | 18.79±2.12a,b | 49.30±5.98 |
in the same column denote significant differences (P<0.05).
A significant increase in the intensity of MTSEA-Texas Red labeling was visually observed in oocytes incubated in the presence of GSNO (
Oocytes from the different experimental groups were submitted to
This study was conducted to ascertain whether the inhibition of NO production during maturation could improve IVF parameters. Many authors have written about the role of NO in oocyte maturation and there is much controversy concerning its function. It has been shown how NO is related to cortical granules, meiotic spindles, aging or degeneration, all factors demonstrated to affect the porcine IVF outcome
During oocyte maturation, hyaluronan is synthesized and accumulated by the CCs, remaining embeddedin a gelatinous matrix
NO has been reported to play a dual role in oocyte meiotic maturation in mice, depending on its concentration, although the mechanism by which it influences oocyte maturation has not been fully clarified. Abbasi et al.
By contrast, in the present paper, the addition of AG to the maturation medium prevented cumulus expansion and also inhibited nuclear maturation. However, L-NAME and to a lesser degree, L-NMMA, only inhibited CC expansion and not nuclear or cytoplasmic maturation, as can be deduced from the later ability to form male pronuclei. Different hypotheses can be proposed to explain these results. First, it is known that during the IVM of porcine oocytes the follicle-stimulating hormone (FSH) increases both prostaglandin (PG) E2 production and the expression levels of EGF-like factors
Second, the fact that the addition of L-NAME to maturation media did not allow cumulus expansion while the oocytes resumed meiosis contradicts previously reported data
It has been shown that ovulated porcine oocytes collected from the oviduct require more time for ZP digestion (hours) than immature or in vitro matured oocytes (minutes), hence the porcine ZP resistance to enzymatic digestion that occurs in the oviduct is independent of fertilization
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are physiologically generated during in vivo oocyte maturation
The lower penetration rate found in the L-NAME group, despite the presence of the three NOS isoforms in porcine oocytes
Nitric oxide has been reported to act on NO-sensitive guanylcyclases
Protein S-nitrosylation regulates the activity of a number of metabolic enzymes, oxidoreductases, proteases, protein kinases and phosphatases in vivo and in vitro, as well as respiratory proteins, receptor/ion channels and transporters, cytoskeletal and structural components, transcription factors, regulatory elements (including G proteins), and others
Despite the above reported studies, there is still a lack of data reporting S-nitrosylation in porcine oocyte maturation. For this reason it was evaluated in this study using a method for selectively labeling S-nitrosylated proteins with a fluorescent tag. It was observed that protein S-nitrosylation only decreased in oocytes matured with AG, the iNOS inhibitor, while the highest value was obtained with the NO donor GSNO. How exactly the decrease in protein nitrosylation is related to maturation is still unknown, but, what is clear from our results is that the inhibition of iNOS during maturation (by AG) produces a lower S-nitrosylation rate in porcine oocytes and that these poorly S-nitrosylated oocytes show a lower ability to resume meiosis. These results agree with Lee et al.
To sum up, our results show that AG inhibits iNOS NO production, decreasing the amount of S-nitrosylated proteins and reducing meiotic resumption; so, it can be concluded that iNOS activity is necessary for proper porcine oocyte maturation. We also demonstrate that NOS inhibition during IVM prevented cumulus expansion and did not improve the IVF system but did reveal the importance of NO in maturation and subsequent fertilization. Furthermore, we report for the first time that protein S-nitrosylation acts as a pathway through which NO exerts its effect on porcine oocyte IVM.
Further studies should be performed to understand the effect of NO production inhibition not only at oocyte level but also at sperm cell level. Gamete protein S-nitrosylation should also be studied in depth to determine the involvement of this posttranslational modification on gamete interaction.
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The authors thank Ms. Rebeca López-Úbeda and Ms. Sonia López for their technical support with the use of NO donor and NOS inhibitors. We also thank Dr. María Teresa Castells and Dr. María Inmaculada García for the image analysis and Mr Juan Antonio Carvajal and Ms Soledad Rodríguez for technical assistance.