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Theriogenology 81 (2014) 556–564

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Theriogenology

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Effect of nitric oxide on the cyclic guanosine monophosphate (cGMP)pathway during meiosis resumption in bovine oocytes

Kátia R. Lancellotti Schwarz a, Pedro R. Lisboa Pires a, Ligia Garcia Mesquita a,Marcos R. Chiaratti b, Cláudia Lima Verde Leal a,*aDepartamento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo,Pirassununga-SP, BrazilbDepartamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos-SP, Brazil

a r t i c l e i n f o

Article history:Received 16 September 2013Received in revised form 6 November 2013Accepted 6 November 2013

Keywords:Guanylate cyclaseNucleotideMaturationPhosphodiesteraseCattle

* Corresponding author. Tel.: þ55 19 3565 4067;E-mail address: clvleal@usp.br (C.L. Verde Leal).

0093-691X/$ – see front matter � 2014 Elsevier Inchttp://dx.doi.org/10.1016/j.theriogenology.2013.11.00

a b s t r a c t

Nitric oxide (NO) is a chemical messenger involved in the control of oocyte maturation. Itstimulates guanylate cyclase to produce cyclic guanosine monophosphate (cGMP), whichin turn activates cGMP-dependent protein kinase (PKG) and some phosphodiesterases thatmay interfere with cAMP levels, a nucleotide also involved in meiosis resumption. The aimof this study was to determine the role played by NO on the cGMP/cAMP pathway duringmeiosis resumption in bovine oocytes. The effects of increasing NO generated by S-nitroso-N-acetylpenicillamine (SNAP; 10�7–10�3 mol/L) and of other drugs that may affect the NO/cGMP pathway (proptoporfirin IX and 8-Br-cGMP) on meiosis resumption were investi-gated in bovine cumulus-oocyte complexes (COCs) matured for 9 hours in a semidefinedmedium (TCM199 þ 3 mg/mL BSA). The COCs matured with 10�7 mol/L SNAP associated ornot with 100 mmol/L oxadiazole-one quinoxaline, a guanylate cyclase inhibitor, also hadtheir cGMP and cAMP levels measured during the first hours of maturation (1, 3, and 6hours). Quantitative polymerase chain reaction was performed by real-time polymerasechain reaction to determine the effects of NO on expression of genes encoding for enzymesof the NO/guanylate cyclase/cGMP and cAMP pathways during the first 9 hours of oocytematuration. Increasing NO levels using 10�7 mol/L SNAP resulted in lower rate of germinalvesicle breakdown (36% germinal vesicle breakdown; P < 0.05) at 9 hours IVM, whereascontrol group and the treatments with 10�9 and 10�8 mol/L SNAP showed about 70%germinal vesicle breakdown (P > 0.05). A temporary increase in cGMP levels was alsoobserved with the same treatment (4.51 pmol/COC) at 1 hour IVM, which was superior tothe control group (2.97 pmol/COC; P < 0.05) and was reversed by inhibiting guanylatecyclase activity with 100 mmol/L oxadiazole-one quinoxaline. Neither cAMP levels nor geneexpression were affected by NO. These results suggest that NO acts via guanylate cyclase/cGMP and that even a temporary increase in cGMP levels leads to a delay in meiosisresumption, even when cAMP levels have declined. Nitric oxide does not act on oocytematuration by affecting cAMP levels or the expression of genes related to the NO/guanylatecyclase/cGMP and cAMP pathways. Also, to our knowledge this is the first report to detectPKG1, PKG2, phosphodiesterase-5A, ADCY3, ADCY6, and ADCY9 transcripts in bovineoocytes.

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K.R. Lancellotti Schwarz et al. / Theriogenology 81 (2014) 556–564 557

1. Introduction

Nitric oxide (NO) is a mediator of local action of hor-mones and neurotransmitters essential in the regulation ofseveral physiological processes involved in reproduction[1]. In mammals, NO is synthesized by NO synthase (NOS)[1], which occurs in three functional isoforms, neuronalNOS (NOS1), inducible NOS (NOS2), and endothelial NOS(NOS3) [2].

In the reproductive system, the NOS/NO system has beenidentified in various tissues. For example, the ovary ex-presses NOS [3], the follicular fluid contains NO [4], andoocytes are capable of its production [5,6]. Inhibiting NOsynthesis in oocytes during IVM results in a decrease in thein vitro production of blastocysts [7] and an increase in thenumber of apoptotic nuclei in embryos [8]. High concen-trations of NO, on the other hand, may impair the meioticprogression tometaphase II and embryonic development [9].Thus, NO has a dubious character on meiosis depending onits concentration, inwhich low concentrations can stimulatematuration and high concentrations can inhibit [10].

Mainly, NO exerts its effects by the activation of thesoluble guanylate cyclase enzyme (sGC), resulting in theproduction of cyclic guanosine monophosphate (cGMP),which usually results in the activation of cGMP-dependentprotein kinase (PKG) and action on some of the enzymes ofthe phosphodiesterase (PDE) family [11].

One of the effects of cGMP is the inhibition of PDE3,resulting in an increase in cAMP levels [12]. Cyclic AMP,produced by adenylate cyclase and degraded by PDEs [13],is a major contributor to the control of meiotic maturationin oocytes [14]. Therefore, a mechanism whereby NO mayparticipate in oocyte maturation involves the regulation oflevels of cyclic nucleotides [15].

The decrease in cAMP levels is required for oocytematuration, because it leads to decreased phosphorylationof certain proteins by cAMP-dependent protein kinase(PKA). Inactivation of PKA in oocytes allows the activationof the catalytic subunit of maturation promoting factor,inducing the resumption of meiosis [16,17]. The drop incGMP levels was also related to meiosis resumption [18],but its role has been less studied than cAMP.

Considering that NO can influence oocyte maturationand that one of its major signaling pathways involves cGMP,we investigated the role of this signaling pathway on theIVM of bovine oocytes and its effect on the cAMP pathway.

To evaluate the effects of this increased availability ofNO during IVM, we assessed meiosis resumption and theinvolvement of GC/cGMP pathway in the process. We alsoevaluated the effects of NO on the levels of cGMP and cAMPand on the relative abundance of transcripts of componentsof the NO, cGMP, and cAMP pathways.

2. Materials and methods

2.1. Media and chemicals

Chemicals were purchased from Sigma Chemical(St Louis, MO, USA), unless otherwise stated. The NO donor,S-nitroso-N-acetylpenicillamine (SNAP; Merck Millipore,Billerica, MA, USA) was prepared as a 10�3 mol/L stock

solution in DMSO. The cGMP analog, 8-bromoguanosine-3 ’,5’cyclicmonophosphate (8-Br-cGMP),was prepared as a 10-mmol/L stock solution in TCM199. The guanylate cyclaseinhibitor, 1H-[1,2,4] oxadiazole [4,3a] quinoxaline-1-one(ODQ), was prepared as a 10-mol/L stock solution inDMSO.All stock solutionswerestoredat�20 �Cuntil thedayof the experiment, when they were diluted in maturationmedium at specific concentrations. Only the drug stimu-lating guanylate cyclase (protoporphyrin IX) was preparedon the day of the experiment, first as a 1mmol/L solution inDMSO and then diluted in maturation medium to thedifferent concentrations used. The 8-Br-cGMP, ODQ, andprotoporphyrin IX were purchased from Biomol ResearchLaboratories (Plymouth Meeting, PA, USA).

2.2. Oocyte collection

Ovaries were collected at a commercial abattoir imme-diately after slaughter and transported in sterile saline so-lution with antibiotics (100 IU/mL penicillin and 100 mg/mLstreptomycin) at 30 �C. In the laboratory, 2- to 8-mm follicleswere aspiratedwith an 18-ga needle attached to a disposable10-mL syringe. The aspirated follicular fluid was placed in50-mL, conical tubes and maintained for 5 minutes forsedimentation. The upper portion of the liquid was removedand the remaining portion was added with 3 to 5 mL ofwashing medium (TCM199 with 25 mmol/L HEPES, 100 IU/mL gentamycin, and 1% fetal calf serum [Gibco-BRL, GrandIsland, NY, USA]). Thematerialwas then transferred to a Petridish (60� 15mm) under a stereomicroscope for selection ofgrade I and II cumulus-oocyte complexes (COCs) [19].

2.3. IVM

For IVM, the COCs selected were cultured in semi-definedmaturationmedium (TCM199with Earle’s salts and20 mmol/L sodium bicarbonate [Gibco], 0.4% albumin BSA,0.2 mmol/L sodium pyruvate, and 10 mg/mL gentamicin).Maturation was carried out in 90-mL droplets overlaid withmineral oil and incubated at 38.5 �C, 5% CO2 in air, andmaximum humidity for up to 9 hours.

2.4. Determining the stage of meiosis in oocytes

The oocytes were denuded by vortexing in PBS with0.1% polyvinyl alcohol (PVA). Denuded oocytes were fixedbetween a slide and coverslip for 24 hours in ethanol andacetic acid (3:1). After fixation, slides were stained with 1%lacmoid and evaluated under a phase contrast microscopeto assess germinal vesicle breakdown (GVBD). The GVBDrate was determined as the percentage of oocytes thatresumedmeiosis and were in GVBD andmetaphase I stagesin relation to the total number of oocytes placed in culture.

2.5. Cyclic AMP and cGMP measurements

Cyclic AMP and cGMP were measured by the enzymeimmunoassay method. Pools of 10 and 30 COCs were usedfor cAMP and cGMP measurements, respectively. Care wastaken to select oocytes with similar size of cumulus in-vestments [10]. Measurements of cGMP and cAMP were

K.R. Lancellotti Schwarz et al. / Theriogenology 81 (2014) 556–564558

made in two independent experiments. The COCs werewashed rapidly in TCM199 with 20 mmol/L HEPES and 0.4%BSA and transferred to 200 mL of 0.1 N HCl for 20 minutes tolyse the cells. After this period, the samples were centri-fuged at 12,000�g for 5 minutes. The supernatant wastransferred to new tubes and stored at �20 �C until sub-jected to the immunoassay according to the instructions ofthe kits (Direct Cyclic AMP and GMP enzyme immunoassaymethod; Enzo Life Sciences, Ann Arbor, MI, USA).

2.6. Quantitative real-time polymerase chain reaction

Groups of 20 COCs were denuded by pippeting inCaþ2-Mgþ2-–freePBS containing0.01%PVA (PBSþPVA). Thepartially denuded oocytes were removed from the solution,which was then centrifuged at 300�g for 15 minutes. Thesupernatant was removed and the cell pellet treated with 1U/mL RNase inhibitor (RNase OUT, Invitrogen, Carlsbad, CA,USA) and stored at �80 �C until use. The partially denudedoocytes were then vortexed for 3 minutes in PBSþPVA toremove any remaining cumulus cells and stored at�80 �C inpools of 20 oocytes in 1 mL of PBS þ PVAwith 1 U/mL RNaseOUT. Total RNA was extracted with TriZol reagent (Invi-trogen), treated with DNase I solution and then reversetranscribed into cDNA using the High Capacity cDNAReverse Transcription kit (Applied Biosystems, Carlsbad, CA,USA) as previously described [20]. Primers were designed

Table 1Primers sequences for analysis of gene expression.

Genes Sequence (50–3

ADCY3adenylate cyclase 3

50 CAACGAGGC50 TCCATGAACC

ADCY6adenylate cyclase 6

50 GACTTGTGC50 CCAACTGCG

ADCY9adenylate cyclase 9

50 TCCTGGTATT50 AGCCCGAGT

GUCY1B3guanylate cyclase 1, soluble, beta 3

50 CTCCCTGGTC50 ATCCAACAA

NOS2nitric oxide synthase 2, inducible

50 AGGTGCACA50 CCGCAGGAT

NOS3nitric oxide synthase 3, endothelial

50 CCTTTGGTGT50 TGGCTTAGG

PDE3Aphosphodiesterase 3A, cGMP-inhibited

50 ATCACCCGG50 CGCATGATG

PDE4Dphosphodiesterase 4D, cAMP-specific

50 ACCCTACAG50 TTCCAGACCG

PDE5Aphosphodiesterase 5A, cGMP-specific

50 TGATCAGTG50 AATGGAGAG

PDE8Aphosphodiesterase 8A

50 CTCCCTGGTC50 ATCCAACAA

PKA1protein kinase, cAMP-dependent, regulatory, type I, alpha

50 CTGCTCAAG50 AGGTTCTGG

PKA2protein kinase, cAMP-dependent, regulatory, type II, beta

50 TTCTGAACGC50 AGAATCAGC

PKG1protein kinase, cGMP-dependent, type I

50 TATGCAGGG50 TCCCAATTAA

PKG2protein kinase, cGMP-dependent, type II

50 AAGCAGCAG50 TTGTCCTTGA

ACTBactin, beta

50 CGCAGAAAA50 TGTCACCTTC

PPIApeptidylprolylisomerase A

50 CCACCGTGTT50 ATCCTTTCTC

GAPDHglyceraldehyde-3-phosphate dehydrogenase

50 CCACTCCCAA50 GCTTCACCAC

using Primer 3 Plus [21] and Autodimer (http://www.cstl.nist.gov; Table 1). Real-time PCR reactions were run in20 mL containing 0.2 mmol/L of each primer (regarding atarget mRNA transcript), 1� SYBR Green PCR Master Mix(Applied Biosystems), 5.5 mL H2O, and 2.5 mL template(eight-fold diluted cDNA) using the AB Step One (AppliedBiosystems). Cycling conditions for amplificationwere 95 �Cfor 15minutes followed by 40 cycles at 95 �C for 20 seconds,57 �C for 20 seconds, and 60 �C for 40 seconds. Pilot exper-iments were run to set up qPCR conditions. Melt-curve an-alyses reported on the specificity of the PCR products thatwere amplified. Data were analyzed using SDS 3.3 softwareand LinRegPCR programs. Gene expression data are pre-sented as relative values to the geometric mean of threemRNA transcripts (ACTB, GAPDH, and PPIA) of housekeepinggenes [22]. Levels of expressionwere consideredaccording tothe Ct (threshold cycle) number, that is, the number of cyclestaken to detect fluorescence signal above background, andclassified as high (þþþ¼ Ct< 25 cycles), medium (þþ¼ 25� Ct < 30 cycles), or low (þ ¼ Ct � 30 cycles) [23].

2.7. Experiments

2.7.1. Experiment 1: Influence of NO on GVBDTo determine the influence of NO on meiosis resump-

tion, COCs were cultured in maturation medium withdifferent concentrations of the NO donor SNAP (10�9, 10�8,

0) GenBank Accession # Fragmentsize (bp)

GCTGCTAGA 30 NM_001206084.1 81GCATGGAC 30

CGTGTACTCCTG 30 NM_0011438771 99GTGCTATGTG 30

CGCCCTGAC 30 NM_001205917 89ATGATTGAAGTTGT30

CGTCCTCAT 30 NM_174641.1 88CCCTTCCTTGC 30

CCGCCTATT 30 NM_001076799. 1 113GTCTTGAACA 30

TTAGGTGAATTTTAG 30 NC_007302.4 159CATCTTAGTAGGTCTC 30

GAAGGACTAAT 30 XM_616776. 4 95ATTCTCCAAGA 30

ACCAGGCACTC 30 XM_003583688.1 100ACTCATTTCAG 30

CCTGATGATCC 30 NM_174417. 2 61GCCACTGAGAA 30

CGTCCTCAT 30 NC_007319-4 88CCCTTCCTTGC 30

GACTCCATCGT 30 NM_001076358. 1 116ATCTGCTTTGC 30

CTGAAAGTGG 30 NM_174649.2 91CGAATCTCCCT 30

ATAATCCATCAGA 30 NM_174436.2 94AGCCCTCAAAC 30

GAGCATGTCTA 30 NM_001144099.1 92AGGTGCGATA 30

CGAGATGAGATTG 30 NC_007326.4 119ACCGTTCCAGT30

CTTCGACATC 30 NM_178320. 2 126TCCAGTGCTCAG 30

CGTGTCTGTT30 NM_001034034.2 84CTTCTTGATCTCATC30

K.R. Lancellotti Schwarz et al. / Theriogenology 81 (2014) 556–564 559

and 10�7 mol/L) and assessed formeiosis stage at the end ofculture. For this experiment, groups of 23 bovine COCswere randomly distributed into droplets of each treatment(100 mL of medium under mineral oil), cultured for 9 hours,and then fixed for evaluation of the proportion of oocytesundergoing GVBD (%GVBD). A control group was culturedwithout the drug and the dose–response test was carriedout in four replicates.

2.7.2. Experiment 2: Influence of cGMP on GVBDTo verify whether the effect of NO on meiosis resump-

tion was given via sGC/cGMP, groups of 23 COC wererandomly allocated into the different treatments, consist-ing of maturation medium (100 mL of medium undermineral oil) supplemented with different concentrations ofa GC stimulator (0, 5, 10, and 50 mmol/L proptoporfirin IX)or a cGMP analog (0, 1, 2, and 4 mmol/L 8-Br-cGMP). After 9hours of culture, oocytes were fixed, stained, and assessedfor %GVBD (meiosis resumption). The effects of protopor-phyrin IX and 8-Br-cGMP were evaluated in independentexperiments. The control group was cultured without drugand the dose–response assays were performed in fourreplicates. The evaluations of the effects of protoporphyrinIX and 8-Br-cGMP were made in independent experiments.

2.7.3. Experiment 3: Effect of NO on intracellular levels of cGMPand cAMP

To determine whether NO may be influencing the levelsof nucleotides to cause the reduction in GVBD rates, cGMP(Fig. 1), and cAMP levels were measured during the earlyhours of IVM. The nucleotide levels were measured inimmature bovine COCs (0 hour) and in those matured inthe presence of the NO donor alone (10�7 mol/L SNAP) orassociated with the sGC inhibitor (100 mmol/L ODQ) for 1, 3,and 6 hours of maturation. The association of ODQ (sGCinhibitor) was performed to evaluate if the effect of NOwould be given via sGC. Nucleotide levels in a control groupcultured without the addition of the drugs were measuredas well. Four replicates were performed for each time ofmaturation and measurements for each nucleotide weredone separately in independent experiments.

Fig. 1. Levels of cyclic guanosine monophosphate (cGMP) in bovinecumulus-oocyte complexes (COCs) matured in vitro for 0, 1, 3, and 6 hours inthe presence of 10�7 mol/L S-nitroso-N-acetylpenicillamine (SNAP; nitricoxide [NO] donor) alone or in combination with 100 mmol/L oxadiazole-onequinoxaline (ODQ; soluble guanylate cyclase enzyme [sGC] inhibitor). Sig-nificant differences between treatments (P < 0.05) are represented bylowercase letters (a,b). Significant differences between time points (P < 0.05)are represented by different uppercase letters (A,B). Results from fourreplicates.

2.7.4. Experiment 4: Effect of NO on the expression of genesinvolved in regulating NO, cGMP, and cAMP levels in oocytesand cumulus cells.

Considering that NO may also affect transcriptionalcontrol of different genes, we evaluated the influence ofNO (10�7 mol/L SNAP) on the gene expression of oocytesand cumulus cells matured in vitro for 9 hours. Theparticipation of the sGC pathway was assessed as well,and an additional group was studied with the associationof the NO donor and the sGC inhibitor (10�7 mol/L SNAPþ 100 mmol/L ODQ). As controls, untreated oocytes wereevaluated. Genes related to the NO signaling pathway,including its synthesizing enzymes (NOS2 and NOS3),genes related to the control of cGMP levels (GUCY1B3,sGC component related to the synthesis of cGMP, andPDE5A, responsible for cGMP degradation) or enzymesactivated by cGMP (PKG1and PKG2) were analyzed. Also,genes related to the control of cAMP levels (ADCY3,ADCY6, ADCY9, involved in the cAMP synthesis, andPDE3A, PDE4D and PDE8A, involved in cAMP degrada-tion) and enzymes activated by cAMP (PKA1 and PKA2)were evaluated. Groups of 20 COCs were matured in vitrofor 9 hours in the different groups (untreated control,SNAP, and SNAPþODQ) and at the end of culture, oocyteswere denuded of cumulus cells as described previouslyand both denuded oocytes and cumulus cells were usedfor gene expression analyses. The experiment was per-formed in seven replicates.

2.8. Statistical analyses

Statistical analyses were performed using the SAS Sys-tem (V9.2; SAS Institute, Inc., Cary, NC). Datawere tested fornormal distribution and homogeneity of variance and weretransformed (arcsine, log, or square root) when thesecriteria were not met. The rate of GVBD was analyzed byone-way ANOVA followed by Bonferroni post hoc test. Theeffects of the treatments on cGMP and cAMP levels wereanalyzed by two-way ANOVA followed by Bonferroni posthoc testing. For gene expression data, samples werecollected from seven replicates and run in duplicate. Thedata from each duplicate were averaged before beinganalyzed by one-way ANOVA followed by Tukey post hoctesting. Differences with probabilities of P < 0.05 wereconsidered significant.

3. Results

3.1. Influence of NO on GVBD

The COCs matured in vitro for 9 hours with differentconcentrations of the NO donor (10�9, 10�8, and 10�7 mol/LSNAP) were evaluated regarding the stage of meiosis todetermine the effect of increased availability of NO on theability of the oocytes to resume meiosis (%GVBD; Fig. 1).

The control group and the treatments with 10�9 and10�8 mol/L SNAP showed about 70% GVBD (P > 0.05), withno effect of these concentrations of SNAP. However, treat-ment with the higher concentration of the NO donor (10�7

mol/L SNAP), reduced the proportion of oocytes resumingmeiosis (36%; P < 0.05).

Fig. 2. Rate of oocyte germinal vesicle breakdown (GVBD) after 9 hours ofIVM in the presence of different concentrations of SNAP (nitric oxide [NO]donor). The bars represent the percentage of cumulus-oocyte complexes(COCs) undergoing GVBD in relation to the total placed in culture. a,bDif-ferent letters indicate significant difference between groups (P < 0.05).Results from four replicates.

K.R. Lancellotti Schwarz et al. / Theriogenology 81 (2014) 556–564560

3.2. Influence of cGMP on GVBD

To verify whether the effect of NO on meiosis resump-tion was given via sGC/cGMP, COCs were matured in vitrofor 9 hours with different concentrations of a GC stimulator(0, 5, 10, and 50 mmol/L proptoporfirin IX) or a cGMP analog(0, 1, 2, and 4 mmol/L 8-Br-cGMP) and assessed for %GVBD.The results are shown in Figure 2.

All proptoporfirin IX concentrations tested (Fig. 3A)caused a decrease (P < 0.05) in meiosis resumption, withGVBD rates varying from 54.4% to 63.8%. The control grouppresented 80% GVBD. The reduction in GVBD induced bythe sGC stimulator (protoporphyrin IX) was also observedwith the use of the cGMP analog (Fig. 3B). All concentra-tions tested reduced the proportion of GVBD (36.6%–51.8%)compared with the control group (62%; P < 0.05).

Because even the lowest concentrations of the drugstested resulted in GVBD reduction, the same drugs andconcentrations were evaluated after 24 hours IVM to verifywhether the effect observed was owing to toxicity or todelayedGVBD.All treatments resulted innormalmaturationrates (proportion of oocytes reaching metaphase II stageafter 24 hours IVM), which were similar to controls. Matu-ration rates varied from 80% to 91% when testing proto-porphorin IX (P > 0.05) and from 94% to 95% when testing8-Br-cGMP (P > 0.05). Therefore, the reduction in GVBDrates were not caused by drug toxicity, but by a temporarydelay inGVBD in treated oocytes. Also, nomorphologic signsof toxicity were observed in these groups.

3.3. Effect of NO on intracellular levels of cGMP and cAMP

To determine whether NO may be influencing the levelsof nucleotides to cause the reduction observed in GVBDrates, cGMP (Fig. 3), and cAMP (Fig. 4) levels weremeasured in COCs treated with the NO donor (10�7 mol/LSNAP) during the first hours of IVM (0, 1, 3, and 6 hours).The participation of sGC in the NO signaling pathway wasevaluated in the group treated with SNAP associated withthe sGC inhibitor (100 mmol/L ODQ).

At the beginning of maturation (immature group;0 hour), cGMP levelswere 5.29 pmol per COC and decreasedsignificantlywithin thefirst hour of culture to 2.97 pmol perCOC (P < 0.05) in the control group. On the other hand,SNAP treatment maintained cGMP levels (4.51 pmol perCOC; P> 0.05) similar to the immature group (0 hours) afterthe first hour of IVM. Therefore, the increase of NO gener-ated by SNAP was able to maintain cGMP levels during thefirst hour of maturation. On the other hand, the associationof SNAP with ODQ resulted in a decrease in cGMP levelsalready after 1 hour IVM (1.54 pmol/COC) compared withthe immature group (P< 0.05), but was similar (P> 0.05) tothe levels observed in the control group at 1 hour IVM,suggesting that the effect of NO on cGMP levels was relatedto sGC. However, cGMP levels remained equally low in allgroups after 3 and 6 hours IVM, ranging from 1.4 to 2.5 and0.07 to 2.1 pmol per CCO, respectively (P > 0.05).

The immature group (0 hour) showed high levels ofcAMP (32.43 fmol/COC), which decreased already duringthe first hour of culture in all groups (P < 0.05) andremained low during the following hours. Levels were 11.7

to 15.9 fmol per COC at 1 hour, 3.3 to 8.0 fmol per COC at 3hours, and 7.4 to 18.3 fmol per COC at 6 hours. The treat-ments caused no difference between the cultured groups(P > 0.05). The addition of SNAP to the culture medium didnot alter cAMP levels; therefore, NO did not affect themeiosis resumption by affecting cAMP pathway under theconditions studied.

3.4. Effect of NO on the expression of genes involved inregulating NO, cGMP, and cAMP levels in oocytes and cumuluscells

Because NO may also affect transcriptional control ofdifferent genes, we evaluated the influence of NO, associ-ated or not with sGC inhibition, on the expression of genesparticipating in the NO/cGMP/cAMP pathways in oocytesand cumulus cells matured in vitro for 9 hours. Genesencoding enzymes synthesizing NO (NOS2 and NOS3),controlling cGMP levels (GUCY1B3 and PDE5A), or activatedby cGMP (PKG1and PKG2) were analyzed (Table 2). Also,genes encoding enzymes controlling cAMP levels (ADCY3,ADCY6, ADCY9, PDE3A, PDE4D, and PDE8A) and activatedby cAMP (PKA1 and PKA2) were evaluated (Table 3).

As expected, NOS2 and NOS3 genes were detected inboth oocytes and in cumulus cells and both isoforms weremore abundant in oocytes than in cumulus cells. Treat-ments did not affect the transcripts of these enzymes whencompared with the control group (P > 0.05). Therefore, NOhad no influence on the transcription of its synthesis en-zymes at the beginning of meiosis.

Regarding the expression of genes related to the controlof cGMP levels, GUCY1B3 was detected in both compart-ments of the COC with predominance in cumulus cells.PDE5A was also detected in both cell types with higherabundance in oocytes. The treatments did not affect theexpression of these genes in the cells studied (P > 0.05).

The two isoforms of the enzyme activated by cGMP, PKG1,andPKG2were studied inboth compartmentsof theCOC, but

Fig. 3. Rate of oocyte germinal vesicle breakdown (GVBD) after 9 hours of IVM in the presence of different concentrations of protoporphyrin IX (sGC stimulator;A) and 8-Br-cGMP (cyclic guanosine monophosphate [cGMP] analog; B). The bars represent the percentage of cumulus-oocyte complexes (COCs) undergoingGVBD in relation to the total placed in culture. a,bDifferent letters indicate significant difference between groups (P < 0.05). Results from four replicates.

K.R. Lancellotti Schwarz et al. / Theriogenology 81 (2014) 556–564 561

theywere only detected in oocytes. Therewere no treatmenteffects (P> 0.5). The PKG2 isoform seems to predominate inrelation to PKG1 in oocytes. The absence of transcripts ofthese genes in cumulus cells indicates that they may not beexpressed in this cell type or are expressed at very low levelsnot detected under our experimental conditions.

The threeADCYisoformsstudiedweredetected inoocytesand cumulus cells. ADCY9was themost expressed isoform inoocytes, followed by ADCY6 and ADCY3. In cumulus cells,ADCY6 predominated over ADCY9 and ADCY3. NO had noeffect on the transcription these genes (P> 0.05.)

PDE3A was detected in oocytes and PDE4D only incumulus cells. PDE8A was expressed in both compart-ments, with lesser abundance in relation to PDE3A in oo-cytes and higher abundance in cumulus cells relative toPDE4D. Expressionwas not affected by the presence of ODQor SNAP during the 9 hours of maturation (P > 0.05).

PKA1 and PKA2 were expressed in both compartments,but in greater amounts in oocytes. In cumulus cells, PKA1was predominant over PKA2. SNAP alone or associatedwith ODQ also did not alter the expression of these genes(P > 0.05).

Fig. 4. Levels of cAMP in bovine cumulus-oocyte complexes (COCs) maturedin vitro for 0, 1, 3, and 6 hours in the presence of 10�7 mol/L S-nitroso-N-acetylpenicillamine (SNAP; nitric oxide [NO] donor) alone or in combinationwith 100 mmol/L oxadiazole-one quinoxaline (ODQ; soluble guanylatecyclase enzyme [sGC] inhibitor). Significant difference (*) between thecontrol group (0 hour) and the other treatments and time points (P < 0.05).Results from four replicates.

4. Discussion

To gather further information on the action of thechemical messenger NO in bovine COCs, the present studyevaluated the effects of NO and of components of the NO/sGC/cGMP pathway on the resumption of nuclear matura-tion in bovine oocytes. The effects of NO on the levels ofcGMP and cAMP and on the expression of genes related tothe control of these nucleotide levels and their actions werealso evaluated.

In the present study, we observed that the increasedavailability ofNOusing 10�7mol/L SNAP resulted in reducedrates of GVBD, causing a delay in the resumption of meiosisobserved at 9 hours IVM. Our results corroborate previousreports using another NO donor (sodium nitroprusside)[10,24] and also with SNAP [9] in cattle, and also in rodents[25–27] and pigs [28].

Because we confirmed the effect of elevated NO onmeiosis resumption and considering that one of the mainmechanisms of action of NO is through increased activity ofsGC with consequent elevation of cGMP, we then evaluatedwhether the stimulation of sGC and the use of a cGMPanalog would mimic the effect of NO, to determine if thispathway was involved in the process. The use of the sGCstimulator (protoporphyrin IX) and the cGMP analog (8-Br-cGMP) also resulted in reduced GVBD rates at 9 hours IVM,consistent with the results achieved with NO in the pre-vious experiment. Cyclic GMP analogs also had similar ef-fect in mouse oocytes [26,29,30]. Thus, the effect ofincreased NO was replicated by stimulating sGC with pro-toporphyrin IX and by the cGMP analog, indicating that theaction of the NO/sCG/cGMP pathway is involved in thedelayed meiosis resumption. Thus, it may be said that this

Table 2Gene expression in oocytes and cumulus cells related to the control ofcyclic guanosine monophosphate levels.

Cell analyzed NOS2 NOS3 GUCY1B3 PDE5A PKG1 PKG2

Oocytes þþþ þþ þ þþ þþ þþþCumulus cell þ þ þþ þ – –

Abbreviations: (þ), gene detected; (�), gene not detected; þþþ, highexpression when Ct <25 cycles; þþ, medium expression when 25 � Ct <30 cycles; þ, low expression when Ct �30 cycles.

Table 3Gene expression in oocytes and cumulus cells related to the control of cAMP levels.

ADCY3 ADCY6 ADCY9 PDE3A PDE4D PDE8A PKA1 PKA2

Oocytes þþ þþ þþþ þþþ � þþ þþþ þþþCumulus

cellþ þþþ þþ � þ þþ þþ þ

Abbreviations: (þ), gene detected; (�), gene not detected;þþþ, high expression when Ct<25 cycles; þþ, medium expression when 25� Ct < 30 cycles; þ,low expression when Ct �30 cycles.

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pathway must be inactivated to allow the progression ofmeiosis, because it seems to be related to the process ofmaintaining the meiosis block in GV until the necessarystimulus to maturation occurs, to remove this inhibitorypathway. In rats, Nakamura et al. [25] suggested thatmeiosis resumption depends on the reduction of NO levelsto relieve meiosis block.

However, contradicting our observations, Bilodeau-Goeseels [10] reported that, although increased NO alsoreduced GVBD in bovine oocytes, such an effect was notvia sGC/cGMP, because when using the same drugs (pro-toporphyrin IX and 8-Br-cGMP) at doses similar to thoseused in the present study, there was no effect of suchtreatments on GVBD rates at 7 hours IVM. The differencesbetween the studies may be owing to differences in cul-ture conditions, such as the maturation medium (unde-fined; TCM199 with 10% fetal calf serum [10] andsemidefined TCM199 with 0.4% BSA in the present study),the evaluation GVBD rates at different times (7 and 9hours, respectively) or different NO donors used (10�5

mol/L sodium nitroprusside and 10�7 mol/L SNAP).To confirm whether treatment with SNAP would actu-

ally increase cGMP levels, the same treatments were testedto measure nucleotide levels. The results showed that theNO donor increased cGMP levels which remained high andsimilar to those observed in immature oocytes prior toculture. Also, when the sGC inhibitor (ODQ) was associatedwith SNAP the effect was abolished, suggesting that theelevation of cGMP was owing to the stimulation of sGC byNO. It is well-known that one of the pathways used by NO isthe stimulation of sGC with subsequent elevation of cGMP[31] and was confirmed to be active in bovine COCs. Theincrease, however, occurred only within the first hour ofIVM, with declining levels of cGMP at 3 and 6 hours ofculture in all groups. Thus, the effect of NO was temporaryand brief (during the first hour), but sufficient to cause adecrease in GVBD evaluated at 9 hours IVM.

Bilodeau-Goeseels [10] also obtained an increase ofcGMP after treatment with NO, and such increase was alsotemporary, but with an increase observed at 3 hours and adecrease at 6 hours. The transient elevation of cGMPinduced by NO may have been caused by the activation ofnegative feedback mechanisms. Cyclic GMP is known to beable to activate PDE5, which is responsible for its own hy-drolysis [32], and transcripts for this enzymewere detectedin oocytes and cumulus cells in our last experiment.Nevertheless, even if temporary, cGMP induced by NO wasable to delay GVBD, so that the effect of NO on cAMP levelswere also addressed, because cGMP can inhibit PDE3 [32],which in turn would result in maintaining higher levels ofcAMP. Decreased cAMP levels are required for meiosis

resumption [33]. Such a mechanism has been implicated inthe control of meiosis in mice [34,35].

However, contrary to our expectations, cAMP levelsdeclined within the first hour of IVM in the presence of NO,even when cGMP levels were also elevated in the sametreatment. These results suggest that cGMP would not beacting on PDE3, but would be delaying meiosis by othermechanisms, different from those described in other spe-cies such as mice [36].

Other actions of cGMP include, for example, transcrip-tional control (activating transcription factors) and activa-tion of ion channels, enzymes, and PKG [37]. When using aPKG inhibitor, no effect was observed on bovine oocytematuration and, similar to our observations, NO did notcause changes in cAMP levels [10]. However, one cannotrule out the possibility of PDE3 inhibition by cGMP inbovine oocytes as also observed in other species such asmice [34,35] and pigs [38].

The lack of effect of NO via cGMP over cAMP could becaused by insufficient levels of cGMP to inhibit PDE3 and/orthe rapid action of cGMP negative feedback on its ownsynthesis, as previously reported in other cell types [39],which could cause rapid loss of the ability to effectivelyinhibit PDE3. Cyclic GMP can activate the enzyme respon-sible for its own destruction (PDE5) and can also inhibit itsenzyme of synthesis (sGC) [32]; therefore, cGMP can in-crease its hydrolysis and reduce its synthesis to decrease itstotal intracellular levels. The enzyme responsible for cGMPsynthesis was also detected in oocytes and cumulus cells.Whether this is the case remains to be determined, and if itis so, it may be necessary to associate a PDE5 inhibitor tomaintain high levels of cGMP for longer periods of time toeffectively inhibit PDE3 and concomitantly increase cAMP.In a study inmouse oocytes, Hanna, et al. [30] observed thatthe increase in cGMP inhibited meiosis, but the associationof a PDE9 inhibitor (another cGMP-specific PDE) to elevatecGMP levels was more efficient.

Nevertheless, the presence of cGMP, evenwithout effecton cAMP, is sufficient to delay meiosis resumption. Thus,not only the reduction in cAMP levels is necessary, but alsothat of cGMP is required to relieve the meiosis block, assuggested in other species [30,35,40–42].

Because both NO [36] and cGMP can affect cellulartranscription [37,43], including that of members of theirown pathways [43,44], transcript levels for enzymesinvolved in NO synthesis (NOS2 and NOS3), cGMP synthesis(GUCY1B3) and degradation (PDE5A), and targets of cGMP(PKG1 and PKG2) were evaluated. Also, genes encodingenzymes involved in cAMP synthesis (ADCY3, ADCY6, andADCY9) and degradation (PDE3A, PDE4D, and PDE8A) andits target enzymes (PKA1 and PKA2) were analyzed as well.

K.R. Lancellotti Schwarz et al. / Theriogenology 81 (2014) 556–564 563

The results indicated that NO does not affect any of thetranscripts studied. Nevertheless, all the members studiedin pathways analyzed were detected and some of them forthe first time in bovine oocytes and cumulus cells. The re-sults indicate that these signaling pathways are present andmust be important for the proper development of theoocyte, and need to be further studied in relation to theirrole in the control of oocyte maturation.

Confirming previous observations, NOS2 and NOS3were expressed in both cell types [45]. Despite reports thatNO can interfere with gene expression, including genes ofthe pathways studied [37], this effect was not observed. Asdescribed in the previous experiments, NO seems to exertits effects on the resumption of meiosis by modulating theactivity of sGC without effects on transcription.

Expression of the sGC enzyme (GUCY1B3 subunit iso-form) was observed in oocytes and cumulus cells. Itsexpression was also detected previously in granulosa cells,theca and CL in sheep [46], and in granulosa cells and oo-cytes of female rats [47] and pigs [48], corroborating ourfindings in cattle. As far as we know, GUCY1B3 expressionhas not been described in bovine COCs, but the detection ofcGMP in bovine COC (this study and [10]) demonstrate thatboth cell types are able to synthesize cGMP.

The control of cGMP levels is given by the balance be-tween synthesis by sGC and degradation by cGMP-specificPDEs [43], which include the PDE5, PDE6, and PDE9 iso-forms [32]. In this study, we detected PDE5 in both celltypes. We also detected PDE5A in porcine COCs [49] and inoocytes and cumulus cells of mice [50].

Among the targets of cGMP are enzymes activated bycGMP, including cGMP-dependent kinases, which weredescribed in two isoforms: PKG1 and PKG2 [32,44]. Theexpression of these enzymes was found in murine gran-ulosa cells and COCs [17], with more expression of PKG2,which was hormonally regulated. Expression of PKG2 wasalso higher than that of PKG1 in the present study.

Cyclic AMP levels are controlled by adenylate cyclase(synthesis) and degradation by PDEs with higher affinityfor cAMP, including isoforms PDE3, PDE4, and PDE8 [33].Cyclic AMP activates PKA, which are described in isoformsPKA1 and PKA2 [51]. There are 10 adenylate cyclase iso-forms [52], and several of them have already been identi-fied in the ovaries of rats.

Our study detected isoforms ADCY3, ADCY6, and ADCY9in oocytes and cumulus cells, corroborating observations inrodent oocytes [53] and bovine cumulus cells [54]. A widevariety of isoforms shows the complexity of cAMP signalingin cells.

The PDEs, which are involved in the reduction of cAMPlevels, are encoded by 11 families of genes [55]. In thisstudy, isoforms PDE3A, PDE4D, and PDE8A were analyzed;PDE3A was detected only in oocytes, PDE4D only in thesomatic compartment, and PDE8A in both cells. Theseresults corroborate previous findings [56] where PDE4Dand PDE8A were detected in bovine cumulus cells. Per-forming a functional study, the authors determined thatthe increased activity of cAMP degradation was given byPDE3A isoform in the oocyte, whereas PDE8A was themajor enzyme degrading cAMP in cumulus cells. Theirfindings reinforce the concept that different isoforms in

different cell types are necessary to control cAMP levelsand their effects.

The PKAs are activated by cAMP to exert their intracel-lular effects. In general, PKA1 has been mostly related toeffects on cell proliferation and apoptosis and PKA2 withthe cell-cycle control and is regulated by mitogens [54]. Inthe present study, PKA1 and PKA2 were detected in oocytesand cumulus cells. In oocytes of female rats, these enzymeswere also expressed and presented an intracellular distri-bution that varied with the stage of maturation and theisoform analyzed, showing differential localization [57].

4.1. Conclusions

Considering the results obtained in the present study, itmay be concluded that NO induces a short-lived increase incGMP levels owing to sCG activation, which is sufficient todelaymeiosis resumption in bovine COCs, but does not affectcAMP levels. Although cAMP levels are reduced, concomitantreduction in cGMP levels is required as well for the propermeiosis resumption. Transcripts for several members of theNO/sCG/cGMP/cAMP pathways are present in oocytes andcumulus cells, some reported for the first time; although notaffected by NO, they show the intricate relationship ofdifferent signaling pathways on meiosis control in bovineCOCs. Additional studies should be conducted to determinethe role of the different members of these pathways on thecontrol of meiosis block and resumption.

Acknowledgments

This work was supported by FAPESP – São Paulo, Brazil.Grant 2008/09321-6.

References

[1] Rettori V, Mc Cann SM. Role of nitric oxide and alcohol on gonad-otropin release in vitro or in vivo. NY Acad Sci 1998;840:185–93.

[2] WC Sessa. The nitric oxide synthase family of proteins. J Vasc Res1994;31:131–43.

[3] Chatterjee S, Gangula PR, Dong YL, Yallampalli C. Immunocyto-chemical localization of nitric oxide synthase-III in reproductiveorgans of female rats during the oestrous cycle. Histochem J 1996;28:715–23.

[4] Basini G, Baratta M, Ponderato N, Bussolati S, Tamanini CI. Is nitricoxide an autocrine modulator of bovine granulosa cell function?Reprod Fertil Dev 1998;10:471–8.

[5] Hattori MA, Nishida N, Takesue K, Kato Y, Fujihara N. FSH sup-pression of nitric oxide synthesis in porcine oocytes. J Mol Endo-crinol 2000;24:65–73.

[6] Hattori MA, Arai M, Saruwatari K, Kato Y. Estrogen regulation ofnitric oxide synthesis in the porcine oocyte. Mol Cell Endocrinol2004;260:13–9.

[7] Matta SG, Caldas-Bussiere MC, Viana KS, Faes MR, Paes deCarvalho CS, Dias BL, et al. Effect of inhibition of synthesis ofinducible nitric oxide synthase-derived nitric oxide by amino-guanidine on the in vitro maturation of oocyte-cumulus complexesof cattle. Anim Reprod Sci 2009;111:189–201.

[8] Schwarz KRL, Pires P, De Bem T, Adona P, Leal CLV. Consequences ofnitric oxide synthase inhibition during bovine oocyte maturation onmeiosis and embryo development. Reprod Domest Anim 2010;45:75–80.

[9] Schwarz KRL, Pires PR, Adona PR, Câmara De Bem T, Leal CLV. Influ-ence of nitric oxide during maturation on bovine oocyte meiosis andembryo development in vitro. Reprod Fertil Dev 2008;20:529–36.

[10] Bilodeau-Goeseels S. Effects of manipulating the nitric oxide/cyclicGMP pathway on bovine oocyte meiotic maturation. Theriogenol-ogy 2007;65:693–701.

K.R. Lancellotti Schwarz et al. / Theriogenology 81 (2014) 556–564564

[11] Denninger JW, Marletta MA. Guanylatecyclase and the NO/cGMPsignaling pathway. Biochem Biophys Acta 1999;1441:334–50.

[12] Richard FJ, Tsafriri A, Conti M. Role of phosphodiesterase type 3A inrat oocyte maturation. Biol Reprod 2001;65:1444–51.

[13] LaforestMF, Pouliot E,GuéguenL, Richard FJ. Fundamental significanceof specific phosphodiesterases in the control of spontaneous meioticresumption in porcine oocytes. Mol Reprod Dev 2005;70:361–72.

[14] Eppig JJ, Downs SM. Chemical signals that regulate mammalianoocyte maturation. Biol Reprod 1984;30:1–11.

[15] Nathan C. Nitric oxide as a secretory product of mammalian cells.Faseb J 1992;6:3051–64.

[16] Han SJ, Vaccari S, Nedachi T, Andersen CB, Kovacina KS, Roth RA,Conti M. Protein kinase B/Akt phosphorylation of PDE3A and its rolein mammalian oocyte maturation. Embryo J 2006;25:5716–25.

[17] Sriraman V, Rudd MD, Lohmann SM, Mulders SM, Richards JS. Cyclicguanosine 50-monophosphate-dependent protein kinase II isinduced by luteinizing hormone and progesterone receptor-dependent mechanisms in granulosa cells and cumulus oocytecomplexes of ovulating follicles. Mol Endocrinol 2006;20:348–61.

[18] Tornell J, Billing H, Hillesjo T. Regulation of oocyte maturation bychanges in ovarian levels of cyclic nucleotides. Hum Reprod 1991;6:411–22.

[19] Loos F, Kastrop P, Van Maurik P, Van Beneden TH, Kruip TA. Het-erologous cell contacts and metabolic coupling in bovine cumulusoocyte complexes. Mol Reprod Dev 1991;28:255–9.

[20] Chiaratti MR, Bressan FF, Ferreira CR, Caetano AR, Smith LC,Vercesi A, et al. Embryo mitochondrial DNA depletion is reversedduring early embryogenesis in cattle. Biol Reprod 2010;82:76–85.

[21] Rozen S, Skalelsky HJ. Bioinformatics methods and protocols. Mo-lecular Biology132. Humana Press; 2000. p. 365–86.

[22] Lonergan P, Rizos D, Gutierrez-Adan A, Fair T, Boland MP. Oocyteand embryo quality: effect of origin, culture conditions and geneexpression patterns. Reprod Domest Anim 2003;38:259–67.

[23] Da Silveira JC, Rao Veeramachaneni DN, Winger QA, Carnevale EM,Bouma GJ. Cell-secreted vesicles in equine ovarian follicular fluidcontain miRNAs and proteins: a possible new form of cell commu-nication within the ovarian follicle. Biol Reprod 2012;86:71:1–10.

[24] Viana KS, Caldas-Bussiere MC, Matta SG, Faes MR, De Carvalho CS,Quirino CR. Effect of sodium nitroprusside, a nitric oxide donor, onthe in vitro maturation of bovine oocytes. Anim Reprod Sci 2007;102:217–27.

[25] Nakamura Y, Yamagata Y, Sugino N, Takayama H, Kato H. Nitric oxideinhibits oocyte meiotic maturation. Biol Reprod 2002;67:1588–92.

[26] Bu S, Xie H, Tao Y, Wang J, Xia G. Nitric oxide influences thematuration of cumulus cell-enclosed mouse oocytes cultured inspontaneous maturation medium and hypoxantine-supplementedmedium through different signaling pathways. Mol Cell Endo-crinol 2004;223:85–93.

[27] Sela-Abramovich S, Edry I, Galiani D, Nevo N, Dekel N. Disruption ofgap junctional communication within the ovarian follicle inducesoocyte maturation. Endocrinology 2006;147:2280–6.

[28] Tao Y, Xie H, Hong H, Chen X, Jang J, Xia G. Effects of nitric oxidesynthase inhibitors on porcine oocyte meiotic maturation. Zygote2005;13:1–9.

[29] Faerge I, Terry B, Kalous J, Wahl P, Lessl M, Ottesen JL, et al.Resumption of meiosis induced by meiosis-activating sterol has adifferent signal transduction pathway than spontaneous resump-tion of meiosis in denuded mouse oocytes cultured in vitro. BiolReprod 2001;65:1751–8.

[30] Hanna CB, Yao S, Wu X, Jensen JT. Identification of phosphodies-terase 9A as a cyclic guanosine monophosphate-specific phospho-diesterase in germinal vesicle oocytes: a proposed role in theresumption of meiosis. Fertil Steril 2012;98:487–95.

[31] Francis SH, Busch JL, Corbin JD, Sibley D. cGMP-dependent proteinkinases and cGMP phosphodiesterases in nitric oxide and cGMPaction. Pharmacol Rev 2010;62:525–63.

[32] Francis SH, Sekhar KR, Ke H, Corbin JD. Inhibition of cyclic nucleo-tide phosphodiesterases by methylxanthines and related com-pounds. Handb Exp Pharmacol 2011;200:93–133.

[33] Eppig JJ.Mammalian oocyte development. In:Ovarian endocrinology.Hoboken, NJ: Blackwell Scientific Publications; 1991. p. 107–31.

[34] Norris RP, Ratzan WJ, Freudzon M, Mehlmann LM, Krall J,Movsesian MA, et al. Cyclic GMP from the surrounding somatic cells

regulates cyclic AMP and meiosis in the mouse oocyte. Develop-ment 2009;136:1869–78.

[35] Vaccari S, Weeks JL, Hsieh M, Menniti FS, Conti M. CyclicGMP signaling is involved in luteinizing hormone-dependentmeioticmaturation of mouse oocytes. Biol Reprod 2009;81:595–604.

[36] Bilodeau-Goeseels S. Cows are not mice: the role of cyclic AMPphosphodiesterases, and adenosine monophosphate-activated pro-tein kinase in the maintenance of meiotic arrest in bovine oocytes.Mol Reprod Dev 2011;78:734–43.

[37] White RE, Han G, Maunz M, Mowafy AM, Barlow RS, Catravas JD,et al. Endothelium-independent effect of estrogen on Ca2þ-activated Kþ channels in human coronary artery smooth musclecells. Cardiovasc Res 2002;53:650–61.

[38] Zaffalon FG, Guimmelette C, Leal CLV, Richard FJ. Cyclic guanosinemonophosphate inhibits phosphodiesterase 3 Activity in porcineoocytes. Reprod Fertil Dev 2013;25:282.

[39] Bryan NS, Bian K, Murad F. Discovery of the nitric oxide signalingpathwayand targets for drugdevelopment. FrontBiosci 2009;1:1–18.

[40] Sela-Abramovich S, Galiani D, Nevo N, Dekel N. Inhibition of ratoocyte maturation and ovulation by nitric oxide: mechanism ofaction. Biol Reprod 2008;78:1111–8.

[41] Törnell J, Billig H, Hillensjö T. Resumption of rat oocyte meiosis isparalleled by a decrease in guanosine 3’,5’-cyclic monophosphate(cGMP) and is inhibited by microinjection of cGMP. Acta PhysiolScand 1990;139:511–7.

[42] Hubbard CJ. The effect in vivo of alterations in gonadotropins andcyclic nucleotides on oocyte maturation in the hamster. Life Sci1983;33:1695–702.

[43] Pilz RB, Broderick KE. Role of cyclic GMP in gene regulation. FrontBiosci 2005;10:1239–68.

[44] Baltrons MA, Pifarré P, Ferrer I, Carot JM, García A. Reducedexpression of NO-sensitive guanylyl cyclase in reactive astrocytes ofAlzheimer disease, Creutzfeldt-Jakob disease, and multiple sclerosisbrains. Neurobiol Dis 2004;17:462–72.

[45] Pires PR, Santos NP, Adona PR, Natori MM, Schwarz KR, De Bem TH,et al. Endothelial and inducible nitric oxide synthases in oocytes ofcattle. Anim Reprod Sci 2009;116:233–43.

[46] Grazul-Bilska AT, Reynolds LP, Redmer DA. Gap junctions in theovaries. Biol Reprod 2006;57:947–57.

[47] Shi F, Stewart RLJ, Perez E, Chen JY, LaPolt PS. Cell-specific expres-sion and regulation of soluble guanylyl cyclase alpha 1 and beta 1subunits in the rat ovary. Biol Reprod 2004;70:1552–61.

[48] Ding W, Zhang W, Hui FM, Zhang YH, Zhang FF, Li XM, et al. Cell-specific expression and immunolocalization of nitric oxide syn-thase isoforms and soluble guanylyl yclase a1 and b1 subunits inthe ovary of fetal, neonatal and immature pigs. Anim Reprod Sci2012;131:172–80.

[49] Sasseville M, Côté N, Gagnon MC, Richard FJ. Up-regulation of 3’5’-cyclic guanosine monophosphate-specific phosphodiesterase in theporcine cumulus-oocyte complex affects steroidogenesis duringin vitro maturation. Endocrinology 2008;149:5568–76.

[50] Wang ZC, SHI FX. Phosphodiesterase 4 and compartmentalization ofcyclic AMP signaling. Chinese Sci Bull 2008;52:34–46.

[51] Skålhegg BS, Taskén K. Specificity in the cAMP/PKA signalingpathway differential expression, regulation, and subcellular locali-zation of subunits of PKA. Front Biosci 1997;2:331–42.

[52] Hanoune J, Defer N. Regulation and role of adenylyl cyclase iso-forms. Annu Rev Pharmacol Toxicol 2001;41:145–74.

[53] Horner K, Livera G, Hinckley M, Trinh K, Storm D, Conti M. Rodentoocytes express an active adenylyl cyclase required for meiotic ar-rest. Dev Biol 2003;258:385–96.

[54] Lastro M, Collins S, Currie WB. Adenylyl cyclases in oocyte matu-ration: a characterization of AC isoforms in bovine cumulus cells.Mol Reprod Dev 2006;73:1202–10.

[55] Ahmad F, Degerman E, Manganiello VC. Cyclic nucleotide phos-phodiesterase 3 signaling complexes. Horm Metab Res 2012;44:776–85.

[56] Sasseville M, Albuz FK, Côté N, Guillemette C, Gilchrist RB,Richard FJ. Characterization of novel phosphodiesterases in thebovine ovarian follicle. Biol Reprod 2009;81:415–25.

[57] Kovo M, Schillace RV, Galiani D, Josefsberg LB, Carr DW, Dekel N.Expression and modification of PKA and AKAPs during meiosis in ratoocytes. Mol Cell Endocrinol 2002;192:105–13.