L-arginine affects the IVM of cattle cumulus-oocyte complexes.

Nitric oxide (NO) is identified as a signaling molecule involved in many cellular or physiological functions, including meiotic maturation of cattle oocytes. This study aimed to evaluate the effect of supplementation of culture medium with the L-arginine (L-arg, NO synthesis precursor) in nuclear maturation of oocytes, concentrations of nitrate/nitrite, progesterone (P4), and 17ß-estradiol (E2) in the culture medium; and the cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) intracellular concentrations in the cumulus-oocyte complexes (COCs) during the first hours of maturation in the presence of hemisections (HSs) of the follicular wall (control -ve). The addition of 5.0-mM L-arg increased (P < 0.05) the percentage of oocytes at the germinal vesicle breakdown stage after 7 hours of cultivation compared with control -ve. All concentrations of L-arg (2.5, 5.0, and 10.0 mM) increased the percentage of oocytes that reached the metaphase I (MI) at 15 hours (P < 0.05) but do not affect the progression from MI to metaphase II (P > 0.05) at 22 hours. All concentrations of L-arg tested increased (P < 0.05) the percentage of cumulus cells with plasma membrane integrity at 22 hours of cultivation. L-arginine did not change (P > 0.05) the nitrate/nitrite, P4, and E2 concentrations in relation to control -ve at any of the times tested. In immature COCs, immediately after being removed from the follicles (0 hours), the intracellular concentration of cGMP in the control -ve and treatment with 5-mM L-arg progressively decreased (P < 0.05) after the first hour of cultivation; however, COCs treated with 5.0-mM L-arg had higher concentrations of cGMP at 1 hour of cultivation (P < 0.05). The cAMP concentration of COCs supplemented or not with 5.0-mM L-arg progressively increased until 3 hours of cultivation and at, 6 hours, decreased (P < 0.05). The results show, in using this system, that (1) the mechanisms that give the oocyte the ability to restart the meiosis until MI after adding 5.0-mM L-arg do not involve changes in the concentration of nitrate/nitrite, P4, and E2 in the culture medium and (2) L-arg acts on a pathway that involves changing the cGMP concentration but does not involve changing cAMP concentration. More studies are needed to assess whether the observed effects of L-arg during IVM using this system are via NO or not and what the role is in increasing the viability of cumulus cells in the resumption and progression of meiosis until MI.

Nitric oxide regulates steroid synthesis by bovine antral granulosa cells in a chemically defined medium.

Nitric oxide (NO) in bovine ovary has been characterized as one of the controllers of granulosa cells' (GC) steroidogenesis and apoptosis. One of the pathways used by NO to have these effects is cGMP. The objectives of the present study were to verify the effect of sodium nitroprusside (SNP), a NO donor, on steroidogenesis, cell viability (mitochondrial activity) and GC cell cycle distribution and if this effect occurs by the NO-cGMP signaling pathway with the addition of SNP with or without 1H-[1,2,3] oxadiaziolo[4,3a]quinoxaline-1-one (ODQ), a selective soluble guanylate cyclase inhibitor. The antral GC from 3 to 5mm diameter cattle follicles was cultured without treatment (control), with ODQ (10(-4)M) and 10(-5), 10(-3) and 10(-1)M SNP with or without ODQ for 24h. Nitrate/nitrite (NO(3)(-)/N0(2)(-)) concentrations were evaluated by Griess method, progesterone (P(4)) and 17beta-estradiol (E(2)) concentrations by chemiluminescence, viability and cell cycle stage by MTT method (3-[4,5-dimethylthiazol-2yl]-2,3 dipheniltetrazolium bromide) and flow cytometry, respectively. Nitrate/nitrite concentration in culture medium increased (P<0.05) in a dose-dependent manner according to SNP concentration added to the culture medium. The GC cultured without treatment, with ODQ and with 10(-5)M SNP in the presence or absence of ODQ developed into cell aggregates and did not vary in cell viability (P>0.05), while GC cultured with 10(-3) and 10(-1)M SNP with or without ODQ presented disorganized GC aggregates or did not develop into cell aggregates and also had substantially decreased cell viability (mitochondrial activity inhibition) and steroids synthesis (P<0.05), and effects were not reversed with us of ODQ. Most GC cultured without treatment (control) or with ODQ, 10(-5) and 10(-3)M SNP with or without ODQ were in the G0/G1 (80-75%) stage and in a lesser proportion (20-25%) in the S+G2/M stage of the cell cycle, while the 10(-1)M SNP treatment resulted in GC in G1 phase arrest. The treatment with 10(-5)M SNP increased (P<0.05) E(2) synthesis and inhibited (P<0.05) progesterone synthesis. The addition of ODQ reversed (P<0.05) the stimulatory effect of 10(-5)M SNP treatment on E(2), but not on P(4) synthesis (P>0.05). These results demonstrated that E(2) synthesis by antral GC from small follicles is modulated by lesser NO concentrations via the cGMP pathway, but not P(4) while steroids inhibition cGMP pathway independent, mitochondrial damage and the interference on cell cycle progression caused by greater NO concentration can lead to cell death.

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.

Effect of sodium nitroprusside, a nitric oxide donor, on the in vitro maturation of bovine oocytes.

Nitric oxide (NO) is a highly reactive free radical involved in intra- and intercellular signaling in various stages of reproduction. The objective of the present study was to evaluate the effect of the addition of sodium nitroprusside (SNP), a NO donor, on nuclear and cytoplasmic in vitro maturation of bovine oocytes. Analysis of variance was conducted and the means were compared by t test at a level of 5%. Low (10(-7) and 10(-9)M) and intermediate (10(-5)M) concentrations of SNP had no significant effect on nuclear maturation, however, when a greater concentration of SNP (10(-3)M) was added, oocytes remained in metaphase I (MI) after 24 h culture (P<0.05) and did not show cumulus expansion. To evaluate if this effect was reversible and if a retardation or inhibition had occurred in the progression from MI to MII, oocytes were cultured in presence of 10(-3)M of SNP for 24 h followed by culture for an additional 24 h in medium with or without SNP. After 48 h, the oocytes remained in MI even when the medium was changed at 24 h with or without SNP. The kinetics of nuclear maturation was assessed to evaluate if there had been or not a retardation in the progression of meiosis with the concentration of 10(-3)M SNP. This concentration delayed germinal vesicle breakdown (VGBD) at 8 h of culture (P<0.05), and at 12 h there was no significant difference between the control and the treated group. The concentrations that did not induce alterations in nuclear maturation were evaluated for cytoplasmic maturation. The concentration of 10(-5)M improved the percentage of peripheral cortical granules (P<0.05), and significantly increased the percentage of blastocysts. These results demonstrate that SNP at greater concentrations (10(-3)M) has a cytotoxic effect, but at intermediate (10(-5)M) concentrations it increases blastocyst rates. NO exhibits a dual effect on bovine oocytes, inhibits (10(-3)M of SNP) nuclear and cytoplasmic maturation or stimulates (10(-5)M of SNP) cytoplasmic maturation, depending on concentration in the culture medium.