Nitric oxide in follicle development and oocyte competence

2017

Dev Growth Differ

Nitric oxides (NO) act as one of the major signal molecules and modulate various cell functions including oocyte meiosis in mammals. The present study was designed to investigate the mechanism of NO action during spontaneous meiotic exit from diplotene arrest (EDA) in rat cumulus oocytes complexes (COCs) cultured in vitro. Diplotene-arrested COCs collected from ovary of immature female rats after 20 IU pregnant mare's serum gonadotropins (PMSG) for 48 h were exposed to various concentrations of NO donor, S-nitroso-N-acetyl penicillamine (SNAP) and inducible nitric oxide synthase (iNOS) inhibitor, aminoguanidine (AG) for 3 h in vitro and downstream factors were analyzed. Our results suggest that SNAP inhibited, while AG induced EDA in a concentration-dependent manner. The iNOS-mediated total NO, cyclic nucleotides and cell division cycle 25B (Cdc25B) levels were reduced significantly. The decreased Cdc25B was associated with the increased Thr14/Tyr15 phosphorylated cyclin-dependent kinase 1 (Cdk1) level and decreased Thr161 phosphorylated Cdk1 as well as cyclin B1 levels leading to maturation promoting factor (MPF) destabilization. The destabilized MPF finally induced spontaneous EDA. Taken together, these results suggest that reduction of iNOS-mediated NO level destabilizes MPF during spontaneous EDA in rat COCs cultured in vitro.

“…Follicular oocytes are capable of generating nitric oxide (NO) sufficient to modulate oocyte physiology (Kuo et al . ; Basini & Grasselli ; Thompson et al . ).…”

Section: Introductionmentioning

confidence: 99%

Reduction of nitric oxide level results in maturation promoting factor destabilization during spontaneous meiotic exit from diplotene arrest in rat cumulus oocytes complexes cultured in vitro

2017

Dev Growth Differ

“…Pituitary gonadotropins modulate expressions of various nitric oxide synthase (NOS) isoforms. 40 Both pregnant mare's serum gonadotropin (PMSG) and hCG have been shown to influence inducible nitric oxide synthase (iNOS) expressions in rat ovary. 41 Recent studies from our laboratory suggest that decreased level of nitric oxide (NO) is associated with meiotic exit from diplotene, as well as M-II arrest, 12 , 17 , 22 while high level of NO induces cell cycle arrest and apoptosis.…”

Section: Introductionmentioning

confidence: 99%

Human Chorionic Gonadotropin Mediated Generation of Reactive Oxygen Species Is Sufficient to Induce Meiotic Exit but Not Apoptosis in Rat Oocytes

BioResearch Open Access

2017

Generation of reactive oxygen species (ROS) is associated with final stages of follicular development and ovulation in mammals. The human chorionic gonadotropin (hCG) mimics the action of luteinizing hormone and triggers follicular development and ovulation. However, it remains unclear whether hCG induces generation of ROS, if yes, whether hCG-mediated increased level of ROS could induce meiotic exit and/or apoptosis in rat oocytes. For this purpose, cumulus–oocyte complexes (COCs) were collected from ovary of experimental rats injected with 20 IU pregnant mare's serum gonadotropin for 48 h followed by 20 IU hCG for 0, 7, 14, and 21 h. The morphological changes in COCs, meiotic status of oocyte, total ROS, hydrogen peroxide (H2O2), inducible nitric oxide synthase (iNOS), nitric oxide (NO), Bax, Bcl-2, cytochrome c, telomerase reverse transcriptase (TERT) expression levels, and DNA fragmentation were analyzed in COCs. Our data suggest that hCG surge increased total ROS as well as H2O2 levels but decreased iNOS expression and total NO level in oocytes. The hCG-mediated increased level of ROS was sufficient to induce meiotic cell cycle resumption in majority of oocytes as evidenced by meiotic exit from diplotene as well as metaphase-II (M-II) arrest and their meiotic status. However, increase of ROS level due to hCG surge was not sufficient to trigger Bax and cytochrome c expression levels and DNA fragmentation in COCs. In addition, increased TERT activity was observed in oocytes collected 21 h post-hCG surge showing onset of oocyte aging. Taken together, these results suggest that hCG induces generation of ROS sufficient to trigger meiotic exit from diplotene, as well as M-II arrest, but not good enough to induce apoptosis in rat oocytes.

“…A moderate increase of intracellular Ca 2+ ([Ca 2+ ]i) level under the physiological range triggers generation of ROS and decreases iNOS expression during spontaneous EDA in rat oocytes cultured in vitro [14,22,[46][47][48]. Our results suggested that increase of ROS level and decrease of iNOS expression induced EDA after 7 h of in vitro culture.…”

Section: Rosmentioning

confidence: 64%

Spontaneous Exit from Diplotene Arrest Associates with the Increase of Calcium and Reactive Oxygen Species in Rat Oocytes Cultured In Vitro

2017

ROS

Several signal molecules modulate the physiology of mammalian oocytes. Changes in the level of various signal molecules and their downstream impact on maturation promoting factor (MPF) during the achievement of meiotic competency remain ill understood. Therefore, the present study aimed to analyze levels of various signal molecules and MPF during the achievement of meiotic competency in rat oocytes. For this purpose, cumulus oocyte complexes (COCs) were collected from animals treated with 20 IU pregnant mare's serum gonadotropin (PMSG) for 48 h, followed by 20 IU human chorionic gonadotropin (hCG) for 14 h. The morphological changes, meiotic status and oocyte competency, cytosolic free calcium (Ca 2+), total reactive oxygen species (ROS), inducible nitric oxide synthase (iNOS), adenosine 3′,5′-cyclic monophosphate (cAMP), guanosine 3′,5′-cyclic monophosphate (cGMP), cell division cycle 25B (Cdc25B), calciumcalmodulin-dependent protein kinase II (CaMKII), Wee1, specific phosphorylation status of cyclin-dependent kinase 1 (Cdk1), and cyclin B1 expression levels were analyzed. Our data suggest that the culture of denuded diplotene-arrested oocytes resulted in spontaneous exit from diplotene arrest (EDA) in a time-dependent manner. The supplementation of hCG (2 IU) did not show any additive effect over spontaneous EDA in vitro. The hCG (20 IU) surge induced achievement of meiotic competency as most of the ovulated oocytes showed metaphase-II (M-II) arrest with extrusion of first polar body (PB-I). The spontaneous EDA was associated with a moderate increase of cytosolic free Ca 2+ as well as total ROS levels. Furthermore, iNOS, cAMP, cGMP, Cdc25B, Thr161 phosphorylated Cdk1, as well as cyclin B1 levels were significantly decreased, while CaMKII, Wee1, and Thr14/Tyr15 phosphorylated Cdk1 levels were increased significantly during spontaneous EDA. These changes were reversed in ovulated oocytes that showed M-II arrest. The above results suggest that the oscillation of signal molecules modulates MPF destabilization during the achievement of meiotic competency in rat oocytes cultured in vitro.