Physiological relevance of nitric oxide in ovarian functions: An overview.

Nitric oxide (NO, nitrogen monoxide), a short-lived, free radical carrying an unpaired electron, is one of the smallest molecules synthesized in the biological system. In addition to its role in angiogenesis, neuronal function and inflammatory response, NO has wide-spread significance in regulation of ovarian function in vertebrates. Based on tissue-specific expression, three different nitric oxide synthase (NOS) isoforms, neuronal (nNOS) or NOS1, inducible (iNOS) or NOS2 and endothelial (eNOS) or NOS3 have been identified. While expression of both inducible (iNOS) and constitutive NOS (eNOS) isoforms varies considerably in the ovary at various stages of follicular growth and development, selective binding of NO with proteins containing heme moieties have significant influence on ovarian steroidogenesis. Besides, NO modulation of ovulatory response suggests physiological significance of NO/NOS system in mammalian ovary. Compared to the duality of NO action on follicular development, steroidogenesis and meiotic maturation in mammalian models, participation of NO/NOS system in teleost ovary is less investigated. Genes encoding nos1 and nos2 have been identified in fish; however, presence of nos3 is still ambiguous. Interestingly, two distinct nos2 genes, nos2a and nos2b in zebrafish, possibly arose through whole genome duplication. Differential expression of major NOS isoforms in catfish ovary, NO inhibition of meiosis resumption in Anabas testudineus follicle-enclosed oocytes and NO/sGC/cGMP modulation of oocyte maturation in zebrafish are some of the recent advancements. The present overview is an update on the advancements made and shortfalls still remaining in NO/NOS modulation of intercellular communication in teleost vis-à-vis mammalian ovary.

Nonylphenol attenuates SOCS3 expression and M1 polarization in lipopolysaccharide-treated rat splenic macrophages.

Endocrine disruptors interfere with normal sexual and reproductive development of numerous organisms. Widely used in several chemical and manufacturing industries, nonylphenol (NP), a potent xenoestrogen, has the potential to perturb immune system. Using rat splenic macrophages (SMΦ) as the model system, NP-modulation of lipopolysaccharide (LPS)-induced inflammatory response has been investigated. Our results demonstrate that NP (0.1-10 µM) attenuates catalase activity, reactive oxygen species (ROS) generation and nitric oxide (NO) synthesis in LPS-treated SMΦ in vitro. NP inhibition of LPS-induced nuclear factor kappa B (NF-κB) activation and pro-inflammatory cytokine gene expression corroborate well with attenuation of suppressor of cytokine signalling 3 (SOCS3). Besides, elevated expression of anti-inflammatory factors reveals inverse correlation with suppression of endotoxin-induced M1 polarization in NP pre-incubated cells. While LPS promotes, NP prevents ERK1/2 (extracellular-signa1-regulated kinase 1/2) phosphorylation and MEK-inhibitor abrogates SOCS3 expression and NO production suggesting involvement of ERK1/2 in NP inhibition of SOCS3 expression. Further, translational inhibitor cycloheximide prevents LPS-induced NF-κB activation indicating functional importance of de novo synthesis of SOCS3, at least in part, in toll-like receptor 4 (TLR4)-mediated inflammatory response. Collectively, present study provides evidence favouring participation of SOCS3 in NP modulation of inflammatory response in rat SMΦ.

Relative importance of phosphatidylinositol-3 kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK3/1) signaling during maturational steroid-induced meiotic G2-M1 transition in zebrafish oocytes.

Participation and relative importance of phosphatidylinositol-3 kinase (PI3K) and mitogen-activated protein kinase (MAPK) signalling, either alone or in combination, have been investigated during 17α,20ß-dihydroxy-4-pregnen-3-one (DHP)-induced meiotic G2-M1 transition in denuded zebrafish oocyte. Results demonstrate that concomitant with rapid phosphorylation (activation) of Akt (Ser473) and MAPK (ERK1/2) at as early as 15 min of incubation, DHP stimulation promotes enhanced an GVBD response and histone H1 kinase activation between 1 and 5 h in full-grown oocytes in vitro. While p-Akt reaches its peak at 60 to 90 min and undergoes downregulation to the basal level by 240 min, ERK1/2 phosphorylation (activation) increases gradually until 120 min and remains high thereafter. Although, priming with MEK1/2 inhibitor U0126 is without effect, PI3K inhibitors, wortmannin or LY294002, delay the GVBD response significantly (P < 0.001) until 3 h but not at 5 h of incubation. Interestingly, blocking PI3K and MEK function together could abrogate steroid-induced oocyte maturation at all time points tested. While DHP stimulation promotes phospho-PKA catalytic (p-PKAc) dephosphorylation (inactivation) between 30-120 min of incubation, simultaneous inhibition of PI3K and MEK1/2 kinases abrogates DHP action. Conversely, elevated intra-oocyte cAMP, through priming with either adenylyl cyclase (AC) activator forskolin (FK) or dibutyryl cAMP (db-cAMP), abrogates steroid-induced Akt and ERK1/2 phosphorylation. Taken together, these results suggest that DHP-induced Akt and ERK activation precedes the onset of meiosis (GVBD response) in a cAMP-sensitive manner and PI3K/Akt and MEK/MAPK pathways together have a pivotal influence in the downregulation of PKA and resumption of meiotic maturation in zebrafish oocytes in vitro.

Expression of two insulin receptor subtypes, insra and insrb, in zebrafish (Danio rerio) ovary and involvement of insulin action in ovarian function.

Present study reports differential expression of the two insulin receptor (IR) subtypes in zebrafish ovary at various stages of follicular growth and potential involvement of IR in insulin-induced oocyte maturation. The results showed that mRNA expression for IR subtypes, insra and insrb, exhibited higher levels in mid-vitellogenic (MV) and full-grown (FG) rather than pre-vitellogenic (PV) oocytes. Interestingly, compared to the levels in denuded oocytes, mRNAs for both insra and insrb were expressed at much higher level in the follicle layer harvested from FG oocytes. Immunoprecipitation using IRß antibody could detect a protein band of desired size (∼95kDa) in FG oocyte lysates. Further, IRß immunoreactivity was detected in ovarian tissue sections, especially at the follicle layer and oocyte membrane of MV and FG, but not PV stage oocytes. While hCG (10IU/ml) stimulation was without effect, priming with insulin (5µM) could promote oocyte maturation of MV oocytes in a manner sensitive to de novo protein and steroid biosynthesis. Compared to hCG, in insulin pre-incubated MV oocytes, stimulation with maturation inducing steroid (MIS), 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) elicited higher maturational response. Potential involvement of insulin-mediated action on acquisition of maturational competence and regulation of oocyte maturation was further manifested through up regulation of 20ß-hydroxysteroid dehydrogenase (20ß-hsd), MIS receptor (mPRα), insulin-like growth factor 3 (igf3) and IGF1 receptor (igf1rb), but not cyp19a expression in MV oocytes. Moreover, priming with anti-IRß attenuated insulin action on meiotic G2-M1 transition indicating the specificity of insulin action and physiological relevance of IR in zebrafish ovary.

Regulation of recombinant human insulin-induced maturational events in Clarias batrachus (L.) oocytes in vitro.

Regulation of insulin-mediated resumption of meiotic maturation in catfish oocytes was investigated. Insulin stimulation of post-vitellogenic oocytes promotes the synthesis of cyclin B, histone H1 kinase activation and a germinal vesicle breakdown (GVBD) response in a dose-dependent and duration-dependent manner. The PI3K inhibitor wortmannin abrogates recombinant human (rh)-insulin action on histone H1 kinase activation and meiotic G2-M1 transition in denuded and follicle-enclosed oocytes in vitro. While the translational inhibitor cycloheximide attenuates rh-insulin action, priming with transcriptional blocker actinomycin D prevents insulin-stimulated maturational response appreciably, albeit in low amounts. Compared with rh-insulin, human chorionic gonadotrophin (hCG) stimulation of follicle-enclosed oocytes in vitro triggers a sharp increase in 17α,20ß-dihydroxy-4-pregnen-3-one (17α,20ß-DHP) secreted in the incubation medium at 12 h. Interestingly, the insulin, but not the hCG-induced, maturational response shows less susceptibility to steroidogenesis inhibitors, trilostane or dl-aminoglutethimide. In addition, priming with phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) or cell-permeable dbcAMP or adenylyl cyclase activator forskolin reverses the action of insulin on meiotic G2-M1 transition. Conversely, the adenylyl cyclase inhibitor, SQ 22536, or PKA inhibitor H89 promotes the resumption of meiosis alone and further potentiates the GVBD response in the presence of rh-insulin. Furthermore, insulin-mediated meiotic maturation involves the down-regulation of endogenous protein kinase A (PKA) activity in a manner sensitive to PI3K activation, suggesting potential involvement of a cross-talk between cAMP/PKA and insulin-mediated signalling cascade in catfish oocytes in vitro. Taken together, these results suggest that rh-insulin regulation of the maturational response in C. batrachus oocytes involves down-regulation of PKA, synthesis of cyclin B, and histone H1 kinase activation and demonstrates reduced sensitivity to steroidogenesis and transcriptional inhibition.

Expression of nitric oxide synthase (NOS) in Anabas testudineus ovary and participation of nitric oxide-cyclic GMP cascade in maintenance of meiotic arrest.

Participation of cyclic nucleotide-mediated signaling in nitric oxide/soluble guanylate cyclase (NO/sGC) regulation of oocyte maturation (OM) in perch (Anabas testudineus) follicle-enclosed oocytes has been investigated. Congruent with sharp decline in follicular cyclic GMP (cGMP) level, nitric oxide synthase (NOS)-inhibitor (L-NAME) attenuates protein kinase A (PKA) phosphorylation but promotes p-ERK1/2 and p-p34Cdc2 (Thr-161) in maturing oocytes. Conversely, NO donor (SNP) prevents OM, potentially through elevated cGMP synthesis. Expression and localization of Nos2 and Nos3 immunoreactivity in perch ovary varied considerably at progressively higher stages of folliculogenesis. While sGC inhibitor (ODQ) alone could induce OM, 8-bromo-cGMP attenuates 17,20ß-P-induced OM indicating functional significance of NO/sGC/cGMP in perch ovary. Interestingly, high NO/cGMP inhibition of OM shows positive relation with elevated cAMP level. MIS induced OM is more susceptible to the oocyte-specific phosphodiesterase (PDE) 3 than PDE4 inhibition. Collectively, high NO/cGMP attenuation of OM potentially involves PDE3 inhibition, cAMP accumulation and PKA activation.

Nitric oxide (NO) inhibition of meiotic G2-M1 transition in Anabas testudineus oocytes: Participation of cAMP-dependent protein kinase (PKA) in regulation of intra-oocyte signaling events.

Nitric oxide (NO) regulation of ovarian function in mammals has been studied extensively. However, relatively less information is available on NO action on meiotic G2-M1 transition in teleost oocytes. In the present study using follicle-enclosed oocytes of Anabas testudineus, NO regulation of intra-oocyte signaling events during meiotic G2-M1 transition were examined. Priming with NO donor, sodium nitroprusside (SNP) prevented 17α,20ß-dihydroxy-4-pregenen-3-one (17,20ß-P)-induced germinal vesicle break down (GVBD) in dose- and duration-dependent manner. Impaired GVBD response in SNP-treated groups corroborated well with reduced p34Cdc2 (Thr161) phosphorylation. Immunoblot analysis revealed that congruent with elevated cAMP-dependent protein kinase (PKA) phosphorylation (activation), NO inhibition of meiotic maturation involves down regulation of Cdc25 activation, Mos synthesis and MAPK3/1 (ERK1/2) phosphorylation. However, priming with PKA inhibitor (H89) could reverse SNP attenuation of oocyte GVBD significantly. Collectively our results indicate that negative influence of NO on meiotic G2-M1 transition in perch oocytes might involve PKA activation.

Cross-talk between insulin signalling and LPS responses in mouse macrophages.

The effect of insulin priming on Il-10 expression, regulation of inflammatory cytokines and participation of intra-cellular signalling events, primarily ERK1/2 and PI3K/Akt, has been investigated in high glucose (HG) and/or lipopolysaccharide (LPS)-induced murine macrophages. Our results demonstrate that congruent with sharp increase in ERK1/2 and CREB phosphorylation, insulin stimulation in vitro promotes significant increase in Il-10 expression in mouse peritoneal macrophage and RAW 264.7 cells, both positive for anti-IRß. Pharmacological inhibition of MEK/MAPK, but not PI3K/Akt cascade, abrogates CREB phosphorylation and Il-10 synthesis indicating functional relevance of insulin action. Conversely, priming with PI3K inhibitor wortmannin prevents insulin attenuation of HG- and/or LPS-induced p38 MAPK and NF-κB activation, Tnf-α, Il-1ß expression as well as NO production. Congruent with reduced Il-10 expression, MEK inhibition abrogates insulin action allowing significant increase in Tlr4 expression and LPS response indicating insulin-induced Il-10 might have pivotal influence in regulation of chronic as well as acute inflammatory response.