Reevaluation of the role of LIP-1 as an ERK/MPK-1 dual specificity phosphatase in the C. elegans germline.

The fidelity of a signaling pathway depends on its tight regulation in space and time. Extracellular signal-regulated kinase (ERK) controls wide-ranging cellular processes to promote organismal development and tissue homeostasis. ERK activation depends on a reversible dual phosphorylation on the TEY motif in its active site by ERK kinase (MEK) and dephosphorylation by DUSPs (dual specificity phosphatases). LIP-1, a DUSP6/7 homolog, was proposed to function as an ERK (MPK-1) DUSP in the Caenorhabditis elegans germline primarily because of its phenotype, which morphologically mimics that of a RAS/let-60 gain-of-function mutant (i.e., small oocyte phenotype). Our investigations, however, reveal that loss of lip-1 does not lead to an increase in MPK-1 activity in vivo. Instead, we show that loss of lip-1 leads to 1) a decrease in MPK-1 phosphorylation, 2) lower MPK-1 substrate phosphorylation, 3) phenocopy of mpk-1 reduction-of-function (rather than gain-of-function) allele, and 4) a failure to rescue mpk-1-dependent germline or fertility defects. Moreover, using diverse genetic mutants, we show that the small oocyte phenotype does not correlate with increased ectopic MPK-1 activity and that ectopic increase in MPK-1 phosphorylation does not necessarily result in a small oocyte phenotype. Together, these data demonstrate that LIP-1 does not function as an MPK-1 DUSP in the C. elegans germline. Our results caution against overinterpretation of the mechanistic underpinnings of orthologous phenotypes, since they may be a result of independent mechanisms, and provide a framework for characterizing the distinct molecular targets through which LIP-1 may mediate its several germline functions.

Organophotocatalyzed Mono- and Bis-Alkyl/Difluoroalkylative Thio/Selenocyanation of Alkenes.

Organophotocatalyzed three-component 1,2-difluoroacetyl/alkyl/perfluoroalkylative thio/selenocyanation of styrene derivatives under stoichiometric, transition metal-, oxidant-, and additive-free, and mild redox-neutral conditions is reported. Organophotocatalyst 4CzIPN operates the overall radical-polar-crossover mechanistic cycle via initial oxidative luminescence quenching, and the key intermediates were experimentally detected. Selective mono-alkylative thiocyanation of alkenes using dibromoalkanes is also demonstrated. This one-pot synthetic methodology is suitable for primary, secondary, and tertiary alkyl halides and also extended for double alkylative thiocyanation of the dibromoalkanes with excellent yields.

An organo-photocatalyzed visible-light-driven multi-component approach for carbothioaryl/alkylation of activated alkenes via C(sp3)-H bond functionalization.

A visible-light-driven organophotocatalyzed multi-component approach for carbothiolation of activated alkenes is demonstrated under environmentally benign and redox-neutral conditions, involving direct C(sp3)-H functionalization followed by electrophilic alkyl/arylthiolation. The three-component difunctionalization reaction is a complete transition-metal and peroxide-free process conducted under milder conditions. In this composite reaction, by employing bench-stable reagents, the formation of two new C(sp3)-C(sp3) and C(sp3)-S bonds is achieved for a wide variety of substrates, showcasing the excellent functional group tolerance and chemoselectivity of the methodology. Furthermore, the scalability and utilization of natural sunlight instead of artificial blue LEDs, along with the use of an inexpensive and easy-to-prepare pyrylium salt as an organo-photocatalyst, make this protocol greener and more energy efficient.

Isotopic Signatures and Outputs of Lead from Coal Fly Ash Disposal in China, India, and the United States.

Despite extensive research and technology to reduce the atmospheric emission of Pb from burning coal for power generation, minimal attention has been paid to Pb associated with coal ash disposal in the environment. This study investigates the isotopic signatures and output rates of Pb in fly ash disposal in China, India, and the United States. Pairwise comparison between feed coal and fly ash samples collected from coal-fired power plants from each country shows that the Pb isotope composition of fly ash largely resembles that of feed coal, and its isotopic distinction allows for tracing the release of Pb from coal fly ash into the environment. Between 2000 and 2020, approx. 236, 56, and 46 Gg Pb from fly ash have been disposed in China, India, and the U.S., respectively, posing a significant environmental burden. A Bayesian Pb isotope mixing model shows that during the past 40 to 70 years, coal fly ash has contributed significantly higher Pb (∼26%) than leaded gasoline (∼7%) to Pb accumulation in the sediments of five freshwater lakes in North Carolina, U.S.A. This implies that the release of disposed coal fly ash Pb at local and regional scales can outweigh that of other anthropogenic Pb sources.

Utilization of dark fermentation effluent for algal cultivation in a modified airlift photobioreactor for biomass and biocrude production.

Developing an efficient photobioreactor (PBR) and reducing freshwater dependence are among the significant challenges for generating 3rd generation biomass feedstock. Addressing these, the present study focused on developing a modified airlift (MoAL) PBR. Its performance was further evaluated and compared with the traditional airlift PBR by cultivating microalgae in dark fermentation spent wash. Lower mixing time and higher interfacial mass transfer coefficient was observed in the MoAL PBR having a perforated draft tube. Experimentally, the MoAL exhibited the maximum biomass concentration of 3.18 g L-1, which was 30% higher than that of the conventional airlift PBR. The semi-continuous operation of the MoAL (with water recycling) achieved the maximum biomass productivity of 0.83 g L-1 d-1, two folds superior to that of batch culture. The comprehensive biomass characterization (proximate, ultimate, and thermochemical) further confirmed its potential for bioenergy application. Considering that, hydrothermal liquefaction of the biomass resulted in a maximum biocrude yield of 31% w/w with a higher heating value (HHV) of 36.6 MJ kg-1. In addition, the biocrude comprised 66.6% w/w lighter fraction (<343 °C), including 21.5% w/w of heavy naphtha, 20.5% w/w of kerosene, and 24.6% w/w of diesel. The results can help develop sustainable technology for simultaneous wastewater remediation and biocrude production.

Regulation of oocyte maturation: Role of conserved ERK signaling.

During oogenesis, oocytes arrest at meiotic prophase I to acquire competencies for resuming meiosis, fertilization, and early embryonic development. Following this arrested period, oocytes resume meiosis in response to species-specific hormones, a process known as oocyte maturation, that precedes ovulation and fertilization. Involvement of endocrine and autocrine/paracrine factors and signaling events during maintenance of prophase I arrest, and resumption of meiosis is an area of active research. Studies in vertebrate and invertebrate model organisms have delineated the molecular determinants and signaling pathways that regulate oocyte maturation. Cell cycle regulators, such as cyclin-dependent kinase (CDK1), polo-like kinase (PLK1), Wee1/Myt1 kinase, and the phosphatase CDC25 play conserved roles during meiotic resumption. Extracellular signal-regulated kinase (ERK), on the other hand, while activated during oocyte maturation in all species, regulates both species-specific, as well as conserved events among different organisms. In this review, we synthesize the general signaling mechanisms and focus on conserved and distinct functions of ERK signaling pathway during oocyte maturation in mammals, non-mammalian vertebrates, and invertebrates such as Drosophila and Caenorhabditis elegans.

Visible-Light-Driven Organophotocatalyzed Multicomponent Approach for Tandem C(sp3)-H Activation and Alkylation Followed by Trifluoromethylthiolation.

A visible-light-driven organophotocatalyzed multicomponent approach has been developed for tandem direct C(sp3)-H activation and alkylation followed by trifluoromethylthiolation in a one-pot operation. We report a completely metal-free, tandem, three-component approach for the difunctionalization of activated alkenes via the photoinduced radical pathway. This protocol allows the formation of two new C(sp3)-C(sp3) and C(sp3)-SCF3 bonds using a bench-stable, easy-to-handle trifluoromethylthiolating reagent under mild reaction conditions. The generosity of this reaction is shown with a library of C(sp3)-H donors and alkenes derivatives. The reaction conditions can tolerate a wide variety of functional groups. Gram-scale synthesis using environmentally benign and straightforward conditions highlights the synthetic advancement of the methodology. Further functionalization of the final product is also successfully demonstrated.

Photofermentative biohydrogen generation from organic acids by Rhodobacter sphaeroides O.U.001: Computational fluid dynamics modeling of hydrodynamics and temperature.

Hydrogen gas is a clean-burning fuel suitable for powering public vehicles. Hydrogen fuel has the highest energy density (143 MJ kg-1 ). This research paper emphasizes three-dimensional hydrodynamics and temperature distribution during photobiohydrogen generation by Rhodobacter sphaeroides strain O.U.001 in a triple-jacketed 1 L photobioreactor (PBR). The fermentation broth has turbulent flow conditions and light gradients among various layers, which affect the light conversion efficiency of the PBR. From the carbon source (malic acid), various organic acids are produced within fermentation (lactate, acetate, and formate). Modeling and simulation studies by computational fluid dynamics confirmed uniform fluid dynamics and heat transfer throughout the annular PBR. The modified Gompertz equation gave good simulated fitting with an experimental value for H2 generation. R. sphaeroides O.U. 001 gave good simulated results for H2 generation with mathematical modeling of substrate consumption kinetics and substrate utilization for biomass.

Visible-Light-Driven Organophotocatalyzed Mono-, Di-, and Tri-C(sp3)-H Alkylation of Phosphoramides.

A photocatalytic metal-free, visible-light-driven, highly atom-economic, direct multiple α-C(sp3)-H alkylation of phosphoramides and thiophosphoramides is demonstrated under environmentally benign conditions. Economically viable and commercially available Eosin-Y is used as an HAT photocatalyst for mono-α-C(sp3)-H alkylation of phosphoramide derivatives. Remarkably, di- and tri-C(sp3)-H alkylation of phosphoramides and thiophosphoramides using an acridinium photocatalyst is reported with good yield and selectivity. Mechanistic studies reveal that monoalkylation of phosphoramides by Eosin-Y follows the HAT mechanism, whereas di- and tri-C(sp3)-H alkylation by the acridinium photocatalyst follows the SET mechanism.

A sensitive 1 H NMR spectroscopic method for the quantification of capsaicin and capsaicinoid: morpho-chemical characterisation of chili land races from northeast India.

INTRODUCTION: Proton (1 H) nuclear magnetic resonance (NMR) spectroscopy based analytical method for the quantification of capsaicin (major pungent principle of chili) has certain advantages including short data acquisition time and better structural authentication. Earlier NMR methods are associated with either of the bottlenecks such as low or lack of information on the sensitivity and scope for the quantification of total capsaicinoid. OBJECTIVE: To develop a sensitive 1 H quantitative NMR (qNMR) technique for capsaicin and total capsaicinoid in dry chili and chili oleoresin and to demonstrate its applicability in a real sample set. METHOD: A 1 H qNMR method was developed using benzene as the internal standard for the quantification of capsaicin (terminal methyl signal) as well as total capsaicinoid (benzyl methylene signal) in dry chili and oleoresin and validated in terms of specificity, linearity, sensitivity, accuracy and precision. RESULTS: The developed 1 H qNMR method was specific, sensitive (limit of detection 4.4 µg/mL and limit of quantitation 14.8 µg/mL), linear in the range 0.083-8.33 mg/mL of capsaicin, accurate and precise. The credibility of the developed method was showcased in the morpho-chemical characterisation of commercially available 15 chili land races from northeast India. The analysis identified the land races with a wide range of capsaicin (trace to 1.49% in the dry fruit and trace to 6.21% in the oleoresin w/w) and oleoresin content (3.35-26.78% w/w). CONCLUSION: The standardized 1 H qNMR method facilitated the findings of chemical basis for the selection of chili land races from this region, capable of producing high-yielding oleoresin with intended degree of pungency.

Ammonium Chloride-Mediated Trifluoromethylthiolation of p-Quinone Methides.

Ammonium chloride-mediated trifluoromethylthiolation of p-quinone methides is reported using inexpensive and bench stable AgSCF3 as a nucleophilic trifluoromethylthiolating (-SCF3) reagent. This method is an efficient strategy for the construction of the benzylic C(sp3)-SCF3 bond to synthesize trifluoromethylthio-diarylmethane derivatives by 1,6-conjugate addition/aromatization under mild reaction conditions without any metal catalyst, oxidants, or additives. This is the first report of trifluoromethylthiolation of p-quinone methides. In addition, di-trifluoromethylthiolation of δ-chloro-p-quinone methide and scalability are demonstrated.

Influence of photobioreactor configuration on microalgal biomass production.

Biodiesel production from microalgae depends on the biomass concentration and lipid content in microalgal cells. Photobioreactors (PBRs) facilitates cultivation of microalgae and renders better process control than open systems. However, reactor configuration and consequential hydrodynamics considerably influence biomass and lipid production from microalgae. Here, four different configurations of PBRs, viz. airlift and bubble column with orifice sparger and newly designed ring sparger, were investigated. Resulting volumetric mass transfer coefficient, mixing time, and shear stress were analyzed at different air flow rates to realize their influence on biomass and lipid production from Neochloris oleoabundans UTEX 1185. Bubble column reactor with ring sparger was observed to exhibit superior performance, which was subsequently simulated using a two-phase Eulerian model to comprehend the influence of air flow rates on mixing time. The developed computational model corroborates well with the experimental findings of optimum air flow rate for maximum biomass yield in bubble column configuration.

A distinctive transformation based diversity oriented synthesis of small ring carbocycles and heterocycles from biocatalytically derived enantiopure α-substituted-ß-hydroxyesters.

A series of structurally novel small ring carbocyclic and heterocyclic molecules were accessed in an enantiopure fashion. The starting materials, α-substituted-ß-hydroxyesters, were achieved through the biocatalytic dynamic kinetic resolution of parent ß-ketoesters in an excellent enantio and diastereocontrolled way. The active functional groups present in the starting precursor, were then tuned sequentially through certain key transformations (functional group interconversions; FGIs) to yield several small molecular scaffolds in a diverse way. Specific transformations such as halocyclization, ene-yne metathesis, dipolar cycloaddition, Mitsunobu cyclization, ring closing metathesis and Pauson-Khand reactions were mainly applied to generate the diversity.

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.

Conserved insulin signaling in the regulation of oocyte growth, development, and maturation.

Insulin signaling regulates various aspects of physiology, such as glucose homeostasis and aging, and is a key determinant of female reproduction in metazoans. That insulin signaling is crucial for female reproductive health is clear from clinical data linking hyperinsulinemic and hypoinsulinemic condition with certain types of ovarian dysfunction, such as altered steroidogenesis, polycystic ovary syndrome, and infertility. Thus, understanding the signaling mechanisms that underlie the control of insulin-mediated ovarian development is important for the accurate diagnosis of and intervention for female infertility. Studies of invertebrate and vertebrate model systems have revealed the molecular determinants that transduce insulin signaling as well as which biological processes are regulated by the insulin-signaling pathway. The molecular determinants of the insulin-signaling pathway, from the insulin receptor to its downstream signaling components, are structurally and functionally conserved across evolution, from worms to mammals-yet, physiological differences in signaling still exist. Insulin signaling acts cooperatively with gonadotropins in mammals and lower vertebrates to mediate various aspects of ovarian development, mainly owing to evolution of the endocrine system in vertebrates. In contrast, insulin signaling in Drosophila and Caenorhabditis elegans directly regulates oocyte growth and maturation. In this review, we compare and contrast insulin-mediated regulation of ovarian functions in mammals, lower vertebrates, C. elegans, and Drosophila, and highlight conserved signaling pathways and regulatory mechanisms in general while illustrating insulin's unique role in specific reproductive processes.

Endocrine and paracrine regulation of meiotic cell cycle progression in teleost oocytes: cAMP at the centre of complex intra-oocyte signalling events.

Participation of major endocrine and/or local autocrine/paracrine factors and potential interplay between apparently disparate intra-oocyte signalling events during maintenance and withdrawal of meiotic prophase arrest has been an area of active research in recent years. Studies on oocyte maturation have contributed substantially in the discovery of some of the most important biochemical and cellular events like functional significance of novel membrane-associated steroid receptors, elucidation of maturation promoting factor (MPF), cytostatic factor (CSF) and other signalling cascades that entrain the cell cycle clock to hormonal stimuli. While follicular estrogen has largely been implicated in maintenance of prophase arrest, involvement of maturational steroid and membrane progestin receptor in resumption of meiotic G2-M1 transition in piscine oocytes has been shown earlier. Moreover, detection of ovarian IGF system, maturational gonadotropin stimulation of IGF ligands and potential synergism between maturational steroid and IGF1 in zebrafish oocytes are most recent advancements. Though endocrine/paracrine regulation of cyclic nucleotide-mediated signalling events in meiotic cell cycle progression is well established, involvement of PI3K/Akt signalling cascade has also been reported in fish, amphibian and mammalian oocytes. The major objective of this overview is to describe how fish oocytes maintain high cAMP/PKA activity and how steroid- and/or growth factor-mediated signalling cascade regulate this pathway for the withdrawal of meiotic arrest. Moreover, special emphasis is placed on some recent findings on interaction of PKA with some of the MPF-regulating components (e.g., synthesis of cyclin B or MEK/MAPK signalling cascade) for the maintenance of prophase arrest.

Maximizing power generation from dark fermentation effluents in microbial fuel cell by selective enrichment of exoelectrogens and optimization of anodic operational parameters.

OBJECTIVE: To selectively enrich an electrogenic mixed consortium capable of utilizing dark fermentative effluents as substrates in microbial fuel cells and to further enhance the power outputs by optimization of influential anodic operational parameters. RESULTS: A maximum power density of 1.4 W/m3 was obtained by an enriched mixed electrogenic consortium in microbial fuel cells using acetate as substrate. This was further increased to 5.43 W/m3 by optimization of influential anodic parameters. By utilizing dark fermentative effluents as substrates, the maximum power densities ranged from 5.2 to 6.2 W/m3 with an average COD removal efficiency of 75% and a columbic efficiency of 10.6%. CONCLUSION: A simple strategy is provided for selective enrichment of electrogenic bacteria that can be used in microbial fuel cells for generating power from various dark fermentative effluents.

Releasing prophase arrest in zebrafish oocyte: synergism between maturational steroid and Igf1.

Binding of 17ß-estradiol (E2) to novel G-protein coupled receptor, Gper1, promotes intra-oocyte adenylyl cyclase activity and transactivates epidermal growth factor receptor to ensure prophase-I arrest. Although involvement of either membrane progestin receptor (mPR) or Igf system has been implicated in regulation of meiosis resumption, possibility of concurrent activation and potential synergism between 17α,20ß-dihydroxy-4-pregnen-3-one (DHP)- and Igf-mediated signalling cascades in alleviating E2 inhibition of oocyte maturation (OM) has not been investigated. Here using zebrafish (Danio rerio) defolliculated oocytes, we examined the effect of DHP and Igf1, either alone or in combination, in presence or absence of E2, on OM in vitro. While priming of denuded oocytes with E2 blocked spontaneous maturation, co-treatment with DHP (3 nM) and Igf1 (10 nM), but not alone, reversed E2 inhibition and promoted a robust increase in germinal vesicle breakdown (GVBD). Although stimulation with either Igf1 or DHP promoted Akt phosphorylation, pharmacological inhibition of PI3K/Akt signalling prevented Igf1-induced GVBD but delayed DHP action till 4-5 h of incubation. Moreover, high intra-oocyte cAMP attenuates both DHP and Igf1-mediated OM and co-stimulation with DHP and Igf1 could effectively reverse E2 action on PKA phosphorylation. Interestingly, data from in vivo studies reveal that heightened expression of igf1, igf3 transcripts in intact follicles corresponded well with elevated phosphorylation of Igf1r and Akt, mPRa immunoreactivity, PKA inhibition and accelerated GVBD response just prior to ovulation. This indicates potential synergism between maturational steroid and Igf1 which might have physiological relevance in overcoming E2 inhibition of meiosis resumption in zebrafish oocytes.

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.