Mating behaviors in ovoviviparous black rockfish (Sebastes schlegelii): molecular function of prostaglandin E2 as both a hormone and pheromone.

Prostaglandins (PGs) are profound hormones in teleost sexual behavior, especially in mating. PGs act as pheromones that affect the olfactory sensory neurons of males, inducing the initiation of a series of mating behaviors. However, the molecular mechanism by which PGs trigger mating behavior in ovoviviparous teleosts is still unclear. In the present study, we employed the ovoviviparous black rockfish (Sebastes schlegelii), an economically important marine species whose reproductive production is limited by incomplete fertilization, as a model species. The results showed that when the dose of PGE2 was higher than 10 nmol/L, a significant (P < 0.05) increase in mating behaviors was observed. Dual-fluorescence in situ hybridization indicated that PGE2 could fire specific neurons in different brain regions and receptor cells in the olfactory sac. After combining with specific neurons in the central nervous system (CNS), a series of genes related to reproduction are activated. The intracerebroventricular administration of PGE2 significantly increased lhb levels (P < 0.05) in both sexes. Moreover, steroidogenesis in gonads was also affected, inducing an increase (P < 0.05) in E2 levels in males and T levels in females. PGE2 levels were also increased significantly (P < 0.05) in both sexes. The present study revealed that PGE2 can activate mating behavior in black rockfish in both hormone and pheromone pathways, leading to variations in sex steroid levels and activation of reproductive behaviors. Our results provide not only novel insight into the onset of mating behaviors in ovoviviparous teleosts but also solutions for the incomplete fertilization caused by natural mating in cage aquaculture. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00214-w.

PGE2 functions in ovoviviparous teleost black rockfish (Sebastes schlegelii): evolutionary status between parturition and ovulation†.

Fish have evolved various reproductive strategies including oviparity, viviparity, and ovoviviparity, which undoubtedly affect the survival of the whole species continuity. As the final step in reproduction, parturition in viviparous vertebrate and ovulation in oviparous teleost seem to share a similar mechanism, when prostaglandins (PGs) act as the trigger to launch the whole process. In the present study, ovoviviparous teleost black rockfish (Sebastes schlegelii) is employed as the research object. Intraperitoneal injection showed that PGE2 (500 µg/kg) could activate the delivery reactions in perinatal black rockfish. RNA-seq data of ovary in perinatal period revealed transcriptional change in cell junction, inflammation, and apoptosis, which is related to mammal parturition and teleost ovulation. Further results proved the positive correlation between ptger EP2 and previous mentioned pathways. Subsequent experiment proved that PGE2 was able to induce the ovulation and spawning in unfertilized individuals, which had a bilayer follicular structure compared to monolayer follicular in perinatal period black rockfish. Both unfertilized and perinatal ovary matrix could response to PGE2 stimulation. In conclusion, the function of PGE2 in activating both parturition and ovulation in a relatively different pathways conserved with viviparity or oviparity provided novel evidence of the evolutionary status of ovoviviparous vertebrates.

FGFs function in regulating myoblasts differentiation in spotted sea bass (Lateolabrax maculatus).

Fibroblast growth factors (FGFs) are a family of structurally related peptides that regulate processes such as cell proliferation, differentiation, and damage repair. In our previous study, fibroblast growth factor receptor 4 (fgfr4) was detected in the most significant quantitative trait loci (QTL), when identified of QTLs and genetic markers for growth-related traits in spotted sea bass. However, knowledge of the function of fgfr4 is lacking, even the legends to activate the receptor is unknown in fish. To remedy this problem, in the present study, a total of 33 fgfs were identified from the genomic and transcriptomic databases of spotted sea bass, of which 10 were expressed in the myoblasts. According to the expression pattern during myoblasts proliferation and differentiation, fgf6a, fgf6b and fgf18 were selected for further prokaryotic expression and purification. The recombinant proteins FGF6a, FGF6b and FGF18 were found to inhibit myoblast differentiation. Overall, our results provide a theoretical basis for the molecular mechanisms of growth regulation in economic fish such as spotted sea bass.

Pro-inflammatory cytokine IL1ß1 participates in promoting parturition related pathways in the ovoviviparous teleost black rockfish (Sebastes schlegelii).

Along with the evolution process, the reproductive strategies evolved including oviparity, viviparity and ovoviviparity, to fit the residential environment maximize the survival rate of the off spring. In mammals, the key to the initiation of parturition is the inflammatory response at the maternal-fetal interface. As a pro-inflammatory cytokine, interleukin 1 beta (IL1ß) plays an important role in the process of human parturition. While less is known about IL1ß1 in teleost parturition, identification of the functions of IL1ß1 in inducing the parturition, black rockfish, an ovoviviparity teleost, which provides over 60% nutrition supply for over 50 000 embryos though a placenta like structure during pregnant, was employed as the research model. In the present study, based on the gene cloning, we detected the expression pattern of both Il1b1 and its receptor perinatal period, as well as the localization to the ovary by in situ hybridization. The different expression genes in transcriptomic data of perinatal primary ovarian cells treated with the recombinant IL1ß1 (rIL1ß1) obtained by prokaryotic expression system were analyzed. Differentially expressed genes, functional enrichment and pathway analysis mainly included immune response, signal transduction and cell death. In summary, our research provides novel insights into the potential role of IL1ß1 in the parturition of ovoviviparity teleost.

Olfactory receptor OR52N2 for PGE2 in mediation of guppy courtship behaviors.

Prostaglandins (PGs) are a type of physiologically active unsaturated fatty acids. As an important sex pheromone, PGs play a vital role in regulating the reproductive behaviors of species by mediating nerve and endocrine responses. In this study, guppy (Poecilia reticulate) was used as the model specie to detect the function of PGE2 in inducing the onset of courtship behaviors. Our results showed that adding PGE2 into the water environment could activate the courtship behavior of male guppy, indicating that the peripheral olfactory system mediated the PGE2 function. Thereafter, the open reading frame (ORF) of olfactory receptor or52n2 was cloned, which was 936 bp in length, coding 311 amino acids. As a typical G protein-coupled receptor, OR52N2 had a conservative seven α-helix transmembrane domains. To confirm the regulatory relationship between OR52N2 and PGE2, dual-luciferase reporter assay was employed to verify the activation of downstream CREB signaling pathways. Results showed that PGE2 significantly enhanced CRE promoter activity in or52n2 ORF transient transfected HEK-293 T cells. Finally, localization of or52n2 mRNA were observed in ciliated receptor cells of the olfactory epithelium using in situ hybridization. Our results provide a novel insight into sex pheromone signaling transduction in reproductive behavior.

Function of secretoneurin in regulating the expression of reproduction-related genes in ovoviviparous black rockfish (Sebastes schlegelii).

Secretoneurin (SN), a conserved peptide derived from secretogranin-2 (scg2), also known as secretogranin II or chromogranin C, plays an important role in regulating gonadotropin in the pituitary, which affects the reproductive system. This study aimed to clarify the mode of action of scg2 in regulating gonad development and maturation and the expression of mating behavior-related genes. Two scg2 cDNAs were cloned from the ovoviviparity teleost black rockfish (Sebastes schlegelii). In situ hybridization detected positive scg2 mRNA signals in the telencephalon and hypothalamus, where sgnrh and kisspeptin neurons were reported to be located and potentially regulated by scg2. In vivo, intracerebral ventricular injections of synthetic black rockfish SNa affected brain cgnrh, sgnrh, kisspeptin1, pituitary lh and fsh and gonad steroidogenesis-related gene expression levels with sex dimorphism. In vitro, a similar effect was found in primary cultured brain and pituitary cells. Thus, SN could contribute to the regulation of gonadal development, as well as reproductive behaviors, including mating and parturition.

Comparative transcriptomic analysis and genome-wide characterization of the Semaphorin family reveal the potential mechanism of angiogenesis around embryo in ovoviviparous black rockfish (Sebastes schlegelii).

To guarantee the quality and survival rate of their offspring, ovoviviparous teleost evolved special characteristics of in vivo fertilization and embryo development. Maternal black rockfish, having over 50 thousand embryos developing within the ovary simultaneously, provided around 40% nutrition throughout oocyte development, while the capillaries around each embryo contributed the rest 60% during pregnancy. Since fertilization, capillaries started to proliferate and developed into a placenta-like structure that covered over half of each embryo. Aimed to characterize the potential mechanism behind, comparative transcriptome analysis of samples collected according to the process of pregnancy. Three important time point in the process, including mature oocyte stage, fertilization and sarcomere period, were chosen for the transcriptome sequencing. Our study identified key pathways and genes involved in the cell cycle as well as DNA replication and repair, cell migration and adhesion, immune, and metabolic functions. Notably, several of the semaphoring gene family members were differently expressed. To confirm the accuracy of these genes, total of 32 sema genes were identified from the whole genome and distinct expression pattern of sema genes was observed in different pregnant stages. Our results revealed a novel insight for further investigating the functions of sema genes in reproduction physiology and embryo processes in ovoviviparous teleost.

TAC3/TACR3 System Function in the Catadromous Migration Teleost, Anguilla japonica.

Neurokinin B (NKB), a member of the tachykinin (TAC) family, plays important roles in mammalian neuropeptide secretion in related to reproduction. However, its potential role in spawning migration teleost is less clear. In the present study, Japanese eel (Anguilla japonica) was employed to study the performance of NKB in regulating reproduction. Results showed that two tac3 and one tacr3 genes were identified in Japanese eel. Sequence analysis showed that two tac3 transcripts, tac3a and tac3b, encode four NKBs: NKBa-13, NKBa-10, NKBb-13, and NKBb-10. However, compared with other species, a mutation caused early termination of TACR3 protein was confirmed, leading to the loss of the 35 amino acid (aa) C-terminal of the receptor. Expression analysis in different tissues showed that both tac3a and tac3b mRNAs were highly expressed in the brain. In situ hybridization localized both tac3a and tac3b mRNAs to several brain regions, mainly in the telencephalon and hypothalamus. Because of the mutation in TACR3 of Japanese eel, we further analyzed whether it could activate the downstream signaling pathway. Luciferase assay results showed the negative regulation of cAMP Response Element (CRE) and Sterol Response Element (SRE) signal pathways by Japanese eel NKBs. Intraperitoneal injection of four different NKB mature peptides at 100 ng/g had negative effect on either gnrh or gth gene expression. However, the high concentration of NKBa-10 and NKBb-13 (1,000 ng/g) upregulated mgnrh and fshb or lhb expression level significantly, which may be mediated by other receptors. In general, the NKBs/NK3Rs system has important functions in regulating eel puberty onset.

Molecular characterization and expression patterns of glucocorticoid receptors in the viviparous black rockfish Sebastes schlegelii.

Glucocorticoid receptors (GRs) are ligand-activated transcription factors associated with anti-inflammation, stress, metabolism and gonadal development. In this study, two gr genes (gr1 and gr2) were cloned and analyzed from a viviparous teleost, black rockfish (Sebastes schlegelii). The phylogenetic analysis of GRs showed that GR1 and GR2 clustered into teleost GR1 and GR2 separately and differed from the GRs of tetrapods or basal ray-finned fishes. Black rockfish GRs possess four modular domains of the nuclear receptor superfamily: an N-terminal domain (NTD), a DNA-binding domain (DBD), a hinge region (HR) and a ligand-binding domain (LBD). Nine conserved amino acid inserts were found in the GR1 DBD, and the ligand cavity-related amino acids of GR1 and GR2 LBD were slightly different. Tissue distribution analysis revealed that grs was widely expressed in various tissues, while cyp11b was mainly expressed in the testis and head kidney. The cyp11b transcripts were localized in the interrenal glands of the head kidney, the main source of cortisol; grs transcripts were detected in oocytes, the follicle layer and the ovarian wall. Histologically, significant blood vessel dilation was observed in the fetal membrane during or after parturition of black rockfish. The highest levels of serum cortisol and ovarian cyp11b mRNA were detected in parturition. In addition, the relative expression level of gr1 was upregulated significantly after delivery, while the levels of gr2 showed no significant change. In addition, in vitro GC treatment inhibited the expression of il1b but significantly upregulated the transcription of il1r1. These data provide evidence that GRs are likely to work as anti-inflammatory factors by inhibiting the functions of pro-inflammatory factors in the parturition of black rockfish.

A theoretical study of hydrogen-bonded molecular clusters of sulfuric acid and organic acids with amides.

Amides, a series of significant atmospheric nitrogen-containing volatile organic compounds (VOCs), can participate in new particle formation (NPF) throught interacting with sulfuric acid (SA) and organic acids. In this study, we investigated the molecular interactions of formamide (FA), acetamide (AA), N-methylformamide (MF), propanamide (PA), N-methylacetamide (MA), and N,N-dimethylformamide (DMF) with SA, acetic acid (HAC), propanoic acid (PAC), oxalic acid (OA), and malonic acid (MOA). Global minimum of clusters were obtained through the association of the artificial bee colony (ABC) algorithm and density functional theory (DFT) calculations. The conformational analysis, thermochemical analysis, frequency analysis, and topological analysis were conducted to determine the interactions of hydrogen-bonded molecular clusters. The heterodimers formed a hepta or octa membered ring through four different types of hydrogen bonds, and the strength of the bonds are ranked in the following order: SOH•••O > COH•••O > NH•••O > CH•••O. We also evaluated the stability of the clusters and found that the stabilization effect of amides with SA is weaker than that of amines with SA but stronger than that of ammonia (NH3) with SA in the dimer formation of nucleation process. Additionally, the nucleation capacity of SA with amides is greater than that of organic acids with amides.

2-Methyltetrol sulfate ester-initiated nucleation mechanism enhanced by common nucleation precursors: A theory study.

Aerosol samples from all over the word contained 2-methyltetrol sulfate ester (MTS). We investigated the role of MTS in new particle formation (NPF) with aerosol nucleation precursors, including sulfuric acid (SA), water (W), ammonia (N), methylamine (MA), dimethylamine (DMA), and trimethylamine (TMA). The analysis was performed using quantum chemical approach, kinetic calculation and molecular dynamics (MD) simulations. The results proved that the molecular interactions in the clusters were mainly H-bonds and electrostatic interaction. The negative Gibbs free energy changes for all the studied MTS-containing clusters indicated that the formation of these clusters was thermodynamically favorable. The stability of the clusters was evaluated according to the total evaporation rate. Here, (MTS)(SA) and (MTS)(W) were the most and least stable cluster, respectively. MD simulations were used for time and spatial analysis of the role of the MTS-SA system. The results indicated that MTS can self-aggregate or absorb SA molecules into clusters, larger than the size of the critical cluster (approximately 1 nm), suggesting that MTS can initiate NPF by itself or together with SA.

Propionamide participating in H2SO4-based new particle formation: a theory study.

Propionamide (PA), an important pollutant emitted into the atmosphere from a variety of sources, is abundant in many areas worldwide, and could be involved in new particle formation (NPF). In this study, the enhancement of the H2SO4 (SA)-based NPF by PA was evaluated through investigating the formation mechanism of (PA) m (SA) n (m = 0-3 and n = 0-3) clusters using computational chemistry and kinetics modeling. Our study proved that the formation of all the PA-containing clusters is thermodynamically favorable. Furthermore, the [double bond, length as m-dash]O group in PA plays an important role in the clusters with more PA than SA, and the basicity of bases exerts a greater influence with an increasing amount of SA. We demonstrate that although the enhancing potential of PA is lower than that of the strongest enhancers of SA-based NPF such as methylamine (MA) and dimethylamine (DMA), PA can enhance the SA-based NPF at the parts per billion (ppb) level, which is typical for concentrations of C3-amides in, for example, urban Shanghai (China). The monomer evaporation is the dominant degradation pathway for the (PA) m (SA) n clusters, which differs from that of the SA-DMA system. The formation rate of PA-containing clusters is comparable to the rate coefficients for PA oxidation by hydroxyl (OH) radicals, indicating that participating in the SA-based NPF is a crucial sink for PA.

The Gas-Phase Formation Mechanism of Dibenzofuran (DBF), Dibenzothiophene (DBT), and Carbazole (CA) from Benzofuran (BF), Benzothiophene (BT), and Indole (IN) with Cyclopentadienyl Radical.

Benzofuran (BF), benzothiophene (BT), indole (IN), dibenzofuran (DBF), dibenzothiophene (DBT), and carbazole (CA) are typical heterocyclic aromatic compounds (NSO-HETs), which can coexist with polycyclic aromatic hydrocarbons (PAHs) in combustion and pyrolysis conditions. In this work, quantum chemical calculations were carried out to investigate the formation of DBF, DBT, and CA from the reactions of BF, BT, and IN with a cyclopentadienyl radical (CPDyl) by using the hybrid density functional theory (DFT) at the MPWB1K/6-311+G(3df,2p)//MPWB1K/6-31+G(d,p) level. The rate constants of crucial elementary steps were deduced over 600-1200 K, using canonical variational transition state theory with a small-curvature tunneling contribution (CVT/SCT). This paper showed that the production of DBF, DBT, and CA from the reactions of BF, BT, and IN with CPDyl involved six elementary steps: the addition reaction, ring closure, the first H shift, C-C cleavage, the second H shift, and elimination of CH3 or H. The cleavage of the C-C bond was regarded as the rate-determining step for each pathway due to the extremely high barrier. The 1-methyl substituted products were more easily formed than the 4-methyl substituted products. The main products were DBF and 1-methyl-DBF, DBT and 1-methyl-DBT, and CA and 1-methyl-CA for reactions of BF, BT, and IN with CPDyl, respectively. The ranking of DBF, DBT, and CA formation potential was as follows: DBT and methyl-DBT formation > DBF and methyl-DBF formation > CA, and methyl-CA formation. Comparison with the reaction of naphthalene with CPDyl indicated that the reactions of CPDyl attacking a benzene ring and a furan/thiophene/pyrrole ring could be inferred to be comparable under high temperature conditions.

Mechanistic and Kinetic Study on Self-/Cross- Condensation of PCTA/DT Formation Mechanisms from Three Types of Radicals of 2,4-Dichlorothiophenol.

Chlorothiophenols (CTPs) are known to be key and direct precursors of polychlorinated thianthrene/dibenzothiophenes (PCTA/DTs). Self/cross-coupling of the chlorothiophenoxy radicals (CTPRs), sulfydryl-substituted phenyl radicals and thiophenoxyl diradicals evolving from CTPs are initial and important steps for PCTA/DT formation. In this study, quantum chemical calculations were carried out to investigate the homogenous gas-phase formation of PCTA/DTs from self/cross-coupling of 2,4-dichlorothiophenoxy radical (R1), 2-sulfydryl-3,5-dichlorophenyl radical (R2) and 3,5-dichlorothiophenoxyl diradical (DR) at the MPWB1K/6-311+G(3df,2p)//MPWB1K/6-31+G(d,p) level. The rate constants of crucial elementary steps were deduced over 600-1200 K, using canonical variational transition state theory with a small curvature tunneling contribution. For the formation of PCTAs, the S•/σ-C• condensation with both thiophenolic sulfur in one radical and ortho carbon in the other radical bonded to single electron is the most efficient sulfur-carbon coupling mode, and the ranking of the PCTA formation potential is DR + DR > R2 + DR > R1 + DR > R1 + R2 > R1 + R1. For the formation of PCDTs, the σ-C•/σ-C• coupling with both ortho carbon in the two radicals bonded to single electron is the energetically favored carbon-carbon coupling mode, and the ranking of the PCDT formation potential is: R2 + DR > R2 + R2 > R1 + DR > R1 + R2 > R1 + R1. The PCTA/DTs could be produced from R1, R2 and DR much more readily than PCDD/DFs from corresponding oxygen substituted radicals.

Quantum Chemical and Kinetic Study on Radical/Molecule Formation Mechanism of Pre-Intermediates for PCTA/PT/DT/DFs from 2-Chlorothiophenol and 2-Chlorophenol Precursors.

Polychlorinated phenoxathiins (PCPTs), polychlorinated dibenzothiophenes (PCDTs), and polychlorinated thianthrenes (PCTAs) are sulfur analogues of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/DFs). Chlorothiophenols (CTPs) and chlorophenols (CPs) are key precursors for the formation of PCTA/PT/DTs, which can react with H or OH to form chloro(thio)phenoxy radical, sulfydryl/hydroxyl-substituted phenyl radicals, and (thio)phenoxyl diradicals. However, previous radical/radical PCTA/DT formation mechanisms in the literature failed to explain the higher concentration of PCDTs than that of PCTAs under the pyrolysis or combustion conditions. In this work, a detailed thermodynamics and kinetic calculations were carried out to investigate the pre-intermediate formation for PCTA/PT/DTs from radical/molecule coupling of the 2-C(T)P with their key radical species. Our study showed that the radical/molecule coupling mechanism explains the gas-phase formation of PCTA/PT/DTs in both thermodynamic and kinetic perspectives. The S/C coupling modes to form thioether-(thio)enol intermediates are preferable over the O/C coupling modes to form ether-(thio)enol intermediates. Thus, although the radical/molecule coupling of chlorophenoxy radical with 2-C(T)P has no effect on the PCDD/PT formation, the radical/molecule coupling of chlorothiophenoxy radical with 2-C(T)P plays an important role in the PCTA/PT formation. Most importantly, the pre-PCDT intermediates formation pathways from the couplings of sulfydryl/hydroxyl-substituted phenyl radical with 2-C(T)P and (thio)phenoxyl diradicals with 2-C(T)P are more favorable than pre-PCTA/PT intermediates formation pathways from the coupling of chlorothiophenoxy radical with 2-C(T)P, which provides reasonable explanation for the high PCDT-to-PCTA ratio in the environment.

Carcinogenic Metabolic Activation Process of Naphthalene by the Cytochrome P450 Enzyme 1B1: A Computational Study.

The metabolic activation and transformation of naphthalene by the cytochrome P450 enzyme (CYP 1B1) plays an important role in its potential carcinogenicity. The process has been explored by a quantum mechanics/molecular mechanics (QM/MM) computational method. Molecular dynamic simulations were performed to explore the interaction between naphthalene and CYP 1B1. Naphthalene involves α- and ß-carbon, the electrophilic addition of which would result in different reaction pathways. Our computational results show that both additions on α- and ß-carbon can generate naphthalene 1,2-oxide. The activation barrier for the addition on ß-carbon is higher than that for the α-carbon by 2.6 kcal·mol-1, which is possibly caused by the proximity between ß-carbon and the iron-oxo group of Cpd I in the system. We also found that naphthalene 1,2-oxide is unstable and the O-C bond cleavage easily occurs via cellular hydronium ion, hydroxyl radical/anion; then it will convert to the potential ultimate carcinogen 1,2-naphthoquinone. The results demonstrate and inform a detailed process of generating naphthalene 1,2-oxide and new predictions for its conversion.

A molecular understanding of the interaction of typical aromatic acids with common aerosol nucleation precursors and their atmospheric implications.

Aromatic acids, which are generated from numerous anthropogenic emissions and secondary transformations, have been considered to play a crucial role in new particle formation. In this study, we performed theoretical calculations at the PW91PW91/6-311++G(3df,3pd) level to investigate the interaction between typical aromatic acids namely benzoic acid (BA), phenylacetic acid (PAA), phthalic acid (PA), isophthalic acid (mPA), and terephthalic acid (PTA) and common atmospheric nucleation precursors namely sulfuric acid (SA), water (H2O), ammonia (NH3), methylamine (MA), dimethylamine (DMA), and trimethylamine (TMA). The geometric analysis, Gibbs free energy analysis, OH/NH-stretching vibrational frequency calculation, and atoms in molecules (AIM) analysis were conducted to determine the interactions in the complexes. The heterodimers formed a six to eight membered ring through four types of hydrogen bond, and the bond strength could be ranked in descending order: SO-H⋯O > O-H⋯O/N > N-H⋯O. The BA/PAA/mPA/PTA-SA complexes had the lowest Gibbs free energy values. PA was more likely to interact with NH3 or amines rather than SA due to an intra-molecular hydrogen bond. Additionally, the aromatic acids have similar ability to interact with SA and NH3 as monocarboxylic/dicarboxylic acid. The formation potential of the heterodimers from aromatic acids with common nucleation precursors in ambient atmosphere was investigated.