Star1 gene mutation reveals the essentiality of 11-ketotestosterone and glucocorticoids for male fertility in Nile Tilapia (Oreochromis niloticus).

Steroidogenic acute regulatory protein (Star) plays an essential role in the biosynthesis of corticosteroids and sex steroids by mediating the transport of cholesterol from the outer to the inner membrane of mitochondria. Two duplicated Star genes, namely star1 and star2, have been identified in non-mammalian vertebrates. To investigate the roles of star genes in fish steriodogenesis, we generated two mutation lines of star1-/- and star1-/-/star2-/- in Nile tilapia (Oreochromis niloticus). Previous studies revealed that deficiency of star2 gene caused delayed spermatogenesis, sperm apoptosis and sterility in male tilapia. Our present data revealed that mutation of star genes impaired male fertility. Disordered seminiferous lobules and spermatic duct obstruction were found in the testis of both types of mutants. Moreover, significant decline in semen volume, sperm abnormality and impaired fertility were also detected in star1-/- and star1-/-/star2-/- males. In star1-/- male fish, lipid accumulation, up-regulation of steroidogenic enzymes, and significant decline of androgens were found. Additionally, hyperplasic interrenal cells, elevated steroidogenic gene expression level and decline of serum glucocorticoids were detected in star1 mutants. Intriguingly, either 11-KT or cortisol supplementation successfully rescued the impaired fertility of the star1-/- mutants. Taken together, these results further indicate that Star1 might play critical roles in the production of both 11-KT and glucocorticoids, which are indispensable for the maintenance of male fertility in fish.

SDF-1/CXCR4 signal is involved in the induction of Primordial Germ Cell migration in a model marine fish, Japanese anchovy (Engraulis japonicus).

Primordial germ cells (PGCs) are pivotal for gonadal development and reproductive success. Though artificial induction of sterility by targeting PGCs are gaining popularity due to its advantages in fish surrogacy and biodiversity management, it is often skill and time intensive. In this study, we have focused on understanding the role of PGCs and the chemotactic SDF-1/CXCR4 signaling on gonad development of Japanese anchovy (JA, Engraulis japonicus), an upcoming marine model organism with eco-commercial values, with an aim to develop a novel, easy, and versatile gonad sterilization method. Our data showed that PGC migration related genes, i.e., sdf-1a, sdf-1b, cxcr4a, cxcr4b and vasa, are phylogenetically closer relatives of respective herring (Clupea harengus) and zebrafish (Danio rerio) homolog. Subsequently, PGC marking and live tracing experiments confirmed that PGC migration in JA initiates from 16 hours post fertilization (hpf) followed by PGC settlement in the gonadal ridge at 44 hpf. We found that overexpression of zebrafish sdf-1a mRNA in the germ cell suppresses cxcr4a and increases cxcr4b transcription at 8 hpf, dose dependently disrupts PGC migration at 24-48 hpf, induces PGC death and upregulates sdf-1b at 5 days post hatching. 48 h of immersion treatment with CXCR4 antagonist (AMD3100, Abcam) also accelerated PGC mismigration and pushed the PGC away from gonadal ridge in a dose responsive manner, and further when grown to adulthood caused germ cell less gonad formation in some individuals. Cumulatively, our data, for the first time, suggests that JA PGC migration is largely regulated by SDF1/CXCR4 signaling, and modulation of this signaling has strong potential for sterile, germ cell less gonad preparation at a mass scale. However, further in-depth analysis is pertinent to apply this methodology in marine fish species to successfully catapult Japanese anchovy into a true marine fish model.

Modeling the SDF-1/CXCR4 protein using advanced artificial intelligence and antagonist screening for Japanese anchovy.

SDF-1/CXCR4 chemokine signaling are indispensable for cell migration, especially the Primordial Germ Cell (PGC) migration towards the gonadal ridge during early development. We earlier found that this signaling is largely conserved in the Japanese anchovy (Engraulis japonicus, EJ), and a mere treatment of CXCR4 antagonist, AMD3100, leads to germ cell depletion and thereafter gonad sterilization. However, the effect of AMD3100 was limited. So, in this research, we scouted for CXCR4 antagonist with higher potency by employing advanced artificial intelligence deep learning-based computer simulations. Three potential candidates, AMD3465, WZ811, and LY2510924, were selected and in vivo validation was conducted using Japanese anchovy embryos. We found that seven transmembrane motif of EJ CXCR4a and EJ CXCR4b were extremely similar with human homolog while the CXCR4 chemokine receptor N terminal (PF12109, essential for SDF-1 binding) was missing in EJ CXCR4b. 3D protein analysis and cavity search predicted the cavity in EJ CXCR4a to be five times larger (6,307 Å³) than that in EJ CXCR4b (1,241 Å³). Docking analysis demonstrated lower binding energy of AMD3100 and AMD3465 to EJ CXCR4a (Vina score -9.6) and EJ CXCR4b (Vina score -8.8), respectively. Furthermore, we observed significant PGC mismigration in microinjected AMD3465 treated groups at 10, 100 and 1 × 105 nM concentration in 48 h post fertilized embryos. The other three antagonists showed various degrees of PGC dispersion, but no significant effect compared to their solvent control at tested concentrations was observed. Cumulatively, our results suggests that AMD3645 might be a better candidate for abnormal PGC migration in Japanese anchovy and warrants further investigation.

UV-A-Induced Photoisomerization and Photodimerization of Curcumin: An Ion Mobility Mass Spectrometry Study.

Curcumin, the bioactive compound present in spice plant turmeric, has been shown to exhibit selective phototoxic activities toward mammalian cancer cells, and it is being used extensively as a photosensitizer (PS) in photodynamic therapies (PDT). However, so far, the fate of curcumin toward photochemical transformations is not well understood. Here we report our findings of a number of novel photochemical reaction channels of curcumin in water-methanol mixture, like photoisomerization, photodimerization, and photooxidation (H2-loss). The reaction was performed by irradiating the curcumin solution with ultraviolet (UV) light of wavelength 350 nm, which is abundant in the earth's troposphere. Product identification and structure elucidation are done by employing an integrated method of drift tube ion mobility mass spectrometry (DTIMS) in combination with high-performance liquid chromatography (HPLC) and collision-induced dissociation (CID) of the mass-selected molecular ions. Two photoisomers of curcumin produced as a result of trans-cis configurational changes about C═C double bonds in the excited state have been identified, and it has been shown that they could serve as the precursors for formation of isomeric dimers via [2 + 2] cycloaddition and H2-loss products. Comparisons of the experimentally measured collision cross-section (CCS) values of the reactant and product ions obtained by the DTIMS method with those predicted by the electronic structure theory are found to be very effective for the discrimination of the produced photoisomers. The observed photochemical reaction channels are potentially significant toward uses of curcumin as a photosensitizer in photodynamic therapy.

Sex Lethal Gene Manipulates Gonadal Development of Medaka, Oryzias latipes, through Estrogenic Interventions.

Germ cells are pivotal for gonadal sexuality maintenance and reproduction. Sex lethal (sxl), the somatic sex determining gene of Drosophila, is the known regulator and initiator of germ cell femininity in invertebrates. However, the role of the Sxl homologue has rarely been investigated in vertebrates. So, we used medaka to clarify the role of sxl in vertebrate gonadogenesis and sexuality and identified two Sxl homologues, i.e., Sxl1a and Sxl1b. We found that sxl1a specifically expresses in the primordial germ cells (PGC), ovary, (early gonia and oocytes), while sxl1b distributions are ubiquitous. An mRNA overexpression of sxl1a accelerated germ cell numbers in 10 DAH XY fish, and sxl1a knockdown (KD), on the other hand, induced PGC mis-migration, aberrant PGC structuring and ultimately caused significant germ cell reduction in XX fish. Using an in vitro promoter analysis and in vivo steroid treatment, we found a strong link between sxl1a and estrogenic germ cell-population maintenance. Further, using sxl1a-KD and erß2-knockout fish, we determined that sxl1 acts through erß2 and controls PGC sexuality. Cumulatively, our study highlights the novel role of sxl1a in germ cell maintenance and sexual identity assignment and thus might become a steppingstone to understanding the commonalities of animal sexual development.

Cyp17a2 is involved in testicular development and fertility in male Nile tilapia, Oreochromis niloticus.

Background: Steroid hormones play an essential role in many reproductive processes of vertebrates. Previous studies revealed that teleost-specific Cyp17a2 (cytochrome P450 family 17 subfamily a 2) might be required for the production of cortisol in the head-kidney and 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) in ovary during oocyte maturation. However, the role of Cyp17a2 in male reproduction remains to be largely unknown. The aim of this study was to explore the essentiality of cyp17a2 gene in male steroidogenesis, spermatogenesis, and male fertility. Methods: A homozygous mutation line of cyp17a2 gene was constructed in tilapia by CRISPR/Cas9 gene editing technology. The expression level of germ cell and meiosis-related genes and steroidogenic enzymes were detected by qRT-PCR, IHC, and Western blotting. EIA and LC-MS/MS assays were used to measure the steroid production levels. And sperm quality was examined by Sperm Quality Analyzer software. Results: In this study, cyp17a2 gene mutation resulted in the significant decline of serum DHP and cortisol levels. On the contrary, significant increases in intermediate products of cortisol and DHP were found in cyp17a2-/- male fish. The deficiency of cyp17a2 led to the arrest of meiotic initiation in male fish revealing as the reduction of the expression of germ cell-related genes (vasa, piwil, oct4) and meiosis-related genes (spo11 and sycp3) by 90 dah. Afterwards, spermatogenesis was gradually recovered with the development of testis in cyp17a2-/- males, but it showed a lower sperm motility and reduced fertility compared to cyp17a2+/+ XY fish. Deletion of cyp17a2 led to the abnormal upregulation of steroidogenic enzymes for cortisol production in the head-kidney. Moreover, unaltered serum androgens and estrogens, as well as unchanged related steroidogenic enzymes were found in the testis of cyp17a2-/- male fish. Conclusion: This study proved that, for the fist time, Cyp17a2 is indispensable for cortisol and DHP production, and cyp17a2 deficiency associated curtailed meiotic initiation and subfertility suggesting the essentiality of DHP and cortisol in the male fertility of fish.

Tautomers and Rotamers of Curcumin: A Combined UV Spectroscopy, High-Performance Liquid Chromatography, Ion Mobility Mass Spectrometry, and Electronic Structure Theory Study.

The structures of tautomers and rotameric forms of curcumin, the bioactive compound present in spice plant turmeric, have been investigated using ion mobility mass spectrometry (IMMS) in conjunction with high-performance liquid chromatography (HPLC) and UV-visible spectroscopy. Two tautomeric forms of this ß-diketone compound, keto-enol and diketo, have been chromatographically separated, and the electronic absorption spectra for these two tautomeric forms in methanol solution have been recorded separately for the first time. The molecular identity of the HPLC-separated solution fractions is established unambiguously by recording the mass and fragmentation spectra simultaneously. The ion mobility spectrum for the deprotonated curcumin anion, [Cur-H]-, corresponding to the diketo tautomer, displays only one peak (P), and the collision cross-section (CCS) value is measured to be 185.9 Å2. However, the ion mobility spectrum corresponding to the HPLC-separated keto-enol tautomer shows three distinctly separated peaks, P, Q, and R, with CCS values of 185.9, 194.8, and 203.4 Å2, respectively, whereby peak R appears to be the most intense one, followed by peaks P and Q. The theoretically calculated CCS values of different isomers of [Cur-H]-, optimized by electronic structure theory methods, display satisfactory correlation with the experimentally observed values, corroborating our assignments. The spectral attributes also indicate the occurrence of structural rearrangements in the electrospray ionization process. With the aid of the electronic structure calculation, low-energy pathways for the occurrence of the structural isomerization to surpass the energy barrier are suggested, which are consistent with the assignments of the peaks observed in the IM spectra.

The role of StAR2 gene in testicular differentiation and spermatogenesis in Nile tilapia (Oreochromis niloticus).

Sex steroids play critical roles in sex differentiation and gonadal development in teleosts. Steroidogenic acute regulatory protein (StAR), transporting cholesterol (the substrate for steroidogenesis) from the outer mitochondrial membrane to the inner membrane, is the first rate-limiting factor of steroidogenesis. Interestingly, two StAR genes (named as StAR1 and StAR2) have been isolated from non-mammalian vertebrates. To characterize the functions of the novel StAR2 gene in the gonadal differentiation and fertility, we generated a StAR2 homozygous mutant line in Nile tilapia (Oreochromis niloticus). StAR2 gene knockout in male tilapia impeded meiotic initiation, associate with the down-regulation of meiosis related gene expressions of vasa, sycp3 and dazl at 90 days after hatching (dah). Meanwhile, cyp11b2 expression and serum 11-KT production significantly declined in StAR2-/- XY fish at 90 dah. From 120-300 dah, spermatogenesis gradually recovered, and so did the expressions of vasa, sycp3 and dazl in StAR2-/- XY fish testes. However, seminiferous lobules arranged disorderly in StAR2-/- XY fish testes at 300 dah. The number of Leydig cells and expressions of downstream steroidogenesis enzymes including cyp11a1, 3ß-HSD-I, 3ß-HSD-II, cyp17a1 and cyp17a2 decreased in StAR2-/- XY fish testes at 300 dah. Serum testosterone and 11-KT levels were significantly lower in StAR2-/- XY fish than that of their control counterparts. Furthermore, significantly elevated ar, fsh and lh expressions in StAR2-deficient XY fish testes and pituitaries were found when compared with the control XY fish. Testes degeneration and spermatogenic cell apoptosis were observed, while no sperm were squeezed out in StAR2-/- XY fish testes at 540 dah. Taken together, our results suggest that StAR2 has a role in testicular development, spermatogenesis and spermiation by regulating androgen production in tilapia, but may not be essential and could be compensated.

Drought dynamics of Northwestern Teesta Floodplain of Bangladesh: a remote sensing approach to ascertain the cause and effect.

Drought is an affliction for a region that primarily depends on agriculture as economic activity. Commonly monitoring and characterizing of drought is performed by only analyzing the meteorological aspect, assuming precipitation as the primary source of water. However, in riverine Bangladesh, this can lead to an erroneous conclusion, as there is a multitude of available water sources. Consequently, in this study, vegetation condition (Standard Vegetation Index), soil moisture (Soil Moisture Index), and precipitation (Standard Precipitation Index) are separately investigated from 2003 to 2019, in the Northwestern Teesta floodplain. Subsequently, statistical regression analysis is performed to determine the relationships between different aspects of drought. In addition, information obtained from field visits and expert opinions has also been assimilated. Analysis of vegetation and soil moisture condition presents a progressively improving scenario. However, SPI shows an incessant decline in meteorological drought conditions, especially after 2007. Evidently, regression analysis does not provide any indication of an interrelationship between the studied agricultural and meteorological parameters. Presumably, this absence is instigated because the study area is highly irrigated as the groundwater table is suitably near the surface and the existence of nearby Teesta river allows for the utilization of surface water. Moreover, the cropping pattern is shifting toward crops that require much less water and to places where soil moisture is scarce. Thus, this study addresses the gap in knowledge regarding the nature of agricultural drought and the dynamics of different aspects of drought which will be invaluable for the water management and agricultural policy in the study area as well as other regions with a similar backdrop.

Sex determination, gonadal sex differentiation, and plasticity in vertebrate species.

A diverse array of sex determination (SD) mechanisms, encompassing environmental to genetic, have been found to exist among vertebrates, covering a spectrum from fixed SD mechanisms (mammals) to functional sex change in fishes (sequential hermaphroditic fishes). A major landmark in vertebrate SD was the discovery of the SRY gene in 1990. Since that time, many attempts to clone an SRY ortholog from nonmammalian vertebrates remained unsuccessful, until 2002, when DMY/dmrt1by was discovered as the SD gene of a small fish, medaka. Surprisingly, however, DMY/dmrt1by was found in only 2 species among more than 20 species of medaka, suggesting a large diversity of SD genes among vertebrates. Considerable progress has been made over the last 3 decades, such that it is now possible to formulate reasonable paradigms of how SD and gonadal sex differentiation may work in some model vertebrate species. This review outlines our current understanding of vertebrate SD and gonadal sex differentiation, with a focus on the molecular and cellular mechanisms involved. An impressive number of genes and factors have been discovered that play important roles in testicular and ovarian differentiation. An antagonism between the male and female pathway genes exists in gonads during both sex differentiation and, surprisingly, even as adults, suggesting that, in addition to sex-changing fishes, gonochoristic vertebrates including mice maintain some degree of gonadal sexual plasticity into adulthood. Importantly, a review of various SD mechanisms among vertebrates suggests that this is the ideal biological event that can make us understand the evolutionary conundrums underlying speciation and species diversity.

Medium-Dependent Crossover from the Red to Blue Shift of the Donor's Stretching Fundamental in the Binary Hydrogen-Bonded Complexes of CDCl3 with Ethers and Ketones.

Mid-infrared spectra for C-D···O hydrogen (H)-bonded binary complexes of CDCl3 with acetone (AC), cyclohexanone (CHN), diethyl ether (DEE), and tetrahydrofuran (THF) have been measured in the vapor phase at room temperature and in an argon matrix at 8 K. Remarkable matrix effect has been observed in each case with respect to the spectral shift of the donor group's stretching fundamental (ΔνC-D). In the case of complexes with AC and CHN, the sign of ΔνC-D changes from a few wavenumbers positive (blue shift) in the vapor phase to a few tens of wavenumbers negative (red shift) in the argon matrix. For the two ether complexes, although no apparent reversal in the sign of ΔνC-D occurs, but the magnitudes of the red shifts in the matrix are manifold larger, and the bands appear with large enhancement in transition intensity. The medium effect has been explained consistently in terms of the local hyperconjugative charge transfer interaction at the H-bonding sites of the complexes and its interplay with the H-bond distance that varies with the physical conditions of the medium. Under the matrix isolation condition, νC-D bands of CHN and THF complexes depict a large number of substructures, which has been interpreted in terms of matrix site effect as well as Fermi resonance enhancement of the fingerprint combination tones and trapping of more than one isomer of the complexes in the matrix sites.

Probing the Endo/Exo Isomeric Variants of a Binary Complex of Cyclopropylamine with p-Fluorophenol by LIF Spectroscopy: A Comparative Study with Ammonia Complex.

Conformational isomers of an O-H···N hydrogen-bonded binary complex between para-fluorophenol (pFP) and a nonrigid primary amine base, cyclopropylamine (CPA), have been probed by means of laser-induced fluorescence (LIF) spectroscopy in a supersonic jet expansion. Two closely spaced electronic origin bands have been identified in the measured LIF excitation spectrum, and their assignments have been corroborated by making comparisons with the spectra of the parent pFP-NH3 complex recorded under the same expansion condition. The observation is consistent with the presence of endo and exo isomeric variants of the complex predicted by electronic structure theory methods, and the endo isomer is stabilized by ∼2 kcal/mol additionally owing to the formation of a C-H···O and a C-H···π type of weak hydrogen bonds between the two moieties. In the fluorescence excitation (FE) spectrum, the low-frequency bands for different intermolecular modes gain substantial intensity, and this spectral feature is in contrast to that of the pFP-NH3 complex. The Franck-Condon intensity of the bands has been simulated invoking Duschinsky rotation scheme, taking into consideration the ground- and excited-state geometries.

Estrogen and estrogen receptors chauffeur the sex-biased autophagic action in liver.

Autophagy, or cellular self-digestion, is an essential cellular process imperative for energy homeostasis, development, differentiation, and survival. However, the intrinsic factors that bring about the sex-biased differences in liver autophagy are still unknown. In this work, we found that autophagic genes variably expresses in the steroidogenic tissues, mostly abundant in liver, and is influenced by the individual's sexuality. Starvation-induced autophagy in a time-dependent female-dominated manner, and upon starvation, a strong gender responsive circulating steroid-HK2 relation was observed, which highlighted the importance of estrogen in autophagy regulation. This was further confirmed by the enhanced or suppressed autophagy upon estrogen addition (male) or blockage (female), respectively. In addition, we found that estrogen proved to be the common denominator between stress management, glucose metabolism, and autophagic action in female fish. To understand further, we used estrogen receptor (ER)α- and ER-ß2-knockout (KO) medaka and found ER-specific differences in sex-biased autophagy. Interestingly, starvation resulted in significantly elevated mTOR transcription (compared with control) in male ERα-KO fish while HK2 and ULK activation was greatly decreased in both KO fish in a female oriented fashion. Later, ChIP analysis confirmed that, NRF2, an upstream regulator of mTOR, only binds to ERα, while both ERα and ERß2 are effectively pulled down the HK2 and LC3. FIHC data show that, in both ER-KO fish, LC3 nuclear-cytoplasmic transport and its associated pathways involving SIRT1 and DOR were greatly affected. Cumulatively, our data suggest that, ERα-KO strongly affected the early autophagic initiation and altered the LC3 nuclear-cytoplasmic translocation, thereby influencing the sex-biased final autophagosome formation in medaka. Thus, existence of steroid responsive autophagy regulatory-switches and sex-biased steroid/steroid receptor availability influences the gender-skewed autophagy. Expectedly, this study may furnish newer appreciation for gender-specific medicine research and therapeutics.

Isomers and Rotamers of DCM in Methanol and in Gas Phase Probed by Ion Mobility Mass Spectrometry in Combination with High Performance Liquid Chromatography.

An integrated method of ion mobility mass spectrometry and high-performance liquid chromatography (HPLC) has been used to investigate the isomeric distribution of a popular fluorescent dye DCM (4-(dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran) in methanol solution. Chromatographic separation of DCM isomers in methanol has been performed by probing the molecular mass (DCMH+), and two distinctly separated peaks are observed at retention times 3.73 (peak-I) and 3.87 (peak-II) min, where the latter one appears nearly twice as intense as the former. However, peak-I appears much weaker compared to peak-II if the chromatogram is recorded by optical probing at the absorption maximum of this dye (467 nm). The ion mobility (IM) spectra of DCMH+ ions corresponding to each of the LC-separated factions show three common peaks A, B, and C, with collision cross-section (CCS) values of 174, 185, and 197 Å2, respectively, but their relative intensities in the two IM spectra appear in opposite sequences. The three IM peaks have been assigned by considering the theoretically calculated CCS values of 13 possible isomers of DCMH+ ions. The IM spectral features also reveal that isomeric interconversions occur during the ESI process. Electronic structure calculations have been used to optimize the geometries of the four isomers of solvated DCM and the corresponding protomeric structures of DCMH+. The isomerization pathways and associated energy barriers have also been calculated. The gas-phase protomers are found to follow a completely different sequence of stability as compared to the neutral isomers. The analysis reveals that peak-I corresponds to one of the cis isomers, whereas peak-II arises due to cumulative contributions of the other three isomers. The absorption spectrum of DCM in methanol is simulated from the computed spectral profiles of the isomers which indicates a distribution of trans1, trans2, cis1, and cis2 isomers as 33.5, 61.5, 2.0, and 3.0%, respectively. The fragmentation behavior of DCMH+ ions in a collision-induced dissociation experiment has been found to be isomer dependent.

Identification of an Emitting Metastable State of p-Fluorophenol-Ammonia 1:2 Complex by Laser-Induced Fluorescence Spectroscopy.

We have demonstrated here, for the first time to our knowledge, the formation of an emitting metastable species upon lowest electronic excitation (S1) of a hydrogen-bonded 1:2 complex of para-fluorophenol (pFP) with ammonia (NH3), which is known to be one of the smallest reactive complexes to undergo excited state H-atom transfer (HAT) reaction to produce •NH4(NH3) radical fragment. The emission spectrum of the species is characterized to be red-shifted, broad, and structureless. From the viewpoint of energy balance, an excited state proton transfer (ESPT) is unfavorable, but according to predicted electronic structure parameters, the metastable state species could be stabilized by charge transfer (CT) interaction at the hydrogen-bonded geometry of the complex. We propose that this species could act as an intermediate to the HAT process in the excited state. The observation of such a state could be valuable to understand the complex dynamics of similar events in biologically relevant systems.

Effects of long term antiprogestine mifepristone (RU486) exposure on sexually dimorphic lncRNA expression and gonadal masculinization in Nile tilapia (Oreochromis niloticus).

Mifepristone (RU486), a clinical abortion agent and potential endocrine disruptor, binds to progestin and glucocorticoid receptors and has multiple functional importance in reproductive physiology. A long-term exposure of RU486 resulted in masculinization of female fish, however, the epigenetic landscape remains elusive. Recent studies demonstrated that long non-coding RNAs (lncRNAs) might play potential roles in epigenetic modulation of sex differentiation, ovarian cancer and germline stem cell survival. To further understand the influence of RU486 exposure on epigenetic regulation, we performed a comparative investigation on sex-biased gonadal lncRNAs profiles using control XX/XY and RU486-induced sex reversed XX Nile tilapia (Oreochromis niloticus) by RNA-seq. In total, 962 sexually differentially expressed lncRNAs and their target genes were screened from the gonads of control and sex reversed fish. In comparison with the control XX group, sex reversal induced by RU486 treatment led to significant up-regulation of 757 lncRNAs and down-regulation of 221 lncRNAs. Hierarchical clustering analysis revealed that global lncRNA expression profiles in RU486-treated XX group clustered into the same branch with the control XY, whereas XX control group formed a separate branch. The KEGG pathway enrichment analysis showed that the cis-target genes between RU486-XX and control-XX were concentrated in NOD - like receptor signaling pathway, Cell adhesion molecules (CAMs) and Biosynthesis of amino acids. Real-time PCR and in situ hybridization experiments demonstrate that lncRNAs showing intense fluctuation during RU486 treatment are also sexually dimorphic during early sex differentiation, which further proves the intimate relationship between lncRNAs and sex differentiation and sexual transdifferentiation. Taken together, our data strongly indicates that a long-term exposure of RU486 resulted in sex reversal of XX female fish and the altered expression of sexually dimorphic lncRNAs might partially account for the sex reversal via epigenetic modification.

Estrogen Receptor ß2 Oversees Germ Cell Maintenance and Gonadal Sex Differentiation in Medaka, Oryzias latipes.

In vertebrates, estrogen receptors are essential for estrogen-associated early gonadal sex development. Our previous studies revealed sexual dimorphic expression of estrogen receptor ß2 (ERß2) during embryogenesis of medaka, and here we investigated the functional importance of ERß2 in female gonad development and maintenance using a transgenerational ERß2-knockdown (ERß2-KD) line and ERß2-null mutants. We found that ERß2 reduction favored male-biased gene transcription, suppressed female-responsive gene expression, and affected SDF1a and CXCR4b co-assisted chemotactic primordial germ cell (PGC) migration. Co-overexpression of SDF1a and CXXR4b restored the ERß2-KD/KO associated PGC mismigration. Further analysis confirmed that curtailment of ERß2 increased intracellular Ca2+ concentration, disrupted intra- and extracellular calcium homeostasis, and instigated autophagic germ cell degradation and germ cell loss, which in some cases ultimately affected the XX female sexual development. This study is expected improve our understanding of germ cell maintenance and sex spectrum, and hence open new avenues for reproductive disorder management.

Modulations of νO-H and νC═O Stretching Frequencies of Difluoroacetic Acid with Internal Rotation of CHF2 Rotor: A Combined Vapor Phase and Matrix Isolation Infrared Spectroscopy Study.

Mid-infrared spectra of difluoroacetic acid (DFAA) have been measured by isolating the molecule in argon and nitrogen matrices at 8 K and also in the vapor phase at room temperature. In argon matrix, the O-H stretching fundamental (νO-H) of -COOH group appears as a doublet with band maxima at 3554 and 3558 cm-1, and a similar doublet for C═O stretching fundamental appears at 1800 and 1810 cm-1. In the vapor phase, the νO-H transition is featured with multiple peaks, and the observed band shape has been deconvoluted as superposition of two transitions both having A-type rotational band contours. We have attributed these transitions to the two internal rotational isomers corresponding to the two distinct minima along -CHF2 torsional coordinate of the molecule. Natural bond orbital (NBO) analysis reveals that these torsional minima are the manifestations of different second order interactions involving bonding and antibonding orbitals corresponding to the rotor -CHF2 and COOH groups of the molecule. By use of the theoretically predicted rotational constants of the rotamers, the band profile for νO-H has been simulated satisfactorily by means of the PGOPHER method, and this has allowed estimating accurately the energy difference between the two rotamers as 0.54 kcal/mol. The predicted energy barrier for interconversion between the rotamers is very small, ∼0.5 kcal/mol from rotamer II to rotamer I, which implies that the molecule could hop almost freely between the two rotameric forms at room temperature. As a result, the frequencies of the key stretching vibrational modes, like νO-H, νC═O, and νC-H, undergo modulation with internal rotation of the rotor -CHF2 group. Such modulation of high frequency modes could be an efficient mechanism for acceleration of rotor-induced IVR (intramolecular vibrational redistribution) well documented in the literature. Furthermore, the spectra measured in matrix isolated environment show signatures for an energetically higher third rotamer, where -OH and -C═O groups are in anti orientation. It has also been shown that DFAA can easily form weak hydrogen bonded dimeric complexes with molecular nitrogen (N2), which causes νO-H to undergo a red shift of ∼30 cm-1 in argon matrix for all three DFAA monomers.

Molecular cloning, characterization and expression analysis of complement components in red sea bream (Pagrus major) after Edwardsiella tarda and red sea bream Iridovirus (RSIV) challenge.

The complement system plays an important role in immune regulation and acts as the first line of defense against any pathogenic attack. To comprehend the red sea bream (Pagrus major) immune response, three complement genes, namely, pmC1r, pmMASP and pmC3, belonging to the classical, lectin and alternative complement cascade, respectively, were identified and characterized. pmC1r, pmMASP, and pmC3 were comprised of 2535, 3352, and 5735 base mRNA which encodes 732, 1029 and 1677 aa putative proteins, respectively. Phylogenetically, all the three studied genes clustered with their corresponding homologous clade. Tissue distribution and cellular localization data demonstrated a very high prevalence of all the three genes in the liver. Both bacterial and viral infection resulted in significant transcriptional alterations in all three genes in the liver with respect to their vehicle control counterparts. Specifically, bacterial challenge affected the pmMASP and pmC3 expression, while the viral infection resulted in pmC1r and pmC3 mRNA activation. Altogether, our data demonstrate the ability of pmC1r, pmMASP and pmC3 in bringing about an immune response against any pathogenic encroachment, and thus activating, not only one, but all the three complement pathways, in red sea bream.

Barrierless Proton Transfer in the Weak C-H···O Hydrogen Bonded Methacrolein Dimer upon Nonresonant Multiphoton Ionization in the Gas Phase.

Intermolecular proton transfer (IMPT) in a C-H···O hydrogen bonded dimer of an α,ß-unsaturated aldehyde, methacrolein (MC), upon nonresonant multiphoton ionization by 532 nm laser pulses (10 ns), has been investigated using time-of-flight (TOF) mass spectrometry under supersonic cooling condition. The mass peaks corresponding to both the protonated molecular ion [(MC)H+] and intact dimer cation [(MC)2]•+ show up in the mass spectra, and the peak intensity of the former increases proportionately with the latter with betterment of the jet cooling conditions. The observations indicate that [(MC)2]•+ is the likely precursor of (MC)H+ and, on the basis of electronic structure calculations, IMPT in the dimer cation has been shown to be the key reaction for formation of the latter. Laser power dependences of ion yields indicate that at this wavelength the dimer is photoionized by means of 4-photon absorption process, and the total 4-photon energy is nearly the same as the predicted vertical ionization energy of the dimer. Electronic structure calculations reveal that the optimized structures of [(MC)2]•+ correspond to a proton transferred configuration wherein the aldehydic hydrogen is completely shifted to the carbonyl oxygen of the neighboring moiety. Potential energy scans along the C-H···O coordinate also show that the IMPT in [(MC)2]•+ is a barrierless process.