Molecular biological, physiological, cytological, and epigenetic mechanisms of environmental sex differentiation in teleosts: A systematic review.

Human activities have been exerting widespread stress and environmental risks in aquatic ecosystems. Environmental stress, including temperature rise, acidification, hypoxia, light pollution, and crowding, had a considerable negative impact on the life histology of aquatic animals, especially on sex differentiation (SDi) and the resulting sex ratios. Understanding how the sex of fish responds to stressful environments is of great importance for understanding the origin and maintenance of sex, the dynamics of the natural population in the changing world, and the precise application of sex control in aquaculture. This review conducted an exhaustive search of the available literature on the influence of environmental stress (ES) on SDi. Evidence has shown that all types of ES can affect SDi and universally result in an increase in males or masculinization, which has been reported in 100 fish species and 121 cases. Then, this comprehensive review aimed to summarize the molecular biology, physiology, cytology, and epigenetic mechanisms through which ES contributes to male development or masculinization. The relationship between ES and fish SDi from multiple aspects was analyzed, and it was found that environmental sex differentiation (ESDi) is the result of the combined effects of genetic and epigenetic factors, self-physiological regulation, and response to environmental signals, which involves a sophisticated network of various hormones and numerous genes at multiple levels and multiple gradations in bipotential gonads. In both normal male differentiation and ES-induced masculinization, the stress pathway and epigenetic regulation play important roles; however, how they co-regulate SDi is unclear. Evidence suggests that the universal emergence or increase in males in aquatic animals is an adaptation to moderate ES. ES-induced sex reversal should be fully investigated in more fish species and extensively in the wild. The potential aquaculture applications and difficulties associated with ESDi have also been addressed. Finally, the knowledge gaps in the ESDi are presented, which will guide the priorities of future research.

High-temperature stress will put the thermo-sensitive teleost yellow catfish (Tachysurus fulvidraco) in danger through reducing reproductivity.

Recently, concerns for species that sex differentiation is influenced by temperature in the context of global warming have increased because disrupted operational sex ratios could threaten population maintenance. In contrast, little attention has been given to the reproductive ability of populations that experienced elevated temperatures. In this study, we demonstrated that high temperature (HT) would decrease population size via three different aspects of reproductive ability for the first time. We show that, in a thermo-sensitive teleost yellow catfish, a short period of HT (+3 °C) exposure during the critical period of sex differentiation leads to a different percentage of masculinization of XX genotypic females (1-23%) in wet-lab and natural water bodies. Combining the results of gonadal appearance, histology, sperm parameters, and fertilization rate, we found that XX pseudo-males induced by HT display significantly discounted fertility and reproductive performance compared to XY normal males. We demonstrate that the survival of the XY genotype is lower than XX genotype under environmental stress, including HT, hypoxia, and parasite infection, and the differential survival seems unrelated to male-biased sexual size dimorphism. The mathematical model predicts that the phenotypic female percent will be stabilized at 50% and the population will be sustainably maintained when masculinizing force is less than 0.5, while HT will put the population in danger when the masculinizing force exceeds 0.5. However, when we combine the real-world data of reproductive ability and mathematic model, our results suggest the population size decreases and the long-term survival of the studied species are threatened under the projected pace of increasing temperature. These findings will be useful for understanding the long-term effects of increasing temperature on sex ratio, reproduction and population maintenance in teleost.

Molecular players involved in temperature-dependent sex determination and sex differentiation in Teleost fish.

The molecular mechanisms that underlie sex determination and differentiation are conserved and diversified. In fish species, temperature-dependent sex determination and differentiation seem to be ubiquitous and molecular players involved in these mechanisms may be conserved. Although how the ambient temperature transduces signals to the undifferentiated gonads remains to be elucidated, the genes downstream in the sex differentiation pathway are shared between sex-determining mechanisms. In this paper, we review recent advances on the molecular players that participate in the sex determination and differentiation in fish species, by putting emphasis on temperature-dependent sex determination and differentiation, which include temperature-dependent sex determination and genetic sex determination plus temperature effects. Application of temperature-dependent sex differentiation in farmed fish and the consequences of temperature-induced sex reversal are discussed.

The controllable and efficient synthesis of two-dimensional metal-organic framework nanosheets for heterogeneous catalysis.

Two-dimensional (2D) MOFs exhibit unique periodicity in surface structures and thus have attracted much interest in the fields of catalysis, energy, and sensors. However, the expanded production scale of 2D MOFs had remained a great challenge in most previous studies. Herein, a controllable and efficient crystallization method for synthesizing 2D MOF nanosheets using high-gravity reactive precipitation is proposed, significantly improving heterogeneous catalysis efficiency. The two-dimensional ZIF-L nanosheets prepared in a rotating packed bed (RPB) reactor show a smaller lateral and lamellar thickness and a higher BET surface area compared to ZIF-L nanosheets prepared in a conventional stirred tank reactor (STR), with a greatly shortened reaction time. Applying the ZIF-L-RPB nanosheets as a catalyst, the catalytic Knoevenagel condensation as a probe reaction displays a high conversion rate of benzaldehyde (99.3%) within 2 h at room temperature, greatly exceeding that displayed by ZIF-L-STR and other reported catalysts. Furthermore, ZIL-L-RPB nanosheets of only 0.2 wt% enhanced the catalytic activity for the glycolysis of poly(ethylene terephthalate) (PET) with a PET conversion and a monomer yield of 90% in a short period of 15 min at 195 °C and almost completely depolymerized PET with a monomer yield of 94% in 30 min, which was far above that achieved by ZIL-L-STR. These results indicate the promising prospects of a high-gravity reactive precipitation strategy with precise size control in an economical way to prepare high-activity 2D MOF nanosheets for a wide range of heterogeneous catalysis.

Out-of-season spawning of largemouth bass in a controllable recirculating system.

Largemouth bass (LMB) production exceeded 0.7 million tons in 2021 and has become one of the most important freshwater aquaculture species in China. The stable and fixed culture cycle led to regular and drastic price fluctuation during the past decade. Strong price fluctuation provides opportunities and challenges for the LMB industry, and out-of-season spawning (OSS) and culture will provide technical support for the opportunities. To induce OSS at a low cost, we established a controllable recirculating system that allows precise thermo-photoperiod manipulation. In the system, four experimental groups were assigned, 18NP (18°C overwintering water temperature, natural photoperiod), 18CP (18°C overwintering water temperature, controlled photoperiod), 16CP (16°C overwintering water temperature, controlled photoperiod), and NTNP (natural water temperature and natural photoperiod), to determine the effects of chilling temperature and photoperiod on spawning performance. OSS was observed in all the experimental groups without significant differences, except NTNP. The manipulated broodstock can re-spawn 3 months later in the next spring in advance. Further analysis of the volume percentage of different stages of oocytes provides a base for excellent regression between the volume percentage of the primary growth stage, cortical alveoli stage, vitellogenesis/maturation stage, and gonadal development/maturation. The results suggest that the volume percentage of oocytes is a better indicator of gonadal development and maturation than the gonadosomatic index. We also found that LMB prefers palm fiber as a spawning nest over gravel. The findings of this work provide important technique guidance for practical OSS of the LMB aquaculture industry and standardization of ovary development and maturation in fish with asynchronous developmental oocytes.

Antibacterial activities of amorphous cefuroxime axetil ultrafine particles prepared by high gravity antisolvent precipitation (HGAP).

In vitro and in vivo antibacterial activities on the Staphylococcus aureus and Escherichia coli of the amorphous cefuroxime axetil (CFA) ultrafine particles prepared by HGAP method were investigated in this paper. The conventional sprayed CFA particles were studied as the control group. XRD, SEM, BET tests were performed to investigate the morphology changes of the samples before and after sterile. The in vitro dissolution test, minimal inhibitory concentrations (MIC) and the in vivo experiment on mice were explored. The results demonstrated that: (i) The structure, morphology and amorphous form of the particles could be affected during steam sterile process; (ii) CFA particles with different morphologies showed varied antibacterial activities; and (iii) the in vitro and in vivo antibacterial activities of the ultrafine particles prepared by HGAP is markedly stronger than that of the conventional sprayed amorphous particles.

Micronization of gemfibrozil by reactive precipitation process.

Ultrafine gemfibrozil (GEM) was prepared by reactive precipitation process in which methyl cellulose (MC) was employed to inhibit the growth and the agglomeration of particles. The impact of NaOH concentrations on bulk GEM consumption was explored. The effects of H2SO4 concentrations and the drying methods on the particle size and morphology were also discussed. The produced ultrafine powders were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, specific surface area analysis and dissolution test. XRD patterns and FT-IR spectra showed that the as-obtained ultrafine GEM was a crystalline powder with the structure and components similar to those of bulk GEM. The ultrafine GEM had a mean particle size of about 1.25 microm with a narrow distribution from 0.6 to 3 microm. The specific surface area reached up to 11.01 m2/g, which was about 6 times as large as that of bulk GEM. In the dissolution tests, about 91.2% of ultrafine GEM was dissolved after 120 min, while there was only 23.6% of bulk GEM dissolved, proving that the dissolution property of ultrafine GEM was significantly enhanced when compared to commercial GEM owing to a decreased particle size and an increased specific surface area.

Effects of Temperature on the Expression of Two Ovarian Differentiation-Related Genes foxl2 and cyp19a1a.

Exposure to stress induces a series of responses and influences a wide range of biological processes including sex differentiation in fish. The present work investigated the molecular and physiological response to thermal stress throughout the early development stage covering the whole period of sex differentiation of bluegill, Lepomis macrochirus. Larvae were treated using three temperatures, 17, 24, and 32°C from 6 to 90 days posthatching (dph) in 30-L round tanks. There is no significant difference of the sex ratio and survival among the three temperature groups in the geographic population used in this study. Two ovarian differentiation-related genes foxl2 and cyp19a1a were detected at 7 dph suggesting that these genes have already played a role prior to sex differentiation. The expression of foxl2 reached the peak and was thermosensitive just prior to the onset of ovarian differentiation at 27 dph. Histological examination displayed that the proliferation of germ cells and ovarian differentiation were delayed at the low-temperature treatment (17°C) at 97 dph compared with higher temperatures. In conclusion, the water temperature regulates the sex differentiation of bluegill through modulation of the expression of foxl2 and cyp19a1a. A comparative study of the expression profile of sex differentiation-related genes in species will shed light on the evolution of sex-determination mechanisms and the impact of stress on sex differentiation.

Preparation of uniform prednisolone microcrystals by a controlled microprecipitation method.

Prednisolone (PDL) microcrystals were successfully prepared by a controlled microprecipitation method. The characterization of PDL microcrystals by SEM and PSD indicated that the hexagonal and tetragonal PDL microcrystals with an average particle size of 1.60 and 1.46 microm could be prepared under a stirring speed of 10,000 rpm at 14 and 4 degrees C, respectively. The morphology and the particle size of PDL could be well controlled, from 1.60 to 6.12 microm for hexagonal microcrystals and 1.46 to 3.90 microm for tetragonal ones, by altering the operating parameters such as temperature, stirring speed and stabilizers. The XRD, TGA-DSC, FT-IR and physical stability studies demonstrated that the as-prepared hexagonal and tetragonal PDL microcrystals with the same pseudopolymorphic form were much more stable in water than the commercial micronized PDL with another crystal form. The dissolution tests showed that the hexagonal and tetragonal PDL microcrystals exhibited significantly enhanced dissolution property when compared to commercial micronized PDL.

Knockdown of E2F3 Inhibits Proliferation, Migration, and Invasion and Increases Apoptosis in Glioma Cells.

E2F3a, as a member of the E2F family, is essential for cell division associated with the progression of many cancers. However, the biological effect of E2F3a on glioma is not understood as well. To investigate the functional mechanism of E2F3a in glioma, we examined the expression of E2F3a in glioma tissue and cell lines. We found that E2F3a was upregulated in glioma tissue compared with adjacent tissue, and this was associated with a poor survival rate. E2F3a was highly expressed in glioma cell lines compared with normal HEB cell lines. Knockdown of E2F3a significantly inhibited cell proliferation, promoted G0/G1 phase arrest, elevated apoptosis rates, and suppressed cell migration and invasion. However, overexpression of E2F3a markedly promoted cell proliferation, migration, and invasion and inhibited apoptosis. Moreover, in vivo studies showed that knockdown of E2F3a expression dramatically inhibited U373 tumor growth in a nude mouse model. Results of real-time PCR and Western blot showed that the depletion of E2F3a upregulated the expression levels of cell apoptosis-related proteins and downregulated migration-related proteins. Conversely, E2F3a overexpression downregulated the expression levels of cell apoptosis-related proteins and upregulated migration-related proteins. In conclusion, our results highlight the importance of E2F3a in glioma and provide new insights into the diagnostics and therapeutics of gliomas.

Expression of Hsp70, Igf1, and Three Oxidative Stress Biomarkers in Response to Handling and Salt Treatment at Different Water Temperatures in Yellow Perch, Perca flavescens.

Stress is a major factor that causes diseases and mortality in the aquaculture industry. The goal was to analyze the expression of stress-related biomarkers in response to different stressors in yellow perch, which is an important aquaculture candidate in North America and highly sensitive to handling in captivity. Three fish groups were established, each having four replicates, and subjected to water temperatures of 14, 20, and 26°C and acute handling stress was performed followed by a salt treatment for 144h at a salinity of 5 ppt. Serum and hepatic mRNA levels of heat shock protein (hsp70), insulin-like growth factor 1 (Igf1), glutathione peroxidase (Gpx), superoxide dismutase 1 (Sod1), and glutathione reductase (Gsr) were quantified at seven times interval over 144 h using ELISA and RT-qPCR. Handling stress caused a significant down-regulation in Hsp70, Gpx, Sod1, and Gsr at a water temperature of 20°C compared to 14 and 26°C. Igf1 was significantly upregulated at 20°C and down-regulated at 14 and 26°C. Salt treatment had a transient reverse effect on the targeted biomarkers in all groups at 72 h, then caused an upregulation after 144 h, compared to the control groups. The data showed a negative strong regulatory linear relationship between igf1 with hsp70 and anti-oxidative gene expressions. These findings could provide valuable new insights into the stress responses that affect fish health and could be used to monitor the stress.

Ribosome RNA Profiling to Quantify Ovarian Development and Identify Sex in Fish.

Terminologies of ovary development, by somewhat subjective describing and naming main changes of oocytes, have been criticized for confusing and inconsistency of terms and classifications, and the incurred consequences impede communication among researchers. In the present work, we developed regression between ovary development and three ribosome RNA (rRNA) indexes, namely 5S rRNA percent, 18S rRNA percent, and 5S-18S rRNA ratio, using close relationship between volume percent of primary growth stage oocytes or gonadosomatic index and rRNA content, demonstrating species-specific quantification of ovary development can be established in species with either synchronous and asynchronous oogenesis. This approach may be extended to any species with primary growth oocytes, e.g. anurans and reptiles, to predict maturity stages in females. We further confirmed that 5S rRNA percent and 5S/18S rRNA ratio can serve as markers to distinguish sexes unambiguously. A micro-invasive sampling method may be invented for non-lethal prediction of ovary development and sex because only a small amount of ovary sample (<50 mg) is needed for the approach established in the current work. Researchers who work with ovary RNA-seq in these taxa should realize that insufficient depletion of rRNA will probably lead to incorrect quantification of gene expression and inaccurate conclusions.

Preparation of amorphous cefuroxime axetil nanoparticles by controlled nanoprecipitation method without surfactants.

Amorphous nanoparticles of cefuroxime axetil (CFA), a poorly water-soluble drug, were produced by the controlled nanoprecipitation method without any surfactants at room temperature. The influence of the operation parameters, such as the types of solvent and anti-solvent, the stirring speed, the solvent/anti-solvent (S/AS) volume ratio, the drug concentration and the precipitation temperature, were experimentally investigated. The results indicated that increasing the stirring speed and the S/AS volume, decreasing the drug concentration and the temperature favored to decrease the particle size from 700 to 900 nm to approximately 300 nm. The XRD analyses confirmed that the as-prepared CFA was amorphous nanoparticles. Furthermore, the amorphous CFA nanoparticles exhibited significantly enhanced dissolution property when compared to the commercial spray-dried product. The results demonstrated that the controlled nanoprecipitation method is a direct and feasible technology which could be utilized for preparation of the poorly water-soluble pharmaceutical nanoparticles.

Sex Determination in Bluegill Sunfish Lepomis macrochirus: Effect of Temperature on Sex Ratio of Four Geographic Strains.

There is increasing evidence that temperature effects on sex ratio in fish species are ubiquitous. Temperature effects on sex ratio could be influenced by parent, strain, and population, whether in fish species with temperature-dependent sex determination or genetic sex determination plus temperature effects. In the present study, effects of genotype-temperature interactions on sex determination in bluegill sunfish were further investigated, based on our previous results, using four geographic strains: Hebron, Jones, Hocking, and Missouri. In the Hebron strain, the two higher-temperature treatment groups (24 °C and 32 °C) produced more males than the low-temperature treatment group (17 °C) from 6 days post-hatching (dph) to 90 dph. In contrast, the low-temperature treatment produced more males than the other two higher-temperature treatments in the Jones strain. No significant effects of temperature on sex ratio were detected in the other two strains. Our results from sex ratio variance in different treatment times suggest that the thermosensitive period of sex differentiation occurs prior to 40 dph. Our results further confirmed that genotype-temperature interactions influence sex determination in bluegill. Therefore, to significantly increase the proportion of males, which grow faster and larger than females, a consumer- and environment-friendly approach may be achieved through selection of temperature sensitivity in bluegill.

Effects of 17α-Methyltestosterone and Aromatase Inhibitor Letrozole on Sex Reversal, Gonadal Structure, and Growth in Yellow Catfish Pelteobagrus fulvidraco.

Monosex populations are in demand in many fish species with sexual dimorphism, e.g., better growth performance, higher gonad value, superior ornamental value. From the point of view of research, a monosex population is one of the best materials for investigating sex-determining mechanisms, sex differentiation, and sex-linked markers. Sex reversal of females (phenotypic reversal from XX female to XX male) is the first step in all-female production in species with an XX/XY system for sex determination. In the present study, masculinization of yellow catfish, a species with XX/XY sex determination, was investigated by oral administration of various doses of 17α-methyltestosterone (MT) or an aromatase inhibitor (AI) letrozole (LZ); effects on survival, growth performance, sex ratio, and changes in gonadal structure were evaluated. Three doses (20, 50, and 100 mg kg(-1) diet) of oral MT or LZ were administered to fry from 10 days post-hatching (DPH) to 59 DPH. Oral administration of MT at all doses did not significantly change the ratio of males (45.8%, 33.3%, and 50.0% respectively) compared to the control group (37.5%), while yielding intersex fish at all doses (4.2% to 8.3%). Oral administration of LZ produced a significantly higher proportion of males in all doses (75.5%, 83.3%, and 75.0%, respectively). Additionally, the lowest dose of LZ improved the growth of treated fish compared to the control, and all doses of LZ enhanced spermatogenesis in treated males.

Dispersion of nanosized aqueous suspensions of barium titanate with ammonium polyacrylate.

The colloidal stability of nanosized barium titanate (BaTiO3) aqueous suspensions with ammonium polyacrylate (PAA-NH4) at different pH values has been investigated by means of zeta potential, adsorption isotherm, sedimentation, and rheology characterization. The isoelectric point of BaTiO3 powders is at pH 2.5 and the value of zeta potential is at its maximum near pH 10. The amount of leached barium ion decreases with increasing pH, but the change decreases with increasing initial pH. Adsorption of PAA-NH4 onto the surface of BaTiO3 decreases its zeta potential. Results show that PAA-NH4 adsorption follows Langmuir monolayer adsorption isotherms and the amount of PAA-NH4 required to stabilize nanosized BaTiO3 suspensions decreases as the pH increases. The mechanism of stabilization of BaTiO3 is shown to be electrosteric under the experimental conditions. Good agreement between zeta potential, sedimentation, and rheological tests is found, which identifies an optimum pH value of about 10 and an optimum dispersant concentration of about 2.0 wt%, independent of the solids volume fraction of suspensions.

Effects of non-steroidal aromatase inhibitor letrozole on sex inversion and spermatogenesis in yellow catfish Pelteobagrus fulvidraco.

The effects of letrozole (LZ), a potent nonsteroidal aromatase inhibitor (AI), on growth performance, sex inversion, and sex changes were investigated in yellow catfish (Pelteobagrus fulvidraco), which display sexual dimorphic growth. Growth performance was promoted significantly in the low-dose LZ treatment, compared with the control. Four LZ treatments produced dose-dependent male proportions that were significantly higher than that of the control. Histological examination of testes treated by LZ displayed a large amount of spermatozoa and enlarged lobule lumens, indicating that LZ treatments can potentially stimulate spermatogenesis. Changes of sex proportions 45 days after the end of the LZ treatments prove that the female germ cells possess a certain degree of bipotentiality. These results suggest that aromatase activity plays a vital role in sex differentiation, as in other teleosts, with inhibition of aromatase activity by AI bringing about sex inversion.

Fabrication of inhalable spore like pharmaceutical particles for deep lung deposition.

An innovative strategy of fabricating uniform spore like drug particles to improve pulmonary drug delivery efficiency was disclosed in the present study. Spore like particles were prepared through combination of high gravity controlled precipitation and spray drying process with insulin as model drug first, showing rough surface and hollow core. The shell of such spore-like particle was composed of nanoparticles in loose agglomerate and could form nanosuspension upon contacting antisolvent. Further characterization confirmed secondary structure and bio-activity was well preserved in spore like particles of insulin. Stable aerosol performance at different dosages with fine powder fraction (FPF) of 80% and comparable FPF (69-76%) for formulated powder were achieved, significantly higher than marketed product Exubera. On the other hand spore like particles of bovine serum albumin, lysozyme and salbutamol sulfate showed similar high FPF of 80%, regardless of different shape of primary nanoparticles, indicating various application of this new process in significant improvement of pulmonary drug delivery.

Preparation of ultrafine fenofibrate powder by solidification process from emulsion.

The solidification process from emulsion, which consisted of emulsifier, water and molten drug as oil phase without use of any organic solvent, was firstly employed to prepare ultrafine fenofibrate (FF) powder. The effects of stirring speed and volume ratios of hot emulsion to cold water on the particle size and morphology were discussed as well as the impacts of different emulsifiers on emulsion. The produced ultrafine powder was characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, specific surface area analysis and a dissolution test. XRD patterns and FT-IR spectra showed that the ultrafine FF was crystalline powder with the structure and the components similar to those of bulk drug. The product had a mean particle size of about 3 microm with a narrow distribution from 1 microm to 5 microm. The specific surface area reached up to 6.23 m(2)/g, which was about 25 folds as large as that of bulk FF. In the dissolution tests, about 96.1% of ultrafine FF was dissolved after 120 min, while there was only 38.1% of bulk drug dissolved, proving that the dissolution property of ultrafine FF was significantly improved when compared to commercial drug.