Droplet Fusion Research Articles

Fisetin Ameliorates Hepatocyte Lipid Droplet Accumulation via Targeting the Rhythmic Protein BMAL1 to Regulate Cell Death-Inducing DNA Fragmentation Factor-α-like Effector C-Mediated Lipid Droplet Fusion.

High fat diet (HFD) induces the enlargement and accumulation of lipid droplets (LDs) in hepatocytes, thereby influencing the homeostasis of lipid metabolism. Cell death-inducing DNA fragmentation factor-α-like effector C (CIDEC), a surface protein of LDs, facilitates their fusion and growth, transforming small LDs into larger ones. Lipophagy, a selective form of autophagy, primarily targets small LDs for degradation. Fisetin (FIS), a natural dietary flavonoid present in various fruits and vegetables, has an unclear mechanism for reducing LD accumulation. In this study, we observed that FIS significantly ameliorated HFD-induced lipid accumulation in the hepatocytes of C57BL/6 mice. In further mechanistic studies, we revealed that FFA enhanced the expression of CIDEC, which promoted the fusion of LDs and caused them to become larger. The enlarged LDs could not be degraded by autophagy, which ultimately led to accumulation of LDs. Conversely, FIS alleviated LD accumulation by inhibiting CIDEC-mediated fusion, resulting in smaller LDs that facilitated lipophagy. Additionally, studies indicated that the dysfunction of circadian rhythms is closely related to lipid metabolism. In our study, we showed that HFD and FFA disrupted circadian rhythm in C57BL/6 mouse hepatocytes and AML12 cells, while FIS modified the rhythm disturbances and increased protein expression of the core clocks BMAL1 and CLOCK. We silenced the BMAL1 protein and revealed that si-BMAL1 upregulated CIDEC proteins. These data suggested that FIS might inhibit CIDEC-mediated LD fusion and enhance hepatocyte lipophagy by promoting the expression of rhythm protein BMAL1, thereby alleviating LD accumulation in C57BL/6 and AML12 cells caused by the HFD and FFA. The present study provided novel insights and potential targets for utilizing functional food factors to mitigate the accumulation of LD in hepatocytes.

CIDEC/FSP27 exacerbates obesity-related abdominal aortic aneurysm by promoting perivascular adipose tissue inflammation

Abstract Abdominal aortic aneurysm (AAA) is strongly correlated with obesity, partially due to the abnormal expansion of abdominal perivascular adipose tissue (PVAT). Cell death-inducing DNA fragmentation factor-like effector C (CIDEC), also known as fat-specific protein 27 (FSP27) in rodents, is specifically expressed in adipose tissue where it mediates lipid droplet fusion and adipose tissue expansion. Whether and how CIDEC/FSP27 plays a role in AAA pathology remains elusive. Here, we show that FSP27 exacerbates obesity and angiotensin II (Ang II)-induced AAA progression. FSP27 deficiency in mice inhibited high-fat diet (HFD)-induced PVAT expansion and inflammation. Both global and adipose tissue-specific FSP27 ablation significantly decreased obesity-related AAA incidence. Deficiency of FSP27 in adipocytes abrogated matrix metalloproteinase-12 (MMP12) expression in aortic tissues. Infiltrated macrophages, which partially colocalize with MMP12, were significantly decreased in the FSP27-deficient aorta. Mechanistically, knockdown of Fsp27 in 3T3-L1 adipocytes inhibited C-C motif chemokine ligand 2 (CCL2) expression and secretion through a c-Jun N-terminal kinase (JNK)-dependent pathway, thereby leading to reduced induction of macrophage migration, while Cidec overexpression rescued this effect. Overall, our study demonstrates that CIDEC/FSP27 in adipose tissue contributes to obesity-related AAA formation, at least in part, by enhancing PVAT inflammation and macrophage infiltration, thus shedding light on its significance as a key regulator in the context of obesity-related AAA.

Establishment of Nano-ITO induced rat model of indium lung disease and exploration of its pathological characteristics

Objective: To study the dynamic pathological characteristics of lung tissue in a Nano-ITO induced rat model of indium lung disease and to guide clinical and basic scientific research to further explore the mechanisms of pulmonary interstitial injury and pulmonary alveolar proteinosis (PAP). Methods: Dose-response (three divided doses) and time-course studies (six exposure periods) were performed to investigate the pulmonary toxicity induced by Nano-ITO. At the end of the experiment, cytokine levels and oxidative stress were analyzed in the bronchoalveolar lavage fluid. Rat lung tissues were also collected for staining with H&E, PAS, Masson's, Oil Red O, and Sirius Red. Ultrastructure of lung tissue cells was observed by transmission electron microscopy. Expression of IL-1β, HO-1, SP-A was observed by immunohistochemistry, and the expression of α-SMA was observed by immunofluorescence. Results: Nano-ITO intratracheal instillation caused pulmonary toxicity by inducing acute inflammation at 3 days, granuloma (nodule) formation and collagen hyperplasia at 14 days, and alveolar proteinosis at 56 days post-exposure. Pathological features of lung tissue included typical alveolar exudates, cellular fibrous nodules, enlarged alveolar fat droplet fusion, cholesterol crystal granuloma and pulmonary alveolar proteinosis. The intra-alveolar eosinophilic material (multilamellated, lattice-shaped, and myelin-like structure) showed abnormal lamellar bodies (features of alveolar type Ⅱ epithelial cells) and abundant rough endoplasmic reticulum and mitochondria (features of fibroblasts) on transmission electron microscopy of the lung tissue from rats exposed to Nano-ITO on the 84th day. Cellular pathology revealed that a large amount of amorphous PAS stain-positive substances appear in BALF at 28 days post-exposure, and pink granular protein-like substances can be seen in alveolar macrophages. Conclusions: There are three characteristic developmental stages in Nano-ITO induced pulmonary injury in rats, acute inflammation, granuloma (nodule) formation and collagen proliferation, and pulmonary alveolar proteinosis, which provide a reference feature model for the pathogenesis of indium lung disease.

Fusion Dynamics and Size-Dependence of Droplet Microstructure in ssDNA-Mediated Protein Phase Separation.

Biomolecular condensation involving proteins and nucleic acids has been recognized to play crucial roles in genome organization and transcriptional regulation. However, the biophysical mechanisms underlying the droplet fusion dynamics and microstructure evolution during the early stage of liquid-liquid phase separation (LLPS) remain elusive. In this work, we study the phase separation of linker histone H1, which is among the most abundant chromatin proteins, in the presence of single-stranded DNA (ssDNA) capable of forming a G-quadruplex by using molecular simulations and experimental characterization. We found that droplet fusion is a rather stochastic and kinetically controlled process. Productive fusion events are triggered by the formation of ssDNA-mediated electrostatic bridges within the droplet contacting zone. The droplet microstructure is size-dependent and evolves driven by maximizing the number of electrostatic contacts. We also showed that the folding of ssDNA to the G-quadruplex promotes LLPS by increasing the multivalency and strength of protein-DNA interactions. These findings provide deep mechanistic insights into the growth dynamics of biomolecular droplets and highlight the key role of kinetic control during the early stage of ssDNA-protein condensation.

Did the exposure of coacervate droplets to rain make them the first stable protocells?

Membraneless coacervate microdroplets have long been proposed as model protocells as they can grow, divide, and concentrate RNA by natural partitioning. However, the rapid exchange of RNA between these compartments, along with their rapid fusion, both within minutes, means that individual droplets would be unable to maintain their separate genetic identities. Hence, Darwinian evolution would not be possible, and the population would be vulnerable to collapse due to the rapid spread of parasitic RNAs. In this study, we show that distilled water, mimicking rain/freshwater, leads to the formation of electrostatic crosslinks on the interface of coacervate droplets that not only suppress droplet fusion indefinitely but also allow the spatiotemporal compartmentalization of RNA on a timescale of days depending on the length and structure of RNA. We suggest that these nonfusing membraneless droplets could potentially act as protocells with the capacity to evolve compartmentalized ribozymes in prebiotic environments.

Chitosan/Silica Hybrid Nanogels by Inverse Nanoemulsion for Encapsulating Hydrophilic Substances

AbstractA strategy for the preparation of a hybrid chitosan/silica nanohydrogel is reported, which combines the gelation of chitosan in a nanoemulsion system with a sol–gel process to produce silica. Chitosan is used as a biopolymer matrix, while silica acts as a structuring additive. Hydrogel nanocapsules are obtained through the ionic interaction of the cationic groups of chitosan with the anionic groups of sodium triphosphate (STP), which is used as a physical cross‐linker. Two alternative preparation methods are compared in this work: in the first one, STP is added to the continuous phase of an inverse emulsion of chitosan; in the second one, the fusion of droplets of two emulsions containing separate chitosan and STP takes place. The size of the obtained nanocapsules ranges from 50 to 200 nm. The efficiency of the formed hydrogel for entrapping a hydrophilic model substance (erioglaucine disodium salt) is investigated for the two systems by studying the release in a neutral aqueous medium. The results indicate that the hydrophilic cargo is efficiently encapsulated by both preparation methods, although the droplet‐fusion method yields more stable suspensions. As a general observation, the release behavior of erioglaucine is systematically retarded when silica is present in the systems.

The Effects of trans-10, cis-12 Conjugated Linoleic Acid on the Production Performance of Dairy Cows and the Expression and Transcription Regulation of Lipid Metabolism-Related Genes in Bovine Mammary Epithelial Cells.

Dietary fatty acids (FAs) determine the quality of dairy products. The trans-10, cis-12 conjugated linoleic acid (t10c12-CLA) is commonly considered an FA factor leading to milk fat depression syndrome (MFDs) in dairy cow. However, its effect on dairy cow performance and involvement in milk fat metabolism have been insufficiently explored. This study administered 136.17 g/day of rumen-protected CLA (RP-CLA) to dairy cows and found a diminution in milk fat percentage and a trend of increasing milk protein percentage on day 21 postpartum. Lactose content, milk yield, and net energy for lactation were unaffected. In the cell experiments, Oil Red O staining showed a notable increase in lipid droplets. Gene and protein expression analysis showed that 300 μM t10c12-CLA upregulated the expression of CD36, DGAT2, and ADRP, while downregulating the expression of ACACA, FASN, SREBP1, FABP3, FATP3, ACSL4, LPIN1, DGAT1, BTN1A1, XDH, SNAP23, and VAMP4. This provides a possible mechanistic pathway for the contradictory phenomenon of t10c12-CLA reducing milk fat while increasing lipid droplets. Overall, t10c12-CLA, as a long-chain fatty acid, can promote lipid droplet synthesis but may reduce milk fat by inhibiting lipid droplet fusion and secretion, FAs de novo synthesis, and triglyceride biosynthesis.

Preparation of Controlled Multicompartmental Gel Microcarriers Based on Aqueous Two-Phase Emulsions for 3D Partitioned Cell Coculture In Vitro.

A facile method was proposed for preparing controllable multicompartment gel microcarriers using an aqueous two-phase emulsion system. By leveraging the density difference between the upper polyethylene glycol solution and the lower dextran-calcium chloride (CaCl2) solution in the collection solution and the high viscosity of the lower solution, controllable fusion of core-shell droplets made by coextrusion devices was achieved at the water/water (w/w) interface to fabricate microcarriers with separated core compartments. By adjusting the sodium alginate concentration, collected solution composition, and number of fused liquid droplets, the pore size, shape, and number of compartments could be controlled. Caco-2 and HepG2 cells were encapsulated in different compartments to establish gut-liver coculture models, exhibiting higher viability and proliferation compared to monoculture models. Notably, significant differences in cytokine expression and functional proteins were observed between the coculture and monoculture models. This method provides new possibilities for preparing complex and functional three-dimensional coculture materials.

Lipid droplets-vacuoles interaction promotes lipophagy in the oleaginous diatom Fistulifera solaris

Autophagy is a highly conserved process for degrading intracellular materials in vacuoles or lysosomes, commonly found in a wide range of eukaryotes. Lipid droplets are intracellular organelles that store neutral lipids, and recent studies have shown that autophagy regulates lipid metabolism by degrading lipid droplets. While autophagy-mediated degradation of lipid droplets, called lipophagy, is recognized as one of the major lipid degradation mechanisms in eukaryotic cells, its characterization in microalgae remains incomplete. Our prior investigations established a potential connection between autophagy and lipid degradation on the oleaginous microalga (diatom) Fistulifera solaris, a candidate for microalgal biofuel production. Inhibition of lipophagy can be a promising strategy to enhance the oleaginous phenotype of this alga. However, the presence of lipophagy in this alga has not been demonstrated because the interaction between vacuoles and lipid droplets has not yet been observed. In this study, we investigated lipophagy in F. solaris by microscopy, transcriptomics, and biochemical assays. Fluorescence microscopy revealed increased vacuolar contact with lipid droplets under conditions promoting lipid degradation, and this process was hindered by autophagy inhibitors. Transmission electron microscopy showed direct fusion of vacuoles and lipid droplets, suggesting that F. solaris might have a novel type of microlipophagy. These data indicate that lipophagy may play an important role in lipid degradation in F. solaris. To our knowledge this study marks the first direct observation of lipophagy in diatoms, contributing to our understanding of lipid metabolism in this organism.

Solvatochromic Buffering Fluorescent Probe Resolves the Lipid Transport and Morphological Changes during Lipid Droplet Fusion by Super-Resolution Imaging.

The varied functions of lipid droplets, which encompass the regulation of lipid and energy homeostasis, as well as their association with the occurrence of various metabolic diseases, are intricately linked to their dynamic properties. Super-resolution imaging techniques have emerged to decipher physiological processes and molecular mechanisms on the nanoscale. However, achieving long-term dynamic super-resolution imaging faces challenges due to the need for fluorescent probes with high photostability. This paper introduces LD-CF, a "buffering probe" for imaging lipid droplet dynamics using structured illumination microscopy (SIM). The polarity-sensitive LD-CF eliminates background fluorescence with a "cyan filter" strategy, enabling wash-free imaging of lipid droplets. In the fluorescent "off" state outside droplets, the probes act as a "buffering pool", replacing photobleached probes inside droplets and enabling photostable long-term SIM imaging. With this probe, three modes of lipid droplet fusion were observed, including the discovery of fusion from large to small lipid droplets. Fluorescence intensity tracking also revealed the direction of lipid transport during the lipid droplet fusion.

Energy Barrier of a Monolayer Stalk Formation during Lipid Droplet Fusion

Energy Barrier of a Monolayer Stalk Formation during Lipid Droplet Fusion

CIDE proteins and their regulatory mechanisms in lipid droplet fusion and growth.

The cell death-inducing DFF45-like effector (CIDE) proteins, including Cidea, Cideb, and Cidec/Fsp27, regulate various aspects of lipid homeostasis, including lipid storage, lipolysis, and lipid secretion. This review focuses on the physiological roles of CIDE proteins based on studies on knockout mouse models and human patients bearing CIDE mutations. The primary cellular function of CIDE proteins is to localize to lipid droplets (LDs) and to control LD fusion and growth across different cell types. We propose a four-step process of LD fusion, characterized by (a) the recruitment of CIDE proteins to the LD surface and CIDE movement, (b) the enrichment and condensate formation of CIDE proteins to form LD fusion plates at LD-LD contact sites, (c) lipid transfer through lipid-permeable passageways within the fusion plates, and (d) the completion of LD fusion. Lastly, we outline CIDE-interacting proteins as regulatory factors, as well as their contribution in LD fusion.

Evaporation characteristics of cis-1,1,1,4,4,4-hexafluoro-2-butene (R1336mzz(Z)) droplet in high pressure and temperature environments

Cis-1,1,1,4,4,4-hexafluoro-2-butene (R1336mzz(Z)) has emerged as an exceptionally promising low-global-warming-potential (GWP) refrigerant, ideal for spray cooling systems in the thermal management of electronic components. Research on the evaporation characteristics of an individual isolated cryogen droplet excludes uncertainties caused by droplet collisions and fusion, thereby laying the foundation for spray cooling. In this paper, a theoretical model for single R1336mzz (Z) droplet evaporation considering the effect of natural convection in a high pressure and temperature environment is proposed. The newly proposed model is validated by comparing the predicted results of the R1336mzz(Z) droplet evaporation with experimental data. Then, the effects of environmental temperature (323–523 K) and pressure (1–20 bar) on the R1336mzz(Z) droplet evaporation are investigated. The results reveal that the effect of increasing the ambient pressure on the droplet lifetime of R1336mzz(Z) undergoes a transition from deceleration to acceleration. Elevated temperature can promote droplet evaporation; however, the promoting effect of increasing the ambient temperature on droplet evaporation will be weakened in high-pressure cases. Increasing the ambient pressure and temperature both can enhance the heat transfer from the environment to the droplet through natural convection, while increasing the pressure greatly inhibits the molecular diffusion during droplet evaporation. Thus, the total evaporation rate depends on the competing effects of these two factors. In addition, the trend of the droplet temperature variation could differ based on droplet initial temperatures, ambient temperatures, and pressures. An increase in the ambient temperature or pressure corresponds to an increase in the droplet equilibrium temperature (Tequ). However, Tequ is almost independent of the droplet initial size and temperature.

The juxtamembrane linker of synaptotagmin 1 regulates Ca2+ binding via liquid-liquid phase separation

Synaptotagmin (syt) 1, a Ca2+ sensor for synaptic vesicle exocytosis, functions in vivo as a multimer. Syt1 senses Ca2+ via tandem C2-domains that are connected to a single transmembrane domain via a juxtamembrane linker. Here, we show that this linker segment harbors a lysine-rich, intrinsically disordered region that is necessary and sufficient to mediate liquid-liquid phase separation (LLPS). Interestingly, condensate formation negatively regulates the Ca2+-sensitivity of syt1. Moreover, Ca2+ and anionic phospholipids facilitate the observed phase separation, and increases in [Ca2+]i promote the fusion of syt1 droplets in living cells. Together, these observations suggest a condensate-mediated feedback loop that serves to fine-tune the ability of syt1 to trigger release, via alterations in Ca2+ binding activity and potentially through the impact of LLPS on membrane curvature during fusion reactions. In summary, the juxtamembrane linker of syt1 emerges as a regulator of syt1 function by driving self-association via LLPS.

Kinetic characterization of low velocity positive collision of double droplets

Droplet positive collision is a complex process involving heat transfer in gas-liquid-solid three-phase flow and droplet collide dynamics. In order to study the kinetic behavior of droplets under positive collision in low velocity (v < 2 m/s), this paper focuses on the spreading, vibration, and fracture characteristics of double droplets by numerical simulation. First, the accuracy of the model is verified by experimental comparison. The effects of droplet diameters, collision velocities and wall contact angles on the spreading process are analyzed, and the spreading factor curves are plotted. Then, the droplet rebound vibration after collision fusion is equated to a single-degree-of-freedom damped vibration system, and the peak vibration height variation curve of the fused droplet is obtained by non-linear fitting. Considering the droplet phase change, the influence law of different conditions on the vibration damping factor and vibration time of the fused droplet is studied. Finally, it is found that rebound fracture and spreading fracture occur after the fusion of double droplets under positive collision, and the critical values of collide velocity required for the occurrence of the aforementioned phenomenon are found. To provide a reliable theoretical basis for the study of heat and mass transfer processes after multiple droplets collide the wall.

Physiology and Mechanism of Milk Fat Globules Secretion in Dairy Animals: A Review

Milk fat globules (MFGs) are a complex compound secreted in the mammary gland, composed of triglycerides; phosphatidylethanolamine (PE) phosphatidylcholine (PC) with unclear mechanism and the fused lipid droplets enclosed by inner layer derived from the endoplasmic reticulum and outer bilayer is directly derived from the apical plasma membrane as the lipid droplets bud out from the cell. The core components of MFGs, triglycerides are synthesized via the de novo fatty acid pathway and LCFA was directly taken from serum. The de novo fatty acid pathway was significantly dependent on the enzymes acetyl-CoA carboxylase (ACACA), fatty acid synthase (FASN) and acyl-CoA synthetase short-chain family member 2 (ACSS2) to synthesize short and medium-chain fatty acids, whereas the bio-hydrogenation pathway is a principal pathway for the synthesis of long-chain fatty acids. Following this, the milk fat globules coated by the membrane are released from the MEC to the lumen with or without other milk components and the membrane that surrounds the fat globule stabilize the milk fat in its dispersed state, prevent flocculation and globule coalescence, and protects against the deleterious effects of lipases. Even though the secretion mechanism of lipid droplets is controversial hormonal and molecular mechanisms are involved. Moreover, the fat globule size and composition are determined by factors such as stage of lactation, physiological state of the animal, Diacylglycerol Acyltransferase1 (DGAT1) activity, and PC/PE ratio along with breed and animal species. While some studies were conducted on milk synthesis and secretion, the molecular mechanism behind lipid droplet fusion, secretion in the apical membrane and how MFGM is formed and the mechanism of MFG synthesis regulation are undiscovered. Therefore, it needs advanced investigation.

Planning method of droplet fusion scheduling based on mixed-integer programming

Planning method of droplet fusion scheduling based on mixed-integer programming

An integrated and multi-functional droplet-based microfluidic platform for digital DNA amplification

Digital DNA amplification is a powerful method for detecting and quantifying rare nucleic acids. In this study, we developed a multi-functional droplet-based platform that integrates the traditional digital DNA amplification workflow into a one-step device. This platform enables efficient droplet generation, transition, and signal detection within a 5-min timeframe, distributing the sample into a uniform array of 4 × 104 droplets (variation <2%) within a chamber. Subsequent in-situ DNA amplification, fluorescence detection, and signal analysis were carried out. To assess the platform's performance, we quantitatively detected the human epidermal growth factor receptor (EGFR) mutation and human papillomavirus (HPV) mutation using digital polymerase chain reaction (dPCR) and digital loop-mediated isothermal amplification (dLAMP), respectively. The fluorescence results exhibited a positive, linear, and statistically significant correlation with target DNA concentrations ranging from 101 to 105 copies/μL, demonstrating the capability and feasibility of the integrated device for dPCR and dLAMP. This platform offers high-throughput droplet generation, eliminates droplet fusion and transition, is user-friendly, reduces costs compared to current methods, and holds potential for thermocycling and isothermal nucleic acid quantification with high sensitivity and accuracy.

Circ_0004535/miR-1827/CASP8 network involved in type 2 diabetes mellitus with nonalcoholic fatty liver disease.

Diagnostic delay in type 2 diabetes mellitus (T2DM) with nonalcoholic fatty liver disease (NAFLD) patients often leads to a serious public health problem. Understanding the pathophysiological mechanisms of disease will help develop more effective treatments. High-throughput sequencing was used to determine the expression levels of circRNAs, and mRNAs in health controls, T2DM patients, and T2DM with NAFLD patients. Differentially expressed genes (DEcircRs, DEmRs) in T2DM with NAFLD were identified by differential analysis. The miRNAs with targeted relationship with the DEcircRs and DEmRs were respectively predicted to construct a ceRNA regulatory network. In addition, enrichment analysis of DEmRs in the ceRNA network was performed. The expression of important DEcircRs was further validated by quantitative real-time PCR (qRT-PCR). The steatosis was detected in glucose treated LO2 cells by overexpressing circ_0004535, and CASP8. There were 586 DEmRs, and 10 DEcircRs in both T2DM and T2DM with NAFLD patients. Combined with predicted results and differential analysis, the ceRNA networks were constructed. The DEmRs in the ceRNA networks were mainly enriched in Toll-like receptor signaling pathway, and apoptosis. Importantly, dual luciferase experiments validated the targeted binding of hsa_circ_0004535 and hsa-miR-1827 or hsa-miR-1827 and CASP8. qRT-PCR experiments validated that hsa_circ_0004535, and CASP8 was downregulated and hsa-miR-1827 was upregulated expression in peripheral blood of T2DM with NAFLD patients. Abnormal cell morphology, and increased lipid droplet fusion were observed in the glucose treated LO2 cells, overexpression of circ_0004535 and CASP8 ameliorated these changes. Our work provides a deeper understanding of ceRNA mediated pathogenesis of T2DM with NAFLD and provides a novel strategy for treatment.

Effects of luteinizing hormone-releasing hormone analog and pimozide on the release of luteinizing hormone and ovulation in artificially matured Japanese eel Anguilla japonica

Pituitary gonadotropins, follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), control oogenesis in all vertebrates. In particular, Lh plays a key role in stimulating the final oocyte maturation and subsequent ovulation. The biosynthesis and secretion of Lh are regulated by several neurohormones, including gonadotropin-releasing hormone (GnRH) and dopamine. GnRH analogs, also known as Lh releasing hormone analogs (LHRHa), and dopamine antagonists are commonly used to induce sexual maturation in teleosts. However, the effects of these reagents differ among fish species. Therefore, in the present study, the effects of LHRHa and pimozide (a dopamine antagonist) on Lh release and ovulation induction were investigated in female eels whose ovarian development was artificially induced by recombinant Fsh with ovaries containing oocytes at the migratory nucleus stage in vitro and/or in vivo. Both LHRHa and pimozide stimulated the release of Lh from pituitary cells in a dose-dependent manner in vitro. Furthermore, the synergistic effects of LHRHa and pimozide were observed in the release of Lh from the pituitary gland. In vivo experiments demonstrated that the administration of pimozide alone or in combination with LHRHa induced the release of Lh and the fusion of oil droplets in oocytes. In addition, 17α-hydroxyprogesterone (OHP) combined with LHRHa and pimozide resulted in complete ovulation at a high rate (87.5% complete ovulation and 12.5% partial ovulation), whereas LHRHa and pimozide without OHP induced partial ovulation at a low rate (0% complete ovulation and 14.3% partial ovulation). These results suggest that combined treatment with LHRHa, pimozide, and OHP could induce ovulation in artificially matured female Japanese eels. However, future studies are needed to evaluate the quality of embryos produced by females induced with LHRHa, pimozide and OHP. Additionally, the pituitary gland obtained from female eels with artificially induced sexual maturation is suitable for investigating the mechanisms of Lh release.