Comparison of Light-Sheet Fluorescence Microscopy and Fast-Confocal Microscopy for Three-Dimensional Imaging of Cleared Mouse Brain.

Whole-brain imaging is important for understanding brain functions through deciphering tissue structures, neuronal circuits, and single-neuron tracing. Thus, many clearing methods have been developed to acquire whole-brain images or images of three-dimensional thick tissues. However, there are several limitations to imaging whole-brain volumes, including long image acquisition times, large volumes of data, and a long post-image process. Based on these limitations, many researchers are unsure about which light microscopy is most suitable for imaging thick tissues. Here, we compared fast-confocal microscopy with light-sheet fluorescence microscopy for whole-brain three-dimensional imaging, which can acquire images the fastest. To compare the two types of microscopies for large-volume imaging, we performed tissue clearing of a whole mouse brain, and changed the sample chamber and low- magnification objective lens and modified the sample holder of a light-sheet fluorescence microscope. We found out that light-sheet fluorescence microscopy using a 2.5× objective lens possesses several advantages, including saving time, large-volume image acquisitions, and high Z-resolution, over fast-confocal microscopy, which uses a 4× objective lens. Therefore, we suggest that light-sheet fluorescence microscopy is suitable for whole mouse brain imaging and for obtaining high-resolution three-dimensional images.

Activation of MAP2K signaling by genetic engineering or HF-rTMS promotes corticospinal axon sprouting and functional regeneration.

Facilitating axon regeneration in the injured central nervous system remains a challenging task. RAF-MAP2K signaling plays a key role in axon elongation during nervous system development. Here, we show that conditional expression of a constitutively kinase-activated BRAF in mature corticospinal neurons elicited the expression of a set of transcription factors previously implicated in the regeneration of zebrafish retinal ganglion cell axons and promoted regeneration and sprouting of corticospinal tract (CST) axons after spinal cord injury in mice. Newly sprouting axon collaterals formed synaptic connections with spinal interneurons, resulting in improved recovery of motor function. Noninvasive suprathreshold high-frequency repetitive transcranial magnetic stimulation (HF-rTMS) activated the BRAF canonical downstream effectors MAP2K1/2 and modulated the expression of a set of regeneration-related transcription factors in a pattern consistent with that induced by BRAF activation. HF-rTMS enabled CST axon regeneration and sprouting, which was abolished in MAP2K1/2 conditional null mice. These data collectively demonstrate a central role of MAP2K signaling in augmenting the growth capacity of mature corticospinal neurons and suggest that HF-rTMS might have potential for treating spinal cord injury by modulating MAP2K signaling.

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents.

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

Single-Step Fast Tissue Clearing of Thick Mouse Brain Tissue for Multi-Dimensional High-Resolution Imaging.

Recent advances in optical clearing techniques have dramatically improved deep tissue imaging by reducing the obscuring effects of light scattering and absorption. However, these optical clearing methods require specialized equipment or a lengthy undertaking with complex protocols that can lead to sample volume changes and distortion. In addition, the imaging of cleared tissues has limitations, such as fluorescence bleaching, harmful and foul-smelling solutions, and the difficulty of handling samples in high-viscosity refractive index (RI) matching solutions. To address the various limitations of thick tissue imaging, we developed an Aqueous high refractive Index matching and tissue Clearing solution for Imaging (termed AICI) with a one-step tissue clearing protocol that was easily made at a reasonable price in our own laboratory without any equipment. AICI can rapidly clear a 1 mm thick brain slice within 90 min with simultaneous RI matching, low viscosity, and a high refractive index (RI = 1.466), allowing the imaging of the sample without additional processing. We compared AICI with commercially available RI matching solutions, including optical clear agents (OCAs), for tissue clearing. The viscosity of AICI is closer to that of water compared with other RI matching solutions, and there was a less than 2.3% expansion in the tissue linear morphology during 24 h exposure to AICI. Moreover, AICI remained fluid over 30 days of air exposure, and the EGFP fluorescence signal was only reduced to ~65% after 10 days. AICI showed a limited clearing of brain tissue >3 mm thick. However, fine neuronal structures, such as dendritic spines and axonal boutons, could still be imaged in thick brain slices treated with AICI. Therefore, AICI is useful not only for the three-dimensional (3D) high-resolution identification of neuronal structures, but also for the examination of multiple structural imaging by neuronal distribution, projection, and gene expression in deep brain tissue. AICI is applicable beyond the imaging of fluorescent antibodies and dyes, and can clear a variety of tissue types, making it broadly useful to researchers for optical imaging applications.

Distinct Firing Activities of the Hypothalamic Arcuate Nucleus Neurons to Appetite Hormones.

The hypothalamic arcuate nucleus (Arc) is a central unit that controls the appetite through the integration of metabolic, hormonal, and neuronal afferent inputs. Agouti-related protein (AgRP), proopiomelanocortin (POMC), and dopaminergic neurons in the Arc differentially regulate feeding behaviors in response to hunger, satiety, and appetite, respectively. At the time of writing, the anatomical and electrophysiological characterization of these three neurons has not yet been intensively explored. Here, we interrogated the overall characterization of AgRP, POMC, and dopaminergic neurons using genetic mouse models, immunohistochemistry, and whole-cell patch recordings. We identified the distinct geographical location and intrinsic properties of each neuron in the Arc with the transgenic lines labelled with cell-specific reporter proteins. Moreover, AgRP, POMC, and dopaminergic neurons had different firing activities to ghrelin and leptin treatments. Ghrelin led to the increased firing rate of dopaminergic and AgRP neurons, and the decreased firing rate of POMC. In sharp contrast, leptin resulted in the decreased firing rate of AgRP neurons and the increased firing rate of POMC neurons, while it did not change the firing rate of dopaminergic neurons in Arc. These findings demonstrate the anatomical and physiological uniqueness of three hypothalamic Arc neurons to appetite control.

Modeling and Experiment for the Diffusion Coefficient of Subcritical Carbon Dioxide in Poly(methyl methacrylate) to Predict Gas Sorption and Desorption.

Several researchers have investigated the phenomenon of polymer-gas mixtures, and a few have proposed diffusion coefficient values instead of a diffusion coefficient model. There is a paucity of studies focused on the continuous change in the diffusion coefficient corresponding to the overall pressure and temperature range of the mixture. In this study, the gas sorption and desorption experiments of poly(methyl methacrylate) (PMMA) were conducted via solid-state batch foaming, and the weight change was measured using the gravimetric method with a magnetic balance. The control parameters were temperature, which ranged from 290 to 370 K, and pressure, which ranged from 2 to 5 MPa in the subcritical regime. Based on the experimental data, the diffusion coefficient of the PMMA was calculated using Fick's law. After calculating the diffusion coefficient in the range of the experiment, the diffusion coefficient model was employed using the least-squares method. Subsequently, the model was validated by comparing the obtained results with those in the literature, and the overall trend was found to be consistent. Therefore, it was confirmed that there were slight differences between the diffusion coefficient obtained using only Fick's equation and the value using by a different method.

Predictive Factors for Skip Lymph Node Metastasis and Their Implication on Recurrence in Papillary Thyroid Carcinoma.

Skip lymph node (LN) metastases in papillary thyroid carcinoma (PTC) belong to N1b classification in the absence of central neck LN involvement. This study aimed to evaluate the predictive factors of skip metastases and their impact on recurrence in PTC patients with pN1b. A total of 334 PTC patients who underwent total thyroidectomy with LN dissection (central and lateral neck compartment) followed by radioactive iodine ablation were included. Patients with skip metastases tended to have a small primary tumor (≤1 cm) and single lateral neck level involvement. Tumor size ≤ 1 cm was an important predictive factor for skip metastases. Univariate analysis for recurrence showed that patients with a central LN ratio > 0.68, lateral LN ratio > 0.21, and stimulated thyroglobulin (Tg) levels > 7.3 ng/mL had shorter RFS (recurrence-free survival). The stimulated Tg level was associated with shorter RFS on multivariate analysis (>7.3 vs. ≤7.3 ng/mL; hazard ratio, 4.226; 95% confidence interval, 2.226-8.022; p < 0.001). Although patients with skip metastases tended to have a small primary tumor and lower burden of lateral neck LN involvement, there was no association between skip metastases and RFS in PTC with pN1b. Stimulated Tg level was a strong predictor of recurrence.

Vibration and Sound Response of Glass-Fiber-Reinforced Polyamide 6 Using Microcellular-Foaming-Process-Applied Injection Molding Process.

In this study, the vibration and sound response characteristics of composites produced via injection molding applied with a microcellular foaming process (MCPs) were improved. The study was conducted using PA6 and glass fiber composites, which are representative thermoplastic engineering plastics. Two types of specimens were used: a plate specimen to confirm the basic sound and vibration characteristics, and a large roof-rack specimen from an actual vehicle with a complex shape. The frequency response function curve was calculated by conducting an impact test, and natural frequency and damping ratio were measured based on the curve. The results confirmed that, in the case of a specimen manufactured through the injection molding process to which MCPs were applied, the natural frequency was lowered, and the damping ratio decreased. The degree of change in the natural frequency and damping ratio was confirmed. To determine the cause of the change in the natural frequency and damping ratio, the mode shape at the natural frequency of each specimen was measured and the relationship was confirmed by measuring the density and the elastic modulus of the composite. In addition, the usability of the specimens to which MCPs were applied was verified by conducting impact strength and tensile strength tests.

MON-2, a Golgi protein, mediates autophagy-dependent longevity in Caenorhabditis elegans.

The Golgi apparatus plays a central role in trafficking cargoes such as proteins and lipids. Defects in the Golgi apparatus lead to various diseases, but its role in organismal longevity is largely unknown. Using a quantitative proteomic approach, we found that a Golgi protein, MON-2, was up-regulated in long-lived Caenorhabditis elegans mutants with mitochondrial respiration defects and was required for their longevity. Similarly, we showed that DOP1/PAD-1, which acts with MON-2 to traffic macromolecules between the Golgi and endosome, contributed to the longevity of respiration mutants. Furthermore, we demonstrated that MON-2 was required for up-regulation of autophagy, a longevity-associated recycling process, by activating the Atg8 ortholog GABARAP/LGG-1 in C. elegans. Consistently, we showed that mammalian MON2 activated GABARAPL2 through physical interaction, which increased autophagic flux in mammalian cells. Thus, the evolutionarily conserved role of MON2 in trafficking between the Golgi and endosome is an integral part of autophagy-mediated longevity.

Effect of Aluminum Flakes on Mechanical and Optical Properties of Foam Injection Molded Parts.

Injection research using aluminum flakes has been conducted to realize metallic textures on the surface of plastic products. Several studies have focused on the effect of the orientation and quality of the flakes when using conventional injection molding methods; however, limited studies have focused on the foam injection molding method. In this study, we examined the orientation of aluminum flakes through foam injection with an inert gas and observed the changes in texture using a spectrophotometer and a gloss meter. The mechanical properties were also studied because the rigidity of the product, which is affected by the weight reduction that occurs during foaming, is an important factor. The results demonstrate that under foam injection molding, reflectance and gloss increased by 6% and 7 GU, respectively, compared to those obtained using conventional injection molding; furthermore, impact strength and flexural modulus increased by 62% and 15%, respectively. The results of this research can be applied to incorporate esthetic improvements to products and to develop functional parts.

A Shift in Tissue Stiffness During Hippocampal Maturation Correlates to the Pattern of Neurogenesis and Composition of the Extracellular Matrix.

Aging changes the mechanical properties of brain tissue, such as stiffness. It has been proposed that the maintenance and differentiation of neural stem cells (NSCs) are regulated in accordance with extracellular stiffness. Neurogenesis is observed in restricted niches, including the dentate gyrus (DG) of the hippocampus, throughout mammalian lifetimes. However, profiles of tissue stiffness in the DG in comparison with the activity of NSCs from the neonatal to the matured brain have rarely been addressed so far. Here, we first applied ultrasound-based shear-wave elasticity imaging (SWEI) in living animals to assess shear modulus as in vivo brain stiffness. To complement the assay, atomic force microscopy (AFM) was utilized to determine the Young's modulus in the hippocampus as region-specific stiffness in the brain slice. The results revealed that stiffness in the granule cell layer (GCL) and the hilus, including the subgranular zone (SGZ), increased during hippocampal maturation. We then quantified NSCs and immature neural cells in the DG with differentiation markers, and verified an overall decrease of NSCs and proliferative/immature neural cells along stages, showing that a specific profile is dependent on the subregion. Subsequently, we evaluated the amount of chondroitin sulfate proteoglycans (CSPGs), the major extracellular matrix (ECM) components in the premature brain by CS-56 immunoreactivity. We observed differential signal levels of CSPGs by hippocampal subregions, which became weaker during maturation. To address the contribution of the ECM in determining tissue stiffness, we manipulated the function of CSPGs by enzymatic digestion or supplementation with chondroitin sulfate, which resulted in an increase or decrease of stiffness in the DG, respectively. Our results illustrate that stiffness in the hippocampus shifts due to the composition of ECM, which may affect postnatal neurogenesis by altering the mechanical environment of the NSC niche.

Mechanical Properties of Biocomposites Using Polypropylene and Sesame Oil Cake.

Sesame oil cakes (SOC) produced during sesame oil production can be classified as plant residues. This study aims to use SOC as a composite material for injection molding. A biocomposite containing polypropylene (PP) and SOC, namely PP/SOC, was developed and its mechanical properties were evaluated. PP/SOC is largely divided into Homo-PP/SOC (HPS) based on Homo-PP and Block-PP/SOC (BPS) based on block-PP. The specimens containing 0-50 wt% SOC were prepared through extrusion and injection molding. As a result of the evaluation, SOC acted as a reinforcement in the matrix, and HPS and BPS showed improved flexural modulus by 36.4% and 37.3% compared to the neat PP, respectively. Tensile strength, on the other hand, decreased by 58% and 55.1%, respectively. To analyze the cause of this, cross-section observation was conducted through scanning electron microscope (SEM), and phase separation and voids were confirmed to be the cause of this. Impact strength of PP/SOC tended to vary depending on the type of matrix. HPS increased by 30.9% compared to neat PP, and BPS decreased by 25%. This tendency difference appears to be the result of SOC inhibiting crystallization of PP, and it has been confirmed through x ray diffraction (XRD) and differential scanning calorimetry (DSC) analysis. Moreover, PP/SOC can be manufactured at a low cost and is environmentally friendly because it utilizes SOC, a plant residue. It can also be applied to commercial products, such as food packaging, owing to its good moldability and improved mechanical properties.

Characteristics of Microcellular Foamed Ceramic Urethane.

Ceramics are non-metallic inorganic materials fabricated from natural or high-purity raw materials through heating and cooling processes. Urethane is a three-dimensional plastic with both elasticity and chemical resistance; moreover, it is used as a rubber substitute. The use of both materials in various applications is gradually increasing. However, as ceramics and urethane have distinctly different properties, this prompted questions regarding the properties of a material that is fabricated using both materials. Therefore, we studied the characteristics of a composite material fabricated through physical foaming using a batch process. The process was conducted with gas saturation, foaming, cooling, and curing. When a specimen of 2 mm thickness was saturated in 5 MPa of CO2 for 2 h, the solubility was 6.43%; when foaming was carried out at a temperature of 150 °C in boiled glycerin, the foaming ratio, cell size, cell density, and void fraction were found to be 43.62%, 24.40 µm, 9.1 × 107 cells/cm2, and 22.11%, respectively. Furthermore, the volume increased by 102.96%, color changed from dark to light gray, hardness decreased by 24%, thermal diffusivity increased by 0.046 mm2/s at 175 °C, and friction coefficient decreased to 0.203. Thus, the microcellular foamed ceramic urethane exhibits a larger volume, lighter weight, and improved thermal conductivity and friction coefficient.

Early escitalopram administration as a preemptive treatment strategy against spasticity after contusive spinal cord injury in rats.

In the majority of spinal cord injury (SCI) patients, spasticity develops in the subacute phase and chronically persists with muscle hypertonia. Among various pathological conditions underlying spasticity, upregulated expression of 5-HT receptors (5-HTR) on the spinal motor neurons due to 5-HT denervation is considered one of crucial factors for hyperexcitability of the spinal circuit. As a 5-HT signal modulator, selective serotonin re-uptake inhibitors (SSRIs) are ordinarily prescribed for diseases associated with 5-HT in the CNS, and are known for their ability to increase 5-HT levels as well as to desensitize 5-HTR. Here, we hypothesized that early SSRI administration as a preemptive treatment strategy would effectively prevent the onset of spasticity. We used a rat model of contusive SCI and administered escitalopram during the first 4 weeks after injury, which is the period required for spasticity development in rodent models. We performed a swimming test to quantify spastic behaviors and conducted the Hoffman reflex test as well as histological analyses for 5-HT2AR and KCC2 expressions. Four weeks of escitalopram administration suppressed spastic behaviors during the swimming test and reduced the population of spasticity-strong rats. Moreover, the treatment resulted in decreased immunoreactivity of 5-HT2AR in the spinal motor neurons. Result of the H-reflex test and membrane expression of KCC2 were not significantly altered. In summary, early escitalopram administration could prevent the onset of spastic behaviors via regulation of 5-HT system after SCI, but could not modulate exaggerated spinal reflex. Our results suggest a novel application of SSRIs for preventative treatment of spasticity.

Consecutive Low Doses of Streptozotocin Induce Polycystic Ovary Syndrome Features in Mice.

Polycystic ovarian syndrome (PCOS) is a common reproductive endocrine disorder in reproductive-age women. Due to its various pathophysiological properties and clinical heterophenotypes, the mechanism of PCOS pathogenesis is still unclear. Several animal models have been used to study PCOS and allow the exploration of the specific mechanism underlying PCOS. We focused on streptozotocin (STZ) to develop a non-steroidal and non-diabetic PCOS model. We administered multiple STZ injections to female C57BL/6 mice (3-4 weeks old) at different concentrations: STZ-15 (15 mg/kg), STZ-30 (30 mg/kg), and STZ-60 (60 mg/kg) treatments. During the experimental period, we analyzed body weight, blood glucose levels, and estrous cycle pattern. Furthermore, five weeks after STZ administration, we examined hormone levels and the morphology of ovarian tissues. Mice in the STZ-15 group did not show differences in body weights, blood glucose level, insulin level, and insulin tolerance compared to wild-type and control groups whereas those in the STZ-60 group presented a typical diabetes phenotype. In the case of the STZ-30 group, only increased blood glucose level was observed. Total testosterone levels were significantly elevated in STZ-15 and STZ-30 groups. Luteinizing hormone (LH) and estradiol levels were not significantly changed in the STZ-treated groups. The number of ovarian antral follicles and atretic follicles significantly increased in the ovary of mice in the STZ-15 and STZ-30 groups. All STZ-treated groups manifested irregular estrus cycles. However, the patterns of estrous cycles were different between mice treated with different STZ concentrations. We found that PI3K-AKT and IRS-1 signaling in the ovary was enhanced by low doses of STZ treatment. Taken together, our finding indicates that multiple injections of STZ at low doses induce PCOS features in mice without induction of diabetes features.

Shrinkage and Warpage Minimization of Glass-Fiber-Reinforced Polyamide 6 Parts by Microcellular Foam Injection Molding.

Shrinkage and warpage of injection-molded parts can be minimized by applying microcellular foaming technology to the injection molding process. However, unlike the conventional injection molding process, the optimal conditions of the microcellular foam injection molding process are elusive because of core differences such as gas injection. Therefore, this study aims to derive process conditions to minimize the shrinkage and warpage of microcellular foam injection-molded parts made of glass fiber reinforced polyamide 6 (PA6/GF). Process factors and levels were first determined, with experiments planned accordingly. We simulated designed experiments using injection molding analysis software, and the results were analyzed using the Taguchi method, analysis of variance (ANOVA), and response surface methodology (RSM), with the ANOVA analysis being ultimately demonstrating the influence of the factors. We derived and verified the optimal combination of process factors and levels for minimizing both shrinkage and warpage using the Taguchi method and RSM. In addition, the mechanical properties and cell morphology of PA6/GF, which change with microcellular foam injection molding, were confirmed.

Mechanical Regulation Underlies Effects of Exercise on Serotonin-Induced Signaling in the Prefrontal Cortex Neurons.

Mechanical forces are known to be involved in various biological processes. However, it remains unclear whether brain functions are mechanically regulated under physiological conditions. Here, we demonstrate that treadmill running and passive head motion (PHM), both of which produce mechanical impact on the head, have similar effects on the hallucinogenic 5-hydroxytryptamine (5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) of rodents. PHM generates interstitial fluid movement that is estimated to exert shear stress of a few pascals on cells in the PFC. Fluid shear stress of a relevant magnitude on cultured neuronal cells induces ligand-independent internalization of 5-HT2A receptor, which is observed in mouse PFC neurons after treadmill running or PHM. Furthermore, inhibition of interstitial fluid movement by introducing polyethylene glycol hydrogel eliminates the effect of PHM on 5-HT2A receptor signaling in the PFC. Our findings indicate that neuronal cell function can be physiologically regulated by mechanical forces in the brain.

Reflectance According to Cell Size, Foaming Ratio and Refractive Index of Microcellular Foamed Amorphous Polymer.

Microcellular foamed plastic has a cell size of approximately 0.1 to 10 microns inside a foamed polymer and a cell density in the range of 109 to 1015 cells/cm3. Typically, the formation of numerous uniform cells inside a polymer can be effectively used for various purposes, such as lightweight materials, insulation and sound absorbing materials. However, it has recently been reported that these dense cell structures, which are induced through microcellular foaming, can affect the light passing through the medium, which affects the haze and permeability and causes the diffused reflection of light to achieve high diffuse reflectivity. In this study, the effects of cell size, foaming ratio and refractive index on the optical performance were investigated by applying the microcellular foaming process to three types of amorphous polymer materials. Thus, this study experimentally confirmed that the advantages of porous materials can be implemented as optical properties by providing a high specific surface area as a small and uniform cell formed by inducing a high foaming ratio through a microcellular foaming process.

MDT-15/MED15 permits longevity at low temperature via enhancing lipidostasis and proteostasis.

Low temperatures delay aging and promote longevity in many organisms. However, the metabolic and homeostatic aspects of low-temperature-induced longevity remain poorly understood. Here, we show that lipid homeostasis regulated by Caenorhabditis elegans Mediator 15 (MDT-15 or MED15), a transcriptional coregulator, is essential for low-temperature-induced longevity and proteostasis. We find that inhibition of mdt-15 prevents animals from living long at low temperatures. We show that MDT-15 up-regulates fat-7, a fatty acid desaturase that converts saturated fatty acids (SFAs) to unsaturated fatty acids (UFAs), at low temperatures. We then demonstrate that maintaining a high UFA/SFA ratio is essential for proteostasis at low temperatures. We show that dietary supplementation with a monounsaturated fatty acid, oleic acid (OA), substantially mitigates the short life span and proteotoxicity in mdt-15(-) animals at low temperatures. Thus, lipidostasis regulated by MDT-15 appears to be a limiting factor for proteostasis and longevity at low temperatures. Our findings highlight the crucial roles of lipid regulation in maintaining normal organismal physiology under different environmental conditions.

Animal Models for Human Polycystic Ovary Syndrome (PCOS) Focused on the Use of Indirect Hormonal Perturbations: A Review of the Literature.

Hormonal disturbances, such as hyperandrogenism, are considered important for developing polycystic ovary syndrome (PCOS) in humans. Accordingly, directly hormone-regulated animal models are widely used for studying PCOS, as they replicate several key PCOS features. However, the pathogenesis and treatment of PCOS are still unclear. In this review, we aimed to investigate animal PCOS models and PCOS-like phenotypes in animal experiments without direct hormonal interventions and determine the underlying mechanisms for a better understanding of PCOS. We summarized animal PCOS models that used indirect hormonal interventions and suggested or discussed pathogenesis of PCOS-like features in animals and PCOS-like phenotypes generated in other animals. We presented integrated physiological insights and shared cellular pathways underlying the pathogenesis of PCOS in reviewed animal models. Our review indicates that the hormonal and metabolic changes could be due to molecular dysregulations, such as upregulated PI3K-Akt and extracellular signal-regulated kinase (ERK) signalling, that potentially cause PCOS-like phenotypes in the animal models. This review will be helpful for considering alternative animal PCOS models to determine the cellular/molecular mechanisms underlying PCOS symptoms. The efforts to determine the specific cellular mechanisms of PCOS will contribute to novel treatments and control methods for this complex syndrome.