3D Bioprinted Liver-on-a-Chip for Drug Cytotoxicity Screening.

Tissues on a chip are sophisticated three-dimensional (3D) in vitro microphysiological systems designed to replicate human tissue conditions within dynamic physicochemical environments. However, the current fabrication methods for tissue spheroids on a chip require multiple parts and manual processing steps, including the deposition of spheroids onto prefabricated "chips." These challenges also lead to limitations regarding scalability and reproducibility. To overcome these challenges, we employed 3D printing techniques to automate the fabrication process of tissue spheroids on a chip. This allowed the simultaneous high-throughput printing of human liver spheroids and their surrounding polymeric flow chamber "chips" containing inner channels in a single step. The fabricated liver tissue spheroids on a liver-on-a-chip (LOC) were subsequently subjected to dynamic culturing by a peristaltic pump, enabling assessment of cell viability and metabolic activities. The 3D printed liver spheroids within the printed chips demonstrated high cell viability (>80%), increased spheroid size, and consistent adenosine triphosphate (ATP) activity and albumin production for up to 14 days. Furthermore, we conducted a study on the effects of acetaminophen (APAP), a nonsteroidal anti-inflammatory drug, on the LOC. Comparative analysis revealed a substantial decline in cell viability (<40%), diminished ATP activity, and reduced spheroid size after 7 days of culture within the APAP-treated LOC group, compared to the nontreated groups. These results underscore the potential of 3D bioprinted tissue chips as an advanced in vitro model that holds promise for accurately studying in vivo biological processes, including the assessment of tissue response to administered drugs, in a high-throughput manner.

Placenta mesenchymal stem cell-derived extracellular vesicles alleviate liver fibrosis by inactivating hepatic stellate cells through a miR-378c/SKP2 axis.

BACKGROUND: Extracellular vesicles derived from mesenchymal stem/stromal cells (MSCs) have shown therapeutic effects on liver fibrosis. This study aimed to evaluate the effects of extracellular vesicles from placenta-derived MSCs (Pd-MSCs-EVs) on liver fibrosis at 3D/2D levels and explore the potential mechanisms. METHODS: The multicellular liver organoids, consisting of hepatocytes, hepatic stellate cells (HSCs), Kupffer cells, and liver sinusoidal endothelial cells, were observed for growth status, morphological changes, and metabolism. Human transformation growth factor- beta 1 (TGF-ß1) was used to induce fibrosis at optimal concentration. The anti-fibrosis effects of Pd-MSCs-EVs were evaluated in liver organoids and HSCs models. Anti-fibrotic content of Pd-MSCs-EVs was identified by multiple experimental validations. RESULTS: TGF-ß1 induced fibrosis in liver organoids, while Pd-MSCs-EVs significantly alleviated fibrotic phenotypes. Following serial verifications, miR-378c was identified as a potential key anti-fibrosis content. In contrast, miR-378c depletion decreased the anti-fibrotic effects of Pd-MSCs-EVs. Additionally, Pd-MSCs-EVs administration repressed TGF-ß1-mediated HSCs activation at 2D or 3D levels. Mechanistically, exosomal miR-378c inactivated HSCs by inhibiting epithelial-mesenchymal transition (EMT) through stabilizing E-cadherin via targeting its E3 ubiquitin ligase S-Phase Kinase Associated Protein 2 (SKP2). CONCLUSION: Pd-MSCs-EVs ameliorated TGF-ß1-induced fibrosis by deactivating HSCs in a miR-378c/SKP2-dependent manner, which may be an efficient therapeutic candidate for liver fibrosis.

Probing prodrug metabolism and reciprocal toxicity with an integrated and humanized multi-tissue organ-on-a-chip platform.

Current drug development techniques are expensive and inefficient, partially due to the use of preclinical models that do not accurately recapitulate in vivo drug efficacy and cytotoxicity. To address this challenge, we report on an integrated, in vitro multi-organoid system that enables parallel assessment of drug efficiency and toxicity on multiple 3D tissue organoids. Built in a low-cost, adhesive film-based microfluidic device, these miniaturized structures require less than 200 µL fluid volume and are amenable to both matrix-based 3D cell culture and spheroid aggregate integration, each supported with an in situ photocrosslinkable hyaluronic acid hydrogel. Here, we demonstrate this technology first with a three-organoid device consisting of liver, cardiac, and lung constructs. We show that these multiple tissue types can be kept in common circulation with high viability for 21 days and validate the platform by investigating liver metabolism of the prodrug capecitabine into 5-fluorouracil (5-FU) and observing downstream toxicity in lung and cardiac organoids. Then we expand the integrated system to accommodate six humanized constructs, including liver, cardiac, lung, endothelium, brain, and testes organoids. Following a 14-day incubation in common media, we demonstrate multi-tissue interactions by metabolizing the alkylating prodrug ifosfamide in the liver organoid to produce chloroacetaldehyde and induce downstream neurotoxicity. Our results establish an expandable, multi-organoid body-on-a-chip system that can be fabricated easily and used for the accurate characterization of drug interactions in vitro. STATEMENT OF SIGNIFICANCE: The use of 3-dimensional (3D) in vitro models in drug development has advanced over the past decade. However, with several exceptions, the majority of research studies using 3D in vitro models, such as organoids, employ single tissue types, in isolated environments with no "communication" between different tissues. This is a significant limiting factor because in the human body there is significant signaling between different cells, tissues, and organs. Here we employ a low-cost, adhesive film-based microfluidic device approach, paired with a versatile extracellular matrix-derived hyaluronic acid hydrogel to support integrated systems of 3 and 6 3D organoid and cell constructs. Moreover, we demonstrate an integrated response to drugs, in which downstream toxicity is dependent on the presence of liver organoids.

Therapeutic effects of a small molecule agonist of the relaxin receptor ML290 in liver fibrosis.

Fibrosis is an underlying cause of cirrhosis and hepatic failure resulting in end stage liver disease with limited pharmacological options. The beneficial effects of relaxin peptide treatment were demonstrated in clinically relevant animal models of liver fibrosis. However, the use of relaxin is problematic because of a short half-life. The aim of this study was to test the therapeutic effects of recently identified small molecule agonists of the human relaxin receptor, relaxin family peptide receptor 1 (RXFP1). The lead compound of this series, ML290, was selected based on its effects on the expression of fibrosis-related genes in primary human stellate cells. RNA sequencing analysis of TGF-ß1-activated LX-2 cells showed that ML290 treatment primarily affected extracellular matrix remodeling and cytokine signaling, with expression profiles indicating an antifibrotic effect of ML290. ML290 treatment in human liver organoids with LPS-induced fibrotic phenotype resulted in a significant reduction of type I collagen. The pharmacokinetics of ML290 in mice demonstrated its high stability in vivo, as evidenced by the sustained concentrations of compound in the liver. In mice expressing human RXFP1 gene treated with carbon tetrachloride, ML290 significantly reduced collagen content, α-smooth muscle actin expression, and cell proliferation around portal ducts. In conclusion, ML290 demonstrated antifibrotic effects in liver fibrosis.-Kaftanovskaya, E. M., Ng, H. H., Soula, M., Rivas, B., Myhr, C., Ho, B. A., Cervantes, B. A., Shupe, T. D., Devarasetty, M., Hu, X., Xu, X., Patnaik, S., Wilson, K. J., Barnaeva, E., Ferrer, M., Southall, N. T., Marugan, J. J., Bishop, C. E., Agoulnik, I. U., Agoulnik, A. I. Therapeutic effects of a small molecule agonist of the relaxin receptor ML290 in liver fibrosis.

Effects of Extracellular Vesicles Derived from Mesenchymal Stem/Stromal Cells on Liver Diseases.

Therapeutic effects of Mesenchymal Stem/Stromal Cells (MSCs) transplantation have been observed in various disease models. However, it is thought that MSCs-mediated effects largely depend on the paracrine manner of secreting cytokines, growth factors, and Extracellular Vesicles (EVs). Similarly, MSCs-derived EVs also showed therapeutic benefits in various liver diseases through alleviating fibrosis, improving regeneration of hepatocytes, and regulating immune activity. This review provides an overview of the MSCs, their EVs, and their therapeutic potential in treating various liver diseases including liver fibrosis, acute and chronic liver injury, and Hepatocellular Carcinoma (HCC). More specifically, the mechanisms by which MSC-EVs induce therapeutic benefits in liver diseases will be covered. In addition, comparisons between MSCs and their EVs were also evaluated as regenerative medicine against liver diseases. While the mechanisms of action and clinical efficacy must continue to be evaluated and verified, MSCs-derived EVs currently show tremendous potential and promise as a regenerative medicine treatment for liver disease in the future.

miR-122 inhibition in a human liver organoid model leads to liver inflammation, necrosis, steatofibrosis and dysregulated insulin signaling.

To investigate the role of miR-122 in the development and regression of non-alcoholic fatty liver disease (NAFLD) in vitro, we used multicellular 3D human liver organoids developed in our laboratory. These organoids consist of primary human hepatocytes, Kupffer cells, quiescent stellate cells and liver sinusoidal endothelial cells. They remain viable and functional for 4 weeks expressing typical markers of liver function such as synthesis of albumin, urea, and alpha-1 p450 drug metabolism. Before mixing, hepatic cells were transduced with lentivirus to inhibit miR122 expression (ABM, CA). Immediately after the organoids were fully formed (day 4) or after 1 or 2 weeks of additional incubation (days 11 or 18), the organoids were analyzed using fluorescent live/dead staining and ATP production; total RNA was extracted for qPCR gene expression profiling. Our results show that miR-122 inhibition in liver organoids leads to inflammation, necrosis, steatosis and fibrosis. This was associated with increase in inflammatory cytokines (IL6, TNF), chemokines (CCL2, CCL3) and increase in a subset of Matrix Metaloproteinases (MMP8, MMP9). An altered expression of key genes in lipid metabolism (i.e LPL, LDLR) and insulin signaling (i.e GLUT4, IRS1) was also identified. CONCLUSION: Our results highlight the role of miR-122 inhibition in liver inflammation, steatofibrosis and dysregulation of insulin signaling. Patients with NAFLD are known to have altered levels of miR-122, therefore we suggest that miR-122 mimics could play a useful role in reversing liver steatofibrosis and insulin resistance seen in patients with NAFLD.

MEX3C interacts with adaptor-related protein complex 2 and involves in miR-451a exosomal sorting.

Some RNA species, especially microRNAs, are non-randomly sorted into exosomes, but how selectivity of RNA exosomal sorting is achieved is unknown. We found that all three variants of RNA-binding ubiquitin E3 ligase (MEX3C)-MEX3C-1, MEX3C-2, and MEX3C-3 -interact with adaptor-related protein complex 2 (AP-2), a cargo adaptor in clathrin-mediated endocytosis. MEX3C's C-terminal RING finger domain and the hnRNP K homology (KH) domain shared by the three MEX3C variants are both necessary for MEX3C/AP-2 interaction. MEX3C associates with the endolysosomal compartment through an endocytosis-like process. siRNA-mediated inhibition of the MEX3C or AP-2 complex substantially decreased exosomal but not cellular microRNA miR-451a expression. Exosomal sorting is ceramide-dependent but not ESCRT-dependent in microRNA miR-451a. That RNA-binding protein associates with membrane trafficking machinery, and that its involvement in exosomal microRNA expression, suggest the existence of a mechanism for specific recruiting of RNA molecules to endosomes for subsequent exosomal sorting.

Multi-tissue interactions in an integrated three-tissue organ-on-a-chip platform.

Many drugs have progressed through preclinical and clinical trials and have been available - for years in some cases - before being recalled by the FDA for unanticipated toxicity in humans. One reason for such poor translation from drug candidate to successful use is a lack of model systems that accurately recapitulate normal tissue function of human organs and their response to drug compounds. Moreover, tissues in the body do not exist in isolation, but reside in a highly integrated and dynamically interactive environment, in which actions in one tissue can affect other downstream tissues. Few engineered model systems, including the growing variety of organoid and organ-on-a-chip platforms, have so far reflected the interactive nature of the human body. To address this challenge, we have developed an assortment of bioengineered tissue organoids and tissue constructs that are integrated in a closed circulatory perfusion system, facilitating inter-organ responses. We describe a three-tissue organ-on-a-chip system, comprised of liver, heart, and lung, and highlight examples of inter-organ responses to drug administration. We observe drug responses that depend on inter-tissue interaction, illustrating the value of multiple tissue integration for in vitro study of both the efficacy of and side effects associated with candidate drugs.

Optical Tracking and Digital Quantification of Beating Behavior in Bioengineered Human Cardiac Organoids.

Organoid and organ-on-a-chip technologies are rapidly advancing towards deployment for drug and toxicology screening applications. Liver and cardiac toxicities account for the majority of drug candidate failures in human trials. Liver toxicity generally produces liver cell death, while cardiac toxicity causes adverse changes in heart beat kinetics. In traditional 2D cultures, beating kinetics can be measured by electrode arrays, but in some 3D constructs, quantifying beating kinetics can be more challenging. For example, real time measurements of calcium flux or contractile forces are possible, yet rather complex. In this communication article, we demonstrate a simple sensing system based on software code that optically analyzes video capture files of beating cardiac organoids, translates these files in representations of moving pixels, and quantifies pixel movement activity over time to generate beat kinetic plots. We demonstrate this system using bioengineered cardiac organoids under baseline and drug conditions. This technology offers a non-invasive, low-cost, and incredibly simple method for tracking and quantifying beating behavior in cardiac organoids and organ-on-a-chip systems for drug and toxicology screening.

Multisensor-integrated organs-on-chips platform for automated and continual in situ monitoring of organoid behaviors.

Organ-on-a-chip systems are miniaturized microfluidic 3D human tissue and organ models designed to recapitulate the important biological and physiological parameters of their in vivo counterparts. They have recently emerged as a viable platform for personalized medicine and drug screening. These in vitro models, featuring biomimetic compositions, architectures, and functions, are expected to replace the conventional planar, static cell cultures and bridge the gap between the currently used preclinical animal models and the human body. Multiple organoid models may be further connected together through the microfluidics in a similar manner in which they are arranged in vivo, providing the capability to analyze multiorgan interactions. Although a wide variety of human organ-on-a-chip models have been created, there are limited efforts on the integration of multisensor systems. However, in situ continual measuring is critical in precise assessment of the microenvironment parameters and the dynamic responses of the organs to pharmaceutical compounds over extended periods of time. In addition, automated and noninvasive capability is strongly desired for long-term monitoring. Here, we report a fully integrated modular physical, biochemical, and optical sensing platform through a fluidics-routing breadboard, which operates organ-on-a-chip units in a continual, dynamic, and automated manner. We believe that this platform technology has paved a potential avenue to promote the performance of current organ-on-a-chip models in drug screening by integrating a multitude of real-time sensors to achieve automated in situ monitoring of biophysical and biochemical parameters.

Three-dimensional testicular organoid: a novel tool for the study of human spermatogenesis and gonadotoxicity in vitro.

Existing methods for evaluating the potential gonadotoxicity of environmental agents and pharmaceutical compounds rely heavily on animal studies. The current gold standard in vivo functional assays in animals are limited in their human predictive capacity. In addition, existing human two-dimensional in vitro models of testicular toxicity do not accurately reflect the in vivo situation. A more reliable testicular in vitro model system is needed to better assess the gonadotoxic potential of drugs prior to progression into clinical trials. The overall goal of this study was to develop a three-dimensional (3D) in vitro human testis organoid culture system for use as both a predictive first tier drug-screening tool and as a model of human testicular function. Multicellular human testicular organoids composed of Spermatogonial Stem Cells, Sertoli, Leydig and peritubular cells were created and evaluated over time for morphology, viability, androgen production and ability to support germ cell differentiation. Enzyme-linked immunosorbent assay measurements confirmed that the organoids produced testosterone continuously with and without hCG stimulation. Upregulation of postmeiotic genes including PRM1 and Acrosin, detected by quantitative-PCR, digital PCR and Immunofluorescence, indicated the transition of a small percentage of diploid to haploid germ cells. As a novel screening tool for reproductive toxicity, 3D organoids were exposed to four chemotherapeutic drugs, and they responded in a dose-dependent manner and maintained IC50 values significantly higher than 2D cultures. This 3D human testis organoid system has the potential to be used as a novel testicular toxicity-screening tool and in vitro model for human spermatogenesis.

A liver-on-a-chip platform with bioprinted hepatic spheroids.

The inadequacy of animal models in correctly predicting drug and biothreat agent toxicity in humans has resulted in a pressing need for in vitro models that can recreate the in vivo scenario. One of the most important organs in the assessment of drug toxicity is liver. Here, we report the development of a liver-on-a-chip platform for long-term culture of three-dimensional (3D) human HepG2/C3A spheroids for drug toxicity assessment. The bioreactor design allowed for in situ monitoring of the culture environment by enabling direct access to the hepatic construct during the experiment without compromising the platform operation. The engineered bioreactor could be interfaced with a bioprinter to fabricate 3D hepatic constructs of spheroids encapsulated within photocrosslinkable gelatin methacryloyl (GelMA) hydrogel. The engineered hepatic construct remained functional during the 30 days culture period as assessed by monitoring the secretion rates of albumin, alpha-1 antitrypsin, transferrin, and ceruloplasmin, as well as immunostaining for the hepatocyte markers, cytokeratin 18, MRP2 bile canalicular protein and tight junction protein ZO-1. Treatment with 15 mM acetaminophen induced a toxic response in the hepatic construct that was similar to published studies on animal and other in vitro models, thus providing a proof-of-concept demonstration of the utility of this liver-on-a-chip platform for toxicity assessment.

Expression of TAT recombinant Oct4, Sox2, Lin28, and Nanog proteins from baculovirus-infected Sf9 insect cells.

Somatic cell reprogramming has generated enormous interest, following the first report of generation of induced pluripotent stem cells (iPSCs) from mouse fibroblasts, but the integration of viral transgenes into the genome is unlikely to be accepted. Given these safety considerations, a method for virus-free transient gene expression from suspension-adapted Sf9 insect cells was developed. Here, we expressed transactivator of transcription (TAT)-fused proteins, Sox2, Oct4, Lin28, and Nanog in Sf9 cells using the baculovirus expression vector system (BEVS). The molecular weights of the TAT-Sox2, TAT-Oct4, TAT-Lin28, and TAT-Nanog fusion proteins were 36kD, 40kD, 24kD, and 36kD, respectively. Further investigation indicated that most of the recombinant proteins remained in the nuclei of the Sf9 insect cells and were therefore unavailable for purification and cellular reprogramming. Once this problem has been solved, it seems likely that protein expressed from baculovirus-infected Sf9 insect cells will be the method of choice for cellular reprogramming.

TALEN-mediated modification of the bovine genome for large-scale production of human serum albumin.

As an initial step towards creating genetically modified cattle as a biopharming source of recombinant human serum albumin (rHSA), we report modification of the bovine albumin (bA) locus by transcription activator-like effector nuclease (TALEN)-stimulated homology-directed repair (HDR). Pedigreed bovine fibroblasts were co-transfected with TALENs and an 11.5-kb human serum albumin (HSA) minigene donor construct, designed to simultaneously disrupt and replace bovine serum albumin (BSA) expression with controlled rHSA expression in both the liver and the milk. Targeted integration of the HSA minigene was confirmed in transfected fibroblasts at a frequency of approximately 11% and transgenic bovine embryos were produced from targeted fibroblasts using somatic cell nuclear transfer (SCNT). The research delineated here lays the foundation for the future generation of transgenic rHSA cattle with the potential to provide a large-scale, reliable, and quality-controlled source of rHSA.

A transgenic insertion on mouse chromosome 17 inactivates a novel immunoglobulin superfamily gene potentially involved in sperm-egg fusion.

Fertilization is the process that leads to the formation of a diploid zygote from two haploid gametes. This is achieved through a complex series of cell-to-cell interactions between a sperm and an egg. The final event of fertilization is the fusion of the gametes' membranes, which allows the delivery of the sperm genetic material into the egg cytoplasm. In vivo studies in the laboratory mouse have led to the discovery of membrane proteins that are essential for the fusion process in both the sperm and egg. Specifically, the sperm protein Izumo1 was shown to be necessary for normal fertility. Izumo1-deficient spermatozoa fail to fuse with the egg plasma membrane. Izumo1 is a member of the Immunoglobulin Superfamily of proteins, which are known to be involved in cell adhesion. Here, we describe BART97b, a new mouse line with a recessive mutation that displays a fertilization block associated with a failure of sperm fusion. BART97b mutants carry a deletion that inactivates Spaca6, a previously uncharacterized gene expressed in testis. Similar to Izumo1, Spaca6 encodes an immunoglobulin-like protein. We propose that the Spaca6 gene product may, together with Izumo1, mediate sperm fusion by binding an as yet unidentified egg membrane receptor.

Lack of nicotinamide mononucleotide adenylyltransferase 2 (Nmnat2): consequences for mouse bladder development and function.

AIMS: To describe the morphological and functional consequences for bladder development and function when nicotinamide mononucleotide adenylyltransferase 2 (Nmnat2) is lacking or reduced. METHODS: The Bloated Bladder (Blad) mouse, lacking Nmnat2, and heterozygotes were utilized for this investigation. Morphology and development of the bladder were studied using immunohistochemistry against urothelial, smooth muscle, and nerve markers. Functional effects were assessed by organ bath experiments and cystometry. RESULTS: Homozygote mutants were malformed and died at birth, whereas heterozygotes survived and morphologically did not differ from wild-type controls. Morphological bladder changes appeared in the Blad mutants as early as embryonic day 15.5 (E15.5) with an extremely distended bladder at E18.5. Staining revealed that all the bladder layers were present and expressed mature markers in all three genotypes. No nerves could be demonstrated by immunohistochemistry in the Blad mutant bladder at E18.5. Organ bath analysis showed that bladders from Blad mutant showed signs of denervation supersensitivity in response to carbachol, and no response to electrical stimulation of nerves at E18.5. Adult heterozygotes, which have a reduced expression of Nmnat2 at E18.5, showed decreased responses to carbachol and electrical stimulation compared to wild-type controls. The latter also retained their ability to empty their bladders, but showed increased micturition pressures compared to controls. CONCLUSIONS: Complete loss of Nmnat2 leads to a mature but distended bladder in utero and is not compatible with survival. Moderate loss of Nmnat2 has no effect on bladder development, survival, and has only modest effects on bladder function later in life.

Nicotinamide mononucleotide adenylyltransferase 2 (Nmnat2) regulates axon integrity in the mouse embryo.

Using transposon-mediated gene-trap mutagenesis, we have generated a novel mouse mutant termed Blad (Bloated Bladder). Homozygous mutant mice die perinatally showing a greatly distended bladder, underdeveloped diaphragm and a reduction in total skeletal muscle mass. Wild type and heterozygote mice appear normal. Using PCR, we identified a transposon insertion site in the first intron of Nmnat2 (Nicotinamide mononucleotide adenyltransferase 2). Nmnat2 is expressed predominantly in the brain and nervous system and has been linked to the survival of axons. Expression of this gene is undetectable in Nmnat2(blad/blad) mutants. Examination of the brains of E18.5 Nmnat2(blad/blad) mutant embryos did not reveal any obvious morphological changes. In contrast, E18.5 Nmnat2(blad/blad) homozygotes showed an approximate 60% reduction of spinal motoneurons in the lumbar region and a more than 80% reduction in the sensory neurons of the dorsal root ganglion (DRG). In addition, facial motoneuron numbers were severely reduced, and there was virtually a complete absence of axons in the hind limb. Our observations suggest that during embryogenesis, Nmnat2 plays an important role in axonal growth or maintenance. It appears that in the absence of Nmnat2, major target organs and tissues (e.g., muscle) are not functionally innervated resulting in perinatal lethality. In addition, neither Nmnat1 nor 3 can compensate for the loss of Nmnat2. Whilst there have been recent suggestions that Nmnat2 may be an endogenous modulator of axon integrity, this work represents the first in vivo study demonstrating that Nmnat2 is involved in axon development or survival in a mammal.

Mex3c mutation reduces adiposity and increases energy expenditure.

The function of MEX3C, the mammalian homolog of Caenorhabditis elegans RNA-binding protein muscle excess 3 (MEX-3), was unknown until our recent report that MEX3C is necessary for normal postnatal growth and enhances the expression of local bone Igf1 expression. Here we report the pivotal role of Mex3c in energy balance regulation. Mex3c mutation caused leanness in both heterozygous and homozygous transgenic mice, as well as a more beneficial blood glucose and lipid profile in homozygous transgenic mice, in both sexes. Although transgenic mice showed normal food intake and fecal lipid excretion, they had increased energy expenditure independent of physical activity. Mutant mice had normal body temperature, Ucp1 expression in brown adipose tissue, and muscle and liver fatty acid oxidation. Mex3c is expressed in neurons and is detectable in the arcuate nucleus, the ventromedial nucleus, and the dorsomedial nucleus of the hypothalamus. Mex3c was not detected in NPY or POMC neurons but was detected in leptin-responsive neurons in the ventromedial nucleus. Mex3c and Leptin double mutant mice were growth retarded and obese and had blood profiles similar to those of ob/ob mice but showed none of the steatosis observed in ob/ob mice. Our data show that Mex3c is involved in energy balance regulation.

Oxidative stress is involved in age-dependent spermatogenic damage of Immp2l mutant mice.

Mitochondrial reactive oxygen species (ROS) have been implicated in spermatogenic damage, although direct in vivo evidence is lacking. We recently generated a mouse in which the inner mitochondrial membrane peptidase 2-like (Immp2l) gene is mutated. This Immp2l mutation impairs the processing of signal peptide sequences from mitochondrial cytochrome c1 and glycerol phosphate dehydrogenase 2. The mitochondria from mutant mice generate elevated levels of superoxide ion, which causes age-dependent spermatogenic damage. Here we confirm age-dependent spermatogenic damage in a new cohort of mutants, which started at the age of 10.5 months. Compared with age-matched controls, protein carbonyl content was normal in testes of 2- to 5-month-old mutants, but significantly elevated in testes of 13-month-old mutants, indicating elevated oxidative stress in the testes at the time of impaired spermatogenesis. Testicular expression of superoxide dismutases was not different between control and mutant mice, whereas that of catalase was increased in young and old mutants. The expression of cytosolic glutathione peroxidase 4 (phospholipid hydroperoxidase) in testes was significantly reduced in 13-month-old mutants, concomitant with impaired spermatogenesis. Apoptosis of all testicular populations was increased in mutant mice with spermatogenic damage. The mitochondrial DNA (mtDNA) mutation rate in germ cells of mutant mice with impaired spermatogenesis was unchanged, excluding a major role of mtDNA mutation in ROS-mediated spermatogenic damage. Our data show that increased mitochondrial ROS are one of the driving forces for spermatogenic impairment.