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Impaired spermatogenesis and male infertility are common manifestations associated with mitochondrial diseases, yet the underlying mechanisms linking these conditions remain elusive. In this study, we demonstrate that mice deficient for the mitochondrial intra-membrane rhomboid protease PARL, a recently reported model of the mitochondrial encephalopathy Leigh syndrome, develop early testicular atrophy caused by a complete arrest of spermatogenesis during meiotic prophase I, followed by degeneration and death of arrested spermatocytes. This process is independent of neurodegeneration. Interestingly, genetic modifications of PINK1, PGAM5, and TTC19 – three major substrates of PARL with important roles in mitochondrial homeostasis – fail to reproduce or modify this severe phenotype, indicating that the spermatogenic arrest arises from distinct molecular pathways. We further observed severe abnormalities in mitochondrial ultrastructure in PARL-deficient spermatocytes, along with prominent electron transfer chain defects, disrupted coenzyme Q (CoQ) biosynthesis, and metabolic rewiring. These mitochondrial defects are associated with a germ cell-specific decrease in GPX4 expression leading arrested spermatocytes to ferroptosis – a regulated cell death modality characterized by uncontrolled lipid peroxidation. Our results suggest that mitochondrial defects induced by PARL depletion act as an initiating trigger for ferroptosis in primary spermatocytes through simultaneous effects on GPX4 and CoQ – two major inhibitors of ferroptosis. These findings shed new light on the potential role of ferroptosis in the pathogenesis of mitochondrial diseases and male infertility warranting further investigation. Up to 9% of men are thought to experience infertility. These individuals may not produce enough healthy sperm cells. The root cause of infertility is often not discovered but, in some cases, it is associated with genetic defects in cell compartments known as mitochondria. Mitochondria are responsible for converting energy from food into a form of chemical energy cells need to power vital processes. However, it remains unclear how defects in mitochondria contribute to male infertility. Leigh syndrome is one of the most prevalent and severe diseases caused by genetic defects in mitochondria. The condition often develops in childhood and affects the nervous system, muscle and other organs, leading to many symptoms including muscle weakness and neurological regression. A previous study found that mutant mice that lack an enzyme, called PARL, display symptoms that are similar to those observed in humans with Leigh syndrome. PARL is found inside mitochondria where it cuts specific proteins to ensure they are working correctly in the cells. Radaelli et al. used extensive microscopy and biochemical analyses to study the fertility of male mice lacking PARL. The experiments revealed that the males were infertile due to a failure to produce sperm: spermatocytes, which usually develop into sperm cells, where much more likely to die in mice without PARL (by a process known as ferroptosis). Further experiments demonstrated that the mitochondria of the mutant mice had a shortage of two crucial molecules, a protein called GPX4 and a lipid called Coenzyme Q, which are required to prevent death by ferroptosis. It appears that this shortage was responsible for the demise of spermatocytes in the male mutant mice affected by infertility. These findings reveal a new role for PARL in the body and provide evidence that mitochondrial defects in living mammals can trigger ferroptosis, thereby contributing to male infertility. In the future, this research may pave the way for new treatments for male infertility and other diseases associated with defects in mitochondria.

BackgroundEngraftment efficiency of human immune cell lineages in super immunodeficient mice varies, and numerous strains have been developed to improve reconstitution of humanized immune system mice. NSG-SGM3 and NOG-EXL mice combine severe immunodeficiency with transgenic expression of human myeloid stimulatory cytokines, resulting in improved expansion of myeloid populations. However, humanized NSG-SGM3 (huNSG-SGM3) mice develop a lethal macrophage activation syndrome (MAS) and mast cell hyperplasia that limit their use in long-term studies, especially those requiring humanization followed by tumor xenotransplantation. It is currently unclear to what extent humanized NOG-EXL (huNOG-EXL) mice suffer from the same conditions observed in huNSG-SGM3 mice. In this study, we aimed to compare the effects of human CD34+ hematopoietic stem cell engraftment in these two mouse strains in an orthotopic glioblastoma patient-derived organoid xenograft model.MethodsNSG-SGM3 mice (n=10) humanized in-house were compared to both NOG-EXL mice (n=10) humanized in-house and to commercially available huNOG-EXL mice (n=12). Mice were euthanized at humane or study endpoints, and a complete pathological assessment was performed. A semiquantitative multiparametric clinicopathological scoring system was developed to characterize the myeloid proliferative disorder.ResultsBoth the NOG-EXL mice humanized in-house and commercially available huNOG-EXL mice survived longer (to experimental endpoint) than huNSG-SGM3 mice (22 vs 17 weeks post engraftment), with significantly less severe MAS and lack of mastocytic proliferation. Major findings included mast cell infiltration of the pancreas and liver (huNSG-SGM3 only), increased eosinophilopoiesis, anemia, and histiocytic infiltration of the spleen (both strains). Engraftment of human lymphocytes, assessed by immunohistochemistry, was similar in the two strains. The longer survival and decreased MAS severity in NOG-EXL mice enabled their use in a patient-derived xenograft transplantation study.ConclusionsHumanized NOG-EXL mice develop a milder myeloid activation syndrome and do not develop mast cell hyperplasia relative to humanized NSG-SGM3 mice. Thus, the NOG-EXL model may be better suited than the NSG-SGM3 model for immuno-oncology studies requiring long-term survival post humanization. Future directions include further assessment of the lymphocyte populations in both strains and application of the humanized NOG-EXL for patient-derived xenograft models.AcknowledgementsThe authors thank the staff of the Penn Vet Histology Lab and the Penn Stem Cell and Xenograft Core for technical assistance.Ethics ApprovalAll the mice included in this study were maintained by the Stem Cell and Xenograft Core at the University of Pennsylvania under the protocol #803506 which has been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). The participant gave informed consent for use of glioblastoma patient-derived organoids, under protocol #832366, approved by the University of Pennsylvania Institutional Review Board (IRB).

The aim of this study was to evaluate the use of cathepsin-activated intraoperative near-infrared (NIR) imaging to detect insulinomas in dogs, a spontaneous large animal model for human disease. A prospective, pilot clinical trial was performed on dogs with naturally occurring insulinomas undergoing exploratory laparotomy. Each dog underwent routine preoperative diagnostic assessment, and a cathepsin-activated fluorophore (VGT-309) was administered intravenously 1-2 days preoperatively. All intraoperative findings with visible light and NIR imaging were recorded and mean NIR fluorescence intensity of tumors and grossly normal pancreas were quantified. Excision of any identified primary tumor and suspected metastatic lesions was performed. All excised tissues underwent histologic evaluation and immunohistochemistry (IHC) for cathepsin B expression. Descriptive statistics were calculated, and differential fluorescence intensity and cathepsin B expression between the pancreatic mass and adjacent grossly normal pancreatic tissue were assessed for statistical significance via paired t tests with p < 0.05 used for significance. Six dogs were enrolled. No adverse events occurred secondary to administration of the imaging agent. In situ, insulinomas had significantly greater mean fluorescence intensities than the surrounding pancreas, and the median tumor to background ratio was 1.906 (range 1.286-2.556). One dog had an occult pancreatic mass that was identified intraoperatively with NIR guidance. Background fluorescence of liver and lymph nodes was observed in all cases, and one dog was diagnosed with nodal and hepatic metastasis. Histologic tumor margins correlated with margins of NIR fluorescence. Cathepsin B expression was determined to be significantly greater in the pancreatic tumor compared to adjacent non-neoplastic pancreas via IHC, and there was no overlap in the range of median IHC-positive proportion values for these tissues. However, there was overlap in the range of IHC-positive proportion values for neoplastic pancreatic samples and lymph node and liver tissues. The findings of this pilot study support further investigation of cathepsin-activated NIR imaging to enhance intraoperative canine insulinoma localization and margin evaluation. Future studies are needed to further characterize and optimize the utility of targeted NIR imaging, particularly to identify metastatic lesions, for canine insulinoma, which may serve as an effective translational model for humans with pancreatic neuroendocrine tumors.