CD-1 Mice Research Articles

A multi-paradigm approach to characterize dominance behaviors in CD1 and C57BL6 male mice.

Social status and dominance are critical factors influencing well-being and survival across multiple species. However, dominance behaviors vary widely across species, from elaborate feather displays in birds to aggression in chimps. To effectively study dominance, it is essential to clearly define and reliably measure dominance behaviors. In laboratory settings, C57BL/6 mice are commonly used to study dominance due to their stable and linear social hierarchies. However, other mouse strains are also used for laboratory research. Despite substantial evidence for strain effects on behavioral repertoires, the impact of strain on dominance in mice remains largely unstudied. To address this gap, we compared dominance behaviors between CD1 and C57BL/6 male mice across four assays: observation of agonistic behaviors, urine marking, tube test, and a reward competition. We found that CD1 mice demonstrate increased fighting, increased territorial marking through urination, and increased pushing and resisting in the tube test. We used unsupervised machine learning and pose estimation data from the reward competitions to uncover behavioral differences across strains and across rank differences between competing pairs. Of the four assays, urine marking and agonistic behaviors showed the strongest correlation with dominance in both strains. Most notably, we found that CD1 dominance rankings based on the tube test negatively correlated with rankings from all three other assays, suggesting that the tube test may measure a different behavior in CD1 mice. Our results highlight that behaviors can be strain-specific in mice and studies that measure social rank should consider assays carefully to promote reproducibility.Significance Statement Recent studies have highlighted that social dominance can significantly impact behavior and the brain. As such, accurately measuring dominance behavior in laboratory settings is crucial in neuroscience research. In this study, we investigated the consistency of four dominance assays for male mice across two common mouse strains. We find that not all assays result in the same dominance rankings and dominance behaviors differ across strains. Our study sheds light on the importance of considering strains for assay selection, rigor, and reproducibility.

Chlorogenic Acid Alleviates Inflammation and Fibrosis in a Murine Model of Bleomycin-Induced Systemic Sclerosis: A Histological Analysis

Background/Objectives: Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by cutaneous and visceral fibrosis, vascular alterations, and a persistent inflammatory response. Despite advances in understanding the pathogenic mechanisms underlying SSc, current therapeutic options remain limited. Chlorogenic acid (CGA) is a polyphenol widely distributed in plants and has shown antioxidant, anti-inflammatory, and antifibrotic properties. However, its therapeutic potential in SSc has not been investigated yet. Methods: A model of SSc was established by administering bleomycin (BLM) at 100 U/kg to CD1 mice via an osmotic minipump. After fourteen days of BLM administration, CGA (60 mg/kg) was intragastric administered on consecutive days until day 20. On day 21, all mice were sacrificed. The effect of CGA was histologically evaluated by hematoxylin and eosin and Masson’s trichrome staining. Results: CGA treatment significantly attenuated dermal fibrosis in the BLM-induced mice model of SSc by reducing histopathological damage, including increased dermal thickness, inflammation, collagen deposition, and SSc-associated pulmonary fibrosis. Conclusions: The evidence shows that CGA attenuates BLM-induced SSc in a mice model and strongly suggests that CGA may be a promising compound for the treatment of SSc.

Pharmacokinetics and fate of free and encapsulated IRD800CW-labelled human BChE intravenously administered in mice

Human butyrylcholinesterase (BChE) is an efficient bioscavenger of toxicants. Highly purified BChE was labelled with the near infrared fluorescent IRDye800CW. The goal was to determine the pharmacokinetics and fate of enzyme in mice. BChE-IRDye800CW was encapsulated in polyethylene glycol−polypropylene sulfide-based spherical polymersome nanoreactors with the following characteristics: 140 nm diameter, ξ = −6 mV, PDI ≤ 0.2, 1 year stability. Encapsulation did not alter the functional properties of BChE. Free and encapsulated enzyme were injected intravenously to CD-1 mice (single dose of enzyme 1.5 mg/kg and PEG-PPS polymersomes 25 mg/kg) and were analyzed for 8 days using an in vivo imaging system. Results showed that the pharmacokinetic distribution a-phase of encapsulated BChE (t1/2 = 17.6 h) was longer than for free enzyme (t1/2 = 6.6 h). The mean half-time for elimination b-phase was 2-time longer for encapsulated enzyme than for free enzyme (150 vs 72 h). Transient changes in infrared fluorescence in organs showed that BChE is eliminated from liver. However, free and encapsulated enzymes were cleared via different pathways. This first study of pharmacokinetics and fate of BChE encapsulated in polymersomes initiates research of new formulations of bioscavengers aimed at increasing the residence time of enzymes in the blood stream.

VP3.15 reduces acute cerebellum damage after germinal matrix-intraventricular hemorrhage of the preterm newborn

Germinal matrix-intraventricular hemorrhage (GM-IVH) is one of the most common complications of the preterm newborn. The pathology of the GM-IVH is not completely understood and even regions distant from the lesion area are severely affected. It has been suggested that cerebellar diaschisis may underlie the neurodevelopmental problems that many of these kids show, including cerebral palsy, attention deficit disorders or hyperactivity. Additionally, GM-IVH has no successful treatment. VP3.15 is a dual action phosphodiesterase 7 (PDE7) and glycogen synthase kinase-3β (GSK-3β) inhibitor that limits neuroinflammation and neuronal loss. Therefore, it might also provide a relevant tool to reduce complications associated with GM-IVH. We have used a murine model of GM-IVH to analyze the short and long-term effects of VP3.15 in brain pathology and behavioral complications. In our hands, the induction of unilateral GM-IVH to P7 CD1 mice results in a short-term (P14) compromise of the cerebellar neuronal population and Purkinje cells arborization, an increase of microglia burden in the nuclei and an overall increase of punctuate cerebellar hemorrhages. Whereas brain alterations are no longer observed in the long term (P110), these animals present overt hyperactivity when analyzed in the adulthood, supporting the long-term behavioral impairment. Also, hyperactivity significantly correlates with ipsi and contralateral cerebellar sizes, neuronal densities and myelin basic protein levels. Importantly, treatment with VP3.15 significantly reduces neuronal loss, Purkinje cells simplification, the presence of cerebellar hemorrhages, as well as hyperactivity. Altogether, our data support the neuroprotective effects of VP3.15 in GM-IVH of the PT.

Anti-Inflammatory and Antinociceptive Properties of the Quercetin-3-Oleate AV2, a Novel FFAR1 Partial Agonist

Free fatty acid receptor 1 (FFAR1) has emerged as the most targeted isoform of the free fatty acid receptors because of its involvement in the modulation of energy balance and its potential role in the control of inflammatory and pain conditions. Quercetin-3-oleate (AV2), recognized as a new FFAR1 partial agonist, was investigated for its ability to modulate inflammation and nociception. Human immortal neuroblastoma SH and the murine macrophagic RAW 264.7 cells were used to evaluate cell viability, the potential cytoprotective activity, and the anti-inflammatory properties of AV2 in vitro. Paw edema, caused by zymosan-A, and the formalin test were used to assess the in vivo anti-inflammatory and antinociceptive effects in CD-1 mice. In vitro, AV2 was devoid of cytotoxicity, significantly reduced ROS in both cell types, and protected RAW 264.7 cells from lipopolysaccharide damage by reducing tumor necrosis factor-α production. Interestingly, AV2 induced a transient elevation of intracellular calcium that was reduced in cells, pre-incubated with the FFAR1 antagonist DC260126. In vivo, AV2 reduced formalin-induced nociception and zymosan A-induced paw edema, and both effects were reversed by the FFAR1 antagonist GW1100. In conclusion, these data strongly support the AV2-mediated antioxidant, anti-inflammatory, and antinociceptive activity. AV2 represents a promising molecule for the clinical management of inflammatory-related pain conditions.

Chronic psychosocial stress affects insulin-like growth factor 1 and its receptors in mouse ovaries.

Context Chronic psychosocial stress negatively affects folliculogenesis and oogenesis. Intraovarian mechanisms mediating these effects are poorly understood. Aims This work aimed to find out how chronic psychosocial stress affects ovarian IGF1 and its receptor (IGF1R), as well as Igf1 and Igf1r gene expression in cumulus-oocyte complexes (COCs). It also aimed to address possible protective effects of gonadotropin stimulation on IGF1 ovarian signalling. Methods Female CD1 mice experienced chronic psychosocial stress of 11-day isolation followed by overcrowding for 10days. To verify the model, blood corticosterone levels and the quality of oocytes were evaluated in stressed females. The levels of IGF1/IGF1R, blood IGF1 concentration, and expression of Igf1 /Igf1r in the ovaries were compared in stressed and unstressed females. Key results Psychosocial stress caused an elevation of corticosterone level, which was alleviated by gonadotropin treatment. The stressed mice showed a decreased IGF1 level in the ovaries and a decreased expression of Igf1 and Igf1r in COCs. In the unstressed females, gonadotropin injection decreased the expression of Igf1 and Igf1r ; in the stressed females, the same treatment increased Igf1r expression. Neither stress nor ovarian stimulation with gonadotropins affected the serum IGF1 level. Conclusions Psychosocial stress suppresses IGF1 signalling in the ovaries. Gonadotropin treatment modulates these effects differently in stressed and unstressed animals. Implications The results may have translational value for human reproduction. Ovarian IGF1 can be considered a candidate for further improvement of IVF results in women under conditions of chronic stress.

Geranylgeranyl diphosphate synthase inhibition impairs osteoclast differentiation, morphology and resorptive activity

Abstract Nitrogen bisphosphonates (NBPs), such as zoledronic acid, target the enzyme farnesyl diphosphate synthase (FDPS) in the isoprenoid biosynthetic pathway (IBP), and are the frontline treatment for osteolytic bone diseases. The strong affinity of these agents for bone limits their distribution out of the skeleton. Geranylgeranyl diphosphate synthase (GGDPS) is directly downstream to FDPS in the IBP and novel GGDPS inhibitors such as RAM2061 have been shown to have key drug-like features including prolonged half-life, metabolic stability, and systemic distribution. Furthermore, RAM2061 exerts anti-neoplastic benefits in mouse models of multiple myeloma and Ewing sarcoma. Therefore, we are interested in determining the potential impact of RAM2061 on osteoclast biology and bone remodeling. Studies utilizing undifferentiated RAW264.7 cells demonstrated that treatment with RAM2061 depletes cells of GGPP, impairs protein geranylgeranylation and induces markers of the unfolded protein response pathway and apoptosis. Differentiation of RAW264.7 cells to mature osteoclasts is disrupted by RAM2061, resulting in decreased numbers of mature osteoclasts, altered morphology and decreased tartrate resistant acid phosphatase activity. Treatment of fully differentiated RAW264.7 cells with RAM2061 led to decreased resorptive activity. Confocal microscopy studies revealed that RAM2061 disrupts Cdc42 localization, inhibiting proper actin ring formation in osteoclasts. No significant impact on bone turnover markers or bone histomorphology was observed following a 3-week treatment of CD-1 mice with RAM2061, although decreased numbers of osteoclasts were observed. Liquid chromatography–tandem mass spectrometry studies confirmed accumulation of RAM2061 in bone from the in vivo studies as well as hydroxyapatite binding in vitro. In conclusion, these studies are the first to demonstrate the anti-osteoclastic activity of GGDPS inhibitor treatment and support future studies exploring the therapeutic benefit of this novel therapy in the setting of pathological bone remodeling.

The contribution of neutrophils to bacteriophage clearance and pharmacokinetics in vivo.

With the increasing prevalence of antimicrobial-resistant bacterial infections, there is interest in using bacteriophages (phages) to treat such infections. However, the factors that govern bacteriophage pharmacokinetics in vivo remain poorly understood. Here, we have examined the contribution of neutrophils, the most abundant phagocytes in the body, to the pharmacokinetics of i.v. administered bacteriophage in uninfected mice. A single dose of LPS-5, a bacteriophage recently used in human clinical trials to treat drug-resistant Pseudomonas aeruginosa, was administered i.v. to both immunocompetent BALB/c and neutropenic CD1 mice. Phage concentrations were assessed in peripheral blood and spleen at 0.25, 1, 2, 4, 8, 12, and 24 hours after administration by plaque assay and qPCR. We observed that the phage clearance was only minimally affected by neutropenia. Indeed, the half-lives of phages in blood in BALB/c and CD1 mice were 3.45 and 3.66 hours, respectively. These data suggest that neutrophil-mediated phagocytosis is not a major determinant of phage clearance. Conversely, we observed a substantial discrepancy in circulating phage levels over time when measured by qPCR versus plaque assay, suggesting that significant inactivation of circulating phages occurs over time. These data indicate that alternative factors, but not neutrophils, inactivate i.v. administered phages.

Supervised machine learning of outbred mouse genotypes to predict hepatic immunological tolerance of individuals

It is essential to elucidate the molecular mechanisms underlying liver transplant tolerance and rejection. In cases of mouse liver transplantation between inbred strains, immunological rejection of the allograft is reduced with spontaneous apoptosis without immunosuppressive drugs, which differs from the actual clinical result. This may be because inbred strains are genetically homogeneous and less heterogeneous than others. We exploited outbred CD1 mice, which show highly heterogeneous genotypes among individuals, to search for biomarkers related to immune responses and to construct a model for predicting the outcome of liver allografting. Of the 36 mice examined, 18 died within 3 weeks after transplantation, while the others survived for more than 6 weeks. Whole-exome sequencing of the 36 donors revealed more than 9 million variants relative to the C57BL/6 J reference. We selected 6517 single-nucleotide and indel variants and performed machine learning to determine whether or not we could predict the prognosis of each genotype. Models were built by both deep learning with a one-dimensional convolutional neural network and linear classification and evaluated by leave-one-out cross-validation. Given that one short-lived mouse died early in an accident, the models perfectly predicted the outcome of all individuals, suggesting the importance of genotype collection. In addition, linear classification models provided a list of loci potentially responsible for these responses. The present methods as well as results is likely to be applicable to liver transplantation in humans.

Distinct Roles of Medial Prefrontal Cortex Subregions in the Consolidation and Recall of Remote Spatial Memories.

It is a common belief that memories, over time, become progressively independent of the hippocampus and are gradually stored in cortical areas. This view is mainly based on evidence showing that prefrontal cortex (PFC) manipulations impair the retrieval of remote memories, while hippocampal inhibition does not. More controversial is whether activity in the medial PFC is required immediately after learning to initiate consolidation. Another question concerns functional differences among PFC subregions in forming and storing remote memories. To address these issues, we directly contrasted the effects of loss-of-function manipulations of the anterior cingulate cortex (aCC) and the ventromedial PFC, which includes the infralimbic (IL) and prelimbic (PL) cortices, before testing and immediately after training on the ability of CD1 mice to recall the hidden platform location in the Morris water maze. We injected an AAV carrying the hM4Di receptor into the PL-IL or aCC. Interestingly, pretest administrations of clozapine-N-oxide (CNO; 3 mg/kg) revealed that the aCC, but not the PL-IL, was necessary to recall remote spatial information. Furthermore, systemic post-training administration of CNO impaired memory recall at remote, but not recent, time points in both groups. These findings revealed a functional dissociation between the two prefrontal areas, demonstrating that both the PL-IL and the aCC are involved in early consolidation of remote spatial memories, but only the aCC is engaged in their recall.

Intestinal distribution of anionic, cationic, and neutral polymer-stabilized nanocarriers measured with a lanthanide (europium) tracer assay

Nanocarriers, more commonly called nanoparticles (NPs), have found increasing use as delivery vehicles which increase the oral bioavailability of poorly water-soluble and peptide therapeutics. Therapeutic bioavailability is commonly assessed by measuring plasma concentrations that reflect the absorption kinetics. This bioavailability is a convolution of the gastrointestinal distribution of the NP vehicle, the release rate of the encapsulated therapeutic cargo, and the absorption-metabolism-distribution kinetics of the released therapeutic. The spatiotemporal distribution of the NP vehicle in the gastrointestinal tract is not well studied and is a buried parameter in PK studies used to measure the effectiveness of an NP formulation. This work is a study of the intestinal distribution and fate of orally dosed NPs in male CD-1 mice over 24 h. NPs have identical hydrophobic cores – composed of poly(styrene) homopolymer, a naphthalocyanine dye, and oleate-coated europium oxide colloids – with one of four different surface stabilizers: neutral poly(styrene)-block-poly(ethylene glycol) (PS-b-PEG), moderately negative hydroxypropyl methylcellulose acetate succinate (HPMCAS), highly negative poly(styrene)-block-poly(acrylic acid) (PS-b-PAA), and highly cationic adsorbed chitosan HCl on PS-b-PAA stabilized NPs. NP hydrodynamic diameters are all below 200 nm, with some variation attributable to the molecular properties of the stabilizing polymer. The encapsulated hydrophobic europium oxide colloids do not release soluble europium ions, enabling the use of highly sensitive inductively coupled plasma mass spectrometry (ICP-MS) to detect NP concentrations in digested biological tissues.Highly anionically-charged PAA and cationically-charged chitosan stabilized NPs showed statistically significant increased retention compared to the neutral PEG-stabilized NPs at p < 0.05 significance and (1-β) > 0.95 power. HPMCAS-stabilized NPs showed statistically insignificant greater retention than PEG-stabilized NPs, and all NP formulations showed clearance from the intestines within 24 h. Different surface charges preferentially reside in different segments of the intestines, where cationic chitosan-stabilized NPs showed increased retention in the small intestines (ileum) and anionic PAA-stabilized NPs in the large intestines (caecum and colon). Modifying the surface charge of a NP can be used to modulate mucoadhesion, total retention, and intestinal segment specific retention, which enables the rational design of delivery vehicles that maximize residence times in appropriate locations.

Differential Regulation of L-Arginine Metabolism through NOS2 and Arginases during Infection with Trypanosoma cruzi.

L-arginine metabolism through arginases and inducible nitric oxide synthase (NOS2) constitutes a fundamental axis for the resolution or progression of Chagas disease. Infection with Trypanosoma cruzi can cause a wide spectrum of disease, ranging from acute forms contained by the host immune response to chronic ones, such as the chronic chagasic cardiomyopathy. Here, we analyzed, in an in vitro model, the ability of two T. cruzi isolates, with different degrees of virulence, to regulate the metabolism of L-arginine through arginase 1 (Arg-1) and NOS2 in macrophages and through arginase 2 (Arg-2) and NOS2 in cardiomyocytes. Stimulation of bone marrow-derived macrophages (BMMΦ), obtained from CD1 mice, with TNF-α + IFN-γ induced their polarization into classically activated macrophages (CAMΦ), which expressed functional NOS2, while stimulation with IL-4 induced their polarization into alternatively activated macrophages (AAMΦ), which expressed functional Arg-1. Interestingly, stimulation of cardiomyocytes, obtained from hearts of CD1 neonatal mice, with TNF-α + IFN-γ or IL-4 also resulted in functional NOS2 and arginase expression, as observed in CAMΦ and AAMΦ, but Arg-2 was the arginase isoform expressed instead of Arg-1. We observed that infection of BMMΦ with the more virulent T. cruzi isolate (QRO) importantly diminished NOS2 expression and nitric oxide (NO) production in CAMΦ, allowing parasite survival, while infection with the less virulent isolate (CI2) did not diminish NOS2 activity and NO production in CAMΦ to a great extent, which resulted in parasite killing. Regarding Arg-1, infection of BMMΦ with the QRO isolate significantly induced Arg-1 expression and activity in AAMΦ, which resulted in a higher parasite load than the one in the unstimulated BMMΦ. Even though infection with CI2 isolate did not increase Arg-1 expression and activity in AAMΦ, the parasite load was higher than the one in the unstimulated BMMΦ but at a lesser magnitude than that observed during infection with the QRO isolate. On the other hand, infection of cardiomyocytes with either QRO or CI2 isolates and further stimulation with TNF-α + IFN-γ inhibited NOS2 expression and NO production, leading to amelioration of infection. Surprisingly, infection of cardiomyocytes with either QRO or CI2 isolates and further stimulation with IL-4 strongly inhibited Arg-2 expression and function, which resulted in parasite loads similar to those observed in unstimulated cardiomyocytes. Our results suggest that T. cruzi isolates that exhibit variable virulence or pathogenicity degrees differentially regulate L-arginine metabolism through Arg-1/2 and NOS2 in macrophages and cardiomyocytes.

The protective effect of irisin against hemorrhagic injury is mediated by PI3K and p38 pathways in hemorrhage/resuscitation.

The objective of this study is to investigate whether PI3kinase (PI3K) and p38 mitogen-activated kinase contributes to the protection of irisin during hemorrhage/resuscitation. Experimental groups were divided by receiving the different treatments during resuscitation: I) Hemorrhage: Adult male CD-1 mice were subjected to hemorrhage at a mean arterial blood pressure of 35~45 mmHg for 60 min followed by 120 min of resuscitation (n=13); II) Hemorrhage + Irisin: receiving irisin (5µg/kg) (n=13); III) Hemorrhage + Irisin + PI3K inhibitor: receiving both Ly294002 (1mg/kg, i.v.) and irisin (n=6); IV) Hemorrhage + Irisin + p38 inhibitor: receiving SB202190 (1mg/kg, i.v.) and irisin (n=6). As compared to hemorrhage/resuscitation control, irisin improved the cardiac function and recovery of hemodynamics in association with the decreased systemic IL-1, IL-6, and TNF-α, which was completely abrogated by PI3K or p38 inhibitions. Furthermore, inhibition of PI3K or p38 abolished irisin-induced reduction of the infiltration of inflammatory cells and TUNEL-positive apoptosis in the cardiac and skeletal muscles. Irisin reduced TNF-α and IL6 expression in cardiac and skeletal muscle, which was abrogated by inhibition of PI3K or p38. Irisin-treated hemorrhage increases the phosphorylation of PI3K and p38 in both cardiac and skeletal muscle, which was mitigated by inhibition of PI3K or p38. Conclusion: PI3K and p38 play a critical role in modulating the protective effect of irisin during the hemorrhage/resuscitation. Significance Statement 1). This study has identified a critical pathway in regulation of trauma/hemorrhage by using a preclinical and reproducible model, in which Irisin, as a hormone factor, stimulates PI3K and p38 pathways to induce the protection against traumatic conditions. 2). The study holds promise to develop a new therapeutic strategy to target irisin and its pathway related to PI3K and p38 to treat trauma and its comorbidities to reduce mortality for clinical implication.

6432 Enriching Testicular Organoid Aggregates to Promote Germ Cell Homing

Abstract Disclosure: A. Patel: None. O.O. Printy: None. C. Joswiak: None. M.M. Laronda: None. The long-term effects of life-saving chemotherapy treatments, such as an increased risk of infertility, are of concern to the growing number of childhood cancer survivors. De novo testicular morphogenesis seeks to generate testicular organoids that are structurally and functionally similar to the native testis. These organoids serve as a tool for investigating patient-specific models for restoring spermatogenesis for prepubertal cancer patients. Our lab has previously developed testicular organoids containing tubule-like structures (TLS) resembling the native seminiferous tubules as assessed by immunohistochemistry of testicular cell-type markers for Leydig cells (HSD3B2), peritubular myoid cells (ACTA1), Sertoli cells (SOX9), germ cells (DDX4), and spermatogonial stem cells (SSCs) (SALL4). Internal architecture demonstrated semi-continuous ring-like structures composed of ACTA1-positive cells encapsulating a population of SOX9-positive cells with HSD3B2-positive cells clustering near the periphery of the organoids. However, organoids lacked native germ cell populations. We hypothesized that murine testicular organoids would merge to form elongated TLS, within which exogenous SSCs would home to existing Sertoli cell niches, when cultured within structures designed from the appropriate dimensions and materials. Organoids were generated from testicular tissue isolated from 5 days-post-partum (dpp) CD1 mice using cell-seeding densities and microenvironmental conditions previously established by our lab. To promote aggregate formation, 48-hour-old organoids were transferred to trough-shaped structures formed from 2% agarose using 3D printed silicone molds. Separately, SSCs were isolated from 5 dpp tdTomato+ mice via magnetic-activated cell sorting of THY1+ cells. Organoids and aggregates were enriched with SSCs by three methods: (1) enrichment of cell suspension during organoid formation, (2) enrichment of culture during aggregate formation, and (3) injection of SSCs into aggregate using 20 µm glass pipette. Organoids transferred to agarose troughs on day 2 of culture merged to form aggregates by day 5 and continued to display dynamic architecture, shortening in length and increasing in width over time. Aggregates were more elongated in shape when cultured in narrow troughs less than 700µm in width. Observations thus far indicate that attempts to inject aggregates resulted in penetrance and inclusion of the SSC suspension; additionally, tdTomato+ cells were visualized within injected aggregates on day 17 of culture. Research is ongoing to define where exogenous SSCs take up residence, how long they survive, and if they proliferate over time within the testicular aggregates. This research expands upon foundational work establishing testicular organoids as an approach to in vitro spermatogenesis with clinical applications in fertility restoration. Presentation: 6/2/2024

7770 Damage of Ovarian Vasculature by Chemotherapies induces Ovarian Dysfunction in Mice

Abstract Disclosure: W. So: None. R. Dong: None. Y. Luan: None. A. Abazarikia: None. S. Kim: None. Background: Chemotherapy demonstrates efficacy to destroying cancer cells; however, it can inadvertently impact normal healthy cells, leading to irreversible side effects, particularly affecting the ovaries in female patients. Female cancer survivors encounter endocrine dysfunction and infertility due to ovarian damage caused by gonadotoxic cancer therapies. The ovary, a vital organ responsible for producing oocytes and endocrine hormones, faces significant challenges following such treatment. A study with premenopausal breast cancer patients reported a significant reduction in ovarian blood flow and volume immediately following chemotherapy. The preservation of ovarian function depends on the ovarian vasculature in supporting the development and maintenance of ovarian follicles. Despite its importance, the relationship between ovarian vasculature and follicle following chemotherapy has not been studied. Results: To investigate the direct impacts of chemotherapy, ovaries from postnatal 5 CD-1 mice were subjected to testing with three chemotherapeutic agents: cisplatin (CDDP), cyclophosphamide (CPA), and doxorubicin (DOXO). The expression of CD31/PECAM-1 (platelet endothelial adhesion molecule) exhibited a significant decrase after 96 hours of culture with 4-HC, an active metabolite of CPA. This reduction implies that ovarian vasculature was also compromised when chemotherapy eliminated immature follicles. In order to evaluate the influence of vascular endothelial growth factor (VEGF) in safeguarding both vasculature and ovarian follicles, ovaries were co-cultured with VEGF165 or VEGF165b in combination with chemotherapy. Interestingly, pre-treatment with VEGF165 before 4-HC led to an increased expression of CD31 in the cortex area, while VEGF165b further diminished CD31 expression compared to treatment with 4-HC alone. Additionally, adolescent CD-1 mice were intraperitoneally injected with CDDP, CPA, or DOXO, and ovaries were harvested one week after injection. Ovaries treated with chemotherapeutic agents exhibited a reduction in ovary weight and decreased CD31 expression compared to control mice. Conclusions: We propose that these chemotherapeutic agents not only induced the loss of ovarian follicles but also affected ovarian vasculature in mice. This suggests a potential decrease in folliculogenesis within the ovary, coupled with the depletion of ovarian reserve, as the delivery of nutrients and oxygen to growing ovarian follicles may be compromised. VEGF165 may play a beneficial role in maintaining ovarian function by safeguarding ovarian vasculature following chemotherapy. Understanding these intricate dynamics is essential for developing strategies to mitigate the negative impact of cancer therapies on reproductive health in female cancer survivors, particularly in preserving endocrine function and reproductive lifespan. Presentation: 6/2/2024

8010 Potent MSB-61 as Type 1 Diabetes (T1D) therapy: mechanism of action in insulin secretion

Abstract Disclosure: N. Ajmal: None. M. Bogart: None. K. Corbin: None. P. Khan: None. M. Shafqat: None. S. Bergmeier: None. G. Gu: None. X. Tong: None. C.S. Nunemaker: None. Type 1 diabetes (T1D) is a chronic autoimmune disorder caused by proinflammatory cytokines (Interleukin 1-beta; IL1-β, Tumor Necrosis Factor-alpha; TNF-α, and Interferon-gamma; IFN-ϒ) from immune cells that destroy insulin-producing beta cells in pancreatic islets and lead to hyperglycemia. The development of possible treatments to cure T1D is needed besides intensive insulin therapy that does not halt disease progression. We have identified a small molecule called MSB-61 that can protect islet cells from endoplasmic reticulum stressors (66% protection) and cytokines (33% protection) as measured by propidium iodide in islets isolated from mice using 50uM MSB-61. Importantly, we observed large increases in insulin secretion during overnight exposure to 50uM MSB-61. Insulin secretion began to increase in response to MSB-61 at ∼4h, reaching a plateau at ∼8h of greater than 4-fold insulin release compared to controls. The Kegg pathway analysis of RNA-seq data on MSB-61 suggested that multiple genes upregulated by MSB-61 are involved in insulin secretion through an amplifying pathway that includes cAMP/PKA and Pi3k/Akt signaling cascades. Based on RNA sequencing data, this study aimed to test MSB-61 for identifying pathway by which it potentiates insulin secretion. Islets from CD-1 mice and mouse insulinoma Min-6 cell lines were used to test amplifying pathways by using cAMP/PKA and Pi3k/Akt analogs and antagonists with and without MSB-61, and overnight insulin secretion and insulin content will be measured by enzyme-linked immunosorbent assay (ELISA). MSB-61 showed no potential effects on insulin secretion when tested for Pi3k/Akt pathway in the presence of analogs and antagonists such as 740-Y-P and wortmannin. It further suggests MSB-61 might potentiate insulin secretion through the cAMP/PKA pathway, which is still under investigation. RNA sequencing analysis of mouse islets showed several genes that belong to Ras family member-2 and have GTPase activity. Genes with GTPase activity will be further investigated to help identify if MSB-61 stimulates insulin secretion through an amplifying pathway involving the cAMP/PKA downstream signaling cascade. Identifying the mechanism of action and signaling pathway by which MSB-61 potentiates insulin secretion will open new ways of identifying T1D therapeutic interventions that would help replace standard insulin therapy. Presentation: 6/3/2024

6432 Enriching Testicular Organoid Aggregates to Promote Germ Cell Homing

Abstract Disclosure: A. Patel: None. O.O. Printy: None. C. Joswiak: None. M.M. Laronda: None. The long-term effects of life-saving chemotherapy treatments, such as an increased risk of infertility, are of concern to the growing number of childhood cancer survivors. De novo testicular morphogenesis seeks to generate testicular organoids that are structurally and functionally similar to the native testis. These organoids serve as a tool for investigating patient-specific models for restoring spermatogenesis for prepubertal cancer patients. Our lab has previously developed testicular organoids containing tubule-like structures (TLS) resembling the native seminiferous tubules as assessed by immunohistochemistry of testicular cell-type markers for Leydig cells (HSD3B2), peritubular myoid cells (ACTA1), Sertoli cells (SOX9), germ cells (DDX4), and spermatogonial stem cells (SSCs) (SALL4). Internal architecture demonstrated semi-continuous ring-like structures composed of ACTA1-positive cells encapsulating a population of SOX9-positive cells with HSD3B2-positive cells clustering near the periphery of the organoids. However, organoids lacked native germ cell populations. We hypothesized that murine testicular organoids would merge to form elongated TLS, within which exogenous SSCs would home to existing Sertoli cell niches, when cultured within structures designed from the appropriate dimensions and materials. Organoids were generated from testicular tissue isolated from 5 days-post-partum (dpp) CD1 mice using cell-seeding densities and microenvironmental conditions previously established by our lab. To promote aggregate formation, 48-hour-old organoids were transferred to trough-shaped structures formed from 2% agarose using 3D printed silicone molds. Separately, SSCs were isolated from 5 dpp tdTomato+ mice via magnetic-activated cell sorting of THY1+ cells. Organoids and aggregates were enriched with SSCs by three methods: (1) enrichment of cell suspension during organoid formation, (2) enrichment of culture during aggregate formation, and (3) injection of SSCs into aggregate using 20 µm glass pipette. Organoids transferred to agarose troughs on day 2 of culture merged to form aggregates by day 5 and continued to display dynamic architecture, shortening in length and increasing in width over time. Aggregates were more elongated in shape when cultured in narrow troughs less than 700µm in width. Observations thus far indicate that attempts to inject aggregates resulted in penetrance and inclusion of the SSC suspension; additionally, tdTomato+ cells were visualized within injected aggregates on day 17 of culture. Research is ongoing to define where exogenous SSCs take up residence, how long they survive, and if they proliferate over time within the testicular aggregates. This research expands upon foundational work establishing testicular organoids as an approach to in vitro spermatogenesis with clinical applications in fertility restoration. Presentation: 6/2/2024

8538 Pancreatic Islet Adaptations to Hyperglycemia Are Prevented by Glycolytic Inhibition and Amplified by Inhibiting Membrane Depolarization

Abstract Disclosure: N.B. Whitticar: None. B.P. List: None. N. Bruce: None. R. Bertram: None. C.S. Nunemaker: None. Long before type 2 diabetes ensues, pancreatic islets undergo functional changes to compensate for increasing insulin resistance. Primarily, the islets increase their ability to secrete insulin by augmenting the glucose-stimulated insulin secretion (GSIS) pathway. As metabolic syndrome worsens, islets become functionally exhausted, leading to cellular dysfunction and apoptosis. To avoid the negative consequences of this compensatory response, various steps of the GSIS pathway can be attenuated to restore normal functional levels before the islets fail. Determining which inhibitors can prevent the earliest adaptations to hyperglycemia may reveal the processes that play a causal role in islet failure. The chemicals mannoheptulose (MH), diazoxide (DZ), and nifedipine (NP) were chosen to inhibit the glucokinase enzyme, KATP-channel closure, and voltage-gated calcium channels, respectively. CD-1 mouse islets were placed in 20 mM glucose to simulate hyperglycemia along with a concentration of each inhibitory drug that reduced insulin secretion by 30-40%. After 48-hour treatment, glucose-stimulated calcium tests were performed to determine if the treatments could prevent the negative adaptation to hyperglycemia. Only islets treated with MH to reduce glycolytic activity maintained a normal calcium response. Islets in the MH group also retained a strong insulin response to glucose and were not depleted of insulin content. Unexpectedly, inhibition of calcium influx and insulin secretion with DZ or NF caused a substantial increase in basal insulin secretion compared to islets in standard culture (P&amp;lt;0.01, n=6; P=0.06, n=6, respectively). These results indicate that the signaling pathway that causes the adaptation to hyperglycemia lies upstream of KATP channels and downstream of glucokinase. Mathematical modeling using the Integrated Oscillator Model was able to replicate our results and suggests that high rates of glycolytic and mitochondrial metabolism are associated with the signal that adapts islets to hyperglycemia, and that reducing intracellular calcium in the face of high glucose further increases basal insulin secretion. Ongoing studies will determine if metabolic acceleration or altered membrane conductance are responsible for the earliest islet adaptations to hyperglycemia. Our results suggest that reducing metabolic activity, but not calcium influx, prevents adaptations to hyperglycemia in mouse islets. These findings can be applied to potentially prevent the progression of obesity and hyperinsulinemia into type 2 diabetes. Presentation: 6/2/2024

8759 Mild Iodine Deficiency During Pregnancy Impairs Hypothalamus-Pituitary-Thyroid Function Programing of the Adult Male Offspring

Abstract Disclosure: M. Oliveira: None. E. Tavares: None. G. Henrique: None. J. Pinto: None. C. Serrano-Nascimento: None. Introduction: Iodine plays a crucial role in the biosynthesis of thyroid hormones and is essential for proper thyroid embryonic development. While severe iodine deficiency during pregnancy and lactation has well-documented deleterious effects, the consequences of mild iodine deficiency (MID) remain not fully understood. MID during pregnancy is a reality in numerous countries, even in regions with seemingly adequate iodine supply in the diet. Previous studies have highlighted the adverse impact of MID on neurological development in offspring, but comprehensive data on thyroid function, particularly in progeny at different developmental stages, are lacking. This study aimed to investigate the repercussions of MID during pregnancy on the programming of thyroid dysfunctions during adulthood. Methods: Female CD1 mice were subjected to iodine-deficient animal chow and iodine-supplemented water, either at normal (NI; 0.2 mg/L) or mild iodine deficiency (MID; 0.8 mg/L) levels during pregnancy. Gene and protein expression in the hypothalamus, pituitary, and thyroid of adult male offspring animals (PND90) were evaluated using qPCR and Western blotting. Additionally, this study explored MID-induced programming of offspring thyroid gene expression through epigenetic mechanisms. Results: Maternal MID exposure led to increased gene and protein expression of thyrotropin-releasing hormone (TRH) and thyroid-stimulating hormone (TSH) in the hypothalamus and pituitary of the offspring animals. Thyroid protein and gene expression of sodium iodide symporter (NIS), TSH receptor (TSHR), thyroid peroxidase (TPO), thyroglobulin (TG), paired box 8 (PAX8), NK2 homeobox 1 (NKX2.1), and monocarboxylate transporter 8 (MCT8) were elevated in the offspring of MID-exposed animals. Epigenetic mechanisms appeared to contribute to the augmented thyroid gene expression in MID-exposed animals, with decreased DNA methyltransferase expression and reduced thyroid DNA global methylation. Additionally, histone 3 hypomethylation and increased acetylation of histones 3 and 4 were observed. Conclusion: The study suggests that MID exposure during pregnancy can be as harmful as severe iodine deficiency in programming the hypothalamus-pituitary-thyroid (HPT) axis, leading to HPT dysfunction during adult life. Consequently, our data suggest that monitoring iodine intake throughout pregnancy is crucial to prevent the occurrence of thyroid disorders in offspring during adulthood. Presentation: 6/1/2024

6579 Neuroendocrine Signatures of Social-Defeated Stress Expressed by Adrenal Hormones in Mice

Abstract Disclosure: J. Noh: None. J. Choi: None. S. Myung: None. M. Kim: None. M. Choi: None. While psychological stress is one of the prevalent contributors to depression, the precise role of metabolic phenotypes in the development of depression remains elusive. Abnormality in hypothalamus-pituitary-adrenal (HPA) axis is a classical feature of depression. Here, an animal model of social defeat stress (SDS) was established with five-week-old C57BL/6J mice to repeated episodes of social defeat for a period of 10 days, facilitated by an aggressive CD-1 mouse. Liquid chromatography-mass spectrometry-based profiling of 29 adrenocortical steroids and 6 medullary amines was employed for serum samples collected at 3, 5, 7, and 10 days during the modeling from both control (n = 28) and SDS (n = 32) group. In response to initial stress, corticosterone (B) and its two precursors were significantly increased (p &amp;lt; 0.03 for all) during 3 days, while 18-hydroxyB was remarkably elevated (p &amp;lt; 0.003 for 3 days and p &amp;lt; 0.005) in SDS mice. In contrast, dopamine (p &amp;lt; 0.03) and metanephrine (p &amp;lt; 0.008) were significantly increased during 5 days in SDS mice, while remarkably higher levels of norepinephrine were also observed during 7 days (p &amp;lt; 0.01). The SDS mice showed the increased levels of normetanephrine for all days of repeated stress exposure (p &amp;lt; 0.002 for all, except 10 days of p &amp;lt; 0.02). In this study, the HPA regulated negative feedback in short period, whereas sympathetic nervous system was continuously stimulated during repeated stress exposure. The present study may provide insight into the neuroendocrine effects of social stress on allostatic overload. Presentation: 6/2/2024