Denise J. Schwahn

REDUCED MALE FERTILITY IS COMMON BUT HIGHLY VARIABLE IN FORM AND SEVERITY IN A NATURAL HOUSE MOUSE HYBRID ZONE

Barriers to gene flow between naturally hybridizing taxa reveal the initial stages of speciation. Reduced hybrid fertility is a common feature of reproductive barriers separating recently diverged species. In house mice (Mus musculus), hybrid male sterility has been studied extensively using experimental crosses between subspecies. Here, we present the first detailed picture of hybrid male fertility in the European M. m. domesticus—M. m. musculus hybrid zone. Complete sterility appears rare or absent in natural hybrids but a large proportion of males (∼30%) have sperm count or relative testis weight below the range in pure subspecies, and likely suffer reduced fertility. Comparison of a suite of traits related to fertility among subfertile males indicates reduced hybrid fertility in the contact zone is highly variable among individuals and ancestry groups in the type, number, and severity of spermatogenesis defects present. Taken together, these results suggest multiple underlying genetic incompatibilities are segregating in the hybrid zone, which likely contribute to reproductive isolation between subspecies.

Top-down Proteomics Reveals Concerted Reductions in Myofilament and Z-disc Protein Phosphorylation after Acute Myocardial Infarction

Heart failure (HF) is a leading cause of morbidity and mortality worldwide and is most often precipitated by myocardial infarction. However, the molecular changes driving cardiac dysfunction immediately after myocardial infarction remain poorly understood. Myofilament proteins, responsible for cardiac contraction and relaxation, play critical roles in signal reception and transduction in HF. Post-translational modifications of myofilament proteins afford a mechanism for the beat-to-beat regulation of cardiac function. Thus it is of paramount importance to gain a comprehensive understanding of post-translational modifications of myofilament proteins involved in regulating early molecular events in the post-infarcted myocardium. We have developed a novel liquid chromatography–mass spectrometry-based top-down proteomics strategy to comprehensively assess the modifications of key cardiac proteins in the myofilament subproteome extracted from a minimal amount of myocardial tissue with high reproducibility and throughput. The entire procedure, including tissue homogenization, myofilament extraction, and on-line LC/MS, takes less than three hours. Notably, enabled by this novel top-down proteomics technology, we discovered a concerted significant reduction in the phosphorylation of three crucial cardiac proteins in acutely infarcted swine myocardium: cardiac troponin I and myosin regulatory light chain of the myofilaments and, unexpectedly, enigma homolog isoform 2 (ENH2) of the Z-disc. Furthermore, top-down MS allowed us to comprehensively sequence these proteins and pinpoint their phosphorylation sites. For the first time, we have characterized the sequence of ENH2 and identified it as a phosphoprotein. ENH2 is localized at the Z-disc, which has been increasingly recognized for its role as a nodal point in cardiac signaling. Thus our proteomics discovery opens up new avenues for the investigation of concerted signaling between myofilament and Z-disc in the early molecular events that contribute to cardiac dysfunction and progression to HF.

Mesenchymoangioblast-derived mesenchymal stromal cells inhibit cell damage, tissue damage and improve peripheral blood flow following hindlimb ischemic injury in mice

BACKGROUND AIMS Existing treatments have limited success in modifying the course of peripheral artery disease, which may eventually lead to limb-threatening ulcers and amputation. Cellular therapies have the potential to provide a new treatment option for this condition, but isolation of cells by conventional means has limitations with respect to reproducibility and scalability. METHODS Induced pluripotent stem cells (iPSCs) were differentiated into precursor cells known as mesenchymoangioblasts (MCAs) and subsequently into mesenchymal stromal cells (MSCs). Hindlimb ischemia in mice was created by ligating both the iliac and femoral arteries of one hindlimb. Immediately after surgery, each animal received intramuscular injections of 5 × 10(6) cells or media in the ischemic limb. Toe necrosis was assessed visually, and hindlimb blood flow was measured by laser Doppler using a set region of interest (ROI) and by tracing the entire foot. Myofiber heterogeneity, nuclear centralization, fatty degeneration, fibrosis and capillary angiogenesis in the gastrocnemius muscle were assessed histologically. RESULTS Blood flow in the MCA-derived MSC-treated animals was higher at each day (P <0.006), and these mice recovered faster than control animals (3.6 vs. 2.5 for set ROI; 7.5 vs. 4.1 foot tracing; slope; P <0.001). There was significantly less myofiber heterogeneity, nuclear centralization, fatty degeneration and fibrosis in MCA-derived MSC-treated animals, indicating less tissue damage. DISCUSSION MCA-derived MSCs improved limb blood flow, reduced necrosis and maintained muscle mass and gross muscle appearance. We conclude that MCA-derived MSCs have a significant and protective effect against ischemic insults.

Cardiopulmonary and histological characterization of an acute rat lung injury model demonstrating safety of mesenchymal stromal cell infusion

BACKGROUND AIMS In the field of cellular therapy, potential cell entrapment in the lungs following intravenous administration in a compromised or injured pulmonary system is an important concern that requires further investigation. We developed a rat model of inflammatory and fibrotic lung disease to mimic the human clinical condition of obliterative bronchiolitis (OB) and evaluate the safety of intravenous infusion of mesenchymal stromal cells (MSCs). This model was used to obtain appropriate safety information and functional characterization to support the translation of an ex vivo-generated cellular product into human clinical trials. To overcome spontaneous recovery and size limitations associated with current animal models, we used a novel multiple dose bleomycin strategy to induce lasting lung injury in rats. METHODS Intratracheal instillation of bleomycin was administered to rats on multiple days. MSCs were intravenously infused 7 days apart. Detailed pulmonary function tests including forced expiratory volume, total lung capacity, and invasive hemodynamic measurements were conducted to define the representative disease model and monitor cardiopulmonary hemodynamic consequences of the cell infusion. Post-euthanasia assessments included a thorough evaluation of lung morphology and histopathology. RESULTS The double dose bleomycin instillation regimen resulted in severe and irreversible lung injury and fibrosis. Cardiopulmonary physiological monitoring reveled that no adverse events could be attributed to the cell infusion process. DISCUSSION Although our study did not show the infusion of MSCs to result in an improvement in lung function or rescue of damaged tissue this study does confirm the safety of MSC infusion into damaged lungs.

Genetic Dissection of Hybrid Male Sterility Across Stages of Spermatogenesis

Hybrids between new species are often sterile. To understand the causes of this reproductive barrier, Schwahn et al. examined testis histology in a large number of hybrids between two nascent species of house mice... Hybrid sterility is a common form of reproductive isolation between nascent species. Although hybrid sterility is routinely documented and genetically dissected in speciation studies, its developmental basis is rarely examined, especially in generations beyond the F1 generation. To identify phenotypic and genetic determinants of hybrid male sterility from a developmental perspective, we characterized testis histology in 312 F2 hybrids generated by intercrossing inbred strains of Mus musculus domesticus and M. m. musculus, two subspecies of house mice. Hybrids display a range of histologic abnormalities that indicate defective spermatogenesis. Among these abnormalities, we quantified decreased testis size, reductions in spermatocyte and spermatid number, increased apoptosis of meiosis I spermatocytes, and more multinucleated syncytia. Collectively, our phenotypic data point to defects in meiosis I as a primary barrier to reproduction. We identified seven quantitative trait loci (QTL) controlling five histologic traits. A region of chromosome 17 that contains Prdm9, a gene known to confer F1 hybrid male sterility, affects multinucleated syncytia and round spermatids, potentially extending the phenotypic outcomes of this incompatibility. The X chromosome also plays a key role, with loci affecting multinucleated syncytia, apoptosis of round spermatids, and round spermatid numbers. We detected an epistatic interaction between QTL on chromosomes 17 and X for multinucleated syncytia. Our results refine the developmental basis of a key reproductive barrier in a classic model system for speciation genetics.

Studies using a porcine model: what insights into human calcium oxalate stone formation mechanisms has this model facilitated?

Animal models are useful in the study of many human diseases. Our current understanding of the biological, physiological, and biochemical aspects of hyperoxaluria and calcium oxalate urolithiasis has been greatly informed by studies using animals. Recently, limitations in the extrapolation to humans of research results derived from laboratory rodents have been identified. The use in biomedical research of a variety of organisms, including large animals, is increasingly encouraged. The purpose of this article is to review the use of pigs in biomedical and stone research, to provide a rationale for using pigs in metabolic stone research, and to describe our 8-year experience in developing a porcine platform for studying hyperoxaluria and calcium oxalate urolithiasis. In this article, we share and review some of the highlights of our findings. We also report results from a recent feeding swine study that demonstrated oxalate-induced renal nephropathy. Finally, we offer ideas for future directions in urolithiasis research using swine.

MP34-01 RENAL HISTOPATHOLOGY AFTER CALCIUM OXALATE STONE INDUCTION: CRYSTALLURIC AND TUBULAR EFFECTS OF HIGH URINARY OXALATE EXCRETION IN A SWINE MODEL

INTRODUCTION AND OBJECTIVES: Swine are good models for stone disease as they closely resemble humans for gastrointestinal and renal morphology and function. We have developed a swine model that manifests hyperoxaluria and calcium oxalate (CaOx) stone formation via two methods: (1) enhanced oxalate (Ox) biosynthesis, from hydroxyproline (HYP) feeding; or (2) enhanced dietary Ox absorption (by feeding sodium oxalate). Histopathology characterized changes in crystalluric and renal tubular parameters in gilts (adult virgin sows) fed either HYP or Ox. These results provide new insights in our swine model of CaOx stone conditions. METHODS: Gilts were randomized to 1 of 4 diets, which were fed for up to 21 days. Diets consisted of (1) 5% HYP mixed with basal diet or (2) 8,100 mg Ox/d with normal calcium (13,000 mg/d) or (3) 8,100 mg Ox/d with low calcium (4,000 mg/d) or (4) control diet with 1,100 mg Ox/d and normal calcium (13,000 mg/d). Kidneys were examined grossly and then formalin-fixed, paraffin-embedded, sectioned, and stained with HE half had only a small number of medullary crystals. Many birefringent sheafshaped crystals (panel A) were observed in gilts fed all three diets, suggesting their composition as CaOx; they were most common in the high Ox/low calcium group. Fibrosis (panel B), cortical inflammation (panel C), and attenuated epithelial cells lining tubules dilated by crystals (panel D) were observed in gilts fed all three experimental diets. Tubular necrosis was observed in gilts fed HYP or high Ox/low calcium. Histologic changes in the kidneys were multiphasic, as expected with a feeding study. Inflammatory foci were identified in HYP, high Ox/low calcium, and control diet groups, making it a poor histologic marker of oxalate-associated disease. CONCLUSIONS: Both HYP and high Ox diets elicit hyperoxaluria, CaOx crystals, and renal morphologic changes consistent with human CaOx diseases. Pathological differences between dietary treatment groups, including crystal formation, fibrosis, inflammation, and tubular necrosis, were observed. Further studies will provide better insight of mechanisms underlying these observations and allow us to identify and delineate the suitability of each treatment for studying the different types of human CaOx-related conditions. Source of Funding: UW-Madison Clinical and Translational Science Award program, NCATS grant UL1TR000427 (awarded to KLP)