Trim37-deficient mice recapitulate several features of the multi-organ disorder Mulibrey nanism

Abstract

Mulibrey nanism (MUL) is a rare autosomal recessive multi-organ disorder characterized by severe prenatal-onset growth failure, infertility, cardiopathy, risk for tumors, fatty liver, and type 2 diabetes. MUL is caused by loss-of-function mutations in TRIM37, which encodes an E3 ubiquitin ligase belonging to the tripartite motif (TRIM) protein family and having both peroxisomal and nuclear localization. We describe a congenic Trim37 knock-out mouse (Trim37(-/-)) model for MUL. Trim37(-/-) mice were viable and had normal weight development until approximately 12 months of age, after which they started to manifest increasing problems in wellbeing and weight loss. Assessment of skeletal parameters with computer tomography revealed significantly smaller skull size, but no difference in the lengths of long bones in Trim37(-/-) mice as compared with wild-type. Both male and female Trim37(-/-) mice were infertile, the gonads showing germ cell aplasia, hilus and Leydig cell hyperplasia and accumulation of lipids in and around Leydig cells. Male Trim37(-/-) mice had elevated levels of follicle-stimulating and luteinizing hormones, but maintained normal levels of testosterone. Six-month-old Trim37(-/-) mice had elevated fasting blood glucose and low fasting serum insulin levels. At 1.5 years Trim37(-/-) mice showed non-compaction cardiomyopathy, hepatomegaly, fatty liver and various tumors. The amount and morphology of liver peroxisomes seemed normal in Trim37(-/-) mice. The most consistently seen phenotypes in Trim37(-/-) mice were infertility and the associated hormonal findings, whereas there was more variability in the other phenotypes observed. Trim37(-/-) mice recapitulate several features of the human MUL disease and thus provide a good model to study disease pathogenesis related to TRIM37 deficiency, including infertility, non-alcoholic fatty liver disease, cardiomyopathy and tumorigenesis.

Keywords: Cardiomyopathy; Fatty liver; Growth failure; Infertility; Mulibrey nanism; Tumorigenesis.

Fig. 1.

Weight and bone parameters in Trim37^−/− mice. (A) Weight development in Trim37^−/− and control mice, age 1 to 18 months; mean±s.d. and group size (n) at given age points 1, 6, 12 and 18 months. M+/+: 15.0±2.9, n=17; 31.4±3.7, n=16; 38.3±5.0, n=4; 41.7±6.3, n=4; M−/−: 15.1±2.5, n=12; 29.3±2.8, n=15; 32.8±7.9, n=6; 32.5±5.3, n=4; F+/+: 12.6±1.5, n=21; 23.1±2.2, n=14; 29.9±4.1, n=8; 32.5±4.8, n=8; F−/−: 12.3±1.6, n=21; 24.3±2.4, n=15; 28.9±3.3, n=8; 29.4±2.6, n=7. Early weight development of Trim37^−/− mice is normal in both males and females. The males start losing weight at the age of 6 months and the females at the age of 12 months. (B) CT scan images of skull and femurs showing the measurement of sagittal (yellow line) and coronal (green line) skull width and femur length (blue and orange lines). (C) Skull size of six-month-old Trim37^−/− mice is significantly smaller compared to control mice: sagittal width 9.6±0.1 mm vs 9.8±0.1 ng/ml and coronal width 13.7±0.2 mm vs 14.0±0.2 mm (mean±s.d.; P=0.01; t-test; n=10/genotype). No difference in the lengths of humeri, ulnae and femurs could be detected between Trim37^−/− and control mice. M, male; F, female.

Fig. 2.

Testicular findings in Trim37^−/− males. (A) Testicles of a 1.5-year-old Trim37^−/− mouse and a wild-type littermate. The size of Trim37^−/− testicles is reduced to approximately 30% of that of wild-type mice. (B) HE-stained sections demonstrating degeneration of germ cells and Leydig cell hyperplasia in Trim37^−/− mouse testis, starting at P13. Total germ cell aplasia is evident in one-month-old mice. (C) Adult Trim37^−/− mice show total germ cell aplasia and Leydig cell hyperplasia that is increasing with age. Leydig cells are marked with white arrowheads. (D) ORO stained testicular sections from 1.5-year-old mice, showing excessive intracellular and intercellular lipid accumulation around the area of Leydig cells in the Trim37^−/− mice testis. Scale bars: 20 µm.

Fig. 3.

Ovarian histology in Trim37^−/− females. (A) HE-stained sections demonstrating reduced number of oocytes in ovaries of Trim37^−/− mice at P30. Scale bars: 20 µm. (B) HE-stained sections of ovaries in adult mice showing germ cell aplasia and hilus cell hyperplasia in the Trim37^−/− mouse. One cyst (C) is visible in the 6-month-old Trim37^−/− female. Scale bar 100 µm. (C) Some Trim37^−/− mice presented with ovarian lipid cell (L) hyperplasia. Scale bars: 20 µm.

Fig. 4.

Non-compaction cardiomyopathy in Trim37^−/− mice. (A) HE-staining of heart sections show hypertrabeculation of the heart in six-month-old Trim37^−/− mice. Scale bars: 500 µm. (B) Endothelial staining (anti-CD34 immunostaining) is present in the trabeculations of the ventricular wall in Trim37^−/− mice indicative of non-compaction cardiomyopathy (arrowheads). Scale bars: 20 µm.

Fig. 5.

Hepatomegaly and fatty liver in Trim37^−/− mice. (A) Enlarged liver and difference in the liver color in a 22-month-old Trim37^−/− female (F) mouse observed in gross pathological examination. (B) HE-staining of liver sections demonstrating sinusoidal dilatation (white arrowheads) and vacuolization of Trim37^−/− liver. (C) Accumulation of neutral lipids in Trim37^−/− liver visualized by ORO staining. Scale bars: 50 µm.

Fig. 6.

Examples of macroscopic findings in pathological gross examination and tumor histology in Trim37^−/− mice. (A) Enlargement of spleen (arrowhead) in an 18-month-old male (M) mouse. The same mouse had lymphoma of the lymph nodes. (B) Enlarged thymus (arrowhead) in a 14-month-old female (F) mouse. The tumor was confirmed to be a neuroendocrine carcinoma in histological examination (J). (C) Enlarged pancreas (arrowhead) showing a granular appearance in an 18-month-old male mouse. In histological examination the pancreatic duct was shown to be cystic, but no tumor formation was present. (D) Abnormal tissue around the ovary (arrowhead) in a 22-month-old female mouse. In histological examination, the ovary was shown to be hemorrhagic and necrotic, possibly presenting ovarian torsion. (E) Ovarian tumor (arrowhead) in an 18-month-old female mouse. The tumor was in histological examination confirmed to be of Sertoli cell type (G). (F–K) HE-staining of tumor sections. Ovarian retiformic Sertoli-cell tumor, female 21 months (F). Ovarian Sertoli cell tumor, female 18 months (G). Ovarian Sertoli cell tumor, female 14 months (H). Primitive neuroendocrine liver tumor, female 21 months (I). Neuroendocrine carcinoma of the thymus, female 14 months (J). Lymphoma found in the abdomen, female 15 months (K). Scale bars: 100 µm. M, male; F, female.