Mutation of the Thap4 gene causes dwarfism and testicular anomalies in rats and mice.

The petit (pet) locus is associated with dwarfism, testicular anomalies, severe thymic hypoplasia, and high postnatal lethality, which are inherited in autosomal recessive mode of inheritance in rats with a Wistar strain genetic background. Linkage analysis localized the pet locus between 98.7 Mb and 101.2 Mb on rat chromosome 9. Nucleotide sequence analysis identified 2 bp deletion in exon 2 of the Thap4 gene as the causative mutation for pet. This deletion causes a frameshift and premature termination codon, resulting in a truncated THAP4 protein lacking approximately two-thirds of the C-terminal side. Thap4 is expressed in various organs, including the testis and thymus in rats. To elucidate the biological function of THAP4 in other species, we generated Thap4 knockout mice lacking exon 2 of the Thap4 gene through genome editing. Thap4 knockout mice also exhibited dwarfism and small testis but did not show high postnatal lethality. Thymus weights of adult Thap4 knockout male mice were significantly higher compared to wild-type male mice. Although Thap4 knockout male mice were fertile, their testis contained seminiferous tubules with spermatogenesis and degenerative seminiferous tubules lacking germ cells. Additionally, we observed vacuoles in seminiferous tubules, and clusters of cells in the lumen in seminiferous tubules in Thap4 knockout male mice. These results demonstrate that spontaneous mutation of Thap4 gene in rats and knockout of Thap4 gene in mice both cause dwarfism and testicular anomalies. Thap4 gene in rats and mice is essential for normal testicular development, maintaining spermatogenesis throughout the entire region of seminiferous tubules.

Molecular evolution of Cypridina noctiluca secretory luciferase for production of spectrum-shifted luminescence-emitting mutants and their application in nuclear receptor-reporter assays.

Luciferase is a popular enzyme used for biological analyses, such as reporter assays. In addition to a conventional reporter assay using a pair of firefly and Renilla luciferases, a simple multicolor reporter assay using multiple firefly or beetle luciferases emitting different color luminescence with a single substrate has been reported. Secretory luciferases have also been used for convenient sample preparation in reporter assays; however, reporter assay using secretory luciferase mutants that emit spectrum-shifted luminescence have not yet been reported. In this study, we generated blue- and red-shifted (-16 and 12 nm) luminescence-emitting Cypridina secretory luciferase (CLuc) mutants using multiple cycles of random and site-directed mutagenesis. Even for red-shifted CLuc mutant, which exhibited relatively low activity and stability, its enzymatic activity was sufficiently high for a luciferase assay (3.26 × 106 relative light unit/s), light emission was sufficiently prolonged (half-life is 3 min), and stability at 37°C was high. We independently determined the luminescence of these CLuc mutants using a luminometer with an optical filter. Finally, we replaced the commonly used reporters, firefly and Renilla luciferases used in a conventional nuclear receptor-reporter assay with these CLuc mutants and established a secretory luciferase-based single-substrate dual-color nuclear receptor-reporter assay.

Muscle volume evaluation using 3DCT for congenital clubfoot.

BACKGROUND: In congenital clubfoot, the lower leg is very thin and the calf muscles are hypoplasic. However, there are few studies reporting real muscle volume. PURPOSE: The purpose of this study is to assay the muscle volume in congenital clubfoot using 3DCT and to quantify the degree of the hypoplasia. MATERIAL AND METHODS: From January 2015 to December 2016, nine consecutive patients, seven male and two female, with unilateral congenital clubfeet were recruited for CT scans. Axial transverse sectional CT scans were acquired from the delineation of the fibular head to the tibial plafond. From the data, we rendered the entire muscle in 3D for muscle volume assay, and further segmented the posterior musculature for comparison between the normal and affected sides. RESULTS: The whole muscle volume on the normal side was 291.23 cm3 (181.23-593.49) and that on the affected side was 225.08 cm3 (120.71-429.08), for an affected side to normal side ratio of 0.79 (0.72-0.9), which was significantly smaller (p < .01). Posterior muscle volume on the normal side was 175.81 cm3 (103.72-376.32) and that on the affected side was 106.52 cm3 (58.3-188.39). The ratio of posterior muscle to whole muscle on the normal side was 0.62 (0.46-0.75), and that on the affected side was 0.48 (0.4-0.55), such that the affected side was significantly smaller (p < .01). CONCLUSION: This study contributes quantitative data supporting the longstanding observations that the posterior calf muscles are significantly smaller on the affected side compared to the normal side in congenital clubfoot, and further underscores the importance of the extending the excursion of these muscles.

Characterization of Enlarged Kidneys and Their Potential for Inducing Diabetes in DEK Rats.

The kidneys participate in the regulation of systemic glucose metabolism via gluconeogenesis, insulin degradation, and the tubular reabsorption of glucose. The present study characterized rats from a strain of a novel type 2 diabetes model with enlarged kidneys (DEK). Histological and biochemical analyses of DEK rats were performed to assess the relationships between their kidneys and hyperglycemia. The kidney weight of diabetic DEK (DEK-DM) gradually increased over time from the onset of diabetes, with the glomerular number being higher in DEK-DM than in normal DEK (DEK-cont). A positive correlation between blood glucose level and kidney weight was observed in DEK-DM. The similar glomerular size and single glomerular creatinine clearance in DEK-cont and DEK-DM indicated that glomerular hypertrophy and hyperfiltration were not involved in the renal enlargement. Uninephrectomy (1/2Nx) in DEK-DM resulted in a reduction in blood glucose level at 7-28 post-operation days, with this concentration remaining lower than in Sham group until 84 days post-operation. 1/2Nx also improved systemic conditions, including reduced body weight gain, polyuria, polydipsia, and hyperphagia. Plasma concentrations of Na, total cholesterol, albumin, and total protein were higher, and urinary excretion of glucose, urea nitrogen, and proteins were lower, in the 1/2Nx than in the Sham group. Remnant kidney weight was two-fold higher in the 1/2Nx than in the Sham group 84 days later. In addition, 1/2Nx resulted in renal tubular dilatation but not in the progression of fibrosis or glomerular lesions. Taken together, these findings indicate that enlarged kidneys were associated with the onset of diabetes and with the resistance to diabetic nephropathy in DEK-DM.

Hyaloklossia Labbé, 1896 (Alveolata: Apicomplexa) in frogs: Description of a new species and proposing a new subfamily to accommodate these enigmatic parasites.

Hyaloklossia Labbé ,1896 (Alveolata: Apicomplexa) is a monotypic genus of renal coccidia found in anurans, particularly in the edible frog Pelophylax kl. esculentus (Amphibia: Anura: Ranidae), distributed in different parts of Europe. Here we propose a new Hyaloklossia species from the Tokyo daruma pond frog, Pelophylax porosus porosus. The coccidium detected in the renal tissue of P. p. porosus shared some morphological characteristics with the type species, Hyaloklossia lieberkuehni (Labbé, 1894), reported from P. kl. esculentus. However, in addition to size differences in several oocyst and sporocyst features between these parasites, phylogenetic analysis of gene fragments from two nuclear ribosomal loci and the mitochondrial cytochrome c oxidase subunit 1, exposed distinct genetic differences between H. lieberkuehni and our new species. Although our analysis validated the monophyly of Hyaloklossia with some members of the Toxoplasmatinae Biocca, 1957, Cystoisosporinae Frenkel et al., 1987, and Eumonosporinae Chou et al., 2021 (Sarcocystidae Poche, 1913), comparison of genetic differences between Hyaloklossia species from P. p. porosus and H. lieberkuehni revealed the presence of a greater number of polymorphisms than that observed when comparing inter-species (Heydornia spp., Besnoisita spp.) or inter-genus (Toxoplasma vs. Neospora, Neospora vs. Hammondia, and Neospora vs. Heydornia) variabilities among members of the Sarcocystidae. This indicates that Hyaloklossia, as re-erected and defined by Modrý et al. (2001, Int. J. Syst. Evol. Microbiol. 51, 767-772), with its homoxenous life cycle, requires placement in its own subfamily. Thus, we propose a new subfamily, Hyaloklossiinae n. subfam., to accommodate two species, H. lieberkuehni from Europe and Hyaloklossia kasumienesis n. sp. which we describe here from P. p. porosus in Japan.

The impact of self-efficacy for physical activity on health-related quality of life in total knee arthroplasty recipients.

BACKGROUND: Some patients with end-stage osteoarthritis of the knee remain unsatisfied after total knee arthroplasty (TKA). We postulated that to increase satisfaction, self-efficacy (SE) for physical activity should receive more attention in rehabilitative intervention, alongside the management of patient expectations, pain, and function. OBJECTIVE: We examined the relative impact of Physical Activity SE on Health-Related Quality of Life (HRQOL) alongside other factors such as pain and physical function which are well-addressed by current interventions. METHODS: One hundred and six first-TKA recipients (15 Male/91 Female, age 73.6 ± 7.2) were evaluated at 3 and 6 months post-operatively using the Medical Outcomes Study 36-Item Health Survey (SF-36v2) for HRQOL, knee extension strength measurement, Timed Up and Go test (TUG), One Leg Standing time test (OLS), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) for pain and function, and an instrument for measuring Physical Activity SE among the frail elderly in Japan. RESULTS: Significant improvement over pre-operative values was found at 3 and 6 months in TUG, OLS, WOMAC Pain and Function, and the 8 subscales of the SF-36v2. Factors found to significantly impact SF-36v2 subscale scores at 6 months post-operatively were found to be knee pain, knee function, and SE for physical activity. CONCLUSION: These results support our postulation that interventions to improve SE for physical activity could have comparable impact alongside interventions for knee pain and knee function, on the advancement of HRQOL among TKA recipients.

Empagliflozin ameliorates symptoms of diabetes and renal tubular dysfunction in a rat model of diabetes with enlarged kidney (DEK).

BACKGROUND: Sodium-glucose cotransporter 2 (SGLT2) inhibitors are widely used to reduce hyperglycemia. The present study investigated the effects of a SGLT2 inhibitor, empagliflozin, on hyperglycemia in a novel rat model of non-obesity type 2 diabetes with enlarged kidney (DEK). METHODS: Male DEK rats with non-fasting blood glucose concentrations ≤300 mg/dl and >300 mg/dl were classified as nondiabetic and diabetic, respectively. Groups of nondiabetic (control) and diabetic (DM-cont) rats were fed standard chow for 12 weeks, whereas another group of diabetic (DM-empa) rats was fed standard chow containing empagliflozin (300 mg/kg/day) for 12 weeks. Blood glucose, body weight, glucose tolerance, food and water intake, urinary volume, plasma and urinary biochemical parameters, and bone mineral density were measured, and their kidneys and pancreas histologically analyzed. RESULTS: Treatment with empagliflozin reduced blood glucose concentration and food intake in diabetic rats, but inhibited loss of adeps renis and led to body weight gain. Empagliflozin attenuated polyuria and polydipsia but increased plasma concentrations of total cholesterol, sodium and total protein toward normal level. Empagliflozin also significantly reduced urinary excretion of proteins and electrolytes and restored bone mineral density and plasma concentrations of valine and isoleucine to normal levels. Moreover, dilation of renal tubules and kidney enlargement were not attenuated in the DM-empa group. CONCLUSION: The response of DEK rats to empagliflozin differed from that of other diabetic animal models, suggesting that DEK rats have unique characters for studying and evaluating the multiple biological effects of SGLT2 inhibitors. These findings also indicted that empagliflozin could ameliorate systemic metabolism and improve renal tubule function in diabetic condition.

Cellular Expression and Subcellular Localization of Wwox Protein During Testicular Development and Spermatogenesis in Rats.

A well-known putative tumor suppressor WW domain-containing oxidoreductase (Wwox) is highly expressed in hormonally regulated tissues and is considered important for the normal development and function of reproductive organs. In this study, we investigated the cellular and subcellular localization of Wwox in normal testes during postnatal days 0-70 using Western blotting and immunohistochemistry. Wwox is expressed in testes at all ages. Immunohistochemistry showed that fetal-type and adult-type Leydig cells, immature and mature Sertoli cells, and germ cells (from gonocytes to step 17 spermatids) expressed Wwox except peritubular myoid cells, step 18-19 spermatids, and mature sperm. Wwox localized diffusely in the cytoplasm with focal intense signals in all testicular cells. These signals gradually condensed in germ cells with their differentiation and colocalized with giantin for cis-Golgi marker and partially with golgin-97 for trans-Golgi marker. Biochemically, Wwox was detected in isolated Golgi-enriched fractions. But Wwox was undetectable in the nucleus. This subcellular localization pattern of Wwox was also confirmed in single-cell suspension. These findings indicate that Wwox is functional in most cell types of testis and might locate into Golgi apparatus via interaction with Golgi proteins. These unique localizations might be related to the function of Wwox in testicular development and spermatogenesis.

Loss of Wwox Perturbs Neuronal Migration and Impairs Early Cortical Development.

Mutations in the WWOX gene cause a broad range of ultra-rare neurodevelopmental and brain degenerative disorders, associated with a high likelihood of premature death in animal models as well as in humans. The encoded Wwox protein is a WW domain-containing oxidoreductase that participates in crucial biological processes including tumor suppression, cell growth/differentiation and regulation of steroid metabolism, while its role in neural development is less understood. We analyzed the exomes of a family affected with multiple pre- and postnatal anomalies, including cerebellar vermis hypoplasia, severe neurodevelopmental impairment and refractory epilepsy, and identified a segregating homozygous WWOX mutation leading to a premature stop codon. Abnormal cerebral cortex development due to a defective architecture of granular and molecular cell layers was found in the developing brain of a WWOX-deficient human fetus from this family. A similar disorganization of cortical layers was identified in lde/lde rats (carrying a homozygous truncating mutation which disrupts the active Wwox C-terminal domain) investigated at perinatal stages. Transcriptomic analyses of Wwox-depleted human neural progenitor cells showed an impaired expression of a number of neuronal migration-related genes encoding for tubulins, kinesins and associated proteins. These findings indicate that loss of Wwox may affect different cytoskeleton components and alter prenatal cortical development, highlighting a regulatory role of the WWOX gene in migrating neurons across different species.

Suture anchor stabilization of symptomatic accessory navicular in adolescents: Clinical and radiographic outcomes.

BACKGROUND: Screw fixation used in modified Kidner procedures to treat persistent symptomatic accessory navicular in adult cases is often challenging in adolescent cases with a small accessory fragment. The present study aimed to document the clinical effect of a suture anchor stabilization technique applicable to such cases where osteosynthesis is considered an ideal outcome. METHODS: Consecutive clinical cases who received this surgical treatment from 2009 to 2016 were retrospectively reviewed. The focus of interest included radiographic union of the accessory bone, changes in symptoms evaluated using a validated clinical outcome scale introduced by the Japanese Society for Surgery of the Foot, and changes in the medial arch bony alignment measured in lateral weight-bearing plain radiographs. RESULTS: Twenty-two feet in 15 individuals (11 females and 4 males, age at surgery 10-16 years) were identified. In 14 feet (64%), radiographic bone union was confirmed within 8 weeks postoperatively. At the final follow-up ranging 12-51 months postoperation, the clinical scores have significantly improved (p < 0.001) to 96 ± 5.71 (mean ± standard deviation, range 87-100), from 54 preoperatively. Radiographic measurements revealed significant postoperative increase of the sagittal talar tilt angle (p < 0.001, increment 4 ± 3°, range 0-11) and the talo-first metatarsal angle (p < 0.001, increment 5 ± 4°, range 0-12). No significant changes were identified in the calcaneal pitch angle, first metatarsal tilt angle, calcaneo-navicular angle, and the navicular height. CONCLUSION: Despite the modest bone union rate, the clinical outcomes suggest distinct symptom-relieving effect, at least in the short- to midterm, while the radiographic measurements suggest positive biomechanical effects. The present suture-anchor stabilization concept appears to be a promising treatment option for persistent symptomatic accessory navicular in adolescent cases.

Factors influencing life-space mobility change after total knee arthroplasty in patients with severe knee osteoarthritis.

[Purpose] The purpose of this study was to identify the factors influencing change in life-space mobility after total knee arthroplasty (TKA) in patients with severe knee osteoarthritis (knee OA). [Participants and Methods] Overall, 58 primary unilateral TKA recipients (9 males and 49 females; age ± SD 74.6 ± 6.5 years) were enrolled. We evaluated Life-Space Assessment (LSA) scores, knee extensor strength, Timed Up and Go test (TUG), one-leg standing time (OLS), Western Ontario and McMaster Universities osteoarthritis Index, and physical activity self-efficacy (SE) before surgery and at 3 months post-operation. [Results] Life space mobility significantly expanded 3 months after surgery compared with preoperative baseline. Preoperatively, walking SE and knee extensor muscle strength on the operative side were found to have strong correlation with LSA scores, while stairs SE and knee extensor muscle strength of the operative side were correlated at 3 months post-operation. [Conclusion] These findings suggest that to expand the life-space mobility of TKA recipients, it is important to enhance self-efficacy for general physical activity in addition to strengthening the quadriceps muscles.

Characterization of Novel Nonobese Type 2 Diabetes Rat Model with Enlarged Kidneys.

A variety of animal models of diabetes mellitus (DM) are required to study the genetics and pathophysiology of DM. We established a novel rat strain showing nonobese type 2 diabetes with enlarged kidneys from the LEA.PET-pet congenic strain and named it Diabetes with Enlarged Kidney (DEK). The body growth of DEK affected rats was similar to that of normal rats before the development of DM but was attenuated with the deterioration of DM. There was a marked difference in the etiology of DEK by gender: DM phenotypes including polyuria, polydipsia, and hyperglycemia (nonfasting blood glucose over 300 mg/dl) were found in male rats aged over 10 weeks but not in female rats. The cumulative incidence of DM in DEK males at the age of 30 weeks was 44.8%. Oral glucose tolerance tests showed glucose intolerance and decreased insulin secretion in response to glucose loading in affected males, features which were exacerbated with age. Affected males exhibited disorganized architecture of pancreatic islets, decreased numbers of ß cells, and markedly decreased expression of insulin, despite no pathological findings of hemorrhage or infiltration of inflammatory cells in the pancreatic islet. Age-related islet fibrosis appeared similar in normal and affected males. Affected males also showed enlarged kidneys with dilation of renal tubules in both the cortex and medulla, but no obvious glomerular lesions typical of diabetic nephropathy (DN) at the age of 30 weeks. Plasma levels of urea nitrogen and creatinine were normal, but hypoalbuminemia was detected. These pathophysiological features in affected males indicated that their renal function was almost maintained despite severe DM. Taken together, these findings indicate that the affected males of the DEK strain are a novel nonobese type 2 diabetes rat model useful for studying the mechanisms underlying ß cell loss and identifying genetic factors protective against DN.

Loss of Wwox Causes Defective Development of Cerebral Cortex with Hypomyelination in a Rat Model of Lethal Dwarfism with Epilepsy.

WW domain-containing oxidoreductase (Wwox) is a putative tumor suppressor. Several germline mutations of Wwox have been associated with infant neurological disorders characterized by epilepsy, growth retardation, and early death. Less is known, however, about the pathological link between Wwox mutations and these disorders or the physiological role of Wwox in brain development. In this study, we examined age-related expression and histological localization of Wwox in forebrains as well as the effects of loss of function mutations in the Wwox gene in the immature cortex of a rat model of lethal dwarfism with epilepsy (lde/lde). Immunostaining revealed that Wwox is expressed in neurons, astrocytes, and oligodendrocytes. lde/lde cortices were characterized by a reduction in neurite growth without a reduced number of neurons, severe reduction in myelination with a reduced number of mature oligodendrocytes, and a reduction in cell populations of astrocytes and microglia. These results indicate that Wwox is essential for normal development of neurons and glial cells in the cerebral cortex.

3D Talar Kinematics During External Rotation Stress Testing in Hindfoot Varus and Valgus Using a Model of Syndesmotic and Deep Deltoid Instability.

BACKGROUND: External rotation stress (ERS) identifies ankle instability after fibular reduction of rotational ankle injuries. Combined hindfoot and ankle motions and an inconsistent starting position could mask differing degrees of instability resulting from syndesmotic and/or deltoid ligament disruption. The goal of this work was to use full 3D talar kinematics to evaluate the effects of hindfoot orientation and foot starting position during ERS on the ability to detect instability caused by ligament disruptions. METHODS: Six cadaveric ankles with metallic fiducial markers were CT scanned in neutral and 3 stress positions: varus hindfoot internal rotation stress (IRS-var), valgus hindfoot ERS (ERS-val), and varus hindfoot ERS (ERS-var). Scans were obtained in stress positions after transecting the deep deltoid ligament (tDDL) and then the syndesmotic ligaments (tDDL+Syn). Talar rotations and translations were computed in the axial, coronal, and sagittal planes in each stress position. Changes in a fixed center of rotation (CoR) relative to the intact sequence were calculated. RESULTS: Axial plane rotation beginning from IRS-var increased significantly for each level of ligamentous instability (P < .05 for all conditions) (10.9 degrees, intact; 14.1 degrees, tDDL; 22.7 degrees, tDDL+Syn during ERS-val; and 16.4 degrees, intact; 23.1 degrees, tDDL; 29.9 degrees, tDDL+Syn during ERS-var). With ERS-val, the talar CoR moved medially (3.6-5.4 mm) and posteriorly (0.5-5.2 mm); ERS-var moved anterior/laterally or posterior/medially depending on the specific ligamentous instability. With tDDL+Syn the ankle became grossly unstable and there were no clear trends in sagittal/coronal rotation or translation. CONCLUSION: An ERS test from internal to external rotation consistently differentiates between normal, tDDL, and tDDL+Syn. Talar CoR moved outside the mortise with ligamentous instability. CLINICAL RELEVANCE: Significant residual deep deltoid instability is likely underrecognized with current practice. The most discriminatory test for detecting such instability in our laboratory was an ERS test performed by internally rotating the foot to a hard, bony endpoint, positioning the hindfoot in varus, and then performing the entire external rotation maneuver while maintaining the varus hindfoot position.

Effects of knockout of the receptor for advanced glycation end-products on bone mineral density and synovitis in mice with intra-articular fractures.

Our group employed the mouse closed intra-articular fracture (IAF) model to test the hypothesis that the innate immune system plays a role in initiating synovitis and post-traumatic osteoarthritis (PTOA) in fractured joints. A transgenic strategy featuring knockout of the receptor for advanced glycation end-products (RAGE -/- ) was pursued. The 42 and 84 mJ impacts used to create fractures were in the range previously reported to cause PTOA at 60 days post-fracture. MicroCT (µCT) was used to assess fracture patterns and epiphyseal and metaphyseal bone loss at 30 and 60 days post-fracture. Cartilage degeneration, synovitis, and matrix metalloproteinase (MMP-3, -13) expression were evaluated by histologic analyses. In wild-type mice, µCT imaging showed that 84 mJ impacts led to significant bone loss at 30 days (p < 0.05), but recovered to normal at 60 days. Bone losses did not occur in RAGE-/- mice. Synovitis was significantly elevated in 84 mJ impact wild-type mice at both endpoints (30 day, p = 0.001; 60 day, p = 0.05), whereas in RAGE-/- mice synovitis was elevated only at 30 days (p = 0.02). Mankin scores were slightly elevated in both mouse strains at 30 days, but not at 60 days. Immunohistochemistry revealed significant fracture-related increases in MMP-3 and -13 expression at 30 days (p < 0.05), with no significant difference between genotypes. These findings indicated that while RAGE -/- accelerated recovery from fracture and diminished synovitis, arthritic changes were temporary and too modest to detect an effect on the pathogenesis of PTOA. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2439-2449, 2018.

Giantin is required for coordinated production of aggrecan, link protein and type XI collagen during chondrogenesis.

Extracellular matrix (ECM) constitutes a proper micro-environment for cell proliferation, migration and differentiation, as well as playing pivotal roles in developmental processes including endochondral ossification. Cartilage ECM is mainly composed of fibrous proteins, including collagen, proteoglycan, and hyaluronan. Because almost all ECM components are transported by intracellular vesicular transport systems, molecules that mediate vesicle transport are also important for endochondral ossification. Giantin, encoded by the Golgb1 gene, is a tethering factor for coatomer 1 (COPI) vesicles and functions in the cis-medial Golgi compartments. An insertion mutation in the Golgb1 gene, resulting in a lack of giantin protein expression, has been detected in ocd/ocd rats that exhibit a pleiotropic phenotype including osteochondrodysplasia. To reveal the function of giantin in chondrogenesis, the present study assessed the effects of loss of giantin expression on cartilage ECM and Golgi morphology. Giantin was expressed in normal, but not in ocd/ocd, chondrocytes in the epiphyseal areas of embryonic femurs, whereas GM130 was expressed in both normal and ocd/ocd chondrocytes. The staining intensities of safranin O and azan (aniline blue) were reduced and enhanced, respectively, in epiphyseal cartilage of ocd/ocd femurs. Immunostaining showed that levels of type II collagen and fibronectin were comparable in normal and ocd/ocd cartilage. Levels of type XI collagen were higher, while levels of aggrecan, link protein and hyaluronan were lower, in ocd/ocd than in normal cartilage, although semi-quantitative RT-PCR showed similar levels of type XI collagen, aggrecan and link protein mRNAs in normal and ocd/ocd cartilage. Isolated chondrocytes of ocd/ocd and normal rats showed similar immunostaining patterns for cis-, medial-, and trans-Golgi marker proteins, whereas monolayers of ocd/ocd chondrocytes showed reduced levels of aggrecan and link protein and increased level of type XI collagen in spite of similar transcripts levels. These findings suggest that giantin plays a pivotal role in coordinated production of aggrecan, link protein and type XI collagen in chondrocytes, and that loss of giantin causes osteochondrodysplasia with disturbance of these ECM components.

Critical roles of Astrin in the mitosis of immature rat Sertoli cells.

Male hypogonadism (hgn/hgn) rats show testicular hypoplasia accompanied by dysplastic development of seminiferous tubules due to loss-of-function mutation of the gene encoding Astrin, which is required for mitotic progression in the division cycle of HeLa cells. In the present study, we examined the cytological base leading to the decrease of Sertoli cells in hgn/hgn testes. In hgn/hgn testes on postnatal day 3, anti-phospho-histone H3 (Ser10) (pH3)-positive mitotic phase and TUNEL-positive apoptosis increased in GATA4-positive Sertoli cells. Isolated immature Sertoli cells from hgn/hgn testes showed increased pH3-assessed mitotic index accompanied by decreased 5-bromo-2'-deoxyuridine-incorporation and increased TUNEL-positive apoptosis, suggesting mitotic delay and cell death. In the visualization of mitotic progression by nocodazole (NOC)-mediated cell cycle arrest and subsequent release, hgn/hgn rat-derived Sertoli cells failed to make the transition from prometaphase to metaphase, and the cells with micronuclei and TUNEL-positive cells gradually increased in a time-dependent manner. Western blot analysis detected ≈142 kDa protein expected as Astrin in extracts of +/+ and +/hgn testes and cultured normal Sertoli cells but not in extracts of hgn/hgn testes. CLASP1 was detected in extracts of both normal and hgn/hgn testes, whereas it was localized in kinetochore of normal mitotic Sertoli cells but diffused in cytoplasm of hgn/hgn Sertoli cells. These results indicate that Astrin is required for normal mitotic progression in immature Sertoli cells and that the most severe type of testicullar dysplasia in hgn/hgn rats is caused by mitotic cell death of immature Sertoli cells due to lack of Astrin.

Progression of renal fibrosis in congenital CKD model rats with reduced number of nephrons.

A congenital reduction in the number of nephrons is a critical risk factor for both onset of chronic kidney disease (CKD) and its progression to end-stage kidney disease (ESKD). Hypoplastic kidney (HPK) rats have only about 20% of the normal number of nephrons and show progressive CKD. This study used an immunohistological method to assess glomerular and interstitial pathogenesis in male HPK rats aged 35-210days. CD68 positive-macrophages were found to infiltrate into glomeruli in HPK rats aged 35 and 70days and to infiltrate into interstitial tissue in rats aged 140 and 210days. HPK rats aged 35 and 70days showed glomerular hypertrophy, loss of normal linear immunostaining of podocine, and increased expression of PDGFr-ß, TGF-ß, collagens, and fibronectin, with all of these alterations gradually deteriorating with age. α-SMA-positive myofibroblasts were rarely detected in glomerular tufts, whereas α-SMA-positive glomerular parietal epithelium (GPE) cells were frequently observed along Bowman's capsular walls. The numbers of PDGFr-ß-positive fibroblasts in interstitial tissue were increased in rats aged 35days and older, whereas interstitial fibrosis, characterized by the increased expression of tubular PDGF-BB, the appearance of myofibroblasts doubly positive for PDGFr-ß and α-SMA, and increased expression of collagens and fibronectin, were observed in rats aged 70 and older. These results clearly indicate that congenital CKD with only 20% of nephrons cause renal fibrosis in rats.

Time-dependent loss of mitochondrial function precedes progressive histologic cartilage degeneration in a rabbit meniscal destabilization model.

The goals of this work were to characterize progression of osteoarthritic cartilage degeneration in a rabbit medial meniscus destabilization (MMD) model and then to use the model to identify pre-histologic disruptions in chondrocyte metabolism under chronically elevated joint contact stresses in vivo. To characterize PTOA progression, 24 rabbits received either MMD or sham surgery. Limb loading was analyzed preoperatively and at regular postoperative intervals using a Tekscan pressure-sensitive walkway. Animals were euthanized 8 (n = 8 MMD; n = 8 sham) or 26 weeks (n = 8 MMD) postoperatively for histological cartilage evaluation by an objective, semi-automated Mankin scoring routine. To examine pre-histologic pathology, MMD was performed on an additional 20 rabbits, euthanized 1 (n = 9) or 4 weeks (n = 10) postoperatively. Chondrocytes were harvested fresh for measurement of mitochondrial function, an intracellular indicator of pathology after mechanical injury. Both MMD and sham surgery caused slight decreases in limb loading which returned to preoperative levels after 2 weeks. Histologically apparent cartilage damage progressed from 8 to 26 weeks after MMD. Changes in chondrocyte respiration were variable at 1 week, but by 4 weeks postoperatively chondrocyte mitochondrial function was significantly reduced. Many human injuries that lead to PTOA are relatively mild, and the cell-level mechanisms leading to disease remain unclear. We have documented PTOA progression in an animal model of subtle joint injury under continued use, and demonstrated that this model provides a realistic environment for investigation of multi-stage cellular pathology that develops prior to overt tissue degeneration and which could be targeted for disease modifying treatments. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:590-599, 2017.