Nathaniel Mills

Effects of Thyroid Hormone on Leydig Cell Regeneration in the Adult Rat Following Ethane Dimethane Sulphonate Treatment

Abstract We tested the effects of thyroid hormone on Leydig cell (LC) regeneration in the adult rat testis after ethane dimethyl sulphonate (EDS) treatment. Ninety-day-old, thyroid-intact (n = 96) and thyroidectomized (n = 5) male Sprague-Dawley rats were injected intraperitoneally (single injection) with EDS (75 mg/kg) to destroy LC. Thyroid-intact, EDS-treated rats were equally divided into three groups (n = 32 per group) and treated as follows: control (saline-injected), hypothyroid (provided 0.1% propyl thiouracil in drinking water), and hyperthyroid (received daily subcutaneous injections of tri-iodothyronine, 100 μg/kg). Testing was done at Days 2, 7, 14, and 21 for thyroid-intact rats and at Day 21 for thyroidectomized rats after the EDS treatment. Leydig cells were absent in control and hyperthyroid rats at Days 2, 7, and 14; in hypothyroid rats at all ages; and in thyroidectomized rats at Day 21. The LC number per testis in hyperthyroid rats was twice as those of controls at Day 21. 3β-Hydroxysteroid dehydrogenase (LC marker) immunocytochemistry results agreed with these findings. Mesenchymal cell number per testis was similar in the three treatment groups of thyroid-intact rats on Days 2 and 7, but it was different on Days 14 and 21. The highest number was in the hypothyroid rats, and the lowest was in the hyperthyroid rats. Serum testosterone levels could be measured in control rats only on Day 21, were undetectable in hypothyroid rats at all stages, and were detected in hyperthyroid rats on Days 14 and 21. These levels in hyperthyroid rats were twofold greater than those of controls on Day 21. Serum androstenedione levels could be measured only in the hyperthyroid rats on Day 21. Testosterone and androstenedione levels in the incubation media showed similar patterns to those in serum, but with larger values. These findings indicate that hypothyroidism inhibits LC regeneration and hyperthyroidism results in accelerated differentiation of more mesenchymal cells into LC following the EDS treatment. The observations of the EDS-treated, thyroidectomized rats confirmed that the findings in hypothyroid rats were, indeed, due to the deficiency of thyroid hormone.

Alternating Magnetic Field Controlled, Multifunctional Nano-Reservoirs: Intracellular Uptake and Improved Biocompatibility

Biocompatible magnetic nanoparticles hold great therapeutic potential, but conventional particles can be toxic. Here, we report the synthesis and alternating magnetic field dependent actuation of a remotely controllable, multifunctional nano-scale system and its marked biocompatibility with mammalian cells. Monodisperse, magnetic nanospheres based on thermo-sensitive polymer network poly(ethylene glycol) ethyl ether methacrylate-co-poly(ethylene glycol) methyl ether methacrylate were synthesized using free radical polymerization. Synthesized nanospheres have oscillating magnetic field induced thermo-reversible behavior; exhibiting desirable characteristics comparable to the widely used poly-N-isopropylacrylamide-based systems in shrinkage plus a broader volumetric transition range. Remote heating and model drug release were characterized for different field strengths. Nanospheres containing nanoparticles up to an iron concentration of 6 mM were readily taken up by neuron-like PC12 pheochromocytoma cells and had reduced toxicity compared to other surface modified magnetic nanocarriers. Furthermore, nanosphere exposure did not inhibit the extension of cellular processes (neurite outgrowth) even at high iron concentrations (6 mM), indicating minimal negative effects in cellular systems. Excellent intracellular uptake and enhanced biocompatibility coupled with the lack of deleterious effects on neurite outgrowth and prior Food and Drug Administration (FDA) approval of PEG-based carriers suggest increased therapeutic potential of this system for manipulating axon regeneration following nervous system injury.

A mouse model for nonalcoholic steatohepatitis

BACKGROUND Nonalcoholic steatohepatitis (NASH) is a hepatic manifestation of the growing metabolic syndrome epidemic that could progress to cirrhosis. Animal models adequately mimicking this condition in humans are scanty. AIM The objective of our study was to investigate whether high-fat diets (HFD) with adequate methionine and choline levels can induce pathophysiological features typical of human NASH in C57BL/6J mice. METHODS Forty C57BL/6J mice, divided into control and high-fat (HF) groups, were fed low-fat diet and HFD, ad libitum respectively for 20 weeks. At the end of 20 weeks, animals were sacrificed and assays were performed for blood biomarkers typical of human NASH. Adipose tissue depots were collected and liver samples were processed for histological examination. RESULTS High-fat feeding led to increased triglyceride accumulation in the liver (8.9 μmol/100 mg liver tissue vs. 2.6 μmol/100 mg for control) and induced histopathological features of human NASH including hepatic steatosis, ballooning inflammation and fibrosis. Expressions of proteins and chemokines predominant in NASH including collagens I, III and IV and platelet derived growth factor (PDGF) A and B were significantly higher in animals fed the HFD. Liver enzymes alanine transaminase, aspartate transaminase and alkaline phosphatase were significantly (P<.05) elevated in the HF group compared to controls. Mice on HFD also developed hyperglycemia, hyperinsulinemia, hypoadiponectinemia along with elevated tumor necrosis factor α, resistin, leptin, free fatty acids, transforming growth factor β and malondialdehyde levels that characterize NASH in humans. CONCLUSION Long-term HF feeding with adequate methionine and choline can induce many of the pathophysiological features typical of human NASH in C57BL/6J mice.

Role of engineered nanocarriers for axon regeneration and guidance: current status and future trends.

There are approximately 1.5 million people who experience traumatic injuries to the brain and 265,000 who experience traumatic injuries to the spinal cord each year in the United States. Currently, there are few effective treatments for central nervous system (CNS) injuries because the CNS is refractory to axonal regeneration and relatively inaccessible to many pharmacological treatments. Smart, remotely tunable, multifunctional micro- and nanocarriers hold promise for delivering treatments to the CNS and targeting specific neurons to enhance axon regeneration and synaptogenesis. Furthermore, assessing the efficacy of treatments could be enhanced by biocompatible nanovectors designed for imaging in vivo. Recent developments in nanoengineering offer promising alternatives for designing biocompatible micro- and nanovectors, including magnetic nanostructures, carbon nanotubes, and quantum dot-based systems for controlled release of therapeutic and diagnostic agents to targeted CNS cells. This review highlights recent achievements in the development of smart nanostructures to overcome the existing challenges for treating CNS injuries.

Excellent biocompatibility of semiconductor quantum dots encased in multifunctional poly(N-isopropylacrylamide) nanoreservoirs and nuclear specific labeling of growing neurons

Quantum dots (QDs) have received attention for labeling biomolecules; however, toxicity of these nanostructures in the intracellular environment has prevented a biomedical breakthrough. Here we report biocompatibility of a QD based multifunctional system on neuronal cells. Moreover, the designed nanostructures bind with high affinity in the cell nucleus. Nucleus specific binding and enhanced biocompatibility, coupled with no deleterious effects on neurite outgrowth, even at high dosages (500 μg/ml sphere conc.) suggest increased therapeutic potential of this system for specific targeting followed by controlled release of drugs in treating neurodegenerative disorders.

Expression and Regulation of Lipocalin-Type Prostaglandin D Synthase in Rat Testis and Epididymis

Abstract Lipocalin-type prostaglandin D synthase (L-PGDS), a bifunctional protein, is expressed in the male reproductive organs of many species. However, the expression and regulation of L-PGDS in rat are still uncertain. The present study investigated the regionalization and regulation of L-PGDS expression in rat testis and epididymis by in situ hybridization and immunohistochemistry under the conditions of sexual maturation, castration, and ethylene dimethane sulfonate (EDS) treatments. In sexually mature rats, L-PGDS mRNA was weakly expressed only in the testicular peritubular cells, whereas L-PGDS immunostaining was highly detected in the Leydig cells by Day 70 postpartum. During sexual maturation, L-PGDS mRNA expression was highly detected in the caput, corpus, and cauda of the epididymis 70 days after birth. Compared with normal L-PGDS expression in adult epididymis, both L-PGDS mRNA expression and protein immunostaining were significantly reduced in the caput, corpus, and cauda epididymis after castration. Testosterone propionate treatment induced a significant increase of L-PGDS expression in the epididymis of castrated rats. Compared with adult rat epididymis, L-PGDS mRNA and protein expression was down-regulated after EDS treatment. Testosterone propionate treatment could induce an increase of L-PGDS mRNA and protein expression in the epididymis of EDS-treated rats. In conclusion, both castration and EDS treatments caused a significant decrease of L-PGDS expression in the epididymis, whereas testosterone propionate treatment could induce an increase of L-PGDS expression in the epididymis of both castrated and EDS-treated rats, indicating that L-PGDS expression in the rat epididymis can be up-regulated by testosterone.

Mevalonate depletion mediates the suppressive impact of geranylgeraniol on murine B16 melanoma cells

The diterpene geranylgeraniol (all trans-3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraen-1-ol) suppresses the growth of human liver, lung, ovary, pancreas, colon, stomach and blood tumors with undefined mechanisms. We evaluated the growth-suppressive activity of geranylgeraniol in murine B16 melanoma cells. Geranylgeraniol induced dose-dependent suppression of B16 cell growth (IC50 = 55 ± 13 µmol/L) following a 48-h incubation in 96-well plates. Cell cycle arrest at the G1 phase, manifested by a geranylgeraniol-induced increase in the G1/S ratio and decreased expression of cyclin D1 and cyclin-dependent kinase 4, apoptosis detected by Guava Nexin™ assay and fluorescence microscopy following acridine orange and ethidium bromide dual staining, and cell differentiation shown by increased alkaline phosphatase activity, contributed to the growth suppression. Murine 3T3-L1 fibroblasts were 10-fold more resistant than B16 cells to geranylgeraniol-mediated growth suppression. Geranylgeraniol at near IC50 concentration (60 µmol/L) suppressed the mRNA level of 3-hydroxy-3-methylglutaryl coenzyme A (HMG‐CoA) reductase by 50%. The impact of geranylgeraniol on B16 cell growth, cell cycle arrest and apoptosis were attenuated by supplemental mevalonate, the product of HMG‐CoA reductase that is essential for cell growth. Geranylgeraniol and d-δ-tocotrienol, a down-regulator of HMG‐CoA reductase, additively suppressed the growth of B16 cells. These results support our hypothesis that mevalonate depletion mediates the tumor-specific growth-suppressive impact of geranylgeraniol. Geranylgeraniol may have potential in cancer chemoprevention and/or therapy.

Fixation Temperature Affects DNA Integrity in the Testis as Measured by the TUNEL Assay

Mature rat testes and liver were fixed with Bouin’s fluid (BF) or modified Davidson’s fixative (mDF) at room temperature (23°C) or 4°C, and DNA integrity was examined by the TUNEL assay. When testes were fixed in BF, TUNEL-stained cells were more prevalent than when fixation occurred in mDF. Independent of fixative, TUNEL-staining was higher when testes were fixed at room temperature relative to 4°C. Significant effects were present for fixative and temperature of fixation, but not their interaction. Relative to BF, mDF also provided for lower TUNEL-staining in liver, but staining was not affected by fixation temperature. Since the TUNEL assay depends on the detection of fragmented DNA strands, harsh fixatives that induce breaks in the DNA can introduce substantial artifacts. Such potential artifacts are especially prevalent in a tissue such as testes with its ongoing division and differentiation activities. Therefore, the current findings lead the authors to conclude that fixation of mature testes in mDF at 4°C minimizes generation of false TUNEL-positive cells.

Mevalonate deprivation mediates the impact of lovastatin on the differentiation of murine 3T3-F442A preadipocytes

The statins competitively inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity and consequently the synthesis of mevalonate. The use of statins is associated with insulin resistance, presumably due to the impaired differentiation and diminished glucose utilization of adipocytes. We hypothesize that mevalonate is essential to adipocyte differentiation and adipogenic gene expression. Adipo-Red assay and Oil Red O staining showed that an eight-day incubation with 0–2.5 µmol/L lovastatin dose-dependently reduced the intracellular triglyceride content of murine 3T3-F442A adipocytes. Concomitantly, lovastatin downregulated the expression of peroxisome proliferator-activated receptor γ (Pparγ), leptin (Lep), fatty acid binding protein 4 (Fabp4), and adiponectin (AdipoQ) as measured by quantitative real-time polymerase chain reaction (real-time qPCR). The expression of sterol regulatory element binding protein 1 (Srebp-1), a transcriptional regulator of Pparγ and Lep genes, was also suppressed by lovastatin. Western-blot showed that lovastatin reduced the level of CCAAT/enhancer binding protein α (C/EBPα) while inducing a compensatory over-expression of HMG CoA reductase. The impact of lovastatin on intracellular triglyceride content and expression of the adipogenic genes was reversed by supplemental mevalonate. Mevalonate-derived metabolites have essential roles in promoting adipogenic gene expression and adipocyte differentiation.

L-prostaglandin D synthase expression and regulation in mouse testis and epididymis during sexual maturation and testosterone treatment after castration.

Lipocalin-type prostaglandin D synthase (L-PGDS) is highly expressed in the adult testis and epididymis of many mammals. The present study was to investigate L-PGDS expression in mouse testis and epididymis during sexual maturation, and the effects of testoster-one replacement on L-PGDS expression in epididymis by in situ hybridization and immunohistochemistry. Both L-PGDS mRNA and protein were highly expressed in the interstitial tissue of adult testis. L-PGDS mRNA was first detected on d 30 after birth and exhibited an abundant signal in adult caput and cauda epididymis. L-PGDS immunostaining was first observed on d 30 after birth. There was a strong level of L-PGDS immunostaining in adult epididymis. Castrated male mice were treated with either vehicle or testosterone propionate following 3 d postcastration. L-PGDS expression steadily declined in a time-dependent fashion in control groups. No L-PGDS mRNA expression or immunostaining was detected in the controls for 12 d. When the castrated mice were treated with testosterone propionate for 5 or 12 d, L-PGDS expression was significantly increased in the whole epididymis. These data suggest that L-PGDS expression in mouse epididymis gradually declined in parallel to the declining concentration of endogenous androgen after castration and increased with the treatment of exogenous testosterone, indicating that L-PGDS expression in mouse epididymis was modulated by androgen levels. However, differential expression in different areas of the epididymis may also be influenced by factors derived from the testis.