Beta-carotene prevents the spermatogenic disorders induced by exogenous scrotal hyperthermia through modulations of oxidative stress, apoptosis, and androgen biosynthesis in mice / 대한수의학회지

We investigated whether β-carotene (β-CA) or ellagic acid (EA), originating from various fruits and vegetables, has a preventive effect against male infertility induced by exogenous scrotal hyperthermia. ICR adult mice were intraperitoneally treated with 10 mg/kg of β-CA or EA daily for 13 days consecutively. During this time, mice were subjected to transient scrotal heat stress in a water bath at 43℃ for 20 min on day 7, and their testes and blood were obtained on day 14 for histopathologic and biochemical analyses. Heat stress induced significant testicular weight reduction, germ cell loss and degeneration, as well as abnormal localization of phospholipid hydroperoxide glutathione peroxidase (PHGPx) and manganese superoxide dismutase (MnSOD) in spermatogenic and Leydig cells. Heat stress also altered the levels of oxidative stress (lipid peroxidation, SOD activity, and PHGPx, MnSOD, and HIF-1α mRNAs), apoptosis (Bax, Bcl-xL, caspase 3, NF-κB, and TGF-β1 mRNAs), and androgen biosynthesis (serological testosterone concentration and 3β-hydroxysteroid dehydrogenase mRNA) in testes. These changes were all improved significantly by β-CA treatment, but only slightly improved by EA treatment. These findings indicate that β-CA, through modulations of oxidative stress, apoptosis, and androgen biosynthesis, is a potent preventive agent against testicular injuries induced by scrotal hyperthermia.

Beta-carotene prevents the spermatogenic disorders induced by exogenous scrotal hyperthermia through modulations of oxidative stress, apoptosis, and androgen biosynthesis in mice

We investigated whether β-carotene (β-CA) or ellagic acid (EA), originating from various fruits and vegetables, has a preventive effect against male infertility induced by exogenous scrotal hyperthermia. ICR adult mice were intraperitoneally treated with 10 mg/kg of β-CA or EA daily for 13 days consecutively. During this time, mice were subjected to transient scrotal heat stress in a water bath at 43℃ for 20 min on day 7, and their testes and blood were obtained on day 14 for histopathologic and biochemical analyses. Heat stress induced significant testicular weight reduction, germ cell loss and degeneration, as well as abnormal localization of phospholipid hydroperoxide glutathione peroxidase (PHGPx) and manganese superoxide dismutase (MnSOD) in spermatogenic and Leydig cells. Heat stress also altered the levels of oxidative stress (lipid peroxidation, SOD activity, and PHGPx, MnSOD, and HIF-1α mRNAs), apoptosis (Bax, Bcl-xL, caspase 3, NF-κB, and TGF-β1 mRNAs), and androgen biosynthesis (serological testosterone concentration and 3β-hydroxysteroid dehydrogenase mRNA) in testes. These changes were all improved significantly by β-CA treatment, but only slightly improved by EA treatment. These findings indicate that β-CA, through modulations of oxidative stress, apoptosis, and androgen biosynthesis, is a potent preventive agent against testicular injuries induced by scrotal hyperthermia.

Long-circulating and target-specific distributions of cyanine 5.5-labeled hyaluronic acid nanoparticles in mouse organs during 28 days after a single administration / 대한수의학회지

Although hyaluronic acid (HA) has been developed as a nanoparticle (NP; 320–400 nm) for a drug delivery system, the tissue targeting efficacy and the pharmacokinetics of HA-NPs are not yet fully understood. After a dose of 5 mg/kg of cyanine 5.5-labeled HA-NPs or HA-polymers was intravenously administrated into mice, the fluorescence was measured from 0.5 h to 28 days. The HA-NPs fluorescence was generally stronger than that of HA-polymers, which was maintained at a high level over 7 days in vivo, after which it gradually decreased. Upon ex vivo imaging, liver, spleen, kidney, lung, testis and sublingual gland fluorescences were much higher than that of other organs. The fluorescence of HA-NPs in the liver, spleen and kidney was highest at 30 min, where it was generally maintained until 4 h, while it drastically decreased at 1 day. However, the fluorescence in the liver and spleen increased sharply at 7 days relative to 3 days, then decreased drastically at 14 days. Conversely, the fluorescence of HA-polymers in the lymph node was higher than that of HA-NPs. The results presented herein may have important clinical implications regarding the safety of as self-assembled HA-NPs, which can be widely used in biomedical applications.

Long-circulating and target-specific distributions of cyanine 5.5-labeled hyaluronic acid nanoparticles in mouse organs during 28 days after a single administration

Although hyaluronic acid (HA) has been developed as a nanoparticle (NP; 320–400 nm) for a drug delivery system, the tissue targeting efficacy and the pharmacokinetics of HA-NPs are not yet fully understood. After a dose of 5 mg/kg of cyanine 5.5-labeled HA-NPs or HA-polymers was intravenously administrated into mice, the fluorescence was measured from 0.5 h to 28 days. The HA-NPs fluorescence was generally stronger than that of HA-polymers, which was maintained at a high level over 7 days in vivo, after which it gradually decreased. Upon ex vivo imaging, liver, spleen, kidney, lung, testis and sublingual gland fluorescences were much higher than that of other organs. The fluorescence of HA-NPs in the liver, spleen and kidney was highest at 30 min, where it was generally maintained until 4 h, while it drastically decreased at 1 day. However, the fluorescence in the liver and spleen increased sharply at 7 days relative to 3 days, then decreased drastically at 14 days. Conversely, the fluorescence of HA-polymers in the lymph node was higher than that of HA-NPs. The results presented herein may have important clinical implications regarding the safety of as self-assembled HA-NPs, which can be widely used in biomedical applications.

Comparative in vivo biodistributions of nanoparticles and polymers of ¹⁷⁷lutetium-labeled hyaluronic acids in mice during 28 days

Hyaluronic acid (HA) has been investigated for biomedical and pharmaceutical applications. This study was conducted to determine the distributions of HA nanoparticles (NPs; size 350–400 nm) and larger HA polymers in mice at intervals after application. ¹⁷⁷Lutetium (Lu)-labeled HA-NPs or HA polymers were intravenously injected (5 mg/kg) into male ICR mice, and radioactivity levels in blood and target organs were measured from 0.25 h to 28 days post-injection. In blood, the radioactivities of HA-NPs and HA polymer peaked at 0.5 h after injection but were remarkably decreased at 2 h; subsequently, they maintained a constant level until 6 days post-injection. HA-NPs and HA polymers were observed in the liver, spleen, lung, kidney, and heart (in ascending order) but were seldom observed in other organs. After 3 days, both the HA-NP and HA polymer levels showed similar steady decreases in lung, kidney, and heart. However, in liver and spleen, the HA-NP levels tended to decrease gradually after 1 day and both were very low after 14 days, whereas the HA polymer level accumulated for 28 days. The results indicate that HA-NPs, with their faster clearance pattern, may act as a better drug delivery system than HA polymers, especially in the liver and spleen.

Temporal and subcellular distributions of Cy5.5-labeled hyaluronic acid nanoparticles in mouse organs during 28 days as a drug carrier

Temporal and subcellular distributions of hyaluronic acid (HA) as a degradable nanoparticle (NP) in animals were investigated to determine if HA-NP could be utilized as an appropriate drug delivery system. After mice were intravenously injected with 5 mg/kg of Cy5.5-labeled HA-NP sized 350–400 nm or larger HA-polymers, the fluorescence intensity was measured in all homogenized organs from 0.5 h to 28 days. HA-NP was greatly detected in spleen, liver and kidney until day 28, while it was maintained at low levels in other organs. HA-polymer was observed at low levels in all organs. HA-NP quantities in spleen and liver were reduced until day 3, but increased sharply between days 3 and 7, then decreased again, while their HA-polymers were maintained at low levels until day 28. In kidneys, both HA-NP and HA-polymer showed high levels after 0.5 h of administration, but steadily decreased until day 28. According to ultrastructural analyses, HA-NP was engulfed in Kupffer cells of liver and macrophages of spleen and kidney at day 1 and was accumulated in the cytoplasm of kidney tubular cells at day 7. Overall, these findings suggest that HA-NP could be considered a desirable drug carrier in the liver, kidney, or spleen.

Anti-atherosclerotic effects of perilla oil in rabbits fed a high-cholesterol diet / 한국실험동물학회지

Anti-atherosclerosis effects of perilla oil were investigated, in comparison with lovastatin, in rabbits fed a high-cholesterol diet (HCD). Hypercholesterolemia was induced in rabbits by feeding the HCD containing 0.5% cholesterol and 1% corn oil, and perilla oil (0.1 or 0.3%) was added to the diet containing 0.5% cholesterol for 10 weeks. HCD greatly increased blood total cholesterol and low-density lipoproteins, and caused thick atheromatous plaques, covering 74% of the aortic wall. Hyper-cholesterolemia also induced lipid accumulation in the liver and kidneys, leading to lipid peroxidation. Perilla oil not only attenuated hypercholesterolemia and atheroma formation, but also reduced fat accumulation and lipid peroxidation in hepatic and renal tissues. The results indicate that perilla oil prevents atherosclerosis and fatty liver by controlling lipid metabolism, and that it could be the first choice oil to improve diet-induced metabolic syndrome.

Hair growth promoting effects of emodin in telogenic C57BL/6 mice

Emodin is an anthraquinone derivative from the roots of Rheum officinale Baill that possesses a variety of biological activities, including inhibition of 5α-reductase and prostaglandin D2. In this study, we investigated whether emodin promotes hair growth. After emodin was topically applied to the shaved dorsal skin of telogenic C57BL/6 N mice, the hair growth rate and morphological analysis were evaluated in dorsal skin for 15 days. After 13 days of treatment, minoxidil or emodin (0.01% or 0.1%)-treated groups showed remarkable regrowth of hairs relative to the vehicle control group. Scoring of the hair growth and rate of hair growth area for 15 days revealed that groups treated with minoxidil and 0.1% emodin were significantly higher than the vehicle control group. Histological examination revealed the emodin and minoxidil groups markedly recovered the number and morphology of hair follicles, including the subcutis depth, relative to the vehicle group. These results suggest that emodin has an excellent promoting effect in hair growth similar to that of minoxidil and might be useful for treatment of baldness or alopecia.

Comparison of teratogenecity induced by nano- and micro-sized particles of zinc oxide in cultured mouse embryos / 대한수의학회지

The increasing uses of zinc oxide nanoparticles (nZnO) in industrial and personal care products raise possible danger of using nZnO in human. To determine whether ZnO induces size-dependent anomalies during embryonic organogenesis, mouse embryos on embryonic day 8.5 were cultured for 2 days under 50, 100, and 150 microg of nZnO (< 100 nm) or micro-sized ZnO (mZnO; 80 +/- 25 microm), after which the morphological changes, cumulative quantity of Zn particles, and expressions of antioxidant and apoptotic genes were investigated. Although embryos exposed to 50 microg of ZnO exhibited no defects on organogenesis, embryos exposed to over 100 microg of ZnO showed increasing anomalies. Embryos treated with 150 microg of nZnO revealed significant changes in Zn absorption level and morphological parameters including yolk sac diameter, head length, flexion, hindbrain, forebrain, branchial bars, maxillary process, mandibular process, forelimb, and total score compared to the same dose of mZnO-treated embryos. Furthermore, CuZn-superoxide dismutase, cytoplasmic glutathione peroxidase (GPx) and phospholipid hydroperoxidase GPx mRNA levels were significantly decreased, but caspase-3 mRNA level was greatly increased in nZnO-treated embryos as compared to normal control embryos. These findings indicate that nZnO has severer teratogenic effects than mZnO in developing embryos.

Comparative anatomical characteristics of cardiac valves in animals / 동물의과학연구지

Although various animals have been used as models of cardiac valvular diseases in humans, the structural characteristics of cardiac valves in animals remain unclear. In this study, we investigated cardiac valves in representative animal models for the purpose of comparative anatomy. Adult hearts from three dogs, four goats, six rabbits, and six fowls were fixed with 10% neutral-buffered formalin and analyzed gross-anatomically. Cardiac appearance was spherical or oval in dogs, goats, and rabbits, whereas it had a long conical shape in fowls. Left atrioventricular (AV) valve was composed of membranous septal and parietal cusps connected to two papillary muscles in all animals. The right AV valve was composed of membranous septal, parietal, and angular cusps with three papillary muscles in dogs and goats, membranous septal and parietal cusps attached to four papillary muscles in rabbits, and a single muscular plate without any papillary muscles and chorda tendinae in fowls. Aortic valves with thin membranous right, left, and septal semilunar cusps in dogs, goats, and rabbits had a thick membrane with a bended free border in fowls. Pulmonary valve (PV) with membranous right, left, and intermediate semilunar cusps made a large central hole by being closely attached to the surrounding wall in dogs, goats, and rabbits, whereas it protruded into half of the lumen as a thick membrane in fowls. The membranous cusp of the PV was composed of several layers in dogs and goats but was a single layer in rabbits and fowls. These findings indicate that even if animals have two completely separated atria and ventricles each, cardiac valves have species-specific morphological characteristics, especially between mammals and fowls.

Expression Patterns of Superoxide Dismutase Genes in the Stage-specific Seminiferous Tubules of Mice Excised by a Laser Capture Microdissection / 한국실험동물학회지

Spermatogenesis is a particularly difficult process to study the unique multiple cellular associations within the seminiferous epithelium. Laser capture microdissection (LCM) is a recently developed technique that enables the isolation of individual cell populations from complex tissues. The superoxide dismutase (SOD) is the first and most important enzyme of antioxidant defense systems against superoxide anion. The aim of this study was to investigate the quantitative changes of SOD gene expression according to the spermatogenic cycle in mouse testes using LCM and real-time polymerase chain reaction (PCR) techniques. Frozen sections (10 micrometer) were obtained from the testes of 8-weeks-old ICR mice. LCM was used to capture all cells in cross-sectioned seminiferous tubules which were grouped into stages I-V, VII-VIII, and IX-XI. The expression level of cytoplasmic Cu, Zn-SOD (SOD1) mRNA was remarkably higher than those of mitochondrial Mn-SOD (SOD2) and extracellular Cu, Zn-SOD (SOD3) mRNAs in mouse testes. During spermatogenesis, the expressions of SOD1 and SOD2 mRNAs were highest on stages I-V, began to decrease after stage VII, and showed a lowest level on stage IX-XI. However, the expression of SOD3 mRNA was highest on stages VII-VIII. These findings suggest that the subtypes of SOD are expressed differentially in mouse testes during spermatogenesis.

Immunohistochemical identification and quantitative analysis of cytoplasmic Cu/Zn superoxide dismutase in mouse organogenesis

Cytoplasmic Cu/Zn superoxide dismutase (SOD1) is an antioxidant enzyme that converts superoxide to hydrogen peroxide in cells. Its spatial distribution matches that of superoxide production, allowing it to protect cells from oxidative stress. SOD1 deficiencies result in embryonic lethality and a wide range of pathologies in mice, but little is known about normal SOD1 protein expression in developing embryos. In this study, the expression pattern of SOD1 was investigated in post-implantation mouse embryos and extraembryonic tissues, including placenta, using Western blotting and immunohistochemical analyses. SOD1 was detected in embryos and extraembryonic tissues from embryonic day (ED) 8.5 to 18.5. The signal in embryos was observed at the lowest level on ED 9.5-11.5, and the highest level on ED 17.5-18.5, while levels remained constant in the surrounding extraembryonic tissues during all developmental stages examined. Immunohistochemical analysis of SOD1 expression on ED 13.5-18.5 revealed its ubiquitous distribution throughout developing organs. In particular, high levels of SOD1 expression were observed in the ependymal epithelium of the choroid plexus, ganglia, sensory cells of the olfactory and vestibulocochlear epithelia, blood cells and vessels, hepatocytes and hematopoietic cells of the liver, lymph nodes, osteogenic tissues, and skin. Thus, SOD1 is highly expressed at late stages of embryonic development in a cell- and tissue-specific manner, and can function as an important antioxidant enzyme during organogenesis in mouse embryos.

Effects of endocrine disrupting chemicals on expression of phospholipid hydroperoxide glutathione peroxidase mRNA in rat testes

Phospholipid hydroperoxide glutathione peroxidase(PHGPx), an antioxidative selenoprotein, is modulated byestrogen in the testis and oviduct. To examine whetherpotential endocrine disrupting chemicals (EDCs) affectthe microenvironment of the testes, the expression patternsof PHGPx mRNA and histological changes were analyzedin 5-week-old Sprague-Dawley male rats exposed to severalEDCs such as an androgenic compound [testosterone (50,200, and 1,000microg/kg)], anti-androgenic compounds [flutamide(1, 5, and 25mg/kg), ketoconazole (0.2 and 1mg/kg), anddiethylhexyl phthalate (10, 50, and 250mg/kg)], andestrogenic compounds [nonylphenol (10, 50, 100, and 250mg/kg), octylphenol (10, 50, and 250mg/kg), and diethyl-stilbestrol (10, 20, and 40microg/kg)] daily for 3 weeks via oraladministration. Mild proliferation of germ cells andhyperplasia of interstitial cells were observed in the testesof the flutamide-treated group and deletion of thegerminal epithelium and sloughing of germ cells wereobserved in testes of the diethylstilbestrol-treated group.Treatment with testosterone was shown to slightly decreasePHGPx mRNA levels in testes by the reverse transcription-polymerase chain reaction. However, anti-androgeniccompounds (flutamide, ketoconazole, and diethylhexylphthalate) and estrogenic compounds (nonylphenol,octylphenol, and diethylstilbestrol) significantly up-regulated PHGPx mRNA in the testes (p<0.05). Thesefindings indicate that the EDCs might have a detrimentaleffect on spermatogenesis via abnormal enhancement ofPHGPx expression in testes and that PHGPx is useful as abiomarker for toxicity screening of estrogenic or anti-androgenic EDCs in testes.

Exposure to genistein does not adversely affect the reproductive system in adult male mice adapted to a soy-based commercial diet

Genistein, a soybean-originated isoflavone, is widely consumed by humans for putative beneficial health effects but its estrogenic activity may affect adversely the development of male reproductive system. Five-week-old ICR mice were purchased and fed with a soybean-based Purina Chow diet until 6 months of age. The animals were exposed by gavage to genistein (2.5 mg/kg/day) or 17beta-estradiol (7.5 microgram/kg/day) for five weeks. Corn oil was used for the negative control. The animals were fed the caseinbased AIN-76A diet throughout the experimental periods. There were no significant differences in body and organ weights of mice among experimental groups. No significant differences in sperm counts and sperm motile characteristics were found between the control and the genistein groups. Treatment of 17beta-estradiol caused a significant decrease in epididymal sperm counts compared to the control (p<0.05). The level of phospholipid hydroxide glutathione peroxidase in the epididymis of mice exposed to genistein was significantly higher than that of the control mice (p<0.05). 17beta-estradiol treatment caused a reduction of germ cells in the testis and hyperplasia of mucosal fold region in the prostate of mice. Genistein treatment did not cause any lesion in the testis, epididymis, and prostate. These results suggest that dietary uptake of genistein at adult stage of life may not affect male reproductive system and functions.