A novel, noncoding-RNA-mediated, post-transcriptional mechanism of anti-Mullerian hormone regulation by the H19/let-7 axis.

In reproductive age women, the pool of primordial follicles is continuously depleted through the process of cyclic recruitment. Anti-Mullerian hormone (AMH) both inhibits the initial recruitment of primordial follicles into the growing pool and modulates the sensitivity of growing follicles to follicle stimulating hormone. Thus, AMH may be an important modulator of female infertility and ovarian reserve; however, the mechanisms regulating AMH remain unclear.To evaluate AMH levels in the absence of H19 lncRNA, H19 knockout (H19KO) mice were evaluated for analysis of ovarian AMH gene expression, protein production, and reproductive function, including assessment of follicle numbers and litter size analysis. To further investigate regulation of AMH by the H19/let-7 axis, let-7 binding sites on AMH were predicted, and in vitro studies of the effect of H19 knockdown/overexpression with let-7 rescue were performed. Lastly, response to superovulation was assessed via oocyte counts and estradiol measurements.The H19KO mouse demonstrates subfertility and accelerated follicular recruitment with increased spontaneous development of secondary, preantral, and antral follicles. Ovaries of H19KO mice have decreased AMH mRNA and protein, and AMH mRNA has a functional let-7 binding site, suggesting a plausible ncRNA-mediated mechanism for AMH regulation by H19/let-7. Lastly, in the absence of H19, superovulation results in higher estradiol and more oocytes, suggesting that H19 functions to limit the number of follicles that mature, produce estradiol, and ovulate. Thus, AMH's inhibitory actions are regulated at least in part by H19, likely via let-7, marking this ncRNA pair as important regulators of the establishment and maintenance of the follicular pool.

Quantifying growing versus non-growing ovarian follicles in the mouse.

BACKGROUND: A standard histomorphometric approach has been used for nearly 40 years that identifies atretic (e.g., dying) follicles by counting the number of pyknotic granulosa cells (GC) in the largest follicle cross-section. This method holds that if one pyknotic granulosa nucleus is seen in the largest cross section of a primary follicle, or three pyknotic cells are found in a larger follicle, it should be categorized as atretic. Many studies have used these criteria to estimate the fraction of atretic follicles that result from genetic manipulation or environmental insult. During an analysis of follicle development in a mouse model of Fragile X premutation, we asked whether these 'historical' criteria could correctly identify follicles that were not growing (and could thus confirmed to be dying). METHODS: Reasoning that the fraction of mitotic GC reveals whether the GC population was increasing at the time of sample fixation, we compared the number of pyknotic nuclei to the number of mitotic figures in follicles within a set of age-matched ovaries. RESULTS: We found that, by itself, pyknotic nuclei quantification resulted in high numbers of false positives (improperly categorized as atretic) and false negatives (improperly categorized intact). For preantral follicles, scoring mitotic and pyknotic GC nuclei allowed rapid, accurate identification of non-growing follicles with 98% accuracy. This method most often required the evaluation of one follicle section, and at most two serial follicle sections to correctly categorize follicle status. For antral follicles, we show that a rapid evaluation of follicle shape reveals which are intact and likely to survive to ovulation. CONCLUSIONS: Combined, these improved, non-arbitrary methods will greatly improve our ability to estimate the fractions of growing/intact and non-growing/atretic follicles in mouse ovaries.

Early Onset of Atherosclerosis of The Carotid Bifurcation in Newborn Cadavers.

INTRODUCTION: The anatomy of arterial bifurcations affects blood flow and has a significant role in the development of vascular disease. Therefore, it is important to know the structural characteristics of the Common Carotid Artery (CCA) and its branches for early onset of atherosclerosis in newborns. AIM: The present study was conducted to evaluate the characteristics of CCA in newborn cadavers. MATERIALS AND METHODS: Eight carotid arteries obtained from newborn cadavers were used. The outflow to inflow area ratios was calculated to evaluate vessel diameters. Additionally, scanning electron and light microscopic investigations were conducted with tissue samples. The brachial artery of each cadaver was used as controls. Correlation between area ratios and atherosclerotic endothelial damage was determined. RESULTS: Light microscopic investigations demonstrated that control group sections showed no positivity for Oil red O staining, while carotid bifurcation regions depicted widespread occurrence of intimal lipid accumulations. Scanning electron microscopic examination of control group sections presented regular endothelial topography, while carotid bifurcation region topography exhibited numerous blood cells and separated endothelial cells. Fibrin accumulation on endothelial surface in low area ratios was another important finding in the examination of its endothelial surface degeneration. The above-mentioned morphological findings seemed to be quite parallel to outflow to inflow area ratio data favouring low area and degeneration. CONCLUSION: The correlation between area ratios and the histological characteristic of cerebral vessels of newborn cadavers indicate that early stages of atherosclerosis began in early embryologic life.

Granulosa cell and oocyte mitochondrial abnormalities in a mouse model of fragile X primary ovarian insufficiency.

STUDY HYPOTHESIS: We hypothesized that the mitochondria of granulosa cells (GC) and/or oocytes might be abnormal in a mouse model of fragile X premutation (FXPM). STUDY FINDING: Mice heterozygous and homozygous for the FXPM have increased death (atresia) of large ovarian follicles, fewer corpora lutea with a gene dosage effect manifesting in decreased litter size(s). Furthermore, granulosa cells (GC) and oocytes of FXPM mice have decreased mitochondrial content, structurally abnormal mitochondria, and reduced expression of critical mitochondrial genes. Because this mouse allele produces the mutant Fragile X mental retardation 1 (Fmr1) transcript and reduced levels of wild-type (WT) Fmr1 protein (FMRP), but does not produce a Repeat Associated Non-ATG Translation (RAN)-translation product, our data lend support to the idea that Fmr1 mRNA with large numbers of CGG-repeats is intrinsically deleterious in the ovary. WHAT IS KNOWN ALREADY: Mitochondrial dysfunction has been detected in somatic cells of human and mouse FX PM carriers and mitochondria are essential for oogenesis and ovarian follicle development, FX-associated primary ovarian insufficiency (FXPOI) is seen in women with FXPM alleles. These alleles have 55-200 CGG repeats in the 5' UTR of an X-linked gene known as FMR1. The molecular basis of the pathology seen in this disorder is unclear but is thought to involve either some deleterious consequence of overexpression of RNA with long CGG-repeat tracts or of the generation of a repeat-associated non-AUG translation (RAN translation) product that is toxic. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Analysis of ovarian function in a knock-in FXPM mouse model carrying 130 CGG repeats was performed as follows on WT, PM/+, and PM/PM genotypes. Histomorphometric assessment of follicle and corpora lutea numbers in ovaries from 8-month-old mice was executed, along with litter size analysis. Mitochondrial DNA copy number was quantified in oocytes and GC using quantitative PCR, and cumulus granulosa mitochondrial content was measured by flow cytometric analysis after staining of cells with Mitotracker dye. Transmission electron micrographs were prepared of GC within small growing follicles and mitochondrial architecture was compared. Quantitative RT-PCR analysis of key genes involved in mitochondrial structure and recycling was performed. MAIN RESULTS AND THE ROLE OF CHANCE: A defect was found in follicle survival at the large antral stage in PM/+ and PM/PM mice. Litter size was significantly decreased in PM/PM mice, and corpora lutea were significantly reduced in mice of both mutant genotypes. Mitochondrial DNA copy number was significantly decreased in GC and metaphase II eggs in mutants. Flow cytometric analysis revealed that PM/+ and PM/PM animals lack the cumulus GC that harbor the greatest mitochondrial content as found in wild-type animals. Electron microscopic evaluation of GC of small growing follicles revealed mitochondrial structural abnormalities, including disorganized and vacuolar cristae. Finally, aberrant mitochondrial gene expression was detected. Mitofusin 2 (Mfn2) and Optic atrophy 1 (Opa1), genes involved in mitochondrial fusion and structure, respectively, were significantly decreased in whole ovaries of both mutant genotypes. Mitochondrial fission factor 1 (Mff1) was significantly decreased in PM/+ and PM/PM GC and eggs compared with wild-type controls. LIMITATIONS, REASONS FOR CAUTION: Data from the mouse model used for these studies should be viewed with some caution when considering parallels to the human FXPOI condition. WIDER IMPLICATIONS OF THE FINDINGS: Our data lend support to the idea that Fmr1 mRNA with large numbers of CGG-repeats is intrinsically deleterious in the ovary. FXPM disease states, including FXPOI, may share mitochondrial dysfunction as a common underlying mechanism. LARGE SCALE DATA: Not applicable. STUDY FUNDING AND COMPETING INTERESTS: Studies were supported by NIH R21 071873 (J.J./G.H), The Albert McKern Fund for Perinatal Research (J.J.), NIH Intramural Funds (K.U.), and a TUBITAK Research Fellowship Award (B.U.). No conflict(s) of interest or competing interest(s) are noted.

Differences between Solution and Membrane Forms of Chitosan on the In Vitro Activity of Fibroblasts.

BACKGROUND: Chitosan, a linear polysaccharide, has been recently used in biomedical applications. In vitro studies have demonstrated its effect on cellular growth and its stimulatory action on cellular layer formation. AIMS: The present study aims to compare the proliferative effects of chitosan in two forms, membranous and solution forms, on Swiss 3T3 mouse embryonic fibroblasts. STUDY DESIGN: In vitro study. METHODS: Three experimental groups were formed: cells were cultured in a normal medium without chitosan (Control Group); cells were cultured either in a medium containing 2.0% chitosan in membranous form (Membrane Group) or chitosan solution at a concentration of 2.0% (Solution Group). Two different methods were used in the experiments: cells cultured on the medium containing chitosan in solution or membranous forms (method 1); and chitosan solution or membranous forms were added into the medium containing previously cultured cells (method 2). RESULTS: Scanning electron microscopic investigations of the experimental groups revealed cells with well-defined cellular projections, intact cellular membranes and tight intercellular junctions. They were especially prominent in the membrane group of method 1 and in the membrane and solution groups of method 2. Mouse monoclonal anti-collagen 1 primary antibody was used to indicate collagen synthesis. Prominent collagen synthesis was detected in the membrane groups on the 10(th) day of culture for both methods. Bromodeoxyuridine (BrdU) and MTT assays were performed in order to assess cellular proliferation and viability, respectively. BrdU labelling tests indicated a higher proliferation index in the membrane group of method 1 on the 5(th) and 10(th) days. For the second method, the membranous form on the 10(th) day and solution form on the 5(th) day were the most effective groups in terms of cellular proliferation. MTT results reflected a high cellular viability in method 1 on the 5(th) day of treatment with the membranous form, whereas cellular viability was highest in the solution form of method 2 on the 5(th) day. CONCLUSION: The membranous form of chitosan induced a significant proliferative effect and increased the ratio of cell-to-cell junctions of Swiss 3T3 mouse embryonic fibroblasts. Conveniently, the solution form also resulted in enhanced cell proliferation and viability compared to the control group. As the solution form is easy to prepare and apply to cells compared to the membrane form, the application of Chitosan directly to media appears to be a convenient alternative for tissue engineering approaches.

Comparision of biocompatibility and cytotoxicity of two new root canal sealers.

The aim of this study was to investigate the remote organ toxicity and connective tissue reaction of two new root canal sealers ("GuttaFlow(®)" and "EndoREZ(®)") and to compare them with zinc oxide eugenol sealer using biochemical and histopathological parameters. A total of 60 white albino Wistar rats were used in the study. 0.1ml of GuttaFlow(®), EndoREZ(®) or Kerr Pulp Canal Sealer(®) were administered subcutaneously into the mid-dorsal thoracic region of rats (15 in each group). Control rats were given saline only. Rats were decapitated after 24h, on day 7 and on day 30 of the experiment and tissue samples from lung, liver, kidney and skin were removed for the determination of malondialdehyde (MDA) and glutathione (GSH) levels. In parallel, tissues were also examined histologically. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) levels, and creatinine and blood urea nitrogen (BUN), concentrations (BUN) were measured to assess liver and kidney functions, respectively. Tumor necrosis factor (TNF) and lactate dehydrogenase (LDH) were also assayed in serum samples. No statistical differences were found among the control and EndoREZ(®), GuttaFlow(®) and Kerr Pulp Canal sealers regarding tissue MDA, GSH levels or serum parameters (p>0.05) at all time points examined. Both of the new root canal sealers showed good compatibility and acceptable tissue toxicity.

Oxytocin or social housing alleviates local burn injury in rats.

BACKGROUND: Thermal injury may cause distant organ inflammation and multiorgan failure. Oxytocin (OT), a nonapeptide, modulates the immune and inflammatory processes. MATERIALS AND METHODS: To investigate the effects of oxytocin on burn-induced tissue injury, Sprague-Dawley rats were subjected to a partial thickness burn. Immediately after burn, half of the burned rats were placed single in the cages, while others were caged in groups. All the rats then were treated with either OT (5 microg/kg, s.c) or saline twice daily for 5 d. The control rats had no burn injury and received no treatments. On day 5, the rats were decapitated, tissue and serum samples were obtained to score the severity of damage and to assay TNF-alpha levels. RESULTS: Burn trauma resulted in oxidative ileal damage, as evidenced by increased apoptotic rate, increased neutrophil recruitment, and enhanced lipid peroxidation. OT treatment depressed the TNF-alpha level and alleviated dermal degeneration, while attenuating ileal damage. Although a higher degree of skin damage was observed in the animals kept isolated following burn injury, keeping the rats in groups did not affect the level of TNF-alpha or the severity of dermal or ileal injury, but abolished the burn-induced elevations in ileal lipid peroxidation and myeloperoxidase activity. Moreover, OT treatment reduced the ileal apoptosis when applied to rats housed in groups, while the treatment did not alter apoptotic ratio in the isolated rats. CONCLUSION: Oxytocin can be considered as a potential agent in treating burn-induced distant organ injury.

Oxytocin ameliorates skin damage and oxidant gastric injury in rats with thermal trauma.

Transient splanchnic vasoconstriction following major burns causes oxidative and/or nitrosative damage in gastrointestinal tissues due to ischemia, which is followed by reperfusion injury. Oxytocin (OT), a hypothalamic nonapeptide, possesses antisecretory and antiulcer effects, facilitates wound healing and is involved in immune and inflammatory processes. To assess the possible protective effect of oxytocin (OT) against burn-induced gastric injury, Sprague-Dawley rats (250-300g) were randomly divided into three groups as control (n=8), OT-treated burn (n=8) and saline-treated burn (n=8) groups. Under anesthesia, the shaved dorsal skin of rats was exposed to 90 degrees C water for 10s to induce burn injury covering 30% of total body surface area in a standardized manner. Either oxytocin (5microg/kg) or saline was administered subcutaneously immediately after and at 24h following burn, and the rats were decapitated at 48h. Serum samples were assayed for TNF-alpha, and stomach was taken for the determination of malondialdehyde (MDA), myeloperoxidase (MPO) activity, DNA fragmentation rate (%) and histopathological examination. MDA and MPO were assayed for products of lipid peroxidation and as an index of tissue neutrophil infiltration, respectively. When compared to control group, burn caused significant increases in gastric MDA and MPO activity and increased microscopic damage scores at 48h (p<0.001). Oxytocin treatment reversed the burn-induced elevations in MDA and MPO levels and reduced the gastric damage scores (p<0.001, p<0.01), while TNF-alpha levels, which were increased significantly at 48thh after injury (p<0.001), were abolished with OT treatment (p<0.001). The results of this study suggest that oxytocin may provide a therapeutic benefit in diminishing burn-induced gastric inflammation by depressing tissue neutrophil infiltration and decreasing the release of inflammatory cytokines, but requires further investigation as a potential therapeutic agent in ameliorating the systemic effects of severe burn.

Pressure ulcer-induced oxidative organ injury is ameliorated by beta-glucan treatment in rats.

Pressure ulcers (PU) cause morphological and functional alterations in the skin and visceral organs. In this study we investigated the role of oxidative damage in PUs and the probable beneficial effect of beta-glucan treatment against this damage. beta-glucan is known to have immunomodulatory effects. Experiments were carried on Wistar albino rats. PU was induced by applying magnets over steel plates that were implanted under the skin, to compress the skin and cause ischemia where removing the magnets cause reperfusion of the tissue. Within the first 12 h, rats were subjected to 5 cycles of ischemia/reperfusion (I/R), followed by 12 h ischemia. This protocol was repeated for 3 days. In treatment groups, twice a day during reperfusion periods, beta-glucan was either applied locally (25 mg/kg) as an ointment on skin, or administered orally (50 mg/kg) as a gavage. At the end of the experimental periods, tissue samples (skin, liver, kidney, lung, stomach, and ileum) were taken for the measurement of malondialdehyde (MDA)--an index of lipid peroxidation--and glutathione (GSH)--a key antioxidant--levels. Neutrophil infiltration was evaluated by the measurement of tissue myeloperoxidase activity, while collagen contents were measured for the evaluation of tissue fibrosis. Skin tissues were also examined microscopically. Liver and kidney functions were assayed in serum samples. Local treatment with beta-glucan inhibited the increase in MDA and MPO levels and the decrease in GSH in the skin induced by PU, but was less efficient in preventing the damage in visceral organs. However, systemic treatment prevented the damage in the visceral organs. Significant increases in creatinine, BUN, ALT, AST, LDH and collagen levels in PU group were prevented by beta-glucan treatment. The light microscopic examination exhibited significant degenerative changes in dermis and epidermis in the PU group. Tissue injury was decreased especially in the locally treated group. Thus, supplementing geriatric and neurologically impaired patients with adjuvant therapy of beta-glucan may have some benefits for successful therapy and improving quality of life.

beta-glucan protects against burn-induced oxidative organ damage in rats.

Thermal injury may lead to systemic inflammatory response, and multiple organ failure. Generation of reactive oxygen radicals and lipid peroxidation play important roles in burn-induced remote organ injury. In the present study, we investigated the putative protective effect of local or systemic beta-glucan treatment on burn-induced remote organ injury. Wistar albino rats were exposed to 90 degrees C bath for 10 s to induce thermal trauma. beta-glucan (3.75 mg/rat locally or 50 mg/kg orally) or saline was administered immediately after the trauma and were repeated twice daily in 48 h groups. Rats were decapitated either 6 or 48 h after burn injury and the skin, lung, liver, ileum and kidney tissues were taken for the measurement of malondialdehyde (MDA)--an index of lipid peroxidation--and glutathione (GSH)--a key antioxidant--levels. Neutrophil infiltration was evaluated by the measurement of tissue myeloperoxidase (MPO) activity, while the tumor necrosis factor-alpha (TNF-alpha) levels were measured in serum samples. Skin tissues were also examined microscopically. Severe skin scald injury (30% of total body surface area) caused significant decreases in GSH levels of the liver and intestinal tissues (p<0.01-<0.001), while MDA levels were significantly (p<0.01-p<0.001) increased at post-burn 6 and 48 h. Both local and systemic beta-glucan treatments significantly reversed (p<0.01-p<0.001) the elevations in MDA levels, while reduced GSH levels were reversed back to control levels (p<0.01-p<0.001); and the raised MPO levels were significantly decreased (p<0.05-p<0.001). The results indicate that both systemic and local administration of beta-glucan were effective against burn-induced oxidative tissue damage in the rat. beta-glucans, besides their immunomodulatory effects, have additional antioxidant properties. Therefore, beta-glucans merit consideration as therapeutic agents in the treatment of burn injuries.

Protective effect of aqueous garlic extract against naphthalene-induced oxidative stress in mice.

The aim of this study was to investigate the possible protective effects of aqueous garlic extract (AGE) against naphthalene-induced oxidative changes in liver, kidney, lung and brain of mice. Balb/c mice (25-30 g) of either sex were divided into five groups each comprising 10 animals. Mice received for 30 days: 0.9% NaCl, i.p. (control); corn oil, i.p; AGE in a dose of 125 mg kg-1, i.p.; naphthalene in a dose of 100 mg kg-1, i.p. (dissolved in corn oil); and AGE (in a dose of 125 mg kg-1, i.p.) plus naphthalene (in a dose of 100 mg kg-1, i.p.). After decapitation, liver, kidney, lung and brain tissues were excised. Malondialdehyde (MDA) and glutathione (GSH) levels and myeloperoxidase activity (MPO) were determined in the tissues, while oxidant-induced tissue fibrosis was determined by collagen content. Tissues were also examined microscopically. Serum aspartate aminotransferase, alanine aminotransferase levels and blood urea nitrogen and creatinine concentrations were measured for the evaluation of hepatic and renal function, respectively. MDA and GSH levels were also assayed in serum samples. In the naphthalene-treated group, GSH levels decreased significantly, while MDA levels, MPO activity and collagen content increased in the tissues (P<0.01-0.001), suggesting oxidative organ damage, which was also verified histologically. In the AGE-treated naphthalene group, all of these oxidant responses were reversed significantly (P<0.05-0.01). Hepatic and renal function test parameters, which increased significantly (P<0.001) following naphthalene administration, decreased (P<0.05-0.001) after AGE treatment. The results demonstrate the role of oxidative mechanisms in naphthalene-induced tissue damage. The antioxidant properties of AGE ameliorated oxidative organ injury due to naphthalene toxicity.