Ultrastructure of coarse granules in the perivitelline space and association with ovulation induction protocols.

OBJECTIVE: To evaluate the origin and ultrastructure of the coarse granules in the perivitelline space (PVS) of oocytes of a group of couples attending assisted reproduction treatment. METHODS: The ultrastructure of five oocytes with coarse granulues in the PVS obtained from three patients were evaluated by transmission electron microscopy (TEM). The influence of the ovulation induction regimen on the formation of granules in the PVS of the oocytes of 214 couples and the developmental capacity of these oocytes presenting granules in the PVS was analyzed retrospectively. RESULTS: In TEM analysis, the microvilli structure was irregular, short, and loosely scattered through the oolemma in the oocytes presenting coarse granules in the PVS. Furthermore, dense lipid droplets were identified within the PVS and the surrounding cumulus cells. In retrospective analysis, the number of oocytes with coarse granules in the PVS was positively correlated with the duration of antagonist administration (r=0.23, p=0.013). Regardless of the type of granule, the presence of coarse or moderately coarse granules in the PVS was positively correlated with low-quality embryos on D3 (r=0.29, p=0.005) and the total number of arrested embryos up to D3 (r=0.33, p<0.001). Furthermore, the presence of coarse granules in the PVS severely exacerbated miscarriage rates. CONCLUSIONS: Our findings suggest that the presence of especially coarse granules in the PVS is correlated with the reduction of further embryonic developmental capacity in post-implantation stages of embryonic development, indicating a negative impact from aggressive ovulation induction protocols on developing oocytes.

Effects of Monobenzyl ether of hydroquinone on 3T3 mouse fibroblast viability and ultrastructure.

Monobenzyl ether of hydroquinone (MBEH) is a topical depigmentation agent used by vitiligo patients to even the skin tone. We aimed to investigate the effects of MBEH on 3T3 mouse fibroblasts. Fibroblasts were treated with 250 µM, 500 µM, and 750 µM MBEH and vehicle (EtOH:DMSO) for 24 hours. Cell numbers of 250 µM, 500 µM, and 750 µM MBEH treated and vehicle groups decreased significantly compared to control group. TUNEL positive cell rate increased with MBEH concentration. In electron microscopic examination, control and vehicle groups showed active cells features, while mitochondrial swelling and cristae loss were seen in 250 µM MBEH-treated group. In cytoplasm of 500 µM MBEH-treated group, there were many multivesicular bodies and autophagic vacuoles. As an indication of apoptosis, cell membrane blebs and reduction in cell size were observed. In 750 µM MBEH-treated group, cells were completely degenerated. Our findings show that MBEH, which is used as a depigmentation agent to lighten the skin by destroying melanocytes, may also have dose-dependent negative effects on the viability of 3T3 mouse fibroblasts, and these may be mediated through autophagic and apoptotic cell death mechanisms.

Investigation of the effects of semaphorin 3A on new bone formation in a rat calvarial defect model.

PURPOSE: This study investigates the effects of semaphorin 3A on new bone formation in an experimental rat model. MATERIALS AND METHODS: Cortical bone defects, 5 mm, were created in the calvaria of 40 Wistar rats, which were then separated into three groups: empty defect (control) group, collagen group, collagen + semaphorin 3A group. The bone blocks were harvested after 4 and 8 weeks. New bone formation was assessed by micro-computed tomography (micro-CT), histology, histomorphometry, transmission electron microscope (TEM) and immunohistochemistry. RESULTS: Increased bone formation was observed in collagen + semaphorin 3A groups both histologically and with micro-CT. In the histomorphometic analysis, the control group had significantly less bone formation compared to both the collagen and collagen + semaphorin 3A group at 4 weeks (p = 0.0001) and 8 weeks (p = 0.0001). The collagen group had significantly less bone formation compared to collagen + semaphorin 3A group both at 4 weeks (p = 0.002) and 8 weeks (p = 0.005). Immunohistochemical analysis revealed that semaphorin 3A inhibited receptor activator of nuclear factor-kB ligand (RANKL) expression and increased the expressions of osteoblastic bone markers at 4 weeks. In TEM analysis, the collagen + semaphorin 3A group had an increased proliferation and bone formation rate at 4 weeks, whereas bone quantity and maturation were enhanced at 8 weeks. CONCLUSION: Locally applied semaphorin 3A increases callus formation at 4 weeks and bone formation at 8 weeks. Semaphorin 3A prevents bone resorption by inhibiting osteoclasts and increases bone formation by inducing osteoblasts.

Mitochondrial activity and cytoskeleton organization in three pronuclei oocytes after intracytoplasmic sperm injection.

SummaryDigyny, the presence of a third pronucleus due to the failure of second polar body extrusion, is problematic after intracytoplasmic sperm injection (ICSI) practices. Mitochondria have critical roles such as production of adenosine triphosphate (ATP) and regulation of Ca2+ homeostasis during oocyte maturation, fertilization and the following development, while the regulation of meiotic spindle formation, chromosome segregation, pronuclear apposition and cytokinesis is closely associated with the cytoskeleton. In this study, mitochondrial membrane potential, distribution of F-actin and γ-tubulin, and the ultrastructure of three pronuclear (3PN) oocytes were investigated. 3PN oocytes after ICSI procedure were taken from patients who were enrolled in assisted reproduction programmes. For mitochondrial membrane potential analysis, fresh oocytes stained with the mitochondrial membrane potential probe JC-1, were evaluated under fluorescence microscopy. The mitochondrial membrane potential of three pronuclear oocytes showed similar results to normal zygotes. γ-Tubulin was stained strongly at the subplasmalemmal domain and microfilaments were localized at the cortical, but not the perinuclear, area. Cytoplasmic halos were moderately or not detected by electron microscopy; lipofuscin granules, degenerated mitochondria, and multilamellated bodies were seen in the ooplasm. Immunohistochemistry and electron microscopic findings suggested that mitochondrial membrane potential has no direct effect on second polar body extrusion. This abnormality can be associated with an altered cytoskeleton due to poor oocyte quality.

Stanozolol administration combined with exercise leads to decreased telomerase activity possibly associated with liver aging.

Anabolic agents are doping substances which are commonly used in sports. Stanozolol, a 17α­alkylated derivative of testosterone, has a widespread use among athletes and bodybuilders. Several medical and behavioral adverse effects are associated with anabolic androgenic steroids (AAS) abuse, while the liver remains the most well recognized target organ. In the present study, the hepatic effects of stanozolol administration in rats at high doses resembling those used for doping purposes were investigated, in the presence or absence of exercise. Stanozolol and its metabolites, 16­ß­hydroxystanozolol and 3'­hydroxystanozolol, were detected in rat livers using liquid chromatography­mass spectrometry (LC­MS). Telomerase activity, which is involved in cellular aging and tumorigenesis, was detected by examining telomerase reverse transcriptase (TERT) and phosphatase and tensin homolog (PTEN) expression levels in the livers of stanozolol­treated rats. Stanozolol induced telomerase activity at the molecular level in the liver tissue of rats and exercise reversed this induction, reflecting possible premature liver tissue aging. PTEN gene expression in the rat livers was practically unaffected either by exercise or by stanozolol administration.

Effects of stanozolol on apoptosis mechanisms and oxidative stress in rat cardiac tissue.

Stanozolol is a widely used 17α-alkylated anabolic androgenic steroid (AAS) derivative. Despite stanozolol's adverse effects, its effect on oxidative stress parameters and mitochondrial apoptosis pathway is not clearly defined. In our study, thirty four male Sprague-Dawley rats were divided into 5 groups as control (C), vehicle control (VC), steroid (ST), vehicle control-exercise (VCE), and steroid-exercise (STE). Animals were subcutaneously administered stanozolol 5 mg/kg in steroid groups and propylene glycol 1 ml/kg in the vehicle-control groups. On the 28th day-after sacrification, oxidative stress (MDA, GSH, PC, SOD, CAT) and apoptosis parameters (TUNEL, Cytochrome-c) in cardiac tissue were evaluated. Also, blood vessel morphology of cardiac tissue was evaluated with Verhoeff-van Giesen staining. It has been demonstrated that stanozolol administration triggers apoptosis by using TUNEL assay and cytochrome-c immunohistochemical staining intensity, while this effect is significantly reduced in the presence of exercise. In conclusion, the present study demonstrated that stanozolol administration induces apoptosis with increasing PC and CAT levels, while GSH, MDA and SOD parameters do not reveal any significant change. Exercise has a protective role in stanozolol induced oxidative stress and apoptosis. According to Verhoeff-van Giesen staining results for blood vessel morphology assessment, it has been seen that exercise has a protective role on cardiac blood vessels. This mechanism needs further investigations with long term exposure studies for clarifying possible pathways.

The effects of retinoic acid on mmp-2 production, proliferation and ultrastructural morphology in rat uterus.

AIMS: Retinoic acid (RA) has a vital importance in order to ensure continuity and morphology in many tissues. Matrix metalloproteinases (MMPs) have significant roles in proliferation, the formation of cancers, and metastasis. In this study the effects of RA on MMP-2 production in cells of rat uterus were investigated. METHODS: Twenty-four adult Spraque Dawley rats were divided into two groups, the experimental group was treated with 40mg/kg/day 13-cis RA for 5days by gavage. Uterine tissue sections were treated with BrdU and MMP-2 antibodies, evaluated using light microscopy. Tissues were fixed with 2.5% glutaraldehyde and evaluated using transmission electron miroscopy. RESULTS: MMP-2 immunoreactivity decreased in the stromal cells compared with the control group and no staining of MMP-2 was observed in glandular epithelium in the experimental group. BrDU labeling of cells showed significant decrease in RA-treated group versus control group cells. Based on the electron microscopy evaluation, the surface epithelial cells of the experimental group showed vacuolization, and an accumulation of lipofuscin bodies was also observed in the gland epithelium. Cells involving autophagic vacuoles contained excess lipid granules in the entire uterus layers especially localized at the border of the endometrium and myometrium. CONCLUSION: RA had negative effects on cell proliferation and cell morphology and inhibited MMP-2 expression.

The effects of titanium dioxide nanoparticles on ultrastructure of zebrafish testis (Danio rerio).

INTRODUCTION: Nanotechnology investigates materials at nanoscale level (0.1-100nm in diameter). There are many commercially nanoproducts such as silver, silicon, titanium, zinc, and gold. They are used in a variety of applications and released to the environment. Titanium dioxide (TiO2) is one of the most commonly used nanoparticles (NP). In this study, the ultrastructural effects of TiO2-NP on zebrafish testis tissue were evaluated. MATERIAL AND METHOD: Zebrafish were divided into four groups (N=60) as one control and 3 experimental groups (1mg/L, 2mg/L and 4mg/L TiO2). Testis tissues were dissected after 5days of the exposure. Tissues were fixed with 2.5% glutaraldehyde at 4°C. After routine electron microscopy tissue processing, the testis were embedded in epon resin. Ultrathin sections were counterstained with 1% uranyl acetate and lead citrate and examined using a transmission electron microscope. RESULTS: Mitochodrial degeneration with swelling and cristae loss were detected in Sertoli cells and spermatogonial cells of TiO2-NP treated groups in a dose-dependent manner. Moreover, autophagic vacuole accumulation were seen in Sertoli cell cytoplasms of the experimental groups. Necrosis was also detected in the 4mg TiO2-NP-treated group. CONCLUSION: TiO2-NP has been used in crop production, food additives, medicine, toothpastes, sunscreens, cosmetics, and in waste water treatment, which contaminated the environment. Our findings showed TiO2-NP-induced autophagy and necrosis at higher doses in Sertoli cells, which consequently negatively affected spermatogenic cells and testicular morphology of Zebrafish. It is important to give much more attention to the use of this NP to minimise the possible effects on nature and organisms.

Determination of DNA damage and telomerase activity in stanozolol-treated rats.

Anabolic androgenic steroids (AAS) are performance-enhancing drugs commonly abused by atheletes. Stanozolol is a synthetic testosterone-derived anabolic steroid. Although it is well known that AAS have several side-effects, there are only few toxicological studies available on the toxic effects and mechanisms of action of stanozolol. The aim of this study was to investigate the genotoxic effects of stanozolol and to determine its effects on telomerase activity in Sprague-Dawley male rats. For this purpose, 34 male rats were divided into 5 groups as follows: i) the control group (n=5); ii) the propylene glycol (PG)-treated group (n=5); iii) the stanozolol-treated group (n=8); iv) the PG-treated group subjected to exercise (n=8); and v) the stanozolol-treated group subjected to exercise (n=8). PG is used as a solvent control in our study. Stanozolol (5 mg/kg) and PG (1 ml/kg) were injected subcutaneously 5 days/week for 28 days. After 28 days, the animals were sacrificed, and DNA damage evaluation (comet assay) and telomerase activity assays were then performed using peripheral blood mononuclear cells (PBMCs). Telomerase activity was measured by using the TeloTAGGG Telomerase PCR ELISA PLUS kit. The results of this study revealed that stanozolol treatment induced DNA damage, while exercise exerted a protective effect. Stanozolol treatment without exercise stimulation was associated with a significant increase in telomerase activity in the PBMCs.

Severe neurodegenerative disease in brothers with homozygous mutation in POLR1A.

In two brothers born to consanguineous parents, we identified an unusual neurological disease that manifested with ataxia, psychomotor retardation, cerebellar and cerebral atrophy, and leukodystrophy. Via linkage analysis and exome sequencing, we identified homozygous c.2801C>T (p.(Ser934Leu)) in POLR1A (encoding RPA194, largest subunit of RNA polymerase I) and c.511C>T (p.(Arg171Trp)) in OSBPL11 (encoding oxysterol-binding protein-like protein 11). Although in silico analysis, histopathologic evidence and functional verification indicated that both variants were deleterious, segregation with the patient phenotype established that the POLR1A defect underlies the disease, as a clinically unaffected sister also was homozygous for the OSBPL11 variant. Decreased nucleolar RPA194 was observed in the skin fibroblasts of only the affected brothers, whereas intracellular cholesterol accumulation was observed in the skin biopsies of the patients and the sister homozygous for the OSBPL11 variant. Our findings provide the first report showing a complex leukodystrophy associated with POLR1A. Variants in three other RNA polymerase subunits, POLR1C, POLR3A and POLR3B, are known to cause recessive leukodystrophy similar to the disease afflicting the present family but with a later onset. Of those, POLR1C is also implicated in a mandibulofacial dysostosis syndrome without leukodystrophy as POLR1A is. This syndrome is absent in the family we present.

Analysis of diabetes-related cerebellar changes in streptozotocin-induced diabetic rats.

BACKGROUND/AIM: Diabetic peripheral neuropathy has been extensively studied and reported, but the number of studies that have investigated diabetes-related changes in the central nervous system are limited, with even fewer studies on the cerebellum. The aim of this experimental study was to perform a histologic analysis of the diabetes-related changes in the cerebellums of diabetic rats. MATERIALS AND METHODS: Twenty Sprague Dawley rats weighing between 200 and 220 g were included in the study. Diabetes was induced in 14 of these rats by a single intraperitoneal injection of 65 mg/kg streptozotocin dissolved in saline, while 6 animals constituted the control group. The induction of diabetes was confirmed by measuring the blood glucose levels in the tail blood with a glucometer. Levels equal to or above 200 mg/dL were considered diabetic. Induction of diabetes failed in 3 animals, who were then excluded from the study. RESULTS: Light and electron microscopic studies revealed that the neurons and glial cells in the diabetic group had degenerative changes, irregularities and disruption in the myelin sheath, disintegration in the presynaptic vesicles, engorged axon terminals, perivascular and mitochondrial swelling, mitochondrial structural changes, and fragmentation of the neurofilaments. CONCLUSION: Ultrastructural alterations are observed in the diabetic rat cerebellum.

Ultrastructural changes in epithelial cells of rats exposed to low concentration of hydrogen sulfide for 50 days.

Hydrogen sulfide (H2S) and other volatile sulfur compounds (VSCs) appear mainly in the oral air of patients with halitosis. It seems that VSCs are directly involved in the pathogenesis of gingival diseases. In previous studies, short-term (7 hours-4 days), high concentrations (5-400 ppm) of H2S applications on periodontal tissues have been evaluated in a culture medium. The aim of the present study was to investigate the potential effects of lower (equivalent to halitosis) concentrations of H2S on rat gingival tissue for longer-term inhalation. The threshold level of pathologic halitosis perceived by humans at 250 ppb of H2S was converted to rat equivalent concentration (4.15 ppm). Rats in the experimental (H2S) group (n=8) were exposed to H2S continuously but not the control rats (n=8). After 50 days, the gingival sulcular tissue samples of each rat were taken and examined using transmission electron microscope. Ultrastructural changes in the sulcular epithelia of the rat gingiva showed deformation of celullar shape, vacuolization, and disintegrity of intercelullar connection by loss of desmosomes and collagen fibrils. No basal membrane damage was observed. Inhalation of low levels of H2S (equivalent of halitosis) in the oral environment causes ultrastructural celullar damages in rat sulcular mucosa. These results suggest that halitosis may be the potential reason for periodontal destruction in humans.

The effects of permethrin on rat ovarian tissue morphology.

All organisms are exposed to chemical agents during their lifetime. One of these agents is a pesticide that is used as fly killer. In this study we investigated the effects of permethrin on rat ovaries using light and electron microscopy. We used 24 Wistar albino female rats and divided them into 3 groups. Dosages 20 and 40 mg/kg/day permethrin were administered by gavage for 14 days. Normal saline was given to control rats. After treatment, ovarian tissues were collected and prepared for light and electron microscopy evaluation. Negative effects of permethrin were detected on follicular and corpus luteum cell morphology in a dose dependent manner when compared with the control group. Picnotic cellular appearance and condensed chromatin were detected as evidence of apoptotic cell death. Furthermore, degenerative changes were seen in the ultrastructure of mitochondria and endoplasmic reticulum. Thus, these findings suggested that permethrin caused degenerative effects on ovarian morphology in a dose dependent manner.

Effects of systemic Thalidomide and intracerebroventricular Etanercept and Infliximab administration in a Streptozotocin induced dementia model in rats.

Tumor necrosis factor-alpha (TNF-α) upregulation enhances amyloid ß (Aß) induced neurotoxicity in Alzheimer's disease (AD). Intracerebroventricular streptozotocin (STZ) administration causes pathological changes and cognitive deficits similar to those seen in AD by causing impairment of brain glucose and energy metabolism. Recent reports indicate a protective role of Thalidomide, Etanercept, and Infliximab, all of which have anti-TNF-α activity, against cognitive and neuropathological changes in experimental and clinical studies. We aimed to investigate the protective effects of Thalidomide, Etanercept, and Infliximab in a rat model of intracerebroventricular STZ-induced dementia. Sprague-Dawley rats (250-300g) were separated to sham (n=6) and STZ (n=24) groups. The STZ group was divided into four groups (STZ, STZ-thalidomide, STZ-etanercept, and STZ-infliximab). Morris's water maze (MWM) and passive avoidance (PA) tests were performed. At the end of the third week, brain tissues were obtained. Histopathological analysis, immunohistochemistry, and electron microscopic examinations were done. The improvement performance of the STZ group was significantly reduced in the MWM test (p<0.001). Compared with the STZ, STZ-thalidomide, STZ-etanercept, and STZ-infliximab groups had significantly better performance (p<0.001, <0.05 and <0.05, respectively) in the MWM test. STZ administration caused a significant decrease in the mean escape latency in PA reflex (p<0.001). Thalidomide, Etanercept, and Infliximab were associated with better PA reflexes compared to the STZ group (p<0.001 for all). Morphological and immunohistochemical results showed increased neurodegenerative changes compared to sham group. Our findings are in line with the findings reported in the literature and encourage further studies with TNF-α antagonists, in particular Thalidomide.