Fine structure of the ductuli efferentes of the hamster (Mesocricetus auratus).

Structurally the ductuli efferentes of the hamster showed 2 distinct segments, a testicular and an epididymal. Both of these segments were lined by a pseudostratified epithelium, which showed basically non-ciliated and ciliated cells. In the testicular segment a 3rd type of oval dark cells was observed. The ultrastructural characteristics of these cells were presented and discussed in this report.

Effect of mating delay in the ovary of Apis mellifera L. (Hymenoptera, Apidae) queens: Histological aspects

This paper reports the effect of mating delay on the queen Apis mellifera ovaries based on a light microscopy analysis. Soon after a queen emerges from the brood cell, oocytes start to differentiate in the ovaries, but if mating does not occur at the correct age (about 6 days after emergence) cell degeneration begins. Ovaries of 15-day-old virgin queens show extensive disorganization with cell death affecting all types of ovariole cells. Types of cell death and the possible causes are discussed.

Localization of myosin-Va in subpopulations of cells in rat endocrine organs

Myosin-Va is a Ca 2+/calmodulin-regulated unconventional myosin involved in the transport of vesicles, membranous organelles, and macromolecular complexes composed of proteins and mRNA. The cellular localization of myosin-Va has been described in great detail in several vertebrate cell types, including neurons, melanocytes, lymphocytes, auditory tissues, and a number of cultured cells. Here, we provide an immunohistochemical view of the tissue distribution of myosin-Va in the major endocrine organs. Myosin-Va is highly expressed in the pineal and pituitary glands and in specific cell populations of other endocrine glands, especially the parafollicular cells of the thyroid, the principal cells of the parathyroid, the islets of Langerhans of the pancreas, the chromaffin cells of the adrenal medulla, and a subpopulation of interstitial testicular cells. Weak to moderate staining has been detected in steroidogenic cells of the adrenal cortex, ovary, and Leydig cells. Myosin-Va has also been localized to non-endocrine cells, such as the germ cells of the seminiferous epithelium and maturing oocytes and in the intercalated ducts of the exocrine pancreas. These data provide the first systematic description of myosin-Va localization in the major endocrine organs of rat. © 2008 Springer-Verlag.

Persistence of experimental Rocio virus infection in the golden hamster (Mesocricetus auratus)

ABSTRACT: Rocio virus (ROCV) is an encephalitic flavivirus endemic to Brazil. Experimental flavivirus infections have previously demonstrated a persistent infection and, in this study, we investigated the persistence of ROCV infection in golden hamsters (Mesocricetus auratus). The hamsters were infected intraperitoneally with 9.8 LD50/0.02 mL of ROCV and later anaesthetised and sacrificed at various time points over a 120-day period to collect of blood, urine and organ samples. The viral titres were quantified by real-time-polymerase chain reaction (qRT-PCR). The specimens were used to infect Vero cells and ROCV antigens in the cells were detected by immunefluorescence assay. The levels of antibodies were determined by the haemagglutination inhibition technique. A histopathological examination was performed on the tissues by staining with haematoxylin-eosin and detecting viral antigens by immunohistochemistry (IHC). ROCV induced a strong immune response and was pathogenic in hamsters through neuroinvasion. ROCV was recovered from Vero cells exposed to samples from the viscera, brain, blood, serum and urine and was detected by qRT-PCR in the brain, liver and blood for three months after infection. ROCV induced histopathological changes and the expression of viral antigens, which were detected by IHC in the liver, kidney, lung and brain up to four months after infection. These findings show that ROCV is pathogenic to golden hamsters and has the capacity to cause persistent infection in animals after intraperitoneal infection.

Expression of fibroblast growth factor 10 and cognate receptors in the developing bovine ovary

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ovarian superstimulation using FSH combined with equine chorionic gonadotropin (eCG) upregulates mRNA encoding proteins involved with LH receptor intracellular signaling in granulosa cells from Nelore cows

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ovary development in honeybee (Apis Mellifera L.) workers under co2 narcosis, caged outside of the colony

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Gene Expression in Bovine Oocytes and Cumulus Cells After Meiotic Inhibition with the Cyclin-Dependent Kinase Inhibitor Butyrolactone I

Contents The aim of this study was to determine the effect of temporary inhibition of meiosis using the cyclin-dependent kinase inhibitor butyrolactone I (BLI) on gene expression in bovine oocytes and cumulus cells. Immature bovine cumulusoocyte complexes (COCs) were assigned to groups: (i) Control COCs collected immediately after recovery from the ovary or (ii) after in vitro maturation (IVM) for 24 h, (iii) Inhibited COCs collected 24 h after incubation with 100 mu m BLI or (iv) after meiotic inhibition for 24 h followed by IVM for a further 22 h. For mRNA relative abundance analysis, pools of 10 denuded oocytes and respective cumulus cells were collected. Transcripts related to cell cycle regulation and oocyte competence were evaluated in oocytes and cumulus cells by quantitative real-time PCR (qPCR). Most of the examined transcripts were downregulated (p < 0.05) after IVM in control and inhibited oocytes (19 of 35). Nine transcripts remained stable (p > 0.05) after IVM in control oocytes; only INHBA did not show this pattern in inhibited oocytes. Seven genes were upregulated after IVM in control oocytes (p < 0.05), and only PLAT, RBP1 and INHBB were not upregulated in inhibited oocytes after IVM. In cumulus cells, six genes were upregulated (p < 0.05) after IVM and eight were downregulated (p < 0.05). Cells from inhibited oocytes showed the same pattern of expression regarding maturation profile, but were affected by the temporary meiosis inhibition of the oocyte when the same maturation stages were compared between inhibited and control groups. In conclusion, changes in transcript abundance in oocytes and cumulus cells during maturation in vitro were mostly mirrored after meiotic inhibition followed by maturation.

Vitellogenin content in fat body and ovary homogenates of workers and queens of Melipona quadrifasciata anthidioides during vitellogenesis

Vitellogenin (Vg) is an egg yolk protein that is produced primarily in the fat body of most female insects. In the advanced social structure of eusocial honeybees, the presence of the queen inhibits egg maturation in the workers ovaries. However in the stingless bee Melipona quadrifasciata, the workers always develop ovaries and lay a certain amount of eggs while provisioning the brood cells with larval food during what is known as the worker nurse phase. The present work is a comparative study of the presence of Vg in homogenates of the fat bodies and ovaries of the nurse workers, and the virgin and physogastric queens of M. quadrifasciata. The presence of Vg was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting using Apis mellifera anti-egg antibody. Vg was not detected in the fat bodies or ovaries of the workers, but it was found in the ovaries of virgin and physogastric queens and in the fat body of physogastric queens. The results are discussed, taking into account the reproductive state of the individuals and the other possible roles of Vg, such as a storage protein for metoabolism of other organs.

Plasma corticosterone elevation inhibits the activation of nuclear factor kappa B (NFKB) in the Syrian hamster pineal gland

We evaluated how the mild stress-induced increase in endogenous corticosterone affected the pineal gland in Syrian hamsters (Mesocricetus auratus). The animals were maintained under constant light for 1 day, instead of a cycle of 14:10-h, to increase the circulating corticosterone levels during the daytime. The nuclear translocation of nuclear factor kappa B (NFKB), which is the pivotal transcription factor for stress and injury, presented a daily rhythm in normal animals. NFKB nuclear content increased linearly from the onset of light [Zeitgeber Time 0 (ZT0)] until ZT11 and decreased after ZT12 when the plasma corticosterone peak was detected in normal animals. However, the 24-h profiles of the two curves were different, and they did not clearly support an exclusive relationship between corticosterone levels and NFKB content. Therefore, we tested the effect of increased endogenous corticosterone through inducing mild stress by maintaining daytime illumination for one night. This stressful condition, which increased daytime corticosterone levels, resulted in a daytime decrease in NFKB nuclear content, and this was inhibited by mifepristone. Overall, this study shows that NFKB has a daily rhythm in Syrian hamster pineal glands and, by increasing endogenous corticosterone with a stressful condition, NFKB activity is regulated. Therefore, this study suggests that the pineal gland in the Syrian hamster is a sensor of stressful conditions.

Norepinephrine stimulates progesterone production in highly estrogenic bovine granulosa cells cultured under serum-free, chemically defined conditions

Background: Since noradrenergic innervation was described in the ovarian follicle, the actions of the intraovarian catecholaminergic system have been the focus of a variety of studies. We aimed to determine the gonadotropin-independent effects of the catecholamine norepinephrine (NE) in the steroid hormone profile of a serum-free granulosa cell (GC) culture system in the context of follicular development and dominance. Methods: Primary bovine GCs were cultivated in a serum-free, chemically defined culture system supplemented with 0.1% polyvinyl alcohol. The culture features were assessed by hormone measurements and ultrastructural characteristics of GCs. Results: GCs produced increasing amounts of estradiol and pregnenolone for 144h and maintained ultrastructural features of healthy steroidogenic cells. Progesterone production was also detected, although it significantly increased only after 96h of culture. There was a highly significant positive correlation between estradiol and pregnenolone production in high E2-producing cultures. The effects of NE were further evaluated in a dose response study. The highest tested concentration of NE (10 (-7) M) resulted in a significant increase in progesterone production, but not in estradiol or pregnenolone production. The specificity of NE effects on progesterone productio n was further investigated by incubating GCs with propranolol (10 (-8) M), a non-selective beta-adrenergic antagonist. Conclusions: The present culture system represents a robust model to study the impact of intrafollicular factors, such as catecholamines, in ovarian steroidogenesis and follicular development. The results of noradrenergic effects in the steroidogenesis of GC have implications on physiological follicular fate and on certain pathological ovarian conditions such as cyst formation and anovulation.

Trypanosoma cruzi invades host cells through the activation of endothelin and bradykinin receptors: a converging pathway leading to chagasic vasculopathy

BACKGROUND AND PURPOSE Independent studies in experimental models of Trypanosoma cruzi appointed different roles for endothelin-1 (ET-1) and bradykinin (BK) in the immunopathogenesis of Chagas disease. Here, we addressed the hypothesis that pathogenic outcome is influenced by functional interplay between endothelin receptors (ETAR and ETBR) and bradykinin B2 receptors (B2R). EXPERIMENTAL APPROACH Intravital microscopy was used to determine whether ETR/B2R drives the accumulation of rhodamine-labelled leucocytes in the hamster cheek pouch (HCP). Inflammatory oedema was measured in the infected BALB/c paw of mice. Parasite invasion was assessed in CHO over-expressing ETRs, mouse cardiomyocytes, endothelium (human umbilical vein endothelial cells) or smooth muscle cells (HSMCs), in the presence/absence of antagonists of B2R (HOE-140), ETAR (BQ-123) and ETBR (BQ-788), specific IgG antibodies to each GPCRs; cholesterol or calcium-depleting drugs. RNA interference (ETAR or ETBR genes) in parasite infectivity was investigated in HSMCs. KEY RESULTS BQ-123, BQ-788 and HOE-140 reduced leucocyte accumulation in HCP topically exposed to trypomastigotes and blocked inflammatory oedema in infected mice. Acting synergistically, ETAR and ETBR antagonists reduced parasite invasion of HSMCs to the same extent as HOE-140. Exogenous ET-1 potentiated T. cruzi uptake by HSMCs via ETRs/B2R, whereas RNA interference of ETAR and ETBR genes conversely reduced parasite internalization. ETRs/B2R-driven infection in HSMCs was reduced in HSMC pretreated with methyl-beta-cyclodextrin, a cholesterol-depleting drug, or in thapsigargin-or verapamil-treated target cells. CONCLUSIONS AND IMPLICATIONS Our findings suggest that plasma leakage, a neutrophil-driven inflammatory response evoked by trypomastigotes via the kinin/endothelin pathways, may offer a window of opportunity for enhanced parasite invasion of cardiovascular cells.

The putative role of ovary removal and progesterone when considering the effect of formaldehyde exposure on lung inflammation induced by ovalbumin

OBJECTIVE: Formaldehyde exposure during the menstrual cycle is known to affect the course of allergic lung inflammation. Because our previous data demonstrated that formaldehyde combined with an ovariectomy reduced allergic lung inflammation, we investigated the putative role of ovary removal and progesterone treatment when considering the effect of formaldehyde on allergic lung inflammation. METHOD: Ovariectomized rats and their matched controls were exposed to formaldehyde (1%, 3 days, 90 min/day) or vehicle, and immediately after exposure, the rats were sensitized to ovalbumin by a subcutaneous route. After 1 week, the rats received a booster by the same route, and after an additional week, the rats were challenged with ovalbumin (1%) by an aerosol route. The leukocyte numbers, interleukin-10 (IL-10) release, myeloperoxidase activity, vascular permeability, ex vivo tracheal reactivity to methacholine and mast cell degranulation were determined 24 h later. RESULTS: Our results showed that previous exposure to formaldehyde in allergic rats decreased lung cell recruitment, tracheal reactivity, myeloperoxidase activity, vascular permeability and mast cell degranulation while increasing IL-10 levels. Ovariectomy only caused an additional reduction in tracheal reactivity without changing the other parameters studied. Progesterone treatment reversed the effects of formaldehyde exposure on ex vivo tracheal reactivity, cell influx into the lungs and mast cell degranulation. CONCLUSION: In conclusion, our study revealed that formaldehyde and ovariectomy downregulated allergic lung inflammation by IL-10 release and mast cell degranulation. Progesterone treatment increased eosinophil recruitment and mast cell degranulation, which in turn may be responsible for tracheal hyperreactivity and allergic lung inflammation

Shikonin directly targets mitochondria and causes mitochondrial dysfunction in cancer cells

Chemotherapy is a mainstay of cancer treatment. Due to increased drug resistance and the severe side effects of currently used therapeutics, new candidate compounds are required for improvement of therapy success. Shikonin, a natural naphthoquinone, was used in traditional Chinese medicine for the treatment of different inflammatory diseases and recent studies revealed the anticancer activities of shikonin. We found that shikonin has strong cytotoxic effects on 15 cancer cell lines, including multidrug-resistant cell lines. Transcriptome-wide mRNA expression studies showed that shikonin induced genetic pathways regulating cell cycle, mitochondrial function, levels of reactive oxygen species, and cytoskeletal formation. Taking advantage of the inherent fluorescence of shikonin, we analyzed its uptake and distribution in live cells with high spatial and temporal resolution using flow cytometry and confocal microscopy. Shikonin was specifically accumulated in the mitochondria, and this accumulation was associated with a shikonin-dependent deregulation of cellular Ca(2+) and ROS levels. This deregulation led to a breakdown of the mitochondrial membrane potential, dysfunction of microtubules, cell-cycle arrest, and ultimately induction of apoptosis. Seeing as both the metabolism and the structure of mitochondria show marked differences between cancer cells and normal cells, shikonin is a promising candidate for the next generation of chemotherapy.

Pioglitazone inhibits androgen production in NCI-H295R cells by regulating gene expression of CYP17 and HSD3B2

Thiazolidinediones (TZDs) such as pioglitazone and rosiglitazone are widely used as insulin sensitizers in the treatment of type 2 diabetes. In diabetic women with polycystic ovary syndrome, treatment with pioglitazone or rosiglitazone improves insulin resistance and hyperandrogenism, but the mechanism by which TZDs down-regulate androgen production is unknown. Androgens are synthesized in the human gonads as well as the adrenals. We studied the regulation of androgen production by analyzing the effect of pioglitazone and rosiglitazone on steroidogenesis in human adrenal NCI-H295R cells, an established in vitro model of steroidogenesis of the human adrenal cortex. Both TZDs changed the steroid profile of the NCI-H295R cells and inhibited the activities of P450c17 and 3betaHSDII, key enzymes of androgen biosynthesis. Pioglitazone but not rosiglitazone inhibited the expression of the CYP17 and HSD3B2 genes. Likewise, pioglitazone repressed basal and 8-bromo-cAMP-stimulated activities of CYP17 and HSD3B2 promoter reporters in NCI-H295R cells. However, pioglitazone did not change the activity of a cAMP-responsive luciferase reporter, indicating that it does not influence cAMP/protein kinase A/cAMP response element-binding protein pathway signaling. Although peroxisome proliferator-activated receptor gamma (PPARgamma) is the nuclear receptor for TZDs, suppression of PPARgamma by small interfering RNA technique did not alter the inhibitory effect of pioglitazone on CYP17 and HSD3B2 expression, suggesting that the action of pioglitazone is independent of PPARgamma. On the other hand, treatment of NCI-H295R cells with mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) enhanced promoter activity and expression of CYP17. This effect was reversed by pioglitazone treatment, indicating that the MEK/ERK signaling pathway plays a role in regulating androgen biosynthesis by pioglitazone.