Effect of Endocrine Disrupters on mRNA Expression of Vitellogenin (VTG) II and Very Low Density Lipoprotein (apoVLDL)II in the Liver of Quail Embryos

The present study was conducted to assess estrogenic activity of nonylphenol (NP) and octylphenol (OP) in quail embryos by determining mRNA levels of liver vitellogenin (VTG) II and very low density lipoprotein (apoVLDL) II. The fertile eggs were treated with a single injection of either NP, OP or ethynyl estradiol (EE) at doses of 10 and 100 nmole/egg in 20μl on day 13 of incubation. In the control group the eggs were treated with the vehicle (corn oil, 20μl/egg). On day 15 of incubation the liver was collected and total RNA was extracted. Both mRNA levels were determined by RT-PCR assay and were expressed in relation to β actin mRNA levels. No expression of VTG II mRNA was detected in the control group, whereas a marked induction of VTG II mRNA was revealed in the EE treatment. A weak but distinct expression of VTG II mRNA was evident in the NP and OP treatment groups. ApoVLDLII transcripts were detected in the control group and induced markedly by the injection of EE with higher expression in females. NP also induced considerable expression in females, whereas no transcripts were detected in males. OP also induced the transcript in females but in males OP at 10 nmole was effective. This study indicates that NP and OP possess estrogenic activity in terms of liver VTG II and apoVLDLII mRNA expression in the quail embryo, and that apoVLDLII expression in female embryo is more sensitive to estrogenic substances

Oxidative Stress Induces Endothelial Cell Senescence via Downregulation of Sirt6

Accumulating evidence has shown that diabetes accelerates aging and endothelial cell senescence is involved in the pathogenesis of diabetic vascular complications, including diabetic retinopathy. Oxidative stress is recognized as a key factor in the induction of endothelial senescence and diabetic retinopathy. However, specific mechanisms involved in oxidative stress-induced endothelial senescence have not been elucidated. We hypothesized that Sirt6, which is a nuclear, chromatin-bound protein critically involved in many pathophysiologic processes such as aging and inflammation, may have a role in oxidative stress-induced vascular cell senescence. Measurement of Sirt6 expression in human endothelial cells revealed that H2O2 treatment significantly reduced Sirt6 protein. The loss of Sirt6 was associated with an induction of a senescence phenotype in endothelial cells, including decreased cell growth, proliferation and angiogenic ability, and increased expression of senescence-associated β-galactosidase activity. Additionally, H2O2 treatment reduced eNOS expression, enhanced p21 expression, and dephosphorylated (activated) retinoblastoma (Rb) protein. All of these alternations were attenuated by overexpression of Sirt6, while partial knockdown of Sirt6 expression by siRNA mimicked the effect of H2O2. In conclusion, these results suggest that Sirt6 is a critical regulator of endothelial senescence and oxidative stress-induced downregulation of Sirt6 is likely involved in the pathogenesis of diabetic retinopathy

Expression of P450arom, AMH and ERα mRNA in Gonads of Turkey, Duck and Goose within One Week of Age

Although many studies have shown that P450aromatase (P450arom), anti-Müllerian hormone (AMH) and estrogen receptor α (ERα) play pivotal roles in sexual differentiation of the gonads during early embryonic development in chickens and quail, few studies have been reported for other domestic birds. Furthermore, little information is available in relation to the mRNA expression in gonads after sexual differentiation in posthatching birds. The present study was conducted to assay mRNA expression of P450arom, AMH and ERα in gonads at day of hatch in turkey and duck and 2 days after hatching in goose using the real-time PCR. The mRNA expression was also determined at one week of age in gonads of these birds. At the time of collection of the left gonads for total RNA extraction, the external appearance of the left and right gonads of male and female was documented by digital camera. Clear asymmetry was observed in the female ovary in which the right ovary was regressed completely by 1-2 days in posthatching turkey, duck and goose. Although gonadal asymmetry was as remarkable as in females, the left testis was larger than the right one in males. Remarkable expression of P450arom mRNA was observed only in females in all the 3 species but substantially no expression was detected in males. Significantly higher expression of AMH mRNA was detected in males than females only in goose but there was no sex difference in turkey and duck at 1-2 days posthatching and one week of age. Weak ERα mRNA expression without a sex difference was detected in the 3 birds. These results suggest that estrogen plays a key role for ovarian development via P450arom mRNA expression after hatching, whereas absence of its expression in males leads to testis development in turkey, duck and goose

Effects of Aromatase Inhibitor (Fadrozole)-Induced Sex-Reversal on Gonadal Differentiation and mRNA Expression of P450arom, AMH and ERα in Embryos and Growth in Posthatching Quail

The present study was conducted to investigate (1) the effective day of treatment with Fadrozole, nonsteroidal aromatase inhibitor (AI), during incubation for sex reversal, (2) the mRNA expression of aromatase (P450arom), anti-Müllerian hormone (AMH) and estrogen receptor α (ERα) in quail gonad and gonadal structures after the AI treatment before hatching and (3) the effects of AI on gonad growth, left-right asymmetry of the gonad and body weight gain between 1 to 7 weeks posthatch. (1) Females AI-treated at 0, 2 and 4 days of incubation had symmetrical gonads at hatch similar to normal males, whereas those treated at 6 and 8 days of incubation had asymmetrical gonads. (2) In females AI-treated at day 0 of incubation, the left gonad became an ovotestis at day 15 of incubation, displaying seminiferous tubules with testicular-like cords and ovarian follicle with ovum. In the control group, P450arom mRNA expression was significantly higher in females than males. However, AI-treated females showed 2 response-patterns of either higher or lower expression. The latter was similar to that of control males. No significant differences in levels of AMH and ERα mRNA between sexes either in control or AI-treated groups was observed. (3) A gradual increase in body weight of the control males and females without sex difference was observed up to 5 weeks of age. However, at 6-7 weeks the average weight of females was heavier than males each in control and treated group. These results indicate that shortly before day 6 of incubation P450arom mRNA expression, at least in a part, is important for asymmetrical formation of gonads in female quail. In AI-treated females, the high responder show similar level of P450arom mRNA in control males and the low responder do not respond to the AI. Body weight is controlled non-gonadally in quail

Profiles of mRNA Expression of FOXL2, P450arom, DMRT1, AMH, P450c17, SF1, ERα and AR, in Relation to Gonadal Sex Differentiation in Duck Embryo

The present study was conducted to show a profile of mRNA expression of sex-differentiation related genes in the duck gonad. Fertile duck eggs (Cherry-valley) were incubated (hatching at day 28 of incubation) and the gonads were collected at days 7, 8, 10, 12 and 15 of incubation after taking photos of the gonads. Left gonad was used for identification of genetic sex and for total RNA extraction. Reverse transcription-polymerase chain reaction (PCR) was performed using appropriate forward and reverse primers for each of FOXL2, P450arom, DMRT1, AMH, P450c17, SF1, ER α and AR genes of the chicken and the corresponding cDNA fragments were sequenced for duck. Subsequently, real-time PCR was performed to detect mRNA expression of those genes in the duck embryo. Male bilateral gonads assumed symmetry in appearance throughout the examined period, whereas the female gonads were noticeably asymmetric at 10 days of incubation with a smaller size of the right. Female asymmetry was distinct by day 15 of incubation. In females, FOXL2 and P450arom mRNA expression began at day 8 of incubation and remained high thereafter, but in males only negligible expression was detected. In males, mRNA expression of DMRT1 and AMH was detected at day 7 of incubation and tended to increase gradually thereafter, whereas in females the expression remained low throughout the examined period. Though the slight expression of P450c17 mRNA started at day 7 only in males, the expression was low throughout the examined period, whereas in female the expression was distinct at day 8 of incubation and remained high thereafter. Expressions of the other genes were variable with no differences in each expression between the sexes. The results indicate that FOXL2 and P450arom play an important role for the ovarian formation

Identification of Spermatogenic Cells Expressing Protamine mRNA in Japanese Quail by RT-PCR

This study was conducted by reverse transcription-polymerase chain reaction (RT-PCR) analysis to determine when the protamine mRNA is expressed in testicular germ cells during spermatogenesis in quail. Ejaculated sperm were obtained from the female cloacal gland after mating. Adult male quail were used for collection of the testicular spermatogenic cells. Twenty cells of ejaculated sperm and those of each mechanically dispersed testicular cells including pachytene spermatocytes, round spermatids and elongated spermatids, were picked up using micropipette under phase-contrast microscope. Total RNA was extracted, reverse-transcribed, and amplified by using either quail protamine or chicken S17 ribosomal protein specific primers. The PCR products were run on TAE gel electrophoresis. There was no protamine mRNA expression in spermatocytes, but the expression was detected in round spermatids and elongated spermatids, as well as in ejaculated sperm. These results suggest that protamine mRNA is expressed at haploid stage of quail spermatogenic cells

Sigma Receptor 1 Modulates Endoplasmic Reticulum Stress in Retinal Neurons

The mechanism by which the molecular chaperone σR1 mediates robust neuroprotection was analyzed in retinal neurons. Under stress, σR1 binds the endoplasmic reticulum protein BiP. The σR1 ligand (+)-pentazocine may exert its effects by dissociating σR1 from BiP

Cystathionine Beta Synthase Expression in Mouse Retina

Purpose: Cystathionine β-synthase (CBS), a key enzyme in the transsulfuration metabolic pathway, converts homocysteine to cystathionine, which is converted to cysteine required for the synthesis of major retinal antioxidant glutathione (GSH). Enzyme activity assays suggest that CBS is present in human and pig retina, however recent studies reported that CBS is not expressed in mouse retina. We found this species difference puzzling. Given the plethora of studies using mouse retina as a model system, coupled with the importance of GSH in retina, we investigated CBS expression in mouse retina at the molecular and cell biological level. Methods: Wildtype (WT) mice or mice lacking the gene encoding CBS (cbs − / − ) were used in these studies. RNA and protein were isolated from retinas and liver (positive control) for the analysis of cbs gene expression by RT-PCR and CBS protein expression by Western blotting, respectively. CBS was analyzed by immunofluorescence in retinal cryosections and primary retinal cells (ganglion, Müller, retinal pigment epithelial). CBS enzyme activity was measured in primary Müller cells. Results: RT-PCR revealed robust cbs expression in WT liver, brain and retina. Western blotting detected CBS in retina, brain and liver of WT mice, but not in cbs − / − mice liver. In immunohistochemical studies, CBS was present abundantly in the ganglion cell layer of retina; it was detected also in primary isolations of Müller, RPE and ganglion cells. CBS activity was detected in Müller cells by fluorescent detection of H 2 S. Conclusions: We have compelling molecular evidence that CBS is expressed in mouse retina at the gene and protein level. Our immunofluorescence data suggest that it is present in several retinal cell types and the data from the enzyme activity assay suggest activity in Müller cells. These findings set the stage to investigate the role of CBS and the transsulfuration pathway in the generation of GSH in mouse retina

Sigma Receptor Ligand, (þ)-Pentazocine, Suppresses Inflammatory Responses of Retinal Microglia

PURPOSE. To evaluate the effects of the r 1 receptor (rR1) agonist, (þ)-pentazocine, on lipopolysaccharide (LPS)-induced inflammatory changes in retinal microglia cells. METHODS. Retinal microglia cells were isolated from Sprague-Dawley rat pups. Cells were treated with LPS with or without (þ)-pentazocine and with or without the rR1 antagonist BD1063. Morphologic changes were assayed. Cell viability was assessed by using MTT assay. Supernatant levels of tumor necrosis factor a (TNF-a), interleukin 10, (IL-10), monocyte chemoattractant protein-1 (MCP-1), and nitric oxide (NO) were determined. Reactive oxygen species (ROS) formation was assayed, and levels of mitogen-activated protein kinases (MAPKs) were analyzed by using Western blot. RESULTS. The rR1 protein was expressed in retinal microglia. Incubation with LPS and/or (þ)-pentazocine did not alter cell viability or rR1 protein levels. Incubation with LPS for 24 hours induced a marked change in microglial morphology and a significant increase in secreted levels of TNF-a, IL-10, MCP-1, and NO. Pretreatment with (þ)-pentazocine inhibited the LPSinduced morphologic changes. Release of TNF-a, IL-10, MCP-1, and NO was reduced with (þ)-pentazocine. Intracellular ROS formation was suppressed with (þ)-pentazocine. Phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) was reduced in the presence of (þ)-pentazocine. The rR1 antagonist BD1063 blocked the (þ)-pentazocine-mediated inhibition of LPS-induced morphologic changes. In addition, BD1063 treatment blocked (þ)-pentazocine-mediated suppression of LPS-induced TNF-a, IL-10, MCP-1, NO, and intracellular ROS release. CONCLUSIONS. Treatment with (þ)-pentazocine suppressed inflammatory responses of retinal microglia and inhibited LPS-induced activation of ERK/JNK MAPK. In neurodegenerative disease, (þ)-pentazocine may exert neuroprotective effects through manipulation of microglia