Cell Journal (Yakhteh)

1-6

Authors: Roya Varmazyar, Ali Noori-Zadeh, Farzad Rajaei, Shahram Darabi, Salar Bakhtiyari

Objective Degeneration of dopaminergic neurons in the substantia nigra of the brain stem is the main pathological aspect of Parkinson’s disease (PD). 17 β-estradiol (E2) has neuroprotective effects on substantia nigra, however, the underlined mechanism is not well-known. In this study, we evaluated the neuroprotective effects of E2 in the ovariectomized 6-hydroxydopamine- (6-OHDA) rat model of PD. Materials and Methods In this experimental study, all animals were ovariectomized to avoid any further bias in E2 levels and then these ovariectomized rats were randomly assigned into three experimental groups (10 rats in each group): ovariectomized control group (OCG), ovariectomized degeneration group receiving 25 μg of 6-OHDA into the left corpus striatum (ODG), and ovariectomized E2 pretreatment group pretreated with 0.1 mgkg-1of 17 β-estradiol for three days prior to the destruction of corpus striatum with 6-OHDA (OE2PTG). The apomorphine behavioral test and Nissl staining were performed in all experimental groups. The expressions of Sequestosome-1 (P62), Unc- 51 like autophagy activating kinase (Ulk1), and microtubule-associated proteins 1A/1B light chain 3B (Lc3) genes were evaluated using reverse transcription- polymerase chain reaction (RT-PCR). Results E2 administration reduced the damages to the dopaminergic neurons of the substantia nigra. The motor behavior, the number of rotations, and histological tests in the treatment group showed the cell survival improvement in comparison with the control groups indicating that E2 can inhibit the neurodegeneration. P62 and Lc3 were expressed in all experimental groups while Ulk1 was not expressed in ODG group. Moreover, Ulk1 was expressed after the treatment with E2 in OE2PTG group. Conclusion E2 prevents neurodegeneration in dopaminergic neurons of the midbrain by over-expression of Ulk1 gene and augmenting the induction of autophagy.

14-26

Authors: Neda Eslahi, Ali Shakeri-Zadeh, Khadijeh Ashtari, Vahid Pirhajati-Mahabadi, Tahereh Tohidi Moghadam, Ronak Shabani, Kamran Kamrava, Zahra Madjd, Chad Maki, Hamid Reza Asgari, Morteza Koruji

Objective The purpose of this study was to evaluate in vitro cytotoxicity of gold nanorods (GNRs) on the viability of spermatogonial cells (SSCs) and mouse acute lymphoblastic leukemia cells (EL4s). Materials and Methods In this experimental study, SSCs were isolated from the neonate mice, following enzymatic digestion and differential plating. GNRs were synthesized, then modified by silica and finally conjugated with folic acid to form F-Si-GNRs. Different doses of F-Si-GNRs (25, 50, 75, 100, 125 and 140 µM) were used on SSCs and EL4s. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) proliferation assay was performed to examine the GNRs toxicity. Flow cytometry was used to confirm the identity of the EL4s and SSCs. Also, the identity and functionality of SSCs were determined by the expression of specific spermatogonial genes and transplantation into recipient testes. Apoptosis was determined by flow cytometry using an annexin V/propidium iodide (PI) kit. Results Flow cytometry showed that SSCs and EL4s were positive for Plzf and H-2kb, respectively. The viability percentage of SSCs and EL4s that were treated with 25, 50, 75, 100, 125 and 140 µM of F-Si-GNRs was 65.33 ± 3.51%, 60 ± 3.6%, 51.33 ± 3.51%, 49 ± 3%, 30.66 ± 2.08% and 16.33 ± 2.51% for SSCs and 57.66 ± 0.57%, 54.66 ± 1.5%, 39.66 ± 1.52%, 12.33 ± 2.51%, 10 ± 1% and 5.66 ± 1.15% for EL4s respectively. The results of the MTT assay indicated that 100 µM is the optimal dose to reach the highest and lowest level of cell death in EL4s and in SSCs, respectively. Conclusion Cell death increased with increasing concentrations of F-Si-GNRs. Following utilization of F-Si-GNRs, there was a significant difference in the extent of apoptosis between cancer cells and SSCs.

35-42

Authors: Fatemeh Hassani, Shahrbanoo Oryan, Poopak Eftekhari-Yazdi, Masood Bazrgar, Ashraf Moini, Nahid Nasiri, Ali Sharifi-Zarchi

Objective The extracellular matrix (ECM) of the cumulus oocyte complex (COC) is composed of several molecules that have different roles during follicle development. This study aims to explore gene expression profiles for ECM and cell adhesion molecules in the cumulus cells of polycystic ovary syndrome (PCOS) patients based on their insulin sensitivity following controlled ovarian stimulation (COS). Materials and Methods In this prospective case-control study enrolled 23 women less than 36 years of age who participated in an intracytoplasmic sperm injection (ICSI) program. Patients were subdivided into 3 groups: control (n=8, fertile women with male infertility history), insulin resistant (IR) PCOS (n=7), and insulin sensitive (IS) PCOS (n=8). We compared 84 ECM component and adhesion molecule gene expressions by quantitative real-time polymerase chain reaction array (qPCR-array) among the groups. Results We noted that 21 of the 84 studied genes differentially expressed among the groups, from which 18 of these genes downregulated. Overall, comparison of PCOS cases with controls showed downregulation of extracellular matrix protein 1 (ECM1); catenin (cadherin-associated protein), alpha 1 (CTNNA1); integrin, alpha 5 (ITGA5); laminin, alpha 3 (LAMA3); laminin, beta 1 (LAMB1); fibronectin 1 (FN1); and integrin, alpha 7 (ITGA7). In the IS group, there was upregulation of ADAM metallopeptidase with thrombospondin type 1 motif, 8 (ADAMTS8) and neural cell adhesion molecule 1 (NCAM1) compared with the controls (P<0.05). Conclusion Downregulation of ECM and cell adhesion molecules seem to be related to PCOS. Gene expression profile alterations in cumulus cells from both the IS and IR groups of PCOS patients seems to be involved in the composition and regulation of ECM during the ovulation process. This study highlights the association of ECM gene alteration as a viewpoint for additional understanding of the etiology of PCOS.

49-56

Authors: Arezoo Khoradmehr, Fahime Mazaheri, Morteza Anvari, Amin Tamadon

Objective Fast Free-of-Acrylamide Clearing Tissue (FACT) is a recently developed protocol for the whole tissue three-dimensional (3D) imaging. The FACT protocol clears lipids using sodium dodecyl sulfate (SDS) to increase the penetration of light and reflection of fluorescent signals from the depth of cleared tissue. The aim of the present study was using FACT protocol in combination with imaging of auto-fluorescency of red blood cells in vessels to image the vasculature of a translucent mouse tissues. Materials and Methods In this experimental study, brain and other tissues of adult female mice or rats were dissected out without the perfusion. Mice brains were sliced for vasculature imaging before the clearing. Brain slices and other whole tissues of rodent were cleared by the FACT protocol and their clearing times were measured. After 1 mm of the brain slice clearing, the blood vessels containing auto-fluorescent red blood cells were imaged by a z-stack motorized epifluorescent microscope. The 3D structures of the brain vessels were reconstructed by Imaris software. Results Auto-fluorescent blood vessels were 3D imaged by the FACT in mouse brain cortex. Clearing tissues of mice and rats were carried out by the FACT on the brain slices, spinal cord, heart, lung, adrenal gland, pancreas, liver, esophagus, duodenum, jejunum, ileum, skeletal muscle, bladder, ovary, and uterus. Conclusion The FACT protocol can be used for the murine whole tissue clearing. We highlighted that the 3D imaging of cortex vasculature can be done without antibody staining of non-perfused brain tissue, rather by a simple auto- fluorescence.

62-69

Authors: Samane Heidarzade, Gholamreza Motalleb, Mohammad Jalil Zorriehzahra

Objective The aim of current study was to provide a proof-of-concept on the mechanism of PLAU and PCDH10 gene expressions and caspases-3, -8, and -9 activities in the apoptotic pathway after treatment of malignant human glioma cell line (U87MG) with cytochalasin H. Materials and Methods In the present experimental study, we have examined cytochalasin H cytotoxic activities as a new therapeutic agent on U87MG cells in vitro for the first time. The cells were cultured and treated with 10-5-10-9M of cytochalasin H for 24, 48 and 72 hours. The assessment of cell viability was carried out by (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazoliumbromide (MTT) assay at 578 nm. The data are the average of three independent tests. mRNA expression changes of PLAU and PCDH10 were then evaluated by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). The fluorometric of caspases-3, -8, and -9 activities were carried out. The morphology changes in the U87MG cells were observed by fluorescence microscope. Results MTT assay showed that cytochalasin H (10-5 M) inhibited the U87MG cancer cells proliferation after 48 hours. Analysis of qRT-PCR showed that the PLAU expression was significantly decreased in comparison with the control (P<0.05). The expression of PCDH10 also showed a significant increase when compared to the control (P<0.001). Fluorescence microscope indicated morphological changes due to apoptosis in U87MG cancer cells, after treatment with cytochalasin H (10-5M, 48 hours). The fluorometric evaluation of caspase-3, -8, and -9 activities showed no significant difference between the caspases and the control group. Conclusion This study shows the effect of caspase-independent pathways of the programmed cell death on the U87MG cancer cell line under cytochalasin H treatment. Further studies are needed to explore the exact mechanism.

78-85

Authors: Monireh Ajami, Mohammad Hadi Sadeghian, Masoud Soleimani, Mohammad Reza Keramati, Mansoureh Ajami, Azadeh Anbarlou, Amir Atashi

Objective The role of epigenetic in regulating the gene expression profile the embryo has been documented. MicroRNAs (miRNAs) are one of these epigenetic mechanisms. Twins are valuable models in determining the relative contributions of genetics and the environment. In this study, we compared differences in the expression levels of 44 miRNAs in hematopoietic stem cells (HSCs) of identical twins to that of fraternal twins as a controls. Materials and Methods In this experimental study, CD133+ HSCs were isolated from cord blood of identical and fraternal twins via magnetic-activated cell sorting (MACS). Variation in of gene expression levels of 44 miRNAs were evaluated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Results Significant differences in expression were observed in both fraternal and identical twins to varying degrees, but variations alteration in expression of the miRNAs were higher in fraternal twins. Conclusion Identical twins had a positive correlation in miRNA expression, while the correlation was not statistically significant in fraternal twins. Altogether, more differences in miRNA expression level in fraternal twins can be attributed to the both genetics and the intrauterine environment. The contribution of the intrauterine environment and genetics to miRNAs expression in HSCs was estimated 8 and 92%, respectively. By comparing of miRNA expression in identical and fraternal twins and identification of their target genes and biological pathways, it could be possible to estimate the effects of genetics and the environment on a number of biological pathways.

92-98

Authors: Fatemeh Rezaei, Seyed Mohammad Moazzeni

Objective Mesenchymal stem cells (MSCs), due to their immunomodulatory functions, are an ideal candidate for the treatment of immune-related diseases. Recurrent spontaneous abortion (RSA) is one of the most common complications of pregnancy which in many cases is related to the immune system disorders. Our previous study has shown that the abortion rate was decreased following the syngeneic MSCs therapy in abortion-prone mice. In this study, the therapeutic effect of syngeneic, allogeneic, and xenogeneic MSCs was compared in a mouse model of RSA. Materials and Methods In this experimental study, MSCs were isolated from adipose tissue (ASCs) of CBA/J and BALB/c mice and human. After characterization, ASCs were injected (IP) at day 4 of gestation to female CBA/J mice following their mating with DBA/2 male mice. In the control group, phosphate-buffered saline (PBS) was injected and CBA/J×BALB/c mating was also used as the normal pregnancy control. On day 14.5 of pregnancy, embryo resorption rate was determined. Results The abortion rate significantly decreased following the ASCs therapy from syngeneic (6.31%), allogeneic (6.54%), and xenogeneic group (12.36%) compared to ASCs non-treated group (34.4%). There was no statistical difference between ASCs treated groups, however syngeneic and allogeneic ASCs reduced the abortion rate more efficiently than xenogeneic ASC. Conclusion The abortion rate was significantly decreased following the intraperitoneal administration of ASCs from various donated sources in abortion-prone mice. These results indicated that the immunogenicity of allogeneic and xenogeneic ASCs is not a contradictory problem for their therapeutic effects on RSA.