Systemic delivery of specific and efficient CRISPR/Cas9 system targeting HPV16 oncogenes using LL-37 antimicrobial peptide in C57BL/6 mice.

Human papillomavirus (HPV) type 16 is the most common sexually transmitted virus related to cervical cancer. Among different types of advanced novel therapies, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas-mediated gene editing holds great promise for cancer treatment. In this research, optimal gRNA sequences targeting HPV16 E5, E6, E7, and p97 promoter for CRISPR/Cas9-mediated genome editing were designed by in silico prediction. After cloning, delivery of the recombinant vectors into C3, TC1 and HeLa tumor cells was evaluated by Lipofectamine 2000, and LL-37 antimicrobial peptide. Then, the levels of cell cycle proteins (p21, p53, and Rb) were investigated after treatment by western blot analysis. Finally, C57BL/6 mice were inoculated with C3 tumor cells, and treated with recombinant vectors and cisplatin. Based on the tumor size reduction and IHC results, the E6 + E7-treated group with a high percentage of cleaved caspase-3 positive cells (45.75%) and low mitotic index of 2-3 was determined as the best treatment among other groups. Moreover, the potential of LL-37 peptide to overcome the CRISPR/Cas9 delivery challenge was shown for the first time. Overall, our study suggests that the CRISPR/Cas9-mediated gene editing of pre-existing tumors is effective, specific and nontoxic, and the outlook for precise gene therapy in cancer patients is very bright.

Detrimental effects of vitrification on integrin genes (α9 and ß1) and in vitro fertilization in mouse oocytes.

OBJECTIVE: Integrins are known as key molecules that importantly involve in fertilization. This study aimed to evaluate effects of vitrification on fertilization rate and expression of integrin genes, α9 and ß1, on mice oocytes in GV and MІІ stages. MATERIALS AND METHODS: From the ovarian tissue and fallopian tube of NMRI mice, germinal vesicle (GV, n = 200) and metaphase II (MII, n = 200) oocytes were obtained. Then, oocytes were distributed into 4 groups including non-vitrified GV, non-vitrified MII, vitrified GV, and vitrified MII. Cryotop method was used for vitrification and oocytes (for 4 weeks) were kept in liquid nitrogen. After that, by using an inverted microscope, the rate of survived oocytes was assessed. Also, in vitro fertilization (IVF) for oocytes, obtained from in vitro maturated MII and mice ovaries (ovulated MII), was done to assess embryos at differenced stages (2-cells, morula, and hatched). Finally, RT-qPCR was performed to investigate the mRNA expression of integrin genes (α9 and ß1). RESULTS: After vitrification, the rate of survived oocytes, 68.65%for GV and 65.07% % for MII, did not show a remarkable difference related to non-vitrified groups, while the fertilization rate in vitrified groups remarkably decrease compared to non-vitrified groups (p < 0.05). Also, the expression of α9 and ß1 genes was significantly altered in vitrified groups when compared to non-vitrified groups (p < 0.05). There was no significant difference in embryo developmental rates for non-vitrified and vitrified groups. CONCLUSION: Cryotop method for vitrification caused an alternation in oocyte quality by reducing fertilization rate and integrin gene expression.

Trehalose and carnosic acid induced LC3I, LC3II ratio, P62 down-regulation and cleaved caspase 3 expression in neural stem cells.

BACKGROUND: Using neural stem cells (NSCs) in cell therapy and regenerative medicine is a growing knowledge. In this study, the protective role of carnosic acid and trehalose against H2O2-induced oxidative stress in autophagy induction and apoptosis inhibition in NSCs was investigated. MATERIAL AND METHODS: The bone marrow stromal cells (BMSCs) were isolated from the femur of the rat and differentiated into NSCs using basic fibroblast and epidermal growth factors (bFGF and EGF), and B27 serum free media. To evaluate the autophagy, the P62 protein was assessed by immunocytochemistry and LC3II / LC3I ratio by Western blotting. Further, we used 3-Methyladenine (3-MA), a widely used autophagy inhibitor to study whether combined treatment of 3-MA with carnosic acid and trehalose modulates autophagy in NSCs. For studying apoptosis, the cleaved caspase-3 protein was evaluated. Carnosic acid and trehalose increased the survival of the NSCs. RESULTS: The H2O2 decreased the autophagy and induced apoptosis with increasing time during 24 hours, however, a pre-treatment with 2 µM carnosic acid and trehalose 3 % induced the autophagy proteins (while increasing the LC3II / LC3I ratio and decreasing the P62) and decreased the apoptosis (while decreasing the expression of the cleaved caspase-3). The results showed that the carnosic acid and trehalose increased the survival of NSCs against the oxidative stress caused by H2O2, decreased apoptosis, and induced autophagy. CONCLUSION: Due to the carnosic acid and trehalose unique properties and its low toxicity, it can be used as an agent in cellular transplantation for reducing oxidative stress and inducing autophagy (Fig. 4, Ref. 37).

Functional differences of Toll-like receptor 4 in osteogenesis, adipogenesis and chondrogenesis in human bone marrow-derived mesenchymal stem cells.

Multipotent human bone marrow-derived mesenchymal stem cells (hMSCs) are promising candidates for bone and cartilage regeneration. Toll-like receptor 4 (TLR4) is expressed by hMSCs and is a receptor for both exogenous and endogenous danger signals. TLRs have been shown to possess functional differences based on the species (human or mouse) they are isolated from therefore, the effects of knockdown of TLR4 were evaluated in humans during the differentiation of MSCs into bone, fat and chondrocyte cells in vitro. We investigated the expression profile of TLR4 during the differentiation of hMSCs into three different lineages on days 7, 14 and 21 and assessed the differentiation potential of the cells in the presence of lipopolysaccharide (LPS, as an exogenous agonist) and fibronectin fragment III-1c (FnIII-1c, as an endogenous agonist). TLR4 expression increased following the induction of hMSC differentiation into all three lineages. Alkaline phosphatase activity revealed that FnIII-1c accelerated calcium deposition on day 7, whereas LPS increased calcium deposition on day 14. Chondrogenesis increased in the presence of LPS; however, FnIII-1c acted as a reducer in the late stage. TLR4 silencing led to decreased osteogenesis and increased adipogenesis. Furthermore, Wnt5a expression was inversely related to chondrogenesis during the late stage of differentiation. We suggest that understanding the functionality of TLR4 (in the presence of pathogen or stress signal) during the differentiation of hMSCs into three lineages would be useful for MSC-based treatments.

The differentiation effect of bone morphogenetic protein (BMP) on human amniotic epithelial stem cells to express ectodermal lineage markers.

Stem cells are a promising tool for treatment of a variety of degenerative diseases. Human amniotic epithelial stem cells (hAECs) have desirable and unique characteristics that make them a proper candidate for cell therapy. In this study, we have investigated the effects of BMP-4 (bone morphogenetic protein-4) and its inhibition on differentiation of AECs into ectodermal lineages. Analysis of AEC-derived ectodermal lineages (neurons and keratinocytes) was performed by using flow cytometry technique for Map2 and ß-tubulin (as neuron markers), Olig2 and MBP (as oligodendrocyte markers), and K14 and K10 (as keratinocyte markers). The results of this study illustrated that noggin (as BMP antagonist), BMP4, and both BMP4 and heparin (together or separately) increased neural and keratinocyte marker expression, respectively. The expression of markers MAP2, olig2, and K14 in hAECs has been significantly decreased 21 days after exposure to differentiation medium (without growth factors) compared with isolation day, which supports the hypothesis that AECs can be dedifferentiated into pluripotent cells. Moreover, activation and inhibition of BMP signaling have no effects on viability of hAECs. The results of this study showed that BMP signaling and its inhibition are the key factors for ectodermal lineage differentiation of amnion-derived stem cells.

Hydrogen sulfide attenuates induction and prevents progress of the 6-hydroxydopamine-induced Parkinsonism in rat through activation of ATP-sensitive potassium channels and suppression of ER stress.

PURPOSE: Studies argue in favor of hydrogen sulfide (H2S) as the next potent therapeutic agent for neurodegenerative diseases. In present study, we investigated the effect of long term treatment with NaHS (as donor of H2S) on induction and progress of the 6-hydroxydopamine (6-OHDA) -induced Parkinsonism in rat. METHODS: The 6-OHDA was injected into medial forebrain bundle of right hemisphere by stereotaxic surgery. Behavioral tests and treatments were carried out to eight weeks after the toxin. Immunohistochemistry and western blotting were carried out to evaluate the survival of tyrosine hydroxylase (TH) -positive neurons in substantia nigra (SN) and also expression of glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP), the markers of endoplasmic reticulum (ER) stress, in striatum and SN. RESULTS: Eight weeks assessment of the behavioral symptoms showed that NaHS especially at dose of 100 µmol/kg attenuates remarkably induction of the Parkinsonism and prevents its progress. NaHS also increased the survival of TH- positive neurons and suppressed 6-OHDA- induced overexpression of GRP78 and CHOP. Blockade of ATP-sensitive potassium (K-ATP) channels with glibenclamide (Glib) prevented markedly the effect of NaHS on both the induction phase and survival of TH- positive neurons. But Glib did not affect the preventing effect of NaHS on the progress phase and its suppressing effect on the overexpression of ER stress markers. CONCLUSION: H2S attenuates induction of the 6-OHDA- induced Parkinsonism and also increases the survival of dopaminergic neurons through activation of K-ATP channels. H2S also prevents progress of the Parkinsonism probably through suppression of ER stress.

Microarray analysis identification of key pathways and interaction network of differential gene expressions during osteogenic differentiation.

BACKGROUND: Adult bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent stem cells that can differentiate into three lineages. They are suitable sources for cell-based therapy and regenerative medicine applications. This study aims to evaluate the hub genes and key pathways of differentially expressed genes (DEGs) related to osteogenesis by bioinformatics analysis in three different days. The DEGs were derived from the three different days compared with day 0. RESULTS: Gene expression profiles of GSE37558 were obtained from the Gene Expression Omnibus (GEO) database. A total of 4076 DEGs were acquired on days 8, 12, and 25. Gene ontology (GO) enrichment analysis showed that the non-canonical Wnt signaling pathway and lipopolysaccharide (LPS)-mediated signaling pathway were commonly upregulated DEGs for all 3 days. KEGG pathway analysis indicated that the PI3K-Akt and focal adhesion were also commonly upregulated DEGs for all 3 days. Ten hub genes were identified by CytoHubba on days 8, 12, and 25. Then, we focused on the association of these hub genes with the Wnt pathways that had been enriched from the protein-protein interaction (PPI) by the Cytoscape plugin MCODE. CONCLUSIONS: These findings suggested further insights into the roles of the PI3K/AKT and Wnt pathways and their association with osteogenesis. In addition, the stem cell microenvironment via growth factors, extracellular matrix (ECM), IGF1, IGF2, LPS, and Wnt most likely affect osteogenesis by PI3K/AKT.

Resistance of human primary mesenchymal stem cells to cytotoxic effects of nutlin-3 in vitro.

BACKGROUND: The small-molecule nutlin-3 was found to be an effective therapeutic compound and p53 activator, and acts as a murine double minute 2 antagonist, although these findings need to be clinically confirmed. The essential components of the bone marrow include mesenchymal stem cells (MSCs), which play a key role in protecting, regenerating, and proliferating hematopoietic stem cells (HSCs). This feature is vital for HSC after exposure to myelotoxic anticancer agents; nevertheless, the effects of nutlin-3 on MSCs remain to be disclosed. The present research study was conducted to examine the antiproliferative and proapoptotic effectiveness of nutlin-3 in bone marrow MSCs (BMSCs). MATERIALS AND METHODS: Human-derived BMSCs were cultured for different durations, that is, 24, 48, and 72 hours, and treated using various concentrations of nutlin-3, including 5, 10, 25, 50, and 100 µΜ. To investigate the effect of nutlin-3 on the apoptosis, cell vitality and proliferation in BMSCs, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), thiazolyl blue tetrazolium bromide, propidium iodide (PI) and annexin V assay, as well as real-time polymerase chain reaction, were used. RESULTS: BMSCs viability significantly decreased (P < .05) in the cells treated at concentrations of 50 and 100 µM for 24 hours and concentrations of 25, 50, and 100 µM for 48 hours and at all concentrations for 72 hours. The apoptosis of BMSCs (TUNEL positive) was significantly more visible at concentrations of 25 and 50 µM compared with that in the controls (P < .05), while this increased through dose-dependent processes. Annexin V/PI staining revealed negligible dose-dependent increases in all the apoptotic cells after 72 hours of incubation, and this apoptosis elevation was significant at 25 and 50 µM (P < .05). CONCLUSION: Resistance to nutlin-3 was observed in human bone marrow-derived MSCs; nevertheless, further clinical data are required to be obtained with long-duration exposure to confirm the present findings.

The effects of Nutlin-3 on morphology, cellular proliferation, and apoptosis in rat primary mesenchymal stem cells.

INTRODUCTION: Nutlin-3 is a powerful antagonist of murine double minute 2/p53 interaction demonstrating promising therapeutic anticancer activity, which has not been clinically approved yet. Mesenchymal stem cells (MSCs) are an important part of the bone marrow niche and support regeneration and proliferation of hematopoietic stem cells after exposure to myelotoxic anticancer agents; however, the effect of Nutlin-3 compounds on MSCs themselves remains to be elucidated. MATERIALS AND METHODS: Rat-derived bone marrow-derived MSCs (BMSCs) were cultured and treated with different concentrations (5, 10, 25, 50, and 100 µM) and times (24, 48, and 72 hr) of Nutlin-3. The microculture tetrazolium test, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and propidium iodide and annexin-V assays, and quantitative real-time reverse-transcription polymerase chain reaction were performed to assess the effects of Nutlin-3 on the cell viability, proliferation, and apoptosis in BMSCs. RESULTS: The viability of BMSCs in the treated cells with concentrations of 100 µM at 24 hr, 50 and 100 µM at 48 hr, and in all concentrations at 72 hr was significantly (p < 0.05) low. The apoptotic index showed that the TUNEL-positive BMSCs were significantly higher in concentrations of 25 and 50 µM in comparison to control group ( p < 0.05) and augmented in a dose-dependent manner. Annexin-V-PI staining showed after 72 hr of incubation, there was a slight dose-dependent increase in total apoptotic cells at 10 and 25 µM of Nutlin-3, but a massive significant increase at 50 µM. CONCLUSION: Here, we show that rat BMSCs are relatively resistant to Nutlin-3; however, further in vivo data with long-term exposure may help to corroborate our findings.

SMER28 Attenuates Dopaminergic Toxicity Mediated by 6-Hydroxydopamine in the Rats via Modulating Oxidative Burdens and Autophagy-Related Parameters.

Parkinson's disease is the second most common neurodegenerative disease that occurs due to cellular autophagy deficiency and the accumulation of alpha-synuclein in the dopaminergic neurons of the substantia nigra pars compacta (SNc) of the brainstem. The SMER28 (also known as 6-Bromo-N-prop-2-enylquinazolin-4-amine) is an autophagy inducer. In this study, the neuroprotective effects of SMER28 were evaluated on autophagy induction, antioxidant system activation, and microgliosis attenuation. The Parkinson's disease model was developed in the male Wistar rats by injection of 6-OHDA into the left striatum. Apomorphine-induced behavior assessment test and SNc cell counting were performed to investigate the neuroprotective effects of SMER28. This study examined the pharmacological roles of SMER28, especially by focusing on the autophagy (p62/ SQSTM1 and LC3II/LC3I ratio where LC3 is microtubule-associated protein 1A/1B-light chain 3), inhibiting free radicals, and activating the antioxidant system. The levels of malondialdehyde (MDA), reactive oxygen species (ROS), glutathione (GSH), GSH/glutathione peroxidase (GPX), superoxide dismutase (SOD) activity and nuclear factor-erythroid 2-related factor-2 (Nrf2) were measured to evaluate the antioxidant activity of SMER28. Moreover, Iba-1 (ionized calcium binding adaptor molecule, indicating microgliosis) and tyrosine hydroxylase immunoreactivities were evaluated in the SNc. In the behavioral assessment, SMER28 (50 µg/kg) attenuated damages to the SNc dopaminergic neurons, characterized by improved motor function. The tissue observations revealed that SMER28 prevented the destruction of SNc neurons and attenuated microgliosis as well. It also reduced MDA and ROS production and increased GSH, GPX, SOD, and Nrf2 activities by inducing autophagy (decreasing p62 and increasing LC3II/LC3I ratio). Consequently, possibly with further studies, it can be considered as a drug for neurodegenerative diseases with proteinopathy etiology.

Molecular alterations contributing to pancreatic cancer chemoresistance.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most common causes of cancer-related death all over the world. This disease is difficult to treat and patients have an overall 5-year survival rate of less than 5%. Although two drugs, gemcitabine (GEM) and 5-fluorouracil (5-FU) have been shown to improve the survival rate of patients systematically, they do not increase general survival to a clinically acceptable degree. Lack of ideal clinical response of pancreatic cancer patients to chemotherapy is likely to be due to intrinsic and acquired chemoresistance of tumor cells. Various mechanisms of drug resistance have been investigated in pancreatic cancer, including genetic and epigenetic changes in particular genes or signaling pathways. In addition, evidence suggests that microRNAs (miRNAs) play significant roles as key regulators of gene expression in many cellular processes, including drug resistance. Understanding underlying genes and mechanisms of drug resistance in pancreatic cancer is critical to develop new effective treatments for this deadly disease. This review illustrates the genes and miRNAs involved in resistance to gemcitabine in pancreatic cancer.

The Effect of bisphenol A and Photobiomodulation Therapy on Autophagy-Related Genes Induction in Adipose Tissue-Derived Stem Cells.

Introduction: As adipose tissue-derived stem cells (ADSCs) can divide rapidly and be prepared non-invasively, they have extensively been used in regenerative medicine. On the other hand, a new method of therapy, known as photobiomodulation (PHT), has been used to treat many diseases, such as inflammatory conditions, wound healing and pain. Besides, exposure to chemical substances such as bisphenol A (BPA), at low levels, can lead to autophagy. This study investigated the effects of BPA and PHT on the expression of autophagy-related genes, including LC3, NRF2, P62, in rat ADSCs as a model. Methods: ADSCs isolation and purification were confirmed by immunocytochemistry (ICC). The cells were then treated with different concentrations of BPA and also subjected to PHT. Reverse transcription polymerase chain reaction (RT-PCR) was used for the evaluation of LC3, NRF2 and P62 gene expressions. Oil red O staining was used for adipogenic vacuole formation. Result: ICC showed that the isolated cells were CD 49-positive but CD 31 and CD 34-negative. The viability test indicated that the number of live cells after 24 hours in the BPA groups at concentrations of 0, 1, 50, 100 and 200 µM was 100%, 93%, 81%, 72%, and 43% respectively. The difference in cell viability between groups 50, 100 and 200 µM was significant as compared with the control groups (P < 0.05). Moreover, in the group with 1 µM concentration of BPA, the expressions of LC3, NRF2 and P62 genes were upregulated. However, in the treatment group at the concentration of 200 µM of BPA, the LC3 gene was expressed, but NRF2 and P62 genes were downregulated. Conclusion: BPA and PHT induce autophagy and adiposeness in ADSCs in a dose-dependent manner.

Effects of Placenta-Derived Human Amniotic Epithelial Cells on the Wound Healing Process and TGF-ß Induced Scar Formation in Murine Ischemic-Reperfusion Injury Model.

BACKGROUND: Pressure ulcers (PUs), a result of ischemic reperfusion (IR) injuries, are prevalent skin problems which show refractoriness against standard therapeutic approaches. Besides, scar formation is a critical complication of ulcers that affects functionality and the skin's cosmetic aspect. The current study aimed to investigate the effects of placenta-derived human amniotic epithelial cells (hAECs), as important agents of regenerative medicine and stem cell therapy, on accelerating the healing of IR ulcers in mice. We also evaluated the effects of these cells on reducing the TGFß-induced scar formation. METHODS: Male Balb/c mice at the age of 6-8 weeks were subjected to three IR cycles. Afterward, the mice were divided into three experimental groups (n = 6 per group), including the control group, vehicle group, and hAECs treatment group. Mice of the treatment group received 100 µL of fresh hAECs 1 × 106 cell/ml suspension in PBS. Afterward, mice were assessed by histological, stereological, molecular, and western blotting techniques at 3, 7, 14, and 21 days after wounding. RESULTS: The histological and stereological results showed the most diminutive scar formation and better healing in the hAECs treated group compared to control group. Furthermore, our results demonstrated that the expression level of Col1A1 on days 3, 14, and 21 in the hAECs treated group was significantly lower than control. Additionally, injection of hAECs significantly reduced the expression level of Col3A1 on days 3, 7, and 21 while increased Col3A1 on the day 14. Otherwise, in the hAECs treated group, the expression levels of VEGFA on days 7 and 14 were higher, which showed that hAECs could promote angiogenesis and wound healing. Also, cell therapy significantly lowered the protein levels of TGF-ß1 on day 14, while the protein level of TGF-ß3 on day 14 was significantly higher. This data could demonstrate the role of hAECs in scar reduction in IR wounds. CONCLUSION: These results suggest that hAECs can promote re-epithelialization and wound closure in an animal model of PU. They also reduced scar formation during wound healing by reducing the expression of TGF-ß1/ TGF-ß3 ratio.

Intercellular adhesion molecule-1 (ICAM-1) gene polymorphism and endometriosis in northern Iran.

PURPOSE: Endometriosis is a chronic pleomorphic disorder with pelvic or systemic manifestations, and is characterized by the presence of endometrial glands and stroma. It is generally considered to involve genetic, environmental, immunological, angiogenic and endocrine processes. The aim of this study was to evaluate the frequency of intercellular adhesion molecule-1 (ICAM-1) polymorphism at position 241 in exon 4 of the ICAM-1 gene in patients with endometriosis and in healthy control subjects in northern Iran. METHODS: A total of 84 unrelated patients and 120 consecutive unrelated healthy controls from the northern Iran were recruited for the study. ICAM-1 codon 241 polymorphism analysis was performed by two independent PCR reactions, based on the two set of oligoprimers specific for Arg (AGG) or Gly (GGG) coding sequence, respectively. RESULTS: Genotype distributions of ICAM-1 gene show significant difference between patients and controls. The R241 allele was detected in 23% of endometriosis patients and in 6% of controls (P = 0.001; odds ratio 4.67; 95% confidence interval 1.81-12.07). Comparing patients with different stage of endometriosis, we found a trend to higher frequency of R241 in patients with stage IV (65 vs 6%; P < 0.0001). CONCLUSION: Our results suggest that ICAM-1 polymorphism in codon 241 is associated with the development of endometriosis susceptibility in the population of northern Iran.

Analysis of DNA fragmentation in mouse embryos exposed to an extremely low-frequency electromagnetic field.

Effects of extremely low-frequency electromagnetic fields (ELF-EMFs) on DNA damage in biological systems are still a matter of dispute. The aim of the present study was to investigate the possible effect of electromagnetic field exposure on DNA fragmentation in cells (blastomers) of mouse blastocysts. Eighty female NMRI mice were randomly divided into 2 groups of 40 animals each. The control group was left unexposed whereas the animals in the EMF-group were exposed to a 50-Hz EMF at 0.5 mT 4 h per day, 6 days a week for a duration of 2 weeks. After the 8(th) day of exposure, the female mice in both groups were superovulated (with injections of pregnant mare serum gonadotropin and human chorionic gonadotropin) and then mated overnight. At approximately 4 days after mating (102 h after the human chorionic gonadotropin treatment), blastocysts were obtained by flushing the uterus horns. The mean numbers of pregnant mice, blastocysts after flushing, blastomers within the blastocysts, and the DNA fragmentation index following staining in both groups were compared using statistical methods (SPSS, the Chi-square test, the Student's t-test and the Mann-Whitney U-test, P < 0.05). The results showed that the mean number of blastocysts after flushing was significantly decreased in the EMF-group compared to that of the control group (P < 0.03). The DNA fragmentation index was significantly increased in the EMF-group compared to control (10.53% vs. 7.14%; P < 0.001). However, there was no significant difference in the mean numbers of blastomers and numbers of pregnant mice between the EMF-exposed and control group. Our findings indicate that the EMF exposure in preimplantation stage could have detrimental effects on female mouse fertility and embryo development by decreasing the number of blastocysts and increasing the blastocysts DNA fragmentation.

Role of sperm apoptosis and oxidative stress in male infertility: A narrative review.

Activation of caspase, externalization of phosphatidyl serine, change in the mitochondrial membrane potential, and DNA fragmentation are apoptosis markers found in human ejaculated spermatozoa. Also, reactive oxygen species (ROS) play a vital role in the different types of male infertility. In this review, data sources including Google Scholar, Scopus, PubMed, and Science Direct were searched for publications with no particular time restriction to get a holistic and comprehensive view of the research. Apoptosis regulates the male germ cells, correct function and development from the early embryonic stages of gonadal differentiation to fertilization. In addition to maintaining a reasonable ratio between the Sertoli and germ cells, apoptosis is one of the well-known quality control mechanisms in the testis. Also, high ROS levels cause a heightened and dysregulated apoptotic response. Apoptosis is one of the well-known mechanisms of quality control in the testis. Nevertheless, increased apoptosis may have adverse effects on sperm production. Recent studies have shown that ROS and the consequent oxidative stress play a crucial role in apoptosis. This review aims to assimilate and summarize recent findings on the apoptosis in male reproduction and fertility. Also, this review discusses the update on the role of ROS in normal sperm function to guide future research in this area.

Effects of extremely low-frequency electromagnetic field on fertility and heights of epithelial cells in pre-implantation stage endometrium and fallopian tube in mice.

OBJECTIVE: To investigate the effects of extremely low-frequency electromagnetic field (ELF-EMF) on fertility and heights of epithelial cells in pre-implantation stage endometrium and fallopian tube in mice. METHODS: Eighty female NMRI mice were randomly divided into 2 groups: control group was not exposed to EMF and experimental group was exposed to 4-hour EMF per day, 6 days a week for 2 weeks to 50 Hz, 0.5 mT EMF. Female mice in two groups were superovulated and mated with male mice over night. At the time of implantation, the blastocysts were obtained from the presumed pregnant mice with vaginal plug by flushing the uterus horns. The samples of uterus horns and fallopian tubes in two groups were taken and were processed for light microscopic studies. RESULTS: The analysis of mean number of the flushed blastocysts in the EMF group showed significant decrease as compared with the control group (P<0.03). Light microscopic study showed that the height of fallopian tube epithelial cells was significantly increased in the EMF group as compared with the control group (P<0.001). However the height of endometrial epithelial cells in the EMF group showed insignificant increase as compared with the control group. CONCLUSION: The results indicate that ELF-EMF has detrimental effect on female reproductive system in mice by decreasing the number of flushed blastocysts and increasing the height of fallopian tube epithelial cells.

Trehalose Neuroprotective Effects on the Substantia Nigra Dopaminergic Cells by Activating Autophagy and Non-canonical Nrf2 Pathways.

Trehalose, as a natural disaccharide, is known as an autophagy inducer. The neuroprotective effects of trehalose in the rat model of Parkinson's disease were the aim of the present study. Parkinson's disease model was induced by injecting 6-hydroxydopamine (6-OHDA) in the striatum of male Wistar rats. Apomorphine-induced behavior and substantia nigra neuronal counts were applied to evaluate the neuroprotective effects of trehalose. The autophagy was studied using the expression of p62 and LC3II/LC3I ratio. In addition, the antioxidant effects of trehalose were assessed by analyzing the levels of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and also glutathione reductase (GR), glutathione peroxidase (GPx) and Catalase (CAT) enzymes. Moreover, the levels of 3, 4-dihydroxyphenylacetic acid (DOPAC) and dopamine (DA) were assessed.The behavioral test showed that trehalose in the treatment group reduced the damage to the substantial nigra dopaminergic neurons, which was characterized by improved motor and reduced rotations in the treatment group as compared with the lesion group. In the histological examinations of the treatment group, trehalose prevented the destruction of dopaminergic neurons. Trehalose treatments increased autophagy (high LC3II/LC3I ratio) and the expression of the p62 protein as well. Through p62-dependent manner, it led to increased nuclear translocation of Nrf2 transcription factor and elevated expression of downstream antioxidant enzymes, such as GR, GPx, and CAT, restoring DA and DOPAC contents of the cells. In the current study, trehalose simultaneously protects substantia nigra dopaminergic cells by activating both non-canonical p62/SQSTM1-Keap1-Nrf2 and autophagy pathways.

17 ß-Estradiol Oxidative Stress Attenuation and Autophagy-Induced Dopaminergic Neuroprotection.

OBJECTIVE: Degeneration of dopaminergic neurons in the substantia nigra of the brain stem is the main pathological aspect of Parkinson's disease (PD). 17 ß-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-1 of 17 ß-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 transcriptionpolymerase 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.

A critical role for miR-184 in the fate determination of oligodendrocytes.

BACKGROUND: New insights on cellular and molecular aspects of both oligodendrocyte (OL) differentiation and myelin synthesis pathways are potential avenues for developing a cell-based therapy for demyelinating disorders comprising multiple sclerosis. MicroRNAs (miRNA) have broad implications in all aspects of cell biology including OL differentiation. MiR-184 has been identified as one of the most highly enriched miRNAs in oligodendrocyte progenitor cells (OPCs). However, the exact molecular mechanism of miR-184 in OL differentiation is yet to be elucidated. METHODS AND RESULTS: Based on immunochemistry assays, qRT-PCR, and western blotting findings, we hypothesized that overexpression of miR-184 in either neural progenitor cells (NPCs) or embryonic mouse cortex stimulated the differentiation of OL lineage efficiently through regulating crucial developmental genes. Luciferase assays demonstrated that miR-184 directly represses positive regulators of neural and astrocyte differentiation, i.e., SOX1 and BCL2L1, respectively, including the negative regulator of myelination, LINGO1. Moreover, blocking the function of miR-184 reduced the number of committed cells to an OL lineage. CONCLUSIONS: Our data highlighted that miR-184 could promote OL differentiation even in the absence of exogenous growth factors and propose a novel strategy to improve the efficacy of OL differentiation, with potential applications in cell therapy for neurodegenerative diseases.