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Abstract Background Leptin, a 167 amino acid peptide hormone, profoundly effects reproduction exerting its biological effects via interaction with the leptin receptor (ObR) which is widely expressed on peripheral tissues. In this study, we have attempted to assess leptin receptor expression in the spermatozoa of fertile males and those diagnosed with male factor infertility; both at the mRNA or protein levels. Methods Semen samples were collected from fertile males and individuals with male factor infertility. In order to evaluate leptin receptor expression several techniques were utilized, including: reverse transcriptase-polymerase chain reaction (RT-PCR), immunostaining, flow cytometry, and western blotting. Mononuclear cells isolated from volunteers' peripheral blood were used as positive controls for leptin receptor expression. Results leptin receptor was noted on mononuclear cells but we were unable to detect this receptor on spermatozoa at the protein level. Leptin receptor expression was detected on peripheral blood mononuclear cells (PBMCs) as positive controls; however it was not detectable on the spermatozoa of both groups by immunofluorescence microscopy or flow cytometry. Furthermore, positive expression of the ObR long isoform as assessed by RT-PCR was observed in the sperm of only four cases, whereas expression of beta-Actin, a house keeping gene, and HspA2, a testis specific gene, was present in all cases. Conclusion The long isoform of leptin receptor may not be present on human sperm. Species difference may be accounted for diverse reproductive physiology which depends on metabolic requirement. Leptin receptor expression at the mRNA level in some individuals may be related to contamination by other cells in semen.

Purpose: The aim of this study was to evaluate expression of Post-Acrosomal WW Binding Protein (PAWP) in infertile men with low and high fertilization post ICSI and also globozoospermic men.Materials and Methods: Semen samples were collected from 18 infertile men with previously failed or low fertili-zation (< 25%) post ICSI, 10 men with high fertilization (>50%) post ICSI, 15 globozoospermic men, and 21 fertile individuals. Then, expression of PAWP was assessed at RNA with quantitative Real Time PCR.Results: Relative expression of PAWP in sperm was significantly (P < .05) lower in infertile men with globozo-ospermia (41.5 ± 5.7) or low fertilization rate (43.3±10.4) compared to fertile (138.8 ± 17.3)or men with high fer-tilization (211.6 ± 75.6). In addition, a significant positive correlation (r = 0.628; P = .001) was observed between percentage of fertilization with the relative expression of PAWP.Conclusion: Considering solid recent evidences regarding PLC? as the main sperm factor involved in oocyte acti-vation, therefore co-localization of PLC? with PAWP in perinuclear theca may account for the above observation and it is likely that PAWP may have other functions and/or it may assist PLC?. [ABSTRACT FROM AUTHOR]

Objective(s): Multiple sclerosis (MS) is considered as a chronic type of an inflammatory disease characterized by loss of myelin of CNS. Recent evidence indicates that Interleukin 17 (IL-17)- producing T helper cells (Th17 cells) population are increased and regulatory T cells (Treg cells) are decreased in MS. Despite extensive research in understanding the mechanism of Th17 and Treg differentiation, the role of microRNAs in MS is not completely understood. Thereby, as a step closer, we analyzed the expression profile of miR-9-5p and miR-106a-5p, and protein level of retinoic acid receptor (RAR)-related orphan receptor C (RORC; Th17 master transcription factor) as direct target of miR-106a-5p and forkhead box P3 (FOXP3; Treg master transcription factor) as indirect target of miR- 9-5p in CD4+ T cells in two groups of relapsing and remitting in our relapsing-remitting MS (RR-MS) patients. Materials and Methods: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was utilized to assess the expression of miRNAs and mRNAs, in 40 RR-MS patients and 11 healthy individuals. Thus, FOXP3 and RAR-related orphan receptor κt (RORκt) was assessed in CD4+T-cells by flow cytometry. We also investigated the role of these miRNAs in Th17/Treg differentiation pathway through bioinformatics tools. Results: An up-regulation of miR-9-5p and down-regulation of miR-106a-5p in relapsing phase of MS patients were observed compared to healthy controls. RORC and FOXP3 were up-regulated in relapsing and remitting phases of MS, respectively. Conclusion: Expression pattern of miR-9-5p and miR-106a-5p and their targets suggest a possible inducing role of miR-9-5p and suppressing role of miR-106a-5p in differentiation pathway of Th17 cells during MS pathogenesis. [ABSTRACT FROM AUTHOR]

Background: Globozoospermia is a rare syndrome with an incidence of less than 0.1% among infertile men. Researchers have recently identified a large deletion, about 200 kbp, encompassing the whole length of DPY19L2 or mutations in SPATA16 and PICK1 genes associated with globozoospermia. The aim of this study was to analyze the DPY19L2 gene deletion using polymerase chain reaction technique for the exons 1, 4- 8, 11 and 22 as well as break point (BP) "a" in globozoospermic men. Materials and Methods: In this experimental study, genome samples were collected from 27 men with globozoospermia (cases) and 36 fertile individuals (controls), and genomic analysis was carried out on each sample. Results: Deletion of DPY19L2 gene accounted for 74% of individuals with globozoospermia. DPY19L2 gene deletion was considered as the molecular pathogenic factor for the onset of globozoospermia in infertile men. By quantitative real-time polymerase chain reaction (qPCR), we genotyped DPY19L2 deletion and identified carriers within the population. Conclusion: This technique may be considered as a method for family counseling and has the potential to be used as a pre-implantation genetic diagnosis, especially in ethnic community with high rate of consanguineous marriages. [ABSTRACT FROM AUTHOR]

Objective: MicroRNAs (miRNA) are a class of non-coding RNAs which play key roles in post-transcriptional gene regulation. Previous studies indicate that miRNAs are dysregulated in patients with multiple sclerosis (MS). Th17 and regulatory T (Treg) cells are two subsets of CD4+ T-cells which have critical functions in the onset and progression of MS. The current study seeks to distinguish fluctuations in expression of CD4+ T-cell derived miR-223 during the relapsing-remitting (RR) phase of MS (RR-MS), as well as the expressions of Th17 and Treg cell markers. Materials and Methods: This experimental study used real-time quantitative polymerase chain reaction (qRT-PCR) to evaluate CD4+ T cell derived miR-223 expression patterns in patients that experienced either of the RR-MS phases (n=40) compared to healthy controls (n=12), along with RNA markers for Th17 and Treg cells. We conducted flow cytometry analyses of forkhead box P3 (FOXP3) and RAR-related orphan receptor γt (RORγt) in CD4+ T-cells. Putative and validated targets of miR-223 were investigated in the miRWalk and miRTarBase databases, respectively. Results: miR-223 significantly upregulated in CD4+ T-cells during the relapsing phase of RR-MS compared to the remitting phase (P=0.000) and healthy individuals (P=0.036). Expression of RORγt, a master transcription factor of Th17, upregulated in the relapsing phase, whereas FOXP3 upregulated in the remitting phase. Additionally, potential targets of miR-223, STAT1, FORKHEAD BOX O (FOXO1) and FOXO3 were predicted by in silico studies. Conclusion: miR-223 may have a potential role in MS progression. Therefore, suppression of miR-223 can be proposed as an appropriate approach to control progression of the relapsing phase of MS. [ABSTRACT FROM AUTHOR]

Background: Peroxisome Proliferator Activated Receptor gamma (PPARγ), a member of nuclear receptor superfamily, comprises two isoforms in mouse. These two isoforms are encoded by different mRNAs, which are arisen by alternative promoter usage. There are two promoter regions upstream of PPARγ1 gene. A 3 kb fragment, containing several transcription factor binding sites, acts as PPARγ1 promoter region. Thus, expression pattern of PPARγ1 isoform is due to the potential transcription factors that could influence its promoter activity. PPARγ, Retinoid X Receptor (RXR) and Vitamin D Receptor (UDR), as nuclear receptors could influence PPARγ gene expression pattern during several differentiation processes. During neural differentiation, PPARγ1 isoform expression reaches to maximal level at neural precursor cell formation. Methods: A vast computational analysis was carried out to reveal the PPARγ1 promoter region. The putative promoter region was then subcloned upstream of an EGFP reporter gene. Then the functionality of PPARγ1 promoter was assessed in different cell lines. Result: Results indicated that Rosiglitazone increased PPARγ1 promoter regulated EGFP expression of neural precursor cells during Embryoid Body (EB) formation. Furthermore vitamin D reduced PPARγ1 promoter regulated EGFP expression of neural precursor cells during EB formation through binding to its receptor. Conclusion: This study suggests that there are potential response elements for PPAR/RXR and UDR/RXR heterodimers in PPARγ1 isoform promoter. Also UDR/RXR heterodimers may decrease PPARγ expression through binding to its promoter. [ABSTRACT FROM AUTHOR]

Objectives: Multiple sclerosis (MS) is considered as a chronic type of an inflammatory disease characterized by loss of myelin of CNS. Recent evidence indicates that Interleukin 17 (IL-17)-producing T helper cells (Th17 cells) population are increased and regulatory T cells (Treg cells) are decreased in MS. Despite extensive research in understanding the mechanism of Th17 and Treg differentiation, the role of microRNAs in MS is not completely understood. Thereby, as a step closer, we analyzed the expression profile of miR-9-5p and miR-106a-5p, and protein level of retinoic acid receptor (RAR)-related orphan receptor C ( RORC ; Th17 master transcription factor) as direct target of miR-106a-5p and forkhead box P3 ( FOXP3 ; Treg master transcription factor) as indirect target of miR-9-5p in CD4 + T cells in two groups of relapsing and remitting in our relapsing-remitting MS (RR-MS) patients., Materials and Methods: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was utilized to assess the expression of miRNAs and mRNAs, in 40 RR-MS patients and 11 healthy individuals. Thus, FOXP3 and RAR-related orphan receptor γt (RORγt) was assessed in CD4 + T-cells by flow cytometry. We also investigated the role of these miRNAs in Th17/Treg differentiation pathway through bioinformatics tools., Results: An up-regulation of miR-9-5p and down-regulation of miR-106a-5p in relapsing phase of MS patients were observed compared to healthy controls. RORC and FOXP3 were up-regulated in relapsing and remitting phases of MS, respectively., Conclusion: Expression pattern of miR-9-5p and miR-106a-5p and their targets suggest a possible inducing role of miR-9-5p and suppressing role of miR-106a-5p in differentiation pathway of Th17 cells during MS pathogenesis.

Objective: MicroRNAs (miRNA) are a class of non-coding RNAs which play key roles in post-transcriptional gene regulation. Previous studies indicate that miRNAs are dysregulated in patients with multiple sclerosis (MS). Th17 and regulatory T (Treg) cells are two subsets of CD4+T-cells which have critical functions in the onset and progression of MS. The current study seeks to distinguish fluctuations in expression of CD4+T-cell derived miR-223 during the relapsing-remitting (RR) phase of MS (RR-MS), as well as the expressions of Th17 and Treg cell markers., Materials and Methods: This experimental study used real-time quantitative polymerase chain reaction (qRT-PCR) to evaluate CD4+ T cell derived miR-223 expression patterns in patients that experienced either of the RR-MS phases (n=40) compared to healthy controls (n=12), along with RNA markers for Th17 and Treg cells. We conducted flow cytometry analyses of forkhead box P3 (FOXP3) and RAR-related orphan receptor γt (RORγt) in CD4+T-cells. Putative and validated targets of miR-223 were investigated in the miRWalk and miRTarBase databases, respectively., Results: miR-223 significantly upregulated in CD4+T-cells during the relapsing phase of RR-MS compared to the remitting phase (P=0.000) and healthy individuals (P=0.036). Expression of RORγt, a master transcription factor of Th17, upregulated in the relapsing phase, whereas FOXP3 upregulated in the remitting phase. Additionally, potential targets of miR-223, STAT1, FORKHEAD BOX O (FOXO1) and FOXO3 were predicted by in silico studies., Conclusion: miR-223 may have a potential role in MS progression. Therefore, suppression of miR-223 can be proposed as an appropriate approach to control progression of the relapsing phase of MS.

Availability of human embryonic stem cells (hESCs) has enhanced the capability of basic and clinical research in the context of human neural differentiation. Derivation of neural progenitor (NP) cells from hESCs facilitates the process of human embryonic development through the generation of neuronal subtypes. We have recently indicated that fibronectin type III domain containing 5 protein (FNDC5) expression is required for appropriate neural differentiation of mouse embryonic stem cells (mESCs). Bioinformatics analyses have shown the presence of three isoforms for human FNDC5 mRNA. To differentiate which isoform of FNDC5 is involved in the process of human neural differentiation, we have used hESCs as an in vitro model for neural differentiation by retinoic acid (RA) induction. The hESC line, Royan H5, was differentiated into a neural lineage in defined adherent culture treated by RA and basic fibroblast growth factor (bFGF). We collected all cell types that included hESCs, rosette structures, and neural cells in an attempt to assess the expression of FNDC5 isoforms. There was a contiguous increase in all three FNDC5 isoforms during the neural differentiation process. Furthermore, the highest level of expression of the isoforms was significantly observed in neural cells compared to hESCs and the rosette structures known as neural precursor cells (NPCs). High expression levels of FNDC5 in human fetal brain and spinal cord tissues have suggested the involvement of this gene in neural tube development. Additional research is necessary to determine the major function of FDNC5 in this process., (Copyright © 2014 Elsevier B.V. All rights reserved.)