Synergistic effects of punicic acid and alpha lipoic acid ameliorate inflammatory and metabolic genes expression in C2C12 myoblast cells under oxidative stress condition.

Inflammation is a reaction of the immune system to infection and injury; in fact, it positioned at the center of metabolic disorders, particularly obesity, type 2 diabetes, and cardiovascular diseases. Thus play a major role not only in their development, but also exerts as a crucial linking factor among those diseases. In this regard, one of the strategies for tackling this problem is application of antioxidants to treat such diseases. The present study was performed to evaluate the synergistic effects of punicic acid (PUA) and alpha-lipoic acid (ALA) as antioxidants and radical scavenging reagents on the expression of some inflammatory and metabolism-related genes under oxidative stress in the muscle cells. The experimental treatments consisted of a range of 20, 40, 80, 160, and 320 µM of PUA, and 5, 25, 50, 100, and 200 µM of ALA with a 200 µM concentration of H2 O2 as an oxidative stress inducer. Accordingly, fatty acid treatments were applied for 24 h, and H2 O2 was treated for 1 h. Our results indicated that the simultaneous treatment of PUA and ALA at optimal concentrations (80 and 50 µM, respectively) decreased the expression of inflammation genes and increased the expression of regulatory genes (Pparγ, Pgc-1α) related to metabolism (p < .05). Unexpectedly, H2 O2 treatment increased the Fndc5 expression (p < .05). Maximal upregulation of Pparγ, Pgc-1α were obtained when fatty acids combination (PUA and ALA) were used in the culture of H2 O2 treated cells (p < .05). Therefore, our findings suggest that the simultaneous use of PUA and ALA fatty acids could reduce oxidative stress, and the expression of inflammatory genes, thereby improving the cell metabolism.

The role of the transsulfuration pathway in spermatogenesis of vitamin D deficient mice.

Vitamin D deficiency is a global health problem and has been linked to defective spermatogenesis and male infertility. In this study, we aimed to investigate the main enzymes involved in the transsulfuration pathway of 1-carbon metabolism, and spermatogenesis function. Therefore, sixteen male C57 mice were addressed to a control (standard diet) or vitamin D deficient (VDD) diet for 14 weeks. The results show that compared to the standard diet, VDD increased final body weight and reduced sperm quality, caused damage to the testicular structure, and decreased the serum levels of testosterone. In addition, serum concentrations of homocysteine, vitamin B12, and sperm oxidative stress markers increased. In testicular tissues, the CBS and CSE protein levels were down-regulated whereas HO-1 was up-regulated at both mRNA and protein expression levels. Within a mice deprivation model, VDD deeply suppressed testosterone and impaired spermatogenesis with oxidative stress-mediated mechanisms. The effects of the deprivation appeared to be at least in part independent of genomic and receptor-mediated vitamin D actions and suggest a specific impairment of the alternative transsulfuration pathway.

Aberrant Expression of TET2 Accounts for DNA Hypomethylation in Varicocele.

OBJECTIVE: Epigenetic modifications such as DNA methylation play a key role in male infertility etiology. This study aimed to explore the global DNA methylation status in testicular spermatogenic cells of varicocele-induced rats and consider their semen quality, with a focus on key epigenetic marks, namely 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC), as well as the mRNA and proteins of ten-eleven translocation (TET) methylcytosine dioxygenases 1-3. MATERIALS AND METHODS: In this experimental study, 24 mature male Wistar rats (8 in each group) were assigned amongst the control, sham, and varicocele groups. Sperm quality was assessed, and DNA methylation patterns of testicular spermatogenic cells were investigated using reverse transcription-polymerase chain reaction (RT-PCR), western blot, and immunofluorescence techniques. RESULTS: Sperm parameters, chromatin and DNA integrity were significantly lower, and sperm lipid peroxidation significantly increased in varicocele-induced rats in comparison with control rats. During spermatogenesis in rat testis, 5-mC and 5-hmC epigenetic marks, and TET1-3 mRNA and proteins were expressed. In contrast to the 5-mC fluorescent signal which was presented in all testicular cells, the 5-hmC fluorescent signal was presented exclusively in spermatogonia and a few spermatids. In varicocele-induced rats, the 5-mC signal decreased in all cells within the tubules, whereas a strong signal of 5-hmC was detected in seminiferous tubules compared to the control group. As well, the levels of TET2 mRNA and protein expression were significantly upregulated in varicocele-induced rats in comparison with the control group. Also, our results showed that the varicocele-induced animals exhibited strong fluorescent signals of TET1-3 in testicular cells, whereas weak fluorescent signals were identified in the seminiferous tubules of the control animals. CONCLUSION: Consequently, we showed TET2 upregulation and the 5-hmC gain at testicular levels are associated with varicocele and sperm quality decline, and therefore they can be exploited as potential biomarkers of spermatogenesis.

Fndc5 knockdown significantly decreased the expression of neurotrophins and their respective receptors during neural differentiation of mouse embryonic stem cells.

Fibronectin type III domain-containing-5 (Fndc5) is a trans-membrane protein which is involved in a variety of cellular events including neural differentiation of mouse embryonic stem cells (mESCs) as its knockdown and overexpression diminishes and facilitates this process, respectively. However, downstream targets of Fndc5 in neurogenesis are still unclear. Neurotrophins including NGF, BDNF, NT-3, and NT-4 are the primary regulators of neuronal survival, growth, differentiation, and repair. These biomolecules exert their actions through binding to two different receptor families, Trk and p75NTR. In this study, considering the fact that neurotrophins and their receptors play crucial roles in neural differentiation of ESCs, we sought to evaluate whether knockdown of Fndc5 decreased neural differentiation of mESCs by affecting the neurotrophins and their receptors expression. Results showed that at neural progenitor stage, the mRNA and protein levels of BDNF, Trk, and p75NTR receptors decreased following the Fndc5 knockdown. In mature neural cells, still, the expression of Trk and p75NTR receptors at mRNA and protein levels and BDNF and NGF expression only at protein levels showed a significant decrease in Fndc5 knockdown cells compared to control groups. Taken together, our results suggest that decreased efficiency of neural differentiation following the reduction of Fndc5 expression could be attributed to decreased levels of NGF and BDNF proteins in addition to their cognate receptors.

New insights into the cellular activities of Fndc5/Irisin and its signaling pathways.

Fndc5, a well-defined myokine and also identified as an adipokine, has a critical role in modulation of metabolism and protection against obesity. These important functions are mediated by irisin, a secretory peptide produced from proteolytic processing of Fndc5. The other beneficial physiological effects of irisin are alleviation of oxidative stress, neuroprotective effects, and anti-inflammatory properties and associated anti-metastatic effects. Fndc5/irisin exerts its biological effects through several intracellular signaling pathways. The major signaling pathway is thought to be MAPK signaling pathways which are involved in neural differentiation, browning of white adipocytes, as well as osteoblast proliferation and differentiation. Other essential functions of Fndc5/irisin are mediated through additional pathways including AMPK pathway, PI3K/AKT, and STAT3/Snail. Thorough understanding of the mechanisms of irisin actions are essential in order to develop Fndc5/irisin for therapeutic purposes. In the present review, we focus on the current knowledge of the signaling pathways that elicit irisin actions.

Positive effects of conjugated linoleic acid (CLA) on the PGC1-α expression under the inflammatory conditions induced by TNF-α in the C2C12 cell line.

The aim of the current study was to evaluate the effects of conjugated linoleic acid (CLA) on the expression of the genes involved in the reduction of inflammatory conditions induced by tumor necrosis factor alpha (TNF-α). To fulfill this, the C2C12 murine myoblast cell line was used. The experimental treatments consisted of 50 and 100 µM of CLA with 10, 15 and 20 ng/mL of TNF-α. Our results indicated that the concentrations of 50, 100 and 150 µM of CLA at 24 and 48 h increased the cell survival rate (p < 0.05), as compared with the control group. The effect of pre-treating CLA before the inflammation induced by TNF-α showed that the amount of cell death caused by TNF-α was inhibited in both 50 and 100 µM concentrations of CLA (p < 0.05). Modulatory effects of CLA on PGC1-α and Fndc5 transcript levels showed that the PGC1-α and Fndc5 expression levels were significantly increased 48 h after treating with 50 and 100 µM of CLA (p < 0.05). Overall, these findings indicate that using CLA could considerably improve the inflammatory condition induced by TNF-α.

A comparative study on the effects of acute and chronic downhill running vs uphill running exercise on the RNA levels of the skeletal muscles PGC1-α, FNDC5 and the adipose UCP1 in BALB/c mice.

The purpose of this study was to investigate the effect of a single bout and 8 weeks of downhill running versus uphill running exercise on expression of PGC1-α, FNDC5 and UCP1 in mice. Forty-eight BALB/c male mice weighing 25-30 g were randomly assigned into 8 groups: 1) acute downhill running (ADR) on a -15° slope; 2) acute uphill running (AUR) on a +15° slope; 3) acute running without inclination (AWI), 4) acute without exercise as control (ACtrl), 5) chronic downhill running (CDR) on a -15° slope; 6) chronic uphill running (CUR) on a +15°slope; 7) chronic running without inclination (CWI), 8) chronic without exercise as control (CCtrl). Twenty four hours after the last training session, the mice were sacrificed and Calf muscles (including soleus and gastrocnemius) and quadriceps muscles (including Rectus femoris and vastus intermedius) were obtained and expression levels of PGC1-α and FNDC5 in crus and quadriceps muscles and UCP1 in visceral and subcutaneous adipose tissues were measured and compared between the groups. PGC-1α and FNDC5 mRNA levels increased after treadmill exercise training in all acute and chronic exercise groups in both skeletal muscle groups. Furthermore mRNA level of UCP1 in subcutaneous adipose tissue but not in visceral adipose tissue increased both after acute and chronic exercise. Collectively, data showed that downhill running exercise to be more effective than other exercises, as downhill running has led to a greater improvement in metabolism may be considered more effective for browning of fat tissue.

PPARγ/Pgc-1α-Fndc5 pathway up-regulation in gastrocnemius and heart muscle of exercised, branched chain amino acid diet fed mice.

BACKGROUND: Previous studies have revealed the inductive effect of branched-chain amino acids (BCAAs) catabolism on fatty acid oxidation and metabolism, especially in muscle cells. In the present investigation, we have attempted to address whether a combination of BCAAs supplement consumption with aerobic exercise could elaborate the expression of PPARγ, Pgc-1α and Fndc5 genes in gastrocnemius muscle and heart tissue of male C57BL/6 mice. METHODS: Thirty-six young male mice with an average weight of 18 ± 2 g were selected. Mice were randomly assigned to 6 groups: 20 mg/mL of BCAAs consumption with simultaneous exercise-training, 60 mg/mL of BCAAs consumption with simultaneous exercise-training, exercise-trained with no BCAAs consumption group, 20 mg/mL BCAAs without exercise-training, 60 mg/mL BCAAs without exercise-training, and untrained mice without BCAAs consumption. RESULTS: The findings showed a combination of 20 mg/mL BCAAs with aerobic exercise significantly increased Fndc5, PPARγ, Pgc-1α gene expression in skeletal muscles although, circulating Irisin levels remained unchanged (p < 0.05). Interestingly, plasma urea and lactate levels were significantly increased in 60 mg/mL BCAAs administrated mice which performed exercised (p < 0.05). Two-way analysis of variance (ANOVA) was used to examine significant difference between groups and sedentary group. CONCLUSIONS: Results showed inductive effect of 20 mg/mL BCAAs on expression levels of Fndc5, PPARγ, Pgc-1α in gastrocnemius muscle similar with counterparts in heart tissue. Of note, higher serum irisin levels were detected after 20 mg/mL BCAAs supplementation coincided with the exercise. GRAPHICAL ABSTRACT: An Overview on supplemantaion of branched chain amoinoacids on metablism of skeletal muscle and heart.

Assessment of PGC1α-FNDC5 Axis in Granulosa Cells of PCOS Mouse Model.

BACKGROUND: Polycystic ovarian syndrome (PCOS) is a metabolic and endocrine disorder which is characterized by hyperandrogenism, anovulation or oligomenorrhea and polycystic ovarian morphology. It is believed that modulation in metabolism of granulosa cells of PCOS patients may lead to infertility. One of the metabolic modulators is FNDC5 and its cleaved form, irisin. The axis of PGC1α-FNDC5 pathway is one of the main factors affecting cellular energy balance the purpose of this study was to evaluate this pathway in granulosa cells derived from PCOS mice model in comparison with control group. METHODS: In the present study, PCOS mouse model was developed by injection of dehydroepiandrosterone (DHEA) hormone in 20 mice for a period of 20 days. Also, 20 uninjected mice were used as the control. Meanwhile, a vehicle group consisted of mice which received daily subcutaneous sesame oil injection (n=20). Relative expressions of PGC1α and FNDC5 in granulosa cells were evaluated by RT-qPCR. Analysis of gene expressions was calculated by the ΔΔCT method and the relative levels of mRNA were normalized to GAPDH transcript levels. Differences in genes expression among three groups were assessed using one-way ANOVA, Tukey's Post Hoc test. RESULTS: Our results showed that expression of FNDC5 was significantly reduced in granulosa cells of DHEA-induced PCOS mice compared with control and vehicle groups (p<0.05), while there was no significant differences in PGC1α expression among different groups. CONCLUSION: Down regulation of FNDC5 transcript level may contribute in metabolic disturbance of granulosa cells derived from PCOS ovary apart from PGC1α levels which remained unchanged.

RNA/Protein Discordant Expression of Fndc5 in Central Nervous System Is Likely to Be Mediated Through microRNAs.

Fibronectin type III domain-containing 5 protein (Fndc5) is responsible for producing a secretory protein termed, "irisin." A modified expression of Fndc5 has been reported in different tissues during development, differentiation processes, as well as in metabolic events such as exercise. One of the important issues to be fixed is whether Fndc5 RNA level and protein content are concerted and modified hand in hand. Therefore, the aim of this study is to assess Fndc5 RNA and protein levels in various tissues of mouse and rat with emphasis on brain. Biopsies from various parts of neonatal and adult mouse and rat tissues were simultaneously assessed for transcript levels of Fndc5 and compared with the respective protein levels at the same time. Data indicated, unlike in muscle, no concerted fluctuations were observed for Fndc5 RNA and protein, especially in brain. Further look at four regions of brain (cerebellum, putamen, hippocampus, and cortex) revealed a similar discrepant expression. To hypothesize whether such discrepancy is arisen by miRNAs, we selected three main miRNAs, which were predicted to target Fndc5 and their expression levels were assessed in central nervous system (CNS) of mouse and hippocampus of rat. miRNA levels showed an antiparallel correlation with protein level of Fndc5, interpreting a putative role in regulating Fndc5 protein content in CNS. This phenomenon may represent the importance of governing Fndc5 content in neural cells, which seems to be crucial for neural function and differentiation.

Fndc5 knockdown induced suppression of mitochondrial integrity and significantly decreased cardiac differentiation of mouse embryonic stem cells.

Fibronectin type III domain-containing 5 protein (Fndc5) is a glycosylated protein with elevated expression in high energy demanded tissues as heart, brain, and muscle. It has been shown that upregulation of Fndc5 is regulated by peroxisome proliferator-activated receptor-γ coactivator-1 alpha (PGC-1α), which is known as a master regulator of mitochondrial function and biogenesis. Also, our group indicated that Fndc5 expression increases gradually during cardiac differentiation of mouse embryonic stem cells (mESCs). In this paper, to clarify the importance of Fndc5 in cardiac differentiation, we south to knock down Fndc5 expression by generation a stably transduced mESC line that derives the expression of a short hairpin RNA (shRNA) against Fndc5 gene following doxycycline (Dox) induction. Knock-down of Fndc5 demonstrated a considerable decrease in expression of cardiac progenitor and cardiomyocyte markers. Considering the fact that mitochondria play a crucial role in cardiac differentiation of ESCs, we investigated the role of Fndc5, as a downstream target of PGC1-α, on mitochondrial indices. Results showed that expression of nuclear encoded mitochondrial genes including PGC1-α, Atp5b, Ndufb5, and SOD2 significantly decreased. Moreover, mitochondrial membrane potential (ΔΨm) and relative ATP content of cardiomyocytes decreased markedly with relative ROS level increase. Together, our results suggest that Fndc5 attenuates process of cardiac differentiation of mESCs which is associated with modulation of mitochondrial function and gene expression.

Diagnosis of genetic defects through parallel assessment of PLCζ and CAPZA3 in infertile men with history of failed oocyte activation.

OBJECTIVES: Phospholipase C ζ (PLCζ) is considered as a nominee for sperm associated oocyte activating factors and is located back-to-back with CAPZA3, an actin-capping protein controlling actin polymerization during spermiogenesis. They contain a common bidirectional promoter. The objective of this study was to identify individuals with parallel low expression of PLCζ and CAPZA3 mRNA, in hope of detecting genetic defects in this bidirectional promoter. MATERIALS AND METHODS: Semen samples were collected from 24 fertile and 59 infertile individuals with total failed, low and high fertilization rate post intra-cytoplasmic sperm injection (ICSI), as well as globozoospermic individuals. Expression of PLCζ and CAPZA3 were assessed by Real time PCR. In addition, PLCζ was assessed by Western blot. RESULTS: Significant correlations between PLCζ with CAPZA3 and also between these two genes with fertilization were observed. Individuals with low fertilization presented significantly lower expression of these two genes. Low expression of PLCζ was also verified by Western analysis. Sequence analysis of bidirectional promoter of these two genes in an individual with parallel low expression of both PLCζ and CAPZA3, revealed a mutation within the CAPZA3 predicted promoter, known as human regulatory factor X4 which is a testis-specific dimeric DNA-binding protein. In the opposite stand, in the same location, the mutation appears to be outside but in the vicinity of PLCζ, in a binding region predicate by Genomatix. CONCLUSION: Parallel assessment of CAPZA3 with PLCζ at mRNA level in individuals with inability to induce oocyte activation may help researcher to identify genetic defects associated with failed fertilization.

Fndc5 overexpression facilitated neural differentiation of mouse embryonic stem cells.

Fndc5 has been recently recognized as a myokine which could be cleaved and secreted into blood stream. It is termed as irisin with an important role in thermogenesis and energy homeostasis. Increased expression of Fndc5 has been reported upon retinoic acid treatment during neural differentiation and its knockdown decreased neural differentiation and neurite outgrowth. This study tries to evaluate the effect of Fndc5 overexpression on rate of neural differentiation in mouse. (Thus, transduced cell line of mouse embryonic stem cell with ability to express Fndc5 under Doxycycline treatment was established. Subsequently, the effect of overexpression of Fndc5 on different stages of neural differentiation was studied). Our study showed an increase enhancement in neuronal precursor markers and mature neuron markers upon overexpression of Fndc5, concluding that Fndc5 facilitates neural differentiation. This effect might be related to increased expression of BDNF following overexpression of Fndc5. Our findings are consistent with recent studies reporting a similar role for Fndc5 in proliferation of neural cells and increase in the expression of neurotrophins like BDNF.

Induced expression of Fndc5 significantly increased cardiomyocyte differentiation rate of mouse embryonic stem cells.

Fibronectin type III domain-containing 5 protein (Fndc5) is an exercise hormone and its transcript profile in mouse showed high degree of expression in heart, skeletal muscle and brain. Our previous studies indicated a significant increase (approximately 10 fold) in mRNA level of Fndc5 when embryonic stem cells were differentiated into beating bodies. As a step closer to identify the involvement of Fndc5 in the process of cardiomyocyte differentiation, we generated a stably inducible transduced mouse embryonic stem cell (mESC) line that overexpressed Fndc5 following Doxycycline induction. Our results indicated that the overexpression of Fndc5 during spontaneous cardiac differentiation significantly increased not only at RNA levels for mesodermal markers but also at the transcriptional levels for cardiac progenitor and cardiac genes. These data suggest that Fndc5 may be involved in cardiomyocyte differentiation. Therefore, a new hope will be arisen for potential application of this myokine for regeneration of damaged cardiac tissues especially in cardiac failure.

An In vitro Study on Chick Somite Ability to Express Cerberus, Chordin, FGF8, Follistatin, and Noggin Transcripts.

BACKGROUND: In vitro simulation of developmental processes is an invaluable tool to shed light on the intrinsic mechanism of developmental biosystems such as central nervous system in mammals. Chick somites have been used to simulate the neural differentiation of human neural progenitor cells. In the present study, we aimed to indicate whether somites have the ability to express required neural differentiation factors at mRNA level. METHODS: Chick embryos were isolated from the yolk surface of the fertilized eggs and somites were subsequently isolated from embryos under a dissecting microscope. Total RNA of the somites was extracted and RT-PCR carried out with specific primers of cerberus, chordin, FGF8, follistatin and noggin. RESULTS: Data showed that five aforementioned factors were co-expressed after 7 days in vitro by somites. CONCLUSION: We concluded that neural induction property of somites appeared by production of required neural differentiation factors including cerberus, chordin, FGF8, follistatin and noggin.

Dual effects of peroxisome proliferator-activated receptor γ on embryonic stem cell self-renewal in presence and absence of leukemia inhibitory factor.

The aim of this study was to evaluate the influence of peroxisome proliferator-activated receptor γ (PPARγ) on self-renewal of mouse embryonic stem cells (mESCs) in the presence and absence of leukemia inhibitory factor (LIF). We demonstrated that in the presence of LIF, the activation of PPARγ by Rosiglitazone led to an increased proliferation of mESCs whereas PPARγ antagonist (GW9662) reversed this effect. Additionally, upon PPARγ activation, LIF increased PPARγ expression and resulted in the degradation of suppressor of cytokine signaling 3 (SOCS3), an important negative regulator of LIF/signal transducers and activators of transcription 3 (STAT3)-pathway. In the absence of LIF, Rosiglitazone decreased proliferation of mESCs. In this state, our results showed that extracellular signal-regulated kinase (ERK) proteins were activated and resulted in the suppression of Nanog expression, an important pluripotency determinant, whereas it did not affect Oct4 expression. These results suggest that the pivotal role of PPARγ on mESC self-renewal depends on the presence and absence of LIF.

Identification of a novel missense mutation of PEX7 gene in an Iranian patient with rhizomelic chondrodysplasia punctata type 1.

Deficiency in the PTS2 protein import pathway due to mutations in PEX7 gene results in the rhizomelic chondrodysplasia punctata (RCDP) type 1. In the present study, we have reported a novel missense mutation, W75R, in the PEX7 gene in an Iranian patient with the RCDP type 1. The inability of PEX7 protein to transport PTS2 containing proteins including peroxisomal 3-ketoacyl-CoA thiolase and PTS2-EGFP protein to the surface of the peroxisomes showed that the W75R mutation in PEX7 gene severely impaired the function of PEX7 protein and was responsible for RCDP type 1 in this patient.

Differentiation of human embryonic stem cell-derived retinal progenitors into retinal cells by Sonic hedgehog and/or retinal pigmented epithelium and transplantation into the subretinal space of sodium iodate-injected rabbits.

Transplantation of retinal cells has recently provided a promising therapeutic approach for retinal degeneration. Here, we differentiated initially retinal progenitors (RPs) from adherent feeder-free human embryonic stem cells (hESCs) with the use of defined media supplemented with a specific combination of growth factors. The differentiated RPs highly (>80%) expressed related molecular features that included Six3 at an early stage in addition to Crx, Rx, Pax6, Otx2, and Chx10 at later stage. Next, we examined the induction of photoreceptors by Shh and/or the coculture of rabbit retinal pigmented epithelium with hESCs-derived RPs. The differentiation of retinal cells was demonstrated by protein and gene expression in all groups. However, S-Opsin, a cone photoreceptor marker, had higher expression in the presence of Shh, whereas expressions of Gli and Hes1 decreased in the same group. Finally, hESC-derived RPs were treated with Shh transplanted into the subretinal space of sodium iodate-injected albino-type adult rabbits and analyzed 4 weeks later. Transplanted retinal cells survived, migrated into retinal layers, and restored a small but significant B-wave. The grafted cells expressed photoreceptor markers, S-Opsin and Rhodopsin. Our results indicate that putative hESC-derived retinal cells express related genes and proteins. Further, our results show that retinal-like cells can be useful replacements for photoreceptors in retinal diseases.

Characterization and Functional Assessment of Mouse PPARγ1 Promoter.

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 PPARγ 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 (VDR), 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. RESULTS: 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 VDR/RXR heterodimers in PPARγ1 isoform promoter. Also VDR/RXR heterodimers may decrease PPARγ expression through binding to its promoter.

Evaluation of the leptin receptor in human spermatozoa.

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.