Effect of smoking on methylation and semen parameters.

One type of epigenetic modification is genomic DNA methylation, which is induced by smoking, and both are associated with male infertility. In this study, the relationship between smoking and CHD5 gene methylation and semen parameters in infertile men was determined. After the MS-PCR of blood in 224 samples, 103 infertile patients (62 smokers and 41 non-smokers) and 121 fertile men, methylation level changes between groups and the effect of methylation and smoking on infertility and semen parameters in infertile men were determined. The results showed that there is a significant difference in the methylation status (MM, MU, UU) of the CHD5 gene between the patient and the control group, and this correlation also exists for the semen parameters (p < .001). The average semen parameters in smokers decreased significantly compared to non-smokers and sperm concentration was (32.21 ± 5.27 vs. 55.27 ± 3.38), respectively. MM methylation status was higher in smokers (22.5%) compared to non-smokers (14.6%). Smoking components affect the methylation pattern of CHD5 gene, and smokers had higher methylation levels and lower semen parameters than non-smokers, which can be biomarkers for evaluating semen quality and infertility risk factors. Understanding the epigenetic effects of smoking on male infertility can be very useful for predicting negative consequences of smoking and providing therapeutic solutions.

CHD5 gene (rs9434741) might be a genetic risk factor for infertility in non-obstructive azoospermia and severe oligozoospermia.

Inability to conceive is one of the health concerns. Chromodomain helicase DNA-binding protein5 (CHD5) gene is a major regulator in the replacement of histone proteins with protamines, the chromatin remodelling in spermatogenesis process. Thus, functional SNPs in this gene can disrupt sperm development. This study aimed to investigate the relationship between CHD5 polymorphism (rs9434741) and male infertility. This case-control study was conducted on 103 infertile and 121 fertile men. CHD5 polymorphism rs9434741 was tested using T-ARMS-PCR to investigate its association with male infertility. The presence of G allele caused 1.52 fold increase (OR = 1.52, 95% CI = 1.09-2.31 and p = 0.019) in infertility susceptibility in the patient group. GG genotype and (AG+GG) were significantly related to the increased risk of infertility (OR = 3.13, 95% CI = 1.26-7.76, p = 0.013; OR = 2.72, 95% CI = 1.35-5.47), respectively. Significant differences were observed between genotypes in NOA and SO groups compared to the control group (p = 0.029). Sperm count and total motility were significantly different among three genotypes in infertile men and the control group (p < 0.001). Analysis of genotypes and alleles frequency indicated statistically significant differences between the patient and control groups (p < 0.05). This study showed that CHD5 polymorphism (rs9434741) could be associated with the risk of male infertility. It is recommended to conduct further studies on different populations.

Effect of Vitex agnus-castus ethanolic extract on hypothalamic KISS-1 gene expression in a rat model of polycystic ovary syndrome.

OBJECTIVE: Polycystic ovary syndrome (PCOS) is an endocrine system disruption that affects 6-10% of women. Some studies have reported the effect of Vitex agnus-castus (Vitagnus) on the hypothalamic-pituitary-gonad axis (HPG). This study was conducted to investigate Vitagnus effect on the expression of kisspeptin gene in a rat model of PCOS. MATERIALS AND METHODS: Thirty-two female rats were distributed into: control, Vitagnus-treatment (365 mg/kg for 30 days), PCOS (Letrozole for 28 days) and PCOS animals treated with Vitagnus (30 days of Vitagnus after PCOS induction). At the end of the treatments, serum and ovaries were collected for analysis. Expression level of KISS-1 gene in the hypothalamus was investigated, using Real-Time-PCR. RESULTS: In the PCOS group compared to control, FSH, progesterone and estradiol levels were decreased, whereas testosterone and LH levels were significantly increased. No significant changes were observed in the Vitagnus-treated animals in compare to control. However, Vitagnus treatment in the PCOS group, resulted in a raise in progesterone, estrogen and FSH levels and a reduction in the levels of testosterone and LH. Quantitative gene expression analysis showed that PCOS induction resulted in over-expression of KISS-1 gene, however, Vitagnus treatment reduced this up-regulated expression to normal level. CONCLUSION: In conclusion, our results indicated that Vitagnus extract inhibited downregulation of KISS-1 gene in the hypothalamus of PCOS rats. Because of the master role of kisspeptin in adjusting the HPG axis, Vitagnus is likely to show beneficial effects in the treatment of PCOS via regulation of kisspeptin expression. This finding indicates a new aspect of Vitagnus effect and may be considered in its clinical applications.

Investigation of cholestasis-related changes in characteristics of spermatogonial stem cells in testis tissue of male Wistar rats.

Paternal metabolic status is an important factor in the health status of offspring. Cholestasis, as a metabolic disorder, significantly disrupts spermatogenesis. Spermatogonial stem cells (SSCs) are considered the dividing germ cells, which maintain spermatogenesis throughout the lifespan. Here, we investigated the in vivo and in vitro effect(s) of cholestasis on SSCs. Cholestasis was induced in rats by bile duct ligation. Four weeks after the cholestasis induction, testicular tissues were analysed using histopathological examinations. The expression of SSC markers, including Plzf and Thy-1, was determined using the immunofluorescent technique. Also, SSCs were isolated from animals, and their proliferation was examined in vitro. The histological examinations revealed that cholestasis caused irregularities in the structure of seminal tubes. Immunostaining showed that the total number of Thy-1- and Plzf-expressing cells was declined in the cholestasis group compared with the control group. In vitro culture of SSCs indicated that the number of SSC colonies and those expressing Plzf were significantly reduced in the culture medium of the cholestasis group. Our results indicated that cholestasis affects the functionality of SSCs and reduces the number and proliferation of them. This finding may be of interest to the effect of metabolic diseases such as cholestasis on spermatogenesis.

Evaluation of dexamethasone treated mesenchymal stem cells for recovery in neurotmesis model of peripheral nerve injury.

OBJECTIVE: Peripheral nerve injuries comprise significant portion of the nervous system injuries. Although peripheral nerves show some capacity of regeneration after injury, the extent of regeneration is not remarkable. The present study aimes to evaluate the regeneration of transected sciatic nerve by a therapeutic value of dexamethasone (DEX) associated with cell therapy (Cell) and biodegradable membrane (Mem) in rat. METHODS: Male Wistar rats (n = 42, 180-200g) were randomly divided into control (Ctrl), Membrane+ Cell, Mem, DEX, DEX+ Cell, DEX+ Mem and DEX+ Cell+ Mem groups. Functional recovery was evaluated at 2, 4, 6, 8 and 12 weeks after surgery using sciatic functional index (SFI), withdrawal reflex latency (WRL) test, electrophysiological and histological analyses. RESULTS: The rats in the DEX+ Cell+ Mem-treated group showed a significant improvement in SFI, WRL and electrophysiological findings during the 2nd to 12th weeks after surgery. In addition, histomorphological findings showed a significant improvement in the DEX+ Cell+ Memtreated group, at 12 weeks after surgery. DISCUSSION: Taken together, use of DEX associated with cell and biodegradable membrane could improve functional and histomorphological properties of the sciatic nerve after injury.

The influence of semen quality on male pronucleus demethylation process during ICSI cycle.

There is growing evidence that the spermatozoon's epigenetic structure is of the utmost importance in the health of the future embryo. Following fertilization, sperm chromatin undergoes epigenetic reprogramming including DNA demethylation and remethylation, which resets gene expression. In some infertile patients, it is inevitable that sperm cells that are not within the range of normal human sperm parameters will be used for intracytoplasmic sperm injection. Understanding the relationship between the human sperm parameters and male pronucleus DNA demethylation seems necessary. We hypothesized that demethylation of the male pronucleus might be altered in zygotes conceived from a spermatozoa obtained from a sample exhibiting an abnormal semen analysis profile. To test the hypothesis, sperm cells from normal and abnormal human semen samples were injected into mouse oocytes. A group of cultured zygotes was fixed before the onset of DNA demethylation and the other group was fixed after DNA demethylation. Both groups were then labeled with a 5 methylcytosine antibody and the level of pronuclei methylation was detected as a function of fluorescent intensity. The level of demethylation was then determined as the difference between 5 methylcytosine fluorescent intensity before and after DNA demethylation. A negative correlation (p<0.05) was observed between sperm motility, morphology, percentage of head defects, protamine deficiency, and DNA demethylation level. However, no correlation was found between the demethylation level and sperm count. In conclusion, these observations suggest that demethylation is altered in the male pronucleus when low quality sperm samples are used.

Pronuclear epigenetic modification of protamine deficient human sperm following injection into mouse oocytes.

Epigenetic abnormalities and abnormal chromatin structure in sperm may lead to male infertility. Protamine deficiency is among the disorders of chromatin structure in sperm. The study of epigenetic changes in male pronuclei is necessary since abnormal sperm is sometimes used to create embryos using assisted reproductive techniques. The present study was carried out to compare epigenetic global marks in male pronuclei derived from normal and protamine deficient sperm cells. To do so, interspecies fertilization was used to obtain the male pronucleus. Normal and protamine deficient sperm cells, which were identified by chromomycin A3 staining, were injected into mouse oocytes. Oocytes were cultured until pronuclear formation and were then labeled with different antibodies (anti 5-methylcytosine, anti 5-hydroxymethylcytosine, and anti acetyl H4K12). Based on the fluorescence intensity, the level of each of these epigenetic factors was determined and they revealed a significant relationship between the level of sperm protamine deficiency and sperm epigenetic factors. Protamine deficiency was found to be associated with an increased methylation (p=0) and decreased hydroxymethylation rate (p=0.015) of the male pronucleus chromatin. However, no association was found between protamine deficiency and the level of H4K12 acetylation (p=0.548). Also, the efficiency of fertilization in protamine deficient sperm cells was less than normal. These results suggest that protamine deficient sperm cells lead to the formation of epigenetically altered pronuclei.

Biological parameters influencing the human umbilical cord-derived mesenchymal stem cells' response to retinoic acid.

Human umbilical cord-derived mesenchymal stem cells (HUCMSCs) are multipotent fetal stem cells that differentiate into various cell lineages. In recent years, they have gained attention for therapeutic applications but very little is known about their sensitivity to chemical agents such as widely used retinoic acid (RA). As a morphogen inducing differentiation of mesenchymal stem cells, RA has for a long time been known to be a potent teratogen promoting craniofacial and limb abnormality in vertebrate embryos. Here, using MTT assay and EB/AO staining as well as TUNEL assay we show that RA in a concentration-dependent manner induces apoptosis through upregulating Caspase expression and increasing Bax/Bcl2 ratio. Moreover, different biological parameters such as initial time seeding, cell density, passage number and duration of RA treatment play a major role in HUCMSCs cytotoxic response to this agent.

Comparative evaluation of cardiac markers in differentiated cells from menstrual blood and bone marrow-derived stem cells in vitro.

In recent years, menstrual blood-derived stem cells (MenSCs) have been introduced as easily accessible and refreshing stem cell source without ethical considerations in the field of regenerative medicine. The aim of this study was to investigate in vitro cardiac differentiation capacity of MenSCs compared to bone marrow-derived stem cells (BMSCs) under two protocols using 5-aza-2'-deoxycytidine (5-aza) and basic fibroblast growth factor (bFGF). Our data revealed that differentiated MenSCs and BMSCs acquired some features of cardiomyocytes; however, degree of differentiation was dependent on the protocol. In a similar manner with BMSCs, differentiated MenSCs showed upper levels of mRNA/protein of late-stage cardiac markers under 5-aza stimulation and continuous treatment with bFGF (protocol 2) compared to those induced by 5-aza alone (protocol 1) evidencing the key role of bFGF in cardiac development of stem cells. Compared to corresponding undifferentiated cells differentiated MenSCs under protocol 2 showed remarkable expression of connexin-43 and TNNT2 at both gene and protein levels, whereas developed BMSCs under the same condition only expressed connextin-43 at the higher level. Superiority of protocol 2 over protocol 1 was confirmed by assessment of LDH and cTnI production by differentiated cells. Based on the accumulative data, our study provided convincing evidence that MenSCs have relatively higher capability to be differentiated toward cardiomyocyte compared with BMSCs. Furthermore, usage of bFGF and 5-aza to induce in vitro cardiac differentiation of MenSCs is highly recommended.

Evaluation of menstrual blood stem cells seeded in biocompatible Bombyx mori silk fibroin scaffold for cardiac tissue engineering.

Recently, silk fibroin scaffolds have been introduced as novel and promising biomaterials in the field of cardiac tissue engineering. This study was designed to compare infiltration, proliferation, and cardiac differentiation potential of menstrual blood-derived stem cells (MenSCs) versus bone marrow-derived mesenchymal stem cells (BMSCs) in Bombyx mori-derived silk scaffold. Our primary data revealed that the fabricated scaffold has mechanical and physical qualities suitable for cardiac tissue engineering. The MenSCs tracking in scaffolds using immunofluorescent staining and scanning electron microscopy confirmed MenSCs attachment, penetration, and distribution within the porous scaffold matrix. Based on proliferation assay using propidium iodide DNA quantification, the significantly higher level of growth rates of both MenSCs and BMSCs was documented in scaffolds than that in two-dimensional culture (p < 0.01). The expression level of TNNT2, a bona fide cardiac differentiation marker, in BMSCs differentiated on silk scaffolds was markedly higher than those cultured in two-dimensional culture indicating the improvement of cardiac differentiation in the silk scaffolds. Furthermore, differentiated MenSCs exhibited higher expression of TNNT2 compared with induced BMSCs. It seems that silk scaffold-seeded MenSCs could be viewed as a novel, safe, natural, and accessible construct for cardiac tissue engineering.

Signaling crosstalk of FHIT, CHK2 and p38 in etoposide induced growth inhibition in MCF-7 cells.

FHIT (Fragile Histidin Triad) is a tumor suppressor gene involved in regulating cell death during DNA damage conditions. The exact mechanism of DNA damage-induced FHIT signaling is not well understood. It is known that p38 kinase and CHK2 kinase are being activated during stress-induced conditions and DNA damage, resulting in cell death. Since both CHK2 and FHIT are being influenced by DNA damage, we have evaluated the interplay of p38, CHK2 and FHIT in response to etoposide-induced cell death. DNA damage was induced by etoposide in MCF-7 cells and viability was examined using MTT. FHIT expression was blocked using siRNA. Protein expression was measured using western blotting. Our results indicated that etoposide induced cytotoxicity in MCF-7. Block of FHIT expression, completely reversed etoposide cytotoxicity. Besides, etoposide induced p38 and CHK2 phosphorylation and reduced FHIT expression in a time-dependent manner. The time-course study indicated that CHK2 had been phosphorylated prior to p38 activation. Knockdown of FHIT expression reduced CHK2 phosphorylation but had no significant effect on p38 activation. Inhibition of p38 kinase and CHK2 prevented etoposide induced alteration in FHIT expression. Furthermore, p38 inhibitors augmented etoposide-induced CHK2 phosphorylation. These results indicate that etoposide lowers FHIT expression and induces cell death via p38 and CHK2 phosphorylation. These results demonstrate a time dependent complex crosstalk of FHIT, p38 and CHK2 pathways in response to etoposide. Moreover, our findings suggest signaling interaction for these pathways which can be targeted for manipulating cell proliferation.

The effect of cholestasis on rewarding and exploratory behaviors induced by opioidergic and dopaminergic agents in mice.

BACKGROUND: Several investigations have indicated that cholestasis decreases opioid receptor expression in the brain following increased opioidergic neurotransmission. The opioidergic system plays an important role in regulation of reward circuits that may be produced via dopamine-dependent mechanisms. It has been suggested that the dopaminergic system of the nucleus accumbens is necessary in conditioned place preference (CPP). The aim of this study is, therefore, to test if cholestasis can alter the reward system and the involvement of opioidergic and dopaminergic systems in this phenomenon.  METHODS: We used CPP and hole-board paradigms to measure the reward effect and exploratory behaviors, respectively, in mice. Cholestasis was induced by ligation of the main bile duct, using two ligatures and transecting the duct between them (BDL mice).  RESULTS: The data showed that morphine (1 and 2 mg/kg), sulpiride (80 mg/kg) and SKF38393 (20 mg/kg) produced CPP, while naloxone (1 mg/kg) and SCH23390 (1mg/kg) produced conditioned place aversion (CPA), whereas quinpirole had no effect in sham-operated mice. However, morphine (2 mg/kg, i.p.), sulpiride (40 mg/kg) and? SKF38393 (10 mg/kg) induced CPP in BDL mice compared to sham-operated mice. Naloxone- or SCH23390-induced CPA was reduced in BDL mice compared with the respective sham-operated mice. Quinpirole tended to induce aversion in BDL mice which was, however, not significant. In addition, quinpirole 1 mg/kg) and SCH23390 (1 mg/kg) increased head-dip exploratory behavior, whereas naloxone (2 mg/kg) caused a decrease in head-dip exploratory behavior in sham-operated mice. Morphine (2 mg/kg), SCH23390 (1 mg/kg) and quinpirole (0.25 and 0.5 mg/kg) induced anxiogenic-like behavior in BDL mice.  CONCLUSION: It can be concluded that cholestasis differentially alters the reward effects of opioidergic and dopaminergic agents.