effect of vitrification on expression and histone marks of Igf2 and Oct4 in blastocysts cultured from two-cell mouse embryos

Objective: vitrification is increasingly used in assisted reproductive technology [ART] laboratories worldwide. In this study the effect of vitrification on the expression and modifications of H3 histones of Igf2 and Oct4 was investigated in blastocysts cultured from vitrified and non-vitrified two-cell embryos

Materials and Methods: in this experimental study, two-cell embryos were cultured in KSOM medium to reach the blastocyst stage. Expression of Igf2 and Oct4 and modifications of H3 histones in regulatory regions of both genes were compared with in vivo blastocysts, which comprise the control group. To gene expression evaluation, reverse transcription-quantitative polymerase chain reaction [RT-qPCR] and the ChIP assay method were carried out to assess expression and histone modifications of the two genes

Results: the expression level of Igf2 was significantly higher in both experimental groups than the control group. In the regulatory region of Igf2, H3K9 methylation decreased whereas H3K9 acetylation increased in the experimental group compared with the control group. In contrast, the expression level of Oct4 was significantly lower in experimental groups. The Oct4 gene promoter showed a significant increase in H3K9 methylation and decrease in H3K9 acetylation [P<0.05]

Conclusion: according to our results, both vitrification and cultivation conditions may lead to changes in expression level and modification of histones in Igf2 and Oct4. However, these effects were the same in vitrified and non-vitrified groups. Indeed, the embryo is most affected by culture environment and in vitro culture. Therefore, vitrification may be used as a low-risk technique for embryo cryopreservation in ART

Association between JMJD1A expression and sperm retrieval in non-obstructive azoospermic patients

Identification of molecular markers which can predict the outcome of sperm retrieval non-invasively in patients with non-obstructive azoospermia [NOA] are valuable in clinical andrology. Jumonji domain-containing 1a [JMJD1A] is a significant epigenetic regulator during spermatogenesis, which plays an important role in the differentiation of post-meiotic germ cells into mature spermatozoa. We therefore aimed to examine the potential association between JMJD1A expression and the outcome of sperm retrieval in patients with NOA. Testicular biopsy specimens from 50 NOA patients with either successful sperm retrieval [sperm+, n=22] or failed sperm retrieval [sperm-, n=28] were collected and then examined for JMJD1A expression by reverse transcription-quantitative polymerase chain reaction [RT-qPCR]. In addition, conventional clinical parameters including luteinizing hormone, follicle-stimulating hormone, testosterone, age, and testicular volume were compared between the two NOA groups. The expression of JMJD1A in the sperm+ group was significantly higher than in the sperm- group [P<0.001], however, no significant difference was observed between the two groups in clinical parameters. The receiver operating characteristic [ROC] curve of JMJD1A expression in predicting the sperm retrieval outcome showed a sensitivity of 90.91% and a specificity of 89.29% with significant discriminatory ability between the sperm+ and sperm- groups [area under the ROC curve [AUC]= 0.91]. This study demonstrates a significant association between the expression of JMJD1A and the success of sperm recovery in patients with NOA, and thus suggests that JMJD1A expression quantification in testicular biopsies may be a valuable biomarker along with conventional parameters in predicting the presence of spermatozoa

Epigenetic aberration of FMR1 gene in infertile women with diminished ovarian reserve

Objective: The diminished ovarian reserve [DOR] is a condition characterized by a reduction in the number and/or quality of oocytes. This primary infertility disorder is usually accompanied with an increase in the follicle-stimulating hormone [FSH] levels and regular menses. Although there are many factors contributing to the DOR situation, it is likely that many of idiopathic cases have genetic/epigenetic bases. The association between the FMR1 premutation [50-200 CGG repeats] and the premature ovarian failure [POF] suggests that epigenetic disorders of FMR1 can act as a risk factor for the DOR as well. The aim of this study was to analyze the mRNA expression and epigenetic alteration [histone acetylation/methylation] of the FMR1 gene in blood and granulosa cells of 20 infertile women

Materials and Methods: In this case-control study, we analyzed the mRNA expression and epigenetic altration of the FMR1 gene in blood and granulosa cells of 20 infertile women. These women were referred to the Royan Institute, having been clinically diagnosed as DOR patients. Our control group consisted of 20 women with normal antral follicle numbers and serum FSH level. All these women had normal karyotype and no history of genetic disorders. The number of CGG triplet repeats in the exon 1 of the FMR1 gene was analyzed in all samples

Results: Results clearly demonstrated significantly higher expression of the FMR1 gene in blood and granulosa cells of the DOR patients with the FMR1 premutation compared to the control group. In addition, epigenetic marks of histone 3 lysine 9 acetylation [H3K9ac] and di-metylation [H3K9me2] showed significantly higher incorporations in the regulatory regions of the FMR1 gene, including the promoter and the exon 1, whereas tri-metylation [H3K9me3] mark showed no significant difference between two groups

Conclusion: Our data demonstrates, for the first time, the dynamicity of gene expression and histone modification pattern in regulation of FMR1 gene, and implies the key role played by epigenetics in the development of the ovarian function

Altered expression of 3´paralogus HOX A-D clusters in endometriosis disease: a case-control study

Background: Endometriosis is a prevalent gynecological disease, with limited known etiology and more researches are required to identify its etiology. In this manner, there is no evidence for expression and function of 3and acute;HOX genes in 4 clusters in the limb and pelvic organs such as the uterus and its disorders [Genes in the HOXA-D clusters are subdivided into 13 paralogous groups]

Objective: This study designed to investigate the expression profile of 5 paralogous [1-5] in four clusters of HOX genes [A, B, C, and D] in ectopic and eutopic tissues of women with endometriosis compared to the normal endometrium

Materials and Methods: Samples were obtained from thirty patients [15 with and 15 without endometriosis] of reproductive age with normal menstrual cycles. The same patient provided both eutopic and ectopic tissues and control women were laparoscopically checked for the absence of endometriosis. The expression profile of these HOX genes was investigated by quantitative real-time polymerase chain reaction technique

Results: We observed significant up-regulation of some members of HOXC and D clusters [HOXD1, HOXD3, HOXC4 and HOXC5] in ectopic and eutopic tissues vs. control. Also, our data showed significant down-regulation of all of HOXA and HOXB paralogous except HOXA1 in ectopic tissues versus control

Conclusion: Our data showed specific cluster dependent modulation of the HOX genes expression in endometriosis [over-expression of some HOX genes in cluster C and D and down-regulation of HOX genes in cluster A and B] in ectopic and eutopic tissues compare to control group. Therefore, it is possible that change of expression level of these genes in endometrium plays a role in the pathogenesis of endometriosis

review on various uses of N-acetyl cysteine

N-acetyl cysteine [NAC], as a nutritional supplement, is a greatly applied antioxidant in vivo and in vitro. NAC is a precursor of L-cysteine that results in glutathione elevation biosynthesis. It acts directly as a scavenger of free radicals, especially oxygen radicals. NAC is a powerful antioxidant. It is also recommended as a potential treatment option for different disorders resulted from generation of free oxygen radicals. Additionally, it is a protected and endured mucolytic drug that mellows tenacious mucous discharges. It has been used for treatment of various diseases in a direct action or in a combination with some other medications. This paper presents a review on various applications of NAC in treatment of several diseases

Expression level of chromodomain Y [CDY]: potential marker for prediction of sperm recovery in non-obstructive azoospermia

Background: The availability of testis specific genes will be of help in choosing the most promising biomarkers for the detection of testicular sperm retrieval in patients with non-obstructive azoospermia [NOA]. Testis specific chromodomain protein Y 1 [CDY1] is a histone acetyltransferase which concentrates in the round spermatid nucleus, where histone hyperacetylation occurs and causes the replacement of histones by the sperm-specific DNA packaging proteins, TNPs and PRMs

Objective: The aim was to evaluate CDY1 gene as a marker for predicting of successful sperm retrieval in NOA patients

Materials and Methods: This research was conducted on 29 patients with NOA who had undergone testicular sperm extraction [TESE] procedure. NOA patients were subdivided into patients with successful sperm retrieval [NOA+, n=12] and patients with unsuccessful sperm retrieval [NOA-, n=17]. Relative expression of CDY1 gene and chromatin incorporation of CDY1 protein were measured by quantitative real-time polymerase chain reaction [qRT-PCR] and ELISA assay, respectively

Results: Quantification of mRNA relative expression and incorporation of CDY1 protein in chromatin showed significant lower expressions and protein levels of CDY1 in testis tissues of NOA- in comparison to NOA+ group

Conclusion: The findings in this study demonstrated a correlation between the low levels of CDY1 function and unsuccessful sperm recovery in the testicular tissues of NOA- compared to NOA+ patients. Therefore, it can be reasonable to consider CDY1 as a potential biomarker for predicting the presence of spermatozoa, although the claim needs more samples to be confirmed

Differential incorporation of beta-actin as a component of RNA polymerase II into regulatory regions of stemness/differentiation genes in retinoic acid-induced differentiated human embryonic carcinoma cells

Objective: Nuclear actin is involved in transcription regulation by recruitment of histone modifiers and chromatin remodelers to the regulatory regions of active genes. In recent years, further attention has been focused on the role of actin as a nuclear protein in transcriptional processes. In the current study, the epigenetic role of nuclear actin on transcription regulation of two stemness [OCT4 and NANOG] and two differentiation] NESTIN and PAX6] marker genes was evaluated in a human embryonal carcinoma cell line [NT2] before and after differentiation induction

Materials and Methods: In this experimental study, differentiation of embryonal cells was induced by retinoic acid [RA], and quantitative real-time polymerase chain reaction [PCR] was used to evaluate differential expression of marker genes before and 3 days after RA- induced differentiation. Chromatin immunoprecipitation [ChIP] coupled with real-time PCR was then undertaken to monitor the incorporation of beta-actin, as a functional component of RNA polymerase II, in the regulatory regions of marker genes

Results: Data showed significant change in nuclear actin incorporation into the promoter regions of NESTIN and PAX6 after RA-induction

Conclusion: We emphasize the dynamic functional role of nuclear actin in differentiation of embryonal cells and its role as a subunit of RNA polymerase II

potential roles of actin in the nucleus

Over the past few decades, actin's presence in the nucleus has been demonstrated. Actin is a key protein necessary for different nuclear processes. Although actin is well known for its functional role in dynamic behavior of the cytoskeleton, emerging studies are now highlighting new roles for actin. At the present time there is no doubt about the presence of actin in the nucleus. A number of studies have uncovered the functional involvement of actin in nuclear processes. Actin as one of the nuclear components has its own structured and functional rules, such as nuclear matrix association, chromatin remodeling, transcription by RNA polymerases I, II, III and mRNA processing. In this historical review, we attempt to provide an overview of our current understanding of the functions of actin in the nucleus

New single nucleotide polymorphism G5508A in the SEPT12 gene may be associated with idiopathic male infertility in Iranian men

Male infertility is a multifactorial disorder, which affects approximately 10% of couples at childbearing age with substantial clinical and social impact. Genetic factors are associated with the susceptibility to spermatogenic impairment in humans. Recently, SEPT12 is reported as a critical gene for spermatogenesis. This gene encodes a testis specific member of Septin proteins, a family of polymerizing GTP-binding proteins. SEPT12 in association with other Septins is an essential annulus component in mature sperm. So, it is hypothesized that genetic alterations of SEPT12 may be concerned in male infertility. The objective of this research is exploration of new single nucleotide polymorphism G5508A in the SEPT12 gene association with idiopathic male infertility in Iranian men. In this case control study, 67 infertile men and 100 normal controls were analyzed for genetic alterations in the active site coding region of SEPT12, using polymerase chain reaction sequencing technique. Fisher exact test was used for statistical analysis and p<0.05 was considered as statistically significant. Genotype analysis indicated that G5508A polymorphic SEPT12 alleles were distributed in three peaks of frequency in both control and diseases groups. Categorization of the alleles into [GG], [GA], [AA] types revealed a significant difference between infertile patients [azoospermic and asthenospermic] and normal controls [p=0.005]. According to our finding we suggest that G5508A polymorphism in SEPT12 gene can affect spermatogenesis in men, the opinion needs more investigation in different populations

Comparative expression analysis of septin 14 in testes of infertile men with normal spermatogenesis and spermatogenic failure

Septins are an evolutionary conserved group of GTP-binding and filament-forming proteins that have diverse cellular roles. An increasing body of data implicates the septin family in the pathogenesis of diverse states including cancers, neurodegeneration, and male infertility. The objective of the study was to evaluate the expression pattern of Septin14 in testis tissue of men with and without spermatogenic failure. The samples retrieved accessible random between infertile men who underwent diagnostic testicular biopsy in Royan institute. 10 infertile men with obstructive azoospermia and normal spermatogenesis and 20 infertile men with non-obstructive azoospermia were recruited for real-time reverse transcription [RT]-PCR analysis of the testicular tissue. Total RNA was extracted with trizol reagent. Comparison of the mRNA level of septin14 revealed that in tissues with partial [n=10] or complete spermatogenesis [n=10], the expression of septin 14 was significantly higher than sertoli cell only tissues. The testicular tissues of men with hypospermatogenesis, maturation arrest and sertoli cell only had lower levels of septin 14 transcripts than normal men. These data indicates that Septin 14 expression level is critical for human spermatogenesis

Epigenetic regulation of osteogenic and chondrogenic differentiation of mesenchymal stem cells in culture

Management of mesenchymal stem cells [MSCs] capabilities to differentiate in to osteogenic and Chondrogenic lineages would be of utmost importance for their future use in difficult to treat cases of destroyed bone and cartilage. Thus, an understanding of the epigenetic mechanisms as important modulators of stem cell differentiation might be useful. Epigenetic mechanism refers to a process that regulates heritable and long-lasting alterations in gene expression without changing the DNA sequence. Such stable changes would be mediated by several mechanisms including DNA methylation and histone modifications. The involvement of epigenetic mechanisms during MSC bone and cartilage differentiation has been investigated during the past decade. The purpose of this review is to cover outstanding research works that have attempted to ascertain the underlying epigenetic changes of the nuclear genome during in vitro differentiation of MSCs into bone and cartilage cell lineages. Understanding such genomic alterations may assist scientists to develop and recognize reagents that are able to efficiently promote this cellular differentiation. Before summarizing the progress on epigenetic regulation of MSC bone and cartilage differentiation, a brief description will be given regarding in vitro conditions that favor MSC osteocytic and chondrocytic differentiation and the main mechanisms responsible for epigenetic regulation of differentiation

Enhancement of glycosaminoglycan-rich matrix production in human marrow-derived mesenchymal stem cell chondrogenic culture by lithium chloride and SB216763 treatment

Cartilage mass produced from mesenchymal stem cell [MSC] differentiation would be a suitable candidate for use in regenerative medicine. Since the proper function of cartilage tissue is largely dependent on matrix glycosaminoglycan [GAG] contents, the objective of this study was to investigate the enhancing effect of two GSK3 inhibitors on the GAG content of cartilage produced by human marrow MSCs in vitro chondrogenesis. MSCs that were used in this experimental study were derived from human marrow aspirates and confirmed using standard assays. Optimal concentrations of Lithium chloride and SB216763 were determined based on the yield of viable cell numbers in MSC cultures treated with varying concentrations of either Lithium chloride or SB216763.Passaged-3 MSCs were then centrifuged into small aggregates and provided with a chondrogenic medium supplemented with either lithium or SB216763 reagent at the optimal concentration determined in the previous experiment. Three weeks after, GAG contents of the culture were quantified and compared to each other and the control. According to our data, the cultures treated with 5 mM Lithium and 1 micro M SB216763 tended to have comparatively more viable cells; therefore these concentrations were used in the differentiation experiments. The addition of either SB216763 or lithium to chondrogenic cultures appeared to significantly enhance cartilage matrix production. In SB216763 and Lithium-treated cultures average GAG concentrations were 6.17 +/- 0.7 and 6.12 +/- 1.1 micro g/ml compared to 2.00 +/- 0.3 micro g/ml in the control [p<0.05]. Using SB216763 and Lithium as supplements in human marrow MSC chondrogenic culture can lead to the production of cartilage mass high in GAG content