Relationship between potential sperm factors involved in oocyte activation and sperm DNA fragmentation with intra-cytoplasmic sperm injection clinical outcomes

Objective: The present study aimed to simultaneously evaluate the association between expression of three potential factors [post-acrosomal sheath WW domain-binding protein [PAWP], phospholipase Czeta [PLCzeta], and truncated form of the kit receptor [TR-KIT]] as candidates of oocyte activation with fertilization rate and early embryonic development

Materials and Methods: In this experimental study, semen samples were collected from 35 intra-cytoplasmic sperm injection [ICSI] candidates and analyzed according to World Health Organization criteria [2010]. Each sample was divided into two parts. The first part was processed for insemination by density-gradient centrifugation [DGC] and the second part was prepared for assessment of sperm morphology [Papanicolaou staining], DNA fragmentation [transferase dUTP nick end labeling [TUNEL]], and three Sperm-borne oocyte-activating factor [s] [SOAFs]-PLCzeta, PAWP, and TR-KIT

Results: Significant positive correlations existed between the percentages of PLCzeta, PAWP, and TR-KIT with fertilization rate. In addition, significant negative correlations existed between the percentage of DNA fragmentation with the percentages of PLCzeta and PAWP. We did not find a relationship between percentages of PLCzeta, PAWP, and TR-KIT with embryo quality and pregnancy rate [P>0.05]. There was a significant negative correlation between percentage of DNA fragmentation with fertilization and embryo quality

Conclusion: Oocyte activation was associated with the studied sperm factors [PAWP, PLCzeta, and TR-KIT]. These factors might hold the potential to be considered as diagnostic factors in the assessment of semen samples to evaluate their potential to induce oocyte activation. In addition, we observed a significant association between DNA fragmentation with fertilization, as well as embryo quality and expression of PAWP and PLCzeta, which indicated that men with high degrees of DNA fragmentation might require artificial oocyte activation. Whether such action should take place, and its cost and benefits should be evaluated in the future

Overexpression of annexin A1 suppresses pro-inflammatory factors in PC12 cells induced by 1-methyl-4-phenylpyridinium

Objective: Annexin A1 [ANXA1] is suggested to have anti-inflammatory function. However, the precise function of ANXA1 has remained unclear. In this study, we therefore examined the potency of ANXA1 in regulating reactive oxygen species [ROS] production and suppressing pro-inflammatory responses in PC12 cells induced by 1-methyl-4-phenylpyridinium [MPP+]

Materials and Methods: In this experimental study, cDNA of ANXA1 was cloned and inserted to the PGL268 pEpi-FGM18F vector to produce a recombinant PGL/ANXA1 vector for transfection into the PC12 cells. ANXA1 transfected cells were then treated with MPP+. Apoptosis and the content of pro-inflammatory factors including ROS, Interlukin-6 [IL-6], inducible nitric oxide synthase [iNOS] and nuclear factor-kappa B [NF-kappaB] were assessed by flow-cytometry, real-time quantitative polymerase chain reaction [RT-qPCR] and western blot in ANXA1-transfected cells and the data were compared with those obtained from mock and control cells

Results: Data revealed that overexpression of ANXA1 is associated with decreased levels of ROS and expression level of IL-6 and iNOS transcripts, and NF-kappaB protein in MPP+ treated PC12 cells

Conclusion: ANXA1 may be considered as an agent for prevention of neurodegenerative or inflammatory conditions

Stem cells from human exfoliated deciduous tooth exhibit stromal-derived inducing activity and lead to generation of neural crest cells from human embryonic stem cells

The neural crest is a transient structure of early vertebrate embryos that generates neural crest cells [NCCs]. These cells can migrate throughout the body and produce a diverse array of mature tissue types. Due to the ethical and technical problems surrounding the isolation of these early human embryo cells, researchers have focused on in vitro studies to produce NCCs and increase their knowledge of neural crest development. In this experimental study, we cultured human embryonic stem cells [hESCs] on stromal stem cells from human exfoliated deciduous teeth [SHED] for a two-week period. We used different approaches to characterize these differentiated cells as neural precursor cells [NPCs] and NCCs. In the first co-culture week, hESCs appeared as crater-like structures with marginal rosettes. NPCs derived from these structures expressed the early neural crest marker p75 in addition to numerous other genes associated with neural crest induction such as SNAIL, SLUG, PTX3 and SOX9. Flow cytometry analysis showed 70% of the cells were AP2/P75 positive. Moreover, the cells were able to self-renew, sustain multipotent differentiation potential, and readily form neurospheres in suspension culture. SHED, as an adult stem cell with a neural crest origin, has stromal-derived inducing activity [SDIA] and can be used as an NCC inducer from hESCs. These cells provide an invaluable resource to study neural crest differentiation in both normal and disordered human neural crest development

CD107a expression and IFN-gamma production as markers for evaluation of cytotoxic CD3+ CD8+ T cell response to CMV antigen in women with recurrent spontaneous abortion

Some evidence has shown a relationship between primary human cytomegalovirus [CMV] infection and pregnancy loss. The impact of CMV infection reactivation during pregnancy on adverse pregnancy outcomes is not completely understood. It is proposed that altered immune response, and therefore, recurrence or reactivation of latent CMV infection may relate to recurrent spontaneous abortion [RSA]; however, few data are available in this regard. To find out about any cell mediated defect and reactivation of latent CMV infection in women with RPL, cellular immunity to the virus has been evaluated by specific cytotoxic T lymphocyte [CTL] response to CMV. In a case control study, CTL CD107a expression and intercellular IFN-gamma production in response to CMV pp65 antigen and staphylococcus enterotoxin B [SEB] in women with RSA were assessed by flow cytometric analysis. Forty-four cases with history of recurrent pregnancy and forty-four controls with history of successful pregnancies were included. The FACSCaliber flow cytometer were used for analysis. No significant difference was observed between CD107a expression and IFN-gamma production in response to CMV PP65 antigen in RPL patients and control group. However, the cytotoxic response to SEB antigen in patients with RPL was significantly lower than control group [p=0.042]. The results of this study show that impaired CD107a expression and IFN-gamma production as CTL response to CMV does not appear to be a major contributing and immune incompetence factor in patients with RPL, but cytotoxic T cell response defect to other antigens requires to be assessed further in these patients

Constructing a mouse Oct4 promoter/EGFP vector, as a whole-cellular reporter to monitor the pluripotent state of cells

The transcription factor Oct-4, is an important marker of undifferentiating level and a key regulating factor for maintenance of pluripotency in cells. Establishment of an Oct-4 promoter-based reporter ystem is an appropriate tool for monitoring the differentiation of embryonic stem cells both in vivo and in vitro. In the present study, we report construction of a recombinant vector, pDB2 Oct4 promoter/EGFP, in which expression of Enhanced Green Fluorescent Protein [EGFP] was controlled by the mouse Oct-4 promoter. In transfected mouse embryonic stem cells with this vector, EGFP was predicted to be specifically expressed in pluripotency state. After transfection, high-level expression of EGFP under the control of Oct-4 promoter was observed in manipulated embryonic stem cells. Thus, our new cellular reporter showed that both the properties of embryonic cells and expression the EGFP could be of great help in studying the differentiating and reprogramming mechanisms of mESCs

Characterization and functional assessment of mouse PPAR[gamma]1 promoter

Peroxisome Proliferator Activated Receptor gamma [PPAR[gamma]], 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 PPAR[gamma] gene. A 3 kb fragment, containing several transcription factor binding sites, acts as PPAR[gamma]1 promoter region. Thus, expression pattern of PPAR[gamma]1 isoform is due to the potential transcription factors that could influence its promoter activity. PPAR[gamma], Retinoid X Receptor [RXR] and Vitamin D Receptor [VDR], as nuclear receptors could influence PPAR[gamma] gene expression pattern during several differentiation processes. During neural differentiation, PPAR[gamma]1 isoform expression reaches to maximal level at neural precursor cell formation. A vast computational analysis was carried out to reveal the PPAR[gamma]1 promoter region. The putative promoter region was then subcloned upstream of an EGFP reporter gene. Then the functionality of PPAR[gamma]1 promoter was assessed in different cell lines. Results indicated that Rosiglitazone increased PPAR[gamma]1 promoter regulated EGFP expression of neural precursor cells during Embryoid Body [EB] formation. Furthermore vitamin D reduced PPAR[gamma]1 promoter regulated EGFP expression of neural precursor cells during EB formation through binding to its receptor. This study suggests that there are potential response elements for PPAR/RXR and VDR/RXR heterodimers in PPAR[gamma]1 isoform promoter. Also VDR/RXR heterodimers may decrease PPAR[gamma] expression through binding to its promoter