Emma L. Marczylo

Smoking induces differential miRNA expression in human spermatozoa: A potential transgenerational epigenetic concern?

Recent work has suggested that environmental chemicals, including those contained in cigarette smoke, can have adverse effects on the exposed individuals as well as their future progeny. The mechanisms underlying transmission of environmentally induced phenotypes through the germ line are not well understood. However, a predominant process appears to be the establishment of permanent heritable epigenetic alterations, and a number of studies have implicated microRNAs in such processes. Here, we show that cigarette smoke induces specific differences in the spermatozoal microRNA content of human smokers compared with non-smokers, and that these altered microRNAs appear to predominantly mediate pathways vital for healthy sperm and normal embryo development, particularly cell death and apoptosis. microRNA-mediated perturbation of such pathways may explain how harmful phenotypes can be induced in the progeny of smokers.

Doxorubicin In Vivo Rapidly Alters Expression and Translation of Myocardial Electron Transport Chain Genes, Leads to ATP Loss and Caspase 3 Activation

Background Doxorubicin is one of the most effective anti-cancer drugs but its use is limited by cumulative cardiotoxicity that restricts lifetime dose. Redox damage is one of the most accepted mechanisms of toxicity, but not fully substantiated. Moreover doxorubicin is not an efficient redox cycling compound due to its low redox potential. Here we used genomic and chemical systems approaches in vivo to investigate the mechanisms of doxorubicin cardiotoxicity, and specifically test the hypothesis of redox cycling mediated cardiotoxicity. Methodology/Principal Findings Mice were treated with an acute dose of either doxorubicin (DOX) (15 mg/kg) or 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) (25 mg/kg). DMNQ is a more efficient redox cycling agent than DOX but unlike DOX has limited ability to inhibit gene transcription and DNA replication. This allowed specific testing of the redox hypothesis for cardiotoxicity. An acute dose was used to avoid pathophysiological effects in the genomic analysis. However similar data were obtained with a chronic model, but are not specifically presented. All data are deposited in the Gene Expression Omnibus (GEO). Pathway and biochemical analysis of cardiac global gene transcription and mRNA translation data derived at time points from 5 min after an acute exposure in vivo showed a pronounced effect on electron transport chain activity. This led to loss of ATP, increased AMPK expression, mitochondrial genome amplification and activation of caspase 3. No data gathered with either compound indicated general redox damage, though site specific redox damage in mitochondria cannot be entirely discounted. Conclusions/Significance These data indicate the major mechanism of doxorubicin cardiotoxicity is via damage or inhibition of the electron transport chain and not general redox stress. There is a rapid response at transcriptional and translational level of many of the genes coding for proteins of the electron transport chain complexes. Still though ATP loss occurs with activation caspase 3 and these events probably account for the heart damage.

Environmentally induced epigenetic toxicity: potential public health concerns

Abstract Throughout our lives, epigenetic processes shape our development and enable us to adapt to a constantly changing environment. Identifying and understanding environmentally induced epigenetic change(s) that may lead to adverse outcomes is vital for protecting public health. This review, therefore, examines the present understanding of epigenetic mechanisms involved in the mammalian life cycle, evaluates the current evidence for environmentally induced epigenetic toxicity in human cohorts and rodent models and highlights the research considerations and implications of this emerging knowledge for public health and regulatory toxicology. Many hundreds of studies have investigated such toxicity, yet relatively few have demonstrated a mechanistic association among specific environmental exposures, epigenetic changes and adverse health outcomes in human epidemiological cohorts and/or rodent models. While this small body of evidence is largely composed of exploratory in vivo high-dose range studies, it does set a precedent for the existence of environmentally induced epigenetic toxicity. Consequently, there is worldwide recognition of this phenomenon, and discussion on how to both guide further scientific research towards a greater mechanistic understanding of environmentally induced epigenetic toxicity in humans, and translate relevant research outcomes into appropriate regulatory policies for effective public health protection.

Anandamide modulates human sperm motility: implications for men with asthenozoospermia and oligoasthenoteratozoospermia

STUDY QUESTION What are the levels of anandamide (N-arachidonoylethanolamide, AEA) in human seminal plasma and how are these related to abnormal spermatozoa? SUMMARY ANSWER Seminal plasma AEA levels were lower in men with asthenozoospermia and oligoasthenoteratozoospermia compared with normozoospermic men. WHAT IS KNOWN ALREADY AEA, a bioactive lipid, synthesized from membrane phospholipids may signal through cannabinoid receptors (CB1 and CB2) to regulate human sperm functions and male reproduction by modulating sperm motility, capacitation and the acrosome reaction in vitro. Local AEA levels are regulated by the synthetic and degradative enzymes, NAPE-PLD and FAAH, respectively. How the deregulation of this endogenous signalling pathway affects human sperm function(s) is not clear. STUDY DESIGN, SIZE AND DURATION This was a cross-sectional study of 86 men presenting at an infertility clinic for semen analysis over a period of 2 years. PARTICIPANTS/MATERIALS, SETTING, METHODS AEA was quantified, by ultra-high performance liquid chromatography-tandem mass spectrometry, in seminal plasma from 86 volunteers. Using qRT-PCR, CB1, CB2, NAPE-PLD and FAAH transcript levels were determined in spermatozoa from men with normozoospermia, asthenozoospermia, oligoasthenoteratozoospermia and teratozoospermia. Normal spermatozoa were exposed in vitro to methanadamide (meth-AEA) to determine its effect on sperm motility, viability and mitochondrial activity. MAIN RESULTS AND THE ROLE OF CHANCE Seminal plasma AEA levels (mean ± SEM) were significantly lower in men with asthenozoospermia (0.080 ± 0.01 nM; P < 0.05) or oligoasthenoteratozoospermia (0.083 ± 0.01 nM; P < 0.05) compared with normozoospermic men (0.198 ± 0.03 nM). In addition, the levels of spermatozoal CB1 mRNA were significantly decreased in men with asthenozoospermia (P < 0.001) or oligoasthenoteratozoospermia (P < 0.001) compared with normozoospermic controls. Supra-physiological levels of meth-AEA decreased sperm motility and viability, probably through CB1-mediated inhibition of mitochondrial activity. LIMITATIONS, REASONS FOR CAUTION The inhibitory effect of meth-AEA was only shown in vitro and may not reflect what happens in vivo. WIDER IMPLICATIONS OF THE FINDINGS As the regulation of the endocannabinoid system appears to be necessary for the preservation of normal sperm function and male fertility, there may be implications for the adverse reproductive consequences of marijuana use. Exocannabinoids, such as Δ(9)-THC, are likely to compete with endocannabinoids at the cannabinoid receptors, upsetting the finely balanced endocannabinoid signalling system. The importance of the endocannabinoid system makes it an attractive target for pharmacological interventions to control male fertility. STUDY FUNDING/COMPETING INTEREST(S) This work was funded in part by miscellaneous educational funds from the University Hospitals of Leicester National Health Services Trust to support the Endocannabinoid Research Laboratory of University of Leicester. The authors declare no competing interests.

Ectopic Pregnancy Is Associated with High Anandamide Levels and Aberrant Expression of FAAH and CB1 in Fallopian Tubes

CONTEXT Ectopic pregnancy is associated with significant morbidity and mortality, but the molecular mechanisms underlying this condition remain unclear. Although the endocannabinoids, N-arachidonoylethanolamine (anandamide), N-oleoylethanolamine, and N-palmitoylethanolamine, are thought to play a negative role in ectopic pregnancy, their precise role(s) within the fallopian tube remains unclear. Anandamide activates cannabinoid receptors (CB1 and CB2) and, together with its degrading [e.g. fatty acid amide hydrolase (FAAH)] and synthesizing enzymes (e.g. N-acyl-phosphatidylethanolamine-specific phospholipase D), forms the endocannabinoid system. High anandamide levels are associated with tubal arrest of embryos in mice and may have a similar role in women. OBJECTIVE The aims were to quantify the levels of the endocannabinoids and evaluate the expression of the modulating enzymes and the cannabinoid receptors in fallopian tubes of women with ectopic pregnancy compared to those of nonpregnant women. DESIGN AND SETTING We conducted a prospective study at the University Hospitals of the Leicester National Health Service Trust. PARTICIPANTS AND METHODS Fallopian tubes collected from women with ectopic pregnancy and nonpregnant women with regular menstrual cycles were used for quantification of endocannabinoids by ultra-HPLC tandem mass spectrometry, were fixed in formalin for immunohistochemistry, and had RNA extracted for RT-quantitative PCR or protein extracted for immunoblotting. RESULTS Anandamide, but not N-oleoylethanolamine and N-palmitoylethanolamine, levels were significantly higher in ectopic fallopian tubes. Endocannabinoid levels from isthmus to ampulla were not significantly different. Cannabinoid receptors and endocannabinoid modulating enzymes were localized in fallopian tube epithelium by immunohistochemistry and showed reduced CB1 and FAAH expression in ectopic pregnancy. CONCLUSION High anandamide levels and reduced expression of CB1 and FAAH may play a role in ectopic implantation.

Assuring safety without animal testing: the case for the human testis in vitro

From 15 to 17 June 2011, a dedicated workshop was held on the subject of in vitro models for mammalian spermatogenesis and their applications in toxicological hazard and risk assessment. The workshop was sponsored by the Dutch ASAT initiative (Assuring Safety without Animal Testing), which aims at promoting innovative approaches toward toxicological hazard and risk assessment on the basis of human and in vitro data, and replacement of animal studies. Participants addressed the state of the art regarding human and animal evidence for compound mediated testicular toxicity, reviewed existing alternative assay models, and brainstormed about future approaches, specifically considering tissue engineering. The workshop recognized the specific complexity of testicular function exemplified by dedicated cell types with distinct functionalities, as well as different cell compartments in terms of microenvironment and extracellular matrix components. This complexity hampers quick results in the realm of alternative models. Nevertheless, progress has been achieved in recent years, and innovative approaches in tissue engineering may open new avenues for mimicking testicular function in vitro. Although feasible, significant investment is deemed essential to be able to bring new ideas into practice in the laboratory. For the advancement of in vitro testicular toxicity testing, one of the most sensitive end points in regulatory reproductive toxicity testing, such an investment is highly desirable.

Variation in Stability of Endogenous Reference Genes in Fallopian Tubes and Endometrium from Healthy and Ectopic Pregnant Women

RT-qPCR is commonly employed in gene expression studies in ectopic pregnancy. Most use RN18S1, β-actin or GAPDH as internal controls without validation of their suitability as reference genes. A systematic study of the suitability of endogenous reference genes for gene expression studies in ectopic pregnancy is lacking. The aims of this study were therefore to evaluate the stability of 12 reference genes and suggest those that are stable for use as internal control genes in fallopian tubes and endometrium from ectopic pregnancy and healthy non-pregnant controls. Analysis of the results showed that the genes consistently ranked in the top six by geNorm and NormFinder algorithms, were UBC, GAPDH, CYC1 and EIF4A2 (fallopian tubes) and UBC and ATP5B (endometrium). mRNA expression of NAPE-PLD as a test gene of interest varied between the groups depending on which of the 12 reference genes was used as internal controls. This study demonstrates that arbitrary selection of reference genes for normalisation in RT-qPCR studies in ectopic pregnancy without validation, risk producing inaccurate data and should therefore be discouraged.

The Effect of Mifepristone (RU486) on the Endocannabinoid System in Human Plasma and First-Trimester Trophoblast of Women Undergoing Termination of Pregnancy

INTRODUCTION High anandamide (AEA) concentrations are detrimental for implantation and early pregnancy. Progesterone, essential for pregnancy, may keep AEA levels low by increasing fatty acid amide hydrolase (FAAH) expression. Here the effect of RU486, a P4 antagonist used to initiate medical termination of pregnancy (MTOP), on plasma AEA concentrations and the endocannabinoid system (ECS) in trophoblasts was examined. OBJECTIVE Quantification of the endocannabinoid concentrations and expression of the ECS in trophoblast tissue of MTOP women and women undergoing surgical termination of pregnancy (STOP). DESIGN AND SETTING A prospective study at the University Hospitals of Leicester National Health Service Trust. PATIENTS AND METHODS AEA, N-oleoylethanolamine (OEA), and N-palmitolylethanolamine (PEA) concentrations in trophoblast tissues and blood samples from 68 MTOP and 15 STOP were analyzed by ultra-high-performance liquid chromatography-tandem mass spectrometry. ECS expression was determined by immunohistochemistry, quantitative RT-PCR, and Western blotting. RESULTS Concentrations of AEA, OEA, and PEA were significantly higher in MTOP than STOP trophoblasts (P = .0062, P = .016, and P = .0029, respectively), whereas no significant differences in plasma AEA, OEA, and PEA concentrations were observed even though plasma AEA and PEA concentrations were significantly (P = .005 and P = .025, respectively) increased the day after RU486 administration in women undergoing MTOP. Changes in the immunohistochemical densities of the AEA modifying enzymes N-acylphophatidylethanolamine-phospholipase D (NAPE-PLD) and FAAH, and the cannabinoid receptors (CB1 and CB2) were observed with increased NAPE-PLD, FAAH, and CB1 expression seen in the trophoblast of MTOP patients. CONCLUSIONS Trophoblast after MTOP demonstrated high AEA concentrations with increased expression of NAPE-PLD, FAAH, and CB1.

Impact of reference gene selection for type 2 cannabinoid receptor gene expression studies in human spermatozoa.

Quantitative real‐time polymerase chain reaction (qRT‐PCR) has been employed to study the gene expression profiles in human spermatozoa, but accurate analysis is dependent upon normalisation of data against an endogenous control. β‐Actin (ACTB) and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) are the most commonly used reference genes for normalisation of gene expression in human spermatozoa, but the expression of these genes in many tissues has considerable variation under different physiological, pathological and experimental conditions which limits their effectiveness in normalisation. The expression stability of a panel of 12 reference genes was studied in normal and pathological human spermatozoa using geNorm and NormFinder software. Although there were some discrepancies in the ranking of reference gene stability, each software program ranked B2M, ACTB, CYC1 and 18S RNA within the top 5 and recommended the combined use of at least two reference genes. Normalisation of qRT‐PCR data for the cannabinoid receptor type 2 in spermatozoa using the different housekeeping genes demonstrated how, without validation, conflicting results are obtained. We recommend that the arbitrary use of reference genes should be avoided and the validation of reference gene stability should be undertaken prior to every study. For normalisation of CB2 expression, we would recommend using the geometric mean of B2M and ACTB.

Differential Expression of the Orphan G-Protein Coupled Receptor Gpr55 in Human Spermatozoa

OBJECTIVE: This study assessed whether endocrine treatment increases the rate of obtaining sperm by ejaculation or surgical retrieval in patients with NOA. DESIGN: Prospective, non-randomized, controlled study. MATERIALS AND METHODS: 612 NOA patients were included. 116 patients chose immediate microsurgical sperm extraction (micro-TESE) and served as the control group. 496 remaining patients were administered clomiphene and were classified and treated based on their response to medication. Patients with an increase in follicle stimulating hormone (FSH) and testosterone continued clomiphene treatment and were the primary study group. For patients with increased FSH and diminishing or no increase in luteinizing hormone (LH) and testosterone, human chorionic gonadotropin (hCG) was added. For patients with no increase in testosterone, LH or FSH, or with decreasing testosterone, clomiphene was replaced by hCG and hMG. After 9 monthly semen analyses, micro-TESE was performed for those who remained azoospermic. Success rates were compared to the control group using the chi-squared test. RESULTS: With medical treatment, 10.9% of patients developed sperm in their semen. Treated patients, who remained azoospermic underwent micro-TESE with a 57% success rate. In total, sperm was made available for 61.7% of medically treated patients compared to 33.6% in the control group (P<0.001), and each treatment group showed a significantly increased rate of sperm retrieval compared to the control group (maximum P1⁄40.01). CONCLUSION: For NOA patients, a course of medical treatment may result in sperm in the ejaculate and increases the likelihood of successful micro-TESE for those remaining azoospermic.