Dicer1 Depletion in Male Germ Cells Leads to Infertility Due to Cumulative Meiotic and Spermiogenic Defects

Background: Spermatogenesis is a complex biological process that requires a highly specialized control of gene expression. In the past decade, small non-coding RNAs have emerged as critical regulators of gene expression both at the transcriptional and post-transcriptional level. DICER1, an RNAse III endonuclease, is essential for the biogenesis of several classes of small RNAs, including microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), but is also critical for the degradation of toxic transposable elements. In this study, we investigated to which extent DICER1 is required for germ cell development and the progress of spermatogenesis in mice.Principal Findings: We show that the selective ablation of Dicer1 at the early onset of male germ cell development leads to infertility, due to multiple cumulative defects at the meiotic and post-meiotic stages culminating with the absence of functional spermatozoa. Alterations were observed in the first spermatogenic wave and include delayed progression of spermatocytes to prophase I and increased apoptosis, resulting in a reduced number of round spermatids. The transition from round to mature spermatozoa was also severely affected, since the few spermatozoa formed in mutant animals were immobile and misshapen, exhibiting morphological defects of the head and flagellum. We also found evidence that the expression of transposable elements of the SINE family is up-regulated in Dicer1-depleted spermatocytes.Conclusions/Significance: Our findings indicate that DICER1 is dispensable for spermatogonial stem cell renewal and mitotic proliferation, but is required for germ cell differentiation through the meiotic and haploid phases of spermatogenesis

Tbx6 Regulates Left/Right Patterning in Mouse Embryos through Effects on Nodal Cilia and Perinodal Signaling

Background: The determination of left/right body axis during early embryogenesis sets up a developmental cascade that coordinates the development of the viscera and is essential to the correct placement and alignment of organ systems and vasculature. Defective left-right patterning can lead to congenital cardiac malformations, vascular anomalies and other serious health problems. Here we describe a novel role for the T-box transcription factor gene Tbx6 in left/right body axis determination in the mouse. Results: Embryos lacking Tbx6 show randomized embryo turning and heart looping. Our results point to multiple mechanisms for this effect. First, Dll1, a direct target of Tbx6, is down regulated around the node in Tbx6 mutants and there is a subsequent decrease in nodal signaling, which is required for laterality determination. Secondly, in spite of a lack of expression of Tbx6 in the node, we document a profound effect of the Tbx6 mutation on the morphology and motility of nodal cilia. This results in the loss of asymmetric calcium signaling at the periphery of the node, suggesting that unidirectional nodal flow is disrupted. To carry out these studies, we devised a novel method for direct labeling and live imaging cilia in vivo using a genetically-encoded fluorescent protein fusion that labels tubulin, combined with laser point scanning confocal microscopy for direct visualization of cilia movement. Conclusions: We conclude that the transcription factor gene Tbx6 is essential for correct left/right axis determination in th

The Adoption of Mediterranean Diet Attenuates the Development of Acute Coronary Syndromes in People with the Metabolic Syndrome

OBJECTIVES: In this work we investigated the effect of the consumption of the Mediterranean diet on coronary risk, in subjects with the metabolic syndrome. METHODS: During 2000–2002, we randomly selected, from all Greek regions, 848 hospitalised patients (695 males, 58 ± 10 & 153 females, 65 ± 9 years old) with a first event of acute coronary syndrome and 1078 frequency matched, by sex, age, region controls, without any suspicious for cardiovascular disease. Nutritional habits were evaluated through a validated questionnaire, while the metabolic syndrome was defined according to the NCEP ATP III criteria. Mediterranean diet was defined according to the guidelines of the Division of Nutrition/Epidemiology, of Athens Medical School. RESULTS: Of the 1926 participants, 307 (36.2%) of the patients and 198 (18.4%) of the controls (P < 0.001) met the ATP III criteria. This was related with 2fold adjusted coronary risk (odds ratio = 2.35, 95% 1.87 – 2.84) in subjects with the metabolic syndrome as compared with the rest of them. No differences were observed concerning the prevalence of the metabolic syndrome and sex of subjects, after adjustment for group of study (P > 0.1). Eighty (26%) of the patients and 70 (35%) of the controls (P < 0.01) with the metabolic syndrome were "closer" to the Mediterranean diet. Multivariate analysis revealed that the adoption of this diet is associated with a 35% (odds ratio = 0.65, 95% 0.44 – 0.95) reduction of the coronary risk in subjects with the metabolic syndrome, after adjusting for age, sex, educational and financial level and the conventional cardiovascular risk factors. CONCLUSION: Consequently, the adoption of Mediterranean diet seems to attenuate the coronary risk in subjects with the metabolic syndrome

Bone mineral density measurement and osteoporosis treatment after a fragility fracture in older adults: regional variation and determinants of use in Quebec

BACKGROUND: Osteoporosis (OP) is a skeletal disorder characterized by reduced bone strength and predisposition to increased risk of fracture, with consequent increased risk of morbidity and mortality. It is therefore an important public health problem. International and Canadian associations have issued clinical guidelines for the diagnosis and treatment of OP. In this study, we identified potential predictors of bone mineral density (BMD) testing and OP treatment, which include place of residence. METHODS: Our study was a retrospective population-based cohort study using data from the Quebec Health Insurance Board. The studied population consisted of all individuals 65 years and older for whom a physician claimed a consultation for a low velocity vertebral, hip, wrist, or humerus fracture in 1999 and 2000. Individuals were considered to have undergone BMD testing if there was a claim for such a procedure within two years following a fracture. They were considered to have received an OP treatment if there was at least one claim to Quebec's health insurance plan (RAMQ) for OP treatment within one year following a fracture. We performed descriptive analyses and logistic regressions by gender. Predictors included age, site of fracture, social status, comorbidity index, prior BMD testing, prior OP treatment, long-term glucocorticoid use, and physical distance to BMD device. RESULTS: The cohort, 77% of which was female, consisted of 25,852 individuals with fragility fractures. BMD testing and OP treatment rates were low and gender dependent (BMD: men 4.6%; women 13.1%; OP treatment: men 9.9%; women 29.7%). There was an obvious regional variation, particularly in BMD testing, ranging from 0 to 16%. Logistic regressions demonstrate that individuals living in long term care facilities received less BMD testing. Patients who had suffered from vertebral fractures, or who had received prior OP treatment or BMD testing, regardless of gender, subsequently received more BMD testing and OP treatments. Furthermore, increasing the distance between a patient's residence and BMD facility precluded likelihood of BMD testing. CONCLUSION: BMD testing rate was extremely low but not completely explained by reduced physical access; gender, age, social status, prior BMD testing and OP treatment were all important predictors for future BMD testing and OP treatment

Pedestrian Injury and Human Behaviour: Observing Road-Rule Violations at High-Incident Intersections

Background Human behaviour is an obvious, yet under-studied factor in pedestrian injury. Behavioural interventions that address rule violations by pedestrians and motorists could potentially reduce the frequency of pedestrian injury. In this study, a method was developed to examine road-rule non-compliance by pedestrians and motorists. The purpose of the study was to examine the potential association between violations made by pedestrians and motorists at signalized intersections, and collisions between pedestrians and motor-vehicles. The underlying hypothesis is that high-incident pedestrian intersections are likely to vary with respect to their aetiology, and thus are likely to require individualized interventions – based on the type and rate of pedestrian and motorist violation. Methods High-incident pedestrian injury intersections in Vancouver, Canada were identified using geographic information systems. Road-rule violations by pedestrians and motorists were documented at each incident hotspot by a team of observers at several different time periods during the day. Results Approximately 9,000 pedestrians and 18,000 vehicles were observed in total. In total for all observed intersections, over 2000 (21%) pedestrians committed one of the observed pedestrian road-crossing violations, while approximately 1000 (5.9%) drivers committed one of the observed motorist violations. Great variability in road-rule violations was observed between intersections, and also within intersections at different observation periods. Conclusions Both motorists and pedestrians were frequently observed committing road-rule violations at signalized intersections, suggesting a potential human behavioural contribution to pedestrian injury at the study sites. These results suggest that each intersection may have unique mechanisms that contribute to pedestrian injury, and may require targeted behavioural interventions. The method described in this study provides the basis for understanding the relationship between violations and pedestrian injury risk at urban intersections. Findings could be applied to targeted prevention campaigns designed to reduce the number of pedestrian injuries at signalized intersections

A Unique Combination of Male Germ Cell miRNAs Coordinates Gonocyte Differentiation

The last 100 years have seen a concerning decline in male reproductive health associated with decreased sperm production, sperm function and male fertility. Concomitantly, the incidence of defects in reproductive development, such as undescended testes, hypospadias and testicular cancer has increased. Indeed testicular cancer is now recognised as the most common malignancy in young men. Such cancers develop from the pre-invasive lesion Carcinoma in Situ (CIS), a dysfunctional precursor germ cell or gonocyte which has failed to successfully differentiate into a spermatogonium. It is therefore essential to understand the cellular transition from gonocytes to spermatogonia, in order to gain a better understanding of the aetiology of testicular germ cell tumours. MicroRNA (miRNA) are important regulators of gene expression in differentiation and development and thus highly likely to play a role in the differentiation of gonocytes. In this study we have examined the miRNA profiles of highly enriched populations of gonocytes and spermatogonia, using microarray technology. We identified seven differentially expressed miRNAs between gonocytes and spermatogonia (down-regulated: miR-293, 291a-5p, 290-5p and 294*, up-regulated: miR-136, 743a and 463*). Target prediction software identified many potential targets of several differentially expressed miRNA implicated in germ cell development, including members of the PTEN, and Wnt signalling pathways. These targets converge on the key downstream cell cycle regulator Cyclin D1, indicating that a unique combination of male germ cell miRNAs coordinate the differentiation and maintenance of pluripotency in germ cells

Dose-Dependent Onset of Regenerative Program in Neutron Irradiated Mouse Skin

Background: Tissue response to irradiation is not easily recapitulated by cell culture studies. The objective of this investigation was to characterize, the transcriptional response and the onset of regenerative processes in mouse skin irradiated with different doses of fast neutrons. Methodology/Principal Findings: To monitor general response to irradiation and individual animal to animal variation, we performed gene and protein expression analysis with both pooled and individual mouse samples. A high-throughput gene expression analysis, by DNA oligonucleotide microarray was done with three months old C57Bl/6 mice irradiated with 0.2 and 1 Gy of mono-energetic 14 MeV neutron compared to sham irradiated controls. The results on 440 irradiation modulated genes, partially validated by quantitative real time RT-PCR, showed a dose-dependent up-regulation of a subclass of keratin and keratin associated proteins, and members of the S100 family of Ca2+-binding proteins. Immunohistochemistry confirmed mRNA expression data enabled mapping of protein expression. Interestingly, proteins up-regulated in thickening epidermis: keratin 6 and S100A8 showed the most significant up-regulation and the least mouse-to-mouse variation following 0.2 Gy irradiation, in a concerted effort toward skin tissue regeneration. Conversely, mice irradiated at 1 Gy showed most evidence of apoptosis (Caspase-3 and TUNEL staining) and most 8-oxo-G accumulation at 24 h post-irradiation. Moreover, no cell proliferation accompanied 1 Gy exposure as shown by Ki67 immunohistochemistry. Conclusions/Significance: The dose-dependent differential gene expression at the tissue level following in vivo exposure to neutron radiation is reminiscent of the onset of re-epithelialization and wound healing and depends on the proportion of cells carrying multiple chromosomal lesions in the entire tissue. Thus, this study presents in vivo evidence of a skin regenerative program exerted independently from DNA repair-associated pathways

Germ Cell-Specific Targeting of DICER or DGCR8 Reveals a Novel Role for Endo-siRNAs in the Progression of Mammalian Spermatogenesis and Male Fertility

Small non-coding RNAs act as critical regulators of gene expression and are essential for male germ cell development and spermatogenesis. Previously, we showed that germ cell-specific inactivation of Dicer1, an endonuclease essential for the biogenesis of micro-RNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), led to complete male infertility due to alterations in meiotic progression, increased spermatocyte apoptosis and defects in the maturation of spermatozoa. To dissect the distinct physiological roles of miRNAs and endo-siRNAs in spermatogenesis, we compared the testicular phenotype of mice with Dicer1 or Dgcr8 depletion in male germ cells. Dgcr8 mutant mice, which have a defective miRNA pathway while retaining an intact endo-siRNA pathway, were also infertile and displayed similar defects, although less severe, to Dicer1 mutant mice. These included cumulative defects in meiotic and haploid phases of spermatogenesis, resulting in oligo-, terato-, and azoospermia. In addition, we found by RNA sequencing of purified spermatocytes that inactivation of Dicer1 and the resulting absence of miRNAs affected the fine tuning of protein-coding gene expression by increasing low level gene expression. Overall, these results emphasize the essential role of miRNAs in the progression of spermatogenesis, but also indicate a role for endo-siRNAs in this process