Regulation of Ovarian Function by the Germ Cell Specific DAZL Gene

The RNA binding protein DAZL (Deleted in Azoospermia) is essential for germ cell survival and subsequent fertility. The transgenic mouse DAZL model has confirmed that knockout (KO) females are infertile as a direct consequence of complete postnatal oocyte ablation. Interestingly, the heterozygous (Het) DAZL females have increased fertility giving rise to significantly more viable offspring, accompanied by significantly reduced plasma FSH and increased inhibin B compared to levels observed in the wildtype (Wt) females. Recent studies to identify putative DAZL mRNA targets suggest that DAZL may have multiple functions and mRNA targets throughout germ cell development. However, how this protein functions within the oocyte and how functional copy number gives rise to increased fertility remains to be fully elucidated.The studies in this thesis sought to identify putative DAZL mRNA targets in addition to molecular mechanisms which may be either affected direct or indirectly as a result of the functional copy number of DAZL (Wt or Het) within the oocyte or follicular unit. Oocytes from Wt and Het were evaluated for their expression of selected oocyte genes and comparative analysis suggests that oocyte gene expression is significantly altered between the genotypes. Genes of interest include Oospl and Hlfoo, both of which are down-regulated in mRNA expression in Het d21 oocytes and dlO ovaries compared to the Wt. Furthermore, an in silico bioinformatics approach was utilised to identify putative DAZL mRNA targets using a consensus DAZL binding sequence. One candidate target, PDCD4, previously identified as a tumour suppressor gene was selected for further investigation. Despite PDCD4 mRNA and protein being highly expressed within the ovary, no difference in mRNA levels between Het and Wt was observed. However, although not ruling out the possibility of being a DAZL target we now have evidence that PDCD4 can function within the steroidogenic cells of the corpus luteum in relation to functional luteolysis. Abstract Indirect actions of DAZL upon local regulation and response of follicle growth in culture were evaluated to investigate follicles at the gonadotrophin dependent stage of growth. Individual follicles from Wt and Het d21 mice were cultured in the presence of FSH at liu, 0.5iu, O.liu and O.Oliu for a six day period. Final follicle size/morphology did not differ between genotypes at liu, 0.5iu and O.liu of FSH, but by d3 at O.Oliu FSH growth o f Wt follicles was significantly (PO.OOl) perturbed compared to the Het. Despite no difference in final size between liu, 0.5iu, O.liu FSH treatments, mRNA analysis of individual follicles demonstrated a significant up-regulation of FSH receptor (P<0.05), aromatase (P<0.05) and inhibin PA (P<0.01) and a significant down-regulation in inhibin PB (P<0.01) expression in the Het follicles compared to the Wt, suggesting an increase in follicle maturity, sensitivity and hence suitability for selection as viable pre-ovulatory follicles. Furthermore, atresia rates from cultured follicles were significantly lower (P<0.05 (liu, O.liu FSH); P<0.01(0,01iu FSH)) in the Het compared to the Wt.These studies provide strong evidence that multiple mechanisms within the oocyte/follicle are directly and indirectly affected as a result of functional copy number of DAZL. Although direct in vivo targets remain to be identified it is clear that DAZL protein potentially targets multiple mRNAs at different stages of development, pre-programming the oocyte to increase the sensitivity of follicle and/or the functioning within a transcription complex regulating development. In conclusion, the beneficial consequences of increased fertility in the Het females is accompanied by a possible acceleration in oocyte and follicle maturation, an increased sensitivity to FSH in vitro with evidence of advanced stages of growth and, a reduction in follicle atresia. These differences support the suggestion that DAZL is having systemic effects on the paracrine communication within the follicle unit between the oocyte and somatic cells altering regulation and subsequent selection, and affecting final ovulation rate and litter size

Regulation of ovarian function by the germ cell specific DAZL gene

The RNA binding protein DAZL (Deleted in Azoospermia) is essential for germ cell survival and subsequent fertility. The transgenic mouse DAZL model has confirmed that knockout (KO) females are infertile as a direct consequence of complete postnatal oocyte ablation. Interestingly, the heterozygous (Het) DAZL females have increased fertility giving rise to significantly more viable offspring, accompanied by significantly reduced plasma FSH and increased inhibin B compared to levels observed in the wildtype (Wt) females. Recent studies to identify putative DAZL mRNA targets suggest that DAZL may have multiple functions and mRNA targets throughout germ cell development. However, how this protein functions within the oocyte and how functional copy number gives rise to increased fertility remains to be fully elucidated. The studies in this thesis sought to identify putative DAZL mRNA targets in addition to molecular mechanisms which may be either affected direct or indirectly as a result of the functional copy number of DAZL (Wt or Het) within the oocyte or follicular unit. Oocytes from Wt and Het were evaluated for their expression of selected oocyte genes and comparative analysis suggests that oocyte gene expression is significantly altered between the genotypes. Genes of interest include Oosp1 and H1foo, both of which are down-regulated in mRNA expression in Het d21 oocytes and d10 ovaries compared to the Wt. Furthermore, an in silico bioinformatics approach was utilised to identify putative DAZL mRNA targets using a consensus DAZL binding sequence. One candidate target, PDCD4, previously identified as a tumour suppressor gene was selected for further investigation. Despite PDCD4 mRNA and protein being highly expressed within the ovary, no difference in mRNA levels between Het and Wt was observed. However, although not ruling out the possibility of being a DAZL target we now have evidence that PDCD4 can function within the steroidogenic cells of the corpus luteum in relation to functional luteolysis. Indirect actions of DAZL upon local regulation and response of follicle growth in culture were evaluated to investigate follicles at the gonadotrophin dependent stage of growth. Individual follicles from Wt and Het d21 mice were cultured in the presence of FSH at 1iu, 0.5iu, 0.1iu and 0.01iu for a six day period. Final follicle size/morphology did not differ between genotypes at 1iu, 0.5iu and 0.1iu of FSH, but by d3 at 0.01iu FSH growth of Wt follicles was significantly (P<0.001) perturbed compared to the Het. Despite no difference in final size between 1iu, 0.5iu, 0.1iu FSH treatments, mRNA analysis of individual follicles demonstrated a significant up-regulation of FSH receptor (P<0.05), aromatase (P<0.05) and inhibin βA (P<0.01) and a significant down-regulation in inhibin βB (P<0.01) expression in the Het follicles compared to the Wt, suggesting an increase in follicle maturity, sensitivity and hence suitability for selection as viable pre-ovulatory follicles. Furthermore, atresia rates from cultured follicles were significantly lower (P<0.05 (1iu, 0.1iu FSH); P<0.01(0.01iu FSH)) in the Het compared to the Wt. These studies provide strong evidence that multiple mechanisms within the oocyte/follicle are directly and indirectly affected as a result of functional copy number of DAZL. Although direct in vivo targets remain to be identified it is clear that DAZL protein potentially targets multiple mRNAs at different stages of development, pre-programming the oocyte to increase the sensitivity of follicle and/or the functioning within a transcription complex regulating development. In conclusion, the beneficial consequences of increased fertility in the Het females is accompanied by a possible acceleration in oocyte and follicle maturation, an increased sensitivity to FSH in vitro with evidence of advanced stages of growth and, a reduction in follicle atresia. These differences support the suggestion that DAZL is having systemic effects on the paracrine communication within the follicle unit between the oocyte and somatic cells altering regulation and subsequent selection, and affecting final ovulation rate and litter size

Influence of oxidative stress, diaphragm fatigue, and inspiratory muscle training on the plasma cytokine response to maximum sustainable voluntary ventilation

The influence of oxidative stress, diaphragm fatigue, and inspiratory muscle training (IMT) on the cytokine response to maximum sustainable voluntary ventilation (MSVV) is unknown. Twelve healthy males were divided equally into an IMT or placebo (PLA) group, and before and after a 6-wk intervention they undertook, on separate days, 1h of (1) passive rest and (2) MSVV, whereby participants undertook volitional hyperpnea at rest that mimicked the breathing and respiratory muscle recruitment patterns commensurate with heavy cycling exercise. Plasma cytokines remained unchanged during passive rest. There was a main effect of time (P < 0.01) for plasma interleukin-1 (IL-1) and interleukin-6 (IL-6) concentrations and a strong trend (P = 0.067) for plasma interleukin-1 receptor antagonist concentration during MSVV. Plasma IL-6 concentration was reduced after IMT by 27 + 18% (main effect of intervention, P = 0.029), whereas there was no change after PLA (P = 0.753). There was no increase in a systemic marker of oxidative stress [DNA damage in peripheral blood mononuclear cells (PBMC)], and diaphragm fatigue was not related to the increases in plasma IL-1 and IL-6 concentrations. A dose-response relationship was observed between respiratory muscle work and minute ventilation and increases in plasma IL-6 concentration. In conclusion, increases in plasma IL-1 and IL-6 concentrations during MSVV were not due to diaphragm fatigue or DNA damage in PBMC. Increases in plasma IL-6 concentration during MSVV are attenuated following IMT, and the plasma IL-6 response is dependent upon the level of respiratory muscle work and minute ventilation

Testing asteroseismology with Gaia DR2: Hierarchical models of the Red Clump

Asteroseismology provides fundamental stellar parameters independent of distance, but subject to systematics under calibration. Gaia DR2 has provided parallaxes for a billion stars, which are offset by a parallax zero-point. Red Clump (RC) stars have a narrow spread in luminosity, thus functioning as standard candles to calibrate these systematics. This work measures how the magnitude and spread of the RC in the Kepler field are affected by changes to temperature and scaling relations for seismology, and changes to the parallax zero-point for Gaia. We use a sample of 5576 RC stars classified through asteroseismology. We apply hierarchical Bayesian latent variable models, finding the population level properties of the RC with seismology, and use those as priors on Gaia parallaxes to find the parallax zero-point offset. We then find the position of the RC using published values for the zero-point. We find a seismic temperature insensitive spread of the RC of ~0.03 mag in the 2MASS K band and a larger and slightly temperature-dependent spread of ~0.13 mag in the Gaia G band. This intrinsic dispersion in the K band provides a distance precision of ~1% for RC stars. Using Gaia data alone, we find a mean zero-point of -41 $\pm$ 10 $\mu$as. This offset yields RC absolute magnitudes of -1.634 $\pm$ 0.018 in K and 0.546 $\pm$ 0.016 in G. Obtaining these same values through seismology would require a global temperature shift of ~-70 K, which is compatible with known systematics in spectroscopy.Comment: Accepted for publication in MNRA

Green Infrastructure for Roadside Air Quality (GI4RAQ) guidance & decision tree: an evidence-based approach to reducing roadside exposure to road transport pollution

Green Infrastructure for Roadside Air Quality, ‘GI4RAQ’, is an initiative by Dr James Levine and Prof Rob MacKenzie at the Birmingham Institute of Forest Research (BIFoR), University of Birmingham, to promote and facilitate evidence-based use of green infrastructure to reduce roadside exposure to road transport pollution. The GI4RAQ Guidance document describes the development of an evidence-based, albeit qualitative, approach to GI4RAQ with Yvonne Brown, Principal Policy Analyst for Air Quality and Climate Change at Transport for London (TfL). It includes essential guidance on the use of the GI4RAQ Decision Tree – a differential diagnostics approach, visualised using a PowerPoint Show with embedded links. Whilst the approach has been developed for TfL and refers to case studies in London, both this guidance and the GI4RAQ Decision Tree are applicable to roads in all towns and cities, and the authors hope that these resources will find widespread use. The GI4RAQ Decision Tree guides the user through a short series of questions to identify the critical characteristics of the street in which they are seeking to reduce roadside exposure to road transport pollution. Subject to these characteristics, 'robustly beneficial' green infrastructure interventions are identified, as well as ones 'potentially beneficial to some at the expense of others'; the terms in inverted commas will be explained in due course. The accompanying guidance, provided here, builds on the “Reduce, Extend, Protect” concept introduced in the Trees & Design Action Group’s guide, ‘First Steps in Urban Air Quality for Built Environment Practitioners’ (Ferranti et al., 2019): first reduce the emissions of pollutants, then extend the distance between people and the sources of these emissions (i.e., vehicles) and, finally, protect those most vulnerable to their health impacts. This guidance is also consistent with, but elaborates on, that recently published by the Greater London Authority, ‘Using Green Infrastructure to Protect People from Air Pollution’ (GLA, 2019). Within TfL, this evidence-based approach to reducing exposure to road transport pollution supports TfL’s Healthy Streets Approach in putting people and their health at the centre of design decisions and the use of public space; it is also integrated into TfL’s Environmental Evaluation Tool, designed to capture and manage the impacts of projects not requiring a full Environmental Impact Assessment under Town and Country Planning Regulations 2017 (MHCLG, 2017). ‘Clean air’, however, is just one of ten positive outcomes sought via TfL’s Healthy Streets Approach, and green infrastructure contributes to a further eight (see ‘Indicators Explained’ section of Healthy Streets Check for Designers spreadsheet). Likewise, whilst this guidance focuses on improving roadside air quality, we recognise that green infrastructure can (simultaneously) deliver further, major benefits; we will highlight the opportunities for co-benefits throughout the document. Improved air quality is just one benefit of – and one consideration in – the planning, planting and investing in green infrastructure for the long term

Exploration of Perceived Psychosocial Benefits of Senior Companion Program Participation Among Urban-Dwelling, Low-Income Older Adult Women Volunteers

Background: As the older adult population increases, it is imperative to increase older adults' opportunities for social involvement, thus maintaining their important roles and contributions to society. While there are known health-related benefits of volunteerism among older adults, a dearth of information exists on the perceived benefits of volunteerism among low-income and ethnic minority older adults. Purpose: To understand the perceived psychosocial benefits of volunteering in the Senior Companion Program and to present findings of focus groups conducted with urban-dwelling, low-income older adult women volunteers. Design and Methods: Inductive content analysis and the Dedoose qualitative data analysis software were used for analyzing data obtained from 59 older adult women Senior Companions who participated in nine focus groups. Results: Content analyses of the focus group transcripts identified four major themes: (1) Reducing social isolation; (2) Improving quality of life; (3) Finding purpose and meaning; and (4) Increasing understanding of aging. The majority of our participants (81%) were African American women, with a mean age of 70 years. Approximately 83.1% had completed high school and 62.7% lived below the poverty line. Discussion and Implications: Findings provided data rich in descriptions of positive psychosocial outcomes, finding meaning and purpose, and a better understanding of aging in urban-dwelling, low-income older women volunteers. The findings also provide support for the need for policies and programs that promote civic engagement in this population

Immunohistochemical evidence for an endocrine/paracrine role for ghrelin in the reproductive tissues of sheep

BACKGROUND: The gut hormone, ghrelin, is involved in the neuroendocrine and metabolic responses to hunger. In monogastric species, circulating ghrelin levels show clear meal-related and body weight-related changes. The pattern of secretion and its role in ruminant species is less clear. Ghrelin acts via growth hormone secretagogue receptors (GHSR-1a) to alter food intake, fat utilization, and cellular proliferation. There is also evidence that ghrelin is involved in reproductive function. In the present study we used immunohistochemistry to investigate the presence of ghrelin and GHSR-1a in sheep reproductive tissues. In addition, we examined whether ghrelin and GHSR-1a protein expression is developmentally regulated in the adult and fetal ovine testis, and whether there is an association with markers of cellular proliferation, i.e. stem cell factor (SCF) and proliferating cell nuclear antigen (PCNA). METHODS: Antibodies raised against ghrelin and its functional receptor, GHSR-type 1a, were used in standard immunohistochemical protocols on various reproductive tissues collected from adult and fetal sheep. GHSR-1a mRNA presence was also confirmed by in situ hybridisation. SCF and PCNA immunoexpression was investigated in fetal testicular samples. Adult and fetal testicular immunostaining for ghrelin, GHSR-1a, SCF and PCNA was analysed using computer-aided image analysis. Image analysis data were subjected to one-way ANOVA, with differences in immunostaining between time-points determined by Fisher's least significant difference. RESULTS: In adult sheep tissue, ghrelin and GHSR-1a immunostaining was detected in the stomach (abomasum), anterior pituitary gland, testis, ovary, and hypothalamic and hindbrain regions of the brain. In the adult testis, there was a significant effect of season (photoperiod) on the level of immunostaining for ghrelin (p < 0.01) and GHSR-1a (p < 0.05). In the fetal sheep testis, there was a significant effect of gestational age on the level of immunostaining for ghrelin (p < 0.001), GHSR-1a (p < 0.05), SCF (p < 0.05) and PCNA (p < 0.01). CONCLUSION: Evidence is presented for the presence of ghrelin and its receptor in various reproductive tissues of the adult and fetal sheep. In addition, the data indicate that testicular expression of ghrelin and its receptor is physiologically regulated in the adult and developmentally regulated in the fetus. Therefore, the ghrelin ligand/receptor system may have a role (endocrine and/or paracrine) in the development (cellular proliferation) and function of the reproductive axis of the sheep

An immunohistochemical study of the localization and developmental expression of ghrelin and its functional receptor in the ovine placenta

<p>Abstract</p> <p>Background</p> <p>Ghrelin is an orexigenic hormone principally produced by the stomach, but also by numerous peripheral tissues including the placenta. Ghrelin acts via growth hormone secretagogue receptors (GHSR-1a) to alter food intake, fat utilization, and cellular proliferation, and has been suggested to play a role in the developmental growth of the fetoplacental unit. The placental expression of ghrelin and its role in ruminant species is not known. We tested the hypotheses that ghrelin and its functional receptor, GHSR-1a, are present in tissues of the ovine placenta, and that their expression is linked to the stage of development.</p> <p>Methods</p> <p>Antibodies raised against ghrelin and GHSR-1a were used in standard immunohistochemical protocols on placental tissues collected from pregnant ewes (n = 6 per gestational time point) at days 50, 80, 100, 128 and 135 of gestation (term ≈ day 145). Immunostaining for ghrelin and GHSR-1a was quantified using computer-aided image analysis. Image analysis data were subjected to one-way ANOVA, with differences in immunostaining between time-points determined by Fisher's least significant difference.</p> <p>Results</p> <p>Positive immunostaining for ghrelin was detected in ovine placentae at all gestational time points, with staining localized to the maternal epithelium, caruncle and trophectoderm. There was a significant effect of gestational age (p < 0.001) on the placental expression of ghrelin, with maximal levels at gestational day 80. GHSR-1a immunostaining was detected in the fetal trophectoderm at all time points. In contrast to the gestational pattern of ghrelin expression, there was no effect of gestational age on placental GHSR-1a immunoexpression.</p> <p>Conclusion</p> <p>Ghrelin and GHSR-1a are both present in the ovine placenta, and ghrelin displays a developmentally-related pattern of expression. Therefore, these data strongly suggest that the ghrelin system may have a role in feto-placental development in sheep.</p