Data on the concentrations of etoposide, PSC833, BAPTA-AM, and cycloheximide that do not compromise the vitality of mature mouse oocytes, parthenogenetically activated and fertilized embryos

AbstractThese data document the vitality of mature mouse oocytes (Metaphase II (MII)) and early stage embryos (zygotes) following exposure to the genotoxic chemotherapeutic agent, etoposide, in combination with PSC833, a selective inhibitor of permeability glycoprotein. They also illustrate the vitality of parthenogenetically activated and fertilized embryos following incubation with the calcium chelator BAPTA-AM (1,2-Bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester)), cycloheximide (an antibiotic that is capable of inhibiting protein synthesis), and hydrogen peroxide (a potent reactive oxygen species). Finally, they present evidence that permeability glycoprotein is not represented in the proteome of mouse spermatozoa. Our interpretation and discussion of these data feature in the article “Identification of a key role for permeability glycoprotein in enhancing the cellular defense mechanisms of fertilized oocytes” (Martin et al., in press) [1]

ID4 levels dictate the stem cell state in mouse spermatogonia

Spermatogenesis is a classic model of cycling cell lineages that depend on a balance between stem cell self-renewal for continuity and the formation of progenitors as the initial step in the production of differentiated cells. The mechanisms that guide the continuum of spermatogonial stem cell (SSC) to progenitor spermatogonial transition and precise identifiers of subtypes in the process are undefined. Here we used an Id4-eGfp reporter mouse to discover that EGFP intensity is predictive of the subsets, with the ID4-EGFPBright population being mostly, if not purely, SSCs, whereas the ID4-EGFPDim population is in transition to the progenitor state. These subsets are also distinguishable by transcriptome signatures. Moreover, using a conditional overexpression mouse model, we found that transition from the stem cell to the immediate progenitor state requires downregulation of Id4 coincident with a major change in the transcriptome. Collectively, our results demonstrate that the level of ID4 is predictive of stem cell or progenitor capacity in spermatogonia and dictates the interface of transition between the different functional states

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p<0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p<0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p<0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP >5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

The role of reactive oxygen species and oxidative stress in post-ovulatory ageing and apoptosis of the mammalian oocyte

Research Doctorate - Doctor of Philosophy (PhD)Following ovulation, the MII stage oocyte awaits fertilization in the oviduct, or, in the case of assistive reproductive technologies (ART), in <i>in vitro</i> culture medium. In the absence of fertilization, however, the oocyte experiences a time-dependent deterioration in quality referred to as post-ovulatory ageing. Post-ovulatory ageing is associated with a decline in fertilization rate, as well as the production of poor quality embryos, an increased risk for post-implantation errors and production of offspring with compromised health. Although the consequences associated with post-ovulatory ageing are well defined, the molecular mechanisms which orchestrate this decline in oocyte quality, or conversely, act to prevent post-ovulatory ageing in the event that timely fertilization has occurred, are not well understood. In this thesis we decipher a critical role for reactive oxygen species (ROS) in the initiation of post-ovulatory ageing and apoptosis of the mammalian oocyte. Using a mouse model, we have characterised a time–dependent accumulation of intracellular ROS following retrieval of the ovulated oocyte. This elevation in ROS was found to instigate a self-perpetuating cycle of lipid peroxidation, electrophilic aldehyde production and mitochondrial damage; resulting in the initiation of an intrinsic apoptotic cascade. The elevation in levels of ROS and electrophilic aldehydes within the oocyte were directly associated with a decreased capacity to participate in fertilization and support embryo development. Importantly, research within this thesis has demonstrated that timely fertilization of the oocyte is associated with an up-regulation of glutathione peroxidase activity, and accelerated DNA repair by the base excision repair (BER) pathway. These post-fertilization changes in oocyte biochemistry aide in circumventing the otherwise inevitable initiation of post-ovulatory ageing by preventing the accumulation of ROS and oxidative DNA damage. In identifying the critical role for ROS and electrophilic aldehydes in post-ovulatory ageing and apoptosis of the oocyte, it was possible to select antioxidant and aldehyde-reactive compounds to attenuate the onset of these processes. Specifically, melatonin was found to significantly improve fertilization rate, embryo formation rate and embryo quality in oocytes aged for 8 and 16 h <i>in vitro</i>, as well as delay the initiation of apoptosis. Similarly, penicillamine was found to prevent the decline in fertilization rate and embryo formation rate associated with elevated levels of electrophilic aldehydes. The adaptation of these supplementation techniques for use in a human ART setting would be advantageous in lengthening the optimal window of time in which oocytes must be inseminated post-retrieval, as well as increasing the viability of re-insemination techniques such as rescue-ICSI; potentially minimising the likelihood that further ovarian stimulation cycles would be necessary following a failure to fertilize by IVF. Collectively, these data provide a significant contribution to the field of knowledge surrounding degeneration and apoptosis of the mammalian oocyte, and provide novel methodologies for attenuating these events in an <i>in vitro</i> setting

Oxidative stress and ageing of the post-ovulatory oocyte

With extended periods of time following ovulation, the metaphase II stage oocyte experiences deterioration in quality referred to as post-ovulatory oocyte ageing. Post-ovulatory ageing occurs both in vivo and in vitro and has been associated with reduced fertilization rates, poor embryo quality, post-implantation errors and abnormalities in the offspring. Although the physiological consequences of post-ovulatory oocyte ageing have largely been established, the molecular mechanisms controlling this process are not well defined. This review analyses the relationships between biochemical changes exhibited by the ageing oocyte and the symptoms associated with the ageing phenotype. We also discuss molecular events that are potentially involved in orchestrating post-ovulatory ageing with a particular focus on the role of oxidative stress. We propose that oxidative stress may act as the initiator for a cascade of events that create the aged oocyte phenotype. Specifically, oxidative stress has the capacity to cause a decline in levels of critical cell cycle factors such as maturation-promoting factor, impair calcium homoeostasis, induce mitochondrial dysfunction and directly damage multiple intracellular components of the oocyte such as lipids, proteins and DNA. Finally, this review addresses current strategies for delaying post-ovulatory oocyte ageing with a particular focus on the potential use of compounds such as caffeine or selected antioxidants in the development of more refined media for the preservation of oocyte integrity during IVF procedures

Functional assessment of spermatogonial stem cell purity in experimental cell populations

Historically, research in spermatogonial biology has been hindered by a lack of validated approaches to identify and isolate pure populations of the various spermatogonial subsets for in-depth analysis. In particular, although a number of markers of the undifferentiated spermatogonial population have now been characterized, standardized methodology for assessing their specificity to the spermatogonial stem cell (SSC) and transit amplifying progenitor pools has been lacking. To date, SSC content within an undefined population of spermatogonia has been inferred using either lineage tracing or spermatogonial transplantation analyses which generate qualitative and quantitative data, respectively. Therefore, these techniques are not directly comparable, and are subject to variable interpretations as to a readout that is representative of a ‘pure’ SSC population. We propose standardization across the field for determining the SSC purity of a population via use of a limiting dilution transplantation assay that would eliminate subjectivity and help to minimize the generation of inconsistent data on ‘SSC’ populations. In the limiting dilution transplantation assay, a population of LacZ-expressing spermatogonia are selected based on a putative SSC marker, and a small, defined number of cells (i.e. 10 cells) are microinjected into the testis of a germ cell-deficient recipient mouse. Using colony counts and an estimated colonization efficiency of 5%; a quantitative value can be calculated that represents SSC purity in the starting population. The utilization of this technique would not only be useful to link functional relevance to novel markers that will be identified in the future, but also for providing validation of purity for marker-selected populations of spermatogonia that are commonly considered to be SSCs by many researchers in the field of spermatogenesis and stem cell biology. Keywords: Spermatogonial stem cell, Limiting dilution, Transplantatio

Testicular Architecture Is Critical for Mediation of Retinoic Acid Responsiveness by Undifferentiated Spermatogonial Subtypes in the Mouse

Summary: Spermatogenesis requires retinoic acid (RA) induction of the undifferentiated to differentiating transition in transit amplifying (TA) progenitor spermatogonia, whereas continuity of the spermatogenic lineage relies on the RA response being suppressed in spermatogonial stem cells (SSCs). Here, we discovered that, in mouse testes, both spermatogonial populations possess intrinsic RA-response machinery and exhibit hallmarks of the differentiating transition following direct exposure to RA, including loss of SSC regenerative capacity. We determined that SSCs are only resistant to RA-driven differentiation when situated in the normal topological organization of the testis. Furthermore, we show that the soma is instrumental in “priming” TA progenitors for RA-induced differentiation through elevated RA receptor expression. Collectively, these findings indicate that SSCs and TA progenitor spermatogonia inhabit disparate niche microenvironments within seminiferous tubules that are critical for mediating extrinsic cues that drive fate decisions. : Lord et al. have demonstrated that, contrary to previous assumptions, spermatogonial stem cells do express a functional complement of retinoic acid and retinoid X receptors (RARs/RXRs) and rely on protection from an undisturbed niche microenvironment to prevent loss of the spermatogenic reservoir to RA-induced differentiation. Keywords: retinoic acid, RAR gamma, spermatogonial stem cells, ID4, GFRA

Dataset supporting the University of Southampton Doctoral thesis &quot;Self-states, attachment and dissociation: relationships and measurement&quot;.

Dataset supporting the University of Southampton Doctoral thesis &quot;Self-states, attachment and dissociation: relationships and measurement&quot; by Clarissa Lord. This data includes an excel file of collected data created by the author as part of their research. </span

Reproductive health research in Australia and New Zealand: highlights from the Annual Meeting of the Society for Reproductive Biology, 2019

International audienceThe 2019 meeting of the Society for Reproductive Biology (SRB) provided a platform for the dissemination of new knowledge and innovations to improve reproductive health in humans, enhance animal breeding efficiency and understand the effect of the environment on reproductive processes. The effects of environment and lifestyle on fertility and animal behaviour are emerging as the most important modern issues facing reproductive health. Here, we summarise key highlights from recent work on endocrine-disrupting chemicals and diet- and lifestyle-induced metabolic changes and how these factors affect reproduction. This is particularly important to discuss in the context of potential effects on the reproductive potential that may be imparted to future generations of humans and animals. In addition to key summaries of new work in the male and female reproductive tract and on the health of the placenta, for the first time the SRB meeting included a workshop on endometriosis. This was an important opportunity for researchers, healthcare professionals and patient advocates to unite and provide critical updates on efforts to reduce the effect of this chronic disease and to improve the welfare of the women it affects. These new findings and directions are captured in this review