Isolation, characterisation and in vitro potential of Oogonial stem cells

The longstanding belief that women are born with a finite ovarian reserve has been debated for over a decade, ever since the discovery, and subsequent isolation, of purported oogonial stem cells (OSCs) from adult mammalian ovaries. This rare cell population has now been reported in the mouse, rat, pig, rhesus macaque monkey and humans and, although a physiological role for the cells has not been proven, they do appear to generate oocytes when cultured in specific environments, resulting in live offspring in rodents. The primary aim of this thesis was to verify independently the existence of OSCs in human ovary and determine whether they could be isolated from a large animal model, the cow. The secondary aim was to investigate the cells’ in vitro potential, both to undergo neo-oogenesis and as a model for germ cell development. Putative bovine and human OSCs were isolated from disaggregated adult ovarian cortex using a previously validated fluorescence-activated cell sorting (FACS)-based technique, with cells sorted for externally expressed DDX4 (VASA). Freshly isolated and cultured cells were characterised by analysing their expression of pluripotency and germline markers, using RT-PCR, immunocytochemistry and Western blotting. The in vitro neo-oogenesis potential of the cells was explored by injecting fluorescently labelled cells into fragments of adult ovarian cortex and by forming aggregated artificial “ovaries” with putative OSCs and fetal ovarian somatic cells. Germ cell model experiments comprised treatment of cultured cells with BMP4 and/or retinoic acid (RA), with subsequent quantitative RT-PCR and immunocytochemistry analysis for downstream BMP4- and RA-response genes, and liposomal-mediated transfection of cells with a DAZL overexpression plasmid to assess their meiosis-related gene response. Scarce populations of putative OSCs were retrieved from 5 human samples (aged 13- 40 years) and 6 bovine samples. The cells were cultured long-term for up to 7 months and demonstrated consistent expression of several pluripotency-associated and germline markers at the mRNA and protein level, including LIN28, NANOG, POU5F1 (OCT4), IFITM3 (fragilis), STELLA, PRDM1 (BLIMP1), and C-KIT, indicating their early germline nature. Investigation of neo-oogenesis potential revealed that putative human OSCs were associated rarely with fetal somatic cells in primordial follicle-like structures, but could not be confirmed to have undergone oogenesis. However, like early germ cells, putative bovine and human OSCs were BMP4 and RA responsive, with both species demonstrating significant upregulation of expression of ID1 and bovine cells exhibiting a significant increase in MSX1, MSX2 and the meiotic marker SYCP3 in response to BMP4 and/or RA treatment. Cells could be successfully transfected to overexpress DAZL; however, no significant downstream gene expression changes were observed. This is the first report of putative bovine OSC isolation and corroborates a previous report showing putative human OSC isolation. Although the expression of both stem cell and germline markers indicates the cells have characteristics of OSCs, their capacity to enter meiosis and form functional oocytes has yet to be determined. Putative bovine OSCs, however, show promise as a novel model for investigating germ cell development. If their potential can be harnessed, then OSCs may have a role in clinical applications, for example in fertility preservation, in the future. Future experiments will examine the neo-oogenesis capabilities of the cells further and explore novel cell delivery systems for clinical use

Habitat selection by vulnerable golden bandicoots in the arid zone

In 2010, vulnerable golden bandicoots (Isoodon auratus) were translocated from Barrow Island, Western Australia, to a mainland predator-free enclosure on the Matuwa Indigenous Protected Area. Golden bandicoots were once widespread throughout a variety of arid and semiarid habitats of central and northern Australia. Like many small-to-medium-sized marsupials, the species has severely declined since colonization and has been reduced to only four remnant natural populations. Between 2010 and 2020, the reintroduced population of golden bandicoots on Matuwa was monitored via capture–mark–recapture data collection, which was used in spatially explicit capture–recapture analysis to monitor their abundance over time. In 2014, we used VHF transmitters to examine the home range and habitat selection of 20 golden bandicoots in the enclosure over a six-week period. We used compositional analysis to compare the use of four habitat types. Golden bandicoot abundance in the enclosure slowly increased between 2010 and 2014 and has since plateaued at approximately one quarter of the density observed in the founding population on Barrow Island. The population may have plateaued because some bandicoots escape through the fence. Golden bandicoots used habitats dominated by scattered shrubland with spinifex grass more than expected given the habitat\u27s availability. Nocturnal foraging range was influenced by sex and trapping location, whereas diurnal refuge habitat, which was typically under a spinifex hummock with minimal overstory vegetation, was consistent across sex and trapping location. Our work suggests that diurnal refuge habitat may be an important factor for the success of proposed translocations of golden bandicoots

Ovarian germline stem cells

It has long been established that germline stem cells (GSCs) are responsible for lifelong gametogenesis in males, and some female invertebrates (for example, Drosophila) and lower vertebrates (for example, teleost fish and some prosimians) also appear to rely on GSCs to replenish their oocyte reserve in adulthood. However, the presence of such cells in the majority of female mammals is controversial, and the idea of a fixed ovarian reserve determined at birth is the prevailing belief among reproductive biologists. However, accumulating evidence demonstrates the isolation and culture of putative GSCs from the ovaries of adult mice and humans. Live offspring have been reportedly produced from the culture of adult mouse GSCs, and human GSCs formed primordial follicles using a mouse xenograft model. If GSCs were present in adult female ovaries, it could be postulated that the occurrence of menopause is not due to the exhaustion of a fixed supply of oocytes but instead is a result of GSC and somatic cell aging. Alternatively, they may be benign under normal physiological conditions. If their existence were confirmed, female GSCs could have many potential applications in both basic science and clinical therapies. GSCs not only may provide a valuable model for germ cell development and maturation but may have a role in the field of fertility preservation, with women potentially being able to store GSCs or GSC-derived oocytes from their own ovaries prior to infertility-inducing treatments. Essential future work in this field will include further independent corroboration of the existence of GSCs in female mammals and the demonstration of the production of mature competent oocytes from GSCs cultured entirely in vitro

Re-implantation of cryopreserved ovarian cortex resulting in restoration of ovarian function, natural conception and successful pregnancy after haematopoietic stem cell transplantation for Wilms tumour

With the improvement of long-term cancer survival rates, growing numbers of female survivors are suffering from treatment-related premature ovarian insufficiency (POI). Although pre-treatment embryo and oocyte storage are effective fertility preservation strategies, they are not possible for pre-pubertal girls or women who cannot delay treatment. In these cases, the only available treatment option is ovarian cortex cryopreservation and subsequent re-implantation. A 32-year-old woman had ovarian cortex cryopreserved 10 years previously before commencing high-dose chemotherapy and undergoing a haematopoietic stem cell transplant for recurrent adult Wilms tumour, which resulted in POI. She underwent laparoscopic orthotopic transplantation of cryopreserved ovarian cortex to the original site of biopsy on the left ovary. She ovulated at 15 and 29 weeks post-re-implantation with AMH detectable, then rising, from 21 weeks, and conceived naturally following the second ovulation. The pregnancy was uncomplicated and a healthy male infant was born by elective Caesarean section at 36(+4) weeks gestation. This is the first report of ovarian cortex re-implantation in the UK. Despite the patient receiving low-risk chemotherapy prior to cryopreservation and the prolonged tissue storage duration, the re-implantation resulted in rapid restoration of ovarian function and natural conception with successful pregnancy

‘Why is it so different now I’m bisexual?’: young bisexual people’s experiences of identity, belonging, self-injury, and COVID19

Bisexual people demonstrate higher rates of Non-Suicidal Self-Injury (NSSI) in comparison to other groups. This study aimed to explore bisexual people’s experiences of sexuality, NSSI and the COVID19 pandemic. Fifteen bisexual people (16–25 years old) with experience of NSSI participated in online qualitative interviews. Thematic analysis was used. Preliminary findings were shared with a subset of participants for member-checking. Participants described experiences of falling between the binary worlds of heterosexuality and homosexuality and described discrimination and invalidation related to this. Lack of access to positive bisexual representation contributed to feelings of self-loathing, with NSSI used to manage emotions or self-punish. The effect of lockdown was not clear cut, depending on personal circumstances and meanings of social interaction for participants. There is a need for greater recognition of significant societal narratives around bisexuality within clinical formulations of mental health difficulties and NSSI within this population

A TSC signaling node at the peroxisome regulates mTORC1 and autophagy in response to ROS

Subcellular localization is emerging as an important mechanism for mTORC1 regulation. We report that the tuberous sclerosis complex (TSC) signaling node, TSC1, TSC2 and Rheb, localizes to peroxisomes, where it regulates mTORC1 in response to reactive oxygen species (ROS). TSC1 and TSC2 were bound by PEX19 and PEX5, respectively, and peroxisome-localized TSC functioned as a Rheb GAP to suppress mTORC1 and induce autophagy. Naturally occurring pathogenic mutations in TSC2 decreased PEX5 binding, abrogated peroxisome localization, Rheb GAP activity, and suppression of mTORC1 by ROS. Cells lacking peroxisomes were deficient in mTORC1 repression by ROS and peroxisome-localization deficient TSC2 mutants caused polarity defects and formation of multiple axons in neurons. These data identify a role for TSC in responding to ROS at the peroxisome, and identify the peroxisome as a signaling organelle involved in regulation of mTORC1

The Glycan Shield of HIV Is Predominantly Oligomannose Independently of Production System or Viral Clade

The N-linked oligomannose glycans of HIV gp120 are a target for both microbicide and vaccine design. The extent of cross-clade conservation of HIV oligomannose glycans is therefore a critical consideration for the development of HIV prophylaxes. We measured the oligomannose content of virion-associated gp120 from primary virus from PBMCs for a range of viral isolates and showed cross-clade elevation (62–79%) of these glycans relative to recombinant, monomeric gp120 (∼30%). We also confirmed that pseudoviral production systems can give rise to notably elevated gp120 oligomannose levels (∼98%), compared to gp120 derived from a single-plasmid viral system using the HIVLAI backbone (56%). This study highlights differences in glycosylation between virion-associated and recombinant gp120

Provision of obstetrics and gynaecology services during the COVID-19 pandemic:a survey of junior doctors in the UK National Health Service

Objective: The COVID-19 pandemic is disrupting health services worldwide. We aimed to evaluate the provision of obstetrics and gynaecology services in the UK during the acute-phase of the COVID-19 pandemic. Design: Interview-based national survey. Setting: Women’s healthcare units in the National Health Service. Population: Junior doctors in obstetrics and gynaecology. Methods: Participants were interviewed by members of the UKARCOG trainees’ collaborative between 28th March and 7th of April 2020. We used a quantitative analysis for closed-ended questions and a thematic framework analysis for open comments. Results: We received responses from 148/155 units (95%), majority of the participants were in years 3-7 of training (121/148, 82%). Most completed specific training drills for managing obstetric and gynaecological emergencies in women with COVID-19 (89/148, 60.1%) and two-persons donning and doffing of Personal Protective Equipment (PPE) (96/148, 64.9%). The majority of surveyed units implemented COVID-19 specific protocols (130/148, 87.8%), offered adequate PPE (135/148, 91.2%) and operated dedicated COVID-19 emergency theatres (105/148, 70.8%). Most units reduced face-to-face antenatal clinics (117/148, 79.1%), and suspended elective gynaecology services (131/148, 88.5%). The two-week referral pathway for oncology gynaecology was not affected in half of the units (76/148, 51.4%), while half reported a planned reduction in oncology operating (82/148, 55.4%). Conclusion: The provision of obstetrics and gynaecology services in the UK during the acute phase of the COVID-19 pandemic seems to be in line with current guidelines, but strategic planning is needed to restore routine gynaecology services and ensure safe access to maternity care on the longterm

Prospective study into the value of the automated Elecsys antimüllerian hormone assay for the assessment of the ovarian growing follicle pool

ObjectiveTo evaluate a new fully automated assay measuring antimüllerian hormone (AMH; Roche Elecsys) against antral follicle count in women of reproductive age.DesignProspective cohort study.SettingHospital infertility clinics and academic centers.Patient(s)Four hundred fifty-one women aged 18 to 44 years, with regular menstrual cycles.Intervention(s)None.Main Outcome Measure(s)AMH and antral follicle count (AFC) determined at a single visit on day 2–4 of the menstrual cycle.Result(s)There was a statistically significant variance in AFC but not in AMH between centers. Both AFC and AMH varied by age (overall Spearman rho −0.50 for AFC and −0.47 for AMH), but there was also significant between-center variation in the relationship between AFC and age but not for AMH. There was a strong positive correlation between AMH and AFC (overall spearman rho 0.68), which varied from 0.49 to 0.87 between centers. An agreement table using AFC cutoffs of 7 and 15 showed classification agreement in 63.2%, 56.9% and 74.5% of women for low, medium, and high groups, respectively.Conclusion(s)The novel fully automated Elecsys AMH assay shows good correlations with age and AFC in women of reproductive age, providing a reproducible measure of the growing follicle pool