All Your Eggs in One Basket: Mechanisms of Xenobiotic Induced Female Reproductive Senescence

The irreplaceable mammalian primordial follicle represents the basic unit of female fertility, serving as the primary source of all developing oocytes in the ovary. These primordial follicles remain quiescent, often for decades, until recruited into the growing pool throughout a woman's adult reproductive years. Once recruited, <1% will reach ovulation, with the remainder undergoing an apoptotic process known as atresia (Hirshfield, 1991). Menopause, or ovarian senescence, occurs when the pool of primordial follicles becomes exhausted

Xenobiotics; effects on female fertility

Research Doctorate - Doctor of Philosophy (PhD)Over the course of the 20th century there has been an increasing trend in western women opting to delay childbirth in the pursuit of social and economic stability. This development has highlighted the need to identify and characterise ovotoxic xenobiotics (foreign chemical compounds) which threaten their fertility. Arguably the most insidious ovotoxic xenobiotics are those which target the irreplaceable primordial follicle pool for destruction, resulting in pre-mature ovarian senescence. Although many of these xenobiotics have been identified, the molecular mechanisms behind their ovotoxicity remain largely unknown. Employing a neonatal mouse model rich in primordial follicles, the studies presented in this thesis were aimed at characterising the mechanisms of ovotoxicity for six xenobiotics known to target immature follicles (4-vinylcyclohexene diepoxide; Methoxychlor; Menadione; Benzo-a-pyrene, 7,12-Dimethylbenz-[a]anthracene; 3-Methylcholanthrene). The effects of short term xenobiotic exposure on long term oocyte viability were also examined to determine whether follicles which survive ovotoxic destruction were still functionally viable. Microarray analysis and quantitative PCR revealed a unique ovarian response to each xenobiotic involving a number of genes linked to follicular growth/development, cell death and tumorigenesis in vitro. Immunohistological and histomorphological analysis confirmed the microarray data, and revealed a consistent mechanism of ovotoxicity involving primordial follicle activation alongside developing follicle atresia in vitro and in vivo. Immunohistological and pharmacological inhibition studies also revealed an essential role for the PI3K/Akt/mTOR signalling pathways in 3-Methylcholanthrene and 7,12-Dimethylbenz-[a]anthracene induced primordial follicle activation and survival. Studies into the effects of short term neonatal exposure on long term female fertility revealed no difference in the number of healthy oocytes ovulated in neonatally treated adults compared to controls. However, comprehensive oocyte viability analysis revealed a decreased capacity for fertilisation caused by oxidative damage to the oolemma membrane due to mitochondrial electron transport chain leakage. The studies conducted in this thesis have identified a common mechanism of xenobiotic induced primordial follicle depletion via a homeostatic mechanism of developing follicle recruitment, with some ovotoxic xenobiotics inducing primordial follicle survival as opposed to atresia. In addition, the contents of this thesis also provide the first documented evidence of short term neonatal exposure causing long term oocyte dysfunction through xenobiotic induced oxidative stress

Contraception targets in mammalian ovarian development

In the human ovary, early in pre-natal life, oocytes are surrounded by pre-granulosa follicular cells to form primordial follicles. These primordial oocytes remain dormant, often for decades, until recruited into the growing pool throughout a woman’s adult reproductive years. Activation of follicle growth and subsequent development of growing oocytes in pre-antral follicles are major biological checkpoints that determine an individual females reproductive potential. In the past decade, great strides have been made in the elucidation of the molecular and cellular mechanisms underpinning maintenance of the quiescent primordial follicle pool and initiation and development of follicle growth. Gaining an in-depth knowledge of the intracellular signalling systems that control oocyte preservation and follicle activation has significant implications for improving female reproductive productivity and alleviating infertility. It also has application in domestic animal husbandry, feral animal population control and contraception in women

Staying alive : PI3k pathway promotes primordial follicle activation and survival in response to 3MC-induced ovotoxicity

3-Methylcholanthrene (3MC) is a potent ovotoxicant capable of causing premature ovarian failure through primordial follicle depletion. Despite 3MCs ovotoxicity having been established for 30 years, relatively little information exists on the mechanisms. In this study, we examined the effects of 3MC exposure on the immature ovarian follicle population. Microarray analysis revealed a complex mechanism of 3MC-induced ovotoxicity involving a number of cellular processes associated with xenobiotic metabolism, ovarian cancer, cell cycle progression, and cell death. 3MC exposure was also found to induce developing follicle atresia and aberrant primordial follicle activation via the stimulation of PI3K/Akt and mammalian target of rapamycin (mTOR) signaling pathways. Inhibition of PI3K/Akt signaling resulted in the severe depletion of the primordial follicle pool, with further analysis identifying increased Akt1-stimulated Bad phosphoinhibition in 3MC-treated primordial follicles. Our results suggest that the primordial follicle pool enters a "prosurvival" state upon 3MC exposure and that its depletion is due to a vicious cycle of primordial follicle activation in an attempt to replace developing follicles undergoing follicular atresia

Adding insult to injury: effects of xenobiotic-induced preantral ovotoxicity on ovarian development and oocyte fusibility

Mammalian females are born with a finite number of nonrenewing primordial follicles, the majority of which remain in a quiescent state for many years. Because of their nonrenewing nature, these ‘‘resting’’ oocytes are particularly vulnerable to xenobiotic insult, resulting in premature ovarian senescence and the formation of dysfunctional oocytes. In this study, we characterized the mechanisms of ovotoxicity for three ovotoxic agents, 4-vinylcyclohexene diepoxide (VCD), methoxychlor (MXC), and menadione (MEN), all of which target immature follicles. Microarray analysis of neonatal mouse ovaries exposed to these xenobiotics in vitro revealed a more than twofold significant difference in transcript expression (p < 0.05) for a number of genes associated with apoptotic cell death and primordial follicle activation. Histomorphological and immunohistological analysis supported the microarray data, showing signs of primordial follicle activation and preantral follicle atresia both in vitro and in vivo. Sperm-oocyte fusion assays on oocytes obtained from adult Swiss mice treated neonatally revealed severely reduced sperm-egg binding and fusion in a dose-dependent manner for all the xenobiotic treatments. Additionally, lipid peroxidation analysis on xenobiotic-cultured oocytes indicated a dose-dependent increase in oocyte lipid peroxidation for all three xenobiotics in vitro. Our results reveal a novel mechanism of preantral ovotoxicity involving the homeostatic recruitment of primordial follicles to maintain the pool of developing follicles destroyed by xenobiotic exposure and to our knowledge provide the first documented evidence of short-term, low- and high-dose (VCD 40–80 mg/kg/day, MXC 50–100 mg/kg/day, MEN 7.5–15 mg/kg/day) neonatal exposure to xenobiotics causing long-term reactive oxygen species-induced oocyte dysfunction

Distinct modes of telomere synthesis and extension contribute to Alternative Lengthening of Telomeres

Summary: Alternative lengthening of telomeres (ALT) is a homology-directed repair mechanism that becomes activated in a subset of cancers to maintain telomere length. One of the defining features of ALT cells is the prevalence of extrachromosomal telomeric repeat (ECTR) DNA. Here, we identify that ALT cells engage in two modes of telomere synthesis. Non-productive telomere synthesis occurs during the G2 phase of the cell cycle and is characterized by newly synthesized internal telomeric regions that are not retained in the subsequent G1, coinciding with an induction of ECTR DNA. Productive telomere synthesis occurs specifically during the transition from G2 to mitosis and is defined as the extension of the telomere termini. While many proteins associated with break-induced telomere synthesis function in both non-productive and productive telomere synthesis, POLH specifically promotes productive telomere lengthening and suppresses non-productive telomere synthesis. These findings delineate the mechanism and cell cycle regulation of ALT-mediated telomere synthesis and extension

Immune regulation of ovarian development: programming by neonatal immune challenge

Neonatal immune challenge by administration of lipopolysaccharide (LPS) produces enduring alterations in the development and activity of neuroendocrine, immune and other physiological systems. We have recently reported that neonatal exposure to an immune challenge by administration of LPS results in altered reproductive development in the female Wistar rat. Specifically, LPS-treated animals exhibited diminished ovarian reserve and altered reproductive lifespan. In the current study, we examined the cellular mechanisms that lead to the previously documented impaired ovulation and reduced follicular pool. Rats were administered intraperitoneally either 0.05mg/kg of LPS (Salmonella Enteritidis) or an equivalent volume of non-pyrogenic saline on postnatal days (PNDs) 3 and 5, and ovaries were obtained on PND 7. Microarray analysis revealed a significant upregulation in transcript expression (2-fold change; p&lt;.05) for a substantial number of genes in the ovaries of LPS-treated animals, implicated in immune cell signalling, inflammatory responses, reproductive system development and disease. Several canonical pathways involved in immune recognition were affected by LPS treatment, such as nuclear factor-κB (NF-kB) activation and LPS-stimulated mitogen-activated protein kinase (MAPK) signalling. Real-time PCR analysis supported the microarray results. Protein expression analysis of several components of the MAPK signalling pathway revealed a significant upregulation in the expression of Toll-like receptor 4 (TLR4) in the neonatal ovary of LPS-treated animals. These results indicate that neonatal immune challenge by administration of LPS has a direct effect on the ovary during the sensitive period of follicular formation. Given the pivotal role of inflammatory processes in the regulation of reproductive health, our findings suggest that early life immune activation via TLR signalling may have significant implications for the programming of ovarian development and fertility

Author Correction: The FANCM-BLM-TOP3A-RMI complex suppresses alternative lengthening of telomeres (ALT).

An amendment to this paper has been published and can be accessed via a link at the top of the paper

RNA binding protein Musashi-2 regulates PIWIL1 and TBX1 in mouse spermatogenesis

RNA-binding proteins (RBP) are important facilitators of post-transcriptional gene regulation. We have previously established that nuclear overexpression of the RBP Musashi-2 (MSI2) during male germ cell maturation is detrimental to sperm cell development and fertility. Herein we determine the genes and pathways impacted by the upregulation of Msi2. Microarray analysis and qPCR confirmed differential gene expression in factors fundamental to the cell cycle, cellular proliferation, and cell death. Similarly, comparative protein expression analysis via iTRAQ, immunoblot, and immunolocalization, identified differential expression and localization of important regulators of transcription, translation, RNA processing, and spermatogenesis. Specifically, the testis-expressed transcription factor, Tbx1, and the piRNA regulator of gamete development, Piwil1, were both found to be targeted for translational repression by MSI2. This study provides key evidence to support a fundamental role for MSI2 in post-transcriptional regulation during male gamete development

Geochemical precursors of the activity of an open-conduit volcano: The Stromboli 2002-2003 eruptive events

Marked increases of CO2, H2 and He dissolved in thermal waters and changes in the dissolved carbon isotopic composition, were observed at Stromboli before the 28 December 2002 eruption and before a violent explosive paroxysm occurred on 5 April 2003. High anomalous CO2 flux values were recorded at the crater rim since a week before the eruption onset. The first anomalies in the thermal waters (dissolved CO2 amount) appeared some months before the eruption, when magma column rose at a very high level in the conduit. High peaks of dissolved H2 and He were recorded a few days before the paroxysm. Carbon isotopic composition indicates a magmatic origin of the dissolved CO2 whose increase, together with those of H2 and He, is attributed to an increasing output of deep gases likely produced by depressurization of a rising batch of a deep gas-rich magma, whose fragments have been emitted during the explosion