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

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

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

Synthesis and Characterisation of Enynyl, Vinyl and Acetylide Complexes of Osmium(II)

Controlled gene regulation during gamete development is vital for maintaining reproductive potential. During the process of gamete development, male germ cells experience extended periods of inactive transcription despite requirements for continued growth and differentiation. Spermatogenesis therefore provides an ideal model to study the effects of posttranscriptional control on gene regulation. During spermatogenesis posttranscriptional regulation is orchestrated by abundantly expressed RNA-binding proteins. One such group of RNA-binding proteins is the Musashi family, previously identified as a critical regulator of testis germ cell development and meiosis in <i>Drosophila</i> and also shown to be vital to sperm development and reproductive potential in the mouse. We focus in depth on the role and function of the vertebrate Musashi ortholog Musashi-1 (MSI1). Through detailed expression studies and utilizing our novel transgenic <i>Msi1</i> testis-specific overexpression model, we have identified 2 unique RNA-binding targets of MSI1 in spermatogonia, <i>Msi2</i> and <i>Erh</i>, and have demonstrated a role for MSI1 in translational regulation. We have also provided evidence to suggest that nuclear import protein, IPO5, facilitates the nuclear translocation of MSI1 to the transcriptionally silenced XY chromatin domain in meiotic pachytene spermatocytes, resulting in the release of MSI1 RNA-binding targets. This firmly establishes MSI1 as a master regulator of posttranscriptional control during early spermatogenesis and highlights the significance of the subcellular localization of RNA binding proteins in relation to their function

Developmental expression of Musashi-1 and Musashi-2 RNA-binding proteins during spermatogenesis: analysis of the deleterious effects of dysregulated expression

Spermatogenesis is a complex developmental process whereby diploid spermatogenic stem cells become haploid and undergo a series of morphological changes to produce physically mature spermatozoa. Crucial to this process are a number of RNA-binding proteins, responsible for the posttranscriptional control of essential mRNAs and particularly pertinent to the two periods of inactive transcription that occur in spermatogenesis. One such group of RNA-binding proteins is the Musashi family, specifically Musashi-1 (MSI1) and Musashi-2 (MSI2), which act as key translational regulators in various stem cell populations and have been linked with the induction of tumorigenesis. In the present study, we examined the differential expression of mammalian MSI1 and MSI2 during germ cell development in the mouse testis. MSI1 was found to be predominately localized in mitotic gonocytes and spermatogonia, whereas MSI2 was detected in meiotic spermatocytes and differentiating spermatids. Extensive examination of the function of Musashi in spermatogenesis was achieved through the use of two transgenic mouse models with germ cell-specific overexpression of full-length isoforms of Msi1 or Msi2. These models demonstrated that aberrant expression of either Msi1 or Msi2 has deleterious effects on normal spermatogenesis, with Msi2 overexpression resulting in male sterility. Studies undertaken on human testicular seminoma tumors provide further insights into the relevance of MSI1 and MSI2 overexpression as diagnostic markers to human stem cell cancers. Overall this study provides further evidence for the unique functions that RNA-binding protein isoforms occupy within spermatogenesis, and introduces the potential manipulation of the Musashi family proteins to elucidate the mechanisms of posttranscriptional gene expression during germ cell development

Maternal smoke exposure impairs the long-term fertility of a female offspring in a murine model

The theory of fetal origins of adult disease was first proposed in 1989 and in the decades since, a wide range of other diseases from obesity to asthma have been found to originate in early development. As mammalian oocyte development begins in fetal life it has been suggested that environmental and lifestyle factors of the mother could directly impact the fertility of subsequent generations. Cigarette smoke is a known ovotoxicant in active smokers, yet disturbingly 13% of Australian and 12% of US women continue to smoke throughout pregnancy. The focus of our investigation was to characterize the adverse effects of smoking on ovary and oocyte quality in female offspring exposed in utero. Pregnant mice were nasally exposed to cigarette smoke for 12 wk throughout pregnancy/lactation and ovary and oocyte quality of the F1 (maternal smoke exposed) generation was examined. Neonatal ovaries displayed abnormal somatic cell proliferation and increased apoptosis leading to a reduction in follicle numbers. Further investigation found that altered somatic cell proliferation and reduced follicle number continued into adulthood, however, apoptosis did not. This reduction in follicles resulted in decreased oocyte numbers, with these oocytes found to have elevated levels of oxidative stress, altered metaphase II spindle and reduced sperm-egg interaction. These ovarian and oocyte changes ultimately lead to subfertility with maternal smoke exposed animals having smaller litters whilst taking longer to conceive. In conclusion our results demonstrate that in utero and lactational exposure to cigarette smoke can have long lasting effects on the fertility of the next generation of females