Two alternative methods for the retrieval of somatic cell populations from the mouse ovary.

Many modern techniques employed to uncover the molecular fundamentals underlying biological processes require dissociated cells as their starting point/substrate. Investigations into ovarian endocrinology or folliculogenesis, therefore, necessitate robust protocols for dissociating the ovary into its constituent cell populations. While in the mouse, methods to obtain individual, mature follicles are well-established, the separation and isolation of single cells of all types from early mouse follicles, including somatic cells, has been more challenging. Herein we present two methods for the isolation of somatic cells in the ovary. These methods are suitable for a range of applications relating to the study of folliculogenesis and mouse ovarian development. First, an enzymatic dissociation utilising collagenase and a temporary, primary cell culture step using neonatal mouse ovaries which yields large quantities of granulosa cells from primordial, activating, and primary follicles. Second, a rapid papain dissociation resulting in a high viability single cell suspension of ovarian somatic cells in less than an hour, which can be applied from embryonic to adult ovarian samples. Collectively these protocols can be applied to a broad array of investigations with unique advantages and benefits pertaining to both.

Damaging legacy: maternal cigarette smoking has long-term consequences for male offspring fertility.

STUDY QUESTION: What are the effects on fertility of cigarette smoke-induced toxicity on male offspring exposed during the gestational/weaning period? SUMMARY ANSWER: Maternal cigarette smoke exposure during the gestational/weaning period causes long-term defects in male offspring fertility. WHAT IS KNOWN ALREADY: Cigarette smoke is a well-known reproductive toxicant which is particularly harmful to both fetal and neonatal germ cells. However, recent studies suggest a significant portion of young mothers in the developed world still smoke during pregnancy. In the context of male reproductive health, our understanding of the effects of in utero exposure on offspring fertility is limited. STUDY DESIGN, SIZE, DURATION: In this study, 27 C57BL/6 5-week-old female mice were exposed via the nose-only to cigarette smoke (treatment) or 27 were exposed to room air (control) for 6 weeks before being housed with stud males to produce litters. In the treatment group, smoke exposure continued throughout mating, pregnancy and lactation until weaning of pups at 21 days post birth. Male offspring were examined at post-natal days 3, 6, 12, 21 and 98 (adult). PARTICIPANTS/MATERIALS, SETTING, METHODS: Approximately 108 maternal smoke-exposed C57BL/6 offspring and controls were examined. Spermatogenesis was examined using testicular histology and apoptosis/DNA damage was assessed using caspase immunohistochemistry and TUNEL. Sertoli cell morphology and fluctuations in the spermatogonial stem cell population were also examined using immunohistochemistry. Microarray and QPCR analysis were performed on adult testes to examine specific long-term transcriptomic alteration as a consequence of maternal smoke exposure. Sperm counts and motility, zona/oolemma binding assays, COMET analysis and mitochondrial genomic sequencing were also performed on spermatozoa obtained from adult treated and control mice. Fertility trials using exposed adult male offspring were also performed. MAIN RESULTS AND THE ROLE OF CHANCE: Maternal cigarette smoke exposure caused increased gonocyte and meiotic spermatocyte apoptosis (P < 0.01) as well as germ cell depletion in the seminiferous tubules of neonatal and juvenile offspring. Aberrant testicular development characterized by abnormal Sertoli and germ cell organization, a depleted spermatogonial stem cell population (P < 0.01), atrophic seminiferous tubules and increased germ cell DNA damage (P < 0.01) persisted in adult offspring 11 weeks after exposure. Microarray analysis of adult offspring testes associated these defects with meiotic germ cell development, sex hormone metabolism, oxidative stress and Sertoli cell signalling. Next generation sequencing also revealed a high mitochondrial DNA mutational load in the testes of adult offspring (P < 0.01). Adult maternal smoke-exposed offspring also had reduced sperm counts with spermatozoa exhibiting morphological abnormalities (P < 0.01), affecting motility and fertilization potential. Odf2, a spermatozoa flagellum component required for coordinated ciliary beating, was also significantly down-regulated (P < 0.01) in maternal smoke-exposed adult offspring, with aberrant localization along the spermatozoa flagellum. Adult maternal smoke-exposed offspring took significantly longer to impregnate control females and had a slight but significant (P < 0.01) reduction in litter size. LIMITATIONS, REASONS FOR CAUTION: This study examined only one species (mouse) using a smoking model which only simulates human cigarette smoke exposure. WIDER IMPLICATIONS OF THE FINDINGS: This study represents the first comprehensive animal model of maternal smoking on male offspring reproductive function, suggesting that exposure during the gestational/weaning period causes long-term defects in male offspring fertility. This is due to a compromised spermatogonial stem cell population resulting from gonocyte apoptosis and impaired spermatogenic development. This results in significant germ cell damage and Sertoli cell dysfunction, impacting germ cell number, tubule organization, DNA damage and spermatozoa in adult offspring. This study strengthens the current literature suggesting that maternal exposure impairs male offspring fertility, which is currently debated due to conflicting studies. STUDY FUNDING/COMPETING INTERESTS: This study was funded by the Australian Research Council, Hunter Medical Research Institute, National Health and Medical Research Council of Australia and the Newcastle Permanent Building Society Charitable Trust. The authors declare no conflict of interest.

Scrambled and fried: cigarette smoke exposure causes antral follicle destruction and oocyte dysfunction through oxidative stress.

Cigarette smoke is a reproductive hazard associated with pre-mature reproductive senescence and reduced clinical pregnancy rates in female smokers. Despite an increased awareness of the adverse effects of cigarette smoke exposure on systemic health, many women remain unaware of the adverse effects of cigarette smoke on female fertility. This issue is compounded by our limited understanding of the molecular mechanisms behind cigarette smoke induced infertility. In this study we used a direct nasal exposure mouse model of cigarette smoke-induced chronic obstructive pulmonary disease to characterise mechanisms of cigarette-smoke induced ovotoxicity. Cigarette smoke exposure caused increased levels of primordial follicle depletion, antral follicle oocyte apoptosis and oxidative stress in exposed ovaries, resulting in fewer follicles available for ovulation. Evidence of oxidative stress also persisted in ovulated oocytes which escaped destruction, with increased levels of mitochondrial ROS and lipid peroxidation resulting in reduced fertilisation potential. Microarray analysis of ovarian tissue correlated these insults with a complex mechanism of ovotoxicity involving genes associated with detoxification, inflammation, follicular activation, immune cell mediated apoptosis and membrane organisation. In particular, the phase I detoxifying enzyme cyp2e1 was found to be significantly up-regulated in developing oocytes; an enzyme known to cause molecular bioactivation resulting in oxidative stress. Our results provide a preliminary model of cigarette smoke induced sub-fertility through cyp2e1 bioactivation and oxidative stress, resulting in developing follicle depletion and oocyte dysfunction.

Streptococcus pneumoniae infection suppresses allergic airways disease by inducing regulatory T-cells.

An inverse association exists between some bacterial infections and the prevalence of asthma. We investigated whether Streptococcus pneumoniae infection protects against asthma using mouse models of ovalbumin (OVA)-induced allergic airway disease (AAD). Mice were intratracheally infected or treated with killed S. pneumoniae before, during or after OVA sensitisation and subsequent challenge. The effects of S. pneumoniae on AAD were assessed. Infection or treatment with killed S. pneumoniae suppressed hallmark features of AAD, including antigen-specific T-helper cell (Th) type 2 cytokine and antibody responses, peripheral and pulmonary eosinophil accumulation, goblet cell hyperplasia, and airway hyperresponsiveness. The effect of infection on the development of specific features of AAD depended on the timing of infection relative to allergic sensitisation and challenge. Infection induced significant increases in regulatory T-cell (Treg) numbers in lymph nodes, which correlated with the degree of suppression of AAD. Tregs reduced T-cell proliferation and Th2 cytokine release. The suppressive effects of infection were reversed by anti-CD25 treatment. Respiratory infection or treatment with S. pneumoniae attenuates allergic immune responses and suppresses AAD. These effects may be mediated by S. pneumoniae-induced Tregs. This identifies the potential for the development of therapeutic agents for asthma from S. pneumoniae.

Bioanalytical utility of sonovoltammetry.

Dopamine dissolved within egg homogenate was used as a model system to study the effects of electrode contamination and its subsequent reactivation through ultrasonically mediated in situ cleaning effects. The merits in conducting electroanalytical investigations under the influence of the ultrasonic field were also appraised. Maintaining the ultrasound field during oxidative measurements was found to yield hydrodynamic profiles that were linear over the concentration range 2-20 microM dopamine. The resulting sonolinear sweep voltammograms were compared with conventional rotating disk measurements, with the former found to provide significantly increased limiting currents that were attenuable through the manipulation of the field intensity. The problem of retaining selectivity in the presence of high concentrations of ascorbate was also assessed with the addition of cupric ion prior to commencing the measurements found to efficiently negate an otherwise substantive interference.

Sonoelectrochemically enhanced determination of 5-aminosalicylic acid.

The electrochemical detection of the anti-inflammatory drug 5-aminosalicylic acid (5-ASA) has been evaluated through the application of linear sweep, square wave and sonolinear sweep voltammetry. The introduction of ultrasound is shown to significantly enhance the oxidation signal intensity thereby enabling the detection of low concentrations with the linear range (1-57 muM) adequate for assessing free drug within physiological samples. Interference from ascorbic acid can be effectively negated through the introduction of cupric ion without any appreciable cost to the voltammetric response to 5-ASA. The efficacy of employing sonolinear sweep voltammetry to the determination of this compound within a compositionally complex tissue culture medium has been assessed with the recovery of 5 muM 5-ASA found to be 102% (RSD=5%, N=3).

Voltammetric investigation of hair dye constituents: application to the quantification of p-phenylenediamine.

The application of electrochemical techniques to the determination of common hair dye constituents was investigated. Cyclic voltammetry was used to probe the electrode response to p-phenylenediamine (PPD), 4-chlororesorcinol and m-aminophenol with square wave voltammetry used to effect quantitative measurements. The nature of the electrode response was characterised and issues of surface fouling resulting from the deposition of electrogenerated reaction products were addressed. A procedure designed to allow the facile determination of PPD in hair care products is presented.