Jessie M. Sutherland

Chlamydia muridarum Infection-Induced Destruction of Male Germ Cells and Sertoli Cells Is Partially Prevented by Chlamydia Major Outer Membrane Protein-Specific Immune CD4 cells

ABSTRACT Chlamydia trachomatis infections are increasingly prevalent worldwide. Male chlamydial infections are associated with urethritis, epididymitis, and orchitis; however, the role of Chlamydia in prostatitis and male factor infertility remains controversial. Using a model of Chlamydia muridarum infection in male C57BL/6 mice, we investigated the effects of chlamydial infection on spermatogenesis and determined the potential of immune T cells to prevent infection-induced outcomes. Antigen-specific CD4 T cells significantly reduced the infectious burden in the penile urethra, epididymis, and vas deferens. Infection disrupted seminiferous tubules, causing loss of germ cells at 4 and 8 wk after infection, with the most severely affected tubules containing only Sertoli cells. Increased mitotic proliferation, DNA repair, and apoptosis in spermatogonial cells and damaged germ cells were evident in atrophic tubules. Activated caspase 3 (casp3) staining revealed increased (6-fold) numbers of Sertoli cells with abnormal morphology that were casp3 positive in tubules of infected mice, indicating increased levels of apoptosis. Sperm count and motility were both decreased in infected mice, and there was a significant decrease in morphologically normal spermatozoa. Assessment of the spermatogonial stem cell population revealed a decrease in promyelocytic leukemia zinc finger (PLZF)-positive cells in the seminiferous tubules. Interestingly, adoptive transfer of antigen-specific CD4 cells, particularly T-helper 2-like cells, prior to infection prevented these effects in spermatogenesis and Sertoli cells. These data suggest that chlamydial infection adversely affects spermatogenesis and male fertility, and that vaccination can potentially prevent the spread of infection and these adverse outcomes.

Intracellular signalling during female gametogenesis.

Female reproductive potential is dictated by the size of the primordial follicle pool and the correct regulation of oocyte maturation and activation--events essential for production of viable offspring. Although a substantial body of work underpins our understanding of these processes, the molecular mechanisms of follicular and oocyte development are not fully understood. This review summarizes recent findings which have improved our conception of how folliculogenesis and oocyte competence are regulated, and discusses their implications for assisted reproductive techniques. We highlight evidence provided by genetically modified mouse models and in vitro studies which have refined our understanding of Pi3k/Akt and mTOR signalling in the oocyte and have discovered a role for Jak/Stat/Socs signalling in granulosa cells during primordial follicle activation. We also appraise a novel role for the metal ion zinc in the regulation of meiosis I and meiosis II progression through early meiosis inhibitor (Emi2) and Mos-Mapk signalling, and examine studies which expand our understanding of intracellular calcium signalling and extrinsic Plcζ in stimulating oocyte activation.

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.

RBM5 Is a Male Germ Cell Splicing Factor and Is Required for Spermatid Differentiation and Male Fertility

Alternative splicing of precursor messenger RNA (pre-mRNA) is common in mammalian cells and enables the production of multiple gene products from a single gene, thus increasing transcriptome and proteome diversity. Disturbance of splicing regulation is associated with many human diseases; however, key splicing factors that control tissue-specific alternative splicing remain largely undefined. In an unbiased genetic screen for essential male fertility genes in the mouse, we identified the RNA binding protein RBM5 (RNA binding motif 5) as an essential regulator of haploid male germ cell pre-mRNA splicing and fertility. Mice carrying a missense mutation (R263P) in the second RNA recognition motif (RRM) of RBM5 exhibited spermatid differentiation arrest, germ cell sloughing and apoptosis, which ultimately led to azoospermia (no sperm in the ejaculate) and male sterility. Molecular modelling suggested that the R263P mutation resulted in compromised mRNA binding. Within the adult mouse testis, RBM5 localises to somatic and germ cells including spermatogonia, spermatocytes and round spermatids. Through the use of RNA pull down coupled with microarrays, we identified 11 round spermatid-expressed mRNAs as putative RBM5 targets. Importantly, the R263P mutation affected pre-mRNA splicing and resulted in a shift in the isoform ratios, or the production of novel spliced transcripts, of most targets. Microarray analysis of isolated round spermatids suggests that altered splicing of RBM5 target pre-mRNAs affected expression of genes in several pathways, including those implicated in germ cell adhesion, spermatid head shaping, and acrosome and tail formation. In summary, our findings reveal a critical role for RBM5 as a pre-mRNA splicing regulator in round spermatids and male fertility. Our findings also suggest that the second RRM of RBM5 is pivotal for appropriate pre-mRNA splicing.

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.

Suppressor of cytokine signaling 4 (SOCS4): moderator of ovarian primordial follicle activation.

Mammalian ovarian primordial follicle activation and regulation is considered as one of the most important stages of folliculogenesis and as such requires exquisite control. Selection of quiescent follicles to enter the growing pool determines the rate of supply of maturing follicles over the female reproductive lifespan. To coordinate this process a range of positive and negative input signals contribute to determine follicle fate. This study demonstrates that the cytokine Leukemia Inhibitory Factor (LIF) activates the Janus Kinase 1/Signal Transducers and Activators of Transcription 3 (JAK1/STAT3) signaling pathway in pre‐granulosa cells and positively regulates primordial follicle activation. Negative regulation of the JAK/STAT pathway is controlled by the suppressor of cytokine signaling 4 (SOCS4) protein, which target members of negative feedback loops, Cardiotrophin like Cytokine (CLC), Poly (rC) Binding Protein 1 (PCBP1), and Cytosolic Malate Dehydrogenase (MDH1) to suppress follicle growth and development. J. Cell. Physiol. 227: 1188–1198, 2012.

The Musashi family of RNA binding proteins: master regulators of multiple stem cell populations.

In order to maintain their unlimited capacity to divide, stem cells require controlled temporal and spatial protein expression. The Musashi family of RNA-binding proteins have been shown to exhibit this necessary translational control through both repression and activation in order to regulate multiple stem cell populations. This chapter looks in depth at the initial discovery and characterisation of Musashi in the model organism Drosophila, and its subsequent emergence as a master regulator in a number of stem cell populations. Furthermore the unique roles for mammalian Musashi-1 and Musashi-2 in different stem cell types are correlated with the perceived diagnostic power of Musashi expression in specific stem cell derived oncologies. In particular the potential role for Musashi in the identification and treatment of human cancer is considered, with a focus on the role of Musashi-2 in leukaemia. Finally, the manipulation of Musashi expression is proposed as a potential avenue towards the targeted treatment of specific aggressive stem cell cancers.

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

ABSTRACT 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. Because 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 and also 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.

Fluorimetric assay of ergotamine.

Studies on the fluorescence properties of ergotamine in water at various pH values, and in several organic solvents are described. An assay procedure for ergotamine, based on its intense fluorescence in ethanol, is presented. Extraction of ergotamine into benzene from basic aqueous solution is followed by transfer of the extract to ethanol for fluorescence determination. The plot of fluorescence intensity vs. concentration is linear up to 5 μg ml-1, and the assay has a limit of detection of 0.002 μg ml-1. Reproducibility data at the 2.5-μg ml-1 level are given.

Developmental Expression of Musashi-1 and Musashi-2 RNA-Binding Proteins During Spermatogenesis: Analysis of the Deleterious Effects of Dysregulated Expression

ABSTRACT 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.