bluuebunny/crossref_metadata_2025 huggingface dataset:
523. PLACENTAL ANTIOXIDANT ENZYMES IN RAT PREGNANCY SHOW ZONE- AND STAGE-DEPENDENT VARIATION
Authors: Jones M. L. , Mark P. J. , Mori T. A. , Waddell B. J.
Published: November 2010

Placental oxidative stress plays a key role in the pathophysiology of placenta-related disorders including preeclampsia. Protection from oxidative stress is provided by antioxidant enzymes which inactivate reactive oxygen species (ROS). The rat placenta consists of two major zones, the junctional (JZ) and labyrinth (LZ), and because only the LZ grows in late gestation we hypothesized it generates more ROS and thus requires greater antioxidant protection. Our previous studies on expression of the antioxidants superoxide dismutase (SOD)-1, SOD-2 and catalase support this hypothesis. Here, we extend these observations to include mRNA expression of SOD-3 and thioredoxin reductases (Txnrd-1, -2, -3) and activities of SOD, hydrogen peroxide (H2O2) scavenging and xanthine oxidase (XO). Placental oxidative damage was assessed by measurement of F2-isoprostanes and TBARS concentrations. We also measured the effects of maternal dexamethasone treatment, since glucocorticoid excess is known to induce oxidative damage in other tissues. Placentas were collected from untreated mothers on days 16 and 22 (term=day 23) and on day 22 after dexamethasone treatment from day 13 (1 μg/ml drinking water). SOD-3, Txnrd-1, -2, and -3 mRNAs were measured in JZ and LZ by qRT-PCR. F2-isoprostanes were measured by GC-MS and kit assays were used to measure TBARS and the activities of SOD, H2O2 scavenging and XO. In both placental zones, expression of SOD-3 and Txnrd-1 mRNAs and H2O2 scavenging activity decreased from day 16 to 22, whereas XO activity increased. Dexamethasone treatment increased H2O2 scavenging in both zones, but had no effect on SOD or XO activities or antioxidant mRNA expression. Despite predicted increases in placental ROS generation in late pregnancy and after dexamethasone, neither F2-isoprostanes nor TBARS were increased. These and our previous data suggest that endogenous protection against oxidative stress is abundant in the rat placenta and provides protection against potential oxidative insults including glucocorticoid excess.
528. THE INTERNALIZATION OF ANTIPHOSPHOLIPID ANTIBODIES INTO TROPHOBLASTS CORRELATES WITH THE EXPRESSION OF MEGALIN
Authors: Chamley L. , Viall C. , Stone P. , Chen Q.
Published: November 2010

Antiphospholipid antibodies (aPL) are autoantibodies that increase the risk of preeclampsia nine fold. We have recently shown aPL increase in the number of syncytial knots shed from placental explants and also change the trophoblast death process towards necrosis. Shedding of necrotic syncytial knots is thought to contribute to the pathogenesis of preeclampsia. Antiphospholipid antibodies but not control antibodies, are internalised into the syncytiotrophoblast suggesting a specific mechanism for internalisation of the aPL. Megalin is known to be an endocytic receptor for the antigen of aPL. We believe that the internalisation of aPL into the syncytiotrophoblast is required to for aPL to affect trophoblast shedding and in this study began to investigate the hypothesis that megalin mediates aPL-internalisation. Monoclonal aPL, IIC5 or ID2, were incubated with monolayers of the trophoblast cell lines, Jar, Jeg 3 or BeWo, or first trimester placental explants for 24 hours. Internalisation of aPL into trophoblasts was determined by fluorescent immuno-staining as was the expression of megalin using an antimegalin antibody (Sigma). Experiments were repeated at least three times. Confocal microscopy demonstrated that the syncytiotrophoblast of explants and BeWo cells, but not Jar or Jeg3 cells, internalised the aPL. Despite treating explants and BeWos with the same amount of aPL the level of aPL internalised by the syncytiotrophoblast of explants was greater than the level internalised by BeWos. Megalin was expressed strongly by the syncytiotrophoblast and weakly by BeWos but was not expressed by Jars or Jeg3 cells. The internalization of aPL into syncytiotrophoblasts may play an important role in regulating trophoblast death leading to aberrant shedding of syncytial knots. This study provides preliminary evidence that megalin expression correlates with the ability of trophoblasts to internalize aPL suggesting it may be the receptor that mediates aPL internalization into trophoblasts making this pathway a potential therapeutic target.
529. PROPROTEIN CONVERTASE 6 PLAYS A CRITICAL ROLE IN MODULATING THE HUMAN ENDOMETRIAL EPITHELIUM FOR RECEPTIVITY AND IMPLANTATION
Authors: Nie G. , Li Y. , Salamonsen L. A. , Simon C. , Quiñonero A. , Horcajadas J. , Rombauts L. , Heng S.
Published: November 2010

Successful embryo implantation is an important step in establishing pregnancy, requiring a healthy embryo and a receptive endometrium. Establishment of endometrial receptivity involves morphological and physiological changes initially in the endometrial epithelium, but the underlying molecular mechanisms are not fully understood. We have previously demonstrated that proprotein convertase 5/6 (PC6), a member of the proprotein convertase (PC) family, is up-regulated in the endometrium specifically at implantation in association with epithelial differentiation, in the human and monkey. PCs convert a range of precursor proteins of important functions into their bioactive forms; they are thus regarded as critical “master switch” molecules. The present study aimed to determine whether PC6 is a critical regulator in the endometrial epithelium for receptivity and implantation. We examined whether endometrial epithelial PC6 dys-regulation is associated with implantation failure in women and whether knockdown of PC6 by siRNA in human endometrial epithelial cells affects embryo adhesion in a cell culture model. Endometrial PC6 expression was assessed by immunohistochemistry in the mid-secretory phase of the menstrual cycle (receptive phase) in two unique clinical cohorts comprising women of known fertility and infertility (with no obvious gynecological disorders, and with fertile males). Endometrial epithelial PC6 levels were significantly lower in infertile vs fertile women in both cohorts. To further establish that PC6 is important for receptivity, a cell model relevant to human implantation was used involving co-culture of uterine epithelial cells with mouse embryos. The epithelial cells were stably transfected with PC6 siRNA and PC6 knock down was confirmed at the levels of mRNA, protein, and activity by real-time RT-PCR, Western blotting and activity assay respectively. Embryos readily adhered to normal epithelial cells, but the adhesion was significantly reduced in the PC6 knockdown epithelial cells. We are currently using proteomics technology to identify the pathways affected by PC6 knockdown. These results strongly suggest that PC6 plays a critical role in modulating the human endometrial epithelium for receptivity and implantation.
002. PIG SPERM EGG INTERACTION AND FORMATION OF A ZONA PELLUCIDA BINDING COMPLEX
Authors: Gadella B. M.
Published: November 2010

In order to achieve fertilization sperm cells first need to successfully interact with the zona pellucida. Before reaching the zona pellucida the sperm cell undergoes extensive remodeling both in the male and female genital tract. These changes serve to mediate optimal recognition of the zona pellucida in the oviduct (primary zona pellucida binding). Optimal sperm-zona interactions are crucial for porcine oocyte fertilization: The zona pellucida- attached sperm cell is triggered to undergo the acrosome reaction and will also become hypermotile. Together these two responses allow the sperm cell to drill through the zona pellucida (secondary zona pellucida binding) this coincides with local sperm zona drilling so that a few sperm can reach the perivitellin space. This delaying strategy allows only one sperm cell in a given time-point to bind and fuse with the oocyte (fertilization) and thus minimizes the risk of polyspermy. The polyspermy block is essentially executed by the fertilized oocyte that immediately secretes its cortical granule content into the perivitellin space. This content blocks sperm-oocyte fusion either by sticking to the oolemma or by the induction of a biochemical reaction of the zona pellucida (zona pellucida hardening). The cortical reaction thus blocks sperm-zona pellucida binding and/or sperm-zona pellucida drilling. Note that zona pellcuida interactions under pig IVF conditions relatively frequently result in polyspermy. It is not clear whether this is also the case in vivo after natural mating. More importantly we do not know how other artificial reproductive technologies affect polyspermy rates. This is especially relevant for new sperm treatments and insemination technologies in which sperm are activated by capacitation media essentially mimicking the IVF media. Therefore, better understanding of sperm activation and of the arrangement of proteins involved in zona pellucida interactions are relevant for designing strategies to further improve pig reproduction.
003. OXIDATIVE STRESS, OSMOTIC STRESS AND APOPTOTIC CHANGES: EFFECTS ON EQUINE SPERMATOZOA
Authors: Ball B. A. , Meyers S.
Published: November 2010

Although considerable progress has been made over the past few years in liquid or frozen storage of equine spermatozoa, there remains a large inter-individual difference in the success of semen preservation for the stallion. Equine spermatozoa undergoing low-temperature storage undergo oxidative damage to membrane phospholipids, proteins and chromatin. Osmotic stress also leads to damage to the plasma membrane and alteration in sperm metabolism. Furthermore, evidence from a number of species suggests that ejaculated spermatozoa undergo apoptotic-like changes as a consequence of cryopreservation. It appears likely that these three processes are interlinked and may impact various compartments in the sperm cell via similar pathways. Therefore, an understanding of these processes and their common metabolic pathways may be important in attempts to obviate adverse affects on equine spermatozoa during storage. Further research should evaluate the molecular pathways which may represent convergence of these stresses on the sperm cell with an aim to reducing their net detrimental effect on sperm during preservation.
Supported by the John P. Hughes Endowment, the UC Davis Center for Equine Health, and the National Center for Research Resources, National Institutes of Health.
040. ARE ENVIRONMENTAL EXPOSURES AFFECTING HUMAN REPRODUCTIVE HEALTH?
Authors: Hunt P.
Published: November 2010

The concern that human reproductive health may be affected by chemicals in our daily environment has grown in recent years with the recognition that: (1) some countries have seen a recognizable decline in sperm counts and an increase in urogenital tract abnormalities among newborn males, (2) the incidence of some cancers has increased precipitously, and (3) the number of infertile couples has increased markedly in many countries. Our laboratory focuses on the oocyte and the factors that cause the production of chromosomally abnormal eggs. We know that the risk of a chromosomally abnormal pregnancy is strongly influenced by maternal age, but there is growing concern that environmental exposures may influence the ability of both the male and female to produce normal gametes. Our laboratory has focused on the effect of exposures to a ubiquitous chemical to which humans are exposed daily, bisphenol A (BPA). BPA is used in a wide variety of consumer products from plastics and resin coatings to eyeglasses and pressure printed receipts. Our studies in mice demonstrate that BPA exposure during fetal development adversely affects female fertility because BPA influences several significant stages of egg development. In the male mouse, we and others have found that prenatal, perinatal, and adult exposures can affect the function of the testis. In current studies we are attempting to determine if effects seen in the mouse are also a feature of BPA exposed primates. We are using a rhesus monkey model to determine how BPA is metabolized in the pregnant and nonpregnant female and how BPA exposure influences the developing fetus. Lastly, in human studies we are evaluating BPA levels in the developing fetus and assessing their effect on the developing fetal ovary.
041. ENDOCRINE DISRUPTING IMPACTS IN RECEIVING WATERS OF THE SYDNEY BASIN
Authors: Lim R.
Published: November 2010

Water reuse for a number of activities including potable water and replacement of environmental flows is becoming more significant due to the prolonged drought Australia has recently experienced. There is also much debate regarding potential impacts of compounds such as steroid endocrine disrupting chemicals (EDCs), pharmaceuticals and personal care products (PPCPs), and persistent organic pollutants (POPs) to environmental and human health. This paper presents an overview of findings on some EDCs in the Sydney Basin to assess the environmental risk they pose. A tiered approach, using a suite of endpoints spanning in vitro (e.g., estrogen receptor binding assay, the 2-hybrid yeast test) to in vivo (using the mosquitofish (Gambusia holbrooki) to assess vitellogenin induction, and morphological and behavioural changes) studies was conducted on aquatic systems receiving urban and treated sewage effluents. In vitro bioassays suggest low levels of estrogenicity in sewage contaminated waterways. Both estradiol (E2) and estrone (E1) were identified in all river water samples, suggesting that sewage contamination is widespread. The synthetic hormone, ethynylestradiol (EE2), was below detection limits in all samples tested. Results indicate that the STPs were not the only source of EDCs in aquatic systems within the Sydney area. Improvements in treatment technologies in STPs have substantially reduced EDC levels in final effluent as indicated by a reduction inendocrine disrupting effects on the mosquitofish over several years of study. In addition, advanced tertiary treatment technology removed EDCs to levels below that measurable by in vitro assays and in vivo fish testing. This tiered weight of evidence approach provided insights to the risks EDCs in sewage effluent produced from current treatment technologies have on the environment.
102. CHARACTERISATION OF THE GTPase DYNAMIN THROUGHOUT MURINE SPERM MATURATION
Authors: Reid A. T. , Roman S. D. , Aitken R. , Nixon B.
Published: November 2010

Throughout sperm maturation distinct remodelling events occur that imbue the cells with both the ability to bind the zona pellucida and undergo the acrosome reaction. Of long standing interest to our laboratory is the elucidation of the molecular mechanisms that underpin the attainment of these key functional attributes. This process begins with a complex range of morphological changes that accompany spermatogenesis, and is continued through post-testicular phases of maturation in both the male (epididymal maturation) and female (capacitation) reproductive tracts. However, among these changes only those occurring during the initial stages of spermatogenesis are intrinsically driven. The fact that the majority of sperm remodelling is extrinsically stimulated, and occurs in the absence of new protein synthesis, highlights the potential importance of processes such as intracellular protein trafficking. This has directed our focus towards the dynamin family of protein traffickers. The GTPase dynamin exists in three isoforms, namely Dnm1, Dnm2 and Dnm3 and is an integral part of the molecular machinery required for vesicle mediated protein translocation. Recent research from our laboratory has demonstrated the presence of these three isoforms in distinct, cell-specific locations during murine spermatogenesis. Immunofluorescence on mouse testis revealed that both Dnm1 and 2 are present within a region corresponding to the developing acrosome in maturing sperm, whilst Dnm3 appears to reside solely within pre-meiotic germ cells. Interestingly, Dnm1 and Dnm2 are both retained within the peri-acrosomal region of the sperm head in mature spermatozoa. Additionally, upon the induction of capacitation in vitro, staining for both Dnm1 and 2 becomes significantly reduced. Collectively these data support the novel hypothesis that dynamin not only participates in sperm remodelling events during spermatogenesis but may also have a previously unappreciated role in capacitation-associated priming of the sperm surface for interaction with the oocyte.
104. LRGUK - A NOVEL GENE INVOLVED IN MALE FERTILITY
Authors: Deboer K. , Borg C. , Jamsai D. , Prawer R. , Adams V. , O'Bryan M.
Published: November 2010

Infertility affects a large number of Australian men. The causative factor in many of these cases is likely to be genetic in origin. As such, the identification and characterisation of novel genes involved in male fertility represents an important area of research. We have undertaken an N-ethyl-nitrosourea (ENU) mutagenesis screen to identify novel genes involved in male fertility. From this screen we have identified a mouse line that we have designated ‘Kaos’. Male mice that are homozygous for the Kaos mutation are infertile as a result of severely disrupted spermatogenesis, with Kaos homozygote males containing only 20% of the normal number of elongated spermatids in their testes, when examined by nuclear resistance to Triton X-100 solubilisation. In contrast, Kaos homozygote females are fertile. To identify the Kaos causal mutation, we used a combination of DNA linkage analysis and candidate gene sequencing. A point mutation was identified in exon 14 of the Leucine-rich Repeats and Guanylate Kinase domain containing (Lrguk) gene, which introduces a pre-mature stop codon into the Lrguk coding sequence. The function of the LRGUK protein is currently unknown. However preliminary expression and phenotype analysis suggests that Lrguk may have a role in spermiogenesis (i.e. haploid germ cell development). The deduced LRGUK protein contains several potentially important domains including a Guanylate Kinase-like (GK) domain and a number of leucine-rich repeats. The GK-like domain has some homology to guanylate kinase, a metabolic enzyme involved in purine nucleotide metabolism. Studies are currently underway to determine whether the LRGUK protein possesses guanylate kinase enzyme activity. In other proteins, the GK-like domain has evolved into mediating protein-protein interactions (1), and current studies are also aimed at examining this possibility. These studies should provide insight into the function of LRGUK and its role in spermatogenesis.
(1) Montgomery J, et al. (2004). MAGUKs in synapse assembly and function: an emerging view. Cell Mol Life Sci. 61(7–8): 911–929.
109. AGGREGATING CLONED WITH IN VITRO FERTILISED EMBRYOS RESULTS IN CHIMAERAS AND IMPROVED FETAL SUVIVAL IN CATTLE
Authors: Oback F. C. , Delaney T. , Oliver J. E. , Berg M. C. , Lee R. S. F. , Wells D. N.
Published: November 2010

Cloning cattle by somatic cell nuclear transfer (NT) results in low survival and high frequencies of abnormal placentation and fetal development. We postulate that such anomalies may be overcome by complementing NT embryos with in vitro fertilised (IVF) embryos to form chimaeras. The gender and germline composition of chimaeras can be experimentally manipulated. Using embryological methods, we aim to produce chimaeric fetuses that are functionally male and produce sperm derived from the somatic NT embryo. Provided sufficient contribution from the IVF embryo, such chimeras should develop more normally than clones. At the 12- to 16-cell stage, individual male NT embryos were aggregated with female IVF embryos derived from X-sorted sperm. Following aggregation, there were no significant difference in blastocyst development between NT/IVF aggregates and disaggregated and re-aggregated IVF and NT controls (86/183 = 47% v. 77/233 = 33% v. 47/109 = 43%, respectively). Suitable quality embryos were transferred individually into synchronised recipient animals. Pregnancy establishment at Day (D) 35 was not significantly different between aggregate, IVF and NT groups (18/57 = 32% v. 11/45 = 24% v. 6/31 = 19%, respectively). Whilst there was no difference in survival between aggregates and IVF controls to ~D100, aggregates survived significantly better than NT controls (16% v. 18% v. 0%; respectively; P < 0.05). In the aggregate group, 7/8 fetuses recovered were phenotypically male. Using RT-PCR, expression of the female-specific mRNA for Xist was detected in 4/5 liver samples, indicating chimaerism. Despite improved survival to ~D100 compared to NT, 3/7 fetuses in the aggregate group still displayed evidence of abnormalities, such as fetal overgrowth. Further studies will explore alternative aggregation strategies and germline transmission of the NT-derived genome in chimaeras.
125. ELEVATED FSH INCREASES PRIMORDIAL FOLLICLE RESERVE WITHOUT INCREASING PRIMORDIAL FOLLICLE FORMATION OR DECREASING OOCYTE QUALITY
Authors: Allan C. M. , McTavish K. J. , Lamb S. , Walters K. A. , Handelsman D. J.
Published: November 2010

Declining ovarian follicle reserve and oocyte quality with age can dictate the mammalian female reproductive lifespan. Elevated circulating levels of follicle-stimulating hormone (FSH) coincides with reproductive ageing in women, and is predicted to accelerate the depletion of ovarian follicle reserve by increasing the recruitment of remaining follicles. Unexpectedly, we found that transgenic expression of human FSH (TgFSH) increased primordial follicle reserve in mature GnRH-deficient hypogonadal (hpg) female mice functionally lacking endogenous gonadotrophin secretion1. Advancing our finding of increased primordial reserve in TgFSH-hpg mice, we find that total primordial follicle numbers is significantly increased (60%) in mature 26 week old non-hpg TgFSH females compared to WT controls. Thus, the FSH-induced increase in primordial reserve is maintained despite increased ovulation rate2 in mature TgFSH females. Embryo transfer experiments showed that embryos derived from 26 week old TgFSH females developed normally in recipient WT females, indicating no significant reduction in the viability of oocytes or early embryos produced by TgFSH mice. In addition, equal primordial numbers in 5 day old TgFSH versus WT ovaries suggested that FSH-induced changes to ovarian reserve was not due to an increased initial primordial follicle pool. Initial analysis of postnatal gene expression (qPCR) found elevated expression of granulosa cell markers (Kit-L, inhibin β) in TgFSH-hpg vs non-Tg hpg ovaries, indicating FSH actions during early follicle development. Therefore, contrary to a classical view that preantral follicles are gonadotrophin-independent, we provide in vivo evidence demonstrating FSH-induced changes to early follicle populations, as well as elevated FSH activity increasing or maintaining primordial reserve without compromising oocyte quality.
(1) Allan CM et al. J Endocrinol. 2006; 188(3): 549–57.(2) McTavish KJ et al. Endocrinology. 2007; 148(9): 4432–9.
129. OVARIAN PHENOTYPE OF THE IKK CONDITIONAL KNOCKOUT MOUSE
Authors: Drummond A. E. , Kuyznierewicz I. , Fuller P. J.
Published: November 2010

Nuclear factor-κB (NFκB) designates a family of transcription factors that has been shown to modulate antiviral, inflammatory and immune responses and promote tumorigenesis. Activation of NFκB is dependent on IKKβ, a component of the IκB kinase (IKK) complex which promotes degradation of IκB inhibitory proteins and allows nuclear translocation of NFκB. Little is known about the role of NFκB signalling in the ovary. We created a gonadal specific IKKβ conditional knockout mouse to explore NFκB action in the ovary. A transgenic mouse line containing floxed IKKβ alleles (M Karin, UCSD) was crossed with a transgenic mouse line expressing cre-recombinase under the control of the anti-Müllerian hormone receptor (AMHR) promoter (M Matzuk, BCM). Female mice arising from this breeding regime will not express IKKβ in granulosa cells and cannot activate NFκB signalling. Ovaries and serum were collected from mice at 7 and 15 weeks of age. Histological analyses were undertaken and the gonadotrophic hormones, follicle stimulating hormone (FSH) and luteinising hormone (LH) were measured. The ovaries of 7 week old IKKβ null mice contained follicles of all developmental stages although corpora lutea were absent indicating that these mice were infertile. Some follicle subtypes may be under-represented and apoptosis may be enhanced; these studies are ongoing. Serum FSH and LH levels were elevated compared to littermate controls. By 15 weeks of age corpora lutea were present. The fertility of the IKKβ conditional knockout is currently being assessed. In summary, IKKβ conditional knockout mice exhibit a reproductive phenotype which includes delayed ovulation. These results validate the hypothesis that ovulation is an inflammatory-like response. This model will be a valuable tool for reproductive research; the subtlety of the phenotype allowing us to tease out the underlying mechanisms and role of NFκB signalling in ovarian function.
136. PROGESTERONE RECEPTOR-REGULATED GENES IN THE PREOVULATORY OVARIAN FOLLICLE AND OVIDUCT
Authors: Akison L. , Russell D. , Robker R.
Published: November 2010

Ovulation is a highly regulated and precisely timed reproductive process but the underlying molecular mechanisms are not well understood. Progesterone receptor (PGR) is a transcription factor highly yet transiently expressed in granulosa cells (GCs) of preovulatory follicles; has low expression in cumulus-oocyte-complexes (COCs); and is abundantly expressed in the oviduct. PR–/– mice validate its essential role in ovulation as they are anovulatory, despite normal growth and development of ovarian follicles and oocytes. Our aim was to use microarray to identify differentially expressed genes in GCs, COCs and oviducts from PR–/– and PR+/– mice, specifically genes potentially involved in oocyte release and transport. GCs, COCs and oviducts were collected from 21d-old mice (n = 5; 3 mice/replicate) at 8h post-hCG/44h post-eCG. Extracted RNA samples were hybridized to Affymetrix Mouse Gene 1.0 ST Arrays and post-experiment processing/analysis performed using Partek Genomics Suite. Gene ontology analysis was performed using Ingenuity Pathway Analysis (IPA). In GCs, 296 genes were differentially expressed (P < 0.05); 78% down-regulated in PR–/–. IPA identified genes involved in cancer migration/invasion, chemotaxis, and adhesion; the chemokine receptor Cxcr4, was >3-fold down-regulated in PR–/–. Proteases were also decreased; Adam8 (3.5-fold) and Adamts1 (2.6-fold) in PR–/–. In oviducts, 1003 genes were differentially expressed at P < 0.05 and 266 genes at P < 0.01; 93% were down-regulated in PR–/–. IPA identified genes involved in cell adhesion, movement/migration, invasion and chemotaxis as well as muscle contraction and vasoconstriction. The most highly down-regulated was Itga8 (>9-fold), one of 11 integrins, well known cellular adhesion receptors, differentially expressed. In COCs, 44 genes were differentially expressed (P < 0.05); 52% down-regulated in PR–/–. IPA identified 18 genes (41%) involved in cancer invasion/migration or adhesion. Thus, this study has identified novel gene targets for PGR regulation, which may have essential roles in the molecular control of oocyte release into the oviduct at ovulation.
149. TRANSLATIONAL CONTROL IN FOLLICULOGENESIS AND OOCYTE DEVELOPMENT: A ROLE FOR RNA-BINDING PROTEIN MUSASHI-1
Authors: Gunter K. M. , Fraser B. A. , Sobinoff A. P. , Pye V. , Siddall N. A. , Hime G. R. , McLaughlin E. A.
Published: November 2010

Control of the maternal mRNA pool during oocyte maturation is crucial to the correct temporal and spatial expression of proteins, particularly during oocyte transcriptional quiescence. We have identified Musashi-1 as being present within the oocyte/ovary, where this RNA-binding protein is believed to act as a translational repressor of target mRNAs. Recent studies in mammalian neural and intestinal systems have identified a number of cell cycle regulators as potential targets of Msi-1. Using Msi-1 protein-RNA immunoprecipitation, we have also identified musashi-2 (msi-2) and c-mos as putative targets in the mouse oocyte. To further study these targets, a transgenic mouse was produced to overexpress Msi-1 exclusively in the oocyte. QPCR analysis, performed on intact ovaries of wild type (WT) and Tg mice, confirmed a 1.5-fold increase in msi-1 expression in tgMsi-1/+ ovaries in excess of WT ovary expression. QPCR analysis of Msi-1 target expression, performed on intact WT and Tg ovaries, in conjunction with transcript obtained from the Msi-1 protein-RNA immunoprecipitation, revealed an overall increase in expression in the tgMsi-1/+ and Msi-1 IP samples, respectively, of p21WAF-1 (~2.5-fold; undetected), cdkn2a (~2-fold; undetected), notch1 (~3-fold;undetected), c-mos (no difference; ~41-fold) and msi-2 (~7-fold; ~10-fold). Immunohistochemical analysis of Msi-2 protein expression in transgenic juvenile mouse ovaries,demonstrated a decrease in expression of Msi-2 in tgMsi-1/+ ovaries, when compared to WT ovary expression, suggesting that Msi-2 mRNA is translationally repressed by Msi-1. Therefore, preliminary analysis suggests that Msi-1 may play a role inregulating transcripts of genes necessary for processes characteristic of meiotic progression and oocyte development.
151. PLACENTAL HtrA3 IS REGULATED BY OXYGEN TENSION AND SERUM LEVELS ARE ALTERED DURING EARLY PREGNANCY IN WOMEN DESTINED TO DEVELOP PREECLAMPSIA
Authors: Nie G. , Li Y. , Puryer M. , Salamonsen L.
Published: November 2010

The pathogenic origin of preeclampsia is defective placental development (placentation) and function. Preeclampsia is not diagnosed until later in pregnancy and reliable early detection is highly desirable. HtrA3 is a recently cloned gene with high expression during placentation in the mouse, rhesus monkey and human. In human 1st trimester placenta, HtrA3 is highly expressed in maternal decidual cells and in certain trophoblast cell types. Placental HtrA3 is secreted into the maternal circulation and clearly detectable in serum of pregnant women in the 1st trimester. The present study examined placental production and serum profile of HtrA3 across gestation in women, the potential molecular mechanisms regulating HtrA3 production, and association between maternal HtrA3 serum levels and preeclampsia. Immunohistochemistry determined HtrA3 expression pattern and cellular localization in 1st, 2nd and 3rd trimester placenta. Maternal serum HtrA3 levels were analysed by Western blotting. Regulation of placental HtrA3 production and secretion by oxygen tension was investigated in 1st trimester placental explants and trophoblast cells. Placental HtrA3 protein was maximally produced in the 1st trimester, then dramatically down-regulated, especially in the syncytiotrophoblast. HtrA3 was secreted into the maternal circulation with a serum profile reflecting placental production. Oxygen tension regulated HtrA3; low oxygen enhanced, while transition from low-to-high oxygen decreased, HtrA3 protein production in syncytiotrophoblast. Maternal serum HtrA3 levels at ~13-14 weeks of gestation were significantly higher in women who subsequently developed preeclampsia. It appeared that HtrA3 down-regulation was delayed in preeclamptic pregnancies. In conclusion, HtrA3 protein production is closely associated with oxygen tension in the placenta. The decline in HtrA3 at the end of 1st trimester may reflect the placental low-to-high oxygen switch. Abnormally high levels of serum HtrA3 at the end of 1st trimester is associated with preeclampsia.
153. COMBINATION METHOTREXATE AND EPIDERMAL GROWTH FACTOR RECEPTOR INHIBITION AS A NOVEL MEDICATION-BASED CURE OF ECTOPIC PREGNANCIES
Authors: Nilsson U. , Johns T. G. , Wilmann T. , Gao Y. , Whitehead C. , Dimitriadis E. , Menkhorst E. , Saglam B. , Greenall S. , Horne A. , Tong S.
Published: November 2010

Ectopic pregnancies are serious gynaecological emergencies that can cause fatal haemorrhage. Most are treated surgically. Given the placenta is heavily reliant on Epidermal Growth Factor Receptor (EGFR) signaling, we set out to develop a medication-based treatment of ectopics using combination gefitinib (EGFR inhibitor) and methotrexate (folate antagonist). Both drugs are well tolerated and available in tablet form. We used in vitro and in vivo approaches to test the ability of gefitinib and methotrexate to regress placental tissue, and to explore molecular mechanisms. In vitro assays included immunohistochemistry (EGFR staining) western blot (EGFR phosphorylation), cell viability assays (Cell-Titre Blue, LDH cytotoxicity assay, xCELLigence system), apoptosis assays (PCR, FACS of M30 antibody) and The Bioplex Platform (phosphorylation of the EGFR pathway). JEG-3 xenografts were used to assess regression of placental tissue in vivo, where serum hCG was also measured (ELISA). EGFR was highly expressed in placentae from ectopic pregnancies. Combination treatment was supra-additive in inducing cell death of placental tissue in vitro, with >70% cell death by 48 hours (Syncytialised BeWos, JEG-3 and 1st trimester trophoblast). This supra-additive effect was demonstrated in both end point assays (cell viability) and regular monitoring using The xCELLigence system. Gefitinib potently blocks EGFR phosphorylation in placental tissues in vitro (Western blot). Both drugs may be converging to inhibit Akt phosphorylation (Bioplex analysis). Combination treatment increases apoptosis (FACS of M30 antibody). In vivo, Gefitinib or MTX as single agents induced significant decrease in xenograft tumour volume in a dose dependent manner (n ≤ 5 mice per treatment). However, combining these drugs was supra-additive in decreasing xenograph tumour volume and weight. Serum hCG in mice was lowest with combination treatment. Combination gefitinib and methotrexate potently regresses placental tissue. It may be a novel therapeutic approach to cure ectopic pregnancies, potentially replacing surgery with tablets.
154. EXTRACELLULAR CALRETICULIN ALTERS ENDOTHELIAL CELL AND TROPHOBLAST CELL FUNCTIONS IN VITRO CONSISTENT WITH PRE-ECLAMPSIA
Authors: Gude N. M. , Crawford K. E. , Stevenson J. L. , Brennecke S. P.
Published: November 2010

Pre-eclampsia is a multisystem disorder of human pregnancy that involves abnormal placentation via insufficient trophoblast cell invasion of the maternal spiral arteries and widespread maternal endothelial cell dysfunction. Factors in plasma of pre-eclamptic women affect both trophoblast and endothelial cell functions during in vitro culture (1). The calcium-binding protein calreticulin is elevated in peripheral blood with pre-eclampsia compared to normotensive pregnancy (2). The aim of this study was to determine the effects of exogenous calreticulin at concentrations relevant to normotensive pregnancy (2 µg/mL) and to pre-eclampsia (5 µg/mL) on human trophoblast cell (HTR8) and microvascular endothelial cell (myometrial) numbers and migratory activity. Cell migration was measured by scratch assay; changes in cell number were measured by MTS assay (Promega). The results showed that calreticulin at 5µg/mL did not affect HTR8 cell number (control 68044+24542 cells, with calreticulin 72810 + 30673 cells, n = 3, P > 0.05) after 48 hours, but significantly inhibited migration of the cells by 48+11% compared to the control at 26 hours (n = 4, P < 0.02). Calreticulin at 5 µg/mL and under conditions that did not change cell number significantly increased migration of the myometrial endothelial cells by 39+7% (n = 4, P < 0.01) at 20 hours. Calreticulin at 5 µg/mL, however, significantly reduced endothelial cell numbers after 3–5 days (control 6213 + 1937 cells, with calreticulin 1937+728 cells, n = 6, P < 0.05). There was no significant change to the functions of either cell type with 2 µg/mL of calreticulin. In conclusion, exogenous calreticulin at a concentration consistent with that found in maternal blood with pre-eclampsia was shown to alter trophoblast and endothelial cell migratory activity and reduce endothelial cell numbers during in vitro culture. These results indicate that elevated circulating calreticulin may contribute to the cellular mechanisms that underlie the development of pre-eclampsia.
(1) Harris et al, Reprod Sci, 2009, 16: 1082–90.(2) Gu et al, Molec Human Repro, 2008, 14: 309–15.
160. THE ROLE OF PHAGOCYTOSIS OF APOPTOTIC SYNCYTIAL KNOTS IN THE PREVENTION OF ENDOTHELIAL CELL ACTIVATION: AN IMPORTANT ADAPTATION FOR NORMAL PREGNANCY
Authors: Chen Q. , Jin H. , Stone P. , Chamley L.
Published: November 2010

Preeclampsia is characterised by an exaggerated inflammatory response and maternal endothelial cell activation. Syncytial knots, dead multinucleated fetal cells shed from the placenta in large numbers during all pregnancies, may be phagocytosed by maternal endothelial cells. Our previous studies showed that phagocytosis of necrotic but not apoptotic syncytial knots led to endothelial cell activation. It is known that phagocytosis of apoptotic cells leads to active tolerance of immune responses and in this study we questioned whether phagocytosis of apoptotic syncytial knots leads to suppression of the endothelial cells ability to be activated. Syncytial knots were harvested from 1st trimester placental explants. Monolayers of endothelial cells were pre-treated with apoptotic syncytial knots for 24 h. After washing, the endothelial cells were treated with the endothelial cell activators LPS, PMA, IL-6, or necrotic syncytial knots for 24 h. In some experiments the inhibitor of phagocytosis, cytochalasin D, was added into the cultures along with apoptotic syncytial knots. Endothelial cell-surface ICAM-1 was measured using cell based ELISAs. Expression of ICAM-1 by endothelial cells that had phagocytosed apoptotic syncytial knots prior to treatment with LPS, PMA, IL-6, or necrotic syncytial knots was significantly (P =/<0.003) reduced, compared to control endothelial cells that had not phagocytosed apoptotic syncytial knots. Inhibiting phagocytosis of apoptotic syncytial knots with cytochalasin D abolished this protective effect. Our data suggest phagocytosis of apoptotic syncytial knots results in the suppression of the ability of endothelial cells to be activated by a number of potent chemical activators, as well as by the physiologically relevant activator, necrotic syncytial knots. This work suggests that the release of apoptotic syncytial knots from the placenta during normal pregnancy may be a mechanism by which the fetus attempts to protect the maternal vasculature against activation.
163. POTENTIAL MARKERS FOR THE PROSPECTIVE ISOLATION OF HUMAN ENDOMETRIAL EPITHELIAL STEM/PROGENITOR CELLS
Authors: Gargett C. E. , Tan C. S. C. , Buhring H.
Published: November 2010

The human endometrium regenerates each month following menstruation, parturition and in post-menopausal women taking hormone replacement therapy. Adult stem/progenitor cells discovered residing in human and mouse endometrium may be responsible for this regenerative capacity. However, assays used to identify these stem/progenitor cells are retrospective. The aim of this study is to identify surface markers for the prospective isolation of human endometrial epithelial progenitor cells using a panel of 22 antibodies. Flow cytometry and was used in the initial screen and immunohistochemistry was used to reveal the location of marker expression. Multi-colour FACS protocols were developed with promising markers in conjunction with EpCAM (epithelial cell marker) and to exclude endothelial (CD31+), leukocytes (CD45+) and stromal (CD90+) cells. Sorted subpopulations were assessed for clonogenicity and self-renewal activity using in vitro cloning assays. Six antibodies were short-listed. 2D1D12 enriched for progenitor cells that formed epithelial clones in culture (n = 2). The 2D1D12+EpCAM+ fraction produced very few colony-forming units (CFU). 2D1D12–EpCAM+ fraction gave rise to small CFU. However the 2D1D12+EpCAM– population showed the greatest progenitor activity, producing many large CFU that could be serially cloned twice, indicating self renewal activity. This preliminary data suggests that 2D1D12+EpCAM–CD90–CD31–CD45– population may enrich for human endometrial epithelial stem/progenitor cells. Importantly, large CFU have previously been reported to exhibit stem cell properties of self-renewal, differentiation and high proliferative potential (1). Future studies will focus on xenografting this population to assess tissue reconstitution ability in vivo. The identification of endometrial epithelial stem/progenitor cell marker(s) will enable their prospective isolation for further characterisation and will assist in the investigation of their potential role in endometrial proliferative disorders such as endometriosis and endometrial cancer.
(1) Gargett CE et al (2009) Biol Reprod 80: 1136–45.
166. EMBRYO IMPLANTATION IS ASSOCIATED WITH SPECIFIC EXPRESSION OF PROPROTEIN CONVERTASE 6 IN THE RABBIT UTERUS
Authors: Li Y. , Nicholls P. K. , Heng S. , Sun Z. , Wang J. , Nie G.
Published: November 2010

Proprotein convertase 5/6 (PC6) is a member of the proprotein convertase family that endoproteolytically cleave latent precursor proteins into their biologically active state. We have previously demonstrated that endometrial PC6 is critical for embryo implantation in mice and primates, including human. PC6 regulates the endometrial physiology specifically at implantation in association with epithelial differentiation during the establishment of endometrial receptivity (in human and monkey) and stromal cell decidualization (in the mouse, human and monkey). PC6 was further confirmed to be a unique PC member that is tightly regulated in the endometrium in relation to implantation. Our further studies (unpublished) suggest that PC6 regulates adhesion molecules in the endometrial epithelium for implantation in women. It is known that between the mouse and human, the endometrial stroma-mediated responses are similar whereas the epithelial cells behave differently. Because PC6 regulates primarily the stromal component (decidualization) in the mouse, in vivo mouse models are critical to investigate the roles of PC6 in decidualization. To address the function of PC6 in endometrial epithelium, non-mouse models relevant to human implantation are required. The rabbit is regarded as an excellent model to study the molecular events of embryo adhesion and attachment. The current study aimed to determine the expression pattern and localisation of PC6 in the rabbit uterus during early pregnancy. Quantitative RT-PCR analysis showed that PC6 mRNA expression was dynamically up-regulated in the rabbit uterus immediately prior to implantation. Western blotting and immunohistochemical analyses demonstrated that PC6 protein was predominantly localised to the basal glands throughout pregnancy, and up-regulated specifically in the epithelium at the embryo attachment site. These findings suggest that PC6 may play an essential role in rabbit implantation, and that the rabbit is a useful animal model to investigate the function of PC6 during embryo attachment.
167. MUSASHI FAMILY OF RNA BINDING PROTEINS: CELL CYCLE REGULATORS IN SPERMATOGENESIS
Authors: McLaughlin E. A. , Fraser B. A. , Pye V. , Bigland M. , Siddall N. A. , Hime G. R.
Published: November 2010

Mammalian meiosis is a tightly regulated process involving specialized cell cycle progression and morphogenetic changes. We have demonstrated that the Musashi family of RNA binding proteins is implicated in the regulation of spermatogonial stem self renewal and germ cell differentiation. Here we describe the novel mechanism by which the Musashi family proteins, Msi1 and Msi2, act to control exit from spermatogonial mitotic amplification and normal entry into meiosis. Gene and protein analysis indicated overlapping Msi1 and Msi2 profiles in enriched populations of isolated germ cells and reciprocal subcellular expression patterns in spermatogonia and pachytene spermatocytes/ round spermatids in testes sections. Recombinant Msi1 protein-RNA pulldown and microarray analysis coupled with in vitro shRNA knockdown studies in spermatogonial culture and subsequent immunoprecipitation and qPCR established that Msi1 targeted Msi2 mRNA for post transcriptional translational repression. Immunoprecipitation of Msi2 target mRNA and subsequent qPCR together with in vitro shRNA knockdown studies inround spermatidculture identified a cell cycle inhibitor protein CDKN1C (p57kip2) as the principal target of Msi2 translational inhibition. Immunolocalisation of CDKN1C protein indicated that expression of this cell cycle regulator coincided with the nuclear import of Msi1 and the appearance of cytoplasmic Msi2 expression in early pachytene spermatocytes. Using a transgenic Msi1 overexpression mouse model in conjunction with quantitative gene and protein expression, we confirmed Msi1 targeting of Msi2 and subsequent Msi2 targeting of CDKN1C for translational repression in vivo. Ectopic overexpression of Msi1 in germ cellsinduces substantial Msi2 downregulation and aberrant CDKN1C expression, resulting in abnormal spermatogenic differentiation, germ cell apoptosis/arrest and sterility. In conclusion, our results indicate a sophisticated molecular switch encompassing cell cycle protein regulation by Musashi family proteins, is required for normal exit from mitotic division, entry into meiosis and post meiotic germ cell differentiation.
169. THE MECHANISM OF SPERMATID MATURATION - A LINK TO TUMOUR SUPPRESSION
Authors: Jamsai D. , Smith S. J. , O'Connor A. E. , Merriner D. J. , Borg C. , Clark B. , Adams V. , Ormandy C. J. , O'Bryan M. K.
Published: November 2010

To comprehensively uncover novel male fertility regulators, we utilised an unbiased forward genetic screen, ENU mutagenesis. Using this approach, we have identified several novel infertile mouse lines including a male-specific infertile line that we designated ‘Joey’. The mutant Joey mice produced no sperm due to an arrest of male germ cells at the round spermatid stage. The mutation was identified in the RNA binding motif 5 (Rbm5) gene that resulted in an arginine to proline substitution within a highly conserved RNA recognition motif of the protein. The substitution of proline is likely to interfere with RNA binding and/or recognition. In humans, the RBM5 gene maps to a region that is frequently deleted in lung cancers. Ex vivo studies have suggested that RBM5 is a tumour suppressor, apoptosis modulator and RNA splicing regulator. To date, the role of Rbm5 has never been liked to male fertility and the Joey line is the only mouse model of Rbm5 dysfunction. Using our RBM5-specific antibody, we showed that RBM5 is expressed in pachytene spermatocytes and round spermatids. Based on the protein localisation, the proposed role of RBM5 in mRNA processing, the onset of the Joey phenotype, and the site of the identified mutation, we hypothesise that the Rbm5 mutant allele results in a hypomorphic protein, and that RBM5 has an essential role in regulating male germ cell mRNA storage, transport and/or translational regulation of mRNAs that are critical for spermatid maturation. Further, we generated mice compound heterozygous of the Joey Rbm5 mutation and Rbm5 null alleles. We showed that the compound heterozygous males are infertile due to spermatid maturation arrest resembling the Joey mutant males. This result further confirmed the identification of the Rbm5 mutation as a cause of infertility in the Joey mice and a crucial role of Rbm5 in male fertility.