bluuebunny/crossref_metadata_2025 huggingface dataset:
003. ENDOMETRIUM: CINDERELLA TISSUE IN A STEM CELL WORLD
Authors: Gargett C. E.
Published: November 2010

Despite human endometrium undergoing more than 400 cycles of regeneration, differentiation and shedding during a woman's reproductive years, and that in non-menstruating species (eg rodents) there are cycles of endometrial growth and apoptosis, endometrial stem/progenitor cells have only recently been identified. Since there are no specific stem cell markers, initial studies using functional approaches identified candidate epithelial and stromal endometrial stem/progenitor cells as colony forming cells/units (CFU) (1). Further evaluation of key stem cell properties of individual CFU demonstrated that rare EpCAM+ epithelial cells and EpCAM- stromal cells underwent self renewal by serial subcloning >3 times and underwent >30 population doublings in culture. Clonally-derived epithelial cells differentiated into cytokeratin+ gland-like structures. Single stromal cells were multipotent as they differentiated into 4 mesodermal lineages; myogenic, adipogenic, osteoblastic and chondrogenic, suggesting that human endometrium contains a rare population of epithelial progenitor cells and mesenchymal stem cells (MSC) (2). Transplantation of freshly isolated human endometrial cells into immunocompromised mice reconstructed endometrial tissue that responded to estrogen and progesterone (3). Endometrial MSC can be prospectively isolated by co-expression of CD146 and PDGFRβ (4), but not Stro-1, a bone marrow MSC marker (5). Currently there are no known markers of endometrial epithelial progenitor cells. Endometrial cancer tissue harbours a small subpopulation of clonogenic, self-renewing, tumour-initiating cells, producing tumours that recapitulate parent tumours in histoarchitecture and differentiation markers (ERα, PR, cytokeratin, vimentin) when xenografted into mice, suggesting they are cancer stem cells. Candidate epithelial and stromal stem/progenitor cells have been identified in mouse endometrium as label retaining cells (LRC) in the luminal epithelium and perivascular cells at the endometrial-myometrial junction, respectively (6). It is likely that endometrial stem/progenitor cells play key roles in the development of gynecological diseases associated with abnormal endometrial proliferation such as endometriosis and endometrial cancer (7).
005. PREPARING FERTILE SOIL: THE IMPORTANCE OF ENDOMETRIAL RECEPTIVITY
Authors: Salamonsen L. A.
Published: November 2010

The human endometrium is receptive for implantation of a blastocyst, for only 4–5 days in each menstrual cycle. Failure of implantation is a major reason for infertility in women, and the inability to achieve endometrial receptivity is responsible for much of the failure of reproductive technologies. Endometrial receptivity requires alterations in the uterine luminal and glandular cells, particularly in terms of their secretory capacity and altered expression of adhesion molecules, along with decidualization of the endometrial stroma, which in women is initiated during the receptive phase, regardless of the presence of a blastocyst. Increased leukocyte numbers are also important. The microenvironments provided by the endometrium during the receptive phase and which support implantation are highly complex and constantly changing. The present review summarizes work from our laboratories and others, regarding these microenvironments, how they impact on receptivity and how they are disturbed in infertile women. Such microenvironments can also be manipulated to provide new contraceptive strategies for women.
007. INFLAMMATORY PATHWAYS IN ENDOMETRIAL CANCER
Authors: Jabbour H. N.
Published: November 2010

Endometrial cancer is the most common gynaecological malignancy and accounts for 5% of cancers in women (http://info.cancerresearchuk.org/cancerstats/). The majority of endometrial cancers occur in post-menopausal women and 80% of patients are diagnosed when the tumour is confined to the uterus (stage 1 disease). Many of the risk factors for developing endometrial cancer are associated with excess exposure to oestrogen unopposed by progesterone. It is well established that local inflammatory pathways contribute to the initiation and progression of endometrial cancers via the release of local mediators to facilitate immune cell recruitment, angiogenesis and neoplastic cell proliferation and metastasis. Prostaglandins are one class of molecules that are important mediators of these processes. Prostaglandins are bioactive lipids produced from arachidonic acid by cyclooxygenase (COX) enzymes and specific terminal prostanoid synthase enzymes. Following biosynthesis, prostaglandins are rapidly transported outside the cell and exert an autocrine/paracrine function by coupling to specific prostanoid G protein-coupled receptors (GPCR). Expression of COX enzyme, synthesis of prostaglandins and expression of various prostaglandin receptors are elevated in pathologies of the endometrium including cancer. Using genome wide array analysis, we have identified target genes that are regulated by prostaglandins such as PGF2α via interaction with its GPCR - FP receptor. Gene ontology analysis have highlighted significant elevation in expression of genes involved in inflammatory and vascular processes which are central to endometrial function. Moreover, using an array of cellular and molecular techniques and in vivo models we have established an important role for PGF2α-FP interaction in regulating inflammatory and vascular functions. To-date, suppression of the activity of prostaglandins in pathology has focussed on using inhibitors of cyclooxygenase enzymes, which are key enzymes in the pathway to prostaglandin synthesis. However, this has been associated with serious cardiovascular side effects. Development of novel drugs that target specific receptors may provide better therapeutic alternatives.
009. KALLIKREIN-RELATED PROTEASES AS NOVEL THERAPEUTIC TARGETS IN PROSTATE AND OVARIAN CANCER
Authors: Clements J. A. , Dong Y. , Loessner D. , Tan O. , Sieh S. , Reichert J. , Burke L. , Stephens C. , Lawrence M. , Stansfield S. , Swedberg J. , Ramsay A. , Hooper J. , Harris J. , Hutmacher D.
Published: November 2010

The kallikrein-related (KLKs) peptidases are implicated in prostate and ovarian cancer invasion/metastasis via activation of growth factors, proteases and extracellular matrix degradation involved in. In our published work, we used cell biology approaches to show novel associations of KLK peptidases with processes indicative of metastasis and the potential of our novel sunflower trypsin inhibitor scaffold-engineered KLK4 inhibitor. Our current studies are directed towards discovering the precise KLK target proteins/substrates and the subsequent signalling pathways involved in these events in order to determine their therapeutic target potential. In this regard, we are using novel tissue engineered biomimetic 3D gel matrices to better mimic the in vivo micro-environment of prostate cancer cells especially in bone metastasis and peritoneal invasion in ovarian cancer. Pilot studies show that PC3 cells cultured on an osteoblast-derived bone matrix undergo an EMT-like change but remain dispersed on the cell surface. In contrast, LNCaP cells cluster aligning with the fibrillar structure as they invade into the bone matrix as typically seen in vivo. KLK4 proteolysis of the osteoblast-derived bone matrix has identified additional novel substrates. In addition, we are exploring the cell biology that underlies the reported high KLK4 or KLK7 levels associated with poorer outcome in women with epithelial ovarian cancer (EOC). Of note, KLK4 or KLK7 transfected SKOV3 EOC cells have increased chemoresistance to taxol and/or cisplastin suggesting a mechanism for this poor outcome. Furthermore, KLK7 transfected SKOV-3 cells form multicellular aggregates (MCA) in agarose suspension (a process indicative of peritoneal tumour cell spread seen in ascites fluid clinically) which can be reversed by a KLK7 blocking antibody indicating the critical role played by KLK7 in this event. These new paradigms are providing novel information on the role of KLK peptidases in prostate and ovarian cancer progression and their potential as novel therapeutic targets.
035. THE ROLE OF THE SERTOLI CELL IN REGULATING SPERMATOGENESIS, IMMUNE RESPONSES AND INFLAMMATORY DISEASE: MULTIPLE FUNCTIONS, COMMON MECHANISMS?
Authors: Hedger M. P. , Muir J. A. , Winnall W. R.
Published: November 2010

There is increasing evidence that the Sertoli cell, in addition to modulating responses to direct antigenic challenges (eg. intratesticular allografts), is central to the response of the testis to inflammation and infection. Systemic inflammation exerts an inhibitory effect on spermatogenesis, which has been attributed to the effects of fever, vascular disturbances, or loss of androgenic support. However, recent studies point to more direct effects of inflammation on spermatogenesis. The discovery that Sertoli cells express Toll-like receptors (TLR), and react to TLR ligands by producing inflammatory cytokines and other mediators, provides a mechanism to account for this direct inhibition. Moreover, the pattern of cytokines produced by the Sertoli cell during inflammation is highly characteristic. For example, when stimulated with TLR ligands the Sertoli cell produces the pro-inflammatory cytokines, interleukin-1α (IL1α) and IL6, and the regulatory cytokine, activin A, but does not produce IL1β and tumour necrosis factor-α, which are major pro-inflammatory products of activated macrophages. The disruptive effects of inflammation on spermatogenesis may be attributed to the elevated production of these cytokines, all of which have stimulatory or inhibitory effects on germ cell mitosis, meiosis and apoptosis and Sertoli cell tight junction formation, In addition, activation of TLR/IL1 mediated inflammatory pathways in the Sertoli cell inhibits its ability to respond to its principal trophic hormone, follicle-stimulating hormone. Studies on the regulation of these interactions will further establish the role of the Sertoli cell in inflammation and infection. However, such studies also have important implications for normal Sertoli cell function, as TLRs can respond to endogenous ligands as well. Consequently, the Sertoli cell may be viewed as a sentinel cell, supporting and protecting spermatogenesis when conditions are optimal, but rapidly shutting down spermatogenesis in the presence of infection or illness. Intriguingly, these apparently disparate roles appear to involve common inflammation-related mechanisms.
036. SEX SPECIFIC FUNCTION OF THE HUMAN PLACENTA: IMPLICATIONS FOR FETAL GROWTH AND SURVIVAL
Authors: Clifton V. , Osei-Kumah A. , Hodyl N. , Scott N. , Stark M.
Published: November 2010

The placenta plays a central role in the development of the fetus by modulating the supply of nutrients and oxygen throughout pregnancy. We have identified that the placenta adapts to the presence of a maternal pathophysiology in a sexually dimorphic manner which results in differences in fetal growth. We have reported that the female fetus reduces her growth in response to chronic maternal asthma which ensures her survival in the presence of an acute asthma exacerbation. Conversely the male fetus continues to grow normally in the presence of maternal asthma but this is associated with a poor outcome in the presence of an acute exacerbation. We propose that the sexually dimorphic response of the fetus is derived from differences in placental adaptation to a pathophysiological condition. In the presence of a female fetus and maternal asthma, we have observed global gene changes in the placenta accompanied by significant alterations in microRNA expression. Downstream of these alterations we have observed differences in protein expression especially in relation to placental cytokines and the glucocorticoid receptor. In the presence of a male fetus there are fewer changes in global placental gene and microRNA expression, and we have observed no alterations in expression of placental cytokines or the glucocorticoid receptor. These differential adaptations ensure increased survival of the female fetus and continued growth of the male fetus in adverse conditions.
037. IMMUNE-LIKE MECHANISMS ASSOCIATED WITH OVULATION
Authors: Richards J.
Published: November 2010

Ovulation is the unique biological process by which a mature oocyte and surrounding somatic cells, the cumulus cell-oocyte complex (COC), are released from the surface of the ovary into the oviduct for transport and fertilization. Ovulation is similar to an inflammatory response: the follicles become hyperemic, produce prostaglandins and synthesize a hyaluronan-rich extracellular matrix. However, this view of ovulation may be too restrictive and need to be broadened to encompass the innate immune cell surveillance response system. This hypothesis is being proposed because ovarian granulosa cells and cumulus cells express and respond to innate immune cell related surveillance proteins (Toll-like receptors 2 and 4) and cytokines such as interleukin 6 (IL6) during ovulation. In addition, recent studies indicate that the ovulation process that is set in motion by the surge of luteinizing hormone (LH) is mediated, in large part, by the EGF-like factors (Amphiregulin, epiregulin and betacellulin) and their critical activation of RAS, most probably KRAS that is expressed at high levels in granulosa cells, and the mitogen activated protein kinases, MAP3/1 (ERK1/2). Mice in which granulosa cells are depleted of ERK1/2 fail to ovulate, oocyte meiosis does not resume, COC expansion is impaired and luteinization is blocked. Thus the global molecular reprogramming of granulosa cell gene expression patterns is completely derailed. Supported, in part by NIH-HD-16229, -16272 and -07495 (SCCPIR).
046. FUTURE PROOFING AUSTRALIA'S MAMMALIAN BIODIVERSITY USING GENOME RESOURCE BANKING AND ART: WHERE ARE WE UP TO?
Authors: Johnston S. D. , Gosalvez J. , Holt W. V.
Published: November 2010

The establishment of a functional genome resource bank for the genetic management and future proofing of Australian native mammals sounds great in theory, but what is the reality of this idea. In order to understand the current rate of progress in this area, we will present an overview of the inherent structural and physiological limitations of non-eutherian mammalian reproduction in terms of gamete biology and ART. For the male, these include (1) an unique mode of spermatid condensation that imparts the need for major structural changes to sperm morphology during epididymal transit, (2) a lack of cysteine protamines and disulphide bonds in the sperm chromatin that predisposes the nucleus to post-thaw chromatin relaxation, (3) an extremely stable acrosome, which to date, has not been possible to experimentally react in vitro, (4) unusual lipid composition in the plasma membrane that potentially makes the sperm cell resistant to cold shock trauma and (5) the need, in some species, for extremely high concentrations of cryoprotectant, that paradoxically, appear to be cytotoxic to the spermatozoon. Female limitations include, (1) the production of a large yolky oocyte and resulting embryo, making it difficult to cryopreserve, (2) a small and technically challenging complex reproductive tract that makes gamete recovery and artificial insemination problematic and (3) a general lack of information on marsupial reproductive physiology and behaviour that has hindered the development of protocols for timed induction of oestrus and ovulation. We shall also identify, socio-political and ethical limitations holding back the application of assisted breeding technology in these species.
047. SPERM COMPETITION, INBREEDING AND THE EVOLUTION OF SUPERIOR EJACULATES
Authors: Fitzpatrick J.
Published: November 2010

The production of viable sperm is essential for male reproductive success. However, because females in many species mate with several males during a single reproductive episode, leading to sperm competition, a male's reproductive success also depends critically on the ability of his sperm to compete efficiently with those from rival males for fertilizations. Therefore, males who regularly encounter sperm competition are expected to produce high quality ejaculates. Here, I will provide an overview of how sperm morphology and performance are influenced by sperm competition, both within and between species, using recent empirical examples. Having established the importance of producing high quality ejaculates in males experiencing sperm competition, I will then examine the reproductive consequences of producing sub-optimal sperm. Given the well known role that inbreeding plays in reducing genetic quality and reproductive success, I will focus in particular on how inbreeding acts to reduce sperm quality. Finally, I will examine the consequences of inbreeding for male reproductive success in species where sperm competition is rampant. Together, these results highlight the evolutionary importance of sperm competition and inbreeding in shaping ejaculate traits.
048. FUNCTIONAL DIFFERENCES BETWEEN SEX-SORTED AND NON-SORTED SPERM
Authors: Evans G. , De Graaf S. P. , Maxwell W. M.C.
Published: November 2010

The development and application of flow cytometric sorting for the pre-selection of sex has progressed at an increasing rate since the first report of live pre-sexed offspring of rabbits (2). The technique has been extended to production of pre-sexed offspring of numerous species and sorted bull semen is now widely available commercially around the world. Due to the stresses involved in the sex-sorting process, sex-sorted sperm may be functionally compromised in terms of reduced motility and viability, and their fertilising lifespan within the female reproductive tract may be reduced. Consequently, fertility in vivo may be compromised. However, improvements to the technology and a greater understanding of its biological impact on the sperm have facilitated recent developments in sheep, and we have demonstrated that sex-sorting is capable of selecting a functionally superior ram sperm population in terms of both in vitro and in vivo function. This has resulted in high fertility after intrauterine insemination of sex-sorted ram sperm (1). Unfortunately, to date, these results have not been matched in other species.
059. POOR GROWTH BEFORE BIRTH IMPAIRS INSULIN SECRETION - WHAT WE HAVE LEARNT ABOUT THE MECHANISMS FROM THE PLACENTALLY-RESTRICTED SHEEP
Authors: Gatford K. L.
Published: November 2010

Diabetes occurs when insulin secretion fails to increase sufficiently to compensate for developing insulin resistance. This implies that the increased risk of diabetes in adults who were small at birth reflects impaired insulin secretion as well as their well-known insulin resistance. More recently, direct evidence has been obtained that adults and children who were growth-restricted before birth secrete less insulin than they should, given their level of insulin resistance. Our research group is using the placentally-restricted (PR) sheep to investigate the mechanisms underlying impaired insulin action (sensitivity and secretion) induced by poor growth before birth. Like the intra-uterine growth-restricted (IUGR) human, the PR sheep develops impaired insulin action by adulthood, but has enhanced insulin sensitivity in infancy, associated with neonatal catch-up growth1, 2. Impaired insulin action begins to develop in early postnatal life, where although basal insulin action is high due to enhanced insulin sensitivity, maximal glucose-stimulated insulin action is already impaired in males3. Our cellular and molecular studies have identified impaired beta-cell function rather than mass as the likely cause of impaired insulin secretion, and we have reported a novel molecular defect in the calcium channels involved in the insulin secretion pathway in the pancreas of these lambs3. Upregulation of IGF-II and insulin receptor are implicated as key molecular regulators of beta-cell mass in the PR lamb3. By adulthood, both basal and maximal insulin action are profoundly impaired in the male lamb who was growth-restricted at birth2. These studies suggest therapies to prevent diabetes in the individual who grew poorly before birth should target beta-cell function, possibly in addition to further increasing beta-cell mass, to improve insulin secretion capacity, and its ability to increase in response to development of insulin resistance. We are now using the PR sheep to test potential therapies, since the timing of pancreatic development and hence exposure to a growth-restricting environment, is similar to that of the human.
060. OVER FEEDING EARLY IN LIFE AND RISK OF OBESITY: INSIGHT FROM THE RODENT
Authors: Morris M. J.
Published: November 2010

While adult lifestyle factors undoubtedly contribute to the incidence of obesity and its attendant disorders, mounting evidence suggests that programming of obesity may occur following over-nutrition during development. As hypothalamic control of appetite and energy expenditure is set early in life and can be perturbed by certain exposures such as under-nutrition and altered metabolic and hormonal signals, in utero exposure to maternal obesity related changes may contribute to programming of obesity in offspring. Data from animal studies indicate both intrauterine and postnatal environments are critical determinants of the development of pathways regulating energy homeostasis. Experimental evidence in rat studies from our laboratory points to an additive detrimental impact of high fat diet consumption after weaning in animals born of obese mothers. Deleterious effects of high fat diet during pregnancy on metabolic profile, adiposity and cardiac hypertrophy were enhanced by postnatal over consumption. Even modest early postnatal overfeeding induced by litter size reduction leads to increased adiposity. Studies are needed to determine to what extent the effect of maternal and early nutritional changes persist. This presentation summarizes recent evidence of the impact of maternal obesity on subsequent obesity risk, paying particular attention to the hypothalamic regulation of appetite, and markers of metabolic control. There is an urgent need to investigate the mechanisms underlying the trans-generational effects of maternal obesity due to an extraordinary rise in the rates of maternal obesity.
061. PATERNAL FACTORS INFLUENCING GESTATIONAL OUTCOME IN OFFSPRING
Authors: Roberts C. T.
Published: November 2010

Fetal programming can often be attributed to sub-optimal, but potentially modifiable, maternal factors such as smoking and poor nutrition. Much of the literature in this field points to factors that cause intrauterine growth restriction (IUGR) and the long term consequences for offspring health. It is not greatly appreciated, however, that other complications that may occur with, or independently of, IUGR predispose offspring, and their mothers, to poor health. These include preeclampsia and gestational diabetes. Elevated maternal BMI increases the risk for most pregnancy complications. Our new data show that paternal obesity (BMI>30) and waist circumference >102cm are associated with IUGR. We have also identified polymorphisms in a number of genes that regulate how the placenta differentiates and invades the maternal decidua, and how the mother adapts to pregnancy, that are associated with adverse pregnancy outcomes. Excitingly, many of these are polymorphisms in the paternal genome. One might reasonably expect that these would be found in imprinted genes expressed only from the paternal allele. However, we have also found several non-imprinted genes in which paternal genotype has a significant influence on pregnancy outcome both on maternal and infant disease states, but also on fetal and placental growth parameters. Furthermore, these genes interact with the maternal environment including diet and smoking to profoundly affect maternal and infant health. Consequently we now propose a complicated model of the control of optimal placental and fetal growth and pregnancy outcome that includes important genetic contributions from both parents to placental genotype that regulate conceptus growth and function. Importantly, paternal genotype can influence placental gene expression and the myriad of placental hormones and growth factors secreted into the maternal circulation that modulate maternal adaptation to pregnancy and, in susceptible women, these interact with maternal genotype, BMI and lifestyle to cause poor maternal and infant health.
062. DEVELOPMENTAL ORIGINS OF TYPE 2 DIABETES: EPIGENETIC MECHANISMS IN BETA CELL FAILURE
Authors: Simmons R.
Published: November 2010

The abnormal intrauterine milieu of intrauterine growth retardation (IUGR) permanently alters gene expression and function of pancreatic b -cells leading to the development of diabetes in adulthood. Expression of the pancreatic homeobox transcription factor Pdx1 is permanently reduced in IUGR and epigenetic modifications are responsible for this decrease. Exendin-4 (Ex-4), a long-acting glucagon-like peptide 1 (GLP-1) analog, given on days 1-6 of life increases Pdx1 expression and prevents the development of diabetes in the IUGR rat. Here we show that Ex-4 increases USF-1 and PCAF association at the proximal promoter of Pdx1, thereby increasing histone acetyl transferase (HAT) activity leading to a permanent increase in histone H3 acetylation and H3K4 methylation. Normalization of these histone modifications precludes DNA methylation thereby preventing silencing of Pdx1 in islets of IUGR animals. These studies demonstrate a novel mechanism whereby a short treatment course of Ex-4 in the newborn period prevents diabetes in adulthood by restoring Pdx1 promoter chromatin structure thus preserving Pdx1 transcription.
101. ISOLATION OF PUTATIVE EMBRYONIC STEM CELLS FROM CLONED PIG EMBRYOS
Authors: Truong K. P. , Vassiliev I. , Beebe L. F.S. , McIlfatrick S. M. , Harrison S. J. , Vassilieva S. , Nottle M. B.
Published: November 2010

The isolation of embryonic stem cells from cloned embryos (NT-ESC) from domestic animals would have a number of biomedical and agricultural applications. Putative ESC lines from in vivo derived and in vitro produced pig embryos were recently established using a new isolation method1. The aim of the current study was to determine whether NT-ESC lines could be isolated from cloned pig embryos using this method. To do this we determined initially whether the treatment of embryos with Trichostatin A (TSA), a histone deacetylase inhibitor, could increase the number of cloned embryos that develop to the blastocyst stage because TSA has been shown to increase blastocyst development and NT-ESC isolation efficiencies in mice2. Cloned embryos were produced as described previously3. Briefly, in vitro matured sow oocytes were enucleated, fused with adult fibroblasts using an electrical pulse and activated about 1.5 hrs later with a second electrical pulse. Reconstructed embryos were then cultured in modified NCSU23 with or without 50nM TSA treatment for the initial 24 hours of culture. Embryo development was assessed on day 6. Treatment with TSA increased the number of cloned embryos that developed to the blastocyst stage (143/471; 30%) compared with control (54/353; 15%; P < 0.0001). Blastocysts were then plated by mechanical depression onto mitotically inactivated mouse embryonic fibroblast feeder layers in a serum-free culture system on day 7. There was no significant difference in the efficiencies of establishment of homogeneous primary outgrowths between TSA treated (17/96; 18%) and control blastocysts (8/43; 19%). Thirteen homogenous outgrowths from the TSA treated group were vitrified at passage 2 or 3. Sublines are currently being characterised to determine their pluripotency.
102. THE EFFECT OF INSULIN ON EMBRYONIC STEM CELL PROGENITOR CELLS IN THE MOUSE BLASTOCYST
Authors: Campbell J. M. , Vassiliev I. , Nottle M. B. , Lane M.
Published: November 2010

Human ESCs are produced from embryos donated at the mid-stage of pre-implantation development. This cryostorage reduced viability. However, it has been shown that this can be improved by the addition of growth factors to culture medium. The aim of the present study was to examine whether the addition of insulin to embryo culture medium from the 8-cell stage of development increases the number of ES cell progenitor cells in the epiblast in a mouse model. In vivo produced mouse zygotes (C57Bl6 strain) were cultured in G1 medium for 48h to the 8-cell stage, followed by culture in G2 supplemented with insulin (0, 0.17, 1.7 and 1700pM) for 68h, at 37 o C , in 5% O2, 6%CO2, 89% N2 . The number of cells in the inner cell mass (ICM) and epiblast was determined by immunohistochemical staining for Oct4 and Nanog. ICM cells express Oct4, epiblast cells express both Oct4 and Nanog. The addition of insulin at the concentrations examined did not increase the ICM. However, at 1.7pM insulin increased the number of epiblast cells (6.6±0.5 cells vs 4.1±0.5, P=0.001) in the ICM, which increased the proportion of the ICM that was epiblast (38.9±3.7% compared to 25.8±3.4% in the control P=0.01). This indicates that the increase in the epiblast is brought about by a shift in cell fate as opposed to an increase in cell division. The effect of insulin on the proportion of cells in the epiblast was investigated using inhibitors of phosphoinositide3-kinase (PI3K) (LY294002, 50µM); one of insulin's main second messengers, and p53 (pifithrin-α, 30µg/ml); a pro-apoptotic protein inactivated by PI3K. Inhibition of PI3K eliminated the increase caused by insulin (4.5±0.3 cells versus 2.2±0.3 cells, P<0.001), while inhibition of p53 increased the epiblast cell number compared to the control (7.1±0.8 and 4.1±0.7 respectively P=0.001). This study shows that insulin increases epiblast cell number through the activation of PI3K and the inhibition of p53, and may be a strategy for improving ESC isolation from human embryos.
105. TRANSPLANTED GERM CELLS CAN COLONIZE THE GONADS OF SEXUALLY COMPETENT FISH AND PRODUCE FUNCTIONAL GAMETES
Authors: Majhi S. K. , Hattori R. S. , Strussmann C. A.
Published: November 2010

Germ cell (GC) transplantation (GCT) has great potential for seed production and conservation of valuable germlines. Currently available approaches to GCT in fish rely on sophisticated equipment and skills for cell transplantation into the blastodisc of embryos and/or the peritoneal cavity of small, sometimes few millimeters long larvae. Moreover, transplanted individuals may take years to grow to maturity, adding to the cost of producing surrogate gametes. In this context, the use of intragonadal GCT into sexually competent recipients that have been experimentally depleted of endogenous GCs might overcome these constraints. Here we demonstrate the feasibility of xenogeneic GCT in sexually competent fish. Spermatogonial cells isolated from pejerrey (Odontesthes bonariensis, Atherinopsidae) donors were implanted surgically into the testes of congeneric Patagonian pejerrey (O. hatcheri) that were severely depleted of endogenous GCs by treatment with Busulfan (40 mg/kg) and elevated water temperature (25°C) (Fig. 1).Donor cell behavior inside the recipient gonads was tracked using fluorescent cell linkers (CFDA-SE and PKH-26) and showed that transplanted spermatogonial cells were able to migrate towards, settle and multiply at the blind ends (cortical region) of the seminiferous lobules. The presence of donor-derived sperm was confirmed by PCR in 20% of the surrogate Patagonian pejerrey fathers at 6 months and fertilization of pejerrey eggs with surrogate sperm produced 1.2-13.3% pure pejerrey offspring (Fig. 2). These findings indicate that transplantation of spermatogonial cells into sexually competent fish can shorten considerably the production time of surrogate gametes and offspring. Ongoing studies are examining (low-tech) refinements in the proposed approach, such as non-surgical transplantation of GCs through genital papilla, and the suitability of GCT for generation of female gametes, for which cryopreservation techniques have not yet been developed. The results obtained so far have been encouraging and these developments will make GCT invaluable for the timely rescue of fish speciesfacing imminent extinction.
106. IL-6 INCREASES THE SHEDDING OF NECROTIC TROPHOBLASTS FROM PLACENTAL EXPLANTS
Authors: Chen Q. , Chen L. , Liu B. , Zhao H. , Stone P. R. , Chamley L. W.
Published: November 2010

Preeclampsia (PE) is characterised by elevated maternal blood pressure, preceded by endothelial cell dysfunction. Dead trophoblasts, shed from the placenta may be one of the factors that trigger PE. Women with PE frequently have elevated serum levels of inflammatory markers such as, IL-6 and TNF a but their functional significance is unclear. In this study we investigated whether these or other cytokines can alter trophoblast shedding from placental explants. Placental explants were treated with 9 different cytokines for 72 hours. Shed trophoblasts then were harvested using our published method1. The numbers of trophoblasts shed were quantified by automated cell counter. Expression of active of caspases 3&7 by the shed trophoblasts was determined using a FLICA kit. The trophoblasts shed from cytokine-treated or control explants were exposed to endothelial cell monolayers and endothelial activation determined by ELISA for cell surface ICAM-1. Treatment of explants with IL-6 caused a 50% increase (p=0.001), while TNF a and TGF b 1, caused smaller significant increases in the numbers of trophoblasts shed. Trophoblasts shed from explants treated with IL-6, TGF b 1, or TGF b 3 expressed significantly less active caspases 3&7 than controls or trophoblasts shed from explants treated with other cytokines. Exposing trophoblasts shed from IL-6- or TGF b 1-treated explants to endothelial cells caused a significant (P<0.001) increase in endothelial activation. Normally trophoblasts shed from the placenta die by an apoptosis-like process and their phagocytosis by endothelial cells is silent but a shift to shedding of necrotic trophoblasts can lead to endothelial cell activation 2. However, it remains unclear what might trigger a shift from apoptotic to necrotic trophoblast death. This study suggests that IL-6 and possibly other cytokines can alter both the number and the nature of shed trophoblasts such that the trophoblast are more necrotic and their phagocytosis by maternal endothelial cells could contribute to the pathogenesis of preeclampsia.
107. INTERLEUKIN 11 AND LEUKEMIA INHIBITORY FACTOR REGULATE CYTOKINE NETWORKS IN HUMAN FIRST TRIMESTER PLACENTA
Authors: Dimitriadis E. , Paiva P. , Manuelpillai U. , Weston G. , Meehan K. , Yap J.
Published: November 2010

Inadequate trophoblast invasion is thought to be involved in the pathogenesis of preeclampsia (PE). Shallow trophoblast invasion has been implicated to lead to placental hypoxia and a localised overproduction of pro-inflammatory cytokines. Interleukin (IL) 11 and leukemia inhibitory factor (LIF) are IL6-type cytokines produced at the maternal-fetal interphase and regulate human trophoblast migration/invasion, but their mechanisms of action are unknown. We aimed to determine the effect of hypoxia on human placental IL11/LIF secretion and the effect of IL11/LIF on placental cytokine secretion. As an in vitro model we cultured human first trimester placental villous explants (gestation weeks 7-9) in serum free conditions under either 2% (hypoxia) or 20% oxygen (normoxia) (N=8/gp) for 48h. Medium was assayed for IL11/LIF by ELISA. The effect of IL11/LIF (N=6/gp) on placental explant cytokine secretion (26 cytokines) was analysed using a quantitative multiplex immunoassay. Data was expressed as pg/mg wet weight and then as % change vs control (100%). IL11 secretion from explants was decreased by 76±5% (p<0.01) under hypoxia vs normoxia while LIF secretion did not differ significantly. Under normoxia the most highly abundant cytokines were IL6, IL8 and MCP-1 while moderate levels of G-CSF, CX3CL1, IP-10, and PGDF were present in conditioned medium. IL11 increased IL10 secretion while it decreased G-CSF, TNFa, IL1receptor antagonist (Ra), IL6 and PDGF secretion (p<0.05) vs control. Similarly LIF decreased G-CSF, TNFa, IL1Ra and additionally IL8 secretion (p<0.05) vs control. IL6 and VEGF secretion were increased (p<0.05) while MCP-1 was reduced (p<0.05) by hypoxia vs normoxia. This study identified for the first time that IL11 was reduced by hypoxia and identified a profile of cytokines secreted by placenta. IL11 and LIF regulated similar and unique anti-inflammatory cytokines in first trimester placenta. Whether placental IL11 or LIF is altered in women with PE remains to be elucidated however this study suggests mechanisms of action and confirms the importance of IL11/LIF in placentation.
108. FUNCTIONAL ROLE OF HtrA3 IN TROPHOBLAST CELL INVASION DURING HUMAN PLACENTAL DEVELOPMENT
Authors: Singh H. , Nie G.
Published: November 2010

Controlled invasion of extravillous trophoblast (EVT) through the maternal decidua is important for placental development and function. Serine protease HtrA3 is highly expressed in the decidual cells in the late secretory phase of the menstrual cycle and throughout pregnancy. It is highly expressed in first trimester in most trophoblast cell types, but not in the invading interstitial trophoblast. HtrA3 and its family members are down-regulated in a number of cancers and are proposed as tumor-suppressors. We hypothesized that HtrA3 is an inhibitor of trophoblast invasion and is down-regulated in invading EVTs, while up-regulation of decidual HtrA3 controls the process. The current study investigated HtrA3 expression in human endometrial stromal cells (HESC) during decidualization in vitro and whether HtrA3 inhibits EVT cell invasion. Stromal cells isolated from human endometrium were decidualized in vitro with estrogen, progesterone and cAMP. Quantitative RT-PCR and western showed HtrA3 mRNA and protein expression was significantly increased in decidualized HESC compared to controls. Indirect immunofluorescence showed homogeneous pattern and increase in intensity of HtrA3 staining in decidualized HESC compared to non-decidualized cells. HTR-8 cells derived from first trimester of pregnancy EVT showed higher levels of HtrA3 mRNA expression compared to other human choriocarcinoma cell lines (AC-1M88, AC-1M32, JEG-3 and BeWo). Both intracellular and extracellular HtrA3 staining was observed in HTR8 cells. Functional role of HtrA3 in cell invasion was determined in HTR-8 cells using an in vitro invasion assay. Exogenous addition of mutant HtrA3 (inhibitor) resulted in a significant increase in HTR-8 cells invading through matrigel coated membrane compared with controls. TGFβ-1 (as positive control) completely inhibited invasion of HTR-8 cells. HtrA3 is tightly regulated during decidualization of HESC in vitro. Inhibition of HtrA3 activity in trophoblastic HTR-8 cells increased invasiveness supporting its functional role during placental development.
109. TROPHOBLAST DEPORTATION IS DEPENDENT UPON CASPASES 3, 8 AND ROCK
Authors: Askelund K. J. , Stone P. , Chamley L. W.
Published: November 2010

Background: Trophoblast deportation is the process whereby multinucleated fragments of the syncytiotrophoblast are shed from the placenta into the maternal blood. It is estimated that 150,000 are shed from the placenta and deported daily in normal pregnancy and that more are shed during preeclampsia1. In normal pregnancy deported trophoblasts are thought to die by apoptosis, which is also increased in villous trophoblast in preeclampsia2. However, experimental confirmation that apoptosis leads to trophoblast shedding is required and it is not clear which components of the apoptotic pathway are involved in trophoblast shedding. Objectives: To determine the effect of inhibiting caspase 3 (executioner), caspases 8 and 9 (initiators), and Rho-associated kinase (ROCK; bleb formation) on the number of trophoblasts shed from first trimester human placentae. Methods : Using an in vitro placental explant model of trophoblast deportation, first trimester placentae were cultured for 72 hours in media containing specific inhibitors of ROCK, caspases 3, 8 or 9. Trophoblasts shed from quintuple explants/inhibitor from five placentae were depleted of contaminating leucocytes and erythrocytes, labelled with trypan blue and the sizes and numbers of shed trophoblasts quantified using a Nexcelom automated counter. Results: The number of trophoblasts that were shed from the explants was significantly increased (p=0.04) when caspase 3 (2.4 fold) and caspase 8 (2.7 fold) were inhibited. There was no significant change following caspase 9 inhibition. The number of shed trophoblasts was significantly decreased when ROCK was inhibited. None of the inhibitors significantly altered the size of the shed trophoblasts. Conclusion: Our data suggest that the apoptosis pathway is involved in trophoblast shedding in vitro from first trimester placentae. That caspase 8 but not caspase 9 affected shedding suggests trophoblasts from normal placentae are induced to die via the extrinsic apoptosis pathway. Aberrant regulation of the apoptosis pathway may contribute to pregnancy pathology.
110. EFFECTS OF cAMP AND SERUM ON HUMAN TROPHOBLAST CELL VIABILITY AND DIFFERENTIATION
Authors: Chen Y. , Allars M. , Abou-Seif C. , Nicholson R. C.
Published: November 2010

The formation of syncytium is a pivotal event for trophoblast cells to interact with the placental bed. While cAMP is regarded as an inducer of syncytialisation, the affect of different culture conditions on this cAMP effect has not been explored. Therefore, the effects of cAMP on cell differentiation and viability in the presence or absence of serum were investigated in the human choriocarcinoma cell lines, BeWo and JEG-3. We observed that in the absence of cAMP, BeWo cells grew best in media containing 10% FCS, followed by media containing charcoal-stripped 10% FCS (10%CCS), and less well in serum-free media. In the presence of cAMP ( 0.25~1.5 mM ), our observations suggest different cellular programmes may be in play. Treatment of BeWo cells with 0.75 mM cAMP for 24h and 48h, in the absence of serum, increased cell viability (MTT assay) by 25.1% and 46.1% respectively, compared to the control cells. Interestingly, this cAMP effect on cell viability was not observed in the JEG-3 cell line. In contrast, BeWo cell viability was decreased by 49.5% and 25.2%, and by 27.5% and 31.1% in JEG-3 cells, when the cAMP stimulated cells were cultured for 48h in 10% CCS and 10% FCS media, respectively. In addition, we observed a change in BeWo, but not JEG-3, cell morphology to a spindle-like shape with pseudopodia when cAMP stimulated cells were cultured in media containing 10% CCS or 10% FCS for greater than 24h. Since the process of syncytialisation may involve apoptotic events, we speculate that the different effects of cAMP on cell viability in trophoblast cells may be related to syncytialising factors contained in serum media. Further study will clarify whether serum promotes syncytium formation, while the lack of serum based factors could switch the cellular programme from one of syncytialisation toward a more proliferative type.
111. LIF REGULATION OF EPITHELIAL CELL FUCOSYLTRANSFERASE EXPRESSION IN MOUSE ENDOMETRIUM DURING EARLY PREGNANCY
Authors: Jasper M. J. , Care A. , Aplin J. D. , Robertson S. A.
Published: November 2010

Fucosyltransferase (FUT) enzymes are key regulators of glycosylated structures mediating embryo attachment to uterine epithelial cells at implantation. The identity of local regulatory signals is unknown. We have previously shown that macrophage co-culture significantly increases epithelial cell FUT2 and FUT4 mRNA expression in vitro, and the effect of co-culture is replicated with macrophage conditioned media. We aimed to define the identity of macrophage-secreted agents active in regulating FUT expression in mouse uterine epithelial cells, and to investigate the importance of macrophages for FUT expression in vivo. FUT1, FUT2, and FUT4 mRNAs were measured by qRT-PCR and data was normalised to β-actin mRNA in mouse uterine epithelial cells after culture with cytokines known to be secreted by macrophages. mRNA was also quantified in luminal epithelium laser-microdissected from mouse uterus on day 4 after mating with intact males or seminal vesicle deficient (SVX) males, to induce normal or depleted uterine macrophage populations respectively. Lectin staining on day 4 pc was quantified using ImageJ software in an alternate model of transient, systemic macrophage ablation following diphtheria toxin administration to CD11b-DTR transgenic mice. Epithelial FUT2 mRNA expression was specifically enhanced in vitro by addition of rLIF (2 ng/ml) (mean relative expression ± SEM, control 100 ± 5.6; rLIF 162.1 ± 11.5). Depletion of macrophages by mating with seminal vesicle deficient males reduced epithelial FUT2 mRNA expression on day 4 pc (intact 100 ± 9.1; SVX 73.5 ± 8.6). Depletion of macrophages in the CD11b-DTR mouse model caused a 30% reduction in the expression of the resulting glycoprotein epitope (α1,2 fucose) as observed by intensity of endometrial epithelial UEA-1 staining (control 100 ± 10; CD11b-DTR 72 ± 9) 24 hr post diphtheria toxin administration. In conclusion, these data demonstrate that endometrial epithelial FUT2 mRNA synthesis in preparation for embryo implantation is mediated via LIF and potentially other factors secreted from macrophages recruited during the inflammatory response to insemination. Uterine macrophage abundance and phenotype may thus be a determinant of receptivity to implantation.
112. DIFFERENTIAL DEVELOPMENT OF SEX-RELATED CHARACTERS OF THE GSP AND PNP/DO CHICKENS AFTER LEFT-OVARIECTOMY
Authors: Valdez M. B. , Mizutani M. , Kinoshita K. , Fujiwara A. , Yazawa H. , Yamagata T. , Shimada K. , Namikawa T.
Published: November 2010

To elucidate the strain difference in the sex reversal of genetic female to phenotypic male, GSP and PNP/DO females were left ovariectomized (ovx) between one day to three days after hatching and the degree of masculinization based on sex-related characters, histological analysis of the right gonad and hormone assay were assessed at one year of age. The GSP and PNP/DO inbred lines were both derived from the Fayoumi breed and are only differentiated based on red blood cell antigens type carried by each of the inbred line. Comb and wattles were found to be significantly bigger (P < 0.05) in the GSP ovx compared to the PNP/DO ovx, although male plumage pattern were more pronounced in the PNP/DO ovx. Spurs were observed both in the GSP and PNP/DO ovx with no significant difference (P > 0.05) in length to the respective male controls and body weight were not significantly different (P > 0.05) to the female controls. The size of the right gonad were significantly bigger (P < 0.05) in the GSP ovx than the PNP/DO ovx. Positive correlations were found in the sex related characters as well as plasma testosterone level and the right gonad weight both in the GSP and PNP/DO ovx except for the spur length which resulted into a negative correlation in the PNP/DO ovx. Histological analysis revealed that the right gonad of PNP/DO ovx are morphologically developed compared to GSP ovx showing more advance stages of spermatogenesis. It could be inferred that PNP/DO females which exhibit hereditary persistent right oviduct, are more responsive to the masculinizing effect of ovariectomy compared to GSP females, suggesting that genetic background may have a possible contribution on the degree of masculinization and subsequent development of sex related characters.