bluuebunny/crossref_metadata_2025 huggingface dataset:
165. PLASTICITY IN IMPRINTING OF BOVINE IGF2R CORRELATES WITH TOTAL EXPRESSION LEVELS IN FETAL TISSUES
Authors: Liu Y. , Fitzsimmons C. , Kruk Z. , Deland M. , Maddocks S. , Kind K. , Hiendleder S.
Published: November 2010

The multifunctional insulin-like growth factor 2 receptor (IGF2R) facilitates endocytosis and subsequent clearance or activation of a variety of ligands involved in cell growth and motility. Thus, the IGF2R gene has a major role in embryonic development and fetal growth. Murine Igf2r is subject to genomic imprinting and maternally expressed in peripheral fetal tissues. However, data on imprinting of IGF2R in human is still controversial with biallelic expression, partial imprinting, and monoallelic expression reported for fetal tissues [1, 2, 3]. Data from additional species may help to understand fetal IGF2R expression. The bovine is similar to human in that it is outbred and monotocous. We have analysed bovine Day 153 fetuses (55% to term, n=40) with Bos primigenius indicus and B. p. taurus genetics to determine the imprinting status of IGF2R in fetal brain, liver and skeletal muscle. Sequencing of PCR amplicons from IGF2R exon 48 revealed a polymorphic microsatellite and 14 SNPs. These were used to identify 15 heterozygous fetuses informative for imprinting analysis. We found biallelic expression of IGF2R in fetal brain and predominantly maternal expression in fetal liver and skeletal muscle. However, we observed considerable plasticity in imprinting in liver and skeletal muscle with paternal expression levels of 7%-21% and 4%-21%, respectively. Fetal liver samples with B. p. indicus maternal genetics showed significantly higher mean paternal expression levels than those with B. p. taurus maternal genetics (P<0.05). Real-time qPCR showed a significant relationship between imprinting and total IGF2R expression level within both tissues (P<0.05). Our data indicate plasticity in imprinting of IGF2R that could fine tune expression levels in fetal tissues.
166. MATERNAL OBESITY IS ASSOCIATED WITH AN INCREASED INCIDENCE OF PROSTATE ABNORMALITIES IN ADULT RAT OFFSPRING
Authors: Chiam K. , Jindal S. , Ryan N. , Moretta S. , De Blasio M. , Kind K. , Tilley W. , Owens J. , Bianco-Miotto T.
Published: November 2010

The World Health Organization has stated that 75% of adults worldwide are overweight, and in Australia nearly 25% of men are obese. Obesity is associated with an increased risk of cardiovascular disease, type 2 diabetes and cancer, with 30 to 40% of the latter possibly preventable by maintaining a healthy weight (The International Association for the Study of Obesity). Prostate cancer is the most commonly diagnosed cancer in men and there is increasing evidence that obesity increases the risk of prostate cancer mortality. High birth weight, an indication of excess nutrition during foetal development, has been associated with an increased risk of childhood and adult obesity, and for cancer. Using an animal model, we investigated whether obese mothers are more likely to have obese sons who are at an increased risk of developing prostate abnormalities and thus prostate cancer, in adulthood. Female rats were fed with either a control diet (4g fat/kg) or high fat diet (100g fat/kg) from before mating and throughout pregnancy. Prostate tissues were collected from the male offspring at 90 days (post-puberty) and 180 days (young adult). Histological analysis of the day 90 prostates identified hyperplasia in 100% of the ventral lobes (VL) and 64% of the dorsolateral lobes (DLP) in offspring of the maternal high fat group compared to 0% in each respectively, in those of the maternal control diet group. The VL is the most hormone sensitive prostate lobe of the rat, while the DLP is considered the equivalent of the human peripheral zone, the region from which the majority of human prostate cancers arise. These results suggest for the first time that maternal high fat diet may induce prostate abnormalities in male offspring that may in turn, predispose to an increased risk of prostate cancer in later life.
167. MUTANT STEROIDOGENIC FACTOR-1 FROM PATIENTS WITH DISORDERS OF SEX DEVELOPMENT SHOW REDUCED ACTIVATION OF THE TESTIS-SPECIFIC ENHANCER OF SOX9
Authors: Ludbrook L. , Fisher B. , Harley V.
Published: November 2010

The orphan nuclear hormone receptor Steroidogenic Factor 1 (SF1; NR5A1) is expressed throughout hypothalamic, pituitary, gonadal and adrenal tissues. Naturally occurring human mutations combined with mouse knockout models have revealed a critical role for SF1 as a transcription factor at multiple stages during gonadal development and during development of the adrenal. Missense mutation or truncation to SF1 in XY humans cause Disorders of Sex Development (DSD) with variable phenotypes. The precise mechanisms of SF1 action that fail in human DSD are not fully determined. This work aimed to utilise naturally occurring DSD-causing mutations in SF1 to increase our understanding of the sex determining function of SF1 in the developing male gonad. Recent work by others (1) identified SOX9 as a key target gene of SF1 during testis determination. SF1 activates Sox9 through a testis-specific enhancer element, termed TES. We tested the abilities of eleven clinical SF1 mutations to activate TES in reporter assays in HEK293T cells. Eight of the eleven SF1 mutants showed considerably reduced activation of TES compared to WT SF1. Furthermore, all mutations causing moderate to severe DSD phenotypes correlated with a more severe impairment of TES activation. In addition, all eleven of the mutants showed reduced synergistic activation of TES in co-transfection with the testis-determining co-factor SRY. Overall, this biochemical analysis of the function of mutant SF1 from DSD patients suggests that a failure of SOX9 up regulation, due to reduced activation of TES during testis development, could be the primary cause of the DSD in some patients with SF1 mutations.
168. IDENTIFICATION AND CHARACTERISATION OF SURFACE PROTEIN COMPLEXES IN HUMAN SPERMATOZOA
Authors: Redgrove K. A. , McLaughlin E. A. , O'Bryan M. K. , Aitken R. J. , Nixon B.
Published: November 2010

Upon leaving the testis mammalian spermatozoa are functionally incompetent and are thus unable to fertilize an oocyte. As the spermatozoa ascend the female reproductive tract, functional maturity is achieved through a complex cascade of biophysical and biochemical changes known as capacitation. An important aspect of this final maturation phase is the remodelling of the sperm surface architecture to enable it to interact with the zona pellucida, a glycoprotein matrix that surrounds the oocyte, and initiate fertilisation. While originally thought to be underpinned by a simple lock and key mechanism, emerging evidence has suggested that this interaction may instead be mediated by a multimeric recognition complex that is formed on the sperm surface during capacitation. However, to date the presence and composition of such a complex has yet to be described. Through the application of Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE), we have provided evidence that human spermatozoa express a number of high molecular weight protein complexes on their surface. Furthermore, the affinity of these surface expressed complexes for the zona pellucida was assessed utilising solubilised human zona pellucida and the technique of Far Western Blotting. Among the complexes that showed affinity for the zona pellucida we identified one comprising 14 subunits of the 20S proteasome. Interestingly, the 20S proteasome has previously been implicated in various aspects of mammalian fertilisation, including zona pellucida penetration and the acrosome reaction, although its precise role in these events has yet to be elucidated. Collectively, these results demonstrate the presence of multimeric protein complexes on the surface of human spermatozoa, and support their putative role in the initial interaction between the sperm and the zona pellucida. Our current research is focused on elucidation of the role of the 20S proteasome in human sperm-zona binding and further investigation of surface expressed protein complexes.
171. IDENTIFICATION OF DECIDUALISATION-INDUCED PROTEIN CHANGES IN HUMAN ENDOMETRIAL STROMAL CELLS BY PROTEOMICS
Authors: Paule S. G. , Kilpatrick L. , Stephens A. N. , Nie G.
Published: November 2010

Decidualisation of human endometrial stromal cells (HESC) is pivotal for successful implantation and can be induced by cAMP analogues such as 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP) and ligands to enhance cellular cAMP levels. The resulting decidualisation HESC is recognised by morphological changes and cellular products such as prolactin or insulin-like growth factors. The regulation of proteins during decidualisation HESC has yet to be clearly identified. The aim of this study was to identify proteins that are altered during decidualisation. HESC were isolated and decidualised with 500μM of 8-Br-cAMP for 72 hours. Decidualisation success was determined by prolactin assay. 2D differential in-gel electrophoresis (DIGE) was used to examine differentially expressed proteins between control and 8-Br-cAMP treated HESC. The proteins of interest were validated by Western blot and/or immunohistochemistry. A total of 88 differentially expressed proteins were identified by matrix assisted laser desorption/ionisation (MALDI) and/or liquid chromatography mass spectrometry (LC-MS). The proteins that were up regulated during decidualisation include insulin growth factor binding protein as well as caldesmon, tropomycin, actin, tubulin, SRC substrate cortactin and calponin-1 which contribute to the re-organisation of cell cytoskeletal restructuring and remodelling. Proteins associated with cellular growth and transformation such as annexin, nuclear migration factor protein and elongation factor 1B were also up regulated. These results suggest that the process of decidualisation is complicated requiring synergy and cross-talk between structural and cell growth proteins and pathways.
172. DEVELOPMENT OF A NON-HORMONAL CONTRACEPTIVE: VAGINAL DELIVERY OF A LIF INHIBITOR: ITS TISSUE DISTRIBUTION AND EFFECT ON IMPLANTATION IN MICE
Authors: Menkhorst E. M. , Zhang J. G. , Morgan P. O. , Metcalf D. , Salamonsen L. A. , Nicola N. A. , Dimitriadis E.
Published: November 2010

Uterine leukaemia inhibitory factor (LIF) is obligatory for fertility in mice and associated with infertility in women. Intraperitoneal injection (IPI) of a long-acting LIF inhibitor conjugated to polyethylene glycol (PEGLA) blocks endometrial LIF action preventing implantation1. Thus, PEGLA is a promising non-hormonal contraceptive. In women, vaginally applied (VA) compounds preferentially localize to the uterus suggesting a desirable delivery method for contraceptive purposes; however this has not been examined in mice. We aimed to compare the effects of VA and IPI PEGLA on tissue distribution and implantation in mice. Non-pregnant or mated female mice (Pregnant day [D] 2 [D0: day of plug], n=3/group) were given 125I-PEGLA by IPI (3x106cpm) or VA (7x105cpm). 125I-PEGLA was measured in blood and tissue at various time points. To block implantation, mated mice (n=4/group) were given PEGLA or control by IPI (500μg/injection) or VA (300μg/injection) at 1200h and 2200h on D2 and 1000h on D3 and the uterus examined for implantation sites on D6. 125 I-PEGLA accumulated in blood and uterus following IPI more rapidly (10min cf 30min), reached a higher concentration (10min, 6h and [blood] 24h; p<0.05) and remained longer (24h cf 6h) compared to VA. The percentage of protein-bound 125I in blood was higher following IPI (79.4+1.9%) than VA (47.5+6.7%) at 6h (p<0.05). Following IPI, 125I-PEGLA accumulated in the liver, gall bladder and stomach (2h) and spleen (24h) compared to control while no specific tissue accumulation was observed following VA. PEGLA prevented implantation following IPI (p<0.05) and reduced the size of implantation sites and decidualization in VA compared to controls. This study demonstrated differences in tissue distribution between VA and IPI PEGLA. It showed that VA PEGLA acted on the uterus in mice albeit to a lesser extent than IPI. It suggests that VA PEGLA is a potential route of delivery for contraceptive purposes.
173. ENDOMETRIAL REPAIR - ENTER THE MATRIX
Authors: Evans J. , Kaitu'u Lino T. , Fernando S. , Salamonsen L.
Published: November 2010

Menstruation and endometrial repair are two opposing functions which occur simultaneously within the menstruating uterus. Whilst the factors that control menstruation are increasingly understood1 the endometrial milieu which governs repair remains elusive. The extracellular-matrix (ECM) plays a dynamic role within the repairing endometrium, with roles suggested for fibronectin and certain integrins2. We have utilised two models of endometrial repair, a mouse menstruation model3, and an endometrial luminal epithelial cell-line (ECC-1)4 with the aim of defining the adhesion and ECM molecules important for endometrial repair. Uterine horns (repairing and non-repairing control from each of 4 mice) were subjected to laser-capture microdissection of the repairing luminal epithelium (LE) and immediate sub-luminal stroma. RNA was extracted and processed for pathway-focused array. ECC-1 cells were grown to over-confluence, wounded and allowed to repair. RNA was extracted on each day during repair and used for oligo-microarray analysis. Menstruating/repairing human endometrium was investigated for expression of ECM molecules by immunohistochemistry.A host of ECM molecules (ADAMTS's, MMP's, cadherins, integrins and matrix proteins) were shown to be altered in the repairing murine endometrium. In agreement with published data2, fibronectin and integrins α5 and β1 were elevated at the time of repair. However, in our models, fibronectin was regulated mainly in the stromal compartment, demonstrated by its absence in the ECC-1 model and concentrated stromal immunolocalization in human endometrium. Both integrins displayed regulated expression in the ECC-1 model and localised to repairing LE of human endometrium. Other noteworthy ECM molecules regulated solely within the murine model include integrins (αM, αL, β2) and selectins expressed on lymphocytes, which may reflect the importance of these cells in endometrial repair5. In conclusion, these data suggest the ECM contributes to the dynamic changes observed during endometrial repair and these models may enable further insight into the roles of each cellular compartment.
175. THE ROLE OF PROPROTEIN CONVERTASE 6 DURING DECIDUALIZATION: REGULATION OF BONE MORPHOGENETIC PROTEIN 2 ACTIVATION
Authors: Heng S. , Hardman B. , Paule S. , Singh H. , Nie G.
Published: November 2010

Proprotein convertase 5/6 (PC6), a member of the proprotein convertase (PC) family, is a critical endometrial factor for implantation. PC6 is up-regulated in the endometrium specifically at implantation in association with epithelial differentiation (in human and monkey) and stromal cell decidualization (in the mouse, human and monkey). PC6 is the only PC member that was significantly up-regulated during decidualization. Knockdown of PC6 inhibits decidualization. PCs function by converting a range of important precursor proteins into their bioactive forms. One group of such proteins is the transforming growth factor beta (TGF-beta) superfamily proteins. They are first synthesized as larger biologically inactive precursors, and then are processed by PCs into their active forms. Bone morphogenetic protein 2 (BMP2) is a TGF-beta superfamily member and demonstrated to be essential for decidualization. We hypothesized that BMP2 is one of the proteins that PC6 activates during decidualization. Freshly isolated stromal cells from human endometrium were decidualized in culture with and without inhibition of PC6 activity. The full-length (precursor, non-active) and processed (activated) forms of BMP2 were determined in cellular lysates and media. The precursor form of BMP2 was reduced whereas the active form was increased during decidualization. Inhibition of PC6 activity inhibited decidualization, and this inhibition was accompanied by a total inhibition of the production of active BMP2. To further confirm the role of PC6 in activating BMP2 in decidualization, active BMP2 was added into cells and the decidualization arrest caused by PC6 inhibition was partially rescued. This study demonstrated that PC6 regulates decidualization by activating molecules such as BMP2 that are essential for decidualization.
176. GDF-9, BMP-15 AND ACTIVIN A CONTRIBUTE TO SEMINAL FLUID SIGNALLING IN HUMAN CERVICAL EPITHELIAL CELLS
Authors: Sharkey D. J. , Gilchrist R. B. , Robertson S. A.
Published: November 2010

We have previously shown that in women, as in animal species, introduction of seminal fluid at coitus induces cytokine expression and a local inflammatory-like response in the female reproductive tract. The response is characterised by induction of mRNAs encoding several pro-inflammatory cytokines including GM-CSF, IL-1α and IL-6, as well as chemokines IL-8, MIP-3α and MCP-1. Recent studies in our laboratory have focused on identifying active signalling agents present in human seminal plasma. To date we have shown that all three mammalian isoforms of TGFβ (TGFβ1, TGFβ2 and TGFβ3) and 19-hydroxy PGE are key regulators of this response. The current study aimed to determine whether other members of the TGFβ superfamily including GDF-9, BMP-15 and Activin A also contribute. To investigate this we utilised immortalised Ect1 ectocervical epithelial cells, which mimic the response of primary ectocervical epithelial cells. Ect1 cells were incubated with increasing doses (0.5, 5.0, 50 or 500 ng/ml) of rGDF-9, rBMP-15 or rActivin A and production of pro-inflammatory cytokines and chemokines was assessed in 24 hour post-treatment supernatants using Luminex microbead technology. BMP-15 was found to stimulate GM-CSF production (2-fold), while GDF-9 and Activin A both stimulated IL-6 production (2.4-fold and 80% increases respectively), all acting in a dose-dependent manner. In contrast, all three factors inhibited IL-8 production by Ect1 cells. These data demonstrate that GDF-9, BMP-15 and Activin A are new seminal fluid signalling agents capable of targeting female reproductive tract epithelial cells and inducing different response profiles compared with TGFβ and 19-hydroxy PGE. The relative bioavailability of these factors in seminal fluid would therefore influence the profile of inflammatory response in the female partner, regulating immune responses to male seminal antigens as well as sexually transmitted pathogens.
177. A MECHANISM UNDERLYING DISORDERS OF SEX DEVELOPMENT CAUSED BY DAX1 DUPLICATION
Authors: Ludbrook L. , Sekido R. , Lovell-Badge R. , Harley V.
Published: November 2010

The DAX1 protein is an orphan nuclear hormone receptor expressed in developing and adult hypothalamic, pituitary, adrenal and gonadal tissues. In humans, duplication of the DAX1 gene at locus Xp21 causes Disorders of Sex Development (DSD), whereby XY individuals develop as females, due to the failure of testicular development. DAX1 acts as a co-factor for nuclear receptor-mediated transcription of steroidogenic genes. In mice, overexpression of a Dax1 transgene causes delayed testis cord formation, a milder phenotype than that seen in human (1). Exactly how DAX1 duplication interferes with typical testicular development is unclear but a ‘window' of DAX1 activity was proposed (2). In order to identify the mechanism of DAX1 action when overexpressed in the developing XY gonad, we have used both in vivo and in vitro approaches. We hypothesised that, when present in excess, DAX1 must repress the action of early testis-forming genes. We investigated the effect of Dax1 over expression, using the Dax1 transgenic mouse line, Dax1812 (1), on expression of Sox9, a critical testis-forming gene. Immunostaining of Dax1812 gonads revealed reduced Sox9 expression, suggesting excess Dax1 antagonises Sox9 upregulation during the early stages of sex determination. To determine whether antagonism of Sox9 was occurring at the transcriptional level we assessed the effect of excess Dax1 on the activity of the Testis-Specific Enhancer of Sox9 (TES), which drives Sox9 transcription in the developing XY gonad (3). In combination, the in vivo and in vitro evidence strongly suggests that Dax1, when present in excess, can repress Sox9 expression through TES and that this repression occurs through inhibition of Steroidogenic Factor-1 activity. With this work we have identified a potential mechanism for disruption of the male-specific sex determination pathway caused by DAX1 duplication and leading to DSD in XY individuals.
178. RAT TESTICULAR MACROPHAGES EXHIBIT AN 'ALTERNATIVELY ACTIVATED' RESPONSE TO LIPOPOLYSACCHARIDE (LPS), INTERFERON-Γ (IFNΓ) AND INTERLEUKIN-4 (IL-4), CONSISTENT WITH IMMUNE PRIVILEGE
Authors: Winnall W. R. , Muir J. A. , Hedger M. P.
Published: November 2010

Testicular macrophages (TMs) are implicated both in the response of the testis to invading pathogens and supporting the immunosuppressive environment that protects developing germ cells (immunoprivilege). Macrophages are classified into two general phenotypes: “classically activated” (M1), which undergo inflammatory responses to LPS and IFNg, and “alternatively activated” (M2), defined by anti-inflammatory activity and regulated by IL-4. Our aim was to establish whether TMs have an M2 phenotype, consistent with immunoprivilege. Rat TMs and bone marrow-derived macrophages (BMMs) were isolated from adult rats, and cultured with LPS, IFNg and/or IL-4 for 2-3h. mRNA expression was measured by real-time RT-PCR and protein production was measured by ELISAs. Compared with BMMs, TMs stimulated with LPS and IFNg, either individually or in combination, expressed low levels of pro-inflammatory cytokines, such as IL-1β, IL-1α and tumour necrosis factor-α, intermediate levels of IL-6, but much greater levels (8-fold) of the anti-inflammatory cytokine, IL-10. TMs also displayed elevated constitutive expression of IL-10 and responded to IL-4, unlike BMMs. However, TMs expressed relatively low levels of another immunoregulatory cytokine, transforming growth factor-β1. After FACS-sorting of TMs using an antibody to CD163, a surface marker associated with M1-M2 progression, CD163+ TMs produced high levels of IL-10 constitutively and after stimulation, whereas CD163- cells produced little or no IL-10. Unexpectedly, both CD163- and CD163+ TMs expressed similar levels of most pro-inflammatory genes. These data indicate polarisation of TMs towards the M2 phenotype, characterised by production of IL-10 and responsiveness to IL-4, although the polarised TMs continue to express pro-inflammatory cytokines, albeit at significantly lower levels than other macrophages. The M2 phenotype is consistent with a role in testicular immunosuppression, but may also contribute to fibrosis, which is associated with testicular responses to vasectomy, cryptorchisism and infertility.
179. CHARACTERISATION OF THE IN VITRO FUNCTION OF ACTIVIN AC
Authors: Gold E. , Harrison C. , Makanji Y. , Risbridger G.
Published: November 2010

Activins are members of the TGF-β superfamily that signal via type II and type I receptor subunits and intracellular Smads1. Activin A stimulates FSH release from the pituitary and is also a potent growth and differentiation factor in many physiological systems2. Over-expression of the activin-βC subunit in vitro leads to a reduction in activin A and an increase in activin AC3. Transgenic mice over-expressing activin-βC show decreased circulating activin A, implying that activin AC may also be formed in vivo4. Recently recombinant activin AC has become available, therefore this study examines the in vitro function and mechanism of action of activin AC. Activin AC stimulates FSH release in LβT2 cells and is a negative growth regulator in LNCaP cells, however the potency of activin AC is 8-10 fold less than activin A. Incubation of LNCaP cells with activin receptor antibodies (ALK4, ActRIIA, ActRIIB) abolishes the growth inhibitory effects of activin AC. Activin AC binds to ActRIIB, however a 20-30 fold decrease in both the potency and affinity of activin AC is evident compared to activin A. In addition, activin AC increases Smad-2 phosphorylation. These results indicate activin AC utilises the same receptors and intracellular signalling molecules as activin A. The activin A antagonists, follistatin and activin C4, also antagonise the growth inhibitory effects of activin AC and reduce Smad-2 phosphorylation and Smad-4 expression. This study shows for the first time that the in vitro function of activin AC is similar to activin A, albeit at a lower potency and provides the impetus to determine the physiological role of activin AC in vivo.
180. CHARACTERIZATION AND FUNCTION OF THE BULL SPERM PROTEIN Spam1: TWO DISTINCT ISOFORMS WITH DISTINCT ROLES
Authors: Morin G. , Sullivan R. , Laflamme I. , Robert C. , Leclerc P.
Published: November 2010

We identified an 80 kDa bull sperm protein (p80) that possesses homology with the Sperm adhesion molecule 1 (Spam1), a GPI-anchored glycoprotein conserved amongst mammals that is required for fertilization. Since bovine Spam1 had not been identified, the aim of this project was to determine if p80 is the bovine Spam1 and to test the hypothesis that it plays a role in gamete interaction during bovine fertilization. Amino acid sequence deduced from 3`/5`Race confirmed that homology between p80 and Spam1 in various species ranged from 47 to 61%.It also revealed specific differences including the absence of a GPI-anchor, the presence of a transmembrane domain, and N- and O- glycosylation sites. By generating and characterising antibodies against p80 N- and C-terminal domains, the protein orientation in the sperm membrane was evaluated. We identified two populations of p80 on the sperm head: one internalised in the anterior region and the second localised on the post-acrosomal region with its hyaluronidase domain exposed to the extracellular environment. Proteomic and immunologic analyses revealed that the p80 post-acrosomal population is a shorter isoform originating from the epididymis while the full length p80 located on the anterior region originates from the testis. Finally, the potential function of p80 during the sperm/zp interaction was evaluated by sperm/zona pellucida (zp) binding assay. The C-terminal extremity of p80 was implicated in sperm binding to the zp by antibody and native protein competition. Furthermore, glycosylation was not required during this interaction since deglycosylated p80 in the incubation medium had the same inhibitory effect on zona binding as the native p80. Collectively, the results demonstrated that p80 is the bovine Spam1, and that two isoforms are present on bull sperm. The hyaluronidase activity of the post-acrosomal isoform is required for cumulus penetration, while the other one participates in sperm/zp binding during fertilization.
181. HEPATOCYTE-NUCLEAR FACTOR 3-ALPHA (HNF-3α) EXPRESSION IN THE DEVELOPING PROSTATE OF THE TAMMAR WALLABY: A MARKER OF PROSTATE DIFFERENTIATION
Authors: Gamat M. , Shaw G. , Renfree M. B.
Published: November 2010

The prostate is the source of about 30% of the seminal fluid. The prostate develops from the urogenital sinus involving epithelial-mesenchymal interactions, and is dependent on androgen secretion for its differentiation. Most studies on prostatic development have focused on the mouse but the tammar may be an alternative model for some aspects of prostatic development due to its structural similarity to the human. The tammar prostate is a cone-shaped secretory gland which consists of a central zone, transition zone and peripheral zone, as in humans. In contrast, the mouse prostate is a multi-lobular organ. Although much is known of the hormonal control of prostatic development in the tammar1,2,3, little is known about the specific markers of its differentiation. HNF-3α is a transcription factor that is expressed in the urogenital epithelium of the prostate in the mouse and human. This study characterised HNF-3α in the developing prostate of the tammar. The full length sequence of the gene was obtained in silico and confirmed by cloning. Tammar HNF-3α is highly conserved, sharing 82% nucleotide identity with the human and 80% nucleotide identity with mouse. We used RT-PCR to examine the temporal expression of HNF-3α, and found that it is expressed throughout prostate development in the male from immediately after birth to day 70, the latest stage examined. It is also expressed in the developing female urogenital sinus during a similar time period. HNF-3α protein has a spatial expression similar to that of the mouse, and it is localised to the nucleus of the urogenital epithelial cells. These results indicate that prostatic differentiation is conserved between the tammar (marsupial) and mouse (eutherian). Characterisation of HNF-3α in the tammar will also provide a useful tool in assessing prostatic differentiation in our ongoing prostatic organ culture experiments.
182. NOVEL SIGNALLING IN MOUSE EMBRYONIC STEM CELLS GENERATES PRIMITIVE ECTODERM-LIKE CELLS
Authors: Morris M. B. , Hamra N. , Lonic A. C. , Felquer F.
Published: November 2010

The phenotypic status of embryonic stem (ES) cells is controlled in part by signalling pathways which translate inputs mediated by extracellular molecules. An important extracellular protagonist in mouse ES cells is LIF (leukaemia inhibitory factor) which interacts with the gp130–LIFR receptor complex to activate a number of downstream signalling pathways, including the STAT3, MEK/ERK and PI3K/Akt. These pathways, together with others, interact in complex and sometimes competing ways to generate the well-known characteristics of mouse ES cells of self-renewal, high rates of proliferation, and pluripotence. The addition of a second molecule, L-proline, to the extracellular environment alters the pluripotent status of mouse ES cells, converting them to a second pluripotent population equivalent to the primitive ectoderm of the pre-gastrulating embryo. This conversion, from ES cells to primitive ectoderm-like cells, primes the latter for directed differentiation to specific cell types (1). Here we show, using inhibitor studies and kinome array analysis, that this small molecule appears to work by (i) changing the balance in activity of signalling pathways already stimulated by LIF and (ii) activating additional signalling pathways. Specifically, L-proline rapidly further activates the LIF-stimulated MEK/ERK pathway, tipping the balance in favour of primitive-ectoderm formation and away from ES-cell self-renewal sustained by LIF-mediated activation of the STAT3 pathway. In addition, L-proline rapidly stimulates other pathways including p38, mTOR and PI3K/Akt each of which contributes, to a greater or lesser extent, to the conversion to primitive ectoderm-like cells. These results indicate that (i) L-proline acts in novel ways to stimulate embryo-like developmental progression in ES cells and (ii) through the addition of small, nontoxic activators and inhibitors of signalling pathways, the differentiation of pluripotent ES cells might be controlled sufficiently well for the homogeneous production of specific cell types suitable for use in animal models of human disease.
501. BACTERIOSPERMIA AND ITS CONTROL IN BUBALINE SEMEN
Authors: Andrabi S. M. H. , Shahab M.
Published: November 2010

The present study was designed to investigate the bacterial species incriminated in bubaline semen and to find out the effectiveness of antibiotics (GTLS; gentamycin, tylosin and linco-spectin or SP; streptomycin and penicillin) in cryodiluent on bacterial control and quality of buffalo bull spermatozoa. For this purpose four experiments were conducted. In experiment 1, a total of 11 bacterial species were identified from buffalo ejaculates. The predominant bacteria were Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa in the ejaculates. In experiment 2, total aerobic bacterial counts in post-thaw samples were lower (P<0.05) in GTLS than in SP or NC (negative control). Fewer bacterial genera were identified in semen samples having GTLS than SP. Majority of the bacterial isolates from ejaculates showed more sensitivity towards GTLS than SP. In experiment 3, motilities (visual and computer-assisted), velocities (straight-line, average path and curvilinear), amplitude of lateral head displacement and plasma membrane integrity in post-thaw semen samples did not differ (P>0.05) due to antibiotics. Spermatozoal abnormalities (acrosome, head, mid-piece and tail) were lower (P<0.05) in GTLS and SP than in NC. In experiment 4, the fertility rates for SP-based vs. GTLS-containing frozen semen of buffalo bull were 42.8 and 55.2%, respectively. The results for GTLS were significantly higher than SP. The fertility rates also differed significantly in the first and second batch of inseminations performed with SP or GTLS-based cryopreserved semen of buffalo bull. In conclusion, a number of bacterial species are isolated from bubaline semen. Bacterial and seminal quality measured by standard laboratory tests and field fertility trials indicate that GTLS is more suitable in extender for cryopreservation of buffalo bull spermatozoa.
503. SUCCESSFUL IN VITRO FERTILIZATION (IVF) AFTER PRIOR FAILED IVF UTILIZING A PROPRIETARY BLEND OF SUPPLEMENTS
Authors: Poursharif B. , Paden J. , Acacio B.
Published: November 2010

Background: To date, little is known about the effect of supplements on the outcome of in vitro fertilization (IVF). The data on this matter is limited to measuring the overall pregnancy rate on a population of women who took a specific supplement, and not on IVF patients. Objectives: To demonstrate the positive role of an investigated supplement in the outcome of patients undergoing IVF. Method: 18 women undergoing IVF treatment were placed on a proprietary combination of vitamins and antioxidants designed to encourage blood flow and improve egg quality. The women were selected for this protocol mostly due to prior poor egg quality and/or large amount of embryo fragmentation .The women took supplementation twice daily for 4–12 weeks prior to transfer. The charts of the patients who used the supplements were used to obtain data. Previous failed IVF was defined as negative pregnancy. Successful IVF outcome was determined by positive chemical pregnancy and clinical pregnancy after one attempt. Range and mean was calculated for patient's age and number of failed previous IVF attempts. The previous IVF attempts were performed in different centers without using this supplement in all patients. Results: Eighteen patients used the supplement before and during their IVF cycles. Patient's age ranged from 28 to 44 with mean of 36.4 years. They had on average, 2 prior failed IVF attempts. Seventeen of 18 patients had successful IVF outcome. The failed patient required frozen testicular extraction of sperm (TESE), prior to IVF. Summary: Seventeen of 18 patients who used our supplements had successful IVF. These patients failed an average of 2 previous IVF attempts without using our supplements. Conclusion: Usage of our supplements is associated with improved rates of success in patients undergoing IVF with a history of prior failed IVF attempts. Larger studies need to be conducted.
505. THE ROLE OF Adamts1 IN BREAST CANCER PROGRESSION AND METASTASIS
Authors: Tan I. A. , Frewin K. M. , Ricciardelli C. , Russell D. L.
Published: November 2010

The extracellular protease Adamts1 is a vital enzyme involved in normal development and has been associated with various processes of extracellular matrix (ECM) remodelling including ovulation and pathologies exhibiting disrupted ECM. In the PyMT-MMTV mouse mammary cancer model we have demonstrated reduced tumour growth and pulmonary metastasis incidence in Adamts1–/– mice. Furthermore, benign DCIS lesions of the mammary gland were significantly more common in Adamts1–/– mice. To better understand the mechanisms surrounding Adamts1 mediated progression from DCIS to invasive cancer we next investigated proliferation, apoptosis, blood vessel density and/or extracellular remodelling. Morphometric comparisons were performed using immunohistochemical markers of proliferation (Ki67) and apoptosis (active caspase3) as well as vasculature (CD34), and known Adamts1 proteolytic target, versican in wildtype and Adamts1-/-PyMT tumours. Our analysis revealed that no significant difference in proliferation between the two genotypes, but Adamts1–/– had increased apoptosis compared with Adamts1+/+ tumours. Contrary to previous reports suggesting Adamts1 is antiangiogenic, no significant difference was found in blood vessel density between Adamts1–/– compared with Adamts1+/+ tumours. Interestingly, versican abundance in peritumoural stroma was lower in Adamts1–/– tumours. This latter finding may provide an explanation for the reduced metastasis incidence found after Adamts1gene inactivation, as many previous studies have associated peritumoral versican abundance to metastasic progression. Our findings increase the understanding of mechanisms whereby Adamts1 promotes breast cancer metastasis by showing its role in increasing cell survival, as well as modulating the stromal ECM environment through which it may promote invasion and metastasis. The involvement of Adamts1 in breast cancer progression presents this protease as a potential novel target for reducing metastasis incidence.
506. MENSTRUAL CYCLE VARIATIONS IN PLASMA microRNA EXPRESSION PROFILES
Authors: Ohlsson Teague E. M. C. , Nisenblat V. , Robertson S. A. , Hull M. L.
Published: November 2010

microRNAs are short, single-stranded RNAs that regulate gene expression at the post-transcriptional level. Plasma and serum microRNAs correlate closely with microRNA profiles of diseased tissue and have been explored as blood-based biomarkers for human diseases, including steroid-driven malignancies. However, reproductive steroid signalling regulates the expression of specific microRNAs and this could impact the utility of microRNA biomarkers in reproductive aged women. We hypothesised that microRNA expression profiles are altered by steroid hormone fluctuations associated with the menstrual cycle. To test this hypothesis, plasma microRNA expression was measured in healthy women at 3 stages of a 28 day menstrual cycle; ie menstrual (day 3-5), follicular (day 9–13) and implantation window/secretory phase (day 18–22). Total RNA was extracted from plasma, multiplex reverse transcription was performed, and the cDNA pre-amplified prior to expression analysis of 667 microRNAs on Taqman low density PCR arrays (n=6 women). Preliminary data shows that up to 200 microRNAs may be detected with this methodology, and that at least 14 of these are differentially expressed (fold change ≥±1.5) at follicular and secretory phase, as compared to menstrual phase. We plan to confirm these findings with standard Taqman microRNA assays (n=10 women). Our findings suggest that plasma miRNA expression profiles change over the menstrual cycle, and that this could confound microRNA-based diagnostic tests. We hope to demonstrate the most appropriate cycle phase for blood-based miRNA profiling, facilitating the development of plasma microRNA-based diagnostic tests and providing valuable information to researchers studying circulating microRNA profiles in reproductive aged women.
507. TGFβ REGULATES ENDOMETRIOSIS-LIKE LESION DEVELOPMENT IN MICE INDEPENDENTLY OF THE RUNX-2/OPN PATHWAY
Authors: Johan M. Z. , Ingman W. V. , Robertson S. A. , Hull M.
Published: November 2010

TGFβ is likely to significantly influence endometriotic lesion development, as TGFβ KO/SCID mice with no host-derived TGFβ activity have smaller human ectopic endometrial lesions than control mice. TGFβ potentially acts via RUNX2, a transcription factor that directly upregulates OPN transcription in osteoblasts, as we have identified RUNX-2 and OPN gene expression at high levels in nude mouse endometriosis-like lesions, in human endometrial stomal cell cultures and in the stroma of endometriotic tissues. We hypothesised that inhibition of RUNX-2 would suppress OPN production and result in reduction of endometriotic lesion formation and size. As P38/MAPK inhibitors suppress TGF-β mediated RUNX-2 transcription, we utilised the nude mouse model to test whether the P38/MAPK inhibitor FR167653 would suppress OPN production in endometriotic tissues resulting in smaller lesions.FR167653 (30 mg/kg twice a day) or placebo was administered for either 10 or 14 days to nude mice (16 in each group) implanted with human endometrial tissue xenografts from 4 different women. The size and weight of the lesions were measured and immunohistochemical analysis of OPN, αSMA (myofibroblasts) and F4/80 (macrophages) was carried out. There was no difference in size or weight of the lesions, and there was no overt difference in any of the staining parameters explored. The inhibition of p38/MAPK did not alter the size of the nude mouse lesions nor OPN staining within these lesions despite being administered at a maximal therapeutic dose. This suggests that TGFβ regulation of endometriotic lesion development is mediated by an alternate molecular pathway.
509. REGULATING MURINE FOLLICLE DEVELOPMENT BY STIMULATION OF THE JAK2/STAT3 SIGNAL TRANSDUCTION PATHWAY
Authors: Keightley R. A. , Nixon B. , Roman S. D. , Russell D. L. , Robker R. L. , McLaughlin E. A.
Published: November 2010

Follicular development requires the recruitment of primordial follicles into the growing follicle pool following initiation of multiple cytokine signalling pathways. Suppression of follicular development is thought to be key to maintaining the population of primordial follicles and allowing for controlled release of these follicles throughout the reproductive lifespan of the female. However, little is known of the processes and signalling molecules that suppress primordial follicle activation and early follicle growth. Our group has identified significant upregulation of the Janus Kinase 2 (JAK2)/ Signal Transducer and Activator of Transcription 3 (STAT3) signalling pathway inhibitor the Suppressor of Cytokine Signalling 4 (SOCS4) that coincides with the initial wave of follicular activation in theneonatal mouse ovary. Further studies by our group have localised the SOCS4 protein to the granulosa cells of activating and growing follicles, suggesting SOCS4 expression may be linked to follicular activation. We have focused on examining protein localisation and gene expression patterns of the eight SOCS family members CIS and SOCS1-7. We have recently demonstrated that co-culture of neonatal ovaries with Kit Ligand (KL) for 2 days increases the mRNA levels of all SOCS genes. We also demonstrated the co-localisation of SOCS2 proteins with the KL receptor c-kit in the mural granulosa cells of antral, and large pre-antral follicles suggesting a significant role for SOCS2 in the later stages of follicular development. We have also shown that culturing ovaries with the potent JAK2 inhibitor AG490 substantially reduces mRNA levels of all SOCS and STAT genes that we have so far measured. We hypothesise a significant role for JAK2/STAT3 signalling in promoting the activation and early growth of ovarian follicles. Our investigations have identified significant roles for JAK2/STAT3 and the SOCS family in the regulation of ovarian follicle development.
510. TRANSFORMING GROWTH FACTOR INDUCED PROTEIN TGFβI PROMOTES OVARIAN CANCER CELL MOTILITY AND ADHESION TO PERITONEAL CELLS
Authors: Ween M. , Hoffmann P. , Rodgers R. J. , Ricciardelli C. , Oehler M. K.
Published: November 2010

Ovarian cancer is characterized by metastases to the peritoneal surface lining the abdominal cavity. It remains unclear which factors promote the implantation of ovarian cancer cells onto the peritoneal lining. We have recently investigated interactions between ovarian cancer cells (OVCAR-5, OVCAR-3, and SKOV-3) and mesothelial cells isolated from omental tissues (LP-9). We conducted a proteomic screen of the conditioned medium of co-cultures of ovarian cancer and mesothelial cells. One of the molecules identified to be modulated is the extracellular matrix adhesion protein, transforming growth factor-beta-induced protein (TGFβI, also known as b ig-H3 or keratoepithelin) which is induced by transforming growth factor-beta in many cell types which has been shown to promote adhesion and migration of hepatoma and astrocytoma cells and enhance colon cancer cell extravasation. In this study we investigated the expression of TGFβI in ovarian cancer tissues and the effects of recombinant TGFβI on ovarian cancer motility and adhesion to peritoneal mesothelial cells. In functional assays, treatment with recombinant TGFβI significantly increased adhesion of all three ovarian cancer cell lines to LP-9 mesothelial cells by up to 25% (P<0.01) and increased motility in OVCAR-5 cells by 62% (P<0.001). Furthermore, addition of a neutralising TGFβI antibody reduced OVCAR-5 adhesion to LP-9 to 79% of control level (P<0.001). TGFβI produced by LP-9 cells was processed to smaller forms when co-cultured with ovarian cancer cell lines by western blotting. MALDI-TOF/TOF mass spectrometry identified TGFβI processing at both the N and C terminal domains. The addition of broad spectrum protease inhibitors blocked the TGFβI processing and reduced OVCAR-5 adhesion to LP-9 cells to 60% of control level (P<0.001). We conclude that although some ovarian cancer cells produce low levels of TGFβI, TGFβI abundantly expressed by peritoneal mesothelial cells can promote ovarian cancer cell adhesion and motility.
511. THE EFFECTS OF HIGH FAT DIET ON LIPID LOCALISATION IN THE PERI-OVULATORY CUMULUS OOCYTE COMPLEX
Authors: Yang X. , Dunning K. R. , Hickey T. E. , Norman R. J. , Liang X. , Robker R. L.
Published: November 2010

Intracellular neutral lipids are stored in discrete droplets that are surrounded by lipid associated proteins, such as adipophilin and perilipin, which control cellular lipid metabolism by regulating the access of lipases. The role of lipids in oocyte maturation is unclear, although they have a potential role as an energy source for the oocyte and early embryo. To elucidate potential mechanisms controlling lipid utilisation in the peri-ovulatory cumulus-oocyte-complex (COC) we 1) localised lipid droplets by immunohistochemistry for adipophilin and perilipin and direct staining of neutral lipids with BODIPY and 2) investigated whether a high fat diet can alter oocyte lipid quantity or localisation. Ovaries were isolated from 21 day old mice before and 10h after the ovulation stimulus hCG. Adipophilin and perilipin were both detected by immunohistochemistry in peri-ovulatory follicles with similar localisation before and after hCG. In separate experiments, adult mice were fed a high fat or control diet for 4 weeks and COCs were isolated from preovulatory follicles prior to hCG or from the oviduct 13h after hCG stimulation followed by BODIPY staining and quantification with confocal microscopy. BODIPY staining showed that COCs possess low levels of lipids evenly distributed in the oocyte before hCG but increased lipid assembled as droplets in the oocyte after ovulation. In mice fed a high fat diet, intracellular lipids were markedly increased in both the cumulus cells and oocytes from preovulatory and ovulated COCs. The ubiquitous expression of lipid droplet proteins in the peri-ovulatory follicle together with the changes in neutral lipid storage concurrent with ovulation suggests that lipid metabolism play an important role in oocyte release, transport and/or developmental competence. Furthermore, the dramatic effect of dietary fat on COC lipid content may contribute to the impaired oocyte quality we have observed in obese mice as well as reduced fertility in obese women