bluuebunny/crossref_metadata_2025 huggingface dataset:
143. DIFFERENCES IN GENE EXPRESSION BETWEEN APICAL AND BASAL CELLS OF THE MEMBRANA GRANULOSA
Authors: Irving-Rodgers H. F. , Lee S. T. , Hatzirodos N. , Hummitzsch K. , Sullivan T. R. , Rodgers R. J.
Published: November 2010

Granulosa cells constitute the ovarian follicular epithelium which at the beginning of folliculogenesis forms a single layer of flattened cells. As the follicle matures the cells acquire a cuboidal morphology, proliferate and differentiate into the cumulus cells surrounding the oocyte, and the mural granulosa cells forming the inner layer of the follicle (the membrana granulosa). Mural granulosa cells may further differ in their functionality depending on whether they are situated apically or basally within the stratified membrana granulosa. Late in folliculogenesis granulosa cells develop the ability to produce oestradiol, and also a specialised extracellular matrix (focimatrix) which is more abundant between apical cells. In order to investigate possible differences between granulosa cells, the expression of genes for oestradiol synthesis (CYP11A1, CYP19A1), focimatrix components (LAMB2, COL4A1, HSPG2), FSH and LH receptors, and cell cycle genes (CCND2, CCNE1, CCNE2, CDKN1B, CDKN2D) were examined in apical and basal granulosa cells from large healthy bovine follicles [n = 18, 14.3 ± 0.3 mm (mean + SEM)] using quantitative RT-PCR. Apical granulosa cells were collected by flushing the follicle with balanced salt solution. The remaining cells were detached from the follicular basal lamina by gently scraping; these are the basal granulosa cells. This collection method resulted in equivalent cell yields of apical and basal cells. Expression for all genes was significantly higher in basal cells in comparison to apical cells (P < 0.05), except for the cycle genes CCND2 and CDKN2D, which did not differ between cell populations. These results suggest that functional heterogeneity exists within the membrana granulosa. How differences between apical and basal cells are established is unknown but may be due to the proximity of the basal cells to the follicular basal lamina. The relevance of this aspect of follicle maturation to the endocrine function of granulosa cells has yet to be determined.
144. MAELSTROM - A PROTEIN THAT IS ESSENTIAL FOR SPERMATOGENESIS AND TRANSPOSABLE REPRESSION IS EXPRESSED IN ADULT OVARY OF MAMMALS AND BIRDS
Authors: Lim S. , Tsend-Ayush E. , Kortschak R. , Ricciardelli C. , Oehler M. , Grutzner F.
Published: November 2010

Maelstrom (MAEL) is a highly evolutionarily conserved protein located at the perinuclear structure of animal germ cells called nuage. The MAEL protein contains HMG and Tudor domains and associates with components of the piRNA and RNAi pathways and chromatin remodelling factors. Recent work has shown that MAEL is required for the differentiation of the germ-line stem cell lineage and for the retroposon repression. In mouse, Mael is expressed in male germ cells and is essential for spermatogenesis and retroposon suppression. We have investigated the evolution of the Mael gene in mammals and birds. As expected the gene is highly conserved in all three mammalian lineages and in chicken. Interestingly, the platypus MAEL has exclusive changes in the DnaQ-H 3’-5’ exonuclease domain and computational modelling suggested that these changes may affect the folding of the protein. Expression analysis revealed that the Mael gene is transcribed in testis but also in adult ovaries of chicken, platypus, mouse and human. In situ hybridisation of the Mael transcript on ovary sections of mouse and platypus shows that gene expression is found in pre-antral and antral follicles. The data so far also showed some differences in the expression pattern between mouse and platypus. In mouse, we detected transcript in oocyte, granulosa cells and cumulus cells whereas in the platypus we only observed expression in oocyte. Earlier work demonstrated that Drosophila Mael mutant ovaries had mislocalisation of the RNAi pathway proteins, Dicer and Argonaute2. It is well known that RNAi pathyway is involved in the repression of transposon in the testis and ovary across animal kingdom. As a key component of the RNAi pathway, MAEL is reported to co-localise and interact with MILI and MIWI proteins. These finding may suggest a role of MAEL in retroposon control in ovary and folliculogenesis.
145. WINGLESS (WNT) 3A INDUCES TROPHOBLAST MIGRATION AND MATRIX METALLOPROTEINASE-2 SECRETION THROUGH CANONICAL WNT SIGNALLING AND PROTEIN KINASE B/AKT ACTIVATION
Authors: Sonderegger S. , Haslinger P. , Sabri A. , Otten J. V. , Leisser C. , Fiala C. , Knofler M.
Published: November 2010

Human placenta and trophoblasts express WNT ligands and WNT receptors suggesting a role for WNT-signalling in placental development. Indeed, a recombinant WNT ligand was recently shown to promote trophoblast migration/invasion, however, the involved signalling cascades and their target genes have not been elucidated. The aim was to investigate signal transduction via canonical WNT-signalling or phosphatidylinositide 3-kinase (PI3K)/AKT-signalling, their cross-talk as well as trophoblast-specific protease expression in trophoblastic SGHPL-5 cells and primary 1st trimester extravillous trophoblasts (EVT). WNT3A-dependent activation/phosphorylation of AKT (pAKT) and the down-stream kinaseglycogen synthase kinase (GSK)-3β were determined by Western blotting (WB). WNT3A-induced canonical WNT-signalling was analysed by luciferase reporter assay (TOPFlash) and nuclear recruitment of β-catenin. Trophoblast migration was studied using transwell assays and villous explant cultures. MMP2 expression/activation was investigated by qRT-PCR and WB/gelatine-zymography of supernatants. All experiments were performed +/– inhibitors of AKT-signalling or canonical WNT-signalling using LY294002 (PI3K inhibitor) or recombinant Dickkopf-1 (DKK1). WNT3A induced pAKT, luciferase expression of the canonical WNT reporter (P < 0.05) as well as accumulation of nuclear β-catenin. Inhibition of PI3K abolished WNT-dependent pAKT, pGSK-3β and cell migration, but did not affect TOPFlash activity or appearance of nuclear β-catenin. Inhibition through DKK1 did not influence pAKT and pGSK-3β, but decreased WNT reporter activity, nuclear β-catenin and cell migration. Both inhibitors decreased WNT3A-induced MMP2 expression in SGHPL-5 cells and pure EVTs (P < 0.05). WNT3A activates PI3K/AKT as well as canonical WNT-signalling through distinct receptors in invasive trophoblasts, since DKK1 did not activate the particular kinase. Although cross-talk between PI3K/AKT and canonical WNT-signalling has been observed in some cell types, these pathways seem to act independently in trophoblasts. However, both pathways promote Wnt-dependent migration and expression of MMP2. This is the 1st study identifying MMP2 as a novel target gene of canonical WNT-signalling in trophoblast.
146. MOLECULAR FILTRATION PROPERTIES OF THE EXPANDED CUMULUS MATRIX: CONTROLLED SUPPLY OF METABOLITES AND EXTRACELLULAR SIGNALS TO CUMULUS CELLS AND THE OOCYTE
Authors: Dunning K. R. , Watson L. N. , Thompson J. G. , Robker R. L. , Russell D. L.
Published: November 2010

Cumulus matrix genes are positively correlated with oocyte competence [1]. Formation of the expanded cumulus matrix during oocyte maturation is well described; however its function remains elusive. We investigated whether cumulus matrix acts as a molecular filter, based on recognised filtration properties of analogous matrices. We found that cumulus matrix controls metabolite supply to the oocyte and retains prostaglandin E2 (PGE2), which is critical in oocyte maturation. The uptake of fluorescently labelled hydrophilic and hydrophobic metabolites showed that cumulus matrix formation significantly impeded diffusion to the oocyte. Expanded in vivo matured cumulus oocyte complexes (COCs, eCG+hCG16h) resisted uptake of glucose and cholesterol compared to unexpanded (eCG44h, P < 0.05), as assessed by confocal microscopy and spatial quantitation of fluorescence (P < 0.05). In vitro maturation (IVM) results in pronounced compositional deficiency of cumulus matrix proteins [2] and poor oocyte quality. Glucose and cholesterol were transported more readily into cumulus cells and the oocyte of IVM COCs (matured in αMEM/5% FCS/50 mIU/mL FSH, 16 h) compared to in vivo matured COCs (P < 0.05 and P = 0.08, respectively). Taking the inverse approach we found that PGE2 synthesised by cumulus cells is retained within the matrix compartment of in vivo matured COCs but IVM COCs did not retain PGE2 and secreted 4.3-fold more into the media. The relationship of retained to secreted PGE2 was significantly higher after in vivo maturation vs IVM COCs (P < 0.0001). This property of the COC matrix reveals a potential mechanism whereby the prostaglandin signal intensifies through a physicochemical mechanism rather than gene regulation. This is the first demonstration that cumulus matrix regulates diffusion toward and secretion from the COC, thus excluding glucose, known to negatively affect oocyte quality, and trapping factors, including PGE2, with critical roles in oocyte maturation and fertilisation. Thus, IVM may reduce oocyte quality due to poor trafficking of metabolites and signalling molecules.
(1) McKenzie LJ, et al. Human cumulus granulosa cell gene expression: a predictor of fertilization and embryo selection in women undergoing IVF. Hum Reprod 2004; 19: 2869–2874.(2) Dunning KR, et al. Altered composition of the cumulus-oocyte complex matrix during in vitro maturation of oocytes. Hum Reprod 2007; 22: 2842–2850.
147. MOLECULAR CHARACTERIZATION OF RENIN - ANGIOTENSIN SYSTEM COMPONENTS IN HUMAN INTRAUTERINE TISSUES AND FETAL MEMBRANES FROM VAGINAL DELIVERY AND CAESAREAN SECTION
Authors: Marques F. Z. , Pringle K. G. , Markus M. , Conquest A. , Hirst J. J. , Sarris M. , Zakar T. , Morris B. J. , Lumbers E. R.
Published: November 2010

The expression of the (pro)renin receptor (ATP6AP2) in late gestational human tissues suggests that the prorenin-angiotensin system (RAS) might influence pregnancy outcome. Here w e characterized the RAS in term fetal membranes (amnion and chorion), decidua and placenta (n = 38) from women undergoing elective cesarean section (non-labouring) or following spontaneous delivery (after labour), and myometrium (n = 16) from elective or emergency cesarean (labouring) deliveries. RT-qPCR was used to quantify prorenin (REN), AGT, ACE, ACE2, AGTR1, AGTR2, ATP6AP2 and MAS1 mRNAs, and immunohistochemistry was used to localize prorenin, AGT, ACE, ACE2 and AGTR1 proteins. In myometrium, mRNAs for downstream signalling proteins (ZBTB16, TGFB1 and PTGS2) were also measured. ACE and AGT mRNA levels were higher in labouring myometrium (P < 0.05), consistent with elevated production of angiotensin II (Ang II), which, by the upregulation of PTGS2 occurring in labour (P = 0.022), could influence labour. In amnion, expression of all RAS component mRNAs, except ATP6AP2, was low. After labour amnion showed lower ACE (P = 0.014) and higher AGTR2 (P = 0.01) mRNA levels. In decidua, RAS components other than AGTR1 and AGTR2 were abundant. Amnion and chorion exhibited higher immunostaining of AGT and prorenin than expected from their low mRNA levels, suggesting that these proteins could have been originated from decidua, where the cognate genes are more active. In placenta, prorenin and AGT were localized to syncytiotrophoblasts and ACE was localized to fetal capillary endothelial cells, while ACE2 distribution was diffuse. AGTR1 mRNA and protein expression was high in the placenta. We propose that ACE in fetal vessels could contribute Ang II to the fetus, while ACE2 in syncytiotrophoblasts might convert placental or maternal circulating Ang II to angiotensin-(1–7), which might then be supplied to the maternal bloodstream. In conclusion, the abundance and distribution of intrauterine RAS components suggest diverse roles for this local RAS in pregnancy.
150. IN VITRO CHARACTERISATION OF BIOFILM FORMATION IN HUMAN FOLLICULAR FLUID
Authors: Pelzer E. S. , Allan J. A. , Allan J. M. , Launchbury T. , Knox C. L.
Published: November 2010

Previous studies have detected bacteria in follicular fluid collected at the time of trans-vaginal oocyte retrieval but this was not shown to adversely affect ART outcomes. The antimicrobial properties of follicular fluid have also been investigated, and follicular fluid was reported to be inhibitory to Gram positive bacteria after four days, but supported the growth of Gram negative bacteria. In this study 36 follicular fluid specimens collected from assisted reproductive technology (ART) patients were tested for the presence of bacteria by culture and 16S rRNA PCR assays. Follicular fluid aliquots (24) were incubated and subcultured for 28 weeks and the ability of these follicular fluids to form biofilms in vitro was assessed. A further 12 follicular fluid specimens were inoculated into broth media with and without the addition of estradiol and progesterone at concentrations reported in follicular fluid of hyperstimulated women. Bacteria in polymicrobial (22%) or singles species populations (78%) were detected in the 24 follicular fluid specimens tested. The most prevalent bacteria detected were Lactobacillus species (18/35 total bacterial isolates, 51%) and Propionibacterium spp. (5/35, 14%). All follicular fluids supported the growth of bacteria in vitro for up to 28 weeks. Bacteria in 18/24 (75%) of follicular fluid specimens, formed biofilms in vitro. The majority of biofilms formed were grade IV 7/18 (39%) or grade III biofilms 7/18 (39%). Lactobacilli were also the most prevalent cultivable species within mature biofilms (51% of total isolates). Whilst high levels of steroid hormones were required for the growth of Bifidobacterium spp. in vitro, the growth of both Streptococcus agalactiae and Escherichia coli was inhibited by these hormones. Follicular fluid is not sterile and is an excellent growth media for bacteria, capable of supporting survival for long periods of time. Bacteria which can survive within follicular fluid over time can form mature biofilms, which enables persistence. Women undergoing ART cycles have elevated concentrations of steroid hormones within their follicular fluid and this can modulate the ability of some bacterial species to proliferate. The impact of these bacteria on the developing oocyte and ART pregnancy outcomes requires further investigation.
152. DEVELOPMENT OF A VAGINALLY APPLIED, NON-HORMONAL CONTRACEPTIVE: THE CONTRACEPTIVE EFFICACY AND IMPACT ON BONE TURNOVER OF PEGLA, A LONG-ACTING LIF ANTAGONIST
Authors: Menkhorst E. M. , Zhang J. G. , Morgan P. O. , Poulton I. J. , Metcalf D. , Salamonsen L. A. , Sims N. A. , Nicola N. A. , Dimitriadis E.
Published: November 2010

The WHO has called for the urgent development of pharmacological, non-hormonal contraceptives. Leukaemia inhibitory factor (LIF) is obligatory for embryo implantation in mice and associated with infertility in women. Injection of a long-acting LIF antagonist (PEGLA) blocks uterine LIF, preventing implantation in mice, making PEGLA a promising non-hormonal contraceptive. LIF and LIFR null mice show decreased bone volume associated with increased osteoclast number and size, suggesting PEGLA may target bone. Vaginally administered PEGLA could be a ‘dual-role’ contraceptive: delivered in a microbicide which blocks the vaginal transmission of sexually transmitted infections. We aimed to establish the contraceptive efficacy of vaginally administered PEGLA and identify non-uterine targets of PEGLA in mice. PEGLA was administered to mated female mice by intraperitoneal (IP) injection or vaginally (n = 4/group) during the peri-implantation period to determine its effect on implantation and bone turnover. The tissue and blood accumulation of 125I-PEGLA or control was identified at various time-points following IP injection (≤120 h) or vaginal administration (≤24 h) (n = 3/group). PEGLA administered via vaginal gel blocked implantation (0.0+0.0 vs 8.5+0.5) at a lower dose (500 μg) than IP injection (1500 μg). PEGLA administered by IP injection resulted in fewer (4.0+0.3% vs 7.7+1.5%; P < 0.05) but larger (20.9+0.9 μm vs 18.1+0.5 μm; P < 0.05) osteoclasts and increased trabecular bone volume (6.8+0.9% vs 3.1+1.1%; P < 0.05) but vaginally administered PEGLA had no effect on bone (P > 0.05). 125 I-PEGLA accumulated more quickly (10 min vs 30 min) and was retained longer (96 h vs 24 h) in blood and tissue following IP injection compared to vaginal administration. This is the first study to show the contraceptive efficacy of a PEGylated compound following vaginal delivery. Local delivery of PEGLA decreased the required dose and eliminated the effect on bone, suggesting that local administration would minimise the non-target effects of PEGLA. Contraceptive trials are now required in non-human primates to progress PEGLA towards human clinical trials.
155. UNDERSTANDING THE CROSSTALK IN THE HUMAN UTERINE CAVITY: ROLES FOR SOLUBLE MEDIATORS DURING EMBRYO IMPLANTATION
Authors: Hannan N. , Paiva P. , Meehan K. L. , Hincks C. , Rombauts L. J. F. , Gardner D. K. , Salamonsen L. A.
Published: November 2010

Embryo implantation requires synchronized dialogue between a receptive endometrium and an activated blastocyst via locally produced soluble mediators. During the mid-secretory (MS) phase of the menstrual cycle there is increased glandular secretion into the uterine lumen. These secretions contain important mediators that modulate the endometrium and support the conceptus during implantation. Analysis of the composition of uterine fluid across the menstrual cycle and in fertile and infertile women will, therefore, provide new insights into uterine receptivity. We hypothesized that multiplex platform analysis of human uterine lavages would identify soluble mediators important for the establishment of pregnancy in humans. Lavages were collected (by flushing the uterine cavity with 5mL of saline) from fertile and infertile women during the MS phase and from fertile women during the mid-proliferative (MP) phase of the menstrual cycle. Comparison of lavages from the three cohorts was performed using quantitative MilliplexTM Luminex® cytokine/chemokine assays containing 42-analytes. Luminex analysis detected a number of cytokines in uterine fluid, revealing 8 soluble mediators previously unknown in the endometrium and present in human uterine fluid including, PDGF-AA, TNFβ, sIL-2Rα, Flt-3 ligand, sCD40L, IL-7, IFNα2 and GRO. Furthermore comparison of the three cohorts revealed VEGF levels were significantly higher in the fertile (MS) fluid when compared to infertile. Functional studies demonstrated that rhVEGF treatment significantly increased the adhesive properties in cells present at the maternal-fetal interface. These findings suggest VEGF plays a role in regulating embryo implantation. Furthermore identifying the soluble mediators in uterine fluid may provide potential markers of endometrial receptivity, insight into the unique microenvironment essential for pregnancy and a profile of maternal factors that influence the implanting blastocyst.
156. IDENTIFICATION OF SCAFFOLDING PROTEINS AS PC6 SUBSTRATES IN THE HUMAN ENDOMETRIAL EPITHELIAL CELLS FOR EMBRYO IMPLANTATION
Authors: Heng S. , Li Y. , Stephens A. N. , Rainczuk A. , Nie G.
Published: November 2010

Successful embryo implantation is an important step in establishing pregnancy, which requires a healthy embryo and a receptive endometrium. Establishment of endometrial receptivity involves morphological and physiological changes initially in the endometrial epithelium, however the underlying molecular mechanisms are not fully understood. We have previously demonstrated that proprotein convertase 5/6 (PC6), a member of the proprotein convertase (PC) family, is up-regulated in the endometrium specifically at implantation in association with epithelial differentiation in the human and monkey. PCs convert a range of precursor proteins of important functions into their bioactive forms, they are thus regarded as critical ‘master switch’ molecules. The aim of this study was to identify target proteins of PC6 in the endometrial epithelial cells important for implantation. We used a HEC1A cell line in which PC6 was stably knocked down by siRNA approach (HEC1A-PC6). HEC1A cells that were similarly transfected with a scrambled siRNA sequence (HEC1A-control) were used as the control. Previous study confirmed that HEC1A-PC6 cells had much reduced capacity to adhere to blastocyst. A proteomic comparison between HEC1A-PC6 treated with or without human recombinant PC6 identified ezrin as a potential PC6 substrate. Ezrin is a cytoplasmic protein which is known to bind to ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) thereby translocating to the plasma membrane.This complex has been associated with cytoskeletal re-organisation and changes in cell polarity. Co-immunoprecipitation of ezrin and EBP50 showed that knockdown of PC6 allowed the binding of ezrin to the C-terminus of EBP50 in HEC1A-PC6, whereas PC6 cleavage of EBP50 in HEC1A-control prevented the binding. This was also confirmed by immunofluorescence showing that ezrin and EBP50 were co-localized to the plasma membrane in HEC1A-PC6. This study thus identified that PC6 regulates scaffolding proteins such as EBP50 and ezrin in the endometrium for embryo implantation.
157. LYMPHATICS IN THE HUMAN PLACENTAL BED AND SURROUNDING THE SPIRAL ARTERIOLES DISAPPEAR DURING ENDOMETRIAL DECIDUALISATION
Authors: Volchek M. , Girling J. E. , Lash G. E. , Cann L. , Kumar B. , Robson S. C. , Bulmar J. N. , Rogers P. A. W.
Published: November 2010

The mammalian placenta plays central roles in maternal tolerance of the semi-allogeneic fetus and fluid balance between maternal and fetal compartments. The lymphatics play a role in both immune function and fluid balance. The aim of this study was to describe the distribution of lymphatic vessels in human placental bed and decidua, with particular focus on the lymphatics that surround the remodelling spiral arteries during decidualisation and trophoblast invasion. Placental bed, non-placental bed and decidual biopsies were obtained from women undergoing elective pregnancy termination (6–18 weeks gestational age) and from women undergoing elective caesarean section at term. Double immunohistochemical labeling was performed on serial sections to identify lymphatic vessels in conjunction with blood vessels, smooth muscle, epithelial and trophoblast cells, or proliferating cells. Using representative photomicrographs, descriptive findings of the organisation of the human placental bed lymphatics were made. Lymphatic vessels positive for podoplanin (D2-40) were abundant in hypersecretory endometrium (lacking stromal decidualisation) at all stages of gestation. By contrast, the decidua was nearly always devoid of lymphatics. In some samples, structures that appeared to be regressing lymphatics were observed at the boundary between hypersecretory and decidual tissues. Lymphatic vessels were notably absent from the vicinity of spiral arteries that were surrounded by decidualised stromal cells. Lymphatic vessels in hypersecretory endometrium appeared larger and more elongated as gestation progressed. Proliferating lymphatic vascular endothelial cells were identified in both large vessels, and in streaks of D2-40 positive cells that could have been newly forming lymphatic vessels. In conclusion endometrial stromal cell decidualisation results in a loss of lymphatics, with this phenomenon being particularly apparent around the spiral arterioles; the functional consequences of this loss have yet to be elucidated.
158. THE UNFOLDED PROTEIN RESPONSE MAY CONTRIBUTE TO GLUCOCORTICOID-INDUCED PLACENTAL GROWTH RESTRICTION IN THE RAT VIA INCREASED PLACENTAL EXPRESSION OF HEAT SHOCK PROTEIN 70
Authors: Mark P. J. , Lewis J. L. , Jones M. L. , Waddell B. J.
Published: November 2010

Perturbations of normal endoplasmic reticulum (ER) physiology occur in a number of pathological conditions, including diabetes and preeclampsia. These pathologies are associated with elevated levels of inappropriately folded proteins and induction of ER stress. Accumulation of misfolded proteins induces the unfolded protein response which increases ER protein folding capacity and promotes ER-associated degradation of unfolded proteins. Glucocorticoids are essential for maturation of fetal organs, however excess exposure during pregnancy retards fetal and placental growth. Glucocorticoids also induce ER stress within macrophages, which reside within the placenta, and activate immune responses which can lead to oxidative stress and subsequent placental dysfunction. We hypothesised that excess glucocorticoid exposure would induce ER stress within the placenta and contribute to restriction of fetal and placental growth. This study compared placentas (n = 6/group) for control (Con) and dexamethasone-exposed pregnancies (Dex; 0.75 μg/mL drinking water from day 13 of gestation) at days 16 and 22 of gestation in the rat (term = 23 days). Placentas were dissected into junctional (JZ) and labyrinth (LZ) zones for separate analysis. Quantitative PCR was used to determine expression of mRNA for markers of ER stress, including heat shock factors (HSF-1 and HSF-2), heat shock proteins (HSP-70 and HSP-90) and C/EBP homologous protein (CHOP10). HSF-1 expression increased 2- to 4-fold from day 16 to 22 in both placental zones, but was not increased by glucocorticoids. Dex-exposure increased HSP-70 expression 2- to 3-fold in the LZ at both days of gestation, indicative of an ER stress response. Similar patterns for JZ expression of HSP-70 were observed. JZ expression of HSP-90 was also upregulated by Dex at day 22 but not day 16. CHOP10 was not induced by Dex-administration in either zone at either gestational time, which suggests that rather than activation of the ATF6/PERK pathway, the activation of ER stress is likely to be via XBP1 induction.
159. EXTRACELLULAR MATRIX DYNAMICS IN SCAR-FREE ENDOMETRIAL REPAIR
Authors: Evans J. , Kaitu'u-Lino T. , Salamonsen L. A.
Published: November 2010

Tissue destruction and repair occur simultaneously within the menstruating uterus. Whilst the factors that control menstruation are increasingly understood (1) 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 integrins (2). We here use both an in vivo mouse model of breakdown and repair (3) and an in vitro model of re-epithelialisation to determine factors involved in repair of this tissue. Murine uterine horns were subjected to laser-capture microdissection of the repairing luminal epithelium (LE) and immediate sub-luminal stroma. Extracted RNA was processed for pathway-focused array that revealed expression of epithelial and leukocyte expressed integrins along with their ligands, proteases and protease inhibitors and basement membrane proteins as elevated in repairing uterine horns. Immunohistochemistry confirmed expression and localisation of integrins a 5 and b 1 in addition to integrin ligands VCAM-1 and fibronectin. Additionally, we demonstrated intimate association of neutrophils with repairing/recently repaired luminal epithelium. To confirm the relevance of these ECM interactions in a human model, human endometrial luminal epithelial cell (ECC-1) monolayers were wounded and treated with inhibitors of MMPs (doxycycline, GM6001) or integrin-fibronectin interactions (RGDS). Significant inhibition of in vitro re-epithelialisation was seen following treatment of wounded ECC-1 monolayers with RGDS, doxycycline or GM6001, suggesting a requirement for these ECM components in endometrial re-epithelialisation. We subsequently demonstrated that menstrual blood derived factors may play a role in the initial endometrial re-epithelialisation. Menstrual blood and peripheral blood were collected on the same day from volunteers. Treatment of wounded ECC-1 monolayers with menstrual blood plasma enhanced the rate of repair compared with peripheral blood plasma from the same woman. Enhanced understanding of the mechanisms governing scar-free repair in the endometrium may help resolve pathological endometrial bleeding disorders and may be applied to aid healing in other tissues.
(1) Jabbour et al, 2006. Endocr Rev. 27(1): 17–46.(2) Cao et al, 2007. Hum Reprod. 22(12): 3223–31(3) Brasted et al, 2003. Biol Reprod. 69(4): 1273–80.
161. PREDICTING GESTATIONAL HYPERTENSION AND PREECLAMPSIA FROM MATERNAL ANGIOTENSIN II AND ANGIOTENSIN 1–7 LEVELS AT 15 WEEKS GESTATION
Authors: Sykes S. D. , Lumbers E. R. , Pringle K. G. , Zakar T. , Dekker G. A. , Roberts C. T.
Published: November 2010

Angiotensin II (AngII) is the main effector peptide of the renin angiotensin system (RAS). The RAS is also involved in the aetiology of hypertension. Angiotensin 1–7 (Ang1–7) acting on the Mas receptor may counteract AngII effects. RAS activity is increased in early gestation. We wanted to determine if maternal plasma AngII and Ang1–7 levels in early gestation predict the onset of hypertension in late gestation. Circulating AngII and Ang1–7 have been measured by RIA (D Casley, Prosearch Pty. Ltd.) in EDTA treated plasma from healthy nulliparous pregnant women at 15 weeks gestation from the Adelaide SCOPE cohort for preeclampsia (PE) (n = 50) and gestational hypertension (GHT) (n = 50), with 131 controls. Log transformation and linear regression showed an inverse, weak (R2 = 0.068), statistically significant relationship (P = 0.003) between AngII and Ang1–7. The predictive capability of these peptides for pregnancy outcome was determined by logistic regression and area under the curve after ROC analysis (AROC). The summaries of these analyses are shown in Table 1. AngII did not increase the predictive strength of either model and was omitted. These models show for the first time that circulating Ang1–7 levels are highly statistically significant predictors of hypertensive diseases of pregnancy as early as 15 weeks gestation, well before diagnosis of disease.
162. FRIEND OR FOE? THE ROLES OF PAF AND p53 DURING EMBRYONIC DIAPAUSE
Authors: Fenelon J. C. , O'Neill C. , Shaw G. , Renfree M. B.
Published: November 2010

In the tammar wallaby, Macropus eugenii, the blastocyst normally remains in embryonic diapause for 11 months without cell division or apoptosis occurring. Progesterone regulates reactivation by inducing active secretion from the endometrium, but the molecular cross-talk between the endometrium and blastocyst is unknown. This process may involve the phospholipid paf. Paf is an embryotrophin that acts as a trophic/survival factor in the early embryo, partly by inactivating (via the PI3K/Akt pathway) the expression of p53, a cell cycle arrest factor (1,2). In vitro, paf production from the tammar endometrium increases after diapause (3). This study examined the expression of the paf receptor (pafr) and p53 in the tammar endometrium and embryo at entry into, during and reactivation from diapause. Both pafr and p53 mRNA were expressed in the endometrium at all stages. However there was no quantitative change in pafr expression. In the endometrium, pafr protein is present on the membrane of the glandular epithelium at all stages examined, but p53 was not expressed in the endometrial nuclei at any stage and hence does not appear to be active. Both pafr and p53 mRNA were also expressed in the embryo from the early cleavage stages, during diapause and in the reactivated blastocyst. Pafr protein was present in the embryo both before and after diapause, but levels were greatly reduced during diapause, indicating it may be necessary for active growth. Unexpectedly, the expression of p53 in the embryo does not appear to depend on the presence or absence of pafr. p53 was expressed in the nuclei of the cleavage stage embryonic cells before diapause, but not during or after diapause. These results suggest that paf and pafr may participate in the molecular control of embryonic diapause in the tammar independent of p53.
(1) Jin XL et al. (2009) Biology of Reproduction 80: 286–294.(2) O’Neill C (2005) Human Reproduction Update 11(3): 215–228.(3) Kojima T et al. (1993) Reproduction Fertility Development 5: 15–25.
164. INTEGRIN CLEAVAGE IS MEDIATED BY PROPROTEIN CONVERTASE 6 IN HUMAN ENDOMETRIAL EPITHELIAL CELLS FOR ENDOMETRIAL RECEPTIVITY AND IMPLANTATION
Authors: Paule S. G. , Nie G.
Published: November 2010

Integrins are transmembrane glycoproteins composed of non-covalently associated α and β chains. Integrins participates in cell–cell interaction and binding to components of the extracellular matrix. Integrin expression changes during the establishment of receptivity for implantation. Integrins α1β1, α4β1 and αvβ3 are expressed in the endometrium during the window of implantation [1, 2] and deficiency in integrin αvβ3 is associated with idiopathic infertility and delayed endometrial maturation [1, 3, 4]. Proprotein convertases (PCs) cleave proproteins at the basic amino acids consensus motif (K/R)-(X)n-(K/R)↓ (where n = 0, 2, 4 or 6 and X is any aa) for activation. Integrins require post-translational cleavage for activation and are known to be cleaved by PCs. PC6 plays a critical role in the establishment of endometrial receptivity. We hypothesized that PC6 cleaves integrins for its activation in the endometrial epithelium for implantation. The aim of this study was to investigate the functional importance of PC6 in integrin cleavage, using a stable PC6-knockdown in HEC1A cells (HEC1A-HP) by siRNA technology.Cells transfected with a scrambled siRNA sequence (HEC1A-control) were used as the control. To determine the amount of functional integrins on the cell surface, HEC1A-PC6 and HEC1A-control were subjected to an integrin monoclonal antibody array. The amount of functional integrins α1, α3, α5, αv, αvβ3, β1, β3, β4 and α5β1 on the cell surfacewas much less in HEC1A-HP than HEC1A-control cells. Western blot analysis confirmed that cleavage of pro-integrin α5 into disulfide-linked heavy chain (110kDa) and light chain (35kDa) was greater in HEC1A-control compared to HEC1A-HP cells, suggesting that knockdown of PC6 affects integrin cleavage. Our studies imply that integrin cleavage is mediated by PC6 in endometrial epithelial cells for the establishment of receptivity for embryo implantation.
(1) Lessey BA, et al., Integrin adhesion molecules in the human endometrium. Correlation with the normal and abnormal menstrual cycle. J Clin Invest, 1992. 90(1): 188–95.(2) Tabibzadeh S, Patterns of expression of integrin molecules in human endometrium throughout the menstrual cycle. Hum Reprod, 1992. 7(6): 876–82.(3) Lessey BA, et al., Further characterization of endometrial integrins during the menstrual cycle and in pregnancy. Fertil Steril, 1994. 62(3): 497–506.(4) Lessey BA, et al., Aberrant integrin expression in the endometrium of women with endometriosis. J Clin Endocrinol Metab, 1994. 79(2): 643–9.
165. LOCATION OF ACTIVE TGFB1 IN THE MAMMARY GLAND DURING DIFFERENT STAGES OF DEVELOPMENT
Authors: Sun X. , Robertson S. A. , Ingman W. V.
Published: November 2010

Development of the mammary gland involves complex interactions between epithelial and stromal cells under the influence of hormones and cytokines. Transforming growth factor beta 1 (TGFB1) is a multi-functional cytokine that we have reported to be essential for normal mammary gland development. TGFB1 is produced and secreted as part of a latent complex, and requires activation at the site of action to have biological effects. In situ hybridisation studies have shown mRNA encoding Tgfb1 is mainly expressed by mammary epithelium; however, the expression pattern of active TGFB1 in the mammary gland during different developmental stages is still unclear. Mammary gland tissue was collected from mice at puberty (5 weeks old), adult diestrus and late pregnancy (day 18 pc). Frozen sections were stained with antibody specifically reactive with active TGFB1 protein (not latent TGFB1 or other TGFB isoforms) for immunofluorescent analysis. Qualitative assessment of the staining revealed different patterns of active TGFB1 localisation depending on developmental stage. The strongest expression of active TGFB1 was observed in the mammary gland at diestrus compared to puberty and pregnancy. At diestrus, active TGFB1 was located around the surface of mammary epithelial cells. The staining was heterogeneous, with distinct zones of active TGFB1 accumulated around some but not all epithelial cells. During puberty, active TGFB1 was observed only within the lumen of the ducts. During late pregnancy, TGFB1 was homogenously distributed within the alveolar epithelium. The different patterns of active TGFB1 observed during puberty, diestrus and pregnancy suggest that TGFB1 has different roles in the mammary gland dependent on developmental stage.
168. TGFβ 2-BETAGLYCAN REGULATE FOETAL TESTIS DEVELOPMENT IN VITRO
Authors: Sarraj M. A. , Umbers A. , Findlay J. K. , Stenvers K. L.
Published: November 2010

Betaglycan is a co-receptor for the TGFβ superfamily, known to modulate TGFβ binding in target cells. We have previously found that betaglycan null murine testes at 12.5-13.5 dpc display poorly delineated seminiferous cords and disrupted Leydig cell development (1). Both TGFβs and inhibins are expressed by the fetal testis and it is currently unclear which regulate its development. We tested the hypothesis that loss of betaglycan compromises the functions of TGFβ2 in the differentiating fetal testis as TGFβ2 is known to bind poorly to its type II receptor in the absence of betaglycan. We tested the effect of TGFβ2 on betaglycan wildtype and null foetal gonad/mesonephros complexes using hanging drop or agar block culture methods. From each embryo, one gonad acted as a control; the other was treated. Gonads were cultured in the presence or absence of TGFβ2 (2.5-5 ng/mL) for 48 hours (n =3 pairs). In both culture methods, development in the absence of exogenous growth factor recapitulated normal cord development in wildtype testis and the disrupted cord phenotype in null testes. TGFβ2-treated cultures, 13.5 dpc wildtype mouse testes displayed a 14-35% reduction in total area compared to untreated cultures. Null testes exhibited significantly smaller reductions in gonadal area (2-13%; P < 0.01), indicating that betaglycan null testes exhibit reduced sensitivity to TGFβ2-mediated growth inhibition. However, preliminary observations suggest that TGFβ2 treatment partly rescued cord formation in two of three betaglycan knockout testes in vitro, with testis morphology confirmed by laminin and AMH immunostaining. These data support the notion that TGFβ2 acts via betaglycan to regulate cord development during foetal testis development.
Supported by the New Investigator NHMRC (AUS) grant #550915 to MS, JKF Fellowship (#441101, #550915, #338516;#241000) and Victorian Government infrastructure funds.
(1) Sarraj et al., 2010. Biol Reprod; 82(1): 153–62.
170. THE ROLE OF MEGALIN IN PROSTATE DEVELOPMENT OF THE MOUSE
Authors: Gamat M. , Shaw G. , Renfree M. B.
Published: November 2010

Prostatic development is dependent on androgens; but the precise mechanism by which androgens mediate their effect is still unclear. Megalin, a cell membrane transporter, may shuttle sex steroids into cells to regulate androgen-responsive genes responsible for prostatic bud induction in the urogenital sinus (UGS). In megalin knockout mice, testicular descent fails and the vagina fails to open in females, both of which are dependent on sex steroid signalling (Hammes et al. 2005) . In this megalin-mediated pathway, SHBG-bound sex steroids bind to megalin, which is internalised. The SHBG-sex steroid complex is released, and the sex steroid is released from SHBG where it can bind to the androgen receptor to regulate androgen responsive genes. Receptor-Associated Protein (RAP) is a molecular chaperone protein that protects newly synthesised megalin from binding to potential ligands in the cytoplasm prior to insertion into the cell membrane. We hypothesised that megalin may shuttle SHBG-bound androgens across the cell membrane. This study characterised the expression and evaluated a possible role for megalin in the development of the mouse prostate. Megalin, SHBG and RAP transcripts were detected in the developing male and female UGS of the mouse from day E14.5 to day E18.5 (when prostatic buds start to form) and in the adult prostate. Megalin, SHBG and RAP protein were localised in the urogenital epithelium. To assess the role of megalin in prostatic development, UGS tissues were incubated with androgens in the presence and absence of RAP. Incubating UGS tissues with RAP did NOT inhibit prostatic bud initiation. Furthermore, in the UGS of megalin knockout mice, prostatic bud formation appeared to be identical to those of wild-type littermates. These results demonstrate that megalin is not involved in prostatic bud initiation. However, the ubiquitous expression of megalin suggests that its role is redundant in the prostate.
(1) Hammes A et al. (2005) Role of endocytosis in cellular uptake of sex steroids. Cell 122(5), 751–62.
171. PARENTAL OBESITY RETARDS EARLY EMBRYONIC DEVELOPMENT AND ALTERS CARBOHYDRATE UTILISATION
Authors: Binder N. K. , Mitchell M. , Gardner D. K.
Published: November 2010

Parental obesity impacts reproductive success and often results in gestational complications. In this study the effects of maternal and paternal obesity on preimplantation embryo quality were investigated through analysis of cell cycle length and carbohydrate utilisation. Zygotes derived from matings of lean or obese C57BL/6J mice were used to evaluate separately maternal and paternal obesity. Embryos were cultured individually, and development monitored with high temporal time-lapse microscopy (every 15 min). After 78 h of culture, glucose consumption and lactate production by expanded blastocysts was determined using ultramicrofluorimetry. Maternal obesity was associated with a significant delay (P < 0.01) in pre-compaction cell cycle length of approximately 1.5 h. Post-compaction there was a significant increase (P < 0.05) in glucose consumption by embryos from obese mothers compared to control embryos, while the glycolytic rate was unchanged. Paternal obesity was associated with a significant cell cycle delay (P < 0.05) of approximately 1h from the second cleavage stage onwards. Resultant blastocysts showed disproportionate changes in carbohydrate metabolism, with a significantly increased (P < 0.05) glycolytic rate compared to control embryos. Metabolic changes were still permissive to blastocyst formation, however cell numbers were significantly reduced (P < 0.001) with both maternal (lean: 54.2 ± 0.8 vs obese: 48.4 ± 1.0) and paternal (lean: 60.5 ± 0.09 vs obese: 50.9 ± 0.09) obesity. These data will help to determine the impact of parental obesity on preimplantation embryo physiology. Slow embryonic development and high glycolytic rate have been linked to reduced implantation rates and are general indicators of compromised embryo quality.
174. ATMOSPHERIC OXYGEN ALTERS THE EMBRYONIC METABOLOME AS QUANTIFIED BY CARBOHYDRATE UPTAKE AND AMINO ACID UTILISATION
Authors: Wale P. L. , Gardner D. K.
Published: November 2010

Oxygen regulates embryo development at both the cleavage and post compaction stages. In this study we investigated the effects of atmospheric oxygen during the preimplantation stages, on the post-compaction embryonic metabolome through quantification of glucose consumption and amino acid utilization. Zygotes obtained from F1 hybrid mice (C57BLxCBA) were randomly allocated to either 5 or 20% oxygen. In the first experiment, following the first 48 h of culture, embryos were cultured individually in 1 μL drops of modified G2 medium (0.5 mM glucose) and moved to fresh drops of medium every 24 h. The glucose concentration in the spent media samples, including controls containing no embryo, was determined by microfluorimetry. In the second experiment, embryos which had developed to the early blastocyst stage after 72 h were cultured for a further 24 h in groups of 10 in 2 μL drops of G2. Analysis of amino acid utilization was performed using liquid chromatography-triple quadrupole mass spectrometry. Glucose consumption by embryos cultured in 5% oxygen was significantly greater on day 4 and day 5 (4.89 ± 0.29 and 6.13 ± 0.41 pmol/embryo/h) compared to embryos cultured in 20% oxygen (2.59 ± 0.40 and 5.09 ± 0.28 pmol/embryo/h; P < 0.05). In contrast amino acid utilisation by embryos cultured in 5% oxygen was significantly less than embryos cultured in 20% oxygen (P < 0.05). The data generated will help to determine the aetiology of oxygen toxicity to the preimplantation embryo. Higher glucose utilisation by embryos in 5% oxygen is consistent with their improved development. Conversely, the increased utilisation of amino acids by blastocysts in 20% oxygen may reflect an adaptation to increased oxidative stress as a result of culture in a non-physiological oxygen concentration. This study demonstrates that atmospheric oxygen during the preimplantation period perturbs the embryonic metabolome which results in a compensatory increase in amino acid utilisation.
175. EFFECTS OF SPECIES DIFFERENCES ON OOCYTE REGULATION OF GRANULOSA CELL PROLIFERATION
Authors: Lin J. , Juengel J. , McNatty K.
Published: November 2010

The role of oocytes in regulating ovulation quota between species is not fully understood. In sheep, the oocyte-derived growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) have profound effects on ovarian follicular development and ovulation-quota. The aim of these studies was to compare the ability of oocytes from sheep (low ovulation-rate) and rat (poly-ovulator) to stimulate radiolabelled thymidine uptake by granulosa cells (GC) both within and between the two species. For these experiments, 32 oocytes denuded of cumulus-cells were co-incubated with 20 000 GC from either species. Rat or sheep oocytes stimulated thymidine uptake by GC from the same species (P < 0.005). Sheep oocytes also stimulated thymidine uptake by rat GC (P < 0.001) but not vice versa. To investigate this further, oocytes and GC were co-incubated with 3.2 µg/mL or 7.6 mg/mL monoclonal antibodies specific to GDF9 or BMP15 respectively and to a hydatids antigen (control). Both sheep and rat oocyte stimulation of thymidine uptake by GC was inhibited with the GDF9 antibody (P < 0.05) but not control, irrespective of species of GC. Sheep oocyte stimulation of rat GC was also inhibited using an antibody to BMP15 (P < 0.05). However, when using the BMP15 antibody to block the effects of rat oocytes on rat GC, the inhibition of thymidine uptake was modest (i.e. ~10%) albeit significant (P < 0.05). The molecular forms of GDF9 and BMP15 in spent media were examined by Western blotting under reducing conditions. For both species, oocyte-secreted GDF9 was present in the mature form. For sheep oocytes, secreted BMP15 was present as promature and monomeric mature forms whereas from rats, trace amounts of mature form was sometimes, but not always, detected. Thus, in sheep both BMP15 and GDF9 are essential for regulating GC proliferation whereas in rats, although responsive to BMP15, GC proliferation is likely regulated primarily by GDF9.