bluuebunny/crossref_metadata_2025 huggingface dataset:
177. ANTIBODIES AGAINST BMPR-IB UNCOVERS A PARACRINE FUNCTION FOR THE RECEPTOR IN MALE MOUSE LEYDIG CELL TESTOSTERONE PRODUCTION IN VITRO
Authors: Ciller I. M. , Ciller U. A. , McFarlane J. R.
Published: November 2010

The male reproductive system is regulated by pituitary gonadotrophins and local testicular factors including the transforming growth factor-β superfamily members which up-regulate and modulate testosterone production respectively. Type I and type II bone morphogenetic protein (BMP) receptors have been identified in both steroidogenic and non-steroidogenic cells in the testis and have been reported to impact steroid production via their effect on steroidogenic enzymes. In this study we investigated if BMPR-IB had an autocrine or paracine role in testosterone production using BMPR-IB antibodies in testis tissue and Leydig cell culture in vitro. Immature (21d) and mature (60d) mice were sacrificed by CO2 asphyxiation and the testis removed, decapsulated and cultured in basal and equine chorionic gonadotrophin (eCG)-conditioned media for three hours at 32 °C in 5 % CO2, in the presence and absence of anti-BMPR-IB. Additionally, Leydig cells were Percoll purified from adult mouse testicular interstitial cells and cultured for three hours with and without anti-BMPR-IB under human chorionic gonadotrophin (hCG) stimulated or unstimulated conditions. After three hours of incubation the culture media was aspirated into labeled vials and assayed for testosterone using a radioimmunoassay. In adult testicular slice culture, treatment with anti-BMPR-IB resulted in a significant decrease in basal and eCG-stimulated testosterone production by 37 % and 41 % respectively, while having no significant effect on basal or eCG-stimulated testosterone production by the immature testis. In purified Leydig cell culture from adult male mice BMPR-IB immunization had no effect on testosterone production in basal or hCG-stimulated conditions. In conclusion, anti-BMPR-IB significantly reduced testosterone production in adult testicular slice culture but not in cell culture, demonstrating that BMP paracrine signalling from the seminiferous tubules is likely to be important in modulating testosterone production by Leydig cells. Additionally, the paracrine signalling appears to be developmentally regulated only occurring in the adult testis.
178. THE CHAPERONIN CONTAINING TCP-1 (CCT/TRiC) MULTISUBUNIT COMPLEX IS INVOLVED IN MEDIATING SPERM - OOCYTE INTERACTIONS
Authors: Dun M. D. , Aitken R. , Nixon B.
Published: November 2010

Mammalian spermatozoa only express their capacity for fertilization following capacitation, a process characterized by a suite of biophysical and biochemical changes that occurs as the cells ascend the female reproductive tract. A key event associated with the attainment of a capacitated state is a dramatic reorganization of the sperm surface architecture to render these cells competent to bind to the protective matrix of the oocyte, the zona pellucida. Our previous analysis of these remodeling events has provided compelling evidence that they include the assembly and/or presentation of multimeric protein complexes on the sperm surface. In addition, we have demonstrated that at least two of these complexes possess strong affinity for solubilized zona pellucida. In our current study we have utilised mass spectrometry analysis to reveal that one of these complexes comprises the eight subunits that form a composite, multimeric structure known as the chaperonin containing TCP-1 (CCT/TRiC) complex. Our collective data suggest that this complex participates indirectly in zona pellucida interaction, possibly through the conveyance of key zona adhesion molecules to the sperm surface during capacitation. Consistent with this notion, we were able to demonstrate that the sperm CCT/TRiC complex releases its bound substrates upon exposure to ATP, and this treatment induced a significant, concomitant reduction in the ability of capacitated sperm to bind to the zona pellucida. Furthermore, the use of immunoprecipitation assays confirmed the interaction of the CCT/TRiC complex with at least one putative zona pellucida receptor candidate, namely zona pellucida binding protein 2 (ZPBP2). Future work is now aimed at identifying additional zona receptors that may reside within this complex and the pathways that regulate its functional assembly.
180. THE CONSEQUENCES OF ACRYLAMIDE EXPOSURE ON THE MALE GERMLINE
Authors: Nixon B. J. , Nixon B. , Roman S. D.
Published: November 2010

Acrylamide is a common industrial compound that has recently been identified in cooked, carbohydrate-rich foods such as potato chips, breads and cereals. Acrylamide has been found to be a reproductive toxin in rodents, eliciting male infertility and transgenerational toxicity through the male germline; thus dietary exposure to the compound may have consequences for male fertility and reproduction in humans. The aim of this project was to elucidate the mechanisms of acrylamide toxicity in male germ cells of mice. Freshly isolated early male germ cells were exposed to acrylamide and assessed for cell viability and aberrant morphology. DNA damage in these cells was also investigated using a modified version of the Comet Assay, which allows for adduct specificity. Significant increases in cell death or aberrant morphology were not observed following acrylamide exposure (1 µM, 18 hours). However, a significant increase in DNA damage (125% increase in mean tail DNA assessed by Comet) was identified; which may originate from either the metabolic conversion of acrylamide to glycidamide, leading to glycidamide adducts, or from oxidative stress. Additionally, the regulation of cytochrome P450 gene expression was measured using real time PCR and early male germ cells were found to upregulate gene expression of cytochrome P450 enzymes in response to acrylamide exposure. Collectively, these results support a genotoxic mode of action of acrylamide toxicity, in addition to potential oxidative damage in male germ cells. However, the mechanism by which acrylamide elicits toxicity in germ cells requires further investigation. Future outcomes of this research may provide insight into factors necessary for the healthy development of offspring and aid in the risk assessment of dietary acrylamide exposure in humans.
181. SIRT6 PROTEIN IS REDUCED IN TESTES AND SPERM FROM OBESE MALE MICE
Authors: Palmer N. O. , Fullston T. , Mitchell M. , Lane M.
Published: November 2010

Obesity in males is associated with altered hormone levels, reduced sperm function and increased sperm DNA damage. However, the underlying molecular mechanism has not been identified. Mammalian SIRT6 protein exhibits caloric intake dependant DNA damage repair in other tissue types. However, a possible role for SIRT6 in male obesity and subfertility has not been considered. Therefore, the aim of this study was to assess the effects of male obesity on SIRT6 in testes and mature sperm. Five week old C57BL6 male mice (n =10 per diet) were fed either a control diet (CD) (6% fat) or a high fat diet (HFD) (21% fat) for 16 weeks before collection of sperm and testes. There was no difference in Sirt6 mRNA levels as determined by qPCR in testes from HFD males. Immunohistochemistry showed SIRT6 localised to the nucleus of transitioningspermatids from late round spermatidsuntil early elongating spermatids. SIRT6 relative fluorescence of these positive cell types was significantly decreased by 22% in males fed the HFD compared to CD (P < 0.05). This was confirmed by a decrease in total SIRT6 protein in testes from HFD males as detected by an immunoabsorbance assay (P < 0.05). Surprisingly, SIRT6 was only present in the acrosome of mature sperm. Acrosomal localisation was confirmed by the loss of SIRT6 staining after an induced acrosome reaction. SIRT6 levels in the acrosome of mature sperm was decreased by 11% in males fed the HFD (P < 0.05). This is the first study to show that SIRT6 is located to the acrosome of mature sperm, specific cells within the testes and is reduced in an obese state. Furthermore, this study suggests a possible role for SIRT6 in the acrosomal reaction and therefore potentially fertilisation, processes which are known to be reduced by male obesity.
301. LEUKEMIA INHIBITORY FACTOR IS A CRITICAL REGULATOR OF DECIDUALIZATION OF ENDOMETRIAL STROMAL CELLS IN HUMANS AND MICE
Authors: Lin L. , Menkhorst E. M. , Dimitriadis E.
Published: November 2010

Decidualization is the differentiation of endometrial stromal cells into decidual cells. It is a critical process in embryo implantation, placentation and the establishment of pregnancy. Inadequate decidualization can lead to infertility, abnormal placentation and recurrent miscarriage. Endometrial leukemia inhibitory factor (LIF) is indispensible in blastocyst implantation in mice and dysregulated in infertile women. LIF is produced by 1st trimester decidual cells but its role in decidualization is not known. This study aimed to examine the role of LIF in human and mouse decidualization. Primary human endometrial stomal cells (HESC) were isolated and decidualized (D) by treatment with estradiol (E) +medroxyprogesterone acetate (MPA) for 14 days. HESC were also treated with E+MPA+/–LIF (0.5, 5, 50, 100 and 200 ng/mL) for 14 days. Prolactin secretion was used to assess the extent of decidualization (n = 6). D and non-D HESC were also treated with LIF (0.5, 5, 50, 100 and 200 ng/mL +/– LIF inhibitor) for 15min and the phosphorylation (p) of signal transducer and activator of transcription (STAT)3/STAT3 abundance was detected by Western blot (n = 4). RNA was isolated for analysis of LIF and LIF receptor (R) mRNA expression during decidualization (n = 4). HESC treated with E+MPA+LIF (50, 100 and 200 ng/mL) secreted more prolactin compared to cells treated with E+MPA alone (P < 0.05). LIF increased pSTAT3/STAT3 abundance in D and non-D cells while LIF+LIF inhibitor abolished pSTAT3/STAT3. LIF mRNA was downregulated while LIF-R mRNA increased during decidualization. In vivo, mated mice (n = 5) were injected intraperitoneally with a unique long acting LIF inhibitor post-implantation at day 4.5 of pregnancy and resulted in reduced decidualization compared to control. This is the first study to demonstrate that LIF promoted decidualization of HESC possibly via pSTAT3. It further suggested that LIF regulated decidualization in mice demonstrating a newly identified critical role for LIF in the establishment of pregnancy.
303. NUTRIENT INTAKE OF BOS INDICUS HEIFERS DURING EARLY AND MID-GESTATION CAUSES CHANGES TO PLASMA CONCENTRATIONS OF TRIIODOTHYRONINE (T3) AND THYROXINE (T4) OF THEIR PROGENY
Authors: Micke G. C. , Sullivan T. M. , Perry V. E. A.
Published: November 2010

Fetal and postnatal growth are mediated by thyroid hormones (TH). Maternal nutrient intake during gestation can program postnatal TH concentrations. This may have significant economic implications for beef cattle production. We investigated the effect of feeding beef heifers high (H = 240%) and low (L = 70%) levels of recommended daily crude protein intake during the first and second trimesters of gestation in a two-by-two factorial design on progeny (n = 68) plasma concentrations of free and total triiodothyronine (FT3 and TT3) and free and total thyroxine (FT4 and TT4) from birth until weaning at 6 mo of age. Exposure to low compared to high protein diets during the second trimester resulted in increased plasma FT3 concentrations relative to TT3 (P = 0.04) at birth. For male progeny, exposure to low compared to high protein diets during the first trimester resulted in greater plasma FT4 concentrations from birth until weaning (P = 0.02). Also for males from birth until weaning, LH had greater plasma TT3 concentrations than HH (P < 0.01). For female progeny, HH had greater plasma TT3 concentrations relative to TT4 than HL from birth until weaning (P = 0.03). Plasma concentrations of FT3 were positively associated with average daily gain relative to birth weight at 1 (r = 0.41; P < 0.01) and 3 mo FT3 (r = 0.41; P < 0.01). Heifer protein intake during the first and second trimesters of gestation has a permanent effect to progeny plasma TH concentrations and these changes are associated with the postnatal growth pathway.
(1) Micke GC, Sullivan TM, Magalhaes RJS, Rolls PJ, Norman ST, Perry VEA. Heifer nutrition during early- and mid-pregnancy alters fetal growth trajectory and birth weight. Anim Reprod Sci. 2010; 117: 1–10.
304. DECIDUAL HtrA3 NEGATIVELY REGULATES TROPHOBLAST INVASION
Authors: Singh H. , Makino S. , Endo Y. , Nie G.
Published: November 2010

Controlled trophoblast invasion cell into the maternal decidua (interstitial invasion) is important for placental development. Abnormalities in the invasion process may lead to pregnancy complications. Decidua secrets many factors to control trophoblast invasion. Serine protease HtrA3 is highly expressed in the decidual cells in the late secretory phase of the menstrual cycle and throughout pregnancy. It is highly expressed in first trimester in most trophoblast cell types, but not in the invading interstitial trophoblast. HtrA3 and its family members are down-regulated in a number of cancers and are proposed as tumor suppressors. We hypothesized that HtrA3 is an inhibitor of trophoblast invasion. The current study aimed to investigate whether HtrA3 secreted by decidual cells regulates trophoblast invasion. Human endometrial stromal cells (HESC) were decidualised with estradial, medroxyprogesterone acetate and cyclic AMP. Real-time RT-PCR, western blotting and immunocytochemistry demonstrated that decidualisation increased HtrA3 mRNA and protein expression. HtrA3 was also detected by western blotting in the conditioned media (CM) of decidualised HESC (96h), confirming its secretory nature. For functional studies, wild type and protease inactive mutant HtrA3 were produced using wheat germ cell-free technology. The mutant has negligible protease activity and significantly inhibited the wild type protease activity, supporting its dominant-negative inhibition and utility as a specific inhibitor of the wild type protein. CM of decidualised HESC suppressed invasion of trophoblast HTR-8 cells, whereas inhibition of HtrA3 in the decidual HESC CM by exogeneous addition of HtrA3 mutant increased trophoblast HTR-8 cell invasion. These results strongly support our hypothesis that decidual HtrA3 negatively regulates trophobalst invasion.
305. OPPOSING ROLES FOR MUCINS 13 AND 15 IN EARLY PREGNANCY
Authors: Poon C. E. , Day M. , Murphy C. R.
Published: November 2010

Successful pregnancy is dependent on the cumulation of numerous changes in both the uterine luminal epithelium (ULE) and the invading blastocyst prior to implantation. The apical surface of the ULE constitutes the first point of contact with the blastocyst; thus forming an important player in the implantation event. A constituent of this surface comprises the Mucin family of proteins, which is suggested to play an important role in implantation. Prior studies have found that some Mucins form an anti-adhesive surface on the uterine epithelium to prevent implantation of the blastocyst. The formation of this anti-adhesive surface relates to their large extracellular domains which act to ‘mask’ adhesive receptors present on the ULE from interaction with corresponding ligands present on the blastocyst surface. This process of contact inhibition has been termed ‘steric hindrance’. This study examined the localisation of previously uncharacterised Mucins 13 and 15 in the uterus and blastocyst during early pregnancy to investigate their role in the implantation event. Western blotting, cell fractionation and immunofluorescence techniques were utilised in this study. It was found that Mucin 13 localised to the apical cell surface of the ULE at time of implantation while conversely Mucin 15 was lost from the apical cell surface. In the blastocyst, both proteins were localised to the trophoblast cells and the inner cell mass, with Mucin 15 additionally present in the zona pellucida. Further, the changes in localisation of these proteins in the ULE corresponded to changes in their glycosylation profiles from pre-implantation to time of implantation. These results demonstrate that Mucins 13 and 15 participate in contrasting roles in implantation. Mucin 13 may promote adhesion between the uterus and blastocyst to facilitate implantation while Mucin 15 may prevent adhesion through the mechanism of steric hindrance, given its absence from the apical cell surface at time of implantation.
306. PREDICTING GESTATIONAL DIABETES 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

Gestational diabetes (GD), a pregnancy complication defined by glucose intolerance with onset during pregnancy, is a condition affecting 5.5%–8.8% of Australian pregnancies1. Untreated GD increases perinatal mortality and babies from GD pregnancies have an increased risk of diabetes and obesity, whilst mothers have an increased risk of type II diabetes later in life1. Circulating levels of angiotensin 1–7 (Ang1–7), a peptide of the renin angiotensin system, have been reported to be reduced in third trimester pregnancies with GD2. The effects of Ang1–7 generally oppose those of angiotensin II (AngII) and it is possible that in early gestation pro-angiogenic functions of AngII are counterbalanced by Ang1–7, causing placental insufficiency and pregnancy complications. We wanted to determine the predictive capability of AngII and Ang1–7, in early gestation, for GD. Healthy nulliparous pregnant women from the Adelaide SCOPE cohort with GD (n = 36) or serving as controls (n = 131) had both peptides measured at 15 weeks using an RIA (D. Casley, Prosearch Pty. Ltd.) on EDTA treated plasma. A predictive model was constructed using logistic regression and area under the curve after ROC analysis (AROC, Table 1). AngII did not change the model and was omitted. This model shows that for every one unit increase of Log (Ang1–7 pg/mL) peptide levels the odds of acquiring GD increase five times, suggesting that Ang1–7 levels in early gestation may be a better disease marker than those seen at late pregnancy.
(1) Hoffman L, Nolan C, Wilson JD, et al., 1998. Gestational diabetes mellitus – management guidelines. The Australasian Diabetes in Pregnancy Society. Med. J. Aust. 169, 93–97.(2) Nogueira AI, Santos RAS, Simoes e Silva AC, et al., 2007. The pregnancy-induced increase of plasma angiotensin-(1–7) is blunted in gestational diabetes. Regul. Pep., 141, 55–60.
307. microRNAs ARE DIFFERENTIALLY EXPRESSED BETWEEN MID AND LATE GESTATION AND IN DIFFERENT FUNCTIONAL ZONES OF THE RAT PLACENTA
Authors: Kong W. , Moretta S. , Grant P. A. , Dziadek M. , Owens J. A.
Published: November 2010

MicroRNAs (miRNAs) are short, single-stranded, non-coding RNAs that regulate translation, by interacting with complementary sites in the 3′UTRs of target mRNAs. Each can target multiple mRNAs and each mRNA can be targeted by many different miRNAs to form regulatory networks. Placental development is characterised by dynamic, co-ordinated changes in expression of regulatory and functional genes that drive invasion, differentiation and growth. These changes may involve miRNAs, but placental miRNA expression during gestation has been little studied in any species to date. MiRNA expression was examined (by microarray) in the rat in mid (day 16) and late (day 20) gestation (term ~22 days) and the two major zones of the placenta, the labyrinth and junctional zones, at and between each gestational age. At day 16, 107 miRNAs were differentially expressed between the labyrinth and junctional zone (P < 0.05); 47% being more highly expressed and the remainder less expressed in the labyrinth compared to the junctional zone. At day 20, 78 miRNAs were differentially expressed between the two zones (P < 0.05); 53% being higher and the remainder lower in the labyrinth compared to the junctional zone. 216 miRNAs were differentially expressed in the labyrinth zone between mid and late gestation; 73% of the differentially expressed labyrinth zone miRNAs were upregulated at day 20, many from a cluster on chromosome 6, a feature of the late gestation mouse placenta as well (Kong et al., unpublished). In the mouse, these same clustered miRNAs are located on chromosome 12 in an imprinted region, with the miRNAs expressed from the maternal allele. Disruption of their expression impairs placental vascular, labyrinth and junctional zone development [1]. Together these findings imply that these clustered miRNAs are likely to play a role in labyrinth zone development and further study is warranted into which placental transcripts are targeted by these imprinted miRNAs.
(1) Georgiades P, et al., Parental origin-specific developmental defects in mice with uniparental disomy for chromosome 12. Development, 2000. 127(21): 4719–28.
308. FOLLISTATIN SPLICE VARIANTS FST288 AND FST315 INCREASE DURING EARLY MOUSE PREGNANCY: REGULATION BY PROGESTERONE?
Authors: Craythorn R. G. , Winnall W. R. , Hedger M. P. , Rogers P. A. W. , De Kretser D. M. , Girling J. E.
Published: November 2010

Follistatin acts by binding and neutralising the activity of activin-A, which has important regulatory roles in development, reproduction and inflammation. There are two isoforms of follistatin comprising 288 and 315 amino acids (FST288 and FST315), resulting from alternative gene splicing. FST288 binds spontaneously to heparan sulphate and is largely bound to cell surface proteoglycans. FST315 is the predominant circulating form and can only bind to heparan sulphate after binding activin-A. The regulation of these splice variants in the female reproductive tract have not been investigated in detail. In this study, our aim was to quantify the expression of FST288 and FST315 mRNA in the mouse uterus during early pregnancy (days 1–4, pre-implantation), and in response to exogenous oestradiol-17b (100 ng × single s.c. injection, dissection after 24 h) and progesterone (1 mg × three daily s.c. injections, dissection 24 h after last injection) in ovariectomised mice. Gene expression was analysed using quantitative RT-PCR. Primers amplifying a product from exon 5 to 6a (unique to FST288) or from exon 5 to 6b (unique to FST315) were used to discriminate the isoforms. In early pregnancy, expression of both FST288 and FST315 increased significantly (approximately 35-fold and 100-fold, respectively) on days 3–5, relative to days 1–2, corresponding with the increase in circulating progesterone levels that occurs at day 3. A significant increase in FST288 and FST315 mRNA expression (both approximately 35-fold) was also observed in ovariectomised mice in response to exogenous progesterone, but there was no increase in response to oestradiol-17β. In contrast to the similar rate of increase in response to exogenous progesterone, FST315 mRNA expression increased more rapidly than FS288 in early pregnancy, indicating that differential regulation of the two isoforms also occurs. We conclude that progesterone regulates both FST288 and FST315 mRNA expression during early pregnancy in the mouse uterus.
309. DEVELOPMENT AND VALIDATION OF AN ELISA FOR HIGH TEMPERATURE REQUIREMENT FACTOR A3 (HtrA3): EARLY DETECTION OF PREECLAMPSIA
Authors: Dynon K. , Nie G.
Published: November 2010

Preeclampsia (PE) is a multisystemic condition in pregnant women that can be life threatening for both mother and baby. PE is a hypertensive disorder that develops concurrently with proteinuria after 20 weeks of gestation. Abnormal placental development during early pregnancy precedes the onset of PE later in gestation. Early diagnosis of PE is essential to reduce PE-related mortality and morbidity. To date there is no clinically useful biochemical diagnostic method that can detect PE during early pregnancy. Our laboratory discovered and cloned the serine protease HtrA3 and has shown that HtrA3 protein levels are intimately involved in placentation (1). Persistently high serum levels of HtrA3 are detected at the end of the first trimester in pregnant women who subsequently develop PE, suggesting that monitoring HtrA3 in maternal blood during early pregnancy may identify women at risk for PE. To develop monoclonal antibodies specific for HtrA3; and to develop an enzyme-linked immunosorbent assay (ELISA) to detect HtrA3 in human sera. Monoclonal antibodies were generated against full length human HtrA3 and small HtrA3 peptides and tested on recombinant HtrA3, human sera and first trimester decidual and villous tissues using western blot, immunoprecipitation and Amplified luminescent proximity homogeneous assay (Alpha)LISA technology. Three antibody pairs were identified that detected either short and/or long isoforms of HtrA3 in sera and placental tissues. Recombinant HtrA3 was detected by AlphaLISA and higher levels of HtrA3 were detected in serum of PE women compared to gestation-matched controls in preliminary testing. These antibody pairs can now be used for the development of specific and high throughput assays. The AlphaLISA will then be used to validate that abnormal serum HtrA3 levels during early pregnancy can predict preeclampsia.
(1) Nie et al, (2006) Biol Reprod 74, 366–374.
310. CAVEOLIN 1 AND 2 IN THE UTERUS DURING EARLY PREGNANCY
Authors: Madawala R. J. , Murphy C. R.
Published: November 2010

Rat uterine epithelial cells undergo many changes during early pregnancy in order to become receptive to blastocyst implantation. These changes include basolateral folding and the presence of vesicles of various sizes which are at their greatest number during the pre-implantation period. The present study investigated the possible role that caveolin 1 and 2 plays in this remodelling specifically days 1, 3, 6, 7, and 9 of pregnancy. Caveolin is a major protein in omega shaped invaginations of the plasma membrane called caveolae that are considered to be specialised plasma membrane subdomains. Caveolae are rich in cholesterol, glycosphingolipids, and GPI anchored proteins and are involved in endocytosis and membrane curvature. Immunofluorescence microscopy has shown caveolin 1 and 2 on day 1 of pregnancy are localised to the cytoplasm of luminal uterine epithelial cells, and by day 6 of pregnancy (the time of implantation), it concentrates basally. By day 9 of pregnancy, expression of both caveolin 1 and 2 in luminal uterine epithelia is cytoplasmic as seen on day 1 of pregnancy. A corresponding increase in protein expression of caveolin 1 on day 6 of pregnancy in luminal uterine epithelia was observed. Interestingly however, caveolin 2 protein expression decreases at the time of implantation as found by western blot analysis. Both caveolin 1 and 2 were localised to blood vessels within the endometrium and myometrium and also the muscle of the myometrium in all days of pregnancy studied. In addition, both caveolin 1 and 2 were absent from glandular epithelium, which is interesting considering that they do not undergo the plasma membrane transformation. The localisation and expression of caveolin 1 and 2 in rat luminal uterine epithelium at the time of implantation suggest possible roles in trafficking of cholesterol and/or various proteins for either degradation or relocation. Caveolins may contribute to the morphology of the basolateral membrane seen on day 6 of pregnancy. All of which may play an important role during successful blastocyst implantation.
311. REGULATION OF THE RENIN ANGIOTENSIN SYSTEM (RAS) IN A TROPHOBLAST CELL LINE BY CYCLIC ADENOSINE MONOPHOSPHATE (cAMP) AND 5'-AZA-2'-DEOXYCYTIDINE (AZA)
Authors: Wang Y. , Pringle K. G. , Chen Y. , Zakar T. , Lumbers E. R.
Published: November 2010

Renin and renin-like activities have been detected in early gestation placentae1,2. To explore what regulates RAS expression in trophoblasts we studied the effects of cAMP and AZA (DNA demethylating agent) on the expression of mRNAs for prorenin receptor (ATP6AP2), prorenin (REN), angiotensinogen (AGT), angiotensin converting enzyme (ACE), ACE2, angiotensin II type 1 receptor (AGTR1), and a downstream target of the prorenin receptor, cyclooxygenase-2 (PTGS2) in the early human trophoblast cell line HTR-8/SVneo. Cells were cultured for 24 or 48 h with vehicle, 0.5 mM cAMP or 15 mM AZA. Messenger RNA abundances were measured relative to Alien RNA using real-time qRT-PCR. All mRNAs were detected except for ACE and ACE2. REN mRNA abundance was increased by cAMP and AZA (P < 0.0001). However, cAMP was more effective than AZA at both 24 and 48 h (P < 0.0001; P < 0.05) at the concentrations employed. AZA significantly increased AGT mRNA levels at 24 h (P = 0.001) and AGTR1 mRNA levels at 48 h (P < 0.0001), while cAMP had no effect. P TGS2 mRNA expression was increased by cAMP at 24 h (P < 0.0001) and by AZA at 48 h (P < 0.0001). cAMP and AZA had no effect on ATP6AP2 abundance. Thus, prorenin may function directly through the prorenin receptor, since cAMP upregulated both REN and its downstream target PTGS2 in HTR-8/SVneo cells, especially considering that ACE and ACE2 were not expressed. Our findings also suggest that expression of the RAS in trophoblast is regulated by DNA methylation as there was sustained over expression of REN, AGT, AGTR1 and PTGS2 with AZA. The ATP6AP2 promoter possesses CpG islands; however, its expression was not affected by AZA. This may suggest that either DNA methylation is not involved in the regulation of this gene, or that the over expressed prorenin binding to its receptor is causing suppression of ATP6AP2 abundance via a previously identified negative feedback pathway.
(1) Pringle et al., Perinatal Society of Australia & New Zealand (PSANZ) 2010, Wellington, New Zealand (Abstract).(2) Itskovitz et al., Journal of Clinical Endocrinology and Metabolism, 1992, 75: 906–10.
312. THE DISTRIBUTION OF PROMININ-1 IN THE RAT UTERUS DURING EARLY PREGNANCY
Authors: Dowland S. N. , Lindsay L. A. , Murphy C. R.
Published: November 2010

Prominin-1 is a recently discovered pentaspan membrane protein present in characteristic cholesterol-based vesicles and associated with microvilli. These vesicles are used to deliver prominin-1 to the apical plasma membrane in a number of cell types. Previous work on uterine epithelial cells has demonstrated a loss of microvilli and the presence of large, cholesterol-based vesicles at the time of implantation. Thus this study aims to determine a role for prominin-1 in rat uterine epithelial cells during early pregnancy. Immunofluorescence microscopy reveals punctate and diffuse prominin-1 staining below the apical plasma membrane on day 1 of pregnancy. At the time of blastocyst implantation (day 6) however, prominin-1 appears concentrated at the apical surface of the cell. Western blotting of isolated uterine epithelial cell lysate revealed a change in prominin-1 glycosylation during early pregnancy. Prominin-1 was determined to be glycosylated on day 1 of pregnancy, but these carbohydrate side chains were lost by the time of attachment. Results seen in the present study indicate that prominin-containing vesicles may be prevented from reaching the apical plasma membrane by the terminal web on day 1 of pregnancy. On day 6, the loss of the terminal web may allow the vesicles to approach and incorporate into the apical plasma membrane, as seen with other uterine vesicles. The deglycosylation of prominin-1 at this time is suggested to allow the protein to bind its ligand and activate downstream signalling pathways that permit implantation. This study constitutes the first reported observation of prominin in endometrial lumenal epithelial cells. These preliminary results, in consideration with previous reports of prominin expression in trophoblast cells, suggest an important role for this protein in early pregnancy.
313. EPIGENETIC REGULATION OF THE CRH GENE PROMOTER INVOLVES SPECIFIC CpG DE-METHYLATION
Authors: Pan X. , Abou-Seif C. , Allars M. , Chen Y. , Nicholson R. C.
Published: November 2010

Corticotropin Releasing Hormone (CRH), is expressed in many regions of the central nervous system and in some peripheral tissues, and plays an important role in determining gestational length. In placenta, a cAMP regulatory site (CRE) is crucial for CRH gene regulation. The promoter of CRH gene has 9 CpG sites, which should be the targets of epigenetic regulation by DNA methylation. The BeWo cell line, derived from human gestational choriocarcinoma, has been widely used as an in vitro model for the placenta. BeWo cells only produce CRH after exposure to cAMP. The DNA methyl transferase (DNMT) inhibitor 5-aza-cytidine stimulates CRH expression 5-fold in camp treated BeWo cells, indicating the CRH promoter as a target of DNMTs. To evaluate methylation differences of the 9 CpG sites in CRH gene promoter in BeWo cells after treatment with cAMP. Genomic DNA was extracted from BeWo cells treated or not with cAMP. Sodium bisulfite conversion was used to modify the genomic DNA. PCR was used to amplify the CRH promoter region with primers that did not contain CpG sites. The PCR products were cloned and sequenced. The CpG methylation status of each sample was obtained by comparing the sequencing results with the original sequence. In non-stimulated cells (control) CpG -4 was methylated in 50% of the clones and CpG -6 was methylated in 75% of the clones, but the other 7 sites were methylated in every clone. In the cAMP treated cells however there was 100% methylation at CpG sites 6 through 9, but only partial methylation at CpG-1 and 3 (60%), CpG-4 and 5 (40%). Most interestingly, there was no methylation found at CpG-2 in any of the clones from cAMP treated cells, indicating that specific CpG de-methylation around the CRE is required for CRH gene expression.
314. PREECLAMPSIA AND SMALL FOR GESTATIONAL AGE BABIES ARE ASSOCIATED WITH DEFECTS IN THE FOLATE-HOMOCYSTEINE METABOLIC PATHWAY
Authors: Furness D. L. F. , Thompson S. D. , Dekker G. A. , Roberts C. T.
Published: November 2010

The folate-homocysteine metabolic pathway plays a critical role in determining the availability of folate and methionine which are essential for placental and fetal development. Defects in this pathway can increase circulating homocysteine concentration and impair DNA synthesis. The purpose of this study was to investigate genetic polymorphisms involved infolate-homocysteine metabolism in association with preeclampsia (PE) and small for gestational age (SGA) babies. The Adelaide SCOPE (Screening for Pregnancy Outcomes) was a prospective study including 1169 nulliparous pregnant couples. Adelaide samples and patient information were collected by SCOPE research midwives. Genotyping was performed by Sequenom MassARRAY. Non-Caucasian couples were excluded from analyses. Pregnancy outcomes were strictly classified: PE (n = 71), SGA (n = 94) PE+SGA (n = 20), controls (n = 450). Chi Square and univariate with post-hoc analyses were performed. Women who developed PE and SGA had a mean BMI of 30.1 and 27.8 respectively, compared to women with healthy pregnancies 25.7 (P <0.001 and P = 0.002). Maternal and fetal MTHFD1 were associated with reduced customised birthweight centiles (P = 0.034 and P = 0.028 respectively). Paternal MTR 2756 genotype was associated with reduced customised birthweight centiles (P = 0.016) and with PE+SGA compared to healthy pregnancies P = 0.006. Neonatal MTR 2756 showed a similar trend with the G allele increased in PE+SGA P = 0.070. In addition, the paternal MTHFR 677 TT genotype was associated with PE+SG (P = 0.026). Variant polymorphisms within folate-homocysteine metabolism genes can slow enzyme activity increasing plasma homocysteine leading to maternal and placental vascular damage associated with both PE and SGA. This study builds on previous work in our laboratory that focused on maternal genotype and circulating factors shown to be associated with PE and SGA including serum folate, vitamin B12 and plasma homocysteine.
315. HOMEOBOX GENES ARE DIFFERENTIALLY EXPRESSED IN PRIMARY VILLOUS AND EXTRAVILLOUS TROPHOBLAST CELL LINEAGES DURING EARLY PREGNANCY
Authors: Murthi P. , Pathirage N. A. , Keogh R. , Cocquebert M. , Segond N. , Brennecke S. P. , Fournier T. , Evain-Brion D. , Kalionis B.
Published: November 2010

During human placental development trophoblast cells differentiate along either the villous cytotrophoblast (VCT) lineage to form the syncytiotrophoblast (ST) or the invasive extravillous cytotrophoblast (EVCT) lineage (1). Abnormalities in early differentiation processes are characteristic of poor placentation, which is associated with fetal growth restriction (FGR) and pre-eclampsia (PE), the major clinical complications of human pregnancy (2). A large family of homeobox gene transcription factors controls “cell-fate decisions” during development (3), but the expression profile and role of homeobox genes in the human trophoblast cell lineages is not well understood. The aim of the study was to determine homeobox gene expression in primary cultures of mononuclear VCT (2h) and EVCT (2 h) obtained from first trimester human chorionic villi of 8–12 weeks of gestation and in vitro differentiated ST (72 h) and invasive EVCT (48 h), respectively. The isolation and characterization of freshly isolated VCT, EVCT and in vitro differentiated ST and invasive EVCT were performed as described previously (1,4). The homeobox gene mRNA profile was performed using PCR arrays in a pooled sample of VCT and EVCT (n = 6 in each group) and further validated by real-time PCR. Homeobox gene expression studies revealed MSX2 mRNA levels were the highest in VCT (2 h) but undetectable in EVCT (2 h). Further comparisons of homeobox gene expression in in vitro differentiated ST to invasive EVCT showed marked increase in MSX2, DLX3, DLX4 and MEIS1 mRNA levels in ST, which are regulators of cellular differentiation in many studies. Homeobox genes HLX and HHEX, which are implicated in regulating cellular proliferation showed decreased mRNA levels in ST compared to invasive EVCT. Our results demonstrated several known placental and novel homeobox genes are differentially expressed in trophoblast cell lineages. Functional studies of these candidate genes will provide a better understanding of the molecular mechanisms of early placental development.
(1) Tarrade et al. (2001) Lab Invest. 81, 1199–1211.(2) LokeYW and King A (1995) Cell Biology and Immunology, Cambridge ed.(3) J Cross et al. (2002) Recent Progress in Hormone Research 57: 221–234.(4) Handschuh et al. (2007) Placenta, 28, 175–184.
316. MATERNAL DIETARY ARGININE SUPPLEMENTATION DURING LATE GESTATION IMPROVES REPRODUCTIVE EFFICIENCY IN PIGS
Authors: De Blasio M. J. , Roberts C. T. , Kind K. , Smits R. , Nottle M. , Owens J. A.
Published: November 2010

Arginine (a non-essential amino acid) and its conversion to nitric oxide (NO) can promote formation of new blood vessels and cause vasodilation. This may reduce resistance and increase blood flow to the uterus and placenta, and the delivery of nutrients for fetal growth and survival. In pregnant rats, dietary arginine deficiency causes IUGR and increases fetal death and perinatal mortality, whereas dietary arginine supplementation reverses this. Human IUGR is associated with impaired NO synthesis, and eNOS activity in umbilical vein endothelial cells, but maternal arginine supplements have produced inconclusive results. We hypothesised that maternal arginine supplementation (MAS) in the pig (a species with naturally occurring IUGR), during late gestation, when placental angiogenesis and vascularity increase, would increase birth and placental weights. Large White (LW) and Landrace (LR) gilts (n = 285) and sows (n = 326), were fed either a control or arginine supplemented (+25 g/d arginine, Nutreco Progenos premix) diet (2.5 kg/d) in late gestation (d75-term at ~114 days). Number born, born alive, still born and mummified, birth weight and d10 weight of progeny were measured. Data were analysed using Univariate ANOVA. MAS in late gestation in gilts and sows reduced the number of still born (Con: 1.17 ± 0.13 piglets/litter; Arg: 0.84 ± 0.09 piglets/litter; P = 0.046). In LW gilts, MAS increased birth weight (Con: 1.21 ± 0.05kg; Arg: 1.34 ± 0.05kg; P < 0.05), and litter birth weight (Con: 13.38 ± 0.72 kg; Arg: 15.27 ± 0.73 kg; P < 0.05). MAS also increased birth weight in LW (Con: 1.17 ± 0.06 kg; Arg: 1.30 ± 0.06 kg; P < 0.05) and LR (Con: 1.47 ± 0.05 kg; Arg: 1.60 ± 0.05 kg; P < 0.05) sows, and reduced still borns in LW sows (Con: 1.12 ± 0.14 piglets/litter; Arg: 0.77 ± 0.09 piglets/litter; P < 0.05). MAS in late gestation improves pregnancy outcomes in terms of piglet survival and birth weight, in LW and LR gilts and sows. MAS during critical periods of placental development may enhance placental-fetal blood flow and nutrient transfer, thereby improving fetal growth and survival.
317. DIFFERENTIAL EFFECTS OF cAMP AND CRH IN CELL VIABILITY AND CELL FUSION OF CYTOTROPHOBLASTIC CELLS
Authors: Chen Y. , Allars M. , Abou-Seif C. , Smith R. , Nicholson R. C.
Published: November 2010

Cell fusion of cytotrophoblasts is critical for a successful pregnancy, and is marked biochemically by proteins such as human chorionic gonadotrophin (hCG) and syncytin, and morphologically as large multi-nucleated cells. Placental corticotrophin releasing hormone (CRH) plays diverse roles during pregnancy and has been linked to the length of gestation. It is well accepted that CRH expression could be upregulated by some hormones, such as hCG via cellular cAMP, and that trophoblastic cell fusion can be induced by Forskolin (increases cAMP). We have shown, by reporter gene assay in human primary placental cells, that 8-Br-cAMP increased CRH gene expression. Thus, it is of interest to determine the roles of 8-Br-cAMP and CRH on trophoblast cell viability and cell fusion. Treatment with 8-Br-cAMP for 72 h significantly inhibited BeWo cell viability (MTT assay) by 29.7% (50 μM) and 54.8% (100 μM), and increased both apoptotic and necrotic cells (FACS analysis). 8-Br-cAMP (100 μM, 48 h) resulted in the appearance of nuclear fragments (DAPI stain) and Fas ligand gene expression by 6.8-fold (real-time RT-PCR). Syncytin 1 mRNA increased by 3.2-fold (real-time RT-PCR) and hCG secretion increased by 4-fold (ELISA assay). The formation of syncytium (CellMask and Hoechst co-stain) could clearly be seen by 72 h. CRH mRNA increased with 8-Br-cAMP treatment of BeWo cells (15-fold at 48 h). Treatment with CRH (100 nM) had a mild inhibitory effect on cell growth (~18%) but no effect on cell viability, up-regulated the expression of syncytin 1 mRNA (2.3-fold at 48 h) and induced cell fusion (72 h), but had no effect on hCG secretion. In summary, we show that 8-Br-cAMP and CRH are both potential inducers of cytotrophoblast cell fusion, and there is a dissociation between morphological and biochemical differentiation. Our data also indicates that the process of cell fusion can be associated with both apoptotic and non-apoptotic events.
318. POLYURIA IN PREGNANCY - CHALLENGES OF DIAGNOSIS!
Authors: Sakthivel A. , Nankervis A. J. , Conn J. , Oats J. , Chitturi S.
Published: November 2010

Increased urinary frequency is a widely experienced symptom in pregnancy; true polyuria is less common and can have a number of causes. It is essential, however that it should be recognized and appropriately investigated, as it may be potentially life threatening to mother and fetus. Diabetes insipidus (DI) is a rare complication of pregnancy. It can be the first presentation of pre-gestational DI, gestational DI or DI associated with acute fatty liver of pregnancy. The latter conditions are illustrated by the following two clinical cases. Osmotic homeostasis can be altered in pregnancy due to resetting of the osmostat and degradation of vasopressin by vasopressinase secreted by the placenta in increasing quantities in the 2nd and 3rd trimesters. Liver failure results in the availability of higher circulating levels of vasopressinase. These cases highlight the difficulties and importance of diagnosis and treatment. Pregnant women often report polyuria during the 3rd trimester. It is important to have a high level of clinical suspicion for the pathological causes, despite the non-specific presentation as underlying conditions can have catastrophic sequelae.
320. THE ROLE OF FIZZY RELATED 1 IN MALE MEIOSIS
Authors: Hopkins L. , Pye V. , Fraser B. , Holt J. , Jones K. , McLaughlin E.
Published: November 2010

Accurate chromosome segregation during mitosis and meiosis is facilitated by a regulatory complex known as the Anaphase Promoting Cyclosome (APC), an ubiquitin ligase complex that tags proteins with ubiquitin. Subsequently targeted proteins are recognised by the 26S proteosome and degraded. In mammalian cells, two temporally regulated co-activators are required for the APC to function; fizzy and fzr1. In studies of female oocyte development fzr1 has been demonstrated to play an important role in maintaining G2 arrest during meiosis by controlling spatial levels of the cell cycle protein Cyclin B1 but the role of Fzr1 in spermatogenesis remains unknown. Germ cell specific conditional knockout fzr1mice were generated using the DDX4-Cre and flox/flox fzr1 mouse lines and initial gross morphological analysis indicated that at 7 weeks of age null mice possessed significantly smaller testes (21.81mg ± 0.23mg) when compared to heterozygote (99.86mg ± 1.58mg) and wildtype littermates (93.06mg ± 1.16mg) n = 3 P < 0.0001. Quantitative gene expression analysis confirmed almost complete absence of fzr1 transcript in testes (20-fold decrease) in comparison to wild-type. Immunoblotting and immunohistochemistry revealed no expression of Fzr1 protein in meiotic and post meiotic germ cells when compared to heterozygote and wild type littermates. Histomorphological analysis of testes tissue sections revealed Fzr1 null males exhibited spermatogenic arrest and a complete absence of round spermatids with concomitant apoptosis in the residual spermatocytes. Epididymal examination confirmed a complete lack of mature spermatozoa in the cauda epididymis of null males. In contrast, both wild type and heterozygote mice displayed normal spermatogenesis and epididymal sperm analysis indicated no distinguishable differences in seminal characteristics with normal motility, morphology and sperm-zona binding capacity. Based on these observations we hypothesise that Fzr1 plays a significant role in the establishment and maintenance of meiosis possibly through regulation of key cell cycle proteins.