bluuebunny/crossref_metadata_2025 huggingface dataset:
035. Circadian rhythms and the early life programming of adult physiological systems
Authors: Kennaway D. J.
Published: November 2010

We are all familiar with the idea that the external environment influences many diverse physiological systems. For example, the level of nutrition can not only influence adult health directly, but also fetal development and subsequently many adult functions in the offspring. Maternal stress can affect fetal outcomes as can the administration of drugs during pregnancy. Until recently, however, the daily changes in environmental light have been considered to really only influence the time that we sleep and in many other species the optimal time to mate. The impact of circadian rhythms on life trajectory has had little attention.
In the last 5 years it has become clear that circadian rhythmicity is entrenched in virtually every cell of our bodies. A suite of clock gene transcription factors that include Clock, Bmal1 and the period and cryptochrome genes, generate a robust daily cycle of transcription and translation of hundreds of proteins. This cellular clock system is synchronised with the external photoperiod through retinal light perception, the hypothalamic suprachiasmatic nucleus (SCN) and neural and hormonal pathways. Most importantly when the clock system in peripheral tissues is disrupted, a growing list of detrimental consequences are being uncovered. As an example, mice with mutations in either Clock or Bmal1 have non-rhythmic peripheral tissues and exhibit mild to severe reproductive failure and metabolic dysfunction. Null per2 mice have a higher incidence of salivary gland hyperplasia, teratomas and increased susceptibility to radiation induced lymphomas. It is also apparent that intrauterine insults (e.g. cocaine administration, poor nutrition and stress) can have long term effects on the central circadian timing system in the SCN. Whether this involves alterations in neural development or gene function is not known. Nevertheless it is time we paid more attention to the temporal nature of our environment as a possible contributor to lifetime disorders and diseases.
037. Impact of glucocorticoids on fetal-placental growth and the postnatal phenotype
Authors: Waddell B. J. , Wyrwoll C. S. , Hewitt D. P. , Mark P. J.
Published: November 2010

Glucocorticoids are recognised as a key fetal programming signal, with excess glucocorticoid exposure in utero linked to various adverse outcomes in offspring including delayed puberty onset, hyperleptinemia and hypertension. Fetal glucocorticoid exposure is controlled by the placental glucocorticoid barrier, whereby two 11β-hydroxysteroid dehydrogenase enzymes regulate transplacental passage of active glucocorticoids (cortisol and corticosterone). Fetal programming by glucocorticoids is likely due to their actions in several fetal tissues, but may also be mediated via effects exerted within the placenta. Indeed, in our model of fetal programming, treatment of pregnant rats with dexamethasone inhibits both fetal and placental growth, and dose–response experiments suggest that the placenta is more susceptible than the fetus to this growth inhibition. Moreover, glucocorticoid treatment stimulates placental apoptosis and reduces expression of several placental gene products, including PPARγ, Muc1 and VEGF. This down-regulation of gene expression occurs specifically within the labyrinth zone, the region of maternal–fetal exchange, and is associated with a marked reduction in placental vascularity. These data indicate that excess placental glucocorticoid exposure is likely to compromise fetal nutrient supply, which in turn could result in adverse fetal programming effects. Subsequent, long-term effects of fetal programming in offspring can either be amplified or attenuated by the postnatal environment. Thus, while programmed hyperphagia and adiposity are exacerbated by a high-energy diet in postnatal life, we have demonstrated that programmed hyperleptinemia and hypertension are prevented by a postnatal diet enriched with omega-3 fatty acids. These effects are mediated, in part, by changes in the adipocyte phenotype, most notably in relation to leptin mRNA expression. In conclusion, fetal programming by glucocorticoids is likely to be mediated, in part, by their detrimental effects on placental growth and vascularity. Postnatally, adverse outcomes of glucocorticoid-induced fetal programming can be prevented by dietary manipulations, thus raising the possibility of preventative, therapeutic interventions.
116. Contribution of claudin-11 to the inter-Sertoli cell tight junction, in vitro
Authors: McCabe M. J. , Stanton P. G.
Published: November 2010

The inter-Sertoli cell tight junction (TJ) forms the blood testis barrier (BTB) between Sertoli cells and is composed of three major transmembrane proteins: claudin-11, occludin and junctional adhesion molecule. Formation of the BTB occurs during puberty associating with an increase in circulating gonadotrophins. Claudin-11 and occludin are hormonally regulated in vitro although their importance to the function of the TJ is unknown. The aim of this study was to investigate the contribution of claudin-11 to the inter-Sertoli cell TJ in vitro by blocking gene expression using RNA interference. Two claudin-11-specific siRNA fragments were designed for this purpose. Sertoli cells in primary culture formed stable TJs within 5 days as measured by transepithelial electrical resistance (TER). The addition of siRNA for 2 days resulted in a significant (P < 0.01) 55% (mean, SD, n = 4 cultures) decrease in TER along with a major reduction in claudin-11 localisation to the TJ as assessed by immunocytochemistry. The specificity of the siRNA was shown by the presence of extensive immunostaining of occludin and of the adherens junction protein β-catenin in the same treatments. Similarly, claudin-11 mRNA expression significantly (P < 0.01) decreased by 71% (mean, SD, n = 3 cultures) in response to both claudin-11 siRNA fragments. Occludin mRNA expression was not affected. It is concluded that claudin-11 contributes at least 55% to the function of the rat Sertoli cell TJ in vitro. It is hypothesised that the remaining 45% of TJ function can be attributed to other integral proteins, such as occludin and junctional adhesion molecule. It is expected that claudin-11 and other TJ proteins play a pivotal role in the function of the BTB in vivo with potential implications in fertility and contraception.
117. Meiosis arrest in a new animal model - a mutation on chromosome 5
Authors: Kennedy C. L. , Ristevski S. , De Kretser D. M. , O'Bryan M. K.
Published: November 2010

Many causes of male infertility are currently unknown and there is a great need for the identification of new genes involved in spermatogenesis. The chemical mutagen, N-ethyl-N-nitrosourea (ENU) was utilized to induce random point mutations in the germline of C57Bl-6J mice in order to generate models of male infertility. Three generation breeding programs produced mice homozygous for ~3–4 point mutations. Following a phenotype-based screen of 1650 G3 males from 155 pedigrees, 15 lines were identified with abnormal male fertility parameters consistent with a recessive mutation. One infertile line, ENU23, exhibited a complete meiosis arrest with no germ cells ever proceeding to become spermatozoa. No infertile females have been observed. Light microscopic examination of Bouin’s fixed paraffin embedded testis sections showed that the arrest occurred post-prophase and prior to the completion of metaphase I, since normal metaphase was not observed. In cells beyond the prophase stage of development, spermatocytes appeared condensed, became enlarged and subsequently underwent cell death by apoptosis confirmed by TUNEL analysis. Electron microscopy visualized normal synaptonemal complexes. The degenerating primary spermatocytes exhibited condensed chromosomes irregularly arranged on a poorly formed microtubular spindle, indicating an abortive attempt to complete metaphase. The percentage of tubules with diplotene/metaphase-like cells was calculated for 3 abnormal and 3 normal ENU23 mice and showed 10.8% compared to 1.37%, respectively, indicating that prior to cell death the cells undergo a lag period. To identify the ENU23 causal mutation, the mutation was bred onto a mixed C57Bl-6J/CBA background and linkage analysis was performed, identifying a region on chromosome 5 between microsatellite markers D5Mit7 and D5Mit32. Candidate genes within this region are currently being sequenced in the search for the ENU-induced mutation causing the ENU23 phenotype. We believe this model of metaphase arrest is unique and will provide insights into the male specific events of meiosis.
118. Regulation of inhibin binding and action via betaglycan expression in mouse Leydig-like TM3 cells
Authors: Escalona R. , Wang Y. , Ooi G. T. , Findlay J. K. , Farnworth P. G.
Published: November 2010

The actions of inhibin and transforming growth factor (TGF)-β2 are enhanced when their respective target cells express the TGF-β/inhibin co-receptor, betaglycan. In the present studies, we investigated the effects of multiple members of the TGF-β superfamily on betaglycan expression, and examined the consequences of such regulation for inhibin binding, and inhibin and TGF-β actions in mouse Leydig-like TM3 cells.
Isoforms of activin (A and B), TGF-β (1 and 2) and BMP (2, 6 and 7) each suppressed the level of betaglycan mRNA in TM3 cells to 43–46%, 26–39% and 50–71% of control, respectively, during overnight treatment. Subsequent inhibin A binding was suppressed to 72–77%, 35–36% and 66–70% of control, respectively, with IC50s of 0.07–0.7, 0.05–0.5 and 0.4–0.6 nM, respectively. The effects of inhibiting betaglycan expression by TM3 cells on their responses to inhibin and TGF-β2 were examined by transfecting cells with a promoter construct that contains three copies of the activin-responsive sequence of the GnRHR promoter (3XpGRAS-PRL-lux) either alone or in the presence of small (21 bp) duplex siRNAs corresponding to the betaglycan gene. Activin A (0.5 nM) stimulated 3XpGRAS-PRL-lux expression 3–4 fold over control in TM3 cells, and inhibin dose-dependently abolished this stimulation, with no interference from the control siRNA (against BF-1 forkhead-like protein). However, inhibin suppression of activin-stimulated activity was antagonized in cells co-transfected with betaglycan siRNA. TGF-β (1 and 2) stimulated 3XpGRAS-PRL-lux expression 5–8 fold over control, and the action of TGF-β2, but not TGF-β1, was attenuated by the betaglycan siRNA.
In summary, activin, TGF-β and BMP isoforms inhibit betaglycan expression by Leydig-like TM3 cells, and inhibin A binding is commensurately reduced. The ‘knock-down’ of betaglycan expression by specific siRNA inhibits TM3 responses to inhibin and TGF-β2. Whether the inhibition of betaglycan expression by activin, TGF-β and BMP has similar consequences for inhibin and/or TGF-β2 action is yet to be determined. These studies raise the possibility that multiple members of the TGF-β superfamily participate in cross-talk via the inhibin/TGF-β co-receptor, betaglycan.
Funded by the NH&MRC of Australia (RegKey 241000 & 198705).
119. Over-expression of activin βC in vivo reveals a role in male fertility
Authors: Gold E. , O'Bryan M. , Meachem S. , McDougall H. , Butler C. , Risbridger G.
Published: November 2010

Introduction: Activin βC subunit antagonises the formation and bioactivity of activin A via intracellular heterodimerisation and decreases activation of the activin signalling pathway (Mellor et al. 2003). Therefore the activin βC subunit heterodimers provide a new mechanism of regulating activin levels. Vedja and colleagues over-expressed the activin βC subunit in malignant liver cell lines, which subsequently displayed inhibition of cell proliferation and induction of apoptosis (Vedja et al. 2003). Conversely, Wada et al. demonstrated that treatment with hr-activin C stimulates growth of a liver cell line (Wada et al. 2004). These recent (and contradictory) reports about the in vitro activity of activin βC have prompted us to examine the in vivo role of activin βC by creating a transgenic mouse over-expressing the βC activin subunit.
Methods: The full-length human cDNA under the control of a CMV promoter was incorporated into the genome of three founder C57/B6 mice. Genotyping was performed by both Southern and PCR. Mice were monitored weekly and culled at 14–16 weeks (adult). Blood was collected by cardiac puncture, organs were weighed and a portion fixed in Bouin’s or frozen for subsequent RNA and protein extraction. Daily sperm production (DSP) was determined by standard methods. Sertoli and germ cell number will be determined using the optical disector (sic) stereological technique in Bouin’s fixed resin sections. Proliferation and apoptosis will be examined using PCNA and TUNEL respectively. Activin A was assessed by ELISA, while FSH, LH, follistatin and total inhibin were determined by RIA.
Results and conclusions: Over-expression of activin-βC resulted in decreased circulating activin A (P < 0.005 TG1, P < 0.05 TG2 and P = 0.08 TG3), a progressive age-related decrease in litter sizes (9.3 WT v. 6.3 TG1, 5.8 TG2 and 4.5 TG3; P < 0.005 v. WT) and testicular DSP (P < 0.05). These data support the hypothesis that βC is a novel in vivo regulator and is the first indication of a role for activin-βC in male fertility. This novel mouse model will significantly advance our understanding of the in vivo role of activin-βC.
(1)Mellor et al. (2003). Endocrinology 144, 4410–4419.(2)Vejda et al. (2003). Carcinogenesis 24, 1801–1809.(3)Wada et al. (2004). Am. J. Physiol. Endocrinol. Metab. 287, E247–E254.
124. Regulation of SOCS3 expression by prostaglandin, prolactin and growth hormone: challenging the Jak/STAT signalling dogma
Authors: Barclay J. L. , Wonisch T. , Anderson S. T. , Waters M. J. , Curlewis J. D.
Published: November 2010

SOCS3 is an inhibitor of various cytokine-receptor signalling pathways and is therefore involved in suppression of cellular responsiveness to these critical regulators. SOCS3 expression is thought to be regulated by a STAT responsive element (SRE). However, our research suggests the involvement of other signalling pathways. In T-47D breast cancer cells, we found that PGE2 induces a 3–5 fold increase in SOCS3 mRNA, as determined by real-time PCR. This effect was not due to phosphorylation of STATs, or inhibited by the Jak2 inhibitor, AG490, but was inhibited by the PI3Kinase inhibitor, LY294002, Akt Inhibitor IV and partially inhibited by the PKA inhibitor, H89. It was not affected by inhibitors of MEK, PDK1, mTOR or p38-MAPK. We concurrently examined PRL-induced SOCS3 expression, and found that although STAT1 and 5 phosphorylation was increased, SOCS3 expression was again inhibited by Akt Inhibitor IV and H89 but unaffected by AG-490. To explore this further, we used a model of GH signalling, BaF3 cells stably expressing GH receptor. GH induced a 15–20 fold increase in SOCS3 mRNA, which was accompanied by increased STAT5 phosphorylation. However the SOCS3 response was not inhibited by AG-490 or H89, but was diminished by Akt Inhibitor IV. Analysis of the SOCS3 promoter revealed a FOXO binding site. When we mutated this site in a mouse SOCS3 promoter–luciferase construct, basal and GH-induced promoter activity was significantly increased. These results are consistent with FOXO acting as a repressor, which is inactivated by Akt. We propose that in T-47D cells, SOCS3 expression involves cross-talk between PI3K/Akt and cAMP/PKA, whereas in BaF3 cells, expression is enhanced by Akt phosphorylation and subsequent FOXO inactivation. These findings contrast with the accepted Jak/STAT regulation of SOCS3 expression.
This work is supported by the Australian Research Council.
125. Identification of a functional binding site on betaglycan for inhibin and TGFβ
Authors: Harrison C. A. , Wiater E. , Lewis K. A. , Gray P. C. , Vale W. W.
Published: November 2010

Betaglycan is a co-receptor that modulates signaling by TGFβ superfamily members including TGFβs and inhibins. Loss of betaglycan expression, or blocking of betaglycan function, has been implicated in several human diseases and in animal disease models. However, characterization of the superfamily ligands and receptors involved in these disease states is complicated because of the pleiotropic nature of betaglycan co-receptor action. Here we report the identification and characterization of a domain within the extracellular region of betaglycan that binds inhibin-A and TGFβ-1. We show that both ligands bind to the membrane proximal region (amino acids 591–760) of the betaglycan extracellular domain. This inhibin/TGFβ-binding region is within the ZP-domain of betaglycan, but is not integral to the conserved ZP motif. Using deletion studies and site-directed mutagenesis, we show that the inhibin and TGFβ binding sites on betaglycan overlap and identify individual amino acids essential for the binding of both ligands. In particular, point mutant V614Y abolishes inhibin and TGFβ binding to the membrane proximal domain of the betaglycan ECD. A full-length betaglycan construct containing this point mutation can still bind TGFβ-1 via a separate N-terminal binding site, but is unable to bind inhibin-A. This mutant is incapable of mediating inhibin’s antagonism of activin or BMP signalling. Mutation of betaglycan V614Y thus separates the co-receptor actions of betaglycan for inhibin and TGFβ. This will allow the clarification of the role of betaglycan in human disease states such as renal cell carcinoma and endometrial adenocarcinoma.
126. Progesterone regulates CXCL14 (macrophage inflammatory protein 2γ) mRNA in human endometrium
Authors: Mokhtar N. M. , Smith S. K. , Charnock-Jones D.
Published: November 2010

The emergence of microarray technology has enabled a thorough study of the level of transcripts in the human body. A high density micoarray analysis revealed a comprehensive list of transcripts, which were significantly different between mid-proliferative and mid-secretory phase endometrium.2 An EST identified from the HG_U95B chip is identical to the 3′UTR of CXCL14 or macrophage inflammatory protein 2γ (MIP 2γ). The level is 19-fold higher in the mid-secretory compared to the mid-proliferative phase of menstrual cycle. This has suggested that the transcript level of CXCL14 may be directly regulated by progesterone. Northern hybridisation and in situ hybridisation confirmed that the transcript level of CXCL14 (MIP 2γ) was high in the mid-to-late secretory endometrium and its mRNA was localised in the glandular epithelium of this tissue.1 In silico analysis has predicted six progesterone response elements (PREs) within 2040 bp upstream from the ATG site. To investigate the possible functions of these PREs, a dual luciferase assay was performed on the ishikawa cell line transfected with five deletion constructs of the gene promoter. Cells were co-transfected with progesterone receptor B (PRB) and treated with 10–6 M progesterone. Luciferase activities of these constructs have localised two fragments that were most likely to contain the active PREs, i.e. PRE1 and PRE2. An electrophoretic mobility shift assay showed that PRE oligonulcleotides within these two regions were able to bind PRB that was synthesised in vitro, although there was a stronger signal seen in the PRE2 region. A dose competition study revealed PRE1/PRB and PRE2/PRB protein binding could be competed with different concentrations of cold wild-type competitor oligonucleotides. Mutagenesis of PRE1 and PRE2 analysed by luciferase reporter assay reduced the inductive effect of progesterone treatment. This study indicates that progestegen induced transcript encoding a chemokine in the human endometrium may likely act as a chemoattractant for leucocytes during the secretory phase of the menstrual cycle.
(1)Mokhtar NM, Smith SK, Charnock-Jones DS. (2003). Characterisation of chemokine macrophage inflammatory protein 2gamma mRNA in human endometrium. 50th Society for Gynaecologic Investigation. Washington DC, USA. March 2003.(2)Borthwick JM, Charnock-Jones DS, Tom BD, Hull ML, Teirney R, Phillip SC, Smith SK. (2003). Determination of the transcript profile of human endometrium. Mol. Hum. Reprod. 9, 19–33.
127. Platelet derived growth factors and receptors contribute toward development of the corpus luteum
Authors: Sleer L. S. , Taylor C. C.
Published: November 2010

In this study the expression of the family of platelet derived growth factors (PDGF) and receptors in the ovarian corpus luteum was identified and characterized, and an effect of their activity on development of the corpus luteum revealed. Gonadotropin-stimulated immature rats were utilized as a model of induced ovulation, luteogenesis and pseudopregnancy, and levels of mRNA for platelet derived growth factors (PDGF-A, PDGF-B, PDGF-C and PDGF-D) and receptors (PDGF-Rα and PDGF-Rβ) in response to gonadotropins were investigated. Intraperitoneal injection of immature rats with pregnant mare’s serum gonadotropin (PMSG) followed 54 h later with human chorionic gonadotropin (hCG) resulted in a significant increase in ovarian mRNA levels for PDGF-Rβ and its ligands, PDGF-B and PDGF-D, as early as 4 h after hCG injection. Gonadotropin regulation of PDGF-B was confirmed by in vitro promoter–reporter assays, which showed a 2–3-fold increase in PDGF-B promoter activity in response to luteinising hormone (LH), and inhibition studies implicated protein kinase A, phosphatidylinositol 3-kinase and mitogen activated protein kinase signaling pathways in the LH-induced upregulation. In the corpus luteum, PDGF-Rα was localized to a subset of luteal steroidogenic cells, and PDGF-Rβ to cells of the luteal microvasculature. PDGF-A, PDGF-B and PDGF-C were also identified in a population of luteal steroidogenic cells. Intraovarian injection of an inhibitor of PDGF receptor activity, the tyrphostin AG1295, prior to injection of hCG in PMSG-primed immature rats resulted in a significant 22.85 ± 10.7% decrease in corpora lutea per treated ovary in comparison to the contralateral vehicle-injected control ovary. In addition, the treated ovary of 3 of 12 rats showed widespread hemorrhage throughout the entire ovary, indicating a possible role for PDGF receptor activity in maintenance of the ovarian vasculature. In summary, these data identify expression of members of the family of platelet derived growth factors and receptors in cells within the corpus luteum and reveal a role during development of the corpus luteum.
201. Effects of relaxin deficiency on matrix metalloproteinase expression in the cervix and vagina of pregnant mice
Authors: McGuane J. T. , Gehring H. M. , Parry L. J.
Published: November 2010

The major functions of relaxin are associated with female reproductive physiology, especially the regulation of biochemical processes involved in the remodelling of the reproductive tract at term. Studies in relaxin deficient mice (Rlx–/–) demonstrate that although females give birth to live young without apparent dystocia, they have abnormal cervices and vaginae. This phenotype is attributed to an increase in stromal collagen, but the mechanism(s) by which relaxin regulates extracellular matrix (ECM) production in reproductive tissues is poorly understood. In this study, we assessed the expression of matrix metalloproteinases (MMPs) in the cervix and vagina of pregnant wild-type (Rlx+/+) and Rlx–/– mice. Tissues were obtained from adult C57/Blk6J Rlx+/+ mice on days 7.5, 14.5, 17.5, 18.5 pc and Rlx–/– littermates on days 7.5, 14.5 and 18.5 pc. Real-time PCR using dual-labelled fluorogenic probes was performed in an Opticon 2 cycler (MJ Research) to quantify MMP-2, -3, -7, -9 and -13 gene expression. In the cervix and vagina of Rlx+/+ mice, only MMP-2 mRNA levels were significantly higher at term compared with earlier stages of gestation. There were significant decreases in MMP-7 and -13 expression at term, but no change in MMP-3 and -9. In contrast, MMP-3, -7, -9 and -13 mRNA levels were significantly higher in the cervix and vagina of late pregnant Rlx–/– mice. The expression of MMP-2 did not differ between Rlx+/+ and Rlx–/– mice at term. Despite the higher expression of the majority of MMPs we examined in Rlx–/– mice, there was no histological evidence of increased ECM degradation in the cervix and vagina in late gestation. Although previous in vitro studies suggest that relaxin positively regulates MMP activity, our data demonstrate that relaxin deficiency does not result in decreased MMP expression in the mouse cervix and vagina in vivo.
203. A differential pattern of follistatin expression in the placenta between spontaneous, induced and non-labouring patient groups
Authors: Rae K. M. , Hollebone K. G. , Meng L. , Clausen D. C. , McFarlane J. R.
Published: November 2010

Follistatin has been identified in human placenta, fetal membranes and fluids, with serum follistatin concentrations rising during pregnancy, particularly near term. Our laboratory has shown follistatin concentrations rise across labour in spontaneous but not induced women.1 As the placenta is a source of follistatin, this study examined placental tissues using immunohistochemistry to determine differences in follistatin localization between groups. Placental tissue was collected immediately following delivery from three groups of women at term, spontaneous onset (n = 4), induction (n = 4) and non-labouring caesarian (n = 4), and immediately formalin fixed. Antigen-retrieval immunohistochemistry using a specific chicken polyclonal antiserum (CK20) raised against a follistatin peptide (AA 121-133) or pre-immune chicken serum was performed. Positive staining of syncytiotrophoblast cells of the chorionic villi was seen in patients undergoing spontaneous labour but not in the induced and caesarian delivery group. The two labouring groups (spontaneous and induced) both showed positive staining for the vascular endothelial cells within the chorionic villi and the stratum basale, whilst the caesarian delivery group was negative for any staining within these vessels. Positive staining of Hofbauer cells was observed in both labouring groups; however, the caesarian group showed infrequent positive staining of these cell types. The differences in expression pattern in the two labouring groups (spontaneous v. induced) may be due to variations in labour lengths (6.5 v. 4.5 h, respectively); however, we would have expected a lower level of expression in the same cell types rather than the complete absence of staining. The positive follistatin staining in the syncytiotrophoblast of spontaneous patients suggests this may be the source of the rising plasma follistatin seen in this group. These differences in staining support our hypothesis that an earlier endocrine signal is absent in the induced and caesarian patient groups.
(1)Rae K, Hollebone K, Clausen DC, Chetty V, McFarlane JR. (2004). A Cross-Sectional Study of Follistatin During Labour in Women. The Endocrine Societies 86th Annual Meeting, New Orleans, 2004.
204. Isolation of CAG repeat containing genes from human placenta and decidua
Authors: Freed K. A. , Brennecke S. P. , Moses E. K.
Published: November 2010

Pre-eclampsia is a serious disorder of pregnancy that manifests clinically in the mother as new-onset hypertension and proteinuria. Although the precise cause remains unknown, the placenta and the decidua play a fundamental role. The worldwide incidence of pre-eclampsia is 2–5% and such a high incidence, in the face of strong negative selection, suggests that the gene(s) involved have a selective advantage and/or a high mutation rate. One class of genetic diseases that involve a high mutation rate are the trinucleotide repeat expansion diseases. In these diseases repeated trinucleotide DNA sequences within specific genes multiply or expand up to 1000-fold. The result of this gene expansion/mutation is altered gene function that confers genetic susceptibility.
Thus, the overall objective of this study was to determine whether there is an association between a trinucleotide (CAG) repeat expansion and pre-eclampsia. The specific aim of this study was to isolate CAG repeat containing genes from human placenta and decidua. An adaptation of the mRNA differential display technique and traditional cDNA library screening was used.
In total, 72 placental and 51 decidual sequences were analyzed using the BLAST nucleotide comparison program. Five cDNAs were analyzed further. The unique sequences surrounding the CAG repeat regions of these five genes will be used to generate primers to ascertain if any of these repeat DNA sequences vary in number in the normal population. If polymorphic genes are identified, the primers will be used on pre-eclamptic pedigrees to determine if pre-eclampsia is associated with a repeat expansion mutation.
205. Fractalkine, HCC-1 and MIP-1β promote human trophoblast migration
Authors: Hannan N. J. , Jones R. L. , Salamonsen L. A.
Published: November 2010

Human embryo implantation is a complex process requiring the attachment of an activated blastocyst to receptive endometrial epithelium and subsequent trophoblast invasion throughout the first trimester of pregnancy. Chemokines, including fractalkine (FKN), MCP-3, HCC-1 and MIP-1β, are produced by human endometrial epithelial and decidual cells with maximal production around the time of implantation/early pregnancy.1,2 Chemokine and receptor expression was characterized in cell types at the human maternal–trophoblast interface. Highly abundant expression of chemokine receptors CX3CR1 and CCR1 was observed in first trimester placenta and in trophoblast cells.3 We hypothesized that CX3CR1 and CCR1 ligands (FKN, MCP-3, HCC-1 and MIP-1β) produced by endometrial epithelial and decidualised stromal cells at the time of implantation promote migration of human trophoblast. We aimed to localize specific chemokine receptors in human first trimester tissue, and to determine whether trophoblast migration could be stimulated by the endometrium and by chemokines. Cellular localisation of specific receptors was assessed by immunohistochemistry in human first trimester implantation sites. Using an in vitro assay, trophoblast migration was assessed in response to human endometrial epithelial (HEEC) and decidualised stromal cells (DESC) (serum-free) conditioned medium and to recombinant human FKN, MCP-3, HCC-1 and MIP-1β. CX3CR1 and CCR1 protein was localised to the vascular extravillous trophoblast (EVTs), but not to the invading interstitial EVTs, with weak staining on the syncytium. Significant migration of cells occurred in response to conditioned media from HEEC and DESC. FKN, MIP-1β and HCC-1, but not MCP-3 also promoted significant trophoblast migration. Neutralizing antibodies for FKN and MIP-1β but not MCP-3 significantly reduced migration to conditioned media, indicating that at least these two chemokines contributed to the effects. These data support a role for endometrial derived chemokines in promoting human trophoblast migration.
(1)Jones et al. (2004). JCEM 89(12), 6155–6167.(2)Hannan et al. (2004). Reprod. Fert. Devel. 16(Suppl.), A225, p. 78.(3)Hannan et al. (2004). JCEM 89(12), 6119–6129.
206. Effect of in vitro embryo culture on placental gene expression in the sheep
Authors: Fletcher C. J. , MacLaughlin S. M. , McMillen I. C. , Walker S. , Sibbons J. , Roberts C. T.
Published: November 2010

In vitro culture (IVC) systems feature commonly in reproductive technologies used in livestock. However, these culture conditions impact on the metabolism and physiology of the developing embryos, as well as on fetal outcome. Culturing rodent embryos in simple defined media results in decreased postimplantation viability and fetal growth rate.1,2 Cytokines and growth factors are present in vivo but are absent from culture and may be causal in the perturbed fetal growth observed. Furthermore, the occurrence of large offspring syndrome (LOS) following embryo IVC in ruminants has been reported and is associated with loss of genetic imprinting.3,4 Abnormal placental development following IVC is also likely to involve perturbed expression of imprinted genes including insulin-like growth factor II (IGF2) and its receptor (IGF2R). The IVC system used for this study included a control embryo transfer group without culture (ET, n = 11), in vitro cultured embryos in serum free defined medium (IVC-NS, n = 10) and in vitro cultured embryos in defined medium with human serum (IVCHS, n = 8). The cultured embryos were transferred to recipient ewes and placentomes were collected at 144–145 days gestation. Fetal weight (kg) was increased in IVCHS (5.15 ± 0.28) compared to ET (4.12 ± 0.24, P = 0.017) and IVC-NS (4.36 ± 0.27). Real-time RT-PCR was used to quantify IGF2 and IGF2R mRNA expression normalized to housekeeper RpP0. Although IGF2 expression was increased in the IVCHS group (2.27 ± 0.44) when compared to ET (1.22 ± 0.37) and IVC-NS (1.17 ± 0.43) groups, this was not significant. In addition, IGF2R expression was increased in the IVCHS (0.008 ± 0.003) group compared to ET (0.003 ± 0.001) and IVC-NS (0.004 ± 0.001) groups, but this was also not significant. IGF2 and IGF2R expression were, however, positively correlated in IVCNS (r = 0.72) and IVCHS (r = 0.95) placentomes, but not control ET placentomes. The presence of serum in IVC promoted fetal growth and increased expression of IGF2 and IGF2R mRNA in placental tissue. Comparison of placental gene expression from IVCHS and naturally mated pregnancies would be valuable to assess the role of serum in placental and fetal development.
(1)Bowman & McLaren, 1970.(2)Kaye & Gardner, 1999.(3)Thompson et al., 1995.(4)Young et al., 1998.
207. The expression of caspase-14 in the human placenta
Authors: Charles A. , Hisheh S. , Kam D. W. R. , Dharmarajan A. M.
Published: November 2010

Pro-apoptotic genes have a role in the differentiation process in the placenta leading to the fusion of cytotrophoblast cells to form the protective syncytiotrophoblast layer. The mechanisms of apoptosis in the human placenta are not clearly understood. However, a major placental apoptotic-signalling pathway is known to involve the caspases. Caspase-14 is the most recently discovered member of the caspase family members and has not previously been examined in the human placenta. The aim of the present study was to detect caspase-14 in the human placenta and study its role in apoptosis. Human placentae were collected from first trimester and term gestation. The study consisted of two parts. In the first part, first trimester and term placentae were assessed for caspase-14 by western blotting and mRNA analysis and localised with immunohistochemical studies. In the second part, apoptosis in first trimester placenta was inhibited in an in-vitro model of explant villi culture with superoxide dismutase (SOD) treatment and the genes assessed. The first study demonstrated caspase-14 to be a cytoplasmic protein localised in the cytotrophoblast cells, the mesenchyme and in the syncytiotrophoblast of the first trimester. In the term placenta, caspase-14 was expressed weakly in the syncytiotrophoblast. The immunostaining data suggest a higher expression of caspase-14 in the first trimester compared to the term placenta, and this observation was later confirmed by western blot analysis. Using the SOD in-vitro explant culture model, no significant difference in the caspase-14 protein levels were seen in either the SOD or control group. This novel study demonstrates for the first time that caspase-14 protein and mRNA are present in the human placenta. The function of caspase-14 in the human placenta is unclear.
208. Exposing necrotic trophoblasts to endothelial cells in vitro causes increased adhesion of monocytes
Authors: Chen Q. , Stone P. , McCowan L. , Chamley L.
Published: November 2010

A number of studies suggest that there is a generalized endothelial cell activation and inflammatory response in preeclampsia, which may be caused by factors released from the placenta including deported trophoblasts. Trophoblasts are the placental cells that are bathed in maternal blood during pregnancy and as they become aged or damaged trophoblasts are shed from the placenta and deported into the maternal circulation. The fate of deported trophoblasts is unknown but we have found that endothelial cells can phagocytose dead trophoblasts. The aim of this study was to examine the effects of phagocytosing dead trophoblasts on endothelial cell–monocyte interactions. Methods: The trophoblast-derived cell lines Jar and Jeg-3 were induced to undergo necrotic death by freeze/thawing or apoptotic death by exposure to UV light. HMEC-1 endothelial cells were labeled with green fluorescent cell tracker stain and then exposed to necrotic or apoptotic trophoblasts for 3 or 24 h. U937 (monocyte) cells were labeled with red fluorescent stain and incubated with the HMEC-1 monolayers for 3 or 24 h. The adhesion of the U937 cells to the HMEC-1 monolayers was quantified by flow cytometry and compared to the adhesion of U937 cells to untreated HMEC-1 monolayers. Results: Exposing the HMEC-1 cells to necrotic, but not apoptotic, trophoblasts induced an approximately two-fold increase in the adhesion of U937 cells to the HMEC-1 monolayers (P = 0.01). The findings were consistent regardless of whether the HEMC-1 cells were exposed to the dead trophoblasts for 3 or 24 h. Conclusions: We have previously shown that endothelial cells phagocytose both apoptotic and necrotic trophoblasts. The results of the current study suggest that shedding necrotic trophoblasts from the placenta could induce endothelial cells to become activated resulting in increased leucocyte adhesion. Thus, dead trophoblasts may be one of the factors released from the placenta that induce preeclampsia.
211. Expression and cellular localization of HTRA3 protease during placental development in mice
Authors: Li Y. , Salamonsen L. A. , Nie G.
Published: November 2010

Placental development in mice involves highly regulated interactions between fetal- and maternal-derived cells. We have previously cloned a novel serine protease (HtrA3) containing an insulin-like growth factor (IGF) binding domain, which was upregulated during pregnancy, especially post-implantation in the mouse uterus.1 The present study examined HtrA3 regulation during placental development in mice, in particular, its expression in the different compartments of the placenta. Expression of mRNA was determined by Northern blot analysis in implantation units containing the decidua, placenta and fetus (day 8.5 to near-term). A specific HtrA3 antibody was generated, affinity-purified and used for Western blot analysis and immunohistochemistry. Both mRNA and protein of HtrA3 were identified specifically in the maternal decidua. In contrast, HtrA3 expression was below detection in trophoblasts, including the giant cells that are in direct contact with the decidua. This pattern persisted from the early stages of placentation to near term. The level of decidual HtrA3 mRNA and its protein gradually decreased as the placenta matured. In the decidua, only the maternal decidual cells, but not blood vessels or uterine NK cells that are present in large numbers, were positive for HtrA3. The specific localization of HtrA3, a protease possessing an IGF binding domain at the maternal–fetal interface, suggests that this protein plays an important role in mediating maternal decidual remodelling and maintenance, probably in association with the IGF system, in placental development and function.
(1)Nie et al. (2003). Mol. Hum. Reprod.
212. Follistatin secretion by the ovary is not directly related to PGF2α induced luteolysis in the ewe
Authors: Ford K. E. , O'Shea T. , McFarlane J. R.
Published: November 2010

Follistatin is a monomeric glycoprotein, expressed ubiquitously, and found in greatest levels in ovarian tissue. Our previous studies have demonstrated that ovarian venous follistatin levels are elevated at the same time as spontaneous luteolysis in merino ewes, but remain constant in circulating and uterine plasma across oestrous. It was hypothesized that the follistatin elevation may be part of functional luteolysis in the ovary.
A cross-sectional study of ovarian venous follistatin during luteolysis was performed using merino ewes, which were synchronized with intravaginal sponges. Luteolysis was induced 10 days after oestrus using a PGF2α (IM) injection. Circulating (jugular and arterial), uterine venous and ovarian venous (luteinized and contralateral) blood samples were taken under general anaesthesia at times 0, 6, 12, 24, 36, 48 and >48 h (54–71 h) after the PGF2α injection. All samples were assayed for progesterone by RIA and follistatin using a specific follistatin antiserum (#204) in a competitive ELISA with rhFS-288 used as standard.
Progesterone concentrations in the venous plasma from the luteinized ovaries plasma were significantly higher than those in the contralateral ovarian and circulating plasma. As expected progesterone concentrations began declining within 6 h and continued until 24 h after PGF2α. Mean circulating plasma follistatin concentrations were similar throughout the experiment with overall means of 9.9 ± 0.5 ng/mL. Both luteinized and contralateral ovarian venous plasma follistatin concentrations (11.8 ± 3.4 ng/mL) were not significantly different from circulating concentrations. However at 48 h ovarian venous follistatin concentrations were significantly elevated (22.5 ± 2.3 ng/mL) and remained elevated for at least 71 h (29.5 ± 7.9 ng/mL).
This experiment confirms our previous work that both luteinized and contralateral ovarian venous follistatin concentrations do elevate concurrently with each other but that the elevation in follistatin is not directly related to functional luteolysis as induced by PGF2α.
213. Regulation of TGFβ superfamily-related genes in the newborn mouse testis by activin
Authors: Mendis S. H. S. , Brown C. , Loveland K. L.
Published: November 2010

Several members of the transforming growth factor-β (TGFβ) superfamily of ligands are known to influence development of male germ cells. In our ongoing investigations of genetically modified mice with different bioactive levels of activin, we noted that testicular germ cell numbers at birth are increased in the absence of activin.1 In this study we compared expression levels of TGFβ-related genes in mice with 0, 1 or 2 copies of the activin βA subunit gene. Candidate genes were identified through hybridisation of total testis RNA from newborn (Day 0) wild type (WT; 2 copies) and activin βA null mice (KO; 0 copies) to a TGFβ SuperArray Simplicity™ membrane. Densitometric analysis of resulting signals revealed several genes that appeared to differ between genotypes. To validate these findings, pooled testes from newborn WT (2 independent groups), heterozygous (het; 2 groups) and activin βA KO (3 groups) mice were collected for RNA extraction. Quantitative real-time PCR analysis was performed using the Roche Light Cycler, with β-actin mRNA level used as the housekeeping reference. The mRNAs selected for analysis were: inhibin α, p15INK4b, Smad5, insulin-like growth factor 1 (IGF-1), ALK6 (BMPRI-B), and tissue inhibitor of metalloproteinase 1 (Timp1). The inhibin α transcript level was significantly decreased in the activin βA KO animal compared with WT, while the het sample showed an intermediate effect. Both Smad5 and IGF-1 transcripts appeared to increase in the KO animals compared with WT; however, the cyclin-dependent kinase inhibitor, p15INK4b, did not change significantly across the genotypes. Similarly, ALK-6 and Timp1 mRNA levels were also unaffected by genotype. These findings illustrate the impact of graded levels of activin A on specific genes in the fetal testis.
(1)Mendis et al. (2004). Reprod. Fertil. Dev. 16(Suppl.), 103.
215. The role of testis specific protein 1 (Tpx-1) and gametogenetin (Ggn) in mammalian spermatogenesis
Authors: Jamsai D. , Hickox D. M. , Gibbs G. M. , De Kretser D. M. , O'Bryan M. K.
Published: November 2010

The sperm tail has a key role in male fertility, since it is responsible for motility, driving sperm toward and into the ovum. In this study, we characterized a sperm tail protein, testis specific protein 1 (Tpx-1), which is exclusively expressed in sperm and is localized to the acrosome and the outer dense fibres (ODF) of the sperm tail. We carried out yeast two-hybrid screening using an adult testis cDNA library to identify interacting protein partners of Tpx-1. A number of putative Tpx-1 interacting proteins were identified including a novel germ cell-specific protein, gametogenetin (Ggn). Ggn is highly expressed in the adult testis, specifically in late pachytene spermatocytes through to round spermatid germ cells. Ggn has more than 10 splice variants giving rise to three proteins namely GGN1, GGN2 and GGN3.1 The biological significance and potential for in vivo interactions between Tpx-1 and Ggn was assessed using Northern blot analysis, immuno-histochemistry and co-immunoprecipitation. To further investigate the nature of Tpx-1 interaction with Ggn, deletion studies were performed in yeast. The cysteine-rich carboxy terminal domain of Tpx-1 was shown to be responsible for binding a region in the last 120 amino acids of Ggn. Since the Ggn clone used for analysis encoded only this carboxy-terminal portion of the Ggn protein, it is also possible that other regions of the protein are involved in the interaction with Tpx-1. Further studies involving full-length clones and/or amino-terminal encoding Ggn clones will be needed to explore the possibility of additional interacting regions. In addition, Tpx-1 and Ggn knockout mice are also being generated. These results provide a greater understanding of the normal processes involved spermatogenesis and may suggest directed means to enhance or suppress male fertility.
(1)Lu B, Bishop CE (2003). Mouse GGN1 and GGN3, two germ cell-specific proteins from the single gene Ggn, interact with mouse POG and play a role in spermatogenesis. J. Biol. Chem. 278, 16 289–16 296.
217. Interaction of cdyl (chromodomain y-chromosome like) with the nuclear transport protein importin α2
Authors: Ly J. D. , Efthymiadis A. , Sarraj M. A. , Ball P. , Loveland K. L. , Jans D. A.
Published: November 2010

Spermatogenesis is a unique, ordered process governed by the precise expression of a specific set of genes at each stage. Progression through successive stages requires the shuttling of proteins and transcription factors into and out of the nucleus to implement changes in gene transcription. Major factors that mediate nucleocytoplasmic transport are members of the importin superfamily, of which there are five and 20 different importin α and β genes, respectively, in mouse. We have previously demonstrated that several importins display distinct mRNA and protein expression patterns in adult mouse testis1 indicating that specific importins carry a specific cargo at discrete stages of spermatogenesis. Identification of importin cargoes in the testis should help describe the potential developmental switches critical to the spermatogenic process. We performed a yeast two-hybrid screen using full length importin α2 as bait and an adult mouse testis library, identifying nine target proteins. Some of these proteins include nuclear components that may be important in eliciting changes in the nuclear structure during spermatogenesis, as well as those involved in cell cycle regulation, homologous chromosome pairing and recombination, transcriptional regulation and guanine nucleotide biosynthesis. One key candidate is CDYL, which has been implicated in male infertility. It is a chromodomain-containing protein that is predominantly expressed during spermiogenesis and has been previously described to participate in hyperacetylation of histone H4, which is believed to facilitate protamine replacement of histones during spermiogenesis. Verification of CDYL-importin α2 interaction was demonstrated using co-immunoprecipation and co-transfection, while immunohistochemical staining of testis sections indicated colocalisation in the same cell types (mainly elongating spermatids). Importantly, preliminary experiments indicated that increasing CDYL nuclear accumulation by over-expressing importin α2 can increase histone H4 acetylation. Our hypothesis is that importin α2 is central in nuclear targeting of CDYL to facilitate its hyperacetylation role during protamine-histone exchange.
(1)Hogarth et al. (2005). Dev. Dynamics (submitted).