bluuebunny/crossref_metadata_2025 huggingface dataset:
295. Peroxisome proliferator activated receptor-alpha is involved in H+-monocarboxylate transporter 2 and catalase protein expression in cultured preimplantation mouse embryos
Authors: Jansen S. , Pantaleon M. , Kaye P. L.
Published: November 2010

Cleavage stage embryos consume pyruvate before switching to glucose as the major energy substrate for blastocyst formation. This switch is conditional, because freshly collected two-cell embryos form blastocysts without glucose by increasing pyruvate consumption. Zygotes cultured without glucose cannot adapt in this way and degenerate, but paradoxically demonstrate upregulation of the H+-monocarboxylate transporter protein, MCT2, in morulae. MCT2 is a high affinity transporter implicated in redox shuttling for peroxisomal beta-oxidation of fatty acids.3 Fatty acids may provide energy for embryos2 but peroxisomal beta-oxidation has not been explored in preimplantation development. Rat oocytes possess a primitive peroxisomal system.1 The possibility therefore exists that MCT2 may also be linked to fatty acid metabolism in embryos. Peroxisome proliferator activated receptor (PPAR)-alpha is a transcriptional regulator of fatty acid transport and beta-oxidation, and controls expression of catalase, a major peroxisomal enzyme. This investigation explores the role of PPAR-α in the glucose-driven control of MCT2 expression in mouse embryos. Zygotes (18 h post-hCG) were cultured in KSOM in the presence or absence of glucose, or KSOM with selective agonists of PPAR-α, fenofibrate and WY 14643. Expression of MCT2 and catalase was analysed by confocal laser scanning immunohistochemistry and western blot. Results confirm the presence of catalase throughout preimplantation development. With glucose, cytoplasmic immunoreactivity for catalase was punctate and diffuse, while MCT2 was localised to apical membranes of outer blastomeres in morulae. Without glucose, catalase and MCT2 expression were increased with notable localisation of catalase to nuclei. This response was reflected in morulae cultured in the presence of glucose and PPAR-α agonists. These data suggest that PPAR-α plays a role in controlling catalase and MCT2 expression in embryos, and that conditions in the absence of glucose are more conducive for PPAR-α activation.
(1)Figueroa C, Kawada ME, Veliz LP, Hidalgo U, Barros C, Gonzalez S and Santos MJ (2000) Peroxisomal proteins in rat gametes. Cell Biochem Biophys 32, 259–268.(2)Hewitson LC, Martin KL and Leese HJ (1996) Effects of metabolic inhibitors on mouse preimplantation embryo development and the energy metabolism of isolated inner cell masses. Mol Reprod Dev 43, 323–330.(3)McClelland GB, Khanna S, Gonzalez GF, Butz CE and Brooks GA (2003) Peroxisomal membrane monocarboxylate transporters: evidence for a redox shuttle system? Biochem Biophys Res Commun 304, 130–135.
296. Characterization of E74 like factor 3 in the murine pre-implantation embryo
Authors: Little E. B. , Stanton J. L. , Green D. P. L.
Published: November 2010

The E26 transformation specific (ETS) family of transcription factors is defined by a highly conserved DNA binding domain containing a hydrophobic core motif 5′-GGA[A/T]-3′. A subfamily of ETS factors, epithelium-specific ETS factor (ESE), includes E74-like factor 3 (Elf3). Elf3 is expressed in many organs including the eye, skin and gastrointestinal tract. By upregulating specific gene transcription, Elf3 is instrumental in causing these organs to become fully differentiated. The presence of Elf3 in the pre-implantation embryo was first reported in 2002.1 Data mining has suggested that the expression of Elf3 increases at the 16-cell embryo and blastocyst stages of pre-implantation development compared to the preceding stages. Reverse transcriptase–polymerase chain reaction (RT-PCR) was used to amplify the 3′ untranslated region (3′UTR) of Elf3. These results suggest that there are two isoforms of Elf3 present in the pre-implantation embryo, which differ by an 88 bp deletion/insertion. The expression and location of these isoforms was investigated using RT-PCR and in-situ hybridisation.
(1)Stanton and Green (2002) Mol. Hum. Rep. 8, 149–166.
298. Dietary protein does not influence mitochondrial distribution in the 2-cell mouse embryo
Authors: Mitchell M. , Lane M.
Published: November 2010

Excess dietary protein can negatively influence fertility. The underlying mechanisms remain to be completely elucidated; however, variations in reproductive tract pH, and ammonium and urea concentrations have been implicated. Mouse embryos cultured in the presence of ammonium showed a shift in mitochondrial distribution away from the nucleus towards the cell cortex, suggestive of reduced mitochondrial activity, ATP production and embryo viability. In this study we determined the effect of dietary protein in vivo, on mitochondrial distribution in the 2-cell mouse embryo. Five-week-old Swiss female mice (n = 10) were fed low (9%), medium (14%) or high (25%) dietary protein for 3weeks; feed intake and body weight were recorded weekly. At 8 weeks of age mice were primed with 5 IU of PMSG, then 5 IU hCG 48 h later, and mated overnight with males of proven fertility. Forty hours post-hCG females were sacrificed, their oviducts collected and flushed with media. The total number of 2-cell embryos and oocytes retrieved were recorded. Active mitochondria were stained in the 2-cell embryos using Mitotracker Green (Molecular Probes), and were visualised using confocal microscopy. Density of perinuclear and cortical staining was determined in Photoshop 7.0, using an established method. Females fed the medium diet consumed significantly less and gained less weight than those fed the low or high diet (Table 1), despite similar final body weights (data not shown). Females fed the low diet tended to have a lower ovulation rate and fewer 2-cell embryos than females consuming the other diets (Table 1, P > 0.05). There was no significant effect of dietary protein on the distribution of mitochondria between the perinuclear and cortical region of the embryo, which may be reflective of lower in vivo ammonium levels compared to those described in culture.
306. Testicular growth factor expression after hemicastration in the neonatal boar
Authors: Wells R. , Scott P. , Harrison D. , Cameron R. , Johnston S. D. , D'Occhio M. J.
Published: November 2010

The molecular mechanisms associated with testicular hypertrophy after hemicastration are poorly understood. Sertoli cells in culture underwent increased proliferation when exposed to fibroblast growth factor-2 (FGF2)1 and transforming growth factor-α (TGFα).2 To determine whether FGF2 and TGFα expression is upregulated during accelerated testicular growth after hemicastration in the boar animals were assigned to (a) control (n = 12), no treatment; (b) hemicastrated (n=14), left testis removed on Day 10 after birth. The right testis was removed from half the control and hemicastrated boars, respectively, on Days 15 and 20 after birth. RNA was extracted from sections of frozen testis, cDNA synthesised using TaqMan® Reverse Transcription, and real-time PCR performed. FGF2 expression was determined using forward (5′GTG TTA CAG ACG AGT GTT TCT TTT TTG3′), internal (5′acg act gga atc taa t3′) and reverse (5′TTC CTC GAC CGG TAA GTA TTG TAG T3′) primers. TGFα expression was similarly determined using forward (5′GGC TGT CCT CAT CAT CAC ATG T3′), internal (5′tgc tga tac act gct gc3′) and reverse (5′CGG CAC CAC TCA CAG TGT TT3′) primers. Data were analysed by ANOVA and LSD test (testis weight) and unpaired two-tailed t-tests assuming equal variance (FGF2, TGFα). There was no difference (P > 0.05) in testis weight between hemicastrated (3.9 ± 0.3 g; mean ± SEM) and control (3.6 ± 0.5 g) boars on Day 5 but on Day 10 hemicastrated boars had a greater (P = 0.01) testis weight (6.2 ± 0.8 g) than controls (4.3 ± 0.4 g). There were no differences (P > 0.05) between control and hemicastrated boars in TGFα or FGF2 expression on Days 5 and 10. It is concluded from the findings that upregulation of TGFα or FGF2 gene expression is not required for testicular hypertrophy subsequent to hemicastration in the neonatal boar.
(1)Biol Reprod (1987); 37: 665–674.(2)Mol Cell Endocrinol (2001); 181: 221–227.
308. Characterization of SIV infection in the male genital tract of juvenile macaques
Authors: Shehu-Xhilaga M. , Dale J. , O'Bryan M. , Hedger M. , Kent S. , De Kretser D.
Published: November 2010

Reproductive organs contribute infected cells and free viral particles to semen in human immunodeficiency type-1 (HIV-1) infected individuals, increasing the risk of infection from the HIV-1 positive male to the mother and ultimately to the offspring. The majority of information gathered with respect to the HIV-1 burden in the male reproductive tract (MGT) have been conducted in tissues obtained on autopsies of testis, prostate and epididymis of individuals that die from AIDS. Therefore, little is known about the progression and pathogenesis of the infection within these organs. Investigating the mechanism of the spread of HIV-1 in the cells and tissues of the MGT, particularly during the asymptomatic stage, remains a critical task.
Infection of macaques with simian immunodeficiency virus (SIV) is a useful animal model for studies of mucosal transmission and viral transmission via breastfeeding. In this study eight juvenile macaques (2.5 yo) were infected with SIVmac for a period of 3–6 months and testis and epididymis tissue were collected in two intervals, 3 and 6 months post-infection. To determine SIV progression and pathogenesis in the MGT we have used EM, immunohistochemistry, confocal microscopy and immunoblotting.
Our preliminary EM obtained via analysis of testis and epididymis tissue of SIV infected macaques show the presence of elongated spermatids in the epididymis. Scattered viral like SIV particles were observed in the testis and epididymal lumen, principally associated with aberrant germ cells. Necrosis of epididymal tissue was also observed, potentially due to the SIV burden in this organ.
The data indicate that SIV infected juvenile macaques are a potential model for studying HIV-1 pathogenesis and its effect in spermatogenesis as well as the immune response of testis in a species that is closely related to humans.
206. Differential regulation of activin and inhibin production by interleukin 1 (IL1), transforming growth factor β1 (TGFβ1) and protein kinase C (PKC) in the Sertoli cell and granulosa cell
Authors: Eede V. , Muir J. A. , O. 'Connor A. E. , Winnall W. R. , Drummond A. E. , Hedger M. P.
Published: November 2010

Activin and inhibin are gonadal regulatory proteins comprising an α-subunit and either a βA-subunit or βB-subunit (inhibin A or B), or two βA-subunits (activin A). Synthesis of the α-subunit, and the inhibins, is regulated by FSH via cAMP/protein kinase A. Regulation of the β-subunits in the gonads is less well defined, but the IL1/MAP kinase, TGFβ /Smad and PKC pathways have been implicated. Sertoli cells and granulosa cells were isolated from 18–22 day-old Sprague-Dawley rats under standard conditions and cultured with IL1, TGFβ1 and the PKC agonists, gonadotrophin releasing hormone (GnRH) or phorbol myristate acetate (PMA). Activin A, inhibin A and inhibin B were measured in culture medium (at 48h) by ELISA. Subunit mRNA expression was measured in cell extracts (at 4 h and 8h) using quantitative RT–PCR. IL1 stimulated βA-subunit and activin A production and inhibited α-subunit and βB-subunit expression and inhibin B production in Sertoli cells, but had no effect in granulosa cells. TGFβ1 stimulated activin A in both cell types, as well as the inhibins in granulosa cells. Surprisingly, TGFβ1 had no effect on Sertoli cell α-subunit or βA-subunit mRNA expression, but did cause a slight reduction of βB-subunit expression. GnRH increased activin A and inhibin A, but not inhibin B, production by granulosa cells and had no effect on Sertoli cells, which lack the GnRH receptor. However, direct activation of PKC by PMA stimulated βA-subunit mRNA expression and activin A production and decreased βB-subunit and inhibin B production by Sertoli cells, with marginal effects on inhibin A. These results indicate that activation of the TGFβ or PKC signalling pathways preferentially stimulates βA-subunit expression and/or translation, leading to increased activin A secretion by Sertoli cells and both activin A and inhibin A secretion by granulosa cells. The ability of IL1 to stimulate activin A is confined to the Sertoli cell.
207. Effect of TGFB1 on mammary gland development is dependent on cellular source of gene expression
Authors: Ingman W. V. , Robertson SA
Published: November 2010

The cytokine TGFB1 is implicated in development of the mammary gland through regulation of epithelial cell proliferation and differentiation during puberty and pregnancy. We have compared mammary gland morphogenesis in virgin Tgfb1+/+, Tgfb1+/− and Tgfb1−/− mice, and transplanted Tgfb1+/+ and Tgfb1−/− epithelium to determine the impact of TGFB1 deficiency on development. When mammary gland tissue was evaluated respective to timing of puberty, invasion through the mammary fat pad of the ductal epithelium progressed similarly irrespective of genotype, albeit fewer terminal end buds were observed in mammary glands from Tgfb1−/− mice. The terminal end buds appeared morphologically normal, and a comparable amount of epithelial proliferation was evident. However, when transplanted into wildtype recipients, Tgfb1−/− epithelium showed accelerated invasion compared with Tgfb1+/+ epithelium. This suggests that the normal rate of ductal extension in Tgfb1−/− null mutant mice is the net result of impaired endocrine or paracrine support acting to limit the consequences of unrestrained epithelial growth. By adulthood, mammary glands in cycling virgin Tgfb1−/− mice were morphologically similar to those in Tgfb1+/+ and Tgfb1+/− animals, a normal branching pattern was observed, and the tissue differentiated into early alveolar structures in the diestrus phase of the ovarian cycle. Transplanted mammary gland epithelium showed a similar extent of ductal branching and evidence of secretory differentiation of luminal cells in pregnancy. These results reveal two opposing actions of TGFB1 during pubertal mammary gland morphogenesis, dependent on the cellular source of gene expression. When expressed in the epithelium, TGFB1 inhibits epithelial ductal growth, when expressed systemically, TGFB1 promotes epithelial ductal growth.
209. The absence of betaglycan affects Sox9 m RNA expression at the time of sex determination in a mouse model
Authors: Sarraj M. A. , Chua H. , Umbers A. , Escalona R. , Loveland K. L. , Findlay J. K. , Stenvers K. L.
Published: November 2010

Betaglycan is a co-receptor that binds both TGF-β and inhibin, and thereby acts as a modulator of the activities of multiple members of the TGF-β superfamily. We have previously shown that the murine betaglycan gene is expressed in somatic cells within the interstitium of the fetal testis from 12.5 dpc-16.5 dpc. Betaglycan protein was predominantly localised to the interstitial cells surrounding the developing seminiferous cords which stained positive for Cyp11a (p450 Scc), a Leydig cell marker. In order to determine the impact of this receptor on fetal Leydig cell biology, RNA was extracted from two independently collected sets of betaglycan knockout and wildtype male and female gonads at 12.5 dpc and 13.5 dpc (n = 4 gonad pairs/set), and quantitative real time PCR was performed to determine changes in the expression levels of key genes involved in fetal Leydig cell differentiation and function. This analysis revealed that the levels of mRNA expression of SF1, Cyp11a and Cyp17a1 were downregulated between 12.5–13.5 dpc in the betaglycan knockout embryos compared with wildtype embryos immediately after the time of sex determination. Interestingly, the expression level of the key Sertoli cell marker SRY-(sex determining region Y)-box 9 (Sox9) was transiently decreased at 12.5 dpc by 50% in the knockout testis in comparison with that of the wildtype testis. No significant change was found one day later at 13.5 dpc. Our data show that betaglycan is predominantly expressed in the fetal Leydig cells of the murine testis and that the presence of this receptor is required for normal fetal Leydig cell differentiation. Furthermore, the transient downregulation of Sox9 expression in null testis suggests that Sertoli cell differentiation may also be affected in betaglycan knockout mice, and that this defect may precede the defect in Leydig cell development.
Supported by: the NHMRC Australia (RegKeys 338516; 241000).
213. Chemokines: key players at the maternal - fetal interface
Authors: Hannan N. J. , Salamonsen L. A.
Published: November 2010

Establishment of pregnancy requires extensive communication at the maternal-fetal interface and involves a plethora of locally acting molecules, including the chemokines. Chemokines are multifunctional molecules initially described for roles in leukocyte trafficking, but since found to participate in many other processes such as differentiation and directed migration. Previously we have shown that the chemokines, CX3CL1 and CCL14, are abundant in human endometrial vasculature, leukocytes, epithelial and decidual cells at the time of implantation and that their receptors, CX3CR1 and CCR1, are present on invading human trophoblast. CX3CL1 and CCL14 directly promote human trophoblast migration. We hypothesised that these endometrial chemokines promote trophoblast migration by regulating adhesion molecules and extracellular matrix (ECM) components on the trophoblast, similar to mechanisms used in leukocyte trafficking. Trophoblast cells (AC1M-88) used previously, showed a marked increase in adhesion to fibronectin following treatment with CX3CL1 and CCL14. Alterations in trophoblast adhesion associated and ECM genes following chemokine stimulation were examined using pathway specific oligo-arrays and quantitative real-time RT–PCR. Over 30 transcripts were affected by CX3CL1 treatment and 15 were regulated by CCL14 treatment. Real-time RT–PCR confirmed significant changes in the mRNA transcripts of α-catenin (CTNNA1), extracellular matrix protein-1 (ECM1), osteopontin (SPP1), integrin α6 (ITGA6), matrix metalloproteinase-12 (MMP12) and integrin β5 (ITGB5) following chemokine treatment. Several of these molecules have previously been implicated in implantation. Immunohistochemistry confirmed the presence of integrin α6, SPP1 and ECM1 protein in first trimester human implantation sites. The temporal and spatial expression of chemokines, their receptors and adhesion related molecules at the maternal-fetal interface emphasises an important role in the controlled directional migration of trophoblast through the maternal decidua. For the first time this study demonstrates direct effects of CX3CL1 and CCL14 on trophoblast adhesion and ECM molecules suggesting mechanisms by which trophoblast cells migrate during early pregnancy.
215. Placental expression of uncoupling protein-2 is reduced by glucocorticoid treatment in late pregnancy: implications for placental oxidative stress
Authors: Jones M. L. , Mark P. J. , Waddell B. J.
Published: November 2010

Placental oxidative stress plays a key role in the pathophysiology of placenta-related disorders in humans, most notably in preeclampsia (PE) and intrauterine growth restriction (IUGR). Protection from oxidative stress is provided by antioxidant enzymes including superoxide dismutase-1 and 2 (SOD-1 and –2) and catalase (CAT), which convert reactive oxygen species (ROS) to inert products. It has also been proposed that uncoupling protein-2 (UCP2) may limit oxidative stress by reducing ROS production, but little is known of UCP2 expression in placenta. Here we measured placental UCP2, SOD-1, SOD-2 and CAT mRNA expression (by qRT–PCR) in normal gestation and after glucocorticoid-induced IUGR. The latter was included because glucocorticoids can increase oxidative stress in other tissues, and placental glucocorticoid exposure is elevated in both PE and IUGR. Placentas were collected on days 16 and 22 of normal pregnancy (term = day 23) and on day 22 after dexamethasone treatment (0.75 mg/mL in drinking water from day 13). The two morphologically-distinct regions of the placenta, the junctional (JZ) and labyrinth (LZ) zones, were analysed separately because effectively all growth occurs in the LZ over this period. Expression of UCP2 in LZ exceeded that in JZ (P < 0.001) and increased in both zones between days 16 and 22 (LZ: 2.0-fold; JZ: 3.2-fold). Dexamethasone treatment reduced UCP2 in LZ (44%; P < 0.05) but not in JZ. SOD1 and SOD2 increased with gestational age in LZ (P < 0.01) and JZ (P < 0.05), but neither were affected by dexamethasone. CAT expression was higher (2.4-fold, P < 0.001) in LZ compared with JZ but did not change with gestational age or dexamethasone. In summary, these data suggest that endogenous protection against oxidative stress increases in the rat placenta during late pregnancy. Moreover, this protection may be compromised by reduced placental UCP2 expression in a model of glucocorticoid-induced IUGR.
221. Localisation of relaxin receptors (Rxfp1) in the uterine artery and the effects of blocking circulating relaxin on passive mechanical wall properties in the uterine artery of late pregnant rats
Authors: Vodstrcil L. A. , Novak J. , Tare M. , Wlodek M. E. , Parry L. J.
Published: November 2010

During pregnancy, the uteroplacental circulation undergoes dramatic alterations to allow for the large increase in blood flow to the feto-placental unit. These alterations are achieved through several mechanisms including structural changes in the uterine artery wall and endothelium-dependent vasodilation. Small renal arteries of relaxin-deficient mice and rats have enhanced myogenic reactivity and decreased passive compliance, and are relatively vasoconstricted (Novak et al. 2001, 2006). To date, no study has identified relaxin receptors (Rxfp1) in arteries or investigated the effects of relaxin deficiency in pregnancy on uterine artery function. The aims of this current study were to: 1) localise Rxfp1 in the uterine arteries, 2) measure myogenic reactivity in small uterine arteries after relaxin treatment, and 3) test the hypothesis that blocking circulating relaxin in late pregnancy will increase uterine artery wall stiffness. We demonstrated that Rxfp1 is expressed in the uterine arteries of pregnant mice and rats. Brightfield immunohistochemistry and immunofluorescence using antibodies specific for rat Rxfp1, α-smooth muscle actin and CD31 localised Rxfp1 protein predominantly to the vascular smooth muscle in the uterine artery of pregnant rats. Administration of recombinant human H2 relaxin (4 ug/h) for 6 h or 5 days in intact and ovariectomised rats reduced myogenic reactivity of small uterine arteries in vitro. Pregnant rats were treated with a monoclonal antibody against circulating relaxin (MCA1) or control (MCAF) for 3 days (Days 17–19) and uterine arteries were mounted on a pressure myograph to assess passive mechanical wall properties. Neutralising circulating relaxin in late pregnancy resulted in a significant increase in uterine artery wall stiffness. These data demonstrate that relaxin acts on the vascular smooth muscle cells in the uterine artery and may be involved in the pregnancy-specific vascular remodelling of uterine arteries to increase vasodilation and blood flow to the uterus and placenta.
(1) Novak J et al. (2001). J Clin Invest 107: 1469–75
(2) Novak J et al. (2006). FASEB J 20: 2352–62
232. Developmentally regulated activin A signal transduction by Sertoli cells is required for normal mouse testis development
Authors: Itman C. M. , Small C. , Griswold M. , Nagaraja A. K. , Matzuk M. M. , Ernst M. , Jans D. A. , Loveland K. A.
Published: November 2010

Activin A, a TGF-β superfamily ligand, is critical for normal mouse testis development and quantitatively normal sperm production. Testicular activin production changes during development, being substantially higher in the immature testis relative to the adult [1, 2]. Activin influences the Sertoli cell, the nurse cell to developing sperm, enhancing proliferation during its immature phase, but not following terminal differentiation [3]. In the Inha−/− mouse, chronic excessive activin production results in Sertoli cell-derived tumours [4] whereas reduced activin bioactivity, in the InhbaBK/BK mouse, delays fertility [5]. Activin signals are transduced by the phosphorylation and nuclear accumulation of the transcription factors SMAD2 and SMAD3. By comparing activin signal transduction in immature v. terminally differentiated Sertoli cells, using quantitative confocal microscopy and western blot analysis of total and phosphorylated SMAD2 and SMAD3, we discovered that mouse Sertoli cells exhibit developmentally regulated activin responses. Activin induces nuclear accumulation of SMAD3, but not SMAD2, in immature cells, although both proteins are phosphorylated. In contrast, terminally differentiated cells exhibit nuclear accumulation of both SMAD2 and SMAD3. We observed that this shift coincides with decreased SMAD3 production at puberty and changes in FSH-induced Smad transcription, which favours Smad3 in immature cells but promotes Smad2 synthesis in terminally differentiated cells. Furthermore, whereas removal of SMAD3 from the Inha−/− mouse rescues the tumour phenotype [6], we demonstrated that insufficient SMAD3 production impairs testis growth. We hypothesised that this developmentally regulated SMAD utilisation drives specific transcriptional outcomes. Using microarray and quantitative PCR, we identified novel activin target genes displaying developmental stage-specific expression patterns coinciding with differential SMAD usage, including Gja1 and Serpina5 which are required for male fertility. These mRNAs are also modulated in vivo, increased 1.5–2 fold in Inha−/− testes and decreased by half in InhbaBK/BK testes, confirming that normal testis development requires carefully regulated activin production and responsiveness.
(1) Buzzard J et al. 2004. Endocrinology 145(7): 3532–3541
(2) Barakat et al. 2008. Reproduction 2008 Epub ahead of print
(3) Boitani C et al. 1995. Endocrinology 136(12): 4538–4544
(4) Matzuk M et al. 1992. Nature 360: 313–319
(5) Brown C et al. 2000. Nature Genetics 25(4): 453–457
(6) Li Q et al. 2007. Molecular Endocrinology 21(10: 2472–2486
235. Activin C antagonises activin A in vitro and over-expression leads to prostate pathologies in vivo
Authors: Gold E. , Jetly N. , Behuria S. , Woodruff T. , Hedwards S. , Risbridger G.
Published: November 2010

Activin A is a well characterised inhibitor of proliferation in most epithelial cells. The actions of activin A on cell growth are mediated through Smad-dependent pathways. Activin A is potent at low levels, therefore its synthesis and bioactivity must be tightly regulated. Follistatin binding or inhibin subunit heterodimerisation block access to the activin receptor and/or receptor activation. We postulate that another mechanism of regulating activin A bioactivity is through the activin-βC subunit. In order to test our hypothesis produced recombinant activin C and mice overexpressing activin-βC. Recombinant activin C abrogated activin A-induced growth inhibition in vitro and the mechanism of action was downregulation of activin A-induced Smad signalling molecules. In the prostate overexpression of activin-βC increased epithelial cell proliferation while there was no significant difference in apoptotic epithelial cells. This imbalance between proliferation and apoptosis led to a significant increase in ventral prostate weight, prostatic hypertrophy and epithelial cell hyperplasia. A significant decrease in nuclear localisation of Smad-2 was associated with activin-βC overexpression in the prostate which implies antagonism of activin signalling also occurs in vivo. This is the first study to provide evidence that activin-βC is an antagonist of activin A in vitro and in vivo and implicates a role for the activin-βC subunit in maintenance of tissue homeostasis in the prostate.
253. Toxic effects of hyperglycaemia arise from induced O-linked glycosylation in early mouse embryos
Authors: Pantaleon M. , Tan H. , Kaye P. L.
Published: November 2010

Glucose flux through the hexosamine biosynthetic pathway (HBP) which is essential for preimplantation development (1) produces uridine 5′-diphospho-N-acetylglucosamine, a donor substrate for multiple glycosylation reactions including O-linked glycosylation. This novel signalling arm of the HBP, known as the hexosamine signalling pathway (HSP) operates via reversible addition of an O-linked β-N-acetylglucosamine (O-GlcNAc) unit to serine and threonine residues of proteins including transcription factors, cytoskeletal components, metabolic enzymes and cellular signalling components. O-linked glycosylation is functionally reciprocal to phosphorylation at the same residues, altering the activity and/or stability of targeted proteins, thus providing a mechanism for modulating cellular physiology in response to glucose availability. The enzymes regulating this O-GlcNAcylation are the β-linked-O-GlcNAc transferase (OGT) and an O-GlcNAc-selective β-N-acetylglucosaminidase (O-GlcNAcase). We hypothesised that the toxicity of hyperglycemia on early embryos arises from increased flux through HBP and increased O-GlcNAcylation of key proteins. Mouse zygotes (18 h post hCG) were cultured under conditions of modified flux through the HSP including hypoglycemia, hyperglycemia or supplemented with glucosamine which feeds exclusively into the HBP to increase downstream O-GlcNAcylation. BADGP was used to inhibit OGT and O-GlcNAcylation. Blastocyst formation, cell proliferation and apoptosis were assessed. Treatments that perturb levels of intracellular protein O-GlcNAcylation inhibited embryo development. Whilst some flux through HBP is required to activate embryonic differentiation (1), excess flux arising from a hyperglycemic environment or glucosamine supplementation reduced cell proliferation and blastocyst formation, confirming the criticality of this novel post-translational signalling pathway. Inhibition of OGT using 2 mM BADGP blocked the negative impact of hyperglycemia on blastocyst formation, cell number and apoptosis supporting our hypothesis that O-GlcNAcylation is a key mechanism used by the embryo to sense and respond to perturbations of glucose in its environment.
(1) Pantaleon M, Scott J and Kaye PL (2008) Biol Reprod, 78(4):595–600
257. Activation of a calcium-activated chloride channel by paf is required for normal preimplantation mouse embryo development in vitro
Authors: Li Y. , Day M. L. , O.'Neill C.
Published: November 2010

Platelet activating factor (paf) is an autocrine survival factor for preimplantation embryo. Binding of paf to its receptor activates PI3kinase, causing an IP3-dependent release of Ca2+ from intracellular stores as well as activation of Ca2+ influx via a dihydropyridine-sensitive Ca2+ channel. These actions result in the generation of a defined intracellular calcium ([Ca2+]i) transient in the 2-cell embryo[1]. By using combined whole-cell patch-clamp and real-time [Ca2+]i analyses, we have shown that paf also induces a concomitant hyperpolarisation of the membrane potential in 2-cell embryos, accompanied by an increased net outward ion current. Both the membrane hyperpolarisation and outward current were dependent upon the occurrence of the paf-induced [Ca2+]i transient[2]. The aim of this study was to investigate the characteristics of the paf-induced outward current in 2-cell embryos and to assess whether it has a role in normal mouse preimplantation development. We show that: (1) removal of extracellular anions or treatment with niflumic acid (NFA, 100 μM, a Ca2+-activated Cl- channel blocker) prevented activation of the outward current by paf but had no effect on the paf-induced [Ca2+]i transient; and (2) The culture of embryos with NFA (100 μM) from the 1-cell to late 2-cell stage significantly reduced their development to the blastocyst stage (P < 0.001), but treatment with NFA from the late 2-cell stage had no effect on development. The results show that paf induces an increase in [Ca2+]i which in turn activates a Ca2+-activated Cl- channel. The activity of this NFA-sensitive channel during the zygote to 2-cell stage is required for normal embryo development.
(1) C. O’Neill (2008) The potential roles of embryotrophic ligands in preimplantation embryo development. Hum Reprod Update 14:275–288.
(2) Y. Li, M.L. Day & C. O’Neill (2007) Autocrine activation of ions currents in the two-cell mouse embryo. Exp Cell Res. 313:2785–2794.
258. Differential expression of H2A and H3 variant histones in mouse preimplantation embryos and R1 ES cells
Authors: Kafer G. R. , Lehnert SA , Kaye P. L. , Moser R. J.
Published: November 2010

Histone variants replace canonical histones in nucleosomes to serve numerous biological processes. This integration alters DNA properties to ultimately regulate gene expression. Previous mouse studies have indicated that some variants (H2AZ and H3.3) are essential for survival, but here we document and correlate histone expression patterns with key developmental events. Using quantitative reverse-transcribed PCR (qRT–PCR) we investigated the expression of 7 genes coding for H2A variants and 4 genes coding for H3 variants in mouse preimplantation embryos and in pluripotent R1 ES cells. Messenger RNA was extracted from pools of 3 embryos flushed from superovulated mice. Embryos were collected at five stages, zygotes, 2-cell embryos, morulae, blastocysts and hatching blastocysts (20 h, 44 h, 68 h, 92 h and 116 h post hCG respectively). The expression of H2A variant genes typically peaked within blastocysts. H2AZ and H2AX expression was 80 – 95% higher in blastocysts than other stages. Conversely, genes coding for H3 variants showed elevated expression in zygotes, where H3.3 expression was 85 – 95% higher and CENPA was ~75% higher than in later preimplantation stages. The expression profiles of histone remodellers SWI/SNF and CAF-1 correlated with the variants they are known to remodel (H2A and H3 variants respectively), suggesting an increased integration of those variants into nucleosomes. We also compared blastocyst and embryonic stem cell (ES cell) expression patterns. R1 ES cells express all histone variants, including H2A.Bbd, H3.1 and H3.2 which were not expressed in preimplantation embryos. Further, expression levels of every histone variant investigated differed significantly between R1 ES cells and hatching blastocysts (ANOVA, P < 0.05, n = 3 experiments). We conclude that histone variant expression reflects preimplantation developmental demands. Further, histone code expression profiles show significant change upon extended cell culture and maintenance of pluripotency as indicated by comparing in vivo hatching blastocysts to the R1 ES cell line.
261. Disruption of mitochondrial function in the blastocyst alters expression of the chromatin remodeler ATRX
Authors: Wakefield S. L. , Filby A. N. , Lane M. , Mitchell M.
Published: November 2010

Exposure of an embryo to suboptimal environments, including poor embryo culture media or inadequate maternal diet, can disrupt fetal and placental development and whilst the exact mechanisms responsible remain unknown, perturbed embryo metabolism has been implicated. We propose that stress applied to an early embryo causes mitochondrial dysfunction, resulting in a permanent epigenetic change. Thus the aim of this study was to determine the affect of directly perturbing mitochondria in the embryo, on development, metabolism and expression of the ATP-dependant chromatin remodelling protein, ATRX. Zygotes collected from gonadotrophin stimulated C57BL/6xCBA mice were cultured to the two-cell stage and then exposed to one of three treatments; control medium (C), medium lacking pyruvate (-P; embryos dependant on the mitochondrial Malate Aspartate Shuttle, MAS) or medium lacking pyruvate plus 5µM amino-oxyacetate (AOA), a specific MAS inhibitor (-P+AOA). Blastocyst development and metabolism were assessed by determining cell number and allocation, glycolysis, and ATP:ADP ratio. Relative gene expression of ATRX, was examined using RT PCR. Embryos dependant on the MAS alone (-P) had significantly decreased blastocyst development (87.1% v. 98.2%, P < 0.05), with a compensatory increase in glycolysis (0.20 v. 0.07 pmol/cell/hr, P < 0.001) despite a decrease in ATP:ADP (0.10 v. 0.13, P < 0.06), relative to the control. Inhibition of the MAS (-P+AOA) further reduced blastocyst development,(77.3%, P < 0.001) and decreased ATP:ADP (0.08, P < 0.004), but there was no change in glycolysis relative to control embryos (0.09 pmol/cell/hr, P = 0.3). Expression of ATRX was significantly increased for –P+AOA embryos relative to the control (1.63 v. 1.0, P < 0.007) but did not differ for –P embryos (1.1). This study demonstrates that direct perturbations of mitochondrial function in the embryo compromises its metabolic regulation and blastocyst development, and the expression of the epigenetic modulator ATRX. Further studies are underway to elucidate the implications of disrupted metabolic control and this epigenetic modulator on pregnancy outcomes.
262. Human cumulus cell gene expression as a marker of clinical embryo grade
Authors: Gebhardt K. M. , Feil D. , Lane M. , Russell D. L.
Published: November 2010

In Australia, Assisted Reproductive Technology (ART) accounts for ~3% of births. However, the success rate remains around 65% for women under 35 years of age, hence multiple embryo transfer is frequently preferred to improve the probabiity of achieving a term pregnancy. A biochemical marker for oocyte and embryo developmental potential would augment successful pregnancy outcomes following IVF/ICSI by optimising oocyte and embryo selection, therefore increasing the number of single embryo transfers (SET) performed in ART cycles. Changes in expression levels in human cumulus cells may reflect the quality of their enclosed oocyte. We investigated cumulus cell gene expression and subsequent embryo development to find a marker of embryo quality. Paired samples of cumulus cells were collected from oocytes that progressed to embryos of either high or low grade from eleven IVF/ICSI patients. Following cumulus oocyte complex retrieval cumulus cells were trimmed from the oocyte, and all oocytes and resulting embryos were cultured and tracked individually. Cumulus cell gene expression was assessed using a real-time RT–PCR assay, measuring expression of cyclooxygenase 2 (COX2; PTGS2), Pentraxin 3 (PTX3), Versican (VCAN), Tumour Necrosis Factor Alpha Induced protein 6 (TNAIFP6; TSG6), Lactate Dehydrogenase A (LDHA), Phosphofructokinase Platelet (PFKP), Gremlin (GREM1), Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) and 18S rRNA. Standard curves using plasmid subclones for each target were run to assess copy numbers of genes. Embryo morphology was assessed by an embryologist and correlated with relative gene expression. Cumulus cell gene expression was altered in cumulus cells from oocytes which subsequently developed into higher quality (Grade 1 and 2) embryos compared with cumulus cells from oocytes which developed into lower quality (Grade 3 and 4) embryos. This may lead to establishment of markers prognostic for developmental outcome, facillitating more reliable selection of higher quality embryos, increasing single embryo transfers and improving health outcomes from ART.
273. The development of an immunoassay to measure progestogen using printed biosensors, and its application to the assessment of ovarian function in the Numbat (Myrmecobius fasciatus)
Authors: Matson P. , Ditcham W. , Palmer C. , Warren K. , Monaghan C. , Kappelle W.
Published: November 2010

A biosensor system using screen printed sensors was developed to measure progesterone as an index of ovarian function, and compared with a standard enzymeimmunoassay (EIA). The sensors were coated with a monoclonal progesterone antibody which cross-reacts with a wide range of progestogens, and incubated in a mixture of sample/standard and progesterone-3-CMO-horseradish peroxidise (Prog/HRP). The endpoint was the change in potential read following the addition of sodium perborate. The assay was optimised in terms of the Prog/HRP concentration, the antibody dilution and incubation times. It was then used to measure progestogen in the urine of five female Numbats (Myrmecobius fasciata). Results were available using the sensors within 20 min compared with the standard EIA protocol of 2 h. The serial dilution of a urine sample taken at the diestrus stage showed parallelism with the serially diluted standard. There was a significant rise in progesterone (mean ± sem) after mating compared with that seen before for both the EIA (1.31 ± 0.20 to 3.70 ± 0.13 ng/mL) and the sensor (1.83 ± 0.33 to 4.02 ± 0.61 ng/mL), and there were no significant differences between the sensor and EIA results at either stage (all P > 0.1). A comparison of the values obtained with the sensors to those obtained with the conventional EIA showed a significant correlation for each of the animals (r = 0.82 to 0.99). It is concluded that the biosensor system is a viable alternative to conventional EIA, and provides the advantage of (a) a shorter assay time and (b) greater potential for use in the field.
276. The technical and biological validation of an LH assay for use with the Western Grey Kangaroo (Macropus fuliginosis) and Black-flanked Rock Wallaby (Petrogale lateralis lateralis)
Authors: Matson P. , Mayberry C. , Willers N. , Blackberry M. A. , Martin G. B.
Published: November 2010

Methods for the measurement of marsupial LH invariably rely upon the similarity of the LH molecule between different species and usually use anti-ovine or anti-bovine LH antibody and an ovine or bovine labelled LH preparation. Initial attempts to measure plasma LH in the Western Grey Kangaroo with assays using antibodies to 4 different isoforms of ovine LH raised in 7 different rabbits were unsuccessful. An enzymeimmunoassay (EIA) developed for the Asian elephant (Zoo Biology 23:45–63) was then applied to the Western Grey Kangaroo and the Black-flanked Rock Wallaby. This EIA has an anti-bovine-LH monoclonal antibody (518B7 provided by Dr Jan Roser, University of California, Davis, USA), biotinylated ovine LH label and bovine LH standard (NIADDK-oLH-26 and NIH-bLH-B10, both provided by Dr Janine Brown and Nicole Abbondanza, Smithsonian Institute, Front Royal, Virginia USA). Technical validation showed that serial dilution down to 1:8 of plasma from 7 individuals of each species showed parallelism to the assay standard curve, and control samples (1.24–5.30 ng/mL) had between-assay coefficients of variation <9%. Biological validation was achieved by challenging animals with intramuscular GnRH (Fertagyl®, 2.5 µg/kg) and measuring LH before and 25 min after the injection. Significant increases in plasma concentrations of LH (mean ± sem; all P > 0.0005) were seen after GnRH for both the Western Grey Kangaroo (from 5.0 ± 0.8 ng/mL to 9.4 ± 1.2 ng/mL; n = 19) and the Black-flanked Rock Wallaby (from 6.0 ± 0.7 ng/mL to 10.6 ± 0.6 ng/mL; n = 28). In conclusion, this assay can be successfully used to measure LH in these two species.
280. INSL3 is a measure of human Leydig cell functionality both during fetal and adult life
Authors: Anand-Ivell R. , Manson J. , Wittert G. , Wohlgemuth J. , Hafen B. , Ivell R.
Published: November 2010

Insulin like factor 3 (INSL3) and testosterone are the two major secretory products of the testis, both produced by the interstitial Leydig cells. The Leydig cells of the testis have two distinct generations, one developing before birth (fetal Leydig cells, FLC) and an adult type (adult Leydig cells, ALC) that become differentiated and functional at puberty. Although these two types of Leydig cells represent distinct populations, rodent studies show that both types produce testosterone and INSL3. Both are presumed to have evolved from a common stem cell pool. We measured INSL3 levels in human amniotic fluids collected at various times of gestation and show for the first time that the human male fetus indeed generates INSL3 at a time appropriate for the first transabdominal phase of testicular descent, which appears to be the primary physiological role for the fetal hormone. INSL3 appears to be independent of androgen production. The adult type Leydig cells (in adult men) secrete INSL3 that can be measured in the peripheral circulation at levels ranging from 0.5 to 2.5 ng/mL. We studied a large randomly recruited cohort of 1183 men from South Australia, comparing serum INSL3 concentrations with age, and a variety of endocrine, cognitive and morphological parameters. INSL3 concentration was observed to decline significantly with age. This however, had no correlation with testosterone or components of the HPG axis. INSL3 is an independent measure of Leydig cell function (quality and number), which appears to be independent of acute control via the HPG axis. Its decline with age reflects a decline in the properties of the Leydig cell population only, and emphasises a gonadal component in the age-related decrease in androgen production.
Research supported by ARC Discovery grant DP0773315.
417. In vivo and in vitro evidence for cancer stem cells in human endometrial cancer
Authors: Hubbard S. , Gargett C. E.
Published: November 2010

Cancer stem cells (CSCs) have been identified in solid human cancers, including breast, colon, and ovary. Recent evidence suggests that the highly regenerative human endometrium harbors rare populations of epithelial stem/progenitor cells1. We hypothesised that CSCs are responsible for the epithelial neoplasia associated with endometrial carcinoma (EC), the most common gynaecological malignancy in women. The aim of this study was to demonstrate that a rare population of EC cells posses CSC properties. Stem cell characteristics were assessed in 25 EC and 2 endometrial hyperplasia tissues obtained from women aged 62 ± 9 yrs. Samples were cultured at clonal densities (100–500 cells/cm2) for 3–5 wks to determine cloning efficiency. Individual clones were serially subcloned (<10 cells/cm2) every 2–4 wks to determine self renewal capacity. Isolated cells in serial dilution (103–106 cells) were placed under the kidney capsule of immunocompromised mice for 12–16 wks to examine for the presence of tumour initiating cells (TIC). Resulting tumours and original parent tumours were examined for markers by immunohistochemistry. Most samples (23/26) contained rare colony forming cells. The cloning efficiency was 0.23% ± 0.28% (n = 11) in G1, 0.78% ± 0.67% (n = 8) in G2, 0.22% ± 0.21% (n = 3) in G3, 0.03% (n = 2) in type II tumours, and 0.14% (n = 2) in hyperplasia samples, and did not differ significantly between grades or between type I EC and normal endometrial epithelial samples 1. Single cell derived clones subcloned 2.5 ± 1.4 (n = 11), 3.2 ± 0.4 (n = 5), 3.5 (n = 2), 3.0 ± 1.7 (n = 3), and 2.5 (n = 2) times in G1, G2, G3, type II tumours and hyperplasia samples respectively, indicating increasing self renewal capacity with increasing tumour grade. Transplanted EC single cell suspensions initiated tumour growth with similar morphology, ERα, PR, EpCAM, cytokeratin, and vimentin expression as the parent tumour, indicating the presence of TIC. This evidence suggests that rare cells possessing the CSC properties of clonogenicty, self renewal, and tumorigenicity, may be responsible for the initiation and progression of EC.
(1) Chan RWS et al. (2004). Biology of Reproduction. 70:1738–1750