bluuebunny/crossref_metadata_2025 huggingface dataset:
274. Brain gene expression changes in MHC Class II genes and Neuropilin 2 associated with the transition from acyclic to cyclic ovarian function in postpartum beef cows
Authors: Flatscher-Bader T. , Suhaimi A. H. M. , Lehnert SA , Reverter A. , Barris W. , Phillips N. , McGowan M. , D. 'Occhio M.
Published: November 2010

The aim was to identify hypothalamic genes associated with transition from acyclic to cyclic ovarian function in postpartum cows. Ovarian status was recorded at slaughter for primiparous Brahman cows (Zebu, Bos indicus) at 27–34 days postpartum and hypothalamic tissue was obtained at the same time. Ovaries were classified as: Ov1 (n = 4), follicles 4–5 mm; Ov2 (n = 4), follicles equal or larger 10 mm without ovulation; Ov3 (n = 4), corpus haemorrhagicum or corpus luteum. Hypothalamic regions were: H1 (SC-POA, APVN, anterior hypothalamic nucleus, anterior portion of the arcuate nucleus, nearby areas of the diagonal band of Broca, and medial septum); H2 (basal hypothalamus-median eminence, ventromedial hypothalamus, posterior portion of the arcuate nucleus, and anterior part of the mammillary body). Gene expression was determined using the Agilent bovine 44k DNA microarray and differential expression (DE) was ascertained by mixed model analysis. Functional clustering of DE genes using DAVID (www.david.abcc.ncifcrf.gov) revealed an enrichment of genes linked with synaptic transmission in H1 and H3 comparing Ov1 and Ov3. Three members of the major histocompatibility complex (MHC) class II gene family (BOLA-DQA1, BOLA-DQA2 and BOLA-DQB) showed a higher expression level in Ov2 than Ov1 in H1 and H2. BOLA-DGA2 and BOLA-DQB were downregulated in Ov3 when comparing Ov2 and Ov3. MHC family members have been associated with plasticity in the brain1. Functional clusters of genes involved in cell-cell adhesion were differentially expressed when comparing Ov1 and Ov2 (H2), and Neutropilin-2 which promotes GnRH neuronal migration2 was upregulated in Ov1. The findings provide preliminary evidence that gene expression related to cellular plasticity within the basal hypothalamus-median eminence is associated with the transition from acyclic to cyclic ovarian function in the postpartum cow.
(1) Håvik B et al. 2007 Neuroscience 148:925–36
(2) Cariboni A et al. 2007 TRENDS in Neurosciences 30:638–44
275. The endocrine disruptor, bisphenol A, alters gene expression in the hypothalamus of the ram lamb but does not alter LH secretion
Authors: Scott C. J. , Clarke I. J. , Wilkins J. , Flatscher-Bader T. , Martin G. B. , Tilbrook A. J.
Published: November 2010

Bisphenol A (BPA) is an endocrine disruptor, which has both oestrogenic and anti-oestrogenic actions. It has profound effects on development, especially of sex organs but also on the brain. The actions on the brain are poorly understood and little is known about males. Most work has been conducted in fish & rodents, but the lifespan of domestic ruminants means that BPA may accumulate in tissues over many years. This study tested the hypothesis that treatment of ram lambs with BPA would alter LH secretion as well as gene activity in various hypothalamic regions. Ram lambs were treated from 1 month old with thrice weekly injections (im) of BPA (3.5 mg/kg) or vehicle (n = 10/group) for 80days. Weekly bodyweights and jugular blood samples were collected and on day 79, blood samples were taken every 15 min for 8 h for LH RIA. The sheep were killed, body & testicular weights collected and the hypothalamus dissected into anterior, dorsal and ventral blocks, then frozen in liquid nitrogen. Bodyweight (Cont. 26.0 ± 0.5 kg v. BPA 25.2 ± 0.7 kg) and paired testicular weight (Cont. 31.6 ± 1.2 g v. BPA 29.3 ± 1.4 g) did not significantly differ (P > 0.05). LH secretion also did not differ, in either mean LH concentrations (Cont 0.31 ± 0.14 v. BPA 0.29 ± 0.12ng/mL) or pulses/8 h (Cont 1.8 ± 0.4 v. BPA 2.0 ± 0.4). Total RNA was extracted from the basal hypothalamus (ventral block) and RNA from 2 treated and 2 control animals was hybridised on 44K Agilent bovine microarrays. Preliminary analysis suggests 118 probes were differentially expressed. Gene ontology analysis of these differentially expressed probes revealed involvement in a broad range of biological processes and cellular components. This study suggests that gene array analysis is a suitable tool with which to study BPA actions in the sheep hypothalamus. Future work will expand this pilot microarray study to include all animals and all hypothalamic regions.
277. Equine chorionic gonadotrophin isoform composition in commercial products compared with isoform composition in pregnant mare plasma
Authors: Ciller U. A. , Ciller I. M. , McFarlane J. R.
Published: November 2010

It is well documented that there is considerable batch to batch variation in the activity of commercial preparations of gonadotrophins. These products are used in a variety of assisted reproductive procedures in the livestock industry, consequently this high degree of variation between products and batches adds to the already considerable between animal variations in response to the treatment. Equine chorionic gonadotrophin (eCG) is a heterodimeric glycoprotein hormone secreted by the placental endometrial cups during the first third of gestation in the horse. Plasma is harvested from pregnant mares between 40–90 days of gestation and the eCG isolated and used to formulate commercial preparations. Previous research has shown that eCG like the other gonadotrophins is a highly heterogeneous molecule with significant differences in bioactivity between isoforms. The aim of this study was to determine whether significant differences in isoform composition exist between various commercial preparations of eCG (n = 15), and how this compares with the isoform composition found in plasma (n = 23). Concentrations of eCG were determined using a competitive eCG ELISA. Liquid phase iso-electric focusing was used to fractionate plasma and the commercial preparations into 10 pH ranges from pH 3.0 to pH 10.0. Data from the 10 fractions were grouped into acidic (pH 3.0–5.1), intermediate (pH 5.2–7.9), or basic (pH 8.0–10.0) isoform categories for analysis. Immunoactivity between commercial eCG products ranged from 44% to 362% of stated bioactivity. Iso-electric focusing showed that the majority of the immunoactivity (92%) of the commercial preparations was found in the acidic fractions (pH 3.0–5.1), and in particular in the pH range 3.0–3.8. This contrasted starkly with isoform profiles found in pregnant mare plasma samples which showed a much greater spread across all 3 pH ranges. In summary, the isolation processes of commercial eCG preparations appears to selectively favour the acidic isoforms of eCG.
278. IGF2 polymorphisms predict pregnancy outcome
Authors: Thompson S. D. , Nowak R. C. , Zhang J. , Dekker G. A. , Roberts C. T.
Published: November 2010

IGF-II is an important determinant of placental trophoblast invasion and subsequent placental function. IGF-II can act in autocrine/paracrine and endocrine fashions to promote placental invasion and differentiation and within both the fetus and placenta to promote fetal growth. We aimed to determine whether polymorphisms in Igf2 are associated with common pregnancy complications associated with uteroplacental insufficiency. Pregnant women were recruited in early pregnancy for a prospective case control study at the Women's and Children's Hospital and Lyell McEwin Health Service, Adelaide, Australia. Buffy coats were retrieved from maternal blood sampled at 15 weeks gestation, paternal blood and cord blood collected following delivery. Pregnancy outcomes were classified into normal (n = 126), preeclampsia (PE, n = 31), gestational hypertension (GH, n = 18), small-for-gestational-age (SGA, n = 50), PE+SGA (n = 16) or preterm birth (PTB, n = 19) by an experienced obstetrician. Three polymorphisms in Igf2, Igf2 ApaI and Igf2 MspI single nucleotide polymorphisms, and INS+2336 deletion/insertion polymorphism were selected for investigation. DNA was extracted from buffy coats and genotyping was performed by PCR followed by High Resolution Melt analyses. Data were analysed by Chi Square and Fisher's Exact Test and Likelihood Ratios (LR) were calculated. In normal pregnancies, all polymorphisms were in Hardy–Weinberg Equilibrium. Igf2 ApaI in the neonate, and hence placenta, was associated with PE (P = 0.016, LR = 7.46) and PTB (P = 0.024, LR = 8.61). Neonatal Igf2 MspI was associated with SGA (P = 0.007, LR = 9.81). Gestational age was associated with maternal Igf2 ApaI (P = 0.0004) and INS+2336 (P = 0.0021), as well as neonatal INS+2336 (P = 0.0046). Birthweight was associated with paternal Igf2 MspI (P = 0.044) when corrected for gestational age. Although this work is ongoing, data thus far suggest polymorphisms in the gene encoding IGF-II, primarily in the placenta, are associated with a range of pregnancy complications which have been associated with impaired placental function. Ongoing research will determine whether these polymorphisms are associated with aberrant placental Igf2 expression.
279. Expression of oestrogen receptor-α and modulators of steroid receptor signalling, proline-rich nuclear receptor-2 and peptidylprolyl isomerade-D, in the hypothalamus of suckled and weaned postpartum beef cows
Authors: Flatscher-Bader T. , Suhaimi A. H. M. , Lehnert SA , Reverter A. , Barris W. , McGowan M. , Phillips N. J. , D.'Occhio M. J.
Published: November 2010

The aim was to characterise gene expression in the hypothalamus of suckled and weaned postpartum beef cows. The hypothalamus was obtained at slaughter from 12 primiparous Brahman cows (Zebu, Bos indicus) at 27 and 34 days postpartum. Six cows were weaned 7 days or 14 days before slaughter. Hypothalamic regions used for gene expression were: H1 (SC-POA, APVN, anterior hypothalamic nucleus, anterior portion of the arcuate nucleus, nearby areas of the diagonal band of Broca, and medial septum); H2 (basal hypothalamus-median eminence, ventromedial hypothalamus, posterior portion of the arcuate nucleus, and anterior part of the mammillary body). Gene expression was determined using the Agilent bovine 44k DNA microarray and differential expression (DE) was ascertained by mixed model analysis. A total of 122 genes were DE in H1 and 84 genes were DE in H2; 41 DE genes were common to H1 and H2. Functional clustering of DE genes using DAVID (www.david.abcc.ncifcrf.gov) revealed DE gene clusters in H1 associated with signalling events and ion binding, and DE gene clusters in H2 associated with hormone activity and ligand-receptor interactions. Of the DE genes, ~25% were linked with oestrogen signalling. This included oestrogen receptor-α (ESR1) that showed lower DE in H2 for weaned cows. Two modulators of steroid receptor signalling, proline-rich nuclear receptor coactivator-2 (PNRC2)1 and peptidylprolyl isomerase D (PPID)2, showed altered expression. In weaned cows, expression level of PNRC2 was lower in H1 and H2, while that of PPID was decreased in H1. The overlapped hypothalamic regions H1 and H2 are known to contain GnRH neuron terminals and kisspeptin neurons. Weaning promotes the resumption of cyclic ovarian function in postpartum cows, and the similar shifts in DE of ESR1, PNRC2 and PPID provided further evidence of a role for oestradiol at the hypothalamus in regulating postpartum reproduction.
(1) Zhou D et al. 2006 Nucleic Acids Res 34:5974–86
(2) Kumar P et al. 2001 Biochem Biophys Res Commun 284:219–25
401. In vivo development of embryos derived from sex-sorted and non-sorted ram sperm
Authors: Beilby K. H. , Grupen C. C. , Maxwell W. M. C. , Evans G.
Published: November 2010

The use of sex-sorted ram sperm results in pregnancy rates similar or superior to that of non-sorted sperm after laparoscopic insemination of synchronised ewes under defined conditions [1, 2]. To further assess the fertility of sex-sorted ram sperm, embryo production and development rates were examined after the insemination of superovulated ewes with either sex-sorted or non-sorted sperm. Merino ewes (n = 30) were synchronised in oestrus using progestagen sponges inserted for 14 days and hormonally stimulated with PMSG (600 IU) on sponge removal (SR), FSH (133 mg) at decreasing doses every 12 h for 4 days before insemination, and GnRH 24 h before insemination. Each ewe was inseminated in the uterus by laparoscopy 42–44 h after SR with 15 × 106 motile X- or Y-chromosome bearing or non-sorted spermatozoa. On day 6 after insemination, antegrade flushing of both uterine horns was performed. Overall, the fertilisation rate was higher using X-chromosome bearing sperm (70%) compared with both Y-chromosome enriched (59%) and non-sorted treatments (64%). Embryo development was more advanced after the insemination of non-sorted sperm with more hatching (hg) and expanded blastocysts (exb) recovered per animal (hg: 3.37 ± 1.19; exb: 5.00 ± 1.68) compared with sorted groups (X hg: 1.71 ± 0.92; exb: 3.28 ± 1.80; Y hg: 1.67 ± 1.67; exb: 1.50 ± 1.02). Moreover, embryos were recovered at earlier developmental stages after insemination with Y- compared with X-chromosome bearing sperm. In conclusion, the use of sex-sorted sperm did not affect the efficiency of embryo production. However, the development of embryos derived from sex-sorted sperm was delayed compared with those from non-sorted sperm.
(1) de Graaf et al. 2007. Reproduction in Domestic Animals 42, 648–653
(2) Beilby et al. 2008. Theriogenology [IN PRESS]
402. Cryopreservation of oocytes and follicular cells of the cane toad Bufo Marinus
Authors: Wooi K. H. , Mahony M. J. , Shaw J. M. , Clulow J.
Published: November 2010

Amphibians are currently the most threatened of all vertebrate groups with more than 30% of all known species in decline, facing extinction or recently extinct. Cryobanking of amphibian germ cells and reproductive tissues could be used to manage threatened species and provide insurance against extinction. However, cryopreservation of fully developed amphibian oocytes and whole embryos has not been achieved due to technical problems freezing such large cellular structures. As an alternative approach, we investigated the feasibility of developing protocols for the slow-cool freezing, storage and retrieval of developmentally competent amphibian ovarian follicles containing Stage I and II oocytes which are much smaller in size than later developmental stages. Ovarian follicles from euthanased Cane Toads were incubated in cryodiluents containing either glycerol or DMSO to assess cryoprotectant toxicity and response to slow cooling freezing protocols. The fluorescent live cell stain SYBR 14 and its counter stain propidium iodide was used to score the proportion of viable follicle cells before and after cryopreservation. Cryoprotectant type, concentration and exposure time all had significant effects (P < 0.05) on the viability of follicle cells, with significant interactions between these variables. Overall, glycerol was less toxic to follicle cells than DMSO. At higher concentrations, glycerol exerted high osmotic stress on oocytes, and there was evidence that DMSO triggered apoptosis in oocytes. The most effective cryopreservation protocol for stage I and II oocyte follicles resulted in a post-thaw recovery of a mean 70% of viable follicular cells. This protocol involved cryopreservation in 15% v/v glycerol, inclusion of seeding and temperature holding periods during cryopreservation, coupled with rapid thawing in a 30°C water bath. The successful cryopreservation of intact follicles in this study indicates the potential to recover functional ovarian tissues post cryopreservation for continuation of amphibian oogenesis in vitro or in vivo.
403. Differential effect of hexoses on sperm metabolism and function in culture
Authors: Bakos H. W. , Lane M.
Published: November 2010

Currently there is lack information regarding how human spermatozoa regulate their energy metabolism. This is surprising considering that carbohydrate metabolism is a vital point for the understanding of sperm function. This coupled with the increased use of assisted reproductive technology and the importance of a well balanced culture media has led us to hypothesise that an imbalance of carbohydrate presence in the media may alter sperm function, particularly in relation to oxidative stress, DNA damage and lipid peroxidation. Sperm samples were obtained from three healthy normospermic donors for this study. Motile sperm were separated from semen samples using density gradient separation. Samples were incubated at different media conditions with varying glucose or fructose concentrations (0, 2.5, 25 mM) for 6–24hrs. Reactive oxygen species (ROS) were measured using 5-(and-6)-carboxy-2', 7'-dichlorofluorescein diacetate (DCFDA). Sperm DNA damage was determined using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling (TUNEL). Lipid peroxidation was assessed using the probe BODIPY (581/591) C11. Carbohydrate uptake from the media was measured using a fluorometric procedure. Statistical differences between treatments were assessed by ANOVA and Bonferroni post-hoc test. No significant motility differences were found following treatments. Results showed an increased level of ROS production as glucose concentration increased (P < 0.05). This was accompanied by an increased number of TUNEL positive cells (P < 0.05). Furthermore, lipid peroxidation of spermatozoa was significantly increased when incubated under high glucose concentrations (P < 0.01). In contrast, increases in fructose concentrations did not alter ROS levels or the number of TUNEL positive cells. Sperm metabolised both glucose and fructose in vitro and the removal of one carbohydrate resulted in a compensatory increase in the metabolism of the other. To our knowledge, this is the first report providing evidence that altered carbohydrate metabolism may induce ROS production, lipid peroxidation and increase the number of sperm exhibiting DNA damage.
404. Repercussions of a transient decrease in pH on embryo viability and subsequent fetal development
Authors: Zander-Fox D. L. , Mitchell M. , Thompson J. G. , Lane M.
Published: November 2010

Changes in the environment to which the preimplantation embryo is exposed can significantly influence fetal outcomes, indicative of ‘embryo programming'. Although many previous in vitro stress models have demonstrated programming changes using high dose stress effects, it is possible to induce similar effects with a physiologically relevant stress model. This study investigates the effect of a subtle transient pH change, during the first cleavage division, on blastocyst viability and fetal outcomes after embryo transfer. Zygotes from F1 hybrid mice (C57BL6xCBA F1) were cultured to the 2-cell stage (19h) in either control G1 medium or in G1 containing a weak acid, 2 mM DMO (5,5-Dimethyl-2,4-oxazolidinedione), then cultured to the blastocyst stage in control media G1/G2 (72h). Exposure to DMO induced a decrease in intracellular pH from 7.25 (control) to 7.10 (DMO). At the blastocyst stage, inner cell mass (ICM) cell number and cellular apoptosis were assessed, or embryos were transferred to pseudopregnant recipients to assess implantation and fetal outcomes. Differences were assessed using Student's t-test or generalised linear modelling followed by post-hoc tests. Exposure to DMO during the first cleavage division significantly reduced total blastocyst cell number from 83.0 ± 6.4 to 63.6 ± 3.8 (P < 0.05), reduced ICM number from 30.6 ± 3.6 to 20.2 ± 1.8 (P < 0.05) and significantly increased the apoptotic cell index from 1.9% to 3.2% (for control verses DMO embryos respectively) (P < 0.05). Blastocyst development was unchanged. Exposure to DMO during the first cleavage division did not alter implantation rates however fetal weight was decreased from 1058.9 mg ± 25.2 (control) to 949.1 mg ± 26.7 (DMO) (P < 0.05) and crown–rump length decreased from 21.9 mm ± 0.4 (control) to 20.6 mm ± 0.5 (DMO) (P < 0.05). In conclusion, this study demonstrates that a transient reduction in pH of only 0.15 units during early preimplantation embryo development significantly reduces resultant blastocyst viability and perturbs fetal growth, indicative of altered embryo programming. The mechanism behind this permanent change however is currently unknown.
405. Isolation and preliminary characterisation of putative sheep embryonic stem cells
Authors: Murray B. M. , O.'Brien C. M. , Johnson J. L. , Conley B. J. , Bello P. , Andronicos N. M. , Hill J. R. , Verma P. J. , Holland M.
Published: November 2010

The establishment of true, fully characterised embryonic stem (ES) cells from livestock, (eg sheep) has yet to be reported. Such cells could make a significant impact on assisted reproductive technologies, vaccine delivery, and animal health and well being in the livestock industries. To date in sheep, there is a single report of putative ES cells which were maintained in an undifferentiated state for only 2 passages. Here we report the isolation and culture of pluripotent ES-like cells from in vivo derived, vitrified, sheep blastocysts. The inner cell mass of blastocysts were isolated by immunosurgery, cultured in Stem Cell Sciences' (SCS) novel inhibitor-based media, on a feeder layer of mouse embryonic fibroblasts (MEFs), resulting in putative ovine ES cells proliferating to at least passage 5. One cell line, BMCOV002, was established out of four thaw-recovered embryos. The colonies formed compact, near homogenous, small cell, multilayered, and well defined dome shaped masses that were morphologically similar to both mouse and human ES cell colonies. A peripheral halo of filamentous differentiated cells was detected in selected colonies from passage 3 to passage 5. The putative ovine ES-like cells were passaged by mechanical excision between days 6–7, and these colonies stained positive for alkaline phosphatase at both passage 3 and passage 5. Expression levels of genes encoding the pluripotent transcription factors OCT4, SOX2, REX1 and NANOG are shown using RT PCR in cells from passage 3. An important first step in studying the properties of ovine ES-like cells is the ability shown here to isolate and culture cells. Our attention now is focussed on maintaining these cells for some months in an undifferentiated state, and on being able to successfully cryopreserve and regenerate these cell lines.
406. Regulation of insulin receptor and its substrates by follicle stimulating hormone in granulosa cells
Authors: Taneja J. , Singh R.
Published: November 2010

Insulin receptor signalling receptor regulates the process of follicular development and maturation, however the regulation of insulin receptor (IR) and its signalling component during folliculogenesis is not well understood. This study demonstrates FSH mediated regulation of insulin receptor isoforms specially IR-B, its substrates and some of the insulin related responses like glycogen synthesis and cell proliferation. There was significant increase in mRNAs expression for insulin receptor (IR) isoforms, IR-A and IR-B in ovary in response to PMSG and in granulosa cells (GCs) in response to FSH. IR protein increased in GCs in response to FSH in a dose and time dependent manner. Interestingly, the expression of mRNAs and proteins for IRS-1 and IRS-2 increased significantly in GCs by FSH. Serine phosphorylation (Ser 636/639) of IRS-1 was decreased by FSH, thus facilitating IRS-1 activation. FSH stimulated glycogen synthesis in a dose dependent manner both by PI 3 kinase dependent and independent pathways. Insulin regulated the amount of FSH stimulated glycogen synthesis by granulosa cells. In contrast, FSH and insulin synergistically stimulated glycogen synthesis GC proliferation which was completely inhibited by LY294002. Knockdown of IRS-1 mRNA by siRNA inhibited FSH stimulated GC proliferation indicating an important role of IRS-1 downstream of FSH. Further research is required to delineate the signalling components involving IRS-1 and IRS-2 in response to FSH and thus involved in the cross-talk between FSH and insulin in GCs. Results thus demonstrate that pituitary FSH regulates insulin receptor and its substrates in rat GCs which might be important for follicular growth and oocyte development.
407. Effects of vitrification and slow-cooling on the structure of lamb and mouse ovarian tissues
Authors: Wang X. , Catt S. L. , Pangestu M. , Temple-Smith P. D.
Published: November 2010

Follicular dynamics of ovarian tissue varies with species and age. The morphology and distribution of primordial, primary and secondary follicles were examined using mouse and lamb ovarian tissue before and after cryopreservation. Ovarian cortical tissue was processed from mouse (C57BL6J × CBA) and abattoir-sourced lamb ovaries and sliced into fragments (1 mm3). The fragments were randomly divided into three experimental groups. For vitrification, the tissue fragments were equilibrated (10% v/v ethylene glycol (EG) and DMSO, 20 min) and transferred to vitrification solution (17% v/v e.g. and DMSO and 0.75M sucrose, 3 min), and loaded onto a Fibreplug (CVM kit, Cryologic). For slow-cooling (SC), up to 10 ovarian fragments were placed in slow cooling solution (10% v/v DMSO and 0.1M sucrose, 5 min, RT), and then loaded in straws before placed in a programmable freezer (Cryologic CL8800i). For histological comparison, fresh and cryopreserved-thawed ovarian tissues were fixed, embedded and sectioned (5µm) and stained with haematoxylin and eosin. Primordial, primary and secondary follicles were evaluated and the normality of follicular structures was scored. Differences between treatment groups were examined using a Chi Square test. In lamb, 83% of follicles assessed were primordial, 16.4% primary and 0.6% secondary whereas in mouse the ratios were 60%, 27% and 13% respectively. In lamb, proportion of follicles with normal morphology after thawing for vitrification (77.2%) and SC (79.3%) was not significantly different from fresh controls (83.1%). Similarly, in mouse, the proportion of normal follicles after warming for vitrification (92.3%) and SC (90.5%) was not significantly different from fresh controls (91.4%). No obvious detrimental effects on morphology of follicles derived from either vitrification or slow-cooling of lamb and mouse ovarian tissue was observed.
408. Liver gene expression in suckled postpartum beef cows maintained on moderate and improved subtropical pasture
Authors: Samadi F. , Nadzir H. M. , Phillips N. J. , McGowan M. M. , Lehnert SA , Loor J. J. , Bionaz M. , D.'Occhio M. J.
Published: November 2010

The liver is the first organ to receive absorbed nutrients from the splanchnic vasculature1 which places the liver at an important juncture between nutrition, metabolic homeostasis and reproductive function. The best described component of this interface is secretion of IGF-1 by the liver and dependence of ovarian follicular maturation on hepatic-derived IGF-12. The aim in the present study was to ascertain the expression of hepatic genes associated with metabolic regulation in postpartum beef cows maintained on moderate or improved pasture, and with contrasting liveweight and body condition. Multiparous Droughtmaster cows (Bos taurus × Bos indicus) were placed on moderate (MP, n = 7) or improved (IP, n = 7) pasture at 6–7 months of gestation and remained on the treatments after calving. Liver biopsies were taken 6 weeks after calving and gene expression was determined by quantitative PCR using SYBR Green3. Week 6 represents attainment of the plateau in nutrient demand for lactation. Data were analysed by ANOVA using SAS STAT. One week after calving, IP cows had greater (P < 0.01) liveweight and body condition score (BCS) than MP cows (585 ± 12 kg and 528 ± 21 kg; 3.7 ± 0.2 BCS and 2.3 ± 0.2 BCS). IP cows had greater (P < 0.05) relative expression of hepatic genes associated with cholesterol biosynthesis (sterol regulatory element binding factor, SREBF2; peroxisome proliferators-activated receptor delta, PPARD), fatty acid biosynthesis (carnitine palmitoyl-transferase, CPT1A; acyl-CoA oxidase, ACOX1; fatty acid desaturase 1, FADS1; sterol regulatory element binding factor 1, SREBF1) and insulin signalling (hepatic insulin receptor substrate 1, IRS1). IP cows resumed cyclic ovarian function between 11–16 weeks postpartum and one MP cow had cycled by 16 weeks. The differences in expression of metabolic genes between MP and IP cows may have partly contributed to differences in reproductive function postpartum.
(1) Drackley J et al. 2006 Journal of Dairy Science 89:1324–36
(2) Lucy MC 2000 Journal of Dairy Science 83:1635–47
(3) Loor JJ et al. 2006 Physiological Genomics 27:29–41
409. Differentially expressed genes in endometroid endometrial carcinoma using microarray: a preliminary report
Authors: Mokhtar N. M. , Saghir F. SA , Dali A. Mohd , Rose I. Mohamed , Jamal A. A.
Published: November 2010

Background: Endometrial cancer is the commonest cancer among women in the western countries. It ranks the fourth in gynaecological cancers incidence in Malaysia. Endometriod carcinoma is the commonest subtype of endometrial cancer. The aetiology of this disease is still not fully understood. Previous theories have suggested the disease is caused by the activation of oncogenes and inactivation of the tumour suppressor genes. The aim of this study is to identify expressing genes involved in the endometrial carcinoma compared with the normal endometrium. Materials and Methods: All endometrial tissues were obtained from patients that undergo total hysterectomy. The pathologist confirms the histolopathological examination. Total RNA was isolated and confirmed using the bioanalyzer 2100. Gene list were profiled using Affymetrix Human Genome Gene Chip 1.0 ST array. The results were analysed using GeneSpring 9.0 GX software. Results: The software analysis showed 237 differentially expressed genes (2-fold change) between normal and tumour. Among of these genes, 28 were found to be upregulated and 209 were downregulated in tumour compared with the normal (P < 0.01). There are several genes that are differentially expressed between endometrial carcinoma and normal tissues and found to be associated with the transforming growth factor-β (TGF-β) pathway. A mong the genes include CD44, CAV1 and TGFβR3. Conclusion: This preliminary finding has suggested the molecular difference of endometrial carcinoma and normal endometrium. These transcripts may be participated in the pathogenesis of this disease.
410. Gene expression analysis in endometriotic lesions using laser capture microdissection
Authors: Fu L. , Girling J. E. , Rogers P. A. W.
Published: November 2010

Previous studies examining gene expression profiles in normal endometrium and endometriotic lesions have used RNA extracted from whole tissue samples. Results from these studies can be difficult to interpret as they reflect expression averaged across several different cell types that may be functionally quite different. The aim of this study was to establish laser capture microdissection (LCM) as a technique to examine gene expression in stromal and epithelial cells from normal and ectopic endometrium. We hypothesised that genes associated with inflammation would be elevated in cells from endometriotic lesions. Full thickness uterine samples were collected during abdominal hysterectomy from normal cycling premenopausal women. Endometriotic lesions were collected during abdominal laparoscopy. Samples were either frozen in OCT or stored in RNAlater for 12 h before freezing. Tissues were immunostained with an antibody against CD10 to identify ectopic endometrial stromal cells before LCM. Endometrial epithelial and stromal cells were collected using the PALM MicroLaser System. RNA quality was accessed using Experion. TGFβ1, MMP1, αSMA, SMAD2 and NFκB mRNA was analysed using real-time RT–PCR. Of the endometriotic samples stored in OCT (n = 58), only 14% (n = 8) had visible endometrial glands. Of these, only 37% (n = 3) had RNA of an acceptable quality for further analysis. However, RNA quality and quantity were dramatically improved in 3 of 5 samples collected in RNAlater. In preliminary studies, expression of TGFβ1 and αSMA mRNA was elevated in endometriotic lesions in comparison to the normal endometrium, whereas NFκB expression did not change. We have shown that RNAlater solution is useful to preserve RNA quality for small clinical endometriotic samples and that immuno-guided LCM-generated homogenous cell populations coupled with real-time RT–PCR can provide valuable insights into cell and disease-specific gene expression in endometriotic lesions.
411. Macrophage abundance and phenotype in the mammary gland fluctuates throughout the estrous cycle
Authors: Chua A. C. L. , Robertson SA , Ingman W. V.
Published: November 2010

Macrophages are versatile cells with roles in the generation and execution of immune responses, tissue breakdown and remodelling and production of regulatory cytokines. These cells are implicated in development of reproductive tract tissues that mature after birth. Descriptive and knockout studies indicates that macrophages play multiple roles in epithelial cell proliferation, phagocytosis and tissue remodelling during pubertal mammary gland development, pregnancy, lactation, and involution. However, the role of macrophages in remodelling over the course of the ovarian cycle has not been previously investigated. Oestrus cycles were monitored in C57/Bl6 mice by daily vaginal smears, and mammary glands were analysed for the abundance and location of macrophages in all four stages of the cycle (n = 6–8 per group). Absolute number of macrophages was quantified by FACs analysis of digested mammary gland tissue stained with macrophage-specific antibody F4/80. The location of macrophages was investigated in fixed, paraffin embedded tissue by immunohistochemistry using F4/80 antibody. H&E staining revealed that the most differentiated epithelial ducts and alveoli structures appeared in diestrus and proestrus. Whilst there was no difference in the absolute number of macrophages at any stage, the location of these macrophages varied. Macrophages accumulated around the more highly differentiated epithelial ducts, leading to significantly more macrophages around the ducts during proestrus compared with metestrus (P < 0.05). These macrophages were in close contact with the epithelium, and displayed long projections and widespread cytoplasm. In contrast, macrophages on the edge of the gland within the collagen-rich connective tissue were characteristically round in shape. These results indicate that macrophages are present in the adult virgin mammary gland, and fluctuate in location and phenotype over the course of the oestrous cycle. Further analysis of the phenotypes of macrophages present in these different locations is required for a more comprehensive understanding of the role of macrophages in mammary tissue homeostasis.
412. The role of macrophages in regulating uterine epithelial cell proliferation
Authors: Care A. S. , Ingman W. V. , Jasper M. J. , Robertson SA
Published: November 2010

During the oestrous cycle, uterine epithelial cells respond to ovarian steroid hormones by producing an array of cytokines and chemokines that cause macrophage recruitment into the uterus and regulate macrophage activation phenotype. In turn, growth factors and cytokines synthesised by macrophages potentially impact epithelial cell proliferation, secretory function and receptivity to embryo attachment. To investigate the hypothesis that uterine macrophages are essential contributors to the proliferation of uterine epithelial cells, we have used an ovariectomy and steroid replacement model in CD11b-DTR ‘Mac-terminator' mice. These mice are engineered for CD11b promoter-driven expression of the monkey diphtheria toxin (DT) receptor, allowing acute systemic ablation of macrophages by administration of human diphtheria toxin (DT). CD11b-DTR mice were ovariectomised, then 2–4 weeks later were primed with E 2, followed by administration of DT (25 ng/g, ip) to effect macrophage depletion, and BrDU to label proliferating cells. Control mice were given PBS instead of DT. Uterine tissues were stained with F4/80 to detect macrophages, and anti-BrDU to detect BrDU+ epithelial cell nuclei. DT treatment was associated with a depletion of >90% of F4/80+ uterine macrophages. However, the numbers of BrDU+ epithelial cells and the architecture of the luminal epithelial surface and abundance of epithelial glands were similar in control and DT-treated uterine tissues. These data suggest that resident macrophages may not be essential for oestrogen-driven uterine epithelial cell proliferation. In ongoing experiments we are assessing the effect of macrophage depletion on epithelial cell expression of functional markers including those involved in regulation of embryo attachment.
413. Genetic diversity of the major histocompatibility complex and response to immunocontraceptives in the brushtail possum
Authors: Holland O. J. , Cowan P. E. , Gleeson D. M. , Chamley L. W.
Published: November 2010

The brushtail possum (Trichosurus vulpecula) is a major invasive pest in New Zealand. One option for its control is the use of an immunocontraceptive vaccine, a method of fertility control that employs the immune system to attack reproductive cells or proteins. Initial trials of immunocontraceptive vaccines have shown individual variation in immune response. Concerns have been raised that the use of such a vaccine on wild populations could result in selection for possums that remain fertile because of low or no response, and subsequently negate the efficacy of the vaccine. Therefore, it is important to establish the basis of this variation. The major histocompatibility complex (MHC) is an important component of the immune system which influences the nature of immune responses. This study aimed to document genetic variation in MHC loci of New Zealand possums, and investigate whether there was a relationship between MHC haplotypes and individual immune responses to immunocontraceptive vaccines. We used known marsupial (possum, red-necked wallaby, tammar wallaby, opossum) MHC sequences to design PCR primers for possum MHC loci. The variability of these loci was screened in populations of possums from locations throughout New Zealand, and between individuals with known responses to immunocontraceptive vaccines. We identified 71 novel class I and class II MHC alleles and observed significant variation in allele identity and frequency between geographically separate possum populations in New Zealand. Comparisons of MHC haplotype and immunocontraceptive vaccine response showed that some of these alleles differ between high-responding and low-responding possums. The considerable variation we have found in the possum MHC, combined with differences in the occurrence of MHC genes at locations around New Zealand, provide a potential basis for the observed variability of individual possum's responses to immunocontraceptive vaccines.
414. The contraceptive potential of a long-acting IL-11 inhibitor
Authors: Menkhorst E. , Salamonsen L. , Robb L. , Dimitriadis E.
Published: November 2010

Interleukin 11 (IL-11) signalling is essential for the establishment of pregnancy in mice, through its action on the differentiation of uterine endometrial stromal cells (decidualisation), a critical process during embryo implantation. IL-11Rα deficient mice are infertile due to defective decidualisation1. IL-11 expression peaks between days (D) 4.5–9.5 of pregnancy (D0: day of plug) in mouse decidua. We examined the effect of administering (intraperitoneal [IP] injection or vaginal gel) a PEGylated IL-11 antagonist (PEGIL-11A) on decidualisation and pregnancy outcome in mice. The sera half-life of PEGIL-11A (IC50 2.8nM) following IP injection was 24h, compared with <1 h for the non-PEGylated antagonist (IC50 0.26nM). Following IP injection, PEGIL-11A localised to decidual cells and blocked the IL-11 decidual target protein, cyclin D3. IP injection of 600µg/application PEGIL-11A (or PEG control) at 1000 h and 1600 h on D3 and 1000 h on D4 (n = 4/group), resulted in smaller implantation sites than controls on D6 due to retarded mesometrial decidual formation. On D10, severe decidual destruction was visible: implantation sites contained regions of haemorrhage and the uterine luminal epithelium had reformed, suggesting a return to oestrous cycling. Following vaginal application in aqueous placebo gel, PEGIL-11A localised to decidual cells. Vaginal application of 200µg/application PEGIL-11A (or control) twice daily from D2 to D5 (n = 4/group), resulted in smaller implantation sites than controls on D6 due to partial inhibition of mesometrial decidual formation. This study demonstrates that PEGIL-11A blocked IL-11 action in the uterus, resulting in total pregnancy loss, equivalent to the IL-11Rα deficient mouse. In women, IL-11 and its receptor are produced by the uterine luminal and glandular epithelium during the period of uterine receptivity2, suggesting that IL-11 may act during initial blastocyst attachment to the luminal epithelium as well as stromal decidualisation. This study provides proof-of-principle for the development of a novel, non-hormonal contraceptive for women.
(1) Robb L et al. Nature Medicine 1998; 4: 303–308.
(2) Dimitriadis E et al. Molecular Human Reproduction 2000; 6: 907–914.
415. Increased expression of an androgen receptor regulated gene, kit ligand, in polycystic ovaries
Authors: Hickey T. , Tuck A. , Butler M. , Jindal S. , Dodd T. , Norman R. , Tilley W.
Published: November 2010

Polycystic ovaries (PCO) are induced by pathological or pharmacological female androgen excess, but the role of the androgen receptor (AR) in the pathogenesis of PCO is unknown. We therefore tested the hypothesis that PCO have increased expression of AR or kit ligand (KITL), a cytokine that was recently identified as a candidate AR-regulated gene in the ovary (1). Immunohistochemical analysis of AR and KITL expression was performed on archival paraffin-embedded sections of 8 morphologically normal and 8 polycystic ovaries from women under the age of 40 years. Stained sections were scanned with a NanoZoomer Digital Pathology System and immunoreactivity was qualitatively assessed using a 0-3+ scale, where 3+ represents the most intense staining. Electronic images of follicles at different stages of folliculogenesis were assessed by two independent observers who were blinded to the morphology of the source ovary. Each individual ovary contributed a minimum of 1 follicle per size class and a minimum of 10 follicles per size class were analysed. AR immunoreactivity was present in granulosa cells at all stages of folliculogenesis, in thecal cells of large antral follicles, and in the ovarian stroma. Staining intensity for AR did not differ between normal and polycystic ovaries. KITL expression, summarised in Table 1, was found to be significantly elevated in the oocytes of primordial and primary follicles and in the granulosa cells of follicles at all stages of folliculogenesis. These results show that AR expression is normal in PCO but expression of an AR-regulated gene is increased, potentially due to an excess of androgen hormone that is characteristic of women with PCO. Based on the roles of KITL established by murine studies, increased expression of KITL could explain many of the features of PCO including follicle excess, hyperthecosis and abnormal androgen secretion.
416. A xenotransplantation model for investigating the role of human endometrial stem/progenitor cells in endometriosis
Authors: Gargett C. E. , Kaitu'u-Lino T. , Chan R. W. S.
Published: November 2010

Endometriosis is a major cause of infertility in women. Recent evidence suggests that stem/progenitor cells are present in human endometrium which may be responsible for its remarkable regenerative capacity.1 In mouse endometrium, candidate epithelial and stromal stem/progenitor cells have been identified as label retaining cells (LRC).2 We hypothesised that endometrial stem/progenitor cells gain access to the peritoneal cavity in menstrual debris where they establish ectopic endometriosis lesions in women who develop endometriosis3. Our aim was to identify LRC in human endometrial tissues transplanted into an ectopic site in immunocompromised mice. Endometrium was dissected into pieces (1x1 mm × depth of endometrium) from hysterectomy tissues from ovulating women (n = 7) and transplanted beneath the kidney capsule of ovariectomised NOD/SCID mice. One week later, mice were administered a single 100 ng oestradiol valerate (E2) injection and 50 ug/g BrdU (6 ip injections over 3 days) to label proliferating endometrial cells with BrdU. E2 injections were given fortnightly to induce weekly cycles of endometrial growth and regression and to chase out the BrdU. Mice were sacrificed after 6–12 weeks chase and the explants examined for LRC and other markers by immunofluorescence. Endometrial explants underwent major remodelling during the BrdU pulse-chase, and cells in both glands and stroma underwent proliferation (PCNA+). Rare LRC were identified in the human endometrial epithelium and stroma. Epithelial LRC were ER-α negative while some stromal LRC were ER-α+ by confocal microscopy. Immunostaining of the LRC for ER-β, α-smooth muscle actin and human endometrial stromal stem/progenitor cell markers (CD146, PDGFR-β)4 will further characterise these candidate stem/progenitor cell populations in ectopic endometrium. This preliminary study suggests that combining the LRC technique with xenotransplantation of human endometrial tissue into a well vascularised ectopic site may provide a novel model for investigating the role of endometrial epithelial and stromal stem/progenitor cells in the pathogenesis of endometriosis.
(1) Chan RWS et al. (2004). Biology of Reproduction. 70:1738–1750
(2) Chan RWS, Gargett CE (2006). Stem Cells 24:1529–1538
(3) Gargett CE (2007) Human Reproduction Update 13:87–101
(4) Schwab KE, Gargett CE (2007) Human Reproduction 22:2903–2911
418. Proteomic analysis of proliferative and secretory phase endometrium
Authors: Chen J. I. C. , Salamonsen L. A. , Hannan N. J. , Stanton P. J. , Nicholls P. , Zhang J. , Robertson D. M. , Stephens A. N.
Published: November 2010

The human endometrium undergoes marked proliferation and differentiation between the proliferative and secretory phases of the menstrual cycle; however the underlying biology is poorly understood. This aims of this study were to identify proteins differentially expressed in the human endometrium between the proliferative and secretory phases of normal menstrual cycles. 2D SDS–PAGE analysis with differential fluorescent Cydye labelling was conducted across the pH range 4–7 on endometrial tissue extracts from mid-proliferative and mid-secretory phases (n = 4/group). Profiles were quantitatively assessed using SameSpots image analysis software. Differentially expressed proteins were identified using MALDI-TOF MS and were used to generate biological network by Ingenuity Pathways Analysis. Protein expression changes for three of the proteins were validated by immunohistochemistry. Of 1017 protein spots detected in the proteome of endometrial tissue, 196 were significantly differentially expressed (P < 0.05) between the proliferative and secretory phases. 157 proteins increased in expression, whilst 39 were decreased compared with the proliferative endometrium. Mass spectrometry identified 76 proteins representing 42 unique gene products. The identity and expression change for 3 proteins (Rho-GD1α, CLIC1, PGRMC-1) was confirmed using immunohistochemical staining of tissue section. Using pathway profiling software, the identified proteins were broadly grouped into seven major functional categories (cell architecture, transcription regulation, transport, membranes, enzymes and regulatory proteins), with the majority showing clear dependence on the Jnk signalling pathway. Comparison of the proteomic data with published mRNA expression array data clearly demonstrated discrepancies between the protein changes and gene expression, suggesting that a large majority of proteomic changes occurring at the transition of proliferative to secretory phase were due to post-translational modifications. These studies enable the understanding of the complex dynamics of protein expression and the possible involvement of post-translational modifications in cyclic changes of human endometrium.