60 research outputs found
The Expression of Prolactin and its Cathepsin D-mediated Cleavage in the bovine Corpus luteum vary with the Oestrous cycle
No full textCopyright © 2007 by the American Physiological Society.In the corpus luteum (CL), blood vessels develop, stabilize, and regress. This depends on the ratio of pro- and anti-angiogenic factors, which change during the ovarian cycle. The present study focuses on the possible roles of 23-K prolactin (PRL) in the bovine CL and its anti-angiogenic N-terminal fragments after extracellular cleavage by cathepsin D (Cath D). Prolactin RNA and protein were demonstrated in the CL tissue, in luteal endothelial cells and steroidogenic cells. Cath D was detected in CL tissue, cell extracts and corresponding cell supernatants. In the intact CL, 23-K PRL levels decreased gradually, whereas Cath D levels concomitantly increased between early to late luteal stages. In vitro, PRL cleavage occurred in the presence of acidified homogenates of CL tissue, cells and corresponding cell supernatants. Similar fragments were obtained with purified Cath D, and their appearance was inhibited by pepstatin A. The aspartic protease specific substrate MOCAc-GKPILF~FRLK(Dnp)-D-R-NH2 was cleaved by CL cell supernatants, providing further evidence for Cath D activity. 16-K PRL inhibited proliferation of luteal endothelial cells accompanied by an increase in cleaved caspase-3. In conclusion: (1) The bovine CL is able to produce PRL and to process it into anti-angiogenic fragments by Cath D activity. (2) PRL cleavage might mediate angioregression during luteolysis.Sabine Erdmann, Albert Markus Ricken, Claudia Merkwitz, Ingrid Struman, Castino Roberta, Katja Hummitzsch, Frank Gaunitz, Ciro Isidoro, Joseph Martial and Katharina Spanel-Borowsk
Pancreatic islet basement membrane loss and remodeling after mouse islet isolation and transplantation: impact for allograft rejection
No full textThe isolation of islets by collagenase digestion can cause damage and impact the efficiency of islet engraftment and function. In this study, we assessed the basement membranes (BMs) of mouse pancreatic islets as a molecular biomarker for islet integrity, damage after isolation, and islet repair in vitro as well as in the absence or presence of an immune response after transplantation. Immunofluorescence staining of BM matrix proteins and the endothelial cell marker platelet endothelial cell adhesion molecule-1 (PECAM-1) was performed on pancreatic islets in situ, isolated islets, islets cultured for 4 days, and islet grafts at 3-10 days posttransplantation. Flow cytometry was used to investigate the expression of BM matrix proteins in isolated islet β-cells. The islet BM, consisting of collagen type IV and components of Engelbreth-Holm-Swarm (EHS) tumor laminin 111, laminin α2, nidogen-2, and perlecan in pancreatic islets in situ, was completely lost during islet isolation. It was not reestablished during culture for 4 days. Peri- and intraislet BM restoration was identified after islet isotransplantation and coincided with the migration pattern of PECAM-1+vascular endothelial cells (VECs). After islet allotransplantation, the restoration of VEC-derived peri-islet BMs was initiated but did not lead to the formation of the intraislet vasculature. Instead, an abnormally enlarged peri-islet vasculature developed, coinciding with islet allograft rejection. The islet BM is a sensitive biomarker of islet damage resulting from enzymatic isolation and of islet repair after transplantation. After transplantation, remodeling of both peri- and intraislet BMs restores β-cell-matrix attachment, a recognized requirement for β-cell survival, for isografts but not for allografts. Preventing isolation-induced islet BM damage would be expected to preserve the intrinsic barrier function of islet BMs, thereby influencing both the effector mechanisms required for allograft rejection and the antirejection strategies needed for allograft survival.H. F. Irving-Rodgers, F. J. Choong, K. Hummitzsch, C. R. Parish, R. J. Rodgers and C. J. Simeonovi
Quantitative elemental analysis of bovine ovarian follicles using X-ray fluorescence imaging
No full textThe X-ray Fluorescence Micro-spectroscopy (XFM) beamline at the Australian Synchrotron was used to image 97 follicle histological sections from 45 different bovine ovaries focusing on healthy antral follicles ranging from small (16 mm) and on antral follicles undergoing atresia. This analysis identified five elements (Cu, Fe, Zn, Se and Br) consistently present within the ovarian tissue with Fe, Zn and Se localised to specific structures. GeoPIXE v6.4g was subsequently used to extract quantitative information pertaining to the elemental concentrations surrounding each of these follicles. Statistical analysis suggested that significant elemental differences were evident between follicle groups sorted according to their health status (Fe and Br), and their size (Se). Se appeared to be the element which most greatly distinguished large antral follicles from smaller counterparts. The ability to use synchrotron radiation to measure trace element distributions in bovine follicles at such high resolutions could have a significant impact on understanding the mechanisms of follicular development. This research is intended to form a baseline study of healthy cycling ovaries which could later be extended to disease states, thereby improving our current understanding of infertility and endocrine diseases involving the ovary.M. J. Ceko, K. Hummitzsch, N. Hatzirodos, R. J. Rodgers and H. H. Harri
Versican induces a pro-metastatic ovarian cancer cell behavior which can be inhibited by small hyaluronan oligosaccharides
No full textThe assembly of pericellular matrix containing hyaluronan (HA) and versican has been shown to be a pre-requisite for proliferation and migration of mesenchymal cells. In this study, we investigated whether treatment with recombinant versican could induce the formation of a pericellular matrix by ovarian cancer cells (OVCAR-3, OVCAR-5, and SKOV-3) and promote their motility, invasion, and adhesion to peritoneal cells in vitro. We also determined whether versican-induced pericellular matrix formation and metastatic cancer cell behavior could be blocked by small HA oligosaccharides. Only combined treatment with recombinant versican and HA resulted in pericellular matrix formation by OVCAR-5 and SKOV-3 but not by OVCAR-3 cells, which lack the HA receptor, CD44. The motility of OVCAR-5 and SKOV-3 cells was significantly increased in scratch wound and chemotaxis assays following treatment with recombinant versican and HA. Versican and HA also promoted invasion of SKOV-3 and OVCAR-5 cells but had no effect on OVCAR-3 cells. We have demonstrated that exogenous HA significantly increased OVCAR-5 and SKOV-3 adhesion to peritoneal cells but adhesion was not further increased by versican treatment. Small HA oligomers (6-10 disaccharides) were able to significantly block formation of pericellular matrix by OVCAR-5 cells, as well as the increased motility and invasion induced by recombinant versican. HA oligomers also significantly blocked OVCAR-5 adhesion to peritoneal cells both in the presence and absence of exogenous HA. The dependence of CD44 for the versican and HA mediated effects were demonstrated by the inhibition of pericellular matrix formation as well as motility and invasion of OVCAR-5 cells following treatment with CD44 neutralizing antibody in the presence of versican and HA. We conclude that the acquisition of a HA/versican pericellular matrix by ovarian cancer cells increases their metastatic potential. HA oligomers can block this mechanism and are promising inhibitors of ovarian cancer dissemination.Miranda P. Ween, Katja Hummitzsch, Raymond J. Rodgers, Martin K. Oehler, Carmela Ricciardell
Trace elements in ovaries: measurement and physiology
No full textTraditionally, research in the field of trace element biology and human and animal health has largely depended on epidemiological methods to demonstrate involvement in biological processes. These studies were typically followed by trace element supplementation trials or attempts at identification of the biochemical pathways involved. With the discovery of biological molecules that contain the trace elements, such as matrix metalloproteinases containing zinc (Zn), cytochrome P450 enzymes containing iron (Fe), and selenoproteins containing selenium (Se), much of the current research focuses on these molecules, and, hence, only indirectly on trace elements themselves. This review focuses largely on two synchrotron-based x-ray techniques: X-ray absorption spectroscopy and x-ray fluorescence imaging that can be used to identify the in situ speciation and distribution of trace elements in tissues, using our recent studies of bovine ovaries, where the distribution of Fe, Se, Zn, and bromine were determined. It also discusses the value of other techniques, such as inductively coupled plasma mass spectrometry, used to garner information about the concentrations and elemental state of the trace elements. These applications to measure trace elemental distributions in bovine ovaries at high resolutions provide new insights into possible roles for trace elements in the ovary.Melanie J. Ceko, Sean O'Leary, Hugh H. Harris, Katja Hummitzsch, and Raymond J. Rodger
X-Ray fluorescence imaging and other analyses identify selenium and GPX1 as important in female reproductive function
No full textCorrected by: Erratum: X-Ray fluorescence imaging and other analyses identify selenium and GPX1 as important in female reproductive function, in Vol. 7, Issue 1, 188. The author H. H. Harris is listed with affiliation ‘c’, however, he should be listed with affiliation ‘a’; i.e. ‘The University of Adelaide’. The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.Studies of selenium (Se) status indicate that Se is necessary for fertility but how precisely is not known. We aimed to show that Se was important in bovine female reproductive function. The elemental distribution in the bovine ovary (n = 45 sections) was identified by X-ray fluorescence (XRF) imaging. Se was consistently localized to the granulosa cell layer of large (>10 mm) healthy follicles. Inductively Coupled Plasma - Mass Spectrometry revealed tenfold higher Se in the bovine follicle wall compared to corpora lutea. Gene expression analysis of selenoprotein genes GPX1, GPX3, VIMP and SELM in bovine granulosa cells revealed that only GPX1 was significantly up-regulated in large healthy follicles compared to the small healthy or atretic follicles (P < 0.05). Western immunoblotting identified GPX1 protein in bovine granulosa cells of large healthy follicles, but not of small healthy follicles. To assess if GPX1 was important in human follicles, cumulus cells from women undergoing IVF/ICSI with single embryo transfer were collected. Oocytes and embryos were cultured and transferred independently in 30 patients undergoing elective single embryo transfer. Gene expression of GPX1 was significantly higher in human cumulus cells from cumulus-oocyte complexes yielding a pregnancy (P < 0.05). We present the first XRF imaging of mammalian ovaries showing that Se is consistently localized to the granulosa cells of large healthy follicles. We conclude that Se and selenoproteins are elevated in large healthy follicles and may play a critical role as an antioxidant during late follicular development.M. J. Ceko, K. Hummitzsch, N. Hatzirodos, W. M. Bonner, J. B. Aitken, D. L. Russell, M. Lane, R. J. Rodgers and H. H. Harri
Maternal periconceptional and first trimester protein restriction in beef heifers: effects on placental parameters and fetal and neonatal calf development
No full textData source: Supplementary material, https://doi.org/10.1071/RD19017Few studies have investigated the effects of nutrition during the periconception and early gestation periods on fetal and placental development in cattle. In this study, nulliparous yearling heifers (n = 360) were individually fed a diet high or low in protein (HPeri and LPeri) beginning 60 days before conception. From 24 to 98 days after conception, half of each treatment group was changed to the alternative high- or low-protein diet (HPost and LPost) yielding four groups in a 2 × 2 factorial design. A subset of heifers (n = 46) was necropsied at 98 days after conception and fetoplacental development assessed. Placentome number and volume decreased in response to LPeri and LPost diets respectively. Absolute lung, pancreas, septum and ventricle weights decreased in LPost versus HPost fetuses, whereas the post-conception diet altered absolute and relative liver and brain weights depending on sex. Similarly, changes in fetal hepatic gene expression of factors regulating growth, glucose output and lipid metabolism were induced by protein restriction in a sex-specific manner. At term, neonatal calf and placental measures were not different. Protein restriction of heifers during the periconception and early gestation periods alters fetoplacental development and hepatic gene expression. These changes may contribute to functional consequences for progeny, but this may not be apparent from gross morphometry at birth.K. J. Copping, K. Hummitzsch, I. C. McMillen, J. L. Morrison, R. J. Rodgers and V. E. A. Perry ... et al
Localization of the trace elements iron, zinc and selenium in relation to anatomical structures in bovine ovaries by X-ray fluorescence imaging
No full textX-ray fluorescence (XRF) was used to image 40 histological cross-sections of bovine ovaries (n=19), focusing on structures including: antral follicles at different stages of growth or atresia, corpora lutea at three stages of development (II-IV), and capillaries, arterioles, and other blood vessels. This method identified three key trace elements [iron (Fe), zinc (Zn), and selenium (Se)] within the ovarian tissue which appeared to be localized to specific structures. Owing to minimal preprocessing of the ovaries, important high-resolution information regarding the spatial distribution of these elements was obtained with elemental trends and colocalizations of Fe and Zn apparent, as well as the infrequent appearance of Se surrounding the antrum of large follicles, as previously reported. The ability to use synchrotron radiation to measure trace element distributions in bovine ovaries at such high resolution and over such large areas could have a significant impact on understanding the mechanisms of ovarian development. This research is intended to form a baseline study of healthy ovaries which can later be extended to disease states, thereby improving our current understanding of infertility and endocrine diseases involving the ovary.Melanie J. Ceko, Katja Hummitzsch, Wendy M. Bonner, Jade B. Aitken, Kathryn M. Spiers, Raymond J. Rodgers and Hugh H. Harri
The short prolactin receptor predominates in endothelial cells of micro- and macrovascular origin
No full textCopyright © 2007 S. Karger AG, BaselBackgroundControversial reports on prolactin receptors (PRL-R), the long and short form, on endothelial cells (EC) may be explained by the choice of EC derived from the micro- and macrovascular bed of either endocrine and non-endocrine organs.MethodsWe studied here PRL-R expression in organs [bovine corpus luteum (CL), umbilical vein, aorta] and in organ-derived EC cultures.ResultsIn the intact CL, both PRL-R forms were present at mRNA and protein level throughout the oestrous cycle stages. The short form prevailed as protein. PRL-R-positive EC were noted by immunofluorescent staining in arterial blood vessels of CL septa, in the umbilical vein and the aorta. In EC cultures of micro- and macrovascular origin, transcripts of both PRL-R forms were shown; again the short-form protein prevailed. Blocking experiments with anti-prolactin (PRL) antibody led to a 60% decrease in cell growth. Treatment with PRL had no effect.ConclusionPRL-R expression in micro- and macrovascular EC is associated with the predominant short form.Albert M. Ricken, Anja Traenkner, Claudia Merkwitz, Katja Hummitzsch, Jens Grosche and Katharina Spanel-Borowsk
Expression of PCOS candidate genes in bovine fetal and adult ovarian somatic cells
No full textPolycystic ovary syndrome (PCOS) is an endocrine metabolic disorder that appears to have a genetic predisposition and a fetal origin. The fetal ovary has two major somatic cell types shown previously to be of different cellular origins and different morphologies and to differentially express 15 genes. In this study, we isolated the somatic gonadal ridge epithelial-like (GREL) cells (n = 7) and ovarian fetal fibroblasts (n = 6) by clonal expansion. Using qRT-PCR, we compared the gene expression levels of PCOS candidate genes with previous data on the expression levels in whole fetal ovaries across gestation. We also compared these levels with those in bovine adult ovarian cells including fibroblasts (n = 4), granulosa cells (n = 5) and surface epithelial cells (n = 5). Adult cell types exhibited clear differences in the expression of most genes. In fetal ovarian cells, DENND1A and ERBB3 had significantly higher expression in GREL cells. HMGA2 and TGFB1I1 tended to have higher expression in fetal fibroblasts than GREL cells. The other 19 genes did not exhibit differences between GREL cells and fetal fibroblasts and FBN3, FSHB, LHCGR, FSHR and ZBTB16 were very lowly expressed in GREL cells and fibroblasts. The culture of fetal fibroblasts in EGF-containing medium resulted in lower expression of NEIL2 but higher expression of MAPRE1 compared to culture in the absence of EGF. Thus, the two fetal ovarian somatic cell types mostly lacked differential expression of PCOS candidate genes.Menghe Liu, Katja Hummitzsch, Nicole A Bastian, Monica D Hartanti, Helen F Irving-Rodgers, Richard A Anderson, and Raymond J Rodger
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