141 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
2019-00136R1_Production_Supplemental_Data_online_supp – Supplemental material for Formation of the Bovine Ovarian Surface Epithelium during Fetal Development
No full textSupplemental material, 2019-00136R1_Production_Supplemental_Data_online_supp for Formation of the Bovine Ovarian Surface Epithelium during Fetal Development by Monica D. Hartanti, Katja Hummitzsch, Wendy M. Bonner, Nicole A. Bastian, Helen F. Irving-Rodgers and Raymond J. Rodgers in Journal of Histochemistry & Cytochemistry</p
Dynamics of extracellular matrix in ovarian follicles and corpora lutea of mice
No full textDespite the mouse being an important laboratory species, little is known about changes in its extracellular matrix (ECM) during follicle and corpora lutea formation and regression. Follicle development was induced in mice (29 days of age/experimental day 0) by injections of pregnant mare's serum gonadotrophin on days 0 and 1 and ovulation was induced by injection of human chorionic gonadotrophin on day 2. Ovaries were collected for immunohistochemistry (n=10 per group) on days 0, 2 and 5. Another group was mated and ovaries were examined on day 11 (n=7). Collagen type IV alpha1 and alpha2, laminin alpha1, beta1 and gamma1 chains, nidogens 1 and 2 and perlecan were present in the follicular basal lamina of all developmental stages. Collagen type XVIII was only found in basal lamina of primordial, primary and some preantral follicles, whereas laminin alpha2 was only detected in some preantral and antral follicles. The focimatrix, a specialised matrix of the membrana granulosa, contained collagen type IV alpha1 and alpha2, laminin alpha1, beta1 and gamma1 chains, nidogens 1 and 2, perlecan and collagen type XVIII. In the corpora lutea, staining was restricted to capillary sub-endothelial basal laminas containing collagen type IV alpha1 and alpha2, laminin alpha1, beta1 and gamma1 chains, nidogens 1 and 2, perlecan and collagen type XVIII. Laminins alpha4 and alpha5 were not immunolocalised to any structure in the mouse ovary. The ECM composition of the mouse ovary has similarities to, but also major differences from, other species with respect to nidogens 1 and 2 and perlecan.Helen F. Irving-Rodgers, Katja Hummitzsch, Lydia S. Murdiyarso, Wendy M. Bonner, Yoshikazu Sado, Yoshifumi Ninomiya, John R. Couchman, Lydia M. Sorokin and Raymond J. Rodger
Transcriptome comparisons identify new cell markers for theca interna and granulosa cells from small and large antral ovarian follicles
No full textIn studies using isolated ovarian granulosa and thecal cells it is important to assess the degree of cross contamination. Marker genes commonly used for granulosa cells include FSHR, CYP19A1 and AMH while CYP17A1 and INSL3 are used for thecal cells. To increase the number of marker genes available we compared expression microarray data from isolated theca interna with that from granulosa cells of bovine small (n = 10 for both theca and granulosa cells; 3-5 mm) and large (n = 4 for both theca and granulosa cells, > 9 mm) antral follicles. Validation was conducted by qRT-PCR analyses. Known markers such as CYP19A1, FSHR and NR5A2 and another 11 genes (LOC404103, MGARP, GLDC, CHST8, CSN2, GPX3, SLC35G1, CA8, CLGN, FAM78A, SLC16A3) were common to the lists of the 50 most up regulated genes in granulosa cells from both follicle sizes. The expression in theca interna was more consistent than in granulosa cells between the two follicle sizes. Many genes up regulated in theca interna were common to both sizes of follicles (MGP, DCN, ASPN, ALDH1A1, COL1A2, FN1, COL3A1, OGN, APOD, COL5A2, IGF2, NID1, LHFP, ACTA2, DUSP12, ACTG2, SPARCL1, FILIP1L, EGFLAM, ADAMDEC1, HPGD, COL12A1, FBLN5, RAMP2, COL15A1, PLK2, COL6A3, LOXL1, RARRES1, FLI1, LAMA2). Many of these were stromal extracellular matrix genes. MGARP, GLDC, CHST8, GPX3 were identified as new potential markers for granulosa cells, while FBLN5, OGN, RAMP2 were significantly elevated in the theca interna.Nicholas Hatzirodos, Katja Hummitzsch, Helen F. Irving-Rodgers, Raymond J. Rodger
Transcriptome profiling of the theca interna from bovine ovarian follicles during atresia
No full textThe theca interna is a specialized stromal layer that envelops each growing ovarian follicle. It contains capillaries, fibroblasts, immune cells and the steroidogenic cells that synthesize androgens for conversion to estradiol by the neighboring granulosa cells. During reproductive life only a small number of follicles will grow to a sufficient size to ovulate, whereas the majority of follicles will undergo regression/atresia and phagocytosis by macrophages. To identify genes which are differentially regulated in the theca interna during follicular atresia, we undertook transcriptome profiling of the theca interna from healthy (n = 10) and antral atretic (n = 5) bovine follicles at early antral stages (<5 mm). Principal Component Analyses and hierarchical classification of the signal intensity plots for the arrays showed primary clustering into two groups, healthy and atretic. A total of 543 probe sets were differentially expressed between the atretic and healthy theca interna. Further analyses of these genes by Ingenuity Pathway Analysis and Gene Ontology Enrichment Analysis Toolkit software found most of the genes being expressed were related to cytokines, hormones and receptors as well as the cell cycle and DNA replication. Cell cycle genes which encode components of the replicating chromosome complex and mitotic spindle were down-regulated in atretic theca interna, whereas stress response and inflammation-related genes such as TP53, IKBKB and TGFB1 were up-regulated. In addition to cell cycle regulators, upstream regulators that were predicted to be inhibited included Retinoblastoma 1, E2 transcription factor 1, and hepatocyte growth factor. Our study suggests that during antral atresia of small follicles in the theca interna, arrest of cell cycle and DNA replication occurs rather than up- regulation of apoptosis-associated genes as occurs in granulosa cells.Nicholas Hatzirodos, Helen F. Irving-Rodgers, Katja Hummitzsch, Raymond J. Rodger
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
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
Is polycystic ovary syndrome a 20th Century phenomenon?
No full textPolycystic ovary syndrome (PCOS) affects around 10% of women of reproductive age and is most common in developed countries. The aetiology of PCOS is not completely understood. Current evidence suggests that the syndrome results from a genetic predisposition interacting with developmental events during fetal or perinatal life that together increase susceptibility in some individuals. This implies that environmental factors influence the initiation of PCOS in the fetus or infant, either directly or via the mother. PCOS is often considered to be an ancient disorder but there is no direct proof of this in the medical or historic record. One of the cardinal features, polycystic ovaries, was first described only in the early 1900s, despite reports of many thousands of autopsies recorded earlier. This conundrum could be explained by postulating that polycystic ovaries were rare before the 1900s and have become more common over the last 100 years. The hypothesis that PCOS is a syndrome of the 20th Century would eliminate the need to explain the paradox of why there exists a genetic predisposition to subfertility syndrome.Raymond J.Rodgers, Larisa Suturina, Daria Lizneva, Michael J.Davies, Katja Hummitzsch, Helen F. Irving-Rodgers, Sarah A.Robertso
Genetic and Fetal Origins of Polycystic Ovary Syndrome
No full textPolycystic ovary syndrome (PCOS) is the most common endocrine disorder that affects women of reproductive age. Although the syndrome affects over 10 percent women worldwide, its cause(s) remains unknown. Numerous studies have focussed on the genetic and fetal origins of the syndrome. However, the outcomes of genome wide association studies and candidate gene studies towards defining PCOS have, arguably, had almost no impact on the definition, diagnosis and treatment of the syndrome. This could be attributed mainly to the divergence of PCOS studies, as these studies have not collectively defined the molecular mechanisms involved in PCOS phenotypes observed. This thesis examined the role of genes in loci associated with PCOS and TGFβ signalling molecules, as well as their upstream regulators and mechanisms in the pathogenesis of PCOS. Genes in loci associated with PCOS (candidate genes) were shown to be dynamically expressed during bovine fetal ovary development. Notably, those genes expressed during the early stages of fetal development such as C8H9orf3, TOX3, FBN3, GATA4, HMGA2 and DENND1A are co-expressed with genes involved in mitochondria function and are regulated upstream by DAP3, MYC, PTEN, HNF4A, ESRRA/G, PSEN1, MYC, mitochondrial LONP1 and TP53. Those expressed in the second trimester or just after mid gestation such as YAP1, INSR, THADA and TGFB1I1 were co-expressed with genes involved in stroma expansion and are regulated upstream by TGF-β signalling molecules including TGFB1, TGFB2, TGFB3 and TGFBR2, coagulation factor II and fibroblast proliferation regulators including FGF2. Candidate genes expressed during the third trimester such as FDFT1, LHCGR, AMH, FSHR, ZBTB16 and PLGRKT are co-expressed with genes involved with folliculogenesis and steroidogenesis and are regulated upstream by SREBF2, INSIGI, TGFB1 and RPTOR among others. Thus, this study infers the possible role of these canonical pathways and upstream regulators in the pathogenesis of PCOS during fetal ovary development. The role of TGFB signalling molecules in the aetiology of PCOS was further examined. These molecules were also found to be dynamically expressed during fetal ovary development and could regulate some PCOS candidate genes in bovine fetal ovary fibroblasts. Across gestation, LTBP1/2/3/4, FBN1, TGFB2/3, TGFBR2/3 and TGFB1I1 expression levels increased, while FBN3, TGFBR3L, TGFBI and TGFB1 decreased and TGFBRAP1, TGFBR1 and FBN2 remained relatively constant. TGFβ1, but not androgens nor AMH, has previously been shown to inhibit expression of candidate genes (AR, INSR, C8H9orf3 and RAD50) but stimulate expression of androgen receptor co-factor, TGFB1I1, in cultured fetal ovarian fibroblasts. Additionally, we showed that expression of PCOS candidate genes ERBB3, NEIL2, IRF1 and ZBTB16 significantly decreased after TGFβ1 treatment in cultured fetal ovarian fibroblasts. These findings further confirm that TGFβ signalling could be involved in the pathogenesis of PCOS or at least in the establishment of polycystic ovaries. The role of candidate genes and TGFβ signalling molecules in various tissues including nonovarian tissues was assessed as PCOS does not only affect the ovary. Both were shown to be dynamically expressed in gonadal (ovary and testis), metabolic (heart, liver and kidney) and brain (brain and cerebellum) tissues during the first half of human fetal development and postnatally until adulthood. More so, some genes were significantly expressed in specific tissues at different time points pre-natally and/or post-natally. Notably, HMGA2, FBN3 and TOX3 were highly expressed during the early stages of fetal development in all tissues but least during adulthood. DENND1A, THADA, MAPRE1, RAB5B, ARL14EP, KRR1, NEIL2 and RAD50 were dynamically expressed in all postnatal tissues studied. Interestingly, correlation between expression of HMGA2/YAP1 and RAD50/YAP1 were significant in at least 5 of the 7 fetal tissues studied. Furthermore, HMGA2, YAP1 and RAD50 correlated significantly with most TGFβ signalling molecules in at least 4 tissues. We further showed that there is certainly a crosstalk within and between PCOS candidate genes and TGFβ signalling molecules during fetal development within each tissue. Considering the fact that HMGA2 and YAP1 are involved in the Hippo signalling pathway as well as epithelial mesenchymal transition (EMT) during embryogenesis through TGFβ signalling, their role in the aetiology of PCOS requires further studies. In conclusion, these findings confirm that genes in loci associated with PCOS and TGFβ signalling molecules are actively involved in the genetic and fetal origins of the syndrome and that there is a possible crosstalk between both in the development of multiple organs. This also infers that dysregulation of PCOS candidate genes and TGFβ signalling molecules during fetal development could initiate/lead to a cascade of molecular events in various tissues accounting for the various phenotypes of PCOS observed in adulthood.Thesis (Ph.D.) -- University of Adelaide, School of Biomedicine, 202
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
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