Molecular basis of oocyte-paracrine signalling that promotes granulosa cell proliferation

Copyright © 2006 Company of BiologistsOocytes regulate follicle growth by secreting paracrine growth factors that act on neighbouring granulosa cells (GCs). Those factors identified to date are mainly members of the transforming growth factor-ß (TGFß) superfamily, but little is known about which specific receptor/signalling system(s) they employ. This study was conducted to determine the requisite pathways utilised by oocytes to promote GC proliferation. We used an established oocyte-secreted mitogen bioassay, where denuded mouse oocytes are co-cultured with mural GCs. Oocytes, growth differentiation factor-9 (GDF9), TGFß1 and activin-A all promoted GC DNA synthesis, but bone-morphogenetic protein 6 (BMP6) did not. Subsequently, we tested the capacity of various TGFß superfamily receptor ectodomains (ECD) to neutralise oocyte- or specific growth factor-stimulated GC proliferation. The BMP type-II receptor (BMPR-II) ECD antagonised oocyte and GDF9 bioactivity dose-dependently, but had no or minimal effect on TGFß1 and activin-A bioactivity, demonstrating its specificity. The TGFßR-II, activinR-IIA and activinR-IIB ECDs all failed to neutralise oocyte- or GDF9-stimulated GC DNA synthesis, whereas they did antagonise the activity of their respective native ligands. An activin receptor-like kinase (ALK) 4/5/7 inhibitor, SB431542, also antagonised both oocyte and GDF9 bioactivity in a dose-dependent manner. Consistent with these findings, oocytes, GDF9 and TGFß1 all activated SMAD2/3 reporter constructs in transfected GC, and led to phosphorylation of SMAD2 proteins in treated cells. Surprisingly, oocytes did not activate the SMAD1/5/8 pathway in transfected GCs although exogenous BMP6 did. This study indicates that oocyte paracrine factors primarily utilise a similar signalling pathway first identified for GDF9 that employs an unusual combination of TGFß superfamily receptors, the BMPR-II and a SMAD2/3 stimulatory ALK (4, 5 or 7), for transmitting their mitogenic actions in GC. This cell-signalling pathway may also have relevance in the hypothalamic-pituitary axis and in germ-somatic cell interactions in the testis.Robert B. Gilchrist, Lesley J. Ritter, Samu Myllymaa, Noora Kaivo-Oja, Rebecca A. Dragovic, Theresa E. Hickey, Olli Ritvos and David G. Mottershea

Green economic development in Lao PDR : a Sustainability Window analysis of Green Growth Productivity and the Efficiency Gap

A novel 'Sustainability Window' (SuWi) approach is applied for simultaneous analysis of the pillars of sustainable development; social, environmental and economic, of Lao PDR. This new method employs a variety of indicators for a comprehensive and holistic analysis of sustainable development and green inclusive economy. The analysis is grounded in the assumption that economic development is required for social development, but that simultaneously development needs to be guarded or limited to protect the environment that underpins it. As all three dimensions of sustainable development are interlinked, a comprehensive analysis requires an analytical approach that is simultaneous. The analyses provide information on minimum levels of economic development that are needed to fulfil social sustainability criteria, in tandem with the maximum economic development that avoids breaching environmental sustainability criteria. If actual economic growth lies between these minima and maxima, we can interpret that development is more sustainable with respect to the relationships embodied by the selected social and environmental indicators. The main source of data is the database of the Sustainable Society Index (SSI) developed by the Sustainable Society Foundation (SSF). The indicators used by SSI have been chosen for the Sustainability Window analysis as they can be used to assess both 'weak' and 'strong' interpretations of sustainability. Weak sustainability is defined operationally as no increase in the environmental or carbon emissions intensity of the economy, while strong sustainability is defined as no increase in absolute emissions. Further, a novel Environmental Efficiency Gap analysis has been included in the Sustainability Window. This provides information about the necessary improvement in GDP production efficiency with respect to environmental emissions. Sustainability Window combined with Environmental Efficiency Gap analysis, provides critical knowledge for planners and decision makers. It provides strategic indications of how to aim for social and environmental sustainability through economic investment and growth targets. These new methods can be used in transdisciplinary research of sustainable development and can also assist in national and regional comparisons. In the case of Lao PDR, the analysis needs to be broadened for more fundamental understanding of the gaps and weaknesses. SuWi can be used to assess the sustainable development needed to address the Sustainable Development Goals by 2030. The SuWi does not provide direct policy recommendations as such, but helps to inform decision makers about the direction of development pathways towards these key goals. (C) 2018 The Authors. Published by Elsevier Ltd.Peer reviewe

Bone morphogenic protein receptor-II is a key receptor for transmitting the actions of oocyte-secreted factors and growth differentiation factor-9 in granulosa cells

Abstract onl

Mouse oocyte-secreted factors and gdf-9 stimulate granulosa cell proliferation via bmpr-ii and activate the smad2/3 pathway

Abstract onlyOocytes regulate follicle growth and development by secreting paracrine growth factors that act on granulosa cells (GC). We have recently determined that growth differentiation factor-9 (GDF-9) accounts for ∼50% of the total mitogenic activity of oocytes, the remaining portion is as yet uncharacterized. This study was conducted to identify the receptor/signalling system utilized by oocytes to promote GC proliferation. We used an established oocyte-secreted mitogen bioassay, where denuded oocytes are co-cultured with primed-mouse mural GC. In this system, oocytes, GDF-9, TGF-b1 and activin-A all promoted GC DNA synthesis in a dose-dependent manner, but bone-morphogenic protein-6 (BMP-6) and BMP-7 did not. The type-II receptor for GDF-9 is BMPRII and using real-time RT-PCR, cumulus cells (CC) and mural GC were found to express equivalent levels of BMPRII mRNA. We tested the capacity of the receptor ectodomain (ECD) to neutralize oocyte-stimulated mural GC proliferation. The BMPRII ECD antagonised both oocyte and GDF-9 bioactivity in a dose-dependent manner, completely abolishing activity of both mitogens at 1 ug/ml. The BMPRII ECD did not antagonize TGF-b and partially antagonized activin-A bioactivity, demonstrating its specificity. The TGFbR-II ECD, ActR-II ECD and ActR-IIB ECD all failed to neutralize oocyte- or GDF-9-stimulated GC DNA synthesis, whereas they did antagonize the activity of their respective ligands. The BMPRII ECD also completely antagonized oocyte-stimulated CC DNA synthesis. Using this oocyte-factor bioassay with mural GC transfected with Smad luciferase reporter constructs, we found that oocytes, GDF-9 and TGF-b (but not BMP-6) activated the Smad2/3 pathway. Consistent with this, oocytes and GDF-9 led to phosphorylation of GC Smad2 molecules as detected by Western blot. Conversely the Smad1/5/8 pathway was activated by BMP-6, but not by GDF-9, TGF-b nor surprisingly by oocytes. This study provides evidence that BMPRII is a key receptor for transmitting the paracrine actions of oocytes in GC. However, oocyte-secreted factors do not activate the BMP intracellular signalling pathway but rather the TGF-b/activin intracellular pathway. Thus oocytes utilize an unusual interaction of the two TGF-b superfamily receptor/signalling systems that are generally considered distinct.http://abstracts.co.allenpress.com/pweb/ssr2004/document/?ID=3851

Immunoneutralization of growth differentiation factor 9 reveals it partially accounts for mouse oocyte mitogenic activity

© 2004 by the Society for the Study of Reproduction, Inc.Paracrine factors secreted by oocytes play a pivotal role in promoting early ovarian follicle growth and in defining a morphogenic gradient in antral follicles, yet the exact identities of these oocyte factors remain unknown. This study was conducted to determine the extent to which the mitogenic activity of mouse oocytes can be attributed to growth differentiation factor 9 (GDF9). To do this, specific anti-human GDF9 monoclonal antibodies were generated. Based on epitope mapping and bioassays, a GDF9 neutralizing antibody, mAb-GDF9-53, was characterized with very low cross-reactivity with related transforming growth factor (TGF)ß superfamily members, including BMP15 (also called GDF9B). Pep-SPOT epitope mapping showed that mAb-GDF9-53 recognizes a short 4-aa sequence, and three-dimensional peptide modeling suggested that this binding motif lies at the C-terminal fingertip of mGDF9. As predicted by sequence alignments and modeling, the antibody detected recombinant GDF9, but not BMP15 in a Western blot and GDF9 protein in oocyte extract and oocyte-conditioned medium. In a mouse mural granulosa cell (MGC) bioassay, mAb-GDF9-53 completely abolished the mitogenic effects of GDF9, but had no effect on TGFß1 or activin A-stimulated MGC proliferation. An unrelated IgG at the same dose had no effect on GDF9 activity. This GDF9 neutralizing antibody was then tested in an established oocyte-secreted mitogen bioassay, where denuded oocytes cocultured with granulosa cells promote cell proliferation in a dose-dependent manner. The mAb-GDF9-53 dose dependently (0–160 µg/ml) decreased the mitogenic activity of oocytes but only by 45% at the maximum dose of mAb. Just 5 µg/ml of mAb-GDF9-53 neutralized 90% of recombinant mGDF9 mitogenic activity, but only 15% of oocyte activity. Unlike mAb-GDF9-53, a TGFß pan-specific neutralizing antibody did not affect the mitogenic capacity of the oocyte, but completely neutralized TGFß1-induced DNA synthesis. This study has characterized a specific GDF9 neutralizing antibody. Our data provide the first direct evidence that the endogenous GDF9 protein is an important oocyte-secreted mitogen, but also show that GDF9 accounts for only part of total oocyte bioactivity.R.B. Gilchrist, L.J. Ritter, M. Cranfield, L.A. Jeffery, F. Amato, S.J. Scott, S. Myllymaa, N. Kaivo-Oja, H. Lankinen, D.G. Mottershead, N.P. Groome, and O. Ritvo