Investigation of the roles of TSG-6 and inter-alpha-inhibitor in female fertility

Lead Research Organisation: University of Manchester
Department Name: Life Sciences

Abstract

Infertility is an issue that affects many families in the UK, with around one in six couples attending fertility clinics. However, for many patients the cause of infertility is unclear. By improving our basic understanding of the molecular processes that occur during ovulation (i.e. the release of an oocyte (or egg) from the ovary) we hope to aid the development of more effective treatments for infertile women. Our research will focus on two proteins called TSG-6 and I-alpha-I. We already know that mice that do not synthesise either one of these proteins cannot produce oocytes that are fertilisable. In a mammalian ovary, each oocyte is contained in a separate follicle and before ovulation the oocyte becomes surrounded by a jelly-like substance. This jelly protects the oocyte during release from the ovary and provides a large surface area that aids sperm binding, which is essential for fertilisation. We have shown that both TSG-6 and I-alpha-I have critical roles in forming the jelly and now have new evidence suggesting that these proteins might also be involved in controlling the breakdown of the ovarian follicle wall to allow oocyte release. We will use various methods to further investigate TSG-6 and I-alpha-I in ovulation. For example, we have developed molecular tools that will allow us to look (e.g. using microscopy) at exactly when and where these proteins are present in mouse ovaries and how they might work together in association with other moelcules. Although studies on the mouse can tell us much about ovulation in general, if our results are to be relevant to the development of treatments for infertility, we must be certain that any processes we identify as being important in mice also occur in humans. A new method called ?in vitro maturation of oocytes?, or IVM, which is just beginning to be used to treat infertile women in the UK will allow us to test this, using material left over from treatments. This is a joint project between researchers at the University of Manchester, who have many years of experience in studying TSG-6 and I-alpha-I and are world-leaders in this field, and clinicians at the Oxford Fertility Unit ? the first centre in the UK to achieve pregnancies using the IVM procedure. The combination of expertise from these two groups represents a unique opportunity to carry out this important study.

Technical Summary

The aim of this work is to investigate fundamental mechanisms that are necessary for female fertility and determine whether processes characterised in the mouse also occur in humans. The proposal focuses on TSG-6 and inter-alpha-inhibitor (IalphaI), proteins that have been shown in mice to be essential for the stabilisation of a nascent matrix that forms around the oocyte (termed cumulus-oocyte complex (COC) expansion) and is necessary for successful ovulation and fertilisation in vivo. The major component of this matrix is the polysaccharide hyaluronan (HA), which is believed to become cross-linked, although, at present this is poorly understood.
We have shown that TSG-6 catalyses the transfer of heavy chains (HC) from IalphaI onto HA to form covalent HC-HA complexes, where interactions between HC have been implicated in matrix cross-linking. To more fully understand COC expansion we will use specific antibodies to determine the spatial/temporal distributions of TSG-6, HA and HCs in mouse ovaries. We will also test whether mutants of recombinant human TSG-6 (defective, e.g. in HA binding and/or HC-HA formation) can rescue the ex vivo expansion of tsg-6-null COCs (as already shown for the WT human protein). Access to in vitro matured (IVM) COCs, from women undergoing treatment for infertility, will provide a unique opportunity to investigate whether mouse and human ovulation involve similar mechanisms. For example, we will measure the release of TSG-6 into the IVM culture medium (by ELISA) and the localisation of TSG-6 and its ligands within the human cumulus matrix by immunofluorescence.
In addition to COC expansion, successful ovulation requires follicle rupture involving proteolytic enzymes, e.g. plasmin. Co-association of TSG-6 with the bikunin chain of IalphaI and certain sulphated glycosaminoglycans substantially enhances the anti-plasmin activity of IalphaI. Our preliminary data indicate that TSG-6 co-localises with anticoagulant heparan sulphate (aHS) in murine ovulating follicles. Reduced TSG-6 immunostaining in Hs3st1-null mouse follicles (which cannot synthesise aHS) suggests that aHS-proteoglycans might sequester TSG-6 or TSG-6/IalphaI complexes and thereby regulate proteolysis during ovualtion. We will investigate whether aHS (from follicular fluids) modulates the anti-plasmin activity of TSG-6/IalphaI and extend our studies on Hs3st1-null follicles to determine the effect of TSG-6 on proteolysis in ovulation.
This study will provide important insights into molecular mechanisms involved in ovulation, while the analysis of patient samples will indicate whether defects in these processes might account for some cases of infertility and potentially lead to improved fertility treatments.

Publications

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Baranova NS (2013) Inter-a-inhibitor impairs TSG-6-induced hyaluronan cross-linking. in The Journal of biological chemistry

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Ievoli E (2011) Implication of the oligomeric state of the N-terminal PTX3 domain in cumulus matrix assembly. in Matrix biology : journal of the International Society for Matrix Biology

 
Description ARUK Programme Grant
Amount £531,022 (GBP)
Organisation Versus Arthritis 
Start 11/2009 
End 05/2015
 
Description ARUK Project Grant (The role of inter-alpha-inhibitor in arthritis: friend or foe?)
Amount £185,377 (GBP)
Funding ID 19489 
Organisation Versus Arthritis 
Start 11/2010 
End 03/2014
 
Description CASE Studentship with Farfield Group Ltd
Amount £75,281 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2010 
End 09/2014
 
Description Doctoral Training Award
Amount £70,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2012 
End 09/2015
 
Description Non-clinical PhD Studentship
Amount £99,832 (GBP)
Funding ID FS/10/52/28678 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2011 
End 09/2014
 
Description Univerity of Manchester and A*STAR PhD Studentship
Amount £127,983 (GBP)
Organisation University of Manchester 
Sector Academic/University
Country United Kingdom
Start 10/2012 
End 09/2016
 
Title Chain specific antibodies for mouse inter-alpha-inhibitor 
Description Antisera raised in rabbits against murine HC1, HC2, HC3 and bikunin (i.e. the protein chains of inter-alpha-inhibitor). 
Type Of Material Antibody 
Year Produced 2010 
Provided To Others? Yes  
Impact Localization of these proteins within the mouse ovary prior to ovulation. Paper in preparation. 
 
Title Detection of TSG-6 protein associated with human IVF and IVM oocytes 
Description We have detected TSG-6 (a protein already established as having a key role in mouse ovulation) within the cumulus matrix surrounding oocytes collected for IVF or cultured in vitro for IVM. This provides novel insights into the mechanism of human ovulation. 
Type Of Material Model of mechanisms or symptoms - human 
Provided To Others? No  
Impact Work is in progress to determine whether TSG-6 could be used as a biomarker to aid with IVF/IVM procedures. Paper in preparation. 
 
Title Human COCs from patients undergoing surgery 
Description Immature COCs recovered from patients undergoing surgery are now available (under ethics). These can be matured in vitro and are allowing us to perform fluorescent microscopy to determine the spatial/temporal distribution of TSG-6, hyaluronan and inter-alpha-inhibitor within the human cumulus matrix. 
Type Of Material Model of mechanisms or symptoms - human 
Provided To Others? No  
Impact This work is allowing us to determine whether the same molecular processes occur in the human and mouse, where the results are likely to aid improvements in IVM. 
 
Title Recombinant heavy chains from human inter-alpha-inhibitor. 
Description Recombinant heavy chains from human inter-alpha-inhibitor and pre-alpha-inhibitor (wild and mutant proteins). 
Type Of Material Technology assay or reagent 
Year Produced 2012 
Provided To Others? Yes  
Impact 1 paper published (Baranova et al., 2013 J. Biol. Chem.) and other papers are in preparation. 
 
Description Structural and functional studies on PTX3 - a key component of cumulus matrix. 
Organisation Humanitas Research Hospital
Country Italy 
Sector Hospitals 
PI Contribution Used reagents provided in our research.
Collaborator Contribution Provision of research reagents.
Impact 3 primary papers in J. Biol. Chem. (2008, 2010, 2014). 1 primary paper in Matrix Biol. (2011). 1 primary paper in Arterioscl. Throm. Vas. (2012). 1 primary paper in Scientific Reports (2018). 1 review article in Cellular & Molecular Life Sciences (2017). Abstract at 36th FEBS Congress, Torino, Italy (2011).
 
Description Structural and functional studies on PTX3 - a key component of cumulus matrix. 
Organisation Sigma-Tau Pharmaceuticals
Country United States 
Sector Private 
PI Contribution Used reagents provided in our research.
Collaborator Contribution Provision of research reagents.
Impact 3 primary papers in J. Biol. Chem. (2008, 2010, 2014). 1 primary paper in Matrix Biol. (2011). 1 primary paper in Arterioscl. Throm. Vas. (2012). 1 primary paper in Scientific Reports (2018). 1 review article in Cellular & Molecular Life Sciences (2017). Abstract at 36th FEBS Congress, Torino, Italy (2011).
 
Description The role of TSG-6 and inter-alpha-inhibitor in female fertility. 
Organisation Geneva University Hospitals
Department Department of Gynaecology and Obstetrics
Country Switzerland 
Sector Hospitals 
PI Contribution Use of reagents, mouse/human tissues and clinical samples in our research.
Collaborator Contribution Provision of clinical samples to enable our research.Provision of mouse tissues and research reagents to support our research.
Impact Abstract at BSMB Autumn meeting (2010). Abstract at Fertility 2011, Dublin, Ireland. Abstract at 2nd Biomedical Imaging Institute Imaging Methods Showcase, Manchester, UK (2011). Abstract at 7th International Conference on Proteoglycans, Sydney, Australia (2011).
 
Description The role of TSG-6 and inter-alpha-inhibitor in female fertility. 
Organisation University of Oxford
Department Nuffield Department of Obstetrics & Gynaecology
Country United Kingdom 
Sector Academic/University 
PI Contribution Use of reagents, mouse/human tissues and clinical samples in our research.
Collaborator Contribution Provision of clinical samples to enable our research.Provision of mouse tissues and research reagents to support our research.
Impact Abstract at BSMB Autumn meeting (2010). Abstract at Fertility 2011, Dublin, Ireland. Abstract at 2nd Biomedical Imaging Institute Imaging Methods Showcase, Manchester, UK (2011). Abstract at 7th International Conference on Proteoglycans, Sydney, Australia (2011).
 
Description The role of TSG-6 in cumulus expansion. 
Organisation University of Rome Tor Vergata
Department Department of Public Health and Cell Biology
Country Italy 
Sector Academic/University 
PI Contribution Provision of research reagents.
Collaborator Contribution Conducting experiments using reagents we provided.
Impact 1 Primary paper in Matrix Biology (2011).1 Primary paper in J. Biol. Chem. (2015). Abstract at 6th International Conference on Proteoglycans (2009). Abstract at BSMB Spring Meeting (2010). Abstract at Gordon Research Conference on Proteoglycans (2010). Abstract at BSMB Autumn Meeting (2010). Abstract at Fertility (2011). Abstract at Fertility 2011.
Start Year 2007
 
Description Understanding the mechanism of cumulus expansion 
Organisation University of Oxford
Department Nuffield Department of Obstetrics & Gynaecology
Country United Kingdom 
Sector Academic/University 
PI Contribution We supplied reagents that were used in this collaboration and helped interpret the results obtained.
Collaborator Contribution The partners used reagents that we provided in their research, which has provided novel insights into the mechanism of ovulation.
Impact 1 paper in Reproduction (2015); 1 paper in preparation.
Start Year 2010
 
Description Use of surface-sensitive techniques to investigate the molecular basis of matrix remodelling. 
Organisation Centre for Cooperative Research in Biomaterials (CIC BiomaGUNE)
Department Biosurfaces Research Unit
Country Spain 
Sector Academic/University 
PI Contribution We have provided reagents and input to the design of experiments and interpretation of results; and used reagents from this collaborator in our own research.
Collaborator Contribution Experiments performed by these collaborators (using reagents we have supplied), and work done in our lab using reagents supplied by this collaborator, have provided novel insights into the molecular interactions that contribute to inflammatory processes and ovulation.
Impact 5 primary papers in J. Biol. Chem. (2010, 2011, 2013, 2014, 2015). Review in Current Opinion in Structural Biology (2017). Abstract at Biological Surfaces and Interfaces, Sant Feliu de Guixols, Spain (2009). Abstract at 6th International Conference on Proteoglycans, Aix les Bains, France (2009). 2 abstracts at 8th International Conference on Hyaluronan, Kyoto, Japan (2010). 2 abstract at ESF-EMBO Symposium on Biosurfaces and Interfaces, Sant Feliu de Guixols, Spain (2011). Abstract at Biophysical Society 58th Annual Meeting, San Francisco, USA (2014). Talk at Gordon Research Conference on Proteoglycans, Andover, USA (2012). Talk at International Society of Hyaluronan Sciences, Oklahoma City, USA (2013). 3 abstracts at International Society for Hyaluronan Sciences HA2013, Oklahoma City, USA (2013) Talk at ESF-EMBO Symposium on Biosurfaces and Interfaces, Sant Feliu de Guixols, Spain (2013). Abstract at Biophysical Society 58th Annual Meeting, San Francisco, USA (2014). Talk at 10th International Meeting on Hyaluronan, Florence, Italy (2015).
Start Year 2006
 
Description School visits. Manchester. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach Local
Primary Audience Schools
Results and Impact Final year undergraduate students (as a part of education projects in the PI and co-I's lab) trialed teaching resources for Key Stage 4 students (14-16 year old) and A level students focused on ovulation and fertilisation at three different Manchester schools and at a museum workshop (~100 pupils in total).

Two of the schools and museum indicated that they will use the resources in future years for their teaching on reproductive biology.
Year(s) Of Engagement Activity 2011,2012
 
Description The official opening of the new Oxford Fertility Unit. 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Regional
Primary Audience Health professionals
Results and Impact One of the co-applicants on this grant presented an overview of research (including our project) as part of the official opening of the Oxford Fertility Unit, following its move to new premises.

It raised awareness of the importance of basic research being done in parallel with the clinical activities of the Fertility Unit.
Year(s) Of Engagement Activity 2009
 
Description Workshops for Manchester schools on inflammation. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Final year undergraduate 'Education' project aimed at GCSE and A level students; workshops on inflammation and inflammatory disease were delivered as part of a Manchester Museum Activity Day for Schools and revision activities on these topics were delivered to GCSE students at a Manchester school. Positive feedback from students and their teachers and interest was stimulated in studying science degrees at university.

Positive feedback to project supervisor on the value of the school visit and the quality/impact of the content delivered.
Year(s) Of Engagement Activity 2014