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.
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.
Organisations
- University of Manchester, Manchester, United Kingdom (Lead Research Organisation)
- University of Oxford, United Kingdom (Collaboration)
- Centre for Cooperative Research in Biomaterials (CIC BiomaGUNE) (Collaboration)
- Humanitas Research Hospital (Collaboration)
- Sigma-Tau Pharmaceuticals (Collaboration)
- Geneva University Hospitals (Collaboration)
- University of Rome II (Tor Vergata), Italy (Collaboration)
Publications

Baranova NS
(2011)
The inflammation-associated protein TSG-6 cross-links hyaluronan via hyaluronan-induced TSG-6 oligomers.
in The Journal of biological chemistry

Baranova NS
(2014)
Incorporation of pentraxin 3 into hyaluronan matrices is tightly regulated and promotes matrix cross-linking.
in The Journal of biological chemistry

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

Briggs DC
(2015)
Metal Ion-dependent Heavy Chain Transfer Activity of TSG-6 Mediates Assembly of the Cumulus-Oocyte Matrix.
in The Journal of biological chemistry

Capp E
(2014)
Modulation of tumor necrosis factor-stimulated gene-6 (TSG-6) expression in human endometrium.
in Archives of gynecology and obstetrics

Higman VA
(2014)
A refined model for the TSG-6 link module in complex with hyaluronan: use of defined oligosaccharides to probe structure and function.
in The Journal of biological chemistry

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

Inforzato A
(2010)
The angiogenic inhibitor long pentraxin PTX3 forms an asymmetric octamer with two binding sites for FGF2.
in The Journal of biological chemistry

Leali D
(2012)
Long pentraxin 3/tumor necrosis factor-stimulated gene-6 interaction: a biological rheostat for fibroblast growth factor 2-mediated angiogenesis.
in Arteriosclerosis, thrombosis, and vascular biology

Lo BKM
(2019)
Oocyte-specific ablation of N- and O-glycans alters cumulus cell signalling and extracellular matrix composition.
in Reproduction, fertility, and development
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 |