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

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.

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.