The Role of Siglecs in Sperm Function and Characterisation of the Interaction of Sperm with Sialic acid on Cervical Mucus

Abstract

Sialic Acid is a carbohydrate with ~ 50 different variants that is involved in several functions including cell recognition and the regulation of immune responses. Previous work within this research group has shown that Sialic Acid is a capping residue on cervical mucus and in the bovine, the Sialic Acid content of cervical mucins change with a relative decrease in Neu5Ac (the most common form) and an increase in Neu5Gc at oestrus. In addition, Sperm movement through non-oestrus cervical mucus can be enhanced by the addition of free Neu5Ac but not Neu5Gc suggesting a competitive binding effect between the sperm surface and cervical mucus Sialic Acid (Alkhodair et al., 2015). Siglecs are Sialic Acid specific receptor proteins and mediate a range of immunological and pathological functions. They have recently been shown to be expressed on both sperm and the epithelium of the Male Reproductive Tract (MRT) and soluble versions of Siglecs have been identified within seminal plasma (Alkhodair, Almhanna et al. 2018). These observations underpin our current hypothesis that: (1) Trans-cervical sperm transit at oestrus is regulated by binding between Siglecs on sperm and Sialic Acid on cervical mucins. (2) In the bovine, sperm bind preferentially to Neu5Ac via Siglecs, and that a switch from Neu5Ac to Neu5Gc on cervical mucin at oestrus is required to reduce sperm Siglec/mucin binding and permit trans-cervical sperm transit. The potential role of Siglecs on sperm in modulating sperm progression could be a physical role interacting with Sialic Acid in mucus and depending on the binding affinity impeding or promoting sperm mucus penetration. These Siglecs may also have a cell signaling role, perhaps sensing the external Sialic Acid signature of the cervical mucus at discrete stages of the oestrous cycle and regulating sperm function such as motility or energy metabolism. In this thesis, the origin of recently identified soluble Siglecs within bovine seminal plasma and within the MRT is characterised and it is suggested that an intact testes-vas deferens axis is important for their maturation. To explore the potential role of Sialic Acid on cervical mucin and Siglecs on Sperm, recombinant versions of two bovine Siglecs, Siglec 5 and Siglec 14, were developed which enable the investigation of their Sialic Acid binding ligands. These were chosen as they showed regional expression on sperm and also in humans they function as paired receptors that mediate opposing immune responses (Ali, Fong et al. 2014). In addition, as previous work had employed human antibodies which were not bovine specific, we developed a bovine antibody specific for Siglec 5. These reagents were employed to analyse Sialic Acid distribution within the MRT and on sperm. The distribution of these Siglecs co-localised with their ligands suggesting a potential regulation of Siglec development and function on sperm. Siglec ligands on cervical mucosa were analysed throughout the oestrous cycle. The ligands for Siglec 5 were present at higher amounts outside of the oestrous period while around oestrus, the ligands for Siglec 14 were expressed. This change in distribution of Sialic Acid may be involved in modulating transcervical sperm movement. Outside of oestrus, Siglec 5 may physically bind to Sialic Acid on cervical mucus inhibiting its movement. At oestrus, the change from Neu5Ac to Neu5Gc may disrupt Siglec 5 binding and release sperm from interacting with cervical mucus. In addition, we suggest that Siglec 14 is activated in the presence of its ligand and this may be involved in the regulation of pro-motility function to enhance sperm movement. It is also possible that these Siglecs may constitute a matched pair of stimulatory and inhibitory Siglecs, similar to that present within the immune system. Future work will focus on the role of each of these Siglecs in sp