Introduction:
Mature spermatozoa recognise and bind hyaluronic acid (HA) in the extracellular matrix via Hyaluronic Acid Binding Proteins (HABPs). HA-binding may be important in supporting the sperms’ progress in the female genital tract. Information on sperm HABPs is limited, however and the current study investigated their expression in human and bovine sperm.
Materials and methods:
The ability of differential density gradient centrifugation (DDGC) processed human sperm to bind HA was evaluated. DDGC sperm populations (90% and 45% fractions) and sperm interacting with HA were assessed for DNA fragmentation and chromatin compaction using acridine orange (AO) and aniline blue (AB) staining, respectively. Additionally, the effect of capacitation on HA-binding was assessed. Expression of HABPs in bovine and human sperm was also investigated alongside changes in their distribution following capacitation and the acrosome reaction. Proteomics was used to monitor changes in the human sperm proteome (including HABPs) following capacitation and the acrosome reaction. Following a protein panning procedure, proteomics was also used to help identify HABPs in both HA-binding and non-binding fractions. The presence of HA-binding motifs (BX7B and Link module, common in several well-known HABPs) was also evaluated in both fractions. Increasing the sensitivity of HABP isolation and detection was attempted using biotinylation of sperm surface proteins alongside HA-affinity chromatography.
Results:
Sperm recovered from 90% fractions had significantly higher HA-binding ability, lower levels of DNA fragmentation and higher levels of chromatin compaction. The reverse was true for sperm from the 45% fractions. HA-binding showed higher efficiency at selecting sperm with higher DNA integrity and chromatin compaction than DDGC. Capacitation enhanced HA binding, which may reflect associated changes in sperm HABPs. Immunocytochemistry localised CD44 to the acrosome and equatorial segment of non-capacitated human sperm and on the anterior acrosome and post-acrosomal sheath of non-capacitated bovine sperm. CD44 was more confined to the equatorial segment following capacitation in human sperm and to the post-acrosomal sheath of bovine sperm. Acrosomal labelling of CD44 was highly reduced after the acrosome reaction. Hyaluronic acid-TRITC (tetramethylrhodamine isothiocyanate) labelled the sperm membrane and the neck region, particularly strongly after capacitation and in acrosome reacted bovine sperm.
Qualitative and quantitative characteristics of 121 proteins altered by capacitation and the acrosome reaction is also reported. Differentially regulated proteins are involved in sperm metabolism, energy production, cell signaling, sperm-oocyte recognition and sperm motility. Following capacitation, mass spectrometry (MS) detected an increase in cilia and flagella associated protein 20, containing two potentially HA-binding BX7B motifs. Analysis of the proteomics data from the panning fractions resulted in the identification of 28 proteins in HA-binding fractions with PDBsum and SAS server highlighting structural similarities between ZPBP2 (found only in HA-binding fractions) and the HA-binding domain of CD44. Data also showed that 50% of binding (including ADAM32 and cilia-and flagella associated protein 20 amongst others) and 34.5% of non-binding proteins contained BX7B motifs, indicating a likely selective enrichment of putative HABPs by the panning technique. Western blot analysis confirmed these results. Isolation of HABPs from bovine sperm using HA affinity chromatography followed by SDS PAGE revealed distinct protein bands of molecular masses 58 and 78 kDa, possibly corresponding to CD44 and RHAMM, respectively, were detected. Other protein bands detected at 16, 27, 38, 42 and 47 may be novel HABPs, including ZPBP2 (molecular mass 38 kDa).
Conclusion:
In conclusion, ZPBP2, ADAM32, Midkine and cilia and flagella associated protein 20 may have HA binding properties. This is also the first study which has brought together compelling evidence for the relationship between excising methods of enriching good quality sperm (DDGC) and HA-binding. Sperm DNA fragmentation and chromatin compaction status, therefore, reflects this relationship and supports claims for a positive sperm quality benefit for HA-based sperm selection particularly for ICSI, where the choice of sperm is more critical. In this regard, the use of more specific, anti-HABP-based methods for sperm selection (initially targeting RHAMM, CD44 and SPAM1) should also be considered.
