Functional role of feline zona pellucida protein 4 trefoil domain: a sperm receptor or structural component of the domestic cat zona pellucida?

The trefoil domain (TFD) is a protein structure characterized by six cysteines, which form a typical three-loop structure by three disulphide bridges. It is assumed that two of these loops generate a hydrophobic groove, which could be a binding site for carbohydrate residues or proteins. The zona pellucida (ZP) protein, ZP4, contains such a TFD. The carbohydrate-/protein-binding property of TFD allows us to assume a potential sperm receptor function of this domain. Additionally, gastrointestinal trefoil peptides are stable against proteases; therefore, a structural role of TFD within the ZP might also be possible. We were able to show that the synthesized and natural folded feline TFD (fTFD) expresses the typical protease resistance that vanished under reducing conditions and after substitution of cysteine residues within the peptide. Furthermore, an antibody directed against the first loop of fTFD was almost unable to bind to intact in vitro mature cat oocytes. Pre-incubation of oocytes in the reducing agent (DDT), however, improved antibody binding substantially. Therefore, we suggest structural masking of the fTFD domain within the intact ZP. An interaction between fTFD and feline sperm cells was examined using several methods, including immunocytochemistry, immunoelectron microscopy, co-immunoprecipitation and far western blot, but we found no indication for an involvement of TFD in the primary sperm binding to the ZP. To summarize, there is increasing evidence that the TFD of fZP4 has a structural rather than a sperm-binding function.

Brilliant cresyl blue staining allows the selection for developmentally competent immature feline oocytes.

The outcome of in-vitro-maturation and in-vitro-fertilization of feline oocytes depends on the selection of high quality oocytes, and is often restricted to morphological criteria. The aim of this study was to test whether the Brilliant cresyl blue (BCB) staining is suitable for pre-selection of feline oocytes before in-vitro-maturation. Cumulus-oocytes-complexes (COC) were released from domestic cat ovaries obtained after ovariectomy and were subjected to BCB staining. BCB+ stained oocytes were characterized by a violet/pale blue staining of the ooplasma, BCB- oocytes remained unstained. Transmission electron microscopy indicated for a slightly advanced stage of BCB- oocytes within the maturation process. After 24 h in-vitro-maturation, almost 75% of BCB+ and 21.5% of BCB- oocytes were able to reach metaphase II. Also, after in-vitro-fertilization, significantly more oocytes developed to morulae (19.2%) if oocytes were preselected for BCB staining, although 8% of unstained COC still reached advanced embryo stages. Prolonged storage of ovaries before COC retrieval for 16-20 h at 4 °C was accompanied by reduced number of BCB+ oocytes (96 of 210, 45.7%) in comparison to freshly isolated COC (151 of 225, 67.1%), and impaired cleavage rate (19.8%) and morula rate (9.4%) of BCB+ oocytes but the rate of embryos which developed to advanced stages remained unchanged (∼50%). To conclude, BCB staining is a very useful tool to preselect immature COC of feline species ensuring higher developmental rates.