Regulation of calcium signaling during bovine fertilization

The generation of a new organism by the union of gametes, dubbed fertilization, is a unifying theme across nearly all living species. Concurrently, divergencies amongst mechanisms of this event exist even within creatures of the same genus, and arguably, within species. The following presents an examination of the mechanisms of fertilization and the subsequent regulation of intracellular calcium ([Ca²⁺]_i) signaling in the eggs of bos taurus (domestic cattle) with specific attention to both conserved and divergent mechanisms from that of the more commonly studied mus musculus (common mouse). Through a multiparametric approach consisting of Ca²⁺ imagery, protein analysis, molecular mutation, and assisted reproductive technologies we examined first the cytologic nature of the release of the sperm-borne egg-activating factor, referred to as sperm factor, during fertilization. We followed by ascertaining the functional properties and minimal requirements of the type I 1,4,5-inositol trisphosphate receptor (IP₃R-1) in bovine eggs during fertilization. Finally, we examined the [Ca²⁺]_i oscillation-inducing ability and cellular localization of the purported sperm factor molecule, phospholipase C&zgr; (PLC&zgr;), during egg activation, with close attention to species specific dynamics of calcium release and molecular determinants of functionality. Our data show that first, although bull sperm contain a cytosolic sperm factor, it is insufficient to trigger [Ca²⁺]_i oscillations in bovine eggs without sperm-egg fusion first occurring. Second, bovine eggs possess a virtually redundant amount of IP₃R-1, capable of mounting near-normal Ca²⁺ responses with only a 20-30% complement of IP₃R-1. Third, bovine PLC&zgr; demonstrates an attenuated enzymatic activity compared to PLC&zgr; from other species, however, remains cytoplasmic as bovine zygotes reach interphase, and appears capable of continued IP ₃ production as determined both by calcium imagery and quantification of IP₃R-1. Additionally, unique sequence elements of bovine PLC&zgr; may be responsible for this decreased enzymatic activity. Lastly, we have determined that bovine eggs possess a unique mechanism to down regulate, but not terminate, interphase stage [Ca²⁺]i oscillations through alteration of IP₃R-1 Ca²⁺ conductivity, independent of IP₃R-1 numbers. The culmination of these results exemplifies how mammals have evolved to adapt components of the same system to achieve the unique requirements of the various respective species.