At fertilization the sperm triggers a series of intracellular calcium oscillations that are pivotal to oocyte activation and development. Although the biological significance of the characteristic intracellular calcium (Ca2+i) oscillations is not fully understood, calcium ions are known to be involved in cortical granule release and in controlling cell cycle progression. Two different hypotheses attempt to explain how sperm initiate (Ca2+i) oscillations in mammalian oocytes. One hypothesis is that spermatozoa interact with a receptor located in the plasma membrane of the oocyte, which results in induction of pathways leading to activation. This receptor is coupled to a GTP-binding protein or to have tyrosine kinase activity and have the ability to induce activation of phospholipase C (PLC). In turn, PLC stimulates the hydrolysis of phosphatidyl inositol (4,5)–bisphosphate (PIP2) to produce diacylglycerol (DAG) and 1,4,5 inositol trisphosphate (IP3), a common Ca2+ releasing compound. Most studies used to develop the mammalian model of oocyte activation have been performed in the mouse. There is a paucity of information from other mammalian models. The predominant mouse model of oocyte activation is that there is a soluble factor (PLC-zeta) delivered to the cytosol after fertilization that induces oocyte activation. However, as data in other mammals is collected, substantial evidence is beginning to support the existence of other more complex oocyte activation pathways in both murine and non-murine systems. Indeed, activation may involve redundant processes, each of which acting alone may be able to induce aspects of oocyte activation. Recent findings demonstrate the involvement of receptors that are known to associate in large, multimeric complexes. This fact leads one to speculate that the process of oocyte activation by the sperm cell is a highly complex and elaborate process that likely involves many more players than perhaps was initially expected.

At fertilization, the sperm triggers intracellular calcium oscillations, which are pivotal to oocyte activation and development. A working hypothesis for the interaction between the sperm and the oocyte is that disintegrin ligands on the inner acrosomal membrane of the sperm bind to integrin receptors on the oocyte vitelline membrane. The aim of these experiments was to find and identify the sperm protein ligands involved in bovine sperm-oocyte interactions. In situ fluorescent labeling of proteins and 2-D gel electrophoresis were used to identify specific sperm membrane proteins that interact with proteins in the oocyte vitelline membrane. Sperm were labeled with a fluorescent dye and used to fertilize zona-free oocytes. Sperm-oocyte complexes were either lysed immediately, or following covalent cross-linking of proteins with dibromobimane. The cross-linking reagent serves the critical function of covalently linking proteins together so that they will remain as a unit through lysis of the cells and 2-D gel analysis, and which can be subsequently identified by mass spectrometry. Lysates were electrophoretically run on the same 2-D gel. The comparison of uncross-linked and cross-linked protein spots revealed that some proteins shifted position based on binding. These spots were picked and proteins identified by mass spectrometry. These results provide a list of specific sperm proteins that interact with oocyte membrane proteins and establish a group of candidate ligands, one or more of which may be responsible for induction of outside-in signaling resulting in oocyte activation and fusion of the gametes.

Integrins facilitate attachment of cells to the extra‐cellular matrix, often binding the arginine‐glycine‐aspartic acid tri‐peptide motif, thus facilitating cell migration, mediating cell–cell adhesion, linking the extracellular matrix (ECM) with cytoskeletal elements, and acting as signaling molecules. Adhesion activates signaling mechanisms that regulate integrin function, cytoskeletal assembly, cell behavior, and protein synthesis. Immunofluorescence was used to determine the presence of integrin α and β subunits on the surface of bovine oocytes using a panel of monoclonal antibodies (mAbs) specific for αL, αM, αX, αV, α2, α4, α6, β1, β2, and β3 antigens, with multiple antibodies for each subunit. Confocal microscopy indicated the presence of αV, α6, α4, α2, ß1, and ß3 integrin subunits on the plasma membrane of bovine oocytes. The presence of these subunits was verified by RT‐PCR analysis using primers designed based on known gene sequences of bovine integrin subunits, or by using sequence information using bovine expressed sequence tags (EST) compared with known human and murine integrin subunit gene sequence information. Previously unpublished sequence information for bovine α6 and β3 integrins was determined. The presence of these integrin subunits on the bovine oocyte vitelline membrane supports the hypothesis that sperm–oocyte interactions in the bovine are mediated by integrins. Mol. Reprod. Dev. 74: 600–607, 2007. © 2006 Wiley‐Liss, Inc.

Due to its economic importance, the production of cattle by nuclear transfer has been a primary research focus for many researchers during the past few years. While many groups have successfully produced cattle by nuclear transfer, and progress in this area continues, nuclear transfer remains a very inefficient technology. This study evaluates the effect of the oocyte source (cow and heifer) on the developmental competence of nuclear transfer embryos. In order for nuclear transfer to be successful, a differentiated donor cell must be reprogrammed and restored to a totipotent state. This reprogramming is probably accomplished by factors within the oocyte cytoplasm. This study indicates that oocytes derived from cows have a greater capacity to reprogram donor cell DNA following nuclear transfer as compared to heifer oocytes based on in vitro development to the 2-cell stage and to the compacted morula/blastocyst stages. Nuclear transfer embryos derived from cow oocytes resulted in significantly higher rates of pregnancy establishment than embryos derived from heifer oocytes and resulted in higher pregnancy retention at 90 and 180 days and a greater number of term deliveries. Following delivery more calves derived from cow oocytes tended to be healthy and normal than those derived from heifer oocytes. The differences in developmental efficiency between nuclear transfer embryos derived from cow and heifer cytoplasts demonstrate that subtle differences in oocyte biology can have significant effects on subsequent development of nuclear transfer embryos.