Characterization of intercellular calcium ion oscillation-inducing sperm factor and its signaling pathway

• Main Author: Kurokawa, Manabu
• Language: ENG
• Published: ScholarWorks@UMass Amherst 2004
• Subjects: Cellular biology|Environmental engineering
• Online Access: https://scholarworks.umass.edu/dissertations/AAI3152721

In mammalian eggs, the fertilizing sperm evokes intracellular Ca 2+ ([Ca2+]i) oscillations that are essential for initiation of egg activation and embryonic development. Although the exact mechanism leading to initiation of [Ca2+]i oscillations still remains unclear, accumulating studies suggest that a presently unknown substance, termed sperm factor, is delivered from the fertilizing sperm into the ooplasm and triggers [Ca2+]i oscillations. Recent evidence shows that sperm/sperm factor stimulates persistent production of inositol 1,4,5-trisphosphate (IP3) and that a sperm-specific phosphoinositide-specific phospholipase C (PLC), PLCζ, may be the long sought-after [Ca 2+]i oscillation-inducing molecule in sperm. In the first study, we show that subtle modification of the sequence of events that lead to fertilization, such as that seen in fertilization achieved by intracytoplasmic sperm injection (ICSI), alters the pattern of [Ca2+]i oscillations and embryonic development, and this alternation is possibly due to the non-physiological release of sperm factor (Chapter 3). Thereafter, we demonstrate that activation of a Src family kinase, which is essential for fertilization-associated Ca2+ release in non-mammalian species, is neither necessary nor sufficient for triggering fertilization-induced [Ca2+]i oscillations in mouse (Chapter 4). Finally (Chapter 5), we demonstrate the complete extraction of the [Ca2+] i oscillation-inducing activity from porcine sperm, and show that regardless of the cellular location or solubility, the same molecule/complex appears to be responsible for [Ca2+]i oscillations induced by injection of porcine sperm extracts (pSEs). Moreover, our results suggest that PLCζ may not be the only active molecule in pSEs. We demonstrate that biotin-labeled peptide A7 (B-A7), a Src kinase inhibitory peptide, specifically binds to and precipitates the active component of sperm factor with avidin beads. Although the proteins that we have isolated and identified thus far are, however, unlikely to be involved in activation of the phosphoinositide pathway when introduced into mouse eggs, future studies using B-A7 should lead to identification of the putative sperm factor molecule.