Functional, biochemical, and chromatographic characterization of the complete [Ca2+]i oscillation-inducing activity of porcine sperm.
A cytosolic sperm protein(s), referred to as sperm factor (SF), is delivered into eggs by the sperm during mammalian fertilization to induce repetitive increases in the intracellular concentration of free Ca2+ ([Ca2+]i) that are referred to as [Ca2+]i oscillations. [Ca2+]i oscillations are essential for egg activation and early embryonic development. Recent evidence shows that the novel sperm-specific phospholipase C (PLC), PLCzeta, may be the long sought after [Ca2+]i oscillation-inducing SF. Here, we demonstrate the complete extraction of SF from porcine sperm and show that regardless of the method of extraction a single molecule/complex appears to be responsible for the [Ca2+]i oscillation-inducing activity of these extracts. Consistent with this notion, all sperm fractions that induced [Ca2+]i oscillations, including FPLC-purified fractions, exhibited high in vitro PLC activity at basal Ca2+ levels (0.1-5 microM), a hallmark of PLCzeta. Notably, we detected immunoreactive 72-kDa PLCzeta in an inactive fraction, and several fractions capable of inducing oscillations were devoid of 72-kDa PLCzeta. Nonetheless, in the latter fractions, proteolytic fragments, presumably corresponding to cleaved forms of PLCzeta, were detected by immunoblotting. Therefore, our findings corroborate the hypothesis that a sperm-specific PLC is the main component of the [Ca2+]i oscillation-inducing activity of sperm but provide evidence that the presence of 72-kDa PLCzeta does not precisely correspond with the Ca2+ releasing activity of porcine sperm fractions.
Kurokawa
Manabu
M
Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003, USA.
Sato
Ken-Ichi
K
Wu
Hua
H
He
Changli
C
Malcuit
Christopher
C
Black
Samuel J
SJ
Fukami
Kiyoko
K
Fissore
Rafael A
RA
D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D002853:Chromatography, Liquid; D015151:Immunoblotting; D008297:Male; D020554:Sperm Injections, Intracytoplasmic; D013094:Spermatozoa / Q000378:metabolism* / Q000502:physiology; D034421:Sus scrofa / Q000378:metabolism*; D010738:Type C Phospholipases / Q000302:isolation & purification*
D010738:Type C Phospholipases; D002118:Calcium