Proteolytic processing of phospholipase Czeta and [Ca2+]i oscillations during mammalian fertilization.
Phospholipase Czeta (PLCzeta) is a sperm-specific PLC capable of causing repetitive intracellular Ca2+ ([Ca2+]i) release ([Ca2+]i oscillations) in mammalian eggs. Accumulating evidence suggests that PLCzeta is the sperm factor responsible for inducing egg activation. Nevertheless, some sperm fractions devoid of 72-kDa PLCzeta showed [Ca2+]i oscillation-inducing and PLCzeta-like PLC activity (Kurokawa et al., (2005) Dev. Biol. 285, 376-392). Here, we report that PLCzeta remains functional after proteolytic cleavage at the X-Y linker region. We found that N-terminal (33 and 37 kDa) and C-terminal fragments (27 kDa), presumably the result of PLCzeta cleavage at the X-Y linker region, were present in fresh sperm as well as in sperm extracts and remained associated as functional complexes. Protease V8 cleaved 72-kDa PLCzeta into 33/37 and 27 kDa fragments, while PLC activity and [Ca2+]i oscillation-inducing activity persisted until degradation of the fragments. Immunodepletion or affinity depletion of these fragments abolished PLC activity and [Ca2+]i oscillation-inducing activity from sperm extracts. Lastly, co-expression of cRNAs encoding residues 1-361 and 362-647 of mouse PLCzeta, mimicking cleavage at the X-Y linker region, induced [Ca2+]i oscillations and embryo development in mouse eggs. Our results support the hypothesis that PLCzeta is the sole mammalian sperm factor and that its linker region may have important regulatory functions during mammalian fertilization.
Kurokawa
Manabu
M
Paige Laboratory, Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003, USA.
Yoon
Sook Young
SY
Alfandari
Dominique
D
Fukami
Kiyoko
K
Sato
Ken-ichi
K
Fissore
Rafael A
RA
D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D002417:Cattle; D002522:Chlorocebus aethiops; D047108:Embryonic Development / Q000187:drug effects; D005260:Female; D005306:Fertilization / Q000187:drug effects / Q000502:physiology*; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D008297:Male; D051379:Mice; D010063:Ovum / Q000166:cytology / Q000187:drug effects; D010446:Peptide Fragments / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology; D054801:Phosphoinositide Phospholipase C / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D018075:RNA, Complementary; D012697:Serine Endopeptidases / Q000494:pharmacology; D013094:Spermatozoa / Q000187:drug effects / Q000201:enzymology; D013347:Subcellular Fractions / Q000187:drug effects; D013552:Swine
D010446:Peptide Fragments; D010455:Peptides; D018075:RNA, Complementary; D054801:Phosphoinositide Phospholipase C; C499234:Plcz1 protein, mouse; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D002118:Calcium