Recombinant human zona pellucida glycoprotein 3 induces calcium influx and acrosome reaction in human spermatozoa.
Recombinant human ZP3 (rhuZP3) generated by Chinese hamster ovary cells transfected with a plasmid containing human ZP3 cDNA was used to study the acrosome reaction (AR) and intracellular calcium fluxes in capacitated human spermatozoa. Conditioned medium containing rhuZP3 significantly induced the AR (P < or = 0.005) in 59.4 +/- 4.7% of spermatozoa (control = 8.5 +/- 3.1%) and caused complete acrosomal loss in a further 17.2 +/- 3.8% of cells (control = 3.7 +/- 0.7%; mean +/- SEM, n = 5). Sperm motility was not affected and acrosomal exocytosis in response to rhuZP3 was also shown to be time-dependent. Basal concentrations of sperm intracellular calcium were measured (82 +/- 7 nM; mean +/- SEM, n = 9). A transient increase in intracellular calcium (typically up to 400-450 nM) occurred within 1 min of rhuZP3 addition and was followed by sustained lower values of calcium (200-400 nM). These responses were dependent on the amount of rhuZP3. This is the first report of zona protein-induced changes in intracellular calcium levels in human spermatozoa. The results support the premise that ZP3 is an agonist of the human sperm AR and that rhuZP3 generated in a eukaryotic cell is effective in this respect.
Brewis
I A
IA
Department of Molecular Biology and Biotechnology, University of Sheffield, UK.
Clayton
R
R
Barratt
C L
CL
Hornby
D P
DP
Moore
H D
HD
D000177:Acrosome / Q000187:drug effects*; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D006224:Cricetinae; D017077:Culture Media, Conditioned; D004527:Egg Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D007424:Intracellular Fluid / Q000378:metabolism; D017136:Ion Transport / Q000187:drug effects; D008297:Male; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D015398:Signal Transduction; D013075:Sperm Capacitation; D013094:Spermatozoa / Q000187:drug effects* / Q000378:metabolism*; D000072216:Zona Pellucida Glycoproteins
D017077:Culture Media, Conditioned; D004527:Egg Proteins; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C000606517:ZP3 protein, human; D000072216:Zona Pellucida Glycoproteins; D002118:Calcium