Oocyte specific oolemmal SAS1B involved in sperm binding through intra-acrosomal SLLP1 during fertilization.
Molecular mechanisms by which fertilization competent acrosome-reacted sperm bind to the oolemma remain uncharacterized. To identify oolemmal binding partner(s) for sperm acrosomal ligands, affinity panning was performed with mouse oocyte lysates using sperm acrosomal protein, SLLP1 as a target. An oocyte specific membrane metalloproteinase, SAS1B (Sperm Acrosomal SLLP1 Binding), was identified as a SLLP1 binding partner. cDNA cloning revealed six SAS1B splice variants, each containing a zinc binding active site and a putative transmembrane domain, with signal peptides in three variants. SAS1B transcripts were ovary specific. SAS1B protein was first detected in early secondary follicles in day 3 ovaries. Immunofluorescence localized SAS1B to the microvillar oolemma of M2 oocytes. After fertilization, SAS1B decreased on the oolemma and became virtually undetectable in blastocysts. In transfected CHO-K1 cells SAS1B localized to the surface of unpermeabilized cells. Recombinant and native SLLP1 co-localized with SAS1B to the microvillar domain of ovulated M2 oocytes. Molecular interactions between mouse SLLP1 and SAS1B were demonstrated by surface plasmon resonance, far-western, yeast two-hybrid, recombinant- and native-co-IP analyses. SAS1B bound to SLLP1 with high affinity. SAS1B had protease activity, and SAS1B protein or antibody significantly inhibited fertilization. SAS1B knockout female mice showed a 34% reduction in fertility. The study identified SAS1B-SLLP1 as a pair of novel sperm-egg binding partners involving the oolemma and intra-acrosomal compartment during fertilization.
Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VA 22908, USA.
Monika
M
Sachdev
Arabinda
A
Mandal
Sabine
S
Mulders
Laura C
LC
Digilio
Subbarayalu
S
Panneerdoss
Viswanadhapalli
V
Suryavathi
Eusebio
E
Pires
Kenneth L
KL
Klotz
Laura
L
Hermens
María Belén
MB
Herrero
Charles J
CJ
Flickinger
Marcel
M
van Duin
John C
JC
Herr
D000177:Acrosome / Q000378:metabolism; D017398:Alternative Splicing; D000818:Animals; D001667:Binding, Competitive; D026481:Blotting, Far-Western; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D005306:Fertilization; D047468:Immunoprecipitation; D007519:Isoantigens / Q000235:genetics / Q000378:metabolism*; D008297:Male; D045726:Metalloproteases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008813:Mice, Inbred ICR; D018345:Mice, Knockout; D009865:Oocytes / Q000378:metabolism*; D011247:Pregnancy; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D029589:Seminal Plasma Proteins / Q000235:genetics / Q000378:metabolism*; D013084:Sperm-Ovum Interactions; D020349:Surface Plasmon Resonance; D020798:Two-Hybrid System Techniques
D007519:Isoantigens; D020033:Protein Isoforms; C506517:SLLP1 protein, mouse; D029589:Seminal Plasma Proteins; C487827:Astl protein, mouse; D045726:Metalloproteases