PLATFORM SESSION 2. MOLECULAR SIGNALING EVENTS IN THE MALE REPRODUCTIVE TRACT
Sunday, August 1, 2004
3:00 PM–5:00 PM
Chair: Jaques Tremblay
Co-Chair: Maureen McAuliff
(10) STUDIES TO UNDERSTAND THE MECHANISM BY WHICH GUINEA PIG FERTILIN IS KEPT IMMOBILIZED WITHIN THE MEMBRANE PRIOR TO CAPACITATION.
Hunnicutt, Gary1, Galatioto, Josephine2, Vargas, Louis3, Kwitny, Susanna1, 1 Population Council, New York, NY2 SUNY Downstate Medical Center, Brooklyn, NY3 Memorial Sloan-Kettering Cancer Center, New York, NY
ABSTRACT- Fertilin is a sperm protein involved in fertilization. It is heterodimeric transmembrane sperm protein composed of an (ADAM 1) and (ADAM 2) subunit and located in the posterior head domain (PHD) of cauda sperm. By FRAP analysis we showed that guinea pig fertilin is restricted from lateral movement suggesting it is anchored. During capacitation, however, fertilin becomes freely mobile within the PHD and stays this way even after the acrosome reaction. We wanted to determine how fertilin is held motionless within the membrane prior to capacitation. Unfortunately, detergent extracts of both cauda and acrosome-reacted sperm similarly resulted in fertilin partitioning to the supernatant fractions. IPs of fertilin from biotinylated sperm also failed to identify a binding partner(s). To determine if immobilized fertilin is trapped within lipid rafts, we isolated rafts but found that fertilin did not co-localize to these fractions. Correspondingly, immunofluorescence for caveolin-1 showed it to be localized to the anterior head and tail of cauda sperm, but not to the PHD. Turning to the yeast two-cell hybrid system, we made and screened a guinea pig testis library using the cytoplasmic tail of fertilin as bait. Because fertilin becomes released during capacitation, we hypothesized that an anchoring partner might have both calcium binding and phosphorylative properties. Of the 155 clones identified, 70 showed compelling bind and were sequenced. Around 50 clones were false positives, leaving ∼20 clones of interest. Based upon its sequence, one clone was similar to a calcium binding, tyrosine phosphorylation regulating protein (CABYR) that becomes active at capacitation, thus fitting our initial hypothesis for a potential fertilin binding partner. Antibodies to the CABYR-like protein are being generated and in vitro protein-protein and co-localization confirming studies are underway. These results will contribute significantly to our understanding of the mechanisms restraining fertilin movement prior to capacitation.
KEY WORDS: fertilin, capacitation, sperm, adam