Chemical N-desulfation of heparin negates its ability to capacitate bovine spermatozoa.
Heparin specifically and saturably binds to bovine spermatozoa and stimulates capacitation as assessed by the ability of spermatozoa to undergo a zona pellucida-induced acrosome reaction (AR) in vitro. However, the structural features of heparin important for capacitation are poorly understood. The purpose of this study was to determine the importance of the sulfate content of heparin for its potency to bind to bull spermatozoa and promote agglutination and capacitation. The pyridine salt of heparin was N-desulfated, which reduced its mean sulfate content from 19.7% to 11.6%. The N-desulfated heparin was then resulfated by incubation with trimethylamine sulfur trioxide for 6, 12, or 24 hr, raising sulfate to original concentrations. Heparin but not N-desulfated heparin competed with [3H]-heparin to bind to spermatozoa. Heparin at 11.6 micrograms/ml reduced [3H]-heparin binding by half when competing with a saturating concentration of the radiolabeled compound (12 micrograms/ml). N-desulfated heparin did not displace [3H]-heparin. Heparin, resulfated 6 hr or 12 hr, was equal to native heparin in binding potency. Heparin at 50, 100, or 250 micrograms/ml caused more than 40% of the cells to head-to-head agglutinate in aggregates of 8 or more. N-desulfated heparin did not cause agglutination. After spermatozoa were incubated with 0, 5, 10, 50, 100, or 250 micrograms/ml of heparin for 4 hr, 100 micrograms/ml of lysophosphatidylcholine (LPC)-induced AR within 20 min in 21.3, 37.7, 27.8, 45.3, 54.2, or 42.5% of the cells, respectively. Sperm exposed to the same concentrations of N-desulfated heparin exhibited AR of 17.7, 27.3, 24.3, 22.5, 27.7, or 33.8%, respectively, following exposure to LPC. Resulfated heparin did not agglutinate or capacitate spermatozoa. In conclusion, N-desulfation of heparin abolished heparin's ability to bind to, agglutinate, and capacitate bovine spermatozoa. Resulfation of N-desulfated heparin restored binding activity but not agglutination or capacitation activity.
Miller
D J
DJ
Endocrinology-Reproductive Physiology Program, University of Wisconsin-Madison 53706.
Ax
R L
RL
D000177:Acrosome / Q000166:cytology; D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006493:Heparin / Q000378:metabolism / Q000494:pharmacology*; D066298:In Vitro Techniques; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D008297:Male; D013075:Sperm Capacitation / Q000187:drug effects*; D013431:Sulfates / Q000378:metabolism*
D008244:Lysophosphatidylcholines; D013431:Sulfates; D006493:Heparin