Heparin and dermatan sulphate induced capacitation of frozen-thawed bull spermatozoa measured by merocyanine-540.
Glycosaminoglycans (GAGs) are present in the oviduct in which the major part of sperm capacitation occurs. In this study we have tested how capacitation of frozen-thawed bull spermatozoa is effected by exposure to different GAGs detectable or possibly present in oviductal fluid; i.e. heparin, hyaluronan, heparan sulphate, dermatan sulphate and chondroitin sulphate. Following exposure of different duration, the spermatozoa were stained with either Chlortetracycline (CTC) or merocyanine-540 and evaluated with epifluorescent light microscopy or flow cytometry, respectively. Heparin elicited a significant increase in the number of alive, capacitated spermatozoa, either expressed as higher merocyanine-540 fluorescence (p < 0.0001) or as B-pattern (p = 0.0021) in the CTC assay, during 4 h of incubation. When comparing the different GAG treatments one by one to the negative control in the flow cytometric study, only heparin and dermatan sulphate were significant (p < 0.0001) higher than the control at 0-30 min of incubation. Duration of incubation did not affect the proportion of capacitated spermatozoa when measured as merocyanine-540 fluorescence or CTC B-pattern, but the length of the incubation did affect the number of dead (Yo-PRO 1 positive) spermatozoa (p < 0.0001). Exposure to zona pellucida proteins significantly increased the proportion of acrosome reacted spermatozoa (p = 0.016). Both heparin and dermatan sulphate induce capacitation of frozen-thawed bull spermatozoa in vitro.
Bergqvist
A-S
AS
Division of Comparative Reproduction, Obstetrics and Udder Health, Department of Clinical Sciences, Faculty of Veterinary Medicine and Animal Science, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden. ann-sofi.bergqvist@kv.slu.se
Ballester
J
J
Johannisson
A
A
Lundeheim
N
N
Rodríguez-Martínez
H
H
D020101:Acrosome Reaction; D000818:Animals; D002417:Cattle; D002751:Chlortetracycline / Q000378:metabolism; D003871:Dermatan Sulfate / Q000494:pharmacology*; D004527:Egg Proteins / Q000378:metabolism; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000032:analysis*; D006493:Heparin / Q000494:pharmacology*; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism; D011744:Pyrimidinones / Q000032:analysis*; D011956:Receptors, Cell Surface / Q000378:metabolism; D013075:Sperm Capacitation / Q000187:drug effects* / Q000502:physiology; D013094:Spermatozoa / Q000187:drug effects* / Q000502:physiology; D000072216:Zona Pellucida Glycoproteins
D004527:Egg Proteins; D005456:Fluorescent Dyes; D008562:Membrane Glycoproteins; D011744:Pyrimidinones; D011956:Receptors, Cell Surface; D000072216:Zona Pellucida Glycoproteins; D003871:Dermatan Sulfate; C003954:merocyanine dye; D006493:Heparin; D002751:Chlortetracycline