Flow cytometric detection of bicarbonate-induced changes in lectin binding in boar and ram sperm populations.
Boar and ram spermatozoa were incubated in Tyrode's medium in the presence or absence of bicarbonate/CO2, a component believed essential for capacitation. At intervals, samples were stained with a range of FITC-lectins to detect changes in surface glycoconjugates, using a rapid staining technique to avoid problems of lectin toxicity. The samples were then analysed directly by flow cytometry, using propidium iodide to distinguish dead cells. In the presence of bicarbonate, a live subpopulation of spermatozoa developed, which in both animal species showed higher binding affinities towards Phaseolus Vulgaris Agglutinin (PHA-E), Sophora Japonica Agglutinin (SJA), and Soybean Agglutinin (SBA), and lower binding affinity towards Erythrina Cristagalli Lectin (ECL). In boar samples, the modified subpopulation reached a maximum after 3 hr incubation, whereas in ram samples it maximized after 1.5 hr. No changes were seen when spermatozoa were incubated in bicarbonate-free medium. The bicarbonate-induced changes in lectin binding were not due to the onset of acrosome reactions, because spermatozoa induced to undergo acrosome reactions with the ionophore A23187 displayed very different lectin-binding patterns. Tested on boar spermatozoa, seminal plasma not only inhibited but reversed the bicarbonate-induced development of the modified subpopulation. EGTA also inhibited development of boar sperm subpopulations; excess Ca2+ was unable to overcome this inhibition, suggesting that multivalent metal ions might be involved in bicarbonate's action. We conclude that bicarbonate causes a loss of surface coating material with affinity for ECL and an unmasking of binding sites for SBA, SJA and PHA-E. A modified subpopulation of live spermatozoa is thereby established, which appears to maximize at a rate in accord with reported capacitation times.
Ashworth
P J
PJ
Department of Veterinary Basic Sciences, Royal Veterinary College, London, United Kingdom.
Harrison
R A
RA
Miller
N G
NG
Plummer
J M
JM
Watson
P F
PF
D000818:Animals; D001639:Bicarbonates / Q000494:pharmacology*; D000001:Calcimycin / Q000494:pharmacology; D002240:Carbohydrate Sequence; D002241:Carbohydrates; D004533:Egtazic Acid / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods; D007700:Kinetics; D037102:Lectins; D008297:Male; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D012661:Semen / Q000502:physiology*; D012756:Sheep; D013075:Sperm Capacitation / Q000187:drug effects / Q000502:physiology*; D013094:Spermatozoa / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D013379:Substrate Specificity; D013552:Swine; D013997:Time Factors
D001639:Bicarbonates; D002241:Carbohydrates; D037102:Lectins; D000001:Calcimycin; D004533:Egtazic Acid