Effect of cryopreservation and oviductal cell conditioned media on Ca2+ flux of equine spermatozoa.
Movement of Ca2+ into spermatozoa is a critically important event for capacitation and the acrosome reaction. In the present study, the nature of Ca2+ movement in fresh equine spermatozoa was established and the effects of oviductal cell conditioned medium (OCM) and cryopreservation on Ca2+ flux were investigated. The ability of fresh and cryopreserved stallion spermatozoa to regulate Ca2+ concentration over time was evaluated in Ca2+ -free PBS. Intracellular Ca2+ concentrations were higher in cryopreserved spermatozoa than in fresh spermatozoa. However, extracellular Ca2+ concentrations were higher in fresh spermatozoa than in cryopreserved spermatozoa. Both fresh and cryopreserved spermatozoa took in 1 mmol exogenous Ca2 l(-1) immediately and rapidly, reaching a plateau in <5 min. The rate of Ca2+ internalization did not differ between fresh and cryopreserved spermatozoa. Oviductal epithelial cells from non-pregnant mares were incubated in TCM-199 and the OCM was harvested after 48 h to evaluate the effect of the OCM on sperm Ca2+ flux. Spermatozoa were incubated in either OCM or TCM-199 for 1 h before microscopic evaluation and Ca2+ concentration determination in PBS. Cryopreserved spermatozoa had higher intracellular and lower extracellular Ca2+ concentrations than did fresh spermatozoa, but incorporated Ca2+ at a slower rate than fresh spermatozoa. OCM incubation increased the relative intracellular Ca2+ concentration in fresh but not cryopreserved spermatozoa compared with spermatozoa incubated in TCM-199 (P < 0.01). In summary, oviductal cell products affect Ca2+ internalization by equine spermatozoa. Cryopreservation strongly affects all aspects of sperm Ca2+ control, regardless of other factors. These results indicate that the high intracellular Ca2+ concentrations of cryopreserved stallion spermatozoa may be one of the reasons why mares must be inseminated very close to ovulation to maximize pregnancy rates using cryopreserved semen.
Leopold
S
S
Equine Research Centre, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Samper
J C
JC
Curtis
E
E
Buhr
M M
MM
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015925:Cryopreservation / Q000662:veterinary*; D017077:Culture Media, Conditioned / Q000494:pharmacology*; D005260:Female; D006736:Horses / Q000502:physiology; D008297:Male; D010057:Oviducts / Q000502:physiology*; D012662:Semen Preservation / Q000662:veterinary*; D013094:Spermatozoa / Q000502:physiology*
D017077:Culture Media, Conditioned; D002118:Calcium