Cryopreservation-induced acrosomal vesiculation in live spermatozoa from cynomolgus monkeys (Macaca fascicularis).
BACKGROUND
Cryopreserved spermatozoa are known to undergo accelerated capacitation and require a shorter incubation time for fertilization. However, details of their acrosomal membranes following cryopreservation remain unclear.


METHODS
Percoll density gradient centrifugation was used to remove dead spermatozoa; thus >90% live spermatozoa were recovered after cryopreservation, and acrosomal status was compared among non-incubated and incubated fresh and cryopreserved spermatozoa.


RESULTS
Transmission election microscopy (TEM) using microwave methods and fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) staining revealed that 21.1 and 61.6% respectively of non-incubated, cryopreserved spermatozoa were intact, whereas 97.6% (TEM) or 91.9% (FITC-PSA) of non-incubated fresh spermatozoa were intact. TEM revealed that 28.8% of the cryopreserved spermatozoa were swollen, and probably included among those counted as intact by FITC-PSA staining. The non-incubated cryopreserved spermatozoa had fused plasma and outer acrosomal membranes, and 36.4% of them had vesiculation when observed by TEM. FITC-PSA staining indicated that 22% of the live spermatozoa were acrosome reacted.


CONCLUSIONS
Acceleration of the acrosome reaction was evident by both TEM and FITC-PSA. Incubation of cryopreserved spermatozoa for 2 h accelerated vesiculation to a state similar to that of fresh spermatozoa that had been incubated for 8 h. These results reveal that in cryopreserved spermatozoa, the process of acrosome reaction begins before incubation.
Okada
A
A
Department of Anatomy, Toho University School of Medicine, 5-21-16 Omori Nishi, Ota-ku, Tokyo 143-8540, Japan. okada2da@med.toho-u.ac.jp
Igarashi
H
H
Kuroda
M
M
Terao
K
K
Yoshikawa
Y
Y
Sankai
T
T
D000177:Acrosome / Q000502:physiology* / Q000648:ultrastructure; D020101:Acrosome Reaction / Q000502:physiology; D000818:Animals; D015925:Cryopreservation; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D037102:Lectins; D008252:Macaca fascicularis; D008297:Male; D008854:Microscopy, Electron; D037121:Plant Lectins; D013094:Spermatozoa / Q000502:physiology* / Q000648:ultrastructure; D013997:Time Factors
D005456:Fluorescent Dyes; D037102:Lectins; D037121:Plant Lectins; C036292:pea lectin; D016650:Fluorescein-5-isothiocyanate