Human Reproduction, Vol. 16, No. 10, 2139-2147,
October 2001
© 2001 European Society of Human Reproduction and Embryology
Cryopreservation-induced acrosomal vesiculation in live spermatozoa from cynomolgus monkeys (Macaca fascicularis)
1 Department of Anatomy, Toho University School of Medicine, Ota-ku, Tokyo and 2 Tsukuba Primate Center, National Institute of Infectious Diseases, Tsukuba, Ibaraki, Japan
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.
Key words: acrosome reaction/cryopreservation/cynomolgus monkey/electron microscopy/spermatozoa
3 Present address: Department of Medical Science, Faculty of Agriculture, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,Tokyo 113-8657, Japan
4 To whom correspondence should be addressed at: Department of Anatomy, Toho University School of Medicine, 5-21-16 Omori Nishi, Ota-ku, Tokyo 143-8540, Japan. E-mail: okada2da{at}med.toho-u.ac.jp
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