title: Cryopreservation-induced acrosomal vesiculation in live spermatozoa from cynomolgus monkeys (Macaca fascicularis).
abstract: 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.
authors.affiliation: 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
authors.forename: A
authors.initials: A
authors.lastname: Okada
authors.forename: H
authors.initials: H
authors.lastname: Igarashi
authors.forename: M
authors.initials: M
authors.lastname: Kuroda
authors.forename: K
authors.initials: K
authors.lastname: Terao
authors.forename: Y
authors.initials: Y
authors.lastname: Yoshikawa
authors.forename: T
authors.initials: T
authors.lastname: Sankai
mesh_terms: 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
chemical_list: D005456:Fluorescent Dyes; D037102:Lectins; D037121:Plant Lectins; C036292:pea lectin; D016650:Fluorescein-5-isothiocyanate