Changes of IZUMOI in bull spermatozoa during the maturation, acrosome reaction, and cryopreservation

To obtain basic information of bull IZUMOI (a sperm protein essential for sperm-egg fusion) and disclose possible causes for the impaired fertilizing ability in bull cryopreserved spermatozoa, we investigated this protein in bull spermatozoa collected from various regions of epididymides, freshly ejaculated spermatozoa, acrosome-reacted spermatozoa, and cryopreserved spermatozoa by Western blotting and the triple staining with the anti-IZUMO1 antibody, fluorescein isothiocyanate-peanut agglutinin, and 4',6-diamidino-2-phenylindole. In the cauda epididymal spermatozoa and freshly ejaculated spermatozoa, bull IZUMOI was detected mainly as a 45-kDa major form. This major form was derived probably from a 52-kDa precursor form in the epididymis. Bull IZUMOI was immunolocalized along the border between the principal and equatorial segments of the acrosomal region (pattern P1 of IZUMOI) in the most of epididymal and freshly ejaculated spermatozoa with normal acrosomes. In the samples after the treatments to induce the acrosome reaction, the percentages of spermatozoa without acrosomes and with IZUMOI in whole equatorial segment (pattern P2 of IZUMOI) significantly increased. These results indicate that bull IZUMOI undergoes maturation-related changes during sperm transit through the epididymis and that it is translocated to the equatorial segment of acrosomal region during the acrosome reaction. On the other hand, severe damages were observed in the acrosomes of 60% of the cryopreserved spermatozoa. Localization of IZUMOI in these spermatozoa was pattern P2 (IZUMOI in whole equatorial segment), P3 (IZUMOI in whole acrosomal region), or P4 (IZUMO was lost). Moreover, after the incubation to compare the stability of acrosomes and IZUMOI localization between cryopreserved spermatozoa and freshly ejaculated spermatozoa, much more spermatozoa lost acrosomes and IZUM01 in the cryopreserved samples compared with freshly ejaculated samples. These findings indicate that impaired fertilizing ability of bull cryopreserved spermatozoa with damaged acrosomes is related partially to the aberrant translocation of IZUMO1 which may be followed by the loss of intact IZUMOI. (C) 2016 Elsevier Inc. All rights reserved.