JOURNAL OF ULTRASTRUCTURE AND MOLECULAR STRUCTURE RESEARCH 96, 12-21 (1986) Localization and Distribution of Actin in Mammalian Sperm Heads C.LORA-LAMIA, L. CASTELLANI-CERESA, F. ANDREETTA, F. COTELLI, AND M. BRIVIO Department of Biology, University of Milan, Italy Received January 7, 1987 Actin was identified in boar and mole spermatozoa by utilizing indirect immunofluorescence, immunoelectron microscopy, and SDS-PAGE, followed by blot and screening with an anti-actin monoclonal antibody. Actin was detected in two places in the sperm head: the equatorial segment of the acrosome and the postacrosomal region. The protein was present in a nonfilamentous form and was localized under the plasma membrane. A small amount of actin was also detected in the sperm tail. The function of actin in the sperm head is discussed. o 1986 Academic PESS, IX. Actin is a ubiquitous protein that also seems to be present in sperm cells from in- vertebrates to mammals, including man. Its role in the acrosome reaction has been dem- onstrated only in some marine inverte- brates, where this protein represents the major constituent of the subacrosomal ma- terial (Tilney, 1975). The presence of actin in mammalian sperm has been demonstrat- ed in several species, including hamster (Clarke and Yanagimachi, 1978; Talbot and Kleve, 1978) dog, rabbit, mouse and guinea pig (Clarke and Yanagimachi, 1978), rat and turkey (Campanella et al., 1979), man (Clarke and Yanagimachi, 1978; Campa- nella et al., 1979; Clarke et al., 1982; Vir- tanen et al., 1984) boar (Tamblyn, 1980) rabbit (Welch and O’Rand, 1985), plains mouse (Flaherty et al., 1983), mole (Cas- tellani-Ceresa et al., 1984, 1986) and boar (Camatini et al., 1986; Castellani-Ceresa et al., 1986) (Table 1). These studies were car- ried out largely with immunofluorescence techniques using poly- and/or monoclonal antibodies, or deoxyribonuclease I. Only a few studies have been performed at the ul- trastructural level. Moreover, in the past few years biochemical techniques involving SDS-PAGE followed by Western blot have been tested on whole sperm or partially sol- ubilized sperm preparations using an anti- actin antibody (Ochs and Wolf, 1985). Al- 12 0889-1605186 $3.00 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved. though these studies confirmed the presence of an actin-like protein in mammalian sperm, the localization of this protein was not conclusive, since the various authors who examined it were not completely in accord about the sperm region to which anti- actin antisera bind. To clarify this situation, we used immu- nofluorescence and immunoelectron mi- croscopy techniques to localize actin in the sperm heads of mole and boar. Further- more, because of the signal found by several authors (using immunofluorescence tech- niques) in the main piece of the tail, we prepared a fraction containing tails only. The fraction obtained was analyzed by SDS- PAGE followed by immunoblotting with an anti-actin antibody, and the presence of ac- tin was revealed by HRP-conjugated IgG. Whereas actin is probably important as a cytoskeletal component during morphoge- netic processes, very little is known about the possible role of this protein during the fertilization process in mammals. MATERIALS AND METHODS Epididymal sperms of mole were obtained according to Castellani-Ceresa et al. (1986). Boar-ejaculated sperms were from Ente Lombard0 Potenziamento Zootecnico (Zorlesco, Milan, Italy). For all techniques used, the sperms were washed twice by suspension in phosphate-buffered saline (PBS) according to Toku-