Abstract. In order to reveal the involvement of the sperm postacrosomal region in the acrosome reaction, we examined the effects of the protein phosphatase inhibitor calyculin A on the postacrosomal protein serine/threonine phosphorylation state and acrosome morphology in boar spermatozoa incubated with a cAMP analog. Proteins were highly phosphorylated on the serine/threonine residues only in the postacrosomal region before incubation. After 90-min incubation without calyculin A, the protein phosphorylation state declined in the postacrosomal region irrespective of the capacitation state while it remained under the detectable level in the other regions of the sperm head. However, addition of calyculin A effectively suppressed the decline in protein phosphorylation state and increased an inactive form of protein phosphatase 1 in the postacrosomal region. On the other hand, this inhibitor had no influence on the protein phosphorylation state in the acrosome and equatorial segment. After incubation without calyculin A for 180 or 360 min, many spermatozoa exhibited acrosomal changes and loss that indicated occurrence of the acrosome reaction. However, addition of calyculin A significantly blocked these events. These results are consistent with our suggestion that postacrosomal serine/threonine-phosphorylated proteins are involved in suppression of the acrosome reaction in boar spermatozoa in vitro. Key words: Acrosome, Boar, Calyculin A, Chlortetracycline, Postacrosomal region, Protein phosphatase (J. Reprod. Dev. 54: [171][172][173][174][175][176] 2008) n mammalian spermatozoa, the head is composed of the acrosome, equatorial segment and postacrosomal region. The acrosome has a unique structure of triple membranes (plasma, outer-acrosomal and inner-acrosomal membranes), and it is generally considered to play pivotal roles in fertilization with oocytes. Briefly, when the acrosome of capacitated spermatozoa interacts with the zona pellucida glycoprotein (ZP3) of oocytes, multiple fusions are induced between the plasma and outer-acrosomal membranes. Consequently, proteases are released from the acrosome and then digest the zona pellucida. This is an exocytosis "acrosome reaction" that enables spermatozoa to penetrate through the zona pellucida. Subsequently, the acrosome-reacted spermatozoa are bound to another zona pellucida glycoprotein (ZP2) to maintain the adhesion to the oocytes [1][2][3]. However, there are several controversial reports that suggest possible involvement of the postacrosomal region in the interaction with the zona pellucida and the intracellular calcium elevation during the acrosome reaction. Specifically, approximately 80% of the ZP3-binding sites have been found in the postacrosomal region of the mouse sperm head, although the remaining sites have been located in the acrosome. Moreover, ZP2-binding sites have only been detected in the postacrosomal region [4]. Investigation with a high-resolution imaging technique using a confocal laser scanning microscope revealed that intracellular calciu...

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