Morphological and structural changes of sperm during the acrosome reaction in the swimming crab Portunus trituberculatus

The acrosome reaction (AR) of sperm from the swimming crab Portunus trituberculatus was induced artificially by treatment with ionophore A23187 (40 μg/ml) or egg-water. Female swimming crabs were collected from Shipu Port (Xiangshan, Zhejiang). The crabs were raised in a pond with aerated fresh sea water and fed with shellfish in March-April 2003. Sperm suspensions were prepared by squeezing and agitating the sperm pellets removed from the seminal receptacles. The sperm suspensions were used for artificial induction of the acrosome reaction. For collecting the egg-water, the ripe ovaries of the female crab which initiate to ovulate were teased into several fragments and soaked in 200 ml of filtered seawater at 4℃ for 24 h. 75.33% and 84.83% of acrosome reaction rate were achieved by treating the sperm with ionophore A23187 and egg-water respectively, and provided a sufficient number of reacted sperm to examine. Egg-water is a more effective and cheaper inducer of the acrosome reaction as compared with ionophore A23187. The morphological and structural changes of sperm during the acrosome reaction in P.trituberculatus were studied in detail with light microscope (LM), scanning electron microscope (SEM) and transmission electron microscope(TEM). The mature sperm in the seminal receptacle has a top-like shape, and consists of an acrosome (A), nuclear cup (N) and (5-10) radial arms (RA) which extend from the nucleus. The spherial acrosome is comprised of acrosomal vesicle (AV) and acrosomal tubule (AT). The acrosomal vesicle surrouding the perforatorium (P) contains inner acrosomal material (IM), outer acrosomal material (OM), apical cap (AC) and subcap zone (SZ), and is limited by acrosomal vesicle membrane (AVM). The umbrella-like apical cap has approximately 70 rays radiating out of the acrosome vesicle membrane. So the apical cap is the only site liable to rupture to form an aperture. The process of acrosome reaction consists of four consecutive stages: (1) swelling of the apical cap and rupturing to form an aperture due to hydration; (2) initial release of the acrosomal material through the aperture and eversion of the acrosomal vesicle; (3) elongation of the perforatorium and subsequent reversal of the acrosomal vesicle membrane; and (4) separation of the acrosomal material, shedding of the acrosomal vesicle membrane at the thinkened ring (TR) and formation of the acrosomal filament (AF). The acrosomal filament consists of the perforatorium being the leading edge of the nail-like reacted sperm and the proximal piece (PP) deriving from the cytoplasmic lamellar complex (LC) of the unreacted sperm. During the acrosome reaction, the most noteworthy difference between the sperm of P.trituberculatus and that of Eriocheir sinensis or Scylla serrata is the behavior of the radial arms. In P.trituberculatus, the contraction of the radial arms can’t be observed until the 4th stage of the acrosome reaction. But in E.sinensis and S.serrata, the acrosome reaction starts with the contraction of the radial arms. Taking the nucleus as the datum line, the net forward movement of the perforatorium is about 2.5 μm during the acrosome reaction. The functions of the radial arm and the forward movement of the perforatorium in the penetration of sperm through the chorion are discussed .