A novel terminal ampullae peptide is involved in the proteolytic activity of sperm in the prawn, Macrobrachium rosenbergii

As the distal part of the crustacean male reproductive tract, terminal ampullae play important roles in sperm development and storage of mature spermatophores. In the present study, the novel gene terminal ampullae peptide (TAP) was cloned from terminal ampullae of the prawn, Macrobrachium rosenbergii. The cDNA sequence consists of 768 nucleotides, with an open-reading frame of 264 nucleotides which encodes a putative 88-amino acid precursor protein with a 17-amino acid residue signal peptide. Western blotting and immunohistochemical analysis revealed that TAP was distributed on terminal ampullae and sperm, and its expression was related to gonad development. To elucidate the functional role of TAP in vivo, we disrupted the TAP gene by RNA interference (RNAi) and evaluated the effect on fertility and several sperm parameters. Although there was no difference in fertility between RNAi-induced prawns and controls, RNAi treatment decreased the sperm gelatinolytic activity and blocked proteolytic activity on the vitelline coat. These data provide evidence that TAP participates in regulating sperm proteolytic activity, and performs a crucial role in sperm maturation and degradation of the vitelline coat during fertilization.

Distribution and Function of Filamentous and Globular Actin in Mitochondrial Translocation During Ascidian Sperm Activation

During fertilization in the sea squirt, Ascidia ceratodes,the penetration process of requires mitochondrial translocation, a process that defines sperm activation in this species, to generate the driving force for passage through the vitelline coat. When a sperm cell come into contact with an egg, the mitochondrion translocates off the head and migrates along the tail via an actimmyosin-dependent process. The presence of actin and myosin on the head and tail has been demonstrated. However, the nature of the relative distribution of filamentous actin (F-actin) and monomeric actin (G-actin) has not been thoroughly studied. And, whereas the signaling cascade that leads to myosin activation has been studied, the events leading to actin polymerization and whether it is required for mitochondrial translocation has not.This project addresses two research questions: What is the distribution of F-actin and G-actin in unactivated and activated sperm cells? And, can preexisting F-actin support mitochondrial translocation or must actin polymerization occur simultaneously with the mitochondrial translocation process?

Three new components contained in the vitelline coat of Tegula pfeifferi

The vitelline coat (VC) lysin of Tegula, a marine molluscan genus, is released from the acrosome of sperm during fertilisation and can lyse the VC of only the same species. The lytic action of this lysin against the VC is not an enzymatic reaction, but a stoichiometric and irreversible one (Haino-Fukushima, 1974).The VC of Tegula pfeifferi consists of glycoproteins containing sulphated polysaccharides, which account for roughly two-thirds of the entire weight of the VC. The presence of a large quantity of polysaccharides in the VC had prevented rapid progress in the analysis of its protein components. Last year, we succeeded in a complete solubilisation of the VC by boiling for a long time in 1% SDS solution, and determined the cDNA sequence coding for a mature 41 kDa glycoprotein, which appears to be the major component of the VC from the results of SDS-polyacrylamide gel electrophoresis (PAGE). The cDNA, referred to as vcp41, comprises 1072 base pairs and contains one open reading frame with a sequence for 319 amino acids containing 19 amino acids of a signal peptide. The deduced amino acid sequence has five N-glycosylation sites and ten cysteine residues. It seems that almost 7 kDa in this 41kDa glycoprotein is polysaccharide constituents (Fan & Haino-Fukushima, 1998).