Motility initiation is a key event during internal fertilization of female-stored sperm, although the underlying mechanisms remain unclear. In internally fertilizing urodeles, quiescent sperm initiate motility on the surface of the egg-jelly, a thick extracellular matrix that accumulates around the egg in oviduct. By immunizing mice with egg-jelly extracts, we successfully generated an α34 monoclonal antibody (mAb) which neutralized sperm motility-initiating activity in the eggjelly of the newt, Cynops pyrrhogaster, in a dose-dependent manner. The α34 mAb recognized an unglycosylated 34 kDa protein in the outermost of the six layers that comprise egg-jelly. Under nonreducing conditions, immunoblotting with α34 mAb produced many bands in addition to the 34 kDa protein, suggesting that the 34 kDa protein associates not only with the jelly matrix itself, but also with additional substances present in the matrix. Our current results are compatible with the supposed features of sperm motility-initiating substance (SMIS), indicating that the 34 kDa protein itself, or a complex consisting of the 34 kDa protein and some other molecules, is the SMIS in C. pyrrhogaster. Immunofluorescence staining further indicated that SMIS was distributed in a dot-like pattern in the outermost jelly layer and was fully covered with acrosome reactioninducing substance (ARIS). Immunocytochemical and scanning electron microscopic examinations of the outermost jelly layer strongly suggests that the 34 kDa protein localized in granules (2 μm) and that ARIS was distributed covering the granules and in the sheet-like structure above the granules. These data suggest that the initiation of sperm motility is mediated by the acrosome reaction.

Localisation of the acrosome reaction inducing activity in egg-jelly was examined in the newt, Cynops pyrrhogaster. The jelly has six layers: the J0, J1, J2, J3, J4 and st layers. Jelly was mechanically dissected and placed on a Millipore filter. When sperm were added from the outer surface side of the jelly, most of them exhibited the acrosome reaction after passing through the jelly. When egg-jelly was divided into four layers, strong activity for the induction of acrosome reaction was detected in the outer layers, J4+st. These findings suggest that the acrosome reaction is induced by a substance in the outer layers of the egg-jelly. Among jelly components separated by SDS-PAGE, a fraction of more than 500 kDa in molecular weight induced the acrosome reaction. Wheat germ agglutinin (WGA), Griffonia simplicifoliar agglutinin 1 (GS-1), Maclura pomifera agglutinin (MPA) and Arachis hypogaea agglutinin (PNA) inhibited the induction of the acrosome reaction by jelly extract, and WGA did so in a dose-dependent manner. Those lectins precipitated some molecules of over 500 kDa. These results suggest that the acrosome reaction is induced by the high molecular-weight components of egg-jelly in C. pyrrhogaster.

Egg-jelly is composed of a network of fibrous components and contains substances regulating the sperm-egg interaction. Many studies on the latter have been conducted, whereas the role of the egg-jelly structure in fertilization has not yet been fully assessed. In this study, we examined the fertilization efficiency in the presence and absence of the structure around the egg of the newt, Cynops pyrrhogaster, using a gelatin gel system. Although de-jellied eggs of C. pyrrhogaster can be fertilized with an adequate number of sperm, the fertilization rate was dramatically increased through the use of the gelatin gel. Sperm showed forward motility with straight morphology in the gel, whereas they swam in circles in solution. This result indicates that the gel structure is significant for sperm guidance to the egg surface, and its presence raises the fertilization efficiency in C. pyrrhogaster. When sperm were entangled in the gel structure, they were immediately folded and never showed any forward motility. Sperm with zigzag morphology were observed in the gelatin gel as well as in the egg-jelly, indicating the elimination of sperm by the gel structure. The effect of sperm elimination on successful fertilization was estimated using gelatin gels of different thickness. Though the variation did not affect the fertilization rate, the rate of normal development gradually increased in the thicker gels. This result indicates that sperm elimination in egg-jelly can function in the fertilization system. The roles of sperm guidance and sperm elimination under the physiological condition of internal fertilization of the newt are discussed.

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This study examined the effect of carbohydrate drink ingestion timing on gastrointestinal tract blood flow and motility after mild cycling exercise. Eight healthy participants were randomly assigned to ingest a liquid solution with 75 g glucose at either 5 min (PE-5) or 30 min (PE-30) after a single bout of leg cycling exercise according to target heart rate (approximately 120 beats/min). As the control trial (Con), participants ingested the same liquid solution without exercise. Celiac artery blood flow (BF), superior mesenteric artery BF, and gastric emptying rate were assessed by ultrasonography before and for 60 min after ingesting the glucose solution. Blood lactate, glucose, and plasma insulin were also measured at baseline and for 60 min after ingesting the glucose solution. Celiac artery BF significantly decreased from resting baseline immediately after exercise in both the PE-5 and PE-30 trials, and then returned to resting baseline just before the ingestion of glucose solution in the PE-30 trial. After ingesting the glucose solution, changes in celiac artery BF, superior mesenteric artery BF, % gastric emptying rate, blood lactate, blood glucose, and plasma insulin were not significantly different among the three trials. The timing of nutrient ingestion after mild exercise does not seem to impact the subsequent gastrointestinal motility, blood flow, and glycemic responses.