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.

No abstract

Spermatocytes of the teleost, Oryzias latipes, at meiotic prophase were cultured without contact with somatic cells. They began to divide, progressing through the meiotic divisions and differentiating into round spermatids within 48 h. The chromosome number in both the primary and secondary spermatocytes at metaphase was n = 24. In spermatids, a single flagellum was formed and the release of residual bodies was observed in vitro. The size and shape of the flagellum were the same as those seen in vivo. The expression of protamine mRNA was detected in round spermatids. This result suggests that gene expression, as well as morphological change, is regulated by the progression of spermatogenesis in cell culture. Furthermore, when the eggs of O. latipes were inseminated with germ cells cultured for 10 days, normal embryos developed and hatched out. These results suggest that the spermatocytes of O. latipes develop into fertile sperm in cell culture.

No abstract

The significance of egg-jelly layers in internal fertilization was evaluated in the newt, Cynops pyrrhogaster. In this species, six egg-jelly layers, J1, J2, J3, J4, J5 and the outermost J6 layers, are accumulated on the surface of the fertilizable eggs in pars convoluta of the oviduct. When a large number of sperm (about 6 x 10(5)) were placed on eggs having different numbers of jelly layers, all the eggs were fully fertilized, although many of the eggs developed abnormally. Upon insemination using about 600 sperm, only eggs with the full set of jelly layers were fertilized at a high rate with normal development. Since around 300 (the range of 48-1,192) sperm were observed on and in the egg-jelly in naturally spawned eggs, we conclude that the J6 layer must be present on the outermost surface of the egg-jelly for successful internal fertilization of the newt. Previous studies have suggested that the J6 layer is a prerequisite for the initiation of sperm motility and the acrosome reaction. In the present study, the fertilization rate decreased in eggs with a full set of jelly layers when inseminated using acrosome-reacted and motile sperm. However, the fertilization rate was high when motile sperm with intact acrosome was used. These results suggest that induction of the sperm acrosome reaction in the J6 layer is an important step in the internal fertilization of the newt.