The behavior of centrioles and ultrastructural changes of the nucleus were observed in maturing oocytes of the starfishes, Asterina pectinifera and Asterias amurensis. Observations were focused on the number and behavior of centrioles during two successive meiotic divisions. Examination of serial sections revealed that in meiosis I each division pole has a pair of centrioles, whereas in meiosis II each has only one centriole, confirming the observations by Sluder et al. (1989) on oocytes of fisaster ocraceus and Asterias forbesi. The first polar body had two centrioles and the second polar body had only one. These results indicate that no duplication of centrioles occurs during the two successive meiotic divisions, and that the egg inherits one centriole from a primary oocyte. IntroductionStarfish oocytes are suitable material for in vitro studies of meiosis. A large number of fullgrown oocytes can be induced to resume meiosis with good synchrony by their treatment with the maturation-inducing hormone, 1 -methyladenine (6). There have been many studies on the physiological and biochemical aspects of the maturation division in starfishes (see 7 for a review). Little is known, however, about the ultrastructural changes associated with meiotic events.Particularly interesting is the behavior of centrioles during meiosis. Longo and Anderson (12) reported that in lMyti/us oocytes, each of the two asters in the first meiotic metaphase contains paired centrioles, whereas each aster of the second meiotic metaphase appears to contain only one centriole. Kuriyama eta/. (10) supposed that centriolar behavior was the same in meiotic division of Spisula oocytes from their observations on artificial activation. In oocytes of some animals, on the other hand, no centrioles have been found in the meiotic spindle pole (22, 26).Fluorescent monoclonal antibodies that recognize centrosomes or centriolar appendages have 1 To whom reprint requests should be addressed. been used as markers for locating the centrioles possibly included in each centrosome (20,21). Unequivocal proof of the presence or absence of centrioles, however, depends upon whether they can be detected by electron microscopy. Here we investigated the process of maturation of starfish oocytes by conventional transmission electron microscopy (TEM) with particular attention to the number of centrioles. We found that in meiosis II each aster contains only one centriole, whereas in meiosis I each aster has a pair of centrioles. During preparation of this paper, we found a recent paper by Sluder et a/. (25) that clearly demonstrates a single centrole in the centrosomes of the second meiotic spindle in starfish oocytes. Our results on oocytes of two other species of starfish thus indicate the generality of the presence of the single centriole in secondary oocytes in starfishes. Part of this work was presented in an abstract form (2001( . Sci., 4, 1071. Materials and MethodsStudies were made on the starfishes, Asterina pectinifera and Asterias arnurensis in their breeding ...

Fertilization in the sea urchin, Anthocidaris crassispina, showed marked temperature dependence; high temperatures (15-30C) were required for fertilization. In contrast, fertilization in Hemicentrotus pulcherrimus occurred over a wide range of temperatures (0-30C). The mechanism of this temperature effect in Anthocidaris was investigated. The number of sperm bound to the egg surface and the rate of the acrosome reaction were markedly reduced by lower temperatures (0-10C). Furthermore, an abnormal elongation of the sperm head tip occurred with higher frequency at lower temperatures. In contrast, the egg activation with calcium ionophore A23187 was not prevented at 10C. The swimming activity measured by distance traveled was also relatively high at 0C, although the activity increased as the temperature rose. These results strongly suggest that temperature exerts a direct influence on fertilization in Anthocidaris by acting on the acrosome reaction. The increased fertilization rate at higher temperatures in Anthocidaris corresponds to the higher temperature observed during the breeding season of this species.

Reactive oxygen species (ROS) cause oxidative stress and act as signal transduction molecules in many cells. Spermatozoa from several mammals generate ROS, which are involved in male infertility and signaling during capacitation. In the present study, we investigated ROS generation by sea urchin spermatozoa at the initiation of motility, during dilution with seawater, and following egg jelly treatment. In seawater containing an ROS indicator, 5-(and 6-)chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H(2)DCFDA), fluorescence increased after the addition of spermatozoa. The ROS generation rate was dependent upon the dilution ratio and respiratory rate of the spermatozoa. Spermatozoa in sodium-free seawater did not increase fluorescence, but fluorescence did increase with the addition of NaCl. Sodium chloride also led to the initiation of sperm motility and respiration. Using the indicator MitoSOX Red, ROS generation was detected from spermatozoa exposed to egg jelly dissolved in seawater, but not in normal seawater. Moreover, the respiratory inhibitor antimycin A prevented CM-H(2)DCFDA-detectable ROS and increased MitoSox-detectable ROS at a higher concentration. These findings revealed that the ROS generated were of different species, possibly hydrogen peroxide (H(2)O(2)) and superoxide anion (O(-)(2)), and their detected levels were altered by egg jelly. We concluded that sea urchin spermatozoa generate at least two species of ROS depending on the physiological conditions to which they are exposed. It is possible that the major ROS from sea urchin spermatozoa changes during the course of fertilization.

Two structurally distinct lectins were purified from the coelomic plasma of holothurian, Stichopus japonicus, by affinity chromatography on a porcine stomach mucin-conjugated agarose column, gel filtration on a Superose 6 column, and ion-exchange chromatography on a HiTrap Q-FPLC. The two lectins showed apparent molecular masses of about 400 kDa (SPL-1) and 60 kDa (SPL-2) on gel filtration, but about 17 kDa on SDS-PAGE under reducing conditions. Both lectins showed hemagglutination activity toward rabbit erythrocytes in the presence of Ca2+ ions. The N-terminal amino acid sequences were highly homologous to but distinct from those of a Ca(2+)-dependent (C-type) lectin named SJL-I purified from the same species. In addition to porcine stomach mucin, the hemagglutination activity of SPL-1 was strongly inhibited by uronic acids such as galacturonic acid, and glucuronic acid, while the activity of SPL-2 was inhibited by GalNAc and galactosides. Both lectins were adsorbed on clotted coelomocytes in the presence of Ca2+ but not in the presence of inhibitory sugars or EGTA, suggesting the presence of an endogenous carbohydrate ligand(s) for plasma C-type lectins in the clot. However, coelomocyte clotting occurred normally even in the presence of inhibitory sugars, but was strongly inhibited by synthetic GRGDSP peptide or EGTA, suggesting the participation of integrin but not the lectin-carbohydrate interaction in the clotting events.

The stimulatory effects of glucose and lactate on protein synthesis by round spermatids (steps 1-8) were further studied. When the cells were incubated with lactate, the response of protein synthesis in round spermatids was closely related to the intracellular level of ATP. The ATP level in spermatids increased to 3.13 +/- 0.20 nmol/10(6) cells from 0.37 +/- 0.02 nmol/10(6) cells after incubation of the cells at 34 degrees C for 60 min in the presence of lactate (20 mM). However, the ATP level fell rapidly to an undetectable level (less than 0.02 nmol/10(6) cells) during incubation for 30 min at 34 degrees C without lactate. The ATP level and the rate of protein synthesis in spermatids increased rapidly when lactate (20 mM) was added to the control cells during incubation. It was also found that incorporation of 32P into ATP was increased by treatment with lactate (20 mM), but glucose (10 mM) had no effect on 32P incorporation into ATP. When the rates of utilization of glucose and lactate by spermatids were examined, using 14C-labeled glucose and lactate, the rate of utilization of lactate was faster than that of glucose. These results suggest that the ATP is probably a major factor in the stimulation of protein synthesis in round spermatids.