It is believed that in most animals only the paternal centrosome provides the division poles for mitosis in zygotes. This paternal inheritance of the centrosomes depends on the selective loss of the maternal centrosome. In order to understand the mechanism of centrosome inheritance, the behavior of all maternal centrosomes/centrioles was investigated throughout the meiotic and mitotic cycles by using starfish eggs that had polar body (PB) formation suppressed. In starfish oocytes, the centrioles do not duplicate during meiosis II. Hence, each centrosome of the meiosis II spindle has only one centriole, whereas in meiosis I, each has a pair of centrioles. When two pairs of meiosis I centrioles were retained in the cytoplasm of oocytes by complete suppression of PB extrusion, they separated into four single centrioles in meiosis II. However, after completion of the meiotic process, only two of the four single centrioles were found in addition to the pronucleus. When the two single centrioles of a meiosis II spindle were retained in the oocyte cytoplasm by suppressing the extrusion of the second PB, only one centriole was found with the pronucleus after the completion of the meiotic process. When these PB-suppressed eggs were artificially activated to drive the mitotic cycles, all the surviving single centrioles duplicated repeatedly to form pairs of centrioles, which could organize mitotic spindles. These results indicate that the maternal centrioles are not equivalent in their intrinsic stability and reproductive capacity. The centrosomes with the reproductive centrioles are selectively cast off into the PBs, resulting in the mature egg inheriting a nonreproductive centriole, which would degrade shortly after the completion of meiosis.
Extreme rigidity of immature starfish oocytes as measured by compression method was found to decline during the early phase of their maturation when induced by 1-methyladenine (I-MeAde). The onset of this decrease in stiffness occurred within 5 to 9 min of 1-MeAde treatment, well before the breakdown of the germinal vesicle, progressively declining to reach a minimum stiffness after 20 min. Dithiothreitol, known as an artificial maturation-inducing agent, caused a similar change. The stiffness is thus expected to serve as a quantitative indicator of the early process of cytoplasmic events, which would induce the breakdown of the germinal vesicle. Cytochalasin B (3 pg/ml) also reduced the stiffness, but unlike the former two agents, the effect was reversible, and did not interfere with the process of maturation. Due to the effect of cytochalasin B, it became possible to enucleate immature oocytes by centrifugal force. Non-nucleate fragments thus obtained still maintained their marked stiffness, which was decreased by the action of 1-MeAde, with a time-course similar to that of intact oocytes.It is known that I-methyladenine, the hormone which initiates maturation of starfish oocytes (9, 1 I ) by acting on the cell surface (10, 18), induces the production of a 'maturationpromoting factor' in the cytoplasm. This in turn triggers the breakdown of the germinal vesicle (15, 16). It is most likely that there are some other accompanying cellular processes initiated also by 1-methyladenine, which interract and result in cytoplasmic maturation of the oocytes.HIRAMOTO and his colleagues (1 9, 22) noticed marked changes in the mechanical properties of the cytoplasm, which occur prior to the breakdown of the germinal vesicle (GVBD). In the present study, detailed investigation of oocyte rigidity, which we postulate as serving as a quantitative indicator of the process of maturation, was carried out. MATERIALS AND METHODS Materialswith cold sea water (15-20°C) until use. Astropecten scoparius and Asterias amurensis were also employed in some experiments. Solutions(DTT) in 0.25 M NaCI, and 2 mg cytochalasin B in ml dimethylsulfoxide, were prepared.Starfishes were collected during their respective breeding seasons, and stored in aquaria supplied Materials mainly used were the oocytes of Asterina pectinifera.As stock solutions, I mM 1-methyladenine (I-MeAde) in de-ionized water, 0.5 M dithiothreitol These were diluted * This paper is dedicated to the memory of the late Professor Jean Clark Dan, 315
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 ...
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