FORMATION OF THE FERTILIZATION MEMBRANE BY INSEMINATION OF IMMATURE STARFISH OOCYTES PRETREATED WITH CALCIUM-FREE SEAWATER

When immature oocytes of the starfish, Asterina pectinifera , were treated with calcium-free seawater for 1 hr and then inseminated in normal seawater, they formed several blisters, indicative of polyspermy, and raised fertilization membranes. These oocytes continued to have intact germinal vesicles. Electron microscopic study revealed that the egg surface remained virtually unchanged after the treatment with calcium-free seawater. Upon insemination, however, the cortical granules broke down and the fertilization membrane was formed. These immature oocytes with ferilization membranes underwent maturation (germinal vesicle breakdown) after treatment with 1-methyladenine.
The treatment with calcium-free seawater seems to bring about some physiological change on the surface of immature oocyte, which bestows some attributes of maturation but is insufficient to mature the oocytes completely.     

CYCLIC SURFACE CHANGES IN THE NON-NUCLEATE EGG FRAGMENT OF XENOPUS LAEVIS

Fertilized uncleaved eggs of Xenopus laevis were divided into nucleate and non-nucleate egg fragments. Both fragments, together with the whole egg of the same batch, were observed by time-lapse cinematography.
Two kinds of cyclic surface changes, (1) rounding-up and relaxing movements and (2) surface contraction waves, accompanying each cleavage in the whole eggs and the nucleate fragments, were also observed even in the non-nucleate fragments although they do not cleave.
Cleavage intervals of the whole egg and the nucleate fragment were nearly equal, but the rounding-up intervals of the non-nucleate fragment were slightly but definitely longer than the cleavage intervals of the nucleate fragment and the whole egg.     

Cytoplasmic Maturity Revealed by the Structural Changes in Incorporated Spermatozoon during the Course of Starfish Oocyte Maturation

Immature oocytes of the starfish, Asterina pectinifera, are polyspermic. Spermatozoa can enter immature oocytes upon insemination, but the changes associated with the fertilization process in oocytes matured with 1-methyladenine (1-MeAde), such as the formation of aster and pronucleus, were not observed. After immature oocytes, previously inseminated, were matured with 1-MeAde, the formation of the sperm monaster was observed during germinal vesicle breakdown (GVBD). Amphiasters and pronuclei were formed after the formation of the second polar body. The acquisition by oocytes of the capacity to undergo the normal process of fertilization, therefore, occurs during the course of oocyte maturation. After injection of the cytoplasm of maturing oocytes into inseminated immature oocytes, the formation of aster and pronucleus was observed, suggesting that maturation-promoting factor (MPF) may be involved in establishing the cytoplasmic conditions (cytoplasmic maturity) necessary for the fertilization process to occur. In contrast, when enucleated, inseminated halves of immature oocytes were treated with 1-MeAde, only monasters were formed, while in the nucleated halves both amphiasters and sperm pronuclei were formed. Thus, germinal vesicle material is required for the formation of amphiaster and sperm pronucleus but not for the formation of monaster. It is possible that the amount of MPF produced in enucleated halves was sufficient only for the formation of the monaster but not for the formation of the amphiaster and pronucleus, since it has been previously established that germinal vesicle material is necessary for the amplification of MPF. The formation of the monaster in the enucleated halves at a time corresponding to GVBD in nucleated controls suggests that the amount of MPF needed for this event is rather small. For the induction of subsequent fertilization process, large amounts of MPF may be required to establish the necessary cytoplasmic conditions, although other possible role of nuclear material is not excluded.     

一种自动控制的温度刺激装置

本文介绍了一种应用于行为实验的温度刺激装置。此装置通过一个直径为1cm、厚约1.2cm 的铜质小盒与皮肤接触。在接触面上装有电热线和热敏电阻。小盒通过塑料管、水泵与恒温水浴槽相通,通过反馈电路对电热线、水泵和恒温水浴槽的控制,使得受刺激部位的皮肤温度在刺激间歇期保持在事先设置的基础水平,在刺激期则迅速上升到预定的数值以产生热刺激和痛刺激。其温度变化可以自动记录。本装置可应用于人体和动物的痛觉和温度觉的实验研究。     

Properties of Invertases in Sugar Storage Tissues of Citrus Fruit and Changes in Their Activities during Maturation

Both acid and alkaline invertases were present in immature juice sacs of satsuma mandarin (Citrus‘Unshu Marcovitch”) fruit, in which sugar content was low. Maturing and mature juice sacs, in which sugar content increased steadily with time, were characterized by the presence of alkaline invertase and the absence of acid invertase. When the immature juice sacs were homogenized with 0.2 M sodium phosphate-citrate buffer (pH 8.0), almost all of the acid invertase activity was found in the solubilized fraction, whereas almost all of the alkaline invertase activity was present in the insoluble fraction. The distribution of alkaline invertase between the solubilized and insoluble fractions changed with the development of fruit. The acid invertase had a molecular weight of 69,000, optimum pH of 4.8–5.3, and K_m value for sucrose of 7.3 mM. The alkaline invertase had a molecular weight of 200,000, pH optimum of 7.2–7.7, and K_m value of 35.7 mM. The hydrolysing activities of both enzymes for raffinose were considerably less than those for sucrose. The alkaline invertase had lower activity for raffinose than the acid invertase.     

Oocyte Maturation in Starfish: Sequential Changes of Oocyte Shape Induced by 1-Methyladenine Associated with Germinal Vesicle Breakdown

Observation of the early events of starfish oocyte maturation revealed a sequential change of the oocyte shape induced by 1-methyladenine (1-MeAde). The lengths of two diameters of the whole oocyte, one parallel to the egg axis (a–v diameter) and the other which is perpendicular to a–v diameter (e diameter) were measured by taking photographs successively. About 10 min after 1–MeAde administration, a sudden decrease of the a–v diameter (shortening of oocyte) occurred followed by a sudden increase (elongation of oocyte). Germinal vesicle breakdown (GVBD) occurred when the a–v diameter suddenly increased. The change of the oocyte shape occurred differentially in the animal and vegital halves i.e. when the egg-axis diameter shortened the surface of the vegital pole side shrank and the surface of the animal pole side expanded. GVBD was suppressed under hypertonic conditions and facilitated under hypotonic conditions. Cytological examination of the process of GVBD revealed (1) separation of the nuclear membrane from the granular cytoplasmic mass, (2) depression of several parts of the nuclear membrane and the increase of amorphous matrix on the outside of the nuclear membrane, and (3) disappearance (fragmentation) of nuclear membrane. The morphological changes were not the same along the egg-axis temporally and spacially. These observations suggest firstly that the cytoplasm, which has been in a certain quiescent state with a certain rigidity before 1–MeAde administration, not only undergoes a decrease in stiffness but also acquires a contractile property after 1–MeAde administration, and secondly that contractile or cytoskeletal components do not evenly function or are not evenly arranged in the animal and vegital halves.     

Crown Architecture and Species Coexistence in Plant Communities

The relationships between crown architecture and species coexistencewere studied using the diffusion model and the canopy photosynthesismodel for multi-species plant communities. The present paperdeals with two species having different crown shapes [conic-canopyplant (CCP) and spheroidal-canopy plant (SCP)], for variousinitial mean sizes at the establishment stage and physiologicalparameter values (photosynthetic rate, etc.). Recruitment processeswere not incorporated into the model, and thus simulations weremade for the effects on the pattern of species coexistence ofeither sapling competition starting from different sapling banksor competition in single-cohort stands with little continualestablishment of species until a stand-replacement disturbance.The following predictions were derived: (1) SCPs can establishlater/slowly in the lower canopy layer even if they are overtoppedby a CCP which established first/rapidly; (2) if SCPs establishedfirst/rapidly and occupy the upper canopy layer, a CCP can rarelyestablish later/slowly in the lower canopy layer; (3) smallest-sizedCCPs can persist well in the lowermost canopy layer overtoppedby a SCP, suggesting a waiting strategy of CCP's saplings inthe understorey of a crowded stand; (4) even if CCPs establishedfirst/rapidly and occupy the upper canopy layer, an SCP canestablish later/slowly in the lower canopy layer. Therefore,the species diversity of SCPs which established first/rapidlyand occupy the upper canopy layer limits the number of CCP specieswhich can establish later/slowly. In contrast, the species diversityof CCPs which established first/rapidly and occupy the uppercanopy layer does not affect the number of SCP species whichcan establish later/slowly. The combination of initial sizesof a CCP and an SCP at the establishment stage (i.e. establishmenttiming) affects the segregation of vertical positions in thecanopy between the two species with different crown shape, andnot only species-specific physiological traits but also crownarchitecture greatly affects the coexistence pattern betweenspecies with different crown architectures. The theoreticalpredictions obtained here can explain coexistence patterns foundin single-cohort conifer-hardwood boreal and sub-boreal forests,pointing to the significance of crown architecture for speciescoexistence. Diffusion equation model; canopy photosynthesis model; conifer-hardwood boreal/sub-boreal forest; sapling establishment; vertical foliage profile     

Allometry and Competition between Saplings of Picea jezoensis and Abies sachalinensis in a Sub-boreal Coniferous Forest, northern Japan

The crown shape and the mode of competition between saplings(<2m in height) of the two conifers,Picea jezoensis andAbiessachalinensis, of a sub-boreal forest, northern Japan, wereinvestigated based on the diffusion model. A model for individualsapling growth considering both inter- and intraspecific competitionwas developed. The effect of species-specific crown shape onthe sapling growth and competition of the two species were examined.PiceajezoensisandAbies sachalinensissaplings had deep conic and shallowflat crowns, respectively.Picea jezoensishad more foliage massthanAbies sachalinensisof the same sapling mass. It was suggestedthat thePicea jezoensissapling has a high cost for assimilation–respirationbalance under dark conditions of closed canopies, whereas theAbiessachalinensissapling maintains effective assimilation even undersuppressed conditions. Widely spaced saplings, such as gap successors,ofPicea jezoensishad a greater relative growth rate (a₀) thanwidely spacedAbies sachalinensis. The crown shape of saplingsof the two species shows different adaptations for efficientpersistence in the sub-boreal forest. Saplings ofPicea jezoensisandAbies sachalinensiswere not uniformlydistributed, but aggregated in different sites as the saplingsgrew, indicating habitat segregation between the two speciesat the sapling stage. Intraspecific sapling competition wasone-sided in each of the two conifers. Interspecific saplingcompetition was one-sided in the direction only fromAbies sachalinensistoPiceajezoensis. Therefore, asymmetric competition prevailed at thesapling stage of the two species. These results contrast withweak symmetric competition or the almost absence of competitionbetween trees (2m in height) of the two species (Kubota andHara,Annals of Botany76: 503–512, 1995). The mode of competitionchanged with the life-history stage from the sapling (intenseand asymmetric) to the tree (weak and symmetric or almost absent). In conclusion (1) asymmetric and intense competition betweensaplings brought about habitat segregation between the dominantspecies,Picea jezoensisandAbies sachalinensis, in the earlystage of life-history; (2) therefore, the coexistence ofPiceajezoensisandAbies sachalinensisof the sub-boreal forest wasdetermined by the boundary conditions for the growth dynamicsof the trees, as segregation of establishment sites resultingfrom asymmetric and intense competition between saplings; (3)then the species composition of the forest was maintained byweak symmetric competition or the almost absence of competitionbetween trees. Crown shape; growth dynamics; species coexistence; habitat segregation; diffusion model     

Effects of Cytochrome c on Liver Functions of Old Rats

WE have shown^1,2 that cytochrome c, when orally administered, is absorbed intestinally. The effects of parenterally administered cytochrome c on the liver have been studied by Drabkin³ and Murano⁴. We have also studied the effects of orally administered cytochrome c on experimental liver injury⁵.     

Nonlethal sampling of DNA from critically endangered diving beetles (Coleoptera: Dytiscidae) using a single antenna

Nonlethal DNA sampling is highly desirable in molecular genetic studies of protected and endangered species. To develop a demonstrably nonlethal method of obtaining DNA from endangered diving beetles (Dytiscus sharpi sharpi Wehncke, Cybister lewisianus Sharp and Cybister brevis Aubé), we amputated the antennae of these endangered diving beetles and investigated the impact of the amputation on reproductive behaviors, egg‐laying and lifespan. Diving beetles with either one or no antennae copulated without delay and laid eggs, comparable to the pairs of intact beetles under breeding conditions. The lifespan of antennae‐amputated D. sharpi sharpi was the same as that of the intact beetles. A single antenna was sufficient to allow polymerase chain reaction (PCR) detection of a mitochondrial DNA gene, cytochrome‐c oxidase subunit I (COI), and the sequence of the COI gene could be determined directly. The PCR‐ready genomic DNA was available both in fresh antennae isolated from living beetles and in old antennae from whole beetles preserved for at least 5–6 years in pure ethanol. These results suggest that an antenna is a good sampling site for isolating genomic DNA from endangered diving beetles without sacrificing and disturbing reproductive behaviors such as mating and egg‐laying, or lifespan.