The insemination of goldfish (Carassium auratus) oocyte maturedin vitro

Full maturation of goldfish oocyte was inducedin vitro by 17 α-hydroxy-20β-dihydroprogesterone. The oocyte maturation involves GV migration to the periphery of the oocyte and germinal vesicle breakdown (GVBD). In the experiment, incubation duration for GVBD varied in different broods of oocytes. Generally, if the duration for GVBD was shorter than 6 h, oocytes would have a better chance to survive after maturation and insemination. The maturation of nucleus (GV) and cytoplasm are not synchronous. Cytoplasm maturation occurs several hs after GVBD. Oocytes inseminated 8–9 h after GVBD have the highest fertilizing and hatching rate. Fertilized ova maturedin vitro can develop to sexually mature adults capable of reproduction. Contribution No 1731 from the Institute of Oceanology, Academia Sinica.     

Cytoplasmic regionalization in starfish oocyte occurrence and localization of cytoplasmic determinants responsible for the formation of archenteron and primary mesenchyme in starfish (asterias amurensis) oocytes

Starfish oocytes with intact germinal vesicles (GVs) were cut along desired planes with glass needles or ligated using silk thread loops into two parts and allowed to mature in vitro, and inseminated. The experimental results showed that (1) only the parts with GVs or partial GV contents (PGVCs) cleaved, those without any GV materials did not; but nucleated and non-nucleated fragments cut from mature eggs were able to divide; (2) the development of animal parts of oocytes containing GVs or PGVCs was like that of animal fragments of matured oocytes with female pronuclei; most of them gave rise to permanent blastulae, and just a few formed ectodermal vesicles with a little primary mesenchyme; (3) a large part of vegetal fragments with GVs or PGVCs, and the vegetal parts of mature eggs without female pronuclei developed into small but normal embryos; (4) the fragments containing GVs or PGVCs obtained from the oocytes along a plane parallel to the animal-vegetal (A-V) axis developed as normally as the halves (with or without female pronuclei) severed from mature eggs along the same axis. Based on the data above, it was concluded that (1) the non-chromatin materials in the oocyte GVs are indispensable for successful fertilization and cleavage of starfish eggs; (2) some factor (s) located asymmetrically in the vegetal hemispheres of starfish oocytes is (are) responsible for formation of the archenteron and primary mesenchyme. It is evident from the above findings that the oocyte cytoplasm of the starfish had already regionalized before the GV break-down. Contribution No. 1722 from the Institute of Oceanology, Academia Sinica     

A new species of ciliates,Hemiophrys polymicronuclei sp. nov. from Donghu Lake,Hubei province

A new species of ciliatesHemiophrys polymicronuclei sp. nov. from Donghu Lake is described. It is characterized by its colorless to yellowish, soft, flat and spindle-shaped body with four micronuclei. The right side of the body is a little more convex than the left side, and more transparent at both anterior and posterior ends. Its slit-like cytostome accounts for 1/4 of the body length on the left side without any trichocyst. There are two rows of trichites near the right anterior end, 6–8 straight bristles on the pellicle opposite the cytostome, 23–25 rows of kinetides on the right side of the body, two ellipsoid macronuclei and many contractile vacuoles in the cytoplasm.     

An experiment on induction of triploidy inEriocheir japonicus hepuensis Dai by heat treatment

Temperature treatment to inhibit extrusion of the polar body of the egg was first used on a lower crustacean,Artemia salina (Gross, 1932), and then was used for inducing triploids of amphibian (Frankhauser and Griffiths, 1939), fish (Svardson, 1945), and mammal (Beatly and Fishchberg, 1949). So far, induction of triploidy has been extensively used to obtain sterile or quick-growth individuals in fish (Swarup, 1956; Lincoln, and Scott, 1983 and Thorgaard, 1986) and mollusk (Stanley et al., 1984), but similar work has not been reported on crab, a higher crustancean.     

Localization of a putative epiboly-determining factor in oocytes of the goldfish (Carassius auratus)

It is unknown whether cytoplasmic determinants in goldfish eggs are asymetrically localized before maturation. In this study fully grown goldfish oocytes with intact germinal vesiles (GVs) were ligated with baby hair loops along desired planes into two parts, matured in vitro, and inseminated. Embryos developed from the animal halves with GV of oocytes ligated along the equator were unable to undergo epiboly, while those developed from the GV-containing parts of oocytes ligated along the animal-vegetal axis were able to. Also, embryos developed from the animal halves obtained from fertilized eggs could undergo epiboly. From these results, it was postulated that the goldfish's premature oocyte possessed a putative factor in the vegetal hemisphere which was responsible for epiboly of the embryonic cells. This work was supported by the NSFC and the “Climb Plan B” (PD-B6-1-1) of the Chinese Commission of Science and Technology. Contribution No. 2915 from the Institute of Oceanology, Chinese Academy of Sciences.     

LOCALIZATION OF A PUTATIVE EPIBOLY-DETERMINING FACTOR IN OOCYTES OF THE GOLDFISH (CARASSIUS AURATUS)

It is unknown whether cytoplasmic determinants in goldfish eggs are asymmetrically localized beforematuration. In this study fully grown goldfish oocytes with infact germinal vesicles (GVs) were ligated withbaby hair loops along desired planes into two parts, mature in vitro, and inseminated. Embryos developedfrom the animal halves with GV of oocytes ligated along the equator un unable to undergoepiboly, while those developed from the GV-containing parts of oocytes ligated along the animal-vegetal axiswere able to. Also, embryos developed from the animal halves obtained from fertilized eggs could undergo epiboly. From there results, it was postulated that the goldfish's premature oocyte possessed a putativefactor in the vegetal hemisphere which was responsible for epiboly of the embryonic cells.     

Comparison of ciliature microtubule organelles in three hypotrichous ciliate species

We examined the structure and spatial organization of ciliature base-associated microtubules (BAM) in three hypotrichous ciliates (Stylonychia mytilus, Pseudourostyla cristata, Euplotes woodruffi) in fluorescence microscopy. The results revealed that BAM, including the anterior (ALM), posterior longitudinal microtubule (PLM) and the transverse microtubule (TM) bands, are composed of tubulin. The respective microtubular bands have cytoplasmic polarization patterns that are significantly asymmetric. The BAM of the midventral files in P. cristata appear cord-shaped compared with the ALM bands of transverse cirri in both S. mytilus and E. woodruffi, which extend to the left anterior side of the cell before converging. The TM bands of the left marginal cirri (MC) in S. mytilus extend to the right side of the cell, while those of the right MC bands extend to the left. Our observations suggest that BAM traits are common in hypotrichous ciliates even though different species possess different microtubule arrangements related to the conserved cirral morphogenetic patterns in the respective species. The differing development of BAM in the three ciliate suggests that the microtubules may be conserved in different hypotrichs. We have also demonstrated that the BAM, which appear polar and asymmetric, are localized in specific cytoskeletal positions and extend in different orientations within the cortex to connect with other ciliature-associated structures and, thus, strengthen the cortex. These BAM features indicate that they are directly associated with cell motion.     

Reproductive cycle and seasonal variations in lipid content and fatty acid composition in gonad of the cockle Fulvia mutica in relation to temperature and food

From March 2004 to February 2005,seasonal variations in lipid content and fatty acid composition of gonad of the cockle Fulvia mutica(Reeve) were studied on the eastern coast of China in relation to the reproductive cycle and environment conditions(e.g.,temperature and food availability).Histological analysis as well as lipid and fatty acid analyses were performed on neutral and polar lipids of the gonad.Results showed that gametogenesis occurred in winter and spring at the expense of lipids previously accumulated in summer and autumn,whereas spawning occurred in summer(20.4-24.6℃).The seasonal variation in lipid content was similar to that of the mean oocyte diameter.In both neutral and polar lipids,the 20:5n-3 and 22:6n-3 levels were relatively higher than saturated fatty acids,and polyunsaturated fatty acids were abundant,with series n-3 as the predominant component.Seasonal variations in the 20:5n-3 and 22:6n-3 levels and the principal n-3 fatty acids were clearly related to the reproductive cycle.The ∑(n-3) and ∑(n-6) values were relatively high during January-May,and the associated unsaturation index was significantly higher than that in other months.The results suggest that fatty acids play an important role in the gametogenesis of F.mutica.     

AN EXPERIMENT ON INDUCTION OF TRIPLOIDY IN ERIOCHEIR JAPONICUS HEPUENSIS DAI BY HEAT TREATMENT

Temperature treatment to inhibit extrusion of the polar body of the egg was first used on a lower crustacean, Anemia salina (Gross, 1932), and then was used for inducing triploids of amphibian (Frankhauser and Griffiths, 1939), fish (Svardson, 1945), and mammal (Beatly and Fishchberg, 1949).So far, induction of triploidy has been extensively used to obtain sterile or quick-growth individuals in fish (Swarup, 1956; Lincoln.and Scott, 1983 and Thorgaard, 1986) and mollusk (Stanley et al., 1984), but similar work has not been reported on crab, a higher crustacean.     

Optimum temperature for the growth performance of juvenile orange-spotted grouper （Epinephelus coioides H.）

Effects of water temperature （17, 21, 25, 30 and 35℃） and body size （14.75-281.41 g initial body weight） on food consumption, growth, feed conversion, and dry matter content in orange-spotted grouper fed to satiation were investigated. The combined effect of temperature （T, ℃） and body weight （W, g） on maximum food consumption （Cmax, g/day） was described as： InCmax=-7.411＋0.828 InW＋0.317T4）.004 7T2, and the optimum feeding temperature was 33.9℃. The combined effect of temperature and body weight on growth （G） was described as： lnG=-4.461-0.2081nW＋0.394T-0.006 3T^2. The optimum growth temperature was 31.4℃, whereas overall growth rates were high at 25, 30 and 35 ℃. Feed conversion efficiencies （FCE, %）, increasing first and then decreasing with increasing temperature, averaged from 1.8 to 2.1 in terms of dry weight of food fish. The optimum temperature for FCE tended to be lower than that for growth or feeding. Dry matter content increased with both increasing water temperature （17, 25, 30 and 35℃） and body weight, and the combined effect of temperature and body weight on dry matter content （DM, %） was described as： lnDM =3.232＋0.01 4 lnW-0.004 4T＋0.001 2TInW.     

Embryonic and larval development in barfin flounder Verasper moseri (Jordan and Gilbert)

Broodstock of Verasper moseri (Jordan and Gilbert) aged 3–4 years old were selected, and reinforced cultivation was conducted to promote maturation under controlled water temperature and photoperiod conditions. Fertilized eggs were obtained by artificial fertilization, and the development of embryos, larvae and juveniles was observed continuously. The results showed that the fertilized eggs of V. moseri were spherical, with transparent yolk and homogeneous bioplasm, and had no oil globule inside. The average diameter of the eggs was 1.77±0.02 mm. The eggs of V. moseri were buoyant in water with salinity above 35. The cleavage type was typical discoidal. Young pigment cells appeared when olfactory plates began to form. Hatching occurred at 187 h after fertilization at a water temperature of 8.5°C. The newly hatched larvae, floating on the water surface, were transparent with an average total length of 4.69±0.15 mm. During the cultivation period, when the water temperature was raised from 9 to 14.5°C, 4-day old larvae showed more melanophores on the body surface, making the larvae gray in color. The pectoral fins began to develop, which enabled the larvae to swim horizontally and in a lively manner. On days 7–8, the digestive duct formed. The yolk sac was small and black. The yolk sac was absorbed on day 11. Larvae took food actively, and body length and body height clearly increased. The rudiments of dorsal and anal fin pterygiophores were discernible and caudal fin ray elements formed on day 19. On day 24, the larval notochord flexed upwards, and the rays of unpaired fins began to differentiate. Pigment cells converged on the dorsal and anal fin rays, and the mastoid teeth on the mandible appeared. On day 29, the left eyes of juveniles began to move upwards. Depigmentation began in some juveniles and they became sandy brown in color on day 37. Most juveniles began to settle on the bottom of the tank. The left eyes of juveniles migrated completely to the right side on day 50, when the average body length attained 2.5±0.18 cm, and juveniles accomplished metamorphosis to young. The embryonic and larval characters of several flounder species are compared.     

Sperm nuclear expansion and meiotic maturation in normal and gynogenetic eggs of the scallop, <Emphasis Type="Italic">Chlamys farreri</Emphasis>

Sperm nuclear expansion, meiosis and the association of the male and female pronuclei leading to the four-cell stage in normal Chlamysfarreri eggs were observed under a fluorescence microscope. The effects of ultraviolet （UV） irradiation on the fertilizing sperm were also examined. Both normal and UV-irradiated sperm nuclei enlarged at three distinct phases （phase A, metaphase I; phase B, polar body formation; and phase C, female pronuclear development and expansion） that were temporally correlated with meiotic process of the maternal chromosomes. Sperm nuclei underwent a rapid, initial enlargement during phase A, but condensed slightly during phase B, then re-enlarged during phase C. The effects of UV irradiation were not apparent during transformation of the sperm nucleus into a male pronucleus, and there was not any apparent effect on meiotic maturation and development of the female pronucleus. However, the rate of expansion of the UV-irradiated sperm nuclei and the size of male pronuclei were reduced apparently. Unlike the female pronucleus, the male pronucleus derived from sperm genome inactivated by UV irradiation did not form chromosomes, but became a dense chromatin body （DCB）. At mitotic anaphase, DCB did not participate in the karyokinesis of the first cleavage as evidenced by chromosomal nondisjunction, demonstrating the effectiveness of using UV irradiation to induce gynogenetic scallop embryos.     

Histological study on the skin of Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

Histological development of Japanese flounder Paralichthys olivaceus larval skin and ultrastructural difference of skin between reared normal and malpigmented Japanese flounder were studied with light microscopy (LM) and transmission electron microscopy (TEM). The results show that the skin develops slowly before the metamorphosis, while at the onset of metamorphosis, the skin develops quickly and becomes complete in structure till about 50d after being hatched. Ultrastructural observation on the normal and malpigmented skins shows that the iridophore and melanophore are adjacent to each other. Profile and structure of the two kinds of pigmcnt cells are more complete in the skin of normal ocular side than in the skin of pigmented blind side. The ultrastructure of typical chloride cell was observed in the skin of Japanese flounder larvae for the first time.     

First record of <Emphasis Type="Italic">Paraonella platybranchia</Emphasis> (Hartman, 1961) (Polychaeta: Paraonidae) from the Yellow Sea

This paper reports for the first time a species of polychaetous family Paraonidae, Paraonella platybranchia (Hartman, 1961), a rare species found previously only in the coast near the boundary between the United States and Mexico, and it was recently discovered in the samples collected in 1982 from Chinese side of the Yellow Sea. This is the first record of the species in China as well as in the Northwest Pacific. Supported by the Knowledge Innovation Program of the Chinese Academy of Sciences (No. O72715)     

The in vitro effects of steroids,human chorionic gonadotropin and cyanoketone on germinal vesicle breakdown of striped mullet (Mugil cephalus L.) oocytes

The in vitro effects of steroids, human chorionic gonadotropin (HCG) and cyanoketone on germinal vesicle breakdown (GVBD) of striped mullet (Mugil cephalus L.) oocytes were investigated. All concentrations of HCG (5,10,50 I.U./ml), progesterone and pregnenolone at the highest concentrations(lug/ml) were moderately effective in inducing GVBD, whereas 17β-estrodiol, cortisol, testosterone, 11β-hydroxyandrostenedione and 11-ketotestosterone did not stimulate GVBD. 17α, 20βdihydroxy-4-pregnen-3-one (17α, 20βdiOHprog) and deoxycorticosterone (DOC) were the most potent steroids in stimulating final oocyte maturation. The results indicate that C21 hydroxylated steroids are potent inducers of final maturation in mullet. Further, co-incubations with 17β-estradiol, cortisol and testosterone did not alter the maturation-inducing effects of HCG or 17α,20βdiOHprog. Cyanoketone, a blocker of 3βHSD activity, was only partially effective in blocking GVBD induced by HCG. This suggests that Δ5 (pregnenolone derived) and Δ4 steroids may be involved in final oocyte maturation in this species.     

Growth,reproduction & population structure of the freshwater crabSinopotamon yangtsekiense bott, 1967, from Zhejiang,China

Monthly investigations were mae on the population of Chinese freshwater crab,Sinopotamon yangtsekiense Bott, 1967 from April, 1984 to March, 1985. The data on 4413 specimens show that the growth was affected mainly by temperature. During the April to November growth period, the crabs' major development occurred from June through October. One year was required for a fine white oocyte to develop into a mature egg. The reproduction period was June–October. Females bearing eggs were taken from June–August, and crabs with young were found from July–October. The females reproduced once a year but could for more than one year. The number of eggs carried by a female varied greatly according to the size of the crab, ranging from 30 to 100 eggs. New-born crabs become mature after 1–2 years. The sex ratio was approximately 1∶1 in the overall population. However, the larger crabs are predominantly male. The age distribution ofS. yangtsekinese was estimated from size frequency histograms. There were more adult crabs (over 70%) from June to October and more immature crabs (over 50%) from November to May.     

Distribution and migration of the common squidTodarodes pacificus in the Yellow Sea

Study of the distribution and migration of the common squid,Todarodes pacificus Steenstrup,basedon the index of important fishing ground(P) and fisheries statistics on the Yellow Sea and northern EastChina Sea during 1980—1991 showed that:1.Its catch in the fishing period(June to November) is 91.77% of the annual yield.The fishingground distributes over the northem and middle Yel1ow Sea and adjacent area of the Changjiang Estuary.2. It over-winters in the northem East China Sea and waters adjacent to Goto Island from De-cember to February and spawns in waters near Haijiao Is1and and west of Kyushu. The main stock mi-grates along 123°30′E to the ChangJiang Estuary, Haizhou Bay. offsea from Shidao to Qingdao,mideastern Yellow Sea, and offsea Weihai and Haiyang Island succesively for feeding after April. The sur-plus stock migrates again to the wintering ground in December.3.The favorable feeding temperature is 6-23℃(optimum of l3-20℃ in the Changjiang Estua-ry and 7-13℃ in the northern and middle Yel     

Effects of base analogues 5-bromouracil and 6-aminopurine on development of zebrafish Danio Rerio

Zebrafish (Danio rerio) genetic screens allow isolation of a wide array of problems in vertebrate biology. The effects of base analogues 5-bromouracil and 6-aminopurine on the development of zebrafish embryos are reported for the first time in this study. The early development of the zebrafish embryos was little affected by 5-bromouracil and 6-aminopurine, while the late development (organogenesis) was significantly impaired. Embryos exposed to 5-bromouracil mainly showed curled tail, wavy body, golden pigmentation and the mouth with protruding lower jaw. 6-aminopurine-treated embryos had defective anterior structures, curled tails and wavy body. RAPD analysis showed that the majority of 5-bromouracil- and 6-aminopurine-treated larvae and fish shared banding patterns in common with the control, suggesting that most mutagenesis induced by these agents are point mutations. However, some fish derived from 5-bromouracil-treated embryos had golden (gol) pigmentation; and RAPD analysis revealed that their band patterns differed from those of the control. Possibly, 5-bromouracil can occasionally cause relatively extensive changes in the fish genome. The results of this study may provide valuable help for detailed studies of mutagenesis. This work was supported by Ministry of Science and Technology (MOST) of China (G 1999012005).     

The role of phosphatidylinositol signaling pathway in regulating serotonin-induced oocyte maturation in Mercenaria mercenaria

Serotonin (5-HT) has been found to stimulate meiotic maturation of oocytes in many molluscs. During maturation, several signaling pathways are involved, especially the phosphatidylinositol and cAMP pathways. In order to examine the possible role of the phosphatidylinositol signaling pathway in regulating oocyte maturation in Mercenaria mercenaria, the effects of the activator/inhibitor of phospholipase (PLC) and protein kinase C (PKC) on serotonin-induced maturation were studied. Results show that high-concentrations of neomycin (inhibitor of PLC) blocked oocyte maturation, while 9, 10-dimethyl-1, 2-benzanthracene (DMBA, activator of PLC) promoted oocyte maturation in the presence of serotonin. It was also found that in the presence of serotonin, phorbol 12-myristate 13-acetate (PMA, activator of PKC) inhibited oocyte maturation, while sphingosine (inhibitor of PKC) stimulated oocyte maturation. These results indicate that serotonin-induced oocyte maturation requires the activation of the phosphatidylinositol pathway. Decrease of PLC inhibited serotonin-induced oocyte maturation, whereas a decrease of PKC stimulated the maturation. Thus, our study indicates that serotonin promotes maturation of M. mercenaria oocytes through PLC stimulated increase in calcium ion concentration via inositol-1, 4, 5-trisphosphate (IP3) but not PKC.