非洲爪蟾胚胎细胞核移入花背蟾蜍成熟卵后的细胞学研究

This paper reports the development of the nuclear transplanted eggs obtained by transplanting Xenopus laevis blastomere nuclei to Bufo raddei. The formation of the donor nuclei is similar to that of the male pronuclei in the normal development egg in the early stage while shows degradation in the late stage after transplanting. The chromosome complement formes in some eggs 30 minutes after transplantation, which is assumed to contribute to the development of the transplanted eggs [Acta Zoologica Sinica 51 （5） ： 924 - 931, 2005].     

In vitro Oocyte Maturation in the Zebrafish Brachydanio rerio and Fertilization and Development of the Mature Egg

Maturation process of zebrafish oocyte was investigated using in vitro incubation.In medium EM-199 containing 0.5 μg/ml of 17α-hydroxyprogesterone incubated under 80％ O_2 and at 25°C,germinal vesicles(GV)of oocytes in stage Ⅳ migrated from midway between the center and theperiphery ofoocytes to the periphery in 40 minutes and the oocytes went into stage V.Half an hourlater,the oocytes underwent germinel vesicle breakdown(GVBD)with a breakdown rate of 59％.Two more hours were needed for such oocytes to complete their final maturation.The mature eggscould not come off from the follicle layer surrounding them by themselves(ovulation).By removingthe follicle and adding active sperms for insemination,we could make the mature eggs fertilized.Thechorion was elevated and blastoderm formed on the animal pole.The cleavage and development ofthese fertilized eggs followed the same course as the naturally matured and fertilized eggs.Usingblastula formation as a marker of successful fertilization of the in vitro matured egg,the fertilizationrate was 78％.This is the first report on the successful in vitro incubation of mature oocytes inzebrafish.The establishment of this in vitro oocyte maturation technology has laid the foundationfor further investigation of the transfer of foreign genes in the germinal vesicles of oocytes.     

多氯联苯对鸡胚的毒性作用

The toxic effects of polychlorinated biphenlys (PCB) on embryonic chickens, especially on liver morphology, were studied. PCB (Aroclor 1 254) was injected into yolk of Hyline fertilized eggs, with doses of 0, 1, 10 and 100 μg/egg, respectively. The survival rate of chicken embryos in each PCB treatment group was significantly lower than that of the control group. The hatching rate of 1 μg/egg group was only 40%. Subcutaneous edema and hydrocephally were found in dead embryos and newly hatched chicks of all treatment groups. Livers of newly hatched chickens were removed for examination of histological structures. After PCB treatment, severe liver injury was apparent. The hepatic structures were damaged and steatosis was observed, together with necrosis and cell vacuolation and pyknosis. There were more Kupffers cells in the hepatic sinusoid. No PAS positive staining was found in the hepatic cytoplasm, indicating that glycogen was exhausted in the hepatic cells. The higher death rate in PCB treated groups may be related to glycogen exhaustion. These findings indicate that PCBs reduced the hatching rate by causing severe subcutaneous edema and liver injury in chicken embryos.     

Electrofusion between Blastula Cells and Unfertilized Egg in Loach (Paramisgurnus dabryanus)

Electrofusion between blastula cells and unfertilized eggs in loach were investigated usingdielectrophoretic field where, under alternating sinusoidal electric field, blastula cells formed beads-like chain in close contact with the unfertilized egg and cell fusion occurred between eggs and thecells in tight contact with them. The nuclei ofblastula cells were brought into the cytoplasm of therecipient eggs, where they promoted the development of the fused eggs just like the zygote nuclei.But the development of the fused eggs was different from that of zygotes. Several nuclei might enterone and the same egg simultaneously and all of them could undergo division, resulting in severalblastomere after the first cleavage of the recipient egg. Before blastula stage, the embryo developingfrom the fused egg showcd irregular shape, but it was soon regulated and developed to a normalblastula which often continued its development into a normal individual. Cell/egg electrofusion cameto its highest fosion rate (80%) 8nd hatching rate (20%), with cell density at 1×10~3 cells/ml, Ca~(++)concentration at 10 mM, mannitol at 0.2 M and when the blastula cells were digested with 100μg/ml pronase E for 6-10 min at 20℃. The mechanism underlying development of electrofused eggsis discussed. As the result indicates, electrofusion might prove to be a promising biotechnology justas nuclear transplantation.     

光周期诱导红腹锦鸡冬季繁殖效果初报

?College of Life Science, Hebei Normal University, Shijiazhuang 050016, China)Golden pheasant ( Chrysolophus pictus) is a monotypic and special species of China.In nature, it breeds once a year in spring summer.From Oct.1998 to Feb.1999, we studied the effects of photo periods on inducing the birds to reproduce in winter and succeeded.In the experiment, 36 adult females and 18 males were divided into three groups.Each group was divided into 6 replicates, each replicate included 2 females and 1 male, and fed in one cage.All the groups were exposed to short day (8L∶16D) for 8 weeks, and then transferred to long day (12L∶12D, 14L∶10D or 16L∶8D). In result, photoperiods significantly affected Golden pheasant reproductive performance in winter.Considered of all of the reproductive performance indicators, the reproductive performance of the group exposure to 14L∶10D was the best, the 16L∶8D group's was less and the 12L∶12D group's was the worst.It may be that the birds' CDL (critical day length) for inducing egg production was about 12 h, and the CDL for optimal egg production was 14L∶10D in winter. The birds exposed to 12L∶12D had no fertilized egg.The other groups had fertilized eggs and the fertility was 15 05% (14L∶10D) and 20 41% (16L∶8D).The hatchability of fertilized egg were 76 47% (14L∶10D) and 80 00% (16L∶8D). The egg laying pattern was affected by the size of photoperiod.The ratio of lay of the birds exposed to 14L∶10D, increased rapidly and sustained in a peak level for 5 weeks, and then fall down.But the other birds (group A and C) increased to the peak at the first week and then fall down quickly.     

Distribution of α-D-mannose residue on zona pellucida and its role(s) in fertilization in pigs

The present study aims to identify the distribution of α-D-mannose residues on zona pellucida (ZP) and their role(s) in fertilization in pigs. In experiment 1, in vitro matured pig oocytes were freed from cu- mulus cells and treated with fluorescein isothiocyanate-labelled Lens culinaris (FITC-LCA), a D-mannose specific binding lectin. After 30 min of treatment, LCA bound evenly throughout the ZP with strong fluorescence. In experiment 2, when LCA-treated oocytes were used for in vitro fertilization, the number of sperm bound to ZP was significantly decreased, and sperm penetration was almost com- pletely blocked. In experiment 3, polysaccharide mannan was added to the in vitro fertilization medium as a competitive inhibitor. Both the number of sperm bound to ZP and the rate of fertilized oocytes were significantly reduced in the mannan-treated group compared with the control group. In experiment 4, spermatozoa were incubated with mannan in vitro. The number of acrosome-reacted spermatozoa was evidently increased in a time-dependent manner during the incubation. These results suggest that α-D-mannose residues presenting on pig ZP might be an important component of sperm receptor and might induce sperm acrosome reaction and thus facilitate the sperm penetration into the ZP.     

Ecdysteroids in the adults and eggs of Opogona sacchari (Bojer), an invasive alien pest

In order to understand the composition and quantitative variation of ecdysteriods in the adults and eggs of Opogona sacchari (Bojer), an invasive alien pest, we analyzed the ecdysteroid composition and titers in the adult and egg of this pest. On day 4 after eclosion, the titer of ecdysteriods in the male adult was 0.080 ng/adult, much lower than 5.978 ng/adult in the female adult. During the development of ovaries, the titer of ecdysteriods was low on the first two days, and high in the late period, with the peak (10.48 ng/ovary) appearing on day 3. During the development of eggs, the titer of ecdysteriods was about 0.010 ng/egg from day 1 to day 3, and then decreased to 0.006 ng/egg on day 4. In both adults and eggs, three main components of ecdysteriods were found by identification of HPLC/RIA. They were 20-hydroxyecdysone, 26-hydroxyecdysone, and an unidentified component.     

Ultrastructure of the Mature Egg and Fertilization in the Fern Ceratopteris thalictroides

The ultrastructure of the mature egg and fertilization in the fern Ceratopteris thafictroides （L.） Brongn. were observed by transmission electron microscopy. The results revealed that the mature egg possesses an obvious egg membrane at the periphery of the egg. Furthermore, a fertilization pore was identified in the upper egg membrane of the mature egg. The structure of the pore is described for the first time. The fertilization experiment indicated that spermatozoids crowd into the cavity above the egg through the neck canal of the archegonium; however, only one of these can penetrate into the egg through the fertilization pore. Immediately on penetration of the spermatozoid, the egg begins to shrink. The volume of the fertilized egg decreases to almost one-half that of the unfertilized egg. As a result, the protoplasm of the fertilized egg becomes dense and opaque, which may lead to a situation where the organelles of both the egg and the fertilizing spermatozoid become indistinguishable. Simultaneously, abundant vesicles containing concentric membranes or opaque materials appear near the fertilization pore in the cytoplasm of the fertilized egg. These vesicles are considered to act as a barrier that prevents polyspermy. The present study provides a new insight into the ultrastructure of the mature egg and the cytological mechanism of fertilization in ferns.     

Effect of antikeratin microinjection on the embryonic development of Xenopus laevis

Anti-keratin monoclonal antibody AF5 was introduced into fertilized eggs of Xenopus laevis.,and its effects on embryonic development were studied.Survival rate of the antikeratin-injected embryos was much lower(only 35.67% at gastrula)than that of the control(74.85% at gastrula),in which embryos were injected with mouse IgG.Most of survivors in the experimental series showed aberrant external appearance.On the other hand,in cleavage stage,ie 2-7h after fertilization,immunohistochemical staining of embryos showed that the expermental embryos were mostly keratin negative,while embryos of the control ones were keratin positive.When introducing this antikeratin into one cell of a 2-cell embryo,only the uninjected half of the embryo continued its development while the other half could not develop at all.These results suggested that intact keratin cytoskeleton in early embryos is indispensable to the embryonic development of Xenopus laevis.     

细胞松弛素B诱导虾夷扇贝多倍体初探

Triploid shellfish are useful for aquaculture because of their sterility,superior growth and improved meat quality.Tetraploid are also valuable for 100% producing triploids through mating with diploid.We tested polyploid induction in Japanese scallop,Patinopecten yessoensis,by inhibiting polar body Ⅰ (PB group) and both polar bodyⅠandⅡ (PPB group) in newly fertilized eggs.Cytochalasin B (0.6 mg/L) was applied at 11～22 min post fertilization (PF),and terminated in PB group when polar body Ⅰ was released about 70% in untreated eggs,in PPB group when polar lobe was observed in control group.The treatment and its control were repeated 5～7 times using different pairs of parents.The ploidy was determined by counting chromosome number at embryo stage,and then was detected by flow cytometry (FCM) at larvae stage and juvenile stage.\;In PB group,aneuploid (31.13%),triploid (3.96%),tetraploid (17.46%) and pentaploid (46.65%) embryos were produced,and in PPB groups,pentaploid embryos became higher (56.2%),triploid and tetraploid were 2.42% and 9.11%.At day 3 PF,the larvae showed tetraploid,pentaploid and aneuploid peaks through checking with FCM in PB group,and showed mainly higher pentaploidy peak in PPB groups.However,at day 14 PF diploids were mainly left,sometimes with small triploid peak.It suggested that most tetraploid,aneuploid and pentaploid larvae were died within the first two weeks PF.At three months PF,a few diploid juveniles were harvested in three control groups.Only 12 juvenile scallops were harvested in one of treated group (PB-7),and 11 of them died accidentally,the alive one in treated group was triploid through checking with FCM.     

中药蟾酥的药理作用研究进展

蟾酥是我国传统中药,是蟾蜍科动物中华大蟾蜍或黑眶蟾蜍的干燥分泌物。蟾酥所含化学成分主要有蟾蜍内酯类、蟾毒色胺类等,现代药理学研究表明其具有抗肿瘤、强心、局麻、镇痛、抗炎等多种作用。本文综述近年来国内外学者对蟾酥的化学成分、药理作用的研究概况。     

韩江下游底栖动物的分布及其对水质的评价

韩江下游底栖动物的分布及其对水质的评价蓝宗辉（韩山师范学院生物系,广东潮州５２１０４１）ＴｈｅＺｏｏｂｅｎｔｈｏｓＤｉｓｔｒｉｂｕｔｉｏｎｉｎｔｈｅＬｏｗｅｒＲｅａｃｈｅｓｏｆＨａｎｊｉａｎｇＲｉｖｅｒａｎｄＡｓｅｓｍｅｎｔｏｆｉｔｓＷａｔｅｒＱｕａ...     

Opiate binding sites and endogenous opioids in Bufo viridis oocytes

Binding sites with high affinity for [3H]naloxone, but not for [3H]morphine and [3H] (D-Ala2, D-Leu5) enkephalin, have been found in membranes of Bufo viridis oocytes. The binding is reversible and saturable. Bound [3H]naloxone is easily displaced both by unlabeled naloxone and bremazocine, much worse by morphine and SKF 10,047; (D-Ala2, D-Leu5) enkephalin and beta-endorphin practically fail to displace [3H]naloxone. Scatchard analysis is consistent with the existence of two classes of binding sites with Kd 15 nM and 10(3) nM. The number of binding sites with high affinity for naloxone is 16 pmol/mg protein of homogenized oocytes which is 20-50-fold higher than in, toad or rat brain. Oocyte extract displaces [3H]naloxone bound with oocytes' membranes and inhibits electrically evoked contractions of the rabbit vas deferens. This inhibition is reversed by naloxone. It is suggested that compounds similar to opiate kappa-agonists exist in oocytes. It cannot be ruled out that they participate via specific receptors in the regulation of oocyte maturation and egg development.     

中国西部地区绿蟾蜍的分类研究

对中国西部（新疆和西藏）原定名为绿蟾蜍Ｂｕｆｏｖｉｒｉｄｉｓ的２７个居群进行了开矿学比较研究,同时与产于邻近地区的Ｂｕｆｏｄａｎａｔｅｎｓｉｓ（土库曼）、Ｂ．ｌｏａｔａｓｔｉｉ和Ｂ．ｓｔｏｍａｔｉｃｕｓ（克什米尔）的３种蟾蜍标本或原始描述作了对比。我们赞同Ｂｏｒｋｉｎ等（１９８８）的研究结果,并将产中地原定名为．ｖｉｒｉｄｉｓ中四倍体的各居群统归为Ｂ．ｄａｎａｔｅｎｓｉｓ。同时将才原各居群分为４个     

Keratin expression during early embryonic development of Bufo bufo gargarizans

Three anti-keratin MAbs were used to identifykeratins expressed in early embryos of Bufo bufogargarizans.MAb AF5 recognized three polypeptides ofkeratin in oocytes,fertilized eggs,up to neurula withMr of 68,65 and 60Kd respectively.At tailbud stage,three other keratins(62,58 and 54Kd)began to expressand could be detected by AF5.MAbs D10 and K12 gavedifferent results,both of them could identify four keratin-like molecules with unusual molecular weights(Mr 98,95,30 and 27 Kd).Moreover,D10 could also detect a 54 Kdkeratin in neurula and tailbud stage embryos,while K12could reveal,beside 54Kd keratin,other four more kera-tins(68,65,62 and 60 Kd).The possible interpretation ofthese results and their implications are discussed.     

长波紫外线照射对花背蟾蜍肾脏结构损伤

为研究长波紫外线对花背蟾蜍(Buforaddei)肾脏的损伤,用波长为365nm的长波紫外线(UVA)以352μW.cm-2的辐射量对花背蟾蜍亚成体进行150、300和450min的连续照射,分别在照射后立即、3、6、9、12和15d取材,常规石蜡切片。结果表明:UVA照射后,3组不同照射时间的花背蟾蜍肾脏整体结构基本完整,但均出现肾小管管壁破裂,管径显著缩小,肾小囊消失,而300min照射组的损伤则极为明显,其后依次为450min照射组与150min照射组,300与450min照射组的肾小体塌陷程度较150min照射组更为严重;经15d的恢复,虽出现明显好转,但大多指标仍与对照组间存在极显著性差异;UVA对花背蟾蜍肾脏有着不可忽视的损伤,虽然具有一定的自我修复的能力,但在野外,紫外辐射的增强和植被的减少可导致两栖类遭受过度的紫外辐射,进而引起两栖类种群的衰减。     

Modulators of Bufo arenarum ovulation

In this study we investigated ovulation in vitro using ovary samples from Bufo arenarum with respect to their response to stimulation with homologous pituitary homogenate (HPH) or with progesterone and prostaglandins (PGF2alpha and PGE1) as intermediates of pituitary action. Ovary samples were obtained from animals captured during the breeding period. Our results demonstrate that the ovulatory response to all different inducers was dose dependent, the highest percentage of ovulated oocytes being obtained with HPH treatment. An important increase in the ovulatory response was obtained by the association of PGF2alpha with either HPH or progesterone at suboptimal doses, indicating that this prostaglandin induced a synergistic potentiating effect. Incubation with cyclooxygenase inhibitors (indomethacin or diclofenac sodium) produced a significant decrease in the ovulation induced by HPH, demonstrating that prostaglandins are involved in the action of the pituitary gland in this process. According to our results, PGE1 not only had no participation in the ovulatory process, but also produced an inhibitory effect on ovulation induced by HPH treatment.     

Lipids during Bufo arenarum oogenesis

The content and composition of phospholipids and triacylglycerols (TAGs) in Bufo arenarum oocytes in stages III and IV of their oogenesis were studied. The total amount of phospholipids in stage IV oocytes is 0.5-fold higher than in stage III oocytes. In both cases, the main phospholipids are phosphatidylcholine (PC) and phosphatidylethanolamine (PE). A striking observation concerns the high level of diphosphatidylglycerol (DPG) in stage III oocytes, which could be indicative of a relatively larger mitochondrial population with respect to other oogenetic stages. A net increase in sphingomyelin content was found during oogenesis. This fact could be related to the role of this phospholipid in the signal transductional pathways. In PC, palmitic (16:0), linoleic (18:2) and oleic (18:1) are the major fatty acids for both types of oocytes, while in PE the main acyl groups are 18:1, 16:0, arachidonic acid (20:4n6) and 18:2. PE is more unsaturated than PC and both phospholipids are more unsaturated in stage III oocytes than in stage IV oocytes. The amount of triacylglycerols is 0.3-fold higher in stage IV oocytes than in stage III oocytes. In both stages, the main fatty acids are 18:2, 18:1 and 16:0. During oogenesis, a significant increase in 18:1 and 18:3n3, and a decrease in 18:2 of TAG were found. The unsaturation index of TAGs from stage IV oocytes is higher than that from stage III oocytes. The TAG increase during oogenesis is consistent with the putative use of these lipids as a source of energy in embryo development.