Embryonic Development in House Fly Eggs Fertilized with Genetically Deficient Sperm

In house flies, Musca domestica L., eggs fertilized with sperm that have chromosome deficiencies and duplications do not hatch, but develop to a stage where a fully differentiated, prehatch larva is formed. Fifteen different chromosome translocations involving most of the 10 arms of the 5 autosomes were studied by crossing male translocation heterozygotes to normal females. Egg hatch was reduced to 36–66% depending on the translocation used. Eggs that did not hatch after 24 hours were fixed, stained, and examined for stage of development. Several translocations involving the right arm of chromosome 4 indicate that the region closest to the centromere contains genes that affect the process of syngamy or early cleavage divisions, but do not reduce the ability of the sperm to compete for egg fertilization. Approximately 70% of the autosomal genes can be absent from sperm (not simultaneously but in different crosses) without inhibiting embryonic development.     

Distribution of Membrane-Associated Calcium in Fertilized Sea Urchin Eggs during Mitosis*

The distribution of membrane-associated calcium in dividing sea urchin eggs was examined with chlortetracycline as a fluorescent chelate probe. The fluorescence of bound chlortetracycline in fertilized eggs was initially evenly distributed, but began to gather around the nucleus in prophase, and formed a dumb-bell shaped condensation enclosing the mitotic apparatus by metaphase. During anaphase and telophase, the fluorescence was observed in kinetochore-to-pole regions of the spindle, with little fluorescence in the interkinetochore region. The astral regions showed intense fluorescence. The distribution of the chlortetracycline-fluorescence coincided with that of ER-like membranes seen in electron micrographs. The distribution of the fluorescence was obscure and the birefringence of spindles disappeared on perfusion on perfusion of the cells in metaphase with 1 mM tetracaine, which is known to displace membrane-bound calcium. These results suggest that intracellular free calcium ions are sequestered in the membrane system associated with the mitotic apparatus during mitosis.     

PKC对小鼠受精卵发育的调控作用

为研究 TPA及 PKC的反义寡核苷酸对 1 -细胞期鼠受精卵发育的影响 ,采用免疫细胞化学法标记 PKC(α及 β亚型 ) ,并用激光扫描共聚焦显微镜测定卵内 PKC荧光强度 ;同时利用显微注射法注射 PKC的反义寡核苷酸 ,观察其对受精卵分裂的影响 . 1 0 0 μg/ L TPA对 1 -细胞期受精卵的发育具有完全抑制作用 .TPA处理 1 2 h后 ,对照组受精卵停留在 1 -细胞期 ,而未经 TPA处理的1 -细胞期卵可以分裂到 2 -细胞期 .共焦激光显示实验组与对照组相比 ,PKC(α、β亚型 )荧光强度均有下降 (P<0 .0 1 ) .显微注射 PKC antisenseα及 antisenseβ的受精卵 ,分别只有 1 4 .2 %和 3.33%的卵可以发育到 2 -细胞期 .与对照组 (注射 M2培养液 )差异显著 (P<0 .0 1 ) .结果表明 ,(1 ) TPA长期处理 1 -细胞期受精卵 ,抑制 1 -细胞期卵分裂到 2 -细胞期 ;(2 ) PKC的反义寡核苷酸 (α及β亚型 )可以抑制小鼠 1 -细胞期卵的发育     

Subcortical Rotation and Specification of the Dorsoventral Axis in Newt Eggs

The specification of the dorsoventral axis in naturally polyspermic eggs of the Japanese newt, Cynops pyrrhogaster , was first examined by studies on the spatial relationship between the dorsal midline of the future body plan and the sperm entrance points (SEPs ¹ ). On local insemination, the dorsal blastopore lip was usually found to be formed opposite the SEPs, as in anuran monospermic eggs. Next the movements of the subcortical layer and the cortex were analyzed. "Subcortical rotation" was observed, similar to that of Xenopus laevis eggs with respect to its timing and extent, and its direction was shown to predict the embryonic axis of the eggs. Thus, the dorsoventral axis was concluded to be determined by essentially the same mechanism in the newt as in Xenopus .
Owing to their large size and long first cell cycle, newt eggs appear to be suitable material for study of subcortical rotation, but their behavior is unique in that subcortical rotation occurs in only the vegetal hemisphere so that the subcortical layer stretches in the future dorsal side. Studies on the movement of Nile blue spots suggested that the cytoplasm under the cortex in newt eggs consists of two layers.     

受精鸡蛋卵黄中DNA和RNA的分布

通过对受精未孵育鸡蛋卵黄的不同部位加以考查,发现DNA和RNA在卵黄中的分布是广泛存在的;采用荧光试剂Hoechst33258和RiboGreen对DNA和RNA进行定量分析,结果表明：DNA在不同部位卵黄中的分布比较均匀,且主要存在于卵黄颗粒中,而RNA的含量在胚下表层卵黄、侧面和植物极中有较大差异.     

An Electrical Model for the Cytoplasmic Calcium Wave in Fertilized Eggs

The calcium wave subsequent to fertilization of the egg is analyzed interms of an electrical equivalent circuit. The circuit consists of aswitch, capacitor, an inductor, and a resistor. The switch symbolizes aseries of chemical reactions initiated by the sperm the lead to thedevelopment of the calcium wave. Its closure signifies the onset of thecalcium wave. The capacitor and inductor represent the endoplasmicreticulum. The capacitive component of the endoplasmic reticulum controlsthe release of calcium ions while the inductive component regulates thesequestration of clacium ions. The resistor represents the inductor andcytoplasm and has very low resistance. The analysis of the circuit showsthat the period of the calcium oscillations is proportional to the size ofthe egg. It agrees with the measurements on various types of eggs.     

Ultracytochemical Modifications of Surface Carbohydrates in Fertilized Eggs of the Common Carp

The surface of the plasma membrane of unfertilized and fertilized carp eggs was examined by four cytochemical techniques, colloidal thorium, colloidal iron, ruthenium red and phosphotungstic acid stainings, to determine the carbohydrate moieties. The surface of the plasma membrane of unfertilized eggs stained only with colloidal iron, which was heterogeneously deposited: no deposits were seen on the plasma membrane near overlying cortical alveoli. In fertilized eggs, the membrane was stained by all four methods. These ultracytochemical modifications of the surface of the plasma membrane may be caused by participation of the limiting membranes of secretory organelles, probably by turnover of the inner surface of the limiting membranes. Neuraminidase treatment of fertilized eggs eliminated the deposits of colloidal iron on the surface of the plasma membrane and caused an increase in stainability with ruthenium red. Treatment with neuraminidase or trypsin prevented the staining with phosphotungstic acid.     

Cdc25B在小鼠受精卵的表达和定位

为阐明细胞分裂周期(Cdc)25B调控小鼠受精卵发育的机制,利用Western印迹检测小鼠受精卵各时期Cdc25B的表达及Cdc2-Tyr15的磷酸化状态。利用间接免疫荧光技术观察Cdc25B在小鼠受精卵的定位。构建pEGFP-Cdc25B融合表达载体并显微注射到受精卵中,观察Cdc25B在受精卵M期的定位变化。结果表明Cdc25B在G1和S期被磷酸化,在G2和M期去磷酸化。Cdc2-Tyr15在G1和S期处于磷酸化状态,G2期只检测到Cdc2-Tyr15轻微的磷酸化信号,M期未检测到任何Cdc2-Tyr15的磷酸化信号。Cdc25B在G1期定位于细胞质和细胞核中,S和G2期定位于细胞质的皮质部分,M期由细胞质转向核区。证明Cdc25B核输出后激活有丝分裂促进因子,从而启动小鼠受精卵的有丝分裂。     

鸡蛋胚下表层卵黄DNA的提取方法

利用Ficoll-400不连续密度梯度离心将受精和未受精鸡蛋的胚下表层卵黄进行纯化, 显微镜观察表明卵黄球形态良好, 没有胚细胞的存在.然后利用较高浓度的蛋白酶K消化,较长时间的酚抽提,最后提取了DNA. 电泳显示DNA条带清晰.该方法简便快速,从每个鸡蛋的胚下表层卵黄可回收10 ng DNA.     

Control of Polar Lobe Formation in Fertilized Eggs of Ilyanassa obsoleta Stimpson

Mechanisms of polar lobe formation in fertilized eggs of Ilyanassaobsoleta are discussed. Data are presented in the context ofa model involving a contractile ring of microfilaments in thecortical cytoplasm of the vegetal hemisphere. The polar lobeneck diameter decreases at two distinct rates during formationof second, third, and fourth polar lobes. During the second,more rapid phase of lobe constriction, eggs contain a band ofmicrofilaments arranged circumferentially in the cortical cytoplasmapposed to the plasma membrane at the base of the polar lobeconstriction. These microfilaments disappear and lobe constrictionsregress in cytochalasin B, but not in colchicine. Colchicineprevents eggs from beginning the second, more rapid phase oflobe constriction. Eggs require the presence of potassium ionsbut not sodium ions for normal polar lobe formation and cleavage.When eggs are placed in isotonic solutions of CaCl₂, within10–15 min they form cytoplasmic blebs which enlarge intolobes. This calcium-induced blebbing is inhibited by cytochalasinB but not by colchicine. Blebbing occurs in the calcium concentrationrange of 0.17 M-0.34 M in the presence of NaCl, MgCl₂, or MnCl₂.Potassium ions actively inhibit the calcium-induced blebbing,however.     

精核蛋白对小鼠1-细胞期受精卵转录的影响

应用一种非常敏感的、新的荧光方法 ,进行了精核蛋白对小鼠 1 细胞期受精卵转录影响的观察 .hCG注射后 18h的受精卵作为精核蛋白抗体显微注射对象 .镜下观察 ,非抗体注射与抗体注射的卵细胞荧光强度有明显差异 .根据荧光分光光度计测得的结果 ,抗体注射卵细胞的平均荧光强度值相当于抗体非注射的 2 2 8%,高 1 3倍 ,经t检验 (n =30 )得P <0 0 1,差别有显著性意义 .而非BSA注射和BSA注射卵细胞的镜下观察 ,则没有多大差异 ;测两组卵细胞的相对荧光强度值分别为 10 0 %和 115 %,t检验 (n =30 )得p >0 0 5 ,差别无统计学意义 .将α 鹅膏蕈碱与精核蛋白抗体等量混合后一起注射 ,卵细胞组间荧光的显著性差别不复存在 .实验结果表明 ,精核蛋白在小鼠 1 细胞期受精卵中起着转录抑制作用 .     

Response to Sperm Penetration of the Cortex of Eggs of the Fish, Plecoglossus altivelis

The response of the egg to sperm penetration was examined in eggs of the fish, Plecoglossus altivelis , by scanning electron microscopy. Eggs responded to sperm penetration by forming a fertilization cone at a "sperm entry site", which is a specialized structure in the egg surface under the micropyle. Within one minute, the fertilization cone showed dramatic morphological changes from its earliest appearance, through full two-storied growth to its marked recession. The sperm entry site in the egg surface is discussed as a morphologically specialized organ responsible for the entrance of a fertilizing spermatozoon. The morphological characteristics of the egg and sperm are also described.     

Sperm Nuclear Transformation and Aster Formation Related to Metabolic Behaviour in Amphibian Eggs

Bufo arenarum oocyte maturation is related to important modifications in the metabolism of carbohydrates. Such changes involve a different relative participation of the main oxidative routes and are under the influence of seasonal variations of the pituitary activity.
Ovulated coelomic oocytes obtained during the winter period, were unable to initiate cleavage after injection of a sperm suspension. The extent of sperm head transformation and aster formation in the cytoplasm of oocytes with a different metabolic behaviour (obtained during the winter and summer periods) were studied.
Our results show that sperm nuclear transformation and DNA synthesis were quite similar in both types of oocytes. In contrast to summer oocytes, in which the pronucleus was sourranded by an aster, no aster structure was formed in winter ooctyes notwithstanding pronucleus formation occurred.
These results suggest that the failure to develop aster may explain the lack of cleaving in winter oocytes. It appears that the metabolic changes, aster formation and the capability to cleave are closely related and could be dissociated from oocyte nuclear maturation in this species.     

小鼠受精卵中蛋白激酶C底物的鉴定

为研究蛋白激酶C（protein kinase C,PKC）在小鼠早期发育中的调节作用,运用超排卵和体外受精技术,采用体外磷酸化和放射自显影的方法,鉴定小鼠1-细胞期受精卵中PKC的底物。经特殊的反复冻融处理,消除卵中内源性蛋白激酶活性。55个受精卵的样品中加入部分纯化的PKC,结合应用较强的PKC抑制剂H-7和星形孢菌素以及促分裂原活化蛋白激酶抑制剂PD098059作为对照,观察到12条PKC底物蛋白的放射自显影带,根据标准蛋白质对值绘制的标准曲线计算,这些磷酸化蛋白的相对分子量分别约为120kDa、100kDa、79kDa、63kDa、59kDa、47kDa、40kDa、34kDa、32kDa、26kDa、24kDa和22kDa。实验结果表明,PKC可通过底物蛋白活性的调节,在小鼠早期发育中发挥重要作用。     

激光微束在两种鱼受精卵上打孔技术研究

在埃及胡子鲶和苏氏芒鲶两种鱼受精卵上,用脉冲ＹＡＧ激光微束打孔技术,成功地导入了外源指示剂和用激光微束打过孔的受精卵孵化出了仔鱼,为今后激光微束打孔植入外源基因和针类转基因研究做了一些有意义的探索工作。     

Intracytoplasmic Sperm Injection Using Cryopreserved, Fixed, and Freeze-dried Sperm in Eggs of Nile Tilapia

Gamete preservation techniques are essential in animal husbandry as well as in assisted reproduction for humans. In this research we attempted to use 3 different sperm preservation techniques in combination with newly developed techniques for intracytoplasmic sperm injection (ICSI) to fertilize eggs of a teleost fish, the Nile tilapia (Oreochromis niloticus). Of 47 eggs injected with fresh sperm, 11 (23%) were fertilized, 5 developed abnormally, and 4 developed normally and hatched; from these, one grew to adulthood. Nuclear DNA content of 4 of the abnormal embryos indicated that they were diploid. Flow cytometric analysis of a blood sample from the surviving ICSI fish collected 2 months after fertilization indicated that the fish was diploid. Of 45 eggs injected with cryopreserved sperm, 9 (20%) developed to the blastula stage. Of 40 eggs injected with sperm preserved in 70% methanol, none were fertilized. No injections were possible with freeze-dried Nile tilapia sperm owing to technical difficulties during manipulation. Although the findings described here are limited, they provide the first steps toward using sperm preservation methods in addition to cryopreservation for fertilization in fishes.     

Escherichia coli gal K和gpt基因注入小鼠受精卵的研究

含有E.coli galK和gpt基因的重组DNA pPB22用显微注射法导入昆明白小鼠受精卵,经发育,最后得到21只成年小鼠。其中1只小鼠的肝脏显示出E.coli gpt酶活性,但未检测出E.coli galK酶活性。印迹分子杂交表明,外源的pPB 22重组DNA已整合到2只小鼠的染色体上,其中包括那只肝脏中显示E.coli gpt酶活性的小鼠。