SITE OF 1-METHYLADENINE RECEPTORS IN THE MATURATION OF STARFISH OOCYTES

Maturation of vitelline coat-free (VCF) oocytes of the starfish, Asterina pectinifera , was studied. When the oocytes, the vitelline coats of which were elevated by adding the ionophorc A-23187, were forced through two sheets of copper mesh, the vitelline coats were completely removed from the oocytes. Although some of the VCF oocytes underwent germinal vesicle breakdown following this mechanical treatment, most of them retained the normal germinal vesicles. These VCF immature oocytes underwent breakdown of germinal vesicles after addition of 1-methyladenine (1-MA). Dose-response curves of VCF oocytes to 1-MA were similar to those of normal oocytes. These results indicate that 1-MA reacts with the plasma membrane and that the presence of the vitelline coat is not prerequisite for inducing oocyte maturation.     

METHIONINE-ACTIVATING ENZYME IN STARFISH FOLLICLE CELLS *

In starfish follicle cells 1-methyladenine is produced under the influence of a gonad-stimulating hormonal peptide (GSS). Since such production of the substance is enhanced by the addition of L-methionine or S-adenosylmethionine in vitro, the presence of methionine-activating enzyme in the follicle cells of the starfish, Asterina pectinifera, was investigated. To detect enzyme activity, the enzyme was partially purified from the supernatant of the follicle-cell homogenate by precipitation with ammonium sulfate followed by gel-filtration on a Sephadex G-150 column. Using such a preparation of the enzyme, the production of S-adenosylmethionine from L-methionine and adenosine triphosphate was clearly demonstrated by thin-layer chromatography. GSS was found to exert no effect on the activity of the methionine-activating enzyme. The hormonal peptide, GSS, is therefore considered to take part in some reaction other than this step in the formation of 1-methyladenine.     

PRODUCTION OF 1-METHYLADENINE INDUCED BY CONCANAVALIN A IN STARFISH FOLLICLE CELLS

Concanavalin A (Con A) was found to induce maturation of oocytes with follicular envelopes in the starfish, Asterina pectinifera . Treating a Con A sample with 85% ethanol and heat revealed that the maturation-inducing activity of the sample was not due to possible contamination with 1-methyladenine, but to Con A itself. However, Con A had little maturation inducing effect on isolated oocytes from which the follicular envelope had been removed, suggesting that its effect is indirect and probably mediated by the follicle cells. When follicle cells were incubated in seawater containing Con A, a maturation-inducing substance was found to have been produced in the incubation medium. This was purified and identified as 1-methyladenine. Therefore it is concluded that Con A has the same capacity as GSS, a gonad-stimulating peptide hormone of neural origin, to induce production of the maturation-inducing substance. Other plant lectins such as phytohemagglutinin P and wheat germ agglutinin had little effect in inducing production of 1-methyladenine in follicle cells.     

EFFECTS OF METHIONINE AND S-ADENOSYLMETHIONINE ON PRODUCTION OF 1-METHYLADENINE IN STARFISH FOLLICLE CELLS

Incubation of follicle cells of the starfish ( Asterias amurensis ) with a gonad-stimulating hormonal peptide (GSS) leads to the production of 1-methyladenine. the trigger of oocyte maturation. Addition of L-methionine to the incubation medium promotes the production of 1-methyladenine. This study shows that S -adenosylmethionine also enhances the production of 1-methyladenine in such an incubation mixture. However, in the absence of GSS, addition of S -adenosylmethionine failed to produce an appreciable amount of 1-methyladenine. When an homogenate of isolated follicle cells was incubated, a certain amount of 1-methyladenine was produced, whether or not GSS was present. Addition of S -adenosylmethionine to the incubation mixture of follicle homogenate enhanced the production of 1-methyladenine. Although addition of adenosine triphosphate (ATP) to such an incubation mixture had little effect in producing 1-methyladenine, it exerted a promoting effect on 1-methyladenine production when S -adenosyl-methionine was present. These results suggest that methionine, through its active form, S -adenosylmethionine, acts as a donor of methyl group in the formation of 1-methyladenine.     

5S RNA AND TRANSFER RNA SYNTHESIS IN GERMINAL VESICLE OOCYTES AND DURING HORMONALLY-INDUCED MEIOTIC MATURATION OF STARFISH OOCYTES

The incorporation of ³H-guanosine as ³H-GMP into 5S RNA and into transfer RNA (tRNA) was examined in isolated large germinal vesicle oocytes, in isolated mature ootids and during and subsequent to hormonally (l-methyladenine)-induced meiotic maturation in the starfish, Asterias forbesii .Purified soluble RNA ¹ preparations at each stage were fractionated by electrophoresis on 10% polyacrylamide gels, while high molecular weight RNAs were resolved by subjecting total RNA samples to electrophoresis on 2.4% acrylamide+0.5% agarose gels. The results showed that large germinal vesicle oocytes, containing a single compact nucleolus, synthesize 5S RNA and tRNA as well as the previously-reported (1, 23-26) nucleolar rRNAs. In contrast, during and subsequent to hormonally-induced meiotic maturation, after germinal vesicle braekdown and nucleolar dissolution, the synthesis of 5S RNA and tRNA continues in the absence of detectable high molecular weight rRNA synthesis.     

水貂卵母细胞发育过程中卵泡细胞的微细结构研究

本文应用光镜,电镜及组织化学方法对水貂卵丘形成过程进行了研究。结果表明,在发育过程中,卵泡细胞内含有一些圆形线粒体,许多单个粗面内质网囊池和丰富的核糖体,高尔基复合体罕见,当卵泡细胞变成单层柱状时,细胞内开始出现成团的微丝及一些直径１－２μｍ的厚壁泡。在卵泡细胞膜上可见到胞吐小泡以及细胞间夹有透明带物质,表明卵泡细胞可能参与透明带的形成,随着卵泡腔的形成,卵泡细胞开始出现含中性脂肪及磷脂的颗粒,卵     

MECHANICAL PROPERTIES OF STARFISH OOCYTES*

The stiffness of the starfish oocyte was determined from the degree of deformation when it was compressed by a definite force between a pair of parallel plates. The deformation of the oocyte increases during continued application of a constant force, indicating visco-elasticity of the cell. A cyclic change in stiffness of the oocyte accompanying meiotic divisions was found: the stiffness of the oocyte decreases during early stage of meiotic division, increases before the onset of the first polar body formation, then decreases, increases again before the onset of the second polar body formation, and decreases thereafter. Deuteration causes increase in stiffness of the oocyte.     

小鼠卵母细胞体外成熟不同阶段对钙的依赖性

本研究旨在探讨小鼠卵母细胞成熟与钙和钙调素的关系。研究发现,20μmol/L W7、50μM BAPTA/AM对GVBD发生没有影响,但阻断了中期Ⅰ的卵母细胞进入中期Ⅱ。通过测定成熟不同阶段细胞内钙的分布,发现GVBD后染色体周围区域有较高水平的钙分布,并且该现象能被加BAPTA/AM而消除。GVBD发生后6h左右高钙分布现象消失。我们还测定了成熟过程中MPF活性的变化,20μmol/L W7、50μmol/L BAPTA/AM对卵母细胞成熟过程中MPF活性的升高没有影响。结果表明:小鼠卵母细胞GVBD的发生不依赖钙和钙调素;钙和钙调素对中期Ⅰ的发育是必需的,并且核周区钙分布可能起着重要作用。     

BINDING OF CONCANAVALIN A TO THE SURFACE OF STARFISH FOLLICLE CELLS AND PRODUCTION OF 1-METHYLADENINE

Starfish follicle cells, treated with concanavalin A (Con A), continued to produce 1-methyl-adenine (1-MeAde), an inducer of starfish oocyte maturation, after rinsing with artificial seawater (ASW). On the other hand, they ceased to produce the substance if treated with methyl α-Dmannoside (αMM). These cells produced again 1-MeAde when re-stimulated with Con A after removal of αMM. An optical study with fluorescein revealed that Con A bound to the cells was not dissociated by rinsing with ASW, but was removed if the cells were treated with αMM. These results suggest that continuous binding of Con A to the surface of the follicle cells is essential for the production of 1-MeAde.     

FINE STRUCTURE OF LOACH OOCYTES DURING MATURATION IN VITRO

The morphological changes during in vitro maturation of Misgurnus anguillicaudatus oocyte are described. The process of oocyte maturation can be divided into three provisional stages based on morphological events. Fully-grown, immature oocytes are opaque yellowish-white. The morphological characteristics of their ooplasm are the existence of annulate lamellae, a mass of long mitochondria and an electron dense layer beneath the vitelline surface. Three hr after a 1 hr exposure to corticosterone, these structures disappear and the cortical ooplasm becomes semi-transparent. In this stage of the maturation process (Stage I), the germinal vesicle, without a nucleolus, moves toward the animal pole, and scattered cytoplasmic inclusions approach the vitelline surface. Six hr after exposure to the hormone (Stage II), the whole ooplasm becomes semi-transparent and large yolk platelets are seen in the animal pole region. Tubular endoplasmic reticula develop throughout the ooplasm and some cortical alveoli (CA) become aligned beneath the vitelline surface. Nine hr after exposure to the hormone (Stage III), the oocyte chorion separates from the follicle cells. Most CA align beneath the vitelline surface and cytoplasm accumulates in the cortical region of the animal hemisphere.     

THE INTRACELLULAR DISTRIBUTION AND HETEROGENEITY OF RIBONUCLEIC ACID IN STARFISH OOCYTES

A study has been made of the content and composition of RNA in cytoplasm, nucleoplasm, and nucleoli from growing oocytes of the starfish Asterias rubens. The determinations were carried out, using ultramicrochemical methods, on units isolated by microdissection from fixed sections. Macrochemical and interferometric control experiments show that RNA can be quantitatively evaluated in this way. The results show that the growing oocyte represents a system in which the relations between the quantities of nucleolar, nucleoplasmic, and cytoplasmic RNA undergo great changes. These changes are continuous for nucleolar and cytoplasmic RNA so that their amounts may be predicted from the size of the cell. Nucleoplasmic RNA, on the other hand, shows great variations among different cells, independent of cell size. Purine-pyrimidine analyses show that each cell component contains an RNA which differs significantly from that of the other two. Cytoplasmic and nucleolar RNA are closely related, the only difference being a slightly higher guanine/uracil quotient for the nucleolar RNA. They are both of the usual tissue RNA type, i.e., they show a preponderance of guanine and cytosine over adenine and uracil. Nucleoplasmic RNA deviates grossly from the RNA of the other two components. Here the concentrations of adenine and uracil are higher than those of guanine and cytosine, respectively. This RNA consequently shows some resemblance to the general type of animal DNA although the purine/pyrimidine ratio is far from unity. Our data favor a nucleolar origin for the stable part of the ribosomal RNA and a nucleoplasmic one for the unstable part (the messenger RNA).     

银鲫卵母细胞体外诱导成熟技术的建立

研究以银鲫为材料, 根据银鲫(Carassius auratus gibelio)卵母细胞生发泡(Germinal vesicle, GV)边移程度及剥离GV中减数分裂前期染色体的凝集状态, 将银鲫Ⅳ时相的卵母细胞分为GV0、GV1、GV2和GV3四个时期; 并进一步比较了分别处于这4个时期银鲫卵母细胞体外诱导培养的成熟率、卵裂率和孵化率。结果表明, GV1期之后的卵母细胞均可有效进行体外诱导成熟, 可正常受精发育, 由于GV1期卵母细胞有较长时间用于显微操作, 因此GV1期卵母细胞被选为进行体外诱导的最早时期的卵母细胞。以GV1期卵母细胞为研究材料, 摸索了银鲫卵母细胞体外诱导成熟的适宜条件: 取GV1期的Ⅳ时相卵母细胞, 放置于pH 8.5、加有1 μg/mL孕酮激素(17α, 20β-dihydroxy-4-pregnen-3-one, DHP)的格氏平衡盐溶液(Gey’s balanced salt solution, GBSS)中, 在23℃培养箱中体外诱导12h后, 将滤泡膜剥离后再进行人工体外授精, 其所获胚胎的孵化率可达55.5%。此外, 将体外转录合成的带GFP标签的h2af1o mRNA注射到GV1期卵母细胞, 发现经显微操作和体外诱导后不仅可以通过GFP绿色荧光信号活体观察GVBD、受精、卵裂和早期胚胎发育的全过程, 而且诱导成熟的卵子仍可正常受精和胚胎发育。研究建立的银鲫卵母细胞体外诱导成熟技术为银鲫和其他鱼类卵母细胞发育过程研究及其相关基因和细胞显微操作提供了技术平台。     

兔卵母细胞体外成熟和体外受精的研究

用贴壁和悬浮生长二种培养系统,分析发情兔血清、滤泡液和激素对体外培养的兔卵母细胞的成熟、原核形成和发育能力的影响,并分析了不同浓度的激素对兔卵母细胞的作用。在贴壁生长的培养系统,滤泡液和激素对卵母细胞有明显的促成熟作用。但用这种卵母细胞体外受精,其原核形成率和发育率都较低。但在体外培养8小时后转移到体内受精,其原核形成和发育率大大提高,三者差别不大。在悬浮培养系统,卵母细胞成熟率、及体外受精后原核形成和发育率都远比贴壁生长的高,尤以原核形成率更甚。兔卵母细胞对激素的耐受力很小,以含FSH(2μg)、LH(1μg)、E_2-17B(1μg)和PRL组合的培波和含低hCG(7IU)的较适宜,高中浓度的FSH、LH和hCG都有促使卵母细胞变性和老化的作用。文中还讨论了二种培养系统不同的机制。     

INDUCTION OF SPAWNING AND OOCYTE MATURATION IN STARFISH BY 1-METHYL-ADENOSINE MONOPHOSPHATE

l-Methyladenosine monophosphate (l-McAMP) induces ovulation and oocyte maturation when applied to isolated ovarian fragments of Asterina pectinifera . However, isolated oocytes fail to mature even in the presence of this substance. When ovarian or testis fragments are incubated with l-McAMP, the supernatant of the incubation mixture acquires the maturation-inducing activity. Also, superantants of gonadal homogenates incubated with l-McAMP have the capacity to convert it to a maturation-inducing substance, suggesting that l-McAMP is decomposed to l-methyladenine, which is believed to be a general inducer of oocyte maturation and ovulation in starfishes. Thus l-MeAMP seems to be an intermediate in l-methyladenine formation when the latter is produced under the influence of the gonad-stimulating hormonal peptide.     

重组人卵泡刺激素在昆虫细胞中的表达

为了研究在昆虫细胞中表达重组人卵泡刺激素,我们以人胎盘组织提取的染色体DNA为模板,利用重叠PCR方法扩增出hFSHβ亚基的cDNA的编码区。将此cDNA克隆入核型多角体病毒(AcNPV)非融合蛋白基因表达载体pVL1393,我们得到了表达载体pVL1393-hFSHβ,然后与BaculoGold~(TM)线性杆状病毒DNA共转染昆虫细胞SF9,经多次扩增后获得高滴度的重组病毒AcNPV-hFSHβ。将此重组病毒感染昆虫细胞,我们得到了在胞浆中表达的hFSHβ亚基,Western blot显示分子量大约为21kDa。以重组病毒AcNPV-hFSHβ与AcNPV-hCGα一同感染昆虫细胞得到了具有分泌性的重组hFSH异二聚体,在非还原的条件下Western blot显示分子量大约为33kDa。     

虹鳟卵母细胞体外成熟与授精的研究

随着基因工程技术的发展,很多学者正致力于转基因鱼的研究。就实验材料而言,鱼类有体外受精、发育,怀卵量多、卵径大、便于显微操作等优点。然而鱼类受精卵的卵黄多、核很小,镜下难以分辨。特别是虹鳟鱼卵膜厚而不透明不可能将外源基因精确地注入到动物极内。因此转化率很低,只有通过大量地注射鱼卵来增加转化个体的数量。这不但要饲养大量实验鱼,而且为受体鱼的检测带来了很大的困难。一般来说通过体外培养成熟的卵母细胞膜薄而透明,核膨大明显可见。因此可以精确地将外源基因注入核区域内从而可以大大地提高转化率。       

蛋白质合成抑制剂亚胺环己酮(CHX)对猪卵母细胞体外成熟的影响

研究了蛋白质合成抑制剂亚胺环己酮 (CHX)对猪卵母细胞体外成熟过程中的GVBD、染色质凝集、MⅡ期成熟及卵丘细胞扩展的作用。结果表明 :( 1)培养液中添加CHX ,可抑制卵母细胞GVBD的发生 ,而且此作用是浓度依赖性的 ,但CHX的抑制效果是完全可逆的 ;( 2 )在含 10 μg/mlCHX液中分别培养 0、 6、 12和 2 4h后转入正常培养液再继续培养至 4 8h ,卵母细胞成熟率分别为 84 1%、 77 1%、 4 8 9%和 2 7 8% ;( 3 )正常培养液中培养 0、 6、 12、 2 4、 3 6和 4 8h后 ,再转入浓度为 10 μg/mlCHX液中继续培养至 4 8h ,卵母细胞成熟率分别为 0、 0、 0、 3 1 3 %、 65 4 %和 79 5 % ;( 4 )CHX对卵丘细胞扩展的影响随培养时间延长而增强 ,在CHX中处理时间为 16h或更长 ,完全抑制卵丘细胞的扩展     

MARKED DECREASE IN THE RIGIDITY OF STARFISH OOCYTES INDUCED BY 1-METHYLADENINE1

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 (1-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 μg/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.     

成熟脱水对种子发育和萌发的作用

成熟脱水是正常性种子发育的末端事件。种子在成熟时胚的脱水耐性增加;当种子萌发时胚变得不耐脱水。当种子获得脱水耐性时,糖、蛋白质和抗氧化防御系统等保护性物质积累;当脱水耐性丧失时,这些物质被降解。成熟脱水是种子从发育过程向萌发过程转变的“开关”,它降低发育的蛋白质和ｍＲＮＡ的合成,终止发育事件和促进萌发事件。顽拗性种子不经历成熟脱水的发育阶段,对脱水高度敏感。