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.     

CHEMICAL STRUCTURAL REQUIREMENTS FOR INDUCTION OF OOCYTE MATURATION AND SPAWNING IN STAREISHES

Effects of various adenine derivatives on oocyte maturation and spawning were studied in the starfishes, Marthasterias glacialis, Astropecten aurantiacus, Patiria miniata, Asterina pectinifera and Asterias forbesi . 1-Methyladenine and 1-ethyladenine were very effective in inducing oocyte maturation and spawning, whereas the following related compounds had no effect: adenine, 3-methyladenine, 7-methyl-adenine, 9-methyladenine, 1-methylguanine, 1-methylhypoxanthine, 6-methylpurine, N₆-methyladenine, N₆-
dimethyladenine, N₆-benzyladenine, N₆-furfuryladenine(kinetin), adenosine, 5' -adenylic acid, adenosine 3',5'-cyclic monophosphate, adenosine triphos-phate, inosine, 5'-inocinic acid, guanine, guanosine, 5'-guanylic acid, hypoxanthine, xanthine, xanthosine, 3-methylcytidine and 5-methylcytosine. 1-Methyladenosine induced oocyte maturation and spawning when isolated ovarian fragments were used as assay material; however, it had little effect in inducing maturation of isolated oocytes. Therefore, this compound seems to active only after its decomposition to 1-methyladenine and ribose. The chemical structure responsible for inducing oocyte maturation and spawning in starfishes is proposed: a short alkyl radical such as methyl or ethyl at N₁ site and an imino radical at C₆ site of the purine nucleus.     

PRESENCE OF 1-METHYLADENINE IN SEA URCHIN GONAD AND ITS RELATION TO OOCYTE MATURATION

A water extract of sea urchin ovary was found to induce maturation of starfish oocytes in vitro . The presence of the active substance was demonstrated in ovaries of the sea urchins, Pseudocentrotus depressus, Anthocidaris crassispina and Hemicentrotus pulcherrimus , and the sand dollars, Clypeaster japonicus and Peronella japonica . The active substance was also contained in the testes of these echinoids. That the content of this substance increases during the reproductive season was demonstrated with Anthocidaris gonads. The active substance present in ovary or testis of the sea urchins was successfully extracted with 85% ethanol and purified with gel-filtrations on Sephadex G-15 columns after washing with chloroform and ether. The purified active substances were the same and were identified as 1-1-methyladenine by thin layer chromatography. 1-Methyladenine was found to be effective in inducing oocyte maturation in Anthocidaris crassispina in vitro . Therefore, 1-methyladenine seems to play an important role in oocyte maturation in echinoids as well as in asteroids.     

CHANGES IN NUCLEOLI AT MATURATION OF NEWT OOCYTE

Changes in the nucleoli of maturing oocytes and the eggs of Cynops pyrrhogaster were studied with light and electron microscopy. Extrachromosomal nucleoli moved toward the center of the germinal vesicle in response to the maturation stimulus, released presumed ribosomal ribonucleoprotein, and further moved toward the center of the nucleus to form an aggregate with chromosomes which behaved in a similar manner. A few. ball-shaped nucleolar masses were formed from this aggregate, leaving the chromosomes and probably the extrachromosomal nucleolar organizer. The chromosomes then proceeded to the first meiotic metaphase. The nucleolar masses were surrounded by a layer of mitochondria and became smaller with formation of pinched-off fragments, which were also surrounded by mitochondria, during the time the egg was moving down the oviduct. Only fragments were observed in the subcortical area of the animal hemisphere of the egg after reaching the lowest part of the oviduct.     

ON THE ROLE OF CALCIUM IONS IN OOCYTE MATURATION IN THE POLYCHAETE CHAETOPTERUS PERGAMENTACEUS*

The germinal vesicle of mechanically released Chaetopterus oocytes disintegrates in natural sea water (NSW), but not in artificial sea water of normal composition (ASW), calcium-free sea water (CaFSW), magnesium-free sea water (MgFSW) or calcium and magnesium-free sea water (CaMgFSW). Several methods of inducing oocyte maturation using chemically well-defined medium have been established. (1) Germinal vesicle breakdown was induced by the treatment of immature oocytes with KCl (60 mM) in ASW or MgFSW. The presence of Ca²⁺ is necessary for inducing oocyte maturation with high potassium concentration. “Differentiation without cleavage” was observed after this treatment. (2) Trypsin (0.3%) induced oocyte maturation in ASW, but not in CaFSW. Oocytes matured in this manner developed to trochophores upon insemination. (3) Immature oocytes, treated with isotonic CaCl₂ for less than 1 min and then transferred to ASW, underwent germinal vesicle breakdown. The oocytes were arrested at the first meiotic metaphase and upon insemination developed to trochophore larvae. (4) Tetracaine (0.4 mM) induced oocyte maturation in the absence of Ca²⁺ in the medium. In ASW, CaFSW or CaMgFSW containing the drug, oocytes were arrested at the first meiotic metaphase, while in MgFSW with tetracaine they developed parthenogenetically up to the 4- and 8-cell stages. The role of calcium in oocyte maturation was established and its importance was discussed based on the results obtained with the different ways of inducing oocyte maturation.     

INDUCTION OF ERYTHROID MATURATION BY DIMETHYL SULFOXIDE IN FRIEND LEUKEMIC CELLS

Enhancement of the erythroid maturation in Friend virus-induced leukemic cells has been examined in vitro by the treatment with dimethyl sulfoxide (DMSO). Although the cell growth was inhibited in the medium containing 2% DMSO, many cells remained viable for a week. By the 3rd day of the culture, the cells treated with DMSO became more strongly agglutinated by phytohemagglutinin than the cells incubated without DMSO. Mouse erythrocyte membrane-specific antigens were also detectable at the 4th day. At the 8th day of the culture hemoglobin synthesis was apparently demonstrated in the cells treated with DMSO, which could not be seen in the untreated cells. Maturation or differentiation along the erythroid pathway in Friend leukemic cells by DMSO is discussed on these markers.     

HORMONAL CONTROL OF OOCYTE MATURATION IN ARENICOLA MARINA L. (ANNELIDA, POLYCHAETA) I. MORPHOLOGICAL STUDY OF OOCYTE MATURATION

In Arenicola marina (Annelida, Polychaeta) the oocytes are arrested in the first prophase stage of mciosis until spawning. Oocyte maturation is under hormonal control: when incubated in vitro in a brain extract oocytes reach the first metaphase at which they remain arrested until fertilization. The meiosis reinitiating substance induces numerous morphological changes in the oocytes: general (shape), cortical (microvilli retraction, plasma membrane flattening), cytoplasmic (cortical granules repartition) and nuclear modifications (germinal vesicle breakdown, chromosome condensation, formation of a meiotic maturation spindle). A kinetic study of these morphological modifications has been performed.     

FORMATION AND BEHAVIOR OF PINOSOMES IN THE SEA URCHIN OOCYTE DURING OOGENESIS

Formation and behavior of the pinosomes at the surface of the oocyte during oogenesis in the 4 species of sea urchins, Anthocidaris crassispina, Temnopleurus toreumaticus, Mespilia globulus and Pseudocentrotus depressus, were studied. The plasma membrane of the oocyte is almost smooth at the early stage of oogenesis, although a small number of cytoplasmic processes appear on it, facing the germinal epithelium. At the beginning of vitellogenetic stage many processes appear on the whole surface of the oocyte. Near the base of the fully grown process, the pinosome designated as the α-pinosome is formed. The α-pinosome may play a part in maturation of the yolk granule. The processes shorten as a whole at the time of the breakdown of the germinal vesicle. Formation of the pinosome designated as the β-pinosome begins just before vitellogenetic stage and continues during this stage. The β-pinosome may be directly concerned with the formation of cortical granules.     

INHIBITORY EFFECT OF RABBIT SERUM FACTOR ON IN VITRO OOCYTE MATURATION OF THE TELEOST FISH,ORYZIAS LATIPES*

The present study indicates that a factor in rabbit serum inhibits the in vitro steroid- and gonadotropin-induced maturation of oocytes of the teleost fish, Oryzias latipes. Such inhibitory activity could not be recognized in the serum of this fish or in the fluids from mammalian follicles. Passage of the serum inhibitor through a cellulose membrane indicated that it has a molecular weight of less than 3,500. The inhibitory activity on steroid-induced oocyte maturation was not destroyed by heating, by repeated freezing and thawing or by treatment with proteolytic enzymes, lipase or amylases. However, its activity could be removed by extraction with charcoal but not with ethyl ether or toluene. The inhibitory action of the heat-stable and dialyzable serum factor was reversible. The factor appears to exert its inhibitory effect upon the oocyte itself in an early step of maturation, rather than upon the follicle cells.     

PHOTOPERIODIC INDUCTION OF OVARIAN MATURATION IN CRAYFISH PROCAMBARUS CLARKII IS MEDIATED BY EXTRARETINAL PHOTORECEPTION

The current study was carried out to investigate whether thephotoperiodic induction of ovarian maturation in crayfish is based on a photosensitiverhythm related to extraretinal photoreceptors. To test this, two batches of61 juvenile crayfish Procambarus clarkiiconsisting of intact organisms and animals lacking retina and lamina were exposed to 24hlight-dark cycles of different photoperiodic schedules based on a night-breakprotocol for 3 months. Both batches of crayfish showed the greatest ovarianmaturation (size, color, degree and size of oocytes) when the light pulseinterrupted the scotophase at 21:00 and 05:00, showing a bimodal photoinduciblerhythm. Results of the current study indicate that crayfish ovarian maturationdepends on a photoinducible rhythm with two possible states that is relatedto the circadian clock of crayfish. This phenomenon is mediated by extraretinalphotoreceptors. Results are interpreted in the light of models of externalcoincidence. (Chronobiology International, 18(3),423–434, 2001)     

内源环化腺苷酸(cAMP)含量变化对卵母细胞成熟的影响

孕酮处理前,冬眠卵内源cAMP平均水平为500 Fmol.左右;处理后cAMP迅速下降,在12小时内下降59%,卵的生殖泡破裂。高温卵或热休克冬眠卵,孕酮刺激后cAMP水平亦下降,生殖泡却未破裂,但在高温卵质中出现依赖“冬眠因子”的促成熟活性物质,而在热休克冬眠卵质中出现不依赖“冬眠因子”的促成熟活性物质。热休克能影响卵的生殖泡破裂,却未影响卵质中MPF的形成。孕酮刺激后引起的卵内cAMP含量下降,只能是卵母细胞成熟的必要条件,而不是唯一的条件。     

蛋白激酶C在小鼠卵母细胞体外成熟和受精中的作用

蛋白激酶是一类重要的丝/苏氨酸蛋白激酶。本实验以小鼠为实验动物,研究了PKC在卵母细胞体外成熟、活化和受精中的可能作用,及两种PKC亚型在卵母细胞中的定位。PKC激活剂PMA可以阻止CV期卵母细胞在体外恢复减数分裂,该作用可被PKC抑制剂calphostin C抵消,但不能被PLCγ抑制剂U73122或PKCδ专一性抑制剂Rottlerin所克服。Western印迹显示PKCα和βⅠ在卵母细胞发育过程中恒量表达。激光共聚焦显微术研究发现,受精或受到活化刺激后PKCα转位到卵母细胞膜上,同时皮质颗粒排放,说明PKCα可能参与调节卵皮质反应。本实验首次在小鼠中研究了PLCγ与受精的关系,发现不存在PKC对PLCγ的正反馈调节。此外,本研究还对小鼠卵巢中对PKCα和βⅠ进行了蛋白定位研究。     

HORMONAL CONTROL OF OOCYTE MATURATION IN ARENICOLA MARINA L. (ANNELIDA, POLYCHAETA)

In Arenicola marina (Annelida, Polychaeta) the oocytes are arrested in the first prophase stage of meiosis until spawning. Oocyte maturation is under hormonal control: when incubated in vitro in a brain extract oocytes reach the first metaphase at which they remain arrested until fertilization. The importance of calcium in oocyte maturation has been investigated by using different drugs known to act on membrane calcium permeability and to modify intracellular free calcium concentration. Tetracaine, procaine, D-600, verapamil (Isoptin), propranolol, oxprenolol and lanthanum chloride, calcium deprivation but not ionophore A23187, are all able to induce oocyte maturation. This suggests that the brain hormone may act on the oocyte by regulating, probably increasing, the intracellular free calcium concentration, as it has been proposed for oocytes of other animals. The importance of -SH/-SS- in meiosis reinitiation is suggested by the fact that dithiothreitol and 2, 3-dimercaptopropanol, two disulfide reducing agents, both induce oocyte maturation.     

HORMONAL CONTROL OF OOCYTE MATURATION IN ARENICOLA MARINA L. (ANNELIDA, POLYCHAETA) II. MATURATION AND FERTILIZATION

In Arenicola marina (Annelida, Polychacta) the oocytes arc arrested in the first prophase stage of meiosis until spawning. Oocyte maturation is under hormonal control: when incubated in vitro in a brain extract oocytes proceed to the first metaphase at which they remain arrested until fertilization. The prophase arrested oocytes can neither be fertilized nor parthcnogenetically activated by ionophore A23187 or 1 M glycerol. On the contrary the metaphase-arrested oocytes can be fertilized and parthenogenetically activated. Fertilizability thus appears during maturation; it seems to be linked to microvilli retraction. A study of spermatozoa capacitation and oocyte fertilization or activation is reported. A scanning electron microscope study of early contact and penetration of spermatozoa is presented.     

INDUCTION OF ASTER FORMATION AND CLEAVAGE IN EGGS OF THE SEA URCHIN HEMICENTROTUS PULCHERRIMUS BY INJECTION OF SPERM COMPONENTS*

Studies were made on which components of sperm were able to induce aster formation and cleavage of eggs of the sea urchin Hemicentrotus pulcherrimus. The sperm components were separated by homogenization and centrifugation into the following 3 fractions: the head-midpiece, midpiece and tail. The head-midpiece fraction was then divided into 2 sub-fractions, the centriole sub-fraction and the centriole-free sub-fractions. Each fraction was injected into unfertilized eggs and after 15–30 min the eggs were inseminated. The ability of a fraction or a sub-fraction to induce aster formation and cleavage was deduced from the frequency of multipolar cleavage. The head-midpiece fraction and the centriole sub-fraction were effective in inducing aster formation and cleavage, but the other fractions were not. It was concluded that isolated centrioles from sea urchin sperm act as division centers in the egg.     

小鼠卵母细胞成熟和受精过程中Ca^2＋分布变化的研究

用焦锑酸钾原位定位法、膜结合Ｃａ＾２＋荧光探针金霉素标记法,分别在电镜和光镜水平对小鼠卵成熟和卵受精过程中结合态Ｃａ＾２＋的分布及其变化进行了研究,发现：１）Ｃａ＾２＋分布于线粒体、胞质、内质网囊泡、微绒毛和透明带等部位,其中以线粒体基质中分布密度为最大;２）减数分裂Ｉ中、后期于纺锤体极区结合有较多的Ｃａ＾２＋;３）生发泡、纺锤体和原核内膜结合态Ｃａ＾２＋含量很少,但纺锤体和原核周围分布较多;４）     

蟾蜍泛素羧基末端水解酶(tUCH)以不依赖其UCH活性的方式参与卵母细胞成熟调控

本实验室已报道中华大蟾蜍卵母细胞的p28蛋白具泛素羧基末端水解酶活性,称作tUCH,它和哺乳类中发现的UCH L1的氨基酸序列具高度同源性,二级结构同源性比较发现,二者可能具类似的功能。本文实验表明:未成熟卵母细胞和成熟卵母细胞的可溶性蛋白中均含有tUCH,约占提取物中蛋白质总量的2%。根据测定所得到的GST-tUCH和GST-UCH L1对底物Ub-AMC的酶动力学参数,说明卵母细胞中tUCH可能与小鼠UCH L1有类似的生物学功能;anti-tUCH单抗可以与原核细胞表达的tUCH和显性失活突变类型tUCH C(90)S特异结合,但不识别小鼠的UCH L1。Anti-tUCH单抗能够和tUCH结合但不能封闭它的UCH活性。当anti-tUCH单抗注入卵母细胞内,则孕酮诱导的生发泡破裂(germinal vesicle breakdown,GVBD)过程受到抑制,足见tUCH参与GVHD调节并不依赖其UCH活性。     

THE PERMEABILITY OF THE AMPHIBIAN OOCYTE NUCLEUS, IN SITU

Ultralow temperature radioautography, suitable for the quantitative localization of diffusible solutes, was used to study the permeability of the nuclear envelope in the intact amphibian oocyte Sucrose-³H solutions were injected into mature oocytes, in volumes of 0 016–0 14% of that of the cell, and the subsequent movement of the solute was recorded. The resultant radioautographs show diffusion gradients in the cytoplasm and nucleus, and concentration gradients across the nuclear envelope Analysis of these gradients discloses that the nuclear envelope is as permeable as a comparable structure composed of cytoplasm, and is about 10⁸ times more permeable than the oocyte plasma membrane The diffusion coefficient of sucrose in cytoplasm is 2 x 10^-6 cm²/sec, or about one-third its diffusivity in pure water. This reduction can probably be accounted for by an effective lengthening of the diffusional path because of obstruction by cytoplasmic inclusions. The nuclear: cytoplasmic sucrose concentration ratio at diffusional equilibrium is about 3 05, or 1.6 times as great as expected from the water content of the two compartments This asymmetry is attributed to an unavailability of 36% of the cytoplasmic water as solvent Finally, sucrose entry into oocytes from a bathing solution was monitored by whole cell analysis and radioautography. These and the microinjection results are consistent with a model in which sucrose entry into the cell is entirely limited by the permeability of the plasma membrane. The results are inconsistent with cell models that hypothesize a short-circuit transport route from the extracellular compartment to the nucleus, and with models in which cytoplasmic diffusion is viewed as limiting the rate of solute permeation.     

ABA、NAA诱导水稻胚性愈伤组织的研究

ABA和NAA联合使用能有效地诱导水稻原生质体再生的愈伤组织向胚性发展。通过液体浅层培养由原生质体得到的愈伤组织,在含ABA和NAA的N_6培养基上培养一段时间,可以诱导原来呈非胚性状态的愈伤组织形成胚性愈伤组织,并在含ZT的N_6分化陪养基上产生绿点。通过对这两种愈伤组织的生化分析,表明二者在游离氨基酸、DNA、RNA、核酸及蛋白质含量等方面,特别是SDS-PAGE谱带存在明显的差异,其细胞的形态与结构也有显著差别,其中经ABA NAA诱导后的愈伤组织其细胞形态与结构特征与来源于种胚的胚性愈伤组织基本类似,所分析的生化指标也大多数相近。结果表明,ABA和NAA联合使用得当,能促进形成胚性愈伤组织。