华东安蕨卵发生的超微结构观察

核心薄囊蕨是蕨类植物中的进化类群,但对受精作用具有显著影响的卵发生研究仍较少,该文利用超微技术对其中蹄盖蕨科的华东安蕨卵发生过程进行了研究,以进一步完善薄囊蕨植物卵发生的科学资料,为理解蕨类植物的有性生殖及演化机制奠定基础。超微结构观察显示:华东安蕨的幼卵和沟细胞在颈卵器中紧密联接;随后,在卵细胞上方出现了分离腔和临时细胞壁,但在卵细胞中间孔区处卵细胞和腹沟细胞始终联接在一起;分离腔中的无定形物质沉积在卵细胞的质膜外形成了1层加厚的卵膜,而在孔区处没有形成卵膜,该位置最后形成了受精孔。在进一步的卵发生过程中,卵细胞核变得高度不规则,形成了大量的核外突和核褶皱。     

普通针毛蕨颈卵器和卵的发育

蕨类植物卵发生是有性生殖研究的重要内容。真水龙骨类II的卵发生尚无人研究, 本文对该类群中的普通针毛蕨(Macrothelypteris torresiana)卵发生过程进行了光镜和透射电镜研究。结果显示: 卵细胞刚形成时, 与腹沟细胞紧密相连, 随着卵进一步发育, 卵细胞和腹沟细胞之间逐渐形成分离腔, 但孔区处卵细胞和腹沟细胞始终相连。随后, 卵细胞上表面有不定型物质堆积在质膜外, 形成一层加厚的卵膜, 孔区处没有卵膜覆盖的位置最后形成受精孔。在卵发育后期, 卵细胞核变得不规则, 近成熟时卵核产生大量核外突。卵发育过程中卵膜的出现、受精孔的产生以及核外突等特征, 与进化的真水龙骨类中其他蕨类植物的卵发生研究结果相似, 与薄囊蕨类中原始基部类群的卵发生现象有显著差别。由此可见, 普通针毛蕨属于进化类型。依据卵发生中卵膜和受精孔等特征推测原始薄囊蕨类经过里白类、桫椤类, 最终演化为水龙骨类。     

阔鳞瘤蕨(水龙骨科)卵细胞发育超微结构观察

水龙骨科是蕨类植物中最进化的类群,该文采用超微技术对水龙骨科的阔鳞瘤蕨(Phymatosorus hainanensis)卵发育过程进行观察,以完善薄囊蕨植物卵发生的资料,为揭示蕨类植物的有性生殖及演化机制奠定基础。结果表明:(1)幼卵、颈沟细胞、腹沟细胞通过胞间连丝紧密连接。(2)发育过程中,卵与腹沟细胞之间细胞壁显著加厚,将卵细胞与腹沟细胞隔离。(3)在壁的下方产生分离腔,内含大量不定形物质,但卵细胞与腹沟细胞在中间处始终相连。(4)分离腔中的不定形物质沉积在卵细胞质膜外形成了一层加厚的卵膜,而在连接区(孔区)处不形成卵膜,该位置最终形成了受精孔。(5)卵细胞核变得高度不规则,近成熟时卵核产生了大量的核外突。     

水蕨卵膜的形成及其超微结构的观察

蕨类植物成熟卵的周围有一层卵膜,但其细微结构和形成过程仍不清楚,本研究应用透射电镜技术对水蕨(Ceratopteris thailictroides)卵细胞发育过程中卵膜的形成及超微结构进行了观察.结果表明水蕨卵细胞在发育中期开始形成卵膜,卵上方的卵膜十分显著,是由多层嗜锇性内质网片层附着于质膜内表面形成的,成熟时卵上方的卵膜中心部分厚,向边缘逐渐变薄,在嗜锇性片层之间填充有嗜锇性物质.比较而言,卵下方及侧面的卵膜薄,由两层紧密连接的嗜锇性膜构成.首次阐明了蕨类植物卵膜形成的超微结构,并对卵膜的一些功能进行了探讨.     

阔叶鳞盖蕨配子体发育及卵发生的研究

采用显微镜和透射电镜对阔叶鳞盖蕨(Microlepia platyphylla)的配子体发育和卵发生过程进行了观察,以阐明其卵发生的细胞学机制,探讨其演化地位。阔叶鳞盖蕨孢子褐色,四面体形,具三裂缝,接种5~10d后孢子萌发,经丝状体和片状体阶段发育为心形原叶体,原叶体发育是铁线蕨型,通常为雌雄异株,精子器产生于不规则配子体的表面,颈卵器产生于心形原叶体生长点的下方,性器官是薄囊蕨型。卵发生研究表明,阔叶鳞盖蕨颈卵器产生于生长点下方表面细胞,经两次分裂形成了顶细胞、初生细胞和基细胞。其中初生细胞再经两次不等分裂产生卵细胞、腹沟细胞和颈沟细胞,此3个细胞通过胞间连丝紧密相连,随发育,腹沟细胞与卵细胞间形成了分离腔,但在孔区处始终通过胞间连丝相连,成熟卵细胞上形成了卵膜和受精孔,卵核表面产生了核外突,通过比较表明阔叶鳞盖蕨卵发生与蕨（Pteridium aquilinum）卵发生相似。     

分株紫萁卵发生的超微结构

用透射电镜对蕨类植物分枝紫其(Osmunda cinnamamae L. var.asiatica Fernald)卵发生进行了超微结构的研究.卵发生过程中,许多泡囊不仅移向细胞周围,而且在细胞质膜内排为一列,并通过胞吐作用聚集在细胞质膜外,它们释放或分泌嗜锇物质.观察到少数泡囊内含片层状结构的嗜饿物质紧贴于细胞质膜,似乎将其冲破.与此同时,在卵细胞和颈卵器壁之间形成分离腔,其宽度大于以往报道的真蕨类,在卵细胞质膜外出现额外的卵膜,其宽度大于蕨属和鳞毛蕨属.造粉体被大型常呈三角状半圆形或近椭圆形的淀粉粒所充满,当卵成熟时逐渐减少.核大型平扁状,核内出现2~3对平行的双层膜,紧贴核膜.未发现核外突.线粒体一度似不发育,最后恢复正常.     

分株紫萁卵发生的超微结构

用透射电镜对蕨类植物分枝紫萁(Osmunda cinnamamae L. var. asiatica Fernald)卵发牛进行了超微结构的研究。卵发生过程中,许多泡囊不仅移向细胞周围,而且在细胞质膜内排为一列,并通过胞吐作用聚集在细胞质膜外,它们释放或分泌嗜锇物质。观察到少数泡囊内含片层状结构的嗜饿物质紧贴于细胞质膜,似乎将其冲破。与此同时,在卵细胞和颈卵器壁之问形成分离腔,其宽度大于以往报道的真蕨类,在卵细胞质膜外出现额外的卵膜,其宽度大于蕨属和鳞毛蕨属。造粉体被大型常呈三角状半圆形或近椭圆形的淀粉粒所充满,当卵成熟时逐渐减少。核大型平扁状,核内出现2-3对平行的双层膜,紧贴核膜。未发现核外突。线粒体一度似不发育,最后恢复正常。     

蕨配子体发育及卵发生的显微结构观察

运用显微观察技术对蕨(Pteridium aquilinum var. latiusculum)配子体发育和卵发生进行了研究。结果表明：(1)蕨孢子黄褐色,四面体形,具三裂缝,接种后3～7 d萌发,经丝状体和片状体阶段发育成原叶体,成熟原叶体雌雄异株或同株。(2)蕨颈卵器产生于生长点下方的表面细胞(颈卵器原始细胞),该细胞经2次分裂形成3层细胞,其上层和下层细胞发育为颈卵器壁细胞,中间细胞为初生细胞,它经2次不等分裂产生3个细胞,分别为卵细胞、腹沟细胞和颈沟细胞;刚产生时,此3个细胞紧贴颈卵器壁,细胞质内液泡较多,随着发育,卵细胞和腹沟细胞之间产生了分离腔,但二者通过孔区相连,在卵细胞上表面可观察到卵膜;此后,颈沟细胞和腹沟细胞逐渐退化,颈卵器壁细胞内具有黑色颗粒物质。连续切片观察发现,成熟卵细胞上表面中央具有受精孔。卵发生的细节尚需超微结构的研究。     

绵马鳞毛蕨精母细胞和游动精子超微结构特征的研究

应用电镜技术对蕨类植物绵马鳞毛蕨(RYOPTERIS CRASSIRHIZOMA Nakai)精母细胞和游动精子的超微结构特征进行了研究。精母细胞为多边形,细胞质内含有丰富的线粒体、质体、内质网、高尔基体等常见的细胞器．在细胞质中还可见到一些同心圆膜状结构,位于质膜的附近或精母细胞的角偶。同心圆膜状结构由双层膜环绕构成,外被l层单位膜。精母细胞与精子器的璧细胞之间形成了分离腔。在精母细胞质膜外形成了嗜锇层,这些结构的形成说明精母细胞已经开始与雄配子体逐渐分离,进入独立发育的阶段。尽管精母细胞之间也有嗜锇层的形成,但嗜锇层是不连续的,其上有一些空隙,精母细胞之间可通过空隙进行物质和信息的交流。成熟的精子细胞外被l层透明的薄膜,里面为游动精子。螺旋状。由环状细胞器环绕3～4圈构成．这些环状细胞器包括多层结卡构、微管带、巨大线粒体、鞭毛带和1个长形浓缩的细胞核。游动精子的后端为一些泡囊化的细胞质．其中包括一些残存的线粒体、造粉质体及大的囊泡等。当成熟的精子细胞排出精子器后。其内的游动精子挣脱透明质膜的束缚,摆脱后端的囊泡,成为1条游动精子。本文还对绵马鳞毛蕨和其它蕨类植物精子的超微结构特征进行了比较。     

地钱卵发育超微结构和细胞化学的研究

采用电镜和细胞化学技术对地钱(Marchantia polymorpha)卵发生过程进行了研究,根据卵发生过程中细胞化学和超微结构特征可将卵发育过程分为幼卵、中期卵和成熟卵3个阶段.幼卵阶段,卵细胞、腹沟细胞及颈沟细胞间有发达的胞间连丝,但卵与腹沟细胞间的胞间连丝很快退化,幼卵细胞内具大量透明的囊泡,均匀分布于细胞质中;卵发育中期,突出特征是卵细胞质内产生嗜锇性的脂滴,位于囊泡中,与此同时,腹沟细胞退化,其细胞质内产生大型囊泡,囊泡内分泌物与卵细胞外的物质类似,呈PAS反应阳性,表明该物质应为多糖类;卵成熟时,腹沟细胞和颈沟细胞完全退化,卵细胞外包被大量粘性多糖类物质,卵细胞核表面不规则,产生明显的核外突,众多的小泡围绕着细胞核,脂滴聚集成簇,卵细胞内其他细胞器不易区分.卵发育过程中,质体不含淀粉粒,线粒体退化,高尔基体相对发达.地钱卵发育的这些特征显著区分于蕨类植物.     

Formation of the Fertilization Pore during Oogenesis of the Fern Ceratopteris thalictroides

The development of the fertilization pore during oogenesis of the fern Ceratopteris thalictroides was followed using transmission electron microscopy. The newly formed egg is appressed closely to the adjacent cells. There are well-developed plasmodesmata between the egg and the ventral canal cell, but none between the egg and the jacket cells of the archegonium. During maturation, a separation cavity is formed around the egg. However, a pore region persistently connects the egg and the ventral canal cell. The extra egg membrane is formed by deposition of sheets of endoplasmic reticulum (ER), but no ER is deposited on the inner surface of the pore region. Thus, a fertilization pore, covered by a layer of plasmalemma, is formed. The ventral canal cell undoubtedly participates the formation of the fertilization pore, probably by absorbing the sheets of ER beneath the pore region. The functional significance of the ventral canal cell in formation of the fertilization pore is discussed. The features of the mature egg include that abundant concentric membranes and osmiophilic vesicles occur in the cytoplasm of the mature egg. The initial, round nucleus of the egg eventually becomes cup-shaped. This investigation gives some new insights about the cells participating oogenesis in ferns.     

中华绒螯蟹成熟卵形态和超微结构的研究

中华绒螯蟹的成熟卵仅有初级卵膜，无次级和三级卵巢，质膜初期厚而多层，且具皱褶，卵核在卵的发育过程中变化很大，未发见中心粒，内质网和高尔基体均始见于蟹卵发育的初期，皮层颗粒先出现于蟹卵深部，随后移到卵的表层，无滋养细胞，蟹卵由卵泡细胞提供物质，形成卵黄，此外，还可直接从血淋巴内摄取卵黄前身物质。     

In vitro reassembly of nuclear envelopes and organelles in Xenopus egg extracts

We reconstituted bilayer nuclear membranes, multilayer membranes, and organelles from mixtures ofXenopus laevis egg extracts and demembranatedXenopus sperm nuclei. Varying proportions of the cytosolic and vesicular fractions from the eggs were used in the reconstitution mixtures. A cytosol：vesicle ratio of 10：1 promoted reassembly of the normal bilayer nuclear membrane with inserted nuclear pore complexes around the decondensed Xenopus sperm chromatin. A cytosol： vesicle ratio of 5：1 caused decondensed and dispersed sperm chromatin to be either surrounded by or divided by unusual multilayer membrane structures with inlaid pore complexes. A cytosol：vesicle ratio of 2.5：1 promoted reconstitution of mitochondria, endoplasmic reticulum networks, and Golgi apparatus. During reassembly of the endoplasmic reticulum and Golgi apparatus, vesicular fragments of the corresponding organelles fused together and changed their shape to form flattened cistemae, which were then stacked one on top of another.     

Glucosylceramide synthesis inhibitors block pharmacologically induced dispersal of the Golgi and anterograde membrane flow from the endoplasmic reticulum: implication of sphingolipid metabolism in maintenance of the Golgi architecture and anterograde membrane flow

PDMP (D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol) and PPMP (D,L-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol), inhibitors of glucosylceramide synthesis, blocked brefeldin A (BFA)- and nordihydroguaiaretic acid-induced dispersal of the Golgi and trans Golgi network, and Golgi-derived vesicles were retained in the juxtanuclear region. PDMP and PPMP did not stabilize microtubules but blocked nocodazole-induced extensive fragmentation and dispersal of the Golgi, and large Golgi vesicles were retained in the juxtanuclear region. PPMP is a stronger inhibitor of glucosylceramide synthesis than PDMP, but PDMP showed a stronger activity against BFA-induced retrograde membrane flow. However, PPMP showed a stronger activity for Golgi disruption and inhibition of anterograde trafficking from the endoplasmic reticulum, and rebuilding of the Golgi architecture. Cumulatively, these results suggest that sphingolipid metabolism is implicated in maintenance of the Golgi architecture and anterograde membrane flow from the endoplasmic reticulum but not in Golgi dispersal induced by BFA.     

Effects of brefeldin A on the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum in a green alga,Scenedesmus acutus

Summary The effects of brefeldin A (BFA) on the structure of the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum (ER), and on the thiamine pyrophosphatase (TPPase) activity in these organelles were examined in a green alga,Scenedesmus acutus, to obtain evidence for the existence of a retrograde transport from the Golgi apparatus to the ER via the nuclear envelope. InScenedesmus, Golgi bodies are situated close to the nuclear envelope throughout the cell cycle and receive the transition vesicles not directly from the ER, but from the nuclear envelope. BFA induced the disassembly of Golgi bodies and an increase in the ER cisternae at the trans-side of decomposed Golgi bodies in interphase cells and multinuclear cells before septum formation. The accumulated ER cisternae connected to the nuclear envelope at one part. TPPase activity was detected in all cisternae of Golgi bodies, but not in the nuclear envelope or the ER in nontreated cells. On the contrary, in BFA-treated cells, TPPase activity was detected in the nuclear envelope and the ER in addition to the decomposed Golgi bodies. When septum-forming cells were treated with BFA, the disassembly of Golgi bodies was less than that in interphase cells, and TPPase activity was detected in the Golgi cisternae but not in the nuclear envelope or the ER. These results suggest mat BFA blocks the anterograde transport from the nuclear envelope to the Golgi bodies but does not block the retrograde transport from the Golgi bodies to the nuclear envelope in interphase and multinuclear cells.Abbreviations BFA brefeldin A - ER endoplasmic reticulum - TPPase thiamine pyrophosphatase     

Brefeldin A induces callose formation in onion inner epidermal cells

Summary The antibiotic fungal toxin brefeldin A (BFA) causes synthesis of additional cell wall material in adult differentiated onion inner epidermal cells at concentrations of 5–30 g/ml. This tertiary wall contains callose and is layered on the secondary cellulosic wall in a time- and dose-dependent manner. Initially, callose is found in pit fields in the form of small vesicular patches. With time and dose, depositions grow in size and form large plugs invaginating into the cell, where the adjacent cytoplasm forms bulky accumulations and contains many organelles including endomembranes. Within the cytoplasm, BFA exerts the characteristic morphological effects on the secretory system including changes of the Golgi stacks, formation of large vesicles, and proliferation of dilated cisternae of the endoplasmic reticulum. Higher concentrations of BFA (60 g/ml) lead to disintegration of the Golgi apparatus; they have no effects on the cell wall, no callose synthesis occurs. We conclude from these observations that BFA has two independent targets in onion cells. BFA acts on the plasma membrane, hence operating as an elicitor of plant defense reactions and thus activates callose synthesis. BFA acts also on the membranes of the secretory system and influences budding and fusion of vesicles at the endoplasmic reticulum and at the dictyosomes. These two mechanisms occur in parallel, suggesting that the secretory system still can play its presumed role in callose synthesis. Only when dictyosomes are completely disintegrated, no more callose is formed.Abbreviations BFA Brefeldin A - PM plasma membrane - GA Golgi apparatus - ER endoplasmic reticulum - GS glucan synthetase Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

Brefeldin A arrests the maturation and egress of herpes simplex virus particles during infection.

Herpes simplex virus (HSV) requires the host cell secretory apparatus for transport and processing of membrane glycoproteins during the course of virus assembly. Brefeldin A (BFA) has been reported to induce retrograde movement of molecules from the Golgi to the endoplasmic reticulum and to cause disassembly of the Golgi complex. We examined the effects of BFA on propagation of HSV type 1. Release of virions into the extracellular medium was blocked by as little as 0.3 microgram of BFA per ml when present from 2 h postinfection. Characterization of infected cells revealed that BFA inhibited infectious viral particle formation without affecting nucleocapsid formation. Electron microscopic analyses of BFA-treated and untreated cells (as in control cells) demonstrated that viral particles were enveloped at the inner nuclear membrane in BFA-treated cells and accumulated aberrantly in this region. Most of the progeny virus particles observed in the cytoplasm of control cells, but not that of BFA-treated cells, were enveloped and contained within membrane vesicles, whereas many unenveloped nucleocapsids were detected in the cytoplasm of BFA-treated cells. This suggests that BFA prevents the transport of enveloped particles from the perinuclear space to the cytoplasmic vesicles. These findings indicate that BFA-induced retrograde movement of molecules from the Golgi complex to the endoplasmic reticulum early in infection arrests the ability of host cells to support maturation and egress of enveloped viral particles. Furthermore, we demonstrate that the effects of BFA on HSV propagation are not fully reversible, indicating that maturation and egress of HSV type 1 particles relies on a series of events which cannot be easily reconstituted after the block to secretion is relieved.     

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

The ultrastructure of the mature egg and fertilization in the fern Ceratopteris thalictroides (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.     

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

The ultrastructure of the mature egg and fertilization in the fern Ceratopteris thalictroides (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.     

Fine structure of the human ovum in the pronuclear stage

A penetrated ovum was recovered from the oviduct of a 33 year old surgical patient who had had sexual intercourse 26 hr before the operation. The ovum was in the pronuclear stage. The ooplasmic organelles were mainly represented by mitochondria, endoplasmic reticulum components, and Golgi elements. Small vesicles were found in the space between the two sheets of the pronuclear envelope. These vesicles appeared to be morphologically similar to the ER vesicles in the ooplasm and were considered to be involved in pronuclear development. Numerous annulate lamellae were seen in the ooplasm as well as in the pronuclei. Ooplasmic crystalloids were also observed. These were thought to represent cytoplasmic yolk. Remnants of the penetrating spermatozoon were found in close relation to one of the pronuclei. The fine structure of the first and second polar body is also described. The nuclear complement of the first polar body consisted of isolated chromosomes, whereas the second polar body contained a membrane-bounded nucleus. In consideration of the possibility that polar body fertilization may take place, these differences in nuclear organization could be of importance. Other recognizable differences between the two polar bodies were presence of dense cortical granules and microvilli in the first polar body, and absence of these structures in the second. These dissimilarities were considered to be related to the organization of the egg cytoplasm at the time of polar body separation.