半滑舌鳎胚胎发育组织学观察

对半滑舌鳎Cynoglossus semilaevis胚胎发育进行了组织学观察,首次描述了半滑舌鳎胚胎发育过程中脊索、眼囊、中胚层、脊髓底板、神经管、肠、耳囊、脑、口咽膜和心管等组织结构.半滑舌鳎眼原基出现后,肌节在胚体后部开始分化.随后神经管前端不断膨大形成脑原基,脑形成之后在后脑的后面形成耳囊.胚体形成后,脊索位于脑的腹面,在胚胎发育过程中脊索细胞空泡化.肠位于脊索腹面.脊索背部有一排立方体细胞,为脊髓底板,脊髓底板位于神经管腹面并延伸到后脑前端.心脏是含有红血球的一个薄壁管,位于胚体头部腹面,且与中脑平行.     

三疣梭子蟹胚胎发育过程中肝胰腺的发生与卵黄物质利用的关系

通过对三疣梭子蟹胚胎进行连续采样和组织切片,系统研究了三疣梭子蟹胚胎发育过程中卵黄囊和肝胰腺的发生与卵黄物质利用的关系。结果表明:(1)三疣梭子蟹胚胎的卵黄岛和卵黄囊结构分别出现在原肠期和无节幼体期,胚胎从原肠期至卵内第一期溞状幼体期,始终存在卵黄岛结构,且卵黄岛中的卵黄物质不断被分解和利用. (2)卵内第二期溞状幼体后,卵黄囊分为两个区域,卵黄囊壁中出现肝胰腺细胞(柱状上皮细胞),此时肝胰腺前体已开始形成,卵黄岛开始融合. (3)卵内第三期溞状幼体阶段,卵黄囊发育成一双肝胰腺,由于肝胰腺中的卵黄物质互相融合,卵黄岛结构消失。此阶段胚胎对卵黄物质的利用加快, 卵黄物质中存在许多空泡状结构;(4)胚胎发育进入孵化前期后,肝胰腺腔内的卵黄物质极少,而初孵溞状幼体肝胰腺腔内卵黄物质已完全消失,肝胰腺为一对囊状结构。这些结果表明在三疣梭子蟹胚胎发育从原肠期到孵化前的过程中,卵黄岛和肝胰腺细胞对于卵黄物质分解和利用起着十分重要的作用。     

半滑舌鳎受精卵、卵黄囊仔鱼和开口仔鱼氨基酸及脂肪酸的变化

采用气相色谱仪和氨基酸分析仪测定了半滑舌鳎(Cynoglossus semilaevis)受精卵、卵黄囊仔鱼和开口仔鱼的氨基酸与脂肪酸组成的变化。结果表明:总氨基酸组成在受精卵和卵黄囊仔鱼之间变化明显,但是在卵黄囊仔鱼和开口仔鱼之间只有细微的变化。开口仔鱼与其摄食的轮虫的总必需氨基酸组成相关。受精卵、卵黄囊仔鱼、开口仔鱼的游离氨基酸含量分别为139 mg/g、3.6 mg/g和2.5 mg/g,占总氨基酸含量的22.3%、3.6%和2.5%。饱和脂肪酸的总量从受精卵到卵黄囊仔鱼明显下降,但是发育到开口仔鱼含量无显著变化。单不饱和脂肪酸和多不饱和脂肪酸的总量在不同发育阶段无显著变化,而EPA和DHA的含量从卵黄囊仔鱼到开口仔鱼有明显下降。这表明在早期发育阶段半滑舌鳎主要利用饱和脂肪酸作为能量代谢的基质,对饱和脂肪酸的利用程度大于单不饱和脂肪酸和多不饱和脂肪酸。半滑舌鳎似乎需要长链的多不饱和脂肪酸如EPA、DHA和ARA。     

黄胫小车蝗卵子发生及卵母细胞凋亡的显微观察

对黄胫小车蝗(Oedaleus infernalis)卵子发生过程和卵母细胞凋亡进行显微观察。结果表明,黄胫小车蝗卵子发生可明显分为3个时期10个阶段,即卵黄发生前期、卵黄发生期和卵壳形成期。第1阶段,卵母细胞位于卵原区,经历减数第一次分裂;第2阶段,卵母细胞核内染色体解体成网状,滤泡细胞稀疏地排列在卵母细胞周围;第3阶段,滤泡细胞扁平状,在卵母细胞周围排成一层;第4阶段,滤泡细胞呈立方形排在卵母细胞周围;第5阶段,滤泡细胞呈长柱形排在卵母细胞周围,滤泡细胞之间、滤泡细胞与卵母细胞之间出现空隙;第6阶段,卵母细胞边缘开始出现卵黄颗粒;第7阶段,卵母细胞中沉积大量卵黄,胚泡破裂;第8阶段,滤泡细胞分泌卵黄膜包围卵黄物质;第9阶段,滤泡细胞分泌卵壳;第10阶段,卵壳分泌结束,卵子发育成熟。卵母细胞发育过程中的凋亡发生在卵黄发生前期,主要表现为滤泡细胞向卵母细胞内折叠,胞质呈团块状等特征。     

凡纳滨对虾卵母细胞卵黄发生的超微结构

利用电镜研究凡纳滨对虾卵母细胞卵黄发生的全过程。结果表明 :凡纳滨对虾卵黄的发生是双源性的。卵黄发生早、中期是内源性卵黄大量合成的阶段 ,卵黄发生中、后期则以外源性卵黄的合成为主。内源性卵黄主要由内质网、线粒体、核糖体、溶酶体、高尔基器等多种胞器活跃参与形成。其中数量众多的囊泡状粗面内质网是形成内源性卵黄粒的最主要的细胞器 ;部分线粒体参与卵黄粒的合成并自身最终演变为卵黄粒 ;丰富的游离核糖体合成了大量致密的蛋白质颗粒并在卵质中直接聚集融合成无膜的卵黄粒 ;溶酶体通过吞噬、消化内含物来形成卵黄粒和脂滴 ,且方式多样 ;高尔基器不直接参与形成卵黄粒。外源性卵黄主要通过卵质膜的微吞饮活动从卵周隙或卵泡细胞中摄取外源物质来形成     

林蛙卵第一次卵裂时卵黄粒的超微结构变化

用水溶性电镜包埋介质等四种不同的方法研究了黑龙江林蛙卵第一次卵裂时卵黄粒的精细结构的变化。观察到在2-细胞期已经有一部分卵黄粒开始降解,这比文献报道卵黄粒在囊胚晚期才开始降解要早。卵黄粒降解步骤:1.从晶形主体边缘脱落晶分子,晶分子进入非晶形区之后电子染色加深,使深色亚区扩大面积。2.在深色亚区中出现“微泡”结构。3.从深色亚区转变成浅色亚区并在后者中出现直径600埃左右的具膜小泡,它们可能是从深色亚区中的“微泡”演化而来。4.具膜小泡随着卵黄粒界膜的破损而释出。     

文昌鱼受精机理研究——受精卵的超显微结构

青岛文昌鱼受精后受精膜即刻明显举起,皮层颗粒以完整形式胞吐到卵周腔中,分散后大部分皮层颗粒物质与卵黄膜结合一起组成三层结构的受精膜,随着受精膜的举起,它由厚变薄。雄性原核以核膜破裂,染色质去浓缩,扩大重建原核膜形成雄性原核,雌性原核形成是由分离的具有双层膜的染色体联合,膨大而成,雄性原核形成早于雌性原核。雄性原核的迁移受微丝控制。文昌鱼卵黄颗粒中存在线行和环形结构,这种亚显微结构电子密度很高,经实验证明,它既不是微管又不是微丝。     

荞麦水合花粉和花粉管中的被膜小泡

荞麦水合花粉粒和生长中的花粉管中内质网潴泡形成的囊袋状结构较少见,但内质网囊袋中含有丰富的被膜小泡,直径约为100－150nm。刚刚形成的花粉管中,被膜小泡主要来自于花粉粒营养细胞的细胞质。生长中的花粉管的被膜小泡可由高尔基体分泌形成。另外还观察到内质网的碎裂也是荞麦花粉管中产生被膜小泡的一种机制。花粉管的被膜小泡中含有花粉管壁的前体物质,与花粉管的壁融合参与花粉管的生长。被膜小泡可能含有与脂体和造粉质体水解有关的酶,参与此类物质的降解。荞麦花柱和柱头细胞内含物的解体物质参与花粉管的生长。     

日本鬼()背鳍棘毒腺中Ⅰ、Ⅱ型毒腺细胞关系的探讨

日本鬼()背鳍棘毒腺中有两种类型毒腺细胞--Ⅰ型细胞和Ⅱ型细胞.两种细胞结构明显不同.本文用形态学方法探讨Ⅰ型与Ⅱ型细胞的关系.结果表明:毒腺组织中Ⅰ型细胞光镜下有的胞质内可见浅染点样结构,并且在不同的细胞内其浅染点状结构的多少有差异;电镜下Ⅰ型细胞膜结构差异较大,有的Ⅰ型细胞的脂质双层膜性结构清楚;有的细胞膜外侧可见膜包小泡;有的细胞内侧面也见小泡形成、融合,使脂质双层膜间隙变宽,其内可见膜包样物质,其电子密度中等或较高,结构类似于Ⅱ型细胞的囊泡样物质.不同的Ⅱ型细胞其胞质内颗粒大小及电子密度不一,囊泡状物多少也不一.含较小而密集颗粒的Ⅱ型细胞胞膜的脂质双层膜的外侧面较规则,与Ⅰ型细胞相似;脂质双层膜的内侧面出现许多扩张的大囊泡,其内含物电子密度高或中等,与胞质内含的颗粒状物质相同;位于扩张的囊泡与胞膜之间的胞质结构有的与I型细胞的胞质内的某些结构相似.含大而稀疏颗粒的Ⅱ型细胞其颗粒数量少、电子密度差异大,并且囊泡样物质增多.推测Ⅱ型细胞可能由Ⅰ型细胞转化而来.     

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。     

Cells released in vitro from the embryonic yolk sac of the stick insect carausius morosus (BR.) (PHASMATODEA : HETERONEMIIDAE) may include embryonic hemocytes

The embryonic yolk sac and the adult dorsal vessel of the stick insect Carausius morosus (Br.) (Phasmatodea : Heteronemiidae) were shown to release a number of cells that appear morphologically similar to circulating adult hemocytes. Like adult hemocytes, these cells reacted positively when tested for both phenoloxidase activity and a monoclonal antibody specifically raised against a vitellin polypeptide. Based on this evidence, it is suggested that yolk sac-released cells behave as potential embryonic hemocytes. A model is thus proposed whereby the yolk sac might host a number of hemopoietic stem cells on their way to the dorsal vessel, and in so doing, it may temporally act as an embryonic hemopoietic organ.     

Vitellin polypeptide pathways in late insect yolk sacs

A panel of monoclonal antibodies was raised against late yolk sacs of the stick insect Carausius morosus and tested by immunoblotting to establish the extent vitellin polypeptides are processed proteolytically during embryonic development. Cryosections of late yolk sacs were also examined by confocal laser microscopy to determine how vitellin cleavage products become spatially distributed amongst yolk granules during the same developmental period. Distinct labelling patterns were obtained on yolk granules depending on: (1) the nature of the proteolytic processing; (2) the origin of vitellin cleavage products; and ultimately (3) their molecular sizes. Monoclonal antibodies raised against vitellin cleavage products resulting from proteolytic processing appeared to label: (1) the entire volume of many yolk granules; (2) their limiting membrane; or (3) a number of small vesicles interposed between larger yolk granules. On the other hand, monoclonal antibodies against vitellin cleavage products that remain invariant throughout development appeared to label either the serosa membrane or the cytosolic space comprised between adjacent yolk granules. Data are interpreted as indicating that vitellin cleavage products may leak out from the yolk granules, gain access to the cytosolic space of the vitellophages and eventually percolate through the serosa membrane enclosing the yolk sac.     

The yolk sac in late embryonic development of the stick insect Carausius morosus (Br.)

Differentiation of the yolk sac was examined ultrastructurally and cytochemically in late embryonic development of the stick insect Carausius morosus. During migration along the yolk sac, endodermal cells form a discontinuous cell epithelium, leaving wide intercellular channels between neighbouring cell clusters. Within the same cell cluster, cells are all joined by septate junctions. In the proximity of the proctodeum region, intercellular channels are filled with numerous cell debris which are shown to derive from vitellophages undergoing cell lysis. Yolk sacs resolved by gel electrophoresis are shown to release a number of vitellin polypeptides into the culture medium. These are equivalent in molecular weight to those present in the vitellophage yolk granules This observation is consistent with the evidence that the basement lamina may act as a course physical filter, retaining particles larger than colloidal thorium dioxide and allowing free percolation of peroxidase. Differentiating endodermal cells form a microvillar striated border along the apical plasma membrane. A number of vesicular criptae were frequently seen in these differentiating endodermal cells. Electron dense granules released by endodermal cells are suggested to play a role in vitellophage lysis and vitellin release from the enclosed yolk granules.     

Yolk utilization in stick insects entails the release of vitellin polypeptides into the perivitelline fluid

This study investigates the developmental fate of vitellin (Vt) polypeptides generated by limited proteolysis in an insect embryo. To this end, a number of polyclonal (pAb) and monoclonal antibodies (mAb) were raised against the yolk sac and the perivitelline fluid of late embryos of the stick insect Carausius morosus. Two dimensional immuno gel electrophoresis and Western blotting demonstrate that polypeptides resulting from Vt processing are present both in the yolk sac and the perivitelline fluid. At the confocal microscope, different labelling patterns were detected in the ooplasm depending on the stage of development attained by the embryo. At early developmental stages, label is associated with large unsegmented portions of the fluid ooplasm. During embryonic development, the fluid ooplasm is gradually transformed into yolk granules by intervention of vitellophages. Prior to dorsal closure, the yolk sac is separated from the perivitelline fluid by interposition of serosa cells (the so called serosa membrane). Several mAbs raised against the perivitelline fluid react specifically with this membrane suggesting that the release of Vt polypeptides from the yolk sac occurs by intracellular transit through the serosa cells. By immunocytochemistry, gold label appears associated with the cell surface and a number of vacuoles of the serosa membrane. These data are interpreted as suggesting that Vt polypeptides resulting from limited proteolysis in stick insect embryos are not exhaustively degraded within the yolk sac, but are instead transferred transcytotically to the perivitelline fluid through the serosa membrane.     

Serine protease P-IIc is responsible for the digestion of yolk proteins at the late stage of silkworm embryogenesis

In silkworms, yolk proteins comprise vitellin, egg-specific protein and 30K proteins, which are sequentially degraded by endogenous proteases strictly regulated during embryogenesis. Although the process has been extensively investigated, there is still a gap in the knowledge about the degradation of silkworm yolk proteins on the last two days of embryonic development. In the present study, we isolated and purified a gut serine protease P-IIc, which demonstrated optimal activity at 25 °C and pH 11. Semi-quantitative RT-PCR combined with western blotting showed that P-IIc was actively expressed and significantly accumulated in the gut on the last two days of embryogenesis. When natural yolk proteins were incubated with P-IIc in vitro, vitellin and ESP were selectively degraded. P-IIc also demonstrated activity towards 30K proteins as evidenced by rapid and complete digestion of BmLP1 and partial digestion of BmLP2 and BmLP3. Furthermore, RNAi knockdown of P-IIc in silkworm embryos significantly reduced the degradation rate of residual yolk proteins on embryonic day 10. Taken together, our results indicate that P-IIc represents an embryonic gut protease with a relatively broad substrate specificity, which plays an important role in the degradation of yolk proteins at the late stage of silkworm embryogenesis.     

Immunocytochemical location of vitellin in the egg of the silkworm,Bombyx mori,during early developmental stages

Summary Vitellin was purified from eggs of the silkworm,Bombyx mori, by a new method in which vitellin was extracted from isolated yolk granules. The purified vitellin had a molecular weight of 540,000. An antibody against purified vitellin was prepared in rabbits. It reacted with the hemolymph vitellogenin as well as with purified vitellin, but not with other proteins in the hemolymph or in the extract from yolk granules. The anti-vitellin IgG was used to immunocytochemically locate vitellin in theBombyx non-diapause egg during early developmental stages. In the egg, just after oviposition, vitellin was located in internal yolk granules and in small yolk granules of the periplasm. During the early developmental stages studied, vitellin was not metabolized uniformly throughout the egg. The vitellin of the internal yolk granules located at the posterior-dorsal part and of the small peripheral yolk granules was utilized in 16 h and 2 days, respectively, after oviposition. A thin, very vitellin-poor layer was located between the periplasm and the vitellin-rich interior in the newly laid egg. it was always in close contact with the periphery where blastoderm and germ-band cells developed.     

Vitamin D and chick embryonic yolk calcium mobilization: identification and regulation of expression of vitamin D-dependent Ca2(+)-binding protein, calbindin-D28K, in the yolk sac

The developing chick embryo acquires calcium from two sources. Until about Day 10 of incubation, the yolk is the only source; thereafter, calcium is also mobilized from the eggshell. We have previously shown that during normal chick embryonic development, vitamin D is involved in regulating yolk calcium mobilization, whereas vitamin K is required for eggshell calcium translocation by the chorioallantoic membrane. We have studied here the biochemical action of 1,25-dihydroxy vitamin D3 in the yolk sac by examining the expression and regulation of the cytosolic vitamin D-dependent calcium-binding protein, calbindin-D28K. Two types of embryos are used for this study, normal embryos developing in ovo and embryos maintained in long-term shell-less culture ex ovo, the latter being dependent solely on the yolk as their calcium source. Our findings are (1) calbindin-D28K is expressed in the embryonic yolk sac, detectable at incubation Days 9 and 14; (2) the embryonic yolk sac calbindin-D28K resembles that of the adult duodenum in both molecular weight (Mr 28,000) and isoelectric point, as well as the presence of E-F hand Ca2(+)-binding structural domains; (3) systemic calcium deficiency caused by shell-less culture of chick embryos results in enhanced expression of calbindin-D28K in the yolk sac during late development; (4) yolk sac calbindin-D28K expression is inducible by 1,25-dihydroxy vitamin D3 treatment in vivo and in vitro; and (5) immunohistochemistry revealed that yolk sac calbindin-D28K is localized exclusively to the cytoplasm of the yolk sac endoderm. These findings indicate that the chick embryonic yolk sac is a genuine target tissue of 1,25-dihydroxy vitamin D3.     

Purification and characterization of the common yolk protein,vitellin, from the estuarine amphipod Leptocheirus plumulosus

Vitellin is a major yolk protein that plays a significant role in the embryonic development of crustacean embryos. This protein was rapidly purified from embryos of the estuarine amphipod, Leptocheirus plumulosus, by subjecting the crude protein homogenate to high affinity column chromatography. SDS-PAGE revealed a single band with an approximate molecular weight of 200,000 daltons. Vitellin was characterized by SDS-PAGE techniques and amino acid composition analysis. L. plumulosus vitellin is a lipoglycophosphoprotein with serine, glutamic acid/glutamine, alanine, and aspartic acid/asparagine accounting for almost 66% of all amino acid residues. Polyclonal antibodies were raised against L. plumulosus vitellin and antibody reactivity was verified by dot-blotting and immuno-fluorescence confocal microscopy. These antibodies are specific for purified vitellin and show little cross-reactivity with other embryonic proteins.     

Changes in vitellin and other yolk proteins during embryonic development in the silkworm, Bombyx mori

Changes in the amounts of vitellin and other yolk proteins of the eggs of the silkworm, Bombyx mori were investigated during embryonic development using polyacrylamide gel electrophoresis and immunotitration techniques. In the newly laid eggs, soluble proteins were separated into at least nine bands after electrophoresis. The major band was identified as vitellin, accounting for about 40% of the total proteins. The four predominant bands including vitellin exhibited the same mobility as the proteins of haemolymph, but one other major band was specific to the eggs, accounting for about 20% of the proteins.During embryonic differentiation 6–7 days after oviposition, the total protein content did not decrease and the banding patterns and their relative concentrations remained unchanged as a whole. However the concentration of the egg specific protein steadily decreased. During subsequent larval differentiation until hatching, the total proteins were utilized to about 50% of the initial levels: the rapid degradation was observed in almost every species of proteins.An immunotitration experiment further demonstrated that vitellin was not utlilized during embryonic differentiation but was consumed markedly during larval differentiation. However, about 30% of initial level was reserved in the newly hatched larvae. Such a prolonged persistence of vitellin is discussed in relation to protein metabolism during embryonic development in silkworms.     

Experimental yolk sac dysfunction as a model for studying nutritional disturbances in the embryo during early organogenesis

Our investigations concerning the importance of cell surface macromolecules during embryonic development led us to the discovery in 1961 that heterologous anti-rat kidney serum produced teratogenesis, growth retardation and embryonic death when injected into the pregnant rat during early organogenesis. It was established that IgG was the teratogenic agent, primarily directed against the visceral yolk sac (VYS) but not the embryo. Heterologous anti-rat VYS serum was prepared which was teratogenic localized in the VYS and served as a model for producing VYS dysfunction and embryonic malnutrition. The role of the yolk sac placenta in histiotrophic nutrition is now recognized to be critical for normal embryonic development during early organogenesis in the rodent. VYS antiserum affects embryonic development primarily by inhibiting endocytosis of proteins by the VYS endoderm, resulting in a reduction in the amino acids supplied to the embryo. Our laboratory has recently developed teratogenic monoclonal yolk sac antibodies (MCA) which can be utilized; to study VYS plasma membrane synthesis and recycling, to compare yolk sac function among different species, and to identify components of the plasma membrane involved in pinocytosis. MCA prepared against certain VYS antigens provide an opportunity to study embryonic nutrition with minimal interference with the nutritional state of the mother. Recent developments in the study of the human yolk sac along with our laboratory's ability to isolate a spectrum of yolk sac antigens, prepare monoclonal antibodies, and perform functional studies, should provide information that will increase our understanding of yolk sac function and dysfunction in the human and determine the relative importance of various amino acids to normal development during mammalian organogenesis.