棉酚甲酸对金鱼精子质膜的影响

利用冷冻断裂-蚀刻制样技术,在超微结构水平上研究了棉酚甲酸对金鱼精子质膜内蛋白颗粒排列图式(包括晶格区)的影响.结果表明,棉酚甲酸能够使金鱼精子质膜内蛋白颗粒分布不匀:或者颗粒聚集成团,或者质膜内出现无颗粒区域;并且还能使质膜内特定部位晶格区的颗粒排列发生混乱.这些变化表明棉酚甲酸能使精子质膜的有序性降低,也即增加了质膜的流动性.     

低温逆境中黄瓜幼苗细胞内钙离子水平变化的细胞化学研究

采用改进的焦锑酸钙沉淀的细胞化学方法,探讨了Ca~(2 )在黄瓜幼苗细胞中超微结构定位分布及在低温逆境条件下Ca~(2 )水平的动态。结果表明:在适宜温度下生长的黄瓜幼苗,其细胞中Ca~(2 )主要定位于液泡及细胞间隙内,说明液泡是植物细胞内的主要钙库;并显示质外体中存在大量的Ca~(2 )分布。当黄瓜幼苗在1℃下冷胁迫28h后,质膜内侧钙沉淀颗粒明显增加,同时观察到液泡内Ca~(2 )分布变得比较集中,并趋向于液泡膜内侧。当幼苗在1℃低温下胁迫40h后,胞内Ca~(2 )水平进一步提高,尤其是质膜内侧及细胞核内出现较大的呈同心圆状的钙沉淀颗粒。作者认为,胞内Ca~(2 )水平的提高,尤其是质膜内侧及细胞核内局部区域Ca~(2 )密集分布,势必会引发一系列代谢过程的紊乱,最终导致幼苗的伤害或死亡。     

甜菊愈伤组织中的质膜内陷:超微结构和酸性磷酸酶细胞化学定位

对在分化条件下的甜菊 (Stevia rebaudiana)愈伤组织分生区域细胞的质膜内陷进行了超微结构和酸性磷酸酶细胞化学研究。结果表明 ,在不同液泡化状态的细胞中均有质膜内陷存在。在原生质浓密的细胞中 ,质膜呈起伏的波纹状 ,某些部位发生明显内陷 ,大小不等 ,多呈圆球状。在部分液泡化细胞中 ,质膜内陷体积增大 ,内含物增多且结构复杂。在液泡化细胞中 ,质膜内陷嵌入中央液泡 ,但彼此间以一膜间隙隔开。质膜内陷中的内含物以小泡和卷绕的膜结构形式存在。酸性磷酸酶活性定位结果显示 ,质膜及其内陷含高的酶活性。推测质膜内陷在功能上与液泡相似 ,构成了这些细胞水解空间的一部分。     

低温胁迫下枇杷幼叶细胞内Ca2+水平及细胞超微结构变化的研究

用焦锑酸钾沉淀的电镜细胞化学方法,研究了低温胁迫下枇杷幼叶细胞内Ｃａ＾２＋水平变化。研究结果表明,枇杷幼叶细胞未经低温处理时,Ｃａ＾２＋定位分布的沉淀颗凿在碍出现在细胞壁、细胞间隙、质膜和液泡;叶绿体、细胞质和细胞核中也有一些Ｃａ＾２＋沉淀颗粒分布。楷杷幼叶经４４ｈ、４℃低温处理后,在质膜和液泡膜上Ｃａ＾２＋沉淀颗粒分布。枇杷幼叶经４４ｈ、４℃低处理后,在质膜和液泡膜上Ｃａ＾２＋的沉淀增多,细胞质     

甜菊愈伤组织细胞中的液泡膜内突和液泡内囊泡

对生长在分化培养基上的甜菊愈伤组织分生区细胞的液泡膜内突和液泡内囊泡,进行了超微结构和酸性磷酸酶细胞化学研究。在不同液泡化时期的细胞中,都存在不同大小和形态的液泡膜内突,它们有的缺乏明显的内含物;有的含有许多小泡或复杂膜系;有的含有一个较大的具许多小泡或复杂膜系的膜束缚囊泡。在液泡内还存在一些游离的液泡内囊泡,它们通常具有两层紧贴的界膜或为多层同心膜,推测它们来自液泡膜内突。ＡｃＰａｓｅ定位结果显     

酿酒酵母GPCR蛋白Ste2亚细胞定位信号探索

G蛋白偶联受体(G protein-coupled receptor,GPCR)家族蛋白在细胞感受各种胞外信号过程中发挥重要作用。Ste2是酵母细胞中GPCR蛋白之一。大量文献报道了Ste2蛋白突变体对其功能和表达的影响,但关于Ste2亚细胞定位的研究相对较少。这项工作的目的在于确定Ste2亚细胞定位,探究Ste2不同跨膜域、胞内外环状结构域和N端、C端对其亚细胞定位的影响。构建了一系列结构域删除或替换突变体,通过荧光显微镜观察判断不同结构区域对Ste2亚细胞定位的影响,并通过与已知的细胞器标记蛋白共定位观察验证亚细胞定位判读结果。结果显示:野生型Ste2荧光信号出现在质膜和液泡内腔; C端缺失突变体荧光信号出现在质膜和内质网。在N端、C端、各环状结构域序列采用动物GPCR蛋白ORI7、OR17-40相应结构域替换的突变体中,C端替换导致液泡内腔信号消失,质膜信号强于野生型; N端和部分环状结构域替换不同程度减弱或消除了质膜定位,液泡腔内信号类似于野生型;部分突变体在胞内出现点状分布的荧光信号。由此推断:Ste2 N端,第一、第二胞外环状结构域和第三胞内环状结构域可能具有影响Ste2运输定位到质膜的功能;而C端则可能在Ste2离开细胞膜进入液泡的过程中发挥作用。初步确定了Ste2的不同结构区域对其定位的影响,为深入研究GPCR蛋白的亚细胞定位机制奠定基础。     

低温胁迫下龙眼(Dimocarpus longana Lour.)幼叶细胞内Ca2+水平及细胞超微结构

用焦锑酸钾沉淀的电镜细胞化学方法研究低温胁迫下龙眼幼叶细胞内Ｃａ＾２＋水平的变化。未经低温处理的幼叶,细胞壁,细胞间隙,质膜和液泡存在大量的Ｃａ＾２＋沉淀颗粒,叶绿体,细胞质和细胞核中也有一些Ｃａ＾２＋沉淀颗粒分布,表明液泡和细胞间隙是植物细胞的钙库。     

甜菊愈伤组织中的质膜内隐：超微结构和酸性磷酸酶细胞化学定位

对在分化条件下的甜菊愈伤组织分生区域细胞的质膜内陷进行了超微结构和酸性磷酸酶细胞化学研究。结果表明,在不同液泡化状态的细胞中均有质膜内陷存在。在原生质浓密的细胞中,质膜呈起伏的波纹状,某些部位发生明显陷,大小不等,多呈圆球状。     

杂交鹅掌楸体胚发生过程中ATP酶活性的超微细胞化学定位

利用透射式电镜,通过胚性细胞的超微切片观察,对杂交鹅掌楸体细胞胚胎发生和发育过程中ATP酶活性进行了超微细胞化学定位.结果表明,非胚性细胞的质膜、液泡膜等膜系统当中存在ATP酶活性,质体、核膜、细胞壁以及细胞间隙上有少许沉积;早期胚性细胞ATP酶反应产物主要沉积于质膜、液泡膜上、淀粉粒、细胞壁加厚处;胚性细胞后期ATP酶活性从质膜逐渐转移入细胞内,细胞质、壁旁体、胞间连丝、细胞膜与细胞间隙、细胞核等处均有ATP酶活性反应.随着胚性细胞的发育及分裂,包裹细胞的厚壁、细胞核、核仁与染色质等处也出现ATP酶活性反应沉淀物.说明杂交鹅掌楸体细胞胚胎发生及发育过程中存在丰富的能量代谢.     

大鼠垂体细胞液泡内存在黄体生成激素（LH）

研究者普遍认为糖蛋白激素存在于促性腺激素（gonadotrophin,GTH)细胞的颗粒内,目前在生殖内分泌领域内对糖蛋白激素形成与释放的研究也主要集中在细胞内颗粒的变化上,我们近年的研究发现,大鼠垂体GTH细胞内黄体生成激素（luteinizing hormone,LH)的分泌与细胞内液泡的形态变化有密切的关系。铁形态也随液泡的形态变化而变化,因而推测“LH的储存与释放可能与液泡有极大的关系”,为进一步揭示垂体细胞的液泡内是否存在LH和探讨哺乳动物垂体细胞的液泡是否具有储存与释放LH的功能,本研究对大鼠垂体细胞的液泡进行了分离和纯化。用SDS－PAGE,Western immunobloting及Con A/HRP等方法分别对纯化的垂体,大脑皮层及肝脏组织的液泡进行了蛋白质,LH及糖蛋白的分析。结果显示：（1）垂体,皮层及肝脏细胞的液泡内均含有丰富的,分子量大小不等的蛋白质成分,不同组织的细胞液泡内蛋白质成分有许多是相似的;（2）垂体组织及其液泡内均含有LH,而且在相同浓度的蛋白量中,两者LH的水平并无明显差异;（3）垂体,皮层和肝脏组织液泡内均有分子量不同的糖蛋白,但只有垂体细胞的液泡内才有与LH位置相同的糖蛋白染色谱带。上述结果表明：虽然哺乳动物不同组织的细胞液泡内含有许多相似的蛋白质成分,但LH是特异性地存在于垂体细胞液泡内。在这些LH分子中,至少有一部分是已经装配了糖基的完整LH分子。因此,垂体细胞的液泡有可能具有储存与释放LH的功能。     

金鱼精子质膜和核膜的区域特异性

Combined SEM and TEM technique including thin sectioning, freeze-fracture and etching as well as cytochemical staining have been used for ultrastructural study on goldfish (Carassius auratus) sperm. It has been shown that primitive sperm plasma membrane and nuclear membrane are differentiated with regional specificity. The results from this study can be summarized as follows: 1. Intercalated protein particles are highly organized in the plasma membrane in the certain region of the head to form crystalline-like structure, in contrast rest of the area is rich in randomly or clustered particles. 2. Many vesicles in different size are often tightly packed in the head, neck and tail regions. Only these plasma membrane covering the vesicles contain almost no protein particles. 3. The vesicles can be densely stained cytochemically, suggesting the existence of glycoprotein. 4. Most of the nuclear membrane have no nuclear pores on it except the area near the neck part where many nuclear pores concentrate.     

Distribution of intramembranous particles and filipin-sterol complexes in mouse sperm membranes: polyene antibiotic filipin treatment

The distribution of intramembranous particles (IMPs) and membrane filipin-sterol complexes (FSC) was examined ultrastructurally in mouse spermatozoa from the male reproductive tract and ejaculates. IMPs were qualitatively analyzed on freeze-fracture replicas of glutaraldehyde-fixed tissue, while membrane FSC were quantitatively analyzed on replicas of filipin-treated cells. The distribution pattern of IMPs of mouse spermatozoa was fundamentally similar to that of other mammalian spermatozoa. 1) In the head, the plasma membrane had a heterogeneous population density, e.g., few IMPs on the acrosomal region, particularly few on the marginal segment, and somewhat regularly arranged IMPs on the postacrosomal region. The acrosomal membrane had many IMPs in hexagonal arrays. The nuclear membrane had many IMPs on the P-face, few IMPs on the variegated E-face, and an intense population density on the P-face of the basal plate. 2) In the neck, the plasma membrane had many IMPs with square arrangements of small IMPs in some areas on the P-face; the redundant nuclear membrane had a few IMPs on both P- and E-faces. 3) In the tail, the plasma membrane had diagonal rows of IMPs in some areas amongst larger IMPs on the middle piece, while it had "zippers" composed of IMPs running parallel to the axis on the principal piece. The distribution of sperm membrane FSC may be summarized as follows: 1) In the head, the acrosomal plasma membrane, which was heavily labeled with filipin, had much more FSC in the equatorial segment than in the marginal segment throughout the study. The postacrosomal plasma membrane generally had no FSC, but some sperm in ejaculates were slightly positive to filipin. The acrosomal membranes (both outer and inner) had no FSC. The nuclear membrane in the main part of the head had less FSC in vas deferens and ejaculated sperm than in the epididymal sperm. The nuclear membrane on the basal plate had no FSC. 2) In the neck, the plasma membrane had little FSC. The redundant nuclear envelope had scattered FSC with a higher incidence in the epididymal sperm than in those from the vas deferens and ejaculates. The membrane scroll, which was elongated from the extreme caudal end of the redundant nuclear envelope, had abundant FSC in the vas deferens and ejaculated sperm. 3) The tail plasma membrane (both middle and principal piece), which was weakly labeled with filipin, had less FSC in sperm from the vas deferens and ejaculates than in those from the epididymis. The limiting membrane covering the mitochondria had no FSC.     

MEMBRANE DIFFERENTIATIONS IN FREEZE-FRACTURED MAMMALIAN SPERM

A correlated thin-sectioning and freeze-fracturing study has been made of guinea pig and rat spermatozoa. In sections, the cell membrane over the acrosome has a concanavalin A and ruthenium red reactive glycocalyx which exhibits an ordered pattern related to the lattice of crystalline domains within the plane of the membrane revealed by freeze-fracturing. The cleaved acrosomal membrane also shows a finer linear periodicity in some areas. The membrane over the equatorial segment of the guinea pig acrosome is marked by a palisade of oblique ridges not observed in the rat. The plasmalemma of the postacrosomal region is rich in membrane intercalated particles, many randomly dispersed, others clustered in rectilinear arrays. A particle-poor zone is found just anterior to the posterior ring. The fold of redundant nuclear envelope posterior to the ring has many nuclear pores in close hexagonal array. The nuclear envelope lining the implantation fossa is devoid of pores. When cleaved it has a particle-free central area surrounded by a broad zone of large, closely packed, hollow particles. The membrane of the mid-piece in the guinea pig (but not the rat) contains linear strands of 6–8-nm particles oriented circumferentially. The membrane investing the principal piece exhibits the usual randomly distributed particles but in addition, a double row of larger (9 nm) particles runs longitudinally within the membrane over outer dense fiber 1. In the corresponding position in thin sections a local thickening of the membrane is discernible. These observations form a basis for further studies on the functional correlates of these regional specializations of the sperm membrane.     

The spermatozoa of ascidians: Acrosome and nuclear envelope

Summary The spermatozoon of Ascidia callosa has a head with a wedge-shaped tip. Between the nuclear envelope and the plasmalemma, at the tip of the head, there are one or two previously undescribed vesicles, 45 to 55 nm in diameter. These vesicles have the characteristics of an acrosome. Their role in the process of fertilization has not been determined. Ultrastructural studies of sperm activation are needed, but claims that the spermatozoa of ascidians do not have an acrosome should be reconsidered.Behind the tip of the sperm there are pores in the nuclear envelope. This part of the envelope also contains a dense band of amorphous material that may have a supportive function. A nearly identical structure, associated with pores has been found in the spermatozoon of Boltenia villosa. An analysis of the nuclear envelope of Ascidia callosa indicates that the same structure has previously been misinterpreted as an acrosome in the spermatozoon of Ascidia nigra.     

The plasama membrane of Xenopus laevis spermatozoon

Xenopus laevis sperm plasma membrane ultrastructure has been studied by means of freeze-fracture, deep-etching, and lectin-gold binding. Xenopus spermatozoa differ from those of other species in that their plasma membrane does not exhibit topographical domains. In fact, no geometric arrangement or characteristic array of particles is present on fractured plasma membrane. Fractures rarely occur in acrosomal or nuclear membranes. Wheat germ agglutinin receptors are distributed homogeneously, on the plasma membrane of the sperm head and tail.     

Capacitation-dependent reorganization of microdomains in the apical sperm head plasma membrane: functional relationship with zona binding and the zona-induced acrosome reaction

For sperm to successfully fertilize an oocyte, it needs to pass through certain steps prior to, during and after initial recognition of the zona pellucida (ZP). During capacitation, the surface of the sperm head becomes remodelled, priming it to bind to the ZP and subsequently to undergo the ZP-induced acrosome reaction. During capacitation, sperm ZP-binding proteins are ordered in functional protein complexes that only emerge at the apical tip of the sperm head plasma membrane; this is also functionally the exclusive sperm surface area involved in primary ZP binding. After primary ZP binding, the same area is probably involved in the induction of the acrosome reaction. A combination of biochemical and proteomic membrane protein techniques have enabled us to dissect and highly purify the apical sperm plasma membrane area from control and capacitated sperm cells. The actual ZP-binding proteins identified predominantly belonged to the sperm membrane-associated family members of spermadhesins (AQN-3) and were present in the aggregating lipid ordered membrane microdomains (lipid rafts) that emerged during in vitro capacitation in the apical ridge area of the sperm head plasma membrane. This clustering of these rafts was dependent on the presence of bicarbonate (involved in protein kinase A activation) and on the presence of albumin (involved in cholesterol removal). Remarkably, cholesterol removal was restricted to the non-raft membrane fraction of the sperm plasma membrane, but did not cause any depletion of cholesterol in the raft membrane fraction. Interestingly, sperm SNARE proteins (both VAMP from the outer acrosomal membrane, as well syntaxin from the apical sperm head plasma membrane) shared lateral redistribution properties, along with the ZP-binding protein complex and raft marker proteins. All of these were recovered after capacitation in detergent-resistant membrane preparations from sperm thought to represent membrane lipid rafts. We inferred that the capacitation-dependent formation of an aggregated lipid ordered apical ridge surface area in the sperm head plasma membrane was not only relevant for ZP-binding, but also for the ZP-induced acrosome reaction.     

ON THE THICKNESS OF THE UNIT MEMBRANE

Peak-to-peak distances between two dense lines of the unit membranes of cell organelles were measured on electron micrographs. These distances were compared with corresponding measurements on the plasma membrane and assigned a percentage value. The comparison between organelle and plasma membrane was always carried out with the same negative, in order to exclude as far as possible errors due to differences in focus or other causes. It was revealed by this study that the membranous structures of the cell can be classified into two groups, one thicker and one thinner. Unit membranes of the thicker group (synaptic vesicles, vesicles and capsules of multivesicular bodies, Golgi vesicles) were not significantly different in thickness from the plasma membrane. Unit membranes of the thinner group (mitochondria, nuclear membranes, Golgi lamellae, endoplasmic reticulum), however, were between 85 and 90 per cent of the thickness of the plasma membrane.     

Boar sperm cytoplasmic droplets: Their ultrastructure,their numbers in the epididymis and at ejaculation and their removal during isolation of sperm plasma membranes

Cytoplasmic droplets of the boar are progressively lost from the flagellum of boar spermatozoa during epididymal transit, at ejaculation and during the nitrogen cavitation technique for isolation of plasma membranes. Apparently very fragile, these structures are broken up in the fluids of the reproductive tract and in the buffer used during the nitrogen cavitation procedure. The maximal potential contamination of cytoplasmic droplet internal vesicular membranes in plasma membrane fractions was determined to be 2.2% of the entire membrane surface area collected. The highly sensitive silver-stained, two-dimensional (2-D) polyacrylamide (PAGE) gels of boar sperm plasma membranes did not reveal cytoplasmic droplet, internal membrane, marker polypeptides, further demonstrating the high purity of plasma membrane preparations. In addition, freeze-fracture demonstrates that the internal membranes of the cytoplasmic droplet show few intramembranous particles and these may contribute little protein to plasma membrane preparations. The presence of two forms of vesicular elements in boar sperm Cytoplasmic droplets (typical vesicles and collapsed vesicles) is described.     

Topographical relations of intramembrane particle distribution patterns in human sperm membranes

The distribution of intramembrane particles in human sperm membranes has been explored with particular reference to the topographical region of the sperm cell and the membranes' fracture face. Conspicuous differences in the size, arrangement, density, and lateral mobility of intramembrane particles between some topographically distinct membrane domains are demonstrated. The greatest regionality is exhibited by the plasma membrane. In sperm head regions, it shows a significant variability and changes its particle distribution during culture in capacitating medium. In contrast, little variability and no changes during the incubation are seen in the acrosomal and nuclear membranes. Striking is the difference in particle distribution on the E face of the outer acrosomal membrane between the acrosomal and equatorial regions. It is suggested that the invariable regional difference in the organization of the outer acrosomal membrane may bear on the different behavior of its two main domains during sperm capacitation and acrosome reaction.