The eggshell of the almond wasp Eurytoma amygdali (Hymenoptera, Eurytomidae) - 2. The micropylar appendage

Micropylar apparatuses in insects are specialized regions of the eggshell through which sperm enters the oocyte. This work is an ultrastructural study and deals with the structure and morphogenesis of the micropylar appendage in the hymenopteran Eurytoma amygdali. The micropylar appendage is a 130 mum long cylindrical protrusion located at the posterior pole of the egg, unlike other insects i.e. Diptera. in which the micropylar apparatus is located at the anterior pole. In mature eggs there is a 0.4 mum wide pore (micropyle) at the tip of the appendage leading to a 6 mum wide micropylar canal. The canal contains an electron-lucent substance, it travels along the whole appendage and finally reaches the vitelline membrane of the oocyte. The vitelline membrane is covered by a wax layer and an electron-lucent layer, whereas the chorion surrounding the canal consists of a granular layer (fine and rough) and a columnar layer. The morphogenesis of the appendage starts in immature follicles: four central cells located at the posterior tip of the oocyte near the vitelline membrane, differing morphologically from the adjacent follicle cells. These central cells degenerate during early chorionic stages, thus assisting in the formation of the micropylar canal. The adjacent, peripherally located cells secrete the electron-lucent substance which fills the canal and at the same time, the fine granular layer is formed starting from the base towards the tip of the appendage. The secretion persists at late chorionic stages and results in the formation of the chorion around the micropylar canal. The extremely long (compared to other insects) micropylar appendage seems to facilitate the egg passage through the very thin and long ovipositor. The structure and morphogenesis of this appendage differs significantly from the micropylar apparatuses studied so far in other insects i.e. Diptera, and may reflect adaptational and evolutionary relationships.     

Ultrastructural features and formation of the micropylar apparatus in the cherry fly Rhagoletis cerasi

The micropylar apparatus (MA) in Rhagoletis cerasi (Diptera, Tephritidae) is located at the anterior pole of the egg and consists of two parts: an outer chorion and an inner vitelline membrane. Sperm entry takes place through the micropylar canal, 2.0–2.5 μm in diameter, which penetrates the micropylar endochorion and terminates in the thick vitelline membrane, thus forming the “pocket.” The pore of the micropylar canal, i.e., the micropyle, is covered by the exochorionic tuft. The formation of the MA is accomplished by 40 micropylar cells during oogenesis. These cells secrete the successive eggshell layers: the vitelline membrane, the wax layer, the innermost chorionic layer, the micropylar endochorion, and the exochorion. Two among 40 micropylar cells differentiate and form two tightly connected projections. The latter contain a bundle of parallel microtubules and participate in the formation of the micropylar canal and the pocket. At the tip of the projections there are two thin extensions full of microfilaments. In late developmental stages the two projections and the extensions degenerate and leave the canal and the pocket behind. We also discuss the structural features of the MA in relation to its physiology among Diptera.     

Eggshell fine structure of Bradysia aprica (Winnertz) (Diptera : Sciaridae)

The eggshell fine structure of the dark-winged fungus-gnat Bradysia aprica (Winnertz) (Diptera : Sciaridae) was investigated by scanning and transmission electron microscopy. At the anterior pole of the ovoid egg is a single micropyle, centrally located in a well-defined micropylar area. The latter is covered by many long drumstick-like chorionic processes that are longer and more numerous than those of the rest of the egg surface. Cross-sections of the eggshell show 3 concentric envelopes: the vitelline envelope, wax layer and chorion. The chorion consists of 3 components with different morphological features: the inner, intermediate and outer chorion. The latter 2 layers, involved in the organization of the drumstick-like processes, have homogeneous features, whereas the former is crystalline and resembles the innermost chorionic layer of other Diptera.     

The micropyle: a sperm guidance system in teleost fertilization

The micropylar region of the Rosy barb, Barbus conchonius, egg consists of 7-10 grooves and ridges, which drain directly into a funnel-shaped vestibule, the only point on the chorion through which sperm-egg contact is achieved during fertilization. Results of time-lapse video microscope study and computer-aided analysis of sperm motility pattern in the micropylar region showed that the fertilizing sperm, usually the first to enter the micropylar region, always travelled preferentially along the grooves into the micropylar pit. Subsequently, 86% of sperm arriving the micropylar region within 30 s travelled preferentially along the grooves into the immediate vicinity of the micropylar pit. The sperm guidance role of the micropylar region was calculated to enhance chances of egg penetration/fertilization by as much as 99.7% once sperm were within the micropylar region, possibly in response to some form of chemo-attractant(s) from the egg. Sperm agglutination post-fertilization was also found to occur preferentially along the grooves. Results of our in vitro fertilization experiments showed association between point of sperm entry and blastodisc formation: the blastodisc formed directly beneath the micropyle in all undisturbed eggs.     

革胡子鲶受精过程的扫描电镜观察

应用扫描电镜观察和描述了革胡子鲶成熟卵和精子的形态、卵壳膜的表面结构和形态、受精孔的位置和结构、精子入卵过程的程序和变化。讨论了精子入卵过程及精孔细胞在解体之后可能转变为一种能够吸引精子在精孔区聚集的“受精素”物质等问题。     

家养鱼类受精生物学的研究 Ⅱ.几种淡水鱼类成熟卵球的精孔器与精子入卵通路的光镜与扫描电镜观察

作者对我国四种淡水养殖鱼类——团头鲂、草鱼、白鲢和花鲢卵球的精孔器作了光学显微镜和扫描电子显微镜的比较描述,在扫描电子显微镜下观察到这几种鱼类的精子均直接经精孔器前庭穿过精孔管进入卵内,并对精孔细胞、受精孔与精子入卵的关系以及精孔的位置进行了讨论。       

Closure of the micropyle during embryonic development of some pelagic fish eggs

The fine structure of the egg envelope and micropyle was studied in unfertilized and developing eggs of the flounder Paralichthys olivaceus (Temminck & Schlegel), the Alaska pollack Theragra chalcogramma (Pallas), the Japanese tilefish Branchiostegus japonicus (Houttuyn) and the porgy Pagrus major (Temminck & Schlegel). The outer envelope surface of the unfertilized egg was wrinkled, while the inner surface was folded. The micropyle of the unfertilized egg consisted of a shallow vestibule and a distinct canal. The micropylar region of the inner surface of the envelope had a conical- or bowl-shaped protrusion. In developing eggs, the thickness of the envelope decreased and showed smooth outer and inner surfaces which indicated that it had been stretched tangentially at the time of the perivitelline space formation. The lumen of the micropylar canal was invariably occupied with envelope material. We postulate that the blockage of the micropylar canal is a result of the stretching of the envelope. The closure of the micropyle inhibits sperm and external pathogens from penetrating into the perivitelline space and seems to be involved in both the permanent prevention of polyspermy and the protection of the developing embryo from bacterial infection.     

金鱼精子入卵过程的扫描电镜观察

本文采用扫描电镜观察了金鱼(Carassius auratus)卵壳膜(chorion)表面结构和精子入卵过程。在壳膜的卵膜孔(micropyle)区有5—10条沟和嵴。位于精孔管下面,卵的质膜为一束较长的微绒毛组成的精子穿入部(sperm entry site)。授精5s,精子头的顶部已附着于精子穿入部,随即两者的质膜发生融合,而围于精子头部四周的微绒毛迅速伸长形成一受精锥,它不断将精子头部包裹。授精110s,精子的头部和颈部已完全进入卵内,受精锥本身也渐趋消失,但精子尾部仍平躺于卵的表面。皮层小泡是在授精30s后才开始破裂并释放其内含物,导致卵子表面呈蜂窝状,并在无膜内表面附着了大量球状物。     

Morphology of the egg shell and the developing embryo of the Red Palm Weevil,Rhynchophorus ferrugineus (Oliver)

The harvested eggs of Rhynchophorus ferrugineus are ovo-cylindrical shaped, averaged 1.09 mm in length and 0.43 mm in width, with ratio of L\W 4.42. The chorionic layer of electron dense material is seen covering the exochorion structure of the eggs. The egg main body chorion exhibits a polygonal pattern and architecture surface of the egg is supported by a system of irregular interconnecting grooves. The micropylar apparatus of the eggs of the Red Palm Weevil, R. ferrugineus is described in the present study for the first time. Two micropylar openings are found closed to the center of the posterior wide pole of the egg. Each micropylar opening presents a single small orifice and its surrounding chorion is porous and densely set with tiny projections allowing the spermatozoa to penetrate the egg. Respiratory aeropyles are distributed on the borders of reticulations in the area chorionic surface of egg capsule. The hatching region is detected on the anterior part at the opposite side of the egg. Changes in the appearance and shape of R. ferrugineus eggs as well as the incidence of embryonic development are observed.     

Ultrastructural changes in micropylar cells and formation of the micropyle during oogenesis in the medaka Oryzias latipes

Ultrastructural changes in differentiating micropylar cells and in the micropyle occur during oogenesis in the medaka, Oryzias latipes. A micropylar cell is not detectable in previtellogenic ovarian follicles. In the early vitellogenic phase, the micropylar cell becomes differentiated from neighboring granulosa cells by its electrondense cytoplasm. The micropylar cell in this phase characteristically displays an increase in rough endoplasmic reticulum, Golgi complexes, and tonofilaments around the nucleus. By the late vitellogenic phase, the enlarged micropylar cell extends a broad cytoplasmic process to the oocyte surface. A conspicuous feature of the process is a large bundle of microtubules oriented perpendicular to the oocyte surface. The inner surface of the micropylar canal has a spiral structure and is covered with the outermost layer of the chorion. In the postvitellogenic phase, the main cell body possesses many tonofilaments, mitochondria, Golgi complexes, and rough endoplasmic reticulum, and the winding cytoplasmic process contains a twisted large bundle of microtubules with a bundle of tonofilaments as its core. The spiral structure of the micropylar vestibule and the micropylar canal reflects the twisting associated with elongation of fibrous bundles of the micropylar cell anchored on the chorion at the animal pole of the oocyte.     

Egg structure of Zorotypus caudelli Karny (Insecta, Zoraptera, Zorotypidae)

The structural features of eggs of Zorotypus caudelli Karny are described in detail. The egg is elliptic with long and short diameters of 0.6 and 0.3 mm respectively, and creamy white. The egg shows a honeycomb pattern on its surface, without any specialized structures for hatching such as an operculum or a hatching line. The fringe formed by a fibrillar substance secreted after the completion of the chorion encircles the lateral surface. The egg layer is composed of an exochorion, an endochorion, and a vitelline envelope. The exochorion and endochorion are electron-dense and homogeneous in structure. The exochorion shows a perforation of numerous branching aeropyles. The exo- and endochorion are connected by numerous small columnar structures derived from the latter. The vitelline envelope is very thin and more electron-dense than the chorion. A pair of micropyles is present at the equator on the dorsal side of the egg. Originating at the micropyle, the micropylar canal runs through the chorion obliquely. The structural features of the eggs of Zoraptera were compared with those of other polyneopteran and paraneopteran orders.     

The fowl tick, <Emphasis Type="Italic">Argas (Persicargas) persicus</Emphasis> (Ixodoidea: Argasidae): Description of the egg and redescription of the larva by Scanning Electron Microscopy

This scanning electron microscopy study revealed that the egg of Argas persicus was covered with chorion which appeared as a wrinkled layer containing regions of three textures. The first had elevated parts of slightly rough surface. The second had irregular smooth elevations; each carried numerous parallel horizontal foldings with vertical ridges. The last region had rough surface with irregularly shaped projections. Following the removal of the chorion, shell was observed to have one polar micropyle and numerous slit like openings. Length (L), width (W) and L/W ratio of the egg were measured. Investigation of larvae revealed extensively folded integument of idiosoma and spherical or elongated tubercles on dorsal plate. Mouth enclosed between ventral hypostome and two dorsal chelicerae. Hypostome carried four longitudinal rows of conical denticles. Each chelicera was made up of two segments; the basal one appeared as a pocket for the distal one. Haller’s organ consisted of an anterior pit containing seven sensilla and a posterior capsule with four apertures. Distribution of chemo- and mechano-sensilla on the body was examined. Measurements of whole body, idiosoma, dorsal plate, capitulum, hypostome, palp and different types of sensilla both on the body and Haller’s organ are also presented.     

The hagfish oocyte at late stages of oogenesis: structural and metabolic events at the micropylar region

The hagfishes (Class Agnatha), primitive vertebrates and massive secretors of mucus, occupy a unique niche in a marine benthic environment and exists in abundance despite an infrequent ovulation of a low number (12-45) of notably large, prolate ellipsoidal (~8 mm x 28 mm. width x length, respectively) eggs. To establish factors that might contribute to survivability of one species (Eptatretus stouti), we examined three groups of mature females and document structural events characteristic of oocytes at late developmental stages: attached to the gonad, after ovulation into the body cavity, and after deposition on aquaria substrata. Quantitative autoradiographic analyses following administration in vivo of [(3)H]-leucine was useful to confirm post-vitellogenic stages and late metabolic events at the micropylar region located at one end of each egg. After egg disassembly, a combination of light microscopy and scanning electron microscopy was used to delineate structural features and 3-D relationships in juxtaposition to the micropylar cup and canal. A thick (~200mum) complex tripartite chorion surrounds a large micropylar cup (~300mum, width at base) consisting of fused polygonal (mainly hexagonal) substructural units (3-4 mum, width) and collectively serves to stabilize the micropylar canal (of size to prevent polyspermy) that penetrates the ooplasm near the germinal vesicle. A tuft composed of numerous chorionic appendages ('anchor filaments', ~2-3 mm length) is enmeshed in a gel matrix and surrounds the cup. When the eggs are deposited, intermediate filament aggregates (IFA) from slime gland mucus become interspersed in the tuft/gel complex. A similar IFA/tuft/gel region found on the vegetal pole-end facilitates firm egg-end attachments and possibly assists localization of egg clutches to a favorable substratum. Although the mode of fertilization is as yet unknown, the collective structural characteristies of the hagfish egg arc indicative of physical strength and are markedly different from eggs of the other group of cyclostomes (lampreys) and from tcleosts. The hagfish produces fewer but larger eggs which are prolate ellipsoidal rather than spherical and have a thicker chorion, a greater chorion to egg width ratio, a much larger micropylar cup with a unique substructure, larger and more localized chorionic appendages, and appendage tufts interspersed with adhesive substances derived not only from the follicular epithelium but also from the slime glands.     

泥鳅精子入卵的动力作用

在扫描电镜下,泥鳅成熟卵卵膜孔外围呈现完整的左涡旋状结构,受精时精子是顺着涡旋的流线进入卵膜孔。涡旋纹理接近对数螺线。本文分析了真骨鱼类的受精因素,除已知的化学因素外,还存在物理因素。也讨论了泥鳅成熟卵卵膜孔形态形成的必然性。     

Ultrastructural features of the egg envelope of silver carp, <Emphasis Type="Italic">Hypophthalmichthys molitrix</Emphasis> (Osteichthyes,Cyprinidae)

Synopsis We investigated the ultrastructure of the egg membrane surface (unfertilized egg) of Hypophthalmichthys molitrix using scanning electron microscopy. The eggs of silver carp like most other teleosts are surrounded by a relatively thick egg membrane, and have a type III micropyle at the animal pole. The micropyle is almost circular in shape and the micropyle canal is located in its center. The micropyle region is not flat. Round or oval accessory pores are also observed in the canal. The surface of zona radiata is wavy and uneven with a uniform distribution of almost round pores with lips. Microvilli like structure are found in the pore opening region.     

Translocation of Uranin within the Living Ovules of Vanilla

In the ovules of Vanilla (Vanilla planifolia Andr.) before fertilization, outer integument surrounded the lower part of ovule. Uranin got into ovule through funiculus, forming, the first center of fluorescence at the chalaza zone of ovule. Then uranin was transported to micropyle end along inner integument, forming the second center of fluorescence at micropyle end of inner integument. Soon, fluorescence appeared in the egg apparatua. After fertilization, the outer integument ovule extended upward, forming micropyle ogerber with inner integument. After getting into ovule through funiculus, uranin spreads to- ward several directions: l. transported to outer integument at the entrance of micropyle; 2. transported downward to chalaza zone along outer integument at the side of funiculus; 3. extended from chalaza zone to the inside and to the outer integument at the side far from funiculus The ovules of Vanilla had no vascular bundles. On transporting in inner integument, however, the cells in inner layer next to the embryo sac appeared to be the major passage. In mature embryo sac, there was cuticle between inner integument and embryo sac at the half of micropyle end. But between embryo sac at the half of chalaza end and nucellus, cuticle was absent. Nutrient could get into embryo sac from chalaza end undoubtedly. As egg apparatus showed the fluorescence after formation of fluorescence center of inner integument at micropylar end, the possibility that nutrient got into embryo sac from micropyle could not be excluded.     

Changes in the chorion and sperm entry into the micropyle during fertilization in the teleostean fish, Oryzias latipes

Specific antibodies against the major chorionic glycoproteins (ZI1 -2 and ZI3) of unfertilized eggs were used to analyze the differences in the chorion and its surrounding constituents before and after fertilization. The glycoproteins in the inner layers of the chorion and its surrounding material were specifically stained by both of the antibodies. Thirty and 60 min after activation, the thickness of the chorion's inner layers was already reduced and the micropylar canal was closed. At the same time, the broadly diluted mucous area (DMA) of glycoproteins on the outermost layer of the chorion in unfertilized eggs was modified to a thin, compact layer. When unfertilized eggs were treated with trypsin, the inner third portion of the micropylar canal closed and the glycoproteins in the DMA were digested. The incidence of sperm entry into the micropyle of these eggs was extremely reduced. These results suggest that in medaka eggs, the chorionic glycoproteins in the DMA on the chorion surface, which have an affinity for spermatozo, play an important role in sperm guidance into the micropyle.     

Factors inducing closure of the micropylar canal in the chum salmon egg

The micropylar canal of the chum salmon egg was almost completely closed following egg activation caused by incubation in a hypotonic salt solution (HSS) for I h. The closure occurred in both inseminated and parthenogenetically activated eggs. Incubation of isolated envelopes from non-activated eggs in HSS or perivitelline fluid (PVF) did not induce any modification in micropylar structure, indicating that normal organization of the egg is essential for inducing closure. To reduce the volume of the perivitelline fluid, the eggs were activated in PVF or HSS containing 8 mM Dextran, Although the envelope showed hardening, closure of the micropyle was not observed in these eggs. The wall of the micropylar canal, however, possessed a slightly rough surface. Following activation in a Ca-free hypotonic salt solution with 10 mM EDTA, hardening of the egg envelope was completely inhibited. Although such eggs possessed an apparent perivitelline space, neither closure of the micropylar canal nor roughening of the canal surface were detected. We conclude that the synergistic action of perivitelline turgor pressure and perivitelline material is responsible for the closure of the micropyle.     

大银鱼卵膜孔结构的电镜观察

扫描电镜下，大银鱼成熟卵卵膜孔区域呈现奇异的放射沟脊状结构。在授精开始的３０秒内，测得卵膜附近约８６％的精子沿着凹沟进入精孔管；还拍摄到了通过卵孔中心的纵切面映象，通过电子计算机模拟，验证了卵膜孔的这种结构，为精子的云集和进入精孔管所提供有利条件，从而，于泥鳅之后又发现了一种真骨鱼，其精子入卵除化学因素外，还存在着不容忽视的机械动力因素。     

Scanning electron microscopy of the eggs of Palingenia longicauda (Olivier) (Ephemeroptera : Palingeniidae)

The eggs of Palingenia longicauda (Ephemeroptera : Palingeniidae) were studied by scanning electron microscopy (SEM), and showed a biconvex shape with the 2 aspects joined along a thick peripheral border. The eggs were completely wrapped by an exochorion that differs in thickness and organization according to the region of the chorionic surface. The thickest part of the exochorion formed a plaque, which also covered part of the peripheral border, and was constituted of a network of filaments. The remaining part of the exochorion was composed of a thin wrinkled sheet. The micropyle, hitherto unknown in Palingeniidae, appeared as a round opening penetrating into the plaque. The fibrillar network surrounding the micropyle dove-tailed with the egg chorion, forming a differentiated raised process. This peculiar interconnection facilitates egg anchoring to the substratum, and is an adaptation of the fibrous coat to the aquatic environment.