Micropylar Canal Research Articles

Structure of the Specialised and Unspecialised Chorion of the Egg in the Stick Insect Malacomorpha cyllarum (Phasmatodea)

The chorion of deposited eggs of the bisexual phasmid Malacomorpha cyllarum (Westwood 1859) was examined by LM, SEM and TEM. The unspecialised chorion consists of three principal layers, from which especially the second one is heavily mineralised. Distinction of sublayers is possible mainly by their different electron density, spongy appearance, and mineralization or simply by phase interfaces, but appears rather arbitrarily in most cases. Specialised regions of the chorion are the operculum and the micropylar plate. The operculum largely shows the same series of layers, but is generally thicker as the unspecialised chorion. Stratification of the highly modified microplyar plate is more complex and characterised by additional spongy modifications, which appear to be derived from the innermost third principal layer. Two micropylar canals, collapsed distally and proximally, run through the micropylar strand. These canals neither open to the exterior nor to the interior of the egg. External and internal closure are achieved by chorion material rather than a secretory plug.

Scanning electron microscope study on the egg chorion of silkmoth, Antheraea assamensis Helf. (Lepidoptera : Saturniidae)

AbstractThe eggshell of Antheraea assamensis Helf. has a highly decorated chorion and its micropylar apparatus is situated at the cephalic end. Scanning electron microscopic investigation on the eggshells of A. assamensis reveals distinct patterns of structural elements viz., central cavity, micropylar rosette encircled the central cavity, and the aeropyles. The micropylar structures are at the anterior pole of the oval shaped eggs opposite to its side of distal pole. Centrally located central cavity leads into eight micropylar openings which further radiate down to the eight micropylar canals. The central cavity (5.28±0.21 μm in diameter) is encircled by a primary rosette of 16-18 cell imprints and each primary cell imprint measured 21.08±3.13 μm in length. The primary petal shaped cell imprints are followed by secondary and tertiary rows of cell imprints. Except the central cavity and micropylar rosette, the entire surface of the chorion is marked by a circular shell imprints which are boarded by aeropyles. Each cell imprint has 4-6 aeropyles and each aeropyle measured 1.06±0.01 μm in diameter. The aeropyles of A. assamensis are covered with a cup shaped like structures and the terminals of these structure has many finger like projections which may help to check the entry of pathogens or dust particles while taking the oxygen from the outside environment to the oocyte.

Two egg-derived molecules in sperm motility initiation and fertilization in the Pacific herring (Clupea pallasi)

Sperm of the Pacific herring are immotile at spawning. Two egg-derived molecules are capable of initiating sperm motility. One is herring sperm activating protein(s) (HSAPs) and the other is sperm motility initiation factor (SMIF). These two motility initiators differ in their location and association with the chorion, and in their isoelectric points and molecular weights. In this study we have investigated the roles of these two inducers with respect to motility and fertilization. Using computer analysis of sperm motility, we found that HSAPs, as well as the C-terminal HSAPs peptide, elicit a linear motility pattern, while SMIF induced a highly circular and asymmetric pattern. HSAPs induced a two-fold increase in intracellular calcium, whereas SMIF induced a four-fold increase of motility initiation. SMIF-exposed sperm, preloaded with BAPTA-AM, showed a more linear motility and this motility trajectory decreased with their fertilizing capability. The difference in intracellular calcium levels between HSAPs and SMIF is consistent with the observed linear and circular motility. In the absence of SMIF, HSAPs do not support fertilization. Fertilization is rescued in these experiments if SMIF is reintroduced. We propose that diffusible HSAPs are not essential for fertilization, but enhance sperm-egg collisions via linear motility. SMIF, which is bound to the micropylar region of the chorion, is required for fertilization and induces circular motility that is a prerequisite for sperm to enter the micropylar canal and fertilize the egg.

Significance of exine shedding in Cupressaceae-type pollen

In conifers, which have non-saccate Cupressaceae-type pollen, the pollen must land on a pollination drop or be picked up by the pollination drop from the surface of the cone near the ovule before it can be taken into the ovule. After contact with the drop, the pollen intine absorbs moisture from the drop, expands and the exine is shed. In this study the significance of the shedding of the exine is interpreted from experiments in which simulated pollination drops and micropyles were used to determine the movement of pollen and other particles in suspension. The non-expanded pollen, which can be observed upon contact with the pollination drop, sheds the exine, which then functions as a non-elastic particle, while the pollen from which the exine was shed swells and functions as an elastic particle because it is enclosed by the flexible intine. Non-elastic particles are not easily transferred through narrow passages (the micropyle and micropylar canal) and tend to plug these passages. However, elastic particles, such as the swollen pollen, are easily transferred along narrow passages even when non-elastic particles are present. The simulated experiments demonstrate that exine shedding is an important feature in getting pollen through the narrow micropyle and micropylar canal to the nucellus of the ovule.

Ultrastructural studies of the morphological variations of the egg surface and envelopes of the African catfish <em>Clarias gariepinus</em> (Burchell, 1822) before and after fertilisation with a discussion of fertilisation mechanism

Much of the existing knowledge of the mechanisms involved in teleost fertilisation is based on a few small model species that have no commercial value. Research is therefore urgently required to address mechanisms involved in fertilisation in species of great commercial value. In this study, the ultrastructural morphological variations in the surface of the egg of Clarias gariepinus were recorded before and after fertilisation by using electron microscopy. The outer surface of the unfertilised egg was smooth, whereas the fertilised egg acquired a network of projections on the vegetal hemisphere. Moreover, different patterns of ornamentation on the egg surface were evident. This pattern of ornamentation varied with the progress of embryonic development. The micropyle of the C. gariepinus egg consisted of a funnel-shaped vestibule, from the bottom of which a cylindrical micropylar canal extended. The micropylar canal decreased in diameter after completion of fertilisation, forming a micropylar disc. The sperm behaviour on the egg surface was oriented towards any depression on the chorion surface. The chorion of ovulated eggs consisted of one layer. After fertilisation the chorion was differentiated into three layers: the double-layered coat, the zona radiata externa and the zona radiata interna. Four protein subunits of the chorion of C. gariepinus were identified by SDS-PAGE. IR-spectra obtained from C. gariepinus chorion revealed that the vibration of chorion proteins exhibited different weak activities in the IR-spectra with minor difference between pre- and post-fertilisation chorion proteins.

Calcium in the micropylar secretion and receptivity of explanted Crocus ovules to intra- and interspecific pollen

The micropylar secrete of the ovules of Crocus vernus ssp. vernus was analyzed for the Ca+2 content by atomic absorption, and its capacity to germinate and attract pollen was tested by pollinating explanted ovules, and incubating in absence of culture medium. The results display a Ca+2 concentration of 28.9 mM in the micropylar secrete. On this secrete both compatible- and incompatible pollen germinates with a mean percentage of 53.7%, and their pollen tubes enter the micropylar canal with percentages of 32.3% to 21.0%. In situ the ovules fail to attract tubes of incompatible pollen. The results are discussed in relation to the ovule receptivity and the guided growth of pollen tubes, substantiating the model of the tropic growth towards increasing calcium concentrations.

Mode of Pollen Tube Growth in Pistils of Eucommia ulmoides (Eucommiaceae, Garryales)

In Eucommia ulmoides, the only extant species of Eucommiaceae, fertilization is delayed for several weeks after pollination. Based on a developmental study of pollen tube growth in the E. ulmoides pistil, we show that during days 11–13 after pollination, pollen tubes grow intermittently in three steps, in close association with the development of ovules: (1) from the stigma to within the placental tissue, (2) from within the placenta through the funiculus into the tissue of an enlarged integumental tip, and (3) from the integumental tip to the mature embryo sac. The number of pollen tubes decreased from many to several during the second step and from several to only one in the third step. Thus, the three‐step pollen tube growth mode plays a role in pollen selection for fertilization. Occasionally, a long micropylar canal promoted the reduction in the number of pollen tubes from two or three to one. A comparison with the diverse modes of pollen tube growth in pistils of species of Fagales, which also demonstrate delayed fertilization, showed that the mode of E. ulmoides is distinct from these modes. This unique mode might have contributed to the survival of this species from the early Eocene.

Comparative morphology of the egg of the castniid palm borer, Paysandisia archon (Burmeister, 1880) (Lepidoptera: Castniidae)

Paysandisia archon (Burmeister, 1880) is an attractive neotropical castniid moth whose presence in Europe was recently reported. Its larvae are endophagous, feeding inside the trunks and branches of several species of palm trees (Arecaceae). The present paper deals with the morphology and biometry of the egg of this moth, comparing them with those of other castniid species. The egg is a typical castniid egg, fusiform, upright sensu Döring, light cream or creamy pink when freshly laid, 4.69 ± 0.37 mm long and 1.56 ± 0.11 mm wide. Larvae emerge by gently splitting the chorion along one of the longitudinal ridges, on the half closer to the micropyle. SEM, TEM and LSCM photographs showing ultrastructural details of the egg are shown for the first time. The micropylar rosette (c. 54 μm in diameter) has generally 14–17 cells; in its centre lies the micropylar pit (c. 6 μm in diameter) which bears 12–16 micropylar canal openings (= micropyles) around its periphery. The pathways followed by those canals through the chorion have been figured. Eggs sampled in the wild (so laid by several females) were found to have a slightly variable number of ridges: most bore seven ridges (68.87%), although a significant portion (30.46%) bore eight and 1 egg (0.67%) bore only six; this against the currently accepted rule of five‐ridged eggs for Castniini (i.e. Neotropical castniids) to which Paysandisia archon belongs. It has also been found that the same female specimen has the capability of producing six‐, seven‐ or eight‐ridged eggs. Five types of egg irregularities affecting the longitudinal ridges are also figured and described. Transverse striae on the egg of P. archon are about 122. Aeropyles (c. 4 μm in diameter) occur on the ridges, at the intersections between the latter and two contiguous (left and right of the ridge) transverse striae, amounting to c. 854 on a seven‐ridged egg and to c. 976 on an eight‐ridged egg. Occasionally minute aeropyles ('microaeropyles') (c. 1.96–3.13 μm in diameter) also occur on transverse striae located close to both egg poles. The chorion of P. archon shows the typical ditrysian fine structure with very thin basal layer (C‐1), 0.3–0.2 μm thick, gas‐filled trabecular layer (C‐2), c. 0.9 μm thick, and lamellar layer (C‐3), its thickness varying between 18.5 and 13 μm due to the bumpy external surface of the chorion. Aeropylar canals, that penetrate layer C‐3, connect the air‐containing inner chorionic meshwork (the trabecular layer C‐2) with the surrounding air; their outer part forms a big bulbous cavity (which opens to the outside through the small opening seen in external SEM images) and, underneath, a narrow canal follows, leading into the trabecular layer (C‐2).

Micropyle in neuropterid insects. Structure and late stages of morphogenesis

The eggshells of five representatives of neuropterid insects belonging to three orders and five families (Neuroptera: Ithonidae and Myrmeleontidae; Raphidioptera: Raphidiidae and Megaloptera: Corydalidae and Chauliodidae) have been analyzed with various microscopic techniques. These studies have revealed that the eggshells of all investigated species are morphologically similar and always equipped with an anteriorly located micropylar apparatus. The eggshell surface is usually uniform, smooth or densely set with small, raised projections. Only in some species ( Euroleon nostras, Oliarces clara) these projections are more regularly arranged and form a characteristic polygonal pattern on the eggshell surface. The shape and size of the micropyle vary from species to species but it always consists of two parts: the core and cortex. In the investigated representatives of Neuropterida the micropyle is pierced by several (about 20) micropylar canals. These canals converge radially in the central part of the core, where they bend and run towards the anterior pole of the egg cell. In the majority of studied species the orifices of the canals are located circumferentially at the base of the micropyle, whereas in the corydalid, Corydalus peruvianus, they have been found on a swollen apical part of the micropyle. Analysis of late choriogenic oocytes have revealed that the micropyle is always formed by two distinct subpopulations of the follicular cells. The micropyle forming cells (MFCs) synthesize and secrete the macromolecules that are subsequently incorporated into the micropyle, while micropylar canal forming cells (MCFCs) send long cytoplasmic projections that mould the micropylar canals. Formation and/or maintenance of these extensions depend on microtubules and/or microfilaments.

Eggshell Fine Structure of Amata passalis F. (Lepidoptera: Amatidae), A Pest of Mulberry

—The eggshell fine structure of a lepidopteran pest of mulberry, Amata passalis F. (Lepidoptera: Amatidae), was investigated using scanning electron microscopy. The micropylar rosette around the micropyle, shell imprints, aeropyles and regional differentiation were studied. The surface of the amatid eggshell had a highly decorated chorion with structural difference at the anterior and posterior poles. The micropylar structure was at the anterior pole of the spherical eggs (488 ± 0.595 mm in diameter), opposite to its side of attachment to the substratum. The micropylar rosette measured 51.20 ± 0.52 mm in diameter and was formed of 15–19 petal-shaped primary cells. The micropylar opening into the micropylar canal was 3.76 ± 0.52 mm in diameter. The entire surface of the chorion had a reticulate pattern of pentagonal and hexagonal cells, each of which was boarded by 3 to 6 aeropyles of similar size (0.36 ± 0.008 mm in diameter).

Anatomically Preserved Williamsonia (Williamsoniaceae): Evidence for Bennettitalean Reproduction in the Late Cretaceous of Western North America

An anatomically preserved ovulate cycadeoid cone has been discovered in Upper Cretaceous (Campanian) sediments of Vancouver Island, British Columbia, Canada. The specimen is preserved by calcareous cellular permineralization and displays diagnostic features of the genus Williamsonia Carruthers. The cone consists of a receptacle from which tightly packed interseminal scales and ovulate sporophylls with terminal ovules diverge over an arc of ca. 300°. Adjacent interseminal scales interdigitate and form a continuous tissue. Seeds are erect and more or less round in cross sections at all levels, and a cupule is not produced. The sarcotesta consists of multicellular peglike projections. The nucellus is attached to the integument only at the chalaza and is vascularized by a shallow cup of tracheids. Apically, the nucellus narrows to a solid finger‐like projection that fits tightly into the base of the micropylar canal. A pollen chamber is not produced. Nucellar cells are often separated from each other and are associated with large, hollow structures that represent pollen tubes similar to those in living conifers. Cellular megagametophytes and immature embryos are also preserved in some seeds. Williamsonia bockii sp. nov. represents the most recent seed cone of the Williamsoniaceae and is the first anatomically preserved reproductive structure of this family to be discovered in western North America. It reveals new features for the family Williamsoniaceae and allows for the interpretation of several additional facets of reproductive biology in the Bennettitales, particularly pollen tube production, pollination biology, and mode of fertilization.

Additional observations on Rhynchosperma quinnii (Medullosaceae): a permineralized ovule from the Chesterian (Upper Mississippian) Fayetteville Formation of Arkansas

New ovules from the Fayetteville Formation (Upper Mississippian) of Arkansas expand our knowledge of the morphology and anatomy of Rhynchosperma and suggest it was produced by a medullosan seed fern. Rhynchosperma has been described as radially symmetrical with a two-layered integument and vascularization in the integument only. The apical portion of the integument is ribbed; the nucellus is fused to the integument and apically differentiated into a dome-shaped pollen chamber. The vascular system is incompletely known and apparently restricted to the base of the integument. The new specimens are like Rhynchosperma in external shape, size, number of ribs, and numerous histological features. However, new data reveal that the nucellus is vascularized by a sheath of tracheids, the integument is vascularized by discrete bundles, the pollen chamber has a nucellar beak, and the nucellus is attached to the integument for a variable distance from the base. In addition, the integument is tripartite with an elaborate apical region; ribs formed by the integument are more pronounced at the apex; and internally open, hollow lobes form a stellate micropylar canal. The presence of a tripartite integument, the nature of the vascular system, the nucellus-integument attachment, the pollen chamber structure, symmetry, and the association with medullosan vegetative remains suggest medullosan affinity for these ovules and strengthens the evidence for the origin of the family before the end of the Lower Carboniferous.

Oogenesis in the swamp eel Synbranchus marmoratus (Bloch, 1795) (Teleostei; synbranchidae). Ovarian anatomy, stages of oocyte development and micropyle structure

Synbranchus marmoratus (Synbranchidae), commonly known as the swamp eel, is a protogynous diandric teleost fish widely distributed throughout South America. The purpose of this work was to study the ovarian anatomy and to describe oocyte developmental stages in the swamp eel, Synbranchus marmoratus. S. marmoratus has a unique sacular ovary. It is covered by a conspicuous muscular wall, probably involved in an egg-releasing system acting as a peristaltic-like mechanism. The internal ovarian anatomy shows a U-shaped ovarian lamella delimiting a dorsal ovarian lumen. The microscopic study shows evidence of the existence of a germinal epithelium in the inner surface of the lamella, which contains germinal cells, pre-follicular cells and epithelial cells. The complete oogenesis process is divided into four stages: oogonia, primary growth, cortical alveoli and vitellogenesis. Besides, the ovulated oocytes, and atretic structures were described. The structure of the micropyle was studied by scanning electron microscopy (MEB). Near the animal pole the vitelline envelope forms crests that fuse together becoming furrow-like structures with a slightly spiraled direction that converge into the micropyle pit where is located the micropylar canal. Although the sex reversal process of Synbranchids has been subject of many studies, this is the first complete description of the ovarian anatomy and oogenesis.

Sperm storage and motility in the ovary of the marine sculpin Alcichthys alcicornis (Teleostei: Scorpaeniformes), with internal gametic association.

Elkhorn sculpin, Alcichthys alcicornis, is a marine teleost with a unique reproductive mode called "internal gametic association," in which sperm introduced into the ovary by copulation enter the micropylar canal of ovulated eggs in the ovarian cavity, but actual sperm-egg fusion does not occur until the eggs have been released into sea water. It is also known that this fish is a multiple spawner, which spawns at intervals of a few days for one month, and the sperm introduced into the ovary at the beginning of the spawning season retain their fertilizability for the entire period. To clarify how the fertilizability of sperm is maintained internally, the ultrastructure of sperm, the morphological characteristics related to sperm storage in the ovary, and the characteristics of sperm motility were investigated. Mature sperm generally have the normal form of teleost sperm, devoid of acrosomal structures. However, it was found that the midpiece is comparatively elongated and has a compact aggregation of many small-size mitochondria. The intraovarian sperm remained floating in the ovarian fluid throughout the spawning season. The sperm showed high motility in isotonic and weak alkaline solution, containing sodium ions, which was similar to the ovarian fluid of this fish. Sperm continued to move in artificial ovarian fluid for 7-14 days. Considering these results together, it is thought that the intraovarian sperm move throughout the spawning season due to the plentiful energy generated by the many mitochondria.

Changes in the Ovary Related to Pollen Tube Guidance

Pollen tube growth in the ovary is examined, paying particular attention to how changes in the sporophytic tissues of the ovary and ovule relate to pollen tube guidance. Once pollen tubes reach the ovary they have to ‘surf’ along the placenta, reach the ovule exostome, enter the micropyle, traverse the nucellus and enter the embryo sac via a synergid. In peach, as in other Prunus species, pollen tube growth in the ovary is not straight and accelerations and decelerations occur along this path. Likewise, while some pollen tubes follow a definite route and achieve fertilization, others lose their course and navigate in a chaotic way. Developmental changes in the ovary and ovule appear to be responsible for this different pollen tube behaviour. Pollen tubes first stop at the obturator, a placental protuberance that lines the pollen tube pathway towards the ovule. Pollen tube growth is not resumed until this structure enters a secretory phase. A close examination of the integumentary domain at the ovule shows that secretion is also required at that point for successful pollen tube penetration. The external integument cells at the exostome, that look no different to their neighbouring cells at anthesis, enter a secretory phase in older ovules; once again pollen tube penetration is concomitant with production of this secretion. A similar situation occurs in the internal integument cells that line the micropylar canal since pollen tubes also require a secretion to traverse this area. However, the production of these secretions does not occur in all ovules, only in those that retain their starch at the time of pollen tube arrival and, thus, are competent to enter the secretory phase.

Structures contributing to the completion of conifer seed germination

The variety of interpretations of the origin and role of the tissues surrounding the emerging radicle of conifer seeds prompted us to study changes during germination. The structures contributing to the opening of the seed coat and protrusion of the radicle in pine and spruce seeds were examined using light microscopy and field emission scanning electron microscopy. The opening of the seed coat was mainly mechanical and primarily affected by the enlargement of the imbibed endotesta cells lining the micropylar canal. The hypertrophied nucellar apex, swelling remnants and mucous substances in the micropyle further enhanced the testa splitting which was reinforced by the straightening of the folded nucellar cap. The expansion of the imbibed megagametophyte and the embryo were conducive to the seed coat opening only at a later stage. This was followed almost immediately by the appearance of the root cap showing an early geotropic response typical for taproots and assuring an immediate anchoring of the emerged embryo to the ground. The translucent tissue composed of elongating cell rows preceded and surrounded the protruding radicle and derived mainly from two origins: the micropylar end of the megagametophyte and the root cap. Observations about the origin of the sheath clarify the reasons for previous interpretations about the putative role of the massive embryo root cap in testa splitting and germination. The protective and adhesive function of the sheath is concluded to be essential to the conifer embryos, which present the epigeal type of germination.

Ovular secretions in the micropylar canal of larches (Larix kaempferi and L. x eurolepis)

In Larix kaempferi (Lamb.) Carr. and L. × eurolepis A. Henry, a secretion fills the micropylar canal of the ovule during a period of archegonial development that ranges from central cell stage until fertilization. Dissection of the ovuliferous scales caused excess fluid to be exuded from the micropylar canal, forming a drop at the tip of the micropyle. This drop was collected, and its production was quantified. Drop volume was recorded, and the percentage of ovules with drops was counted. The maximum volume of 217 nL far exceeded the volume of the micropyle, which ranged between 18 ± 8 and 28 ± 7 nL (mean ± SD). Removal of drops resulted in further drops being secreted. No drops were produced in ovules that had aborted megagametophyte development.Key words: Larix, micropylar secretions, ovule development.

Micropylar exudates in Douglas fir - timing and volume of production

In Pseudotsuga menziesii, a secretion fills the micropylar canal about 7 weeks postpollination until fertilization. Micropylar volumes were measured and found to show variation. Dissection of the ovuliferous scales caused excess fluid to be exuded from the micropylar canal, forming a drop at the tip of the micropyle. This drop was collected, and its production quantified in three trees. Volume and percentage of ovules with drops were greatest when the archegonia in the female gametophyte were at central cell and/or egg cell stage. The volume of exuded drops far exceeded that of the micropyle. Production of subsequent drops by the ovules further confirms that the fluid is actively produced upon dissection

Studies on fertilization in the teleost. III. The relationship between nuclear behavior and the histone H1 kinase activity in anesthetized medaka eggs

To investigate the mechanisms of fertilization in the teleostean egg, the relationship between the nuclear behavior and the activity of histone H1 kinase was examined in medaka, Oryzias latipes, eggs that were anesthetized at sperm penetration. Inseminated in the anesthetized state, most eggs failed to undergo the propagative waves of increase in cytoplasmic Ca(2+) and exocytosis of cortical alveoli (CABD). The sperm-penetrated eggs that exhibited no or partial CABD only around the animal pole underwent a transient contraction of the cortical cytoplasm toward the animal pole region and were designated nonactivated eggs. Temporary compaction of the second meiotic metaphase (MII) chromosomes was accompanied by contractile movement of the cortical cytoplasm, but not by completion of the second meiotic division. The activity of histone H1 kinase in nonactivated eggs remained high, although it decreased slightly concurrent with sperm penetration. Cyclin B and cdc2 levels remained unchanged as well. The nonactivated eggs began to transform the penetrated sperm nucleus into metaphase chromosomes in the cortical cytoplasm facing the inner end of micropylar canal within 20 min postinsemination (PI). Two figures of typical metaphase chromosomes were found in the animal pole area at </=40 min PI. Chromosome condensation in nonactivated eggs was not inhibited by actinomycin D, nor was the high activity of histone H1 kinase reduced. In the presence of cycloheximide or 6-dimethylaminopurine (6-DMAP), however, the compact sperm nucleus and the MII chromosomes transformed to interphase nuclei without CABD or extrusion of the polar body, although the activity of histone H1 kinase remained high. These results suggest that in the fish egg, transformation of MII chromosomes to an interphase nucleus may not be caused by loss of MPF activity, but rather than by the loss of activity of a short-lived protein kinase(s), sensitive to 6-DMAP that is independent of CABD in the cascade reactions triggered by increased cytoplasmic calcium. Copyright 1999 Wiley-Liss, Inc.

Surface ultrastructure of paddlefish eggs before and after fertilization

The surface ultrastructure of eggs of the paddlefish Polyodon spathula was investigated by scanning electron microscopy. Mature eggs of paddlefish possess four to 12 micropyles in the animal polar region. There are sperm entry sites in the egg surface under the micropyles which consist of tufts of microvilli. Five to nine sperm entry sites were observed on mature eggs. Probably, the number of sperm entry sites corresponds to the number of micropyles. In a few eggs, 1 min after fertilization the ball‐like enlarged top of a cytoplasmic process (probably a full‐grown fertilization cone) had reached the external aperture or the canal of several micropyles. In other micropyles of the same egg, a few smaller cytoplasmic processes or flocculent material were found in the micropylar canal. With one exception, no sperm tails were found there. The formation of the full‐grown cytoplasmic process is possibly initiated before the cortical reaction has started in an area of the animal hemisphere. Three, 10 and 20 min after fertilization, the uneven surface of the cortical cytoplasm in the animal polar region rose gently where microvilli were much less than the in other area and together with a secondary polar body at the latter stage. Taken together, paddlefish eggs may have sperm entry sites corresponding to the number of micropyles and respond to the stimulus of fertilization by forming a few cytoplasmic processes–fertilization cones (larger and smaller). Sperm penetration into the egg may be achieved at an earlier stage of fertilization (sperm‐egg contact), as inferred from the fact that a secondary polar body was formed at the 20‐min stage irrespective of the exceptional finding of the sperm tail.