Starfish sperm-oocyte jelly binding triggers functional changes in cortical granules. A study using acid phosphatase and ruthenium red ultrastructural histochemistry

Starfish oocytes were examined before fertilization, immediately after insemination, and during the cortical reaction by means of acid phosphatase and ruthenium red ultrastructural histochemistry. Oocyte cortical granules are composed of a lamellar body and a surrounding matrix which is subdivided into dense and light portions. In unfertilized oocytes cortical granules are not stained by ruthenium red but show a weak acid phosphatase activity in the light portion of the granule matrix. Immediately after the adhesion of the spermatozoon to the oocyte jelly coat, the light matrix portion of cortical granules appears stained by ruthenium red and shows a strong acid phosphatase activity. During the cortical reaction, cortical granules are released into the perivitelline space and the lamellar body, surrounded by the stained matrix, fuses with the fertilization envelope. Our data suggest that membrane permeability changes and enzyme activation occur in the egg when the spermatozoon binds to the oocyte jelly coat.     

Starfish sperm-oocyte jelly binding triggers functional changes in cortical granules

Summary Starfish oocytes were examined before fertilization, immediately after insemination, and during the cortical reaction by means of acid phosphatase and ruthenium red ultrastructural histochemistry. Oocyte cortical granules are composed of a lamellar body and a surrounding matrix which is subdivided into dense and light portions. In unfertilized oocytes cortical granules are not stained by ruthenium red but show a weak acid phosphatase activity in the light portion of the granule matrix. Immediately after the adhesion of the spermatozoon to the oocyte jelly coat, the light matrix portion of cortical granules appears stained by ruthenium red and shows a strong acid phosphatase activity. During the cortical reaction, cortical granules are released into the perivitelline space and the lamellar body, surrounded by the stained matrix, fuses with the fertilization envelope. Our data suggest that membrane permeability changes and enzyme activation occur in the egg when the spermatozoon binds to the oocyte jelly coat.     

Ultrastructure comparée de la spermiogenèse et du spermatozoïde de trois espèces de Paravortex (Rhabdocoela, "Dalyellioida", Graffillidae), Turbellariés parasites intestinaux de Mollusques

Three species of the genus Paravortex were studied. Spermiogenesis involves progressive lengthening of the spermatid. No centriole nor axoneme were found. Thc mature spermatozoon is threadlike, 30μm long and aflagellate. It contains a nucleus, two longitudinal parallel mitochondria, numerous dense bodies limited by a membrane, a row of cortical microtubules and granules. The spatial arrangement of the microtubules wah deduced from a statistical analysis of their number in transverse section. The microtubules are continuous along the length of the spermatozoon and are arranged along a long cone. The granules, not limited by a membrane, are about 25 nm in diameter. They are arranged in a semicircle and represent a new structure for sperm of Platyhclminthes. A statistical comparison demonstrated that two species may be distinguished by the number of their cortical sperm microtubules. This is the tirst ultrastructural study of an aflagellate spermatozoon in the Rhabdocoela. A rickettsia was found in P. cardii .     

THE SURFACE EVENTS AT FERTILIZATION OF THE SEA URCHIN EGG I. EVENTS ON THE SURFACE OF THE VITELLINE COAT1

Sperm-egg interaction during normal fertilization in the sea urchins, Strongylocentrotus intermedius and Hemicentrotus pulcherrimus, was studied by scanning and transmission electron microscopy. Several seconds after insemination, acrosome-reacted spermatozoa were found attached to the surface of the vitelline coat on each egg. Soon, several bulges of the vitelline coat appeared surrounding the fertilizing spermatozoon. These bulges then spread over the surface increasing in number, while they became fewer and disappeared around the sperm head. Thin sections of the bulging areas revealed discharging cortical granules. As the bulging vitelline coat was elevated, the sperm head was incorporated into the perivitelline space, passing through a small hole in the coat that resulted from penetration of the sperm acrosomal process immediately before fusion of the gametes. When the spermatozoon disappeared beneath the fertilization membrane, a hole was left in the membrane and the cortical reaction had finished on the other hemispheric surface. Mechanical removal of the membrane at that time exposed a spermatozoon protruding perpendicularly from the egg plasma membrane surface. The anterior tip of the sperm head was smoothly connected with the egg surface, and neither microvillous projections nor cytoplasmic covering of the egg cytoplasm could be found around the spermatozoon.     

The penetration of the spermatozoon through the sea urchin egg surface at fertilization : Observations from the outside on whole eggs and from the inside on isolated surfaces

The external and cytoplasmic surfaces of the sea urchin egg at fertilization have been examined with the scanning electron microscope (SEM). The outside events were documented by glueing eggs to polylysine coated glass plates, adding sperm and fixing rapidly. To reveal the inner aspects of the surface as the sperm travels through it to reach the egg cytoplasm, the fertilized egg surface was isolated in 0.3 M KC1, 0.35 M glycine, 2 mM MgCl₂, 2 mM EGTA, pH 7.5, glued onto a polylysine-coated plate and processed for the SEM. The events of spermatozoon attachment, membrane fusion, sperm entry, rotation and detachment into the egg cytoplasm as well as the associated cortical changes are described. The egg cortex is revealed to be a uniform network of fibrous bundles.The spermatozoon initially attaches to the egg surface by the acrosomal filament. As membrane fusion occurs between the gametes, the plasma membrane of the egg engulfs the sperm, the cortical granules start to discharge and a spreading surface deformation, possibly caused by a cortical contraction, is initiated. The perpendicularly entering spermatozoon is surrounded by a cluster of elongate microvilli which appear to have 235 nm vesicles associated with their bases. The sperm is prevented by the cortex from directly entering the egg cytoplasm and lies upon the egg surface between the plasma membrane and the matrix of cortical fibers. It is subsequently rotated additionally to enter the egg cytoplasm with the posterior end first. A scar is left in the cortex where the spermatozoon penetrated. The egg cortex is shown to consist of 50–200 nm uniformly arranged fibers, and its thickness ranges from 0.2 to 0.5 μm. It is speculated that this structure may be contractile.     

Spermatozoon ultrastructure of Aponurus laguncula (Digenea: Lecithasteridae), a parasite of Aluterus monoceros (Pisces: Teleostei)

The mature spermatozoon of Aponurus laguncula, a parasite of the unicorn leatherjacket Aluterus monoceros, was studied by transmission electron microscopy. The spermatozoon possesses 2 axonemes of the 9 + “1” trepaxonematan pattern, attachment zones, a nucleus, a mitochondrion, external ornamentation of the plasma membrane and cortical microtubules. The major features are the presence of: 1) external ornamentation in the anterior part of the spermatozoon not associated with cortical microtubules; 2) one mitochondrion; and 3) cortical microtubules arranged as a single field in the ventral side. The maximum number of microtubules is in the nuclear region. The extremities of the axonemes are characterized by the disappearance of the central core and the presence of microtubule doublets or singlets. This study is the first undertaken with a member of the Lecithasteridae and exemplifies the sperm ultrastructure for the superfamily Hemiuroidea.     

The ultrastructural characters of the mature spermatozoon of Opechona bacillaris (Molin, 1859) (Digenea,Lepocreadiidae) a parasite of Scomber colias Gmelin, 1789 (Scombridae) off the coast of Dakar (Senegal)

This study presents the ultrastructure of the mature spermatozoon of Opechona bacillaris, a digenean belonging to the family Lepocreadiidae. The sperm cell of O. bacillaris exhibits the general pattern described in most of the Lepocreadioidea: two axonemes of the 9 + ‘1’ pattern of the Trepaxonemata, mitochondria, a cortical mitochondrion, a nucleus, electron‐dense material in the anterior extremity of the spermatozoon, external ornamentation of the plasma membrane with associated spinelike bodies, and granules of glycogen. However, particularities of O. bacillaris are the simultaneous presence in the anterior extremity of the spermatozoon of the electron‐dense material, a mitochondrion, and the absence of cortical microtubules. In the Lepocreadiidae, we describe for the first time in O. bacillaris spinelike bodies associated with the external ornamentation of the plasma membrane and two mitochondria. The first mitochondrion is moniliform and composed of a mitochondrial cord with joined mitochondrial bulges. The second mitochondrion shows a regular form. The posterior tip of the spermatozoon has only singlets to owing to the disorganization of the second axoneme and granules of glycogen as occurs in Hypocreadium caputvadum, the other studied species of the family Lepocreadiidae.     

Sperm characters of the digenean Nephrotrema truncatum (Troglotrematidae): a kidney parasite of Crocidura russula (Soricidae) and their phylogenetic significance

ABSTRACT

Spermatological characteristics of the troglotrematid digenean Nephrotrema truncatum, a parasite of the shrew Crocidura russula, have been investigated by means of transmission electron microscopy. The ultrastructural study reveals that the mature spermatozoon of N. truncatum exhibits many ultrastructural characters previously described in most gorgoderoideans. These are two axonemes of the 9+‘1‘ trepaxonematan pattern, four attachment zones, a lateral expansion, an external ornamentation of the plasma membrane associated with spine-like bodies and cortical microtubules, and in the posterior part of the anterior spermatozoon region, two bundles of parallel cortical microtubules with the maximum number located in the anterior part of the spermatozoon, a nucleus, two mitochondria, and granules of glycogen. The obtained results are compared with those of other digeneans, particularly the Gorgoderoidea. The sperm cells gorgoderoideans are of type IV, characterised by a 9+‘1‘ pattern of axonemes, the presence of an external ornamentation associated with cortical microtubules and located in the posterior area of the anterior extremity, the presence of two bundles of cortical microtubules, the maximum number of cortical microtubules located in the anterior region of the spermatozoon, and the presence of generally two mitochondria. However, dicrocoeliids and troglotrematids have spermatozoa with ornamentation of the plasma membrane and lateral expansions.     

Fertilization in a crab. II. Cytological aspects of the cortical reaction and fertilization envelope elaboration

At fertilization, the egg of Carcinus maenas undergoes cortical vesicle exocytosis, in response to the first contacts between the spermatozoon and the egg plasma membrane. This process was observed in vitro and may be connected with a cortical reaction. Carcinus maenas eggs display two populations of cortical vesicles which, during the reaction, successively release two different exudates: a fine granular material and a mass of ring-shaped granules. During the first steps of exocytosis, the two superimposed vitelline envelopes are detached from the egg surface, and the inner one gradually changes. Thus a new coating, derived from the coalescence of the secreted ring-shaped granules, is progressively elaborated under the vitelline envelopes. These events occur over a 7–8 hr period. The morphological uniqueness of the cortical vesicle exudates and the complexity of the related events are discussed in terms of the cortical reaction and of the formation of the fertilization envelope in Carcinus maenas.     

The effect of nicotine on sperm-Egg interaction in the sea urchin: Polyspermy and electrical events

We have studied some of the effects of nicotine on sea urchin eggs, spermatozoa, and their interaction using electrical recording techniques and fertilization-rate experiments. Pretreating eggs with nicotine enhances the fertilization rate, whereas this drug has an inhibitory effect on spermatozoa. Pulse-treated eggs or eggs fertilized in the presence of nicotine give rise to attenuated step depolarizations, which may be attributed to a decrease in membrane resistance (Rm) of the egg or, in the latter case, to an alteration to the spermatozoon. Concurrently, with the change in the step depolarization there is a reduction in amplitude of the fertilization potential (FP) suggesting that the cortical reaction is in some way altered. Nicotine has no effect on the Rm of fertilized eggs or oocytes, where there are no cortical granules. We suggest that nicotine alters the cortex of sea urchin eggs–possibly by causing a partial dissolution of cortical granules–which renders the eggs more receptive to spermatozoa. The reductions in amplitude of the step depolarization and the FP are consequences of this alteration.     

Ultrastructural Observations on the Spermatozoon,Oocyte and Fertilization Process in Gonapodasmius,a Gonochoristic Trematode (Trematoda: Digenea: Didymozoidae)

Observations were performed in the uterus of a female Gonapodasmius sp., a gonochoristic didymozoid Trematode. The oocyte is a round cell 6 μm in diameter, which shows a ‘nucleolus-like cytoplasmic body’ and cortical granules. The spermatozoon is filiform, mobile and about 50 μm long. There is no acrosome. The anterior tip of the spermatozoon contains two centrioles made up of singlets and cortical microtubules with associated glycocalyx. The centrioles are continued as two axonemes of the classical 9 + ‘1’ pattern of flatworms, accompanied by a mitochondrion and a short row of cortical longitudinal microtubules. It is the posterior part of the sperm cell which contains the nucleus. At the outset of fertilization, the anterior part of the spermatozoon coils around the oocyte and penetrates it by lateral fusion. The posterior region of the spermatozoon, with the nucleus, is the last part to enter the oocyte, after passing through a perforation in the forming eggshell. The whole spermatozoon thus penetrates the female cell.     

Ultrastructure of the spermatozoon of Bothriocotyle sp. (Cestoda: Bothriocephalidea), a parasite of Schedophilus velaini (Sauvage, 1879) (Perciformes: Centrolophidae) in Senegal

The mature spermatozoon of Bothriocotyle sp. is filiform and tapered at both extremities. It possesses 2 axonemes of unequal length, showing the 9 + "1" pattern of Trepaxonemata. The anterior extremity exhibits a crest-like body. Thereafter, the crest-like body disappears, and the first axoneme is surrounded by a ring of cortical microtubules (about 27 units) that persist until the appearance of the second axoneme. This ring of cortical microtubules is characteristic only for species of Bothriocephalidea and represents a very useful phylogenetic character. The spermatozoon cytoplasm is slightly electron-dense and contains numerous electron-dense granules of glycogen in several regions. The anterior and posterior extremities of the spermatozoon lack cortical microtubules. The posterior extremity of the spermatozoon of Bothriocotyle sp. possesses a nucleus and a disorganized axoneme, which also characterizes spermatozoa of the Echinophallidae studied to date.     

Ultrastructural study of the male gamete of Glossobothrium sp. (Cestoda: Bothriocephalidea: Triaenophoridae) a parasite of Schedophilus velaini (Perciformes: Centrolophidae) in Senegal

This paper describes the ultrastructure of the male gamete of Glossobothrium sp. (Bothriocephalidea: Triaenophoridae). The mature spermatozoon of Glossobothrium sp. is filiform and possesses two axonemes, a single helicoidal crested body, a parallel nucleus, parallel cortical microtubules and granules of glycogen. In Glossobothrium sp. we describe for first time a 200-250 nm thick crest-like body in the Bothriocephalidean. The anterior part of the spermatozoon exhibits a ring of 27 electron-dense cortical microtubules encircling the first axoneme. This structure persists until the appearance of the second axoneme. When the ring of electron-dense cortical microtubules disappears, the spermatozoon exhibits two bundles of thin cortical microtubules. The posterior part of the spermatozoon contains the posterior extremity of the second axoneme, the posterior extremity of the nucleus and few cortical microtubules. Soon nucleus disappears and the axoneme is disorganized. Thus the posterior extremity of the spermatozoon of Glossobothrium sp. exhibits only singlets produced by the disorganization of the doublets of the second axoneme and few cortical microtubules. This type of posterior extremity of the mature spermatozoon has never been described previously in the Triaenophoridae.     

Ultrastructural study of the male gamete of Pleurogonius truncatus Prudhoe, 1944 (Platyhelminthes, Digenea, Pronocephalidae) parasite of Eretmochelys imbricata (Linnaeus, 1766)

In Pronocephaloidea, the spermatozoa of only two species have been studied today. Because of this, we present in this work data concerning to a third specie, Pleurogonius truncatus Prudhoe, 1944. The mature spermatozoon of P. truncatus possesses two axonemes with the 9+"1" pattern typical of Trepaxonemata, mitochondrion, nucleus, parallel cortical microtubules, spinelike bodies, cytoplasmic expansion and an external ornamentation of the plasma membrane. A particularity of the spermatozoon of P. truncatus is in the ultrastructure of the anterior spermatozoon extremity with only cortical microtubules and ornamentation of the plasma membrane. This type of anterior extremity has never been described until today in Pronocephaloidea. On the other hand, the ultrastructure of the posterior extremity of the spermatozoon confirms that already described in Pronocephalidae.     

Ultrastructure of spermiogenesis and the spermatozoon of Mesocestoides litteratus (Cestoda, Mesocestoididae).

This paper constitutes the first ultrastructural study of spermiogenesis and the spermatozoon of a cestode belonging to the family Mesocestoididae, Mesocestoides litteratus. Spermiogenesis in M. litteratus is characterised by a flagellar rotation and a proximodistal fusion. The zone of differentiation presents striated roots associated with the centrioles and also an intercentriolar body. The most interesting ultrastructural feature found in the mature spermatozoon of M. litteratus is the presence of parallel cortical microtubules. The spermatozoon also exhibits a single crest-like body and granules of glycogen. The pattern of spermiogenesis and the parallel position of cortical microtubules reveal the lack of concordance between M. litteratus and cyclophyllidean species studied to date in spermiogenesis and in the ultrastructural organisation of spermatozoon. This study provides new spermatological data and calls into question the validity of the current systematic position of mesocestoidids.     

Ultrastructure of the spermatozoon of Centroderma spinosissima (Stossich, 1886) (Digenea: Mesometridae) and its phylogenetic potential

The Mesometridae includes only five genera and eight species. The available data on the ultrastructure of sperm cells of mesometrid species referred to two species only, Elstia stossichianum and Wardula capitellata. The present study revealed the ultrastructure of the spermatozoon of a third genus and third species of Mesometridae, Centroderma spinosissima. The mature spermatozoon of C. spinosissima presents two axonemes with different lengths of the Ehlers’ 9 + ‘1’ trepaxonematan pattern, a nucleus, two mitochondria, two bundles of parallel cortical microtubules, external ornamentation of the plasma membrane, a lateral expansion, spine-like bodies, cytoplasmic ornamented buttons and granules of glycogen. The spermatozoon of C. spinosissima is similar to those of the previously studied mesometrids. However, some peculiarities such as the presence of two mitochondria, the disposition of the external ornamentation of the plasma membrane and the morphology of the posterior spermatozoon extremity, characterize the male gamete of C. spinosissima. Moreover, the presence of cytoplasmic ornamented buttons is a characteristic found only in the mature spermatozoon of mesometrids and it probably represents an autapomorphy for this family.     

Ultrastructural characters of the spermatozoon of the digenean Hypocreadium caputvadum Kacem et al., 2011 (Lepocreadioidea: Lepocreadiidae), an intestinal parasite of Balistes capriscus in Tunisia

The ultrastructural organization of the spermatozoon of the digenean Hypocreadium caputvadum (Lepocreadioidea: Lepocreadiidae) is described. Live digeneans were collected from Balistes capriscus (Teleostei: Balistidae) from the Gulf of Gabès, Tunisia (Eastern Mediterranean Sea). The mature spermatozoon of H. caputvadum shows several ultrastructural characters such as two axonemes of different lengths exhibiting the classical 9 + “1” trepaxonematan pattern, a nucleus, two mitochondria, granules of glycogen, external ornamentation of the plasma membrane and two bundles of parallel cortical microtubules. Moreover, in the anterior extremity, the second axoneme is partly surrounded by a discontinuous and submembranous layer of electron-dense material. Our study provides new data on the spermatozoon of H. caputvadum in order to improve the understanding of phylogenetic relationships in the Digenea, particularly in the superfamily Lepocreadioidea. In this context, the electron-dense material surrounding one of the axonemes in the anterior spermatozoon extremity constitutes the unique distinguishing ultrastructural character of lepocreadioideans, and it is present in spermatozoa of lepocreadiids, aephnidiogenids and gyliauchenids.     

Spermiogenesis and the spermatozoon ultrastructure of Robphildollfusium fractum (Digenea: Gyliauchenidae), an intestinal parasite of Sarpa salpa (Pisces: Teleostei)

Spermiogenesis in Robphildollfusium fractum begins with the formation of a differentiation zone containing: two centrioles, each bearing striated rootlets, nucleus, several mitochondria and an intercentriolar body constituted by seven electron-dense layers. The two centrioles originate two free flagella growing orthogonally to the median cytoplasmic process. Later, the free flagella rotate and undergo proximodistal fusion with the median cytoplasmic process. Nuclear and mitochondrial migrations occur before this proximodistal fusion. Finally, the young spermatozoon detaches from the residual cytoplasm after the constriction of the ring of arched membranes. The spermatozoon of R. fractum exhibits two axonemes of different length of the 9 + ‘1’ trepaxonematan pattern, nucleus, two mitochondria, two bundles of parallel cortical microtubules, external ornamentation of the plasma membrane, spine-like bodies and granules of glycogen. Additionally, a shorter axoneme, which does not reach the nuclear region, the presence of an electron-dense material in the anterior spermatozoon extremity and the morphologies of both spermatozoon extremities characterize the mature sperm of R. fractum.     

Ultrastructure of the spermatozoon of Anomotaenia quelea (Mettrick, 1961) (Cestoda,Cyclophyllidea, Dilepididae), an intestinal parasite of Quelea quelea (Aves,Ploceidae) in Senegal

The mature spermatozoon of Anomotaenia quelea exhibits an apical cone of electron-dense material and two helicoidal crest-like bodies. The apical cone near its base is surrounded by a lucent cytoplasm and a spiraled layer of cortical microtubules. The crest-like bodies are of different lengths, spiraled and make an angle of 30–40° to the hypothetical spermatozoon axis. The axoneme is of the 9 + ‘1’ trepaxonematan pattern and is surrounded by a periaxonemal sheath of electron-dense material. The cytoplasm contains in regions III and IV numerous electron-dense granules situated between the periaxonemal sheath and the cortical microtubules. The posterior extremity of the spermatozoon of A. quelea exhibits a nucleus and a disorganized axoneme and cortical microtubules. This type of posterior extremity of the mature spermatozoon has never been described previously in a Dilepididae. Similarly, two crest-like bodies have not been observed before in a dilepidid cestode.     

Ultrastructure of the spermatozoon of Anomotaenia quelea (Mettrick, 1961) (Cestoda,Cyclophyllidea, Dilepididae), an intestinal parasite of Quelea quelea (Aves,Ploceidae) in Senegal

The mature spermatozoon of Anomotaenia quelea exhibits an apical cone of electron-dense material and two helicoidal crest-like bodies. The apical cone near its base is surrounded by a lucent cytoplasm and a spiraled layer of cortical microtubules. The crest-like bodies are of different lengths, spiraled and make an angle of 30–40° to the hypothetical spermatozoon axis. The axoneme is of the 9 + ‘1’ trepaxonematan pattern and is surrounded by a periaxonemal sheath of electron-dense material. The cytoplasm contains in regions III and IV numerous electron-dense granules situated between the periaxonemal sheath and the cortical microtubules. The posterior extremity of the spermatozoon of A. quelea exhibits a nucleus and a disorganized axoneme and cortical microtubules. This type of posterior extremity of the mature spermatozoon has never been described previously in a Dilepididae. Similarly, two crest-like bodies have not been observed before in a dilepidid cestode.