Ultrastructure and Phylogenetic Significance of Notaspidean Spermatozoa (Mollusca, Gastropoda, Opisthobranchia)

Spermatozoa of five notaspidean opisthobranchs [Berthellina citrina, Berthella ornata, Pleuro-branchus peroni, Pleurobranchaea maculata, Umbruculum sinicum] were examined using TEM. In all five species, the acrosome (sensu lato) consists of an apical vesicle (the acrosomal vesicle) and acrosomal pedestal. The acrosomal pedestal overlaps the nuclear apex, and in P. peroni (and possibly B. ornata) is periodically banded—-the first reported incidence of this type of substructure in any euthyneuran acrosome. Although sperm nuclei of P. peroni, B. ornata and B. citrina differ in length and also the number of keels present (nucleus 7 μm long with four/five keels present in Pleurobranchus; 17 μm long with one keel in Berthella; 15 μm long with a very weak keel in Berthellina), the basal invagination to which the centriolar derivative, axoneme and coarse fibres are attached is always poorly developed, and very little overlap between nucleus and midpiece occurs. In P. maculata and U. sinicum, the nucleus forms a helical cord around the axoneme and mitochondrial derivative such that it is not possible to recognize exclusively ‘nuclear’ and ‘midpiece’ regions of the spermatozoon. In all notaspideans investigated, (1) the axoneme, coarse fibres and glycogen helix are enclosed by the paracrystalline and matrix components of the mitochondrial derivative and (2) a dense ring structure (attached to the plasma membrane) and glycogen piece are observed. While the glycogen piece is very short (0.85–1.43 μm) with a very degenerate axoneme in B. citrina, B. ornata and P. peroni, this region of the spermatozoan is well developed (30–35 μm long) in U. sinicum and exhibits a fully intact 9 + 2 axoneme. The ‘glycogen piece’(or its presumed homologue) in P. maculata spermatozoa is very short (0.65 μm), devoid of any axonemal remnant and constructed of a hollow, internal cylinder attached to an outer (incomplete) shell, and contains scattered (glycogen) granules. Spermatozoal structure supports a close relationship between the genera Berthellina, Berthella and Pleurobranchus. These three genera have more distant links with Pleurobranchaea, while Umbraculum maintains an isolated, specialized position within the Notaspidea.     

Spermatozoon structure and spermiogenesis in Nassarius kraussianus (Gastropoda,Prosobranchia, Nassariinae)

Summary

The testis of Nassarius kraussianus (Nassariinae) produces two types of spermatozoa, a motile euspermatozoon and a non-motile paraspermatozoon. The euspermatozoon is filiform and about 95/μm long. The elongated head (40 μm long) is comprised of a slender nucleus (about 0.5 μm diameter) which is penetrated throughout by an intranuclear canal housing the anterior portion of the axoneme. A short (about 2 μm long) conical acrosome surmounts the nucleus anteriorly. The mid-piece (23 μm in length) consists of six to seven modified mitochondria which are helically arranged around the axoneme. Posterior to the mid-piece the tail is composed of a short glycogen piece and an end piece. The paraspermatozoon is spindle-shaped (about 50 μm long) and contains multiple (16–20) axonemes the basal bodies of which fuse anteriorly. Posteriorly, numerous small mitochondria and electron-dense bodies lie between the axonemes. Structural changes during eu- and paraspermiogenesis mirror those described for other species of gastropod mollusc with dimorphic spermatozoa. However unlike other molluscs, the cytoplasmic bridges which connect developing spermatids contain well developed stacks of endoplasmic reticulum which form a continuum with that in the cytoplasm of the spermatids. These structures may in some way facilitate the synchronous development of the spermatozoa.     

Ultrastructure of the sperm and spermatogenesis and spermiogenesis of Dina lineata (hirudinea,erpobdellidae)

The mature sperm of Dina lineata is of the modified type. The sperm are 48 μm long and 0.3 μm wide. The sperm are filiform and helicoidal cells with a distinct head, a midpiece, and a tail. There are two distinct regions in the head: the acrosome and the posterior acrosome, each with its own characteristic morphology. The midpiece is the mitochondrial region and has a single mitochondrion. Two distinct portions can be observed in the tail: the axonematic region and the terminal piece. In the process of spermatogenesis the early spermatogonia divide to form a poliplast of 512 spermatic cells. In the spermiogenesis the following sequential stages can be distinguished: elongation of the flagellum; reciprocal migration of mitochondria and Golgi complex; condensation of chromatin and formation of the posterior acrosome; spiralization of nuclear and mitochondrial regions; and, finally, formation of the anterior acrosome. The extreme morphological complexity of the Dina spermatozoon is related to the peculiar hypodermal fertilization which characterizes the erpobdellid family. Correlation between sperm morphology and fertilization biology in the Annelida is revised.     

Spermatogenesis in the archaic hydrothermal vent bivalve,Bathypecten vulcani,and comparison of spermatozoon ultrastructure with littoral pectinids

Summary

Bathypecten vulcani is considered a relict species from the Paleozoic, based on shell characteristics such as the presence of calcite prisms. To date, it is the only pectinid species reported from hydrothermal ecosystems. Histological and ultrastructural studies show that spermatogenesis is identical to that of littoral pectinids. The spermatozoon has a 2.7 μm long pyriform head and a 40 μm flagellum. The four mitochondria of the mid-piece are about 1.2 μm in diameter. The nucleus contains dense chromatin fibres and possesses a wide, shallow (0.1 μm) anterior fossa and a narrow, deeper (0.2 μm) posterior nuclear fossa. Comparison of the ultrastructural characteristics of the spermatozoon of B. vulcani with those of littoral pectinids shows that they can be used as a diagnostic feature of this species. In particular, its acrosome characters will be a useful complement to the shell characters in the study of the phylogenetic position of this species in relation to other pectinids.     

An ultrastructural analysis of the mature spermatozoon of Anguispira alternata (say) (pulmonata,stylommatophora)

Mature spermatozoa from the hermaphroditic duct of adult snails were examined using various techniques of light microscopy as well as scanning and transmission electron microscopy. The sperm are approximately 557 μm in length including a dextrally spiral head approximately 13 μm long. The head consists of an electron-dense nucleus sculptured into a double-ridged spiral and an acrosome projecting approximately 0.45 μm beyond the apex of the nucleus. The acrosome consists of a membrane-bound vesicle approximately 0.1 μm in diameter and a column of homogeneous material which extends along one side of the terminal spiral of the nucleus. This material is separated from the nucleus by the nuclear envelope. The neck region, though similar to that found in other pulmonates, possesses a unique coiled structure surrounding the central doublet of the axoneme. The midpiece axoneme possesses a 9+9+2 configuration anteriorly grading into a 9+2 pattern for the majority of its length. There are three mitochondrial helices – one primary and two secondary – in the midpiece. Only the primary helix persists throughout the midpiece.     

Ultrastructure of Spermatozoa and Spermatids in Bonellia viridis and Hamingia arctica (Echiura) With Some Phylogenetic Considerations

Spermatozoa of the echiurans Bonellia viridis and Hamingia arctica show a similar ultrastructure. They are of a modified type. The head consists of a roughly cylindrical nucleus, which has a cover of electron-dense material. The acrosome is very large and consists of an acrosomal vesicle and a rod-shaped perforatorium or acrosomal rod. In close association with the nucleus, one or two mitochondria are found forming an irregular ring around the posterior tip of the nucleus and the centriolar apparatus. There are two centrioles, the proximal one with the conventional triplet microtubular structure. The tail flagellum is about 50 μm long and has the 9+2 axonemal structure. The oblique attachment of the acrosome to the anterior part of the nucleus gives the spermatozoon a bilateral symmetry. However, in the nuclear morphology, the arrangement of electron-dense material around the nucleus, in the mitochondria, and in the attachment of the tail flagellum, the spermatozoon shows asymmetric organization. The sperm structure in bonelliids is unique but its genesis and the morphology of the mitochondrial midpiece support the theory that the echiurans are related to the annelids. The main results of the study are summarized in Fig. 11.     

Chromatin condensation and acrosome development during spermiogenesis of Ensis ensis (Mollusca,Bivalvia)

We describe chromatin condensation and acrosome development during spermiogenesis of Ensis ensis. The overall shape of the mature spermatozoon corresponds to the primitive type. The nucleus is oval and on its superior pole there is an elongated acrosome; the middle piece contains four mitochondria around the centriolar complex. The condensation of the nuclei seems to occur in three steps: first the diameter of chromatin fibers increases slightly from 17 to 20 nm; second, in midspermatids fiber pairs coalesce; and third, the coalescence continues by addition of other fibers until the nuclei become highly compacted. Chromatin changes are related with nuclear protein composition. Small proacrosomal vesicles show two regions of different electron density. At a later stage they fuse to give a single, spherical vesicle in round spermatids, which migrates to the upper pole and transforms into a tapered acrosome (18 μm long) with a central channel filled with finely fibrous material. © 1994 Wiley-Liss, Inc.     

Spermiogenesis and spermatozoa ultrastructure of Aonides oxycephala (Annelida: Spionidae) from the Sea of Japan

Adults of Aonides oxycephala, common inhabitants of shallow boreal waters in the Atlantic and Pacific Oceans, release gametes into the water where fertilization and lecithotrophic larval development occur. During spermiogenesis, the acrosomal vesicle migrates from the posterior to the anterior end of the spermatid and the number of mitochondria reduces from six in early spermatids to four in mature spermatozoa. Each spermatozoon has an ovoid head with the acrosome 1.4?±?0.1?µm long and 1.6?±?0.1?µm wide and the nucleus 1.7?±?0.1?µm long and 2.3?±?0.1?µm in diameter, four spherical mitochondria, two centrioles oriented perpendicular to each other, putative glycogen in the shape of dense granules in the midpiece, and a flagellum with 9?×?2?+?2 organization of microtubules. The acrosome is a complex heterogeneous structure with five ordered layers of different electron densities, lying in a shallow depression on the anterior end of the nucleus. The nucleus is barrel-shaped (truncated ovoid) with the centriolar fossa housing the distal and proximal centrioles. Spermiogenesis and ultrastructure of spermatozoa of A. oxycephala are similar to those of another free spawning spionid, Marenzelleria viridis. Aonides and Marenzelleria have not, however, been considered as closely related taxa; thus, similarity in the morphology of their sperm might result from convergence or parallelism.     

Complex spermatozoon of the live-bearing half-beak,Hemirhamphodon pogonognathus (Bleeker): Ultrastructural description (Euteleostei,Atherinomorpha, Beloniformes)

The spermatozoon of Hemirhamphodon pogonognalhus shows modifications that are frequent though not obligate in internally fertilizing sperm, notably elongation of the nucleus and extension of the mitochondria of the midpiece as an elongate sheath around the proximal region of the axoneme. These similarities to poecilid and jenynsid sperm are considered homoplasic. As in the mature sperm of all but one investigated teleost, an acrosome is absent. The elongate, blade-shaped, electron-dense nucleus has a mean length of 3.2 μm; its basal implantation fossa, less than one-tenth of the length of the nucleus, houses the anterior half of the distal and only centriole (of triplet construction with satellite rays), a centriolar plug, and a mass connecting the centriole to the wall of the fossa. A unilateral putative centriole adjunct is present. The anterior region of the axoneme is surrounded by a mitochondrial sleeve, and internal to this, separated by a cisterna, by a submitochondrial sleeve. The mitochondrial sleeve unites posteriorly with the submitochondrial sleeve. Between the submitochondrial sleeve and the axoneme is a space, the cytoplasmic canal, that is open to the exterior posteriorly. The discrete, cristate mitochondria, in their sleeve, are unique in investigated atherinomorph sperm in being bilateral, grouped on only two opposing sides of the axoneme, with an arc-shaped ‘intermitochondrial link’ between. The 9 + 2 flagellum is unique for the Animalia in having 23 radial subplasmalemmal rods, repeated longitudinally (periodicity 0.025 pm) in a quasicrystalline array. Internal fertilization is deduced to have arisen in the Exocoetoidei independently of that in the Cyprinidcntiformes.     

Formation of the cylindrical structure during the acrosome reaction of abalone spermatozoa

Spermatozoa of abalone Haliotis discus were examined before and during the acrosome reaction with special regard to one of the newly formed structures: a cylindrical structure surrounding a part of the elongated acrosomal process near the opening of the acrosomal vesicle. The structure, about 0.2 μm in diameter and about 1 μm in length, was revealed to be composed of a tightly coiled, fine tubular structure about 20 nm in diameter. In the course of the acrosome reaction, a triple-spiral structure appeared in the anterior part of the acrosomal vesicle. Since this spiral structure was also composed of a tightly coiled 20 nm tubule(s), it was concluded that this structure was transformed into the single-walled cylindrical structure by simple stretching in the direction of its longitudinal axis. In the clumps of spermatozoa that underwent acrosome reaction in suspension, the cylindrical structures were frequently found in contact with each other and/or other structures, indicating that they are very sticky.     

Secretion patterns and effect of prostate-derived granules on the sperm acrosome reaction of rabbit buck

There is increasing evidence that the particulate fraction of seminal plasma plays an important role in reproduction of several mammalian species. However, the origin and role of these granules in the physiology of rabbit spermatozoa is partially unknown. The aim of this study was to investigate the implication of prostate gland in the production and secretion of granules into the rabbit semen and the role of prostate-derived granules in the sperm acrosome reaction. Light and electron microscopy of the prostate gland showed that the anterior and middle tracts of the prostate (namely the proprostate and prostate, respectively) are chiefly implicated in the secretion of granules of different size: smaller granules (SG; 0.5 μm) and large granules (LG; 4 μm). Two major patterns of secretion were identified, based on electron microscope views: storage granules (large granules) seem to empty inner smaller granules directly into the duct by exocytosis, or the storage vesicle itself is released in toto into the ducts (diacytosis). In vitro experiments using granules from vasectomized rabbits, to exclude testicular origin of granules, showed that granules reduce the acrosome reaction of Percoll-selected spermatozoa, independently of the size. Interestingly, spermatozoa incubated with heat-treated granules showed a higher sperm acrosome reaction rate, suggesting a potential role of granule-derived proteins in this process. Inhibition of the acrosome reaction is a crucial event in rabbit reproduction; ejaculated spermatozoa have to wait for a long time (8-16 h) for egg availability in the female tract after mating. Taking together, our results demonstrate that prostate granules secreted either by exocytosis or diacytosis can preserve spermatozoa fertilizing ability, by preventing sperm acrosome reaction. The type of granule-derived proteins or other macromolecules implicated in this process should be further investigated.     

Spermiogenesis in the Oligochaetoid Polychaete Questa (Annelida, Questidae)

The spermatids are connected to a central cytophore by cytoplasmic bridges and are polarized in the sequence: "empty cytoplasm"; uncondensed nucleus; mitochondria which surround the distal region of the nucleus and the centrioles; axoneme; posterolateral to the base of the axoneme, the Golgi apparatus and (when secreted) the acrosomal rudiment. The dome-shaped acrosome vesicle elongates progressively as it migrates to the tip of the elongating and condensing nucleus; subacrosomal material gives rise to an almost equally long, tubular, thick-walled perforatorium. After the acrosome has greatly elongated, the mitochondria are reduced to two, which lose their rounded form and invest the growing axoneme to give a very elongate midpiece. Transfer of materials from nucleus to mitochondria is discussed. Microtubules surrounding the acrosome and nucleus disappear by maturity, but those internal to the mitochrondria apparently persist as the accessory microtubules, unique in the Annelida, which surround the 9 + 2 axoneme. Microvilli of the egg envelope, which have tetrads of terminal branches (epivitelline projections) resembling epicuticular projections, are less than 1 μm long, whereas the mature acrosome exceeds 5 μm. This suggests that the correlation seen in oligochaetes is absent.     

Ultrastructure of the sperm of Dolania americana Edmunds and Traver (Ephemeroptera : Behningiidae)

The ultrastructure of the mature sperm of the mayfly, Dolania americana Edmunds and Traver (Ephemeroptera : Behningiidae), is described from scanning and transmission electron microscopy. The head is 0.7–1 μm wide and 4.6–6.9 μm long, rodlike, and topped by a short, rounded acrosome 0.4 μm long and 0.6 μm wide. The flagellum is 5–6 times the head length and is flattened, except for a thin, tubelike terminal portion. The axoneme pattern is 9-9-1 (9 outer singlet microtubules, 9 doublet microtubules, and a central dark element) and is new for Ephemeroptera. The inner dynein arms are conspicuous and outer arms are lacking, and radial spokes and a central sheath are prominent. A densely-staining and bi-lobed accessory body lies adjacent to the axoneme. A mitochondrial derivative with regularly arranged transverse-to-oblique cristae lies adjacent to the accessory body.     

Spermatogenesis in the endosymbiont-bearing bivalve Loripes lucinalis (Veneroida: Lucinidae)

In the endosymbiont-bearing bivalve Loripes lucinalis, spermatogenesis is similar to that described for numerous bivalve species and leads to the formation of an aquasperm. The head and midpiece measure 10.5 ± 1.5 μm in length. The head is made up of a cylindrical nucleus slightly tapered apically and capped by a short conical acrosome. The nucleus lacks both an anterior and posterior nuclear invagination. The acrosome is 1.0 ± 0.1 μm long and consists of an acrosomal cone containing a diffuse subacrosomal material and an apical electron-lucent vacuole. There is no axial rod. The midpiece is made up of four mitochondrial spheres that surround the distal and proximal centrioles. The base of the distal centriole is joined to the plasmic membrane by the pericentriolar complex made up of nine radial arms. A cytoplasmic collar is observed that sheaths the flagellum as it emerges from the distal centriole. The spermatozoa present in mature acini are grouped into characteristic rings, which may have a nutritive function, with the acrosome oriented toward the centre of these ring formations. Also present within the gonad are somatic cells that seem to play a nutritive role in relation to the germinal cells. These nutritive cells undergo a cycle of development and lysis that corresponds to the spermatogenic cycle of the bivalve. These cells are large and rich in glycogen and lipid inclusions. In-depth examination of nutritive cells and gametes reveals that the male gonad is devoid of microorganisms in either a vegetative or cryptic form, suggesting that a vertical transmission through paternal gonadal inheritance is a very unlikely means of symbiont transmission in L. lucinalis. © 1996 Wiley-Liss, Inc.     

A comparison of the structure of the spermatozoa and spermatogenesis of 16 species of patellid limpet (Mollusca: Gastropoda: Archaeogastropoda)

Light and transmission electron microscopy of the spermatozoa and spermatogenesis of 16 species (in three genera, Patella, Helcion, Cellana) of patellid limpet have shown that head lengths of the sperm range from 3 to 13 μm, and each species has a sperm with a unique morphology, indicating that the spermatozoa can be used as a taxonomic character. Although spermatozoon structure is species specific, five types can be recognized, based on the size, shape, and structure of the nucleus and acrosome. The occurrence of five morphological types of sperm, one of which (Cellana capensis) is particularly different from other patellids, suggests that the taxonomy of the family Patellidae be re-examined. The morphological changes that occur during spermatogenesis are very similar in all species, although two patterns of chromatin condensation are found. Those species with sperm that have short squat nuclei (length:breadth < 3.5:1) have a granular pattern of condensation. Species with sperm that have more elongate nuclei (length:breadth > 5:1) have an initial granular phase followed by the formation of chromatin fibrils. These fibrils become organized along the long axis of the elongating nucleus. The absence of a manchette suggests that nuclear elongation is brought about from within the nucleus.     

Sperm ultrastructure in Kellia suborbicularis (Bivalvia: Galeommatoidea: Kellidae)

The sperm cells of Kellia suborbicularis are narrow with a short bullet‐shaped acrosome, a 5.0–5.5 µm long and 0.4–0.6 µm broad nucleus, and a short midpiece with a ring of five mitochondria. The disposition of the subacrosomal substance into a coronet‐like formation is unique, and the sperm structure offers no clue to the relationship between Kellia and other galeommatoidean genera. The possible significance of narrow elongate sperm for their entry into the brood pouch is discussed.     

Collection and evaluation of epididymal sperm in captive agoutis (Dasyprocta aguti)

The objective was to establish a protocol for the collection and evaluation of epididymal sperm in agoutis. Eight males (1-2 y old) underwent left orchidectomy and epididymal sperma were collected by retrograde flush. Average values were flush volume 32 μL, pH 6.9, sperm concentration 748 x 10⁶ sperm/mL, with motility 86.5% and vigor 4.6. Viable sperm were present in all flush samples; 66% of sperm were alive, and 41.9% of sperm responded positively to the hypoosmotic test (using distilled water). There were 21.1% morphologically abnormal sperm, of which 2.0 and 19.1% were primary and secondary defects, respectively. The acrosome was intact in 99.5% of sperm. The sperm head was 4.89 ± 0.41 μm long and 3.13 ± 0.35 μm wide, with an area of 13.01 ± 2.01 μm². Midpieces were 5.33 ±0.44 μm long and 0.98 ± 0.13 wide, sperm tails were 29.91 ± 2.29 μm, and overall sperm length was 40.12 ± 2.44 μm. In conclusion, epididymal sperm collection from agoutis was satisfactory; the collected sperm has the potential to be stored, facilitating development of other reproductive biotechnologies for this species.     

Ultrastructure of the spermatozoa and eggs of the ocean pout (Macrozoarces americanus L.), an internally fertilizing marine fish

Ultrastructure of sperm and eggs of the ocean pout (Macrozoarces americanus L.), an internally fertilizing marine teleost, was examined by scanning and transmission electron microscopy. The results showed that the sperm do not have an acrosome but have a very long mid-piece (one to two times the sperm head length) containing numerous well-developed elongated mitochondria. The sperm also have two tails (is biflagellate) each consisting of nine peripheral and one central pair (9 ± 2) of microtubules. This long mid-piece and the biflagellate nature of the sperm appear to be associated with the long life-span of the sperm and with sperm dispersal in the ovary to fertilize the eggs internally. The ocean pout eggs are enveloped by a porous chorionic membrane similar to that found in other teleosts but have two micropyles, a condition likely related to a mechanism of egg fertilization which increases the egg fertlity in the presence of low sperm numbers. Following insemination, some biochemically undefined excretions appeared on the surface of fertilized eggs and led to the acquisition of adherent capability of the eggs which formed a tightly associated egg mass in sea water. © 1995 wiley-Liss, Inc.     

An Electron Microscopic Study of Spermatogenesis in Marenzelleria viridis (Verrill, 1873) (Polychaeta; Spionidae)

Abstract Spermatogenesis in Marenzelleria viridis was studied by ultrastructural investigation. The testes are formed on the greatly ramified nephridial blood vessel and are enveloped by a thin layer of peritoneal cells. The spermatogonia vary in shape, are about 10 μm in diameter and are not linked by intercellular bridges. Pairs or tetrads of spermatocytes connected by intercellular bridges float freely in the coelomic cavity. A complex acrosome is produced by a Golgi complex. The acrosome consists of four to five different structures, forms cisternae and, in the mature spermatozoon, lies deep in an invagination of the nucleus. Two centrioles are also situated in a deep centriolar fossa, the proximal centriole being perpendicular to the distal one. The mature spermatozoon is an ect-aquasperm measuring about 5 μm in length and 2.5 μm in width. The midpiece consists of five spherical mitochondria arranged around the axoneme behind the nucleus. The axoneme is connected to the plasma membrane by a satellite complex. The microtubules of the flagellum are arranged in a typical 9 × 2 + 2 configuration. The spermatogenesis and the sperm morphology of M. viridis were compared with those of other members of the family Spionidae. Copyright © 1996 The Royal Swedish Academy of Sciences. Published by Elsevier Science Ltd.     

Male reproductive system in the South American catfish Conorhynchus conirostris

The testes of the catfish Conorhynchus conirostris ( n  = 67) from the São Francisco River, Minas Gerais, Brazil were of the fringed type, similar to those of some Pimelodidae. The germ, Sertoli and Leydig cells showed characteristics which are general for all vertebrates although the spermatozoa had a peculiar morphology, with an ovoid head without an acrosome, inverted U‐shaped nucleus, a short midpiece and a long tail, typical of teleosts showing external fertilization. The spermatic duct and genital papilla performed a secretory function.