The ultrastructure of spermatozoa and spermiogenesis in pyramidellid gastropods,and its systematic importance

Ultrastructural observations on spermiogenesis and spermatozoa of selected pyramidellid gastropods (species ofTurbonilla, Pyrgulina, Cingulina andHinemoa) are presented. During spermatid developement, the condensing nucleus becomes initially anterio-posteriorly compressed or sometimes cup-shaped. Concurrently, the acrosomal complex attaches to an electrondense layer at the presumptive anterior pole of the nucleus, while at the opposite (posterior) pole of the nucleus a shallow invagination is formed to accommodate the centriolar derivative. Midpiece formation begins soon after these events have taken place, and involves the following processes: (1) the wrapping of individual mitochondria around the axoneme/coarse fibre complex; (2) later internal metamorphosis resulting in replacement of cristae by paracrystalline layers which envelope the matrix material; and (3) formation of a glycogen-filled helix within the mitochondrial derivative (via a secondary wrapping of mitochondria). Advanced stages of nuclear condensation (elongation, transformation of fibres into lamellae, subsequent compaction) and midpiece formation proceed within a microtubular sheath (‘manchette’). Pyramidellid spermatozoa consist of an acrosomal complex (round to ovoid apical vesicle; column-shaped acrosomal pedestal), helically-keeled nucleus (short, 7–10 μm long, shallow basal invagination for axoneme/coarse fibre attachment), elongate helical midpiece (composed of axoneme, coarse fibres, paracrystalline and matrix materials, glycogen-filled helix), glycogen piece (length variable, preceeded by a dense ring structure at junction with midpiece). The features of developing and mature spermatozoa observed in the Pyramidellidae are as observed in opisthobranch and pulmonate gastropods indicating that the Pyramidelloidea should be placed within the Euthyneura/Heterobranchia, most appropriately as a member group of the Opisthobranchia.     

Spermatozoal ultrastructure of four Sparidae fishes: Acanthopagrus berda, Acanthopagrus australis, Lagodon rhomboids and Archosargus probatocephus

The mature spermatozoa of two Taiwan protandrous hermaphrodite Sparidae Acanthopagrus berda and Acanthopagrus australis are investigated and compared with those of other two Sparidae (Lagodon rhomboids and Archosargus probatocephus) from the Western hemisphere. Ultrastructurally the spermatozoon of these four species has a spherical, homogeneously electron-dense nucleus with an axial nuclear fossa. The midpiece contains one to four spherical mitochondria and encircles the basal body of the flagellum. The mature spermatozoa of the four species are of the primitive or ect-aquasperm form and conform to the teleostean type I spermatozoon with the flagellar axis inserts perpendicular and medial to the nuclear fossa. Variation in the depths of the nuclear fossa and mitochondria number is substantial in these four Sparidae species. This study provide useful systematic characters to the existing knowledge of comparative spermatology of Sparidae.     

A review of spermatozoan morphology in Cetacea with new data for the genus Kogia

There have been few studies on the morphology of spermatozoa of the Cetacea. Here we present a review of the existing published material and present new data for spermatozoa of Kogia breviceps, Kogia sima and Delphinus delphis . In contrast to the usual spiral arrangement of mitochondria in the midpiece of mammalian sperm, cetaceans exhibit a unique arrangement of spherical mitochondria arranged in tiers and columns along the midpiece, which is primitive in appearance. In addition, the midpiece of the cetacean spermatozoon is much shorter than that of other mammalian orders. Detailed knowledge of the morphology of the spermatozoa of cetaceans may help elucidate aspects of male reproduction and the fertilization process in this group.     

Adhesion between plasma membrane and mitochondria with linking filaments in relation to migration of cytoplasmic droplet during epididymal maturation in guinea pig spermatozoa

High-resolution microscopy has been used to investigate the mechanism of the migration of cytoplasmic droplets during epididymal maturation of guinea pig spermatozoa. On testicular spermatozoa, droplets are located at the neck and, after passage through the middle cauda epididymidis, migrate only as far as the center of the midpiece. Initially, the space between the plasma membrane and outer mitochondrial membranes outside the droplet is 30.8±11.0 nm, whereas on mature spermatozoa, it significantly (P<0.01) narrows to a more consistent 15.9±1.3 nm. This is accompanied by the appearance of thin filaments cross-linking the two membranes above and below the droplet. Changes also occur in the arrangement of intramembranous particles (IMPs) in the plasma membrane overlying the midpiece. At the spermatid stage, linear arrays of IMPs are absent but appear on immature spermatozoa, where they are short with an irregular orientation, in the epididymis. On mature spermatozoa, numerous parallel linear arrays are present at the region where the plasma membrane adheres to the mitochondria. The membrane adhesion process can thus be observed two-dimensionally. The initial migration of the droplet from the neck is probably attributable to diffusion, with the formation of cross-linking filaments between the two membranes in the proximal midpiece preventing any backward flow and squeezing the droplet distally until it is arrested at the central midpiece by the filaments formed in the distal midpiece. The filaments might also stabilize the flagellum against hypo-osmotic stress encountered during ejaculation and within the female tract.     

Sperm mitochondria of patients with normal sperm motility and with asthenozoospermia: morphological and functional study

Studies were performed on ejaculated human spermatozoa (32 subjects with normal sperm motility and 25 subjects with low sperm motility). Morphology of sperm midpiece was evaluated in light, fluorescent and transmission or scanning electron microscope. Changes in mitochondrial membrane potential (delta(psi)m) and mass of mitochondria were analysed by flow cytometry using mitochondrial specific probes JC-1 and Mito Tracker Green FM. Moreover, oxidoreductive capability of sperm mitochondria was assessed using cytochemical reaction for NADH-dependent dehydrogenases. In flow cytometry analysis of JC-1-stained spermatozoa, two asthenozoospermic subpopulations were distinguished: patients with a high percentage (76 +/- 11%, 13 subjects) and patients with a low percentage (29 +/- 14%,12 subjects) of spermatozoa with functional-polarized mitochondria with high delta(psi)m. Our microscopic investigations of spermatozoa of seven asthenozoospermic patients reveal that the deformed and unusually thickened sperm midpieces (50-70% of cells), occasionally with persistent cytoplasmic droplet, contain supernumerary mitochondria with normal substructure, full oxidoreductive capability and high delta(psi)m. The midpiece deformations cause nonprogressive movement or immotility. They can also appear in smaller number of spermatozoa (5-35% of cells) in patients with normal sperm motility. Moreover, in three cases of asthenozoospermia midpiece malformations were accompanied by abnormal morphology of outer dense fibers and axoneme. The cytochemical, fluorescence and SEM studies showed the absence of midpieces in many (60-80%) spermatozoa in some other cases of asthenozoospermia. The morphological observations corresponded with flow cytometry analysis of Mito Tracker Green FM-stained spermatozoa. Our results suggest that in some cases of asthenozoospermia the sperm mitochondria can be functionally active and display high delta(psi)m in large number of cells. The results may suggest that asthenozoospermia does not necessarily result from energetic disturbances of sperm mitochondria. The low sperm motility may be associated with deformations of the mitochondrial sheath containing functional mitochondria. The combination of fluorescence microscopy and flow cytometry with electron microscopic investigations is a sensitive, precise and comprehensive examination which helps discover sperm abnormalities responsible for asthenozoospermia.     

KLC3 is involved in sperm tail midpiece formation and sperm function

Kinesin light chain 3 (KLC3) is the only known kinesin light chain expressed in post-meiotic male germ cells. We have reported that in rat spermatids KLC3 associates with outer dense fibers and mitochondrial sheath. KLC3 is able to bind to mitochondria in vitro and in vivo employing the conserved tetratrico-peptide repeat kinesin light chain motif. The temporal expression and association of KLC3 with mitochondria coincides with the stage in spermatogenesis when mitochondria move from the spermatid cell periphery to the developing midpiece suggesting a role in midpiece formation. In fibroblasts, expression of KLC3 results in formation of large KLC3 aggregates close to the nucleus that contain mitochondria. However, the molecular basis of the aggregation of mitochondria by KLC3 and its role in sperm tail midpiece formation are not clear. Here we show that KLC3 expression from an inducible system causes mitochondrial aggregation within 6h in a microtubule dependent manner. We identified the mitochondrial outer membrane porin protein VDAC2 as a KLC3 binding partner. To analyze a role for KLC3 in spermatids we developed a transgenic mouse model in which a KLC3ΔHR mutant protein is specifically expressed in spermatids: this KLC3 mutant protein binds mitochondria and causes aggregate formation, but cannot bind outer dense fibers. Male transgenic mice display significantly reduced reproductive efficiency siring small sized litters. We observed defects in the mitochondrial sheath structure in a number of transgenic spermatids. Transgenic males have a significantly reduced sperm count and produce spermatozoa that exhibit abnormal motility parameters. Our results indicate that KLC3 plays a role during spermiogenesis in the development of the midpiece and in the normal function of spermatozoa.     

Spermiogenesis and spermatozoa ultrastructure in Salminus and Brycon, two primitive genera in Characidae (Teleostei: Ostariophysi: Characiformes)

In Salminus, spermiogenesis is cystic and gives origin to a type I aquasperm. Spermatid differentiation is characterized by chromatin condensed into thick fibres, nuclear rotation, nuclear fossa formation, cytoplasmic channel formation, mitochondrial fusion producing long and ramified mitochondria, and the presence of several membranous concentric rings around the plasma membrane that encircles the cytoplasmic channel. In Salminus and Brycon, spermatozoa are very similar. They exhibit a spherical nucleus and chromatin condensed into fibre clusters, and a deep nuclear fossa. They show a long midpiece with few elongate mitochondria at the initial region and a cytoplasmic channel completely encircled by one or two membranous concentric rings. The flagellar axis is perpendicular to the nucleus and exhibits the classic axoneme (9 + 2). The very strong similarity observed between Salminus and Brycon spermatozoa supports the hypothesis that these subfamilies are likely to have a monophyletic origin.     

Morphology and fine structure of Barbus barbus (Teleostei: Cyprinidae) spermatozoa

Morphology and fine structure of Barbus barbus L 1758 spermatozoa were studied using scanning (SEM) and transmission (TEM) electron microscopy. The results confirm that spermatozoa exhibit morphological features typical to all teleost fishes. They are differentiated into a head, a midpiece and a flagellum with the typical '9 + 2' pairs of microtubules. Both dynein arms are present in the flagellum. The spermatozoa have spherical nuclei, 4–6 mitochondria located in the postnuclear cytoplasmic region and centriolar complex (proximal and distal centrioles). Total length, head width, length of midpiece and length of flagellum were measured to be 56.35 ± 7.42, 1.80 ± 0.06, 0.48 ± 0.14 and 54.30 ± 6.97 μm, respectively. Highly significant linear correlation was observed between posterior and anterior width of midpiece (P < 0.01). Principal component analysis (PCA) was used to explore which parameters can explain the individual variation of sperm morphology. About 44% of the total accumulated variance was absorbed by the analysis of the two first components, distinguishing different groups of parameters related to head and midpiece. The lengths of flagellum and head are more isolated; indicating that the individual variation of sperm morphology depends on these two parameters. Comparing the results of this study with information on cyprinids spermatozoa reveals that the number of mitochondria and the length of the flagellum are good characters to characterize spermatozoa of the Cyprinidae in a phylogenetic arrangement.     

Ultrastructure of Thunnus thynnus and Euthynnus allettevatus spermatozoa

The spermatozoa of Thunnus thynnus and Euthynnus alletteratus consist of an acrosome-less head (comprising the ovoid nucleus and the short midpiece) and a long flagellar tail that contains the conventional 9 + 2 axoneme and lacks lateral fins. The centrioles are arranged at approximately right angles and lie outside of a shallow nuclear groove. The flagellum inserts laterally on the nucleus, therefore the spermatozoon is asymmetrical. The midpiece contains a few mitochondria which are separated from the axoneme by the cytoplasmic canal; they are spherical in T. thynnus and elongate, somewhat irregular in E. alletteratus . Although the main ultrastructural characteristics of the spermatozoa appear to indicate a great homogeneity in the sperm morphology within the family Scombridae, small species-specific divergences may be of use in systematics.     

Comparative ultrastructural study of the cuticle and spermatozoa in Propappus volki Michaelsen, 1916 (Annelida: Clitellata)

The ultrastructure of the cuticle and mature spermatozoa of the oligochaete Propappus volki Michaelsen, 1916 is described with the aim of providing additional data for clarifying the systematic position of the taxon. P. volki is a fresh-water species living in streams, and is easily recognized by its proboscis on the pre-segmental prostomium and, in mature specimens, by a clitellum covering the segments XII–XIV. The cuticle is composed of a proximal fibre zone and a distal layered epicuticle covered with membrane-bound epicuticular projections. The fibre zone consists of collagenous fibres in a matrix, arranged in either densely packed parallel layers with the fibres oriented in the same direction, or with more loosely distributed fibres, although with the same main orientation. The epicuticular projections are pyramidal with the base leaning on the outer surface of the epicuticle. The cuticle covering the proboscis differs in morphology from that of the rest of the worm; the fibre zone is composed of thin and short fibrils running in all directions, and the epicuticular projections are longer and more narrow than the projections in other regions of the worm.

The spermatozoa are filiform cells formed, in sequence, by an acrosome, an elongated nucleus, a long midpiece, and a flagellum. The acrosomal tube is short and straight with a completely external acrosomal vesicle. Following the acrosome is a apically corkscrew-shaped and basally straight nucleus. The midpiece is twisted and formed by five mitochondria. The flagellum shows a prominent central sheath arrangement.

A comparison with ultrastructurally described cuticles and spermatozoa from other clitellate species reveals most similarities with enchytraeids.     

Failure of the expression of phospholipid hydroperoxide glutathione peroxidase in the spermatozoa of human infertile males

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) was intensely expressed in mitochondria in the midpiece of human spermatozoa by immunostaining with anti-PHGPx monoclonal antibodies. The PHGPx not only reduced phospholipid hydroperoxide but also scavenged hydrogen peroxide in human spermatozoa. We found a dramatic decrease in the level of expression of PHGPx in the spermatozoa of some infertile males by immunoblotting with anti-PHGPx monoclonal antibodies. These individuals accounted for about 10% of the group of 73 infertile males that we examined. All seven patients with PHGPx-defective spermatozoa were classified as suffering from oligoasthenozoospermia, a defect in which both the number and the motility of spermatozoa are significantly below normal. Males with PHGPx-defective spermatozoa accounted for 26% of the 27 infertile males with oligoasthenozoospermia. No defects in expression of PHGPx in spermatozoa were observed in 31 fertile volunteers. After a 3-h incubation, the relative number of motile spermatozoa with low-level expression of PHGPx was significantly lower than that of spermatozoa with normal expression of PHGPx. The PHGPx-defective spermatozoa failed to incorporate rhodamine 123, revealing a loss of mitochondrial membrane potential. Ultrastructual analysis of mitochondria by electron microscopy demonstrated that the morphology of mitochondria in PHGPx-defective spermatozoa was abnormal. The results suggest that failure of the expression of mitochondrial PHGPx in spermatozoa might be one of the causes of oligoasthenozoospermia in infertile men.     

Spermatozoa morphology and ultrastructure in Nile perch,Lates niloticus (Linnaeus, 1758)

Lates niloticus is a valuable commercial fish species with good potential for aquaculture. However, there is limited information on the type and structure of the Nile perch spermatozoon, which could potentially aid in culture of this species. Here, we describe the spermatozoon ultrastructure in L. niloticus using transmission and scanning electron microscopy. The spermatozoon had a round head-shape, medio-laterally flat, no acrosome, a short midpiece located laterally to the nucleus, uniflagella with one wing. The head of the spermatozoon contained the nucleus, centriolar system, proximal part of the flagellum, and cytoplasmic channel. Centrioles were arranged at an angle of 90° to each other, forming a T-shape, parallel to the nucleus. The midpiece was cylindrical, loaded with cytoplasm, five to seven spherical mitochondria; and the flagellum’s plasma membrane extended to form one lateral wing. The spermatozoa were classified as type II spermatozoa. L. niloticus spermatozoon differed from that of its Australian congener L. calcarifer, especially in the centriole arrangement and nuclear shape, length of the midpiece and the number of mitochondria and lateral wings.     

The action of gossypol on rat germinal cells

Adult male Sprague-Dawley rats were fed daily with 25 mg/kg of gossypol acetic acid for 55 days. The examination of the testis revealed the constant integrity of the blood-testis barrier. The earliest damage to the germinal cells appeared on the 14th day of treatment in spermatids in stage 18-19, and consisted of mitochondrial swelling and cristae disorganization, which was manifested in an altered assembly of the mitochondrial helix and possible axonemal alterations. It appeared that the target cell was a 30–37-day-old spermatid that showed the first alterations 6–14 days later, when it reached stage 18. The mitochondrial and eventual axonemal damage was conserved after the migration into the epididymis, affecting 6% of the total sperm population on the 14th day of treatment and reaching the maximum (100%) on the 25th day of treatment. On the 5th day of treatment, epididymal spermatozoa revealed, moreover, the presence of other tail alterations: lack of half of the axonemal components (also in sperm with unaffected mitochondria), loss of midpiece plasma membrane, and breaking of the outer accessory fibers. The conclusion is that a direct action of gossypol on epididymal spermatozoa is superimposed on the action earlier exerted on the spermatids.     

Ultrastructure of Pagrus major and Rhabdosargus sarba spermatozoa (Perciformes: Sparidae: Sparinae)

Transmission and scanning electron microscopy were used to investigate the ultrastructure of spermatozoa in two Sparinae species Pagrus major and Rhabdosargus sarba. Ultrastructurally, the spermatozoa of P. major and R. sarba both consist of a spherical, homogeneously electron-dense nucleus with a deep axial nuclear fossa, and an unusual notch, in the nuclear region. The midpiece contains two spherical mitochondria in R. sarba and one in P. major. The comparison of spermatozoal ultrastructure of these two species of Sparidae shows that they closely resemble one another and suggests that they are closely related. Variation in the geometry and dimensions of the mitochondrion and nucleus is substantial in these two Sparidae species. It is concluded that the spermatozoa of both species are of primitive type, and they are distinguished by several unique features which may provide useful systematic characteristics.     

Spermatozoa of the Anomalodesmata (Bivalvia, Mollusca) with special reference to relationships within the group

Sperm ultrastructure in the marine bivalve order Anomalodesmata is considered in the light of new information for Australian Myochama anomioides and Cuspidaria latesulcata . In M. anomioides , the acrosomal complex lies posterior to the nucleus, in contact with the asymmetrical midpiece mitochondria – an unusual configuration reported from most of the studied Anomalodesmata. Spermatozoa of M. anomioides resemble those of Myadora brevis (both in family Myochamidae). Myochamid spermatozoa are considerably less modified than those of the Lyonsiidae or Laternulidae, and may prove a basal type within the non-septibranch anomalodesmatans. In contrast, C. latesulcata differs from other examined anomalodesmatans in having an anterior acrosomal complex and radial midpiece mitochondria (classic aquasperm features). Sperm data for the Anomalodesmata are limited but congruent with the most recent phylogenetic analyses that recognize distinct 'thraciid' and 'lyonsiid' clades. Results for C. latesulcata suggest septibranch origins before the development of a posteriorly positioned acrosomal complex and mitochondrial asymmetry.     

Morphological evidence of correlational selection and ecological segregation between dextral and sinistral forms in a polymorphic flatfish, Platichthys stellatus

Phenotypic polymorphisms in natural systems are often maintained by ecological selection, but only if niche segregation between morphs exists. Polymorphism for eyed-side direction is rare among the approximately 700 species of flatfish (Pleuronectiformes), and the evolutionary mechanisms that maintain it are unknown. Platichthys stellatus (starry flounder) is a polymorphic pleuronectid flatfish exhibiting large, clinal variation in proportion of left-eyed (sinistral) morphs, from 50% in California to 100% in Japan. Here I examined multiple traits related to swimming and foraging performance between sinistral and dextral morphs of P. stellatus from 12 sites to investigate if the two morphs differ in ways that may affect function and ecology. Direction of body asymmetry was correlated with several other characters: on an average, dextral morphs had longer, wider caudal peduncles, shorter snouts and fewer gill rakers than sinistral morphs. Although the differences were small in magnitude, they were consistent in direction across samples, implying that dextral and sinistral starry flounder may be targeting different prey types. Morphological differences between morphs were greatest in samples where the chances of competitive interactions between them were the greatest. These results suggest that the two morphs are not ecologically identical, may represent a rare example of divergent selection maintaining polymorphism of asymmetric forms, and that correlational selection between body asymmetry and other characters may be driven by competitive interactions between sinistral and dextral flatfish. This study is one of very few that demonstrates the ecological significance of direction in a species with polymorphic asymmetric forms.     

A new technique for the precise location of lactate and malate dehydrogenases in goat, boar and water buffalo spermatozoa using gel incubation film.

Gel incubation film, which contained gelatin to prevent the diffusion of enzyme during chemical reaction and phenazine methosulfate to operate as a hydrogen acceptor between NADH and tetrazolium, was used and light microscopy revealed that lactate dehydrogenase was located in the head and tail of the spermatozoa as well as in the midpiece, whereas malate dehydrogenase was confined to the midpiece in spermatozoa of the animals examined. In goat spermatozoa, lactate dehydrogenase was associated mainly with the inner acrosomal membrane in the head, the mitochondrial matrix in the midpiece and with flagellar fibrils in the tail, whereas malate dehydrogenase was present only in the mitochondrial matrix.     

Observations of spermiogenesis and epididymal sperm maturation in the rufous hare wallaby, Lagorchestes hirsutus (Metatheria, Mammalia)

Acrosomal development in the early spermatid of the rufous hare wallaby shows evidence of formation of an acrosomal granule, similar to that found in eutherian mammals, the Phascolarctidae and Vombatidae. Unlike the other members of the Macropodidae so far examined, the acrosome of this species appears to be fully compacted at spermiation and extends evenly over 90% of the dorsal aspect of the nucleus. During spermiogenesis, the nucleus of the rufous hare wallaby spermatid showed evidence of uneven condensation of chromatin; this may also be related to the appearance of unusual nucleoplasm evaginations from the surface of the fully condensed spermatid. This study was unable to find evidence of the presence of Sertoli cell spurs or nuclear rotation during spermiogenesis in the rufous hare wallaby. The majority of spermatozoa immediately before spermiation had a nucleus that was essentially perpendicular to the long axis of the sperm tail. Nuclei of spermatozoa found in the process of being released or isolated in the lumen of the seminiferous tubule were rotated almost parallel to the long axis of the flagellum; complete parallel alignment occurred during epididymal maturation. At spermiation spermatozoa have characteristically small cytoplasmic remnants compared to those of other macropods. Unlike the majority of macropodid spermatozoa so far described, the spermatozoa of the rufous hare wallaby showed little evidence of morphological change during epididymal transit. There was no formation of a fibre network around the midpiece or of plasma membrane specializations in this region; the only notable change was a distinctive flattening of midpiece mitochondria and scalloping of the anterior mitochondrial sheath to accommodate the sperm head. Preliminary evidence from spermiogenesis and epididymal sperm maturation supports the classification of the rufous hare wallaby as a separate genus but also indicates that its higher taxonomic position may need to be re‐evaluated.