Ultrastructural immunogold localization of major sperm protein (MSP) in spermatogenic cells of the nematode Acrobeles complexus (Nematoda,Rhabditida)

The nematode spermatozoa represent a highly modified (aberrant) type of male gametes that lack a flagellum but for which the process of spermatogenesis culminates in the production of a crawling spermatozoon on the basis of the cytoskeletal component known as “major sperm protein”, or MSP. MSP is also known as an important hormone triggering oocyte maturation and ovulation in the model nematode Caenorhabditis elegans, where this protein was first identified. However, direct evidence of MSP localization and of its fate in nematode spermatogenic cells is rare. In this study, the spermatogenesis and sperm structure in the rhabditid nematode Acrobeles complexus (Rhabditida: Tylenchina: Cephalobomorpha: Cephaloboidea: Cephalobidae) has been examined with electron microscopy. Morphological observations were followed by high-pressure freezing and freeze-substitution fixation which allows post-embedding immunogold localization of MSP in all stages of sperm development using antibodies raised for MSP of C. elegans. In spermatocytes, synthetic activity results in the development of specific cellular components, fibrous bodies (FB) and membranous organelles (MO), which appear as FB-MO complexes where the filamentous matter of FB has been MSP-labeled. The spermatids subdivide into a residual body with superfluous cytoplasm, and a main cell body which contains nucleus, mitochondria and FB-MO complexes. These complexes dissociate into individual components, MO and FB, with the MSP being localized in FB. Immature spermatozoa from testes are opaque cells where a centrally located nucleus is surrounded by mitochondria, MO and FB clustered together, the MSP still being localized only in FB. Cytoplasm of mature spermatozoa from spermatheca is segregated into external pseudopods lacking organelles and a central cluster of mitochondria with intact MO surrounding the central nucleus. The FB ultimately disappear, and the MSP labeling becomes concentrated in the filamentous content of pseudopods and cytoplasm of the main cell body. Although the spermatogenesis and sperm structure of A. complexus is similar to that of many other rhabditid nematodes, their intact MO makes the morphology of the mature spermatozoa distinct from the “rhabditid pattern” and may be considered as a synapomorphy. The MSP localization in spermatogenic cells of A. complexus also follows the “rhabditid pattern” described in C. elegans and Ascaris spp. Our results and techniques of MSP labeling of A. complexus spermatogeneous cells reveal new possibilities to elucidate different research questions on MSP localization in nematodes related to C. elegans. Furthermore, the laboratory-cultured A. complexus strains can be used as a new and fascinating model to study MO and MSP functions in nematode reproduction.     

Ultrastructure of germ cells,Sertoli cells and mitochondria during spermatogenesis in mature testis of the Chinese Taihang black goats (Capra hircus)

The objective of this study was to describe the ultrastructure of germ cells, Sertoli cells and mitochondria in mature testis of the Chinese Taihang black goat. The characteristics of germ cell nucleus and mitochondria changing during spermatogenesis were investigated by transmission electron microscopy (TEM). The results showed that the spermatogonium was elliptical, and its nucleus was about 4–5 μm. The round mitochondria can be observed throughout the cytoplasm around the nucleus. Small patches of heterochromatin were distributed throughout the nucleus. Spermatocyte was oval-shaped with a nucleus of about 4–4.5 μm in diameter. The heterochromatin began to attach to the inner surface of the nuclear membrane. Spermatid was about 4 μm and oval in shape. Its nucleus was oval or round and approximately 2–3 μm in diameter. The borderline between nucleus membrane and karyoplasm was distinct. During spermiogenesis, spermatid nucleus was condensed and elongated, and chromatin reached the highest condensation in the mature spermatozoon. The mid-piece was surrounded by mitochondria at the neck region. The sperm tail showed the typical “9 + 2″ structure, contained axoneme and central singlet microtubules. The nuclei of the Sertoli cells were irregular shaped and showed indentations in the membrane. In the mature testes of goat bucks, abundant mitochondria were around the germ cells and Sertoli cells. The scattered mitochondria were aggregated around the base of the flagellum (axoneme) during the spermatid differentiation stage. In conclusion, the present study showed that the spermatogenic process of Taihang black goat followed the pattern of mammals with some specific.     

Effect of rapid freezing–thawing techniques on the sperm parameters and ultrastructure of Chinese Taihang black goat spermatozoa

Supercooling sperm in liquid nitrogen vapour is a feasible and economic technique for the practical production. The study aimed to reveal the negative effects of this rapid freezing and thawing processes on Taihang black goat spermatozoa and to find out the changing of spermatozoa motility and ultrastructure by using CASA and TEM. Qualified semen samples, which collected from twenty Chinese Taihang black goats using artificial vagina were pooled and investigated the kinematics parameters and ultrastructural morphology. The results showed that freezing–thawing caused a significant reduction in the spermatozoon total motility (P < 0.001), in rapid and medium cell numbers (P < 0.001) and motility parameters (VAP, VSL, VCL, ALH and BCF) (P < 0.01). Immotile spermatozoa number was increased significantly after freezing–thawing (P < 0.001). In the ultrastructural analysis, the shape with a sperm nucleus characterized by ruptures, bend and deformity was observed. The plasma membranes were broken, and nucleoplasm erupted. The mitochondria in the middle piece were disturbed by partial absence or additional accumulations. Swelling, coiling, vacuolization and structural disorganization of mitochondria were also observed. In conclusion, Freezing–thawing procedure has a detrimental effect on motility, membrane integrity and mitochondria of goat spermatozoa. Transmission electron microscopy provides an intuitive observation to investigate deformity spermatozoa.     

Comparative sperm ultrastructure of three species in Siniperca (Teleostei: Perciformes: Sinipercidae)

Siniperca chuatsi, Siniperca kneri, and Siniperca scherzeri are three of the most economically important sinipercid species. The ultrastructure and morphology of the mature spermatozoa of them are examined using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The sperm consists of an acrosome-less head, a short midpiece and a long flagellum. Ultrastructurally, it has a homogeneously electron-dense nucleus in a granular pattern with nuclear lucent and a nuclear fossa excluding the centriolar complex. One to four mitochondria with lamellar cristae encircle the basal body of the flagellum in the midpiece. The cytoplasm surrounding the centrioles and the cylindric cytoplasmic channel contains glycogen granules and vesicles. Comprising the conventional 9 + 2 axoneme, vesicles and lateral fins, the sperm flagellum is inserted laterally on the nucleus, therefore the spermatozoon is asymmetrical. All of the spermatozoa of the three species are of the primitive or ect-aquasperm form and conform to the teleostean type II spermatozoa instead of the previously supposed type I. Variations in the shape of the heads, angles between the two centrioles, location of the cytoplasmic vesicles, mitochondrial number and structural characteristics of the lateral fins are notable among the three species. S. chuatsi is a sister-group of the other two species and is the most differentiated. The present study provides fresh insights to the comparative spermatology of Siniperca fishes and will be useful to the existing knowledge of the sinipercid fishes in systematic characters, biodiversity conservation and reproduction.     

Fine structure of the spermatozoon of Perinereis macropus (Claparède, 1870) (polychaeta,Nereidae)

The mature spermatozoa of Perinereis macropus were investigated by transmission electron microscopy. The spermatozoon is composed with a large anterior part (head), a short middle piece and a long flagellum. The head contains a large acrosomal complex with a convex acrosomal vesicle, a subacrosomal space, a fibrillar crown and an acrosomal rod which penetrates into the nucleus invagination. The later is U shaped (in longitudinal section). The short middle piece contains about nine to eleven mitochondria and a centriole associated with the flagellum. This centriole, slightly eccentric to the sperm axis, anchors to the plasma membrane by nine satellite rays of the pericentriolar complex. The axoneme has a “9 + 2″ arrangement of microtubules. In cross section, the flagellar membrane extends in two lateral protuberances, aligned with the axis formed by the two central microtubules of the axoneme. The spermatozoon of P. macropus conforms to the primitive type with an acrosomal extension. Nevertheless, the acrosome complex ultrastructure shows noticeable modifications from the basic form. This finding agrees with the previously observed reproductive pattern (broadcast spawning – free-swimming larvae), and may be helpful to classify the sperm type of P. macropus.     

Actin localisation and the effect of cytochalasin D on the osmotic tolerance of cauda epididymidal kangaroo spermatozoa

This study examined the hypothesis that filamentous actin associated with the complex cytoskeleton of the kangaroo sperm head and tail may be contributing to lack of plasma membrane plasticity and a consequent loss of membrane integrity during cryopreservation. In the first study, the distribution of G and F actin within Eastern Grey Kangaroo (EGK, Macropus giganteus) cauda epididymidal spermatozoa was successfully detected using DNAse-FITC and a monoclonal F-actin antibody (ab205, Abcam), respectively. G-actin staining was most intense in the acrosome but was also observed with less intensity over the nucleus and mid-piece. F-actin was located in the sperm nucleus but was not discernable in the acrosome or sperm tail. To investigate whether cytochalasin D (a known F-actin depolymerising agent) was capable of improving the osmotic tolerance of EGK cauda epididymal spermatozoa, sperm were incubated in hypo-osmotic media (61 and 104 mOsm) containing a range of cytochalasin D concentrations (0-200 microM). Cytochalasin D had no beneficial effect on plasma membrane integrity of sperm incubated in hypo-osmotic media. However, when EGK cauda epididymidal sperm were incubated in isosmotic media, there was a progressive loss of sperm motility with increasing cytochalasin D concentration. The results of this study indicated that the F-actin distribution in cauda epididymidal spermatozoa of the EGK was surprisingly different from that of the Tammar Wallaby (M. eugenii) and that cytochalasin-D does not appear to improve the tolerance of EGK cauda epididymidal sperm to osmotically induced injury.     

Acrosome reaction in Octopus tankahkeei induced by calcium ionophore A23187 and a possible role of the acrosomal screw

The octopod sperm is unique especially in two aspects: the screw-shaped acrosome and its inner layered substructure (striation). The present study aims to investigate morphological changes of Octopus tankahkeei spermatozoa during the acrosome reaction (AR) and to pursue functions of the internal substructures revealed by inducing AR with the calcium ionophore A23187. Gradual changes of the spermatozoa were traced using fluorescence and electron microscopy. The AR process included the bulging, vesiculation, and dehiscence of the plasma membrane around the acrosome and the nucleus, as well as the vesiculation of the mitochondrial sheath. Membrane vesiculation outside the nucleus has never been reported in the order Octopoda. The rigid screw and the inner striation of the acrosome remained intact surmounting the nucleus, suggesting that these two structures have potential functions during fertilization. In addition, the detachment of the sperm head and the tail was commonly observed in this study, both in intact and acrosome-reacted sperm. Fluorescence microscopy revealed that the detached mitochondrial sheath usually gave weaker and more dispersive signals than the joint ones. This phenomenon implied that the intense energy release might promote the detachment of the mitochondrial sheath.     

Ultrastructure of the spermatozoa of Cicadella viridis (Linnaeus) and its bearing on the phylogeny of Auchenorrhyncha

The ultrastructure of mature spermatozoa of the leafhopper Cicadella viridis (Linnaeus) was investigated using light and transmission electron microscopy. The spermatozoon is composed of a head containing an acrosome and an elongated nucleus, and a long tail, which consists of a flagellum. The acrosome is conical and invaginated to form a subacrosomal space, and the acrosomal contents are filled with electron-dense tubular substructures. The nucleus is linear and filled with homogeneously condensed chromatin. The centriolar adjunct is parallel to the nucleus and connects the nucleus with the mid-piece/flagellum. The flagellum is formed by a 9+9+2 axoneme, two mitochondrial derivatives and two accessory bodies. The mitochondrial derivatives with an orderly array of peripheral cristae are symmetrical. The accessory bodies are small and slightly elliptical. The end of the axoneme shows progressive loss of microtubules. Comparison of sperm ultrastructure of C. viridis with those of other Auchenorrhyncha families supports the major relationships within Cicadomorpha as (Membracoidea (Cicadoidea, Cercopoidea)).     

Spermatozoon of the freshwater rotifer Brachionus calyciflorus (Rotifera,Monogononta): Advances in morphological and ultrastructural studies

The morphological and ultrastructural features of the spermatozoon in Brachionus calyciflorus are described using light, fluorescence and transmission electron microscopy (TEM). The mature spermatozoon, which appears to be thread-like, is composed of a slightly expanded anterior of cell body region and a flagellum region without acrosome. The cell body region and flagellum region are respectively 16–27 μm and 20–33 μm in length (n = 60). The spermatozoon is characterized by a mass of dense tubular materials, which occupy most of the cell. Some mitochondria are distributed around the nuclear region in the anterior of the cell body region, while in the posterior portion of cell body, the chromatin often contains a single lobated nucleus arranged at the center of cell. The flagellum contains the classic axoneme (9 × 2 + 2) and possesses lateral undulating membrane. Mature B. calyciflorus males have no germ cell stages earlier than the spermatids in the testis. TEM examination reveals rigid rods as well as predominant typical spermatozoon in the testis. Observations, based on successive photographs and videos, enabled a first-time recording of the unique inverted movement of the spermatozoon, which indicated that the movement of the spermatozoon is driven by the flagellum. Our study also provides further supplementary insights into the phylogenetic systematics of the Rotifera.     

The evidence of Tobacco rattle virus impact on host plant organelles ultrastructure

Tobraviruses, like other (+) stranded RNA viruses of plants, replicate their genome in cytoplasm and use such usual membranous structures like endoplasmic reticulum. Based on the ultrastructural examination of Tobacco rattle virus (TRV)-infected potato and tobacco leaf tissues, in this work we provide evidence of the participation of not only the membranous and vesicular ER structures but also other cell organelles during the viral infection cycle. Non-capsidated TRV PSG particles (potato isolate from the Netherlands) (long and short forms) were observed inside the nucleus while the presence of TRV capsid protein (CP) was detected in the nucleus caryolymph and within the nucleolus area. Both capsidated and non-capsidated viral particles were localized inside the strongly disorganized chloroplasts and mitochondria. The electron-dense TRV particles were connected with vesicular structures of mitochondria as well as with chloroplasts in both potato and tobacco tissues. At 15–30 days after infection, vesicles filled with TRV short particles were visible in mitochondria revealing the expanded cristae structures. Immunodetection analysis revealed the TRV PSG CP epitope inside chloroplast with disorganized thylakoids structure as well as in mitochondria of different tobacco and potato tissues. The ultrastructural analysis demonstrated high dynamics of the main cell organelles during the TRV PSG–Solanaceous plants interactions. Moreover, our results suggest a relationship between organelle changes and different stages of virus infection cycle and/or particle formation.     

Morphology of the male reproductive system and spermatozoa in Centris Fabricius, 1804 (Hymenoptera: Apidae, Centridini)

The genera Centris and Epicharis constitute the Centridini and are widely distributed in the Neotropical region. Centris is also found in the southern portion of the Neartic region, although both genera are more abundant in the humid tropical regions. To describe the structure of the male reproductive system and spermatozoa, light and transmission electron microscopy were used. The male reproductive system of Centris sp. is formed by a pair of testes, a pair of deferent ducts, a pair of seminal vesicles, a pair of accessory glands and an ejaculatory duct connected to the external genitalia, the aedeagus. In this species, testes and the pre-vesicular deferens ducts as well as the seminal vesicles are encapsulated in a single conjunctive capsule, the scrotal membrane. Each testis consists of four testicular follicles, made up of cysts with up to 64 germinative cells. Histologically, the seminal vesicles are formed by a simple cylindrical epithelium, basal membrane and muscular tunic. The spermatozoa of Centris analis, C. fuscata, C. tarsata and Centris sp. are morphologically similar. They have two easily distinguishable regions: the head and flagellum. The head is formed by the two-layer acrosome, the linear nucleus and the flagellum, the centriole adjunct, the axoneme of pattern 9+9+2 microtubules, two asymmetric mitochondrial derivatives and two accessory bodies. These Centris species share various morphological characteristics of the male reproductive system and spermatozoa with the other bees previously described, indicating that several characteristics are synapomorphic for the family Apidae. Studies on the morphology of the male reproductive system and spermatozoa in Hymenoptera have demonstrated the diversity of the information provided by these reproductive structures, which can be used in taxonomy studies and the phylogeny of this important group of insects.     

Cellular organisation of the mature testes and stages of spermiogenesis in Danio rerio (Cyprinidae; Teleostei)--structural and ultrastructural studies

The male gonads of Danio rerio occupy a position typical of the Teleostei species. The structure of the testes corresponds to the anastomosing tubular type with unrestricted spermatogonia and represents a cystic type of spermatogenesis. The results of this study indicate that four distinct stages of cell differentiation can be identified during spermiogenesis. These stages are characterised by chromatin condensation, the development of flagellum, nuclear rotation, the formation of nuclear fossa and the elimination of excess cytoplasm. A round head and the absence of an acrosome characterise the differentiated sperm. The midpiece is short and large, and C-shaped mitochondria form a ring surrounding the initial region of the flagellum. The axoneme shows a 9 + 2 pattern. In the D. rerio spermatozoa the flagellar axis is at an angle of 110° to the nucleus diameter running through the centriole.     

Ultrastructural analysis of spermiogenesis in the Eastern Fence Lizard,Sceloporus undulatus (Squamata: Phrynosomatidae)

Studies on reptilian sperm morphology have shown that variation exists at various taxonomic levels but studies on the ontogeny of variation are rare. Sperm development follows a generalized bauplan that includes acrosome development, nuclear condensation and elongation, and flagellar development. However, minute differences can be observed such as the presence/absence of manchette microtubules, structural organization during nuclear condensation, and presence/absence of a nuclear lacuna. The purpose of this investigation was to examine sperm development within the Sceloporus genus. The process begins with the development of an acrosomal complex from Golgi vesicles followed by nuclear condensation and elongation, which results in the presence of a nuclear lacuna. As the acrosomal complex differentiates, flagellar development commences with elongation of the distal centriole. Spermatid development culminates in a mature spermatid with a highly differentiated acrosomal complex, a condensed nucleus with a nuclear lacuna, and a differentiated flagellum. Although the overall developmental pattern is consistent with other squamate species, minute differences are observed, even within the same genus. For example there is variation in the presence/absence of an endoplasmic reticulum complex during acrosome development, presence/absence of a nuclear lacuna, and presence/absence of manchette microtubules within the three species of Sceloporus studied to date. Future studies concerning sperm morphology in closely related species will aid in our understanding of variation in sperm development and may prove to be useful in testing phylogenetic and evolutionary hypotheses.     

Spermiogenesis and sperm ultrastructure of Machilontus sp (Insecta: Archaeognatha) with phylogenetic consideration

The sperm structure of the jumping bristletail Machilontus sp has been described. The species shares several sperm characteristics with other genera of the same order Archaeognatha. During late spermiogenesis the spermatid bends at half of its length with the two limbs closely apposed within the same plasma membrane. The sperm has a helicoidal bi-layered acrosome with a filamentous perforatorium and a long nucleus. The elongated flagellum consists of an axoneme with 9 accessory microtubules external to the 9+2, two rows of conventional mitochondria and two accessory bodies. The accessory bodies are located lateral to the axoneme and are probably responsible for the shifting of the accessory tubules in two opposite groups of 5 and 4 tubules, respectively. These sperm characteristics represent common traits of all Archaeognatha.     

Ca2+ and cAMP regulations of microtubule sliding in hyperactivated motility of bull spermatozoa

To reach and fertilize the egg, mammalian spermatozoa change their flagellar movement in the female reproductive tract, named hyperactivation. The biochemical analyses of the hyperactivated movement using demembranated spermatozoa defined the factors inducing this peculiar movement; namely, large asymmetrical flagellar movement observed in the early stage of the hyperactivation was induced with a high Ca²⁺ concentration while large symmetrical flagellar movement in the late stage of the hyperactivation was generated with low Ca²⁺ and high cAMP concentrations. Under these conditions, the microtubule sliding of bull sperm flagella was investigated by disintegrating the sperm flagella with MgATP²⁻ after extracting their plasma membrane and mitochondria. The large asymmetrical flagellar movement was caused by a long sliding displacement of a fiber of the doublet microtubules. On the other hand, the large symmetrical flagellar movement was generated by a large amount of microtubule sliding by many doublet microtubules.     

Ultrastructure of the mature testicular vesicle of Pseudonannolene tocaiensis Fontanetti, 1996 (Diplopoda, Pseudonannolenidae)

The mature testicular vesicles of the species Pseudonannolene tocaiensis have three portions: a peripheral portion; a central portion that is typically secretory and between these two an intermediary portion, a lumen filled with spermatozoa. Both secretory and peripheral portions present the same type of cells. The ultrastructural analysis of the peripheral portion suggests it is involved in hormone synthesis. The secretory portion probably contributes to the production of spermatic fluid, compensating for the absence of male accessory glands in this species.     

Comparative ultrastructural study of spermatozoa in some oyster species from the Asian-Pacific Coast

Sperm organization in the oysters Crassostrea gigas, Crassostrea nippona, Crassostrea cf. rivularis and Saccostrea cf. mordax inhabiting Asian Pacific coast was studied. The spermatozoa of all studied species had a number of common morphological characters such as a cup-like acrosome with heterogeneous matrix on its top, an axial rod in the subacrosomal space, a barrel-shaped nucleus, four mitochondria in the midpiece, pericentriolar complexes, and a 9+2-organized flagellum. The spermatozoa of C. cf. rivularis differed from the other species by having cytoplasm processes in the midpiece region. Such structures have never been described in the Ostreidae. Additionally, each species could be identified by the shape and size of sperm compartments (acrosome, nucleus, anterior nuclear fossa). The most significant interspecific difference was found in the size of an anterior nuclear fossa. The smallest anterior nuclear fossa was found in C. cf. rivularis (about 0.24 μm in length reaching about 22% of the nuclear length) while the biggest in C. gigas from the Sea of Japan (about 0.53 μm in length reaching about 46% of the nuclear length). The spermatozoa of C. gigas collected from the Sea of Japan and Taiwan Strait differed significantly in almost all the studied parameters. Since sperm morphology has been successfully used for species differentiation, this suggests the existence of two species rather than two populations. The data obtained indicate the species-specific difference in the sperm ultrastructure within the Ostreidae, which may be identified both ultrastructurally and morphometrically.     

Ultrastructure of the mature spermatozoon of the bivalve Scapharca broughtoni (Mollusca: Bivalvia: Arcidae)

The ultrastructure of mature spermatozoa of the giant clam bivalve Scapharca broughtoni was investigated by transmission electron microscopy for the first time. The mature spermatozoon consists of a head which is composed of a cone-shaped acrosome, a round nucleus, and a tail region. A subacrosomal space contains an axial rod and a basal plate, the latter lying between the acrosome and the nucleus. Although the nucleus lacks an anterior invagination, an inverted shallow V-shaped posterior invagination is present within the nucleus. Within the middle portion of the spermatozoon lie five spherical mitochondria while the long whip-like end portion is composed of an axoneme with the typical 9 + 2 structure. Our conclusion is that the spermatozoon of S. broughtoni is of the type I anacrosomal “aquasperm”, and the morphology of acrosome and nucleus are an adaptation to external fertilization.     

The effects of different cryoprotectants and the temperature of addition on the survival of red deer epididymal spermatozoa

With the aim of finding an ideal cryoprotectant in a suitable concentration for red deer epididymal spermatozoa conservation, we evaluated the effects of four most commonly used cryoprotectants (CPAs), Glycerol (G), Ethylene glycol (EG), Propylene glycol (PG), and Dimethyl sulfoxide (DMSO), on the sperm survival. Besides, the effects of two temperatures of CPA addition--22 degrees C (ambient temperature) and 5 degrees C--on sperm quality were also tested. For each temperature tested, sperm samples were evaluated after 0, 15, 30 and 60 min of spermatozoa exposition to CPAs. Thus, sperm quality was in vitro judged by microscopic assessments of individual sperm motility (SMI), and of plasma membrane (Viability) and acrosome (NAR) integrities. Overall, DMSO showed the highest toxicity for red deer epididymal spermatozoa, and glycerol the lowest. Thus, at 60 min of incubation SMI results showed that the toxicity to red deer epididymal spermatozoa of the four CPAs are in the following sequence: G approximately = EG approximately = PG < DMSO ('less than' symbol means P < 0.05, and approximate symbol means P = 0.08). Furthermore, our results also showed a differential response of acrosome membrane to temperature of CPAs addition. Regardless of the CPA used, statistically significant variations (P < 0.05) were found between the two temperatures of addition of CPAs for acrosome integrity, the best being 22 degrees C (NAR = 83.8% vs. 69.8%). These data indicate that sperm quality of red deer epididymal spermatozoa, in addition to be affected by the cryoprotectant, can also be influenced by the temperature at which CPAs are added prior to freezing.