Spermatogenesis in a Free-Living Marine Nematode Neochromadora poecilosoma (Chromadorida,Chromadoridae) Studied Using TEM

Spermatogenesis and the structure of mature spermatozoa were studied using TEM in a free-living marine chromadorid nematode Neochromadora poecilosoma from the Sea of Japan. In spermatocytes, fibrous bodies (FB) develop; in spermatids, the synthetic apparatus lies in the residual body, while the nucleus, mitochondria, and FB are located in the main cell body (MCB). The nucleus consists of a diffuse chromatin of fibrous structure, which is not enclosed in a nuclear envelope. In the spermatid stage, the development of FB is completed, and immature spermatozoa from the proximal region of the testis do not show any structural differences from the MCB of spermatids. The mature spermatozoa are polarized cells. They attach to the uterus wall by a pseudopod filled with filaments of the cytoskeleton; in the MCB of spermatozoon, there is a nucleus surrounded by mitochondria and osmiophilic bodies. The spermatozoa of N. poecilosoma show typical ultrastructure features of sperm cells found in most studied nematodes (amoeboid nature and the absence of axoneme, acrosome, and nuclear envelope). However, no aberrant organelles characteristic of nematode spermatozoa were found throughout sperm development in N. poecilosoma and other chromadorids.     

Spermatozoal ultrastructures of two marine perciform teleost fishes, the goatfish, Paraupeneus spilurus (Mullidae) and the rabbitfish, Siganus fuscescens (Siganidae) from Taiwan

Mature spermatozoa of two perciform teleost fishes, Paraupeneus spilurus (Mullidae) and Siganus fuscescens (Siganidae) from Taiwan were examined using transmission and scanning electron microscopy. Despite the fact that spermatozoa of both species are of the primitive type, the results of the present study highlight the potential application of spermatozoal morphology in studies of fish phylogenetic relationships. To our knowledge, the flattened nucleus observed in P. spilurus spermatozoa is reported for the first time. Several features common to Sigandae spermatozoa-the unusual almost parallel situation of the centrioles, the arrangement of mitochondria and the near absence of shallow nuclear fossa-are significantly different from other common teleost sperm types. These unique features may be synapomorphies for the Siganidae and Mullidae and evidently contribute to the study of phylogenetic relationships in teleosts.     

The phylogenetic significance of sperm morphology in the Platyhelminthes

The phylogenetic significance of flatworm sperm morphology is discussed against the background of general spermatology. The modified type of spermatozoon of the Nemertodermatida, a group of primitive flatworms, indicates that the Platyhelminthes evolved from forms characterized by the primitive type of metazoan sperm and by the primitive mode of fertilization, implying the release of sperm freely into sea water.The occurrence of aberrant types of spermatozoa in most platyhelminths is obviously a consequence of early evolution of the internal mode of fertilization, which characterizes all true members of this group. It can be concluded, from the ultrastructure of these aberrant spermatozoa that higher metazoans cannot have evolved from seriated flatworms related to the recent Seriata (Proseriata and Tricladida). Even the seemingly primitive Acoela have such aberrant spermatozoa that evolution of higher metazoans from acoels related to the recent Acoela seems highly improbable.The ultrastructure of the spermatozoa of the parasitic groups of flatworms (Monogenea, Digenea, Cestoda) is very similar to that found in the Kalyptorhynchia, a further indication that the parasitic groups are related to the rhabdocoel turbellarians.     

An ultrastructural study of spermatogenesis and sperm morula breakdown inArenicola marina (L.) (Annelida: Polychaeta)

Spermatogenesis in the lugwormArenicola marina, in common with other members of Arenicolidae, occurs in the coelomic fluid and results in the formation of discs of mature spermatozoa known as a morulae. Within a morula, individual spermatozoa are connected by a common mass of cytoplasm called the cytophore and therefore make up a syncitium. Immediately prior to spawning, and in response to an endocrine substance known as “Sperm Maturation Factor” (SMF), the structure of the sperm morulae breaks down and free spermatozoa are liberated. These are subsequently spawned from the body cavity. The investigation described here uses transmission electron microscopy to investigate the ultrastructural changes, which accompany spermatogenesis and the breakdown of sperm morulae in response to SMF in vitro. The study demonstrates that the cytophore appears to have a key role both during spermatogenesis and during sperm morula breakdown. The ultrastructure of sperm morulae and of mature spermatozoa is described. The structure of spermatozoa is shown to be primitive with a single flagellum which appears to be coiled at its distal end. The phagocytosis of free spermatozoa by coelomocytes is also described and it is suggested that these may play a role in the resorption of unspawned gametes in vivo.     

A Fine Structural Study of the Fertilization Process of the Jellyfish Cladonema uchidai

This study described the fertilization process of the jellyfish Cladonema uchidai by means of transmission electron microscopy. Female pronucleus was situated in close vicinity to the animal pole of the spawned egg, where the surface of the egg was flat or slightly depressed. Microvilli were observed except on the surface at the animal pole. The egg was entirely covered with a coat composed of fibrous materials. The spermatozoon was of the primitive type, and the proacrosomal vesicles were found immediately beneath the plasma membrane of the antero-lateral region of the sperm head. Within 15 sec after insemination, spermatozoa were incorporated in the egg cytoplasm only at the microvilli-free surface at the animal pole. Neither opening of the proacosomal vesicles nor formation of the acrosomal process was observed. No appreciable changes of cortical cytoplasm could be detected, although the egg became sticky after fertilization. Decondensation of the incorporated sperm nucleus occurred without breakdown of the original nuclear envelope. Within 10 min after insemination, the sperm nucleus still under the process of its decondensation fused with the female pronucleus. These findings were discussed in comparison with the fertilization process of higher metazoans as well as of other cnidarians.     

The location of the major protein in Caenorhabditis elegans sperm and spermatocytes

Using an affinity-purified antibody to the major sperm protein (MSP) in Caenorhabditis elegans sperm we have shown by immunofluorescence that the MSP is localized in the fibrous bodies of spermatocytes and early spermatids, in the cytoplasm of late spermatids, and in the pseudopods of spermatozoa. The MSP can also form crystalline inclusions in mutant and wild-type sperm. The function of this protein is still unknown, but its ability to form filaments and its localization in the pseudopod, together with the lack of actin in these sperm suggest that the MSP may be required for amoeboid motility.     

Cauda epididymal spermatozoa of the rufous hare wallaby, Lagorchestes hirsutus (Metatheria, Mammalia) imaged by electron and confocal microscopy

The ultrastructure of mature Lagorchestes hirsutus spermatozoa is described for the first time, revealing unusual aspects of sperm structure in macropodid species. The sperm head is ovoid rather than cuneiform, lacks a ventral nuclear groove and has an acrosomal distribution over approximately 85–90% of its dorsal surface. Immediately adjacent to the nuclear membrane the peripheral nucleoplasm in most spermatozoa form an irregular series of distinctive evaginations previously not described in the spermatozoa of any other marsupial. The midpiece is extremely thickened and short, containing no helical network or peripheral plasma membrane specializations. Axonemal structure is unspecialized with no connecting lamellae; dense outer fibres are closely adherent to axonemal doublets. The sperm morphology of this species is highly aberrant in comparison to other macropod taxa and supports the retention of Lagorchestes as a distinctive genus. In light of this new information, skeletal and serological data should be re‐evaluated to determine the true taxonomic and phylogenetic position of this species.     

Calcium signaling and the MAPK cascade are required for sperm activation in Caenorhabditis elegans

In nematode, sperm activation (or spermiogenesis), a process in which the symmetric and non-motile spermatids transform into polarized and crawling spermatozoa, is critical for sperm cells to acquire fertilizing competence. SPE-8 dependent and SPE-8 independent pathways function redundantly during sperm activation in both males and hermaphrodites of Caenorhabditis elegans. However, the downstream signaling for both pathways remains unclear. Here we show that calcium signaling and the MAPK cascade are required for both SPE-8 dependent and SPE-8 independent sperm activation, implying that both pathways share common downstream signaling components during sperm activation. We demonstrate that activation of the MAPK cascade is sufficient to activate spermatids derived from either wild-type or spe-8 group mutant males and that activation of the MAPK cascade bypasses the requirement of calcium signal to induce sperm activation, indicating that the MAPK cascade functions downstream of or parallel with the calcium signaling during sperm activation. Interestingly, the persistent activation of MAPK in activated spermatozoa inhibits Major Sperm Protein (MSP)-based cytoskeleton dynamics. We demonstrate that MAPK plays dual roles in promoting pseudopod extension during sperm activation but also blocking the MSP-based, amoeboid motility of the spermatozoa. Thus, though nematode sperm are crawling cells, morphologically distinct from flagellated sperm, and the molecular machinery for motility of amoeboid and flagellated sperm is different, both types of sperm might utilize conserved signaling pathways to modulate sperm maturation.     

Nup358 integrates nuclear envelope breakdown with kinetochore assembly

Nuclear envelope breakdown (NEBD) and release of condensed chromosomes into the cytoplasm are key events in the early stages of mitosis in metazoans. NEBD involves the disassembly of all major structural elements of the nuclear envelope, including nuclear pore complexes (NPCs), and the dispersal of nuclear membrane components. The breakdown process is facilitated by microtubules of the mitotic spindle. After NEBD, engagement of spindle microtubules with chromosome-associated kinetochores leads to chromatid segregation. Several NPC subunits relocate to kinetochores after NEBD. siRNA-mediated depletion of one of these proteins, Nup358, reveals that it is essential for kinetochore function. In the absence of Nup358, chromosome congression and segregation are severely perturbed. At the same time, the assembly of other kinetochore components is strongly inhibited, leading to aberrant kinetochore structure. The implication is that Nup358 plays an essential role in integrating NEBD with kinetochore maturation and function. Mitotic arrest associated with Nup358 depletion further suggests that mitotic checkpoint complexes may remain active at nonkinetochore sites.     

Principes de préparation et de congélation des spermatozoïdes testiculaires humains

The advantages and feasibility of human testicular spermatozoa cryoconservation for intracytoplasmic sperm injection (ICSI) have now been clearly demonstrated. However, the freezing protocol is based on empirical knowledge obtained from freezing of ejaculated spermatozoa. Testicular spermatozoa may not be fully mature gametes and may also be retrieved in only limited quantities. Little research has been conducted to determine whether they have the same cryobiological requirements as ejaculated spermatozoa. A better understanding of their cryobiological features and assessment of possible subcellular changes after thawing would help to optimize testicular preparations for cryopreservation (whole biopsies, seminiferous tubules, shredded suspension, single spermatozoa, etc.), freezing-thawing procedure, freezing media, and storage. Finally, there is a growing need for welldefined criteria (nuclear quality, etc.) to evaluate the tolerance of testicular spermatozoa to freezing-thawing procedure for ICSI     

COMPARATIVE SPERMATOLOGY OF THREE SPECIES OF DONAX (BIVALVIA) FROM SOUTH AFRICA

The fine structure of the sperm and spermatogenesis in threespecies of Donax (D. madagascariensis, D. sordidus and D. serra)are described. Although the morphology of the sperm of all speciesis very similar, each has unique features. Donax madagascariensisand D. sordidus reportedly hybridize in regions of sympatryand their spermatozoa are morphologically closer to one anotherthan to D. serra. All sperm are of the primitive type with ahead(about 2 µmu; long), mid-piece of four mitochondria andtail. The head comprises a barrel-shaped nucleus which is cappedby a small, complex acrosome. The structure of the acrosomeis typical of heterodont bivalves. During spermatogenesis thepattern of nuclear chromatin condensation is granular. Glycogenfirst appears in the cytoplasm of spermatids, and in the maturesperm is sited in the mid-piece and base of the acrosome. (Received 15 May 1989; accepted 25 June 1989)     

Examination of living and fixed gametes and early embryos stained with supravital fluorochromes (Hoechst 33342 and 3,3′-dihexyloxacarbocyanine iodide)

We have examined living and fixed gametes and early embryos of surf clams, sea urchins, and hamsters stained with the supravital dyes Hoechst 33342 for DNA and 3,3′-dihexyloxacarbocyanine iodide (DIOC₆) for mitochondria and endoplasmic reticulum. Hoechst staining (10 μM) was confined exclusively to egg and sperm chromatin and, in living marine specimens, did not interfere with sperm motility, fertilization, or nuclear activity during meiosis or early embryogenesis. Although Hoechst staining did not appear to affect the motility of hamster sperm, only zonae-free eggs inseminated. Because chromatin retained Hoechst 33342 stain during fertilization, the paternally and maternally derived chromosomes of living and fixed preparations fluoresced and their number, organization, and location within the zygote cytoplasm could be determined. Hence, polyspermy and other nuclear abnormalities were amenable to examination in these stained preparations. DIOC₆ staining (8.7 μM) was restricted primarily to the mitochondria of spermatozoa. Eggs stained with DIOC₆ (0.87 to 8.7 μM) were brightly fluorescent because of their size and the presence of large numbers of mitochondria and other DIOC₆-positive organelles. Sea urchin and surf clam sperm stained with DIOC₆ fertilized unstained eggs and the location of the incorporated sperm mitochondrion up to first cleavage was followed. Although hamster sperm stained with DIOC₆ were less motile than unstained sperm, they were capable of inseminating only zonae-free eggs. These observations demonstrate that staining with supravital fluorochromes provides a rapid and useful method to analyze macromolecular and organelle changes in a variety of living and fixed gametes and embryos.     

龙洞山溪鲵精子的超微结构

应用透射电镜和扫描电镜对龙洞山溪鲵(Batrachuperuslongdongensis)精子的超微结构进行观察和研究,探讨山溪鲵属以及小鲵科物种精子的结构特征,并探讨有尾两栖类精子结构特征的演化及其与生殖进化的关系。结果表明:1)龙洞山溪鲵的精子具小鲵科物种精子的共同特征,即精子无顶体钩,顶体呈三叶草状,尾部无线粒体,轴纤维粗大呈圆柱状等特征;2)龙洞山溪鲵精子核脊的形态结构与小鲵科其它属物种以及有尾类其它科物种精子的核脊存在明显的差别,由此可见核脊形态结构具有属或种的特异性。此外,与已有报道的有尾两栖类物种精子的结构特征进行分析比较表明:1)有尾类精子特征的分化与其受精方式的变化是一致的;2)精子结构特征支持隐鳃鲵超科为单系的推测     

Resorption of unemitted gametes in Lithognathus mormyrus (Sparidae,Teleostei): a possible synergic action of somatic and immune cells

The resorption of unemitted gametes during the post-spawning period of the male and female reproductive cycles in Lithognathus mormyrus was studied by histochemical, histological and cytological methods. The resorption of residual spermatozoa involved the phagocytotic activity of Sertoli cells bounding the seminiferous cysts of spermatozoa, and those associated with spermatogonia lining the lobular lumen. Spermatozoa remaining in the sperm duct were phagocytozed by the lining epithelial cells. Eosinophilic granulocytes and macrophages were identified in the vicinity of residual spermatozoa. The remnants of oocytes underwent an atretic phenomenon in which follicle cells were firstly involved, inducing a progressive fragmentation of the oocyte cytoplasm. Subsequently, eosinophilic granulocytes invaded oocyte degenerative areas and clung to the remaining vitelline inclusions ensuring their biotransformation into waste products (brown bodies). The analogy of the resorption processes of both male and female unemitted gametes during the post-spawning period of natural reproductive cycle, involving first the enveloping somatic cells and then immune cells, is emphasized.     

Reproduction in a carnivorous sponge: the significance of the absence of an aquiferous system to the sponge body plan

Sponges usually produce, release, and capture gametes via the aquiferous system, and so the absence of both choanocytes and an aquiferous system in the carnivorous sponge Asbestopluma occidentalis has led to unusual characteristics of development for this Phylum. Sperm are highly specialized elongate cells tightly packed into spermatic cysts in the peripheral tissue of the sponge. Mature spermatozoa have proacrosomal vesicles at the anterior end and a ciliary pit surrounding the flagellum. Clusters of four to five oocytes are in synchronous stages of cleavage, suggesting that fertilization is synchronous. All stages of embryos occur in the same individual. Early cleavage was holoblastic and equal; blastomeres in two-, four- and eight-cell embryos were compact and 16-cell stage embryos were bi-layered. Late-stage embryos show three cellular regions along the anterior-posterior axis: the anterior hemisphere with heterogeneous cells, a mid-region with cells lying perpendicular to the A-P axis in a collagenous matrix, and small cells at the posterior pole. Unusually for Porifera, multiciliated cells cover all but the posterior pole. It is inferred that fertilization occurs by capture of intact spermatic cysts whose surrounding forceps spicules become trapped in the anisochelae of neighboring sponges. The elongate shape of sperm may be designed to penetrate the loose collagenous mesohyl, such that the arrival of a packet of sperm would lead to simultaneous fertilization of oocytes in a cluster. Loss of the water canal system in carnivorous sponges has allowed the evolution of features that are highly specialized for the habitat of this animal, but such modifications were not necessarily a prerequisite for the subsequent evolution of metazoans. Given the extremely versatile mechanisms of gametogenesis, embryogenesis, and tissue/body structure in sponges, generalizations regarding basal metazoan reproduction, development, and structure must be approached with caution.     

Fertilization of eggs of zebrafish, Danio rerio, by intracytoplasmic sperm injection

To evaluate the potential for fertilization by sperm injection into fish eggs, sperm from zebrafish, Danio rerio, were microinjected directly into egg cytoplasm of two different zebrafish lines. To evaluate physiological changes of gametes on the possible performance of intracytoplasmic sperm injection (ICSI), four different combinations of injection conditions were conducted using activated or nonactivated gametes. From a total of 188 zebrafish eggs injected with sperm in all treatments, 31 (16%) developed to blastula, 28 (15%) developed to gastrula, 10 (5%) developed abnormally to larval stages, and another 3 (2%) developed normally and hatched. The highest fertilization rate (blastodisc formation) was achieved by injection of activated spermatozoa into nonactivated eggs (35%). Injections were most effective when performed within the first hour after egg collection. Flow cytometric analysis of the DNA content of the developing ICSI embryos revealed diploidy, and the use of a dominant pigment marker confirmed paternal inheritance. Our study indicates that injection of a single sperm cell into the cytoplasm of zebrafish eggs allows fertilization and subsequent development of normal larvae to hatching and beyond.     

Yolk protein is expressed in the insect testis and interacts with sperm

Background  

Male and female gametes follow diverse developmental pathways dictated by their distinct roles in fertilization. While oocytes of oviparous animals accumulate yolk in the cytoplasm, spermatozoa slough off most of their cytoplasm in the process of individualization. Mammalian spermatozoa released from the testis undergo extensive modifications in the seminal ducts involving a variety of glycoproteins. Ultrastructural studies suggest that glycoproteins are involved in sperm maturation in insects; however, their characterization at the molecular level is lacking. We reported previously that the circadian clock controls sperm release and maturation in several insect species. In the moth, Spodoptera littoralis, the secretion of glycoproteins into the seminal fluid occurs in a daily rhythmic pattern. The purpose of this study was to characterize seminal fluid glycoproteins in this species and elucidate their role in the process of sperm maturation.     

Bovine sperm chromatin is not protected from the effects of ultrasmall gold nanoparticles

The response of ejaculated bovine spermatozoa to gold nanoparticles was studied by the standard method of nuclear chromatin decondensation in vitro. After the treatment of semen samples with a hydrosol containing gold nanoparticles with an average diameter of ～3.0 nm and a concentration of 1 × 10¹⁵ particles/mL, the ability of sperm nuclei to decondense in the presence of sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) dramatically changed compared to the control. The frequencies of gametes with nondecondensed (“intact”), partially decondensed, and completely decondensed nuclei correlated as 40: 32: 28% and 0: 36: 64% in the experiment and the control, respectively. Moreover, the appearance of a sufficiently large number of gametes with destructed and almost completely destroyed nuclei was noticed in the spermatozoa treated with gold nanoparticles. This article suggests the putative mechanisms of action of ultrasmall gold nanoparticles on the structural and functional integrity of the deoxyribonucleoprotein (DNP) complex of mature male gametes.