Partial formation of sperm dimorphism from spermatocytes of the cottoid fish, Hemilepidotus gilberti in cell culture

Polymorphism of sperm is considered to be significant for the reproductive strategy in some animal species. The phenomenon is thought to occur in the species-specific stage of spermatogenesis, but how the identical germ cells are differentiated towards polymorphic sperm remains unknown. We here performed a germ cell culture in the cottoid fish, Hemilepidotus gilberti, whose sperm exhibit dimorphism with fertilizable eusperm and unfertilizable parasperm. In the culture, germ cells, which were obtained with an identical morphology, a spherical shape of 5-7 microm in diameter, differentiated into smaller spherical cells with a single nucleus, a moving flagellum and localized mitochondria. In addition, large retroflex-shaped cells with two elongated nuclei were also observed in the cell culture. Germ cells that had each morphological feature were histologically also observed in some cysts of the spermatogenetic testis, suggesting that the former type of cell corresponded to developing eusperm and the latter corresponded to developing parasperm. When BrdU was incorporated into germ cells in the culture, it was detected in both cells with eusperm-like and those with parasperm-like morphologies. These findings suggest that DNA-duplicating spermatocytes are potent to autonomously progress a part of spermatogenesis to form dimorphic sperm.     

Obstructive Role of the Dimorphic Sperm in a Non-copulatory Marine Sculpin, Hemilepidotus gilberti, to Prevent Other Males' Eusperm from Fertilization

In many species of animals, males normally produce parasperm (dimorphic sperm) along with eusperm (normal sperm) during spermatogenesis. In the present study, to clarify the role of parasperm of the non-copulatory sculpin Hemilepidotus gilberti, whose reproduction is characterized by polyandrous oviposition involving sneaking by neighboring territorial males, we observed the movements of parasperm. Parasperm could not move by themselves, but they were transported in solutions by passive movement due to collisions with actively swimming eusperm. In the viscous ovarian fluid (OF), which isolates eggs from seawater by covering them during spawning, parasperm did not exhibit any movement. However, they could be transported by eusperm movement in solutions with dissolved OF, partly because the viscosity of the fluid become lower. And then, in some solutions parasperm formed lumps. Lump formation of parasperm was also observed at the boundary surface of an egg mass where OF contacted seawater. Eusperm added experimentally to a solution in which parasperm were forming lumps were engulfed in the lumps and never escaped. Thus, lump formation of parasperm would be obstacles for the later arriving eusperm. Although lumps formed against both kin and non-kin eusperm, parasperm are thought to be available to overcome sperm competition which would occur during spawning that involves sneaking being almost concurrent with lump formation. The territorial male eusperm reach the eggs while his parasperm hinder other males' eusperm from reaching the eggs. Thus, we concluded that parasperm of H. gilberti play a role on protection of paternity by blocking rival eusperm physically from approaching eggs.     

Sex and a snail's sperm: on the transport,storage and fate of dimorphic sperm in Littorinidae

Summary

Littorinid parasperm develop from a distinctive lineage of germ cells which exhibit a process of nuclear destruction that has apototic characteristics. Fragments of DNA and other nuclear breakdown products are incorporated into secretion granules in parasperm that ultimately find their way to the female bursa copulatrix. Spermatozeugmata are stored in the seminal vesicles and, if unused during the breeding season, they are recycled by phagocytosis. Attachment between eusperm and parasperm is facilitated by an electrostatic interaction of proteins. Detachment, caused by alkaline prostate fluid, occurs by the time the ejaculate reaches the tip of the penis. Thus transport of eusperm by parasperm to the female is unlikely. parasperm are sterile cells that may function in defense against rival eusperm as suggested by the presence of lysosomes, or they may act as nuptial gifts as they are packed with glycoprotein nutrients. Differences in the reactivity of different parasperm to specific lectins may enable separation of dimorphic sperm by lectin affinity chromatography, thereby facilitating future studies on individual parasperm. In female Littorinidae, sperm are stored incapacitated in storage organs, or rarely in the ovary itself. In L. littorea serotonin caused spawning of unencapsulated eggs, which, in the presence of activated sperm, became polyspermic.     

The double sperm line in Isochaetides (Annelida,Clitellata, Tubificidae)

Sperm morphology and spermatogenesis were examined in the oligochaete annelid Isochaetides arenarius, a species belonging to the subfamily Tubificinae inhabiting the sediments of Lake Baikal. As all tubificines, Isochaetides produces two types of spermatozoa, named eusperm and parasperm. The eusperm are the fertilizing male gametes and consist, in sequence, of an acrosome, a nucleus, a mitochondrial mid-piece, and a tail. The parasperm have the same general architecture, but differ in cytological details: the acrosome is shorter, devoid of a perforatorium, and the acrosome vesicle has a different, simpler, shape. The nucleus is much shorter and rectilinear (the eusperm nucleus is twisted). The mid-piece mitochondria are less numerous but their overall volume is larger. The flagellum has a plasma membrane largely separated from the axoneme, and is devoid of glycogen granules. After mating, the two sperm types gather in the spermathecae to form spermatozeugmata; in these structures the parasperm form an external sheath involving the centrally located eusperm and their tails are connected by conspicuous septate junctions. Parasperm nuclei are produced through a process of fragmentation of the 'spermatocytes', whereas the flagellar basal bodies are produced by a process similar to that giving rise to basal bodies in ciliated epithelia.     

Changes in nuclear structure during eupyrene spermatogenesis in Murex brandaris

Changes in nuclear structure during eupyrene spermatogenesis of Murex brandaris have been studied using light and electron microscopy. In the first phases, spermatogonia show round nuclei, with several electrodense masses of chromatin and a thin layer of heterochromatin associated with the nuclear membrane. Primary spermatocytes possess larger nucleii, with less condensed chromatin, and the synaptonemal complexes are apparent. During spermiogenesis, chromatin becomes lamellar, and the nucleus twists about its principal axis while it elongates. The nuclear twisting is accompanied by a progressive chromatin condensation, which causes a highly electrodense nucleus at the end of the process.     

Identification of a novel sperm class and its role in fertilization in Drosophila

In many species, males have evolved to produce a sterile sperm (parasperm) in conjunction with fertilizing sperm (eusperm). Here, we document evidence of males depositing two morphologically distinct types of parasperm (1 and 2) into the female reproductive tract in Drosophila pseudoobscura. These parasperm differ in length, shape, amount produced, amount in long‐term storage and may have separate roles in ensuring male fertilization success. Although both parasperm types protect eusperm from female spermicides, only parasperm 2, which has a corkscrew shape, is associated with sperm competition. Increased production of parasperm 2 is also negatively correlated with the eusperm and parasperm 1 production. Thus, selection may be acting on parasperm production in the presence of sperm competition. Our findings show how both sperm competition and cryptic female choice may be acting in conjunction to influence the evolution of ejaculate composition. Our identification and characterization of two distinct parasperm morphs will enhance the ability for further evaluation of parasperm's role in fertilization.     

Dimorphic Sperm Influence Semen Distribution in a Non-copulatory Sculpin Hemilepidotus Gilberti

We used an artificial semen emission test to examine the semen transporting role of parasperm (unflagellated sperm), which are produced along with eusperm (normal sperm) by an incomplete meiosis in the marine sculpin Hemilepidotus gilberti. After separation of contents of semen (eusperm, parasperm, and seminal plasma) by centrifugation, the distance traveled by semen discharged vertically from a syringe into seawater was compared among various test semen; eusperm semen (eusperm re-mixed with seminal plasma), parasperm semen (parasperm re-mixed with seminal plasma), normal semen (eusperm and parasperm re-mixed with seminal plasma), and natural semen (unmodified semen). Parasperm semen traveled more than 1.5 times as far as the eusperm semen. The lateral dispersion width of normal semen after emission was significantly narrower than that of eusperm semen. These findings indicate that parasperm can reduce the lateral dispersion and prolong the distance semen travels. The eusperm ratio in the lower portion of the emitted semen did not differ from that in the upper portion, indicating that eusperm evenly distribute within the ejaculate and reach the lower portion of semen. Since males cannot closely approach eggs that are deposited in the narrow space between the female's belly and the spawning substrate, restraint of lateral dispersion and prolongation of the distance semen traveled would increase the number of eusperm arriving at eggs, despite reduction of eusperm in the ejaculate.     

Morphological changes of sperm nuclei during spermatogenesis in the brown alga Cystoseira hakodatensis (Fucales, Phaeophyceae)

Morphological changes and chromatin condensation of sperm nuclei were observed during spermatogenesis in the fucalean brown alga Cystoseira hakodatensis (Yendo) Fensholt. Ultrastructural studies have shown that the mature spermatozoid has an elongated and concave nucleus with condensed chromatin. The morphological changes and the chromatin condensation process during spermatogenesis was observed. Nuclear size decreased in two stages during spermatogenesis. During the first stage, spherical nuclei decreased in size as they were undergoing meiotic divisions and the subsequent mitoses within the antheridium. During the second stage, the morphological transformation from a spherical into an elongated nucleus occurred. Afterwards, chromatin condensed at the periphery in each nucleus, and chromatin‐free regions were observed in the center of the nucleus. These chromatin‐free regions in the center of nucleus were compressed by the peripheral chromatin‐condensed region. As the result, the elongated and concave nucleus of the mature sperm consisted of uniformly well‐condensed chromatin.     

Ectaquasperm-like parasperm in an internally fertilizing gastropod

Abstract. Pomacea canaliculata is an internally fertilizing gastropod that produces, besides fertilizing sperm (=eusperm), a large number of unfertile sperm (=parasperm) that have no chromatin, are fusiform, and have three to five flagella. Here, we report that this snail also produces another type of parasperm, which results from a peculiar spermiogenesis including an anterior cytoplasmic migration. The mature oligopyrene parasperm has: (1) a rounded head including a partly lysed nucleus, (2) a conical mid-piece with eight large mitochondrial structures, and (3) a single flagellum (∼20 μm). These characteristics, although not found in any other gastropod parasperm, are shared with the externally fertilizing "ectaquasperm" and with the early spermiogenic stages of internally fertilizing "introsperm" found among the Annelida and basal Mollusca. There are indications that this sperm type may be produced by a truncation of euspermiogenesis, as proposed by Buckland-Nicks & Scheltema for the expression of ectaquasperm in bilaterian evolution.     

Temporal pattern of the double sperm line production in Tubifex tubifex (Annelida,Oligochaeta)

Laboratory cohort cultures of the tubificid Tubifex tubifexwere carried out at 21 °C in homogeneous conditions. Our aim was to study the temporal pattern of spermatogenesis of the two sperm lines which characterise this species. Starting from the second week after cocoon laying, we analysed the seminal vesicle content. We counted the cysts in the vesicles and arranged them into three classes: premeiotic, paraspermatids and euspermatids. Our results show that spermatogenesis begins early, at week 3, but cysts of spermatids are not found until week 5 for the parasperm line and week 6 for the eusperm line. These data are derived from a preliminary study of complex population dynamics. Nonetheless, they allow us to formulate some hypothesis regarding the mechanism which regulate the production of the two types of sperm.     

A phylogenetic analysis of Tubificinae and Limnodriloidinae (Annelida, Clitellata, Tubificidae) using sperm and somatic characters

Marotta, R., Ferraguti, M. & Erséus, C. (2003). A phylogenetic analysis of Tubificinae and Limnodriloidinae (Annelida, Clitellata, Tubificidae) using sperm and somatic characters. — Zoologica Scripta , 32 , 255–278.
The spermatozoa and the sperm aggregates of 13 species belonging to four genera ( Smithsonidrilus, Limnodriloides, Thalassodrilides, Doliodrilus ) in the tubificid subfamily Limnodriloidinae (Annelida, Oligochaeta) were studied and compared with the spermatozoal patterns already described in the subfamily Tubificinae. Two characters considered exclusive for the Tubificinae were found in the more spermatologically variable Limnodriloidinae: the production of two kinds of spermatozoa, eusperm and parasperm, and the presence, in the spermathecae, of sperm aggregates formed by a combination of the two sperm types. A parsimony analysis was performed on the spermatozoal data of the species examined and compared with that based on the somatic characters of the same species: a critical revision of the already codified eusperm characters was carried out and the ultrastructure of parasperm was used as a new subset of spermatozoal characters. In a total evidence approach, a further parsimony analysis was run using a matrix combining both sets of characters. This analysis suggested that the double sperm line and the sperm aggregates composed of both eusperm and parasperm may well be homologous in tubificines and limnodriloidines. It thus supported the previous notion that Tubificinae and Limnodriloidinae are closely related and indicated that these subfamilies may be sister taxa.     

Spermicide, cryptic female choice and the evolution of sperm form and function

Sperm competition and cryptic female choice profoundly affect sperm morphology, producing diversity within both species and individuals. One type of within-individual sperm variation is sperm heteromorphism, in which each male produces two or more distinct types of sperm simultaneously, only one of which is typically fertile (the "eusperm"). The adaptive significance of nonfertile "parasperm" types is poorly understood, although numerous sperm-heteromorphic species are known from many disparate taxa. This paper examines in detail two female-centred hypotheses for the evolution and maintenance of this unconventional sperm production strategy. First, we use game theoretical models to establish that parasperm may function to protect eusperm from female-generated spermicide, and to elucidate the predictions of this idea. Second, we expand on the relatively undeveloped idea that parasperm are used by females as a criterion for cryptic female choice, and discuss the predictions generated by this idea compared to other hypotheses proposed to explain sperm heteromorphism. We critically evaluate both hypotheses, suggest ways in which they could be tested, and propose taxa in which they could be important.     

Ultrastructural analysis of spermatozoa of Korscheltell us lupulinus L. (Lepidoptera : Hepialidae) and Micropterix calthella L. (Lepidoptera : Micropterigidae)

In Korscheltellus lupulinus (Lepidoptera : Hepialidae), both eusperm and parasperm can be found, whereas Micropterix calthella (Lepidoptera : Micropterigidae) produces only eusperm. The eupyrene sperm in K. lupulinus is 250 μm long. Spermatozoa found in K. lupulinus are very similar to those found in higher Lepidoptera, but the eusperm of M. calthella do not possess similar features. As in other Lepidoptera, the axoneme pattern of K. lupulinus is 9 + 9 + 2. Anteriorly, the axoneme is replaced by a fibrous stem similar to an acrosomal structure; posteriorly it becomes progressively uneven. The sperm of K. lupulinus has other original features. The anterior region of the sperm is rigid and contains the nucleus, which has a longitudinal groove adjacent to the axoneme. The 2 mitochondrial derivatives extend from the posterior end of the nucleus. There are no accessory bodies. Many peripheral cytoplasmic vesicles are present. A thin sheath surrounds the cell. It is thick in the parasperm and made of concentric or spiralling lamellae; it does not have any lacinate appendages.The axoneme of the sperm of M. calthella extends over the entire length of the cell and spirals in a helix around the nucleus. The nucleus is also almost as long as the cell. The 2 mitochondrial derivatives, flanked by accessory bodies, follow this spiralling. The axoneme has a 9 + 2 pattern. The plane, which contains the 2 central microtubules, is at an angle with the median plane of the nucleus.     

Nuclear fragmentation characterises paraspermiogenesis in Tubifex tubifex (Annelida, Oligochaeta)

It is known that tubificine oligochaetes produce two types of spermatozoa: eusperm, fertilizing sperm with regular haploid DNA content; and parasperm, with a much lower DNA content, protecting and carrying the eusperm. Whereas mature spermatozoa and spermatids of the two lines are easily recognized by their morphology and DNA content, little is known about the first steps of differentiation of the two lines. This subject is addressed here in two ways: we have measured DNA content by a new method based on confocal laser microscopy and found that the total DNA content of parasperm cysts is extremely variable and equal or lower than total DNA content of eusperm cysts. Then we focused on the spermatocytes, and we found that the cells which will form paraspermatids undergo a peculiar kind of nuclear fragmentation which differ greatly from a regular cell division. During fragmentation DNA is distributed unevenly among the spermatids and this gives rise to a great and variable number of parasperm with variable DNA content. Immunocytochemical assays revealed that a proper meiotic spindle is never formed during fragmentation and that actin may play an important role in the chromatin division. Electron micrographs showed that the centrioles undergo a phenomenon of mass reproduction similar to that found in ciliated epithelia which supplies each of the numerous paraspermatids of its basal body. Mol. Reprod. Dev. 59: 442-450, 2001.     

Integrated and independent evolution of heteromorphic sperm types

Sperm are a simple cell type with few components, yet they exhibit tremendous between-species morphological variation in those components thought to reflect selection in different fertilization environments. However, within a species, sperm components are expected to be selected to be functionally integrated for optimal fertilization of eggs. Here, we take advantage of within-species variation in sperm form and function to test whether sperm components are functionally and genetically integrated both within and between sperm morphologies using a quantitative genetics approach. Drosophila pseudoobscura males produce two sperm types with different functions but which positively interact together in the same fertilization environment; the long eusperm fertilizes eggs and the short parasperm appear to protect eusperm from a hostile female reproductive tract. Our analysis found that all sperm traits were heritable, but short sperm components exhibited evolvabilities 10 times that of long sperm components. Genetic correlations indicated functional integration within, but not between, sperm morphs. These results suggest that sperm, despite sharing a common developmental process, can become developmentally and functionally non-integrated, evolving into separate modules with the potential for rapid and independent responses to selection.     

Paraspermatogenesis in Ceratostoma foliatum (Neogastropoda): confirmation of programmed nuclear death

Sperm dimorphism, where ejaculates contain both fertile "eusperm" and sterile "parasperm", has been implicated in sperm competition in animals. Most parasperm lack an acrosome, eliminate the nucleus and develop a complex cytoplasm, swollen with secretory vesicles. This study of parasperm in the foliate whelk Ceratostoma foliatum, focuses on characterizing the process of nuclear breakdown and the nature of cytoplasmic secretions. Testis squash preparations were treated with a Promega apoptosis detection [terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling-TUNEL] assay (Cedar Lane) to detect nuclear apoptosis; acridine orange was used to localize lysosomes. Parasperm were isolated on Percoll gradients and their proteins characterized by running electrophoresis of lysed, purified fractions on polyacrylamide gels. Results show that the process of nuclear breakdown follows the hallmarks of apoptosis in ultrastructure, which is confirmed by the presence of 3'-OH overhangs of DNA fragments. This research implicates snail parasperm as models for elucidating the nuclear events of apoptosis.     

Parasperm: morphological and functional studies on nonfertile sperm

Sperm polymorphism, a phenomenon in which more than one type of sperm is produced within a species, occurs widely in animals from invertebrates to vertebrates. Sperm in this phenomenon can be categorized into fertile sperm and nonfertile sperm on the basis of fertilization ability. Nonfertile sperm can be further classified into parasperm and aberrant deformed sperm. Parasperm are sperm produced through a constant developmental process along with normal fertile eusperm, and they are readily distinguished from deformed sperm, which are irregularly crippled by unpredictable errors at certain stages during spermatogenesis. Sperm identified as parasperm occur widely in invertebrates but are presently quite limited in vertebrates. This may be the result of the deficiency both of studies on parasperm and of clear criteria to identify parasperm in vertebrates. Some vertebrates show unique spermatogenesis, such as symplastic spermatid and semicystic spermatogenesis. Thus, parasperm must be identified by comparing cells in each cyst and in semen, because irregularly shaped cells in the seminal duct could be either parasperm or normal spermatids. Although parasperm are identified by clear criteria in vertebrates, only in cottoid fishes to date, it is possible for parasperm to be discovered in other vertebrates. Recently, roles related to sperm competition have been reported in several species (e.g., the marine cottoid fish Hemilepidotus gilberti), and, with some of them, parasperm production is influenced by an intraspecific factor such as a sex ratio or the density of a population. Sperm competition is one of the important candidates for influencing evolution of parasperm functions, but not all parasperm seem to have a relation to sperm competition. Parasperm function may relate to the ecological conditions of each species that produces parasperm. Studies on parasperm function will be advanced by an ecological approach concerning male fertilization success as well as cytological investigation for parasperm. An erratum to this article is available at .     

Nuclear matrix involvement in sperm head structural organization

Summary— In the sperm nuclei the DNA is packaged into a highly condensed form and is not organized into nucleosome and solenoid but is bound and stabilized mainly by the protamines that arrange the DNA in an almost crystalline state. As demonstrated for somatic cells, the sperm DNA has been reported to be organized in loop domains attached to the nuclear matrix structures. However, the possible role of the sperm head matrix in maintaining the loop organization in absence of a typical nucleosomal structures has not been fully elucidated. By using in situ nick translation at confocal and electron microscope level, we analyzed the organization of the DNAprotamine complex and its association with the sperm nuclear matrix. The data obtained indicate that the chromatin organization in sperm nuclei is maintained during the sperm condensation by means of interactions with the nuclear matrix at fixed sites. The fine stucture of sperm nucleus and of sperm nuclear matrix, investigated on sections and replicas of freeze-fractured specimens, suggests that the lamellar array, observed by freeze-fracturing in the sperm nuclei, could depend on the inner matrix which presents a regular organization of globular structures possibly involved in the maintenance of chromatin domains in highly condensed sperm nuclei also.     

WHAT USE IS AN INFERTILE SPERM? A COMPARATIVE STUDY OF SPERM‐HETEROMORPHIC DROSOPHILA

Sperm size and number are important determinants of male reproductive success. The genus Drosophila exhibits a remarkable diversity of sperm production strategies, including the production of multiple sperm morphs by individual males, a phenomenon called sperm heteromorphism. Sperm-heteromorphic Drosophila species in the obscura group produce large numbers of infertile "parasperm" in addition to fertile eusperm. Parasperm have been hypothesized to perform a number of roles in place of fertilization, predominantly focused on their potential function in postcopulatory sexual selection. However, the evolutionary significance of parasperm remains unknown. Here we measured several male and female morphological, behavioral, and life-history traits in 13 obscura group species to test competing hypotheses of parasperm function using comparative methods. We found that parasperm size was unrelated to female reproductive tract morphology but was negatively related to our two indices of sperm competition, suggesting that postcopulatory sexual selection may indeed have shaped the evolution of parasperm. We also found abundant coevolution between male and female reproductive traits. Some of these relationships have been found in both sperm-monomorphic and sperm-heteromorphic taxa, but others are dissimilar. We discuss the significance of our results to the evolution of reproductive traits and the elusive function of Drosophila parasperm.     

Reorganization of chromatin in Xenopus egg extracts: electron microscopic studies.

The structural basis of mitotic condensation of chromosomes is one of the problems of cell biology yet to be elucidated. A variety of approaches have been used to study this problem and a large number of hypotheses have been proposed to explain the different levels of compaction of chromatin. Xenopus egg extracts, now widely used to study various aspects of cell biology, provide a valuable tool to study mitotic condensation of chromosomes. No detailed study has however yet been reported on the submicroscopic organization of condensed chromosomes in vitro in egg extracts. We present here the results of our electron microscopic studies on the organization of condensed chromosomes in vitro, using demembranated sperm nuclei and mitotic (CSF-arrested) extracts of Xenopus laevis eggs, clarified by high speed centrifugation. Upon introduction of sperm nuclei in egg extracts, the nuclei swell and the chromatin undergoes a rapid decondensation; at this stage the chromatin is formed of 10 nm fibrils. After longer incubation, the chromatin condenses, and by 2 h chromosomal structures can be visualized by staining with DAPI or Hoechst 33258. Our results on the organization of chromosomes in different stages of condensation are discussed in relation to the different hypotheses proposed to explain the process of mitotic condensation of chromosomes. Finally, this study demonstrates the feasibility of high-resolution analysis of the process of chromosome condensation.