The Ca increase by the sperm factor in physiologically polyspermic newt fertilization: Its signaling mechanism in egg cytoplasm and the species-specificity

The newt, Cynops pyrrhogaster, exhibits physiological polyspermic fertilization, in which several sperm enter an egg before egg activation. An intracellular Ca²⁺ increase occurs as a Ca²⁺ wave at each sperm entry site in the polyspermic egg. Some Ca²⁺ waves are preceded by a transient spike-like Ca²⁺ increase, probably caused by a tryptic protease in the sperm acrosome at the contact of sperm on the egg surface. The following Ca²⁺ wave was induced by a sperm factor derived from sperm cytoplasm after sperm–egg membrane fusion. The Ca²⁺ increase in the isolated, cell-free cytoplasm indicates that the endoplasmic reticulum is the major Ca²⁺ store for the Ca²⁺ wave. We previously demonstrated that citrate synthase in the sperm cytoplasm is a major sperm factor for egg activation in newt fertilization. In the present study, we found that the activation by the sperm factor as well as by fertilizing sperm was prevented by an inhibitor of citrate synthase, palmitoyl CoA, and that an injection of acetyl-CoA or oxaloacetate caused egg activation, indicating that the citrate synthase activity is necessary for egg activation at fertilization. In the frog, Xenopus laevis, which exhibits monospermic fertilization, we were unable to activate the eggs with either the homologous sperm extract or the Cynops sperm extract, indicating that Xenopus sperm lack the sperm factor for egg activation and that their eggs are insensitive to the newt sperm factor. The mechanism of egg activation in the monospermy of frog eggs is quite different from that in the physiological polyspermy of newt eggs.     

SPERM COMPETITION AND THE EVOLUTION OF NUPTIAL FEEDING BEHAVIOR IN THE CRICKET,GRYLLODES SUPPLICANS (WALKER)

The pattern of sperm predominance in doubly mated female crickets, Gryllodes supplicans, was investigated using a radiation-sterility technique. Female G. supplicans made significant use of sperm from both males in fertilizing eggs; overall, first males to mate enjoyed a small advantage, fertilizing about 60% of the offspring produced subsequent to the second mating. The combined use of the sperm of both males in fertilizing eggs occurred soon after the second mating; evidently, mixing of ejaculates within a female's spermatheca does occur. Male G. supplicans provide females with a nuptial gift, the spermatophylax, which influences the time at which a female removes the externally attached sperm-ampulla; this in turn determines the quantity of sperm that is transferred. Moreover, the degree of sperm precedence achieved by a male may be positively related to the time at which the female removes his sperm ampulla. Thus males, by feeding females, ensure not only that a sufficient number of sperm are transferred to fertilize all of a female's eggs, but also may increase the certainty of their paternity. In mating systems in which females control sperm transfer and paternity is influenced by numbers of sperm (i.e., numerical sperm competition), an increase in prezygotic investment in females may be an adaptive male response.     

Serological identification of sperm antigens specified by lethalt-alleles in the mouse

Cytotoxic antisera were prepared by immunizing wild-type recipient mice with sperm from donors carrying different recessive lethal alleles at theT locus (T/t ⁰,T/t ^{w 1} ,T/t ^{w 5} , andT/t ^{w 32} ). After removal of sperm autoantibody by absorption with sperm of recipient type, each antiserum reacted only with sperm from males whose genotype contained at least one of the immunizing alleles. Cytotoxicity was high against sperm populations in which both immunizing alleles were represented and was lower when only one was present. Thus, each allele at theT locus which has so far been tested serologically is recognizable as a discrete antigen on the surface of sperm.This paper is dedicated to Professor Hans Grüneberg, F.R.S. on the occasion of his retirement from the chair of Animal Genetics at University College London, in appreciation of the fact that it was he who first suggested that surface properties played an important role in the origin of abnormalities associated with mutants at theT-locus.     

Gamete interaction in the white sturgeon Acipenser transmontanus: a morphological and physiological review

Synopsis Sturgeon gametes differ from those of most fish in that the sperm possess acrosomes that undergo exocytosis and filament formation while the eggs possess numerous micropyles. Acipenser transmontanus eggs are encased by multilayered envelopes that consist of outer adhesive jelly coats and three structured layers interior to the jelly. The glycoprotein jelly layer only becomes adhesive upon exposure to freshwater. The layer interior to the jelly, layer 3, is the other carbohydrate-containing component of the egg envelope. This layer consists of a water-insoluble glycoprotein that, upon freshwater exposure, is hydrolyzed by a trypsinlike protease to yield a water-soluble, lower molecular weight carbohydrate-containing component. This component can be identified in the surrounding medium when unfertilized eggs are incubated in freshwater. This egg water component elicits acrosome reactions only in homologous sperm. The A. transmontanus sperm acrosome reaction is a Ca⁺⁺ and/or Mg⁺⁺ dependent event that includes the formation of a 10 μ long fertilization filament. A. transmontanus fertilization can occur at low sperm per egg ratios; however, crossfertilization of A. transmontanus eggs with lake sturgeon, A. fluvescens, sperm results in a very low number of fertilized eggs, even at high sperm per egg ratios. The morphological, physiological, and biochemical phenomenon reviewed in this paper are related to the environment in which they occur. Also, the possible role of the acrosome and the presence of numerous micropyles are discussed.     

Experimental evidence that high levels of inbreeding depress sperm competitiveness

The effects of inbreeding on sperm quantity and quality are among the most dramatic examples of inbreeding depression. The extent to which inbreeding depression results in decreased fertilization success of a male’s sperm, however, remains largely unknown. This task is made more difficult by the fact that other factors, such as cryptic female choice, male sperm allocation and mating order, can also drive patterns of paternity. Here, we use artificial insemination to eliminate these extraneous sources of variation and to measure the effects of inbreeding on the competitiveness of a male’s sperm. We simultaneously inseminated female guppies (Poecilia reticulata) with equal amounts of sperm from an outbred (f = 0) male and either a highly (f = 0.59) or a moderately inbred (f = 0.25) male. Highly inbred males sired significantly fewer offspring than outbred males, but share of paternity did not differ between moderately inbred and outbred males. These findings therefore confirm that severe inbreeding can impair the competitiveness of sperm, but suggest that in the focal population inbreeding at order of a brother–sister mating does not reduce a male’s sperm competitiveness.     

THE GENETICS OF ASYMMETRICAL MALE STERILITY IN DROSOPHILA MOJAVENSIS AND DROSOPHILA ARIZONENSIS HYBRIDS: INTERACTIONS BETWEEN THE Y-CHROMOSOME AND AUTOSOMES

Crosses between Drosophila mojavensis and D. arizonensis produce fertile females, but the males from the cross ♂ D. mojavensis × ♀ D. arizonensis are sterile. The chromosomal basis of sperm immotility was studied in these hybrids. Interspecific crossing-over was avoided by crossing hybrid males to pure-species females, and chromosomal identification in backcross progeny was possible by means of electrophoretic markers. The main findings are as follows. The Y-chromosome and two autosomes are involved in the determination of sperm motility. The other autosomes, with the exception of the sixth which was not tested, appear to have no effect. The effect of the D. arizonensis X-chromosome was not examined, but it is established that the D. mojavensis X-chromosome has no effect on sperm motility in males carrying the D. arizonensis Y-chromosome and any combination of autosomes. The Y-chromosome and the two autosomes interact with each other in a simple and predictable way, so that certain combinations of these chromosomes always produce motile sperm and others immotile sperm. Thus, asymmetrical male hybrid sterility may have a simple genetic basis. In contrast to ethological isolation, the genetic basis for this postmating isolating mechanism does not appear to vary among conspecific populations, an observation which suggests that postmating isolation antedates ethological isolation in these species.     

The need for sperm selection may explain why termite colonies have kings and queens,whereas those of ants,wasps and bees have only queens

Hymenoptera have haploid males, which produce sperm by cloning. Sperm selection theory predicts that because termites have diploid males that produce genetically diverse sperm, they may profit from a high sperm surplus (large K), whereas Hymenoptera (ants, bees and wasps) should produce few sperm per fertilization (low Κ). Male reproductive “kings”, which continuously provide spermatozoa during the whole life of the queen, allow for a large K. Available empirical evidence confirms the existence of a large difference in K between diploid insects, especially Blattodea (Isoptera) (K > 1,000), and haplo-diploids such as Hymenoptera (K < 10). The available data suggest that sperm selection may be an important evolutionary force for species with diploid, but not haploid males.     

INDIVIDUAL VARIATION IN SPERM COMPETITION SUCCESS OF YELLOW DUNG FLIES,SCATOPHAGA STERCORARIA

Intraspecific variation in the proportion of offspring sired by the second male to mate with a female (P₂) is an aspect of sperm competition that has received little attention. We examined variation in the sperm competition success of individual male dung flies, Scatophaga stercoraria. In unmanipulated matings, copula duration was dependent on male size with smaller males copulating for longer. A principal component analysis was used to generate uncorrelated scores based on a male's size and copula duration. Using these scores demonstrated that P₂ values were dependent both on the relative size and copula durations of competing males. When copula duration was held constant, the success of an individual male increased as his body size, relative to the first male, increased. We interrupted copulations of “large” and “small” second males and fitted the resultant P₂ values to a linear model of sperm competition with unequal ejaculates. The data fit well to a model of sperm displacement in which sperm mix quickly on introduction to the sperm stores. Furthermore, they show that “large” males have a greater rate of sperm displacement than “small” males. The levels of prey availability during testis maturation may influence a male's success in sperm competition although his immediate mating history does not. We show why an understanding of variation in sperm competition success is important for understanding the mechanisms and evolutionary significance of sperm competition.     

Daily rhythm in myogenic contractions of vas deferens associated with sperm release cycle in a moth

In the gypsy moth, Lymantria dispar, release of sperm bundles from the testis into the upper vas deferens (UVD) and subsequent transfer of sperm bundles into the seminal vesicles (SV) occurs in a daily rhythm. The UVD undergoes different types of contractions despite the fact that its musculature appears to receive no innervation. Patterns of the UVD movements were recorded throughout the daily sperm release and transfer cycle. In males kept in light-dark cycles, transfer of sperm from the UVD to the SV was accompanied by a characteristic pattern of UVD contractions of high frequency and amplitude. In males kept in constant light, which fail to transfer sperm, this contraction pattern was absent. It is concluded that the vas deferens muscles undergo daily changes in contraction pattern in phase with the light-dark cycle. The increased muscular contractions appear to be a causal factor in the gated sperm transfer from the UVD to the SV.Abbreviations LD light-dark - LL constant light - SV seminal vesicle - UVD upper vas deferens     

Interspecific Variation in Structural Organisation of the Spermatozoon in the Asian Bandicoot Rats,Bandicota Species (family Muridae)

The structural organisation of the spermatozoon from two species of bandicoot rats Bandicota bengalensis and Bandicota indica was investigated by light and electron microscopy together with the effect of incubation in Triton-X 100 and sodium dodecyl sulphate. The sperm head of B. bengalensis is invariably falciform, has a uniform electron-dense nucleus capped by an acrosome with a posteriolateral equatorial segment, a subacrosomal cytoskeleton with a large rostral perforatorium, and a sperm tail, attached to the lower concave surface of the sperm head, with typical coarse fibres and fibrous sheath. By contrast, the sperm head shapes of B. indica are generally conical or bulbous, the nucleus contains a few large vacuoles, the acrosome lacks an equatorial segment, no recognisable perforatorium occurs, and the sperm tail, which is attached basally, is very short with only modest development of coarse fibres and fibrous sheath. These results indicate that, within the genus Bandicota, huge interspecific differences in morphology of the spermatozoon have evolved. The spermatozoa of B. bengalensis are similar to those of Rattus and many other murids and thus presumably represent the ancestral condition, whereas those of B. indica (and B. savilei) are unlike spermatozoa from any other eutherian mammal so far described. © 1998 The Royal Swedish Academy of Sciences. Published by Elsevier Science Ltd. All rights reserved     

Quantitative cytology of the sperm cells of Brassica campestris and B. oleracea

Pollen grains of Brassica campestris L. var. acephala DC and B. oleracea L. were serially sectioned and examined using transmission electron microscopy to determine the three-dimensional organization of sperm cells within the microgametophyte and the quantity of membrane-bound organelles occurring within each cell. Sperm cells occur in pairs within each pollen grain, but are dimorphic, differing in size, morphology and mitochondrial content. The larger of the two sperm cells (S_vn) is distinguished by the presence of a blunt evagination, which in B. oleracea wraps around and lies within shallow furrows on the vegetative nucleus and in B. campestris can penetrate through internal enclaves of the vegetative nucleus. This sperm cell contains more mitochondria in both species than the second sperm cell (S_ua). This latter cell is linked to the first by a common cell junction with the S _vn, but is not associated with the vegetative nucleus and lacks a cellular evagination. Such differences are indicative of a system of cytoplasmic heterospermy in which sperm cells possess significantly different quantities of mitochondria.Abbreviations mtDNA mitochondrial DNA - S_ua sperm cell unassociated with the vegetative nucleus - S_vn Sperm cell physically associated with the vegetative nucleus     

Changes in microtubule structures during the first cell cycle of physiologically polyspermic newt eggs

The unfertilized egg of the newt, Cynops pyrrhogaster, has a second meiotic spindle at the animal pole and numerous cortical cytasters. After physiologically polyspermic fertilization, all sperm nuclei incorporated into the egg develop sperm asters, and the cortical cytasters change into bundles of cortical microtubules. The size of the sperm asters in the animal hemisphere is ∼5.6-fold larger than that in the vegetal hemisphere. Only one sperm nucleus moves toward the center of the animal hemisphere to form a zygote nucleus with the egg nucleus. This movement is inhibited by nocodazole, but not by cytochalasin B. The centrosome in the zygote nucleus divides into two parts to form a bipolar spindle for the first cleavage synchronously with the nuclear cycle, but centrosomes of accessory sperm nuclei in the vegetal hemisphere remained to form monopolar interphase asters and subsequently degenerate around the first cleavage stage. The size of sperm asters in monospermically fertilized Xenopus eggs was ∼37-fold larger than those in Cynops eggs. Since sperm asters that formed in polyspermically fertilized Xenopus eggs exclude each other, the formation of a zygote nucleus is inhibited. Cynops sperm nuclei form larger asters in Xenopus eggs, whereas Xenopus sperm nuclei form smaller asters in Cynops eggs compared with those in homologous eggs. Since there was no significant difference in the concentration of monomeric tubulin between those eggs, the size of sperm asters is probably regulated by a component(s) in egg cytoplasm. Smaller asters in physiologically polyspermic newt eggs might be useful for selecting only one sperm nucleus to move toward the egg nucleus. Mol. Reprod. Dev. 47:210–221, 1997. © 1997 Wiley-Liss, Inc.     

Migration of sperm cells during pollen tube elongation in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>: behavior during transport,maturation and upon dissociation of male germ unit associations

The promoter sequence of sperm-expressed gene, PzIPT isolated from the S_vn (sperm associated with the vegetative nucleus) of Plumbago zeylanica, was fused to a green fluorescent protein (GFP) reporter sequence and transformed into Arabidopsis thaliana to better visualize the live behavior of angiosperm sperm cells. Angiosperm sperm cells are not independently motile, migrating in a unique cell-within-a-cell configuration within the pollen tube. Sperm cells occur in association with the vegetative nucleus forming a male germ unit (MGU). In Arabidopsis, GFP was expressed equally in both sperm cells and was observed using a spinning disk confocal microscope, which allowed long duration observation of cells without bleaching or visible laser radiation damage. Pollen activation is reflected by conspicuous movement of sperm and pollen cytoplasm. Upon pollen germination, sperm cells enter the forming tube and become oriented, typically with a sperm cytoplasmic projection leading the sperm cells in the MGU, which remains intact throughout normal pollen tube elongation. Maturational changes, including vacuolization, general rounding and entry into G2, were observed during in vitro culture. When MGUs were experimentally disrupted by mild temperature elevation, sperm cells no longer tracked the growth of the tube and separated from the MGU, providing critical direct evidence that the MGU is a functional unit required for sperm transmission.     

Functional association between female sperm storage organs and male sperm removal organs in calopterygid damselflies

Female damselflies in the family Calopterygidae have two sperm storage organs: a spherical bursa copulatrix and a tubular spermatheca. Male flies have a peculiar aedeagus with a recurved head with which to remove bursal sperm, and lateral spiny processes to remove spermathecal sperm. The lateral processes differ among species and populations in terms of their width relative to the spermathecal duct: the narrower processes are physically able to access spermathecal sperm, while the wider ones are not. In the present study, sperm storage patterns and aedeagal structures were compared between two calopterygid species with different spermathecal structures –Calopteryx cornelia and Mnais pruinosa– with respect to not only sperm quantity (number) but also sperm quality (viability), by using a recently developed method based on live/dead dual fluorescence. Calopteryx cornelia is a typical spermathecal sperm remover. In this species, viability was similar between bursal and spermathecal sperm. In contrast, in M. pruinosa, the spermatheca was much smaller than the bursa and often contained no sperm. Even when the spermatheca of this species did contain sperm, a high percentage of it was dead. Although the spermatheca of M. pruinosa has such atrophic tendencies, males have nevertheless developed long and spiny lateral processes similar to those of C. cornelia, suggesting the processes have functions other than spermathecal sperm removal. They possibly function as stoppers or guides for manipulating the aedeagal head to remove the sperm mass from the bursa.     

Variation in the post‐mating fitness landscape in fruit flies

Sperm competition is pervasive and fundamental to determining a male's overall fitness. Sperm traits and seminal fluid proteins (Sfps) are key factors. However, studies of sperm competition may often exclude females that fail to remate during a defined period. Hence, the resulting data sets contain fewer data from the potentially fittest males that have most success in preventing female remating. It is also important to consider a male's reproductive success before entering sperm competition, which is a major contributor to fitness. The exclusion of these data can both hinder our understanding of the complete fitness landscapes of competing males and lessen our ability to assess the contribution of different determinants of reproductive success to male fitness. We addressed this here, using the Drosophila melanogaster model system, by (i) capturing a comprehensive range of intermating intervals that define the fitness of interacting wild‐type males and (ii) analysing outcomes of sperm competition using selection analyses. We conducted additional tests using males lacking the sex peptide (SP) ejaculate component vs. genetically matched (SP⁺) controls. This allowed us to assess the comprehensive fitness effects of this important Sfp on sperm competition. The results showed a signature of positive, linear selection in wild‐type and SP⁺ control males on the length of the intermating interval and on male sperm competition defence. However, the fitness surface for males lacking SP was distinct, with local fitness peaks depending on contrasting combinations of remating intervals and offspring numbers. The results suggest that there are alternative routes to success in sperm competition and provide an explanation for the maintenance of variation in sperm competition traits.     

Participation of sperm proteasome in fertilization of the phlebobranch ascidian Ciona intestinalis

Sperm proteasomes are thought to be involved in sperm binding to and in sperm penetration through the vitelline coat of the eggs of the stolidobranch ascidian Halocynthia roretzi. However, it is not known whether they are involved in the fertilization of eggs of other ascidians. Therefore, we investigated whether sperm proteasomes are also involved in the fertilization of the eggs of the primitive phlebobranch ascidian Ciona intestinalis. Fertilization of the eggs of C. intestinalis was potently inhibited by the proteasome inhibitors MG115 and MG132 but not by the cysteine protease inhibitor E-64-d. On the other hand, neither fertilization of the vitelline coat-free eggs nor sperm binding to the vitelline coat was inhibited by the two proteasome inhibitors at a concentration sufficient to inhibit fertilization of intact eggs. These results indicate that the proteasome plays an essential role in sperm penetration through the vitelline coat rather than in sperm binding to the coat or in sperm-egg membrane fusion. The proteasome activity, which was detected in the sperm extract using Suc-Leu-Leu-Val-Tyr-MCA as a substrate, was strongly inhibited by both MG115 and MG132, and was weakly inhibited by chymostatin, whereas neither leupeptin nor E-64-d inhibited the activity. The molecular mass of the enzyme was estimated to be 600-kDa by Superose 12 gel filtration, and the activity in sperm extract was immunoprecipitated with an anti-proteasome antibody. These results indicate that the proteasome present in sperm of C. intestinalis is involved in fertilization, especially in the process of sperm penetration through the vitelline coat, probably functioning as a lysin. Mol. Reprod. Dev. 50:493–498, 1998. © 1998 Wiley-Liss, Inc.     

Ultrastructural observations on the spermatozoa of a species of <Emphasis Type="Italic">Lepidodasys</Emphasis> (Gastrotricha,Macrodasyida)

The ultrastructure of the spermatozoon of a species in the marine gastrotrich genus Lepidodasys is described. The filiform cell is composed of a cork-screw acrosome, a long single mitochondrion surrounded by a helical nucleus, and a flagellum with a 9 × 2 + 2 axonemal arrangement. The structure of the sperm of this species from Denmark appears closely similar to those of the other two species of Lepidodasys studied so far from Italy and Florida (US). Peculiar features (cylindrical nucleus, absence of a periaxonemal sheath) place this genus far from the others in the family Lepidodasyidae. The absence of synapomorphies between Lepidodasys and other genera of Lepidodasyidae suggests that the family is polyphyletic. The sperm ultrastructure fully fits the species of Lepidodasys into the marine order Macrodasyida, with the sperm ground plan of which its sperm shares a number of details.     

A scanning electron microscopical study of sperm development and activation in Arenicola marina L. (Annelida: Polychaeta)

Abstract

The lugworm, Arenicola marina L. has an annual cycle of reproduction with epidemic spawning and external fertilisation. The spermatozoa of Arenicola are unusual in that they are held immotile (as plates of several hundred cells known as morulae) in the coelomic fluid until activated just prior to spawning. Activation of Arenicola sperm is brought about by a sperm maturation factor (SMF) from the prostomium and can be carried out in vitro using an assay technique developed by Bentley (1985). Scanning electron microscopy is used here to examine the changes which occur during in vitro activation. This revealed that the bundles of flagella of inactive sperm become disorganised as flagella beating commences but the flagella at this stage are still bound together at their tips. The sperm heads then become separated from the cytophore and finally the distal binding of the flagella is broken to give free-swimming spermatozoa. Coelomocytes present in the coelomic fluid resorb unspawned gametes prior to the initiation of the next gametogenic phase.     

Interspecific variation of sperm morphology in the Australian rodent genus Zyzomys

The sperm head morphology and tail length of two species of Australian rock rats, Zyzomys argurus and Zyzomys pedunculatus, are presented. In Z. argurus the sperm head has an apical hook together with two ventral processes extending from the upper concave surface that are largely composed of cytoskeletal material, and the sperm tail is about 135 µm in length. By contrast, in Z. pedunculatus the sperm head is paddle‐shaped with the nucleus capped by an acrosome that has a large apical segment and is surrounded by a thin layer of cytoskeletal material, and the sperm tail is only around 85 µm in length. Since the structure of the spermatozoon of Z. argurus is similar to that of most of the old endemic Australian rodents it is presumed to be the ancestral condition within the Zyzomys genus with that of Z. pedunculatus being highly derived and showing convergence with the sperm structure in some other orders of mammals.