Evolution of octopod sperm I: comparison of nuclear morphogenesis in Eledone and Octopus

Morphogenesis of the Eledone cirrhosa sperm nucleus, as studied by electron microscopic techniques, is compared with that of Octopus vulgaris. Both species of cephalopods belong to the family Octopodidae. The results indicate that extensive nuclear helicoidization during E. cirrhosa spermiogenesis is brought about by modifications of the function of structural components already present in the late steps of O. vulgaris spermiogenesis. In particular, changes in the regulation of perinuclear microtubule contraction in E. cirrhosa spermatids, as well as a decrease in basicity of protamines, promote nuclear helicoidization. Disulphide bond formation between protamine molecules fixes the completely helicoidal shape of the nucleus in mature sperm of E. cirrhosa.     

Evolution of octopod sperm II: comparison of acrosomal morphogenesis in Eledone and Octopus

The first stages of acrosome development during Eledone cirrhosa spermiogenesis are similar to that in Octopus vulgaris, and comprise the initial elongation of both organelles. However, the acrosome in E. cirrhosa does not continue its elongation as it does in O. vulgaris. Instead, its length remains fixed and it undergoes a process of helicoidization that includes the entire organelle. In each spermatid, helicoidization of the E. cirrhosa acrosome occurs simultaneously with helicoidization of the nucleus. The acrosome is associated with special structures that probably are involved in the helical torsion of the organelle. We propose a hypothesis to explain the evolutionary relationship between the acrosomes of O. vulgaris and E. cirrhosa, particularly as it is influenced by nucleomorphogenesis and microtubular contraction.     

The cardiac innervation of Eledone cirrhosa (Lamarck) (Mollusca: Cephalopoda)

The innervation to the cardiac organs and vessels of the octopods Eledone cirrhosa, E. moschata and Octopus vulgaris is described from vitally stained fresh material and wax-embedded sections. This innervation arises from the paired visceral nerves and includes two main peripheral ganglia (fusiform and cardiac) on each side. Several new details of the innervation are reported. Nerves supplying the lateral venae cavae arise from the ventricular nerves at the level of the ventricle. Nerve fibres run to the efferent branchial vessels from the cardiac ganglia. A small ganglion, lying on the auriculo-ventricular nerve, is described for some specimens of both species of Eledone, and is named the auricular ganglion. Commissural strands linking the right and left ventricular nerves of either side are found in Eledone, comparable to those previously described from Octopus. The detailed branching pattern of the innervation shows considerable individual variation and consistent interspecific differences. In E. cirrhosa the fine fibres innervating the inner and outer muscle layers of the auricle show distinct differences in their configuration. Innervation at the surface of the ventricular lumen and around the coronary arterial vessels shows evidence of specialization. The muscle of the branchial heart, particularly the valve leaflets at the junction of the heart and the lateral vena cava, is abundantly innervated. The observations are discussed in relation to other cephalopods and to their probable physiological significance. It is suggested that they provide evidence for a greater degree of neural influence in the control of the cardiac organs than is usually supposed and that they support the idea that the lateral venae cavae have a significant role in the generation of circulatory pressures.     

Hole boring of crustacean prey by the octopus Eledone cirrhosa (Mollusca, Cephalopoda)

Eledone cirrhosa has been found to make a borehole in the carapace of a high proportion of its crustacean prey. This is the first account of drilling in crustaceans by octopus. The frequency of incidence of the boring behaviour varied between prey species from 17 to 93% of those killed. The incidence of boreholes was higher in crabs killed by small octopuses. Using crabs mainly of the genera Cancer, Carcinus, Corystes and Macropipus , the distribution and orientation of the boreholes was recorded. The boreholes occurred in any part of the carapace but the great majority were found close to the mid-line and in the posterior half. The long axis of the oval penetration was usually aligned with the anteroposterior axis of the crab. The Mediterranean species Eledone moschata was also found to bore crabs, and newly hatched juveniles of this species are capable of boring as early as their second killing of a live crab. No evidence was found that Octopus vulgaris normally bores the carapace of crabs.     

Crab and mollusc shell drilling by Octopus vulgaris (Mollusca: Cephalopoda) in the Ria de Vigo (north-west Spain)

Octopus vulgaris living in the Ria de Vigo, north-west Spain, sometimes drills a cavity in the carapace of crabs and shells of molluscs. These holes have features with the same characteristics as those made in molluscs by O. vulgaris and in crabs by Eledone cirrhosa when isolated in aquaria. This is the first report of drilling the carapace of crabs by Octopus vulgaris .     

Optic gland implants and their effects on the gonads of Octopus.

1. Optic glands transplanted from one Octopus vulgaris into another cause enlargement of the gonads and ducts of the recipient. 2. Enlargement occurs whether or not the gland was secreting when implanted and regardless of the sex of the donor or recipient. 3. Glands derived from Eledone moschata or Octopus macropus implanted into O. vulgaris are as effective as glands derived from O. vulgaris. 4. Implants derived from Sepia officinalis or Loligo vulgaris appear to be ineffective.     

Chromatin condensation,cysteine-rich protamine,and establishment of disulphide interprotamine bonds during spermiogenesis of Eledone cirrhosa (Cephalopoda)

During spermiogenesis in Eledone cirrhosa a single protamine substitutes for histones in nuclei of developing spermatids. This protein displays a peculiar primary structure. It contains 22.6 mol% cysteine residues (19 cysteines in 84 residues). This makes it the most cysteine-rich protamine known. The proportion of basic residues is relatively low (arginine 36.9 mol%, lysine 19.0 mol%). The protamine of E. cirrhosa condenses spermiogenic chromatin in a pattern which comprises fibres with a progressively larger diameter and lamellae that finally undergo definitive coalescence. We have also performed a study that estimates the number of interprotamine disulphide bonds formed during the process of spermiogenic chromatin condensation by means of sequential disappearance of MMNA (monomaleimido-nanogold) labelling. During the first step of spermiogenesis, protamines are found spread over very slightly condensed chromatin with their cysteines in a reactive state (protamine-cys-SH). From this stage the interprotamine disulphide bonds are established in a progressive way. First they are formed inside the chromatin fibres. Subsequently, they participate in the mechanism of fibre coalescence and finally, in the last step of spermiogenesis, the remaining free reactive -SH groups of cysteine form disulphide bonds, thus promoting a definitive stabilization of the nucleoprotein complex in the ripe sperm nucleus.     

Phospholipids in Mediterranean cephalopods

Polar lipids of the cephalopods Eledone moschata, Sepia officinalis and Todarodes sagittatus mantle, represent 50.5%, 66.1% and 74.2% of wet tissue respectively. On the other hand the polar lipids of these three species of cephalopods constitute of 80.8%, 94.8% and 93.7% of phospholipids, respectively. The main phospholipids identified were phosphatidylcholine (52.2, 51.3 and 58.4% of total phospholipids respectively in the above mentioned species), phosphatidylethanolamine (18.1, 19.7 and 23.9%), sphingomyelin (10.7, 15.2 and 6.7%), lyso-phosphatidylcholine (3.1, 3.8 and 1.8%) and the unusual lipid ceramide aminoethylphosphonic acid (15.9, 10 and 9.2%). The 56.8% of phosphatidylcholine in Eledone moschata, the 46% in Sepia officinalis and the 74.1% in Todarodes sagittatus refer to the structure of 1,2-diacyl-glycerocholine and the remaining percentage refer to the structure of 1-o-alkyl-2-acyl-glycerocholine or 1-o-alkyl-1-enyl-2-acyl-glycerocholine. The 87.2% of phosphatidylethanolamine in Eledone moschata, the 81% in Sepia officinalis and the 90.7% in Todarodes sagittatus refer to the structure of 1,2-diacyl-glyceroethanolamine and the remaining percentage refer to the structure of 1-o-alkyl-2-acyl-glyceroethanolamine or 1-o-alkyl-1-enyl-2-acyl-glyceroethanolamine. The major saturated fatty acids in phosphatidylcholine and phosphatidylethanolamine were C16:0 (30.3-67.5% and 23.2-54.5%) and C18:0 (3.6-17% and 15.4-28%), respectively, while the major unsaturated fatty acids in these lipids were C18:1n-9, n-7 (1.0-7.3% and 5.3-10.5%), C20:5n-3 (1.5-9.8% and 4,5-15.8%) and C22:6n-3 (12.5-42.0% and 7.0-11.3%), respectively.     

Biochemical dynamics of spermatogenesis and oogenesis in Eledone cirrhosa and Eledone moschata (Cephalopoda: Octopoda)

The effect of spermatogenesis and oogenesis on protein, lipid, glycogen, cholesterol and energy contents, total amino acid and fatty acid profiles of Eledone cirrhosa and Eledone moschata tissues (gonad, digestive gland and muscle) was investigated. A significant (p<0.05) increase in the amino acids and protein content of the gonad throughout sexual maturation (namely in oogenesis) was observed, but the allocation of these nitrogen compounds from the digestive gland and muscle was not evident. The major essential amino acids (EAA) in the three tissues were leucine, lysine and arginine. The major nonessential amino acids (NEAA) were glutamic acid, aspartic acid and alanine. A significant increase in lipid and fatty acid contents of gonad and digestive gland was observed. There was also little evidence of accumulated lipid storage reserves being used for egg production. It seems that for egg production Eledone species use energy directly from food, rather than from stored products. Most of saturated fatty acid (SFA) content of the three tissues was presented as 16:0 and 18:0, monounsaturated fatty acid (MUFA) content as 18:1 and 20:1 and polyunsaturated fatty acid (PUFA) content as 20:4n-6, 20:5n-3 and 22:6n-3. Cholesterol and glycogen contents significantly increased in gonad and digestive gland throughout maturation while the muscle revealed no obvious pattern. If Eledone's component sterols are of a dietary origin, a considerable variation in the cholesterol content between species might be expected on the basis of the sterol composition of their prey. Although spermatogenesis and oogenesis had a significant effect (p<0.05) in gonad and digestive gland energy content, the biochemical composition of digestive gland and muscle may not be primarily influenced by sexual maturation, but rather by other biotic factors such as feeding activity, food availability, spawning and brooding.     

GLUTAMATE METABOLISM IN OCTOPUS BRAIN IN VIVO;ABSENCE OF A WAELSCH EFFECT

Abstract— The metabolism of [U-¹⁴C]glutamate was followed in vivo in the octopus Eledone cirrhosa following intracranial injection, and compared with that in the mammalian brain.
By contrast with the rat brain, the specific activity of glutamine recovered from Eledone optic and vertical lobes was lower than that of glutamate at short time intervals after injection. Thus the Waelsch effect was not apparent in this species. Again, in contrast with the rat brain, radioactivity could be found in alanine but not in GABA following [U-¹⁴C]glutamate injection. This was compatible with observations made previously in vitro.
The significance of these intraspecies differences in metabolism and compartmentation is discussed.     

Wound healing and the haemocyte response in the skin of the Lesser octopus Eledone cirrhosa (Mollusca: Cephalopoda)

The general structure of the skin of the Lesser octopus, Eledone cirrhosa is described. The component regions and cell types being (1) the epidermis, composed of columnar cells with a border of microvilli on their external surface, mucous cells and secretory cells of an unknown type, abutting a non cellular basal lamina; (2) the dermis, containing the elements of the colour change system, vascular and neural processes and an a cellular matrix.
A sequential, morphological study of wound closure revealed three major mechanisms in the healing response; muscular contraction, a dermal cellular reaction and epidermal migration. The process of wound closure in Eledone is significantly slower than that seen, for example, in teleost fish (30 h rather than 30 min for the initiation of epidermal migration). The significance of these results in relation to the osmotic balance between seawater and the internal milieu of Eledone is discussed, together with their relevance to resistance against infection by pathogens.     

Absorption of D(+)glucose by the intestine of the eledone (Eledone moschata Lamarch): effect of phloridzoside]

"The in vivo intestinal absorption of D(+)glucose by Eledone (Eledone moschata Lamarck), a cephalopod mollusc, is decreased by 2.10(-4)M phlorizin and increased by 2.10(-2)M phlorizin. It is noted that phlorizin is dissolved in the glucose solution before its introduction into the intestinal tube. The first result is similar to that known in vertebrates; the second differs."     

An electron microscopic radioautographic study of the uptake of tritiated serotonin by nerve fibres in the posterior salivary duct and gland of cephalopods

In the posterior salivary duct and gland of Octopus vulgaris and of Eledone cirrhosa, the duct secretory nerve trunks and their ramifications in the gland tubules include many fibres that incorporate labelled serotonin. However, there are also unlabelled secretory fibres, which cannot be discriminated from incorporating fibres on morphological grounds. Neuroglandular junctions are not apparently established by incorporating fibres. In the duct, the motor nerve trunks contain a small number of labelled fibres, and nerve bundles supplying the duct muscle contain, in variable proportions, serotonin incorporating fibres. Both labelled and unlabelled nerve fibres reach the duct muscle fibres, but neuromuscular junctions involve only unlabelled presynaptic fibres. The nerve fibres which join the gland muscle are usually unlabelled, and the small quota of incorporating fibres in the motor trunks apparently supply only duct tissues. Both secretory and motor trunks, originating from different ganglia, can be considered to contain heterogeneous fibres, releasing different neurotransmitters at the terminals. Certain of these fibres could be serotoninergic.     

Spermatozoa of the deep-sea cephalopod Vampyroteuthis infernalis Chun: ultrastructure and possible phylogenetic significance

Sperm ultrastructure in the rare deep-sea cephalopod Vampyroteuthis infernalis is described, based on formalin-fixed material held in the Australian Museum (Sydney). The species is the sole member of the coleoidean order Vampyromorpha, which represents a level of organization intermediate between that of the Sepioidea-Teuthoidea and the Octopoda. Spermatozoa of Vampyroteuthis, the simplest observed in any cephalopod, exhibit the following features: (1) a spheroidal acrosome lacking any complex substructure; (2) a short (8.5 microns) fusiform nucleus with a deep (2.2-2.5 microns) basal invagination (containing an extensive plug of dense material); (3) two triplet centrioles arranged parallel to the sperm longitudinal axis; (4) a short (1 micron) midpiece composed of a triangular cluster of mitochondria surrounding the centrioles; and (5) a tail (length 130-135 microns) that is continuous with one of the centrioles (here considered as a 'distal' centriole). An annulus and membranous skirt are absent, though the coarse fibres do fuse into a ring at the tail-midpiece junction). These cells show some resemblance to sperm or spermatids of sepioids and teuthoids (spheroidal acrosome, short nucleus) but are also remarkably similar to mid-spermatids of Octopus (with the exception of the uncondensed nucleus in Octopus spermatids). Sperm morphology supports the current assignment of Vampyroteuthis to a separate coleoidean order--Vampyromorpha--and also suggests that a close link exists between the Vampyromorpha and Octopoda.     

Localization of the Rho GTPases and some Rho effector proteins in the sperm of several mammalian species

The acrosome reaction is a fundamental event in the biology of the sperm and is a prerequisite to fertilization of the egg. Members of the Rho family of GTPases and their effectors are present in the cytoplasm and/or plasma membrane overlying the acrosome of porcine sperm. We have implicated the Rho family of GTPases and the Rho-activated kinase, ROCK-1, in mediating the zona-pellucida-induced acrosome reaction. Others have implicated the Rho GTPase in regulating the ionophore-induced acrosome reaction in the sperm of several mammalian species as well as in motility of bovine sperm. In this study, the localization of the Rho GTPases (RhoA, RhoB, Rac1 and Cdc42) as well as the effectors RhoGDI, PI(4)P5K and ROCK-1, was determined in boar, human, rat, ram, bull and elephant sperm. The four GTPases were each present in the sperm head of all species examined. RhoGDI was expressed in the head and tail of sperm from all species except pig, where it was present only in the head. PI(4)P5K was expressed in both head and tail of sperm from all species, but expression was typically weaker in the tail. Finally, ROCK-1 was expressed in the heads and tails of all sperm except that of the boar, where it was present only in the acrosomal region. These observations taken together suggest that the expression of Rho GTPases in sperm has been conserved throughout mammalian evolution, most likely due to the role of these GTPases in regulating acrosomal exocytosis.     

Energy balance and cold adaptation in the octopus Pareledone charcoti

A complete energy balance equation is calculated for the Antarctic octopus Pareledone charcoti at 0 degrees C. Energy used in respiration, growth, and excretion of nitrogenous and faecal waste, was recorded along with the total consumption of energy through food, for three specimens of P. charcoti (live weights: 73, 51 and 29 g). Growth rates were very slow for cephalopods, with a mean daily increase in body weight of only 0.11%. Assimilation efficiencies were high, between 95.4 and 97.0%, which is consistent with previous work on octopods. The respiration rate in P. charcoti was low, with a mean of 2.45 mg O(2) h(-1) for a standard animal of 150 g wet mass at 0 degrees C. In the North Sea octopus Eledone cirrhosa, respiration rates of 9.79 mg O(2) h(-1) at 11.5 degrees C and 4.47 mg O(2) h(-1) at 4.5 degrees C for a standard animal of 150 g wet mass were recorded. Respiration rates between P. charcoti and E. cirrhosa were compared using a combined Q(10) value between P. charcoti at 0 degrees C and E. cirrhosa at 4.5 degrees C. This suggests that P. charcoti are respiring at a level predicted by E. cirrhosa rates at 4.5 and 11.5 degrees C extrapolated to 0 degrees C along the curve Q(10)=3, with no evidence of metabolic compensation for low temperature.     

Analysis of terminal sugar moieties and species-specificities of acrosome reaction-inducing substance in Xenopus (ARISX)

The acrosome reaction of Xenopus sperm is triggered by the acrosome reaction-inducing substance in Xenopus (ARISX), an oviductal pars recta-derived, sugar-rich substance decorated on the entire surface of the vitelline envelope (VE) during ovulation. Here we addressed the functional importance of the sugar moiety in ARISX. Among various lectins examined, soybean agglutinin and Dolichos biflorus agglutinin were shown to abolish the acrosome reaction-inducing activity of ARISX present in pars recta extract or on the VE, indicating the importance of the terminal alpha-N-acetylgalactosamine residue for the function of ARISX. Consistently, the acrosome reaction-inducing activity was not affected by proteinase K digestion, in spite of the simultaneous shift of ARISX to a smaller molecular weight. Indirect immunofluorescence microscopic examinations showed that ARISX was distributed as two types of structures on VE; thick fiber-like materials and thin filamentous materials, and that a new structure appeared on the fertilization envelope instead of the thin filamentous materials. Sperm from several amphibian species were subjected to an in vitro assay during induction of the acrosome reaction with ARISX. The resulting limited population of sperm from a non-Xenopus species underwent acrosome reaction, implying a weak species-specificity of ARISX.     

Jelly coat and induction of the acrosome reaction in echinoid sperm

In a previous study we established that sperm from four species of echinoids differ in their specificity for induction of the acrosome reaction by heterotypic jelly coat and we presented evidence that there were only small compositional differences in the active component of the jelly coat, a polysaccharide composed of fucose sulfate units. In the current report we present additional studies related to the species specificity of jelly coat with respect to Ca²⁺ uptake (or exchange), which occurs concomitantly with the acrosome reaction, and activation of phospholipase activity, which appears to occur subsequent to the acrosome reaction. The specificity of jelly coat in inducing these processes is the same as that previously observed in induction of the acrosome reaction. Binding of jelly coat to sperm has been demonstrated, and has been shown to be species specific. This finding raises the possibility that a receptor for jelly coat exists on the surface of the sperm. Finally, based on chemical and physical-chemical studies, evidence is presented that establishes that, despite compositional similarities, the fucose sulfate polysaccharides from the four species of eggs differ in structure.     

Acrosome reaction is subfamily specific in sea star fertilization

In the fertilization process of sea stars, sperm is activated to go through the acrosome reaction before cell fusion. We focused on induction of the acrosome reaction as a key process in fertilization. Six species of sea stars were used in this study: Asterias amurensis, Asterias rubens, Asterias forbesi, Aphelasterias japonica, Distolasterias nipon, and Asterina pectinifera. Acrosome reaction assays indicate that the acrosome reaction can be induced across species within Asteriinae subfamily. However, cross-fertilization assays indicate that sea stars have species specificity in fertilization. Therefore, steps after the acrosome reaction are responsible for the species specificity. To explain acrosome reaction subfamily specificity at the molecular level, the sugar components of egg jelly were examined and analyzed by principal component analysis. A. amurensis and A. forbesi belong to the same induction group of the acrosome reaction. D. nipon and An. pectinifera are in a unique group. Enzyme-linked immunosorbent assays indicate that Asteriinae subfamily share a common glycan structure, the Fragment 1 of Acrosome Reaction-Inducing Substance from A. amurensis. Fragment 1 plays an important role in the subfamily specificity of acrosome reaction induction. In addition, A. amurensis sperm activating peptide was recognized by sperm from the same superorder. These results demonstrate that the specificity of acrosome reaction induction is present at the subfamily level in sea stars.     

Sperm ultrastructure in representatives of six bivalve families from Peter the Great Bay, Sea of Japan

The ultrastructure of sperm in seven species of bivalves, the representatives of six families, Arcidae (Anadara broughtonii, Arca boucardi), Anomiidae (Pododesmus macrochisma), Tellinidae (Macoma tokyoensis), Ostreidae (Crassostrea gigas), Myidae (Mya japonica) and Trapezidae (Trapezium liratum) is described. All the studied sperm were typical tail sperm, adapted to external insemination, which, however, had a specific structure. Differences were revealed in the form of head, acrosome structure and number of mitochondria. The studied species of the above families had their specific morphology, the Arcidae species had a bullet- or barrel-shaped head with four or five mitochondria in the middle part; the Anomiidae had conic head, the acrosome with periacrosome material and four mitochondria (a basic feature of sperm is the axial core entering periacrosome material and consisting of bundle of actin filaments); the Myidae had a curved conic head and four mitochondria; in the Tellinidae the head was bullet-shaped, the periacrosome material contained a fibril component and four mitochondria; the Trapezidae had sperm of a conic form with spherical acrosome. The spherical sperm of C. gigas were similar to sperm of Saccostrea commercialis and Crassostrea virginica, but with some distinctions in the acrosome substructure. The morphology of sperm testified to the correct attribution of the Crassostreidae family as a synonym to the Ostreidae family.