Sperm nucleomorphogenesis in the cephalopod Sepia officinalis

Sperm nucleomorphogenesis in the cephalopod Sepia officinalis is the product of the interaction between perinuclear microtubules and condensing chromatin. This interaction occurs during spermiogenesis and is established through the nuclear membrane. As in other cephalopod species, the perinuclear microtubules are transient structures. In the case of S. officinalis, they begin to appear in the basal area of the early spermatid and progress from there, establishing contact with the external nuclear membrane and follow a defined, but not symmetric, geometry. Thus, the microtubules accumulate preferentially in one area of the nuclear membrane which we refer to here as the "dorsal zone". Later, the microtubules will be eliminated before the mature spermatid migrates to the epidydimis. The chromatin is condensed within the nucleus following a complex pattern, beginning as fibro-granular structures until forming fibres of approximately 45 nm diameter (patterning phases). From this stage on, an increase in the chemical basicity of DNA-interacting proteins is produced, and chromatin fibres coalesce together, being recruited to the dorsal zone of the membrane, where there is a higher density of microtubules. This last step (condensation phases) allows the chromatin fibres to be arranged parallel to the axis of the elongating nucleus, and more importantly, is deduced to cause a lateral compression of the nucleus. This lateral compression is in fact a recruitment of the ventral zone toward the dorsal zone, which brings about an important reduction in nuclear volume. The detailed observations which comprise this work complement previous studies of spermiogenesis of Sepia and other cephalopods, and will help to better understand the process of cellular morphology implicated in the evolution of sperm nuclear shape in this taxonomic group.     

Allometry of thermal limitation in the cephalopod Sepia officinalis

Cuttlefish (Sepia officinalis) routine metabolic rate was determined in response to acute thermal changes at a rate of 1 degrees C h(-1) for a variety of animal sizes (15-496 g wet mass, laboratory reared at 15 degrees C). In a thermal frame of 11 to 23 degrees C, oxygen consumption rates (MO(2), in mumol O(2) g(-1) min(-1)) were observed to rise with increasing temperature (T, in degrees C) and to decline with increasing body mass (m, in g), according to the formula: ln MO(2)=-3.3+0.0945T-0.215 ln m (R(2)=0.93). Outside the above thermal window, animals were not able to increase MO(2) at similar rates, indicating a beginning oxygen limitation of metabolism. Large animals (>100 g body mass) already displayed lower than expected MO(2) values at 8 and 26 degrees C, while smaller animals (15 g wet mass) were characterized by a wider thermal window (MO(2) values deviated from expected rates at 5 and 29 degrees C). Morphometric data of cuttlefish mantle skin area was obtained to discuss size - related effects of skin respiration potential on thermal tolerance. Cuttlefish growth was observed to be isometric, as constant 'Vogel numbers' of 4.2 indicated (animal body masses: 11 to 401 g). In the same mass range, specific mantle surface area declined three-fold from 10.7 (0.24) (means+/-SD) to 3.3 (0.52) cm(2) g(-1). Thus, increased thermal tolerance in smaller animals may be enabled by a higher skin respiration potential due to higher specific skin surface areas. An elevated fraction of MO(2) provided by means of skin respiration in small animals could relieve the cardiovascular system, which previously has been found a major limiting component during acute thermal stress in cuttlefish.     

Localization of an extracerebral NSV-ganglion in the cephalopod, Sepia officinalis

The neurosecretory system of the vena cava (NSV) of Sepia officinalis contacts the palliovisceral lobe with some axons that run parallel to the visceral nerves. Distal to the palliovisceral lobe the NSV- system widens to form a hitherto unknown extracerebral ganglion containing approximately 4200 perikarya. This is regarded as the primary origin of neurotransmitters. Distal to this nucleus area the volume and the number of cell bodies in the NSV-system is reduced. It contains neurosecretory nerve cells, two glial cell types, large cell accumulations, connective tissue and capillaries. Histochemical, immunohisto- and immunocytochemical examinations show the existence of catecholamines, and the coexistence of serotonin and FMRFamide in the NSV-System. A HPLC analysis differentiated between dopamine, noradrenaline, and adrenaline.     

Hemocyanin re-uptake in the renal and branchial heart appendages of the coleoid cephalopod Sepia officinalis

The renal and branchial heart appendages of Sepia officinalis L. were investigated in order to elucidate a possible involvement of their excretory epithelia in hemocyanin metabolism. Immunocytochemical findings and tracer experiments indicate that after passing the barrier of ultrafiltration the hemocyanin molecules are taken up by the epithelial cells of the renal and branchial heart appendages and are subsequently carried back to the circulatory system, suggesting a mechanism of hemocyanin recycling. Apart from a function in maintaining constant hemocyanin levels, the present study indicates that the renal and branchial heart appendages are also sites of temporary hemocyanin storage.     

Ovarian jelly-peptides (OJPs), a new family of regulatory peptides identified in the cephalopod Sepia officinalis

In marine invertebrates, numerous water-borne peptides involved in reproductive behavior have been characterized. In this study, we focused on three ovarian water-borne peptides, released by full-grown oocytes (FGO) in the genital coelom and in the lumen of the oviduct in the cuttlefish Sepia officinalis. The first one (DQVKIVL), was characterized by the monitoring of HPLC purified fraction using a myotropic bioassay. Subsequently, a peptidomic approach consisting of a mass spectrometry comparative screening performed between the peptide content of FGO with that of FGO-conditioned medium, led to the identification of two additional water-borne peptides. The second peptide identified (DEVKIVL) was characterized by MS/MS and the primary structure of the third one (DEVKIVLD) was elucidated by a combination of Edman degradation, acid hydrolysis and MS/MS analysis. Sequence homology, tissue mapping and bioactivity demonstrate that these peptides belong to the same family. DQVKIVL-related-peptides strictly localized in the female genital tract modulate the whole female genital tract and the main nidamental gland contractions. Furthermore, these peptides form a jelly, when resuspended in water. This particular property could play an important role in the kinetics of peptide diffusion in the external medium. Thus, these regulatory peptides were named ovarian jelly-peptides (OJPs).     

Temperature-dependent oxygen extraction from the ventilatory current and the costs of ventilation in the cephalopod Sepia officinalis

Earlier work found cuttlefish (Sepia officinalis) ventilatory muscle tissue to progressively switch to an anaerobic mode of energy production at critical temperatures (T _c) of 7.0 and 26.8°C. These findings suggested that oxygen availability limits thermal tolerance. The present study was designed to elucidate whether it is the ventilatory apparatus that sets critical temperature thresholds during acute thermal stress. Routine metabolic rate (rmr) rose exponentially between 11 and 23°C, while below (8°C) and above (26°C) this temperature range, rmr was significantly depressed. Ventilation frequency (f _V) and mean mantle cavity pressure (MMP) followed an exponential relationship within the entire investigated temperature range (8–26°C). Oxygen extraction from the ventilatory current (EO₂) decreased in a sigmoidal fashion with temperature, falling from > 90% at 8°C to 32% at 26°C. Consequently, ventilatory minute volume (MV_V) increased by a factor of 20 from 7 to 150% body weight min⁻¹ in the same temperature interval. Increases in MMP and MV_V resulted in ventilatory muscle power output (P _out) increasing by a factor of > 80 from 0.03 to 2.4 mW kg⁻¹ animal. Nonetheless, costs for ventilatory mechanics remain below 1.5% rmr in the natural thermal window of the population (English Channel, 9–17°C), owing to very low MMPs of < 0.05 kPa driving the ventilatory stream, and may maximally rise to 8.6% rmr at 26°C. Model calculations suggest that the ventilatory system can maintain high arterial PO₂ values of > 14 kPa over the entire temperature interval. We therefore conclude that the cuttlefish ventilation system is probably not limiting oxygen transfer during acute thermal stress. Depression of rmr, well before critical temperatures are being reached, is likely caused by circulatory capacity limitations and not by fatigue of ventilatory muscle fibres.     

Nitric oxide: a vasodilatatory mediator in the cephalic aorta of Sepia officinalis (L.) (Cephalopoda)

Evidence is presented that nitric oxide (NO) may regulate blood pressure in cephalopod molluscs. In vitro tests performed on the cephalic aorta of Sepia officinalis (L.) (Cephalopoda) showed that the NO releasers (glyceroltrinitrate, sodium nitroprusside, 3-morpholinylsydnoneimine chloride and KNO(2)) induced concentration-dependent vasodilatation of vessel segments (without the tunica adventitia/periadventitia) precontracted by dopamine. These vasodilatatory actions could be totally blocked by oxadiazolo[4,3-a] quinoxalin-1-one, an inhibitor of the NO-sensitive guanylyl cyclase, and partially mimicked by the cyclic guanosine monophosphate (cGMP) analogue 8-bromo cGMP and by the phosphodiesterase inhibitor, zaprinast. The NO-precursor, L-arginine, showed vasodilatatory effects only on segments of the aorta in which the layers containing nerves (tunica adventitia/periadventitia) had been left intact, suggesting that NO synthase may be located within peripheral nerves.     

Nitric oxide: a vasodilatatory mediator in the cephalic aorta of Sepia officinalis (L.) (Cephalopoda)

Evidence is presented that nitric oxide (NO) may regulate blood pressure in cephalopod molluscs. In vitro tests performed on the cephalic aorta of Sepia officinalis (L.) (Cephalopoda) showed that the NO releasers (glyceroltrinitrate, sodium nitroprusside, 3-morpholinylsydnoneimine chloride and KNO₂) induced concentration-dependent vasodilatation of vessel segments (without the tunica adventitia/periadventitia) precontracted by dopamine. These vasodilatatory actions could be totally blocked by oxadiazolo[4,3-a] quinoxalin-1-one, an inhibitor of the NO-sensitive guanylyl cyclase, and partially mimicked by the cyclic guanosine monophosphate (cGMP) analogue 8-bromo cGMP and by the phosphodiesterase inhibitor, zaprinast. The NO-precursor, l-arginine, showed vasodilatatory effects only on segments of the aorta in which the layers containing nerves (tunica adventitia/periadventitia) had been left intact, suggesting that NO synthase may be located within peripheral nerves. Accepted: 11 August 1998     

Peptidergic control of egg-laying in the cephalopod Sepia officinalis: involvement of FMRFamide and FMRFamide-related peptides.

The peptidergic control of egg-laying was investigated in Sepia officinalis by using a myotropic bioassay. Three myotropic high-performance liquid chromatography fractions were obtained from optic lobe extracts. In the first fraction, FMRFamide (FMRFa) and FLRFa were isolated and sequenced. FMRFa-related peptides then were sought by dotting immunobinding of optic lobes extracts. The four immunoreactive fractions detected revealed the occurrence of FMRFa, FLRFa, FIRFa, and ALSGDAFLRFa predicted by the precursor already cloned from the optic lobes of S. officinalis (J Exp Biol 200:1483-9;1997). These peptides clearly appeared to be involved in the regulation of oocyte transport through the oviduct: the tetrapeptides FMRFa and FLRFa stimulated the contractions, whereas FIRFa and ALSGDAFLRFa lowered the tonus, the frequency, and the amplitude of the contractions. The occurrence of FaRPs in the nervous endings of the accessory sex glands suggested that this peptide family is involved in the regulation of secretory processes of the egg capsule. Indeed, FMRFa modulates the contractions of the main nidamental glands in vitro and, thus, should induce mechanical release of the secretion in vivo during ovulation. These results show that the FaRPs could play an important role in the synchronization of ovulation and egg capsule coating.     

Mechanisms of protein degradation in mantle muscle and proposed gill remodeling in starved Sepia officinalis

Cephalopods have relatively high rates of protein synthesis compared to rates of protein degradation, along with minimal carbohydrate and lipid reserves. During food deprivation on board protein is catabolized as a metabolic fuel. The aim of the current study was to assess whether biochemical indices of protein synthesis and proteolytic mechanisms were altered in cuttlefish, Sepia officinalis, starved for 7 days. In mantle muscle, food deprivation is associated with a decrease in protein synthesis, as indicated by a decrease in the total RNA level and dephosphorylation of key signaling molecules, such as the eukaryote binding protein, 4E-BP1 (regulator of translation) and Akt. The ubiquitination-proteasome system (UPS) is activated as shown by an increase in the levels of proteasome β-subunit mRNA, polyubiquitinated protein, and polyubiquitin mRNA. As well, cathepsin activity levels are increased, suggesting increased proteolysis through the lysosomal pathway. Together, these mechanisms could supply amino acids as metabolic fuels. In gill, the situation is quite different. It appears that during the first stages of starvation, both protein synthesis and protein degradation are enhanced in gill. This is based upon increased phosphorylation of 4E-BP1 and enhanced levels of UPS indicators, especially 20S proteasome activity and polyubiquitin mRNA. It is proposed that an increased protein turnover is related to gill remodeling perhaps to retain essential hemolymph-borne compounds.     

Fine structure and function of the digestive cell of Sepia officinalis (Mollusca: Cephalopoda)

The post–embryonic development of the digestive gland has been studied by light and electron microscopy on animals reared in the laboratory. The gland reaches its "adult" structure around the end of the first month after hatching.
The infrastructural changes of the digestive gland have been followed during digestion at various ages (5,10,20,30 days and "adult"); ferritin was used for a tracer to study absorption.
These experiments have produced evidence of endocytosis intake of large proteins and further intracellular digestion inside the digestive cell. The residues of digestion are excreted in a "brown body" while the metabolites are used in new syntheses for the cell itself. These results demonstrate that "ancestral" digestive processes have been kept in at least one cephalopod.     

Adenosine triphosphatase localization in the branchial heart of Sepia officinalis L. (Cephalopoda)

Sodium- and potassium-dependent adenosine triphosphatase (Na+--K+-ATPase) is demonstrated in the branchial heart of Sepia officinalis L. by biochemical, cytochemical and autoradiographical methods. The biochemical data indicate the presence of Na+--K+-ATPase, shown by potassium and magnesium dependency and inhibition by ouabain. Cytochemically and autoradiographically, the enzyme is localized in the sarcolemma of the muscle cells. The positive reaction of the transparent cells (type I cells) is due to activity of alkaline phosphatases. The dark cells (type II cells) react negatively. In addition to the Na+--K+-ATPase, a magnesium-activated adenosine triphosphatase (Mg2+-ATPase) and a bicarbonate-stimulated ATPase (HCO3(-)-ATPase) are localized in the mitochondria.     

Adenosine triphosphatase localization in the branchial heart appendage of Sepia officinalis L. (Cephalopoda).

Sodium- and potassium-dependent adenosine triphosphatase (Na+--K+-ATPase) has been demonstrated in the branchial heart appendage (pericardial gland) of Sepia officinalis L. by biochemical, cytochemical and autoradiographical methods. The biochemical data indicate the presence of Na+--K+-ATPase, judging from the potassium dependency and, with some restrictions, the inhibition by ouabain. Cytochemically and autoradiographically, the enzyme could be localized on the cytoplasmic surfaces of the lateral plasma membranes and the basal membrane infoldings (basal labyrinth) of the folded epithelium of the branchial heart appendage. The pdocytes of the peripheral zone of the organ reacted negatively. In addition to the Na+--K+-ATPase, a magnesium-activated adenosine triphosphatase (Mg2+-ATPase) was demonstrated in the folded epithelium, localized mainly in the mitochondria but also at the brush border and in the apical intercellular space, whereas a bicarbonate-stimulated ATPase (HCO-3-ATPase) was present only in the mitochondria.