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.     

Identification of SepCRP analogues in the cuttlefish Sepia officinalis: a novel family of ovarian regulatory peptides

In the cuttlefish, Sepia officinalis, the ovary appears to be one of the main sources of regulatory peptides involved in the successive steps of egg-laying. Following the identification of the SepCRP-1, which is a peptide extracted from ovary and involved in egg capsule secretion, investigations were focused on the identification of related peptides. Seven related-Sepia Capsule Releasing Peptides (R-SepCRPs) were identified by means of mass spectrometry and characterized using MS/MS spectra and/or Edman degradation. Finally, primary structures were verified by the comparison of MS/MS spectra from endogenic and synthetic peptides. This new ovarian peptide family exhibits a conserved SLXKD tag involved in the biological activity. LC-MS/MS screening clearly demonstrates that R-SepCRPs are restricted to the female genital tract. Expressed during vitellogenesis, they are released by vitellogenic follicles and full-grown oocytes (FGO) in the genital coelom. Biological activities suggest that R-SepCRPs would be responsible for the storage of FGO before mating and would take part in the mechanical secretion of egg capsule products, as previously described for SepCRP-1.     

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.     

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.     

Egg capsule secretion in invertebrates: a new ovarian regulatory peptide identified by mass spectrometry comparative screening in Sepia officinalis

Mass spectrometry comparative screening was used to identify ovarian regulatory peptides involved in the successive steps of egg-laying in the cuttlefish Sepia officinalis. The peptide content of full-grown oocytes (FGO) was compared with that of oocyte-conditioned medium, which resulted in the detection of peptides that were present in both samples. These peptides, which are suspected of being released by the oocyte in the genital tract, were submitted to a structural analysis. This strategy led to the characterization of a new ovarian regulatory peptide (EISLDKD) able to inhibit the contractions of the whole female genital tract and of the main nidamental glands (MNG). As EISLDKD appeared to be the first regulatory peptide directly involved, at physiological concentrations, in the secretion of the egg capsule by the main nidamental glands, it was named SepCRP for Sepia Capsule Releasing Peptide. Mass spectrometry analysis clearly demonstrated that SepCRP was expressed during vitellogenesis by the ovarian follicles and released by the FGO in the lumen of the female genital tract. In association with the ovarian 5-HT, SepCRP would be responsible for the storage of FGO to avoid the spawning of unfertilized oocytes before mating. Released by the distal oviduct in the mantle cavity, SepCRP probably in association with a cocktail of ovarian regulatory factors targets the MNG to regulate the egg capsule secretion. Thus, the ovary appeared to be one of the main sources of regulatory peptides involved in the successive steps of egg-laying in the cephalopod mollusk S. officinalis.     

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.     

Electron microscopical and histochemical studies of differentiation and function of the cephalopod gill (Sepia officinalis L.)

Summary Phase contrast and electron microscopical investigations on the gill ofSepia officinalis L., from the stage shortly before hatching to the adult stage, demonstrate (1) peripheral respiratory epithelial areas and (2) highly folded epithelia rich in mitochondria in the concave recesses of the gill lamellae. Enzyme-histochemical and cytochemical findings, and in particular the GOT proof, suggest by analogy with the chloride cells of the teleosts that the cells of this second type of epithelium are capable of active transport, and that they probably secrete ammonia. Apparently the gill of cephalopods, too, serves both respiratory and excretory functions.This study was supported by the Deutsche Forschungsgemeinschaft     

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.     

Food imprinting, new evidence from the cuttlefish Sepia officinalis

Imprinting provides precocial offspring with an efficient means to optimize their subsequent behaviours. We discovered food imprinting using a sophisticated invertebrate model: the cuttlefish. We showed that early juveniles preferred the prey to which they have been visually familiarized, when the amount of information was sufficient and only if such familiarization occurred during a short sensitive period. We also demonstrated that the effects of visual food imprinting overcame those of the first food ingested. Our study shows that visual imprinting is a critical process in animals, surpassing more direct reward experiences that occur outside the critical exposure period.     

A new collagen from the extracellular matrix of Sepia officinalis cartilage

Guanidinium chloride treatment of Sepia officinalis cartilage solubilized a component that contained hydroxyproline. Electron-microscopy observation of rotary-shadowed preparations of this component revealed it to consist of rod-like units themselves consisting of filaments. Dialysis of an acetic acid solution against ATP afforded polymeric aggregates consisting of a succession of two or three thick sections showing transverse electron-opaque banding, separated by thinner sections without banding. Electrophoresis produced a main band of about 140 kDa sensitive to bacterial collagenase. After reduction with mercaptoethanol, electrophoresis afforded a 40-kDa band. Pepsin digestion resulted in additional electrophoretic bands. These data suggest the presence of a collagen in Sepia cartilage with characteristics unlike those of any known collagen.     

The SepOvotropin: a new ovarian peptide regulating oocyte transport in Sepia officinalis

In the cuttlefish Sepia officinalis, the successive steps of egg laying are controlled by multiple neuropeptides. Recent experiments led us to suppose that there was possible involvement of a second regulation pathway by the release of ovarian regulatory peptides in the genital tract. Using HPLC fractionation and an in vitro biological test, a C-terminal amidated peptide modulating the motility of the Sepia officinalis oviduct was isolated from an extract of vitellogenic ovarian follicles. The mass of this peptide as determined by MALDI-TOF (1501.8 Da) and analysis by Edman degradation led to the following sequence: Pro-Lys-Asp-Ser-Met-Leu-Leu-Leu-Gln-Val-Pro-Val-Tyr-amide. The peptide mapping performed by LC/MS revealed a distribution restricted to the follicles, the full grown oocytes and the eggs. This new peptide, called SepOvotropin, modulated contractions of the whole genital tract in physiological conditions from a threshold concentration between 10(-20) and 10(-19) M, demonstrating for the first time the occurrence of a specific peptidergic control of egg-laying in cephalopods.     

Identification and tissue mapping of APGWamide-related peptides in Sepia officinalis using LC-ESI-MS/MS

This paper demonstrates for the first time the occurrence of tetrapeptides related to APGWamide in the mollusk cephalopod Sepia officinalis. LC-ESI-MS/MS analysis allowed the identification of the APGWamide-related peptides predicted by the two genes cloned previously in Lymnaea stagnalis and in Mytilus edulis, as well as the dipeptide GWamide released from the processing of the tetrapeptides by a dipeptidyl aminopeptidase (DPAP). TPGWamide and GWamide appeared to be exclusively located in the CNS, and the APGWamide in both the CNS and the nerve endings. The RPGWamide and the KPGWamide were not detected by LC-ESI-MS/MS suggesting they could be totally processed into GWamide. The in vitro processing of the tetrapeptides into GWamide by optic lobe extract revealed a differential processing for each, with APGWamide (44.7%)>RPGWamide(24.3%)>KPGWamide(19.3%)>TPGWamide (11.7%). The tissue mapping results, together with the processing efficiency data suggest that the GWamide is mainly produced from the M. edulis gene products RPGWamide and KPGWamide.     

Melanogenesis in the ink gland of Sepia officinalis

Among the various melanin-producing systems, the ink gland of the cuttlefish (Sepia officinalis) has traditionally been regarded as a most convenient model system for the studies of melanogenesis. The ink gland is a highly specialized organ with immature cells in the inner portion, from where the cells gradually mature, migrate towards the outer portion of the gland and become competent to produce melanin giving rise to particulate melanosomes. When cell maturation is complete, melanin is secreted into the lumen of the gland, accumulated into the ink sac and ejected on demand. Biochemical studies carried out over the past two decades have shown that the ink gland contains a variety of melanogenic enzymes, including tyrosinase, a peculiar dopachrome rearranging enzyme (which catalyses the rearrangement of dopachrome to 5,6-dihydroxyindole) and a peroxidase (presumably involved in the later stages of melanin biosynthesis). These enzymes are functionally interactive in close subcellular compartments of ink gland cells and appear to act in a concerted fashion during the process of melanogenesis in the mature portion of the gland. More recent studies have revealed that ink production and ejection are affected and modulated by the N-methyl-D-aspartate (NMDA)-nitric oxide (NO)-cyclic GMP (cGMP) signalling pathway. Glutamate NMDA receptor and NO synthase, the enzyme responsible for the synthesis of NO, have been detected by biochemical and immunohistochemical techniques in immature ink gland cells. Stimulation of NMDA receptors caused a marked elevation of cGMP levels, activation of tyrosinase and increased melanin synthesis in the mature portion of the gland, via the NO-guanylyl cyclase interaction. This signalling is also present in different regions of the nervous system in Sepia and in certain neural pathways controlling contraction of the ink sac sphincters and wall muscle in the ejection mechanism. Overall, these and other findings allowed elaboration of an improved model of melanin formation in Sepia, which underscores the complex interplay of melanogenic enzymes and regulatory factors, highlighting both the similarities and the differences with melanogenesis in mammals.     

Arm regeneration in two species of cuttlefish Sepia officinalis and Sepia pharaonis

To provide quantitative information on arm regeneration in cuttlefish, the regenerating arms of two cuttlefish species, Sepia officinalis and Sepia pharaonis, were observed at regular intervals after surgical amputation. The third right arm of each individual was amputated to ~10–20 % starting length. Arm length, suction cup number, presence of chromatophores, and behavioral measures were collected every 2–3 days over a 39-day period and compared to the contralateral control arm. By day 39, the regenerating arm reached a mean 95.5 ± 0.3 % of the control for S. officinalis and 94.9 ± 1.3 % for S. pharaonis. The process of regeneration was divided into five separate stages based on macroscopic morphological events: Stage I (days 0–3 was marked by a frayed leading edge; Stage II (days 4–15) by a smooth hemispherical leading edge; Stage III (days 16–20) by the appearance of a growth bud; Stage IV (days 21–24) by the emergence of an elongated tip; and Stage V (days 25–39) by a tapering of the elongated tip matching the other intact arms. Behavioral deficiencies in swimming, body postures during social communication, and food manipulation were observed immediately after arm amputation and throughout Stages I and II, returning to normal by Stage III. New chromatophores and suction cups in the regenerating arm were observed as early as Stage II and by Stage IV suction cup number equaled that of control arms. New chromatophores were used in the generation of complex body patterns by Stage V. These results show that both species of cuttlefish are capable of fully regenerating lost arms, that the regeneration process is predictable and consistent within and across species, and provide the first quantified data on the rate of arm lengthening and suction cup addition during regeneration.     

Small-scale rearing of cuttlefish (Sepia officinalis) for research purposes

We report a methodology refined over 20 years to culture and maintain a small colony of Sepia officinalis for research year-round. Wild-caught eggs were obtained annually from England and reared in semi-closed natural seawater systems of large volume. Constant temperature (15 °C) and day length (12L/12D) delayed sexual maturation and reproductive behavior. This extended life cycle by roughly 50%, thus providing small research animals for about 18 months from every annual batch of eggs. A novel live food – gammarid crustaceans collected from washed up seagrass– was provided to hatchlings and juveniles. Juveniles were then trained to take thawed shrimp thereafter, thus reducing the expense of live foods. Typical survival to one year was >65%. With these methods, healthy sexually immature cuttlefish were available year-round for behavioral and physiological studies without the confounding influences of hormonally-induced fighting, mating, and egg laying that typically occur within six months.