A. Di Cosmo

Effects of estradiol and progesterone on the reproduction of the freshwater crayfish Cherax albidus.

In this study we have investigated the role of 17β-estradiol and progesterone in the reproduction of the crayfish Cherax albidus by using vitellogenin (VTG) as a biomarker. Early-vitellogenic (EV), full-vitellogenic (FV), and non-vitellogenic (NV) females of Cherax albidus were treated with 17β-estradiol, progesterone, or both for 4 weeks. Levels of VTG mRNA in the hepatopancreas were detected by RT-PCR. The PCR product was sequenced and showed 97% homology with Cherax quadricarinatus VTG. 17β-estradiol was more effective than progesterone and 17β-estradiol plus progesterone in increasing the vitellogenin transcript in the hepatopancreas of EV and FV females. On the contrary, progesterone was more effective than 17β-estradiol and 17β-estradiol plus progesterone in increasing the vitellogenin concentration in the hemolymph of EV and FV females. Hepatopancreas histology and fatty acid composition of females injected with hormones showed major modifications. No effects were registered in NV females. In conclusion, 17β-estradiol and progesterone influence VTG synthesis, although our data indicate that they act through different pathways and are not effective until the proper hormonal environment is established, as demonstrated by their inefficacy in NV females.

Conservative nature of oestradiol signalling pathways in the brain lobes of octopus vulgaris involved in reproduction, learning and motor coordination.

Oestradiol plays crucial roles in the mammalian brain by modulating reproductive behaviour, neural plasticity and pain perception. The cephalopod Octopus vulgaris is considered, along with its relatives, to be the most behaviourally advanced invertebrate, although the neurophysiological basis of its behaviours, including pain perception, remain largely unknown. In the present study, using a combination of molecular and imaging techniques, we found that oestradiol up‐regulated O. vulgaris gonadotrophin‐releasing hormone (Oct‐GnRH) and O. vulgaris oestrogen receptor (Oct‐ER) mRNA levels in the olfactory lobes; in turn, Oct‐ER mRNA was regulated by NMDA in lobes involved in learning and motor coordination. Fluorescence resonance energy transfer analysis revealed that oestradiol binds Oct‐ER causing conformational modifications and nuclear translocation consistent with the classical genomic mechanism of the oestrogen receptor. Moreover, oestradiol triggered a calcium influx and cyclic AMP response element binding protein phosphorylation via membrane receptors, providing evidence for a rapid nongenomic action of oestradiol in O. vulgaris. In the present study, we demonstrate, for the first time, the physiological role of oestradiol in the brain lobes of O. vulgaris involved in reproduction, learning and motor coordination.

L-Glutamate and its Ionotropic Receptors in the Nervous System of Cephalopods

In several species of cephalopod molluscs there is good evidence for the presence of L-glutamate in the central and peripheral nervous system and evidence for both classes of ionotropic receptor, AMPA/kainate and NMDA.The best evidence for glutamate being a transmitter in cephalopods comes from pharmacological, immunohistochemical and molecular investigations on the giant fibre system in the squid stellate ganglion. These studies confirm there are AMPA/kainate-like receptors on the third-order giant axon. In the (glial) Schwann cells associated with the giant axons both classes of glutamate receptor occur.Glutamate is an excitatory transmitter in the chromatophores and in certain somatic muscles and its action is mediated primarily via AMPA/kainate-like receptors, but at some chromatophores there are NMDA-like receptors.In the statocysts the afferent crista fibres are also glutamatergic, acting at non-NMDA receptors.In the brain (of Sepia) a neuronal NOS is activated by glutamate with subsequent production of nitric oxide and elevation of cGMP levels. This signal transduction pathway is blocked by D-AP-5, a specific antagonist of the NMDA receptor.Recently immunohistochemical analysis has demonstrated (in Sepia and Octopus) the presence of NMDAR2A /B - like receptors in motor centres, in the visual and olfactory systems and in the learning system. Physiological experiments have shown that glutamatergic transmission is involved in long term potentation (LTP) in the vertical lobe of Octopus, a brain area involved in learning. This effect seems to be mediated by non-NMDA receptors. Finally in the CNS of Sepia NMDA-mediated nitration of tyrosine residues of cytoskeletal protein such as alpha-tubulin, has been demonstrated.

Quantitative dynamic imaging of biological processes with solid state radiation detectors

We have developed a technique and a prototype for /spl beta/-rays and /spl gamma/-rays quantitative imaging of biological samples which have been radioactively labeled. The prototype is based on a solid state pixel detector, bump-bonded to a low threshold, single particle counting chip: the detector we used is 200-/spl mu/m thick GaAs, with pixel size 170 /spl mu/m. In this paper we describe an experiment that shows the capability of monitoring a dynamic process. Specifically, [/sup 14/C]L-Leucine uptake by eggs of mollusk cephalopod Octopus vulgaris and the consequent migration of marker into the ooplasm was followed.

Real time /spl beta/-imaging with silicon hybrid pixel detectors: kinetic measurements with C-14 amino acids and P-32 nucleotides

We present some additional results obtained with the BETAview system, a digital system for real time /spl beta/-imaging based on semiconductor pixel detectors and on a single photon counting read-out chip. We describe the system assembled with a Si detector, 300-/spl mu/m thick, segmented into 64 /spl times/64 square pixels with 170-/spl mu/m pitch. The solid-state pixel array detector is bump-bonded pixel by pixel to a low threshold, single particle counting electronics, the photon counting chip (PCC) developed by the Medipix1 Collaboration for biomedical applications. Each cell (corresponding to a detector pixel) of the front-end chip has a maximum count rate of 2 MHz, a minimum nominal threshold of 1400 e/sup -/ (corresponding about 5 keV in Si), and contains a 15-bit counter. The detector sensitive area is about 1 cm/sup 2/, but several detectors could be assembled in arrays. The measured background counts were about 3.5/spl times/10/sup -2/ cps/cm/sup 2/, which implies a very high sensitivity of the device. The detection threshold used in this experiment was /spl sim/15 keV. We show the results obtained in the real-time monitoring of two biological dynamic processes: an amino acid uptake by living cells and the thermal denaturation process of oligonucleotides. Specifically, we have followed over time the accumulation of a marked amino acid ([/sup 14/C]L-Leucine) into Octopus vulgaris eggs cells. The results, described in Section III-B, show that after about 20 min from the beginning of the uptake process, the radioactively marked eggs become clearly visible over the background culture radioactive medium. In Sections III-C and III-D, we also describe the successful performance of the /spl beta/-imaging system in genetic studies involving the real time reconstruction of denaturation and kinetics curves for two different /sup 32/P-radiolabeled nucleotides.