Conservative Nature of Oestradiol
Conservative Nature of Oestradiol Signalling Pathways in the Brain Lobes of Octopus vulgaris Involved in Reproduction, Learning and Motor Coordination E. De Lisa*, M. Paolucci� and A. Di Cosmo* *Department of Structural and Functional Biology, University of Napoli ‘Federico II’, Napoli, Italy.
�Department of Biological, Geological and Environmental Sciences, University of Sannio, Benevento, Italy.
Octopus vulgaris demonstrates sophisticated behaviours as a result
of two main evolutionary events. First, advanced encephalisation of
the ganglionic masses associated with hierarchical organisation of
function and, second, the development of advanced cognitive capa-
bilities (1). The central nervous system (CNS) comprises a central
part, encircling the oesophagus, and paired optic lobes laterally
connected by a distinct optic tract. The central part is divided into
suboesophageal and supraoesophageal lobes, linked by the perio-
esophageal magnocellular lobes. A combination of anatomical,
imaging, electrical and chemical stimulation, lesioning and neuro-
physiological recording techniques have demonstrated the hierar-
chical organisation of the Octopus CNS and regionalisation of
functions (2,3). The degree of encephalisation and functional orga-
nisation of the Octopus CNS shows similarities to the mammalian
and insect brains, and the convergence in the organisation of the
brain areas associated with learning is remarkable (4,5). In addition,
the demonstration of the conservative nature of molecular mecha-
nisms underlying learning and memory across phyla has led
recently to the proposal of a general theory of chronic pain,
according to which the mechanisms of learning based on neuronal
plasticity are similar to the molecular mechanisms of chronic pain
(6). The mounting evidence that oestradiol modulates chronic pain
in vertebrates (7) and the demonstration of the antinociceptive
effects of neuroactive steroids in the land snail, Cepaea nemoralis,
strongly support the existence in mollusks of modulatory mechan-
isms analogues to analgesia in vertebrates (8). In O. vulgaris, our
group has demonstrated the conservative nature of neural and
neuroendocrine control mechanisms. Indeed, the olfactory lobes
control sexual maturity through gonadotrophin-releasing hormone
(GnRH) neurones (9); in the olfactory lobes, NMDA stimulation
increases GnRH mRNA levels, probably through a gluta-
mate ⁄ NMDA ⁄ nitric oxide signal transduction pathway (10); NMDA
Journal of Neuroendocrinology
Correspondence to:
A. Di Cosmo, Department of
Structural and Functional Biology,
University of Napoli, ‘Federico II’, via
Cinthia, 80126 Napoli, Italy (e-mail:
dicosmo@unina.it).
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 neurophysiologi-
cal 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 bind-
ing 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, learn-
ing and motor coordination.
Key words: neurosteroids, GnRH, learning, Octopus vulgaris, motor coordination, fluorescence
resonance energy transfer.
doi: 10.1111/j.1365-2826.2011.02240.x
Journal of Neuroendocrinology 24, 275–284
ª 2011 The Authors. Journal of Neuroendocrinology ª 2011 Blackwell Publishing Ltd
Journal of Neuroendocrinology From Molecular to Translational Neurobiology
receptors have been found in many, but not all lobes of Octopus
and Sepia officinalis CNS, including those involved in learning and
memory (vertical superior frontal system) (11,12). Furthermore, ver-
tebrate-like steroids and steroidogenic activity have been identified
in the gonads (13) and in the brain of O. vulgaris and its relative
S. officinalis (14). The activity of two key steroidogenic enzymes,
3b- and 17b-hydroxysteroid dehydrogenase, demonstrated that neurosteroids are synthesised in loco in the CNS of both cephalo-
pods. 3b-hydroxysteroid dehydrogenase activity was localised in specific brain lobes that are involved in learning and memory, as
well as in the control of the movement. 17b-hydroxysteroid dehy- drogenase activity was detected in both the brain and optic lobe.
Previously, an oestrogen receptor was characterised in the repro-
ductive system of O. vulgaris, suggesting a possible involvement in
the regulation of reproduction further sustained by 17b-oestradiol fluctuation throughout the reproductive cycle (15). Evidence for an
oestrogen receptor has been reported in O. vulgaris (16) and other
mollusks (17–21), although limited physiological data on this recep-
tor are available. In the present study, for the first time, we
have mapped the expression of oestrogen receptor in the lobes of
O. vulgaris CNS associated with learning, motor coordination and
reproductive behaviour. In addition, the findings of the present
study demonstrate both genomic and nongenomic actions of 17b- oestradiol. In this respect, the Octopus promises to be a valuable
model system for understanding how oestradiol modulates neural
plasticity.
Materials and methods
Animals
Females of O. vulgaris (N = 15; weighing 1.0–1.5 kg) were captured in the
bay of Naples, Italy, and maintained in aquarium tanks (80 · 60 · 50 cm). According to Di Cosmo et al. (15), the ovary was in the previtellogenic
phase, when the 17b-oestradiol level is very low. Water temperature was 16 �C (light ⁄ dark cycle 8 : 16 h). There are no specific legal or ethical regu- lations relating to experimental work with octopuses in Italy. Our research
using octopuses conforms with the ethical principles of Reduction, Refine-
ment and Replacement (22). Specific attention was paid to avoiding and
minimising any suffering in accordance with Directive 2010 ⁄ 63 ⁄ EU. Animals were deeply anaesthetised and sacrificed as reported by Piscopo et al. (23).
3H-17b-oestradiol binding assay
Preparation of cytosol and nuclear extract of O. vulgaris CNS was performed
as described previously (14). [2,4,6,7-3H]-17b-oestradiol (specific activity 95 Ci ⁄ mmol) was purchased from Amersham Biosciences (Piscataway, NJ, USA). Nuclear extract or cytosol were used for Scatchard analysis. Aliquots
of 200 ll of sample were incubated with 0.3–5 nM 3H-17b-oestradiol (total binding) and 0.3–5 nM 3H-17b-oestradiol plus 100-fold unlabelled 17b-oes- tradiol (nonspecific binding) for 16 h at 4 �C. After incubation, 0.6 ml of dextran-charcoal suspension was added. The mixture was vortexed and kept
on ice for 5 min, followed by centrifugation at 800 g for 10 min at 4 �C. The supernatant was decanted in counting vials with 5 ml of Maxifluor scin-
tillation fluid (Packard, Milan, Italy). Radioactivity was measured in a liquid
scintillation counter (Beckman Coulter, Fullerton, CA, USA) at 30% counting
efficiency. For binding specificity evaluation, cytosol and nuclear extract
samples were incubated with 5 nM of 3H-17b-oestradiol with or without
increasing concentrations of various unlabelled steroids (1, 10 and 100-fold
excess). Radioinert reagents used were: 17b-oestradiol, progesterone, testos- terone, diethylstilboestrol, oestrone and oestriol. All reagents were obtained
from Sigma-Aldrich (St Louis, MO, USA).
Production of digoxigenin-labelled RNA probes and in situ hybridisation (ISH)
pcDNA3 vector containing O. vulgaris oestrogen receptor (Oct-ER) gene
(GenBank accession number DQ533956) was provided by Thornton’s labora-
tory and forward (5¢-AAATGCAGAGGTGCGACGAT-3¢) and reverse (5¢- TCAAGTGCCCATTCCAATAACATC-3¢) primers were used to amplify a 300-bp cDNA by polymerase chain reaction (PCR). Amplification product was cloned
into pGEM-T Easy vector (Promega, Madison, WI, USA) and sequenced
(Primm, Milan, Italy). Sense and antisense digoxigenin-labelled Oct-ER RNA
probes were generated by in vitro transcription using the DIG-RNA Labelling
Kit (SP6 ⁄ T7) (Roche Applied Sciences, Laval, QC, Canada) in accordance with the manufacturer’s instructions. Brain was removed and fixed in 4% para-
formaldehyde in phosphate-buffered saline (PBS) for 24 h at 25 �C. Tissues were dehydrated and embedded in paraffin. Horizontal sections (4 lm) were placed on Superfrost plus slides (Menzel-Gläser, Braunschweig, Germany)
and treated with 10 lg ⁄ ml of Proteinase K. Sections were prehybridised at 60 �C for 1 h in prehybridisation buffer containing 20% formamide, 4 · sal- ine-sodium citrate, 1 · Denhardt’s solution, 500 lg ⁄ ll yeast tRNA and 500 lg ⁄ ll salmon sperm DNA. Hybridisation was performed at 60 �C for approximately 16 h with 15 ng ⁄ ll probe in prehybridisation buffer. RNAse A treatment was performed for 30 min at 37 �C. Sections were then incubated in blocking reagent for 20 min at 50 �C under shaking. Anti-digoxigenin Fab fragments conjugated to alkaline phosphatase 1 : 2000 were used for detec-
tion with Nitro blue tetrazolium chloride ⁄ 5-bromo-4-chloro-3-indolyl phos- phate. All reagents were obtained from Roche Applied Sciences unless
otherwise specified. Negative control experiments were carried using sense
probe. Images of the sections were digitally captured with a charge-coupled
device video camera mounted on a Nikon Eclipse E400 microscope (Nikon,
Tokyo, Japan).
17b-oestradiol and NMDA in vitro stimulation of olfactory lobe
Whole olfactory lobes were dissected from optic tracts and employed in in
vitro stimulation experiments in the presence of artificial sea water (ASW)
and incubated for 2 h. 17b-oestradiol was added at a concentration in the range 10 pM to 10 nM. NMDA was added at a concentration of 50 lM and 2-amino-5-phosphonopentanoic acid (D-APV), an antagonist of NMDA-type
glutamate receptors (24), was used at concentration of 100 lM (10). The same experiments were also performed in the presence of 50 lM NMDA in Ca2+-free ASW. All reagents were obtained from Sigma-Aldrich.
Real-time PCR
After in vitro stimulation experiments, total RNA was extracted from olfac-
tory lobes of O. vulgaris using EZNA Mollusk RNA kit (Omega Bio-Tek, Inc.,
Norcross, GA, USA) in accordance with the manufacturer’s instructions. Total
RNA (5 lg) of each sample was reverse transcribed using the SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) with 250 ng of random
primers in accordance with the manufacturer’s instructions. All samples were
pretreated with 50 U of RNase-free DNase (Promega). For real-time PCR,
complementary DNA was synthesised and PCR was carried out using SYBR
PCR master kit (Applied Biosystems, Inc., Foster City, CA, USA), in accordance
with the manufacturer’s instructions using 50 nM of both sense (5¢-GCA- CAAAACTACCACTTTAGCAATG-3¢) and antisense (5¢-TCTGAAG TGACACTGAA
276 E. De Lisa et al.
ª 2011 The Authors. Journal of Neuroendocrinology ª 2011 Blackwell Publishing Ltd, Journal of Neuroendocrinology, 24, 275–284
