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ORIGINAL ARTICLE

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:

[email protected]).

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- 3 H]-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

3 H-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 3 H-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

TGTCTGAGA-3¢) primers for O. vulgaris GnRH (Oct-GnRH) and 50 nM of both sense (5¢-AAA TGCAGAGGTGCGACGAT-3¢) and antisense (5¢-TGGGCAGTGT- CAATCAATGC-3¢) primers for Oct-ER. All the reactions were performed on an ABI 7300 Real-Time PCR System (Applied Biosystems). Each reaction was run

in triplicate. Accurate amplification of the target amplicon was checked by

performing a melting curve. Relative levels of expression of the gene targets

were calculated using the cycle-threshold method, and comparative analyses

were conducted using O. vulgaris 18S rRNA as reference values. Primers were

chosen using Primer Express 2.0 software (Applied Biosystems). Data were

analysed in accordance with the relative quantification method.

Western blot analysis

After 12% sodium dodecylsulphate (SDS)-polyacrylamide gel electrophoresis,

proteins were transferred on Immobilon polyvinylidene fluoride membrane

(Millipore, Billerica, MA, USA). Membrane was incubated for 1 h in blocking

solution (5% nonfat dry milk in PBS containing Tween 20 0.1%) followed by

incubation in primary and secondary antibody at the appropriate dilution.

Immunopositive bands were visualised using the SuperSignal� West Pico

Chemiluminescent Substrate in accordance with the manufacturer’s instruc-

tions (Pierce Biotechnology, Inc., Rockford, IL, USA) using a Chemidoc EQ

System (Bio-Rad Labs, Hercules, CA, USA). To quantify the cyclic AMP

response element binding protein (CREB) and phosphorilated CREB (p-CREB)

ratio, after detection of pCREB protein, membrane was stripped for 20 min

at 60 �C using a buffer containing 62,5 mM Tris-HCl (pH 6.8), 100 mM b-mercaptoethanol and 2% (w ⁄ v) SDS. After stripping, the membrane was re-probed with antibodies to CREB and ⁄ or b-actin.

Antibodies

Primary antibodies were employed at the dilutions: polyclonal anti-human

ER-a, 1 : 500; monoclonal anti-Hsp90 (mouse IgG2b isotype) (Sigma- Aldrich), 1 : 500; polyclonal anti-CREB, 1 : 500; polyclonal anti-p-CREB,

1 : 500; and monoclonal anti-actin, 1 : 1000. Secondary antibodies used

were: goat polyclonal anti-rabbit and goat polyclonal anti-mouse IgG,

horseradish peroxidase conjugated (Pierce Biotechnology, Inc.) 1 : 10000.

Antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA)

unless otherwise specified.

Construction of fusion protein EGFP-Oct-ER-DsRED

We used the enhanced green fluorescent protein (EGFP) and the red fluores-

cent protein (DsRED) to generate the fusion protein EGFP-Oct-ER-DsRED.

The DsRed coding sequence was obtained by BamHI and XbaI (New England

Biolabs, Beverly, MA, USA) restriction enzyme cleavage of the vector pDs-

Red-Express-N1 (BD Biosciences Clontech, Palo Alto, CA, USA) and cloned

downstream from the multicloning site of the vector for enhanced GFP,

pEGFP-C1 (BD Biosciences Clontech). The generated fluorescence resonance

energy transfer vector (pFRET), encoding the fusion construct GFP-linker-

DsRED, was sequenced and cleaved by BglII (New England Biolabs) and

BamHI. The full-length gene for Oct-ER was amplified from a pcDNA3 ⁄ Oct- ER vector with specific primers containing BglII and BamHI sites as adapters.

The Oct-ER PCR product was digested at BglII and BamHI sites, cloned in

pFRET vector to generate pFRET-Oct-ER and sequenced (Primm).

HeLa culture and EGFP-Oct-ER-DsRED fusion protein expression

Oestrogen receptor deficient HeLa cells were grown according to the ATCC

specific requirement. For the FRET analysis, cells were maintained for 1 week

in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich) containing 10% foe-

tal bovine serum charcoal-treated (Invitrogen). HeLa cells were grown in

Gibco Opti-MEM I Reduced-Serum Medium (Invitrogen) for 24 h and tran-

sient transfected with pFRET-Oct-ER vector using Lipofectamine 2000 (Invi-

trogen) in accordance with the manufacturer’s instructions. Forty-eight

hours after transfection, cells were stimulated with 5 nM 17b-oestradiol in the absence or in presence of tamoxifen (25) at a concentration of 50 nM

and 500 nM (added 10 min before 17b-oestradiol). Tamoxifen is an anti-oes- trogen and competes with 17b-oestradiol for oestrogen receptor binding. Images of living cells were acquired with an Inverted Research Nikon Micro-

scope ECLIPSE Ti for qualitative and quantitative FRET analysis. Transfected

cells were lysed in PBS, NP40 1%, phenylmethanesulphonyl fluoride protease

inhibitors and proteins were quantified in Bradford assays.

Calcium measurement

For calcium measurement, HeLa cells were transiently co-transfected with

pcDNA3-Oct-ER and YC3.60 ⁄ pcDNA3 (26), a calcium sensor (kindly provided by Professor Atsushi Miyawaki, RIKEN Brain Science Institute, Wako City,

Japan) using Lipofectamine 2000 Reagent as described above. Intracellular

fluorescence measurement was performed on Victor II plate reader (Perkin-

Elmer Life Sciences, Boston, MA, USA) equipped with filters set including

CFPex 425 ⁄ 20, YFPex 495 ⁄ 10, CFPem 480 ⁄ 40 and YFPex 525 ⁄ 20 at 25�C. The intracellular calcium concentration was calculated upon excitation at

457 nm and emission at 535 nm (FRET channel): [Ca2+]=Kd Q(R – Rmin) ⁄ (Rmax – R) where Rmin and Rmax are, respectively, the R values at low (5 mM

ethylene glycol tetraacetic acid) and saturating (1 lM ionomycin, 10 mM CaCl2) concentrations and Q is the ratio F0 ⁄ F saturating at 440 nm (26). Experiments were carried out in presence of 5 nM 17b-oestradiol, 5nM 17b- oestradiol bovine serum albumin (BSA) conjugated, 5 nM 17b-oestradiol BSA conjugated plus 500 nM tamoxifen and 5 nM 17b-oestradiol BSA conjugated plus 10 lM thapsigargin. Thapsigargin, a potent inhibitor of sarco-endoplas- mic reticulum Ca2+-ATPases, was used at different concentrations (1, 10,

100 lM) as a positive control. The concentration of 10 lM thapsigargin has been shown to reduce the Ca2+ binding activity significantly (27). Transfect-

ed cells were lysed as above. All reagents were obtained from Sigma-

Aldrich.

Statistical analysis

Statistical analyses were performed using GRAPHPAD PRISM, version 3.03 (Graph-

Pad Software Inc., San Diego, CA, USA). Data are presented as the mean � SEM. Statistical significance was calculated using Kruskal–Wallis test for

initial intergroup comparison followed by Mann–Whitney test a as post-hoc

test.

Results

3H-17b-oestradiol binding assay and specificity

To evaluate the presence of 17b-oestradiol binding molecules in O. vulgaris CNS, we performed a 3H-17b-oestradiol binding assay. 3H-17b-oestradiol binding activity was found in both the cytosol and nuclear extract of the CNS of O. vulgaris. When the concentra-

tion of total proteins was limited to 2 mg ⁄ ml, saturation occurred at approximately 5 nM of

3 H-17b-oestradiol. Scatchard analysis dis-

played only one binding component with an apparent Kd of 0.3 � 0.02 nM for the nuclear extract (Fig. 1A) and of 0.2 � 0.03 nM

for the cytosol (Fig. 1B). The specificity of the 3H-17b-oestradiol

Oestradiol signalling pathways in Octopus brain 277

ª 2011 The Authors. Journal of Neuroendocrinology ª 2011 Blackwell Publishing Ltd, Journal of Neuroendocrinology, 24, 275–284

binding moiety in the cytosol and nuclear extract of the CNS was

determined by competition assay in the presence of 1, 10 and 100-

fold of several different unlabelled competitors. At a concentration

of 100-fold, 17b-oestradiol was the most potent competitor, fol- lowed by diethylstilboestrol, oestrone, oestriol, progesterone and

testosterone (Fig. 1C). In summary, binding assay analysis revealed

the presence of 17b-oestradiol binding molecules with high affinity and specificity for the ligand in both the cytosol and nuclear

extract of the CNS of O. vulgaris.

Expression analysis of Oct-ER mRNA in horizontal sections throughout the middle and posterior suboesophageal masses and throughout supraoesophageal mass of O. vulgaris CNS

To evaluate the presence and the distribution of Oct-ER mRNA in

horizontal sections throughout the middle and posterior suboesoph-

ageal masses and throughout supraoesophageal mass of O. vulgaris

CNS, we performed ISH analysis. The distribution of Oct-ER mRNA-

expressing lobes in the CNS is summarised in Table 1. In the middle

suboesophageal mass, Oct-ER mRNA was expressed in all pedal

lobes. In the posterior lateral pedal lobe, large pear-shaped neuro-

nes expressed Oct-ER mRNA (Fig. 2B). In the posterior pedal lobe,

large motor neurones and small neurones expressing Oct-ER mRNA

can be seen (Fig. 2C, D). In the posterior suboesophageal mass, Oct-

ER mRNA was expressed in the palliovisceral and magnocellular

lobes. The palliovisceral lobe showed large and small neurones

expressing Oct-ER mRNA (Fig. 2E). In the magnocellular ventral lobe,

many small neurones mixed with large ones showed Oct-ER-mRNA

(Fig. 2F). Oct-ER-mRNA was expressed in both cytoplasm and nuclei

of neurones. When, in the control experiments, sense probe was

used, no staining was observed in any of the lobes (Fig. 2A). In the

supraoesophageal mass, Oct-ER mRNA was expressed in neurones

of all lobules of the vertical lobe. In the median and lateral vertical

lobules, Oct-ER mRNA was expressed in the cytoplasm of many lay-

0.5

0.6

(A) (B) (C)

0.4

0.30.3

0.2 0.2

0.1

B /FB /F

0.00.0 0 1 2 3 4 50

Bound 3H-oestradiol [nM] Bound 3H-oestradiol [nM] [3H] Oestradiol -17b (nM × 102)

[3 H

] o es

tr ad

io l -

1 7 b

B o u n d ( %

)

1 2 3 4 5 0

Oestradiol Oestrone

Oestriol DES Progesterone

Testosterone

1 10 100 0

100

Fig. 1. 3H-17b-oestradiol (3H-17b-E2) binding assays. Scatchard plot of 3H-17b-E2 binding in the nuclear extract (A) and cytosol (B) of the central nervous

system of Octopus vulgaris. Only the specific binding is shown. (C) Octopus oestrogen receptor specificity in the nuclear extract. The cytosol gave similar

results. Specificity was determined by competition assay in the presence of 1- 10- and 100-fold of several competitors. Data are reported as percentage of the

inhibition of binding with 3 H-17b-E2. Data are representative of three separate experiments. DES: diethylstilboestrol.

Table 1. Octopus vulgaris Brain Regions Expressing Octopus Oestrogen

Receptor (Oct-ER) mRNA.

Brain regions

Neurones size (lm)

Large Small

Subesophageal lobes

Brachial lobe ) Anterior pedal lobe + 15–20

Lateral pedal lobe ++ 15–20

Posterior pedal lobe ++ 20 5–10

Palliovisceral lobe ++ 15–20 > 10

Magnocellular lobes

Ventral magnocellular lobe ++ 15–20 5–10

Dorsal magnocellular lobe ++ 10–15 5–10

Posterior magnocellular lobe ++ 10–15 5–10

Supraesophageal lobes

Median basal lobe ++ 10–15 5

Lateral basal lobe ++ 10–15 5

Vertical-superior frontal system

Lateral superior frontal lobe ++ 10 5

Vertical lobe ++ 5

Subvertical lobe + 10 5

Precomminsural lobe + 10 5

Optic lobe

Outer granular layer + 5

Plexiform layer No cells

Inner granular layer + 5

Medulla ++ 20–25

Optic tract lobes

Olfactory lobe ++ 20 5–10

Peduncle lobe + 10

Optic gland )

), Lobes with no Oct-ER mRNA-expressing neurones; +, lobes with sparse positive Oct-ER mRNA-expressing neurones; ++, lobes with extensive posi-

tive Oct-ER mRNA-expressing neurones.

278 E. De Lisa et al.

ª 2011 The Authors. Journal of Neuroendocrinology ª 2011 Blackwell Publishing Ltd, Journal of Neuroendocrinology, 24, 275–284

ers of amacrine cells (Fig. 2G, H). When, in the control experiments,

sense probe was used, no staining was observed in any of the lob-

ules (Fig. 2I). Neither glial cells, nor nerve fibres were labelled

according to the technique employed. In summary, in situ hybridi-

sation analysis revealed that the expression of Oct-ER mRNA was

present in the lobes of the CNS involved in the control of repro-

duction, learning and motor coordination.

Effect of 17b-oestradiol on Oct-GnRH and Oct-ER mRNA expression in the olfactory lobe

After having analysed the Oct-ER expression by ISH in the olfac-

tory lobes (Table 1), we assessed the effect of 17b-oestradiol on transcriptional activity of GnRH neurones that control O. vulgaris

reproductive behaviour. Quantitative (q)PCR analysis was per-

formed to determine the relative expression of Oct-GnRH mRNA

after stimulation of the olfactory lobes with 17b-oestradiol at a concentration in the range 10 pM to 10 nM. The results obtained

are shown in Fig. 3(A). Significant changes in Oct-GnRH expression

were observed in the presence of all concentrations of 17b-oes- tradiol employed. Oct-GnRH mRNA expression increased by

approximately three-fold in the presence of 1 nM 17b-oestradiol and approximately 2.5-fold in the presence of 10 nM 17b-oestra- diol with respect to the control (U = 0, P = 0,0091). qPCR analysis

was performed to determine the relative expression of Oct-ER

mRNA after stimulation of the olfactory lobes with 17b-oestradiol at a concentration in the range 10 pM to 10 nM. The results

obtained indicated that Oct-ER mRNA relative expression increased

by approximately 2.5-fold with respect to the control after stimu-

lation with 17b-oestradiol at a concentration of both 1 nM and 10 nM (U = 0, P = 0,0102). No significant changes in Oct-ER

mRNA expression were observed in the presence of 10–100 pM

17b-oestradiol with respect to the control (Fig. 3B). In summary, 17b-oestradiol up-regulates the transcriptional activity of both Oct-GnRH and Oct-ER genes in the olfactory lobe.

Effect of NMDA on Oct-ER mRNA expression in the supraoesophageal mass

After having analysed the Oct-ER expression by ISH in suprao-

esophageal mass (Fig. 2 and Table 1), we assessed the effect of

NMDA on Oct-ER mRNA level. qPCR analysis was performed to

(A) (B) (C)

(D) (E) (F)

(G) (H) (I)

Fig. 2. In situ hybridisation (ISH) of Octopus vulgaris oestrogen receptor (Oct-ER) mRNA expression. Horizontal sections throughout the middle and posterior

suboesophageal masses. (A) Control showing no staining. (B) Posterior lateral pedal lobe: Oct-ER mRNA expression was confined to many large tapering neuro-

nes (15–20 lm). (C) Posterior pedal lobe: both large (20 lm) and small (10 lm) Oct-ER mRNA expressing neurones can be seen. (D) Higher magnification of the lobe in (C) showing Oct-ER mRNA expression in both cytoplasm and nucleus. (E) Palliovisceral lobe with large (15–20 lm) and small (5–10 lm) neurones expressing Oct-ER mRNA. (F) Magnocellular ventral lobe. Both small (5–10 lm) and large (15–20 lm) neurones showed Oct-ER-mRNA. Scale bars = 25 lm (A, B, C, E, F); 15 lm (D). Horizontal sections throughout supraoesophageal mass. (G) Median and lateral vertical lobules where Oct-ER mRNA was confined to many layers of amacrine cells (5 lm). (H) Higher magnification of lateral vertical lobule showing Oct-ER mRNA in the cytoplasm of amacrine cells. (I) Control showing no staining. Scale bars = 35 lm (G, H, I). Arrowheads indicate the neuropil.

Oestradiol signalling pathways in Octopus brain 279

ª 2011 The Authors. Journal of Neuroendocrinology ª 2011 Blackwell Publishing Ltd, Journal of Neuroendocrinology, 24, 275–284

determine the relative expression of Oct-ER mRNA after stimulation

of the CNS with 50 lM of NMDA. Oct-ER mRNA levels increased by approximately three-fold with respect to the control (Fig. 3C). The

stimulation with 50lM NMDA in Ca2+ free ASW, decreased by approximately two-fold Oct-ER mRNA levels with respect to the

stimulation with 50 lM NMDA. In the presence of 100 lM D-APV, an antagonist of NMDA receptors, Oct-ER mRNA levels were similar

to the control (U = 0, P = 0.0156). In summary, Oct-ER mRNA is

up-regulated by NMDA.

Expression of Oct-ER in HeLa cells: intracellular FRET experiments

To investigate the conformational Oct-ER folding in response to

interaction with 17b-oestradiol, we performed FRET analysis. HeLa cells were transfected with pFRET vector carrying the Oct-ER gene

at the N-terminal of DsRED sequence and at the C-terminal of GFP

sequence. Expression of Oct-ER protein was detected by live

microscopy imaging. Both GFP and DsRED signals were found in

the cytoplasm upon excitation at 488 nm and 568 nm, respectively.

In the presence of 5 nM 17b-oestradiol and upon excitation at 488 nm and emission at 650 nm, a FRET signal was observed both

in the nucleus and in the cytoplasm (Fig. 4A, B). After 12 h of incu-

bation with 5 nM 17b-oestradiol, a FRET signal was detected mainly in the nucleus (Fig. 4C, D). We measured FRET intensity (N = 10)

(Fig. 4E–I) according to Gordon’s method (28). In the presence

of 5 nM 17b-oestradiol, FRET intensity increased by approximately 2.5-fold with respect to the control (absence of 17b-oestradiol). In the presence of tamoxifen at a concentration of 50 nM, relative

FRET intensity diminished by approximately 1.8-fold with respect to

the treatment with 17b-oestradiol at concentration of 5 nM (U = 0, P = 0,0273). At a concentration of 500 nM tamoxifen, no FRET sig-

nal was observed (FRET value was < 1) (Fig. 4I). In summary, the

FRET signal moved from the cytosol to the nucleus after 12 h of

incubation with 17b-oestradiol, confirming the classical model of sex steroid binding.

Western blot analysis of Oct-ER transfected HeLa cells lysates

To evaluate the dimerisation dynamic of Oct-ER, we performed

western blotting on lysate of pFRET transfected HeLa cells in the

presence of 17b-oestradiol and tamoxifen. In the pFRET-transfected HeLa cells lysate, in the absence of 17b-oestradiol, we found a band of approximately 56-kDa immunoreactive to hERa antibody (Fig. 4J, lane 1). In the presence of 5 nM 17b-oestradiol, a band of approximately 112-kDa immunoreactive to hERa antibody appeared. The 56-kDa immunoreactive band was still present (Fig. 4J, lane 2).

In the presence of 50 nM tamoxifen, only a 56-kDa immunoreactive

band to hERa antibody was present (Fig. 4J, lane 3). In the absence of 17b-oestradiol, we found two bands of approximately 140 and 90-kDa immunoreactive to anti-Hsp90 antibodies (Fig. 4K, lane 3).

The 140-kDa band also cross-reacted against anti-hERa antibody (Fig. 4K, lane 1). In the presence of 17b-oestradiol, only the 90-kDa immunoreactive band was present when antibodies against hHsp90

were used (Fig. 4K, lane 2). The 90-kDa immunoreactive band was

also found in untransfected HeLa cell lysates (data not shown). In

summary, Oct-ER homodimerised and heterodimerised with Hsp90.

Expression of Oct-ER in HeLa cells: calcium measurement and CREB phosphorylation

To assess the existence of calcium signalling triggered by Oct-ER,

we performed calcium FRET analysis and evaluated CREB phosphor-

ylation by western blotting. CREB is a transcription factor target for

calcium-regulated signalling pathways. HeLa cells were cotransfect-

ed with a calcium FRET sensor and pcDNA3 ⁄ Oct-ER. The calcium sensor expression was detected by live microscopy and Oct-ER

expression was confirmed by western blotting. Relative FRET

intensity (N = 10) was measured in the presence of both 5 nM of

17b-oestradiol and 5 nM 17b-oestradiol BSA conjugated (Fig. 5A). Calcium concentration increased in a time-dependent manner. The

first increase in calcium concentration was observed within the first

4 ** **

* *

Ct r

10 p

10 0

pM 1

nM 10

n M Ct

r Ct

50 µ

M N

M DA

50 µ

M N

M DA

A SW

Ca 2+

fr ee

NM DA

5 0

µM +

D -A

PV 1

00 µ

10 p

10 0

pM 1

nM 10

n M

O ct

-G n R H

/1 8 S

rR N

O ct

-E R /1

8 S

rR N

O ct

-E R /1

8 S

rR N

A(A) (B) (C)

Fig. 3. Relative levels of Octopus vulgaris gonadotrophin-releasing hormone (Oct-GnRH) mRNA (A) and O. vulgaris oestrogen receptor (Oct-ER) mRNA (B) in

the olfactory lobe. Data are presented as the mean � SEM (N = 6). *P < 0.05. *Significant versus control (Ctr). Relative levels of Oct-ER mRNA (C) in the su- praoesophageal mass. Data are presented as the mean � SEM (N = 6). *P < 0.05. *Significant versus control (Ctr) and versus 50 lM NMDA + 100 lM 2-amino-5-phosphonopentanoic acid (D-APV). ASW, artificial sea water. 18S rRNA was used as normaliser gene.

280 E. De Lisa et al.

ª 2011 The Authors. Journal of Neuroendocrinology ª 2011 Blackwell Publishing Ltd, Journal of Neuroendocrinology, 24, 275–284

25 s. In the presence of 5 nM 17b-oestradiol and after incubation for 15 min with 10 lM thapsigargin, a decrease in the calcium FRET ratio was observed. After incubation with tamoxifen (500 nM), FRET

intensity was at baseline level. To investigate the 17b-oestradiol effect on the kinase signalling cascade, western blotting was per-

formed on cotransfected HeLa cells lysate. An increase in CREB

phosphorylation in a time-dependent manner was observed. The

quantification of CREB phosphorylation is showed in Fig. 5(B, C). In

summary, 17b-oestradiol BSA conjugated stimulated an increase in calcium concentration, which in turn induced a rapid phosphoryla-

tion of the CREB, indicating a nongenomic mechanism of action for

17b-oestradiol signaling.

Discussion

The present study clarifies, for the first time, the role of 17b-oestra- diol in the nervous system of O. vulgaris and extends the knowl-

edge of the physiological mechanisms underlying the control of

reproductive function. Given the expression of Oct-ER mRNA in

brain lobes implicated in learning and motor coordination centres,

we discuss the probable involvement of 17b-oestradiol in the regu- lation of aspects of the complex behaviour of this cephalopod.

Using a physiological range of 17b-oestradiol concentrations, as determined by our previous studies on sex steroid hormone fluctua-

tions during the annual reproductive cycle (15), we observed a

–112 kDa

–56 kDa –90 kDa

–140 kDa

3 (I)

(A) (B) (E) (F)

(J) (K)

R el

at iv

e FR

ET in

te n

si ty

2 3 1

–

–– –

–

–– +

+hERa

hERa hHsp90

tamoxifen

17b -E217b -E2

17 b-

E 2

17 b-

E 2 +

50 n

M ta

m ox

ife n

17 b-

E 2 +

50 0

nM ta

m ox

ife n

+ +++ +

HeLa HeLa

2 3

Fig. 4. Heterologous expression assays of Octopus vulgaris oestrogen receptor (Oct-ER) in HeLa cells. (A) Pseudocoloured fluorescence resonance energy trans-

fer (FRET) ratio live images of HeLa cells expressing Oct-ER upon incubation with 5 nM 17b-oestradiol (17b-E2) and (B) nuclei counterstained with 4¢,6-diamidi- no-2-phenylindole (DAPI). (C) Pseudocoloured FRET ratio live images HeLa cells expressing Oct-ER after 12 h of incubation with 5 nM 17b-E2 and nuclei counterstained with DAPI; (D) the same cells observed in brightfield. FRET intensity analysis. Live cells imaging. The excitation (kex) and emission (kem) wave- length was: (E) kex488 nm ⁄ kem588 nm; (F) kex568 nm ⁄ kem670 nm; (G) kex488 nm ⁄ kem670 nm; (H) kex488 nm ⁄ kem670 nm. Nuclei counterstained with DAPI. (I) Relative FRET intensity of HeLa cells expressing Oct-ER in presence of 5 nM 17b-E2, 5 nM 17b-E2 + 50 nM tamoxifen and 5 nM 17b-E2 + 500 nM tamoxifen. Values are the mean � SEM (N = 10). *P < 0.05. *Significant versus 17b-E2 + 50 nM tamoxifen and versus 17b-E2 + 500 nM tamoxifen. Western blotting anal- ysis of pFRET transfected HeLa cells lysate expressing Oct-ER. (J) Lane 1: immunoreactive band to hERa antibody, in the absence of 17b-E2; lane 2: immunore- active bands to hERa antibody, in the presence of 5 nM 17b-E2; lane 3: immunoreactive band to hERa antibody, in the presence of 5 nM 17b-E2 and 50 nM tamoxifen. (K) Lane 1: immunoreactive band to hERa antibody, in the absence of 17b-E2; lane 2: immunoreactive band to HSP90, in the presence of 5 nM 17b-E2; lane 3: immunoreactive bands to HSP90 antibody, in the absence of 17b-E2.

Oestradiol signalling pathways in Octopus brain 281

ª 2011 The Authors. Journal of Neuroendocrinology ª 2011 Blackwell Publishing Ltd, Journal of Neuroendocrinology, 24, 275–284

concentration-dependent increase of Oct-ER mRNA levels in the

olfactory lobes of O. vulgaris CNS, demonstrating that Oct-ER

mRNA expression was up-regulated by 17b-oestradiol. Moreover, the stimulation of the olfactory lobes with 17b-oestradiol increased the Oct-GnRH mRNA confirming the existence of a strong coupling

between 17b-oestradiol and the transcriptional activity of Oct- GnRH neurones. The discovery of the role played by 17b-oestradiol in the modulation of the transcriptional activity of both Oct-GnRH

and Oct-ER genes in the olfactory lobes is in agreement with the

existence of an axis ‘area subpedunculate-olfactory lobe-optic gland’

responsible for the reproductive behaviour of O. vulgaris, in which

the activity of Oct-GnRH neurones is not only under the control of

glutamatergic neurones (10), but also is regulated by 17b-oestra- diol. In situ hybridisation analysis confirmed that the expression of

Oct-ER mRNA was present to the lobes of the CNS directly involved

in the control of reproduction. Interestingly, the Oct-ER mRNA

expression was observed in both cell bodies and nuclei, where clas-

sic binding assay analysis revealed the presence of 17b-oestradiol binding molecules with high affinity and specificity for the ligand.

FRET analysis further clarified the conformational Oct-ER folding in

response to interaction with 17b-oestradiol. The addition of 17b-oestradiol to HeLa cells transfected with Oct-ER generated a FRET signal corresponding to active Oct-ER configuration. Moreover,

the FRET signal moved from the cytosol to the nucleus after 12 h

of incubation with 17b-oestradiol, consistent with the classical model of sex steroid binding receptors stating that the receptor is

present in the cytosol as a monomer that dimerises and translo-

cates into the nucleus upon binding with ligand. In the presence of

the oestrogen receptor antagonist tamoxifen, the FRET signal in the

nucleus was very faint, confirming that the receptor activation was

oestradiol-dependent. Oct-ER dimerisation, after 17b-oestradiol exposure, is a crucial step for ER gene-transcription activity (29). In

the present study, we observed that Oct-ER homodimerised and

also induced the dissociation of Oct-ER-Hsp90 complex. Further-

more, because Oct-ER mRNA was expressed in lobes associated with

learning (vertical superior frontal system in the supraoesophageal

mass) and motor coordination (pedal, palliovisceral and magnocellu-

lar lobes in the suboesophageal mass), it is not unexpected that

17b-oestradiol elicits rapid signalling mediated by calcium via plasma membrane Oct-ER. Indeed, 17b-oestradiol BSA conjugated stimulated an increase in calcium concentration that induced a

rapid phosphorylation of the CREB. These findings are in agreement

with the hypothesis of an ancient nongenomic mechanism for oes-

trogen signalling (16,30). In the present study, we clearly demon-

strate that Oct-ER is activated by 17b-oestradiol and exhibits both the fast (nongenomic) and classical genomic actions. Interestingly,

we have noted that the Oct-ER mRNA was expressed in the same

lobes where we previously observed NMDA receptor immunoreactiv-

ity (11). The presence of both Oct-ER and NMDA receptors in the

‘learning centres’ strongly suggests that 17b-oestradiol may modu- late the glutamatergic neurones involved in the control of learning

and motor coordination (31). This suggestion is further supported

by the results showing the increase of Oct-ER mRNA levels after

stimulation with NMDA. However, significant discrepancies in the

physiological role of oestrogen receptor have been highlighted in

the species belonging to the classes of gastropods, bivalves and

cephalopods. Among gastropods, the oestrogen receptor of Aplysia

californica and Thais clavigera does not specifically bind oestradiol

in vitro, exhibiting a constitutive activity (17,18). By contrast, in

Marisa cornuarietis (32) and Nucella lapillus (21), in vivo effects of

endocrine disruptors on the reproductive cycle, support the exis-

tence of oestrogen signalling pathways mediated by oestrogen

receptor. In all species of bivalve studied, the oestrogen receptor

was activated by oestradiol (19,30). In O. vulgaris, oestrogen recep-

tor has been reported to possess constitutive activity (16). Most

importantly, our evidence of evolutionary conservation of oestradiol

150 200 Time (s)

5 nM 17b -E2-BSA

17b -E2-BSA

HeLa 0 – + + + + +

pCREB

CREB

b -action

0 50 100 150 200 250 300 350 Time (s)

p C

R EB

/C R

0.0

0.5

1.0

1.5

10’30’60’180’360’

5 nM 17b -E2 500 nM tamoxifen + 5 nM 17b -E2-BSA

10 µM thapsigargin + 5 nM 17b -E2-BSA

10050

[C a2

+ ]

n M

150

100

(A) (B) (C)

Fig. 5. Relative calcium fluorescence resonance energy transfer (FRET) intensity of HeLa cells expressing Octopus vulgaris oestrogen receptor (Oct-ER). (A) Cal-

cium FRET ratio of HeLa cells expressing Oct-ER in the presence of 5 nM 17b-oestradiol (17b-E2), 5 nM 17b-E2 bovine serum albumin (BSA) conjugated (17b- E2-BSA), 500 nM tamoxifen + 5 nM 17b-E2-BSA, 10 lM thapsigargin + 5 nM 17b-E2-BSA. Data are presented as the mean � SEM (N = 10). Western blotting analysis of cyclic AMP response element binding protein (CREB) phosphorylation in cotransfected HeLa cells lysate in the presence of 5 nM 17b-E2-BSA. b-actin was used a reference protein. (B) Immunoblot analysis of phosphorilated cAMP response element-binding (p-CREB, top panel). After detection of pCREB protein, the same membrane was stripped and re-probed with CREB (middle panel) and b-actin (lower panel) antibodies. (C) The graph shows the percentage of CREB phosphorylation over time.

282 E. De Lisa et al.

ª 2011 The Authors. Journal of Neuroendocrinology ª 2011 Blackwell Publishing Ltd, Journal of Neuroendocrinology, 24, 275–284

signalling pathways mediated by Oct-ER, as reported in the present

study, calls for a reassessment of the phylogenetic reconstruction

of the oestrogen receptors based on significant differences exerted

by mollusks (16,19,21,32–34). In recent years, numerous studies

have demonstrated the involvement of oestradiol in nociceptive

responses in vertebrates (7,35,36). Oestradiol increases peripheral

nociceptor activity also in human acute pain patients (37). Never-

theless, the oestradiol modulation of pain perception mechanisms

remains largely unclear. The demonstration of the conservative nat-

ure of oestradiol signalling pathways, along with the recent

advances in knowledge of the striking similarities in the behavioural

and cellular responses to sense pain across gastropods to mammals

(6–8), lead us to propose the cephalopod O. vulgaris as new model

system for further understanding the ancestral, conserved molecular

mechanisms and developing ethical scientific procedures that could

help the legislative framework (EU directive 86 ⁄ 609 and revisions) currently covering research using invertebrates (38).

Acknowledgements

We thank Professor John Thornton (University of Oregon) for providing

pcDNA3-Oct-ER vector; Professor Atsushi Miyawaki (RIKEN Brain Science

Institute, Japan) for providing a YC3.60 ⁄ pcDNA3 vector; and Dr Salvatore Carotenuto for its technical assistance in ISH experiments. This research was

supported by the Italian FIRB (2003–2006) project and National Scientific

Research Project (PRIN 2002–2004) grant.

Received 20 May 2011,

revised 15 September 2011,

accepted 7 October 2011

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