BEHAVIORAL AND NEURAL EFFECTS IN BEHAVIORAL AND NEURAL EFFECTS IN QUAIL EXPOSED TO XENOESTROGENS QUAIL EXPOSED TO XENOESTROGENS DURING EMBRYONIC DEVELOPMENTDURING EMBRYONIC DEVELOPMENT

PanzicaPanzica G.C.*, Mura E., VigliettiG.C.*, Mura E., Viglietti--Panzica C.*Panzica C.*

UNIVERSITY OF TORINO, ITALYUNIVERSITY OF TORINO, ITALYDeptDept. . AnatomyAnatomy, , Pharmacology Pharmacology and and Forensic Forensic MedicineMedicine

*NIT, INN, GRIDES*NIT, INN, GRIDES

ACKNOWLEDGEMENTSACKNOWLEDGEMENTSThese studies have been performed with the support of other reseThese studies have been performed with the support of other research arch groupsgroups

Jacques Balthazart(Liege, Belgium)

Mary Ann Ottinger(College Park, USA)

Supported by Regione Piemonte, Fondazione CRT, and University of Torino

ISS‐Roma 15/10/2008

Hormones and Brain

Gonadal, adrenal, and thyroid hormones affect the brain directly through hormone receptors located in discrete populations of neurons.

Gonadal, adrenal, and thyroid hormones affect the brain directly through hormone receptors located in discrete populations of neurons.

The secretion of hormones is also under the control of neural and pituitary factors. Therefore the brain-endocrine axis has a delicately balanced state that can be altered in various ways, and any agent that interfere with normal hormone secretion can alter brain functions.

The secretion of hormones is also under the control of neural and pituitary factors. Therefore the brain-endocrine axis has a delicately balanced state that can be altered in various ways, and any agent that interfere with normal hormone secretion can alter brain functions.

ISS‐Roma 15/10/2008

Gender differences in the brain

• Gender differences are present in the brain of all vertebrates:– Differences in gross anatomy– Differences in the volume of brain nuclei– Differences in neural connectivity– Differences in cell size or shape– Differences in enzyme expression, in neurotrasmitters and

neuropeptides

• Sexually dimorphic circuits may be related or not to the control of sexually dimorphic functions

• Gender differences are present in the brain of all vertebrates:– Differences in gross anatomy– Differences in the volume of brain nuclei– Differences in neural connectivity– Differences in cell size or shape– Differences in enzyme expression, in neurotrasmitters and

neuropeptides

• Sexually dimorphic circuits may be related or not to the control of sexually dimorphic functions

ISS‐Roma 15/10/2008

Brain Sexual Differentiation

• Brain sexually dimorphic circuits and functions are organized during the embryonic period (in birds) or immediately after birth (in mammals).

• Alterations of hormonal balance during this period generally result in permanent alterations of neural circuits and of related functions, including behavior.

• This phase of the development is defined “critical period”.

• The critical period is specie-specific and hormone-specific.

• Brain sexually dimorphic circuits and functions are organized during the embryonic period (in birds) or immediately after birth (in mammals).

• Alterations of hormonal balance during this period generally result in permanent alterations of neural circuits and of related functions, including behavior.

• This phase of the development is defined “critical period”.

• The critical period is specie-specific and hormone-specific.

ISS‐Roma 15/10/2008

Panzica et al., 2007Panzica et al., 2007

•Endocrine disrupting chemicals (EDCs) can interact with living beings during all the phases of the life.•Endocrine disrupting chemicals (EDCs) can interact with living beings during all the phases of the life.

•During embryonic or early postnatal life they can, in particular, interfere with the mechanisms leading to brain sexual differentiation

•Alteration of brain sexual differentiation may heavily impact development of neural circuits controlling several behaviors, in particular those involved in the control of reproduction and social behaviors

•During embryonic or early postnatal life they can, in particular, interfere with the mechanisms leading to brain sexual differentiation

•Alteration of brain sexual differentiation may heavily impact development of neural circuits controlling several behaviors, in particular those involved in the control of reproduction and social behaviors

ISS‐Roma 15/10/2008

BRAIN EFFECTS OF EDCs

• Several nervous circuitries in vertebrates are affected by precocious exposure to EDCs :– The rat SDN-POA– The hippocampus– The catecholaminergic system (Locus coeruleus, AVPV)– Several peptidergic circuits

• These effects may explain functional alterations that have been observed in animals exposed to EDCs during development.

• Several nervous circuitries in vertebrates are affected by precocious exposure to EDCs :– The rat SDN-POA– The hippocampus– The catecholaminergic system (Locus coeruleus, AVPV)– Several peptidergic circuits

• These effects may explain functional alterations that have been observed in animals exposed to EDCs during development.

ISS‐Roma 15/10/2008

Animal models • Several studies investigated cellular effects of EDCs

in in vitro systems or in lower vertebrates.

• In recent years, several laboratories, including our, started to investigate the effects of precocious exposure to EDCs on both behavior and neural circuits in higher vertebrates.

• In particular, we used two different experimental approaches for birds and mammals, trying to mimic the real mode of exposure to EDCs that may happens during pre- and postnatal development.

• Several studies investigated cellular effects of EDCsin in vitro systems or in lower vertebrates.

• In recent years, several laboratories, including our, started to investigate the effects of precocious exposure to EDCs on both behavior and neural circuits in higher vertebrates.

• In particular, we used two different experimental approaches for birds and mammals, trying to mimic the real mode of exposure to EDCs that may happens during pre- and postnatal development.

ISS‐Roma 15/10/2008

Exogenous Estradiol or Testosterone administered during embryonic criticalperiod (before day 12) irreversibly destroymale copulatory behavior

CRITICAL PERIOD FOR SEXUAL DIFFERENTIATION OF BEHAVIOR IN BIRDS

ISS‐Roma 15/10/2008

THE SEXUALLY DIMORPHIC PARVOCELLULAR VT SYSTEM

Panzica et al. 2001Panzica et al. 2001

BST and POM project to Lateral Septum, in addition POM is projecting to several brainstem regions showing sexually dimorphic AVT innervationBST and POM project to Lateral Septum, in addition POM is projecting to several brainstem regions showing sexually dimorphic AVT innervation

ISS‐Roma 15/10/2008 24th Conference of European Comparative Endocrinologists

• Embryonic administrationof E2 demasculinizes the male VT system

• Embryonic administrationof an inhibitor of aromatase masculinizesthe female VT system

• These effects are in parallelwith irreversible effects on the differentiation of male copulatory behavior

• Embryonic administrationof E2 demasculinizes the male VT system

• Embryonic administrationof an inhibitor of aromatase masculinizesthe female VT system

• These effects are in parallelwith irreversible effects on the differentiation of male copulatory behavior

ISS‐Roma 15/10/2008

VASOTOCIN AND CONTROL OF REPRODUCTION IN VASOTOCIN AND CONTROL OF REPRODUCTION IN JAPANESE QUAILJAPANESE QUAIL

• AVT is found in regions related to the control of sexual activities (POM controlling male copulatory behavior)

• AVT-ir fibers are found in close contact with aromatase-ir and GnRH-irelements

• AVT i.cv. administration inhibits male copulatory behavior

• AVT parvocellular sexually dimorphic system is under the control of estradiol for its differentiation during the embryonic life and expression in the adult

• The sexually dimorphic AVT system in quail may therefore represent a model to test the estrogenic activity of pollutants that affect male quail sexual behavior

• AVT is found in regions related to the control of sexual activities (POM controlling male copulatory behavior)

• AVT-ir fibers are found in close contact with aromatase-ir and GnRH-irelements

• AVT i.cv. administration inhibits male copulatory behavior

• AVT parvocellular sexually dimorphic system is under the control of estradiol for its differentiation during the embryonic life and expression in the adult

• The sexually dimorphic AVT system in quail may therefore represent a model to test the estrogenic activity of pollutants that affect male quail sexual behavior

Balthazart et al., 1997Balthazart et al., 1997

Panzica et al., 1999Panzica et al., 1999

Castagna et al., 1998Castagna et al., 1998

Panzica et al., 1998Panzica et al., 1998

Panzica et al., 2002Panzica et al., 2002

ISS‐Roma 15/10/2008

XENOESTROGENS AND AVTXENOESTROGENS AND AVT

• The paradigm is that xenoestrogens may alter the animal physiology through their binding to estrogen receptor.

• In quail model this means that a xenoestrogenadministered during embryonic life, should reduce or abolish male copulatory behavior and interfere with the differentiation of the AVT system

• The paradigm is that xenoestrogens may alter the animal physiology through their binding to estrogen receptor.

• In quail model this means that a xenoestrogenadministered during embryonic life, should reduce or abolish male copulatory behavior and interfere with the differentiation of the AVT system

ISS‐Roma 15/10/2008

EXPERIMENTAL PROCEDUREEXPERIMENTAL PROCEDURE

3 17Incubation day

Injection

7           8Postnatal weeks

Hatching sacrifice

Behavioral tests

ISS‐Roma 15/10/2008

We used three compounds

• Diethylstilbestrol (DES), a synthetic compound, formerly used as an abortion preventing agent, which is now present as a pollutant in the environment.

• Genistein, a phytoestrogenfound in the soy. It binds mainly to ERbeta

• Ethylene,1,1-dichloro-2,2-bis-p-chlorophenyl (DDE) a metabolite of DDT with antiandrogenic action

We used three compounds

• Diethylstilbestrol (DES), a synthetic compound, formerly used as an abortion preventing agent, which is now present as a pollutant in the environment.

• Genistein, a phytoestrogenfound in the soy. It binds mainly to ERbeta

• Ethylene,1,1-dichloro-2,2-bis-p-chlorophenyl (DDE) a metabolite of DDT with antiandrogenic action

DES

DES

GENISTEIN

DDE

ISS‐Roma 15/10/2008

Effects of EDCs on Male copulatory behavior

Embryonic treatments with DES,  Genistein or  DDE  significantly decrease male copulatory behaviorEmbryonic treatments with DES,  Genistein or  DDE  significantly decrease male copulatory behavior

Viglietti‐Panzica et al, 2005 Viglietti‐Panzica et al, 2005 

Viglietti‐Panzica et al, 2007 Viglietti‐Panzica et al, 2007 

Mura et al, submittedMura et al, submitted

ISS‐Roma 15/10/2008

Effects of EDCs on VT systemViglietti‐Panzica et al, 2005 Viglietti‐Panzica et al, 2005 

Viglietti‐Panzica et al, 2007 Viglietti‐Panzica et al, 2007 

Mura et al, submittedMura et al, submitted

ISS‐Roma 15/10/2008

POMPOM

BSTBSTVTVT

MALE COPULATORY BEHAVIORMALE COPULATORY BEHAVIOR

POMPOM

BSTBSTVTVT

MALE COPULATORY BEHAVIORMALE COPULATORY BEHAVIOR

E2

DES

DDE

GEN

X

X

*****

*** **

***

****

* ERαERβ

ISS‐Roma 15/10/2008

Summary• The sexually dimorphic AVT-parvocellular system is very

sensitive, during embryonic development, to xenoestrogens

• Anatomical alterations of this system are parallel to the alteration of the male copulatory behavior

• Both indicators are, therefore, very useful to detect estrogenic potentialities in different types of putative endocrine disruptors

• XE may deeply interfere with the organization and differentiation of that parts of the central nervous system that are organizing at the time of exposure and of the related behaviors

• The sexually dimorphic AVT-parvocellular system is very sensitive, during embryonic development, to xenoestrogens

• Anatomical alterations of this system are parallel to the alteration of the male copulatory behavior

• Both indicators are, therefore, very useful to detect estrogenic potentialities in different types of putative endocrine disruptors

• XE may deeply interfere with the organization and differentiation of that parts of the central nervous system that are organizing at the time of exposure and of the related behaviors

ISS‐Roma 15/10/2008

CONCLUSIONS

•In the majority of studies involving high vertebrates and xenoestrogens (XE) the doses were very high and the administration route largely artificial (i.e. intraperitoneal injection)

•Our studies demonstrated that XE may interfere with the differentiation of the central nervous system, even when they are administered at levels comparable to the environmental doses and through more natural routes, as accumulation within the eggs (quails) or treatments of the mothers (mice).

•Limited exposure and low doses may result in alterations of neural circuits that profoundly impact reproduction or social behavior, as the NO-circuits (involved in the regulation of dopamine system), the kisspeptin system and the vasotocin system of birds

•Data collected in our and other laboratories should stimulate a critical reanalysis of the way to determine the “safe” exposure levels to EDCs for wild species as well as for human, considering among the factors to be analyzed the behavior and related neural circuits.

•In the majority of studies involving high vertebrates and xenoestrogens (XE) the doses were very high and the administration route largely artificial (i.e. intraperitoneal injection)

•Our studies demonstrated that XE may interfere with the differentiation of the central nervous system, even when they are administered at levels comparable to the environmental doses and through more natural routes, as accumulation within the eggs (quails) or treatments of the mothers (mice).

•Limited exposure and low doses may result in alterations of neural circuits that profoundly impact reproduction or social behavior, as the NO-circuits (involved in the regulation of dopamine system), the kisspeptin system and the vasotocin system of birds

•Data collected in our and other laboratories should stimulate a critical reanalysis of the way to determine the “safe” exposure levels to EDCs for wild species as well as for human, considering among the factors to be analyzed the behavior and related neural circuits.

ISS‐Roma 15/10/2008

5th International Meeting onSTEROIDS AND NERVOUS SYSTEM

Torino (Italy) February 13 - 18 2009

Organizers GC Panzica (Torino) RC Melcangi (Milano)

http://www.dafml.unito.it/anatomy/panzica/neurosteroids/index.html

Deadline December 15th

ISS‐Roma 15/10/2008

alpha beta

QuickTime™ e undecompressore

sono necessari per visualizzare quest'immagine.

ERα ERβ

17th day old embryos

Different expression of ERα and β in quail embryo. ERβ is more expressed than ERα

ISS‐Roma 15/10/2008

Estrogen Receptor Alpha or Beta?•Two estrogen receptors (α and β) are present in BST, SL and POM

•β-receptor is largely diffused within these nuclei during the embryonic life, whereas α-receptor appears only later at the end of the development.

•PPT is an agonist of a-receptor. Quail embryos exposed to PPT don’t have alterations of both sexual behavior and VT-immunoreactivity

•EE2 is an agonist for both receptors

•Two estrogen receptors (α and β) are present in BST, SL and POM

•β-receptor is largely diffused within these nuclei during the embryonic life, whereas α-receptor appears only later at the end of the development.

•PPT is an agonist of a-receptor. Quail embryos exposed to PPT don’t have alterations of both sexual behavior and VT-immunoreactivity

•EE2 is an agonist for both receptors

CONTROL PPT EE2

ISS‐Roma 15/10/2008

THE RAT VASOPRESSIN SYSTEM

The magnocellularsystem (PVN and SON) sends axons to the neurohypophysis

The magnocellularsystem (PVN and SON) sends axons to the neurohypophysis

SCN

PVN

SON

The parvocellularsystem of SCN is notsexually dimorphicand sends axons to telencephalic and thalamic regions

The parvocellularsystem of SCN is notsexually dimorphicand sends axons to telencephalic and thalamic regions

BST

MA

The parvocellular system of BST and MA is sexually dimorphic and sends axons to several extrahypothalamic regions including the lateral septum,the hippocampus and the brainstem

The parvocellular system of BST and MA is sexually dimorphic and sends axons to several extrahypothalamic regions including the lateral septum,the hippocampus and the brainstem

ISS‐Roma 15/10/2008

THE RAT VASOPRESSIN SYSTEM

De Vries et al. 1985

BST

MA

MALE FEMALE + T

MALE FEMALE + T

VP-ir neurons and fibers are denser in males than in females even if females are treated with T in adulthood. This sex dimorphismcan be reversed by early postnatal treatments.

VP-ir neurons and fibers are denser in males than in females even if females are treated with T in adulthood. This sex dimorphismcan be reversed by early postnatal treatments.

ISS‐Roma 15/10/2008

VASOTOCIN AND CONTROL OF VASOTOCIN AND CONTROL OF REPRODUCTIONREPRODUCTION

• AVT is found in regions related to the control of sexual activities (POM controlling male copulatory behavior)

• AVT-ir fibers are found in close contact witharomatase-ir and GnRH-ir elements

• AVT i.cv. administration inhibits male copulatorybehavior

ISS‐Roma 15/10/2008

Post-mortem DiI in SL retrogradelylabels BSTm and POM neurons

Balthazart et al., 1994

BSTmBSTm and POM neurons project to SL in quailand POM neurons project to SL in quail

ISS‐Roma 15/10/2008

VASOTOCIN AND CONTROL OF VASOTOCIN AND CONTROL OF REPRODUCTIONREPRODUCTION

• AVT is found in regions related to the control of sexual activities (POM controlling male copulatory behavior)

• AVT-ir fibers are found in close contact witharomatase-ir and GnRH-ir elements

• AVT i.cv. administration inhibits male copulatorybehavior

ISS‐Roma 15/10/2008

AromataseAromatase--producingproducing neuronsneurons

GnRHGnRH--II producingproducing elementselements

Balthazart et al., 1997

Panzica et al., 1999

ISS‐Roma 15/10/2008

VASOTOCIN AND CONTROL OF VASOTOCIN AND CONTROL OF REPRODUCTIONREPRODUCTION

• AVT is found in regions related to the control of sexual activities (POM controlling male copulatory behavior)

• AVT-ir fibers are found in close contact witharomatase-ir and GnRH-ir elements

• AVT i.cv. administration inhibits male copulatorybehavior

ISS‐Roma 15/10/2008

AVT and COPULATORY AVT and COPULATORY BEHAVIORBEHAVIOR

• Central administration of AVT inhibits male copulatory behavior

• Administration of AVT receptor antagonist stimulates male copulatory behavior

• Administration of AVT receptor antagonistprevents the VT-inducedinhibition of sexualbehavior

Castagna et al. 1998

ISS‐Roma 15/10/2008

THE SEXUALLY DIMORPHIC PARVOCELLULAR THE SEXUALLY DIMORPHIC PARVOCELLULAR VASOTOCIN SYSTEM OF THE JAPANESE QUAILVASOTOCIN SYSTEM OF THE JAPANESE QUAIL

EFFECTS OF GONADAL STEROIDSEFFECTS OF GONADAL STEROIDS

• In the adult male the expression of AVT-ir elements and AVT mRNA is under the control of Testosterone (T) or its estrogenicmetabolite Estradiol (E2)

• Sexual differentiation of AVT system is dependent by the absence of E2 during the embryonic development