Regulacion Neuroendocrina Reproduccion Peces Teleosteos

Regulacin Neuroendocrina de la Reproduccin en Peces Telesteos

Jos A. Muoz Cueto

Departamento de Biologa. Facultad de Ciencias del Mar y Ambientales.

Universidad de Cdiz

Espaa

III CONGRESO DE ACUACULTURA EN AGUAS CONTINENTALES. ESPE 2012

Sangolqu, Ecuador

REGULACIN NEUROENDOCRINA DE LA REPRODUCCIN EN

PECES

De todos los factores cerebrales implicados en el control de la

secrecin de gonadotrofinas, la hormona liberadora de

gonadotrofinas (GnRH) ha sido considerada como uno de los

ms importante


PECES

GnRH en peces telesteos


PECES

! Existencia sospechada al principio de la dcada de los 70 (Breton et al. 1972)

! Aislada en 1983 por Nancy Sherwood y

colaboradores a partir de hipotlamos de salmn

sGnRH pGlu-His-Trp-Ser-Tyr-Gly-Trp-Leu-Pro-Gly-NH2

mGnRH pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 Roger Guillemin (Amoss et al., 1971) y Andrew V. Schally (Matsuo et al., 1971)

GnRH estimula la liberacin de FSH y LH


PECES

0

10

20

0 2 6 24 48hours p.i.

pla

sma

GT

H 1

(n

g/m

l)

0

20

40

0 2 6 24 48hours p.i.

pla

sma

GT

H 2

(n

g/m

l)Salino GnRH (5 mg/kg) GnRH (25 mg/kg)

Breton et al., 1998

La familia GnRH: 14 isoformas en vertebrados


PECES

GnRH 1 2 3 4 5 6 7 8 9 10 Mamfero (m) Pollo-I (c-I) Dorada (sb) Rana (f) Cobaya (gp) Salmn (s) Corgono (wh) Pez gato (cf) Arenque (hr) Tiburn (dg) Pollo-II (c-II) Medaka (md) Lamprea III (l-III) Lamprea I (l-I) Tunicado I (tu-I) Tunicado II (tu-II) Pulpo (oc)

His - - -

Tyr - - - - - - - - - - - -

Trp - - - - - - - - - - - -

Tyr - -

Phe

Ser - - - - - - - - - - - - - - - -

Tyr - - - - - -

His His His His Phe His Leu

Asp Leu Asn

Gly - - - - - - - - - - -

Asp Glu

Tyr Cys

-

Leu - - -

Val Trp

- Met - -

Trp Trp

- Trp Trp

Phe His Trp

Arg Gln Ser Trp

- Leu Asn Asn Ser Leu Tyr Ser Lys Lys

Lys Ala His

Pro - - - - - - - - - - - - - - - -

pGlu - - - - - - - - - - - - - - - - Asn-Tyr

GlyNH2 - - - - - - - - - - - - - - - -

Existen dos sistemas GnRH distintos en todos los grupos de vertebrados


PECES

anterior ?

dfGnRH mGnRH mGnRH cfGnRH sGnRH mGnRH cGnRH-I cGnRH-I mGnRH

posterior ? Powell et al., 1996 cGnRH-II DAntonio et al., 1995 cGnRH-II Leprtre et al., 1993 cGnRH-II Montero et al., 1994 cGnRH-II Zandbergen et al., 1995 cGnRH-II Amano et al., 1991 cGnRH-II Conlon et al., 1993 cGnRH-II Tsai and Licht, 1993 cGnRH-II van Gils et al., 1993 cGnRH-II Lescheid et al., 1997

referencia

Clase agnatos tiburones esturin anguila pez gato salmn anfibios reptiles aves mamferos

Tres isoformas de GnRH en perciformes


PECES


PECES

Cales son las funciones de las distintas formas de GnRH?

? ?

Las 3 formas de GnRH estimulan la secreccion de gonadotrofinas


PECES

0

5

10

15

20

25

0 h 2 h 4 h 8 h

sal.sbGnRHsGnRHcGnRH II

LH ng/ml

y estn presentes en la hipfisis (?)

anti sbGnRH o GnRH-3

anti cGnRH-II o GnRH-2

Anti sGnRH o GnRH-1

La inervacin GnRH en la hipfisis de lubina Foto F. Confente

anti cGnRH-II o GnRH-2

anti sGnRH o GnRH3

anti sbGnRH o GnRH1

La familia GnRH: 14 isoformas en vertebrados


PECES

GnRH 1 2 3 4 5 6 7 8 9 10 Mamfero (m) Pollo-I (c-I) Dorada (sb) Rana (f) Cobaya (gp) Salmn (s) Corgono (wh) Pez gato (cf) Arenque (hr) Tiburn (dg) Pollo-II (c-II) Medaka (md) Lamprea III (l-III) Lamprea I (l-I) Tunicado I (tu-I) Tunicado II (tu-II) Pulpo (oc)

His - - -

Tyr - - - - - - - - - - - -

Trp - - - - - - - - - - - -

Tyr - -

Phe

Ser - - - - - - - - - - - - - - - -

Tyr - - - - - -

His His His His Phe His Leu

Asp Leu Asn

Gly - - - - - - - - - - -

Asp Glu

Tyr Cys

-

Leu - - -

Val Trp

- Met - -

Trp Trp

- Trp Trp

Phe His Trp

Arg Gln Ser Trp

- Leu Asn Asn Ser Leu Tyr Ser Lys Lys

Lys Ala His

Pro - - - - - - - - - - - - - - - -

pGlu - - - - - - - - - - - - - - - - Asn-Tyr

GlyNH2 - - - - - - - - - - - - - - - -

Los sistemas GnRH de la lubina


PECES

Sistemas GnRH1 o sbGnRH


PECES

Gonzlez-Martnez et al. JCN 2001, 2002

Sistemas GnRH3 o sGnRH


PECES

Retina

TNgc

reas sensoriales y sensorimotoras

Servili et al., GCE 2012 Gonzlez-Martnez et al. JCN 2001, 2002


PECES

Pineal

Sistemas GnRH2 o cGnRH-II

Servili et al., Endocrinology 2010

Gonzlez-Martnez et al. JCN 2001, 2002

Estos resultados sugieren un papel de las formas GnRH2 (cGnRH-II) y GnRH3 (sGnRH) en la modulacin de las funciones de la pineal y la retina, respectivamente.


PECES

POA sbGnRH

TNgc sGnRH Syn cGnRH-II

Pineal

Pit Retina

? ?

La situacin es an ms compleja porque no slo existen distintos tipos de GnRH sino distintos tipos de receptores de GnRH con diferencias de expresin y afinidad por las distintas formas de GnRH


PECES

1A

1C

1B

2B

2A

Tipo II

Tipo I

Slo el receptor GnRH-R1A, expresado fuertemente en clulas LH y en menor medida en clulas FSH, posee cierta sensibilidad por la GnRH-1,

la ms abundante en la hipfisis


PECES

sbGnRH-R1A

10 -23 10 -21 10 -19 10 -17 10 -15 10 -13 10 -11 10 -9 10 -7 10 -5 -10 0 10

20 30 40 50 60 70 80 90

100 110 120

cGnRH II (GnRH-2) sGnRH (GnRH-3) sbGnRH (GnRH-1)

Ligand concentrations (M)

%cG

nRH

sbGnRH

cGnRH II sGnRH sbGnRH 1,22x10-12 1,06x10-12 1,28x10-10

EC50 (M)

Servili et al., Endocrinology 2010 From Gonzalez-Martinez et al., 2004 Gonzlez-Martnez et al. BOR 2004

El receptor GnRH-R2b, expresado fuertemente en el rgano pineal, slo posee sensibilidad por la forma GnRH-2

la nica que alcanza la pineal de la lubina


PECES



PECES

Dopamina


PECES

En peces, el principal factor cerebral

responsable de la inhibicin de la secrecin de gonadotrofinas es la

dopamina


PECES

La primera evidencia de la existencia de un factor inhibidor de la secrecin de gonadotrofinas se

obtuvo en hembras de carpn dorado

Peter y Paulencu, 1980


PECES

Las lesiones en ciertas reas cerebrales determinaban un aumento en la secrecin de gonadotrofinas y la ovulacin en hembras de

carpn dorado

Estas lesiones deban destruir algn factor

cerebral inhibidor

Peter y Paulencu, 1980 Kah et al., 1987

Peter y Paulencu, 1980


PECES

La dopamina inhibe la secrecin de gonadotrofinas en el carpn dorado

Chang et al., 1983

Kah et al., 1986


PECES

La dopamina tambin inhibe la secrecin de

gonadotrofinas en otras especies como la trucha

Vacher et al., 2000

FSH

LH

Dopamina Dopamina Dopamina

Dopamina Dopamina Dopamina


PECES

dorada

lubina

Striped bass

Holland et al., 1998b Zohar and Mylonas, 2001 Prat et al., 2001

. pero en muchos otros peces telesteos no se ha podido demostrar una inhibicin dopaminrgica


PECES

Neuropptido Y


PECES

! Un pptido de 36 aminocidos aislado por primera vez a partir del cerebro de cerdos

Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Leu-Ala-Arg-Tyr

Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH2 Tatemoto, 1982

Y

Y


PECES

El NPY est presente en la hipfisis de peces y estimula la secrecin in vivo e in vitro de gonadotrofinas

Rodrguez-Gmez et al., 2001

Cerd-Reverter et al., 1999 In vitro

In vivo


PECES

Adems de los efectos directos, el NPY tiene tambin efectos indirectos en la secrecin de gonadotrofinas,

mediante la estimulacin de las neuronas GnRH

Y2

Y2 NPY

Trudeau, 1997

Las neuronas GnRH reciben inervacin NPY


PECES

El Neuropptido Y: un nexo de unin entre la reproduccin y metabolismo

invierno verano

GSI Ingesta

Muchas especies de peces cesan su ingesta durante la estacin reproductiva


PECES

La expresion del Neuropeptido Y en el rea preptica del salmn aumenta

durante el ayuno

Silverstein et al., 1998


PECES

y en la lubina, el efecto del Neuropptido Y sobre la liberacin de LH es tambin mayor en individuos en ayuno

Cerda-Reverter et al., 1999

Alimentados Ayuno


PECES

cido -amino butrico (GABA)

GABA


PECES

GABA estimula la liberacin de gonadotrofinas en el carpn dorado

Trudeau et al., 2000; Blazquez et al., 1998

Fibra GABA

LH cell

Photo O. Kah

Fibra GABA

LH cell

Photo O. Kah

Fibra GABA


PECES

GABA tiene efectos tanto directos como indirectos en la secrecin de gonadotrofinas, y sus efectos dependen del ambiente esteroideo

Trudeau, 1997

Testo Blanco

GTHII (ng/ml)

40

E2

c c c

b b

a

Trudeau et al., 1993

Control

GABA (100mg/g)


PECES

.y del momento del ciclo reproductivo

O. Kah Ancona-4 2007

SalineGABA (10mg/g)

Oct Nov Feb Aug

**

GTHng/ml

GABA stimulates GTH releasein goldfish

from Kah et al., 1992

*

Kah et al., 1992


PECES

Fraser et al., 2002 hembras machos

La tasa de sntesis del GABA depende de la temperatura


PECES

El control neuroendocrino de la rproduccin

Y2

Y2 NPY

Trudeau, 1997


PECES

Nuevos factores neuroendocrinos de inters aplicado en acuicultura: kisspeptinas

Tena-Sempere et al., GCE 2012

anatomical association of gpr54 with the GnRH system [47].Thereafter, two distinct kisspeptin receptor transcripts (gpr54-1band gpr54-2b) have been isolated in a variety of fish species andits expression examined by quantitative PCR. The detection ofgpr54-1b and gpr54-2b mRNAs in brain and gonads at differentreproductive stages reinforces their potential reproductive role,although their functional relevance may vary among gender andspecies [1,45,74].

The neuroanatomical distribution of the pair kiss/kisspeptinreceptor has been recently reviewed in a variety of fish species[1,45,74]. Fish inhabit a wide range of environmental niches andthus they have developed a great variety of reproductive strategies.Moreover fish investigators have used different experimental ap-proaches to study the kiss/kisspeptin receptor system(s). Thismakes it difficult to provide a unified overview of all studies con-ducted so far. In this section, we will review only the informationrelated to the neuroanatomical organization gained by in situhybridization (ISH) and immunocytochemical studies. ISH studiesin medaka [21] identified for the first time in fish neuronal kiss1cell bodies in two hypothalamic nuclei, the nucleus posteriorisperiventricularis (NPPv) and the nucleus ventralis tuberis (NVT).The number of neurons of NVT was larger in breeding than innon-breeding fish, and more numerous in males than in females.From these results, it was concluded that kiss1 system is pivotalfor the regulation of reproduction in that species. Afterwards,Kitahashi et al. [22] cloned a novel kisspeptin gene (kiss2) in thezebrafish and medaka, and by using ISH and laser capture micro-dissection coupled with real-time PCR identified kiss1 mRNAexpression in the ventromedial habenula and the periventricularhypothalamic nucleus, and kiss2 expressing neurons in the poster-

ior tuberal nucleus and the periventricular hypothalamic nucleus.Almost simultaneously, kiss1 and kiss2 genes were cloned in a mar-ine teleost, the sea bass [17]. Neuroanatomical distribution of theirexpression has been characterized recently by ISH assays, whichpoint out that the mediobasal hypothalamus, and specially the nu-cleus of the lateral recess, is an important area of expression ofboth kiss1 and kiss2 genes. Furthermore, kiss1 expressing cells werealso found at the level of the habenular region in this fish species[12].

Of note, studies conducted in zebrafish on brain expression ofkiss genes during development, and on the effects of kiss1 andkiss2 decapeptide administration suggested that the habenularkiss1 and the hypothalamic kiss2 are potential regulators of repro-duction, and that kiss2 is the predominant regulator of gonadotro-pin synthesis [22]. In the same vein, Felip et al. observed that thekiss2 decapeptide robustly elicited LH release, both in prepubertaland adult male sea bass, therefore reinforcing the important role ofthe kiss2 system in central regulation of the fish reproductive axis[17].

Two kisspeptin receptors genes have been characterized in thesea bass [4], and interestingly gpr54-1b and gpr54-2b-expressingcells have been shown to be mostly located in the same regionsas their cognate ligands [12]. Additionally, these authors observedthat GnRH1-expressing neurons co-express gpr54-2b indicatingthat they are target for kisspeptins via gpr54, in line with the func-tional observations of potent LH-releasing effects of kiss2 in thisspecies [17].

The information on the targets of the axonal projections of kiss1and kiss2 neurons in fish has been hindered mainly due to the dif-ficulty in obtaining specific antibodies that selectively recognize

kiss1 kiss2 -2a -2b-1a -1b

Theria

striped bassgrouper

human

fathead minnow

atlantic croaker

AgnathaChondrichthyes

Osteichthyes

Actinopterygii

Teleostei

Monotremata

Mammalia

Reptilia

Amniota

Tetrapoda

AmphibiaSarcopterygii

mouseopossumplatypus

Aves

Squamatachicken

lizardfrog

lampreyelephant shark

takifugutetraodonsticklebacksoleatlantic halibutmedakagrey mullet

european eel

goldfishzebrafishbluefin tunatilapia

sea basscobia

Vertebrata

Gnathostomata

gpr54

A

B

C

D

E

F

2

chub mackerel

2

1

****

*

*

**

**

*********

**

**

****

* *

*

**

********************

*** ***** * *

kiss1 kiss2 -2a -2b-1a -1b

Theria

striped bassgrouper

human

fathead minnow

atlantic croaker

AgnathaChondrichthyes

Osteichthyes

Actinopterygii

Teleostei

Monotremata

Mammalia

Reptilia

Amniota

Tetrapoda

AmphibiaSarcopterygii

mouseopossumplatypus

Aves

Squamatachicken

lizardfrog

lampreyelephant shark

takifugutetraodonsticklebacksoleatlantic halibutmedakagrey mullet

european eel

goldfishzebrafishbluefin tunatilapia

sea basscobia

Vertebrata

Gnathostomata

gpr54

A

B

C

D

E

F

2

chub mackerel

2

1

****

*

*

**

**

*********

**

**

****

* *

*

**

********************

*** ***** * *

Fig. 1. A schematic representation of the known kiss and gpr54 genes during vertebrate evolution is presented. The kiss and gpr54 genes probably originated from a commonancestral gene and diversified through gene or genome duplication, and gene modification or deletion. The symbol (d) indicates whole-genome duplications, whereas theasterisk () indicates the presence of gene(s). The number (1) indicates the identification of a second kisspeptin receptor-like gene in platypus whose sequence displays a lowdegree of conservation when it is compared with the available sequences of four different forms of gpr54. The number (2) indicates the characterization of a third kiss gene hasbeen reported in the Western clawed frog [24]. The letters AF indicate different orders in Teleostei: (A) tetraodontiformes, (B) gasterosteiformes, (C) pleuronectiformes, (D)perciformes, (E) cypriniformes and (F) anguilliformes.

M. Tena-Sempere et al. / General and Comparative Endocrinology 175 (2012) 234243 237

Kisspeptina 1 YNWNSFGLRF Humanos y primates YNWNSFGLRY Mamferos y anfibios YNLNSFGLRY Peces

Kisspeptina 2 FNFNPFGLRF Anfibios y algunos peces FNYNPFGLRF Peces

B

M K1 K2 R2 G 18S K1 K2 R2 G 18S K1 K2 R2 G 18S

NRLv (A) NRLv (B) NRLd

100 bp200 bp

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Zmora et al. Figure 1.REGULACIN

NEUROENDOCRINA DE LA REPRODUCCIN EN

PECES


Distribucin reas centrales:

Hipotlamo, telencfalo, tlamo, tegmento mesenceflico, bulbo y tractos olfativos, techo y nervios pticos, mdula, cerebelo, hipfisis (PCR).

Habnula (ISH) Hipotlamo mediobasal (ISH)

Tejidos perifricos: Testculos, ovario, corazn, msculo,

estmago, intestino, bazo, hgado, rion, tejido adiposo, pncreas, branquias, ojo, piel (PCR).

B

M K1 K2 R2 G 18S K1 K2 R2 G 18S K1 K2 R2 G 18S

NRLv (A) NRLv (B) NRLd

100 bp200 bp

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Zmora et al. Figure 1.Zmora et al. Figure 2:

a b c

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Zmora et al., BOR 2012


PECES


Funciones: Diferenciacin sexual del cerebro Pubertad Estimulacin de las neuronas GnRH Control de la secrecin de gonadotrofinas Mediacin de los efectos

retroalimentadores positivos de los estrgenos

Generacin del pico preovulatorio de gonadotrofinas

Regulacin metablica de la fertilidad Control ambiental de la reproduccin

(fotoperiodo)


PECES


Funciones: Diferenciacin sexual del cerebro Pubertad Estimulacin de las neuronas GnRH Control de la secrecin de gonadotrofinas Mediacin de los efectos retroalimentadores

positivos de los estrgenos Generacin del pico preovulatorio de

gonadotrofinas Regulacin metablica de la fertilidad Control ambiental de la reproduccin

(fotoperiodo)

Filby et al., BOR 2008

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Pimephales promelas


PECES





(fotoperiodo)

Zmora et al. Figure 4

Juv PP Maturekiss

1 m

RN

A (c

opie

s/Xg

tota

l RN

A)

0

2e+4

4e+4

6e+4

8e+4

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A B

C D

**



PECES





(fotoperiodo)


A B

C D

Kisspeptin (nmole/Kg BW) Kiss1 Kiss2

*

*

***

* ** **

** * **

*****


A B

C D

Kisspeptin (nmole/Kg BW) Kiss1 Kiss2

*

*

***

* ** **

** * **

*****



PECES





(fotoperiodo)

Oka, JoN 2009 Zhao and Wayne, PLoS ONE 2012


PECES





(fotoperiodo)


DC

A B


DC

A B


DC

A B


DC

A B



PECES

Nuevos factores neuroendocrinos de inters aplicado en acuicultura: kisspeptinas 68 A. Felip et al. / Molecular and Cellular Endocrinology 312 (2009) 6171

Fig. 5. Analysis of the effects of intramuscular administration of sbsKiSS-1 andsbsKiSS-2 peptides on LH (A) and FSH (B) secretion over a 240-min period in pre-pubertal 1-year-old sea bass. (C) LH secretion over a 240-min period in pubertal2-year-old male sea bass. In all cases, vehicle-injected groups (PBS) served as con-trols. Hormonal values are the mean SEM of 510 independent determinations.LH levels are presented as percentage of the basal levels of animals at 0min beforeinjection. FSH levels are presented as percentage of RLU over unstimulated cells.*P


PECES





(fotoperiodo)

unpublished data). Thus, as already suggested in tilapia(25), GnRH neurons are likely to be a target of Kiss2acting through kiss2r receptors. In agreement, zebrafishkiss2r havemore affinity than kiss1r for Kiss2 ligand (16).This is also consistent with the fact that Kiss2 is moreefficient thanKiss1 in regulating the synthesis or release ofgonadotrophins (17, 20). Furthermore, Kiss2, but notKiss1 neurons, appeared regulated by estrogens in the ze-brafish (47). This set of data strongly suggests that kiss2neurons in zebrafish are involved in the neuroendocrine

control of gonadotrophin release through the GnRHsystem.

Kiss2 neurons are sensitive to estradiolWhile estradiol treatment of juvenile zebrafish caused

increased expression of kiss1 messengers, the effects onkiss2 were much more pronounced and also visible by insitu hybridization because estrogen treatment caused theappearance of cells in the ventral and caudal hypothala-mus. Interestingly, the medaka exhibits in the same loca-

FIG. 8. AD, In situ hybridization of kiss2 in control (A and C) and E2-treated fish (B and D) showing the increase in the number of neuronsexpressing kiss2 in the dorsal (Hd) or caudal (Hc) hypothalamus and at the periphery of the anterior tuberal nucleus (ATN).: Scale bar, 20 !m (Aand B); 40 !m (C and D). E, Estradiol (10!8 M) treatment causes a significant increase in both kiss1 and kiss2 mRNA expression in the brain of 2-month-old juvenile zebrafish. The effect is more pronounced on kiss2 mRNAs. E2 also causes an increase in kiss2r receptors messengers. F,Schematic representation of the organization of the Kiss1 and Kiss2 systems in the brain of zebrafish. Kiss1 neurons are restricted to the habenula(Hb). They express also kiss1r and project only into the IPN and the superior raphe (SR). Kiss2 neurons are located in the dorsal (Hd), lateral, andventral hypothalamus (Hv). These neurons send extensive projections toward the subpallium, the entopeduncular nucleus (EN), the preoptic region(POA), the thalamus, the ventral and caudal hypothalamus, and the torus semicircularis (TS). In all these regions, kiss2r are widely expressed. Kiss2fibers make direct contacts with GnRH3 neurons, which project to the pituitary. In contrast, no Kiss fibers were seen in the pituitary (P), whereasKiss2-positive cells are present in the pars intermedia. CC, Crista cerebelli; LX, vagal lobe; MO, medulla oblongata; OB, olfactory bulb; OC, opticchiasma; Pal, palium; TeO, optic tectum.

Endocrinology, April 2011, 152(4):15271540 endo.endojournals.org 1537

unpublished data). Thus, as already suggested in tilapia(25), GnRH neurons are likely to be a target of Kiss2acting through kiss2r receptors. In agreement, zebrafishkiss2r havemore affinity than kiss1r for Kiss2 ligand (16).This is also consistent with the fact that Kiss2 is moreefficient thanKiss1 in regulating the synthesis or release ofgonadotrophins (17, 20). Furthermore, Kiss2, but notKiss1 neurons, appeared regulated by estrogens in the ze-brafish (47). This set of data strongly suggests that kiss2neurons in zebrafish are involved in the neuroendocrine

control of gonadotrophin release through the GnRHsystem.

Kiss2 neurons are sensitive to estradiolWhile estradiol treatment of juvenile zebrafish caused

increased expression of kiss1 messengers, the effects onkiss2 were much more pronounced and also visible by insitu hybridization because estrogen treatment caused theappearance of cells in the ventral and caudal hypothala-mus. Interestingly, the medaka exhibits in the same loca-

FIG. 8. AD, In situ hybridization of kiss2 in control (A and C) and E2-treated fish (B and D) showing the increase in the number of neuronsexpressing kiss2 in the dorsal (Hd) or caudal (Hc) hypothalamus and at the periphery of the anterior tuberal nucleus (ATN).: Scale bar, 20 !m (Aand B); 40 !m (C and D). E, Estradiol (10!8 M) treatment causes a significant increase in both kiss1 and kiss2 mRNA expression in the brain of 2-month-old juvenile zebrafish. The effect is more pronounced on kiss2 mRNAs. E2 also causes an increase in kiss2r receptors messengers. F,Schematic representation of the organization of the Kiss1 and Kiss2 systems in the brain of zebrafish. Kiss1 neurons are restricted to the habenula(Hb). They express also kiss1r and project only into the IPN and the superior raphe (SR). Kiss2 neurons are located in the dorsal (Hd), lateral, andventral hypothalamus (Hv). These neurons send extensive projections toward the subpallium, the entopeduncular nucleus (EN), the preoptic region(POA), the thalamus, the ventral and caudal hypothalamus, and the torus semicircularis (TS). In all these regions, kiss2r are widely expressed. Kiss2fibers make direct contacts with GnRH3 neurons, which project to the pituitary. In contrast, no Kiss fibers were seen in the pituitary (P), whereasKiss2-positive cells are present in the pars intermedia. CC, Crista cerebelli; LX, vagal lobe; MO, medulla oblongata; OB, olfactory bulb; OC, opticchiasma; Pal, palium; TeO, optic tectum.

Endocrinology, April 2011, 152(4):15271540 endo.endojournals.org 1537



PECES





(fotoperiodo)

Carnevali et al., PLoS ONE 2012

Kiss1

Kiss2

Melatonin (dose)


PECES

Control neuroendocrino de la reproduccin en peces

GnRH Dopamina

- +

?

RFamide peptide family. Since the discovery of FMRFamide as acardioexcitatory molecule from the central nervous system of thebivalve mollusc (Price and Greenberg, 1977), numerous RFamidepeptides have been identified in various invertebrate phyla,including cnidarians, nematodes, annelids, molluscs, and arthro-pods (Greenberg and Price, 1992). Invertebrate RFamide peptidesproduced within the nervous system can act as neurotransmittersand neuromodulators. These neuropeptidesmay also act viscerallythrough the endocrine system to control a variety of behavioraland physiological processes. Interestingly, immunohistochemicalstudies using the antiserum against FMRFamide suggested thatvertebrate nervous systems also possess some unknown RFamidepeptides (Raffa, 1988; Rastogi et al., 2001). In fact, over the pastdecade a variety of RFamide peptides have been identified in thebrains of vertebrates. In addition to the group of GnIH and itshomologous peptides, LPXRFamide peptides, to date, four moregroups in the RFamide peptide family have been documented invertebrates based on the structures of vertebrate RFamide peptides(Fig. 3).

7.1. PQRFamide peptide group

Among these groups of the RFamide peptide family, morphine-modulatory peptides, such as neuropeptide FF (NPFF), neuropep-tide AF (NPAF) and neuropeptide SF (NPSF) (Yang et al., 1985; Yangand Martin, 1995; Bonnard et al., 2001; Burlet-Schiltz et al., 2002;Bonnard et al., 2003) were purified from the central nervoussystem of several mammals. These peptides share a common

C-terminal Pro-Gln-Arg-Phe-NH2 motif (i.e., PQRFamide peptidegroup).

NPFF and NPAF were isolated from bovine brain by affinitypurification with the antiserum against FMRFamide. Both peptideswere found to decrease tail-flick latency in rats (Yang et al., 1985).These two RFamide peptides are derived from a single gene inseveral mammalian species (Perry et al., 1997; Vilim et al., 1999).The mature endogenous forms that correspond to NPFF and NPAFwere identified in several mammals (Bonnard et al., 2001; Burlet-Schiltz et al., 2002). Two research groups independently con-tributed to the elucidation of the biological functions of NPFF inpain and analgesia (Panula et al., 1996, 1999; Roumy and Zajac,1998). In addition to its involvement in painmechanisms, NPFF hasbeen implicated in several different physiological processes, suchas memory (Kavaliers and Colwell, 1993), cardiovascular regula-tion (Roth et al., 1987), osmoregulation (Majane and Yang, 1991;Sunter et al., 2001; Kalliomaki and Panula, 2004), and food intake(Murase et al., 1996; Sunter et al., 2001). Furthermore, NPFF hasbeen found to stimulate PRL release (Aarnisalo et al., 1997) and toinhibit aldosterone secretion (Labrouche et al., 1998). Thelocalization of NPFF in the brain has been extensively examinedvia immunohistochemistry and in situ hybridization (Panula et al.,1996; Vilim et al., 1999). NPFF was mainly expressed in the rat inthe supraoptic nucleus (SON), the PVN, the nucleus of the solitarytract (NTS) in themedulla oblongata, the spinal trigeminal nucleus,and the dorsal horn of the spinal cord. Earlier reports showed thatthere were NPFF-ir cells between the dorsomedial nucleus (DMN)and the ventromedial nucleus (VMN) of the hypothalamus that

Fig. 3. Phylogenetic tree of the RFamide peptide family. Extensive studies over the past decade have demonstrated that vertebrate brains produce a variety of RFamidepeptides. To date, five groups of the RFamide peptide family have been documented as follows: (1) PQRFamide peptide group, (2) PRL-releasing peptide (PrRP) group, (3) GnIH(LPXRFamide peptide) group, (4) kisspeptin group, (5) pyroglutamylated RFamide peptide (QRFP)/26RFamide group. GnIH, a newly discovered avian hypothalamicneuropeptide, and its mammalian homologous peptide RFRP inhibit gonadotropin secretion and share a common C-terminal LPXRFamide (X = L or Q) motif. By contrast, thenewly identified neuropeptide kisspeptin acts to stimulate the reproductive axis. Mammalian kisspeptin possesses a C-terminal RFamide or RYamide motif. See Ukena andTsutsui (2005) and Tsutsui and Ukena (2006).

K. Tsutsui / Progress in Neurobiology 88 (2009) 7688 83


PECES

Nuevos factores neuroendocrinos de inters aplicado en acuicultura: GnIH

hormona inhibidora de gonadotrofinas

transfected with an orphan 7-transmembrane G-proteincoupled receptor (GPCR) named OT7T022 [7]. This is theGnIH receptor (herein termed GnIH-R) and is expressed ata high level in the hypothalamus, with moderate levels ofexpression in the thalamus, mid-brain, medulla, eye andtestis. This receptor is one of two previously named neuro-peptide FF (NPFF) receptors. NPFF1 is the receptor forGnIH, whereas NPFF2 is the receptor for NPFF, a RFa-mide peptide family member initially proposed to modu-late the actions of morphine [26,27]. GnIH-R was alsoidentified by Dockray [28] as GPR147 (termed RFR-2).The receptor has now been identified in a range of species,including the rat [29], human [29] and sheep [15]. In thesheep brain, GnIH-R is found in the suprachiasmatic,supraoptic nucleus and periventricular nuclei, and thepars tuberalis. GnIH-R is found in the hypothalamus,the cerebrum, mesencephalon, spinal cord and pituitaryin birds [30], and is expressed by GnRH cells in the avianbrain [10,31]. To our knowledge, expression by GnRH-expressing cells has not yet been shown in mammals,but it will be important to do so in order to ascribe aphysiological role. Regulation of the receptor at this levelcould be an important means of controlling reproductivefunction. Regarding the expression of GnIH-R in otherregions of the brain, histological data identifying cell typesare lacking and functional data are required to ascertainthe full range of actions of GnIH. Such studies might revealnovel functions for GnIH.

In spite of the early indications that GnIH was presentin the brains of mammals and that it negatively regulatesreproductive function [7], evidence that it is a significantregulatory peptide in mammals has only been forthcomingrecently. The description of the presence of GnIH ortholo-gues and GnIH-R in the brains of a range of mammalianspecies and the projections of GnIH neurons to relevantneuroendocrine cells strongly suggest a role for GnIH inthe regulation of reproduction and food intake. Functionaldata now substantiate its significance.

GnIH regulates GnRH-expressing neurons andgonadotropes in mammalsIntracerebroventricular (ICV) injection of GnIH-3 reducedplasma LH secretion in ovariectomized (OVX) hamsters [8]and gonad-intact rats [12,32], which is presumed to be areflection of action on GnRH-expressing cells (Figure 2).Other work showed that ICV administration of GnIH-3was not effective in reducing plasma LH levels in OVXestrogen-treated rats [33] or OVX rats [22]. Using a well-characterized estradiol/progesterone surge-induction pro-tocol in OVX rats, Anderson and colleagues showed thatGnIH reduced the activation of GnRH neurons (assessedby detection of the immediate early gene c-Fos) (Figure 2)and also reduced neuronal activation in the anteroventralperiventricular region, which provides stimulatory input toGnRH neurons [33]. Surprisingly, the LH surge was onlyslightly (non-significantly) reduced inmagnitude. Action ofGnIH at this level is in accord with the GnIH-R expressionin GnRH cells, although this has only been shown in birdsand not yet in mammals [10,31]. The functional data areconsistent with the observed projections of GnIH cells toGnRH cells [17]. Further evidence of function was obtained

by electrophysiology, showing that GnIH treatment inhi-bits the firing rate of GnRH neurons in brain slices fromboth male and female mice [19,34] (Figure 2). Interest-ingly, whereas 43% of cells showed reduced firing, 9%showed an increased firing rate [34]. Thus, in mice at least,GnIH-3 acts on GnRH cells, substantiating the in vivo dataobtained in rats and hamsters. Nevertheless, more exten-sive studies of how GnIH acts on GnRH cells are requiredto understand the full importance of the peptide in thecontrol of reproduction.

Evidence of a central role for GnIH has been also forth-coming from studies showing that ICV administration ofGnIH antisense oligonucleotide to pre-pubertal ratsreduced immunochemical detection of GnIH cells in thebrain and increased plasma LH levels [32]. Given this, itwas surprising that the antisense treatment did not causeadvancement of puberty. Further evidence of a central rolein the regulation of GnRH cells has been obtained inseasonal breeders.

In terms of a peripheral role for GnIH acting at thepituitary, intravenous (IV) administration of GnIH-3 toOVX rats had no effect on basal LH secretion and onlyminimal (but significant) effects on GnRH-stimulatedsecretion [13](Figure 2), which was taken as evidence ofthere being no major effect at the gonadotrope level. How-ever, Murakami et al [22] reported a contrary result,showing a reduction in plasma LH levels in OVX rats2 h after IV administration, with lack of effect of ICVadministration. Accordingly, they concluded that the pep-tide acts as a hypophysiotropic peptide (Figure 2). Convin-cing effects of systemically administered GnIH-3 were seen

Figure 2. Inhibitory effects of GnIH on the hypothalamic pituitary gonadal axis.Effects are shown for both intracerebroventricular (icv) and intravenous (iv)administration in vivo, including effects on GnRH neurons (which express theGnIH-R in birds, at least) and LH secretion from the pituitary gonadotropes (whichexpress GnIH-R). In vitro effects of GnIH on primary pituitary cell cultures are alsosummarized, including a decline in calcium (Ca) induction, LH release and LHbFSHb subunit expression in response to GnRH treatment. POA, preoptic area, OC,optic chiasm, ME, median eminence. Relevant references are indicated.

Review Trends in Endocrinology and Metabolism Vol.21 No.4

257

Tsutsui, PiN 2009 Smith and Clarke, TEM 2010


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hormona inhibidora de gonadotrofinas

Modificado de Tsutsui, PiN 2009


PECES


! Los efectos de la hormona inhibidora de gonadotrofinas (GnIH) descritos en peces, varan en funcin del estado de desarrollo gonadal y de la especie

Thus, gfLPXRFamide peptides may be a novel factorregulating pituitary hormone secretion in fish. It has beenreported that fGRP-RP-2 stimulates not only GH, butalso PRL release both in vitro and in vivo in the bullfrog(Ukena et al. 2003b). Therefore, it is considered thatLPXRFamide peptides regulate the release of pluralpituitary hormones in frogs as well as fish.Considering that GnIH, an avian LPXRFamide pep-

tide, inhibits the release of GTHs (Tsutsui et al. 2000,Osugi et al. 2004), and fGRP and fGRP-RP-2, frogLPXRFamide peptides, stimulate the release of GH andGH/PRL, respectively (Ukena et al. 2003b), it is likelythat gfLPXRFa-1, -2 and -3, fish LPXRFamide peptides,are also involved in the regulation of pituitary hormonesecretion. The present study indicates that gfLPXRFa-1,-2 and -3 stimulate the release of GTHs and GH,suggesting that these fish LPXRFamide peptides functionlike GnRH and GH-releasing hormone (GHRH). Insockeye salmon, salmon GnRH (sGnRH), which is dis-tributed from the olfactory nerve through the hypothala-mus, is involved in the release of GTH (Amano et al.1998). Furthermore, salmon GHRH (sGHRH) wasidentified in sockeye salmon (Parker et al. 1993) and astimulatory effect of sGHRH on GH release from thepituitary of coho salmon Oncorhynchus kisutch was reported(Parker et al. 1997). There is no report indicating physio-logical changes in plasma FSH, LH and GH levels insockeye salmon. To understand the physiological roles ofgfLPXRFamide peptides in fish reproduction andgrowth, we need to examine physiological changes incirculating FSH, LH and GH levels and the effects of

Figure 3 Reversed-phase HPLC profile of gfLPXRFa-immunoreactivityin the pituitary. The retained material was loaded onto areversed-phase HPLC column with a linear gradient of 1531%acetonitrile (CH3CN) containing 01% trifluoroacetic acid for40 min at a flow rate of 05 ml/min. gfLPXRFa-immunoreactivitiesin the fractions obtained from HPLC were evaluated by thecompetitive ELISA and are shown by solid columns. Arrowsindicate the retention times of synthetic gfLPXRFa-1, -2 and -3.

Figure 4 Changes in released levels of (A) FSH, (B) LH, (C) GH,(D) PRL and (E) SL. Each column and vertical line represents themean!S.E. of determinations (n=57). *P


PECES


PECES

opsin

Estudio morfofuncional del rgano pineal de la lubina y el lenguado

Confente et al., Aquaculture 2008 Herrera-Prez et al., JCN 2011


PECES

Estudio de las vas de salida de la informacin del rgano pineal


PECES

Conexiones del rgano pineal y la retina en la lubina y el lenguado: DiI

Servili et al., BBE 2011


PECES

Estudio de las vas de salida de la informacin del rgano pineal


PECES

La melatonina media en la transduccin de la informacin ambiental

Reloj

Calendario


PECES

La melatonina se produce y/o tiene dianas a lo largo de todo el eje reproductivo: pineal

Bayarri et al., P&B 2004 Herrera-Prez et al., JCN 2011

2100 0300 0900 1500

Mel

aton

in (p

g/m

l)

1800 0000 0600 1200

a a a a

c

ab

bc bc

0

20

40

60

80

100

120

Plasma Pineal


PECES

La melatonina se produce y/o tiene dianas a lo largo de todo el eje reproductivo: cerebro

Sauzet et al., GCE 2008 Herrera-Prez et al., JCN 2010

MelR MT1

Expresin de MT1 en reas neuroendocrinas

Hypothalamic MT1 receptors appeared in cells of the ventral (NLTv) and dorsal (NLTd) subdivisions of the lateral tuberal nucleus.

Telencephalic expression of the sea bass MT1 receptor was observed in the ventral (Vv, Vs) and dorsal (Dd, Dm2) telencephalon

The expression of the sea bass MT1 receptor in the preoptic area (POA) was evident in the parvocellular part of the parvocellular preoptic nucleus (NPOpc) and in the suprachiasmatic nucleus (NSC).


PECES

La melatonina se produce y/o tiene dianas a lo largo de todo el eje reproductivo: hipfisis

Confente et al., GCE 2010

MelR MT1 MelR MT2 MelR Mel1c


PECES

La melatonina se produce y/o tiene

dianas a lo largo de todo el eje reproductivo

incluida la gnada


PECES

Efectos de la melatonina sobre el eje reproductivo El papel de la melatonina en la reproduccin ha sido

investigado en diferentes especies de peces

las dosis de melatonina usadas, las condiciones de fotoperiodo, la estacin y la edad de los animales...

Pinealectoma

Resultados contradictorios y muy variables en funcin de

Niveles plasmticos e hipofisarios de gonadotrofinas, GnRH, hormonas gonadales, maduracin gonadal

Administracin de melatonina


PECES

Efectos de la melatonina sobre el eje reproductivo El papel de la melatonina en la reproduccin ha sido

investigado en diferentes especies de peces

Administracin de melatonina

Khan and Thomas, 1996 In vivo In vitro


PECES

Control neuroendocrino de la reproduccin

MUCHAS GRACIAS POR SU ATENCIN

Regulacion Neuroendocrina Reproduccion Peces Teleosteos

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