Grzegorz GOSIEWSKI, Miroslawa SOKOLOWSKA-MIKOLAJCZYK, Jaroslaw CHYB, and Magdalena SOCHA

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1 PL-ISSN (print), ISSN (online) Foli iologic (Krków), vol. 63 (2015), No 1 Institute of Systemtics nd Evolution of nimls, PS, Krków, 2015 doi: /f63_1.25 Preliminry Results Concerning the Influence of Humn Kisspeptin on LH Secretion in Prussin Crp (Crssius gielio) Femles t the Stge of Ovrin Recrudescence nd Spwning Seson* Grzegorz GOSIEWSKI, Miroslw SOKOLOWSK-MIKOLJCZYK, Jroslw CHY, nd Mgdlen SOCH ccepted Decemer 15, 2014 GOSIEWSKI G., SOKOLOWSK-MIKOLJCZYK M., CHY J., SOCH M Preliminry results concerning the influence of humn kisspeptin on LH secretion in Prussin crp (Crssius gielio) femles t the stge of ovrin recrudescence nd spwning seson. Foli iologic (Krków) 63: The im of the experiments ws to evlute the influence of humn kisspeptin on LH secretion of Prussin crp (Crssius gielio) femles during ovrin recrudescence nd spwning seson. For the experiments, humn kisspeptin KISS1 (0.1 mg kg -1 of ody weight.w.), GnRH nlogue (Des Gly 10, D-l 6 ) GnRH- (20 g kg -1.w.) nd dopmine ntgonist (pimozide) (5 mg kg -1.w.) were used lone or in comintions. t 3, 6, 12, 24 hours fter injection(s) lood smples were collected from ll fish. LH levels were mesured in plsm with the use of the ELIS method. KISS1 did not show ny significnt effects on spontneous LH secretion in oth tested sesons. t 12 hours smpling time (oth stges of gond mturity) comintion of tested compounds (GnRH-+KISS1) significntly incresed LH relese in comprison with the control. In the stge of gond recrudescence KISS1 significntly incresed LH secretion evoked y pimozide t 24 hours. comintion of three components: KISS1, GnRH-, nd pimozide significntly decresed LH secretion in comprison to LH secretion evoked y GnRH- nd pimozide during stge of gond recrudescence. These results suggest tht kisspeptin is involved in sesonl control of reproduction in Prussin crp. The possile interction of kisspeptin nd the dopminergic system is lso discussed. Key words: Kisspeptin, LH, fish, Crssius gielio, seson, reproduction. Grzegorz GOSIEWSKI, Miroslw SOKOLOWSK- MIKOLJCZYK, Jroslw CHY, Mgdlen SOCH, University of griculture in Krków, Deprtment of Ichthyoiology nd Fisheries, T. Spiczkow 6, Krków, Polnd. E-mil: grzechu272@gmil.com Puerty nd reproduction, crucil events in the life history of ll nimls, re controlled y environmentl fctors (temperture, photoperiod) s well s endogenous fctors such s hormones, neurohormones, neurotrnsmitters or steroids (ZOHR et l. 2010). In fish, gond mturtion (ended y spermition or ovultion) is regulted y the hypothlmopituitry-gondl (HPG) xis. mjor role in this process is plyed y gondolierin (GnRH) produced in the hypothlmus. GnRH controls the relese of gondotropins (LH nd FSH) from the pituitry glnd (PETER & YU 1997; YU et l. 1997). Gondotropins ct on gonds to stimulte the synthesis of sex steroid hormones nd gondl development. Sex steroids ffect GnRH nd gondotropin relese y positive or negtive feedck effects depending on the stge of gond mturity (CRIM & EVNS 1983; TRUDEU 1997; ROU et l. 2007). In mny fish species, the dopminergic system is responsile for inhiition of GnRH nd LH secretion (KEIN & CLNE 1979; CRDINUD et l. 1998; NIEUWENHUYS et l. 1998). It ws shown tht the strength of dopmine (D) inhiition on LH secretion differs etween species nd could depend on the stge of gond mturity nd seson (SOKOLOWSK et l. 1985; PETER et l. 1991). Strong dopmine inhiition ws oserved in juvenile eel in which D is puerty gtekeeper. In cyprinids wek inhiitory effect of D ws oserved during the initil stge of gmetogenesis nd strong inhiition ws found during the pre-ovultory LH surge nd ovultion (SOKOLOWSK et l. 1985; VIDL et l. 2004; PRK et l. 2007). *Supported y Ntionl Science Centre grnt numer DEC-2011/01/N/NZ4/01159 nd DS-3202/KiIR.

2 26 G. GOSIEWSKI et l. The discovery of kisspeptin in 2001 (KOTNI et l. 2001; MUIR et l. 2001; OHTKI et l. 2001) incresed our knowledge of endocrine control of puerty nd mturtion. Kisspeptin is neurohormone controlling the HPG xis vi the GnRH system in ll vertertes, including fish (MESSGER et l. 2005; VN ERLE et l. 2008; ZOHR et l. 2010; TEN-SEMPERE 2012; MECHLY et l. 2013; OGW & PRHR 2013). Injections of kisspeptin increse LH secretion in goldfish, Crssius urtus (LI et l. 2009), Europen se ss, Dicentrrchus lrx (FELIP et l. 2009), Yellowtil Kingfish, Seriol llndi (NOCILLDO et l. 2012) nd Striped ss, Morone sxtilis (ECK et l. 2012; ZMOR et l. 2012). lso, in vitro studies demonstrte the ility of kisspeptins to induce LH relese from cultured pituitry cells (YNG et l. 2010; CHNG et l. 2012). Moreover, it ws shown tht kisspeptin is le to modulte ctivity of the HPG xis; therefore kisspeptin my ply role s puerty gtekeeper in juvenile eel (PSQUIER et l. 2011). Kisspeptin receptor expression ws loclized close to GnRH neurons in cichlid fish (PRHR et l. 2004) nd ws greter in fish which were t the eginning of puerty thn t pre- nd postpuerty stges. These results demonstrted tht GnRH cells re direct trgets for kisspeptin in fish nd tht kisspeptin induces GnRH relese, s lredy shown in mmmls (DHILLO et l. 2005, 2007; MESSGER et l. 2005; PLNT et l. 2006; CRTY & FRNCESCHINI 2008). lso, dopmine ffects LH relese in juvenile fish (VIDL et l. 2004; PRK et l. 2007) which is very well documented t the time of finl mturtion (ovultion nd spermition) (OMELJNIUK et l. 1989; DULK et l. 1992; TRUDEU et l. 1993; TRUDEU 1997). Since the reltion etween GnRH nd dopmine s well s etween GnRH nd kisspeptin systems hs een demonstrted in fish, the crucil prolem is the existence of possile reltionship etween kisspeptin nd the dopminergic system. ccording to our knowledge there is no dt showing direct reltionship etween these two systems in fish: however in experiments conducted on ewes it ws shown tht interction etween kisspeptin nd dopmine is involved in sesonl chnges in reproductive function, mesured y the vrition in GnRH nd LH secretion (GOODMN et l. 2012). lso, in mice it ws demonstrted tht one of the su-popultions of kisspeptin neurons is responsile for synthesizing D nd projecting it to GnRH neurons (CLRKSON & HERISON 2011). Therefore the im of our experiments ws to exmine the effects of kisspeptin on spontneous nd GnRH-stimulted LH relese in Prussin crp (Crssius gielio loch, 1782) with ctive nd locked dopminergic systems t different stges of gond development. Mteril nd Methods ll experiments were pproved y the First Locl Ethicl Committee on niml Testing in Krków 41/2011. Fish were collected from the ponds of the Fisheries Reserch Sttion of the University of griculture in Krków. Two yer old femles of Crssius gielio of verge ody weight (.w.) 83 ± 17 g nd gondosomtic index (GSI) (4.30 ± 2.06 % stge of gond recrudescence; ± 3.47 % mture fish) were trnsferred to glss tnks with erted wter. fter 2 dys of cclimtistion to experimentl conditions fish were divided into 8 groups of 14 nimls ech. During the winter experiment (Jnury) wter temperture ws 12±1 C nd winter photoperiod (L:D = 8:16) ws mintined, wheres during summer experiments (My) wter temperture ws 20±1 C nd summer photoperiod (L:D = 16:8) ws estlished. In oth sesons, fish were given intrperitonel (ip) injections of metstin (45-54) mide, humn (Sigm-ldrich, US) kisspeptin (KISS1) t dose of 0.1 mg kg -1 of.w.; pimozide (Pim) (Sigm-ldrich, US) dopmine ntgonist t dose of 5 mg kg 1 of.w. nd (Des Gly 10, D-l 6 ethylmide) LH-RH (Sigm-ldrich, US) GnRH nlogue (GnRH-) t dose of 20 g kg -1 of.w. lone or in comintions. Drugs were dissolved in 0.6 % sline solution (KISS1 nd GnRH-) or soluilized in cidified ethylene glycol (pimozide). Smples of lood (200 l) were collected from cudl veins of ech fish with the use of heprinized syringes efore injection (time 0) nd 3, 6, 12 or 24 hours fter injection. For plsm seprtion smples were centrifuged for 3 minutes t g. LH levels were mesured in plsm with the use of the enzyme-linked immunosorent ssy method ELIS (KH et l.1989). The otined LH concentrtions were nlysed using GrphPd Prism sttisticl softwre (version 5 GrphPd Softwre, US). ll dt were expressed s the percent of pre-tretment vlue (mesured t time 0) nd presented s men ± SEM. nonprmetric Mnn-Whitney test (U-test) with onferroni correction ws performed. Differences etween groups were considered significnt t P<0.05. Results The effects of KISS1 on the spontneous or GnRH- stimulted LH secretion in fish t the stge of gond recrudescence or in mture fish (Fig. 1).

3 Humn Kisspeptin in Fish Reproduction Stte of gond mturity: LH (% of pre-tretment) ,c,c,c, * recrudescent mture ** 0 GnRH - GnRH - + GnRH - GnRH - + GnRH - GnRH hours 12 hours 24 hours Fig. 1. Plsm LH levels expressed s the percentge of pretretment (men S.E.M) levels in Prussin crp injected with KISS1 (0.1 mg kg -1.w.) or GnRH- (20 g kg -1.w.) or their comintion t the time of gond recrudescence or in mture fish. Different letters ove the rs represent significnt differences (P<0.05) etween groups within smpling time (lower cse letters concern recrudescent fish nd cpitl letters concern mture fish). sterisks ove squre rckets represent significnt differences etween recrudescent nd mture fish. Numer of fish per group: n = 14. t 3 hours fter injection there were no significnt chnges in LH levels etween ll tested drugs (dt not shown). t ll smpling times (3, 6, 12 nd 24 hours fter injection) there were no significnt chnges in spontneous LH secretion in KISS1 treted fish, neither t the time of gond recrudescence nor in mture fish. GnRH- lone stimulted LH secretion in comprison with the control group only t 6 hours fter injection (in oth stges of gond mturity). t 12 hours smpling time (oth stges of gond mturity) the comintion of tested gents (GnRH-+KISS1) significntly incresed LH relese in comprison with control, despite the fct tht ech of them lone did not significntly chnge LH secretion. t 12 hours smpling time the potentition of GnRH- ction y KISS1 ws oserved in mture fish: secretion of LH ws higher in comined tretment thn in fish receiving only GnRH-. Comprison of LH secretion in mture fish nd those hving gonds t the stge of recrudescence showed tht only comined tretment (GnRH-+KISS1) t 12 nd 24 hours post tretment cused higher stimultion of LH secretion in fish t the time of gond recrudescence (P<0.0158; P<0.0046, respectively). The effects of KISS1 on LH secretion in fish treted with pimozide t the stge of gond recrudescence or in mture fish (Fig. 2). t ll smpling times in mture fish pimozide lone significntly incresed LH relese in comprison to the control group. The comintion of KISS1 with pimozide ws very effective in stimultion of LH secretion in comprison to sline (control group): the differences were highly significnt. This comined tretment fter 24 hours evoked significntly higher LH relese in recrudescent fish thn pimozide lone, demonstrting the potenti-

4 28 G. GOSIEWSKI et l Stte of gond mturity: recrudescent mture LH (% of pre-tretment) ,c,c,c *,c,c 0 Pim Pim + Pim Pim + Pim Pim + 6 hours 12 hours 24 hours Fig. 2. Plsm LH levels expressed s the percentge of pretretment (men S.E.M) levels in recrudescent nd mture Prussin crp injected with KISS1 (0.1 mg kg -1.w.) or pimozide (Pim, 5 mg kg 1.w.) or their comintion. Different letters ove the rs represent significnt differences (P<0.05) etween groups within smpling time (lower cse letters concern recrudescent fish nd cpitl letters concern mture fish). sterisk ove squre rckets represent significnt differences etween recrudescent nd mture fish. Numer of fish per group: n = 14. tion effect of KISS1 on pimozide ction. In mture fish this potentition ws not oserved. The only significnt difference etween fish in different stges of mturity ws found t 24 hours fter tretment with pimozide+kiss1: in mture fish LH secretion ws lower thn in fish with gonds during recrudescence. The effects of KISS1 on LH secretion in fish treted with comintion of GnRH- nd pimozide in fish t the stge of gond recrudescence or in mture fish (Fig. 3). LH secretion in fish treted with GnRH-+pimozide or GnRH-+pimozide+KISS1 ws extremely high (severl thousnd percent in reltion to pre-tretment) nd ws significntly higher thn in control fish t oth investigted stges of gond mturity. t 6 hours smpling time KISS1 significntly reduced the response to the comintion of GnRH-+pimozide in fish with recrudescing gonds, ut not in mture fish.

5 Humn Kisspeptin in Fish Reproduction * Stte of gond mturity: 5000 recrudescent mture c GnRH - + Pim GnRH - + Pim + GnRH - + Pim GnRH - + Pim + GnRH - + Pim GnRH - + Pim + LH (% of pre-tretment) 6 hours 12 hours 24 hours Fig. 3. Plsm LH levels expressed s the percentge of pre-tretment (men S.E.M) levels in recrudescent nd mture Prussin crp injected with KISS1 (0.1 mg kg -1.w.), GnRH- (20 g kg -1.w.) or pimozide (Pim, 5 mg kg 1.w.) or their comintion. Different letters ove the rs represent significnt differences (P<0.05) etween groups within smpling time (lower cse letters concern recrudescent fish nd cpitl letters concern mture fish). sterisk ove squre rckets represent significnt differences etween recrudescent nd mture fish. Numer of fish per group: n = 14. Discussion To dte, most of the evidence for the role of kisspeptins in the onset of puerty nd the ctivtion of the HP xis in fish comes from gene expression nlyses. There re however, studies showing tht in vivo exogenous kisspeptin dministrtion ccelertes gondl development (ECK et l. 2012; ZMOR et l. 2012). The mechnism nd the site of ction (hypothlmus nd/or pituitry glnd) in this process re not well known. There re dt showing tht kisspeptin induces GnRH relese from the hypothlmus nd lso cts directly t the pituitry (NOCILLDO et l. 2007; YNG et l. 2010; ZOHR et l. 2010; CHNG et l. 2012). ccording to our knowledge there is no informtion on the interction of kisspeptin with other systems except for GnRH involved in the control of LH secretion, especilly with the dopminergic pthwy hving crucil role in gondotropin relese inhiition in fish. In our experiments on Prussin crp, intrperitonel injections of humn kisspeptin t dose of 0.1 mg kg -1.w. showed no effect of KISS1 on the spontneous secretion of LH, neither in mture nor recrudescing nimls (Fig. 1) despite the fct tht

6 30 G. GOSIEWSKI et l. erlier pilot experiments demonstrted tht kisspeptin (t 0.01 nd 0.1 mg kg -1.w.) significntly stimulted spontneous secretion of LH in fish with gonds t the stge of recrudescence (dt not shown). full explntion of this lck of response in the present study in comprison to erlier tested doses of this hormone is not possile t the moment nd hopefully the next series of experiments will confirm the ility of this type of kisspeptin to ffect spontneous gondotropin relese in Prussin crp. comprehensive ssessment of the effects of different kisspeptins on LH secretion in fish is not possile ecuse only limited dt from in vivo experiments re ville. Existing results showed tht different kisspeptins ffected some elements of the HP xis: stimultion of LH relese y goldfish kisspeptin 1 ws oserved y LI et l. (2009) or incresed expression of kiss1r coding gene in fthed minnow y mmmlin kisspeptin (FILY et l. 2008). Humn kisspeptin, used in our experiments, could hve lower ility to stimulte LH secretion in Prussin crp thn the species-specific peptide, possily cusing the non-significnt effect on spontneous LH relese. ccording to our knowledge there is no other dt on the use of humn kisspeptin in fish. dmittedly, chnges of the spontneous relese of LH under the influence of humn kisspeptin were not demonstrted in oth tested sesons ut yet n dditive effect of kisspeptin nd GnRH- on LH relese ws found: 12 hours fter injection of oth hormones, LH concentrtions were significntly higher in comprison to the control while GnRH- or kisspeptin given lone hd no sttisticlly significnt effect on LH secretion (Fig. 1). The stimultory effect of KISS1+GnRH- on LH relese ws oserved in mture fish s well s in those with recrudescing gonds nd the only difference ws tht in recrudescent fish this dditive effect lsted longer, up to 24 hours fter injection. It should e noted tht GnRH- given lone stimulted significnt LH relese in oth sesons, ut this stimultion ws oserved only up to 6 hours post-injection (Fig. 1). From the dt otined in mmmls or in fish it is cler tht kisspeptin my ffect LH secretion directly, t the pituitry level (GUTIÉRREZ-PSCUEL et l. 2007; SUZUKI et l. 2008; YNG et l. 2010; CHNG et l. 2012) s well s indirectly, vi other hypothlmic fctors, such s the GnRH system (IRWIG et l. 2004; MESSGER et l. 2005; ZOHR et l. 2010; TEN-SEMPERE 2012; MECHLY et l. 2013; OGW & PRHR 2013). In our experiments (oth sesons) the potentiting effect of kisspeptin on GnRH- ction ws lso found: LH levels in groups receiving oth drugs were significntly higher thn in fish treted only with GnRH-, t 12 hours in mture fish or t 24 hours t the time of gond recrudescence (Fig. 1). sed on our results we cnnot explin t which level (hypothlmic or pituitry) this potentition occurred. Interestingly, this potentition ws found regrdless of the seson: in oth mture nd recrudescent fish the secretion of gondotropin, expressed s the percent of pretretment, ws very similr nd the differences etween sesons were smll, which is quite unusul ecuse the sesonlity of the reproductive xis sensitivity to hormonl tretment is well recognized in fish (CHNG & PETER 1983; SOKOLOWSK et l. 1985; PETER et l. 1986; PETER 1991; KIM et l. 2011). To test the possiility of indirect ction of kisspeptin on LH secretion, i.e., vi different pthwy thn the GnRH system, pimozide (dopmine receptor ntgonist) ws dministered to Prussin crp receiving kisspeptin. Extremely strong stimultion of LH relese (Fig. 2), s consequence of locking of D receptors, ws shown during spwning seson. Pimozide given lone evoked lmost n 8-fold increse of LH secretion, lsting up to 24 hours, when compred to the control group. This response ws even stronger thn to GnRH- lone (Fig. 1). The dt on fish (CHNG & PETER 1983; SOKOLOWSK et l. 1985; PETER et l. 1986; SOKOLOWSK-MIKOLJCZYK et l. 2002; 2002) show tht differentil response of LH secretion to exogenous drugs or hormones (pimozide, GnRH nlogues) exists t different stges of gond mturity: usully weker effects re oserved t the time of gond recrudescence. This ws not the cse in the present investigtion. This oservtion is indirectly supported y findings otined in mmmls. SZWK et l. (2010) demonstrted tht kisspeptin inhiited D neurons in ovriectomized (OVX) rts treted with estrdiol (E2). Moreover, the comintion of pimozide nd kisspeptin cused strong stimultion of LH relese in comprison to the control group, especilly evident in mture fish (Fig. 2). This ws likely due to the strong ction of pimozide lone. In recrudescent fish this stimultion t 24 hours post injection ws two-fold higher thn in fish receiving only pimozide, showing tht kisspeptin ws le to still potentite the LH relesing ility of pimozide despite its strong ction. ecuse such n effect ws found in recrudescent fish only, it my suggest tht kisspeptin is more importnt for ccelertion of the mturtion process in the new seson thn for finl oocyte mturtion. The fct tht kisspeptin cn potentite the stimultory effects of GnRH- or pimozide on LH secretion ut t the sme time ws not effective when given lone, my suggest tht the ction of humn kisspeptin is rther indirect, through the dopminergic system. The elimintion or diminution of do-

7 Humn Kisspeptin in Fish Reproduction 31 pmine inhiition on LH secretion y pimozide is sometimes crucil to demonstrte the potentil of other hormones involved in gondotropin secretion regultion in fish (PETER et l. 1986). In the conditions of our experiment this reduction enled kisspeptin, inctive when given lone, to mrk its stimultory potentil in the control of LH relese (significntly importnt stimultion of KISS1 dministered with pimozide in comprison to pimozide lone). However, in the cse of the comined tretment with GnRH- nd kisspeptin in fish with locked dopminergic system y pimozide, the response to kisspeptin ws reversed: t 6 hours post tretment in recrudescent fish sttisticlly significnt reduction of LH secretion ws oserved when compred with pimozide nd GnRH- injected fish (Fig. 3). sed on our results it is not possile to explin this chnge in the direction of kisspeptin ction on LH relese, especilly since kisspeptin cts in vertertes (lso in fish) s centrl processor/meditor of severl inputs including photoperiod, energy lnce sttus of the orgnism or steroid hormone feedck nd the finl LH secretion is the result of the response to ll these fctors (DUNGN et l. 2006; ZMOR et l. 2012). ccording to our knowledge there is no evidence on dopmine nd kisspeptin interction in GnRH (nd LH) relese in fish, however the reltionships etween kisspeptin nd GnRH nd lso etween dopmine nd GnRH (nd LH) were lredy estlished (see Introduction) which is why dopmine involvement in these processes (dopminekisspeptin-gnrh ) is highly likely. esides the inhiitory ction of kisspeptin on D neurons (SZWK et l. 2010), there is informtion tht in mmmls (nestrous ewes) dopminergic neurons ct vi kisspeptin neurons to inhiit GnRH (nd LH) secretion (GOODMN et l. 2010; 2012). lso, in mice, CLRKSON nd HERISON (2011) descried supopultion of kisspeptin neurons synthesizing dopmine nd projecting to GnRH neurons. It is possile then tht in fish, kisspeptin neurons my integrte mny hormonl nd environmentl signls nd tht dopmine inhiition on GnRH relese is prtly direct (t the level of GnRH producing neurons) nd prtly indirect through diminution of stimultory ction of kisspeptin on LH secreting cells. Our results re still preliminry nd need to e verified y further in vivo nd lso in vitro experiments. References ERLE VN R., KILLE P., LNGE., TYLER C.R Evidence for the existence of functionl Kiss1/Kiss1 receptor pthwy in fish. Peptides 29: ROU S., WELTZEIN F.., LE ELLE N., DUFOURS S Development of rel-time RTPCR ssys for eel gondotropins nd their ppliction to the comprison of in vivo nd in vitro effects of sex steroids. Gen. Comp. Endocrinol. 153: ECK.H., FULLER S.., PETMN E., MCENTIRE M., DRWISH., FREEMN D Chronic exogenous kisspeptin dministrtion ccelertes gondl development in sses of the genus Morone. Comp. iochem. Physiol. Prt Mol. Integr. Physiol. 162: CRTY., FRNCESCHINI I sic spects of the control of GnRH nd LH secretions y kisspeptin: potentil pplictions for etter control of fertility in femles. Reprod. Dom. nim. 43: CRDINUD., GILERT J.M., LIU F., SUGMORI K.S., VINCENT J.D., NIZNIK H.., VERNIER P Evolution nd origin of the diversity of dopmine receptors in vertertes. dv. Phrmcol. 42: CHNG J.P., PETER R.E Effects of pimozide nd des Gly 10, [D-l 6 ] luteinizing hormone-relesing hormone ethylmide on serum gondotropin concentrtions, germinl vesicle migrtion, nd ovultion in femle goldfish, Crssius urtus. Gen. Comp. Endocrinol. 52: CHNG J.P., MR., WLSICHUK M., WONG.O Kisspeptin-1 directly stimultes LH nd GH secretion from goldfish pituitry cells in C(2+)-dependent mnner. Gen. Comp. Endocrinol. 179: CLRKSON J., HERISON.E Dul phenotype kisspeptin-dopmine neurones of the rostrl periventriculr re of the third ventricle project to gondotrophin-relesing hormone neurones. J. Neuroendocrinol. 23: CRIM L.W., EVNS D.M Influence of testosterone nd/or luteinizing hormone relesing hormone nlogue on precocious sexul development in the juvenile rinow trout. iol. Reprod. 29: DHILLO W.S., CHUDHRI O.., PTTERSON M., THOMPSON E.L., MURPHY K.G., DMN M.K., MCGOWN.M., MER V., PTEL S., GHTEI M.., LOOM S.R Kisspeptin-54 stimultes the hypothlmic-pituitry gondl xis in humn mles. J. Clin. Endocrinol. Met. 90: DHILLO W.S., CHUDHRI O.., THOMPSON E.L., MURPHY K.G., PTTERSON M., RMCHNDRN R., NIJHER G.K., MER V., KOKKINOS., DONLDSON M., GHTEI M.., LOOM S.R Kisspeptin-54 stimultes gondotrophin relese most potently during the preovultory phse of the menstrul cycle in women. J. Clin. Endocrinol. Met. 92: DULK J.G., SLOLEY.D., STCEY N.E., PETER R.E reduction in pituitry dopmine turnover is ssocited with sex pheromone-induced gondotropin secretion in mle goldfish. Gen. Comp. Endocrinol. 86: DUNGN H., CLIFTON D., STEINER R Kisspeptin neurons s centrl processors in the regultion of GnRH secretion. Endocrinology 147: FELIP., ZNUY S., PINED R., PINILL L., CRRILLO M., TEN-SEMPERE M., GOMEZ Evidence for two distinct KiSS genes in non-plcentl vertertes tht encode kisspeptins with different gondotropin-relesing ctivities in fish nd mmmls. Mol. Cell. Endocrinol. 312: FILY.L., VN ERLE R., DUITMN J., TYLER C.R The kisspeptin/gondotropin relesing hormone pthwy nd moleculr signling of puerty in fish. iol. Reprod. 78: GOODMN R.L., JNSEN H.T., ILLINGS H.J., COOLEN L.M., LEHMN M.N Neurl systems mediting sesonl reeding in the ewe. J. Neuroendocrinol. 22: GOODMN R.L., MLTY M.J., MILLR R.P., HILEMN S.M., NESTOR C.C., WHITE., TSENG.S., COOLEN L.M., LEHMN M.N Evidence tht dopmine cts vi kisspeptin to hold GnRH pulse frequency in check in nestrous ewes. Endocrinology 153:

8 32 G. GOSIEWSKI et l. GUTIÉRREZ-PSCUEL E., MRTÍNEZ-FUENTES.J., PINILL L., TEN-SEMPERE M., MLGÓN M.M., CSTÑO J.P Direct pituitry effects of kisspeptin: ctivtion of gondotrophs nd somtotrophs nd stimultion of luteinising hormone nd growth hormone secretion. J. Neuroendocrinol. 19: IRWIG M.S., FRLEY G.S., SMITH J.T., COHIDO.V., POP S.M., CUNNINGHM M.J., GOTTSCH M.L., CLIFTON D.K., STEINER R Kisspeptin ctivtion of gondotropin relesing hormone neurons nd regultion of KiSS-1 mrn in the mle rt. Neuroendocrinology 80: KH O., PONTET., NUNEZ R.J., CLS., RETON Development of n enzyme-linked immunosorent ssy for goldfish gondotropin. iol. Reprod. 41: KEIN J.W., CLNE D Multiple receptors for dopmine. Nture 277: KIM D-J., SUZUKI Y., ID K The control mechnism of gondotropin-relesing hormone nd dopmine on gondotropin relese from cultured pituitry cells of rinow trout Oncorhynchus mykiss. Fish qut. Sci. 14: KOTNI M., DETHEUX M., VNDENOGERDE., COMMUNI D., VNDERWINDEN J.M., LE POULE E., REZILLON S., TYLDESLEY R., SUREZ-HUERT N., VNDEPUT F., LNPIN C., SCHIFFMNN S.N., VSSRT G., PRMENTIER M The metstsis suppressor gene KiSS-1 encodes kisspeptins, the nturl lignds of the orphn G protein coupled receptor GPR54. J. iol. Chem. 276: LI S., ZHNG Y., LIU Y., HUNG X., HUNG W., LU D., ZHU P., SHI Y., CHENG C.H., LIU X., LIN H Structurl nd functionl multiplicity of the kisspeptin/gpr54 system in goldfish (Crssius urtus). J. Endocrinol. 201: MECHLY.S., VINS J., PIFERRER F The kisspeptin system genes in teleost fish, their structure nd regultion, with prticulr ttention to the situttion in Pleuronectiformes. Gen. Comp. Endocrinol. 188: MESSGER S., CHTZIDKI E.E., M D., HENDRICK.G., ZHN D., DIXON J., THRESHER R.R., MLINGE I., LOMET D., CRLTON M.., COLLEDGE W.H., CRTY., PRICIO S Kisspeptin directly stimultes gondotropin-relesing hormone relese vi G protein-coupled receptor 54. Proc. Ntl. cd. Sci. US 102: MUIR.I., CHMERLIN L., ELSHOURGY N.., MICHLOVICH D., MOORE D.J., CLMRI., SZEKERES P.G., SRU H.M., CHMERS J.K., MURDOCK P., STEPLEWSKI K., SHON U., MILLER J.E., MIDDLETON S.E., DRKER J.G., LRMINIE C.G.C., WILSON S., ERGSM D.J., EMSON P., FULL R., PHILPOTT K.L., HRRISON D.C XOR12, novel humn G protein-coupled receptor, ctivted y the peptide KiSS-1. J. iol. Chem. 276: NIEUWENHUYS R., DONKELR H.J., NICHOLSON C The Centrl Nervous System of Vertertes (In: Comprtive Verterte Neurontomy: Evolution nd dpttion. utler.., Hodos W. eds, Springer, erlin, New Jersey): NOCILLDO J.N., IRN J., LEE Y.Y., LEVVI-SIVN., MECHLY.S., ZOHR Y., ELIZUR The Kiss2 receptor (Kiss2r) gene in Southern luefin Tun, Thunnus mccoyii, nd in Yellowtil Kingfish, Seriol llndi functionl nlysis nd isoltion of trnscript vrints. Mol. Cell. Endocrinol. 362: NOCILLDO J.N., LEVVI-SIVN., CRRICK F., ELIZUR Temporl expression of G-protein-coupled receptor 54 (GPR54), gondotropin-relesing hormones Mugil cephlus. Gen. Comp. Endocrinol. 150: OGW S., PRHR I.S ntomy of the kisspeptin systems in teleosts. Gen. Comp. Endocrinol. 15: OHTKI T., SHINTNI Y., HOND S., MTSUMONTO H., HORI., KNEHSHI K., TERO Y., KUMNO S., TKTSU Y., MSUD Y., ISHISHI Y., WTNE T., SD M., YMD T., SUENG M., KITD C., USUKI S., KUROKW T., OND H., NISHIMUR O., FUJINO M Metstsis supresor gene KiSS-1 encodes peptide lignd of G-protein coupled receptor. Nture 411: OMELJNIUK R.J., HII H.R., PETER R.E ltertions in pituitry GnRH nd dopmine receptors ssocited with the sesonl vrition nd regultion of gondotropin relese in the goldfish (Crssius urtus). Gen. Comp. Endocrinol. 74: PRHR I.S., OGW S., SKUM Y Lser-cptured single digoxigenin-leled neurons of gondotropinrelesing hormone types revel novel G proteincoupled receptor (Gpr54) during mturtion in cichlid fish. Endocrinology 145: PRK W., LEEC.H., LEEC.S., KIMD.J., KIMJ.H., TMRU C.S., SOHN Y.C Effects of gondotropin-relesing hormone nlog comined with pimozide on plsm sex steroid hormones, ovultion nd egg qulity in freshwter-exposed femle chum slmon (Oncorhynchus ket). quculture 27: PSQUIER J., LFONT.G., LEPRINCE J., VUDRY H., ROUSSEU K., DUFOUR S First evidence for direct inhiitory effect of kisspeptins on LH expression in the eel, nguill nguill. Gen. Comp. Endocrinol. 173: PETER R.E., CHNG J.P., NHORNIK C.S., OMELJNIUK R.J., SOKOLOWSK M., SHIH S.H., ILLRD R Interctions of ctecholmines nd GnRH in regultion of gondotropin secretion in teleost fish. Recent. Prog. Horm. Res. 13: PETER R.E., TRUDEU V.L., SLOLEY.D., rin regultion of reproduction in teleosts. ull. Inst. Zool., cdemi Sinic, Monogrph. 16: PETER R.E., YU K.L Neuroendocrine regultion of ovultion in fishes: sic nd pplied spects. Rev. Fish iol. Fish. 7: PLNT T.M., RMSWMY S., DIPIETRO M.J Repetitive ctivtion of hypothlmic G protein-coupled receptor 54 with intrvenous pulses of kisspeptin in the juvenile monkey (Mcc multt) elicits sustined trin of gondotropin-relesing hormone dischrges. Endocrinology 147: SOKOLOWSK-MIKOLJCZYK M., SOCH M., MIKOLJ- CZYK T., CHY J., EPLER P The effects of nltrexone, n opioid receptor ntgonist, on plsm LH levels in common crp (Cyprinus crpio L.). Comp. iochem. Physiol. C Toxicol. Phrmcol. 131: SOKOLOWSK-MIKOLJCZYK M., SOCH M., MIKOLJ- CZYK T., CHY J., EPLER P Sesonl short-term effects of nltrexone on LH secretion in mle crp (Cyprinus crpio L.). Comp. iochem. Physiol. C Toxicol. Phrmcol. 131: SOKOLOWSK M., PETER R.E., NHORNIK C.S., CHNG J.P Sesonl effects of pimozide nd des Gly10 [D-l6] LH-RH ethylmide on gondotrophin secretion in goldfish. Gen. Comp. Endocrinol. 57: SZWK R.E., RIERIO.., LEITE C.M., HELEN C.V., FRNCI C.R., NDERSON G.M., HOFFMN G.E., NSELMO-FRNCI J Kisspeptin regultes prolctin relese through hypothlmic dopminergic neurons. Endocrinology 151: SUZUKI S., KDOKW H., HSHIZUME T Direct kisspeptin-10 stimultion on luteinizing hormone secretion from ovine nd porcine nterior pituitry cells. nim. Reprod. Sci. 103: TEN-SEMPERE M., FELIP., GÓMEZ., ZNUY S., CRRILLO M Comprtive insights of the kisspeptin/kisspeptin receptor system: lessons from nonmmmlin vertertes. Gen. Comp. Endocrinol. 175: TRUDEU V.L Neuroendocrine regultion of gondotrophin II relese nd gondl growth in the goldfish, Crssius urtus. Rev. Reprod. 2:

9 Humn Kisspeptin in Fish Reproduction 33 TRUDEU V.L., SLOLEY.D., WONG.O., PETER R.E Interctions of gondl steroids with rin dopmine nd gondotropin-relesing hormone in the control of gondotropin-ii secretion in the goldfish. Gen. Comp. Endocrinol. 89: VIDL., PSQULINI C., LE ELLE N., HOLLND M.C., SIHI M., VERNIR P., ZOHR Y., DUFOUR S Dopmine inhiits luteinizing hormone synthesis nd relese in the juvenile Europen eel: neuroendocrine lock for the onset of puerty. iol. Reprod. 71: YNG., JING Q., CHN T., KO W.K., WONG.O Goldfish kisspeptin: moleculr cloning, tissue distriution of trnscript expression, nd stimultory effects on prolctin, growth hormone nd luteinizing hormone secretion nd gene expression vi direct ctions t the pituitry level. Gen.Comp. Endocrinol. 165: YU K.L., LIN X.W., D CUNH STOS J., PETER R.E Neurl regultion of GnRH in teleost fishes. (In: PRHR I.S., SKUM Y. (Eds.), GnRH Neurons: Gene to ehvior. rin Shuppn, Tokyo) ZMOR N., STULEFIRLD J., ZULPERI Z., IRN J., LEVVI-SIVN., MUÑOZ-CUETO J.., ZOHR Y Differentil nd gond stge-dependent roles of Kisspeptin1 nd Kisspeptin2 in reproduction in the modern teleosts. Morone species. iol. Reprod. 86: 177. ZOHR Y., MUÑOZ-CUETO J.., ELIZUR., KH O Neuroendocrinology of reproduction in teleost fish. Gen. Comp. Endocrinol. 165: